-----BEGIN PRIVACY-ENHANCED MESSAGE----- Proc-Type: 2001,MIC-CLEAR Originator-Name: webmaster@www.sec.gov Originator-Key-Asymmetric: MFgwCgYEVQgBAQICAf8DSgAwRwJAW2sNKK9AVtBzYZmr6aGjlWyK3XmZv3dTINen TWSM7vrzLADbmYQaionwg5sDW3P6oaM5D3tdezXMm7z1T+B+twIDAQAB MIC-Info: RSA-MD5,RSA, SlQxUJdMzhxcq4mDDmIHiLefE7wfeky5kFX3ckb0JXDinFHc4KaJZRL2+hK9JpkJ xGM88VrznYhWovBb0Ai1xg== 0000891618-98-001013.txt : 19980309 0000891618-98-001013.hdr.sgml : 19980309 ACCESSION NUMBER: 0000891618-98-001013 CONFORMED SUBMISSION TYPE: 10-K PUBLIC DOCUMENT COUNT: 4 CONFORMED PERIOD OF REPORT: 19971231 FILED AS OF DATE: 19980306 SROS: NASD FILER: COMPANY DATA: COMPANY CONFORMED NAME: CERUS CORP CENTRAL INDEX KEY: 0001020214 STANDARD INDUSTRIAL CLASSIFICATION: BIOLOGICAL PRODUCTS (NO DIAGNOSTIC SUBSTANCES) [2836] IRS NUMBER: 680262011 STATE OF INCORPORATION: DE FISCAL YEAR END: 1228 FILING VALUES: FORM TYPE: 10-K SEC ACT: SEC FILE NUMBER: 333-11341 FILM NUMBER: 98559335 BUSINESS ADDRESS: STREET 1: 2525 STANWELL DRIVE SUITE 300 CITY: CONCORD STATE: CA ZIP: 94520 BUSINESS PHONE: 5106039071 MAIL ADDRESS: STREET 1: 2525 STANWELL DRIVE STREET 2: STE 300 CITY: CONCORD STATE: CA ZIP: 94520 FORMER COMPANY: FORMER CONFORMED NAME: CERUS TECHNOLOGIES INC DATE OF NAME CHANGE: 19960731 10-K 1 FORM 10-K 1 ================================================================================ SECURITIES AND EXCHANGE COMMISSION WASHINGTON, D.C. 20549 FORM 10-K [X] ANNUAL REPORT PURSUANT TO SECTION 13 OR 15(D) OF THE SECURITIES EXCHANGE ACT OF 1934 FOR THE FISCAL YEAR ENDED DECEMBER 31, 1997 or [ ] TRANSITION REPORT PURSUANT TO SECTION 13 OR 15(D) OF THE SECURITIES EXCHANGE ACT OF 1934 COMMISSION FILE NUMBER 0-21937 CERUS CORPORATION (Exact name of registrant as specified in its charter) DELAWARE 68-0262011 (State or other jurisdiction of (IRS Employer Identification incorporation or organization) Number) 2525 STANWELL DR., SUITE 300 94520 CONCORD, CALIFORNIA (Zip Code) (Address of principal executive offices) (510) 603-9071 (Registrant's telephone number, including area code) Securities registered pursuant to Section 12(b) of the Act: NONE Securities registered pursuant to Section 12(g) of the Act: COMMON STOCK, PAR VALUE $.001 PER SHARE (Title of Class) Indicate by check mark whether the registrant (1) has filed all reports required to be filed by Section 13 or 15(d) of the Securities Exchange Act of 1934 during the preceding 12 months (or for such shorter period that the registrant was required to file such reports), and (2) has been subject to such filing requirements for the past 90 days. Yes X No Indicate by check mark if disclosure of delinquent filers pursuant to Item 405 of Regulation S-K is not contained herein, and will not be contained, to the best of registrant's knowledge, in definitive proxy or information statements incorporated by reference in Part III of this Form 10-K or any amendment to this Form 10 - K. [ ] The approximate aggregate market value of the Common Stock held by non-affiliates of the registrant, based upon the closing price of the Common Stock reported on the Nasdaq National Market on February 27, 1998, was $84,215,505. As of February 28, 1998, there were 9,228,497 shares of the registrant's common stock outstanding. DOCUMENTS INCORPORATED BY REFERENCE Certain portions of the registrant's definitive proxy statement, to be filed not later than 120 days after December 31, 1997 in connection with the registrant's 1998 Annual Meeting of Stockholders, is incorporated by reference into Part III of this Form 10-K. ================================================================================ 2 TABLE OF CONTENTS
PAGE PART I Item 1. Business....................................................................... 1 Item 2. Properties..................................................................... 20 Item 3. Legal Proceedings.............................................................. 20 Item 4. Submission of Matters to a Vote of Security Holders............................ 20 PART II Item 5. Market for the Registrant's Common Equity and Related Stockholder Matters...... 21 Item 6. Selected Financial Data........................................................ 22 Item 7. Management's Discussion and Analysis of Financial Condition and Results of Operations..................................................................... 23 Item 8. Financial Statements and Supplemental Data..................................... 26 Item 9. Changes in and Disagreements with Accountants on Accounting and Financial Disclosure..................................................................... 26 PART III Item 10. Directors and Executive Officers of the Registrant............................. 27 Item 11. Executive Compensation......................................................... 27 Item 12. Security Ownership of Certain Beneficial Owners and Management................. 27 Item 13. Certain Relationships and Related Transactions................................. 27 PART IV Item 14. Exhibits, Financial Statement Schedules and Reports on Form 8-K................ 27
i 3 PART I This report contains forward-looking statements within the meaning Section 21E of the Securities Exchange Act of 1934 which are subject to the "safe harbor" created by those sections. These forward-looking statements include, but are not limited to, statements concerning the Company's plans to continue development of its current product candidates; conduct clinical trials with respect to its product candidates; seek regulatory approvals; address certain markets; engage third-party manufacturers to supply its clinical trial and commercial requirements; continue to rely on a third party for a marketing, sales and distribution capability; and evaluate additional product candidates for subsequent clinical and commercial development. These forward-looking statements may be found under the captions "Business" and "Management's Discussion and Analysis of Financial Condition and Results of Operations." Forward-looking statements not specifically described above also may be found in these and other sections of this report. Actual results could differ materially from those discussed in the forward-looking statements as a result of certain factors, including those discussed herein. ITEM 1. BUSINESS OVERVIEW Cerus Corporation ("Cerus" or the "Company") is developing systems designed to improve the safety of blood transfusions by inactivating infectious pathogens in blood components (platelets, plasma and red blood cells) used for transfusion and inhibiting the leukocyte activity that is responsible for certain adverse immune and other transfusion-related reactions. Preclinical studies conducted by the Company have indicated the ability of its systems to inactivate a broad array of viral and bacterial pathogens that may be transmitted in blood component transfusions and to inhibit leukocyte activity. The Company believes that, as a result of the mechanism of action of its proprietary technology, its systems also have the potential to inactivate many new pathogens before they are identified and before tests have been developed to detect their presence in the blood supply. Because the Company's systems are being designed to inactivate rather than merely test for pathogens, these systems also have the potential to reduce the risk of transmission of pathogens that would remain undetected by testing. The Company has completed four Phase 1 and Phase 2 clinical trials of its platelet pathogen inactivation system in healthy subjects, and a Phase 2c pilot patient study currently is underway. The Company has completed a Phase 1 clinical trial of its plasma pathogen inactivation system in healthy subjects, with a Phase 2 study in healthy subjects currently underway. The Company's red blood cell pathogen inactivation system is in preclinical development. The Company's product development and commercialization programs are being conducted pursuant to two agreements (the "Agreements") with Baxter Healthcare Corporation ("Baxter") providing for development, manufacture and marketing of pathogen inactivation systems for platelets, plasma and red blood cells. The Agreements provide for Baxter and the Company to share development expenses, for Baxter's exclusive right and responsibility to market the systems worldwide and for the Company to receive a share of the gross profits from the sale of the systems. BACKGROUND Blood transfusions are required to treat a variety of medical conditions, including anemia, low blood volume, surgical bleeding, trauma, acquired and congenital bleeding disorders and chemotherapy-induced blood deficiencies. Worldwide, over 90 million whole blood donations occur each year. Approximately 39 million of those donations occur in North America, Western Europe and Japan, the major geographical markets for the Company's products. The Company estimates the combined production in these regions of platelets, FFP and red blood cells in 1995 to have been approximately 4 million, 9 million and 31 million, respectively. Whole blood is composed of plasma, the liquid portion of blood containing essential clotting proteins, and three cellular blood components: platelets, red blood cells and white blood cells (leukocytes). Platelets are essential to coagulation, while red blood cells carry oxygen to tissues and carbon dioxide to the lungs. Leukocytes play a 1 4 critical role in immune and other defense systems, but can cause harmful immune transfusion-related reactions in, or transmit disease to, transfusion recipients. Patients requiring transfusions typically are treated with the specific blood component required for their particular deficiency, except in cases of rapid, massive blood loss, in which whole blood may be transfused. Platelets often are used to treat cancer patients following chemotherapy or organ transplantation. Red blood cells frequently are administered to patients with trauma or surgical bleeding, acquired chronic anemia or genetic disorders, such as sickle cell anemia. Plasma used for transfusions is stored in frozen form and is referred to as fresh frozen plasma, or FFP. FFP generally is used to control bleeding. Plasma also can be separated, or "fractionated," into different parts that are used to expand blood volume, fight infections or treat diseases such as hemophilia. Despite recent improvements in donor screening and in the testing and processing of blood, patients receiving transfusions of blood components face a number of significant risks from blood contaminants, as well as adverse immune and other transfusion-related reactions induced by leukocytes. Viruses such as hepatitis B (HBV), hepatitis C (HCV), human immunodeficiency virus (HIV), cytomegalovirus (CMV) and human T-cell lymphotropic virus (HTLV) can present life-threatening risks. In addition, bacteria, the most common agents of transfusion-transmitted disease, can cause complications such as sepsis, which can result in serious illness or death. Although donor screening and diagnostic testing of donated blood have been successful in reducing the incidence of transmission of many pathogens, diagnostic testing has a number of limitations, such as the inability of most tests to detect pathogens prior to the generation of antibodies, ineffectiveness in detecting genetic variants of viruses, and the risk of human error. In addition, emerging or unidentified pathogens for which no tests exist represent a threat to the blood supply. Because of the continuing risk of transmission of serious diseases through transfusion of contaminated blood components from both known and unknown pathogens, together with the limitations of current approaches to providing a safe blood supply, there remains a need for an approach to blood-borne pathogen inactivation that is safe, easy to implement and cost-effective. TECHNOLOGY AND PRODUCTS The Company is developing pathogen inactivation systems employing its proprietary small molecule compounds, which act by preventing the replication of nucleic acid (DNA or RNA). Platelets, FFP and red blood cells do not contain nuclear DNA or RNA. When the inactivation compounds are introduced into the blood components for treatment, they cross bacterial cell walls or viral membranes, then move into the interior of the nucleic acid structure. When subsequently activated by an energy source, such as light, the compounds bind to the nucleic acid of the viral or bacterial pathogen, preventing replication of the nucleic acid. A virus, bacteria or other pathogenic cell must replicate in order to cause infection. The Cerus compounds react in a similar manner with the nucleic acid in leukocytes. This interaction inhibits the leukocyte activity that is responsible for certain adverse immune and other transfusion-related reactions. These compounds are designed to react with nucleic acid only during the pathogen inactivation process and not after the treated blood component is transfused. The systems are also designed to reduce the amount of unbound, or residual, inactivation compound and breakdown products of the inactivation process prior to transfusion. CLINICAL DEVELOPMENT The Company believes that, in deciding whether a pathogen inactivation system is safe and effective, the United States Food and Drug Administration (the "FDA") and foreign regulatory authorities are likely to take into account whether it adversely affects the therapeutic efficacy of blood components as compared to the therapeutic efficacy of blood components not treated with the system, and that the FDA will weigh the system's safety, including potential toxicities of the inactivation compounds, and other risks against the benefits of using the system in a blood supply that has become safer in recent years. No assurance can be given that any of the Company's development programs will be successfully completed, that any further investigational new drug application ("IND") or investigational device exemption application ("IDE") will become effective or that additional clinical trials will be allowed by the FDA or other regulatory authorities, that clinical trials will commence or be completed as anticipated, that required United States or foreign regulatory approvals will be obtained on a timely basis, if at all, or that any products for which approval is obtained will be commercially successful. 2 5 Based on discussions with the FDA, the Company believes that it will be required to provide data from human clinical trials to demonstrate the safety of treated blood components and their therapeutic comparability to untreated components, but that only data from in vitro and animal studies, not data from human clinical disease transmission studies, will be required to demonstrate the system's efficacy in inactivating pathogens. In light of these criteria, the Company's clinical trial programs are different from typical Phase 1, Phase 2 and Phase 3 clinical studies. There can be no assurance, however, that the Company's program for demonstrating safety and efficacy will ultimately be acceptable to the FDA or that the FDA will continue to believe that this clinical plan is appropriate. See "Additional Business Risks - Uncertainty Associated with Clinical and Preclinical Testing" and " -- Government Regulation." PRODUCTS IN DEVELOPMENT The Company is developing treatment systems to inactivate infectious pathogens in platelets, FFP and red blood cells and to inactivate leukocytes to reduce the risk of certain adverse transfusion-related reactions. The following table identifies the Company's product development programs:
THERAPEUTIC CERUS PRODUCT IN INACTIVATION DEVELOPMENT PROGRAM INDICATION DEVELOPMENT COMPOUND STATUS - -------------------- ------------------ -------------------- ---------------- ------------------ Platelets Surgery, cancer Platelet Pathogen S-59 Phases 1a, 1b, chemotherapy, Inactivation System 2a and 2b transplantation, Clinical Trials bleeding completed disorders Phase 2c Clinical Trial commenced in November 1997 Plasma (FFP) Surgery, FFP Pathogen S-59 Phase 1 Clinical transplantation, Inactivation System Trial completed bleeding disorders Phase 2 Clinical Trial commenced in January 1998 Red Blood Cells Surgery, Red Blood Cell S-303 Preclinical transplantation, Pathogen Development; anemia, cancer Inactivation lead compound chemotherapy, System selected trauma
PLATELET PROGRAM Platelets are cellular components of blood that are an essential part of the clotting mechanism. Platelets facilitate blood clotting and wound healing by adhering to damaged blood vessels and to other platelets. Platelet transfusions are used to prevent or control bleeding in platelet-deficient (thrombocytopenic) patients, such as those undergoing cancer chemotherapy or organ transplant. Transfusion units of platelets are obtained either by combining the platelets from four to six whole blood donations (pooled random donor platelets), or in an automated procedure in which a therapeutic dose of platelets is obtained from a single donor (apheresis or single donor platelets). The Company's platelet pathogen inactivation system applies a technology that combines light and the Company's proprietary inactivation compound, S-59, which is a synthetic small molecule from a class of compounds known as psoralens. S-59 was selected from over 100 psoralen derivatives synthesized by the Company, following preclinical studies conducted by the Company to assess safety and ability to inactivate pathogens and leukocytes while preserving platelet function. 3 6 When illuminated, S-59 undergoes a specific and irreversible chemical reaction with nucleic acid. This chemical reaction renders the genetic material of a broad array of pathogens and cells incapable of replication. A virus, bacteria or other pathogenic cell must replicate in order to cause infection. A similar reaction with leukocyte nucleic acid inhibits the leukocyte activity that is responsible for certain adverse immune and other transfusion-related reactions. Most of the S-59 is converted to breakdown products during and after the inactivation reaction. Studies conducted by the Company with preclinical models have indicated that, following transfusion, the unbound S-59 and its unbound breakdown products are rapidly metabolized and excreted. As a further safety measure, the system under development employs a removal process designed to reduce the amount of residual S-59 and unbound breakdown products prior to transfusion (the S-59 reduction device or "SRD"). The Company's platelet pathogen inactivation system, developed with Baxter, has been designed for use in the blood center setting. The system consists of a disposable processing set, containing the S-59 compound and the SRD, and an illumination device to deliver light to initiate the inactivation reaction. The Company believes that, in order to manufacture the SRD for its platelet pathogen inactivation system on a commercial scale, it will need to modify the SRD configuration and the related manufacturing process. The Company currently is pursuing such modification. There can be no assurance that use of a reconfigured SRD will not cause the Company to have to conduct additional clinical studies or otherwise to experience delays in the approval process. Human clinical trials of the platelet pathogen inactivation system are currently being pursued by the Company. Baxter is the sponsor of such trials. To date, the Company has completed studies in healthy subjects that have demonstrated the safety and tolerability and the recovery and lifespan of platelets treated with the platelet pathogen inactivation system. Based on the results of its Phase 2a clinical trial, the Company submitted a protocol to the FDA for a Phase 3 randomized clinical study of treated apheresis platelets in approximately 100 patients requiring platelet transfusion. The Company reviewed this protocol with the FDA and agreed to perform a pilot study in 15 patients prior to initiating the larger Phase 3 trial. This Phase 2c trial, currently underway, is a double blind, controlled cross-over study in which double dose platelet transfusions are given to thrombocytopenic patients and bleeding time correction and post-transfusion platelet count increment are measured. The results of this pilot Phase 2c clinical trial, given its small size, cannot satisfactorily be evaluated statistically. Thus, non-statistically significant variations in bleeding times, or other measures of the trial, may result in the FDA requiring additional patients to be enrolled in the Phase 2c trial. Due to the nature of the trial, its implementation and its assessment of bleeding times, enrollment of a significant number of additional patients would be impracticable to accomplish on a timely basis and could significantly delay completion of this trial and, as a result, commencement of a Phase 3 clinical trial in the United States. This Phase 2c trial currently is not expected to be completed until at least the third quarter of 1998. In addition, there can be no assurance that, upon completion of the pilot study, the FDA will permit initiation of a Phase 3 trial in the United States. The Company has submitted a protocol to the ethical committees in five European countries for a Phase 3 clinical trial of treated apheresis and pooled random donor platelets in approximately 120 patients requiring platelet transfusion. Approval to proceed has been obtained for four of the five study sites, but patient enrollment has not yet commenced. The primary endpoint in these Phase 3 studies will be the increase in post-transfusion platelet count adjusted for platelet dose and patient size (the "corrected count increment"). The Phase 3 European clinical trial is a randomized study designed to assess the therapeutic efficacy of platelets treated with the pathogen inactivation system for apheresis platelets and pooled random donor platelets. The Phase 3 United States clinical trial is being designed to assess the therapeutic efficacy of platelets treated with the pathogen inactivation system for apheresis platelets, not pooled random donor platelets. If the Company decides to seek FDA approval of the platelet pathogen inactivation system for use in treating pooled random donor platelets, the Company may be required by the FDA to conduct additional clinical studies. FFP PROGRAM Plasma is a noncellular component of blood that contains coagulation factors and is essential for maintenance of intravascular volume. Plasma is either separated from collected units of whole blood or collected directly by apheresis. The collected plasma is then packaged and frozen to preserve the coagulation factors. Some of the frozen plasma is made available for fractionation, while some is designated for use as FFP. FFP is a source of all blood clotting factors except platelets and is used to control bleeding in patients who require clotting factors, such as 4 7 patients undergoing transplants or other extensive surgical procedures, patients with chronic liver disease or certain genetic clotting factor deficiencies. The Company's pathogen inactivation system for FFP will use the same S-59 psoralen compound and an SRD and illumination device similar to those being used by the Company in its clinical trials for its platelet pathogen inactivation system. The FFP pathogen inactivation system under development has been designed for use in the blood center setting and is compatible with plasma collected either manually or by apheresis. In vitro studies conducted by the Company to date have indicated the efficacy of the FFP pathogen inactivation system for the inactivation in FFP of a broad array of viral pathogens. Because of the mechanism of action of its FFP pathogen inactivation system, the Company believes that its system may also inactivate protozoans and inhibit leukocyte activity. Although bacterial contamination in FFP is typically not as significant a problem as in platelets, the Company believes that the FFP pathogen inactivation system will inactivate bacteria at the levels typically found in FFP. To date, the Company has conducted no studies on protozoans or to detect inhibition of leukocyte activity in FFP and only limited studies on bacteria in FFP, and there can be no assurance that the Company's FFP pathogen inactivation system would effectively inactivate protozoans, leukocytes or bacteria. The Company has assessed the impact of S-59 photochemical treatment on the function of plasma proteins. Plasma derived from whole blood or apheresis must be frozen within six to eight hours of collection to meet the standard as "fresh frozen plasma." After freezing, plasma may be stored for up to one year, thawed once, and must be transfused within four hours of thawing. The Company has measured the in vitro coagulation function activity of various clotting factors in FFP after photochemical treatment, SRD treatment, freezing and thawing. These in vitro results are not necessarily indicative of coagulation function that may be obtained in vivo, and there can be no assurance that the FDA or foreign regulatory authorities would view such levels of coagulation function as adequate. The Company has completed a clinical study in healthy subjects that has demonstrated the safety and tolerability of FFP treated with the pathogen inactivation system as well as the comparability of post-transfusion coagulation factors between subjects transfused with treated and untreated FFP. A Phase 2 clinical trial commenced in January 1998 and is presently underway at three trial sites. In this study, 30 healthy subjects will donate plasma, with one-half being prepared and frozen as standard FFP and one-half being treated with the Company's pathogen inactivation system and frozen as S-59 FFP. After plasma collection, subjects will be treated with an oral anticoagulant to reduce the levels of clotting factors II, VII, IX, and X. Following anticoagulant therapy, subjects will be transfused with a therapeutic dose of either standard FFP or S-59 FFP in random order. Following transfusion, the levels of clotting factors II, VII, IX, and X will be measured and compared between S-59 and standard FFP transfusions. The Phase 2 trial is designed to serve as a transitional therapeutic efficacy study into a Phase 3 trial to evaluate the therapeutic efficacy of S-59 FFP for several major therapeutic indications, including chronic liver disease with minor surgery, liver transplant surgery, and thrombotic thrombocytopenic purpura with multiple whole blood volume plasma exchange. While the Company has presented this clinical trial plan to the FDA, there can be no assurance that the FDA will concur with this trial plan. RED BLOOD CELL PROGRAM Red blood cells are essential components of blood that carry oxygen to tissues and carbon dioxide to the lungs. Red blood cells may be transfused as a single treatment in surgical and trauma patients with active bleeding or on a repeated basis in patients with acquired anemia or genetic disorders, such as sickle cell anemia, or in connection with chemotherapy. The Company has developed a system for pathogen inactivation in red blood cells using a compound that binds to nucleic acid in a manner similar to that of S-59-based systems, but does not require light. The Company's method for inactivating pathogens in red blood cells is based on a proprietary ALE compound, S-303, a small molecule synthesized by the Company. The selection of S-303 was based on preclinical studies of over 100 ALE compounds synthesized by the Company to assess safety, stability and ability to inactivate pathogens, while preserving red blood cell survival and function. 5 8 In vitro studies by the Company have indicated the efficacy of the ALE process for the inactivation of a broad array of viral and bacterial pathogens with preservation of red blood cell function. Because of the mechanism of action of its red blood cell ALE treatment system, the Company believes that its system may also inactivate protozoans and inhibit leukocyte function. However, the Company has conducted no studies on protozoans or to detect inhibition of leukocyte activity in red blood cells, and there can be no assurance that the Company's red blood cell system would be effective to inactivate protozoans or leukocytes. The Company is currently conducting good laboratory practice (GLP) toxicology and pathogen inactivation validation studies on its red blood cell pathogen inactivation system. The Company has not filed to commence a Phase 1 clinical trial in red blood cells. There can be no assurance as to whether Phase 1 trials, if commenced, will be successful or as to the timing of these studies or the acceptance of the design of these or any later studies by the FDA. FUTURE PRODUCT DEVELOPMENT The Company believes that the technology it has developed for treatment of platelets, FFP and red blood cells may have application in treating other blood products, including plasma fractions, such as Factor VIII and Factor IX clotting factors, and recombinant equivalents of plasma derivatives. The Company also believes that the compounds and processes it has developed for inactivation of pathogens and leukocytes may have other medical applications in which reactions with nucleic acid may serve to prevent or control the activities of cells or microorganisms. ALLIANCE WITH BAXTER The Agreements with Baxter provide for the development, manufacture and marketing of pathogen inactivation systems for platelets, FFP and red blood cells. The Agreements generally provide for Baxter and the Company to share development expenses equally, subject to mutually agreed budgets established from time to time, and for Baxter's exclusive right and responsibility to market the systems worldwide. Under the Agreements, the Company is to receive between 26.8% and 30.7% of revenues from sales of inactivation system disposables for platelets after deducting from such revenues the amount by which Baxter's and the Company's cost of goods for the inactivation system disposables exceeds certain dollar amounts specified in the agreement (the "Premium"). The percentage of Premium to be received by the Company will be determined on the basis of the market price of the disposables; in no event, however, will the amount to be received per inactivation system disposable be less than $8.50, plus 2.2% of the Premium, nor more than $20.00, plus 2.2% of the Premium. For red blood cells and FFP, the Company and Baxter are to share equally in gross profits from sales of inactivation system disposables, after deducting from such gross profits a specified percentage allocation to be retained by the marketing party for marketing and administrative expenses. However, the revenue sharing under this agreement is subject to adjustment upon the occurrence of certain events, including any adjustments in the relative sharing by the parties of development expenses. For red blood cells and FFP, the Company and Baxter are also to receive their respective costs of goods for compounds and components supplied for inactivation system disposables and the depreciation of certain instruments used in the systems. For platelets, red blood cells and FFP, Baxter will retain revenues from the sales of any related instruments, such as the illumination devices used to activate S-59. The Company is obligated to supply the inactivation compound for the systems, with Baxter supplying the remaining components. Through December 31, 1997, Baxter paid the Company up-front license fees and milestone and development payments totaling $14.8 million and invested $17.5 million in the capital stock of the Company. Baxter currently owns 1,457,830 shares of the Company's common stock, or approximately 15.9% of the Company's common stock currently outstanding. Baxter is obligated to make additional equity investments, at 120% of the market price at the time of each investment, subject to the achievement of certain milestones as follows: (i) either $5 million, upon the achievement of both (a) the mutual determination by the Company and Baxter that there is sufficient data to conclude that Phase 3 platelet trials are likely to satisfy specified criteria (the "Interim Platelet Determination") and (b) the filing of a regulatory application with the FDA to begin a Phase 1 study under the red blood cell program or comparable filing in Europe under such program, or separate equity investments of $2 million upon the Interim Platelet Determination, and $3 million upon the approval of a regulatory application by the FDA under the red blood cell program or comparable approval in Europe under such program; and (ii) $5 million, upon the achievement of both (a) the approval by the FDA to commence a Phase 3 study in the United States or comparable approval in 6 9 Europe under the red blood cell program and (b) the approval by the FDA of an application to market products developed under the platelet program or comparable approval in Europe under such program. Baxter has agreed that it will not at any time, nor will it permit any of its affiliates to, own capital stock of the Company having 20.1% or more of the outstanding voting power of the Company. Such restrictions on stock purchases will not apply in the event a third party makes a tender offer for a majority of the outstanding voting securities of the Company or if the Board of Directors of the Company determines to liquidate or sell to a third party substantially all of the assets or a majority of the voting securities of the Company or to approve a merger or consolidation in which the Company's stockholders will not own a majority of the voting securities of the surviving entity. In March 1998, the Company and Baxter entered into an amendment to one of the Agreements providing that, to the extent the approved spending for 1998 for the red blood cell project exceeds $7.3 million, Cerus will fund all expenses for the red blood cell project in 1998 in excess of such amount, up to the amount of the approved budget. To compensate Cerus for such excess expenditures, Baxter will fully fund the first expenditures under the approved budget for the red blood cell project for 1999 in an amount equal to such excess expenditures, after which the parties shall equally share the expenses of the red blood cell project. If there is for any reason not an approved budget for the red blood cell project for 1999, Baxter will fully fund the first expenditures for 1999 under the approved budget for such other Cerus-Baxter program or programs as Cerus shall designate in an amount equal to the Cerus 1998 excess expenditures. If by July 1, 1999, however, there is not an approved budget for such other Cerus-Baxter program or programs that is at least equal to such excess expenditures, Baxter will promptly pay to Cerus one-half of the amount by which the excess expenditures exceed the amount of expenditures to be funded by Baxter. Cerus anticipates that the expenditures for 1998 for the FFP program will exceed the previously approved budget. Cerus and Baxter are discussing each company's level of funding in 1998 for the FFP program. There can be no assurance that the parties will agree on a budget that would permit the FFP program to proceed in accordance with the Company's plans. Baxter has certain discretion in decisions concerning the development and marketing of pathogen inactivation systems. There can be no assurance that Baxter will not elect to pursue alternative technologies or product strategies or that its corporate interests and plans will remain consistent with those of the Company. The Company is aware that Baxter is developing an alternative pathogen inactivation system for FFP, based on a compound known as methylene blue. The Agreements contain restrictions on the Company's ability to develop and market pathogen inactivation systems for blood components outside the Agreements. The Company is entitled, however, to enter into development and licensing agreements with third parties for pathogen inactivation technology for plasma derivatives and recombinant equivalents of plasma derivatives. Such development and licensing agreements are free of any rights of Baxter, except that the Company must offer Baxter the right to license such technology on terms no less favorable than the terms offered to other plasma derivative manufacturers. The development programs under the Agreements may be terminated by Baxter or the Company on 90 days' notice. If either party so terminates as to a program, the other party will gain exclusive development and marketing rights to the program, and the terminating party's sharing in program revenues will be reduced significantly. See "Additional Business Risks - Reliance on Baxter." MANUFACTURING AND SUPPLY The Company has in the past utilized, and intends to continue to utilize, third parties to manufacture and supply the psoralen and ALE inactivation compounds for its systems and to rely on Baxter to supply system components for use in clinical trials and for the potential commercialization of its products in development. The Company has no experience in manufacturing products for commercial purposes and does not have any manufacturing facilities. Consequently, the Company is dependent on contract manufacturers for the production of compounds and on Baxter for other system components for development and commercial purposes. Under the Agreements, the Company is responsible for developing and delivering its proprietary compounds for effecting pathogen inactivation to Baxter for incorporation into the final system configuration. Baxter is responsible for manufacturing or supplying the disposable units, such as blood storage containers and 7 10 related tubing, as well as any device associated with the inactivation process. This arrangement applies both to the current supply for clinical trials and, if applicable regulatory approvals are obtained, the future commercial supply. In order to provide the inactivation compounds for its platelet and FFP pathogen inactivation systems, the Company has contracted with two manufacturing facilities for large-scale synthesis of S-59, although only one currently is performing such synthesis and currently has a stock of compound sufficient to support the anticipated remaining clinical trials planned for the platelet and FFP pathogen inactivation systems. The Company has not contracted with a manufacturer to produce large-scale quantities of S-303. There can be no assurance that the Company will be able to contract for the manufacturing of products and compounds for its pathogen inactivation systems in the future on reasonable terms, if at all. The Company purchases certain key components of its compounds from a limited number of suppliers. While the Company believes that there are alternative sources of supply for such components, establishing additional or replacement suppliers for any of the components in the Company's compounds, if required, may not be accomplished on a timely basis and could involve significant additional costs. See "Additional Business Risks - Reliance on Third Party Manufacturing; Dependence on Key Suppliers." MARKETING, SALES AND DISTRIBUTION If appropriate regulatory approvals are received, Baxter will be responsible for the marketing, sales and distribution of the Company's pathogen inactivation systems for blood components worldwide. The Company does not currently maintain, nor does it intend to develop, its own marketing and sales organization but instead expects to rely on Baxter to market and sell its pathogen inactivation systems. There can be no assurance that the Company will be able to maintain its relationship with Baxter or that such marketing arrangements will result in payments to the Company. Revenues to be received by the Company through any marketing and sales arrangement with Baxter will be dependent on Baxter's efforts, and there can be no assurance that the Company will benefit from Baxter's present or future market presence or that such efforts will otherwise be successful. If the Agreements were terminated or if Baxter's marketing efforts were unsuccessful, the Company's business, financial condition and results of operations would be materially adversely affected. COMPETITION The Company expects to encounter competition in the sale of products it may develop. If regulatory approvals are received, the Company's products may compete with other approaches to blood safety currently in use, as well as with future products developed by biotechnology and pharmaceutical companies, hospital supply companies, national and regional blood centers, and certain governmental organizations and agencies. Many companies and organizations that may be competitors or potential competitors have substantially greater financial and other resources than the Company and may have greater experience in preclinical testing, human clinical trials and other regulatory approval procedures. The Company's ability to compete successfully will depend, in part, on its ability to develop proprietary products, develop and maintain products that reach the market first, are technologically superior to and/or are of lower cost than other products on the market, attract and retain scientific personnel, obtain patent or other proprietary protection for its products and technologies, obtain required regulatory approvals, and manufacture, market and sell any product that it develops. In addition, other technologies or products may be developed that have an entirely different approach or means of accomplishing the intended purposes of the Company's products, or that might render the Company's technology and products uncompetitive or obsolete. Furthermore, there can be no assurance that the Company's competitors will not obtain patent protection or other intellectual property rights that would limit the Company's ability to use the Company's technology or commercialize products that may be developed. Several companies are developing technologies which are, or in the future may be, the basis for products that will directly compete with or reduce the market for the Company's pathogen inactivation systems. A number of companies are specifically focusing on alternative strategies for pathogen inactivation or removal in various blood components. Although no commercial processes are currently available to eliminate or inactivate pathogens in platelets and red blood cells, a number of pathogen inactivation methods are used commercially in Europe for FFP, including treatment with solvent-detergent or methylene blue. Because the solvent-detergent process combines hundreds of units of plasma into large pools, there is increased risk of transmission of pathogens not inactivated by the process, such as parvovirus B19. In December 1996, the Blood Products Advisory Committee, an advisory panel 8 11 to the FDA, unanimously recommended that solvent-detergent be approved for use in treating FFP. Although recommendations of advisory committees are not binding, unanimous recommendations generally are followed by the FDA. If approved by the FDA, the treatment of FFP by solvent-detergent may become a widespread practice prior to any commercialization of the Company's FFP pathogen inactivation system, making such commercialization more difficult. Other groups are developing synthetic blood product substitutes or products to stimulate the growth of platelets. If any of these technologies is successfully developed, it could have a material adverse effect on the Company's business, financial condition and results of operations. The Company believes that the primary competitive factors in the market for pathogen inactivation systems will include the breadth and effectiveness of pathogen inactivation processes, ease of use, the scope and enforceability of patent or other proprietary rights, product price, product supply and marketing and sales capability. In addition, the length of time required for products to be developed and to receive regulatory and, in some cases, reimbursement approval are important competitive factors. The Company believes it competes favorably with respect to these factors, although there can be no assurance that it will be able to continue to do so. The biopharmaceutical field is characterized by rapid and significant technological changes. Accordingly, the Company's success will depend in part on its ability to respond quickly to medical and technological changes through the development and introduction of new products. Product development involves a high degree of risk, and there can be no assurance that the Company's product development efforts will result in any commercially successful products. Under the Agreements, Baxter has reserved the right to market competing products not within the field of psoralen or ALE inactivation. Baxter is conducting several independent product development efforts in blood collection and processing that may improve blood component quality and safety. The Company is aware that Baxter is developing an alternative pathogen inactivation system for FFP, based on methylene blue. The development and commercialization of the Company's pathogen inactivation systems could be materially adversely affected by competition with Baxter or by Baxter's election to pursue alternative strategies or technologies in lieu of those of the Company. See "Additional Business Risks -- Rapid Technological Change; Significant Competition." PATENTS, LICENSES AND PROPRIETARY RIGHTS The Company's success depends in part on its ability to obtain patents, to protect trade secrets, to operate without infringing upon the proprietary rights of others and to prevent others from infringing on the proprietary rights of the Company. The Company's policy is to seek to protect its proprietary position by, among other methods, filing United States and foreign patent applications related to its proprietary technology, inventions and improvements that are important to the development of its business. As of December 31, 1997, the Company owned 33 issued or allowed United States patents and 14 issued or allowed foreign patents. The Company's patents expire at various dates between 2003 and 2015. In addition, the Company has 29 pending United States patent applications and has filed 26 corresponding patent applications under the Patent Cooperation Treaty, three of which are currently pending in Europe, Japan, Australia and Canada. Proprietary rights relating to the Company's planned and potential products will be protected from unauthorized use by third parties only to the extent that they are covered by valid and enforceable patents or are effectively maintained as trade secrets. There can be no assurance that any patents owned by, or licensed to, the Company will afford protection against competitors or that any pending patent applications now or hereafter filed by, or licensed, to the Company will result in patents being issued. In addition, the laws of certain foreign countries do not protect the Company's intellectual property rights to the same extent as do the laws of the United States. The patent positions of biopharmaceutical companies involve complex legal and factual questions and, therefore, their enforceability cannot be predicted with certainty. There can be no assurance that any of the Company's patents or patent applications, if issued, will not be challenged, invalidated or circumvented, or that the rights granted thereunder will provide proprietary protection or competitive advantages to the Company against competitors with similar technology. Furthermore, there can be no assurance that others will not independently develop similar technologies or duplicate any technology developed by the Company. Because of the extensive time required for development, testing and regulatory review of a potential product, it is possible that, before any of the Company's products can be commercialized, any related patent may expire or remain in existence for only a short period following commercialization, thus reducing any advantage of the patent, which could adversely affect the 9 12 Company's ability to protect future product development and, consequently, its operating results and financial position. Because patent applications in the United States are maintained in secrecy until patents issue and since publication of discoveries in the scientific or patent literature often lag behind actual discoveries, the Company cannot be certain that it was the first to make the inventions covered by each of its issued or pending patent applications or that it was the first to file for protection of inventions set forth in such patent applications. There can be no assurance that the Company's planned or potential products will not be covered by third-party patents or other intellectual property rights, in which case continued development and marketing of such products would require a license under such patents or other intellectual property rights. There can be no assurance that such required licenses will be available to the Company on acceptable terms, if at all. If the Company does not obtain such licenses, it could encounter delays in product introductions while it attempts to design around such patents, or could find that the development, manufacture or sale of products requiring such licenses is foreclosed. Litigation may be necessary to defend against or assert such claims of infringement, to enforce patents issued to the Company, to protect trade secrets or know-how owned by the Company or to determine the scope and validity of the proprietary rights of others. In addition, interference proceedings declared by the United States Patent and Trademark Office may be necessary to determine the priority of inventions with respect to patent applications of the Company. Litigation or interference proceedings could result in substantial costs to and diversion of effort by the Company, and could have a material adverse effect on the Company's business, financial condition and results of operations. There can be no assurance that these efforts by the Company would be successful. The Company is a licensee under a worldwide exclusive license agreement with Miles, Inc. and Diamond Scientific Corporation with respect to two United States patents covering inventions pertaining to psoralen-based photochemical decontamination treatment of whole blood or blood components and four United States patents relating to vaccines, as well as related foreign patents. Whether the Company's psoralen-based pathogen inactivation systems practice either of the photochemical decontamination patents depends on an interpretation of the scope of the patent claims. If such systems practice such patents, the license provides for Cerus to make milestone payments and pay royalties on revenues Cerus receives from sales of such systems. The license also calls for minimum annual royalty payments in order to maintain certain exclusive rights. The manner in which any such milestone payments and royalties would be shared by Baxter, if at all, has not been determined. The Company may rely, in certain circumstances, on trade secrets to protect its technology. However, trade secrets are difficult to protect. The Company seeks to protect its proprietary technology and processes, in part, by confidentiality agreements with its employees and certain contractors. There can be no assurance that these agreements will not be breached, that the Company will have adequate remedies for any breach, or that the Company's trade secrets will not otherwise become known or be independently discovered by competitors. To the extent that the Company's employees or its consultants or contractors use intellectual property owned by others in their work for the Company, disputes may also arise as to the rights in related or resulting know-how and inventions. In August 1996, the Company received correspondence from Circadian Technologies, Inc., an Australian entity, alleging that unspecified trade secrets and know-how jointly owned by Circadian and the Auckland Division Cancer Society of New Zealand were, without the consent of Circadian, used in the development by the Cancer Society and the Company of unspecified compounds for the Company's red blood cell program. Such claims do not relate to the Company's platelet or FFP programs. In subsequent correspondence, Circadian has indicated that it is seeking compensation in the form of royalties or a lump sum payment. Based on its investigation of the matter to date, the Company does not believe that the claims are meritorious. Any future litigation involving these allegations, however, would be subject to inherent uncertainties, especially in cases where complex technical issues are decided by a lay jury. There can be no assurance that, if a lawsuit were commenced, it would not be decided against the Company, which could have a material adverse effect upon the Company's business, financial condition and results of operations. 10 13 GOVERNMENT REGULATION The Company and its products are comprehensively regulated in the United States by the FDA and, in some instances, by state and local governments, and by comparable governmental authorities in other countries. The FDA regulates drugs, medical devices, and biologics under the Federal Food, Drug and Cosmetic Act and other laws, including, in the case of biologics, the Public Health Service Act. These laws and implementing regulations govern, among other things, the development, testing, manufacturing, record keeping, storage, labeling advertising, promotion and premarket clearance or approval of products subject to regulation. The Company believes its pathogen inactivation systems will be regulated by the FDA as devices. It is also possible, however, that the FDA will decide to regulate the pathogen inactivation systems as biologics, as drugs, as combination products including drugs or biologics and one or more medical devices, or as drugs or biologics with one or more medical devices (i.e., the blood bags and light source) requiring separate approval or clearance. Whether the FDA regulates the pathogen inactivation systems as devices or as one or more of the other alternatives, it is likely that the FDA's Center for Biologics Evaluation and Research will be principally responsible for regulating the pathogen inactivation systems. Before a medical device may be marketed in the United States, the FDA must clear a pre-market notification (a "510(k)") or approve a pre-market approval application ("PMA") for the product. Before a new drug may be marketed in the United States, the FDA must approve an NDA for the product. Before a biologic may be marketed in the United States, the FDA must approve a Biologic License Application ("BLA"). Before a combination product can be marketed in the United States, it must have an approved NDA, BLA or PMA, depending on which statutory authority the FDA elects to use. Despite the multiplicity of statutory and regulatory possibilities, the steps required before approval are essentially the same whether the product is ultimately regulated as a medical device, biologic, drug, a combination product, or a combination thereof. The steps required before a medical device, drug or biologic may be approved for marketing in the United States pursuant to a PMA, BLA or NDA, respectively, generally include (i) preclinical laboratory and animal tests, (ii) submission to the FDA of an investigational device exemption ("IDE") (for medical devices) or an IND (for drugs or biologics) for human clinical testing, which must become effective before human clinical trials may begin (iii) appropriate tests to show the product's safety, (iv) adequate and well-controlled human clinical trials to establish the product's efficacy for its intended indications, (v) submission to the FDA of a PMA, BLA or NDA, as appropriate and (vi) FDA review of the PMA, BLA or NDA in order to determine, among other things, whether the product is safe and effective for its intended uses. In addition, the FDA inspects the facilities at which the product is manufactured and will not approve the product unless compliance with current Good Manufacturing Practices ("cGMP") requirements is satisfactory. The steps required before a medical device may be cleared for marketing in the United States pursuant to a 510(k) are generally the same, except that instead of conducting tests to demonstrate safety and efficacy, data, including clinical data if necessary, must be obtained to show that the product is substantially equivalent to a legally marketed device, and the FDA must make a determination of substantial equivalence rather than a determination that the product is safe and effective. The Company believes that, in deciding whether a pathogen inactivation system is safe and effective, the FDA is likely to take into account whether it adversely affects the therapeutic efficacy of blood components as compared to the therapeutic efficacy of blood components not treated with the system, and that the FDA will weigh the system's safety, including potential toxicities of the inactivation compounds, and other risks against the benefits of using the system in a blood supply that has become safer in recent years. There can be no assurance that the FDA will not require further toxicology studies of the Company's products. Based on discussions with the FDA, the Company believes that it will be required to provide data from human clinical studies to demonstrate the safety of treated platelets and their therapeutic comparability to untreated platelets, but that only data from in vitro and animal studies, not data from human clinical studies, will be required to demonstrate the system's efficacy in inactivating pathogens. In light of these criteria, the Company's clinical trial programs for platelets and FFP will consist of studies that differ from the usual Phase 1, Phase 2 and Phase 3 clinical studies. 11 14 There can be no assurance, however, that these means of demonstrating safety and efficacy will ultimately be acceptable to the FDA or that the FDA will continue to believe that this clinical plan is appropriate. Moreover, even if the FDA considers these means of demonstrating safety and efficacy to be acceptable in principle, there can be no assurance that the FDA will find the data submitted sufficient to demonstrate safety and efficacy. In particular, although the Company anticipates that the FDA will consider in vitro and animal data an appropriate means of demonstrating efficacy in pathogen inactivation, there can be no assurance that the FDA will so conclude, and any requirement to provide other than in vitro and animal data would adversely affect the timing and could affect the success of the Company's efforts to obtain regulatory approval. Even if regulatory approval or clearance is granted, it could include significant limitations on the indicated uses for which a product could be marketed. For example, the Company does not believe that it will be able to make any labeling claims that the Company's pathogen inactivation systems may inactivate any pathogens for which it does not have in vitro, and in certain cases animal, data supporting such claims. The testing and approval/clearance process requires substantial time, effort and financial resources, and is generally lengthy, expensive and uncertain. The approval process is affected by a number of factors, including the availability of alternative treatments and the risks and benefits demonstrated in clinical trials. Additional animal studies or clinical trials may be requested during the FDA review period and may delay marketing approval. After FDA approval for the initial indications, further clinical trials may be necessary to gain approval for the use of the product for additional indications. The FDA may also require post-marketing testing to monitor for adverse effects, which can involve significant expense. Later discovery of previously unknown problems with a product may result in restrictions on the product, including withdrawal of the product from the market. In addition, the policies of the FDA may change, and additional regulations may be promulgated which could prevent or delay regulatory approval of the Company's planned products. There can be no assurance that any approval or clearance will be granted on a timely basis, if at all. Any failure to obtain or delay in obtaining such approvals or clearances, and any significant limitation on their indicated uses, could have a material adverse effect on the Company's business, financial condition and results of operations. A medical device, biologic or drug, its manufacturer, and the holder of the PMA or 510(k), BLA or NDA for the product are subject to comprehensive regulatory oversight, both before and after approval or clearance is obtained. Violations of regulatory requirements at any stage, including during the preclinical and clinical testing process, during the approval/clearance process or after the product is approved/cleared for marketing, could result in various adverse consequences, including the FDA's requiring that a clinical trial be suspended or halted, the FDA's delay in approving/clearing or refusing to approve/clear a product, withdrawal of an approved/cleared product from the market and the imposition of criminal penalties. For example, the holder of a PMA or 510(k), BLA or NDA is required to report certain adverse reactions to the FDA, and must comply with certain requirements concerning advertising and promotional labeling for the product. Also, quality control and manufacturing procedures must continue to conform to cGMP regulations after approval or clearance, and the FDA periodically inspects manufacturing facilities to assess compliance with cGMP. Accordingly, manufacturers must continue to expend time, monies and efforts on regulatory compliance, including cGMP compliance. In addition, new government requirements may be established that could delay or prevent regulatory approval or clearance of the Company's products under development or otherwise alter the applicable law. There can be no assurance that the FDA will determine that the facilities and manufacturing procedures of Baxter or any other third-party manufacturer of the Company's planned products will conform to cGMP requirements. In addition to the regulatory requirements applicable to the Company and its products, there are also regulatory requirements applicable to the Company's prospective customers, which are primarily entities that ship blood and blood products in interstate commerce. Such entities are regulated by the FDA pursuant to the Food, Drug and Cosmetic Act and the Public Health Service Act and implementing regulations. Blood centers and others that ship blood and blood products interstate will likely be required to obtain approved license supplements from the FDA before shipping products processed with the Company's pathogen inactivation systems. This requirement and/or FDA delays in approving such supplements may deter some blood centers from using the Company's products, and blood centers that do submit supplements may face disapproval or delays in approval that could provide further disincentives to use of the systems. The regulatory impact on potential customers could have a material adverse effect on the Company's business, financial condition and results of operations. 12 15 The Phase 3 European clinical trial is being designed to assess the therapeutic efficacy of the platelet pathogen inactivation system for use in treating apheresis platelets and pooled random donor platelets. The Phase 3 United States clinical trial is being designed to assess the therapeutic efficacy of the platelet pathogen inactivation system for use in treating apheresis platelets, not pooled random donor platelets, which represent 55% and 45% of the market, respectively. If the Company decides to seek FDA approval of the platelet pathogen inactivation system for use in treating pooled random donor platelets, the Company may be required by the FDA to conduct additional clinical studies. In addition, there currently are three principal manufacturers of automated apheresis collection equipment used in the United States, including Baxter. The equipment of each manufacturer collect platelets into plastic disposables designed for that equipment; thus, a pathogen inactivation system designed for disposables used by one manufacturer will not necessarily be compatible with other manufacturers' collection equipment. The Company intends initially to seek FDA approval of a platelet pathogen inactivation system configured for Baxter's aphersis collection equipment. If the Company determines that compatibility with other equipment is desirable, it will need to develop additional processing procedures. Although the Company believes that the FDA would accept the clinical data from the original system for platelets collected using other equipment and procedures and would require only limited additional studies to show comparability, there can be no assurance that it would do so. Because of the risk of bacterial growth, current FDA rules require that platelets may not be stored for more than five days after collection from the donor. The rules also require that pooled platelets be transfused within four hours of pooling and, as a result, most pooling occurs at hospitals. However, the Company's platelet pathogen inactivation system is being designed to be used at blood centers, not at hospitals, and requires a processing time of approximately eight hours. Therefore, in order for the Company's platelet pathogen inactivation system to be effectively implemented and accepted at blood centers as planned, the FDA-imposed limit on the time between pooling and transfusion would need to be lengthened or eliminated for blood products treated with the Company's systems, which are being designed to inactivate bacteria that would otherwise contaminate pooled platelets. If the Company were to pursue the pooled random donor platelet market, it would need to work with the FDA during the approval/clearance process to obtain the necessary changes in these limitations. There can be no assurance, however, that the FDA would change this requirement and, if such a change were not made, the Company's business, financial condition and results of operations would be materially adversely affected. The Company is developing a European investigational plan based on the platelet and FFP treatment systems using S-59 being categorized as Class III devices under European Union regulatory authorities. However, there can be no assurance that this approach will be accepted by European authorities. The European Union has promulgated rules that require that medical devices receive by mid-1998 the right to affix the CE Mark, an international symbol of adherence to quality assurance standards and compliance with applicable European medical device directives. Failure to receive CE Mark certification will prohibit the Company from selling its products in the European Union. The Company is subject to federal, state and local laws, rules, regulations and policies governing the use, generation, manufacture, storage, air emission, effluent discharge, handling and disposal of certain materials, biological specimens and wastes. There can be no assurance that the Company will not be required to incur significant costs to comply with environmental and health and safety regulations in the future. The Company's research and development involves the controlled use of hazardous materials, including certain hazardous chemicals and radioactive materials. Although the Company believes that its safety procedures for handling and disposing of such materials comply with the standards prescribed by state and federal regulations, the risk of accidental contamination or injury from these materials cannot be eliminated. In the event of such an accident, the Company could be held liable for any damages that result and any such liability could exceed the resources of the Company. EMPLOYEES As of February 28, 1998, the Company had 76 employees, 59 of whom were engaged in research and development and 17 in finance and other administration. The Company also had consulting arrangements with nine individuals. No employee of the Company is covered by collective bargaining agreements, and the Company believes that its relationship with its employees is good. 13 16 ADDITIONAL BUSINESS RISKS The Company's business is subject to the following risks in addition to those discussed above and elsewhere in this report. Early Stage of Product Development. The Company's pathogen inactivation systems for blood transfusion components are in the research and development stage and will require additional preclinical and clinical testing prior to submission of any regulatory application for commercial use. The Company has not commenced the large-scale clinical trials that would, if successful, lead to filing product approval applications with the FDA. The Company's products are subject to the risks of failure inherent in the development of medical device, pharmaceutical and biological products and products based on new technologies. These risks include the possibility that the Company's approach to pathogen inactivation will not be safe or effective, that the Company's products will not be easy to use or cost-effective, that third parties will develop and market superior or equivalent products, that any or all of the Company's products will fail to receive any necessary regulatory approvals, that such products will be difficult or uneconomical to manufacture on a commercial scale, that proprietary rights of third parties will preclude the Company from marketing such products and that the Company's products will not achieve market acceptance. As a result of these risks, there can be no assurance that the Company's research and development activities will result in any commercially viable products. Uncertainty Associated with Preclinical and Clinical Testing. The regulatory process includes preclinical and clinical testing of each product to establish its safety and efficacy, and may include post-marketing studies requiring expenditure of substantial resources. The results from preclinical studies and early clinical trials conducted by the Company may not be predictive of results obtained in later clinical trials, and there can be no assurance that clinical trials conducted by the Company will demonstrate sufficient safety and efficacy to obtain the requisite approvals or that marketable products will result. For example, at the request of the FDA, the Company designed and commenced a pilot Phase 2c clinical trial in 15 thrombocytopenic patients to assess the effect of treated platelets on post-transfusion bleeding time correction and platelet count increment. The results of this pilot Phase 2c clinical trial, given its small size, cannot satisfactorily be evaluated statistically. Thus, non-statistically significant variations in bleeding times, or other measures of the trial, may result in the FDA requiring additional patients to be enrolled in the Phase 2c trial. Due to the nature of the trial, its implementation and its assessment of bleeding times, enrollment of a significant number of additional patients could be impracticable to accomplish on a timely basis and could significantly delay completion of this trial and, as a result, commencement of the proposed Phase 3 clinical trial. This Phase 2c trial currently is not expected to be completed until at least the third quarter of 1998. The rate of completion of the Company's clinical trials may be delayed by many other factors, including slower than anticipated patient enrollment or any other adverse event occurring during the clinical trials. Completion of testing, studies and trials may take several years, and the length of time generally varies substantially with the type, complexity, novelty and intended use of the product. Data obtained from preclinical and clinical activities are susceptible to varying interpretations, which could delay, limit or prevent regulatory approval. In addition, delays or rejections may be encountered based upon many factors, including changes in regulatory policy during the period of product development. The Company's products under development require significant additional research and development efforts. No assurance can be given that any of the Company's development programs will be successfully completed, that any further INDs or IDEs will become effective or that additional clinical trials will be allowed by the FDA or other regulatory authorities, that clinical trials will commence as planned, that required United States or foreign regulatory approvals will be obtained on a timely basis, if at all, or that any products for which approval is obtained will be commercially successful. Due to the uncertain nature of clinical trial programs, there can be no assurance that the proposed schedules for IND or IDE and clinical protocol submissions to the FDA, initiations of studies and completions of clinical trials can be maintained. Any delays in the Company's clinical trials or failures to obtain required regulatory approvals would have a material adverse effect on the Company's business, financial condition and results of operations. Reliance on Baxter. Under the terms of the Agreements, the Company relies on Baxter for significant funding, product development support, the manufacture and supply of certain system components and the marketing of its planned products. The Company anticipates that, prior to commencement of product sales, if any, the Company's principal source of revenue will continue to be payments under the Agreements. 14 17 The development programs under the Agreements may be terminated by Baxter on 90 days' notice. If the Agreements were terminated, if Baxter failed to provide the committed funding or if Baxter's product development efforts were unsuccessful, the Company may need to obtain additional funding from other sources and would be required to devote additional resources to the development of its products, delaying the development of its products. Any such delay would have a material adverse effect on the Company's business, financial condition and results of operations. There can be no assurance that disputes will not arise in the future with respect to the Agreements. Possible disagreements between Baxter and the Company could lead to delays in the research, development or commercialization of certain planned products or could require or result in time-consuming and expensive litigation or arbitration and would have a material adverse effect on the Company's business, financial condition and results of operations. Under the terms of the Agreements, Baxter is responsible for manufacturing the disposable units, such as blood storage containers and related tubing, as well as any devices associated with the inactivation processes. If the Agreements were terminated or if Baxter otherwise failed to deliver an adequate supply of components, the Company would be required to identify other third-party component manufacturers. There can be no assurance that the Company would be able to identify such manufacturers on a timely basis or enter into contracts with such manufacturers on reasonable terms, if at all. Any delay or change in the availability of devices or disposables from Baxter or its suppliers could adversely affect the timely submission of products for regulatory approval or the market introduction and subsequent sales of such products and would have a material adverse effect on the Company's business, financial condition and results of operations. Moreover, the inclusion of components manufactured by others could require the Company to seek new approvals from government regulatory authorities, which could result in delays in product delivery. There can be no assurance that the Company would receive any such required regulatory approvals. Any such delay would have a material adverse effect on the Company's business, financial condition and results of operations. If appropriate regulatory approvals are received, Baxter will be responsible for the marketing, sales and distribution of the Company's pathogen inactivation systems for blood components worldwide. The Company does not currently maintain, nor does it intend to develop, its own marketing and sales organization but instead expects to rely on Baxter to market and sell its pathogen inactivation systems. There can be no assurance that the Company will be able to maintain its relationship with Baxter or that such marketing arrangements will result in payments to the Company. Revenues to be received by the Company through any marketing and sales arrangement with Baxter will be dependent on Baxter's efforts, and there can be no assurance that the Company will benefit from Baxter's present or future market presence or that such efforts will otherwise be successful. If the Agreements were terminated or if Baxter's marketing efforts were unsuccessful, the Company's business, financial condition and results of operations would be materially adversely affected. The Agreements provide for key decision-making regarding development and commercialization by a management board comprised of equal representation from Baxter and the Company and, in the case of FFP and red blood cells, one independent member. There can be no assurance that such board's decisions will be consistent with the Company's interests, that Baxter will not elect to pursue alternative technologies or product strategies or that its corporate interests and plans will remain consistent with those of the Company. In the Agreements, Baxter agreed to certain limited restrictions on its ability to independently develop and market products that compete with the products under the Agreements. There can be no assurance that these provisions will prevent Baxter from developing or marketing competing products. The Company is aware that Baxter is developing an alternative pathogen inactivation system for FFP based on methylene blue. The development and commercialization of the Company's pathogen inactivation systems could be materially adversely affected by competition with Baxter or by Baxter's election to pursue alternative strategies or technologies in lieu of those of the Company. Government Regulation. All of the Company's products under development and anticipated future products are or will be subject to extensive and rigorous regulation by the federal government (principally the FDA) and state, local, and foreign governments. Such regulations govern, among other things, the development, testing, manufacturing, labeling, storage, pre-market clearance or approval, advertising, promotion, sale and distribution of such products. The process of obtaining regulatory approvals or clearances is generally lengthy, expensive and uncertain. To date, none of the Company's products under development has been approved for sale in the United States or any foreign market. Satisfaction of pre-market approval or clearance or other regulatory requirements of the FDA, or similar requirements of foreign regulatory agencies, typically takes several years, and may take longer, 15 18 depending upon the type, complexity, novelty and intended use of the product. There can be no assurance that the FDA or any other regulatory agency will grant approval or clearance for any product being developed by the Company on a timely basis, if at all. If regulatory approval of a product is granted, such approval may impose limitations on the indicated uses for which a product may be marketed. Further, even if regulatory approval is obtained, later discovery of previously unknown problems with a product may result in restrictions on the product, including withdrawal of the product from the market. The policies of the FDA and foreign regulatory bodies may change, and additional regulations may be promulgated, which could prevent or delay regulatory approval of the Company's planned products. Delay in obtaining or failure to obtain regulatory approvals could have a material adverse effect on the Company's business, financial condition and results of operations. Among the conditions for FDA approval of a pharmaceutical, biologic or device is the requirement that the manufacturer's quality control and manufacturing procedures conform to cGMP requirements, which must be followed at all times. The FDA enforces cGMP requirements through periodic inspections. There can be no assurance that the FDA will determine that the facilities and manufacturing procedures of Baxter or any other third-party manufacturer of the Company's planned products will conform to cGMP requirements. Blood centers and others that ship blood and blood products interstate will likely be required to obtain approved license supplements from the FDA before shipping products processed with the Company's pathogen inactivation systems. This requirement and/or FDA delays in approving such supplements may deter some blood centers from using the Company's products, and blood centers that do submit supplements may face disapproval or delays in approval that could provide further disincentives to use the systems. The regulatory impact on potential customers could have a material adverse effect on the Company's business, financial condition and results of operations. No Assurance of Market Acceptance; Concentrated Market. The Company believes that market acceptance of the Company's pathogen inactivation systems will depend, in part, on the Company's ability to provide acceptable evidence of the safety, efficacy and cost-effectiveness of its products, as well as the ability of blood centers to obtain FDA approval and adequate reimbursement for such products. The Company believes that market acceptance of its pathogen inactivation systems also will depend upon the extent to which physicians, patients and health care payors perceive that the benefits of using blood components treated with the Company's systems justify the systems' additional costs and processing requirements in a blood supply that has become safer in recent years. While the Company believes that its pathogen inactivation systems are able to inactivate pathogens in excess of concentrations that the Company believes typically are present in contaminated blood components when the blood is donated, there can be no assurance that contamination levels will never exceed the capacity of the Company's pathogen inactivation systems. The Company does not expect that its planned products will be able to inactivate all known and unknown infectious pathogens, and there can be no assurance that the inability to inactivate certain pathogens will not affect the market acceptance of its products. There can be no assurance that the Company's pathogen inactivation systems will gain any significant degree of market acceptance among blood centers, physicians, patients and health care payors, even if clinical trials demonstrate safety and efficacy and necessary regulatory approvals and health care reimbursement approvals are obtained. In the United States, approximately 55% of platelets are collected by apheresis. The Company's Phase 3 United States clinical trial protocol for its platelet pathogen inactivation system has been designed to assess the therapeutic efficacy of such system in treating apheresis platelets only. There can be no assurance that the market share for apheresis platelets will increase. If such market share does not increase, the Company may decide to conduct additional clinical studies in order to obtain FDA approval of the system for use in treating random donor platelets. In addition, there currently are three principal manufacturers of automated apheresis collection equipment used in the United States, including Baxter. The equipment of each manufacturer collect platelets into plastic disposables designed for that equipment; thus, a pathogen inactivation system designed for disposables used by one manufacturer will not necessarily be compatible with the other manufacturers' collection equipment. The Company intends initially to seek FDA approval of a platelet pathogen inactivation system configured for Baxter's apheresis collection equipment. If the Company determines that compatibility with other equipment is desirable, it will need to develop additional processing procedures. Although the Company believes that the FDA would accept the clinical data from the original system for platelets collected using other equipment and procedures and would require only limited additional studies to show comparability, there can be no assurance that it would do so. 16 19 The Company's target customers are the limited number of national and regional blood centers, which collect, store and distribute blood and blood components. The failure to penetrate even a small number of these customers could have a material adverse effect on the Company's business, financial condition and results of operations. Rapid Technological Change; Significant Competition. The biopharmaceutical field is characterized by rapid and significant technological change. Accordingly, the Company's success will depend in part on its ability to respond quickly to medical and technological changes through the development and introduction of new products. Product development involves a high degree of risk, and there can be no assurance that the Company's product development efforts will result in any commercially successful products. Technological developments may result in the Company's products becoming obsolete or non-competitive before the Company is able to generate any significant revenue. Any such occurrence would have a material adverse effect on the Company's business, financial condition and results of operations. The Company expects to encounter competition in the sale of products it may develop, and its success will be dependent upon its ability to compete. Many companies and organizations that may be competitors or potential competitors have substantially greater financial and other resources than the Company and may have greater experience in preclinical testing, human clinical trials and other regulatory approval procedures. The Company's ability to compete successfully will depend, in part, on its ability to develop proprietary products, develop and maintain products that reach the market first, are technologically superior to and/or are of lower cost than other products on the market, attract and retain scientific personnel, obtain patent or other proprietary protection for its products and technologies, obtain required regulatory approvals, and manufacture, market and sell any product that it develops. In addition, other technologies or products may be developed that have an entirely different approach or means of accomplishing the intended purposes of the Company's products, or that might render the Company's technology and products uncompetitive or obsolete. Furthermore, there can be no assurance that the Company's competitors will not obtain patent protection or other intellectual property rights that would limit the Company's ability to use the Company's technology or commercialize products that may be developed. Dependence on Key Employees. The Company is highly dependent on the principal members of its management and scientific staff. The loss of the services of one or more of these employees could have a material adverse effect on the Company's business, financial condition and results of operations. The Company believes that its future success will depend in large part upon its ability to attract and retain highly skilled scientific and managerial personnel. Competition for such personnel is intense. There can be no assurance that the Company will be successful in attracting and retaining such personnel and the failure to do so could have a material adverse effect on the Company's business, financial condition and results of operations. In addition, a substantial portion of the stock options currently held by many of the Company's key employees are vested and may be fully vested over the next several years before the Company achieves significant revenues or profitability. The Company intends to grant additional options and provide other forms of incentive compensation to attract and retain such key personnel, although there can be no assurance such objective will be achieved. Patent and License Uncertainties. The Company's success depends in part on its ability to obtain patents, to protect trade secrets, to operate without infringing upon the proprietary rights of others and to prevent others from infringing on the proprietary rights of the Company. There can be no assurance that any patents owned by, or licensed to, the Company will afford protection against competitors or that any pending patent applications now or hereafter filed by, or licensed to, the Company will result in patents being issued. In addition, the laws of certain foreign countries do not protect the Company's intellectual property rights to the same extent as do the laws of the United States. There can be no assurance that the Company's planned or potential products will not be covered by third-party patents or other intellectual property rights, in which case continued development and marketing of such products would require a license under such patents or other intellectual property rights. There can be no assurance that such required licenses will be available to the Company on acceptable terms, if at all. If the Company does not obtain such licenses, it could encounter delays in product introductions while it attempts to design around such patents, or could find that the development, manufacture or sale of products requiring such licenses is foreclosed. Litigation may be necessary to defend against or assert claims of infringement, to enforce patents issued to the Company, to protect trade secrets or know-how owned by the Company or to determine the scope and validity of the proprietary rights of others. In addition, interference proceedings declared by the United States Patent and Trademark Office may be necessary to determine the priority of inventions with respect to patent applications of the 17 20 Company. Litigation or interference proceedings could result in substantial costs to and diversion of effort by the Company, and could have a material adverse effect on the Company's business, financial condition and results of operations. The Company may rely, in certain circumstances, on trade secrets to protect its technology. However, trade secrets are difficult to protect. The Company seeks to protect its proprietary technology and processes, in part, by confidentiality agreements with its employees and certain contractors. There can be no assurance that these agreements will not be breached, that the Company will have adequate remedies for any breach, or that the Company's trade secrets will not otherwise become known or be independently discovered by competitors. Limited Operating History; History of Losses and Expectation of Future Losses. The Company's net losses in fiscal years 1997, 1996 and 1995 were $14.7 million, $10.2 million and $2.4 million, respectively. As of December 31, 1997, the Company had an accumulated deficit of approximately $34.9 million. The Company has not received any revenues from product sales, and all revenues recognized by the Company to date have resulted from the Agreements and federal research grants. All of the Company's planned pathogen inactivation systems are in the research and development stage. The Company will be required to conduct significant research, development, testing and regulatory compliance activities on these products that, together with anticipated general and administrative expenses, are expected to result in substantial losses in future periods. The Company expects that the amount of such losses will fluctuate from quarter to quarter as a result of differences in the timing of expenses incurred and potential revenues from Baxter under the Agreements, and such fluctuations may be significant. The Company's ability to achieve a profitable level of operations will depend on successfully completing development, obtaining regulatory approvals and achieving market acceptance of its pathogen inactivation systems. There can be no assurance that the Company will ever achieve a profitable level of operations. Reliance on Third-Party Manufacturing; Dependence on Key Suppliers. The Company has no experience in manufacturing products for commercial purposes and does not have any manufacturing facilities. Consequently, the Company is dependent on contract manufacturers for the production of compounds and on Baxter for other system components for development and commercial purposes. There can be no assurance that existing manufacturers or any new manufacturer will be able to provide sufficient quantities of the compounds needed for the Company's pathogen inactivation systems in the future or that the Company will be able to enter into arrangements for the commercial-scale manufacture of such compounds on reasonable terms, if at all. In the event that the Company is unable to obtain or retain third-party manufacturing, it will not be able to commercialize its products as planned. Failure of any third-party manufacturer to deliver the required quantities of products on a timely basis and at commercially reasonable prices would materially adversely affect the Company's business, financial condition and results of operations. In addition, inclusion of components manufactured by other third parties could require the Company to seek new approvals from government regulatory authorities, which could result in delays in product delivery. There can be no assurance that such approval would be obtained. In the event the Company undertakes to establish its own commercial manufacturing capabilities, it will require substantial additional funds, manufacturing facilities, equipment and personnel. The Company purchases certain key components of its compounds from a limited number of suppliers. While the Company believes that there are alternative sources of supply for these components, establishing additional or replacement suppliers for any of the components in the Company's compounds, if required, may not be accomplished on a timely basis and could involve significant additional costs. Any failure by the Company to obtain any of the components used to manufacture the Company's compounds from alternative suppliers, if required, could limit the Company's ability to manufacture its compounds and would have a material adverse effect on the Company's business, financial condition and results of operations. Risk of Product Liability. The testing, marketing and sale of the Company's products will entail an inherent risk of product liability, and there can be no assurance that product liability claims will not be asserted against the Company. The Company intends to secure limited product liability insurance coverage prior to the commercial introduction of any product, but there can be no assurance that the Company will be able to obtain product liability insurance on acceptable terms or that insurance subsequently obtained will provide adequate coverage against any or all potential claims. Any product liability claim against the Company, regardless of its merit or eventual outcome, could have a material adverse effect upon the Company's business, financial condition and results of operations. 18 21 Environmental Regulation; Use of Hazardous Substances. The Company is subject to federal, state and local laws, rules, regulations and policies governing the use, generation, manufacture, storage, air emission, effluent discharge, handling and disposal of certain materials, biological specimens and wastes. There can be no assurance that the Company will not be required to incur significant costs to comply with additional environmental and health and safety regulations in the future. The Company's research and development involves the controlled use of hazardous materials, including certain hazardous chemicals and radioactive materials and pathogens. Although the Company believes that its safety procedures for handling and disposing of such materials comply with the standards prescribed by state and federal regulations, the risk of accidental contamination or injury from these materials cannot be eliminated. In the event of such an accident, the Company could be held liable for any damages that result and any such liability could exceed the resources of the Company. Uncertainty Regarding Health Care Reimbursement and Reform. The future revenues and profitability of biopharmaceutical and related companies as well as the availability of capital to such companies may be affected by the continuing efforts of the United States and foreign governments and third-party payors to contain or reduce costs of health care through various means. In the United States, given recent federal and state government initiatives directed at lowering the total cost of health care, it is likely that the Congress and state legislatures will continue to focus on health care reform and the cost of pharmaceuticals and on the reform of the Medicare and Medicaid systems. While the Company cannot predict whether any such legislative or regulatory proposals will be adopted, the announcement or adoption of such proposals could have a material adverse effect on the Company's business, financial condition and results of operations. The Company's ability to commercialize its products successfully will depend in part on the extent to which appropriate reimbursement levels for the cost of the products and related treatment of blood components are obtained from governmental authorities, private health insurers and other organizations, such as health maintenance organizations ("HMOs"). Third-party payors are increasingly challenging the prices charged for medical products and services. The trend toward managed health care in the United States and other countries and the concurrent growth of organizations such as HMOs, which could control or significantly influence the purchase of health care services and products, as well as legislative proposals to reform health care or reduce government insurance programs, may all result in lower prices for the Company's products. The cost containment measures that health care payors and providers are instituting and the effect of any health care reform could materially adversely affect the Company's ability to operate profitably. Volatility of Stock Price. The trading price of the Company's Common Stock is subject to significant fluctuations. Factors such as the announcements of scientific achievements or new products by the Company or its competitors; governmental regulation; health care legislation; developments in patent or other proprietary rights of the Company or its competitors, including litigation; fluctuations in the Company's operating results; comments made by analysts, including changes in analysts' estimates of the Company's financial performance; and market conditions for health care stocks in general could have significant impact on the future price of the Common Stock. In addition, the stock market has from time to time experienced extreme price and volume fluctuations, which may be unrelated to the operating performance of particular companies. In the past, securities class action litigation has often been instituted following periods of volatility in the market price for a company's securities. Such litigation could result in substantial costs and a diversion of management attention and resources, which could have a material adverse effect on the Company's business, financial condition and results of operations. 19 22 EXECUTIVE OFFICERS OF THE REGISTRANT The executive officers of the Company and their ages as of February 27, 1998 are as follows:
NAME AGE POSITION ---- --- -------- Stephen T. Isaacs ............... 49 President, Chief Executive Officer and Director David S. Clayton ................ 54 Vice President, Finance and Chief Financial Officer Laurence M. Corash .............. 53 Vice President, Medical Affairs John E. Hearst .................. 62 Vice President, New Science Opportunities
STEPHEN T. ISAACS founded the Company in September 1991 and has served as President, Chief Executive Officer and a member of the Board of Directors since that time. Mr. Isaacs was previously President and Chief Executive Officer of HRI, a research and development company, from September 1984 to December 1996. From 1975 to 1986, Mr. Isaacs held a faculty research position at the University of California at Berkeley. DAVID S. CLAYTON has been Chief Financial Officer of the Company since May 1996 and Vice President, Finance of the Company since July 1996. From 1992 to May 1996, Mr. Clayton was a financial consultant to various companies, including the Company. From 1989 through May 1992, Mr. Clayton was the Executive Vice President of Trans Ocean Ltd., a company engaged in leasing of international maritime shipping containers. LAURENCE M. CORASH, M.D., a co-founder of the Company, has been Vice President, Medical Affairs of the Company since July 1996. From July 1994 until he assumed his current position, Dr. Corash was Director of Medical Affairs. Dr. Corash was a consultant to the Company from 1991 to July 1994. Dr. Corash has been a Professor of Laboratory Medicine at the University of California, San Francisco since July 1985 and Chief of the Hematology Laboratory for the Medical Center at the University of California, San Francisco since January 1982. Dr. Corash has served as a consultant to the FDA Advisory Panel for Hematology Devices since 1990. JOHN E. HEARST, PH.D., D.SC., a co-founder of the Company, was elected Vice President, New Science Opportunities in July 1996. From January 1996 until July 1996, Dr. Hearst served as Director, New Science Opportunities. He has served as a member of the Board of Directors of the Company since January 1992. Dr. Hearst has been a Professor of Chemistry at the University of California at Berkeley since 1972. In 1984, Dr. Hearst co-founded HRI. ITEM 2. PROPERTIES The Company leases approximately 17,400 square feet for its main facility and approximately 9,900 square feet for an additional facility, both of which contain laboratory and office space, in Concord, California. The lease of the main facility extends through June 1999 with two five-year renewal options. The lease of the additional facility extends through January 1999, with renewal options for up to six years. The Company also has a short-term lease for approximately 4,500 square feet at a facility located near its main facility in Concord. The Company believes that its facilities will be adequate to meet its needs for the foreseeable future. ITEM 3. LEGAL PROCEEDINGS Not applicable. ITEM 4. SUBMISSION OF MATTERS TO A VOTE OF SECURITY HOLDERS Not applicable. 20 23 PART II ITEM 5. MARKET FOR THE REGISTRANT'S COMMON EQUITY AND RELATED STOCKHOLDER MATTERS (a) The Company's Common Stock is traded on the Nasdaq National Market under the symbol "CERS." The Company completed the initial public offering of its Common Stock on January 30, 1997. The following table sets forth, for the periods indicated, the high and low sales prices for the Common Stock as reported by the Nasdaq National Market:
HIGH LOW ---- --- Fiscal 1997: First Quarter (from January 30) . $ 12 3/8 $ 7 3/4 Second Quarter .................. 12 3/4 8 1/2 Third Quarter ................... 18 8 Fourth Quarter .................. 25 1/2 16 7/8
On February 27, 1998, the last reported sale price of the Company's Common Stock on the Nasdaq National Market was $16 1/8 per share. At February 27, 1998, the Company had approximately 282 holders of record of its Common Stock. (b) On January 30, 1997, the Company sold 2,000,000 shares of Common Stock in an initial public offering for which the Company received net proceeds of $21,070,000. Of such proceeds, the Company has applied $10,650,000 to contracts and consultants, $5,450,000 to employee salaries and related expenses, $1,440,000 to supplies and office expenses, $1,390,000 to professional fees, $1,360,000 to facilities and the purchase and installation of equipment, $460,000 to travel and entertainment, $250,000 to insurance, $70,000 to repayment of indebtedness. 21 24 ITEM 6. SELECTED FINANCIAL DATA The following table summarizes certain selected financial data for the fiscal year ended December 31, 1997. The information presented should be read in conjunction with the financial statements and notes included elsewhere herein. The selected financial data for the periods prior to the financial statements included herein are derived from audited financial statements.
YEARS ENDED DECEMBER 31, ----------------------------------------------------------------------- 1997 1996 1995 1994 1993 ----------- ----------- ----------- ----------- ----------- (IN THOUSANDS, EXCEPT PER SHARE DATA) STATEMENT OF OPERATIONS DATA: Revenue ......................... $ 6,851 $ 3,609 $ 6,799 $ 4,796 $ 230 Operating expenses: Research and development ...... 19,569 12,080 8,125 5,680 2,485 General and administrative .... 3,163 2,200 1,517 1,194 1,210 ----------- ----------- ----------- ----------- ----------- Total operating expenses .. 22,732 14,280 9,642 6,874 3,695 ----------- ----------- ----------- ----------- ----------- Loss from operations ............ (15,881) (10,671) (2,843) (2,078) (3,465) Other income (expense), net ..... 1,217 464 483 278 (50) ----------- ----------- ----------- ----------- ----------- Net loss ........................ $ (14,664) $ (10,207) $ (2,360) $ (1,800) $ (3,515) =========== =========== =========== =========== =========== Net loss per share-basic and diluted(1) .................... $ (1.76) $ (5.98) $ (1.67) $ (1.24) $ (2.35) Shares used in computing net loss per share-basic and diluted(1) 8,351,872 1,705,821 1,413,969 1,456,336 1,492,785
DECEMBER 31, ----------------------------------------------------------------------- 1997 1996 1995 1994 1993 --------- --------- --------- --------- ---------- (IN THOUSANDS) BALANCE SHEET DATA: Cash and cash equivalents . $ 11,604 $ 6,002 $ 9,659 $ 7,802 $ 6,076 Working capital ........... 21,374 2,653 7,263 5,865 3,884 Total assets .............. 27,315 8,812 11,349 9,684 6,807 Capital lease obligations, less current portion .... 43 92 32 94 -- Accumulated deficit ....... (34,870) (20,206) (9,999) (7,639) (5,838) Total stockholders' equity (deficit) ............... 22,475 4,839 8,663 5,439 (516)
(1) See Note 1 of Notes to Financial Statements for a description of the method used in computing the net loss per share. The net loss per share amounts prior to 1997 have been restated as required to comply with Statement of Financial Accounting Standards No. 128, "Earnings Per Share" (Statement No. 128) and the Securities and Exchange Commission's Staff Accounting Bulletin No. 98, (SAB 98). For further discussion of net loss per share and the impact of Statement No. 128 and SAB 98, see the notes to the consolidated financial statements. 22 25 ITEM 7. MANAGEMENT'S DISCUSSION AND ANALYSIS OF FINANCIAL CONDITION AND RESULTS OF OPERATIONS The following discussion of the financial condition and results of operations of the Company should be read in conjunction with the Financial Statements and the Notes thereto included elsewhere in this report. This report contains forward-looking statements that involve risks and uncertainties. The Company's actual results could differ significantly from those discussed in these forward-looking statements as a result of certain factors, including those set forth under "Business" and elsewhere herein. OVERVIEW Cerus is developing systems designed to improve the safety of blood transfusions by inactivating infectious pathogens in blood components used for transfusion (platelets, plasma and red blood cells) and inhibiting the leukocyte (white blood cell) activity that is responsible for certain adverse immune and other transfusion-related reactions. The Company's platelet and plasma pathogen inactivation systems are in Phase 2 clinical trials in the United States, and its red blood cell pathogen inactivation system is in preclinical development. Since its inception in 1991, Cerus has devoted substantially all of its efforts and resources to the research, development and clinical testing of techniques and systems for inactivating pathogens in blood transfusion components. The Company has been unprofitable since inception and, as of December 31, 1997, had an accumulated deficit of approximately $34.9 million. All of the Company's pathogen inactivation systems are in the research and development stage. The Company will be required to conduct significant research, development, testing and regulatory compliance activities on these products that, together with anticipated general and administrative expenses, are expected to result in substantial losses at least until commercialization of its products under development. The Company's ability to achieve a profitable level of operations in the future will depend on its ability to successfully complete development, obtain regulatory approvals and achieve market acceptance of its pathogen inactivation systems. As a result, there can be no assurance that the Company will ever achieve a profitable level of operations. Further, a significant portion of the Company's development funding is provided by Baxter Healthcare Corporation ("Baxter") under the agreements described below. There can be no assurance that such agreements will not be modified or terminated as provided therein. In December 1993, Cerus entered into a development and commercialization agreement with Baxter to develop a system for inactivation of pathogens in platelets. The agreement provides for Baxter to make an equity investment and certain up-front license and milestone payments. The agreement further provides for Baxter and the Company to generally share system development costs equally, subject to mutually agreed budgets established from time to time. The agreement also provides for a sharing of revenue from sales of inactivation system disposables, after each party is reimbursed for its cost of goods above a specified level. In January 1997, the Company and Baxter amended the agreement to provide that the Company would receive an additional 2.2% of the adjusted product revenue from the sale of the platelet pathogen inactivation system disposables in return for payment by the Company to Baxter of $5.5 million in 1997 in four equal quarterly installments for development costs. In January and July 1995, Cerus received approximately $2.6 million from Baxter in connection with interim funding agreements related to the development of pathogen inactivation systems for plasma for transfusions (fresh frozen plasma or "FFP") and red blood cells. In April 1996, Cerus entered into a second development and commercialization agreement with Baxter, principally focused on the FFP and red blood cell pathogen inactivation systems. The agreement provides for Baxter to make certain equity investments, including two future milestone-based investments of $5 million each at 120% of the market price at the time of the investment. The agreement also provides for Baxter and the Company to generally share development costs of the systems equally, subject to mutually agreed budgets established from time to time. The agreement further provides for the Company and Baxter to share gross profits from the sale of inactivation system disposables, after deducting from such gross profits a specified percentage allocation to be retained by the marketing party for marketing and administration expenses. 23 26 Through December 31, 1997, Baxter has paid the Company up-front license fees and milestone and development payments totaling $14.8 million and has invested $17.5 million in the capital stock of the Company under the agreements described above. In March 1998, the Company and Baxter entered into an amendment to the April 1996 agreement providing that, to the extent the approved spending for 1998 for the red blood cell project exceeds $7.3 million, Cerus will fund all expenses for the red blood cell project in 1998 in excess of such amount, up to the amount of the approved budget. To compensate Cerus for such excess expenditures, Baxter will fully fund the first expenditures under the approved budget for the red cell project for 1999 in an amount equal to such excess expenditures, after which the parties shall equally share the expenses of the red cell project. If for any reason there is not an approved budget for the red cell project for 1999, Baxter will fully fund the first expenditures for 1999 under the approved budget for such other Cerus-Baxter program or programs as Cerus shall designate in an amount equal to the Cerus 1998 excess expenditures. If by July 1, 1999, however, there is not an approved budget for such other Cerus-Baxter program or programs that is at least equal to such excess expenditures, Baxter will promptly pay to Cerus one-half of the amount by which the excess expenditures exceed the amount of expenditures to be funded by Baxter. Cerus anticipates that the expenditures for 1998 for the FFP program will exceed the previously approved budget. Cerus and Baxter are discussing the level of funding each company will support in 1998 for the FFP program. There can be no assurance that the parties will agree on a budget that would permit the FFP program to proceed in accordance with the Company's Plans. To date, the Company has not received any revenue from product sales and it will not derive revenue from product sales unless and until one or more planned products receives regulatory approval and achieves market acceptance. The Company anticipates that its sources of revenue until product sales occur will be limited to payments under development and commercialization agreements with Baxter in the area of blood component pathogen inactivation, payments from the United States government under research grant programs, payments from future collaboration agreements, if any, and interest income. Under the agreements, all research, development, preclinical and clinical costs of the pathogen inactivation projects are shared by Cerus and Baxter. Because more of such research and development is typically performed internally at Cerus than at Baxter and because Cerus is generally responsible for engaging third parties to perform certain aspects of these projects, the Company's research and development expenses have exceeded its share of expenses. As a result, the Company has recognized revenue from Baxter, giving rise to a receivable due from Baxter and corresponding periodic balancing payments to the Company. At December 31, 1997, the amount of the Baxter receivable was approximately $4.4 million. On February 13, 1998, Baxter paid Cerus approximately $4.5 million in satisfaction of such receivable and anticipated adjustments. Through December 31, 1997, the Company had recognized approximately $19.2 million in revenue under its agreements with Baxter, including the license fee and milestone amounts described above, and approximately $3.1 million under United States government grants. RESULTS OF OPERATIONS YEARS ENDED DECEMBER 31, 1997, 1996, AND 1995 Revenue. Revenue earned under the Agreements for the years ending December 31, 1997, 1996 and 1995 was $6.2 million, $2.9 million and $6.0 million and accounted for 90%, 79% and 89% of the Company's total revenue, respectively. Revenue from Baxter increased in 1997 from 1996, as the Company recognized milestone and license fee revenue related to the platelet program of approximately $1.7 million and recognized increased development revenue primarily relating to its FFP and red blood cell programs. Revenue from Baxter decreased to approximately $2.9 million in 1996 from 1995, as no milestone or license fee revenue relating to the platelet program was recognized, as compared with approximately $1.7 million in milestone and related license fee revenue recognized during 1995. In addition, FFP and red blood cell development revenue decreased in 1996 by approximately $1.2 million, principally reflecting lower revenue during 1996 under the interim funding agreements. Government grant revenue, generally unchanged among the periods, was approximately $660,000, $758,000 and $751,000 for the years 1997, 1996 and 1995, respectively. Research and Development Expenses. Research and development expenses for the years ending December 31, 1997, 1996 and 1995 were $19.6 million, $12.1 million and $8.1 million, respectively. The increases in 1997 and 1996 were due principally to third party costs, particularly toxicology studies, compound manufacturing development and initiation of clinical trials relating to the platelet and plasma programs, as well as to increased 24 27 activity at the Company in the FFP and red blood cell programs. A significant portion of the increase was the result of increased payroll and other personnel expenses, related laboratory supplies, equipment and facilities expansion. In addition, as described above, under an amendment to the 1993 platelet agreement, Cerus made payments to Baxter in 1997 of $5.5 million for development costs in return for an additional 2.2% share of platelet pathogen inactivation system adjusted product revenue. General and Administrative Expenses. General and administrative expenses were approximately $3.2 million in 1997, $2.2 million in 1996 and $1.5 million in 1995. The increases were primarily attributable to increased personnel levels associated with the expansion of the Company's operations. Other Income (Expense). Interest income was approximately $1.2 million in 1997, $482,000 in 1996 and approximately $500,000 in 1995. The increase from 1996 to 1997 was attributable primarily to increased average cash balances related to proceeds from the Company's initial public offering and private placement to Baxter (see Liquidity and Capital Resources below). The increase from 1995 to 1996 was attributable primarily to increased average cash balances related to financings and funding under the Baxter platelet agreement. Interest expense was relatively unchanged and was approximately $15,000, $18,000 and $17,000 for the years 1997, 1996 and 1995, respectively. LIQUIDITY AND CAPITAL RESOURCES From inception to December 31, 1997, Cerus has financed its operations primarily through private placements of preferred and common equity securities, an initial public offering of common stock totaling approximately $60.1 million and project funding provided by Baxter totaling $19.2 million. During that period, the Company received approximately $3.1 million under United States government grants and approximately $2.6 million in interest income. At December 31, 1997, the Company had cash and cash equivalents of approximately $11.6 million and short-term investments of approximately $10.0 million. Net cash used in operating activities for 1997, 1996, and 1995 was approximately $16.6 million, $8.9 million and $3.4 million, respectively, resulting primarily from net losses. From inception through December 31, 1997, net cash used in investing activities of approximately $12.3 million resulted from short-term cash investments, purchases of furniture and equipment, and leasehold improvements. At December 31, 1997, the Company's net operating loss carryforwards were approximately $27.8 million and $14.1 million for federal and state income tax purposes, respectively. The Company's federal research and development tax credit carryforwards were approximately $1.2 million and $400,000 for federal and state income tax purposes, respectively, at December 31, 1997. The federal net operating loss and tax credit carryforwards expire at various dates from 2007 to 2012. The California state net operating loss expires in 2001 and 2002. The Tax Reform Act of 1986 and state tax statutes contain provisions relating to changes in ownership that may limit the utilization in any given year of available net operating loss carryforwards and research and development credits. See Note 6 of Notes to Financial Statements. The Company's future capital requirements and the adequacy of its available funds will depend on many factors, including progress of the platelet and FFP programs and the related clinical trials, progress of the red blood cell program, achievement of milestones leading to equity investments, regulatory approval and successful commercialization of the Company's pathogen inactivation systems, costs related to creating, maintaining and defending the Company's intellectual property position, and competitive developments. The Company believes that its available cash balances, together with anticipated cash flows from existing Baxter and grant arrangements, will be sufficient to meet its capital requirements for at least the next 12 months. In the event that additional capital is required, the Company may seek to raise that capital through public or private equity or debt financings or through additional collaborative arrangements or government grants. Future capital funding transactions may result in dilution to stockholders. There can be no assurance that such capital will be available on favorable terms, if at all. 25 28 IMPACT OF THE YEAR 2000 Computer programs using two rather than four digits to identify the year in a date field may cause computer systems to malfunction in the year 2000. Any computer programs that have time-related software may determine a date using "00" as the year 1900 rather than the year 2000. This could result in a system failure or miscalculations causing disruptions of operations, including, among other things, a temporary inability to engage in specific business activities. Based on a recent assessment, the Company has determined that it will be required to upgrade or replace a portion of its software so that its computer systems will function properly with respect to dates in the year 2000 and thereafter. The Company believes that, with upgrades of existing software and/or conversions to new software, the year 2000 issue will not pose significant operational problems for its business activities. The Company has initiated communications with its significant suppliers to determine the extent to which the Company's operations are vulnerable to those third parties' failure to solve their own year 2000 issues. The Company anticipates that its costs associated with the upgrade and/or conversion of existing computer software relating to the year 2000 issue is less than $100,000. There can be no assurance that the systems of other companies on which the Company relies will be converted on a timely basis and will not have an adverse effect on the Company's operations. Estimated costs were derived utilizing numerous assumptions of future events, including the continued availability of certain resources and other factors. However, there can be no assurance that these estimates will be achieved, and actual results could differ materially from those anticipated. Specific factors that might cause such material differences include, but are not limited to, the availability and cost of personnel trained in this area, the ability to locate and correct all relevant computer codes, and similar uncertainties. ITEM 8. FINANCIAL STATEMENTS AND SUPPLEMENTAL DATA The Company's financial statements, together with related notes and report of Ernst & Young, LLP, independent auditors, are listed in Item 14(a). ITEM 9. CHANGES IN AND DISAGREEMENTS WITH ACCOUNTANTS ON ACCOUNTING AND FINANCIAL DISCLOSURE Not applicable. 26 29 PART III ITEM 10. DIRECTORS AND EXECUTIVE OFFICERS OF THE REGISTRANT Information required by this item, insofar as it relates to directors, will be contained under the captions "Election of Directors" and "Compliance with Section 16(a) of the Securities Exchange Act of 1934" in the Company's definitive proxy statement with respect to the Company's 1998 Annual Meeting of Stockholders (the "Proxy Statement"), and is hereby incorporated by reference thereto. The information relating to executive officers of the Company is contained in Part I of this report. ITEM 11. EXECUTIVE COMPENSATION The information required by this item will be contained in the Proxy Statement under the caption "Executive Compensation," and is hereby incorporated by reference thereto. ITEM 12. SECURITY OWNERSHIP OF CERTAIN BENEFICIAL OWNERS AND MANAGEMENT The information required by this item will be contained in the Proxy Statement under the caption "Security Ownership of Certain Beneficial Owners and Management," and is hereby incorporated by reference thereto. ITEM 13. CERTAIN RELATIONSHIPS AND RELATED TRANSACTIONS The information required by this item will be contained in the Proxy Statement under the caption "Certain Relationships and Related Transactions," and is hereby incorporated by reference thereto. ITEM 14. EXHIBITS, FINANCIAL STATEMENT SCHEDULES AND REPORTS ON FORM 8-K The following documents are being filed as part of this report on Form 10-K: (a) Financial Statements. Report of Independent Auditors .........................................29 Balance Sheets as of December 31, 1997 and 1996.........................30 Statements of Operations for the three years ended December 31, 1997...........................................31 Statements of Stockholders' Equity for the three ended December 31, 1997.......................................32 Statements of Cash Flows for the three years ended December 31, 1997.................................................33 Notes to Financial Statements...........................................34
Other information is omitted because it is either presented elsewhere, is inapplicable or is immaterial as defined in the instructions. (b) No reports on Form 8-K were filed during the quarter ended December 31, 1997 27 30 (c) Exhibits
EXHIBIT NUMBER. DESCRIPTION OF EXHIBIT ------- ---------------------- 3.1(1) Registrant's Amended and Restated Certificate of Incorporation. 3.2(1) Registrant's Bylaws. 10.1(1) Form of Indemnity Agreement entered into between the Registrant and each of its directors and executive officers. 10.2(1) 1996 Equity Incentive Plan. 10.3(1) Form of Incentive Stock Option Agreement under the 1996 Equity Incentive Plan. 10.4(1) Form of Nonstatutory Stock Option Agreement under the 1996 Equity Incentive Plan. 10.5(1) 1996 Employee Stock Purchase Plan Offering. 10.7(1) Warrant Agreement, dated May 11, 1992, between the Registrant and Comdisco, Inc. to purchase Series A Preferred Stock. 10.8(1) Warrant Agreement, dated July 12, 1993, between the Registrant and Comdisco, Inc. to purchase Series B Preferred Stock. 10.9(1) Warrant Agreement, dated May 25, 1994, between the Registrant and Comdisco, Inc. to purchase Series C Preferred Stock. 10.10(1) Warrant Agreement, dated April 25, 1995, between the Registrant and Comdisco, Inc. to purchase Series D Preferred Stock. 10.11(1) Form of Warrant to purchase shares of Series B Preferred Stock of the Registrant. 10.12(1) Form of Warrant to purchase shares of Series C Preferred Stock of the Registrant. 10.13(1) Series D Preferred Stock Purchase Agreement, dated March 1, 1995, between the Registrant and certain investors. 10.14(1) Series E Preferred Stock Purchase Agreement, dated April 1, 1996, between the Registrant and Baxter Healthcare Corporation. 10.15(1) Common Stock Purchase Agreement, dated September 3, 1996 between the Registrant and Baxter Healthcare Corporation. 10.16(1) Amended and Restated Investors' Rights Agreement, dated April 1, 1996, among the Registrant and certain investors. 10.17+(1) Development, Manufacturing and Marketing Agreement, dated December 10, 1993 between the Registrant and Baxter Healthcare Corporation. 10.18+(1) Development, Manufacturing and Marketing Agreement, dated April 1, 1996, between the Registrant and Baxter Healthcare Corporation. 10.21(1) Industrial Real Estate Lease, dated October 1, 1992, between the Registrant and Shamrock Development Company, as amended on May 16, 1994 and December 21, 1995. 10.22(1) Real Property Lease, dated August 8, 1996, between the Registrant and S.P. Cuff. 10.23(1) Lease, dated February 1, 1996, between the Registrant and Holmgren Partners. 10.24(1) First Amendment to Common Stock Purchase Agreement, dated December 9, 1996, between the Registrant and Baxter Healthcare Corporation. 10.25+(1) Amendment, dated as of January 3, 1997, to the Agreement filed as Exhibit 10.17. 10.26(1) Memorandum of Agreement, dated as of January 3, 1997, between the Registrant and Baxter Healthcare Corporation. 10.27* License Agreement, dated as of November 30, 1992, by and among the Company, Miles Inc. and Diamond Scientific Corporation. 23.1 Consent of Ernst & Young LLP, Independent Auditors. 27.1 Financial Data Schedule.
- -------------- + Certain portions of this exhibit are subject to a confidential treatment order. (1) Incorporated by reference from the Company's Registration Statement on Form S-1 (File No. 333-11341) and amendments thereto. * Confidential treatment has been requested for certain portions of this exhibit. 28 31 Report of Ernst & Young LLP, Independent Auditors The Board of Directors and Stockholders Cerus Corporation We have audited the accompanying balance sheets of Cerus Corporation as of December 31, 1997 and 1996, and the related statements of operations, stockholders' equity, and cash flows for each of the three years in the period ended December 31, 1997. These financial statements are the responsibility of the Company's management. Our responsibility is to express an opinion on these financial statements based on our audits. We conducted our audits in accordance with generally accepted auditing standards. Those standards require that we plan and perform the audit to obtain reasonable assurance about whether the financial statements are free of material misstatement. An audit includes examining, on a test basis, evidence supporting the amounts and disclosures in the financial statements. An audit also includes assessing the accounting principles used and significant estimates made by management, as well as evaluating the overall financial statement presentation. We believe that our audits provide a reasonable basis for our opinion. In our opinion, the financial statements referred to above present fairly, in all material respects, the financial position of Cerus Corporation at December 31, 1997 and 1996, and the results of its operations and its cash flows for each of the three years in the period ended December 31, 1997 in conformity with generally accepted accounting principles. Ernst & Young LLP Walnut Creek, California January 27, 1998, except for Note 2 as to which the date is March 6, 1998 29 32 CERUS CORPORATION BALANCE SHEETS
DECEMBER 31, 1997 1996 ------------ ------------ ASSETS Current assets: Cash and cash equivalents .............................................. $ 11,603,596 $ 6,002,050 Short-term investments ................................................. 9,977,178 -- Accounts receivable from a related party ............................... 4,376,141 325,515 Other current assets ................................................... 214,687 206,015 -------------------------------- Total current assets ..................................................... 26,171,602 6,533,580 Furniture and equipment at cost: Laboratory and office equipment ........................................ 1,309,950 900,280 Leasehold improvements ................................................. 1,440,863 1,440,863 -------------------------------- 2,750,813 2,341,143 Less accumulated depreciation and amortization ......................... 1,718,984 1,156,918 -------------------------------- Net furniture and equipment .............................................. 1,031,829 1,184,225 Deferred financing costs ................................................. -- 969,142 Other assets ............................................................. 111,725 124,663 -------------------------------- Total assets ............................................................. $ 27,315,156 $ 8,811,610 ================================ LIABILITIES AND STOCKHOLDERS' EQUITY Current liabilities: Accounts payable ....................................................... $ 1,298,868 $ 391,267 Accrued compensation and related expenses .............................. 931,991 631,380 Accrued third-party toxicology and development expenses ................ 896,000 854,300 Accrued financing costs ................................................ -- 413,000 Other accrued expenses ................................................. 1,600,502 514,848 Deferred revenue ....................................................... -- 981,523 Current portion of capital lease obligations ........................... 69,816 94,130 -------------------------------- Total current liabilities ................................................ 4,797,177 3,880,448 Capital lease obligations, less current portion .......................... 43,002 92,127 Commitments and contingencies Stockholders' equity: Preferred stock, $.001 par value; 5,000,000 shares authorized: issuable in series; none issued and outstanding at December 31, 1997, 3,001,630 at December 31, 1996; aggregate liquidation preference of $24,485,277 at December 31, 1996 .. -- 3,002 Common stock, $.001 par value; 50,000,000 shares authorized: 9,183,629 and 1,928,807 shares issued and outstanding at December 31, 1997 and 1996, respectively ............... 9,184 1,929 Additional paid-in capital ............................................. 57,476,775 25,425,529 Deferred compensation .................................................. (140,937) (310,387) Notes receivable from stockholders ..................................... -- (75,206) Accumulated deficit .................................................... (34,870,045) (20,205,832) -------------------------------- Total stockholders' equity ............................................... 22,474,977 4,839,035 -------------------------------- Total liabilities and stockholders' equity ............................... $ 27,315,156 $ 8,811,610 ================================
See accompanying notes. 30 33 CERUS CORPORATION STATEMENTS OF OPERATIONS
YEARS ENDED DECEMBER 31, 1997 1996 1995 ------------------------------------------ Revenue: Licenses, milestones and development funding from a related party ............................................ $ 6,190,781 $ 2,851,236 $ 6,047,579 Government grants ........................................... 660,066 758,305 751,356 ------------------------------------------ Total revenue ................................................. 6,850,847 3,609,541 6,798,935 Operating expenses: Research and development .................................... 19,569,469 12,080,445 8,125,311 General and administrative .................................. 3,163,012 2,200,018 1,517,152 ------------------------------------------ Total operating expenses ...................................... 22,732,481 14,280,463 9,642,463 ------------------------------------------ Loss from operations .......................................... (15,881,634) (10,670,922) (2,843,528) Other income (expense): Interest income ............................................. 1,232,322 482,384 500,028 Interest expense ............................................ (14,901) (18,347) (16,821) ------------------------------------------ Total other income (expense) .................................. 1,217,421 464,037 483,207 ------------------------------------------ Net loss ...................................................... $(14,664,213) $(10,206,885) $ (2,360,321) ========================================== Net loss per share - basic and diluted ........................ $ (1.76) $ (5.98) $ (1.67) ========================================== Shares used in computing net loss per share - basic and diluted 8,351,872 1,705,821 1,415,969 ==========================================
See accompanying notes. 31 34 CERUS CORPORATION STATEMENTS OF STOCKHOLDERS' EQUITY
PREFERRED STOCK COMMON STOCK ADDITIONAL ------------------------------------------------------ PAID-IN SHARES AMOUNT SHARES AMOUNT CAPITAL --------------------------------------------------------------------- Balances at December 31, 1994 ....... 2,091,593 $ 2,092 1,413,272 $ 1,413 $ 13,154,907 Exercise of stock options ......... -- -- 4,623 5 1,681 Issuance of Series D convertible preferred stock, net of issuance costs of $60,806 ..... 529,084 529 -- -- 5,494,047 Issuance of warrants to purchase Series D preferred stock ...... -- -- -- -- 87,500 Net loss .......................... -- -- -- -- -- --------------------------------------------------------------------- Balances at December 31, 1995 ....... 2,620,677 2,621 1,417,895 1,418 18,738,135 Exercise of stock options ......... -- -- 510,912 511 258,411 Issuance of Series E convertible preferred stock, net of issuance costs of $100,777 .............. 380,953 381 -- -- 5,898,768 Payment on notes receivable ....... -- -- -- -- -- Deferred compensation ............. -- -- -- -- 530,215 Amortization of deferred compensation ................... -- -- -- -- -- Net loss .......................... -- -- -- -- -- --------------------------------------------------------------------- Balances at December 31, 1996 ....... 3,001,630 3,002 1,928,807 1,929 25,425,529 Public offering of common stock, net of expenses of $2,945,259 ......... -- -- 2,000,000 2,000 21,052,741 Issuance of common stock ............ -- -- 714,080 714 10,542,502 Conversion of preferred stock ....... (3,001,630) (3,002) 4,412,243 4,412 (3,118) Issuance of common stock under stock option and employee stock purchase plans and warrant exercises........ -- -- 129,664 130 459,754 Common shares reacquired ............ -- -- (1,165) (1) (633) Payment on notes receivable ......... -- -- -- -- -- Amortization of deferred compensation ................... -- -- -- -- -- Net loss ............................ -- -- -- -- -- --------------------------------------------------------------------- Balances at December 31, 1997 ....... -- $ -- 9,183,629 $ 9,184 $ 57,476,775 ===================================================================== NOTES RECEIVABLE TOTAL DEFERRED FROM ACCUMULATED STOCKHOLDERS' COMPENSATION STOCKHOLDERS DEFICIT EQUITY ---------------------------------------------------------- Balances at December 31, 1994 ....... $ -- $ (80,588) $ (7,638,626) $ 5,439,198 Exercise of stock options ......... -- -- -- 1,686 Issuance of Series D convertible preferred stock, net of issuance costs of $60,806 ..... -- -- -- 5,494,576 Issuance of warrants to purchase Series D preferred stock ...... -- -- -- 87,500 Net loss .......................... -- -- (2,360,321) (2,360,321) ---------------------------------------------------------- Balances at December 31, 1995 ....... -- (80,588) (9,998,947) 8,662,639 Exercise of stock options ......... -- -- -- 258,922 Issuance of Series E convertible preferred stock, net of issuance costs of $100,777 .............. -- -- -- 5,899,149 Payment on notes receivable ....... -- 5,382 -- 5,382 Deferred compensation ............. (530,215) -- -- -- Amortization of deferred compensation ................... 219,828 -- -- 219,828 Net loss .......................... -- -- (10,206,885) (10,206,885) ---------------------------------------------------------- Balances at December 31, 1996 ....... (310,387) (75,206) (20,205,832) 4,839,035 Public offering of common stock, net of expenses of $2,945,259 ......... -- -- -- 21,054,741 Issuance of common stock ............ -- -- -- 10,543,216 Conversion of preferred stock ....... -- -- -- (1,708) Issuance of common stock under stock option and employee stock purchase plans and warrant exercises -- -- -- 459,884 Common shares reacquired ............ -- -- -- (634) Payment on notes receivable ......... -- 75,206 -- 75,206 Amortization of deferred compensation ................... 169,450 -- -- 169,450 Net loss ............................ -- -- (14,664,213) (14,664,213) ---------------------------------------------------------- Balances at December 31, 1997 ....... $ (140,937) $ -- $(34,870,045) $ 22,474,977 ==========================================================
See accompanying notes. 32 35 CERUS CORPORATION STATEMENTS OF CASH FLOWS
YEARS ENDED DECEMBER 31, 1997 1996 1995 ------------------------------------------ OPERATING ACTIVITIES Net loss ........................................................... $(14,664,213) $(10,206,885) $ (2,360,321) Adjustments to reconcile net loss to net cash used in operating activities: Depreciation and amortization .................................... 562,066 477,551 369,267 Amortization of deferred compensation ............................ 169,450 219,828 -- Changes in operating assets and liabilities: Accounts receivable from a related party ....................... (4,050,626) (325,515) -- Other current assets .......................................... (8,672) 52,568 72,220 Other assets .................................................. 12,938 36,706 42,184 Accounts payable .............................................. 907,601 133,657 (246,115) Accrued compensation and related expenses ..................... 300,611 275,869 191,911 Accrued third-party toxicology and development expenses ....... 41,700 854,300 (586,383) Other accrued expenses ........................................ 1,085,654 472,500 31,582 Deferred revenue .............................................. (981,523) (918,981) (933,241) ------------------------------------------ Net cash used in operating activities .............................. (16,625,014) (8,928,402) (3,418,896) INVESTING ACTIVITIES Purchases of furniture and equipment ............................... (378,645) (164,960) (124,359) Purchases of short-term investments ................................ (31,977,178) -- -- Sale of short-term investments ..................................... 1,000,000 -- -- Maturities of short-term investments ............................... 21,000,000 -- -- ------------------------------------------ Net cash used in investing activities .............................. (10,355,823) (164,960) (124,359) FINANCING ACTIVITIES Net proceeds from sale of preferred stock .......................... -- 5,899,149 5,494,576 Proceeds from issuance of common stock ............................. 32,613,983 258,922 1,686 Payment of fractional shares on preferred stock conversion ......... (1,708) -- -- Repurchase of common stock ......................................... (634) -- -- Deferred financing costs ........................................... -- (556,142) -- Payments on notes receivable from shareholders ..................... 75,206 5,382 -- Payments on capital lease obligations .............................. (104,464) (170,916) (96,265) ------------------------------------------ Net cash provided by financing activities .......................... 32,582,383 5,436,395 5,399,997 ------------------------------------------ Net increase (decrease) in cash and cash equivalents ............... 5,601,546 (3,656,967) 1,856,742 Cash and cash equivalents, beginning of period ..................... 6,002,050 9,659,017 7,802,275 ------------------------------------------ Cash and cash equivalents, end of period ........................... $ 11,603,596 $ 6,002,050 $ 9,659,017 ========================================== Supplemental disclosures: Interest paid .................................................... $ 14,901 $ 18,347 $ 16,821 ========================================== Supplemental schedule of noncash investing and financing activities: Issuance of preferred stock warrants in connection with an operating lease line ........................................... $ -- $ -- $ 87,500 ========================================== Capital lease obligations incurred ............................... $ 31,025 $ 226,936 $ 79,624 ========================================== Deferred compensation related to stock option grants ............. $ -- $ 530,215 $ -- ========================================== Conversion of preferred stock to common stock .................... $ 24,534,998 $ -- $ -- ==========================================
See accompanying notes. 33 36 CERUS CORPORATION NOTES TO FINANCIAL STATEMENTS DECEMBER 31, 1997 1. THE COMPANY AND ITS SIGNIFICANT ACCOUNTING POLICIES BASIS OF PRESENTATION Cerus Corporation (the "Company") (formerly Steritech, Inc.), incorporated in California on September 19, 1991, is developing systems designed to improve the safety of blood transfusions by inactivating infectious pathogens in blood components used for transfusion (platelets, fresh frozen plasma ("FFP") and red blood cells) and inhibiting the leukocyte (white blood cell) activity that is responsible for certain adverse immune and other transfusion-related reactions. The Company has entered into two development and commercialization agreements with Baxter Healthcare Corporation ("Baxter") to develop, manufacture and market these pathogen inactivation systems (see Note 2). The Company has not received any revenues from product sales, and all revenues recognized by the Company to date have resulted from the Company's agreements with Baxter and federal research grants. The Company will be required to conduct significant research, development, testing and regulatory compliance activities on its pathogen inactivation systems that, together with anticipated general and administrative expenses, are expected to result in substantial additional losses in future periods. The Company's ability to achieve a profitable level of operations will depend on successfully completing development, obtaining regulatory approvals and achieving market acceptance of its pathogen inactivation systems. There can be no assurance that the Company will ever achieve a profitable level of operations. USE OF ESTIMATES The preparation of financial statements in conformity with generally accepted accounting principles requires management to make estimates and assumptions that affect the reported amounts of assets and liabilities at the date of the financial statements and the reported amounts of revenues and expenses during the reporting period. Actual results could differ from those estimates. REVENUES AND RESEARCH AND DEVELOPMENT EXPENSES Revenues related to the cost reimbursement provisions under development contracts are recognized as the costs on the project are incurred. Revenues related to milestones specified under development contracts are recognized as the milestones are achieved. Prepaid license fees, included in deferred revenue, are recognized as revenues upon achievement of milestones. Research and development costs are expensed as incurred. The Company receives certain United States government grants which support the Company's research effort in defined research projects. These grants generally provide for reimbursement of approved costs incurred as defined in the various grants. Revenues associated with these grants are recognized as costs under each grant are incurred. CASH, CASH EQUIVALENTS, AND SHORT-TERM INVESTMENTS The Company considers all highly liquid investments with original maturities less than three months to be cash and cash equivalents. Cash equivalents consist principally of short-term money market instruments and commercial paper. 34 37 CERUS CORPORATION NOTES TO FINANCIAL STATEMENTS (CONTINUED) 1. THE COMPANY AND ITS SIGNIFICANT ACCOUNTING POLICIES (CONTINUED) CASH, CASH EQUIVALENTS, AND SHORT-TERM INVESTMENTS (CONTINUED) In accordance with Statement of Financial Accounting Standards No. 115, "Accounting for Certain Investments in Debt and Equity Securities," the Company has classified all debt securities as available-for-sale at the time of purchase and re-evaluates such designation as of each balance sheet date. The available-for-sale securities recorded at December 31, 1997 totaled $21,580,375. Unrealized gains and losses at December 31, 1997 and 1996 and realized gains and losses for the years then ended were not material. Accordingly, the Company has not made a provision for such amounts in its balance sheets. The cost of securities sold is based on the specific identification method. Substantially, all of the Company's cash, cash equivalents, and short-term investments are maintained by three major financial institutions. FURNITURE AND EQUIPMENT Furniture and equipment are stated at cost less accumulated depreciation and amortization. Depreciation on furniture and equipment is calculated on a straight-line basis over the estimated useful lives of the assets (principally five years for laboratory equipment and furniture and three years for office equipment). Leasehold improvements are amortized on a straight-line basis over the shorter of the lease term or the estimated useful lives of the improvements. STOCK-BASED COMPENSATION In October 1995, the Financial Accounting Standards Board issued Statement of Financial Accounting Standards No. 123, "Accounting for Stock-Based Compensation" ("FAS 123"). The Company adopted FAS 123 in 1996. The Company accounts for employee stock options in accordance with Accounting Principles Board Opinion No. 25 and has adopted the "disclosure only" alternative described in FAS 123. INCOME TAXES The Company accounts for income taxes based upon Financial Accounting Standards Board Statement No. 109, "Accounting for Income Taxes." Under this method, deferred tax assets and liabilities are determined based on differences between the financial reporting and tax bases of assets and liabilities and are measured using the enacted tax rates and laws that will be in effect when the differences are expected to reverse. NET LOSS PER SHARE - BASIC AND DILUTED In February 1997, the Financial Accounting Standards Board issued Statement No. 128, "Earnings Per Share" ("FAS 128"), which is required to be adopted for the period ended December 31, 1997. FAS 128 replaced the calculation of primary and fully diluted net income (loss) per share with basic and diluted net income (loss) per share. Unlike primary net income (loss) per share, basic net income (loss) per share excludes any dilutive effects of options, warrants and convertible securities. 35 38 CERUS CORPORATION NOTES TO FINANCIAL STATEMENTS (CONTINUED) 1. THE COMPANY AND ITS SIGNIFICANT ACCOUNTING POLICIES (CONTINUED) NET LOSS PER SHARE - BASIC AND DILUTED (CONTINUED) In February 1998, Staff Accounting Bulletin No. 98 ("SAB 98") was issued and amends the existing Securities and Exchange Commission staff guidance primarily to give effect to FAS 128. Topic 4.D of SAB 98 revises the instructions regarding the dilutive effects of stock issued for consideration below the initial public offering ("IPO") price or options and warrants to purchase common stock with exercise prices below the IPO price, previously referred to as cheap stock. The new guidance highlights the treatment that should be given to the dilutive effect of common stock or options and warrants to purchase common stock issued for nominal consideration. All net loss per share amounts for all periods have been presented, and where appropriate, restated to conform to the FAS 128 and SAB 98 requirements. Common stock equivalent shares from convertible preferred stock and from stock options and warrants are not included as the effect is anti-dilutive. PRO FORMA NET LOSS PER SHARE Pro forma net loss per share is computed as described above and also gives effect, even if anti-dilutive, to common equivalent shares from convertible preferred shares that automatically converted to common shares upon the closing of the Company's initial public offering (using the as-if-converted method):
YEARS ENDED DECEMBER 31, 1997 1996 1995 ------------------------------------------ Net loss ...................................................... $(14,664,213) $(10,206,885) $ (2,360,321) ========================================== Weighted average common shares outstanding .................... 8,351,872 1,705,821 1,415,969 Adjustment to reflect the effect of the assumed conversion of convertible preferred stock ................................ -- 4,202,396 3,722,770 ------------------------------------------ Shares used in computing pro forma basic and diluted net loss per share .................................................. 8,351,872 5,908,217 5,138,739 ========================================== Pro forma basic and diluted net loss per share ................ $ (1.76) $ (1.73) $ (0.46) ==========================================
Common stock equivalents are excluded from the diluted net loss per share calculation, as the effect is anti-dilutive. RECLASSIFICATION OF PRIOR YEAR BALANCES Certain balances in the 1996 financial statements have been reclassified to conform to the current year financial statement presentation. 36 39 CERUS CORPORATION NOTES TO FINANCIAL STATEMENTS (CONTINUED) 1. THE COMPANY AND ITS SIGNIFICANT ACCOUNTING POLICIES (CONTINUED) IMPACT OF RECENTLY ISSUED ACCOUNTING STANDARDS In June 1997, the Financial Accounting Standards Board issued Statement No. 130, "Reporting Comprehensive Income" ("FAS 130"), and Statement No. 131, "Disclosure about Segments of an Enterprise and Related Information" ("FAS 131"). The Company is required to adopt these statements in fiscal year 1999. FAS 130 establishes new standards for reporting and displaying comprehensive income and its components. FAS 131 requires disclosure of certain information regarding operating segments, products and services, geographic areas of operation and major customers. Adoption of these statements is not expected to have a significant impact on the Company's consolidated financial position, results of operations or cash flows. 2. LICENSING AGREEMENTS WITH BAXTER HEALTHCARE CORPORATION, A RELATED PARTY OF THE COMPANY In December 1993, Cerus entered into a development and commercialization agreement with Baxter Healthcare Corporation ("Baxter") to develop a system for inactivation of pathogens in platelets. The agreement provides for Baxter to make an equity investment and certain up-front license and milestone payments. The agreement further provides for Baxter and the Company to generally share system development costs equally, subject to mutually agreed budgets established from time to time. The agreement also provides for a sharing of revenue from sales of inactivation system disposables, after each party is reimbursed for its cost of goods above a specified level. In January 1997, the Company and Baxter amended the agreement to provide that the Company would receive an additional 2.2% of the adjusted product revenue from the sale of the platelet pathogen inactivation system disposables in return for payment by the Company to Baxter of $5.5 million in 1997 in four equal quarterly installments for development costs. In January and July 1995, Cerus received approximately $2.6 million from Baxter in connection with interim funding agreements related to the development of pathogen inactivation systems for plasma for transfusions (fresh frozen plasma or "FFP") and red blood cells. In April 1996, Cerus entered into a second development and commercialization agreement with Baxter, principally focused on the FFP and red blood cell pathogen inactivation systems. The agreement provides for Baxter to make certain equity investments, including two future milestone-based investments of $5 million each at 120% of the market price at the time of the investment. The agreement further provides for Baxter and the Company to generally share development costs of the systems equally, subject to mutually agreed budgets established from time to time. The agreement also provides for the Company and Baxter to share gross profits from the sale of inactivation system disposables, after deducting from such gross profits a specified percentage allocation to be retained by the marketing party for marketing and administration expenses. Through December 31, 1997, Baxter has paid the Company up-front license fees and milestone and development payments totaling $14.8 million and has invested $17.5 million in the capital stock of the Company under the agreements described above. 37 40 CERUS CORPORATION NOTES TO FINANCIAL STATEMENTS (CONTINUED) 2. LICENSING AGREEMENTS WITH BAXTER HEALTHCARE CORPORATION, A RELATED PARTY OF THE COMPANY (CONTINUED) Under the agreements, all research, development, preclinical and clinical costs of the pathogen inactivation projects are shared by Cerus and Baxter. Because more of such research and development is typically performed internally at Cerus than at Baxter and because Cerus is generally responsible for engaging third parties to perform certain aspects of these projects, the Company's research and development expenses have exceeded its share of expenses. As a result, the Company has recognized revenue from Baxter, giving rise to a receivable due from Baxter and corresponding periodic balancing payments to the Company. At December 31, 1997, the amount of the Baxter receivable was approximately $4.4 million. On February 13, 1998, Baxter paid Cerus approximately $4.5 million in satisfaction of such receivable and anticipated adjustments. Through December 31, 1997, the Company had recognized approximately $19.2 million in revenue under its agreements with Baxter, including the license fee and milestone and development funding amounts described above. In March 1998, the Company and Baxter entered into an amendment to the April 1996 agreement providing that, to the extent the approved spending for 1998 for the red blood cell project exceeds $7.3 million, Cerus will fund all expenses for the red blood cell project in 1998 in excess of such amount, up to the amount of the approved budget. To compensate Cerus for such excess expenditures, Baxter will fully fund the first expenditures under the approved budget for the red blood cell project for 1999 in an amount equal to such excess expenditures, after which the parties shall equally share the expenses of the red blood cell project. If for any reason there is not an approved budget for the red blood cell project for 1999, Baxter will fully fund the first expenditures for 1999 under the approved budget for such other Cerus-Baxter program or programs as Cerus shall designate in an amount equal to the Cerus 1998 excess expenditures. If by July 1, 1999, however, there is not an approved budget for such other Cerus-Baxter program or programs that is at least equal to such excess expenditures, Baxter will promptly pay to Cerus one-half of the amount by which the excess expenditures exceed the amount of expenditures to be funded by Baxter. Cerus anticipates that the expenditures for 1998 for the FFP program will exceed the previously approved budget. Cerus and Baxter are in discussion regarding the level of funding each company will support in 1998 for the FFP program. As of December 31, 1997, Baxter owned 1,457,830 shares of the common stock of the Company, representing approximately 15.9% of the outstanding common stock of the Company. Baxter has agreed that it will not at any time, nor will it permit any of its affiliates, to own capital stock of the Company having 20.1% or more of the outstanding voting power of the Company. Such restrictions on stock purchases will not apply in the event a third party makes a tender offer for a majority of the outstanding voting securities of the Company or if the Board of Directors of the Company determines to liquidate or sell to a third party substantially all of the assets or a majority of the voting securities of the Company or to approve a merger or consolidation in which the Company's stockholders will not own a majority of the voting securities of the surviving entity. Revenue relating to licenses, milestones and development funding for all periods presented are as a result of agreements with Baxter. 38 41 CERUS CORPORATION NOTES TO FINANCIAL STATEMENTS (CONTINUED) 3. INVESTMENTS Investments classified as available-for-sale as of December 31, 1997, were as follows: Money market mutual funds ...................... $ 4,584,900 U.S. government obligations .................... 5,092,388 Commercial paper ............................... 11,903,086 ------------ Total investments .............................. 21,580,374 Less: amounts classified as cash equivalents ... (11,603,196) ============ Short-term investments ......................... $ 9,977,178 ============
4. COMMITMENTS AND CONTINGENCIES The Company leases its office facilities and certain equipment under non-cancelable operating leases with initial terms in excess of one year which require the Company to pay operating costs, property taxes, insurance and maintenance. These facility leases generally contain renewal options and provisions adjusting the lease payments. Capital lease obligations represent the present value of future rental payments under capital lease agreements for laboratory and office equipment. The original cost and accumulated amortization on the equipment under capital leases is $444,994 and $269,941, respectively, at December 31, 1997 and $413,969 and $161,472, respectively, at December 31, 1996. Future minimum payments under capital and operating leases are as follows:
CAPITAL OPERATING YEAR ENDING DECEMBER 31, LEASES LEASES ------------------ 1998 ...................................... $ 78,648 $529,009 1999 ...................................... 32,541 172,205 2000 ...................................... 8,389 -- 2001 ...................................... 4,893 -- ------------------ Total minimum lease payments .............. 124,471 $701,214 ======== Amount representing interest ............... 11,653 -------- Present value of net minimum lease payments 112,818 Current portion ............................ 69,816 -------- Long-term portion .......................... $ 43,002 ========
Rent expense for office facilities and certain equipment was $677,501, $732,302 and $801,632 for the years ended December 31, 1997, 1996 and 1995, respectively. 39 42 CERUS CORPORATION NOTES TO FINANCIAL STATEMENTS (CONTINUED) 4. COMMITMENTS AND CONTINGENCIES (CONTINUED) TRADE SECRET MATTER In August 1996, an Australian entity, alleged that its unspecified trade secrets and know-how were used in the development by the Company of unspecified compounds for the Company's red blood cell program without its consent. This entity has indicated that it is seeking compensation in the form of royalties or a lump-sum payment. Based on its investigation of the matter to date, the Company believes that the claims are without merit. However, any future litigation involving these allegations would be subject to inherent uncertainties, especially in cases where complex technical issues are decided by a lay jury. There can be no assurance that, if a lawsuit were commenced, it would not be decided against the Company, in which case, settlement of this claim could have a material adverse effect upon the Company's business, financial condition and results of operations. 5. STOCKHOLDERS' EQUITY COMMON STOCK On January 30, 1997, the Company completed an initial public offering of 2,000,000 shares of common stock at $12.00 per share. The Company received net proceeds of $21.1 million. In conjunction with the initial public offering, the Company sold an additional 496,878 shares of its common stock to Baxter for an aggregate purchase price of approximately $5.5 million. Additionally, at the time of the initial public offering, 33,315 warrants to purchase 47,605 shares of common stock were exercised for an aggregate price paid to the Company of $183,000 and Series A, B, C, D and E preferred stock converted into 4,412,243 shares of common stock. 1992 STOCK OPTION PLAN In January 1992, the Company's Board of Directors approved the 1992 Stock Option Plan (the "Plan"), which provides for the grant of stock options to purchase up to 588,000 of the Company's common stock. On May 18, 1996, the Company's Board of Directors approved an increase in the number of shares of common stock reserved for issue under the Plan from 588,000 to 1,029,000. Under the Plan, two types of options may be granted: Incentive Stock Options ("ISOs") and Non-Qualified Stock Options ("NQSOs"). The ISOs may be granted at a price per share not less than the fair market value at the date of grant. The NQSOs may be granted at a price per share not less than 85% of the fair market value at the date of grant. The option term is 10 years. Vesting, as determined by the Board of Directors, generally occurs ratably over four years. In the event option holders cease to be employed by the Company, except in the event of death or disability or as otherwise provided in the option grant, all unvested options are forfeited and all vested options must be exercised within a three-month period, otherwise the options are forfeited. Options granted are immediately exercisable and unvested (but issued) shares are subject to repurchase by the Company if the holder is no longer employed by the Company. As of December 31, 1997, 84,173 shares were subject to this repurchase provision. On July 24, 1996, the Company adopted the 1996 Equity Incentive plan (the "Incentive Plan") (approved by the stockholders in January 1997) as an amendment and restatement of the Company's 1992 Stock Option Plan, and reserved an additional 441,000 shares of common stock for issuance thereunder. The Incentive Plan provides for grants of incentive stock options to employees and non statutory stock options, restricted stock purchase awards, stock appreciation rights and stock bonuses to employees, directors and consultants of the Company. 40 43 CERUS CORPORATION NOTES TO FINANCIAL STATEMENTS (CONTINUED) 5. STOCKHOLDERS' EQUITY (CONTINUED) 1992 STOCK OPTION PLAN (CONTINUED) The Company has elected to follow Accounting Principles Board Opinion No. 25, "Accounting for Stock Issued to Employees" ("APB 25") and related interpretations in accounting for its employee stock options because, as discussed below, the alternative fair value accounting provided for under FAS 123 requires the use of option valuation models that were not developed for use in valuing employee stock options. "As adjusted" information regarding net loss and net loss per share is required by FAS 123, and has been determined as if the Company had accounted for its employee stock options and grants under its employee stock purchase plan described below under the fair value method of that Statement. The fair value for these options and shares was estimated at the date of grant using a Black-Scholes option pricing model with the following weighted-average assumptions:
YEARS ENDED DECEMBER 31, 1997 1996 1995 --------------------------------- Expected dividend yield .......... 0% 0% 0% Expected volatility .............. .7424 .5925 .5925 Risk-free interest rate .......... 6.13% 5.09% 5.35% Expected life of the option ...... 2.6 years 5 years 5 years
The Black-Scholes option valuation model was developed for use in estimating the fair value of traded options which have no vesting restrictions and are fully transferable. In addition, option valuation models require the input of highly subjective assumptions including the expected stock price volatility. Because the Company's employee stock options and purchased shares have characteristics significantly different from those of traded options, and because changes in the subjective input assumptions can materially affect the fair value estimate, in management's opinion, the existing models do not necessarily provide a reliable single measure of the fair value of its employee stock options. For purposes of "as adjusted" disclosures, the estimated fair value of the options is amortized to expense over the options' vesting period. The effects of applying FAS 123 on "as adjusted" net loss are not likely to be representative of the effects on reported net loss/income for future years. The Company's reported and "as adjusted" information at December 31 follows:
1997 1996 1995 ---------------------------------------------------- Net loss, as reported ............................... $(14,664,213) $(10,206,885) $(2,360,321) Net loss, "as adjusted" ............................. (14,966,213) (10,509,885) (2,365,321) Net loss per share - basic and diluted, as reported . (1.76) (5.98) (1.67) Net loss per share - basic and diluted, "as adjusted" (1.79) (6.16) (1.67)
41 44 CERUS CORPORATION NOTES TO FINANCIAL STATEMENTS (CONTINUED) 5. STOCKHOLDERS' EQUITY (CONTINUED) 1992 STOCK OPTION PLAN (CONTINUED) Activity under the Plan is set forth below:
OUTSTANDING OPTIONS ---------------------------------- WEIGHTED AVERAGE NUMBER OF PRICE EXERCISE PRICE OF SHARES PER SHARE SHARES UNDER PLAN --------------------------------------------------------- Balances at December 31, 1994................................... 498,256 $ .262- .544 Granted....................................................... 75,705 .714 $ .714 Cancelled..................................................... (63,290) .262- .544 .376 Exercised..................................................... (4,623) .262- .544 .365 --------------------------------------------------------- Balances at December 31, 1995................................... 506,048 .262- .714 .670 Granted....................................................... 418,126 .714- 10.200 2.533 Cancelled..................................................... (5,879) .714 .714 Exercised..................................................... (510,912) .262- 2.721 .344 --------------------------------------------------------- Balances at December 31, 1996................................... 407,383 .262- 10.200 2.523 Granted....................................................... 51,300 10.625- 23.875 14.925 Cancelled..................................................... (10,184) .544- 4.082 3.240 Exercised..................................................... (61,229) .262- 2.721 1.045 --------------------------------------------------------- Balances at December 31, 1997................................... 387,270 $ .262- 23.875 $ 4.380 =========================================================
The weighted average fair value of options granted during 1997, 1996 and 1995 is $8.107, $6.005 and $1.024 per share, respectively. Options to purchase 507,116 shares of common stock were available for future grant. The following table summarizes information concerning outstanding and vested options as of December 31, 1997:
OPTIONS OUTSTANDING OPTIONS VESTED ---------------------------------------------------- --------------------------------------- WEIGHTED AVERAGE WEIGHTED NUMBER OF REMAINING WEIGHTED AVERAGE NUMBER OF AVERAGE RANGE OF EXERCISE PRICES SHARES CONTRACTUAL LIFE EXERCISE PRICE SHARES EXERCISE PRICE - ------------------------------ --------------- ----------------- ------------------ --------------------------------------- $0.262-0.7143............. 47,033 6.82 $ 0.611 38,247 $ 0.598 $2.721-4.082.............. 274,605 8.38 $ 2.814 106,879 $ 2.801 $8.163-14.000............. 53,932 9.28 $ 11.952 9,962 $ 10.877 $19.000-23.875............ 11,700 9.87 $ 21.375 - - =============== ================= ================== ======================================= 387,270 8.36 $ 4.380 155,088 $ 2.776 =============== ================= ================== =======================================
42 45 CERUS CORPORATION NOTES TO FINANCIAL STATEMENTS (CONTINUED) 5. STOCKHOLDERS' EQUITY (CONTINUED) The Company recognized deferred compensation of $530,215 for the difference between the exercise price and deemed fair value of certain stock options granted during the year ended December 31, 1996. This amount is being amortized by periodic charges to operations over the four year vesting periods of the individual options. Amortization expense related to deferred compensation totaled $169,450 and $219,828 for the years ended December 31, 1997 and 1996, respectively. EMPLOYEE STOCK PURCHASE PLAN On July 24, 1996, the Company's Board of Directors approved the Employee Stock Purchase Plan (the "Purchase Plan") (approved by the stockholders in January 1997) covering an aggregate 220,500 shares of common stock. The Purchase Plan is intended to qualify as an employee stock purchase plan within the meaning of Section 423(b) of the Code. Under the Purchase Plan, the Board of Directors may authorize participation by eligible employees, including officers, in periodic offerings following the adoption of the Purchase Plan. The offering period for any offering will be no more than 27 months. During 1997, employees purchased 20,830 shares under the Purchase Plan. At December 31, 1997, 199,670 shares were available for issuance. WARRANTS The Company had the following warrants issued in connection with an operating lease line, outstanding at December 31, 1997 to purchase shares of common stock, all of which expire in February 2002:
NUMBER EXERCISE DATE OF SHARES PRICE PER SHARE ISSUED --------- --------------- ------ 30,545 $2.62 May 1992 5,805 $3.45 July 1993 9,187 $5.44 May 1994 6,615 $7.14 April 1995 ============== 52,152 ==============
43 46 CERUS CORPORATION NOTES TO FINANCIAL STATEMENTS (CONTINUED) 5. STOCKHOLDERS' EQUITY (CONTINUED) REINCORPORATION AND STOCK SPLIT On January 14, 1997, the Company effected a stock split of all outstanding shares of common stock such that each share of common stock will be split into 1.47 shares of common stock. All common shares in the accompanying financial statements have been retroactively adjusted to reflect the stock split. In connection with the stock split, the conversion and exercise provisions of the outstanding shares of preferred stock, stock options and warrants have been adjusted accordingly. At the same time, the Board authorized the Company to proceed with the reincorporation of the Company into Delaware. Upon the reincorporation, the authorized stock of the Company became 5,000,000 shares of preferred stock, par value $.001 per share, and 50,000,000 shares of common stock, par value $.001 per share. Also upon reincorporation, the Board of Directors received the authority, without further action by the stockholders, to issue up to 5,000,000 shares of preferred stock in one or more series and to fix the designations, powers, preferences, privileges, and relative participating, optional or special rights and the qualifications, limitations, or restrictions thereof, including dividend rights, conversion rights, voting rights, and terms of redemption and liquidation preferences, any or all of which may be greater than the rights of the common stock. 6. INCOME TAXES Deferred income taxes reflect the net tax effects of temporary differences between the carrying amounts of assets and liabilities for financial reporting purposes and the amounts used for income tax purposes. Significant components of the Company's deferred tax assets are as follows:
DECEMBER 31, 1997 1996 ----------------------------- Net operating loss carryforward .......................... $ 10,200,000 $ 6,400,000 Research and development credit carryforward ............. 1,600,000 800,000 Certain expenses not currently deductible for tax purposes 2,200,000 -- Deferred revenue ......................................... -- 400,000 Capitalized research and development ..................... 900,000 400,000 Other .................................................... 300,000 200,000 ----------------------------- Gross deferred tax assets ................................ 15,200,000 8,200,000 Valuation allowance ...................................... (15,200,000) (8,200,000) ----------------------------- Net deferred tax assets .................................. $ -- $ -- =============================
44 47 CERUS CORPORATION NOTES TO FINANCIAL STATEMENTS (CONTINUED) 6. INCOME TAXES (CONTINUED) The valuation allowance increased by $7,000,000 and $4,000,000 for the fiscal years ended in 1997 and 1996, respectively. The increase is primarily attributable to the increase in the net operating loss and tax credit carryforwards. The Company believes that, based on a number of factors, the available objective evidence creates sufficient uncertainty regarding the realizability of the deferred tax assets such that a full valuation allowance has been recorded. These factors include the Company's history of net losses since its inception, the need for FDA approval of the Company's products prior to commercialization, expected near-term future losses, the nature of the Company's deferred tax assets, the lack of firm sales backlog, no significant excess of appreciated asset value over the tax basis of the Company's net assets and the absence of taxable income in prior carryback years. The valuation allowance at December 31, 1997 includes $400,000 related to deferred tax assets arising from tax benefits associated with stock option plans. This benefit, when realized, will be recorded as an increase in stockholders' equity rather than as a reduction in the income tax provision. Although management's operating plans assume, beyond the near-term, taxable and operating income in future periods, management's evaluation of all available information in assessing the realizability of the deferred tax assets in accordance with Financial Accounting Standards Board Statement No. 109, "Accounting for Income Taxes," indicates that such plans are subject to considerable uncertainty. Therefore, the valuation allowance has been increased to fully reserve the Company's deferred tax assets. The Company will continue to assess the realizability of the deferred tax assets based on actual and forecasted operating results. At December 31, 1997, the Company had net operating loss carryforwards of approximately $27,800,000 for federal and $14,100,000 for state income tax purposes. The Company also had research and development tax credit carryforwards of approximately $1,200,000 for federal income tax purposes and approximately $400,000 for state income tax purposes at December 31, 1997. The federal net operating loss and tax credit carryforwards expire between the years 2007 and 2012. The state net operating loss carryforward expires in 2001 and 2002. Utilization of the Company's net operating losses and credits are subject to a substantial annual limitation due to the ownership change limitations provided by the Internal Revenue Code. The annual limitation may result in the expiration of net operating losses and credits before utilization. 7. RETIREMENT PLAN The Company maintains a defined contribution savings plan (the "401(k) Plan") that qualifies under the provisions of Section 401(k) of the Internal Revenue Code and covers all employees of the Company. Under the terms of the 401(k) Plan, employees may contribute varying amounts of their annual compensation. The Company may contribute a discretionary percentage of qualified individual employee's salaries, as defined, to the 401(k) Plan. Company contributions of $3,500 and $2,700 were charged to operations in 1996 and 1995, respectively, in order to cover certain costs of the 401(k) Plan. The Company did not contribute to the 401(k) Plan in 1997. 45 48 SIGNATURES Pursuant to the requirement of Section 13 or 15(d) of the Securities Exchange Act of 1934, the Registrant has duly caused this report to be signed on its behalf by the undersigned, thereunto duly authorized, in the City of Concord, State of California, on the 6th day of March, 1998. CERUS CORPORATION By: /s/ Stephen T. Isaacs -------------------------------------- Stephen T. Isaacs President and Chief Executive Officer Pursuant to the requirements of the Securities Act of 1933, this report has been signed below by the following persons in the capacities and on the dates indicated.
SIGNATURE TITLE DATE --------- ----- ---- /s/ Stephen T. Isaacs President, Chief Executive Officer and March 6, 1998 - --------------------------------- Stephen T. Isaacs Director (Principal Executive Officer) March 6, 1998 /s/ David S. Clayton Vice President, Finance and Chief Financial March 6, 1998 - --------------------------------- David S. Clayton Officer (Principal Financial and Accounting Officer) March 6, 1998 /s/ B. J. Cassin Chairman of the Board - --------------------------------- B. J. Cassin /s/ John E. Hearst Director March 6, 1998 - --------------------------------- John E. Hearst /s/ Peter H. McNerney Director March 6, 1998 - --------------------------------- Peter H. McNerney /s/ Dale A. Smith Director March 6, 1998 - --------------------------------- Dale A. Smith /s/ Henry E. Stickney Director March 6, 1998 - --------------------------------- Henry E. Stickney
49 EXHIBIT INDEX
EXHIBIT NUMBER. DESCRIPTION OF EXHIBIT ------- ---------------------- 3.1(1) Registrant's Amended and Restated Certificate of Incorporation. 3.2(1) Registrant's Bylaws. 10.1(1) Form of Indemnity Agreement entered into between the Registrant and each of its directors and executive officers. 10.2(1) 1996 Equity Incentive Plan. 10.3(1) Form of Incentive Stock Option Agreement under the 1996 Equity Incentive Plan. 10.4(1) Form of Nonstatutory Stock Option Agreement under the 1996 Equity Incentive Plan. 10.5(1) 1996 Employee Stock Purchase Plan Offering. 10.7(1) Warrant Agreement, dated May 11, 1992, between the Registrant and Comdisco, Inc. to purchase Series A Preferred Stock. 10.8(1) Warrant Agreement, dated July 12, 1993, between the Registrant and Comdisco, Inc. to purchase Series B Preferred Stock. 10.9(1) Warrant Agreement, dated May 25, 1994, between the Registrant and Comdisco, Inc. to purchase Series C Preferred Stock. 10.10(1) Warrant Agreement, dated April 25, 1995, between the Registrant and Comdisco, Inc. to purchase Series D Preferred Stock. 10.11(1) Form of Warrant to purchase shares of Series B Preferred Stock of the Registrant. 10.12(1) Form of Warrant to purchase shares of Series C Preferred Stock of the Registrant. 10.13(1) Series D Preferred Stock Purchase Agreement, dated March 1, 1995, between the Registrant and certain investors. 10.14(1) Series E Preferred Stock Purchase Agreement, dated April 1, 1996, between the Registrant and Baxter Healthcare Corporation. 10.15(1) Common Stock Purchase Agreement, dated September 3, 1996 between the Registrant and Baxter Healthcare Corporation. 10.16(1) Amended and Restated Investors' Rights Agreement, dated April 1, 1996, among the Registrant and certain investors. 10.17+(1) Development, Manufacturing and Marketing Agreement, dated December 10, 1993 between the Registrant and Baxter Healthcare Corporation. 10.18+(1) Development, Manufacturing and Marketing Agreement, dated April 1, 1996, between the Registrant and Baxter Healthcare Corporation. 10.21(1) Industrial Real Estate Lease, dated October 1, 1992, between the Registrant and Shamrock Development Company, as amended on May 16, 1994 and December 21, 1995. 10.22(1) Real Property Lease, dated August 8, 1996, between the Registrant and S.P. Cuff. 10.23(1) Lease, dated February 1, 1996, between the Registrant and Holmgren Partners. 10.24(1) First Amendment to Common Stock Purchase Agreement, dated December 9, 1996, between the Registrant and Baxter Healthcare Corporation. 10.25+(1) Amendment, dated as of January 3, 1997, to the Agreement filed as Exhibit 10.17. 10.26(1) Memorandum of Agreement, dated as of January 3, 1997, between the Registrant and Baxter Healthcare Corporation. 10.27* License Agreement, dated as of November 30, 1992, by and among the Company, Miles Inc. and Diamond Scientific Corporation. 23.1 Consent of Ernst & Young LLP, Independent Auditors. 27.1 Financial Data Schedule.
- -------------- + Certain portions of this exhibit are subject to a confidential treatment order. (1) Incorporated by reference from the Company's Registration Statement on Form S-1 (File No. 333-11341) and amendments thereto. * Confidential treatment has been requested for certain portions of this exhibit. EX-10.27 2 LICENSE AGREEMENT DATED NOVEMBER 30, 1992 1 EXHIBIT 10.27 ***TEXT OMITTED AND FILED SEPARATELY CONFIDENTIAL TREATMENT REQUESTED UNDER 17.C.F.R. Sections 200.80(B)(4), 200.83 AND 240.24B-2 LICENSE AGREEMENT THIS Agreement, effective November 30, 1992 by and between STERITECH, INC., a California corporation, having a principal office at 2341 Stanwell Drive, Concord, California 94520 ("STERITECH") and MILES INC., an Indiana corporation, Mobay Road, Pittsburgh, Pennsylvania 15205 ("MILES"), and DIAMOND SCIENTIFIC CORPORATION, a ________ corporation, ("DIAMOND"). WITNESSETH THAT: WHEREAS, United States Patent No. 4,545,987, entitled: "PSORALEN INACTIVATED DOUBLE-STRANDED RNA VIRAL VACCINES", was issued on October 8, 1985, a copy of which is attached hereto as Exhibit A; WHEREAS, United States Patent No. 4,693,981, entitled: "PREPARATION OF INACTIVATED VIRAL VACCINES", was issued on September 15, 1987, a copy of which is attached hereto as Exhibit B; WHEREAS, United States Patent No. 4,727,027, entitled: "PHOTOCHEMICAL DECONTAMINATION TREATMENT OF WHOLE BLOOD OR BLOOD COMPONENTS", was issued on February 23, 1988, a copy of which is attached hereto as Exhibit C; WHEREAS, United States Patent No. 4,748,120, entitled: "PHOTOCHEMICAL DECONTAMINATION TREATMENT OF WHOLE BLOOD OR BLOOD COMPONENTS", was issued on May 31, 1988, a copy of which is attached hereto as Exhibit D; WHEREAS, United States Patent No 4,791,062, entitled: "FVR VACCINE", was issued on December 13, 1988, a copy of which is attached hereto as Exhibit E; WHEREAS, United States Patent No. 5,106,619, entitled: "PREPARATION OF INACTIVATED VIRAL VACCINES", was issued on April 21, 1992, a copy of which is attached hereto as Exhibit F; WHEREAS, DIAMOND, which is a wholly owned subsidiary of MILES, is the owner by assignment of U.S. Patent Nos.: 4,545,987; 4,693,981; 4,727,027; 4,748,120; 4,791,062; and 5,106,619 and has the right to grant licenses under these patents; 1 2 WHEREAS, DIAMOND, which is a wholly owned subsidiary of MILES, is the owner of the foreign patents and patent applications that correspond to the above patents and are identified in Exhibit G and has the right to grant licenses under the foreign patents and patent applications. WHEREAS, STERITECH desires to obtain [...***...] license to the inventions and discoveries embodied in U.S. Patent Nos.: 4,545,987; 4,693,981; 4,727,027; 4,748,120; 4,791,062; and 5,106,619 Related Patents and Patent Applications, and the Foreign Patents and Patent Applications (identified in Exhibit G) in the field of [...***...]; WHEREAS, MILES is willing to grant STERITECH such [...***...] in the field of [...***...], subject to rights reserved by the U.S. government, to U.S. Patent Nos.: 4,545,987; 4,693,981; 4,727,027; 4,748,120; 4,791,062; and 5,106,619; Related Patents and Patent Applications, and the Foreign Patents and Patent Applications (identified in Exhibit G); WHEREAS, MILES is willing to grant STERITECH a license, subject to rights reserved by the U.S. government, to U.S. Serial No. 07/350,335, and its related foreign applications (identified in Exhibit H) which applications are jointly owned by DIAMOND and the Regents of the University of California at San Francisco. NOW THEREFORE, the parties agree as follows: 1. DEFINITIONS The following definitions will apply throughout this Agreement: LICENSED PATENTS shall mean U.S. Patent Nos.: 4,545,987; 4,693,981; 4,727,027; 4,748,120; 4,791,062; and 5,106,619 as well as the Foreign Patents identified in Exhibits G and H; RELATED PATENTS AND PATENT APPLICATIONS shall mean any continuation, reissue, reexamination, continuation-in-part, extension or divisional application of a Licensed Patent and/or any letters patent that issue thereon; FOREIGN PATENT APPLICATIONS shall mean the foreign patent applications identified in Exhibits G and H or that may be filed in the future as foreign counterparts of the Licensed Patents and/or any letters patent that issue thereon; LICENSED PRODUCT shall mean any product for use in the field of [...***...] that is covered by a claim of a Licensed Patent in the country in which it is sold and/or any product utilizing a process covered by a claim of a Licensed Patent in the country in which it is used; and AFFILIATES OF STERITECH shall mean all organizations that are at least 50% owned or controlled by STERITECH or any organization owning at least 50% of or controlling STERITECH or subsidiaries of such organizations owning at least 50% of or controlling STERITECH. - ---------- *CONFIDENTIAL TREATMENT REQUESTED 2 3 NET SALES shall mean the gross revenues received by STERITECH and its Affiliates from the sale of Licensed Products less sales, use and/or value added taxes actually paid, import and/or export duties, tariffs and other excise taxes actually paid, transportation prepaid or allowed, and amounts allowed or credited due to returns, rebates, discounts and the like (not to exceed the original billing or invoice amount). For cases in which the Licensed Product is a product covered by a claim of a Licensed Patent, Net Sales shall be computed on the revenues received by STERITECH and its Affiliates from sales of such product. For cases in which the process covered by the claim of a Licensed Patent is used (e.g., [...***...]), Net Sales shall be computed on the revenues received by STERITECH and its Affiliates from the sales of the materials that perform such process. Where such materials are sold as components of a system (e.g., [...***...]) the royalty base on which Net Sales will be computed shall be the [...***...] that permit performance of the licensed process, rather than [...***...]. 2. GRANT MILES and DIAMOND hereby grant to STERITECH, and its Affiliates, a [...***...] license in the field of [...***...], including [...***...], subject to rights reserved by the U.S. government, under the Licensed Patents, Related Patents and Patent Applications, and Foreign Patent Applications, for [...***...], with the right to make, have made, use, and sell products covered by a claim thereunder and practice any process covered by a claim thereunder, including the right to sub-license others to make, have made, use, or sell any such product or practice any such process. This license is [...***...] with respect to U.S. Serial No. 07/350,335 only with respect to MILES' rights under this application, which application is jointly owned by DIAMOND and The Regents of the University of California at San Francisco. 3. CONSIDERATION AND ROYALTIES 3.1 PAYMENTS MADE IN CONSIDERATION OF THE EXECUTION OF THE LICENSE AGREEMENT. As consideration for the licenses granted herein, STERITECH will pay a non-refundable license fee, upon execution of this Agreement, of $[...***...]. 3.2 ADVANCED ROYALTIES TO BE PAID DURING LICENSE. In addition to the fee specified in Paragraph 3.1, STERITECH will pay a non-refundable milestone payment of $[...***...] upon [...***...] for the [...***...] Licensed Product and a non-refundable milestone payment of $[...***...] upon [...***...] for the [...***...] Licensed Product. These milestone payments will be credited against royalties. 3.3 ROYALTY. STERITECH will pay MILES a royalty equal to [...***...] of Licensed Products. Only [...***...] shall be paid under this Agreement for each Licensed Product sold regardless of [...***...] of Licensed Patents that are applicable thereto. No royalty shall be payable on [...***...] is purchasing the Licensed Product for use in its own commercial activities, - ---------- *CONFIDENTIAL TREATMENT REQUESTED 3 4 excluding use solely for [...***...]. Royalties shall otherwise be payable on the [...***...] of STERITECH. 3.4 CESSATION OF OBLIGATION TO PAY ROYALTY. STERITECH's obligation to pay a royalty for a Licensed Product which is made, used or sold in any given country shall cease: (a) if every one of the claims directed to Licensed Product which would be infringed but for the license granted herein and all of the claims directed to the process(es) of making and using that Licensed Product which would be infringed but for the license granted herein are held invalid and unenforceable by a court of competent jurisdiction in a final, unappealable decision; or (b) upon expiration of the last to expire of the Licensed Patents; or (c) if no claim of any Licensed Patent covers Licensed Product in the country where the product is sold and no claim of any Licensed Patent covers Licensed Product in the country in which Licensed Product is produced or used. 3.5 MINIMUM ROYALTIES. STERITECH shall pay the following minimum royalties, inclusive of royalties payable under Section 3.3:
AMOUNT YEAR $[...***...] [...***...] $[...***...] [...***...] $[...***...] [...***...] $[...***...] [...***...]
The first year to which minimum royalty requirement shall apply shall be the earlier of [...***...] or the [...***...] for the first Licensed Product. Each twelve months period thereafter shall constitute a year for the purpose of calculating minimum royalties. In the event minimum royalties are not paid as provided above for any year, MILES may, by notice to STERITECH given within [...***...] of the end of the respective year: (a) terminate this License Agreement, if STERITECH is not then exercising reasonable good faith efforts to develop and/or market Licensed Products, or (b) [...***...]. 4. PAYMENT - ---------- *CONFIDENTIAL TREATMENT REQUESTED 4 5 4.1 ACCOUNTS. STERITECH shall keep a complete and correct account of the number of Licensed Products sold in sufficient detail to determine the amounts due to MILES. STERITECH shall keep such account for at least three years after making the royalty payment. STERITECH's records shall be available upon written request for inspection at reasonable times during regular business hours by an independent certified public accountant selected by MILES to whom STERITECH has no reasonable objection for the purpose of verifying royalty statements and payments made by STERITECH under this Agreement. This accountant shall not disclose to MILES any information other than the quantities and payments required to be reported hereunder. MILES shall hold all such information in confidence. 4.2 PAYMENT. Within [...***...] after the end of each of STERITECH's operating quarters, STERITECH shall send MILES a written statement, setting forth all sales of Licensed Product and a computation of royalties on these sales to MILES in accordance with this Agreement. Such statements shall be accompanied by payment of the total amount of royalty due. To the extent no royalty is due because of credits for previous milestone payments, this shall be reported by STERITECH. 5. LITIGATION 5.1 NOTIFICATION. MILES agrees to notify STERITECH in writing if the validity, infringement, or priority of invention of any of the Licensed Patents is put in issue by any person not a party hereto. 5.2 INFRINGEMENT. MILES agrees to use all reasonable measures to enforce the Licensed Patents against infringers. Upon learning of infringement of a Licensed Patent, STERITECH shall promptly notify MILES of the infringement and provide MILES with such notice concerning such infringement. MILES shall have [...***...] from the date of STERITECH's notice to abate the infringement or to file suit against the infringer. 5.3 LAWSUITS. If MILES brings suit against an infringer of a Licensed Patent, it shall give notice of such suit to STERITECH and STERITECH shall have [...***...] in which to elect to join MILES in the prosecution of the suit. 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Roy By: /s/ Mark Yogman --------------------------------- ------------------------------------- Title: Vice President, Strategic Planning Date: November 19, 1992 ATTEST: DIAMOND SCIENTIFIC CORPORATION By: /s/ Kathryn L. Johns By: /s/ Spencer J. Nunley --------------------------------- ------------------------------------- Title: Secretary Date: November 20, 1992 ATTEST: STERITECH, INC. By: /s/ D. Hall By: /s/ Stephen T. Isaacs --------------------------------- ------------------------------------- Title: Chief Executive Officer Date: November 30, 1992 9 10 EXHIBIT A United States Patent [19] [11] Patent Number: 4,545,987 Giles et al. [45] Date on Patent: Oct. 8, 1985 - -------------------------------------------------------------------------------- [54] PSORALEN INACTIVATED DOUBLE-STRANDED RNA VIRAL VACCINES [75] Inventors: Richard E. Giles, Alameda; David R. Stevens, Fremont; Gary P. Wiesehahn, Alameda, all of Calif. [73] Assignee: Advanced Genetics Research Institute, Oakland, Calif. [21] Appl. No.: 563,939 [22] Filed: Dec. 20, 1983 [51] Int. CL4. . . . . . . . . . . . . . . . . A61K 39/12 [52] U.S. CL . . . . . . . . . . . . . . . . . 424/89; 435/235; 435/238 [58] Field of Search . . . . . . . . . . . . . 435/235, 238; 424/89 [56] References Cited PUBLICATIONS Theiler, Vet. J., (1980), 64:600-607. Kemeny and Drehle, Am. J. Vet. Res. (1961), 22:921-925. Alexander and Haig, Onderstepoort J. Vet. Res., (1951), 25:3-15. Parker et al., Vet. Rec., (1975), 96:284-287. Isaacs et al., Biochemistry, (1977), 16:1058-1064. Hearst and Thiry, Nucleic Acids Res., (1977), 4:1339-1347. Hanson et al., J. Gen. Virol., (1978), 40:345-358. Talib and Banerjee, Virology, (1982), 118:430-438. Hanson, Medical Virology II, Proceedings of the 1982 International Symposium on Medical Virology, de la Maza and Peterson, eds., New York: Elsevier Biomedical, 1983, pp. 45-75. J. Parker et al., (1975), Vet. Rec. 96:284-287. J. L. Stott, et al., (1979), Proc. Annu. Meet US Anim Health Assoc., 177:55-62. B. I. Osburn et al., and J. L. Stott et al., (1979), Fed. Proc. 38 (3 part 1) 1091 Coden: FEPRA. Primary Examiner -- Shep K. Rose Attorney, Agent or Firm -- Townsend & Townsend [57] ABSTRACT Novel vaccines of double-stranded RNA viruses are prepared by psoralen inactivation under mild conditions in an inert atmosphere, optionally in the presence of a mild singlet oxygen scavenger. The resulting inactivated virus can be used as a vaccine for inoculation of hosts to provide for the stimulation of the immune system to the virus. 11 Claims, No Drawings 11 4,545,987 1 PSORALEN INACTIVATED DOUBLE-STRANDED RNA VIRAL VACCINES BACKGROUND OF THE INVENTION 1. Field of the Invention The disease Bluetongue is a systemic viral infection of ruminants, such as sheep and cattle. The Bluetongue virus (BTV) is transmitted by small biting flies and is known to occur in twenty serotypic variants that do not provide cross-protection immunologically. The Bluetongue virus is the prototype orbivirus and is composed of ten double-stranded RNA genomic segments. Bluetongue virions have an inner capsid of five polypeptides and a diffuse non-enveloped outer layer containing two polypeptides. It is found that variable amino acid sequences in P2, the major surface polypeptide, are responsible for immunologic serotype specificity. A core protein, P7, is detected by the complement fixation assay and determines cross-reacting group specificity. In the United States, the primary serotypes observed are 11 and 17, with serotypes 2, 10 and 13 being observed less frequently. The first vaccine for BTV was an attenuated live virus vaccine, which has been utilized over forty years in South Africa. Other modified live virus Bluetongue vaccines have also been reported. These attenuated live virus vaccines induce teratogenic lesions in fetuses and may also result in the emergence of recombinant virus strains. There is, therefore, need for an effective vaccine against Bluetongue, which provides protection to an inoculated mammalian host, without the hazards observed with attenuated live Bluetongue virus. 2. Description of the Prior Art Theiler, Vet. J. (1908) 64:600-607 describes an attenuated live Bluetongue virus vaccine. Kemeny and Drehle, Am. J. Vet. Res. (1961) 22:921-925 describe a tissue culture-propagated BTV for vaccine preparation. Alexander and Haig, Onderstepoort J. Vet. Res. (1951) 25:3-15 describe the use of attenuated BTV in the production of a polyvalent vaccine for sheep. Parker et al, Vet. Rec. (1975) 96:284-287 describe an inactivated vaccine against Bluetongue. Isaacs et al, Biochemistry (1977) 16:1058-1064, describe the synthesis of several psoralen derivatives and their photoreactivity with double-stranded RNA. Hearst and Thiry, Nucleic Acids Research (1977) 4:1339-1347; Hanson et al, J. Gen. Virol. (1978) 40:345-358; and Talib and Banerjee, Virology (1982) 118:430-438, describe the photoreactivity of various psoralen derivatives with animal viruses. Hanson, in Medical Virology II, Proceedings of the 1982 International Symposium on Medical Virology, de la Maza and Peterson, eds., New York: Elsevier Biomedical, 1983, pp. 45-75, has cited unpublished data on the inactivation of Bluetongue virus utilizing psoralen photochemistry. SUMMARY OF THE INVENTION Vaccines are provided for inoculation against Bluetongue virus, which inactivated vaccines are prepared by irradiating the virus suspension with light in the presence of psoralen in an inert atmosphere for a time sufficient to completely inactivate the virus. The resulting inactivated virus suspension may then be stored for subsequent use. 2 DESCRIPTION OF THE SPECIFIC EMBODIMENTS Vaccines are provided for inoculation of ruminants against Bluetongue. The vaccines are prepared by inactivation of one or more serotypes of Bluetongue virus (BTV), a multisegmented double-stranded RNA orbivirus. The BTV is inactivated by combining a suspension of the BTV in an appropriate medium with a sufficient amount of a psoralen to provide for complete inactivation of BTV upon irradiation with long wavelength ultraviolet light (UVA), while maintaining an inert atmosphere. The resulting inactivated virus preparation may be stored until used for inoculation. Inoculated ruminants react to vaccination with the subject vaccine by producing neutralizing antibodies. Any of the serotypes of BTV may be inactivated by the subject method. Serotypes of particular interest include 2,10,11,13 and 17, which are the serotypes observed most frequently in the United States, but the other serotypes prevalent in other geographic areas can also be employed. In preparing the vaccine, the BTV is grown in cultured mammelian cells. Illustrative cells include Vero cells, monkey kidney cells, CCL 10 hamster cells, LMTK-cells, or other cells permissive for BTV which can be grown in vitro as monolayer cultures or in suspension culture. The host cells are grown to nominally 80% saturation density, and infected with BTV at a lower multiplicity of infection (MOI) generally less than about 0.05, and more than about 0.005, preferably about 0.01. After absorbing the viral inoculum to the cells by incubation for a limited period of time at a temperature in the range of about 35 degrees to 40 degrees C., an appropriate mammalian cell growth or maintenance medium is added and the cells incubated at a temperature in the range of about 35 degrees to 40 degrees C., in the presence of about 5% carbon dioxide in air for sufficient time to observe that at least 50% of the cell culture exhibits cytopathic effect (CPE). The CPE is characterized by cell rounding (in monolayers), cell detachment (from monolayers) and cell degeneration. The crude cell lysate is allowed to incubate overnight at a temperature in the range from about 0 degrees to 5 degrees C. The material is harvested and collected by low speed centrifugation. The resulting pellet is extracted several times in an appropriate buffer, at a pH in the range from about 8 to 9.5, preferably about 8.5 to 9. The extracted pellet suspension is centrifuged at low speed and the supernatant containing the virus is collected. The pellet may be extracted repeatedly with buffer to enhance the total yield of virus. The virus-containing liquid is then clarified by low speed centrifugation, retaining the virus suspended in the liquid, which may then be stored at 4 degrees C. Tris buffer (2 mM pH 8.8) may be used as the extraction and storage buffer, although other appropriate buffers which will not interfere with the subsequent processing may be used. The particular medium which is used for the growth of the cells will be a conventional mammalian cell culture medium, such as Eagle's Minimum Essential Medium or Medium 199, usually supplemented with additives such as broth prepared from dehydrated standard microbial culture media, fetal bovine serum, calf serum, or the like. The compounds which are used for viral inactivation are furocoumarins. These compounds are primarily 12 4,545,987 3 illustrated by the class of compounds referred to as psoralens, which includes psoralen and derivatives thereof, where the substituents will be: alkyl, particularly of from 1 to 3 carbon atoms, e.g., methyl; alkoxy, particularly of from 1 to 3 carbon atoms, e.g., methoxy; and substituted alkyl, of 1 to 6, more usually 1 to 3 carbon atoms having from 1 to 2 heteroatoms, which will be oxy, particularly hydroxy or alkoxy of from 1 to 3 carbon atoms, e.g., hydroxymethyl and methoxymethyl; or amino, including mono- and diakyl amino or aminoalkyl having a total of from 0 to 6 carbon atoms, e.g., aminomethyl. There will be from 1 to 5, usually 2 to 4 substituents, which will normally be at the 4,5,8,4' and 5' positions, particularly at the 4'-position. Illustrative compounds include 5-methoxypsoralen; 8-methoxypsoralen (8-MOP); 4, 5', 8-trimethylpsoralen (TMP); 4'-hydroxymethyl-4,5'8-trimethylpsoralen (HMT); 4'-aminomethyl-4,5', 8-trimethylpsoralen (AMT); 4-methylpsoralen; 4,4'-dimethylpsoralen; 4,5'-dimethylpsoralen; 4' 8-dimethylpsoralen; and 4'-methoxymethyl-4,5', 8-trimethylpsoralen. Of particular interest is AMT. The furocoumarins may be used individually or in combination. Each of the furocoumarins may be present in amounts ranging from about 0.01 (greek mu)g/ml to 1 mg/ml, preferably from about 0.5 (greek mu)g/ml to 100 (greek mu)g/ml, there not being less than about 1 (greek mu)g/ml nor more than about 1 (greek mu)mg/ml of furocoumarins. In carrying out the invention the furocoumarin(s), in an appropriate solvent which is substantially inert and sufficiently polar to allow for dissolution of the furocounarin(s), is (are) combined with the viral suspension, conveniently a viral suspension in an aqueous buffered medium, such as used for storage. The amount of virus will generally be about 1x10(6) to 10(10), more usually about 1x10(7) to 10(9) and preferably about 1x10(8) to 5x10(8) pfu/ml. The furocoumarin will be at a concentration of about 0.001 mg/ml to 0.5 mg/ml, more usually about 0.05 mg/ml to 0.2 mg/ml. The amount of solvent which is used to dissolve the furocoumarin will be sufficiently small so as to readily dissolve in the aqueous viral suspension and have little, if any, effect on the results. The psoralen may be added to the viral suspension in a signal addition or in multiple additions, where the virus is irradiated between additions. Usually, the number of additions will be from about 1 to 5, more usually from about 1 to 4, and preferably from about 2 to 4. The total amount of psoralen which will be added will be sufficient to provide a concentration of at least about 0.01 mg/ml to about 1 mg/ml, usually not more than about 0.75 mg/ml and preferably not more than about 0.5 mg/ml. Since a substantial proportion of the psoralen will have reacted with the RNA between additions, the total concentration of psoralen in solution will generally not exceed about 0.1 mg/ml. The total time for the irradiation will vary depending upon the light intensity, the concentration of the psoralen, the concentration of the virus, and the manner of irradiation of the virus, where the intensity of the irradiation may vary in the medium. The total time will usually be at least about 2 hrs. and not more than about 60 hrs., generally ranging from about 10 hrs. to 50 hrs. The times between additions of psoralen, where the psoralen is added incrementally, will generally vary from about 1 hr. to 24 hrs., more usually from about 2 hrs. to 20 hrs. 4 The temperature for the irradiation is preferably under 25 degrees C., more preferably under 20 degrees C. and will generally range from about - 10 degrees to 15 degrees C., more usually from about 0 degrees to 10 degrees C. The irradiation is normally carried out in an inert atmosphere, where all or substantially all of the air has been removed. Inert atmospheres include nitrogen, helium, argon, etc. The light which is employed will generally have a wavelength in the range from about 300 nm to 400 nm. The intensity will generally range from about 0.1 mW/cm(2) to about 5 W/cm(2). Optionally, a small amount of a singlet oxygen scavenger may be included during the virus inactivation. Singlet oxygen scavengers include ascorbic acid, dithioerythritol, sodium thionite, glutathione, etc. The amount of scavenger will generally be at a concentration of about 0.001 M to 0.5 M, more usually at about 0.05 M to 0.2 M, where the addition may be made in a single or multiple additions. During irradiation, the medium may be maintained still, stirred or circulated and may be either continuously irradiated or be subject to alternating periods of irradiation and non-irradiation. The circulation may be in a closed loop system or in a single pass system ensuring that all of the sample has been exposed to irradiation. It may be desirable to remove the unexpended furocoumarin and/or its photobreakdown products from the irradiation mixture. This can be readily accomplished by one of several standard laboratory procedures such as dialysis across an appropriately sized membrane or through an appropriately sized hollow fiber system after completion of the irradiation. Alternatively, one could use affinity columns for one or more of the low molecular weight materials to be removed. The inactivated vaccine may then be formulated in a variety of ways for use for inoculation. The concentration of the virus will generally be from about 10(6) to 10(9) pfu/ml, as determined prior to inactivation. The vaccine may include cells or may be cell-free. It may be in an inert physiologically acceptable medium, such as ionized water, phosphate-buffered saline, saline, or the like, or may be administered in combination with a physiologically acceptable immunologic adjuvant, including but not limited to mineral oils, vegetable oils, mineral salts and immunopotentiators, such as muramyl dipeptide. The vaccine may be administered subcutaneously, intramuscularly, or intraperitoneally. Usually, a specific dosage at a specific site will range from about 0.1 ml to 4 ml, where the total dosage will range from about 0.5 ml to 8 ml. The number of injections and their temporal spacing may be highly variable, but usually 1 to 3 injections at 1, 2 or 3 week intervals are effective. The following examples are offered by way of illustration and not by way of limitation. EXPERIMENTAL Virus Growth and Tissue Culture Hamster cells [BHK-21 (C-13), American Type Culture Collection, (CCL 10)] are grown as monolayers in plastic cell culture vessels in Eagle's Minimum Essential Medium with Earle's Salts (MEM) and non-essential amino acids (MEN) supplemented with 10% heat inactivated calf serum (C) and 10% tryptose phosphate broth (Tp, Difco 0060). Cell cultures are used to produce live BTV from master seed virus obtained from 13 4,545,987 5 Dr. T.L. Barber, USDA, Denver, Colorado. Cells are grown in culture vessels to 80% to 100% confluency (approximately 1 times 10(5) to 2 times 10(5) cells/ cm(2) of growth surface area) using standard mammalian cell culture techniques. Generally, Corning plastic roller bottles (Corning No. 25140-850) with a growth surface area of 850 cm(2), containing 100 ml of MEN supplemented with 10% C(1) and 10% Tp and 1 times 10(8) to 2 times 10(8) CCL 10 cells per bottle are used for virus production . The cell cultures are initiated by seeding approximately 1 times 10(6) to 5 times 10(7) cells into 100 ml growth medium in a roller bottle containing about 5% CO(2) in air and incubating the roller bottle on a roller bottle rotator at 1 to 5 rpm at 35 degrees C. to 38 degrees C. The cultures are grown to 80% to 100% confluency over a 7 to 14 day period with a 100% medium change every 2 to 4 days. When the monolayers are 80% to 100% confluent the culture medium is removed and the monolayer is infected with approximately 1 times 10(6) to 2 times 10(6) plaqueforming units (pfu) of STV in 20 ml of MEN with 2% heat-inactivated fetal bovine serum (F(i)). The multiplicity of infection (MOI) is approximately 0.01. The virus inoculum is adsorbed to the cells by incubation at 35 degrees C. to 38 degrees C. for 1 hr. at 1 to 5 rpm. One hundred milliliters of MEN containing 10% C(i) and 10% TP is added per roller bottle. The post-infection incubation is at 35 degree C. to 38 degrees C. in 5% CO(2) in air with rotation. Two to four days post-infection, BTV cytopathic effect (CPE) is evident. The CPE is characterized by cell rounding, cell detachment, and cell degeneration. When at least 50% of the cell monolayer exhibits CPE the contents of the roller bottle are swirled or scraped with a rubber policeman to remove loosely attached materials from the roller bottle walls. The roller bottles and contents are incubated at 4 degrees C. overnight. The harvest material is decanted into sterile contrifuge bottles. The virus, cells, and cell debris are pelleted by centrifugation at 2,000 times g for 60 min., at 4 degrees C. The pellet is resuspended aseptically in 8 ml of 2 mM Tris-HCl, pH 8.8, for each original roller bottle. The suspension is mixed vigorously on a vortex mixer, and/or sonicated at 4 degrees C. for 1 min., and centrifuged at 1,400 times g for 30 min. at 4 degrees C. The virus-containing supernatant is collected and the pellet is extracted twice more with 8 ml/roller bottle aliquots of 2 mM Tris-HCl, pH 8.8 The virus-containing supernatants are pooled and clarified by centrifugation at 4,000 times g for 30 min. at 4 degrees C. The clarified supernatant is stored at 4 degrees C. Virus Assay Confluent monolayers of LMTK - or Vero (ATCC CCL 81) cells are prepared in 6 cm diameter mammalian cell culture plastic petri dishes (Corning #25010) or other convenient cell culture vessel. The growth medium used for LMTK - cell is alpha-modified Eagle's Minimum Essential Medium, Earle's Salts ((greek alpha)ME) + 10% F(i) and the growth medium used for Vero cells is MEN + 5% F(i). Ten-fold serial dilutions of virus samples are made by adding 0.5 ml of the virus sample to 4.5 ml of phosphate buffered saline (PBS), pH 7.2 to 7.4 + 2% F(i) in a screw cap tube. The growth medium is removed from a 6 cm culture dish cell monolayer, 0.1 ml virus sample (undiluted or diluted) is added, and the virus is absorbed to the monolayer for 1 to 2 hrs. at 35 degrees C. to 38 degrees C. Two or more dishes are used for each sample. Five ml of overlay medium is added per 6 cm culture dish. The overlay medium is prepared by mixing equal parts of solution A (100 ml 2 times MEM with L- 6 glutamine. GIBCO #320-1935, + 10 ml F(i) and 1.8% to 2% Noble Agar (Difco 0142) in deionized H(2)O at 44 degrees C. to 45 degrees C. The cultures are incubated at 35 degrees C. to 38 degrees C. in 5% CO(2) in air for 5 days. A second overlay containing Neutral Red at a final concentration of 0.005% is added on day 5. Plaques are counted on a day 6 or day 7 post-infection. The virus liter in pfu/ml is calculated by multiplying the average number of plaques per dish by the reciprocal of the dilution. The pfu/ml is the value used to determine the amount of virus needed to infect cells at a MOI of approximately 0.01. The pfu/ml in a virus preparation prior to inactivation is used to determine the vaccine dose. Inactivation Protocol Twenty-five ml of BTV serotype 11 (1.5 times 10(8) pfu/ml) is mixed with 0.25 ml of 4'-aminomethyl 4,5', 8-trimethylpsoralen (AMT; 1 mg/ml in DMSO). The mixture is placed in a 150 cm(2) tissue culture flask (T-150; Corning #25120). The viral suspension in the flask is placed in an argon atmosphere for 10 min. and then a stream of argon gas is blown over the viral suspension for an additional 2 min. The flask is tightly capped and the suspension is irradiated for 3.25 hours at 4 degrees C. using GE BLB fluorescent bulbs at an intensity of 1.5 mW/cm(2). An additional 0.25 ml of AMT is then added to the viral suspension, the suspension is transferred by pipet to a new T-150 flask, and the solution is again flushed with argon. The flask is irradiated for an additional 14.75 hrs. at 4 degrees C. under the same long wavelength UV light source. After this irradiation an additional 0.25 ml of AMT solution is added to the suspension and it is again transferred to a new T-150 flask. The solution is flushed with argon as before and irradiated for an additional 5.5 hrs. at 4 degrees C. The inactivated BTV is stored at 4 degrees C. Assessment of Inactivation by Blind Passage CCL 10 cells are grown to confluency in 850 cm(2) roller bottles using standard cell culture procedures as described above. The culture medium is removed from the roller bottle and 2.0 ml of the inactivated virus preparation, mixed with 18 ml of medium containing 2% F(i), is adsorbed to the roller bottle cell monolayer for 60 min at 35 degrees C. to 38 degrees C. with rotation at 1 to 5 rpm. After adsorption the unabsorbed inoculum is removed and 100 ml of growth medium (MFN with 10% C(i) and 10% Tp) is added and the roller bottle culture incubated at 35 degrees C. to 38 degrees C. for 7 days with daily observation for viral CPE (see plaque assay above for description of CPE). The roller bottle culture should receive a 100% medium change every 2 to 3 days. If no CPE is observed during the first roller bottle passage, the cell monolayer is chilled at 4 degrees C. for 12 to 24 hrs. The cells are scraped into the medium which is then decanted into a centrifuge bottle. The cells are pelleted by centrifugation at 4 degrees C. at 2,000 times g for 30 min. and resuspended in 2.0 ml of 2 mM Tris-HCl (pH 8.8) by vigorous mixing using a vortex mixer. The resuspended material is centrifuged at 2,000 times g for 20 min. at 4 degrees C. The supernatant is added to 18 ml of growth medium containing 2% F(i) and used to infect a new confluent roller bottle culture of CCL 10 cells as described immediately above. The second roller bottle blind passage is observed for 7 days and fed every 2 to 3 days. If no CPE is observed during the second roller bottle blind passage, a third roller bottle blind passage is performed. If no CPE has been 14 4,545,987 7 observed by the end of the third roller bottle blind passage the virus preparation is considered inactivated. EXAMPLE I Four New Zealand white rabbits were randomly assigned to 2 groups, designated A and B. Both groups were given 4 immunizations at two week intervals. The first immunization consisted of 1 ml of vaccine (10(8) pfu BTV serotype 11) and 1 ml of Freund's Complete Adjuvant. The second through fourth immunizations utilized 1 ml of vaccine (10(8) pfu BTV serotype 11) and 1 ml of Freund's Incomplete Adjuvant. All immunizations were given intramuscularly (TM). The vaccine given to Group A (Vaccine #1) was inactived with AMT-UVA in the presence of 0.01 M ascorbic acid. Vaccine #1 was dialyzed for 12 hours against 2 mM Tris,ph 8.6. The vaccine given to Group B (Vaccine #2) was inactivated with AMT-UVA without ascorbic acid and sonicated three times (2 minutes each time) using a cup horn probe (Heat Systems Model 431A) at a power setting of 3 (Heat Systems Model W220). Both Vaccine #1 and Vaccine #2 were deemed inactivated since no live virus was detected during blind passage. Inactivated vaccine was also tested for safety by chicken embryo inoculation. Egg deaths attributable to live virus were not encountered. Both rabbit groups were bled via auricular venipuncture one week following the second, third, and fourth immunizations. Serum from each rabbit was pooled with that of its groupmate, and the pooled sera were tested for anti-BTV antibodies by two standard serologic assays, serum neutralization (Jochim and Jones, Am, J. Vet. Res. (1976) 37:1345-1347) and agar gel precipitation (Jochim et al., Am. Assoc. Vet. Lab, Diag., 22nd Proceed: 463-471, 1979). Pre-immunization rabbit serum was used as the negative control; BTV immune sheep serum was used as the positive control of both immunologic procedures. Pooled sera from Groups A and B reduced the number of viral plaques (serum neutralization) greater than eighty percent when the sera were diluted 1:40, which was the highest dilution examined. Negative and positive control sera behaved as expected.
TABLE 1 - ------------------------------------------------------------------------------ Serum Neutralization Data From Rabbits Vaccinated with AMT-UVA-inactivated Bluetongue Virus Vaccines. -------------------------------------- TITER*: ---------------------------------------------- GROUP 1 5 40 - ------------------------------------------------------------------------------ A + + + B + + + Normal Rabbit Serum + - - BTV-Immune Sheep Serum + + + - ------------------------------------------------------------------------------
* Reciprocal of serum dilution neutralizing 80 percent of BTV plaque activity on BHK cells. The data are from post-second immunization serum samples. Pooled post-immunization sera from Groups A and B precipitated BTV antigen in immunodiffusion plates when tested at dilutions up to 1:16. Normal rabbit serum did not precipitate the standard BTV antigen. BTV-immune sheep serum did precipitate the BTV antigen, but not at dilutions greater than 1:2. Of the two immunologic procedures utilized, serum neutralization is predictive for immunity to live BTV challenge in the target species. EXAMPLE II Each of two adult sheep, known to be susceptible to BTV, were inoculated subcutaneously (SQ) with 2 ml of AMT-UVA inactivated BTV plus adjuvant (1:1; 8 vaccine to aluminum hydroxide adjuvant). The vaccine contained approximately 10(8) pfu/ml of BTV prior to inactivation. A third sheep was inoculated SQ with 6 ml of the identical vaccine without adjuvant. Seven weeks later the three sheep were given identical inoculations SQ that consisted of 5 ml of vaccine and aluminum hydroxide adjuvant (2:1 vaccine to adjuvant; 10(8) pfu BTV/ml of vaccine). The three sheep were monitored for clinical evidence of BTV, including daily body temperature recording and bi-daily virus isolation attempts. No evidence of BTV was observed, indicating that the vaccine was inactivated. Serum was collected weekly for serum neutralization and agar gel precipitation testing. Normal sheep sera and BTV-immune sheep sera were used for negative and positive control samples in the serologic tests. The first vaccine inoculations induced precipitating anti-BTV antibody in all three sheep. Their pre-exposure sera were uniformly negative for anti-BTV precipitating antibody. Modest neutralizing anti-BTV anti-body titers (1:5) were elicited in two of three sheep following one immunization. The second immunization elicited a distinct immunologic anamnestic response, inducing neutralizing titers of 1:40, 1:80, or 1,1600 in the three sheep.
TABLE 2 - ------------------------------------------------------------------------------ Serum Neutralization Data From Sheep Immunized with an AMT-UVA Inactivated BTV Vaccine. ------------------------------------- TITERS* Sheep No.: ---------------------------------------------- 1 2 3 - ------------------------------------------------------------------------------ Pre-First Immunization ------------------------ Day 0 <5 <5 <5 Post-First Immunization ------------------------ Day 21 5 5 <5 Post-Second Immunization ------------------------ Day 7 80 160 40 Day 14 80 40 40 Day 21 80 80 40 Day 42 80 80 80 Post-Challenge -------------- Day 7 160 160 80 Day 14 320 160 80 - ------------------------------------------------------------------------------
* Reciprocal of highest 2-fold dilution reducing BTV plaque activity on BHK cells by 80 percent. The sheep were challenged by SQ syringe inoculation 10(5) egg lethal doses for BTV serotype 11. The three sheep remained clinically normal during the BTV challenge period, indicating that the vaccine was efficaceous. It is evident from the above results that the BTV which is psoralen-inactivated retains its immunogenicity, particularly as to those sites which elicit an immune response which is effective in protecting a host against subsequent BTV-infection. Thus, the psoralen inactivation can be carried out under conditions which do not modify the immunogenic sites of the virus, so as to elicit an immunogenic response which will be effective against the live BTV. Furthermore, the BTV RNA virus is efficiently inactivated under mild conditions to the point of complete inactivation, whence it may be safely administered to a host. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious 15 4,545,987 9 that certain changes and modifications may be practiced within the scope of the appended claims. What is claimed is: 1. A vaccine useful for inoculation of a mammalian host susceptible to infection by Bluetongue virus (BTV), which comprises at least one furocoumarin-inactivated BTV serotype in from about 10(6) to 10(9) pfu/ml, wherein said inactivation is as a result of irradiation of BTV in the presence of an inactivating furocoumarin with long wavelength ultraviolet light at a temperature below about 40 degrees C. for a time sufficient to inactivate said BTV to a non-infectious degree, and an immunologic adjuvant. 2. A vaccine according to claim 1, wherein said furocoumarin is 4'-aminomethyl-4,5',8-trimethylpsoralen. 3. A vaccine according to claim 2, wherein said BTV is of serotype 11. 4. A vaccine according to claim 1, wherein said BTV is inactivated in the presence of a singlet oxygen scavenger. 5. A vaccine according to claim 1, wherein said inactivation is performed in the substantial absence of oxygen. 6. A vaccine according to claim 1, wherein said BTV is grown in substantially confluent monolayers of cells immediately prior to inactivation. 7. A vaccine useful for inoculation of a mammalian host susceptible to infection by Bluetongue virus (BTV), which comprises BTV serotype 11 inactivated with 4'-aminomethyl-4,5',8-trimethylpsoralen by irradiation with long wavelength ultraviolet light at a temperature in the range of about -- 10 degrees to 25 degrees C. for a time sufficient to inactivate said BTV to become non-infectious, said BTV being present in an amount of about 10(6) to 10(9) pfu/ml, and an immunologic adjuvant. 10 8. A vaccine useful for inoculation of a mammalian host susceptible to infection by Bluetongue virus (BTV), which comprises at least one furocoumarin-inactivated BTV serotype in from about 10(6) to 10(9) pfu/ml, wherein said inactivation is as a result of irradiation of BTV in the presence of an inactivating furocoumarin with long wavelength ultraviolet light at a temperature below about 40 degrees C. for a time sufficient to inactivate said BTV to a non-infectious degree. 9. A vaccine useful for inoculation of a mammalian host susceptible to infection by Bluetongue virus (BTV), which comprises BTV serotype 11 inactivated with 4'-aminomethyl-4,5',8-trimethylpsoralen by temperature in the range of about -- 10 degrees to 25 degrees C. for a time sufficient to inactivate said BTV to become non-infectious, said BTV being present in an amount of 10(6) to 10(9) pfu/ml. 10. A method for producing a vaccine for inoculation of a mammalian host susceptible to infection by bluetongue virus (BTV), which method comprises inactivating at least one BTV serotype by exposure to long wavelength ultraviolet light in the presence of a furocoumarin at a temperature below about 40 degrees C. for a time sufficient to inactivate said BTV to a non-infectious degree, and combining said inactivated BTV with an appropriate adjuvent. 11. A method for producing a vaccine for inoculation of a mammalian host susceptible to infection by bluetongue virus (BTV), which method comprises exposure of at least one BTV serotype to long wavelength ultraviolet light in the presence of 4'-aminomethyl-4,5',8-trimethylpsoralen at a temperature in the range from about -- 10 degrees C to 25 degrees C. for a time sufficient to inactivate the BTV to a non-infectious degree, and combining said inactivated BTV with a suitable adjuvant. * * * * * 16 EXHIBIT B UNITED STATES PATENT [19] [11] PATENT NUMBER: 4,693,981 WIESEHAHN ET AL. [45] DATE OF PATENT: Sep. 15, 1987 - -------------------------------------------------------------------------------- [54] PREPARATION OF INACTIVATED VIRAL VACCINES [75] Inventors: Gary P. Wiesehahn, Alameda; Richard P. Creagan, Alta Loma; David R. Stevens, Fremont; Richard Giles, Alameda all of Calif. [73] Assignee: Advanced Genetics Research Institute, Oakland, Calif. [*] Notice: The portion of the term of this patent subsequent to Oct. 8, 2002 has been disclaimed. [21] Appl. No.: 785,354 [22] Filed Oct. 7, 1985 RELATED U.S. APPLICATION DATA [63] Continuation-in-part of Ser. No. 563,939. Dec. 20, 1983, Pat. No. 4,545,987, and a continuation-in-part of Ser. No. 592,661, Mar. 23, 1984, abandoned. [51] Int. Cl(4).......................................................A61K 39/12 [52] U.S. Cl....................................................435/238; 424/89; 424/90 [58] Field of Search........................................424/89, 90; 435/235, 435/238 [56] REFERENCES CITED U.S. PATENT DOCUMENTS 4,124,598 11/1978 Hearst.......................................260/343.21 4,169,204 9/1979 Hearst..........................................546/270 4,196,281 4/1980 Hearst...........................................536/28 4,545,987 10/1985 Giles et al......................................424/89 4,568,542 2/1986 Kronenberg.......................................424/90 FOREIGN PATENT DOCUMENTS 0066886 12/1982 European Pat. Off. OTHER PUBLICATIONS Hearst & Thiry, Nucleic Acids Research, 1977, 4:1339-1347. Talib and Banerjee, Virology 118, 1982, 430-438. Carl V. Hanson, Medical Virology II, de la Maza & Peterson, eds., Elsevier Biomedical, New York, pp. 45-79. deMol and van Henegouwen (1981) Photochem. Photobiol. 33:815-819. deMol et al. (1981) Photochem. Photobiol. 34:661-666. Joshi and Pathak (1983) Biochem. Biophys. Res. Comm. 112:638-646. Grossweiner (1982) NCI Monograph No. 66, 47-54. Rodighiero and Dall'Acqua (1982) NCI Monograph No. 66, 31-40. deMol et al. (1981) 95:74462k p. 74467 Chem. Interactions. Primary Examiner -- Shep K. Rose Attorney, Agent, or Firm -- Bertram I. Rowland [57] ABSTRACT Vaccines employing inactivated viruses having improved retention of antigenic characteristics are prepared by psoralen-inactivation of the live virus in a non-oxidizing atmosphere. By excluding oxygen and other oxidizing species from the inactivation medium, degradation of the antigen characteristics resulting from irradiation with ultraviolet light is largely prevented. The resulting inactivated viruses are employed in vaccine preparations for the inoculation of susceptible hosts to inhibit viral infection. 9 CLAIMS, NO DRAWINGS 17 4,693,981 1 PREPARATION OF INACTIVATED VIRAL VACCINES This application is a continuation-in-part of application Ser. No. 563,939, filed on Dec. 20, 1983, now U.S. Pat. No. 4,545,987, and application Ser. No. 592,661, filed on Mar. 23, 1984, abandoned. BACKGROUND OF THE INVENTION 1. Field of the Invention. The present invention relates to the preparation of inactivated viral vaccines. More particularly, the invention relates to psoralen inactivation of viral particles under conditions which limit antigenic degradation of the viral particles caused by the inactivation. Vaccination against both bacterial and viral diseases has been one of the major accomplishments of medicine over the past century. While effective vaccines have been developed for a large number of diseases, development of safe and effective vaccines for a number of other diseases remains problematic. The use of inactivated or killed microbial agents as a vaccine, although generally safe, will not always be effective if the immunogenic characteristics of the agent are altered. Indeed, the preferential degradation of certain antigens on the inactivated microorganisms might produce an immune response which allows for an immunopathological response when the host is later challenged with the live microorganism. In contrast, the preparation of live, attenuated microbial agents as a vaccine will often provide improved immunologic reactivity, but increases the risk that the vaccine itself will be infectious, e.g., as a result of reversion, and that the organism will be able to propagate and provide a reservoir for future infection. Thus, one must generally choose between improved effectiveness and greater degree of safety when selecting between the viral inactivation and viral attenuation techniques for vaccine preparation. The choice is particularly difficult when the virus is resistant to inactivation and requires highly rigorous inactivation conditions which are likely to degrade the antigenic characteristics. It is therfore desirable to provide improved methods for inactivating viruses, which methods are capable of inactivating even the most resistant viruses under conditions which do not substantially degrade the antigenic structure of the viral particles. In particular, the inactivated viruses should be useful as vaccines and free from adverse side effects at the time of administration as well as upon subsequent challenge with the live virus. 2. Description of the Prior Art The reactivity of psoralen derivatives with viruses has been studied. See, Hearst and Thiry (1977) Nuc. Acids Res. 4:1339-1347; and Talib and Banerjee (1982) Virology 118:430-438. U.S. Pat. No. 4,124,598 and 4,196,281 to Hearst el al. suggest the use of psoralen derivatives to inactivate RNA viruses, but include no discussion of the suitability of such inactivated viruses as vaccines. U.S. Pat. No.4,169,204 to Hearst el al. suggests that psoralens may provide a means for inactivating viruses for the purpose of vaccine production but presents no experimental support for this proposition. European patent application 0 066 886 by Kronenberg teaches the use of psoralen inactivated cells, such as virus-infected mammalian cells, for use as immunological reagents and vaccines. Hanson (1983) in: Medical 2 Virology II, de la Maza and Peterson, eds., Elsevier Biomedical, New York, pp. 45-79, reports studies which have suggested that oxidative photoreactions between psoralens and proteins may occur. SUMMARY OF THE INVENTION The present invention provides for the production of furocoumarin- inactivated viral vaccines under conditions which substantially preserve the antigenic characteristics of the inactivated viral particles. It has been recognized by the inventors herein that the inactivation of viruses by exposure to ultraviolet radiation in the presence of furocoumarin compounds can degrade the anitgenic structure of the viral particle. While such degradation can be limited by employing less rigorous inactivation conditions, certain recalcitant viruses require relatively harsh inactivation conditions in order to assure that all residual infectivity is eliminated. The inactivation conditions required to eliminate substantially all infectivity in such recalcitrant viruses can so degrade the viral particle that it is unsuitable for use as the immunogenic substance in a vaccine. Even if the degradation is not so complete, partial degradation of the antigenic characteristics may render the vaccine less effective than would be desirable. That is, the vaccine may require higher concentrations of the inactivated viral particles in each inoculation, and/or the vaccination program may require additional inoculations in order to achieve immunity. According to the present invention, vaccines are prepared by treatment with furocoumarins and long wavelength ultraviolet (UVA) light under conditions which limit the availability of oxygen and other reactive, particularly oxidizing, species. It has been found that such conditions allow for the inactivation of even recalcitrant viral particles without substantial degradation of the antigenic characteristics of those particles. Thus, viruses which have heretofore been resistant to furocoumarin-inactivation may now be inactivated without loss of the desired immunogenicity, and viruses which have previously been successfully inactivated may now be inactivated under conditions which better preserve their antigenic characteristics, making them more efficient immunogenic substances for use in vaccines. DESCRIPTION OF THE SPECIFIC EMBODIMENTS According to the present invention, vaccines useful for the inoculation of mammalian hosts, including both animals and humans, against viral infection are provided. The vaccines are prepared by inactivation of live virus in an inactivation medium containing an amount of an inactivating furocoumarin sufficient to inactivate the virus upon subsequent irradiation with long wave-length ultraviolet radiation. Degradation of the antigenic characteristics of the live virus is reduced or eliminated by limiting the availability of oxygen and other oxidizing species in the inactivation medium. Suitable vaccines may be prepared by combining the inactivated viruses with a physiologically-acceptable carrier, typically an adjuvant, in an appropriate amount to elicit an immune response, e.g., the production of serum neutralizing antibodies, upon subsequent inoculation of the host. The present invention is suitable for producing vaccines to a wide variety of viruses, including human viruses and animal viruses, such as canine, feline, bo- 18 4,693,981 3 vine, porcine, equine, and ovine viruses. The method is suitable for inactivating double stranded DNA viruses, single-stranded DNA viruses, double-stranded RNA viruses, and single-stranded RNA viruses, including both enveloped and nonenveloped viruses. The following list is representative of those viruses which may be inactivated by the method of the present invention. - -------------------------------------------------------------------------------- Viruses which may be inactivated ------------------------------------------- Representative Viruses - -------------------------------------------------------------------------------- dsDNA ------ Adenoviruses Adenovirus canine adenovirus 2 Herpesviruses Herpes simplex viruses, Feline Herpes 1 Papovaviruses Polyoma, Papilloma Poxviruses Vaccinia ssDNA ------ Parvovirus Canine parvovirus, Feline panleukopenia dsRNA ------ Orbiviruses Bluetongue virus Reoviruses Reovirus ssRNA ------ Calicivirus Feline calicivirus Coronavirus Feline infectious peritonitis Myxovirus Influenza virus Paramyxovirus Measles virus, Mumps virus, Newcastle disease virus, Canine distemper virus, Canine parainfluenza 2 virus Picornavirus Polio virus, Foot and mouth disease virus Retrovirus Feline leukemia virus, Human T-cell lymphoma virus, types I, II and III. Rhabdovirus Vesicular stomatitis virus rabies Togavirus Yellow fever virus, Sindbis virus, Encephalitis virus - -------------------------------------------------------------------------------- Of particular interest are those viruses for which conventional vaccine approaches have been unsuccessful or marginally successful. For such viruses, inactivation procedures which are sufficiently rigorous to assure the total loss of infectivity often result in partial or complete destruction of the antigenic characteristics of the virus. With such loss of antigenic characteristics, the viruses are incapable of eliciting a protective immunity when administered to a susceptible host. While it would be possible to utilize less rigorous inactivation conditions in order to preserve the antigenic integrity of the virus, this approach is not desirable since it can result in incomplete inactivation of the virus. In preparing the subject vaccines, sufficient amounts of the virus to be inactivated may be obtained by growing seed virus in a suitable mammalian cell culture. Seed virus, in turn, may be obtained by isolation from an infected host. Suitable mammalian cell cultures include primary or secondary cultures derived from mammalian tissues or established cell lines such as Vero cells, monkey kidney cells, BHK21 hamster cells, LMTK(31) cells, and other cells permissive for the desired virus and which may be grown in vitro as monolayer or suspension cultures. The cell cultures are grown to approximately 80% saturation density, and infected with the virus at a low multiplicity of infection (MOI), usually between about 0.05 and 0.005, preferably at about 0.01. After absorbing the viral inoculum to the cells by incubation for a limited period of time at a temperature in the range from 35 degrees C. to 40 degrees C., an appropriate growth or maintenance medium is added. The cells are further incubated at about the same temperature, in the pres- 4 ence of about 5% carbon dioxide in air, until a sufficient amount of virus has been produced. The growth and maintenance medium will usually be a conventional mammalian cell culture medium, such as Eagle's Minimum Essential Medium or Medium 199, usually supplemented with additives such as broth prepared from dehydrated standard microbial culture media, fetal bovine serum, fetal calf serum, or the like. The furocoumarins useful for inactivation are primarily illustrated by the class of compounds referred to as psoralens, including psoralen and substituted psoralens where the substituents may be alkyl, particularly having from one to three carbon atoms, e.g., methyl; alkoxy, particularly having from one to three carbon atoms, e.g., methodoxy: and substituted alkyl having from one to six, more usually from one to three carbon atoms and from one to two heteroatoms, which may be oxy, particularly hydroxy or alkoxy having from one to three carbon atoms, e.g., hydroxy methyl and methoxy methyl, or amino, including mono- and dialkyl amino or aminoalkyl, having a total of from zero to six carbon atoms, e.g., aminomethyl. There will be from 1 to 5, usually from 2 to 4 substituents, which will normally be at the 4, 5, 8, 4' and 5' positions, particularly at the 4' position. Illustrative compounds include 5-methoxypsoralen; 8-methoxypsoralen (8-MOP); 4.5', 8-trimethylpsoralen (TMP); 4'-hydroxymethyl-4.5', 8-trimethylpsoralen (HMT); 4'-aminomethyl-4.5', 8-trimethylpsoralen (AMT); 4-methylpsoralen; 4.4'-dimethylpsoralen; 4.5'-dimethylpsoralen; 4', 8-dimethylpsoralen; and 4'-methoxymethyl-4.5', 8-trimethylpsoralen. Of particular interest are AMT and 8-MOP. The furocoumarins may be used individually or in combination. Each of the furocoumarins may be present in amounts ranging from about 0.01 (greek mu)g/ml to 1 mg/ml, preferably from about 0.5 (greek mu)g/ml to 100 (greek mu)g/ml, there not being less than about 1 (greek mu)g/ml nor more than about 1 mg/ml of furocoumarins. In carrying out the invention the furocoumarin(s), in an appropriate solvent which is substantially inert and sufficiently non-polar to allow for dissolution of the furocoumarin(s), are combined with the viral suspension, conveniently a viral suspension in an aqueous buffered medium, such as used for storage. The amount of virus will generally be about 1x10(6) to 10(11), more usually about 1x10(7) to 10(9) and preferably about 1x10(8) to 5x10(8) pfu/ml. The furocoumarin(s) will be at a concentration of about 0.001 mg/ml to 0.5 mg/ml, more usually about 0.05 mg/ml to 0.2 mg/ml. The amount of solvent which is used to dissolve the furocoumarin will be sufficiently small so as to readily dissolve in the aqueous viral suspension. Although viral inactivation according to the present invention will normally be carried out in an inactivation medium as just described, in some cases it may be desirable to introduce furocoumarins to the virus by addition to a cell culture medium in which the virus is grown. The inactivation is then carried out by separating the live viral particles from the culture medium, and exposure of the particles to ultraviolet light in an inactivation medium which may or may not contain additional furocoumarins. This method of inactivation is useful where the virus is resistant to inactivation when the furocoumarin is added to the inactivation medium only. When employing furocoumarins with limited aqueous solubility, typically below about 50 g/ml, it has been found useful to add an organic solvent, such as 19 4,693,981 5 dimethyl sulfoxide (DMSO), ethanol, glycerol, polyethylene glycol (PEG) or polypropylene glycol, to the aqueous treatment solution. Such furocoumarins having limited solubility include 8-MOP, TMP, and psoralen. By adding small amounts of such organic solvents to the aqueous composition, typically in the range from about 1 to 25% by weight, more typically from about 2 to 10% by weight, the solubility of the furocoumarin can be increased to about 200 greek mu g/ml. or higher. Such increased furocoumarin concentration may permit the use of shorter irradiation times. Also, inactivation of particularly recalcitrant microorganisms may be facilitated without having to increase the length or intensity of ultraviolet exposure, and the addition of an organic solvent may be necessary for inactivation of some viruses with particular furocoumarins. The ability to employ less rigorous inactivation conditions is of great benefit in preserving the antigenicity of the virus during inactivation. At times, it may be desirable to employ organic solvents, particularly DMSO, with all furocoumarins regardless of solubility. For some microorganisms, particularly viruses having tight capsids, the addition of the organic solvent may increase the permeability of the outer coat or membrane of the microorganism. Such increase in permeability would facilitate penetration by the furocoumarins and enhances the inactivation of the microorganism. The furocoumarin may be added to the viral suspension in a single addition or in multiple additions, where the virus is irradiated between additions, or may be added continuously during the entire treatment period, or a portion thereof. Usually, the number of additions will be from about 1 to 50, more usually from about 10 to 40, and preferably from about 2 to 4. The total amount of furocoumarin which will be added will be sufficient to provide a concentration of at least about 0.01 mg/ml to about 1 mg/ml. usually not more than about 0.75 mg/ml. and preferably not more than about 0.5 mg/ml. Since a substantial proportion of the furocoumarin will have reacted with the nucleic acid between additions, the total concentration of furocoumarin in solution will generally not exceed about 0.1 mg/ml. In cases where the furocoumarin(s) employed are particularly unstable, it may be beneficial to add the furocoumarin solution continuously during the irradiation procedure. In order to preserve the antigenic characteristics of the virus, irradiation is carried out in the substantial absence of oxygen and other oxidizing species. This is particularly important when employing psoralens that generate more singlet oxygen on a molar basis. For example, AMT generates more singlet oxygen than 8-MOP. Conveniently, oxygen and other gases may be removed from the inactivation medium by maintaining the medium in a non-oxidizing gas atmosphere, e.g., hydrogen, nitrogen, argon, helium, neon, carbon dioxide, and the like. The inactivation medium may be held in an enclosed vessel, and the space above the liquid medium surface filled with the non-oxidizing gas. Oxidizing species initially in the medium will be exchanged for the non-oxidizing gases according to gas-liquid equilibrium principles. Preferably, the space above the inactivation medium will be flushed with non-oxidizing gas to remove the oxidizing species and further lower their equilibrium concentration in the liquid medium. Depending on the volume of the inactivation medium, the flushing should be continued for at least 1 minute, pref- 6 erably at least 2 minutes, usually being in the range from about 3 to 30 minutes. Flushing may be continued during the irradiation period, but need not be so long as the oxidizing species have been substantially removed and the vessel remains sealed to prevent the intrusion of air. Optionally, a single oxygen scavenger may be added to the inactivation medium prior to irradiation to further prevent interaction of oxygen with the furocoumarin and the virus. Suitable oxygen scavengers include ascorbic acid, dithioerythritol, sodium thionate, glutathione, and the like. The scavenger will be present at a concentration sufficient to block active oxygen species, usually being between 0.001M and 0.5M. more usually being between about 0.005M and 0.02M, where the addition may be in single, multiple, or continuous additions. The concentration of dissolved oxygen may be reduced through the use of enzyme systems, either in solution or immobilized on a solid substrate. Suitable enzyme systems include glucose oxidase or catalase in the presence of glucose and ascorbic acid oxidose in the presence of ascorbate. Such enzyme systems may be employed alone or together with the other methods for oxygen reduction discussed above. The total time for the irradiation will vary depending upon the light intensity, the concentration of the furocoumarin, the concentration of the virus, and the manner of irradiation of the virus, where the intensity of the irradiation may vary in the medium. The time of irradiation necessary for inactivation will be inversely proportional to the light intensity. The total time will usually be at least about 2 hrs. and not more than about 60 hrs., generally ranging from about 10 hrs. to 50 hrs. The times between additions of furocoumarin, where the furocoumarin is added incrementally, will generally vary from about 1 hour to 24 hrs., more usually from about 2 hrs. to 20 hrs. The light which is employed will generally have a wavelength in the range from about 300 nm to 400 nm. Usually, an ultraviolet light source will be employed together with a filter for removing UVB light. The intensity will generally range from about 0. 1mW/cm(2) to about 5W/cm(2), although in some cases, it may be much higher. The temperature for the irradiation is preferably under 25 degrees C., more preferably under 20 degrees C. and will generally range from about -10 degrees C. to 15 degrees C., more usually from about 0 degrees to 10 degrees C. During irradiation, the medium may be maintained still, stirred or circulated and may be either continuously irradiated or be subject to alternating periods of irradiation and non-irradiation. the circulation may be in a closed loop system or in a single pass system ensuring that all of the sample has been exposed to irradiation. It may be desirable to remove the unexpended furocoumarin and/or its photobreakdown products from the irradiation mixture. This can be readily accomplished by one of several standard laboratory procedures such as dialysis across an appropriately sized membrane or through an appropriately sized hollow fiber system after completion of the irradiation. Alternatively, one could use affinity methods for one or more of the low molecular weight materials to be removed. The inactivated virus may then be formulated in a variety of ways for use as a vaccine. The concentration of the virus will generally be from about 10(6) to 10(9) pfu/ml, as determined prior to inactivation, with a total 20 4,693,981 7 dosage of at least 10(5) pfu/dose, usually at least 10(6) pfu/dose, preferably at least 10(7) pfu/dose. The total dosage will usually be at or near about 10(9) pfu/dose, more usually being about 10([ILLEGIBLE]) pfu/dose. The vaccine may include cells or may be cell-free. it may be an inert physiologically acceptable medium, such as ionized water, phosphate-buffered saline, saline, or the like, or may be administered in combination with a physiologically acceptable immunologic adjuvant, including but not limited to mineral oils, vegetable oils, mineral salts and immunopotentiators, such a muramyl dipeptide. The vaccine may be administered subcutaneously, intramuscularly, intraperitoneally, orally, or nasally. Usually, a specific dosage at a specific site will range from about 0.1 ml to 4 ml. where the total dosage will range from about 0.5 ml to 8 ml. The number of injections and their temporal spacing may be highly variable, but usually 1 to 3 injections at 1, 2, or 3 week intervals are effective. The following examples are offered by way of illustration, not by way of limitation. EXPERIMENTAL Materials and Methods A. Virus Growth and Tissue Culture Hamster cells [BHK-21(C-13), American Type Culture Collection (ATCC), CCL 10] were grown as monolayers in plastic cell culture vessels in Eagle's Minimum Essential Medium (MEM) with Earle's salts and nonessential amino acids (MEN) supplemented with 10% heat inactivated calf serum (C(i)) and 10% tryptose phosphate broth (Tp. e.g., Difco 0060). Cell cultures were used to produce live vesicular stomatitis virus. New Jersey serotype (VSV-NJ) from master seed virus originally obtained from the ATCC (VR-159), and live blue-tongue virus (BTV) from master seed virus originally obtained from Dr. T. L. Barber, USDA, Denver, Colorado. Cells were grown in culture vessels to 80% to 100% confluency (approximately 2 x 10(5) cells per cm(2) of growth surface area) using standard mammalian cell culture techniques. Corning plastic roller bottles (Corning No. 25140-850) with a growth surface area of 850 cm(2)-containing 100 ml of MEN supplemented with 10% C(i) and 10% Tp and 1 x 10(8) to 2 x 10(8) CCL 10 cells/bottle were used for virus production. The cell cultures were initiated by seeding approximately 1 x 10(6) to 5 x 10(7) cells into 100 mls of growth medium in a roller bottle containing 5% CO(2) in air on a roller bottle rotator at 1 to 5 rpm at 35 degrees C. to 38 degrees C. The cultures were grown to 80% to 100% confluency over a six to fourteen day period with a medium change every two to four days. When the monolayers reached 80% to 100% confluency, the culture medium was removed and the monolayer was infected with approximately 1 x 10(6) to 2 x 10(6) plaque forming units (pfu) of VSV or BTV in 20 mls of MEN, with 2% heat-inactivated fetal bovine serum (F(i)) added for BTV. The multiplicity of infection (MOI) was approximately 0.01. the MOI may range from 0.001 pfu/cell to 0.033 pfu/cell. The virus inoculum was adsorbed to the cells by incubation at 35 degrees C. to 38 degrees C. for one hour at 1 to 5 rpm. One hundred mls of MEN containing 10% YELP supplement (v/v) for VSV, or 10% C(1) and 10% Tp for BTV, was added per roller bottle. YELP supplement contains: yeast extract BBL 11929, 5 g/liter; lactalbumin hydrolysate GIBCO 670-1800, 25 g/liter; and Bacto-Peptone (Difco 0118), 50 g/liter. The post-infection incubation was carried out 8 at 35 degrees C. to 38 degrees C. in 5% CO(2)/95% air with rotation. Sixteen to forty-eight hours post-infection, VSV cytopathic effect (CPE) was evident, while BTV CPE became apparent from 2 to 4 days post infection. The CPE was characterized by cell rounding, cell detachment, and cell degeneration. When visual or microscopic examination indicated that at least 50% of the cell monolayer exhibited CPE, the contents of the roller bottle were swirled to remove loosely attached materials from the roller bottle walls. The harvest material was decanted from the roller bottles into centrifuge bottles. The crude VSV harvest was clarified by centrifugation at 500 to 1000 x g for 20 minutes, at 4 degrees C. The BTV harvest was centrifuged at 2,000 x g for 60 minutes at 4 degrees C. The clarified VSV preparations were concentrated by ultrafiltration using a Pellicon cassette system (Millipore XX42ASY60) with a cassette having a nominal exclusion limit of 10(5) daltons (Millipore PTHK 000C5). The Pellicon cassette system was sterilized by filling the assembled unit with IN NaOH and incubating the unit 12 to 24 hours at room temperature. The NaOH solution was pumped out of the cassette system and the system was flushed with two to four liters of sterile H(2)O. The assembly and operation of the Pellicon system were in accordance with the instructions furnished by the manufacturer. All steps in the concentration were performed aseptically. The clarified VSV was concentrated 15 to 40 fold, dimethylsulfoxide (Sigma D-5879) added to a final concentration of 7.5% v/v, and suitable aliquots of the virus stored frozen at -80 degrees C. to -100 degrees C. For BTV, the pellet resulting from centrifugation was resuspended aseptically in 8 ml of 2mM Tris-HCl, pH 8.8, for each original roller bottle. The suspension was mixed vigorously on a vortex mixer, and/or sonicated at 4 degrees C for 1 min., and centrifuged at 1,400 times g for 30 min. at 4 degrees C. The virus-containing supernatant was collected and the pellet was extracted twice more with 8 ml/roller bottle aliquots of 2mM Tris-HCl, pH 8.8. The virus-containing supernatants were pooled and clarified by centrifugation at 4,000 x g for 30 min. at 4 degrees C. The clarified supernatant was stored at 4 degrees C. Feline herpes I virus (FVR, the infective agent of feline viral rhinotracheitis) was grown as follows. Cat cell lines AKD (ATCC CCL150) or Fc3Tg (ATCC CCL176) were grown as monolayers in plastic cell culture vessels in a standard defined culture medium consisting of MEN; F12K; MEM; or alpha MEM. Medium was supplemented with 2% to 15% inactivated fetal calf serum (F(i)) or 2% to 20% YELP. Cell cultures were used to produce live Feline Herpes I virus from master seed virus derived from Feline Herpes I virus (ATCC VR636). Cells were grown in culture vessels to 80% to 100% confluency (approximately 1 x 10(5) to 2 x 10(5) cells per cm(2) of growth surface area) using standard mammalian cell culture techniques as follows. Corning plastic roller bottles containing 50 to 100 ml of MEN supplemented with 10% F(i) and 1 x 10(8) to 2 x 10(8) AKD or Fc3Tg cells/bottle were used for Feline Herpes I virus production. The cell cultures were initiated by seeding approximately 1 x 10(6) to 5 x 10(6) cells into 50 to 100 mls of growth medium in a roller bottle containing about 5% CO(2) in air and incubating the roller bottle on a roller bottle rotator at 1 to 5 rpm at 35 degrees C. to 38 degrees C. The cultures were grown to 80% to 21 4,693,981 9 100% confluency over a 7 to 14 day period with a 100% medium change every 3 to 5 days. When the monolayers were 80% to 100% confluent, the culture medium was removed and the monolayer was washed with 20 to 50 mls of phosphate buffered saline (PBS) pH 7.2 to 7.4 (NaCl 8 g+KCl 0.2 g+Na(2)NPO(4) 1.14 g+KH(2)PO(4)0.2 g). The PBS wash was discarded, and the roller bottle was infected by the addition of approximately 1 x 10(7) to 2 x 10(7) plaque forming units (pfu) of Feline Herpes 1 virus in 10 mls of PBS containing 2% F(i). The multiplicity of infection (MOI) was approximately 0.1. The virus inoculum was adsorbed to the cells by incubation at 35 degrees C. to 38 degrees C. for one hour at 1 to 5 rpm. The inoculation fluid was removed and 50 mls of MEN containing 10% F(i) was added per roller bottle. The post-infection incubation was at 35 degrees C. to 38 degrees C. in 5% CO(2) in air with rotation. Herpesvirus cytopathic effect (CPE) was evident forty to forty-eight hours post-infection. The CPE was characterized by cell rounding, cell detachment, and cell degeneration. The contents of the roller bottle were swirled 48 hours post-infection to remove loosely attached materials from the roller bottle walls, and the contents of the roller bottles were decanted into centrifuge bottles. The virus, cells, and cell debris were pelleted by centrifugation at 10,000 x g for 30 minutes. Cell associated (CA) Feline Herpes I virus was prepared by: 1. resuspending the 10,000 x g pellet in approximately 5 ml of a resuspension medium containing 80 parts F12K, 10 parts F(i) and 10 parts dimethylsulfoxide (DMSO) for each original roller bottle; 2. freezing the resuspended CA virus at -20 degrees C. for 1.5 to 2 hours, and 3. transferring the CA virus frozen at -20 degrees C. to temperatures ranging from -80 degrees C. to -100 degrees C. Cell free (CF) Feline Herpes I virus was prepared by: 1. resuspending the 10,000 x g pellet in F12K; 2. freezing and thawing the resuspended material 3 times; 3. clarifying the freeze-thawed material by centrifugation at 10,000 x g for 30 minutes; and 4. freezing the clarified supernatant (CF virus) at temperatures ranging from -80 C. to -100 C. CF or CA virus was thawed by gentle agitation at 37 degrees C. in a water bath. B. Virus Assay Confluent monolayers of LMTK-, Vero (ATCC CCL 81), Fc3Tg, or AKD cells were prepared in 6 cm diameter mammalian cell culture plastic petri dishes (Corning #25010) or other convenient cell culture vessels. The growth medium used for LMTK- cells was alpha ME (alpha modified Eagles Minimum Essential Medium, Earle's Salts)+10% F(i). The growth medium used for Vero cells was MEN+5% F(i). The growth medium used for Fc3Tg cells was MEN+10% F(i) and the growth medium used for AKD cells was F12K+15% F(i) (VSV and BTV were titered on LMTK- or Vero cells. Feline Herpes I was titered on Fc3Tg or AKD cells). Ten fold serial dilutions of virus samples were made by adding 0.5 ml of the virus sample to 4.5 mls of diluent (phosphate buffered saline, pH 7.2 to 7.4, plus 2% F(i) in a screw cap tube. The growth medium was removed from a 6 cm culture dish cell monolayer, 0.1 ml of virus sample (undiluted or diluted) was added, and the virus was adsorbed to the mono- 10 layer for 1 to 2 hours at 35 degrees C. to 38 degrees C. Two or more monolayers were used for each sample. Five ml of overlay medium was added per 6 cm culture dish, except for Feline Herpes I, where the unadsorbed inoculum was removed, and 4 mls of overlay medium was added per 6 cm culture dish. The overlay medium for BTV or VSV was prepared by mixing equal parts of solution A (100 ml 2X MEM with L-glutamine. GIBCO #320-1935, +10 ml F(i) and 1.8% to 2% Noble Agar (Difco 0142)in deionized H(2)0 at 44 degrees C. to 45 degrees C. The overlay medium for Feline Herpes I was prepared by mixing equal parts solution A and 1% methyl cellulose (4,000 centriposes) in deionized H(2)0 (Fisher M-281 sterilized by autoclaving). The virus infected cultures were incubated at 35 degrees C. to 38 degrees C. in 5% CO(2) in air. Twenty-four hours before plaques were counted, a second overlay containing Neutral Red at a final concentration of 0.005% was added. Plaques were counted on day 2 or day 3 post-infection for VSV, on day 2 to 4 for FVR and on day 6 or 7 for BTV. The virus titer in pfu/ml was calculated by multiplying the average number of plaques per dish by the reciprocal of the dilution. The pfu/ml was the value used to determine the amount of virus needed to infect cells at a MOI of approximately 0.01. The pfu/ml in a virus preparation prior to inactivation was used to determine the immunizing dose. C. Virus Inactivation 1. VSV Inactivation The thawed stock of VSV was pipetted into sterile T-150 tissue culture flasks (nominally 25 ml into each of four flasks). To each flask was added 0.25 ml of 4'-aminomethyl-4.5', 8-trimethylpsoralen (AMT) stock solution (stock solution is 1 mg/ml AMT dissolved in sterile, deionized water). Each flask was allowed to equilibrate in an argon atmosphere for at least 10 minutes. After equilibration, a stream of argon gas was directed into each flask for a least two minutes. The flasks were then tightly capped and placed under a long wavelength ultraviolet (320 nm to 400 nm) light source (GE BLB fluorescent bulbs) at a temperature between 0 degree C. and 20 degrees C. for approximately 11 hours. The incident light intensity was approximately lmW/cm(2) (measured by a J-221 long wavelength UV meter). After the irradiation was completed, the flasks were removed from the light source and an additional 0.25 ml of AMT stock solution was mixed into each flask. The contents of each flask were pipetted into new, sterile T-150 flasks, and the flasks were again flushed with argon and irradiated for an additional 11 hours. This procedure was repeated three more times until five additions (a total of approx. 50 (greek mu)g/ml) of AMT had been performed, the virus sample had been irradiated for at least 55 hours, and at least four flask changes had been performed. After all of the irradiations had been completed, the contents of the flasks were aseptically transferred to a common sterile container and stored at -85 degrees C. 2. BTV Inactivation Twenty-five ml of BTV serotype 11 (1.5 x 10(8) pfu/ml) was mixed with 0.25 ml of 4'-aminomethyl 4,5', 8-trimethylpsoralen (AMT; 1 mg/ml in DMSO). The mixture was placed in a 150cm(2) tissue culture flask (T-150; Corning #25120). The viral suspension in the flask was placed in an argon atmosphere for 10 min., 22 4,693,981 11 and a stream of argon gas as then blown over the viral suspension for an additional 2 min. The flask was tightly capped and the suspension irradiated for 3.25 hrs. at 4 degrees C. using GE BLB fluorescent bulbs at an intensity of 1.5mW/cm(2). An additional 0.25 ml of AMT was then added to the viral suspension, the suspension transferred by pipette to a new T-150 flask, and the solution again flushed with argon. The flask was irradiated for an additional 14.75 hours at 4 degrees C. under the same long wavelength UV light source. After this irradiation an additional 0.25 ml of AMT solution was added to the suspension, and it was again transferred to a new T-150 flask. The solution was flushed with argon as before and irradiated for an additional 5.5 hours at 4 degrees C. The inactivated BTV was stored at 4 degrees C. 3. Feline Herpes I Inactivation a. Cell Free Virus Nineteen mls of CF-FVR (1.9 x 10(7) pfu/ml) were mixed with 0.4 ml of hydroxymethyltrioxsalen (HMT: 1 mg/ml in DMSO) and 1.9 ml of sodium ascorbate (0.1 M in H(2)O). The mixture was prepared in 150 cm(2) tissue culture flasks (T-150, Corning No. 25120) that were subsequently deaerated for 2 minutes with pure argon gas. The virus-containing flasks were irradiated for 55 minutes at 4 degrees C. using G.E. BLB fluorescent bulbs at an intensity of 1.5 mW/cm(2). The FVR/HMT/ascorbate mixture was then transferred by pipet into a second T-150 flask, which was deaerated for 2 minutes using pure argon gas. The second T-150 flask was irradiated for an additional 28 minutes at 4 degrees C. under the same long wavelength UV light source. The CFV-FVR preparation was stored at -100 degrees C. in a REVCO freezer. Subsequently the CF-FVR preparation was thawed and placed into a T-150 flask. The flask was deaerated with pure argon gas for 2 minutes and irradiation was continued as described above for an additional 15 hours and 40 minutes. b. Cell Associated Virus Cells from 10 roller bottles (about 1x10(8) to 2x10(8) cells/roller bottle) were resuspended in 28 mls of cell culture media. Twenty mls of the suspension were placed into a T-150 flask. To this flask was added 2 ml of freshly prepared sterile 0.1 M sodium ascorbate and 0.4 ml HMT (1 mg/ml in DMSO). The flask was deaerated with pure argon gas for 2 minutes, and the flask was irradiated at 4 degrees C. using G.E. BLB fluorescent bulbs at an intensity of 1.5 mW/cm(2) for 75 minutes. The viral suspension was then transferred by pipet from the T-150 flask into a second T-150 flask and again deaerated with pure argon gas for 2 minutes. Irradiation was continued for an additional 95 minutes. The CA-FVR preparation was adjusted to 10% DMSO and the suspension was frozen at -20 degrees C. for 1 hour and then stored at -100 degrees C. in a REVCO freezer. The stored frozen CA-FVR preparation was subsequently thawed, and the cells were pelleted in a clinical centrifuge. The cells were resuspended in 21 mls of serum-free medium to which 2.1 mls of freshly prepared 0.1 M sodium ascorbate and 0.4 ml of HMT (1 mg/ml in DMSO) were added. The sample was transferred by pipet to a T-150 flask, and irradiation was continued for an additional 15 hours and 40 minutes. 12 Results A. Bluetongue Virus 1. Assessment of Inactivation by Blind Passage CCL 10 cells were grown to confluency in 850 cm(2) roller bottles using standard cell culture procedures, as described above. The culture medium was removed from the roller bottle, and 2.0 ml of the inactivated virus preparation mixed with 18 ml of medium containing 2% F(i) was adsorbed to the roller bottle monolayer for 60 min at 35 degrees C. to 38 degrees C. with rotation at 1 to 5rpm. After adsorption, the residual unabsorbed inoculum was removed, and 100 ml of growth medium (MEN with 10% C(i) and 10% Tp) was added and the roller bottle culture incubated at 35 degrees C. to 38 degrees C. for 7 days with daily observation for viral CPE. The roller bottle culture received a 100% medium change every 2 to 3 days. If no CPE was observed during the first roller bottle passage, the cell monolayer was chilled at 4 degrees C. for 12 to 24 hrs. The cells were scraped into the medium which was then decanted into a centrifuge bottle. The cells were pelleted by centrifugation at 4 degrees C. at 2,000 x g for 30 min. and resuspended in 2.0 ml of 2mM Tris-HCl (pH 8.8) by vigorous mixing using a vortex mixer. The resuspended material was centrifuged at 2,000 x g for 20 min. at 4 degrees C. The supernatant was added to 18 ml of growth medium containing 2% F(i) and used to infect a new confluent roller bottle culture of CCL 10 cells, as described immediately above. The second roller bottle blind passage was observed for 7 days and fed ever 2 to 3 days. If no CPE was observed during the second roller bottle blind passage, a third roller bottle blind passage was performed. If no CPE had been observed by the end of the third roller bottle blind passage the virus preparation was considered inactivated and suitable for in vivo testing. 2. Immunization of Rabbits with Psoralen-inactivated BTV Vaccine a. Example 1 Four New Zealand white rabbits were randomly assigned to 2 groups, designated A and B. Both groups were given 4 immunizations at two week intervals. The first immunization consisted of 1 ml of vaccine (10(8) pfu BTV serotype 11) and 1 ml of Freund's Complete Adjuvant. The second through fourth immunizations utilized 1 ml of vaccine (10(6) pfu BTV serotype 11) and 1 ml of Freund's Incomplete Adjuvant. All immunizations were given intramuscularly (IM). The vaccine given to Group A (Vaccine #1) was inactivated with AMT-UVA in the presence of 0.01M ascorbic acid. Vaccine #1 was dialyzed for 12 hours against 2mM Tris, pH 8.6. The vaccine given to Group B (Vaccine #2) was inactivated with AMT-UVA without ascorbic acid and sonicated three times (2 minutes each time) using a cup horn probe (Heat Systems Model 431A) at a power setting of 3 (Heat Systems Model W220). Both Vaccine #1 and Vaccine #2 were deemed inactivated since no live virus was detected during blind passage. Inactivated vaccine was also tested for safety by chicken embryo inoculation. Egg deaths attributable to live virus were not encountered. Both rabbit groups were bled via auricular venipuncture one week following the second, third, and fourth immunizations. Serum from each rabbit was pooled with that of its groupmate, and the pooled sera were tested for anti-BTV antibodies by two standard 23 4,693,981 13 serologic assays, serum neutralization (Jochim and Jones, Am. J. Vet. Res. (1976) 37:1345-1347) and agar gel precipitation (Jochim et al., Am. Assoc. Vet. Lab. Diag., 22nd Proceed.: 463-471, 1979) Pre-immunization rabbit serum was used as the negative control; BTV immune sheep serum was used as the positive control for both immunologic procedures. Pooled sera from Groups A and B reduced the number of viral plaques (serum neutralization) greater than eighty percent (arbitrarily selected end point) when the sera were diluted 1:40, which was the highest dilution examined. Negative and positive control sera behaved as expected.
TABLE 1 - -------------------------------------------------------------------------------- Serum Neutralization Data From Rabbits Vaccinated with AMT-UVA-inactivated Bluetongue Virus Vaccines --------------------------------------- Titer* ----------------------------------- Group 1 5 40 - -------------------------------------------------------------------------------- A + + + B + - + Normal Rabbit Serum - - - BTV-Immune Sheep Serum + + plus or minus - -------------------------------------------------------------------------------- * Reciprocal of serum dilution neutralizing [ILLEGIBLE] percent of BTV plaque activity on BHA cells. The data are from the post-second immunization serum samples.
Pooled post-immunization sera from Groups A and B precipitated BTV antigen in immunodiffusion plates when tested at dilutions up to 1:16. Normal rabbit serum did not precipitate the standard BTV antigen. BTV-immune sheep serum did precipitate the BTV antigen, but not at dilutions greater than 1:2. Of the two immunologic procedures utilized, serum neutralization is considered predictive for immunity to live BTV challenge in the target species. b. Example 2 Twelve New Zealand white rabbits were randomly assigned to six groups, A-F, two rabbits per group. An additional four rabbits were assigned to group G. These sixteen rabbits were vaccinated twice subcutaneously with the AMT-UVA inactivated Bluetongue virus vaccines described in Table 2. Preinactivation titer was approximately 10(8) pfu for each serotype. The vaccines were formulated with 20% (v/v) aluminum hydroxide adjuvant, and were given with a three week interval between the first and second inoculations. The sixteen rabbits were bled by auricular venipuncture on days 0, 14 and 35. Each serum was heat-inactivated and tested against BTV serotypes 10, 11, 13 and 17 for serum neutralizing antibody. All vaccinated rabbits developed SN titers against the homologous vaccine serotypes (Table 3). These data demonstrated the immunopotency of a multivalent AMT-UVA inactivated Bluetongue virus vaccine.
TABLE 2 - -------------------------------------------------------------------------------- Serotype Composition of Inactivated Bluetongue Virus Vaccines Tested in Rabbits ---------------------------------------------- BTV Serotype Group Rabbit # Composition - -------------------------------------------------------------------------------- A 1, 2 10 B 3, 4 11 C 5, 6 13 D 7, 8 17 E 9, 10 11, 17 F 11, 12 10, 11, 17 G 13, 14, 15, 16 10, 11, 13, 17 - --------------------------------------------------------------------------------
14 TABLE 3 - -------------------------------------------------------------------------------- Serum Neutralizing Data from Rabbits Vaccinated with AMT-UVA Single and Multi-Serotypes Bluetongue Virus Vaccines --------------------------------------------------- SN Titer* Against ------------------------------------------------------ Group Rabbit BTV-10 BTV-11 BTV-13 BTV-17 - -------------------------------------------------------------------------------- A 1 1:160 1:10 1:10 1:10 2 1:320 1:10 1:10 1:10 B 3 1:10 1:320 1:10 1:10 4 1:10 1:80 1:10 1:10 C 5 1:20 1:20 1:160 1:10 6 1:20 1:10 1:40 1:10 D 7 1:10 1:10 1:10 1:320 8 1:10 1:10 1:10 1:320 E 9 1:20 1:160 1:20 1:160 10 1:20 1:160 1:20 1:160 F 11 1:160 1:160 1:20 1:160 12 1:40 1:40 1:20 1:80 G 13 1:160 1:160 1:80 1:160 14 1:160 1:160 1:80 1:160 15 1:160 1:160 1:40 1:160 16 1:80 1:160 1:160 1:160 - -------------------------------------------------------------------------------- * Reciprocal of serum dilution neutralizing [ILLEGIBLE] of BTV plaque activity on [ILLEGIBLE] cells. The data are from the post-second immunization sera (Day 35). Negative and positive control sera behaved as expected in the SN assay.
3. Immunization of Sheep with Psoralen-inactivated BTV Vaccine a. Example 1 Each of two adult sheep, known to be susceptible to BTV, was inoculated subcutaneously (SQ) with 2 ml of AMT-UVA inactivated BTU plus adjuvant (1:1; vaccine to aluminum hydroxide adjuvant). The vaccine contained approximately 10(8) pfu/ml of BTV prior to inactivation. A third sheep was inoculated SQ with 6 ml of the identical vaccine without adjuvant. Seven weeks later the three sheep were given identical inoculations SQ that consisted of 5 ml of vaccine and aluminum hydroxide adjuvant (2:1 vaccine to adjuvant; 10(8) pfu BTV/ml of vaccine). The three sheep were monitored for clinical evidence of BTV, including daily body temperature recording and bi-daily virus isolation attempts. No evidence of BTV was observed, indicating that the vaccine was inactivated. Serum was collected weekly for serum neutralization and agar gel precipitation testing. Normal sheep sera and BTV-immune sheep sera were used for negative and positive control samples in the serologic tests. The first vaccine inoculations induced precipitating anti-BTV antibody in all three sheep. Their pre-exposure sera were uniformly negative for anti-BTV precipitating antibody. Modest neutralizing anti-BTV antibody titers (1:5) were elicited in two of three sheep following one immunization. The second immunization elicited a distinct immunologic anamnestic response, inducing neutralizing titers of 1:40, 1:80, or 1:160 in the three sheep.
TABLE 4 - -------------------------------------------------------------------------------- Serum Neutralization Data From Sheep Immunized with an AMT-UVA Inactivated BTV Vaccine ---------------------------------------------- TITERS* Sheep No.: ----------------------------------- 1 2 3 - -------------------------------------------------------------------------------- Pre-First Immunization Day 0 <5 <5 <5 Post-First Immunization Day 21 5 5 <5 Post-Second Immunization - --------------------------------------------------------------------------------
24 4,693,981 15
TABLE 4-continued - ------------------------------------------------------------------------------- Serum Neutralization Data From Sheep Immunized with an AMT-UVA Inactivated BTV Vaccine. - ------------------------------------------------------------------------------ TITTERS* Sheep No.: --------------------------------------- 1 2 3 - ------------------------------------------------------------------------------- Day 7 80 160 40 Day 14 80 40 40 Day 21 80 80 40 Day 42 80 80 80 Post-Challenge ----------- Day 7 160 160 80 Day 14 320 160 80 - ------------------------------------------------------------------------------- *reciprocal of highest 2-fold dilution reducing BTV plaque activity on BHA cell by 80 percent
The sheep were challenged by SQ syringe inoculation of 10(5) egg lethal doses of BTV serotype 11. The three sheep remained clinically normal during the BTV challenge period, indicating that the vaccine was efficaceous. It is evident from the above results that the BTV which is psoralen-inactivated retains its immunogenicity, particularly as to those sites which elicit an immune response which is effective in protecting a host against subsequent BTV-infection. Thus, the psoralen inactivation can be carried out under conditions which do not modify the immunogenic sites of the virus, so as to elicit an immunogenic response which will be effective against the live BTV. Furthermore, the BTV RNA virus is efficiently inactivated under mild conditions to the point of complete inactivation, whence it may be safely administered to a host. b. Example 2 Eight experimental and four control sheep, known to be Bluetongue Virus susceptible, were housed together in an insect-proof facility. The experimental sheep were inoculated twice subcutaneously with AMT-UVA inactivated BTV Serotype 11 vaccine. Each vaccinate received approximately 3 x 10(8) pfu BTV-11 formulated with twenty-five percent (v/v) aluminum hydroxide adjuvant. Three weeks elapsed between immunizations. Control sheep were inoculated with tissue culture fluid in 25% percent (v/v) aluminum hydroxide. Serum samples were collected prior to vaccination, following vaccinations, and following challenge, and tested for SN antibodies. All sheep were challenged by subcutaneous inoculation of 2 x 10(5) ELD(50) BTV-11 four weeks post-second vaccination. Virus isolation was performed twice weekly post-challenge for six weeks. Virus isolation from sheep blood was done by intravenous chicken embryo inoculation, followed by specific BTV serotype identification by neutralization in vitro. Five of the eight vaccinated sheep developed SN titers of 1:20 post-second vaccination. All eight vaccinates resisted subcutaneous challenge with 2 x 10(5) ELD(50) BTV-11, whereas the four control sheep developed uniform viremia as assessed by egg inoculation. Sheep data are given in Table 5.
TABLE 5 - ------------------------------------------------------------------------------- Serum Neutralization and Virus Isolation Data from Sheep Vaccinated with AMT-UVA Inactivated BTV-11 Vaccine and Subsequently Challenged with 2 x 10(5) ELD(50) of Live BTV-11 - ------------------------------------------------------------------------------- SN Titer Virus Isolation Sheep Base- Post-Second Post- Post-Challenge Day ------------------ No. line Vaccination Challenge 4 11 15 18 - -------------------------------------------------------------------------------
16
TABLE 5-continued - ------------------------------------------------------------------------------- Serum Neutralization and Virus Isolation Data from Sheep Vaccinated with AMT-UVA Inactivated BTV-11 Vaccine and Subsequently Challenged with 2 x 10(5) ELD(50) of Live BTV-11 - ------------------------------------------------------------------------------- SN Titer Virus Isolation Sheep Base- Post-Second Post- Post-Challenge Day ------------------ No. line Vaccination Challenge 4 11 15 18 - ------------------------------------------------------------------------------- Experimental - ----------- 650 neg 1:20 1:160 - - - - 651 neg 1:20 1:40 - - - - 652 neg 1:20 1:160 - - - - 653 neg 1:20 1:40 - - - - 656 neg 1:10 1:160 - - - - 658 neg 1:10 1:40 - - - - 659 neg 1:20 1:160 - - - - 660 neg 1:10 1:160 - - - - Controls - -------- 654 neg neg 1:10 - + + + 655 neg neg neg + + + + 661 neg neg 1:40 + + + + 662 neg neg 1:160 - - - - - -------------------------------------------------------------------------------
B. Feline Herpes Virus I 1. Assessment of Inactivation by Blind Passage Fc3Tg or AKD cells were grown to confluency in 850 cm(2) roller bottles using standard cell culture procedures as described above. The culture medium was removed from the roller bottle, and 2.0 mls of the inactivated virus preparation, mixed with 18 mls of medium containing 2% F(i), were absorbed to the roller bottle cell monolayer for 60 minutes at 35 degrees C. to 38 degrees C. with rotation at 1 to 5 rpm. After adsorption, the inoculum was removed and 150 ml of maintenance medium (MEN or F12K with 2% F(i)) added. The roller bottle culture was then incubated at 35 degrees C. to 38 degrees C. for 7 days with daily observation for viral CPE. The roller bottle culture received a 100% medium change after 3 to 5 days. If no CPE was observed during the first roller bottle passage, the cell monolayer was scraped into the maintenance medium which was then decanted into a centrifuge bottle. The cells were pelleted by centrifugation at room temperature at 1,000 x g for 15 minues, resuspended in 20 ml of fresh maintenance medium, and passed to a new confluent roller bottle culture of Fc3Tg or AKD cells as described above. The second roller bottle blind passage was observed for 7 days and fed once on day 3 to 5. If no CPE was observed during the second roller bottle behind passage, a third roller bottle blind passage was performed. If no CPE was observed by the end of the third roller bottle passage, the virus preparation was considered inactive. 2. Administration Procedure for Psoralen-inactivated FVR Vaccines Photochemically inactivated FVR was inoculated via syringe into cats by various routes, including but not limited to intravenously (IV), subcutaneously (SQ), intramuscularly (IM), or intraperitoneally (IP). The vaccine was administered in various volumes (0.5 to 3.0 ml) and in various concentrations (10(6) to 10(8) pfu; either CF, CA or in combination). In the following examples, the vaccine was administered in combination with aluminum hydroxide as an immunologic adjuvant. The number of injections and their temporal spacing was as set forth in each example. 25 4,693,981 17 3. Immunization with Psoralen-inactivated CF-FVR Vaccine The experimental group consisted of four specific pathogen free kittens (2 males, 2 females) four months old (Liberty Laboratories, Liberty Corner, N.J.). The control group consisted of two similar female kittens. The experimental group was inoculated IM with 3x10(7) pfu (3 mls) of HMT inactivated CF-FVR on days 0 and 21, and again inoculated with 3x10(7) pfu HMT inactivated with an equal amount of 2% aluminum hydroxide [AI(OH)(3)] adjuvant on day 61. Controls were vaccinated at eight weeks and at thirteen weeks of age with a commercial FVR vaccine using the manufacturer's recommended procedure. Sireum samples were collected weekly and tested for anti-FVR neutralizing antibodies. Following live virus challenge (10(6) pfu intranasally and intraconjunctivally), a numerical scoring system (Table 6) was used to assess the clinical response of both experimental and control cats. TABLE 6
- ------------------------------------------------------------------------------- Scoring System for Clinical Effects of Herpesvirus Challenge in Cats Factor Degree Score - ------------------------------------------------------------------------------- Fever 101 to 102 degrees F. 0 102 to 103 1 103 to 104 3 greater than 104 5 Depression slight 1 moderate 3 severe 5 Sneezing occasional 1 moderate 3 paroxysmal 5 Lacrimation serous 1 mucoid 3 purulent 5 Nasal Discharge serous 1 mucoid 3 purulent 5 Appetite normal, eats all food 0 fair, eats more than 1 1/2 of food poor; eats less than 3 1/2 of food none; eats nothing 5 - -------------------------------------------------------------------------------
Three of four experimental cats developed serum neutralizing anti-FVR antibody (SN) titers of 1:2 that were detected between day 42 and day 58. Following the third immunization (day 61), four of four experimental cats had SN titers of 1:4 (day 80). Baseline SN antibody titers on the experimental cats were negative. The control cats die not develop detectable SN antibody titers during the pre-challenge period. All cats were exposed to 10(6) pfu of live FVR by intraconjunctival and intranasal exposure on day 91. Each cat was monitored twice daily for the absence, presence and degree of severity of factors given in Table 6. A composite clinical score was derived for each cat after a 15 day observation period. Three of four experimental cats demonstrated mild temperature elevation and serous ocular or nasal discharge along with mild intermittent depression and appetite suppression. Their composite scores were 39, 42, and 35 respectively for the 15 day observation period. The fourth experimental cat was more severely affected (composite score = 84) by moderate, but transient, sneezing and mucoid nasal discharge. Both control cats were severely affected by live virus challenge. Severe purulent nasal and ocular discharge and lack of 18 appetite were apparent. The control cats had composite scores of 133 and 253. Three weeks following live FVR challenge, all cats were tested for SN antibody titers against FVR. Three of four experimental cats had SN antibody titers of 1:16 while the fourth cat had a 1:8 titer. One of the control cats had an SN antibody titer of 1:4 while the second control lacked an SN antibody titer against FVR. 4. Immunization with Psoralen-inactivated CA-FVR Vaccine Nine age-matched specific pathogen free kittens, 4 months old (Liberty Laboratories, Liberty Corner, N.J.), were randomly assigned to three experimental groups designated A, B, and C. Group A (controls) was inoculated twice with 1 ml tissue culture fluid and 1 ml aluminum hydroxide adjuvant. Group B was inoculated twice with a commercial FVR vaccine according to the manufacturer's recommendation. Group C was inoculated three times with 10(7) HMT-inactivated CA-FVR in aluminum hydroxide (total volume = 2 ml; 1:1 vaccine to adjuvant). All injections were given IM at three week intervals. Live FVR virus (10(6) pfu intransally and intraconjunctivally) was given on day 63 and a numerical scoring system (Table 6) was used to assess the kittens' clinical response for a 15 day post-challenge period. Serum samples were collected from all kittens prior to vaccination, prior to the second and third immunizations, prior to live FVR challenge, and at 15 days post-challenge. The sera were utilized to assess neutralizing antibody titers by standard procedures. The control kittens (Group A) maintained SN antibody titers less than 1:2 (negative) throughout the pre-challenge period. Fifteen days following live FVR challenge Group A kittens uniformly had SN antibody titers of 1:2. Kittens in Groups B and C lacked detectable anti-FVR antibody titers pre-immunization, but all kittens in Groups B and C had SN antibody titers of 1:2 or 1:4 after two immunizations. The third immunization in Group C kittens did not significantly alter their SN antibody titers. Following a 15 day post-challenge period, kittens in Groups B and C demonstrated an anamnestic immunologic response, with SN antibody titers ranging from 1:16 to 1:64. Clinically, Group A kittens were severely affected by live FVR challenge, whereas kittens in Groups B and C were significantly protected by their respective vaccines. The composite clinical scores for Group A were 125, 141, and 128 for the 15 day post-challenge period. The composite clinical scores for Group B were 25, 20, and 64, while Group C had composite clinical scores of 21, 15, and 34. The clinical signs evident were characteristic of FVR. From the SN data and clinical scoring, it is evident that kittens immunized with the experimental HMT-inactivated FVR vaccines (cell-free or cell associated) in the above examples were significantly immune to the clinical effects of severe FVR challenge. C. Vesicular Stomatitis Virus 1. Assessment of Inactivation by Intracerebral Inoculation of Mice Suckling mice (0 to 10 days old) were inoculated intracerebrally with 0.02 ml of the psoralen-inactivated 26 4,693,901 19 VSV-NJ using a tuberculin syringe and a 28 or 30 gauge needle. Each vaccine lot was tested in four to nine suckling mice. The mice were observed three times daily for a minimum of seven days. Residual low-level live VSV kills suckling mice in two to five days. The sensitivity of this assay is approximately 1 to 5 pfu of live VSV per intracerebral dose. Inactivated VSV-NJ vaccine was considered safe (inactivated) if all inoculated suckling mice survived the seven day observation period. The VSV-NJ vaccine batches used hereinafter each passed the suckling mouse safety test prior to use. 2. Virus Neutralization in Mice Immunized with Psoralen-inactivated VSV-NJ Vaccine Groups of ten adult white mice each were injected using three immunological adjuvants (aluminum hydroxide gel, incomplete Freund's, or oil emulsion) with one of three psoralen-inactivated VSV-NJ vaccine doses (10(9), 10(8), or 10(7) pfu/dose). The oil emulsion was prepared as described by Stone et al. (1978) Avian Dis. 22:666-674. All mice were injected IP once each, on day 0 and day 21. Serum samples were collected from the orbital sinus on day 20 and on day 33 and pooled serum samples were assessed for serum neutralization (SN) activity by standard procedures. See, Castaneda et al. (1964) Proc. US Livestock San. Assoc. 68:455-468. Serum samples were negative for neutralizing antibodies to VSV-NJ prior to vaccination. The vaccine with oil emulsion adjuvant induced the highest SN titers after one injection. All three vaccine doses, regardless of adjuvant, induced SN titers of at least 1:2000 after two injections. Serum dilutions were tested for SN activity only to 1:2560. The results are set forth in Table 7.
TABLE 7 - -------------------------------------------------------------------------------- Virus Neutralization Indices* of Mouse Sera After One and Two Injections of Psoralen- Inactivated VSV-NJ Vaccine - -------------------------------------------------------------------------------- Log(10) of Vaccine Concentration (pfu/dose) No. of -------------------------------------- Adjuvant Injections 7 8 9 - -------------------------------------------------------------------------------- Aluminum hydroxide gel 1 67* 905 905 Aluminum hydroxide gel 2 >2560 2560 >2560 Freund's Incomplete 1 226 57 905 Freund's Incomplete 2 2033 >2560 >2560 Oil Emulsion 1 >2560 >2560 >2560 Oil Emulsion 2 >2560 >2560 >2560 - -------------------------------------------------------------------------------- *Virus neutralization index is the reciprocal of the serum dilution that neutralized 32 TCID(30) of VSV-NJ
3. Virus Neutralization in Hamsters Vaccinated with Psoralen-inactivated VSV-NJ Vaccine Groups of five MHA hamsters each were injected with either 10(9), 10(8), or 10(7) pfu psoralen-inactivated VSV-NJ per dose, with or without aluminum hyroxide adjuvant (1:1). All hamsters were injected intramuscularly (IM) once each, on day 0 and again on day 21. Pooled serum samples were collected on day 21 and on day 34 for serum neutralization testing by standard procedures. Serum neutralizing antibodies were elicited by all three vaccine doses tested, with or without aluminum hydroxide adjuvant. SN titers are given in Table 8. 20
TABLE 8 - -------------------------------------------------------------------------------- Virus Neutralization Indices* of Hamster Sera After One and Two Injections of Psoralen-Inactivated VSV-NJ Vaccine - -------------------------------------------------------------------------------- Log(10) of Vaccine Concentration (pfu/dose) No. of -------------------------------------- Adjuvant Injections 7 8 9 - -------------------------------------------------------------------------------- None 1 134* 134 1076 None 2 >1280 1810 >2560 Aluminum hydroxide gel 1 538 538 >2560 Aluminum hydroxide gel 2 >1810 1920 2560 - -------------------------------------------------------------------------------- *Virus neutralization index is the reciprocal of the serum dilution that neutralized 32 TCID(30) of VSV-NJ
4. Live VSV-NJ Challenge of Mice Vaccinated with Psoralen-inactivated VSV-NJ Vaccine Three groups of fourteen, sixteen and seventeen adult white mice each were injected with either 10(7), 10(6), or 10(5) pfu psoralen-inactivated VSV-NJ per dose, respectively, using oil emulsion adjuvant with all injections. Each mouse was injected once IP (day 0). Pooled serum samples were collected on day 0 and again on day 21, and these samples were tested for SN antibody titers by standard procedures. The results are set forth in Table 9.
TABLE 9 - -------------------------------------------------------------------------------- Virus Neutralization Indices* of Mouse Sera After One Injection with Psoralen- Inactivated VSV-NJ Vaccine, Using Oil Emulsion Adjuvant - -------------------------------------------------------------------------------- Log(10) of Vaccine Concentration (pfu/dose) - -------------------------------------------------------------------------------- Day 5 6 7 - -------------------------------------------------------------------------------- 0 --* -- -- 21 -- -- 40 - -------------------------------------------------------------------------------- * Virus neutralization index is the reciprocal of the serum dilution that neutralized 56 TCID(30) of VSV-NJ
Each group of white mice was subdivided into three groups of about five mice each. Each mouse group was challenged with either 1, 10 or 100 minimum lethal doses (MLD) of live VSV by intracerebral inoculation on day 33. Two of five mice that were immunized with 10(6) pfu psoralen-inactivated VSV-NJ survived a one MLD VSV challenge but five of five mice that were immunized with 10(7) pfu psoralen-inactivated VSV-NJ vaccine survived both a 1 or 10 MLD VSV challenge. One of four mice that were vaccinated at 10(7) pfu/dose psoralen-inactivated VSV-NJ survived a 100 MLD VSV challenge. The results (no. dead/no. challenged) are set forth in Table 10.
TABLE 10 - -------------------------------------------------------------------------------- Live VSV-NJ Challenge of Mice Injected with Psoralen-Inactivated VSV-NJ - -------------------------------------------------------------------------------- Challenge Dilution Dose Psoralen- ------------------------------------------------------------- Inactivated 10-(5) 10-(4) 10-(3) VSV-NJ Vaccine (1 MLD) 10 (MLD) (100 MLD) - -------------------------------------------------------------------------------- 10(7) pfu 0/5* 0/5 3/4 10(6) pfu 3/5 4/5 3/6 10(5) pfu 5/5 4/5 7/7 - -------------------------------------------------------------------------------- *Number dead/number challenged
27 4,693,981 21 5. Virus Neutralization in Cattle Immunized with Psoralen-inactivated VSV-NJ Vaccine Four groups of six mature beef cattle each were injected with either 10(6) or 10(7) pfu/dose psoralen-inactivated VSV-NJ vaccine, with or without aluminum hydroxide adjuvant (1:1). Each cow was vaccinated subcutaneously (SQ) on day 0 and again on day 21. A control group consisted of an additional six cattle that were inoculated only with adjuvant on day 0 and again on day 21. All cattle were bled on days 0, 14, 21, and 35. Serum from each animal was tested for SN antibodies to VSV-NJ by standard procedures. The aluminum hydroxide adjuvant was required to elicit significant SN titers in cattle, and 10(8) pfu/dose induced the highest responses. The results are set forth in Table 11. A VSV-NJ virus neutralization index greater than 1000 has been reported to represent protection against 10(6) ID(50) of live VSV by intralingual challenge in cattle. See, Castaneda et al. (1964) Proc. US Livestock San Assoc. 68:455-468.
TABLE 11 - ------------------------------------------------------------------------------ Virus Neutralization Indices* From Cattle Injected With Psoralen-Inactivated VSV-NJ Vaccine ------------------------------------------ Day Serum Collected ------------------------------ Group Treatment Animal 0** 14 21** 15 - ------------------------------------------------------------------------------ A 10(8) pfu/dose 310 -- 16 16 256 + Al(OH) (3) 731 -- -- -- >16 911 -- 128 64 2048 921 -- 8 8 1024 943 -- 16 32 1024 944 -- 32 32 512 B 10(7) pfu/dose 303 -- -- -- 256 + Al(OH) (3) 304 -- -- -- 64 308 4 4 8 512 542 -- -- -- 8 914 -- 16 4 512 1670 -- -- -- >128 C Controls 305 -- -- -- -- 309 -- -- -- -- 314 -- -- -- -- 315 -- -- -- -- 316 -- -- -- -- 318 -- -- -- -- D 10(8) pfa/dose 302 -- -- -- 4 without adjuvant 611 -- -- -- 4 714 -- -- -- 8 732 -- -- -- 4 747 -- -- -- -- 996 -- -- -- 32 E 10(7) pfa/dose 101 -- -- -- -- without adjuvant 312 -- -- -- 4 616 -- -- -- -- 721 -- -- -- -- 722 -- -- -- -- 1944 -- -- -- -- - ------------------------------------------------------------------------------ * Virus neutralization nodes in the required of the serum dilutions that neutralized 32 TCID (50) of VSV-NJ ** Immunization Days
6. Live VSV-NJ Challenge of Cattle Vaccinated with Psoralen-inactivated VSV-NJ Vaccine Ten mature cattle were divided into two groups of five animals each. Group I was designated experimental and Group II was designated control. All ten cattle were clinically normal and lacked evidence of previous VSV exposure; that is, they were negative for serum neutralizing (SN) antibody. Group I cattle were vaccinated subcutaneously with 10(8) pfu (prior to inactivation) psoralen-inactivated VSV twice with a three week interval. Vaccine volume was 2 ml. containing aluminum 22 hydroxide adjuvant. Group II cattle were not exposed to the psoralen-inactivated VSV. Approximately two weeks post-second vaccination, the cattle of both Groups I and II were challenged intradermalingually with 0.1 ml live VSV in log dilutions of 5.6 pfu to 5.6 x 10(5) pfu/injection site. Thus each animal's tongue received six separate 0.1 ml injections, representing a quantitative challenge system. Serum neutralizing titers for cattle in each group measured before and after challenge are presented in Table 12.
TABLE 12 - ------------------------------------------------------------------------------- Serum Neutralization Titers From Cattle Vaccinated With Psoralen-Inactivated VSV-NJ Vaccine ------------------------------------------- After After Day of Post Arrival 1st vacc 2nd vacc[ILLE- Challenge[ILLE- Challenge[ILLE- GIBLE] GIBLE] GIBLE] Animal Day No. 0 18 35 42 60 - ------------------------------------------------------------------------------- Group I - ------- 4009-V neg* 1:160 1:1280 1:1280 1:1280 4383-V neg 1:80 1:1280 1:1280 1:2560 4389-V neg 1:80 1:640 1:2560 ND 6153-V neg 1:80 1:1280 1:1280 (equal 1:20480 6244-V neg 1:320 1:1280 1:1280 to or ND GROUP II greater - -------- than) 3780-C neg neg neg neg 1:10240 3781-C neg neg neg neg 1:10240 3784-C neg neg neg neg 1:10240 4007-C neg neg neg neg 1:10240 7912-C neg neg neg neg 1:10240 - ------------------------------------------------------------------------------- * 100 TCID (50) of VSV-NJ 8 = 1000 TCDI (50) OF VSV-NJ [ILLEGIBLE] 37 TCID (50) of VSV-NJ * negative at 1:20, the lowest dilution tested ND = not done
Vaccinated animals had a fifty percent reduction in lesion number, and lesions on vaccinates were fifty percent smaller and healed faster than on controls. Control animals developed lesions at both earlier and later time points. On post-challenge day eighteen, all five controls had lesions, whereas four of five vaccinates were normal. The fifth vaccinate's lesions were milder than those of any control animal on post-challenge day eighteen. Using the Mann-Whitney modification of Wilcoxon's two sample test, the vaccinates were significantly protected against live VSV challenge (P=.075). On the average, vaccinated cattle were protected against 25 times the minimum infectious dose required to produce lesions in control animals. According to the present invention, viruses inactivated with furocoumarins and ultraviolet radiation in the substantial absence of oxygen and other oxidizing species retain their immunogenicity and are suitable as the immunogenic substance in vaccines against a number of virally-induced diseases. The inactivated viruses of the present invention are non-infectious and safe when administered to a host for vaccination, yet display enhanced antigenic integrity when compared to vaccines inactivated in the presence of oxygen. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims. What is claimed is: 28 4,693,981 23 1. A method for inactivating a live virus without substantially degrading their antigenic characteristics, said method comprising: exposing the virus to a preselected intensity of long wavelength ultraviolet radiation and a preselected concentration of an inactivating furocoumarin for a time period sufficiently long to render the virus non-infectious but less than that which would result in degradation of its antigenic characteristics, wherein said exposure is performed in the substantial absence of oxygen and other oxidizing species. 2. A method as in claim 1, wherein the furocoumarin is added to an inactivation medium containing the live virus. 3. A method as in claim 1, wherein the furocoumarin is introduced to the live virus by addition to a cell culture medium in which the virus is grown. 24 4. A method as in claim 1, wherein the inactivation medium is maintained under a non-oxidizing gas atmosphere. 5. A method as in claim 4, wherein the inactivation medium is flushed with the non-oxidizing gas. 6. A method as in claim 4, wherein the non-oxidizing gas is selected from the group consisting of hydrogen, nitrogen, argon, helium, neon, carbon dioxide, and mixtures thereof. 7. A method as in claim 1, wherein an oxygen scavenger is added to the inactivation medium. 8. A method as in claim 7, wherein the oxygen scavenger is sodium ascorbate. 9. An improved method for inactivating viruses, said method being of the type wherein the virus is inactivated by exposure to long wavelength ultraviolet radiation in the presence of an inactivating furocoumarin, said improvement comprising performing said exposure to ultraviolet radiation in the substantial absence of oxygen and other oxidizing species. * * * * * 29 EXHIBIT C United States Patent [19] [11] Patent Number 4,727,027 Wiesehahn et al. [45] Date of Patent: Feb. 23, 1988 [54] PHOTOCHEMICAL DECONTAMINATION TREATMENT OF WHOLE BLOOD OR BLOOD COMPONENTS [75] Inventors: Gary P. Wiesehahn, Alameda; Richard P. Creagan, Alta Loma, both of Calif. [73] Assignee: Diamond Scientific Co., Des Moines, Iowa [21] Appl. No.: 785,356 [22] Filed: Oct. 7, 1985 Related U.S. Application Data [63] Continuation-in-part of Ser. No. 928,841, Oct. 20, 1986, which is a continuation of Ser. No. 490,681, May 2, 1983, abandoned. [51] Int. Cl.(4) ................C12N 13/00; C07K 13/00; A61K 39/00; C07G 7/00 [52] U.S. Cl. ......................435/173; 530/380; 530/381; 530/382; 530/385; 530/386; 530/387; 530/388; 530/392; 530/393; 530/347; 514/2; 514/6; 424/88; 424/89; 424/92; 424/101; 422/24; 422/28; 422/29; 426/234; 426/318; [58] Field of Search.........435/173, 183, 188, 236, 435/238, 269, 800, 814, 172.1; 260/112 R, 112 B, 121; 424/89, 90, 101, 88, 92; 514/2, 6, 8; 530/350, 363, 380-394, 412-414, 427 [56] References Cited U.S. PATENT DOCUMENTS 4,124,598 11/1978 Hearst et al. ........................260/343.21 4,169,204 9/1979 Hearst et al. ...........................546/270 4,321,919 3/1982 Edelson................................128/214 R 4,327,086 4/1982 Fukushima et al. ....................... 424/177 4,568,542 2/1986 Kronenberg............................... 424/90 4,595,653 6/1986 Kronenberg................................ 435/5 OTHER PUBLICATIONS deMol and van Henegouwen (1981) Photochem. Photobiol. 33:815-819. deMol et al. (1981) Photochem. Photobiol, 34:661-666. Joshi and Pathak (1983) Biochem. Biophys. Res. Comm., 112:638-646. Grossweiner (1982) NCI Monograph No. 66, 47-54. Rodighiero and Dall'Acqua (1982), NCI Monograph No. 66, 31-40. deMol et al. (1981) 95:74462k,p. 74467 Chem. Interactions. Hyde and Hearst (1978) Biochemistry 17:1251-1257. Hanson et al. (1978), J. Gen. Virol. 40-345-358. Swanstrom et al. (1981), Virol. 113:613-622. Redfield et al.(1981), Infec. and Immun. 32:1216-1226. Hanson "Inactivation of Viruses for Use as Vaccines ... Med. Virol, II, de La Maza & Peterson, eds. Cremer et al., (1982) J. Clin, Microbiol., 15:815-823. Veronese, F. M., et al., (1981), Photochem. Photobiol. 34:351. Veronese et al. (1982), Photochem. Photobiol. 36:25. Singh and Vadasz (1978) Photochem. Photobiol. 28:539-545. Musajo et al., Experentia, vol. XXI, pp. 22-24, "Photosensitizing Furocovmanhs-Interaction with DNA and Photoinactivation of DNA Containing Viruses". Primary Examiner--Thomas G. Wiseman Assistant Examiner--Robin Lyn Tieskin Attorney, Agent, or Firm--Zarley McKee, Thomte, Voorhees & Sease [57] ABSTRACT Biological compositions are decontaminated by treatment with furocoumarin derivatives and irradiation under particular conditions in which the proteins retain their original physiological activities and any pathogenic microorganisms and polynucleotide fragments thereof are rendered inactive. It has been found that reduction of the amount of dissolved oxygen in the treatment solution substantially inhibits denaturation of the proteins. 22 Claims, No Drawings 30 4,727,027 1 PHOTOCHEMICAL DECONTAMINATION TREATMENT OF WHOLE BLOOD OR BLOOD COMPONENTS This application is a continuation-in-part of application Ser. No. 928,841, filed Oct. 20, 1986, which is a continuation of application Ser. No. 490,681, filed on May 2, 1983 now abandoned. BACKGROUND OF THE INVENTION 1. Field of the Invention Recipients of blood and blood components risk acquiring infections from pathogenic microorganisms, either pre-existing in the blood at the time of collection or transmitted to the blood product during manipulation. Medical personnel who are in contact with collected human blood or clinical samples also have a significant chance of being exposed to potentially lethal blood-borne or sample-borne organisms. Blood components today are obtained from blood donors and frequently involve pooled lots, where one or more of the donors may be harboring a viral, bacterial or other infection. Since the blood or blood components are required to provide physiological functions in a mammalian host, normally a human host, these functions must not be impaired by the decontamination treatment of the biological composition. In addition, the blood or blood components may not be modified in such a way as to make them immunogenic which could result in an adverse immune response. Finally, any treatment should not leave residues or products detrimental to the health of the host or such residues or products should be readily removable. 2. Description of the Prior Art U.S. Pat. No. 4,327,086 describes a method for heat treating an aqueous solution containing human blood coagulation factor XIII. U.S. Pat. No. 4,321,919 proposes extracorporeal treatment of human blood with 8-methoxypsoralen (8-MOP) and ultraviolet light. Hyde and Hearst, Biochemistry (1978) 17: 1251-1257, describe the binding of two psoralen derivatives to DNA and chromatin. Musajo et al., Experientia (1965) XXI, 22-24, describe photo-inactivation of DNA-containing viruses with photosensitizing furocoumarins. See also, Hanson et al. (1978) J. Gen. Virol. 40: 345-358; Swanstrom et al. (1981) Virol. 113: 613-622; Redfield et al. (1981) Infec. and Immun. 32: 1216-1226; Hanson (1983) "Inactivation of viruses for Use as Vaccines and Immunodiagnostic Reagents" in Medical Virology II, de al Maza and Peterson, eds, and Cremer et al. (1982) J. Clin. Microbiol. 15: 815-823, each of which describe viral inactivation by exposure to ultraviolet radiation in the presence of furocoumarins. Some data showing substantial impairment of the biological function of certain enzyme proteins using furocoumarins are published in the scientific literature (see for example, Veronese, F. M. et al., Photochem. Photobiol. 34: 351 (1981); Veronese, F. M. et al., Photochem. Photobiol. 36: 25 (1982)). Singh and Vadasz (1978) Photochem. Photobiol. 28: 539-545 attribute the photoinactivation of E. coli ribosomes by ultraviolet radiation in the presence of furocoumarins to the presence of singlet oxygen. SUMMARY OF THE INVENTION Methods and compositions are provided for the decontamination of biological compositions, such as blood 2 31 and blood products, by inactivating microorganisms and polynucleotide fragments thereof capable of causing a pathological effect in mammalian hosts. The biological compositions are decontaminated by treatment with furocoumarins and long wavelength ultraviolet (UVA) light under conditions which limit the availability of oxygen and other reactive species. It has been found that such conditions allow for inactivation of even recalcitrant viral pathogens without degrading biologically active proteins, such as Factor VIII, which are present in the composition. DESCRIPTION OF THE SPECIFIC EMBODIMENTS In accordance with the subject invention, biological compositions which may harbor microorganisms capable of causing harmful physiological effects in a host are combined with furocoumarin compositions and treated with UVA light under predetermined conditions, whereby the microorganisms and polynucleotide fragments thereof are inactivated while the physiological activities of non-nucleic acid components of the compositions are retained. The treatment conditions are selected to minimize the likelihood that biologically active non-nucleic acid components of the compositions, such as proteins, are degraded. In particular, precautions are taken to reduce the level of dissolved oxygen and other reactive species in the composition during exposure to the ultraviolet light. As used hereinafter and in the claims, the term "microorganisms" should be understood to mean (1) prokoryotic, eukaryotic and viral microorganisms containing nucleic acids (either DNA or RNA), and (2) nucleic acid genomes or sub-genomic fragments from microorganisms. Various biological compositions may be decontaminated by the methods of the present invention, particularly aqueous compositions containing biologically active proteins derived from blood or blood components. Whole blood, packed red cells, platelets, and plasma (fresh or fresh frozen plasma) are exemplary of such compositions. Blood components of particular interest include plasma, protein portion, antihemophilic factor (AHF, Factor VIII); Factor IX and Factor IX complex (Factors II, VII, IX and X); fibrinogens, Factor XIII, prothrombin and thrombin (Factor II and IIa); immunoglobulins (e.g. IgA, IgD, IgE, IgG and IgM and fragments thereof e.g., Fab, F(ab')(2), and Fc); hyper-immune globulins as used against tetanus and hepatitis B; cryoprecipitate; albumin; interferons; lymphokines; transfer factors; etc. Other biological compositions include vaccines, recombinant DNA produced proteins, oligopeptide ligands, etc. The protein concentration in the acqueous biological compositions will generally range from about 1 (greek mu)g/ml to 500 mg/ml, more usually from about 1 mg/ml to 100 mg/ml. The pH will normally be close to physiologic pH (-7.4), generally in the range of about 6 to 9, more usually about 7. Other components may be present in the compositions, such as salts, additives, buffers, stabilizers, or the like. These components will be conventional components, which will be added for specific functions. Furocoumarins useful for inactivation include psoralen and derivatives, where the substituents will be: alkyl, particularly of from 1 to 3 carbon atoms, e.g. methyl; alkoxy, particularly of from 1 to 3 carbon atoms, e.g., methoxy; and substituted alkyl, of 1 to 6, more usually 1 to 3 carbon atoms having from 1 to 2 32 4,727,027 3 heteroatoms, which will be oxy, particularly hydroxy or alkoxy of from 1 to 3 carbon atoms, e.g. hydroxymethyl and methoxymethyl, or amino, including mono- and dialkyl amino having a total of from 1 to 6 carbon atoms, e.g., aminomethyl. There will be from 1 to 5, usually 2 to 4 substituents, which will normally be at the 4, 5, 8, 4' and 5'-positions, particularly at the 4'-position. Illustrative compounds include 5-methoxypsoralen, 8-methoxypsoralen (8-MOP), 4,5',8-trimethylpsoralen (TMP), 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT), 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT), 4-methylpsoralen, 4,4'-dimethylpsoralen, 4,5'-dimethylpsoralen, 4',8-dimethylpsoralen, and 4'-methoxymethyl-4,5',8-trimethylpsoralen. When employing furocoumarins with limited aqueous solubility, typically below about 50 (greek mu)g/ml, it has been found useful to add an organic solvent, such as dimethyl sulfoxide (DMSO), ethanol, glycerol, polyethylene glycol (PEG), or polypropylene glycol to the aqueous treatment solution. Such furocoumarins having limited solubility include 8-MOP, TMP, and psoralen. By adding small amounts of such organic solvents to the aqueous composition, typically in the range from about 1 to 25% by weight, more typically from about 2 to 10% by weight, the solubility of the furocoumarin can be increased to about 200 (greek mu)g/ml, or higher. Such increased furocoumarin concentration may permit the use of shorter irradiation times. Also, inactivation of particularly recalcitrant microorganisms may be facilitated without having to increase the length or intensity of ultraviolet exposure, and the addition of an organic solvent may be necessary for inactivation of some viruses with particular furocoumarins. The ability to employ less rigorous inactivation conditions is of great benefit in preserving the biologic activity of blood proteins during decontamination. At times, it may be desirable to employ organic solvents, particularly DMSO, with all furocoumarins regardless of solubility. For some microorganisms, particularly viruses having tight capsids, the addition of the organic solvent may increase the permeability of the outer coat or membrane of the microorganism. Such increase in permeability would facilitate penetration by the furocoumarins and enhances the inactivation of the microorganism. The subject furocoumarins are active with a wide variety of pathogenic microorganisms, viruses, and polynucleotide fragments thereof, DNA or RNA, whether single stranded or double stranded. Illustrative viruses include: adenovirus, arenavirus, bacteriophage, bunyavirus, hepatitis viruses, including types A, B and non-A, non-B (also designated type C), herpesvirus, retroviruses such as human T-lymphtropic viruses (HTLV), including HTLV types I, II and III, orthomyxovirus, papovavirus, paramyxovirus, picornavirus, poxvirus, reovirus, rhabdovirus, and togavirus. Additional pathogenic microorganisms include bacteria, chlamydia, mycoplasma, protozoa, rickettsia and other unicellular microorganisms. This inactivation method will also be effective against uncharacterized infectious agents which contain nucleic acids, either DNA or RNA. The furocoumarins may be used individually or in combination. Each of the furocoumarins may be present in amounts ranging from about 0.01 (greek mu)g/ml to 1 mg/ml, preferably from about 0.5 (greek mu)g/ml to 100 (greek mu)g/ml, there not being less than about 1 (greek mu)g/ml nor more than about 1 mg/ml of furocoumarins. 4 The furocoumarins may be added to the biological composition by any convenient means in a manner substantially assuring the uniform distribution of the furocoumarins in the composition. Such addition may be made in a single dose, in a series of doses over time, or continuously during the entire treatment period or a portion thereof. The composition may be irradiated under conditions ensuring that the entire composition is exposed to sufficient irradiation, so that the furocoumarins may react with any polynucleotide present to inactivate the polynucleotide. Depending upon the nature of the composition, particularly its opacity, as in the case of blood, the depth of the solution subject to irradiation will vary widely. Usually, the depth will be not less than about 0.025 millimeter, but may be a centimeter or more. With whole blood, the depth will generally range from about 0.025 millimeter to 2.5 millimeters. The light which is employed will generally have a wavelength in the range of about 300 nm to 400 nm. Usually, an ultraviolet light source will be employed together with a filter for removing UVB light. The intensity will generally range from about 0.1 mW/cm(2) to about 5 W/cm(2), although in some cases it may be much higher. The medium being irradiated may be irradiated while still, stirred or circulated, and may either be continuously irradiated or be subject to alternating periods of irradiation and non-irradiation. The circulation may be in a closed loop system or it may be in a single pass system ensuring that all of the sample has been exposed to irradiation. The total time for irradiation will vary depending upon the nature of the sample, the furocoumarin derivative used, the intensity and spectral output of the light source and the nature of the polynucleotides which may be present. The time of irradiation necessary for inactivation will be inversely proportional to the light intensity. Usually, the time will be at least 1 min. and not more than about 20 hrs., more usually from about 15 mins. to about 2 hrs. When circulating the solution, the rate of flow will generally be in the range of about 0.1 ml/min to 50 liters/min. In order to inhibit denaturation of biologically active proteins, it is desirable to reduce the availability of dissolved oxygen and other reactive species in the biological composition before or during the exposure to ultraviolet radiation. A variety of steps to reduce the oxygen availability may be taken, either individually or in combination. Oxygen scavengers, such as ascorbate, glutathione, sodium thionate, and the like, may be added which combine with singlet oxygen and other reactive oxygen species to prevent reaction with the proteins. Physiologically acceptable proteins, such as human or bovine serum albumin (BSA), and the like, may also be added. Such large proteins act both to bind metals which catalyze reactions involving oxygen as well as by preferentially binding the oxygen and other reactive radicals. The biological composition may also be flushed with inert or less reactive gases, such as hydrogen, helium, neon, carbon dioxide, nitrogen, argon, and the like, to reduce the concentration of oxygen and other dissolved gases in the biological composition by equilibrium exchange (mass transfer) with the flushing gas. Flushing may be accomplished by passing the inert gas over or through the biological composition, for a predetermined minimum amount of time, usually at least 30 minutes, more usually at least one hour, prior to exposure to the ultraviolet radiation. The concentration of dissolved oxygen may also be reduced through the use of enzyme systems either in 33 4,727,027 5 solution or immobilized on a solid substrate. Suitable enzyme systems include glucose oxidase or catalase in the presence of glucose and ascorbic acid oxidase in the presence of ascorbate. Such enzyme systems may be employed alone or together with the other methods for oxygen reduction discussed above. To further inhibit denaturation of the biologically active proteins, the temperature of the biological composition should be maintained below about 60 degrees C., preferably below 40 degrees C., more preferably in the range from - -10 degrees C. to 30 degrees C., during exposure to the ultraviolet radiation. It may be desirable to remove the unexpended furocoumarin and/or its photobreakdown products from the irradiation mixture. This can be readily accomplished by a variety of conventional separation techniques, such as dialysis across an appropriately sized membrane or through an appropriately sized hollow fiber system after completion of the irradiation. It may be desirable in certain applications to remove bound or unbound furocoumarins using affinity methods (e.g., magnetic beads) or using antibodies, including monoclonal antibodies, either in solution or attached to a substrate. Enzymes, either in solution or attached to a substrate, could be used to convert the furocoumarins to nontoxic unreactive products. Alternatively, desirable components such as factor VIII could be removed by precipitation or affinity methods by leaving the furocoumarins in solution. The following examples are offered by way of illustration and not by way of limitation. EXPERIMENTAL The following experiments were performed in order to demonstrate the ability of psoralen photoreaction to destroy microbial contaminants contained in whole blood and blood products without destroying the biological activity of blood proteins. Since whole blood exhibits very high optical density for longwave UV light (320 nm to 380 nm), the blood was irradiated through a suitably short optical path length. In Example 1, blood was pumped through polyethylene capillary tubing of 0.875 millimeter inside diameter. The tubing was coiled around a 1.27 centimeter diameter tube and immersed in water which was maintained at 18 degrees C. The blood was continuously circulated through the tubing by means of a peristaltic pump. The blood required approximately 2.5 minutes for a complete cycle through the capillary tubing and was in the light beam for approximately 20% of the stated irradiation time. The light source was a low pressure mercury lamp filtered through a cobalt glass filter. The filter transmits light of approximately 320 nm - 380 nm. with peak transmittance at 360 nm. The incident intensity at the sample was approximately 40 mW/cm(2). The apparatus employed for Examples II through XI consisted of an upper and lower bank of lamps emitting longwave ultraviolet light (e.g. GE F20T 12 BLB bulbs, 320 - 400 nm). Samples were placed on plate glass between the light sources. Irradiation times and intensities were as described for each Example. EXAMPLE I Inactivation of feline rhinotracheitis Feline rhinotracheitis virus, a member of herpes-virus family, was added to heparinized whole rabbit blood in an amount that would give a final concentration of approximately 2 x 10(7) PFU/ml. 4'-hydroxymeth- 6 yl-4,5',8-trimethylpsoralen (HMT) was added to a portion of the rabbit blood and aliquots were irradiated for various periods of time. To test for remaining live virus, duplicate plaque assays were performed using cultured feline cells (Fc3Tg) (ATCC CCL 176), with a methyl-cellulose overlay. Virus titers were obtained as the arithmetical mean of viral plaques observed in duplicate assays cultures 72 hours after exposure to test samples. The blood aliquot that received HMT only and no irradiation gave a titer of 5.3 x 10(6)PFU/ml. The aliquot that received HMT and five minutes of irradiation exhibited a titer of 4.5 x 10(6)PFU/ml. In the aliquot that received psoralen plus one hour of irradiation there was not detectable live virus remaining. The sensitivity of this assay should have permitted detection of residual virus at titers (greater than or equal to) 1.0 x 10(1) PFU/ml. A blood sample which had received HMT and one hour of irradiation also showed no apparent damage to the red blood cells as judged by phase contrast microscope analysis and by absence of visible hemolysis. These data therefore demonstrate that high virus titers present in whole blood can be inactivated by psoralen plus light treatment which leaves the red cell component of the blood intact. EXAMPLE II Protective effect of inert gas flushing on Factor VIII activity Eight samples of pooled normal plasma were prepared and treated as follows. Samples 5-8 were continuously flushed with argon. AMT (20 (greek mu)g/ml) and TMP (5 (greek mu)g/ml) were added to samples 2, 4, 6, and 8. Samples 3, 4, 7, and 8 were exposed to UVA radiation (4.2 mW/cm(2), 320-400nm). Factor VIII activity was determined for each sample after six hours of such treatment. The results are set forth in Table 1.
TABLE 1 - ------------------------------------------------------------------------------- FVIII Activity Sample Argon UVA Drugs units/ml % Retained - ------------------------------------------------------------------------------- 1 0 0 0 0.86 Control 2 0 0 + 0.84 98 3 0 + 0 0.58 67 4 0 + + 0.10 12 5 + 0 0 0.75 87 6 + 0 + 0.64 74 7 + + 0 0.58 67 8 + + + 0.45 52 - -------------------------------------------------------------------------------
These results demonstrate that argon flushing to reduce the level of dissolved oxygen in the treatment solution substantially enhances the retention of Factor VIII activity. EXAMPLE III Protective effect of ascorbate on Factor VIII activity Three samples of Factor VIII concentrate were prepared with 3% BSA added. Test samples 2 and 3 were flushed with argon prior to UVA exposure. Nothing further was added to the first sample. AMT (180 (greek mu)g/ml) was added to the second and third samples, while 5 mM ascorbate was added to the third sample only. The second and third samples were exposed to UVA (4.2 mW/cm(2), 320-400 nm) radiation for the four hour period, while the first sample was kept in the dark (control). Factor VIII activity of all samples was measured after the four hour test period and the retained activity was determined. The results are summarized in Table 2. 34 4,727027 7
TABLE 2 - ------------------------------------------------------------------------------------- FVIII ACTIVITY SAMPLE UVA AMT ASCORATE UNITS/ML - -------------------------------------------------------------------------------------- 1 0 0 0 0.81 2 + + 0 0.38 3 + + + 0.59 - --------------------------------------------------------------------------------------
These results demonstrate that the addition of ascorbate to the treatment solution substantially enhances the retention of Factor VIII activity in samples treated with psoralens and exposed to ultraviolet radiation. EXAMPLE IV Protective effect of BSA on Factor VIII activity Six samples of Factor VIII concentrate were flushed with argon and then exposed for three hours to UVA radiation (4.2 mW/cm2, 320-400 nm). Selected amounts of AMT and/or BSA were added to certain samples prior to irradiation. Factor VIII activity was measured before and after irradiation, and the percentage of retained activity determined. The results are set forth in Table 3.
TABLE 3 - ------------------------------------------------------------------- RETAINED SAMPLE AMT ((greek mu)g/ml) BSA(%) FVIII ACTIVITY(%) - ------------------------------------------------------------------- 1 0 0 88 2 0 10 110 3 30 1 77 4 30 5 98 5 30 10 86 6 60 10 84 - -------------------------------------------------------------------
These results demonstrate that BSA enhances the retention of Factor VIII activity when exposed to UVA radiation, both in the presence and absence of AMT. EXAMPLE V Inactivation of Mycoplasma species Acholeplasma laidlawii with 8-MOP Culture: Six days old and approximately 10(7) cells/ml. Irradiation: Irradiated samples were exposed to UVA (approximately 4.2 mW/cm(2)) for three exposure periods of two hours each. Samples were transferred to a new vessel for each period of UVA treatment. Furocoumarin: 8 metoxypsoralen (8-MOP) in DMSO was used at the concentrations given in Table 4. After each two hour period of irradiation, a fresh aliquot of furocourmarin was added to restore the minimal drug concentration to the level indicated in Table 4. Additives: Additional DMSO and/or sodium ascorbate (ASC) were added to samples as indicated in Table 4. Assay: Residual live mycoplasma were assayed using the standard microbiological culture tests prescribed by U.S. Department of Agriculture in 9CFR part 113.28.
TABLE 4 - --------------------------------------------------------------- MYCOPLASMA GROWTH TEST -------------------------------------- % DMSO DIRECT BULK FINAL TREATMENT OF SAMPLES PLATING BROTH (V/V) - --------------------------------------------------------------- UVA only + + 0 DMSO (6% v/v) + + 6 ASC (10 mM) + + 0 UVA + ASC + + 0 UVA + DMSO + ASC + + 6
8
TABLE 4 - continued - --------------------------------------------------------------- MYCOPLASMA GROWTH TEST -------------------------------------- % DMSO DIRECT BULK FINAL TREATMENT OF SAMPLES PLATING BROTH (V/V) - --------------------------------------------------------------- ASC + DMSO + + 6 UVA + ASC + 8-MOP (100 (greek mu)g/ml) - + 2.3 UVA + DMSO + ASC + 8-MOP - + 8.2 (100 (greek mu)g/ml) UVA + ASC + 8-MOP (200 (greek mu)g/ml) - - 4.6 UVA + DMSO + ASC 8-MOP - - 10.6 (200 (greek mu)g/ml) - ---------------------------------------------------------------
+ = growth - - = no growth These results demonstrate that the decontamination method of the present invention is useful for the inactivation of bacterial species. EXAMPLE IV Inactivation of Mycoplasma species Mycoplasma orale with AMT and TMP without additives Culture: Seven days old and approximately 2.9 X 10(6) cells/ml. Irradiation: Same as for Example V except irradiated samples were only exposed for one treatment period of the duration indicated in Table 5. Furocoumarins: 4' aminomethyl-4,5', 8-trimethlylpsoralen (AMT) 4,5',8-trimethylpsoralen(TMP) Additives: None Assay: Same as for Example V.
TABLE 5 - --------------------------------------------------------------- MYCOPLASMA GROWTH TEST ---------------------------- DIRECT BULK TREATMENT OF SAMPLES PLATING BROTH - --------------------------------------------------------------- No treatment + + UVA (1 hour) + ND UVA (3 hours) - ND AMT (30 (greek mu)g/ml) + ND AMT (30 (greek mu)g/ml) = UVA (1 hour) - + AMT (30 (greek mu)g/ml) = UVA (3 hours) - - TMP (7.5 (greek mu)g/ml) = UVA (1 hour) - - - ---------------------------------------------------------------
ND = not done, + = growth, - = no growth These results further confirm the efficacy of the present invention in inactivating bacterial species. EXAMPLE VII Inactivation of vesicular stomatitis virus and retention of Factor VIII activity Treatment samples comprising vesicular stomatitis virus (3.3 X 10(8) pfu/ml) were prepared in PBS-diluted AHF concentrate. The VSV was inactivated in two samples by the addition of AMT (180 (greek mu)g/ml), 1% BSA, 10mM ascorbate, and exposure to UVA (6.4 mW/cm(2), 320-400 nm) for approximately nine hours. The samples were continuously flushed with argon. Inactivation was confirmed by plaque assay on LM(TK-) mouse cells any by injection of 20 (greek mu)l into suckling mouse brains. The mouse brain assay will detect 10 pfu/ml. Both samples were shown to be non-infective. Factor VIII activity was monitored in the treated samples as well as a control sample which was not irradiated using a modified APTT assay. The effect on the Factor VIII activity is shown in Table 6. 35 4,727,027 9
TABLE 6 - -------------------------------------------------------------------------------- Elapsed VSV FVIII Sample Time (pfu/ml) Activity (U/ml) - -------------------------------------------------------------------------------- 1 0 10(8) 7.2 9 0 6.6 2 0 10(8) 6.25 2 0 ND 9 0 5.6 CONTROL 0 10(8) * 9 10(8) * - --------------------------------------------------------------------------------
ND: Not done *No significant loss of activity These results demonstrate that a virally infected biological composition may be decontaminated by the method of the present invention without substantial loss of biological activity of a biologically-active protein. EXPERIMENT VIII Inactivation of non-A, non-B hepatitis virus A study was undertaken to evaluate the effects of furocoumarin and UVA on the virus which causes non-A, non-B hepatitis. This virus is believed to be the major cause of post-transfusion hepatitis in the United States. The only suitable animal model for this virus is the chimpanzee model. Samples of Non-A, non-B hepatitis virus were inactivated, as described below, and injected into chimpanzees. The samples were injected intravenously into chimpanzees anesthetized with ketamine. These animals were naive with respect to non-A, non-B hepatitis, had been followed for an extended period with normal liver enzymes (SGPT, SGOT), and had at least two normal liver biopsies examined by light and electron microscopy in the two month period prior to inoculation. During the trial, liver enzymes were checked weekly and periodic liver biopsies were done. Results through 26 weeks post-inoculation indicate that there were no significant liver enzyme elevations, and liver biopsies were negative. Inactivation was as follows. Four coded samples were obtained which contained from 100 to 100,000 chimpanzee infectious doses (CID(50)) of the Hutchinson strain of non-A, non-B hepatitis virus in 1.0 ml of fetal calf serum. These samples were treated under code as follows. Each sample was diluted with phosphate buffered saline to a total of 10.0 ml containing a final concentration of the following: 1% Bovine serum albumin 5 mM Sodium ascorbate 20 (greek mu)g/ml AMT 0.5 (greek mu)g/ml TMP Each 10.0 ml sample was added to a T-75 flask (Corning) prerinsed with 5% BSA, flushed with argon and incubated in the dark overnight at room temperature (21 degrees C.). The flasks were then irradiated at an average of 5.0 mW/cm(2) under black light bulbs emitting UVA light (G.E. BLB F20T12). At 1 hour intervals an additional 20 (greek mu)g/ml AMT and 0.5 (greek mu)g/ml TMP were added and the flasks reflushed with argon. At three hour intervals the samples were transferred to fresh BSA-rinsed flasks. Parallel flasks with 10(8) pfu/ml vesicular stomatitis virus (VSV) in place of the non-A, non-B virus (inactivation control) were prepared and irradiated as above. Samples were taken for testing at 0, 1, 3 and 6 hours. Parallel flasks with 10% factor VIII concentrate (Koate, Cutter Biological) were prepared and irradiated as above. Samples were taken at time 0 and at 3 hour intervals and frozen at -80 degrees C. After 9 hours the experiment 10 was stopped temporarily and resumed the next morning. Samples were kept at room temperature (21 degrees C.) during this time. Conditions for the second 9 hours were the same as for the first 9 hours except that a second parallel VSV sample was prepared with 10(8) VSV/ml. Aliquots from this second VSV sample were removed at 2, 4, and 5 hours for subsequent assays. The VSV aliquots were assayed for residual viral activity by plaque assay on LM(TK-) cells and by injection into suckling mouse brains. Factor VIII activity in the concentrate samples was determined by a one stage clotting test. At the conclusion of the second 9 hours, the samples containing non-A, non-B hepatitis were sent under code to Southwest Foundation for Research and Education (SFRE), now known as Southwest Foundation for Biomedical Research (SFBR), for inoculation of chimpanzees. Chimpanzees received the following doses of inactivated non-A, non-B hepatitis virus:
- -------------------------------------------------------------------------------- Chimp No. Inactivated Virus (CID(50)) - -------------------------------------------------------------------------------- 72 100 83 1,000 80 10,000 97 100,000 - --------------------------------------------------------------------------------
All four chimps remained negative for non-A, non-B hepatitis infection during six months of clinical observation. Following the six month observation period, chimp no. 97 who had received the highest dose (approx. 100,000 CID(50)) of inactivated non-A, non-B hepatitis virus, was inoculated with approximately 33 CID(50) live virus. This challenge dose was prepared from a reserved aliquot of the original sera from which the inactivated viruses had been obtained. Chimp no. 97 developed symptoms of infection 10 weeks after inoculation, thus demonstrating the chimp's susceptibility to the virus. This experiment demonstrated that the inactivation procedures used were capable of killing at least 10(3.5) CID(50) virus. In the parallel experiments, VSV at 3.4 X 10(8) pfu/ml (average of 2 experiments) was reduced to non-detectable levels in plaque assays after two hours of the inactivation procedure. No residual infectivity was detected by the more sensitive suckling mouse brain assay in inocula subjected to four hours of inactivation (Table 7).
TABLE 7 - -------------------------------------------------------------------------------- Fucoumarins/UVA VSV Plaques Suckling Mice (hours) (pfu/ml) (days to death) - -------------------------------------------------------------------------------- 0 3.4 x 10(8) 2, 2, 2, 2, 2 1 7.2 x 10(2) NT 2 0 NT 3 0 NT 4 0 no deaths 5 0 no deaths 6 0 no deaths - --------------------------------------------------------------------------------
NT = not tested In the second set of parallel experiments, handling and sample manipulation in the T-75 tissue culture flasks produced greater loss of factor VIII activity than was caused by the inactivation procedure (Table 8). After 18 hours of treatment, activity in the sample containing fucoumarins and exposed to UVA was 98% of that remaining in the shielded handling control which contained no fucoumarins. These results (Table 8)demon- 36 4,727,027 11 strated that the activity of a highly labile protein can be preserved under conditions capable of inactivating high titers of non-A, non-B hepatitis virus.
TABLE 8 - ------------------------------------------------------------------------------- Factor VIII Activity (units/ml) ------------------------ Fucoumarins/UVA Handling % Activity Retained (hours) Test Control (Test/Control) x 100 - ------------------------------------------------------------------------------- 3 0.91 1.02 89 6 0.79 0.82 96 9 0.69 0.89 79 12 0.74 0.79 94 13 0.66 0.73 90 18 0.59 0.60 98 - -------------------------------------------------------------------------------
EXPERIMENT IX Inactivation of non-A, non-B hepatitis and hepatitis B viruses in combination Two samples, each of which contained about 10(4.5) CID(50) of MS-2 (ayw) strain of hepatitis B virus (HBV) and 10(4) CID(50) of the Hutchinson stain of non-A, non-B hepatitis virus (NANB), were prepared for inactivation. The diluent for one sample was reconstituted AHF concentrate (Factor VIII). The diluent for the other sample was phosphate-buffered saline (PES). Each sample contained an aliquot of bacteriophaze R17 as an internal control. The HBV and NANB viruses were portions of National Institutes of Health stock materials diluted in fetal calf serum (FCS) or 1% bovine serum albumin (BSA). Heparin (1 unit/ml) was included in sample preparation to control any activated clotting factors present in the calf serum. 8-Methoxypsoralen was dissolved in dimethyl sulfoxide (DMSO) and added to each sample at final concentration of 300 micrograms per ml. The DMSO was present as 6% of the total sample volume of 5 ml. Samples containing vesicular stomatitis virus (VSV), feline leukemia virus (FeLV), and bacteriophages fd and R17 were prepared in factor VIII diluent and inactivated in parallel with the hepatitis virus samples to serve as external controls. Experimental and control samples were mixed in 50 ml polypropylene conical vials, then pipetted gently into silanized glass medicine bottles (250 cc) prior to inactivation. Sample bottles were capped with cuffed rubber stoppers fitted with blunt cannulas. Prior to inactivation, samples were flushed with a mixture of 4% hydrogen in pre-purified nitrogen for 1 hour. The oxygen level throughout the flushing cycle was below 1 ppm of oxygen as measured by a Couloximeter (Chemical Sensor Development, Torrance, CA). Samples were irradiated at approximately 5 mW/cm(2). After five hours irradiation, the hepatitis samples (Nos. 5 and 6) were transferred to fresh bottles, a second aliquot of R17 was added to the hepatitis samples, and the bottles were flushed for 30 minutes with the hydrogen/nitrogen mixture. These samples were then irradiated for an additional five hours. Results of the assays for infectivity of control viruses are presented in Table 9.
TABLE 9 - --------------------------------------------------------------------------------------------------- Hours UVA (pfu/ml or ffu/ml) Sample ----------------------------------------------------------------------------- (virus) 0 1 2 2.5 3 4 5 7.5 10 - --------------------------------------------------------------------------------------------------- No. 5 [-10(6)] -- -- -- -- -- 0 -- 0 (R17)(*) No. 6 [-10(6)] -- -- -- -- -- 0 -- 0 (R17)(*)
12
TABLE 9 - continued - --------------------------------------------------------------------------------------------------- Hours UVA (pfu/ml or ffu/ml) Sample ----------------------------------------------------------------------------- (virus) 0 1 2 2.5 3 4 5 7.5 10 - --------------------------------------------------------------------------------------------------- VSV-2(**) 1.83 x 10(2) -- -- 0 -- -- 0 0 0 FeLV-2 2 x 10(4) -- -- 0 -- -- 0 0 0 R17-2 6.1 x 10(8) 2.5 x 10(4) 0 -- 0 0 0 -- -- fd-2 6.6 x 10(10) <10 0 -- 0 0 0 -- -- - ---------------------------------------------------------------------------------------------------
Safety tests for endotoxins were performed by injecting 0.2 ml crude R17 filtrate into the ear vein of one rabbit and 0.2 ml of 10([ILLEGIBLE]) dedication into the ear vein of another rabbit. No reactions were seen during the two-week observation period following the injection. "An aliquot of this sample inactivated for 10 hours was tested by suckling mouse brain assay for residual infectivity. 10 suckling mice <14 days old were injected intracerebrally with 20 (greek mu)l of the VSV sample. Two died of trauma, while the remaining 8 were alive and well at 14 days. As seen in Table 9, the internal control virus (R17), a single stranded RNA bacteriophage, was completely inactivated in both samples No. 5 and No. 6 at the 5 hour time point. Initial titer was 10(8) pfu/ml. A second aliquot containing 10(8) fu/ml was added at five hours. After 10 hours this second aliquot was also completely inactivated. Parallel samples containing factor VIII concentrate were prepared and irradiated as described above. Results are shown in Table 10. No loss of factor VIII activity was observed after 10 hours of treatment.
TABLE 10 - ------------------------------------------------------------------------------- Sample -------------------------- No. 1 No. 2 - ------------------------------------------------------------------------------- 8-MOP 0 0.06 ml (300 (greek mu)g) UVA (hours) 0 10 Barbital Buffer 0.06 ml 0 Koate 0.84 ml 0.84 R.17/fd* 0.10 0.10 Viral activity (pfu/ml) 1.2 x 10(10) 0 Factor VIII activity units/ml 19.4 19.4 - ------------------------------------------------------------------------------- *Virus prepared as 1:10 dilution in 5% BSA
At Southwest Foundation for Biomedical Research, chimp No. 64 was inoculated with sample No. 5 and chimp No. 216 was inoculated with sample No. 6. These chimps had been on baseline evaluation for several months and had no elevations in liver enzymes on weekly testing. The animals were bled weekly, and the samples were tested for SGPT, SGOT, and HBsAg, anti-Hbs and anti-HBc. Liver biopsies were obtained at weeks 5, 7, 9, 11, 13, 15, 20, and 26. Biopsy material was examined by light microscopy immunofluorescence and electron microscopy for changes characteristic of hepatitis infection. During the 26-week observation period following inoculation, enzyme levels remained low, histology examinations were normal, and no HBV markers were detected. EXPERIMENT X Inactivation of Simian AIDS (SAIDS) Virus (an RNA virus) with AMT. Vacutainer tubes (10cc) were prepared with 0.5 ml of the fresh sterile solutions indicated in Table 11 and stored overnight at 4 degrees C. 0.5 ml of SAIDS virus suspension was added sterilely to each tube. Samples were irradiated with approximately 5 mW/cm(2) UVA for the times indicated in Table 11. Non-irradiated samples were stored in the dark at 4 degrees C. All samples were added to Raji cells and observed fior syncytia induction over a 10 day period. Cultures were then expanded and observed for an additional 10 days. Samples that had been positive (1, 3, 5, 8, 9) were expanded into flasks and 37 4,727,027 13 supernatants from the flasks were filtered through a 0.45 (greek mu)g filter. The filtrates were added to fresh Raji cells and observed for syncytia induction. Results are show in Table 11. The initial syncytial induction in samples 1, 8 and 9 may have been due to the presence of inactivated virus. The samples which remained positive after expansion were the ones which received no UVA treatment (samples 3, 5, 9 and the untreated control).
TABLE 11 - -------------------------------------------------------------------------------- AMT Ascorbate PBS-A Syncytia Formation Sample 1* 1** 1 UVA Initial Subculture - -------------------------------------------------------------------------------- 1 - 50 450 2 hr + - 2 20 - 480 2 hr - - 3 - - 500 - + + 4 - - 500 2 hr - - 5 20 50 430 - + + 6 20 50 430 15 min - - 7 20 50 430 30 min - - 8 20 50 430 1 hr + - 9 20 50 430 2 hr + - untreated - - - - + + - -------------------------------------------------------------------------------- * AMT, 5 mg/ml in dH(2)O (filter sterilized) ** Sodium ascorbate, 200 mM in dH(2)O (filter sterilized)
EXPERIMENT XI Inactivation of Feline Leukemia Virus (FeLV-A) (an RNA virus) with 8-MOP in DMSO. Two-ml aliquots of FeLV-A at 2x10(7) FFU/ml in F-12K medium were placed in vacutainer tubes. 8-MOP was dissolved in DMSO and added to the virus-containing samples to a final concentration of 50 (greek mu)g/ml as shown in Table 14. All samples were flushed with argon for 30 min. Irradiated samples were exposed to UVA at approximately 4 mW/cm(2) for the times shown in Table 14. The unirradiated controls were stored in the dark at 4 degrees C. The two 25-hour samples received additions of 8-MOP at 0, 5, 10, 15 and 20 hours. After each addition the tubes were flushed with argon for 30 min. Assessment of inactivation was by Clone 81 focus assay for all samples and blind passage on AK-D cells for 6 weeks for sample H. Results of the focus assays are given in Table 12. No live virus was detected in any of the experimental samples following 2 hr UVA irradiation.
TABLE 12 - -------------------------------------------------------------------------------- 8-MOP UVA Tube Cl-81 Focus Assay Sample (greek mu)g/ml (hrs) Changes Titer (FFU/ml) - -------------------------------------------------------------------------------- A 0 0 3 1.71 x 10(7) B 0 25 3 6.45 x 10(6) C 250* 0 3 1.8 x 10(7) D 50 1 0 9.71 x 10(2) E 50 2 0 0 F 50 4 0 0 G 50 6 0 0 H 250* 25 3 0 - -------------------------------------------------------------------------------- * (50 (greek mu)g/ml) x 5 additions
EXPERIMENT XII Effect of Oxygen Levels on Factor VIII Exposed to Furocoumarins and UVA The following experiment was conducted to determine the effect of different levels of molecular oxygen on factor VIII exposed to furocoumarins and UVA light. Furocoumarins used for this experiment were 8-methyoxypsoralen (8-MOP) and 4'-aminomethyl-4,5',8- 14 trimethylpsoralen (AMT). Samples of factor VIII concentrate (Koate, Cutter Biological) were flushed with gas containing various levels of molecular oxygen for a time sufficient to reach equilibrium (equal to or greater than 1 hour). The samples were contained in 10 ml red-top vacutainer tubes (Becton-Dickinson). 8-MOP or AMT was added to give a final concentration of 0.2 mM furocoumarin (8-MOP: 43.2 (greek mu)g/ml; AMT: 58.6 (greek mu)g/ml). Total sample volume was 1.0 ml. The sample tubes were irradiated at approximately 2.5 mW/cm(2) for 10 hours. Results of factor VIII assays are given in Table 13.
TABLE 13 - -------------------------------------------------------------------------------- Factor VIII Activity Oxygen level (units/ml) --------------------------------------------------------- (parts per) 8-MOP AMT Control million and UVA and UVA no UVA - -------------------------------------------------------------------------------- 1 20.4 20.0 - 54 15.2 8.8 - 988 10.0 5.3 18.8* 210,000 3.4 0.3 20.0** (Room air) - -------------------------------------------------------------------------------- * 8-MOP added, flushed ** no drug, no flushing
Decreasing the oxygen level has a protective effect on factor VIII exposed to furocoumarin and UVA light. There was no discernible loss of factor VIII activity at the lowest level of oxygen used (approx. 1 ppm). This oxygen effect was seen for both 8-MOP and for AMT, although the loss of factor VIII activity at the higher levels of oxygen was more marked for AMT. This is much more active on a molar basis than 8-MOP as a singlet oxygen generator. It is evident from the above results, and in accordance with the subject invention, that polynucleotides in biochemical compositions can be inactivated to provide a safe composition of administration to a mammalian host. The proteins present in the composition retain their physiological activity, so that they can fulfill their physiological function in a mammalian host. The method is simple, rapid, and can be expanded to treat large samples. The small amount of chemical reagent required will not generally be harmful to the host. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims. What is claimed is: 1. A method for decontaminating a blood clotting factor containing composition of viral contaminants, in a manner which substantially maintains the biological activity of the blood clotting factors, said method comprising adding to the blood clotting factor containing composition at least one furocoumarin, and irradiating the furocoumarin containing composition under U-V light, wherein the amount of furocoumarin and irradiation conditions are sufficient to inactivate substantially all the viral contaminants, and wherein the concentration of dissolved oxygen is reduced to a level sufficient to substantially inhibit the denaturation of the blood clotting factors. 2. A method as in claim 1, wherein the level of dissolved oxygen is reduced by addition of an oxygen scavenger to the composition. 3. A method as in claim 1, wherein the level of dissolved oxygen is reduced by equilibrium exchange with an inert or less reactive gas. 38 4,727,027 15 4. A method as in claim 1, wherein the level of dissolved oxygen and other reactive species is reduced by addition of a physiologically-acceptable protein. 5. A method as in claim 4, wherein the physiologically acceptable protein is human or bovine serum albumin. 6. A method as in claim 1, wherein the solubility of the furocoumarin in the aqueous composition is increased by the addition of from about 1% to 25% by weight of an organic solvent. 7. A method as in claim 6, wherein the organic solvent is selected from the group consisting of dimethyl sulfoxide, ethanol, glycerol, polyethylene glycol, and propylene glycol. 8. A method according to claim 1, wherein at least two furocoumarins are present. 9. A method according to claim 1 wherein any unreacted furocoumarin(s) or photobreakdown products thereof are selectively removed. 10. A method according to claim 1 wherein furocoumarins or biological components which have reacted with the furocoumarin(s) are selectively removed by antibodies to those modified components. 11. A method for decontaminating a blood clotting factor containing composition of viral contaminants in a manner which substantially maintains the biological activity of the blood clotting factors, said method comprising adding to the blood clotting factor containing composition at least one furocoumarin such that the total furocoumarin concentration is at least 1 (greek mu)g/ml and not more than 300 (greek mu)g/ml, and irradiating the furocoumarin containing composition under U-V light which wavelengths are in the range of about 300 nm to 400 nm and at an intensity of about 0.1 mw/cm(2) to 5 w/cm(2) and at a depth of at least 0.025 millimeters for a total irradiation time of about 5 minutes to about 12 hours, and wherein the level of dissolved oxygen in the blood clotting factor containing composition is substan- 16 tially reduced to substantially inhibit the denaturation of the blood clotting factors. 12. A method as in claim 11, wherein the level of dissolved oxygen is reduced by addition of an oxygen scavenger to the composition. 13. A method as in claim 11, wherein the level of dissolved oxygen is reduced by equilibrium exchange with a less reactive gas. 14. A method as in claim 11, wherein the level of dissolved oxygen and other reactive species is reduced by addition of a physiologically-acceptable protein. 15. A method as in claim 14, wherein the physiologically acceptable protein is human or bovine serum albumin. 16. A method as in claim 11, wherein the solubility of the furocoumarin in the aqueous composition is increased by the addition of from about 1% to 25% by weight of an organic solvent. 17. A method as in claim 16, wherein the organic solvent is selected from the group consisting of dimenthyl sulfoxide, ethanol, glycerol, polyethylene glycol, and propylene glycol. 18. A method according to claim 11, wherein two furocoumarins are added to said composition. 19. A method according to claim 18, wherein said two furocoumarins are 4'-hydroxymethyl-4,5',8-trimethylpsoralen and 4'-aminomethyl-4,5',8-trimethylpsoralen. 20. A method according to claim 11, wherein the viral contaminants comprise at least one of Hepatitis A, Hepatitis B, and Non-A Non-B Hepatitis viruses. 21. A method according to claim 11 wherein the viral contaminants comprise a virus which causes Acquired Immune Deficiency Syndrome (AIDS). 22. A method according to claim 11 wherein the furocoumarin added is 8-methoxypsoralen. * * * * * 39 EXHIBIT D UNITED STATES PATENT [19] [11] PATENT NUMBER: 4,748,120 Wiesehahn [45] DATE OF PATENT: *May 31, 1988 - -------------------------------------------------------------------------------- [54] PHOTOCHEMICAL DECONTAMINATION TREATMENT OF WHOLE BLOOD OR BLOOD COMPONENTS [75] Inventor: Gary P. Wiesehahn, Alameda, Calif. [73] Assignee: Diamond Scientific Co., Des Moines, Iowa [* ] Notice: The portion of the term of this patent subsequent to Feb. 23, 2005 has been disclaimed. [21] Appl. No.: 928,841 [22] Filed: Oct. 20, 1986 RELATED U.S. APPLICATION DATA [63] Continuation of Ser. No. 490,681, May 2, 1983, abandoned. [51] Int. Cl.(4) .................................. C12N 13/00; A61K 39/00; A61K 35/14; A61K 35/48 [52] U.S. Cl. ..................................... 435/173; 424/85; 424/89; 424/90; 424/101; 514/2; 514/6; 530/380; 530/381; 530/383; 530/387; 530/389; 530/829 [58] Field of Search .............................. 435/172.1, 173, 183, 435/188, 236. 238, 269, 800, 814; 424/89, 90, 101, 85; 514/2; 530/350, 363, 380-388, 412-414, 427 [56] REFERENCES CITED U.S. PATENT DOCUMENTS 4,124,598 11/1978 Hearst et al. ............ 260/343.21 4,169,204 9/1979 Hearst et al. ............ 546/270 4,321,919 3/1982 Edelson .................. 128/214 R OTHER PUBLICATIONS Musajo et al, Experentia, vol. XXI, pp. 22-24, "Photo-sensitizing Furocoumanns: Interaction with DNA and Photo-Inactivation of DNA Containing Viruses". Veronese et al, Photochem Photobiol, vol. 36, pp. 25-30, "Photoinactivation of Enzymes by Linear and Angular Furocoumanns". De Mol et al., Chem. Abst., vol. 95, No. 74462k, p. 197, 1981, "On the Involvement of Singlet Oxygen in Mutation Induction by 8-Methoxypsoraten and UVA Radiation in Escherichia coli K-12". De Mol et al, Photochem Photobiol, vol. 33, pp. 815-819, 1981, "Relation Between Some Photobiological Properties of Furocoumanns and their Extent of Singlet Oxygen Formation". deMol et al. (1981) Photochem. Photobiol. 34:661-666. Joshi and Pathak (1983) Biochem. Biophys. Res. Comm., 112:638-646. Grossweiner (1982) NCI Monograph, No. 66, 47-54. Rodighiero and Dall'Acqua (1982) NCI Monograph, No. 66, 31-40. Primary Examiner - Thomas G. Wiseman Assistant Examiner - Robin Lyn Teskin Attorney, Agent, or Firm - Zarley, McKee, Thomte, Voorhees & Sease [57] ABSTRACT Biological compositions are freed of functional polynucleotides by treatment of the biological composition with psoralen derivatives under irradiation conditions in which the proteins retain their original physiological activities and any polynucleotide present is rendered inactive. 32 CLAIMS, NO DRAWINGS 40 4,748,120 1 PHOTOCHEMICAL DECONTAMINATION TREATMENT OF WHOLE BLOOD OR BLOOD COMPONENTS This is a continuation of application Ser. No. 490,681, filed May 2, 1983, now abandoned. BACKGROUND OF THE INVENTION 1. Field of the Invention Recipients of blood and blood components risk acquiring infections from foreign biological organisms, either pre-existing in the blood at the time of collection or transmitted to the blood product during manipulation. Medical personnel who are in contact with collected human blood or clinical samples also have a significant chance of being exposed to potentially lethal blood-borne or sample-borne biological organisms. Blood components today are obtained from blood donors and frequently involve pooled lots, where one or more of the donors may be harboring a viral, bacterial or other infection. Since the blood or blood components are required to provide physiological functions in a mammalian host, normally a human host, these functions must not be impaired by the decontamination treatment of the biological composition. In addition, the blood or blood components may not be modified in such a way as to make them immunogenic which could result in an adverse immune response. Finally, any treatment should not leave residues or products detrimental to the health of the host or such residues or products should be readily removable. 2. Description of the Prior Art U.S. Pat. No. 4,327,086 describes the method for heat treating an aqueous solution containing human blood coagulation factor XIII. U.S. Pat. No. 4,321,919 proposes extracorporeal treatment of human blood with 8-methoxypsoralen (8-MOP). Hyde and Hearst, Bio-chemistry (1978) 17:1251-1257, describe the binding of two psoralen derivatives to DNA and chromatin. Musajo et al., Experientia (1965) XXI, 22-24, describe proto-inactivation of DNA-containing viruses with photosensitizing furocoumarins. U.S. Pat. Nos. 4,350,594, 4,348,283 and 4,350,156 describe filtration methods for selective removal of blood components based on molecular weight. U.S. Pat. No. 4,329,986 describes extracorporal treatment of blood with a chemotherapeutic agent which is subsequently removed by dialysis. The July/August 1982 issue of Genetic Engineering News proposed the use of psoralens to sterilize "clinical or commercial reagents or instruments." Some data showing substantial impairment of the biological function of certain enzyme proteins using furocoumarins are published in the scientific literature (see for example, Veronese, F.M. et al., Photochem. Photobiol. 34:351(1981); Veronese, F.M. et al., Photochem. Photobiol. 36:25 (1982)). SUMMARY OF THE INVENTION Methods and compositions are provided for decontamination of biological compositions, permanently inactivating polynucleotides capable of having pathological effect in a mammalian host. Particularly, furocoumarin comparisons are employed for inactivating polynucleotides, such as viral genomes, capable of infectious replication in a mammalian host. Compositions for use in a mammalian host may be decontami- 2 nated by treatment with furocoumarins and long wave-length ultraviolet (UVA) light. DESCRIPTION OF THE SPECIFIC EMBODIMENTS In accordance with the subject invention, compositions to be employed with mammalian hosts, which may harbor polynucleotides capable of detrimental physiological effects in a host, are combined with furocoumarin compositions and treated with UVA light under predetermined conditions, whereby the physiological activities of the non-nucleic acid components are retained. (Wherever the term "polynucleotide" is used in this application it should be understood to mean: (1) microorganisms containing nucleic acids (either DNA or RNA), (2) nucleic acid genomes or sub-genomic fragments from microorganisms, from procaryotes (lower life forms) or from eucaryotes (higher life forms), or (3) any other nucleic acid fragments.) In decontaminating the biological composition, an aqueous medium containing the biological preparation is combined with an appropriate amount of the furocoumarin composition and irradiated with ultraviolet light under conditions where all of the polynucleotide is inactivated, while the components other than nucleic acid retain their normal physiological activities. Various biological compositions may be employed, particularly protein compositions involving blood or blood components. Whole blood, packed red cells, platelets, and plasma (fresh or fresh frozen plasma) are of interest. Other blood components of interest include plasma protein portion, antihemophilic factor (AHF, Factor VIII); Factor IX and Factor IX complex (Factors II, VII, IX and X); fibrinogens, Factor XIII, prothrombin and thrombin (Factor II and IIa); immunoglobulins (e.g. IgA, IgD, IgE, IgG and IgM and fragments thereof e.g. Fab, F(ab')(2), Fc); hyper-immune globulins as used against tenanus and hepatitis B; cryoprecitate; albumin; interferons; lymphokines; transfer factors; etc. Other biological compositions include vaccines, recombinant DNA produced proteins, oligopeptide ligands, etc. the protein concentration in the aqueous medium will generally range from about 1 (greek mu)g/ml to 500 mg/ml, more usually from about 1 mg/ml to 100 mg/ml. The pH will normally be close to physiologic pH(~7.4), generally in the range of about 6 to 9, more usually about 7. Other components may be present in the medium, such as salts, additives, buffers, stabilizers, or the like. These components will be conventional components, which will be added for specific functions. The furocoumarins will include psoralen and derivatives, where the substitutents will be: alkyl, particularly of from 1 to 3 carbon atoms, e.g. methyl; alkoxy, particularly of from 1 to 3 carbon atoms, e.g. methoxy; and substituted alkyl, of 1 to 6, more usually 1 to 3 carbon atoms having from 1 to 2 heteroatoms, which will be oxy, particularly hydroxy or alkoxy of from 1 to 3 carbon atoms, e.g. hydroxymethyl and methoxymethyl, or amino, including mono- and dialkyl amino having a total of from 1 to 6 carbon atoms, e.g. aminomethyl. There will be from 1 to 5, usually 2 to 4 substituents, which will normally be at the 4, 5, 8, 4' and 5' positions, particularly at the 4'-position. Illustrative compounds include 5-methoxypsoralen, 8-methoxypsoralen (8-MOP), 4, 5',8-trimethylpsoralen (TMP), 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT), 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT), 4-methylpsoralen, 4,4'-dimethylpsoralen, 4,5'-dimethylp- 41 4,748,120 3 sorslen, 4'8-dimethylpsoralen, and 4'-methoxymethyl-4,5',8-trimethylpsoralen. The subject furocoumarins are active with a wide variety of viruses and other polynucleotides, DNA or RNA, whether single stranded or double stranded. Illustrative viruses include: adenovirus, arenavirus, bacteriophage, bunyavirus, herpesvirus, orthomyxovirus, papovavirus, paramyxovirus, picornavirus, poxvirus, reovirus, retrovirus, rhabdovirus, and togavirus. Additional pathogenic microorganisms include bacteria, chlamydia, mycoplasma, protozoa, rickettsia and other unicellular microorganisms. Furocoumarins may also be effective in inactivating Hepatitis B and Non-A Non-B Hepatitis viruses. This inactivation method may also be used against uncharacterized infectious agents which may contain nucleic acid (such as the agent which causes Acquired Immune Deficiency Syndrome). In addition to the furocoumarins, additives may be included which scavenge for singlet oxygen or other highly reactive oxygen containing species. Such additives include ascorbate, glutathione, sodium thionine, etc. In some instances these additives may have adverse effects, so that in each instance, their use will be determined empirically. Where such additives are present, they will be present in amounts ranging from about 20 (greek mu)g to 20 mg per ml. The furocoumarins may be used individually or in combination, preferably in combination. Each of the furocoumarins may be present in amounts ranging from about 0.01 (greek mu)g/ml to 1 mg/ml, preferably from about 0.5 (greek mu)g/ml to 100 (greek mu)g/ml, there not being less than about 1 (greek mu)g/ml nor more than about 1 mg/ml of furocoumarins. For RNA, the preferred furocoumarins are AMT and HMT. For DNA, the preferred furocoumarin is TMP. For mixtures of DNA- and RNA-containing polynucleotides, or for inactivation of infectious agents or possibly infectious agents of unknown or uncertain nucleic acid classification, or for protection against infections of unknown etiology, preferably TMP and AMT are used in combination. In carrying out the invention, the furocoumarins may be added to the biological composition by any convenient means in a manner substantially assuring the uniform distribution of the furocoumarins in the composition. The composition may then be irradiated under conditions ensuring that the entire composition is exposed to sufficient irradiation, so that the furocoumarins may react with any polynucleotide present to inactivate the polynucleotide. Depending upon the nature of the medium, particularly its opacity, as in the case of blood, the depth of the solution subject to irradiation will vary widely. Usually, the depth will be not less than about 0.025 millimeter, but may be a centimeter or more. With whole blood, the depth will generally range from about 0.025 millimeter to 2.5 millimeters. The light which is employed will generally have a wavelength in the range of about 300 nm to 400 nm. The intensity will generally range from about 0.1 mW/cm(2) to about 5 W/cm(2). In order to prevent denaturation, the temperature should be maintained below about 60 degrees C., preferably below about 40 degrees C., usually from about -10 degrees C. to 30 degrees C. The medium being irradiated may be irradiated while still, stirred or circulated, and may either be continuously irradiated or be subject to alternating periods of irradiation and non-irradiation. The circulation may be in a closed loop system or it may be in a single pass system ensuring that all of the sample has been exposed to 4 irradiation. The total time for irradiation will vary depending upon the nature of the sample, the furocoumarin derivative used, the intensity and spectral output of the light source and the nature of the polynucleotides which may be present. Usually, the time will be at least 1 min. and not more than about 6 hrs., more usually from about 15 mins. to about 2 hrs. When circulating the solution, the rate of flow will generally be in the range of about 0.1 ml/min to 50 liters/min. It may be desirable to remove the unexpended psoralen and/or its photobreakdown products from the irradiation mixture. This can be readily accomplished by dialysis across an appropriately sized membrane or through an appropriately sized hollow fiber system after completion of the irradiation. It may be desirable in certain applications to remove bound or unbound furocoumarins using antibodies, including monoclonal antibodies, either in solution or attached to a substrate. The following examples are offered by way of illustration and not by way of limitation. EXPERIMENTAL The following experiments were performed in order to demonstrate the ability of the psoralen photoreaction to destroy microbial contaminants contained in whole blood and blood products. (1) Feline rhinotracheitis virus, a member of the herpesvirus family, was added to heparinized whole rabbit blood in an amount that would give a final concentration of approximately 2 x 10(7)PFU/ml.4'-hydroxymethyl-4,5',8- trimethylpsoralen (HMT) was added to a portion of the rabbit blood and aliquots were irradiated for various periods of time. To test for remaining live virus, duplicate plaque assays were performed using cultured feline cells (Fc3Tg)(ATCC CCL 176), with a methylcellulose overlay. Virus titers were obtained as the arithmetical mean of viral plaques observed in duplication assay cultures 72 hours after exposure to test samples. The results are as follows: The blood aliquot that received HMT only and no irradiation gave a titer of 5.3 X 10(6)PFU/ml. The aliquot that received HMT and five minutes of irradiation exhibited a titer of 4.5 X 10(6)PFU/ml. In the aliquot that received psoralen plus one hour of irradiation there was no detectable live virus remaining. The sensitivity of this assay should have permitted detection of residual virus at titers greater than or equal to 1.0 x 10(1)PFU/ml. A blood sample which had received HMT and one hour of irradiation also showed no apparent damage to the red blood cells as judged by phase contrast microscope analysis and by absence of visible hemolysis. These data therefore demonstrate that high virus titers present in whole blood can be inactivated by psoralen plus light treatment which leaves the red cell component of the blood intact. (2) In the second experiment Blue Tongue Virus (Serotype 11), a member of the reovirus family, and Feline Rhinotracheitis Virus, and Simian Virus 40 were added to a solution of Profilate (a commercial preparation of human clotting factor VIII produced by Alpha Therapeutics). The lyophilized preparation of Profilate (180 units) was dissolved in 10 ml of sterile water included with the commercial preparation. This solution was further diluted with barbital buffer (11.75 g sodium barbital and 14.67 g NaCl dissolved in 2 liters of de-ionized water and filtered through a 0.22 micron filter) to a final concentration of 5 units per milliliter. One portion (2 ml) was set aside at room temperature in the dark. This was sample 190 1. A second 2 ml portion was 42 4,748,120 5 pumped through the apparatus described below for 1 hour with irradiation. This was sample #2. Through addition of appropriate amounts of reagents a third 2 ml portion was adjusted to contain 10 (greek mu)g/ml AMT and 10 (greek mu)g/ml HMT and was also irradiated for 1 hour. This was sample #3. The fourth 2 ml portion was adjusted to 10 (greek mu)g/ml AMT, 10 (greek mu)g/ml AMT, and 10 mM sodium ascorbate and was also irradiated for 1 hour. This was sample #4. All the samples were kept at 20 degrees C. throughout the manipulations. The total elapsed time from dissolving of the lyophilized preparation to the completion of the clotting factor VIII assays was 6 and one-half hours. The clotting factor VIII assays were performed at a variety of dilutions (ranging from 1:5 to 1:100) for each sample and were compared with the activity in normal human serum and with pooled normal human serum. The results are summarized in Table 1.
TABLE 1 - -------------------------------------------------------------------------------- Effect of Photochemical Inactivation Procedure and Its Components* on in vitro Activity of Factor VIII(+) ----------------------------------------- Sample -------------------------------------- #1 #2 #3 dilution normal pool F(-),UVA(-) F(-),UVA(+) F(+),UVA(+) - -------------------------------------------------------------------------------- 1:5 97 108 225 150 186 1:10 102 102 245 155 186 1:20 93 92 280 176 196 1:50 101 95 265 190 232 1:100 -- 100 255 196 263 --- --- --- --- --- Average 98 99 254 173 213 - -------------------------------------------------------------------------------- *F -- Furocoumarins; UVA -- long wavelength ultraviolet light; (+) Factor VIII activity expressed in % of normal activity. 100% -- IU/ml of Factor VIII activity.
The sample that was subjected to the psoralen inactivation protocol (sample #3) retained 84% of the factor VIII activity that was present in the control sample (#1). This was higher than the product activity retained by the sample that was only irradiated (68% retained for sample #2) and indicates that the psoralen photochemistry has little or no effect on the activity of factor VIII. Samples otherwise identical to sample 1, 2, and 3 above were seeded with 2X10(6)PFU/ml of Feline Rhinotracheitis Virus (FeRT), 1X10(7)PFU/ml of Blue Tongue Virus (BTV), and 4X10(8)PFU/ml of Simian Virus 40 (SV-40). Table 2 shows the results of the plaque assays on those samples.
TABLE 2 - -------------------------------------------------------------------------------- Effect of Photochemical Inactivation Procedure and Its Components* on Infectivity of Virus in Factor VIII preparation.(+) ----------------------------------------------- Sample 1 Sample 2 Sample 3 F(-),UVA(-) F(-),UVA(+) F(+),UVA(+) - -------------------------------------------------------------------------------- FeRT Titer 8.6 X 10(5) 3.5 X 10(5) 0.0 BTV Titer 3.8 X 10(7) 1.4 X 10(7) 1.1 X 10(2) SV-40 Titer 2.5 X 10(8) 1.6 X 10(8) 1.2 X 10(3) - -------------------------------------------------------------------------------- *F -- Furocoumarins; UVA -- long wavelength ultraviolet light; (+) Infectivity determined by plaque assays in tissue culture.
In the case of FeRT the number of detectable virus particles was reduced by more than five orders of magnitude to beneath the limit of detection in the plaque assay. The BTV infectivity was reduced by about five orders of magnitude to 110 PFU/ml. The SV40 infectivity was reduced to a titer of 1.2X10(3). Thus, it is shown that multiple, widely distinct types of virus can be simultaneously inactivated by at least five orders of 6 magnitude in the presence of factor VIII, using the simple, convenient, brief process described above, with retention of at least 84% of factor VIII activity. Based on the above observations, it is predictable that by extending, repeating or modifying the treatment, the probability of an infectious virus particle remaining can be reduced to an arbitrarily low value. In this manner suitable safety margins can be achieved for any of the cited applications. APPARATUS AND SYSTEM Since whole blood exhibits very high optical density for longwave UV light (absorption is high for visible light in the 400 nm to 500 nm range), blood was irradiated through a suitably short optical path length. In this experiment blood was pumped through polyethylene capillary tubing of 0.875 millimeter inside diameter. The tubing was coiled around a 1.27 centimeter diameter tube and immersed in water which was maintained at 18 degrees C. The blood was continuously circulated through the tubing by means of a peristaltic pump. The blood required approximately 2.5 minutes for a complete cycle through the capillary tubing and was in the light beam for approximately 20% of the stated irradiation time. The light source was a low pressure mercury lamp filtered through a cobalt glass filter. The filter transmit light of approximately 320 nm-380 nm, with peak transmittance at 360 nm. The incident intensity at the sample was approximately 40 mW/cm(2). It is evident from the above results, and in accordance with the subject invention, that polynucleotides in biochemical compositions can be inactivated to provide a safe composition for administration to a mammalian host. The proteins present in the composition retain their physiological activity, so that they can fulfill their physiological function in a mammalian host. The method is simple, rapid, and can be expanded to treat large samples. The small amount of chemical reagent required will not generally be harmful to the host. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims. What is claimed is: 1. A method for decontaminating blood components suspected of containing viruses, said blood components being selected from the group consisting of red blood cells, platelets, blood clotting factors, plasma and immunoglobulins, without substantial impairment of the physiological activities of the treated blood components, said method comprising: (a) adding to a blood component selected from the group consisting of red blood cells, platelets, blood clotting factors, plasma and immunoglobulins at least one psoralen compound in an amount sufficient to inactivate substantially all contaminating viruses prevent; and thereafter (b) irradiating said psoralen treated blood component with long wavelength ultraviolet light under operating conditions which maintain the concentrations of reactive oxygen species at levels which do not substantially impair the physiological activity of the treated blood component, and wherein said irradiation is conducted for a time sufficient to inactivate substantially all contaminating viruses present. 43 4,748,120 7 2. A method according to claim 1 wherein the conditions which maintain the concentration of reactive oxygen species at levels which do not substantially impair the physiological activity of the treated blood component comprise the addition of an oxygen scavenger. 3. A method according to claim 2 further comprising selectively removing any unreacted psoralen(s) or photobreakdown products thereof by ultrafiltration of dialysis. 4. A method according to claim 1, wherein at least two psoralens are present. 5. A method according to claim 1, wherein said component is immunoglobin. 6. A method according to claim 1, wherein said blood component is red cells. 7. A method according to claim 1, wherein said blood component is a clotting factor. 8. A method according to claim 1, wherein said blood component is platelets. 9. A method according to claim 1, wherein said blood component is plasma. 10. A method according to claim 1, wherein said psoralen has at least one substituent which is alkyl of from 1 to 3 carbon atoms, alkoxy of from 1 to 3 carbon atoms, or substituted aklyl of from 1 to 6 carbon atoms having 1 to 2 heteroatoms which are oxy or amino. 11. A method according to claim 1, wherein said psoralen has at least one substitutent which is alkoxy of from 1 to 3 carbon atoms. 12. A method according to claim 11, wherein said psoralen is 5-methoxypsoralen (5-MOP), 8-methoxypsoralen (8-MOP) or 4'-methoxymethyl-4,5', 8-trimethylpsoralen. 13. A method according to claim 1, wherein said psoralen has at least one substituent which is alkyl of from 1 to 3 carbon atoms. 14. A method according to claim 13, wherein said psoralen is 4,5',8-trimethylpsoralen (TMP), 4-methylpsoralen, 4,4'-dimethylpsoralen, 4,5'-dimethylpsoralen or 4',8-dimethylpsoralen. 15. A method according to claim 1, wherein said psoralen has at least one substituent which is alkyl of from 1 to 6 carbon atoms having from 1 to 2 heteroatoms which are oxy or amino. 16. A method according to claim 15, wherein said psoralen is 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) or 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT). 17. A method for decontaminating blood components suspected of containing viruses, said blood components being selected from the group consisting of red blood cells, platelets, blood clotting factors, plasma and immunoglobulins, without substantial impairment of the physiological activity of the treated blood components, said method comprising: (a) adding to a blood component selected from the group consisting of red blood cells, platelets, blood clotting factors, plasma and immunoglobulins at least one psoralen compound in a total psoralen 8 concentration of at least 1 ug/ml and not more than 300 ug/ml; and thereafter (b) passing said psoralen treated blood component through a light beam with a wavelength in the range of 300 nm to 400 nm at an intensity of about 0.1 mw/cm(2) to 5 W/m(2) at a depth of at least 0.025 mm for a total radiation time of about 5 minutes to about 12 hours, wherein said irradiation is conducted under operating conditions which maintain the concentrations of reactive oxygen species at levels which do not substantially impair the physiological activity of the treated blood component. 18. A method according to claim 17 wherein the conditions which maintain the concentrations of reactive oxygen species at levels which do not substantially impair the physiological activity of the treated blood component comprise the addition of an oxygen scavenger. 19. A method according to claim 18 further comprising selectively removing any unreacted psoralen(s) or photobreakdown products thereof by ultrafiltration or dialysis. 20. A method according to claim 17, wherein at least two psoralen are present. 21. A method according to claim 17, wherein said blood component is red cells. 22. A method according to claim 17, wherein said blood component is platelets. 23. A method according to claim 17, wherein said blood component is plasma. 24. A method according to claim 17, wherein said blood component is a clotting factor. 25. A method according to claim 17, wherein said blood component is an immunoglobin. 26. A method according to claim 17, wherein said psoralen has at least one substituent which is alkyl of from 1 to 3 carbon atoms, alkoxy of from 1 to 3 carbon atoms, or substituted alkyl of from 1 to 6 carbon atoms having 1 to 2 heteroatoms which are oxy or amino. 27. A method according to claim 17, wherein said psoralen has at least one substituent which is alkoxy of from 1 to 3 carbon atoms. 28. A method according to claim 27, wherein said psoralen is 5-methoxypsoralen (5-MOP), 8-methoxypsoralen (8-MOP) or 4'-methoxymethyl-4,5',8-trimethylpsoralen. 29. A method according to claim 17, wherein said psoralen has at least one substituent which is alkyl of from 1 to 3 carbon atoms. 30. A method according to claim 29, wherein said psoralen is 4,5',8-trimethylpsoralen (TMP), 4-methylpsoralen, 4,4'-dimethylpsoralen, 4,5'-dimethylpsoralen or 4',8-dimethylpsoralen. 31. A method according to claim 17, wherein said psoralen has at least one substituent which is alkyl of from 1 to 6 carbon atoms having from 1 to 2 heteroatoms which are oxy or amino. 32. A method according to claim 31, wherein said psoralen is 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) or 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT). * * * * * 65 44 EXHIBIT E UNITED STATES PATENT [19] [11] Patent Number: 4,791,062 Wiesehahn et al. [45] Date of Patent: Dec. 13, 1988 - -------------------------------------------------------------------------------- [54] FVR VACCINE [75] Investors: Gary P. Wiesehahn, Alameda; Richard E. Giles, Union City; David R. Stevens, Fremont, all of Calif. [73] Assignee: Diamond Scientific Co., Des Moines, Iowa [21] Appl. No.: 20,201 [22] Filed: Jul. 6, 1987 Related U.S. Application Data [63] Continuation of Ser. No. 707,102, Feb. 28, 1985, abandoned. [51] Int. Cl.(4).................A61K 39/12; A61K 39/245 [52] U.S. Cl. ..........................435/238; 424/89; 435/236 [58] Field of Search.........................424/89, 90; 435/235-239 [56] References Cited U.S. PATENT DOCUMENTS 4,522,810 6/1985 Pedersen.........................435/235 4,545,987 10/1985 Giles et al. ....................435/235 4,556,556 12/1985 Wiesehahn et al. ................ 424/90 4,693,981 9/1987 Wiesehahn et al. ................435/238 4,727,027 2/1988 Wiesehahn et al. ................ 424/89 4,748,120 5/1988 Wiesehahn et al. ................ 424/89 Primary Examiner--Shep K. Rose Attorney, Agent, or Firm--Zarley, McKee, Thomte, Voorhees & Sease [57] ABSTRACT Novel vaccines for feline viral rhinotracheitis are prepared by psoralen inactivation of live Feline Herpesvirus I by exposure to ultraviolet radiation in the presence of an inactivating furocoumarin. The resulting inactivated viruses are suitable as the immunogenic substances in vaccines, which vaccines are useful for inoculation of hosts susceptible to feline virus rhinotracheitis. 10 CLAIMS, NO DRAWINGS 45 4,791,062 1 FVR VACCINE This is a continuation of copending application Ser. No. 707,102 filed on Feb. 28, 1985 now abandoned. BACKGROUND OF THE INVENTION 1. Field of the Invention Vaccination against both bacterial and viral diseases has been one of the major accomplishments of modern medicine. While effective vaccines have been developed for a large number of animal and human diseases, development of safe and effective vaccines for a number of other diseases remains problematic. In preparing suitable vaccines, the primary objectives are eliciting an immunogenic response which provides immunity against the disease of interest while assuring that the vaccine itself is non-pathogenic. In preparing vaccines, a number of general approaches have been developed. The use of killed microbial agents as a vaccine, although generally save, will not always be effective if the immunogenic characteristics of the agent are altered. In contrast, the preparation of live attenuated microbial agents as a vaccine will often provide improved immunologic reactivity, but will increase the risk that the vaccine itself will become pathogenic, e.g., as a result of reversion. Thus, although much experience has been gained over the years relating to the preparation of vaccines, the successful preparation of an effective vaccine against a particular infectious agent can never be assured, even when employing techniques which were previously successful for other microorganisms. Feline viral rhinotracheitis (FVR) is species specific and enzootic in cat populations worldwide. The causative agent is a Herpesvirus (Feline Herpes I), and transmission is by direct contact or by infectious aerosols. Infection affects the nasal and ocular mucous membranes initially. Clinical signs, which commence within 2 to 10 days post infection, may include sneezing, coughing, lacrimation (excessive tearing), serous to mucopurulent nasal discharge, conjunctivitis, rhinitis, anorexia, dehydration, dyspnea, and severe depression. Cutaneous, ocular, nasal or oral ulcers and abortions may also be encountered. Pyrexia, up to 105 degrees F. (40.5 degrees C.), and a mild to moderate neutraphilic leukocytosis or mild anemia may be present. However, pyrexia and neutraphilia are mainly associated with secondary bacterial infection. The course is often 1 to 3 weeks, but more prolonged systemic disease such as pneumonia or hepatitis may occur, especially in kittens. In fatal cases the course may extend to 4 or 5 weeks. Individual cats may die from the more severe manifestations or from secondary complications. At necropsy, respiratory tract lesions are most consistently encountered. These include hyperemic nasal and respiratory passages often covered with fibrinous or mucopurulent exudate. Secondary bacterial pneumonia may result in widely disseminated bacterial emboli. Microscopically, intranuclear inclusions, if present, occur most often in respiratory epithelium. Prior to the introduction of vaccines, 15% to 20% of isolated cat populations were reported to be asymptomatic carriers of FVR, providing a continuous reservoir for infection. Urban cat populations have carrier rates of 50% to 80%. 2. Description of the Prior Art 2 Feline viral rhinotracheitis was first recognized as a disease entity by Crandell and Mauer (1958) Proc. Soc. Expt. Bio. Med. 97:487-490. Experimental FVR infection results in low serum neutralizing antibody titers (e.g., 1:4 to 1:10). See Crandall et al. (1961) J.A.V.- M.A., 138:191-196; and Hoover et al. (1970) Am. J. Path. 58:269-282. Individual cats may be resistant to reinfection with FVR, although they have little or no detectable serum antibody against FVR (Bartholomew et al. (1968) Cornell Vet. 58:248-265). Infection immunity is short-lived, and cats may be reinfected six months following a primary infection. Reinfection elicits mild clinical signs and reduced viral shedding (Walton and Gillespie (1970) Cornell Vet. 60:232-239). Previous attempts at vaccine development for FVR have included formalin inactivation (Fisher, et al. (1966) VM/SAC 61:1182-1189; Tan et al. (1971) N.Z. Vet. J. 19:12-15; and Povey et al. (1978) Feline Pract. 8:36-42); temperature sensitive mutants (Slater et al. (1976) Develop. Biol. Stand. 33:410-416); and tissue culture attenuated live virus isolates (Bittle et al. (1974) VM/SAC 69:1503-1505; Bittle et al. (1975) Am. J. Vet. Res. 36:89-91; F. Scott (1975) Feline Practice Jan.-Feb.:-17-22; and Edwards et al. (1977) VM/SAC Feb:2-05-209). Chemically inactivated FVR vaccines failed to induce immunity (Fisher et al. (1966) VM/SAC 61:1182-1189), although later trials with formalin inactivated FVR vaccines were somewhat successful (Tan et al. (1971) N.Z. Vet. J. 19:12-15 and Povey et al. (1978) Feline Pract. 8:36-42). Formalin inactivated FVR vaccines are critically dependent on the incorporation of a suitable immunologic adjuvant such as mineral oil. One FVR vaccine production method utilized DNA inhibitors to select biochemically uncharacterized FVR mutants that were subsequently inactivated by UV irradiation (Davis et al. (1976) VM/SAC Oct: 1405-1410). Inactivation was less than 100%, and the remaining live virus was cloned at 30 degrees C. ((plus or minus)2 degrees C.) and subjected to repeated cycles of the same process, resulting in an attenuated virus strain. See, U.S. Pat. No. 4,031,204. U.S. Pat. No. 4,287, 178 discloses that temperature sensitive FVR mutants can be utilized as an attenuated live virus vaccine for FVR. Attenuated live FVR vaccines are efficacious, but may induce clinical disease or abortions. Combination vaccines have been described in which FVR and other feline pathogens are incorporated (Bittle et al. (1975) Feline Practice Nov. - Dec:- 13-15 and Edwards et al. (1977) Feline Practice July:4-5-50). These vaccines are also produced by standard procedures known to the art. The preparation of psoralens and their use in inactivating viruses are described in U.S. Pat. Nos. 4,196,281 and 4,124,598. SUMMARY OF THE INVENTION Vaccines for inoculation against feline viral rhinotracheitis are prepared by irradiating live Feline Herpes I virus, the etiologic agent which causes FVR, with light in the presence of an inactivating furocoumarin compound for a time sufficient to render the virus completely non-infectious. It has been found that inactivated Feline Herpes I virus retains immunogenicity, and that inoculation of a susceptible host with such inactivated viruses elicits the production of serum neutralizing antibody and protects the host against subsequent challenge with live, infectious Feline Herpes I virus. The inactivated Feline Herpesvirus I may be combined with a physiologically-acceptable carrier or 46 4,791,062 3 adjuvant, usually at from about 10(6) to 10(9) pfu/ml, to form the vaccine. DESCRIPTION OF THE SPECIFIC EMBODIMENTS Vaccines useful for the inoculation of feline hosts against feline viral rhinotracheitis are provided. The vaccines are prepared by inactivation of live Feline Herpes I virus in an appropriate medium with a sufficient amount of an inactivating furocoumarin to provide for inactivation of the virus upon subsequent irradiation with long wavelength ultraviolet (UV) radiation. The resulting inactivated virus may be stored until used for inoculation. Prior to inoculation, the inactivated virus will usually be combined with a physiologically-acceptable carrier or immunologic adjuvant. Any of the isolates of Feline Herpes I virus, or combinations thereof, may be inactivated and utilized to prepare a vaccine according to the present invention. Such live, virulent viruses can be obtained from cats suffering from feline viral rhinotracheitis according to conventional techniques. See, e.g., Crandell and Mauer, supra.; Bittle et al. (1960) Amer. J. Vet. Res. 21:547; and Ditchfield and Grinyer (1965) Virology 26:504. Generally, virus obtained from the nasal and conjunctival membranes of infected cats are used to infect suitable feline cells grown in tissue culture. The viruses are replicated and isolated by serial passage following well known techniques. Alternatively, the virus may be derived from generally available sources, such as Feline Herpes 1 virus available from the American Type Culture Collection under designation VR636. A specific method for growing virus from seed virus is set forth in the Experimental section hereinafter. In preparing the subject vaccines, the desired virus is grown in mammalian cell culture. Suitable cell lines include the AKD cell line (ATCC CCL 150) and Fc3Tg (ATCC CCL 176), and other cell lines permissive for Feline Herpes I virus and which can be grown in vitro as monolayer cultures or as suspension cultures. The cell cultures are grown to approximately 80% saturation density and infected with the feline herpesvirus at a multiplicity of infection (MOI), usually between about 0.03 and 0.3, preferably about 0.1. After adsorbing the viral inoculum to the cells by incubation for a limited period of time at a temperature in the range from about 35 degrees c. to 40 degrees C., an appropriate growth or maintenance medium is added. The cells are incubated at temperatures in the range from about 35 degrees C. to 40 degrees C., in the presence in the range from about 35 degrees C. to 40 degrees C., in the presence of about 5% carbon dioxide in air until at least about 50% of the cell culture exhibits cytopathic effect (CPE). CPE is characterized by cell rounding (in monolayers) and cell degeneration. The culture vessel is shaken to detach loosely adhering cells and cellular debris, and the contents of each vessel are aseptically decanted into centrifuge bottles. The crude virus preparation is centrifuged at 10,000 X g for 30 minutes and the supernatant is discarded. The pellet is resuspended in one-twentieth the original volume of maintenance medium containing 7 to 10% (v/v) dimethly sulfoxide (Sigma Chemical Co., St. Louis, MO 63178, cat. no. D 5879). For cell-associated virus preparations, the foregoing suspension is stored frozen at or below -80 degrees c. Cell-free virus preparations are produced from the foregoing suspension by freezing and thawing the suspension three times, centrifuging the resulting lysate at 10,000Xg or 30 minutes, and collect- 4 ing and storing the virus-containing supernatant at or below -80 degrees C. Cell-free virus may also be prepared from a suspension which lacks dimethyl sulfoxide. The particular growth and maintenance medium may be a conventional mammalian cell culture medium, such as Eagle's Minimum Essential Medium or Medium 199, usually supplemented with additives such as fetal bovine serum, fetal calf serum, broth prepared from dehydrated standard microbial culture media, or the like. The furocoumarins useful for inactivation are primarily illustrated by the class of compounds referred to as psoralens, which includes psoralens and substituted psoralens where the substitutents will be: alkyl, particularly having from 1 to 3 carbon atoms, e.g., methyl; alkoxy, particularly having from 1 to 3 carbon atoms, e.g., methoxy; and substituted alkyl having from 1 to 6, more usually from 1 to 3, carbon atoms and from 1 to 2 heteroatoms, which will be oxy, particularly hydroxy or alkoxy having from 1 to 3 carbon atoms, e.g., hydroxy methyl and methoxy methyl, or amino, including mono- and dialkyl amino or aminoalkyl, having a total of from 0 to 6 carbon atoms, e.g., aminomethyl. There will be from 1 to 5, usually from 2 to 4 substituents, which will normally be at the 4, 5, 8, 4' and 5' positions, particularly at the 4' position. Illustrative compounds include 5-methoxypsoralen; 8-methoxypsoralen (8-MOP); 4,5',8-trimethylpsorlen (TMP); 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT); 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT); 4-methylpsoralen; 4,4'-dimethylpsoralen; 4,5'-methyoxymethyl-4,5'8-trimethylpsorlen. Of particular interest are HMT, AMT and 8-MOP. The furocoumarins may be used individually or in combination. Each of the furocoumarins may be present in amounts ranging from about 0.01 (greek mu)g/ml to 1 mg/ml, preferably from about 5 (greek mu)g/ml to 300 (greek mu)g/ml, there not being less than about 1 g/ml nor more than about 1 mg/ml of furocoumarins. In carrying out the invention the furocoumarin(s), in an appropriate solvent which is substantially inert and sufficiently polar to allow for dissolution of the furocoumarin(s), are combined with the viral suspensions, conveniently a viral suspension in an aqueous buffered medium, such as used for storage. The amount of virus will generally be about 1 X 10(6) to 10(11), more usually about 1 X 10(7) to 10(9) and preferably about 1 X 10(8) to 5 X 10(8) pfu/ml. The furocoumarin will be at a concentration of about 0.001 mg/ml to 0.5 mg/ml, more usually about 0.02 mg/ml to 0.3 mg/ml. The amount of solvent which is used to dissolve the furocoumarin will be sufficently small so as to readily dissolve in the aqueous viral suspension and have little, if any, effect on the results. The furocoumarin may be added to the viral suspension in a single addition or in multiple additions, where the virus is irradiated between additions. Usually, the numer of additions will be from aboutr 1 to 50, more usually from about 10 to 40, and preferably from about 20 to 40. The total amount of furocoumarin which will be added will be sufficient to provide a concentration of at least about 0.01 mg/ml to about 1 mg/ml, usually not more than about 0.75 mg/ml. Since a substantial proportion of the furocoumarin will have reacted with the nucleic acide between additions, the total concentration of furocoumarin in solution will generally not exceed about 0.3 mg/ml. 47 4,791,062 5 The total time for the irradiation will vary depending upon the light intensity, the concentration of the furocoumarin, the concentration of the virus, and the manner of irradiation of the virus, where the intensity of the irradiation may vary in the medium. The total time will usually be at least about 2 hrs. and not more than about 80 hrs., generally ranging from about 10 hrs. to 50 hrs. The times between additions of furocoumarin, where the furocoumarin is added incrementally, will generally vary from about 30 min. to 24 hrs., more usually from about 1 hr. to 3 hrs. The temperature for the irradiation is preferably under 25 degrees C., more preferably under 20 degrees C. and will generally range from about -10 degrees C. to 15 degrees C., more usually from about 0 degrees to 10 degrees C. The irradiation is normally carried out in an inert atmosphere, where all or substantially all of the oxygen has been removed. Inert atmospheres include nitrogen, helium, argon, etc. The light which is employed will generally have a wavelength in the range from about 300 nm to 400 nm. The intensity will generally range from about 0.1 mW/cm(2) to about 5W/cm(2). Optionally, a small amount of a singlet oxygen scavenger may be included during the virus inactivation. Singlet oxygen scavengers include ascorbic acid, dithioerythritol, sodium thionite, glutathione, etc. The amount of scavenger will generally be at a concentration of about 0.001M to 0.5M, more usually at about 0.01M to 0.1M, where the addition may be made in a single or multiple additions. During irradiation, the medium may be maintained still, stirred or circulated and may be either continuously irradiated or be subject to alternating periods of irradiation and non-irradiation. The circulation may be in a closed loop system or in a single pass system ensuring that all of the sample has been exposed to irradiation. It may be desirable to remove the unexpended furocoumarin and/or its photobreakdown products from the irradiation mixture. This can be readily accomplished by one of several standard laboratory procedures such as dialysis across an appropriately sized membrane or through an appropriately sized hollow fiber system after completion of the irradiation. Alternatively, one could use affinity columns for one or more of the low molecular weight materials to be removed. The inactivated virus may then be formulated in a variety of ways for use for inoculation. The concentration of the virus will generally be from about 10(6) to 10(9) pfu/ml, as determined prior to inactivation, with a total dosage of at least 10(5) pfu/dose, usually at least 10(6) pfu/dose, preferably at least 10(7) pfu/dose. The total dosage will usually be at or near about 10(8) pfu/dose, more usually being about 10(6) to 10(7) pfu/dose. The vaccine may include cells or may be cell-free. It may be an inert physiologically acceptable medium, such as ionized water, phosphate-buffered saline, saline, or the like, or may be administered in combination with a physiologically acceptable immunologic adjuvant, including but not limited to mineral oils, vegetable oils, mineral salts and immunopotentiators, such as muramyl dipeptide. The vaccine may be administered subcutaneously, intramuscularly, or intraperitoneally. Usually, a specific dosage at a specific site will range from about 0.1 ml to 4 ml, where the total dosage will range from about 0.5 ml. to 8 ml. The number of injections and their temporal 6 spacing may be highly variable but usually 1 to 3 injections at 1, 2 or 3 week intervals are effective. The following examples are offered by way of illustration, not by way of limitation. EXPERIMENTAL Materials and Methods A. Virus Growth Cat cell lines AKD (ATCC CCL150) or Fc3Tg (ATCC CCL176) were grown as monolayers in plastic cell culture vessels in a standard defined culture media consisting of Minimum Essential Medium (MEM) and Earles salts with non-essential amino acids (MEN); F12K; MEM; or alpha MEM. Medium was supplemented with 2% to 15% inactivated fetal calf serum (F(i)) or 2% to 20% YELP (YELP consists of: yeast extract 5 g; lactalbumin hydrolysate 25 g; Bacto-peptone 50 g; deionized H(2)0 1000 mls sterilized by autoclaving or filtration). Cell cultures were used to produce live Feline Herpes I virus from master seed virus derived from Feline Herpes I virus (ATCC VR636). Cells were grown in culture vessels to 80% to 100% confluency (approximately 1 x 10(5) to 2 x 10(5) cells per cm(2) of growth surface area) using standard mammalian cell culture techniques as follows: Corning plastic roller bottles (Corning No. 25140-850) with a growth surface area of 850 cm(2) containing 50 to 100 ml of MEN supplemented with 10% F(i) and 1 x 10(8) to 2 x 10(8) AKD or Fc3Tg cells/bottle were used for virus production. The cell cultures were initiated by seeding approximately 1 x 10(6) to 5 x 10(6) cells into 50 to 100 mls of growth medium in a roller bottle containing about 5% CO(2) in air and incubating the roller bottle on a roller bottle rotator at 1 to 5 rpm at 35 degrees C. to 38 degrees C. The cultures were grown to 80% to 100% confluency over a 7 to 14 day period with a 100% medium change every 3 to 5 days. When the monolayers were 80% to 100% confluent, the culture medium was removed and the monolayer was washed with 20 to 50 mls of phosphate buffered saline (PBS) pH 7.2 to 7.4 (NaCl 8 g + KCl 0.2 g + Na(2)HPO(4) 1.14 g + KH(2)PO(4) 0.2 g). The PBS wash was discarded, and the roller bottle was infected by the addition of approximately 1 x 10(7) to 2 x 10(7) plaque forming units (pfu) of Feline Herpes I virus in 10 mls of PBS containing 2% F(i). The multiplicity of infection (MOI) was approximately 0.1. The virus inoculum was adsorbed to the cells by incubation at 35 degrees C. to 38 degrees C. for one hour at 1 to 5 rpm. The inoculation fluid was removed and 50 mls of MEN containing 10% F(i) was added per roller bottle. The post-infection incubation was at 35 degrees C. to 38 degrees C. in 5% CO(2) in air with rotation. Herpesvirus cytopathic effect (CPE) was evident forty to forty-eight hours post-infection. The CPE was characterized by cell rounding, cell detachment, and cell degeneration. The contents of the roller bottle were swirled 48 hours post-infection to remove loosely attached materials from the roller bottle walls, and the contents of the roller bottles were decanted into centrifuge bottles. The virus, cells, and cell debris were pelleted by centrifugation at 10,000 x g for 30 minutes. Cell associated (CA) virus was prepared by: 1. resuspending the 10,000 x g pellet in approximately 5 ml of a resuspension medium containing 80 parts F12K, 10 parts Fi, and 10 parts dimethylsulfoxide (DMSO) for each original roller bottle; 48 4,791,062 7 2. freezing the resuspended CA virus at -20 degrees C. for 1.5 to 2 hours; and 3. transferring the CA virus frozen at -20 degrees to temperatures ranging from -80 degrees C. to -100 degrees C. Cell free (CF) virus is prepared by: 1. resuspending the 10,000 x g pellet in F12K; 2. freezing and thawing the resuspended material 3 times; 3. clarifying the freeze-thawed material by centrifugation at 10,000 x g for 30 minutes; and 4. freezing the clarified supernatant (CF virus) at temperatures ranging from -80 degrees C. to -100 degrees C. CF or CA virus was thawed by gentle agitation at 37 degrees C. in a water bath. B. Virus Assay Confluent monolayers of FC3Tg or AKD cells were prepared in 6 cm diameter mammalian cell culture plastic petri dishes (Corning No. 25010). The growth medium used for Fc3Tg cells was MEN + 10% F(i) and the growth medium used for AKD cells was F12K + 15% F(i). Ten fold serial dilutions of virus samples were made by adding 0.5 ml of the virus sample to 4.5 mls of PBS + 2% F(i) in a screw cap tube. The growth medium was removed from a 6 cm culture dish cell monolayer, 1.00 ml of virus sample (undiluted or diluted) was added, and the virus was adsorbed to the monolayer for 2 hours at 35 degrees C. to 38 degrees C. Two or more dishes were used for each sample. The unadsorbed inoculum was removed, and 4 mls of overlay medium was added per 6 cm culture dish. The overlay medium was prepared by mixing equal parts solution A (100 ml 2 x MEM with L-glutamine, GIBCO #320-1935, +4 ml F(i)) and 1% methyl cellulose (4,000 centipoises) in deionized H2O (Fisher M-281 sterilized by autoclaving). After the overlay was added the cultures were incubated at 35 degrees C. to 38 degrees C. in 5% CO(2) in air for at least 48 hours and examined for virus plaques which appeared as either open circular areas in the monolayer with rounded cells at the edge of the open area or as foci of multinucleated syncytial cells. The virus titer in pfu/ml was calculated by multiplying the average number of plaques per dish by the reciprocal of the dilution. The pfu/ml was the value used to determine the amount of virus needed to infect cells at a MOI of approximately 0.1. The pfu/ml in a virus preparation prior to inactivation was used to determine the immunizing dose. C. Inactivation of Cell Free Virus (CF-FVR) Nineteen mls of CF-FVR (1.9 x 10(7) pfu/ml) were mixed with 0.4 ml of hydroxymethyltrioxsalen (HMT; 1 mg/ml in DMSO) and 1.9 ml of sodium ascorbate (0.1 M in H(2)O). The mixture was prepared in 150 cm(2) tissue culture flasks (T-150, Corning No. 25120) that were subsequently deaerated for 2 minutes with pure argon gas. The virus-containing flasks were irradiated for 55 minutes at 4 degrees C. using G.E. BLB fluorescent bulbs at an intensity of 1.5 mW/cm(2). The FVR/HMT/ascorbate mixture was then transferred by pipet into a second T-150 flask, which was deaerated for 2 minutes using pure argon gas. The second T-150 flask was irradiated for an additional 28 minutes at 4 degrees C. under the same long wavelength UV light source. The CF-FVR preparation was stored at -100 degrees C in a REVCO freezer. Subsequently the CF-FVR preparation was thawed and placed into a T-150 flask. The flask was deaerated with pure argon gas for 2 minutes and 8 irradiation was continued as described above for an additional 15 hours and 40 minutes. D. Inactivation of Cell Associated Virus (CA-FVR) Cells from 10 roller bottles (about 1 x 10(8) to 2 x 10(8) cells/roller bottle) were resuspended in 28 mls of cell culture media. Twenty mls of the suspension were placed into a T-150 flask. To this flask was added 2 ml of freshly prepared sterile 0.1 M sodium ascorbate and 0.4 ml HMT (1 mg/ml in DMSO). The flask was deaerated with pure argon gas for 2 minutes, and the flask was irradiated at 4 degrees C. using G.E. BLB fluorescent bulbs at an intensity of 1.5 mW/cm(2) for 75 minutes. The viral suspension was then transferred by pipet from the T-150 flask into a second T-150 flask and again deaerated with pure argon gas for 2 minutes. Irradiation was continued for an additional 95 minutes. The CA-FVR preparation was adjusted to 10% DMSO and the suspension was frozen at -20 degrees C. for 1 hour and then stored at -100 degrees C. in a REVCO freezer. The stored frozen CA-FVR preparation was subsequently thawed, and the cells were pelleted in a clinical centrifuge. The cells were resuspended in 21 mls of serum-free medium to which 2.1 mls of freshly prepared 0.1 M sodium ascorbate and 0.4 ml of HMT (1 mg/ml in DMSO) were added. The sample was transferred by pipet to a T-150 flask, and irradiation was continued for an additional 15 hours and 40 minutes. E. Assessment of Inactivation by Blind Passage Fc3Tg or AKD cells were grown to confluency in 850 cm(2) roller bottles using standard cell culture procedures as described above. The culture medium was removed from the roller bottle, and 2.0 mls of the inactivated virus preparation, mixed with 18 mls of medium containing 2% F(i), were adsorbed to the roller bottle cell monolayer for 60 minutes at 35 degrees C. to 38 degrees C. with rotation at 1 to 5 rpm. After adsorption, the inoculum was removed and 150 ml of maintenance medium (MEN or F12K with 2% F(i)) added. The roller bottle culture was then incubated at 35 degrees C. to 38 degrees C. for 7 days with daily observation for viral CPE (see plaque assay above for description of CPE). The roller bottle culture received a 100% medium change after 3 to 5 days. If no CPE was observed during the first roller bottle passage, the cell monolayer was scraped into the maintenance medium which was then decanted into a centrifuge bottle. The cells were pelleted by centrifugation at room temperature at 1,000 x g for 15 minutes, resuspended in 20 ml of fresh maintenance medium, and passed to a new confluent roller bottle culture of Fc3Tg or AKD cells as described above. The second roller bottle blind passage was observed for 7 days and fed once on day 3 to 5. If no CPE was observed during the second roller bottle blind passage, a third roller bottle blind passage was performed. If no CPE was observed by the end of the third roller bottle passage, the virus preparation was considered inactive. F. Administration Procedure Photochemically inactivated FVR is inoculated via syringe into cats by either single or multiple routes, including but not limited intravenously (IV), subcutaneously (SQ), intramuscularly (IM), or intraperitoneally (IP). The vaccine is administered in various volumes (0.5 to 3.0 ml) and in various concentrations (10(6) to 10(8) pfu; either CF, CA or in combination). In the following examples the vaccine was administered in combination 49 4,791,062 9 with aluminum hydroxide as an immunologic adjuvant. The number of injections and their temporal spacing was as set forth in each example. RESULTS A. Inoculation with CF-FVR The experimental group consisted of four specific pathogen free kittens (2 males, 2 females) four months old (Liberty Laboratories, Liberty Corner, N.J.). The control group consisted of two similar female kittens. The experimental group was inoculated IM with 3x10(7) pfu (3 mls) of HMT inactivated CF-FVR on days 0 and 21, and again inoculated with 3x10(7) pfu HMT inactivated with an equal amount of 2% aluminum hydroxide [A1(OH)(3)] adjuvant on day 61. Controls were vaccinated at eight weeks and at thirteen weeks of age with a commercial FVR vaccine using the manufacturer's recommended procedure. Serum samples were collected weekly and tested for anti-FVR neutralizing antibodies. Following live virus challenge (106 pfu intranasally and intraconjunctivally), a numerical scoring system (Table 1) was used to assess the clinical response to both experimental and control cats.
TABLE 1 - ------------------------------------------------------------------------------- Scoring System for Clinical Effects of Herpesvirus Challenge in Cats ----------------------------------------- FACTOR DEGREE SCORE - ------------------------------------------------------------------------------- Fever 101 to 102 degrees F. 0 102 to 103 1 103 to 104 3 greater than 104 5 Depression slight 1 moderate 3 severe 5 Sneezing occasional 1 moderate 3 paroxysmal 5 Lacrimation serous 1 mucoid 3 purulent 5 Nasal Discharge serous 1 mucoid 3 purulent 5 Appetite normal; eats all food 0 fair; eats more than 1/2 of food 1 poor; eats less than 1/2 of food 3 none; eats nothing 5 - -------------------------------------------------------------------------------
Three of four experimental cats developed serum neutralizing anti-FVR antibody (SN) titers of 1:2 that were detected between day 42 and day 58. Following the third immunization (day 61), four of four experimental cats had SN titers of 1:4 (day 80). Baseline SN antibody titers on the experimental cats were negative. The control cats did not develop detectable SN antibody titers during the pre-challenge period. All cats were exposed to 10(6) pfu of live FVR by intraconjunctival and intranasal injection on day 91. Each cat was monitored twice daily for the absence, presence and degree of severity of factors given in Table 1. A composite clinical score was derived for each cat after a 15 day observation period. Three of four experimental cats demonstrated mild temperature elevation and serous ocular or nasal discharge along with mild intermittent depression and appetite suppression. Their composite scores were 39, 42, and 35 respectively for the 15 day observation period. The fourth experimental cat was more severely 10 affected (composite score = 84) by moderate, but transient, sneezing and mucoid nasal discharge. Both control cats were severely affected by live virus challenge. Severe purulent nasal and ocular discharge and lack of appetite were apparent. The control cats had composite scores of 133 and 253. Three weeks following live FVR challenge, all cats were tested for SN antibody titers against FVR. Three of four experimental cats had SN antibody titers of 1:16 while the fourth cat had a 1:8 titer. One of the control cats had an SN antibody titer of 1:4 while the second control lacked an SN antibody titer against FVR. B. Inoculation with CA-FVR Nine age-matched specific pathogen free kittens, 4 months old (Liberty Laboratories, Liberty Corner, N.J.), were randomly assigned to three experimental groups designated A, B, and C. Group A (controls) was inoculated twice with 1 ml tissue culture fluid and 1 ml aluminum hydroxide adjuvant. Group B was inoculated twice with a commercial FVR vaccine according to the manufacturer's recommendation. Group C was inoculated three times with 10(7) HMT-inactivated CA-FVR in aluminum hydroxide (total volume = 2 ml; 1:1 vaccine to adjuvant). All injections were given IM at three week intervals. Live FVR virus (10(6) pfu intranasally and intraconjunctivally) was given on day 63 and a numerical scoring system (Table 1) was used to assess the kittens' clinical response for a 15 day post-challenge period. Serum samples were collected from all kittens prior to vaccination, prior to the second and third immunizations, prior to live FVR challenge, and at 15 days post-challenge. The sera were utilized to assess neutralizing antibody titers by standard procedures. The control kittens (Group A) maintained SN antibody titers less than 1:2 (negative) throughout the pre-challenge period. Fifteen days following live FVR challenge Group A kittens, uniformly had SN antibody titers of 1:2. Kittens in Group B and C lacked detectable anti-FVR antibody titers pre-immunization, but all kittens in Groups B and C had SN antibody titers of 1:2 or 1:4 after two immunizations. The third immunization in Group C kittens did not significantly alter their SN antibody titers. Following a 15 day post-challenge period, kittens in Groups B and C demonstrated an anamnestic immunologic response, with SN antibody titers ranging from 1:16 to 1:64. Clinically, Group A kittens were severely affected by live FVR challenge, whereas kittens in Groups B and C were significantly protected by their respective vaccines. The composite clinical scores for Group A were 125, 141, and 128 for the 15 day post-challenge period. The composite clinical scores for Group B were 25, 20, and 64, while Group C had composite clinical scores of 21, 15, and 34. The clinical signs evident were characteristic of FVR. From the SN data and clinical scoring, it is evident that kittens immunized with the experimental HMT-inactivated FVR vaccines (cell-free or cell associated) in the above examples were significantly immune to the clinical effects of severe FVR challenge. According to the present invention, furocoumarin-inactivated feline herpesvirus I retains its immunogenicity and is suitable as the immunogenic substance in a vaccine against feline viral rhinotracheitis. The inacti- 50 4,791,062 11 vated virus of the present invention is non-infectious and is safe when administered to a host for vaccination. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims. What is claimed is: 1. A method of making a vaccine useful for inoculation of a feline host susceptible to feline virus rhinotracheitis, said method comprising: (1) inactivating at least one feline Herpesvirus I isolate by (a) adding to said feline Herpesvirus I isolate a small but inactivating effective amount of a furocoumarin; and thereafter (b) exposing said feline Herpesvirus I to ultraviolet light having a wavelength within the range of from about 300 nm to 400 nm and an intensity of from about 0.1 mW/cm(2) to 5 W/cm(2) at a temperature below about 40 degrees C. for a time sufficient to render said virus noninfectious without destroying the characteristic immunologenic response of said feline Herpesvirus I isolate. 2. The method according to claim 1 wherein said furocoumarin is 4'-hydroxymethyl-4,5', 8-trimethylpsoralen. 3. The method of claim 1 wherein said inactivating occurs in the substantial absence of oxygen. 12 4. The method according to claim 1 wherein said virus is inactivated in the presence of a singlet oxygen scavenger. 5. The method according to claim 1 wherein said virus is grown in substantially confluent monolayers of host cells immediately prior to said inactivating procedure. 6. A method of preparing a vaccine useful for inoculation of a feline host susceptible to feline virus rhinotracheitis, said method comprising: subjecting at least one inactivated feline Herpesvirus I isolate to ultraviolet light having a wavelength within the range of from about 300 nm to 400 nm and an intensity of from about 0.1 mW/cm(2) to 5W/cm(2) at a temperature below about 40 degrees C. in the presence of an inactivating furocoumarin for a time sufficient to render said virus noninfectious without destroying its characteristic immunogenic response. 7. The method according to claim 6 wherein said furocoumarin is 4'-hydroxymethyl-4,5', 8-trimethylpsoralen. 8. The method of claim 6 wherein said inactivating occurs in the substantial absence of oxygen. 9. The method according to claim 6 wherein said virus is inactivated in the presence of a singlet oxygen scavenger. 10. The method according to claim 6 wherein said virus is grown in substantially confluent monolayers of host cells immediately prior to said inactivating procedure. * * * * * 51 EXHIBIT F UNITED STATES PATENT [19] [11] PATENT NUMBER: 5,106,619 WIESEHAHN ET AL. [45] DATE OF PATENT: APR. 21, 1992 - -------------------------------------------------------------------------------- [54] PREPARATION OF INACTIVATED VIRAL VACCINES [75] Inventors: GARY P. WIESEHAHN, Alameda; RICHARD P. CREAGAN, Alta Loma; DAVID R. STEVENS, Fremont; RICHARD GILES, Alameda, all of Calif. [73] Assignee: DIAMOND SCIENTIFIC CO., Des Moines, Iowa [21] Appl. No.: 463,081 [22] Filed: JAN. 10, 1990 RELATED U.S. APPLICATION DATE [60] Continuation of Ser. No. 69,117, Jul. 2, 1987, abandoned, which is a division of Ser. No. 785,354, Oct. 7, 1985, Pat. No. 4,693,981, which is a continuation-in-part of Ser. No. 592,661, Mar. 23, 1984, Pat. No. 4,556,556, which is a continuation-in-part of Ser. No. 563,939, Dec. 20, 1983, Pat No. 4,545,987. [51] Int. Cl.(5) .................................... A61K 39/12; C12N 7/04 [52] U.S. Cl. ............................................. 424/89; 424/90; 435/236; 435/238; 546/270 [58] Field of Search ..................................424/89, 90; 435/236, 435/238; 546/270 [56] REFERENCES CITED U.S. PATENT DOCUMENTS 4,036,952 7/1977 Bauer et al. ......................424/89 Primary Examiner -- Johnnie R. Brown Assistant Examiner -- Abdel A. Mohamed Attorney, Agent, or Firm -- Joseph C. Gil; Lyndanne M. Whalen [57] ABSTRACT Vaccines employing inactivated viruses having improved retention of antigenic characteristics are prepared by psoralen-inactivation of the live virus in a non-oxidizing atmosphere. By excluding oxygen and other oxidizing species from the inactivation medium, degradation of the antigen characteristics resulting from irradiation with ultraviolet light is largely prevented. The resulting inactivated viruses are employed in vaccine preparations for the inoculation of susceptible hosts to inhibit viral infection. 12 CLAIMS, NO DRAWINGS 52 5,106,619 1 PREPARATION OF INACTIVATED VIRAL VACCINES This application is a continuation of application Ser. No. 07/069,117, filed Jul. 2, 1987, now abandoned, which is a divisional of Ser. No. 06/785,354, filed Oct. 7, 1985 (U.S. Pat. No. 4,693,981), which is a continuation-in-part of Ser. No. 06/592,661, filed Mar. 23, 1984 (U.S. Pat. No. 4,556,556), which is a continuation-in-part of Ser. No. 06/563,939, filed Dec. 20, 1983 (U.S. Pat. No. 4,545,987). BACKGROUND OF THE INVENTION 1. Field of the Invention The Present invention relates to the preparation of inactivated viral vaccines. More particularly, the invention relates to psoralen inactivation of viral particles under conditions which limit antigenic degradation of the viral particles caused by the inactivation. Vaccination against both bacterial and viral diseases has been one of the major accomplishments of medicine over the past century. While effective vaccines have been developed for a large number of diseases, development of safe and effective vaccines for a number of other diseases remains problematic. The use of inactivated or killed microbial agents as a vaccine, although generally safe, will not always be effective if the immunogenic characteristics of the agent are altered. Indeed, the preferential degradation of certain antigens on the inactivated microorganisms might produce an immune response which allows for an immunopathological response when the host is later challenged with the live microorganism. In contrast, the preparation of live, attenuated microbial agents as a vaccine will often provide improved immunologic reactivity, but increases the risk that the vaccine itself will be infectious, e.g., as a result of reversion, and that the organism will be able to progate and provide a reservoir for future infection. Thus, one must generally choose between improved effectiveness and greater degree of safety when selecting between the viral inactivation and viral attenuation techniques for vaccine preparation. The choice is particularly difficult when the virus is resistant to inactivation and requires highly rigorous inactivation conditions which are likely to degrade the antigenic characteristics. It is therefore desirable to provide improved methods for inactivating viruses, which methods are capable of inactivating even the most resistant viruses under conditions which do not substantially degrade the antigenic structure of the viral particles. In particular, the inactivated viruses should be useful as vaccines and free from adverse side effects at the time of administration as well as upon subsequent challenge with the live virus. 2. Description of the Prior Art The reactivity of psoralen derivatives with viruses has been studied. See, Hearst and Thiry (1977) Nuc. Acids Res. 4:1339-1347; and Talib and Banerjee (1982) Virology 118:430-438. U.S. Pat. Nos. 4,124,598 and 4,196,281 to Hearst et al. suggest the use of psoralen derivatives to inactivate RNA viruses, but include no discussion of the suitability of such inactivated viruses as vaccines. U.S. Pat. No. 4,169,204 to Hearst et al. suggests that psoralens may provide a means for inactivating viruses for the purpose of vaccine production but presents no experimental support for this proposition. 2 European patent application 0 066 886 by Kronenberg teaches the use of psoralen inactivated cells, such as virus-infected mammalian cells, for use as immunological reagents and vaccines. Hanson (1983) in: Medical Virology II, de la Maza and Peterson, eds., Elsevier Biomedical, New York, pp. 45-79, reports studies which have suggested that oxidative photoreactions between psoralens and proteins may occur. SUMMARY OF THE INVENTION The present invention provides for the production of furocoumarin-inactivated viral vaccines under conditions which substantially preserve the antigenic characteristics of the inactivated viral particles. It has been recognized by the inventors herein that the inactivation of viruses by exposure to ultraviolet radiation in the presence of furocoumarin compounds can degrade the antigenic structure of the viral particle. While such degradation can be limited by employing less rigorous inactivation conditions, certain recalcitrant viruses require relatively harsh inactivation conditions in order to assure that all residual infectivity is eliminated. The inactivation condition required to eliminate substantially all infectivity in such recalcitrant viruses can so degrade the viral particle that it is unsuitable for use as the immunogenic substance in a vaccine. Even if the degradation is not so complete, partial degradation of the antigenic characteristics may render the vaccine less effective than would be desirable. That is, the vaccine may require higher concentrations of the inactivated viral particles in each inoculation, and/or the vaccination program may require additional inoculations in order to achieve immunity. According to the present invention, vaccines are prepared by treatment with furocoumarins and long wavelength ultraviolet (UVA) light under conditions which limit the availability of oxygen and other reactive, particularly oxidizing, species. It has been found that such conditions allow for the inactivation of even recalcitrant viral particles without substantial degradation of the antigenic characteristics of those particles. Thus, viruses which have heretofore been resistant to furocoumarin-inactivation may now be inactivated without loss of the desired immunogenicity, and viruses which have previously been successfully inactivated may now be inactivated under conditions which better preserve their antigenic characteristics, making them more efficient immunogenic substances for use in vaccines. DESCRIPTION OF THE SPECIFIC EMBODIMENTS According to the present invention, vaccines useful for the inoculation of mammalian hosts, including both animals and humans, against viral infection are provided. The vaccines are prepared by inactivation of live virus in an inactivation medium containing an amount of an inactivating furocoumarin sufficient to inactivate the virus upon subsequent irradiation with long wavelength ultraviolet radiation. Degradation of the antigenic characteristics of the live virus is reduced or eliminated by limiting the availability of oxygen and other oxidizing species in the inactivation medium. Suitable vaccines may be prepared by combining the inactivated viruses with a physiologically-acceptable carrier, typically an adjuvant, in an appropriate amount to elicit an immune response, e.g., the production of serum neutral- 53 5,106,619 3 izing antibodies, upon subsequent inoculation of the host. The present invention is suitable for producing vaccines to a wide variety of viruses, including human viruses and animal viruses, such as canine, feline, bovine, porcine, equine, and ovine viruses. The method is suitable for inactivating double stranded DNA viruses, single-stranded DNA viruses, double-stranded RNA viruses, and single-stranded RNA viruses, including both enveloped and non-enveloped viruses. The following list is representative of those viruses which may be inactivated by the method of the present invention. - -------------------------------------------------------------------------------- Viruses which may be inactivated --------------------------------------------- Representative Viruses - -------------------------------------------------------------------------------- dsDNA ----- Adenoviruses Adenovirus, canine adenovirus 2 Herpesviruses Herpes simplex viruses, Feline Herpes I Papovaviruses Polyoma, Papilloma Poxviruses Vaccinia ssDNA ----- Parvovirus Canine parvovirus, Feline panleukopenia dsRNA ----- Orbiviruses Bluetongue virus Reoviruses Reovirus ssRNA ----- Calicivirus Feline calicivirus Coronavirus Feline infectious peritonitis Myxovirus Influenza virus Paramyxovirus Measles virus, Mumps virus, Newcastle disease virus, Canine distemper virus, Canine parainfluenza 2 virus Picornavirus Polio virus, Foot and mouth disease virus Retrovirus Feline leukemia virus, Human T-cell lymphoma virus, types I, II and III Rhabdovirus Vesicular stomatitis virus, rabies Togavirus Yellow fever virus, Sindbis virus, Encephalitis virus - -------------------------------------------------------------------------------- Of particular interest are those viruses for which conventional vaccine approaches have been unsuccessful or marginally successful. For such viruses, inactivation procedures which are sufficiently rigorous to assure the total loss of infectivity often result in partial or complete destruction of the antigenic characteristics of the virus. With such loss of antigenic characteristics, the viruses are incapable of eliciting a protective immunity when administered to a susceptible host. While it would be possible to utilize less rigorous inactivation conditions in order to preserve the antigenic integrity of the virus, this approach is not desirable since it can result in incomplete inactivation of the virus. In preparing the subject vaccines, sufficient amounts of the virus to be inactivated may be obtained by growing seed virus in a suitable mammalian cell culture. Seed virus, in turn, may be obtained by isolation from an infected host. Suitable mammalian cell cultures include primary or secondary cultures derived from mammalian tissues or established cell lines such as Vero cells, monkey kidney cells, BHK21 hamster cells, LMTK- cells, and other cells permissive for the desired virus and which may be grown in vitro as monolayer or suspension cultures. The cell cultures are grown to approximately 80% saturation density, and infected with the virus at a low multiplicity of infection (MOI), usually between about 0.05 and 0.005, preferably at about 0.01. 4 After adsorbing the viral inoculum to the cells by incubation for a limited period of time at a temperature in the range from 35 degrees C. to 40 degrees C., an appropriate growth or maintenance medium is added. The cells are further incubated at about the same temperature, in the presence of about 5% carbon dioxide in air, until a sufficient amount of virus has been produced. The growth and maintenance medium will usually be a conventional mammalian cell culture medium, such as Eagle's Minimum Essential Medium or Medium 199, usually supplemented with additives such as broth prepared from dehydrated standard microbial culture media, fetal bovine serum, fetal calf serum, or the like. The furocoumarins useful for inactivation are primarily illustrated by the class of compounds referred to as psoralens, including psoralen and substituted psoralens where the substituents may be alkyl, particularly having from one to three carbon atoms, e.g., methyl; alkoxy, particularly having from one to three carbon atoms, e.g., methoxy; and substituted alkyl having from one to six, more usually from one to three carbon atoms and from one to two heteroatoms, which may be oxy, particularly hydroxy or alkoxy having from one to three carbon atoms, e.g., hydroxy methyl and methoxy methyl, or amino, including mono- and dialkyl amino or aminoalkyl, having a total of from zero to six carbon atoms, e.g., aminomethyl. There will be from 1 to 5, usually from 2 to 4 substituents, which will normally be at the 4, 5, 8, 4' and 5' positions, particularly at the 4' position. Illustrative compounds include 5-methoxypsoralen; 8-methoxypsoralen (8-MOP); 4,5',8-trimethylpsoralen (TMP); 4'-hydroxymethyl-4,5',8-trimethyl- psoralen (HMT); 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT); 4-methylpsoralen; 4,4'-dimethylpsoralen; 4,5'-dimethylpsoralen; 4',8-dimethylpsoralen; and 4'-methoxymethyl-4,5',8-trimethylpsoralen. Of particular interest are AMT and 8-MOP. The furocoumarins may be used individually or in combination. Each of the furocoumarins may be present in amounts ranging from about 0.01 (greek mu)/ml to 1 mg/ml, preferably from about 0.5 (greek mu)/ml to 100 (greek mu)/ml, there not being less than about 1 (greek mu)/ml nor more than about 1 mg/ml or furocoumarins. In carrying out the invention the furocoumarin(s), in an appropriate solvent which is substantially inert and sufficiently non-polar to allow for dissolution of the furocoumarin(s), are combined with the viral suspension, conveniently a viral suspension in an aqueous buffered medium, such as used for storage. The amount of virus will generally be about 1 x 10(6) to 10(11), more usually about 1 x 10(7) to 10(9) and preferably about 1 x 10(8) to 5 x 10(8) pfu/ml. The furocoumarin(s) will be at a concentration of about 0.001 mg/ml to 0.5 mg/ml, more usually about 0.05 mg/ml to 0.2 mg/ml. The amount of solvent which is usually to dissolve the furocoumarin will be sufficiently small so as to readily dissolve in the aqueous viral suspension. Although viral inactivation according to the present invention will normally be carried out in an inactivation medium as just described, in some cases it may be desirable to introduce furocoumarins to the virus by addition to a cell culture medium in which the virus is grown. The inactivation is then carried out by separating the live viral particles from the culture medium, and exposure of the particles to ultraviolet light in an inactivation medium which may or may not contain additional furocoumarins. This method of inactivation is useful 54 5,106,619 5 where the virus is resistant to inactivation when the furocoumarin is added to the inactivation medium only. When employing furocoumarins with limited aqueous solubility, typically below about 50 (greek mu)g/ml, it has been found useful to add an organic solvent, such as dimethyl sulfoxide (DMSO) ethanol, glycerol, polyethylene glycol (PEG) or polypropylene glycol, to the aqueous treatment solution. Such furocoumarins having limited solubility include 8-MOP, TMP, and psoralen. By adding small amounts of such organic solvents to the aqueous composition, typically in the range of about 1 to 25% by weight, more typically from about 2 to 10% by weight, the solubility of the furocoumarin can be increased to about 200 (greek mu)g/ml, or higher. Such increased furocoumarin concentration may permit the use of shorter irradiation times. Also, inactivation of particularly recalcitrant microorganisms may be facilitated without having to increase the length or intensity of ultraviolet exposure, and the addition of an organic solvent may be necessary for inactivation of some viruses with particular furocoumarins. The ability to employ less rigorous inactivation conditions is of great benefit in preserving the antigenicity of the virus during inactivation. At times, it may be desirable to employ organic solvents, particularly DMSO, with all furocoumarins regardless of solubility. For some microorganisms, particularly viruses having tight capsids, the addition of the organic solvent may increase the permeability of the outer coat or membrane of the microorganism. Such increase in permeability would facilitate penetration by the furocoumarins and enhances the inactivation of the microorganism. The furocoumarin may be added to the viral suspension in a single addition or in multiple additions, where the virus is irradiated between additions, or may be added continuously during the entire treatment period, or a portion thereof. Usually, the number of additions will be from about 1 to 50, more usually from about 10 to 40, and preferably from about 2 to 4. The total amount of furocoumarin which will be added will be sufficient to provide a concentration of at least about 0.01 mg/ml to about 1 mg/ml, usually not more than about 0.75 mg/ml and preferably not more than about 0.5 mg/ml. Since a substantial proportion of the furocoumarin will have reacted with the nucleic acid between additions, the total concentration of furocoumarin in solution will generally not exceed about 0.1 mg/ml. In cases where the furocoumarin(s) employed are particularly unstable, it may be beneficial to add the furocoumarin solution continuously during the irradiation procedure. In order to preserve the antigenic characteristics of the virus, irradiation is carried out in the substantial absence of oxygen and other oxidizing species. This is particularly important when employing psoralens that generate more singlet oxygen on a molar basis. For example, AMT generates more singlet oxygen than 8-MOP. Conveniently, oxygen and other gases may be removed from the inactivation medium by maintaining the medium in a non-oxidizing gas atmosphere, e.g., hydrogen, nigrogen, argon, helium, neon, carbon dioxide, and the like. The inactivation medium may be held in an enclosed vessel, and the space above the liquid medium surface filled with the non-oxidizing gas. Oxidizing species initially in the medium will be exchanged for the non-oxidizing gases according to gas-liquid equilibrium principles. Preferably, the space above the inac- 6 tivation medium will be flushed with non-oxidizing gas to remove the oxidizing species and further lower their equilibrium concentration in the liquid medium. Depending on the volume of the inactivation medium, the flushing should be continued for at least 1 minute, preferably at least 2 minutes, usually being in the range from about 3 to 30 minutes. Flushing may be continued during the irradiation period, but need not be so long as the oxidizing species have been substantially removed and the vessel remains sealed to prevent the intrusion of air. Optionally, a singlet oxygen scavenger may be added to the inactivation medium prior to irradiation to further prevent interaction of oxygen with the furocoumarin and the virus. Suitable oxygen scavengers include ascorbic acid, dithioerythritol, sodium thionate, glutathione, and the like. The scavenger will be present at a concentration sufficient to block active oxygen species, usually being between 0.001M and 0.5M, more usually being between about 0.005M and 0.02M, where the addition may be single, multiple, or continuous additions. The concentration of dissolved oxygen may be reduced through the use of enzyme systems, either in solution or immobilized on a solid substrate. Suitable enzyme systems include glucose oxidase or catalase in the presence of glucose and ascorbic acid catalase in the presence of ascorbate. Such enzyme system may be employed alone or together with the other methods for oxygen reduction discussed above. The total time for the irradiation will vary depending upon the light intensity, the concentration of the furocoumarin, the concentration of the virus, and the manner of irradiation of the virus, where the intensity of the irradiation may vary in the medium. The time of irradiation necessary for inactivation will be inversely proportional to the light intensity. The total time will usually be at least about 2 hrs. and not more than about 60 hrs., generally ranging from about 10 hrs. to 50 hrs. The times between additions of furocoumarin, where the furocoumarin is added incrementally, will generally vary from about 1 hour to 24 hrs., more usually from about 2 hrs. to 20 hrs. The light which is employed will generally have a wavelength in the range from about 300 nm to 400 nm. Usually, an ultraviolet light source will be employed together with a filter for removing UVB light. The intensity will generally range from about 0.1 mW/cm(2) to about 5 W/cm(2), although in some cases, it may be much higher. The temperature for the irradiation is preferably under 25 degrees C., more preferably under 20 degrees C. and will generally range from about -10 degrees C. to 15 degrees C., more usually from about 0 degrees C. to 10 degrees C. During irradiation, the medium may be maintained still, stirred or circulated and may be either continuously irradiated or be subject to alternating periods of irradiation and non-irradiation. The circulation may be in a closed loop system or in a single pass system ensuring that all of the sample has been exposed to irradiation. It may be desirable to remove the unexpended furocoumarin and/or its photobreakdown products from the irradiation mixture. This can be readily accomplished by one of several standard laboratory procedures such as dialysis across an appropriately sized membrane or through an appropriately sized hollow fiber system after completion of the irradiation. Alter- 55 5,106,619 7 natively, one could use affinity methods for one or more of the low molecular weight materials to be removed. The inactivated virus may then be formulated in a variety of ways for use as a vaccine. The concentration of the virus will generally be from about 10(6) to 10(9) pfu/ml, as determined prior to inactivation, with a total dosage of at least 10(5) pfu/dose, usually at least 10(6) pfu/dose, preferably at least 10(7) pfu/dose. The total dosage will usually be at or near about 10(9) pfu/dose, more usually being about 10(8) pfu/dose. The vaccine may include cells or may be cell-free. It may be an inert physiologically acceptable medium, such as ionized water, phosphate-buffered saline, saline, or the like, or may be administered in combination with a physiologically acceptable immunologic adjuvant, including but not limited to mineral oils, vegetable oils, mineral salts and immunopotentiators, such as muramyl dipeptide. The vaccine may be administered subcutaneously, intramuscularly, intraperitoneally, orally, or nasally. Usually, a specific dosage at a specific site will range from about 0.1 ml to 4 ml, where the total dosage will range from about 0.5 ml to 8 ml. The number of injections and their temporal spacing may be highly variable, but usually 1 to 3 injections at 1, 2 or 3 week intervals are effective. The following examples are offered by way of illustration, not by way of limitation. EXPERIMENTAL Materials and Methods A. Virus Growth and Tissue Culture Hamster cells [BHK-21(C-13), American Type Culture Collection (ATCC), CCL 10] were grown as monolayers in plastic cell culture vessels in Eagle's Minimum Essential Medium (MEM) with Earle's salts and non-essential amino acids (MEN) supplemented with 10% heat inactivated calf serum (C(i)) and 10% tryptose phosphate broth (Tp, e.g., Difco 0060). Cell cultures were used to produce live vesicular stomatitis virus, New Jersey serotype (VSV-NJ) from master seed virus originally obtained from the ATCC (VR-159), and live bluetongue virus (BTV) from master seed virus originally obtained from Dr. T. L. Barber, USDA, Denver, Colo. Cells were grown in culture vessels to 80% to 100% confluency (approximately 2 x 10(5) cells per cm(2) of growth surface area) using standard mammalian cell culture techniques. Corning plastic roller bottles (Corning No. 25140-850) with a growth surface area of 850 cm(2) containing 100 ml of MEN supplemented with 10% C(i) and 10% Tp and 1x10(8) to 2x10(8) CCL 10 cells/bottle were used for virus production. The cell cultures were initiated by seeding approximately 1x10(6) to 5x10(7) cells into 100 mls of growth medium in a roller bottle containing 5% CO(2) in air on a roller bottle rotator at 1 to 5 rpm at 35 degrees C. to 38 degrees C. The cultures were grown to 80% to 100% confluency over a six to fourteen day period with a medium change every two to four days. When the monolayers reached 80% to 100% confluency, the culture medium was removed and the monolayer was infected with approximately 1x10(6) to 2x10(6) plaque forming units (pfu) of VSV or BTV in 20 mls of MEN, with 2% heat-inactivated fetal bovine serum (F(i)) added for BTV. The multiplicity of infection (MOI) was approximately 0.01. The MOI may range from 0.001 pfu/cell to 0.033 pfu/cell. The virus inoculum was absorbed to the cells by incubation at 35 degrees C. to 38 degrees C. for one hour at 1 to 5 rpm. One hundred mls of MEN 8 containing 10% YELP supplement (v/v) for VSV, or 10% C(i) and 10% Tp for BTV, was added per roller bottle. YELP supplement contains: yeast extract BBL 11929, 5 g/liter; lactalbumin hydrolysate GIBCO 670-1800, 25 g/liter; and Bacto-Peptone (Difco 0118), 50 g/liter. The post-infection incubation was carried out at 35 degrees C. to 38 degrees C. in 5% CO(2)/95% air with rotation. Sixteen to forty-eight hours post-infection, VSV cytopathic effect (CPE) was evident, while BTV CPE became apparent from 2 to 4 days post infection. The CPE was characterized by cell rounding, cell detachment, and cell degeneration. When visual or microscopic examination indicated that at least 50% of the cell monolayer exhibited CPE, the contents of the roller bottle were swirled to remove loosely attached materials from the roller bottle walls. The harvest material was decanted from the roller bottles into centrifuge bottles. The crude VSV harvest was clarified by centrifugation at 500 to 1000 x g for 20 minutes, at 4 degrees C. The BTV harvest was centrifuged at 2,000xg for 60 minutes at 4 degrees C. The clarified VSV preparations were concentrated by ultrafiltration using a Pellicon cassette system (Millipore XX42ASY60) with a cassette having a nominal exclusion limit of 10(5) daltons (Millipore PTHK 000C5). The Pellicon cassette system was sterilized by filling the assembled unit with 1N NaOH and incubating the unit 12 to 24 hours at room temperature. The NaOH solution was pumped out of the cassette system and the system was flushed with two to four liters of sterile H(2)O. The assembly and operation of the Pellicon system were in accordance with the instructions furnished by the manufacturer. All steps in the concentration were performed aseptically. The clarified VSV was concentrated 15 to 40 fold, dimethylsulfoxide (Sigma D-5879) added to a final concentration of 7.5% v/v, and suitable aliquots of the virus stored frozen at - -80 degrees C. to -100 degrees C. For BTV, the pellet resulting from centrifugation was resuspended aseptically in 8 ml of 2 mM Tris-HCl, pH 8.8, for each original roller bottle. The suspension was mixed vigorously on a vortex mixer, and/or sonicated at 4 degrees C. for 1 min., and centrifuged at 1,400 x g for 30 min. at 4 degrees C. The virus-containing supernatant was collected and the pellet was extracted twice more with 8 ml/roller bottle aliquots of 2 mM Tris-HCl, 8.8. The virus-containing supernatants were pooled and clarified by centrifugation at 4,000 x g for 30 min. at 4 degrees C. The clarified supernatant was stored at 4 degrees C. Feline herpes I virus (FVR, the infective agent of feline viral rhinotracheitis) was grown as follows. Cat cell lines AKD (ATCC CCL150) or Fc3Tg (ATCC CCL176) were grown as monolayers in plastic cell culture vessels in a standard defined culture medium consisting of MEN; F12K; MEM; or alpha MEM. Medium was supplemented with 2% to 15% inactivated fetal calf serum (F(i)) or 2% to 20% YELP. Cell cultures were used to produce live Feline Herpes I virus from master seed virus derived from Feline Herpes I virus (ATCC VR636). Cells were grown in culture vessels to 80% to 100% confluency (approximately 1x10(5) to 2x10(5) cells per cm2 of growth surface area) using standard mammalian cell culture techniques as follows. Corning plastic roller bottles containing 50 to 100 ml of MEN supplemented with 10% F(i) and 1x10(8) to 2x10(8) AKD or Fc3Tg cells/bottles were used for Feline Herpes I virus production. The cell cultures were 56 5,106,619 9 initiated by seeding approximately 1 x 10(6) to 5 x 10(6) cells into 50 to 100 mls of growth medium in a roller bottle containing about 5% CO(2) in air and incubating the roller bottle on a roller bottle rotator at 1 to 5 rpm at 35 degrees C. to 38 degrees C. The cultures were grown to 80% to 100% confluency over a 7 to 14 day period with a 100% medium change every 3 to 5 days. When the monolayers were 80% to 100% confluent, the culture medium was removed and the monolayer was washed with 20 to 50 mls of phosphate buffered saline (PBS) pH 7.2 to 7.4 (NaCl 8 g + KCl 0.2 g + Na(2)HPO(4) 1.14 g + KH(2)PO(4)0.2 g). The PBS wash was discarded, and the roller bottle was infected by the addition of approximately 1 x 10(7) to 2 x 10(7) plaque forming units (pfu) of Feline Herpes I virus in 10 mls of PBS containing 2% F(i). The multiplicity of infection (MOI) was approximately 0.1. The virus inoculum wase adsorbed to the cells by incubation at 35 degrees C. to 38 degrees C. for one hour at 1 to 5 rpm. The inoculation fluid was removed and 50 mls of MEN containing 10% F(i) was added per roller bottle. The post-infection incubation was at 35 degrees C. to 38 degrees C. in 5% CO(2) in air with rotation. Herpes virus cytopathic effect (CPE) was evident forty to forty-eight hours post-infection. The CPE was characterized by cell rounding, cell detachment, and cell degeneration. The contents of the roller bottle were swirled 48 hours post-infection to remove loosely attached materials from the roller bottle walls, and the contents of the roller bottles were decanted into centrifuge bottles. The virus, cells, and cell debris were pelleted by centrifugation at 10,000 x g for 30 minutes. Cell associated (CA) Feline Herpes I virus was prepared by: 1. resuspending the 10,000 x g pellet in approximately 5 ml of a resuspension medium containing 80 parts F12K, 10 parts F(i), and 10 parts dimethylsulfoxide (DMSO) for each original roller bottle; 2. freezing the resuspended CA virus at -20 degrees C. for 1.5 to 2 hours; and 3. transferring the CA virus frozen at -20 degrees C. to temperatures ranging from -80 degrees C. to -100 degrees C. Cell free (CF) Feline Herpes I virus was prepared by: 1. resuspending the 10,000 x g pellet in F12K; 2. freezing and thawing the resuspended material 3 times; 3. clarifying the freeze-thawed material by centrifugation at 10,000 x g for 30 minutes; and 4. freezing the clarified supernatant (CF virus) at temperatures ranging from -80 degrees C. to -100 degrees C. CF or CA virus was thawed by gentle agitation at 37 degrees C. in a water bath. B. Virus Assay Confluent monolayers of LMTK-, Vero (ATCC CCL 81), Fc3Tg, or AKD cells were prepared in 6 cm diameter mammalian cell culture plastic petri dishes (Corning #25010) or other convenient cell culture vessels. The growth medium used for LMTK- cells was alpha ME (alpha modified Eagles Minimum Essential Medium, Earle's Salts) + 10% F(i). The growth medium used for Vero cells was MEN + 5% F(i). The growth medium used for Fc3Tg cells was MEN + 10% F(i), and the growth medium used for AKD cells was F12K + 15% F(i) (VSV and BTV were titered on LMTK- or Vero cells. Feline Herpes I was titered on Fc3Tg or AKD cells). Ten fold serial dilutions of virus samples were made by adding 0.5 ml of the virus sample 10 to 4.5 mls of diluent (phosphate buffered saline, pH 7.2 to 7.4, plus 2% F(i)) in a screw cap tube. The growth medium was removed from a 6 cm culture dish cell monolayer, 0.1 ml of virus sample (undiluted or diluted) was added, and the virus was adsorbed to the monolayer for 1 to 2 hours at 35 degrees C. to 38 degrees C. Two or more monolayers were used for each sample. Five ml of overlay medium was added per 6 cm culture dish, except for Feline Herpes I, where the unadsorbed inoculum was removed, and 4 mls of overlay medium was added per 6 cm culture dish. The overlay medium for BTV or VSV was prepared by mixing equal parts of solution A (100 ml 2X MEM with L-glutamine, GIBCO #320-1935, + 10 ml F(i)) and 1.8% to 2% Noble Agar (Difco 0142) in deionized H(2)O at 44 degrees C. to 45 degrees C. The overlay medium for Feline Herpes I was prepared by mixing equal parts solution A and 1% methyl cellulose (4,000 centriposes) in deionized H(2)O (Fisher M-281 sterilized by autoclaving). The virus infected cultures were incubated at 35 degrees C. to 38 degrees C. in 5% CO(2) in air. Twenty-four hours before plaques were counted, a second overlay containing Neutral Red at a final concentration of 0.005% was added. Plaques were counted on day 2 or day 3 post-infection for VSV, on day 2 to 4 for FVR and on day 6 or 7 for BTV. The virus titer in pfu/ml was calculated by multiplying the average number of plaques per dish by the reciprocal of the dilution. The pfu/ml was the value used to determine the amount of virus needed to infect cells at a MOI of approximately 0.01. The pfu/ml in a virus preparation prior to inactivation was used to determine the immunizing dose. C. Virus Inactivation 1. VSV Inactivation The thawed stock of VSV was pipetted into sterile T-150 tissue culture flasks (nominally 25 ml into each of four flasks). To each flask was added 0.25 ml of 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT) stock solution (stock solution is 1 mg/ml AMT dissolved in sterile, deionized water). Each flask was allowed to equilibrate in an argon atmosphere for at least 10 minutes. After equilibration, a stream of argon gas was directed into each flask for at least two minutes. The flasks were then tightly capped and placed under a long wavelength ultraviolet (320 nm to 400 nm) light source (GE BLB fluorescent bulbs) at a temperature between 0 degrees C. and 20 degrees C. for approximately 11 hours. The incident light intensity was approximately 1 mW/cm(2) (measured by a J-221 long wavelength UV meter). After the irradiation was completed, the flasks were removed from the light source and an additional 0.25 ml of AMT stock solution was mixed into each flask. The contents of each flask were pipetted into new, sterile T-150 flasks, and the flasks were again flushed with argon and irradiated for an additional 11 hours. This procedure was repeated three more times until five additions (a total of approx. 50 (greek mu) g/ml of AMT had been performed, the virus sample had been irradiated for at least 55 hours, and at least four flask changed has been performed. After all of the irradiations had been completed, the contents of the flasks were aseptically transferred to a common sterile container and stored at - -85 degrees C. 2. BTV Inactivation Twenty-five ml of BTV serotype 11 (1.5x10(8) pfu/ml) was mixed with 0.25 ml of 4'-aminomethyl 4,5',8-trimethylpsoralen (AMT; 1 mg/ml in DMSO). 57 5,106,619 11 The mixture was placed in a 150 cm(2) tissue culture flask (T-150; Corning #251201). The viral suspension in the flask was placed in an argon atmosphere for 10 min., and a stream of argon gas was then blown over the viral suspension for an additional 2 min. The flask was tightly capped and the suspension irradiated for 3.25 hrs. at 4 degrees C. using GE BLB fluorescent bulbs at an intensity of 1.5 mW/cm(2). An additional 0.25 ml of AMT was then added to the viral suspension, the suspension transferred by pipette to a new T-150 flask, and the solution again flushed with argon. The flask was irradiated for an additional 14.75 hours at 4 degrees C. under the same long wavelength UV light source. After this irradiation an additional 0.25 ml of AMT solution was added to the suspension, and it was again transferred to a new T-150 flask. The solution was flushed with argon as before and irradiated for an additional 5.5 hrs. at 4 degrees C. The inactivated BTV was stored at 4 degrees C. 3. Feline Herpes I Inactivation a. Cell Free Virus Nineteen mls of CF-FVR (1.9 x 10(7) pfu/ml) were mixed with 0.4 ml of hydroxymethyltrioxsalen (HMT; 1 mg/ml in DMSO) and 1.9 ml of sodium ascorbate (0.1M in H(2)O). The mixture was prepared in 150 cm(2) tissue culture flasks (T-150, Corning No. 25120) that were subsequently deaerated for 2 minutes with pure argon gas. The virus-containing flasks were irradiated for 55 minutes at 4 degrees C. using G.E. BLB fluorescent bulbs at an intensity of 1.5 mW/cm(2). The FVR/HMT/ascorbate mixture was then transferred by pipet into a second T-150 flask, which was deaerated for 2 minutes using pure argon gas. The second T-150 flask was irradiated for an additional 28 minutes at 4 degrees C. under the same long wavelength UV light source. The CF-FVR preparation was stored at -100 degrees C. in a REVCO freezer. Subsequently the CF-FVR preparation was thawed and placed into a T-150 flask. The flask was deaerated with pure argon gas for 2 minutes and irradiation was continued as described above for an additional 15 hours and 40 minutes. b. Cell Associated Virus Cells from 10 roller bottles (about 1 x 10(8) to 2 x 10(8) cells/roller bottle) were resuspended in 28 mls of cell culture media. Twenty mls of the suspension were placed into a T-150 flask. To this flask was added 2 ml of freshly prepared sterile 0.1M sodium ascorbate and 0.4 ml HMT (1 mg/ml in DMSO). The flask was deaerated with pure argon gas for 2 minutes, and the flask was irradiated at 4 degrees C. using G.E. BLB fluorescent bulbs at an intensity of 1.5 mW/cm(2) for 75 minutes. The viral suspension was then transferred by pipet from the T-150 flask into a second T-150 flask and again deaerated with pure argon gas for 2 minutes. Irradiation was continued for an additional 95 minutes. The CA-FVR preparation was adjusted to 10% DMSO and the suspension was frozen at -20 degrees C. for 1 hour and then stored at -100 degrees C. in a REVCO freezer. The stored frozen CA-FVR preparation was subsequently thawed, and the cells were pelleted in a clinical centrifuge. The cells were resuspended in 21 mls of serum-free medium to which 2.1 mls of freshly prepared 0.1M sodium ascorbate and 0.4 ml of HMT (1 mg/ml in DMSO) were added. The sample was transferred by pipet to a T-150 flask, and irradiation was continued for an additional 15 hours and 40 minutes. 12 Results A. Bluetongue Virus 1. Assessment of Inactivation by Blind Passage CCL 10 cells were grown to confluency in 850 cm(2) roller bottles using standard cell culture procedures, as described above. The culture medium was removed from the roller bottle, and 2.0 ml of the inactivated virus preparation mixed with 18 ml of medium containing 2% F(i) was adsorbed to the roller bottle cell monolayer for 60 min. at 35 degrees C. to 38 degrees C. with rotation at 1 to 5 rpm. After adsorption, the residual unabsorbed inoculum was removed, and 100 ml of growth medium (MEN with 10% C(i) and 10% Tp) was added and the roller bottle culture incubated at 35 degrees C. to 38 degrees C. for 7 days with daily observation for viral CPE. The roller bottle culture received a 100% medium change every 2 to 3 days. If no CPE was observed during the first roller bottle passage, the cell monolayer was chilled at 4 degrees C. for 12 to 24 hrs. The cells were scraped into the medium which was then decanted into a centrifuge bottle. The cells were pelleted by centrifugation at 4 degrees C. at 2,000 x g for 30 min. and resuspended in 2.0 ml or 2 mM Tris-HCl (pH 8.8) by vigorous mixing using a vortex mixer. The resuspended material was centrifuged at 2,000 x g for 20 min. at 4 degrees C. The supernatant was added to 18 ml of growth medium containing 2% F(i) and used to infect a new confluent roller bottle culture of CCL 10 cells, as described immediately above. The second roller bottle blind passage was observed for 7 days and fed every 2 to 3 days. If no CPE was observed during the second roller bottle blind passage, a third roller bottle blind passage was performed. If no CPE had been observed by the end of the third roller bottle blind passage the virus preparation was considered inactivated and suitable for in vivo testing. 2. Immunization of Rabbits with Psoralen-inactivated BTV Vaccine a. Example 1 Four New Zealand white rabbits were randomly assigned to 2 groups, designated A and B. Both groups were given 4 immunizations at two week intervals. The first immunization consisted of 1 ml of vaccine (10(8) pfu BTV serotype 11) and 1 ml of Freund's Complete Adjuvant. The second through fourth immunizations utilized 1 ml of vaccine (10(8) pfu BTV serotype 11) and 1 ml of Freund's Incomplete Adjuvant. All immunizations were given intramuscularly (IM). The vaccine given to Group A (Vaccine #1) was inactivated with AMT-UVA in the presence of 0.01M ascorbic acid. Vaccine #1 was dialyzed for 12 hours against 2 mM Tris, pH 8.6. The vaccine given to Group B (Vaccine #2) was inactivated with AMT-UVA without ascorbic acid and sonicated three times (2 minutes each time) using a cup horn probe (Heat Systems Model 431A) at a power setting of 3 (Heat Systems Model W220). Both Vaccine #1 and Vaccine #2 were deemed inactivated since no live virus was detected during blind passage. Inactivated vaccine was also tested for safety by chicken embryo inoculation. Egg deaths attributable to live virus were not encountered. Both rabbit groups were bled via auricular venipuncture one week following the second, third, and fourth immunizations. Serum from each rabbit was pooled with that of its groupmate, and the pooled sera were tested for anti-BTV antibodies by two standard serologic assays, serum neutralization (Jochim and Jones Am. J. Vet. Res. (1976) 37:1345-1347) and agar 58 5,106,619 13 gel precipitation (Jochim et al., Am. Assoc. Vet. Lab. Diag., 22nd Proceed.: 463-471, 1979). Pre-immunization rabbit serum was used as the negative control; BTV immune sheep serum was used as the positive control for both immunologic procedures. Pooled sera from Groups A and B reduced the number of viral plaques (serum neutralization) greater than eighty percent (arbitrarily selected end point) when the sera were diluted 1:40, which was the highest dilution examined. Negative and positive control sera behaved as expected.
TABLE 1 - -------------------------------------------------------------- Serum Neutralization Data From Rabbits Vaccinated with AMT-UVA inactivated Bluetongue Virus Vaccines. --------------------------------------- Titer* ------------------------------------- Group 1 5 40 - ----- ------ ------ ------ A + + + B + + + Normal Rabbit Serum - - - BTV-Immune Sheep Serum + + + - - -------------------------------------------------------------- *Reciprocal of serum dilution neutralizing 80 percent of BTV plaque activity on BHK cells. The data are from the post-second immunization serum samples.
Pooled post-immunization sera from Groups A and B precipitated BTV antigen in immunodiffusion plates when tested at dilutions up to 1:16. Normal rabbit serum did not precipitate the standard BTV antigen. BTV-immune sheep serum did precipitate the BTV antigen, but not at dilutions greater than 1:2. Of the two immunologic procedures utilized, serum neutralization is considered predictive for immunity to live BTV challenge in the target species. b. Example 2 Twelve New Zealand white rabbits were randomly assigned to six groups, A-F, two rabbits per group. An additional four rabbits were assigned to Group G. These sixteen rabbits were vaccinated twice subcutaneously with the AMT-UVA inactivated Bluetongue virus vaccines described in Table 2. Preinactivation titer was approximately 10(8) for each serotype. The vaccines were formulated with 20% (v/v) aluminum hydroxide adjuvant, and were given with a three week interval between the first and second inoculations. The sixteen rabbits were bled by auricular venipuncture on days 0, 14, and 35. Each serum was heat-inactivated and tested against BTV serotypes 10, 11, 13 and 17 for serum neutralizing antibody. All vaccinated rabbits developed SN titers against the homologous vaccine serotypes (Table 3). These data demonstrated the immunopotency of a multivalent AMT-UVA inactivated Bluetongue virus vaccine.
TABLE 2 --------------------------------------- Serotype Composition of Inactivated Bluetongue Virus Vaccines Tested in Rabbits --------------------------------------- BTV Serotype Group Rabbit# Composition - ------- --------- -------------- A 1,2 10 B 3,4 11 C 5,6 13 D 7,8 17 E 9,10 11,17 F 11,12 10,11,17 G 13,14,15,16 10,11,13,17 - ------------------------------------------------
14
TABLE 3 - --------------------------------------------------- Serum Neutralizing Data From Rabbits Vaccinated with AMT-UVA Single and Multi-Serotype Bluetongue Virus Vaccines - --------------------------------------------------- SN Titer* Against: --------------------------------------- Group Rabbit BTV-10 BTV-11 BTV-13 BTV-17 - ----- ------- ------ ------ ------ ------ 1 1:160 1:10 1:10 1:10 A 2 1:320 1:10 1:10 1:10 3 1:10 1:320 1:10 1:10 B 4 1:10 1:80 1:10 1:10 5 1:20 1:20 1:160 1:10 C 6 1:20 1:10 1:40 1:10 7 1:10 1:10 1:10 1:320 D 8 1:10 1:10 1:10 1:320 9 1:20 1:160 1:20 1:160 E 10 1:20 1:160 1:20 1:160 11 1:160 1:160 1:20 1:160 F 12 1:40 1:40 1:20 1:80 13 1:160 1:160 1:80 1:160 14 1:160 1:160 1:80 1:160 G 15 1:160 1:160 1:40 1:160 16 1:80 1:160 1:160 1:160 - --------------------------------------------------- *Reciprocal of serum dilution neutralizing 80% of BTV plaque activity on Vero cells. The data are from the post-second immunization sera (Day 35). Negative and positive control sera behaved as expected in the SN assay.
3. Immunization of Sheep with Psoralen-inactivated BTV vaccine. a. Example 1 Each of two adult sheep, known to be susceptible to BTV, was inoculated subcutaneously (SQ) with 2 ml of AMT-UVA inactivated BTV plus adjuvant (1:1, vaccine to aluminum hydroxide adjuvant). The vaccine contained approximately 10(8) pfu/ml of BTV prior to inactivation. A third sheep was inoculated SQ with 6 ml of the identical vaccine without adjuvant. Seven weeks later the three sheep were given identical inoculations SQ that consisted of 5 ml of vaccine and aluminum hydroxide adjuvant (2:1 vaccine to adjuvant; 10(8) pfu BTV/ml of vaccine). The three sheep were monitored for clinical evidence of BTV, including daily body temperature recording and bi-daily virus isolation attempts. No evidence of BTV was observed, indicating that the vaccine was inactivated. Serum was collected weekly for serum neutralization and agar gel precipitation testing. Normal sheep sera and BTV-immune sheep sera were used for negative and positive control samples in the serologic tests. The first vaccine inoculations induced precipitating anti-BTV antibody in all three sheep. Their pre-exposure sera were uniformly negative for anti-BTV precipitating antibody. Modest neutralizing anti-BTV anti-body titers (1:5) were elicited in two of three sheep following one immunization. The second immunization elicited a distinct immunological anamnesic response, inducing neutralizing titers of 1:40, 1:80, or 1:160 in the three sheep.
TABLE 4 - -------------------------------------------------------------- Serum Neutralization Data From Sheep Immunized with an AMT-UVA Inactivated BTV Vaccine - -------------------------------------------------------------- Titers* Sheep No.: ------------------------------------- Group 1 2 3 - ----- ------ ------ ------ Pre-First Immunization Day 0 <5 <5 <5 Post-First Immunization Day 21 5 5 <5 Post-Second Immunization
59 5,106,619 15
TABLE 4 - continued - -------------------------------------------------------------------------------- Serum Neutralization Data From Sheep Immunized with an AMT-UVA Inactivated BTV Vaccine. ---------------------------------------------- TITERS* Sheep No.: ----------------------------------------- 1 2 3 - -------------------------------------------------------------------------------- Day 7 80 160 40 Day 14 80 40 40 Day 21 80 80 40 Day 42 80 80 80 Post-Challenge Day 7 160 160 80 Day 14 320 160 80 - -------------------------------------------------------------------------------- *Reciprocal of highest 2-fold dilution reducing BTV plaque activity on BHK cells by 80 percent.
The sheep were challenged by SQ syringe inoculation of 10(5) egg lethal doses of BTV serotype 11. The three sheep remained clinically normal during the BTV challenge period, indicating that the vaccine was efficaceous. It is evident from the above results that the BTV which is psoralen-inactivated retains its immunogenicity, particularly as to those sites which elicit an immune response which is effective in protecting a host against subsequent BTV-infection. Thus, the psoralen inactivation can be carried out under conditions which do not modify the immunogenic sites of the virus, so as to elicit an immunogenic response which will be effective against the live BTV. Furthermore, the BTV RNA virus is efficiently inactivated under mild conditions to the point of complete inactivation, whence it may be safely administered to a host. b. Example 2 Eight experimental and four control sheep, known to be Bluetongue Virus susceptible, were housed together in an insect-proof facility. The experimental sheep were inoculated twice subcutaneously with AMT-UVA inactivated BTV Serotype 11 vaccine. Each vaccinate received approximately 3 x 10(8) pfu BTV-11 formulated with twenty-five percent (v/v) aluminum hydroxide adjuvant. Three weeks elapsed between immunizations. Control sheep were inoculated with tissue culture fluid in 25% percent (v/v) aluminum hydroxide. Serum samples were collected prior to vaccination, following vaccinations, and following challenge, and tested for SN antibodies. All sheep were challenged by subcutaneous inoculation of 2 x 10(5) ELD(50) BTV-11 four weeks post-second vaccination. Virus isolation was performed twice weekly post-challenge for six weeks. Virus isolation from sheep blood was done by intravenous chicken embryo inoculation, followed by specific BTV serotype identification by neutralization in vitro. Five of the eight vaccinated sheep developed SN titers of 1:20 post-second vaccination. All eight vaccinates resisted subcutaneous challenge with 2 x 10(5) ELD(50) BTV-11, whereas the four control sheep developed uniform viremia as assessed by egg inoculation. Sheep data are given in Table 5.
TABLE 5 - -------------------------------------------------------------------------------- Serum Neutralization and Virus Isolution Data from Sheep Vaccinated with AMT-UVA Inactivated BTV-11 Vaccine and Subsequently Challenged with 2 x 10(5) ELD(50) of Live BTV-11 ------------------------------------------------------------- Virus Isolation SN Titer Post-Challenge Sheep Post-Second Post- Day No. Baseline Vaccination Challenge --------------- 4 11 15 18 - -------------------------------------------------------------------------------- Experi- mental - ------- 650 neg 1:20 1:160 -- -- -- --
16
TABLE 5 - continued - -------------------------------------------------------------------------------- Serum Neutralization and Virus Isolution Data from Sheep Vaccinated with AMT-UVA Inactivated BTV-11 Vaccine and Subsequently Challenged with 2 x 10(5) ELD(50) of Live BTV-11 ------------------------------------------------------------- Virus Isolation SN Titer Post-Challenge Sheep Post-Second Post- Day No. Baseline Vaccination Challenge --------------- 4 11 15 18 - -------------------------------------------------------------------------------- Experi- mental - ------- 651 neg 1:20 1:40 -- -- -- -- 652 neg 1:20 1:160 -- -- -- -- 653 neg 1:20 1:40 -- -- -- -- 656 neg 1:10 1:160 -- -- -- -- 658 neg 1:10 1:40 -- -- -- -- 659 neg 1:20 1:160 -- -- -- -- 660 neg 1:10 1:160 -- -- -- -- Controls - -------- 654 neg neg 1:10 + + + + 655 neg neg neg + + + + 661 neg neg 1:40 + + + + 662 neg neg 1:160 + + + - - --------------------------------------------------------------------------------
B. Feline Herpes Virus I 1. Assessment of Inactivation by Blind Passage Fc3Tg or AKD cells were grown to confluency in 850 cm(2) roller bottles using standard cell culture procedures as described above. The culture medium was removed from the roller bottle, and 2.0 mls of the inactivated virus preparation, mixed with 18 mls of medium containing 2% F(i), were adsorbed to the roller bottle cell monolayer for 60 minutes at 35 degrees C. to 38 degrees C. with rotation at 1 to 5 rpm. After adsorption, the inoculum was removed and 150 ml of maintenance medium (MEN or F12K with 2% F(i)) added. The roller bottle culture was then incubated at 35 degrees C. to 38 degrees C. for 7 days with daily observation for viral CPE. The roller bottle culture received a 100% medium change after 3 to 5 days. If no CPE was observed during the first roller bottle passage, the cell monolayer was scraped into the maintenance medium which was then decanted into a centrifuge bottle. The cells were pelleted by centrifugation at room temperature at 1,000 x g for 15 minutes, resuspended in 20 ml of fresh maintenance medium, and passed to a new confluent roller bottle culture of Fc3Tg or AKD cells as described above. The second roller bottle blind passage was observed for 7 days and fed once on day 3 to 5. If no CPE was observed during the second roller bottle blind passage, a third roller bottle blind passage was performed. If no CPE was observed by the end of the third roller bottle passage, the virus preparation was considered inactive. 2. Administration Procedure for Psoralen-inactivated FVR Vaccines Photochemically inactivated FVR was inoculated via syringe into cats by various routes, including but not limited to intravenously (IV), subcutaneously (SQ), intramuscularly (IM), or intraperitoneally (IP). The vaccine was administered in various volumes (0.5 to 3.0 ml) and in various concentrations (10(6) to 10(8) pfu; either CF, CA or in combination). In the following examples, the vaccine was administered in combination with aluminum hydroxide as an immunologic adjuvant. The number of injections and their temporal spacing was as set forth in each example. 3. Immunization with Psoralen-inactivated CR-FVR Vaccine The experimental group consisted of four specific pathogen free kittens (2 males, 2 females) four months old (Liberty Laboratories, Liberty Corner, N.J.). The 60 5,106,619 17 control group consisting of two similar female kittens. The experimental group was inoculated IM with 3 x 10(7) pfu (3 mls) of HMT inactivated CF-FVR on days 0 and 21, and again inoculated with 3 x 10(7) pfu HMT inactivated with an equal amount of 2% aluminum hydroxide [(Al(OH)(3)] adjuvant on day 61. Controls were vaccinated at eight weeks and at thirteen weeks of age with a commercial FVR vaccine using the manufacturer's recommended procedure. Serum samples were collected weekly and tested for anti-FVR neutralizing antibodies. Following live virus challenge (10(6) pfu intranasally and intraconjunctivally), a numerical scoring system (Table 6) was used to assess the clinical response of both experimental and control cats.
TABLE 6 - -------------------------------------------------------------------------------- Scoring System for Clinical Effects of Herpesvirus Challenge in Cats ---------------------------------------- Factor Degree Score - -------------------------------------------------------------------------------- Fever 101 to 102 degrees F. 0 102 to 103 1 103 to 104 3 greater than 104 5 Depression slight 1 moderate 3 severe 5 Sneezing occasional 1 moderate 3 paroxysmal 5 Lacrimation serous 1 mucoid 3 purulent 5 Nasal Discharge serous 1 mucoid 3 purulent 5 Appetite normal; eats all food 0 fair; eats more than 1 1/2 of food poor; eats less than 3 1/2 of food none; eats nothing 5 - --------------------------------------------------------------------------------
Three of four experimental cats developed serum neutralizing anti-FVR antibody (SN) titers of 1:2 that were detected between day 42 and day 58. Following the third immunization (day 61), four of four experimental cats had SN titers of 1:4 (day 80). Baseline SN antibody titers on the experimental cats were negative. The control cats did not develop detectable SN antibody titers during the pre-challenge period. All cats were exposed to 10(6) pfu of live FVR by intraconjunctival and intranasal exposure on day 91. Each cat was monitored twice daily for the absence, presence and degree of severity of factors given in Table 6. A composite clinical score was derived for each cat after a 15 day observation period. Three of four experimental cats demonstrated mild temperature elevation and serous ocular or nasal discharge along with mild intermittent depression and appetite suppression. Their composite scores were 39, 42, and 35 respectively for the 15 day observation period. The fourth experimental cat was more severely affected (composite score=84) by moderate, but transient, sneezing and mucoid nasal discharge. Both control cats were severely affected by live virus challenge. Severe purulent nasal and ocular discharge and lack of appetite were apparent. The control cats had composite scores of 133 and 253. Three weeks following live FVR challenge, all cats were tested for SN antibody titers against FVR. Three of four experimental cats had SN antibody titers of 1:16 while the fourth cat had a 1:8 titer. One of the control 18 cats has an SN antibody titer of 1:4 while the second control lacked an SN antibody titer against FVR. 4. Immunization with Psoralen-inactivated CA-FVR Vaccine. Nine age-matched specific pathogen free kittens, 4 months old (Liberty Laboratories, Liberty Corner, N.J.), were randomly assigned to three experimental groups designated A, B, and C. Group A (controls) was inoculated twice with 1 ml tissue culture fluid and 1 ml aluminum hydroxide adjuvant. Group B was inoculated twice with a commercial FVR vaccine according to the manufacturer's recommendation. Group C was inoculated three times with 10(7) HMT-inactivated CA-FVR in aluminum hydroxide (total volume=2 ml; 1:1 vaccine to adjuvant). All injections were given 1M at three week intervals. Live FVR virus (10(6) pfu intranasally and intraconjunctivally) was given on day 63 and a numerical scoring system (Table 6) was used to assess the kittens' clinical response for a 15 day post-challenge period. Serum samples were collected from all kittens prior to vaccination, prior to the second and third immunizations, prior to live FVR challenge, and at 15 days post-challenge. The sera were utilized to assess neutralizing antibody titers by standard procedures. The control kittens (Group A) maintained SN anti-body titers less than 1:2 (negative) throughout the pre-challenged period. Fifteen days following live FVR challenge Group A kittens uniformly had SN antibody titers of 1:2. Kittens in Groups B and C lacked detectable anti-FVR antibody titers pre-immunization, but all kittens in Groups B and C had SN antibody titers of 1:2 or 1:4 after two immunizations. The third immunization in Group C kittens did not significantly alter their SN antibody titers. Following a 15 day post-challenge period, kittens in Groups B and C demonstrated an anamnestic immunologic response, with SN antibody titers ranging from 1:16 to 1:64. Clinically, Group A kittens were severely affected by live FVR challenge, whereas kittens in Groups B and C were significantly protected by their respective vaccines. The composite clinical scores for Group A were 125, 141, and 128 for the 15 day post-challenge period. The composite clinical scores for Group B were 25, 20, and 64, while Group C had composite clinical scores of 21, 15, and 34. The clinical signs evident were characteristic of FVR. From the SN data and clinical scoring, it is evident that kittens immunized with the experimental HMT-inactivated FVR vaccines (cell-free or cell associated) in the above examples were significantly immune to the clinical effects of severe FVR challenge. C. Vesicular Stomatitis Virus 1. Assessment of Inactivation by Intracerebral Inoculation of Mice Sucking mice (0 to 10 days old) were inoculated intracerebrally with 0.02 ml of the psoralen-inactivated VSV-NJ using a tuberculin syringe and a 28 or 30 gauge needle. Each vaccine lot was tested in four to nine suckling mice. The mice were observed three times daily for a minimum of seven days. Residual low-level live VSV kills suckling mice in two to five days. The sensitivity of this assay is approximately 1 to 5 pfu of live VSV per intracerebral dose. Inactivated VSV-NJ vaccine was considered safe (inactivated) if all inoculated suckling 61 5,106,619 19 mice survived the seven day observation period. The VSV-NJ vaccine batches used hereinafter each passed the suckling mouse safety test prior to use. 2. Virus Neutralization in Mice Immunized with Psoralen-inactivated VSV-NJ Vaccine Groups of ten adult white mice each were injected using three immunological adjuvants (aluminum hydroxide gel, incomplete Freund's, or oil emulsion) with one of three psoralen-inactivated VSV-NJ vaccine doses (10(9), 10(8), or 10(7) pfu/dose). The oil emulsion was prepared as described by Stone et al. (1978) Avian Dis. 22:666-674. All mice were injected IP once each, on day 0 and day 21. Serum samples were collected from the orbital sinus on day 2 and on day 33 and pooled serum samples were assessed for serum neutralization (SN) activity by standard procedures. See, Castaneda et al. (1964) Proc. US Livestock San. Assoc. 68:455-468. Serum samples were negative for neutralizing antibodies to VSV-NJ prior to vaccination. The vaccine with oil emulsion adjuvant induced the highest SN titers after one injection. All three vaccine doses, regardless of adjuvant, induced SN titers of at least 1:2000 after two injections. Serum dilutions were tested for SN activity only to 1:2560. The results are set forth in Table 7.
TABLE 7 - ------------------------------------------------------------------------------------------- Virus Neutralization Indices* of Mouse Sera After One and Two Injections of Psoralen- Inactivated VSV-NJ Vaccine ------------------------------------------- Log(10) of Vaccine Con- centration (pfu/dose) No. of --------------------------------------------------------- Adjuvant Injections 7 8 9 - ------------------------------------------------------------------------------------------- Aluminum hydroxide gel 1 67* 905 905 Aluminum hydroxide gel 2 > 2560 2560 > 2560 Freund's Incomplete 1 226 57 905 Freund's Incomplete 2 2033 > 2560 > 2560 Oil Emulsion 1 > 2560 > 2560 > 2357 Oil Emulsion 2 > 2560 > 2560 > 2560 - ------------------------------------------------------------------------------------------- *Virus neutralization index is the reciprocal of the serum dilutions that neutralized 32 TCID(50) of VSV-NJ.
3. Virus Neutralization in Hamsters Vaccinated with Psoralen-inactivated VSV-NH Vaccine Groups of five MHA hamsters each were injected with either 10(9), 10(8), or 10(7) pfu psoralen-inactivated VSV-NJ per dose, with or without aluminum hydroxide adjuvant (1:1). All hamsters were injected intramuscularly (IM) once each, on day 0 and again on day 21. Pooled serum samples were collected on day 21 and on day 34 for serum neutralization testing by standard procedures. Serum neutralizing antibodies were elicited by all three vaccine doses tested, with or without aluminum hydroxide adjuvant. SN titers are given in Table 8.
TABLE 8 - ------------------------------------------------------------------------------------------- Virus Neutralization Indices* of Hamster Sera After One and Two Injections of Psoralen-Inactivated VSV-NJ Vaccine ------------------------------------------- Log(10) of Vaccine Con- centration (pfu/dose) No. of -------------------------------------------------------- Adjuvant Injections 7 8 9 - ------------------------------------------------------------------------------------------- None 1 134* 134 1076 None 2 1280 1810 > 2560 Aluminum hydroxide gel 1 538 538 > 2560 Aluminum hydroxide gel 2 1810 1920 2560 - ------------------------------------------------------------------------------------------- *Virus neutralization index is the reciprocal of the serum dilution that neutralized 32 TCID(50) of VSV-NJ.
20 4. Live VSV-NJ Challenge of Mice Vaccinated with Psoralen-inactivated VSV-NJ Vaccine Three groups of fourteen, sixteen and seventeen adult white mice each were injected with either 10(7), 10(6) or 10(5) pfu psoralen-inactivated VSV-NJ per dose, respectively, using oil emulsion adjuvant with all injections. Each mouse was injected once IP (day 0). Pooled serum samples were collected on day 0 and again on day 21, and these samples were tested for SN antibody titers by standard procedures. The results are set forth in Table 9.
TABLE 9 - -------------------------------------------------------------------------------- Virus Neutralization Indices* of Mouse Sera After One Injection With Psoralen- Inactivated VSV-NJ Vaccine, Using Oil Emulsion Adjuvant ------------------------------------------- Log(10) of Vaccine con- centration (pfu/dose) ------------------------------------------ Day 5 6 7 - -------------------------------------------------------------------------------- 0 --* -- -- 21 -- -- -- - -------------------------------------------------------------------------------- *Virus neutralization index is the reciprocal of the serum dilution that neutralized 56 TCID(50) of VSV-NJ
Each group of mice was subdivided into three groups of about five mice each. Each mouse was challenged with either 1, 10 or 100 minimum lethal doses (MLD) of live VSV by intracerebral inoculation on day 33. Two of five mice that were immunized with 10(6) pfu psoralen-inactivated VSV-NJ survived a one MLD VSV challenge but five of five mice that were immunized with 10(7) pfu psoralen-inactivated VSV-NJ vaccine survived both a 1 or 10 MLD VSV challenge. One of four mice that were vaccinated at 10(7) pfu/dose psoralen-inactivated VSV-NJ survived a 100 MLD VSV challenge. The results (no, dead/no. challenged) are set forth in Table 10.
TABLE 10 - -------------------------------------------------------------------------------- Live VSV-NJ Challenge of Mice Injected with Psoralen-Inactivated VSV-NJ ------------------------------------------- Challenge Dilution Dose Psoralen- -------------------------------------------------- Inactivated 10-(5) 10-(4) (10-(3) VSV-NJ Vaccine (1 MLD) (10 MLD) (100 MLD) - -------------------------------------------------------------------------------- 10(7) pfu 0/5* 0/5 3/4 10(6) pfu 3/5 4/5 3/6 10(5) pfu 5/5 4/5 7/7 - -------------------------------------------------------------------------------- *Number dead/number challenged
5. Virus Neutralization in Cattle Immunized with Psoralen-inactivated VSV/NJ vaccine. Four groups of six mature beef cattle each were injected with either 10(8) or 10(7) pfu/dose psoralen-inactivated VSV-NJ vaccine, with or without aluminum hydroxide adjuvant (1:1). Each cow was vaccinated subcutaneously (SQ) on day 0 and again on day 21. A control group consisted of an additional six cattle that were inoculated only with adjuvant on day 0 and again on day 21. All cattle were bled on days 0, 14, 21, and 35. Serum from each animal was tested for SN antibodies to VSV-NJ by standard procedures. The aluminum hydroxide adjuvant was required to elicit significant SN titers in cattle, and 10(8) pfu/dose induced the highest responses. The results are set forth in Table 11. A VSV-NJ virus neutralization index greater than 1000 has been reported to represent protection against 10(6)ID(50) of live VSV by intralingual challenge in cattle. See, Castaneda et al. (1964) Proc. US Livestock San Assoc. 68:455-468. 62 21
TABLE 11 - ----------------------------------------------------------------------------------------- Virus Neutralization Indices* From Cattle Injected with Psoralen-Inactivated VSV-NJ Vaccine ----------------------------------------- Day Serum Collected ----------------------------------------------------- Group Treatment Animal 0** 14 21** 35 - ----------------------------------------------------------------------------------------- A 10(8) pfu/ 310 - 16 16 256 dose + 731 - - - > 16 A1(OH)(3) 911 - 128 64 2048 921 - 8 8 1024 943 - 16 32 1024 944 - 32 32 512 B 10(7) pfu/ 303 - - - 256 dose + 304 - - - 64 A1(OH)(3) 308 4 4 8 512 542 - - - 8 914 - 16 4 512 1670 - - - > 128 C Controls 305 - - - - 309 - - - - 314 - - - - 315 - - - - 316 - - - - 318 - - - - D 10(8) pfu/ 302 - - - 4 dose 611 - - - 4 without 714 - - - 8 adjuvant 732 - - - 4 747 - - - - 996 - - - 32 E 10(7) pfu/ 101 - - - - dose 312 - - - 4 without 616 - - - - adjuvant 721 - - - - 722 - - - - 1944 - - - - - ----------------------------------------------------------------------------------------- *Virus neutralization index is the reciprocal of the serum dilution that neutralized 32 TCID(50) of VSV-NJ. **Immunization Days
6. Live VSV-NJ Challenge of Cattle Vaccinated with Psoralen-inactivated VSV-NJ Vaccine Ten mature cattle were divided into two groups of five animals each. Group I was designated experimental and Group II was designated control. All ten cattle were clinically normal and lacked evidence of previous VSV exposure; that is, they were negative for serum neutralizing (SN) antibody. Group I cattle were vaccinated subcutaneously with 10(8) pfu (prior to inactivation) psoralen-inactivated VSV twice with a three week interval. Vaccine volume was 2 ml, containing aluminum hydroxide adjuvant. Group II cattle were not exposed to the psoralen-inactivated VSV. Approximately two weeks post-second vaccination, the cattle of both Groups I and II were challenged intradermalingually with 0.1 ml live VSV in log dilutions of 5.6 pfu to 5.6 times 10(5) pfu/injection site. Thus each animal's tongue received six separate 0.1 ml injections, representing a quantitative challenge system. Serum neutralizing titers for cattle in each group measured before and after challenge are presented in Table 12.
TABLE 12 - ------------------------------------------------------------------------------------------------------- Serum Neutralization Titers From Cattle Vaccinated With Psoralen-Inactivated VSV-NJ Vaccine ------------------------------------------- Animal Ar- After After Day of Post No. rival 1st Vacc(a) 2nd Vacc(b) Challenge(a) Challenge(c) Day 0 18 35 42 60 - ------------------------------------------------------------------------------------------------------- Group I: - ------ 4009-V neg* 1:160 1:1280 1:1280 1:1280 4383-V neg 1:80 1:1280 1:1280 1:2560 4389-V neg 1:80 1:640 1:2560 ND 6153-V neg 1:80 1:1280 1:1280 (greater than or equal to) 1:20480
22
TABLE 12 - continued - ------------------------------------------------------------------------------------------------------- Serum Neutralization Titers From Cattle Vaccinated With Psoralen-Inactivated VSV-NJ Vaccine ------------------------------------------- Animal Ar- After After Day of Post No. rival 1st Vacc(a) 2nd Vacc(b) Challenge(a) Challenge(c) Day 0 18 35 42 60 - ------------------------------------------------------------------------------------------------------- 6246-V neg 1:320 1:1280 1:1280 ND Group II: - ------ 3780-C neg neg neg neg 1:10240 3781-C neg neg neg neg 1:10240 3784-C neg neg neg neg 1:10240 4007-C neg neg neg neg 1:10240 7912-C neg neg neg neg 1:10240 - ------------------------------------------------------------------------------------------------------- (a) 100 TCID(50) of VSV-NJ (b) greater than 1000 TCID(50) of VSV-NJ (c) 37 TCID(50) of VSV-NJ * negative at 1:20, the lowest dilution tested ND = not done
Vaccinated animals had a fifty percent reduction in lesion number, and lesions on vaccinates were fifty percent smaller and healed faster than on controls. Control animals developed lesions at both earlier and later time points. On post-challenge day eighteen, all five controls had lesions whereas four of five vaccinates were normal. The fifth vaccinate's lesions were milder than those of any control animal on post-challenge day eighteen. Using the Mann-Whitney modification of Wilcoxon's two sample test, the vaccinates were significantly protected against live VSV challenge (P=0.075). On the average, vaccinated cattle were protected against 25 times the minimum infectious dose required to produce lesions in control animals. According to the present invention, viruses inactivated with furocoumarins and ultraviolet radiation in the substantial absence of oxygen and other oxidizing species retain their immunogenicity and are suitable as the immunogenic substance in vaccines against a number of virally-induced diseases. The inactivated viruses of the present invention are non-infectious and safe when administered to a host for vaccination, yet display enhanced antigenic integrity when compared to vaccines inactivated in the presence of oxygen. Although the foregoing invention has been described in some detail by way of illumination and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims. What is claimed is: 1. A viral vaccine produced by exposing a live virus to a preselected intensity of long wavelength ultraviolet radiation and a preselected concentration of an inactivating furocoumarin for a time period sufficiently long to render the virus non-infectious but not long enough to degrade its antigen characteristics, wherein said exposure is performed in the substantial absence of oxygen and other oxidizing species. 2. A viral vaccine as in claim 1, wherein the inactivation medium is maintained under a non-oxidizing gas atmosphere. 3. A viral vaccine as in claim 1, wherein the inactivation medium is flushed with the non-oxidizing gas. 4. A viral vaccine as in claim 2, wherein the non-oxidizing gas is selected from the group consisting of nitrogen, argon, helium, neon, carbon dioxide, and mixtures thereof. 63 5. A viral vaccine as in claim 1, wherein an oxygen scavenger is added to the inactivation medium. 6. A viral vaccine as in claim 5, wherein the oxygen scavenger is sodium ascorbate. 7. A viral vaccine as in claim 1, wherein the virus is exposed to the furocoumarin by adding said furocoumarin to an inactivation medium containing the live virus. 8. A viral vaccine as in claim 1, wherein the furocoumarin is introduced to the live virus by addition to a cell culture medium in which the virus is grown. 9. A viral vaccine comprising in combination: an inactivated live virus and a physiologically acceptable vaccine carrier; said virus being further characterized as inactivated by exposure to long wavelength ultraviolet radiation and an amount of a furocoumarin sufficient to inactivate the virus, which together render the virus non-infectious without destroying its antigen characteristics. 10. The vaccine in claim 9 wherein the furocoumarin is a psoralen. 11. The vaccine in claim 9 wherein the furocoumarin is 5-methoxypsoralen (5-MOP). 12. The vaccine in claim 11 wherein the furocoumarin is 8-methoxypsoralen. (8MOP). * * * * * 64 EXHIBIT G 1/3/1 (Item 1 from file: 351) 009031974 WPI Acc No: 92-159335/19 XRAM Acc No: C92-073563 Inactivated viral vaccines - prepd. by psoralen inactivation of live virus in non-oxidising atmos. used for inoculation against DNA and RNA viral Patent Assignee: (DIAM-) DIAMOND SCIENTIFIC Author (Inventor): CREAGAN R P; GILES R; STEVENS D R; WIESEHAHN G P Patent Family: CC Number Kind Date Week US 5106619 A 920421 9219 (Basic) Priority Data (CC No Date): US 563939 (831220); US 592661 (840323); US 785354 (851007); US 69117 (870702); US 463081 (900110) 1/3/2 (Item 2 from file: 351) 008533075 WPI Acc No: 91-037138/06 XRAM Acc No: C91-015912 Decontaminating blood components to destroy viruses - by adding psoralen cpd(s)., irradiating with long wavelength UV, and adding glucose and/or post-treatment gassing Patent Assignee: (DIAM-) DIAMOND SCIENTIFIC Author (Inventor): WIESEHAHN G P; CORASH L Patent Family: CC Number Kind Date Week CA 2015315 A 901111 9106 (Basic) Priority Data (CC No Date): US 350335 (890511) Applications (CC, No, Date): CA 15315 (900424) 1/3/3 (Item 3 from file: 351) 007742402 WPI Acc No: 89-007514/01 XRAM Acc No: C89-003599 Vaccine for feline virus rhinotracheitis - prepd. by exposing feline herpes virus I to ultraviolet radiation in the presence of furocoumarin Patent Assignee: (DIAM-) DIAMOND SCIENTIFIC Author (Inventor): WISEHAHN G P; GILES R E; STEVENS D R Patent Family: CC Number Kind Date Week US 4791062 A 881213 8901 (Basic) FI 8805120 A 900508 9032 Priority Data (CC No Date): US 707102 (850228); US 70201 (870706) 1/3/4 (Item 4 from file: 351) 007443445 WPI Acc No: 88-077379/11 Related WPI Accession(s): 84-277589 XRAM Acc No: C88-034732 Photochemical viral inactivation in blood clothing factor compsns. - by adding furocoumarin cpd(s)., reducing dissolved oxygen concn. and UV irradiating Patent Assignee: (DIAM-) DIAMOND SCIENTIFIC Author (Inventor): WIESEHAHN G P; CREAGAN R P Patent Family: CC Number Kind Date Week US 4727027 A 880223 8811 (Basic) Priority Data (CC No Date): US 785356 (851007); US 490681 (830502); US 65 Page 2 928841 (861020) 1/3/5 (Item 5 from file: 351) 007280343 WPI Acc No: 87-277350/39 Related WPI Accession(s): 85-210121; 85-323311 XRAM Acc No: C87-117850 Prepn. of inactivated viral vaccines - by furo-coumarin-inactivation in non-oxidising atmos. Patent Assignee: (ADGE-) ADVANCED GENETICS Author (Inventor): WIESEHAHN G P; CREAGAN R P; STEVENS D R; GILES R Patent Family: CC Number Kind Date Week US 4693981 A 870915 8739 (Basic) Priority Data (CC No Date): US 785354 (851007); US 563939 (831220); US 592661 (840323) 1/3/6 (Item 6 from file: 351) 004496433 WPI Acc No: 85-323311/51 Related WPI Accession(s): 87-277350 XRAM Acc No: C85-139975 Vaccine against vesicular stomatitis virus infection contg. virus inactivated by irradiating with UV light in presence of furocoumarin Patent Assignee: (ADGE-) ADV GENETICS RES Author (Inventor): WIESEHAHN G P; GILES R E Patent Family: CC Number Kind Date Week US 4556556 A 851203 8551 (Basic) Priority Data (CC No Date): UF 592661 (840323); US 785354 (851007) 1/3/7 (Item 7 from file: 351) 004383243 WPI Acc No: 85-210121/35 Related WPI Accession(s): 87-277350 XRAM Acc No: C85-091582 Bluetongue virus vaccine contg. bluetongue virus inactivated by irradiation in presence of furocoumarin Patent Assignee: (ADGE-) ADV GENETICS RES; (ADGE-) ADV GENETICS RES LT Author (Inventor): GILES R E; STEVENS D R; WIESEHAHN G P Patent Family: CC Number Kind Date Week AU 8435693 A 850627 8535 (Basic) ZA 8408857 A 850515 8535 US 4545987 A 851008 8543 Priority Data (CC No Date): US 563939 (831220); US 785354 (851007) Applications (CC, No, Date): AU 8435693 (841120); ZA 848857 (841114) 1/3/8 (Item 8 from file: 351) 004132049 WPI Acc No: 84-277589/45 Related WPI Accession(s): 88-077379 XRAM Acc No: C84-117678 XRPX Acc No: N84-207210 Decontamination of biological protein-contg. compsns. by addn. of furo-coumarin and irradiation to inactivate polynucleotide(s) Patent Assignee: (ADGE-) ADV GENETICS RES; (DIAS-) DIAMOND SCIENTIFIC; (DIAM-) DIAMOND SCI CO Author (Inventor): WIESEHAHN G P Patent Family: 66 Page 3 CC Number Kind Date Week EP 124363 A 841107 8445 (Basic) AU 8427521 A 841108 8501 ZA 8403270 A 841025 8508 JP 60016930 A 850128 8510 CA 1224622 A 870728 8734 US 4748120 A 880531 8824 EP 124363 B 901219 9051 DE 3483751 G 910131 9106 Priority Data (CC No Date): US 490681 (830502); US 785356 (851007); US 928841 (861020) Applications (CC,No,Date): EP 84302845 (840427); ZA 843270 (840502); JP 8486273 (840501) 1/3/9 (Item 1 from file: 350) 002274367 WPI Acc No: 79-73577B/40 XRAM Acc No: C79-B73577 Psoralen derivs. contg. cyclic aminomethyl gp. -- to enhance solubility and ability to react wih nucleic acids; PSORIASIS CHEMOTHERAPEUTIC Patent Assignee: (REGC) UNIV OF CALIFORNIA Author (Inventor): HEARST J E; RAPOPORT H; ISAACS S Patent Family: CC Number Kind Date Week US 4169204 A 790925 7940 (Basic) Priority Data (cc No Date): US 937292 (780828); US 734031 (761020) 1/3/10 (Item 2 from file: 350) 002017192 WPI Acc No: 78-30223A/17 XRAM Acc No: C78-A30223 Substd. trimethyl psoralen(s) -- for treatment of vitiligo and psoriasis, and for virus inactivation Patent Assignee: (REGC) UNIV OF CALIFORNIA Patent Family: CC Number Kind Date Week BE 859912 A 780419 7817 (Basic) DE 2746942 A 780427 7818 NL 7711539 A 780424 7819 JP 53053699 A 780516 7825 FR 2376859 A 780908 7841 US 4124598 A 781107 7846 GB 1556307 A 791121 7947 US 4196281 A 800401 8015 CH 635346 A 830331 8315 IT 1087022 B 850531 8623 DE 2760392 A 861009 8642 JP 87000153 B 870106 8704 JP 6201795 A 870121 8709 DE 2746942 C 880526 8821 JP 88026119 B 880527 8825 Priority Data (CC No Date): US 734031 (761020); US 937292 (780828); US 938277 (780831) 67 EXHIBIT H file: MASTER RECORD QUERY donePrintHypertextLast-screenUpdate[OTHER] next-screen 3301 ============================================================================== MONO[ 3301 ] #[ ] ATTY[ LW ] DIV[ ] ASSN[ DIAMOND SCIENTIFIC ] TITL[ PHOTOCHEMICAL DECONTAMINATION TREATMENT OF WHOLE BLOOD AND BLOOD COMPONENTS ] INVN[ GARY P. WIESEHAHN ] USSN[ 06/490,681 ] USFL[ 05/02/83 ] CASN[ 453,232 ] CAFL[ 05/01/84 ] FORN[ AU - 563,557, EPO - 84/302,845, (124,363) JP - 86273/84, ZA - 84/3270 ep 124,363 ] USP#[ ] USIS[ ] CAP#[ 1,224,622 ] CAIS[ 07/28/87 ] NOTE[ DIAMOND SCIENTIFIC NO. 2-2 ABANDONED CONT. FILED ] ] -MORE- file: MASTER RECORD QUERY donePrintHypertextLast-screenUpdate[OTHER] next-screen 3301 ============================================================================== MONO[ 3301 CONT. ] #[ ] ATTY[ LW ] DIV[ ] ASSN[ DIAMOND SCIENTIFIC ] TITL[ PHOTOCHEMICAL DECONTAMINATION TREATMENT OF WHOLE BLOOD AND BLOOD COMPONENTS ] INVN[ GARY P. WIESEHAHN ] USSN[ 06/928,841 ] USFL[ 10/20/86 ] CASN[ ] CAFL[ ] FORN[ ] USP#[ 4,748,120 ] USIS[ 5/31/88] CAP#[ ] CAIS[ ] 68 file: MASTER RECORD QUERY donePrintHypertextLast-screenUpdate[OTHER] next-screen 3301 ============================================================================== MONO[ 3301 CONT. II ] #[ ] ATTY[ LW ] DIV[ ] ASSN[ DIAMOND SCIENTIFIC] TITL[ PHOTOCHEMICAL DECONTAMINATION TREATMENT OF WHOLE BLOOD AND BLOOD COMPONENTS ] INVN[ GARY P. WIESEHAHN ] USSN[ 07/164,915 ] USFL[ 03/07/88 ] CASN[ ] CAFL[ ] FORN[ ] USP#[ ] USIS[ ] CAP#[ ] CAIS[ ] NOTE[ DIAMOND SCIENTIFIC ] --More-- ] file: MASTER RECORD QUERY donePrintHypertextLast-screenUpdate[OTHER] next-screen 3301 ============================================================================== MONO[ 3301 CONT. II/CIP ] #[ ] ATTY[ LW ] DIV[ ] ASSN[ DIAMOND SCIENTIFIC ] TITL[ IMPROVED METHOD OF BLOOD COMPONENT DECONTAMINATION BY GLUCOSE ADDITION AND POST-DECONTAMINATION GASING ] INVN[ GARY P. WIESEHAHN AND LAURENCE CORASH ] USSN[ 07/350,335 ] USFL[ 05/11/89 ] CASN[ 2,015,315 ] CAFL[ 04/24/90 ] FORN[ ] USP#[ ] USIS[ ] CAP#[ ] CAIS[ ] NOTE[ DIAMOND SCIENTIFIC ] --MORE-- EX-23.1 3 CONSENT OF ERNST & YOUNG LLP 1 EXHIBIT 23.1 CERUS CORPORATION CONSENT OF INDEPENDENT AUDITORS We consent to the incorporation by reference in the Registration Statement on Form S-8 No. 333-27097 pertaining to the 1996 Equity Incentive Plan and Employee Stock Purchase Plan of Cerus Corporation of our report dated January 27, 1998, except for Note 2 as to which the date is March 6, 1998, with respect to the financial statements of Cerus Corporation included in the Annual Report (Form 10-K) for the year ended December 31, 1997. Ernst & Young LLP Walnut Creek, California March 6, 1998 EX-27.1 4 FINANCIAL DATA SCHEDULE
5 1 YEAR DEC-31-1997 JAN-01-1997 DEC-31-1997 11,603,596 9,977,178 4,376,141 0 0 26,171,602 2,750,813 1,718,984 27,315,156 4,797,177 0 0 0 9,184 22,465,793 27,315,156 0 6,850,847 0 0 22,732,481 0 14,901 (14,664,213) 0 (14,644,213) 0 0 0 (14,664,213) (1.76) (1.76)
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