THE PRODUC'HQN OF RA3:ES EMMUNE SERUM Thai: for flu Degree of M. S. MiCHSGAN SWEET COLLEGE Everafi' A. Naésm‘: i954 {THESIS This is to certify that the thesis entitled The Production of Babies Immune Serum presented bg Everett A. N 31 son has been accepted towards fulfillment of the requirements for _M._§.__ degree in Jaciemology flfW M 3’01 profi ssor Date MarCh 12; 19511 THE PRODUCTION OF RABIES IMMUNE SERUM by Everett A. Nelson A THESIS Submitted to the School of Graduate Studiee of Michigan State College of Agriculture and Applied Science in partial ful- fillment of the requirements for the degree of MASTER OF SCIENCE Department of Bacteriology 195h (4‘1- 31/ C“. II. III. IV. V. TABLE OF CONTENTS Acknowledgment Introduction Historical background Experimental methods A. B. C. D. E. F. Preparation of immunizing antigen Inoculation of rabbits Bleeding of rabbits. In vitro neutralization tests Complement fixation Preparation of refined serum Emperimental results A. B. In vitro neutralization tests 1. Fixed virus 2. Street virus Complement fixation Summary and conclusions Bibliography 3.1341532! ACKNOWLEDGMENT The author wishes to express his sincere appreciation to the Michigan Department of Health for making this study possible. He is also greatly indebted to Dr. Henrik J. Stafseth of the Michigan State College for his personal understanding and counsel; and to Dr. Serge Lensen of the Michigan Department of Health for his helpful guidance and for the performance of the tests in that part of this study dealing with rabies street virus. The author extends his sincere thanks to Mrs. Jean W. Glassen of the Michigan Department of Health for her valuable advice and criticism. I. INTRODUCTION The observations by Pasteur that the infective agent in rabies could be made non-infective for dogs by serial passage in rabbits, and that bacterial cultures become non-infective by desiccation, led him to apply these principles to the preparation of a vaccine for human prophylaxis. The first known attempt to produce an immunity to rabies in humans was in 1885 when a child, bitten by a rabid dog, was treated with a suspension of desiccated spinal cord from.an infected rabbit. The treatment was successful, and the method came into general use. The vaccines in use today, however, are not generally of this type, but of the Semple (1) type containing phenol inactivated brain virus or the Habel (2), Levinson (3), Hodes (A) type containing ultra- violet inactivated virus. The disadvantage in the use of a brain tissue vaccine is that a series of injections (seven to twenty-one) must be administered and sensitivity to the brain tissue may develop. Neuro-paralytic accidents occur in one of every 600 patients (5,6) and thus it would seem advisable to decrease the number of treatments and to supplement them with an agent less likely to cause an unfavor- able reaction. Antirabies serum might be used as such a supplementary agent. It would also serve to neutralize virus immediately, whereas it takes ten to fourteen days for antibodies to appear in individuals being treated with vaccine. During this period virus may reach the central nervous system and no longer be accessible to the antibody. 2. II. REVIEW OF THE LITERATURE As early as 1889 Babes and Lepp (7) conjectured as to the feasibility of using the fluids and cells of rabies refractive animals for the prophylactic treatment of dogs exposed to rabies virus. Remlinger (8) reported differences in the antirabies serum titers from.one rabies-immunized sheep to another and therefore advised careful titration of the serum from.each bleeding. The first clinical trials noted were those of Harie (9), who reported satisfactory results with serumrvaccine therapy in 300 humans bitten on the face and the extremities. Koprowski (10) reported on clinical trials in Georgia with twenty individuals who received serum and vaccine. None of them showed any signs of rabies at the time the article was published. He also reported on an individual bitten by a guinea pig with active street virus in its saliva.. Another indi- vidual was bitten on the hands by her own dog which proved to be rabid by the presence of Negri bodies in its brain. Both persons were treated with serum followed by vaccine. These patients developed neither rabies nor any ill effects from the treatment. Koprowski and Cox (11) reported on another clinical survey conducted in 19h8 in the United States in which practitioners returned questionnaires to the authors on individuals treated with serum concentrates. None of the forty-eight patients included in the survey had shown any signs of rabies at the time of publication. Some of these treated cases 3. received a sheep serum and others a rabhfi'serum. There were some unfavorable side effects in patients who received the sheep serum but none in those receiving the rabbit serum. The experiments by Hoyt gt g1. (12) and Yen (13) using mice as test animals indicated a higher percentage of survival among the serum-treated animals than in the untreated controls. Hoyt gt‘gl. (14) were of the opinion that the blood-brain barrier must be broken in order for treatment to be successful. Habel (15) also reported on extensive studies using mice, guinea pigs and monkeys as test animals. His findings indicated that serum.administered at the site of infection within seventyatwo hours was the most effective. Also a much higher percentage of animals survived, if the serum prophylaxis was followed with a series of daily injections of phenolized vaccine. Most workers agree that in order to be effective the serum.concen- trates must be given not later than three to five days after exposure, followed by a series of vaccine treatment, with the number of treat- ments dependent upon the severity and location of the exposure. The illustrated publication of Blatt £2.§l0 (16) serves a good purpose in showing the anguish and suffering of patients infected with rabies. The number of such cases could probably be lessened with wider use of antirabies immune serum. A. III. EXPERIMENTAL METHODS A. PREPARATION OF IMMUNIZING ANTIGEN Rabbits were infected with the Michigan Department of Health (MDH) strain of rabbit adapted fixed rabies virus. This strain is one re- portedly fixed by Pasteur and has had continuous rabbit passage in these laboratories since 1932. The animals were inoculated intracere- brally with 0.2 ml of a 1:100 dilution of the centrifuged supernatant fluid of rabbit brain suspension. Symptoms of fixed virus rabies ap- peared four days after infection. The rabbits were sacrificed, using air embolism, within forty-eight hours after exhibiting symptoms and the brains removed aseptically. The brains were weighed and stored at ~55°C until the time of emulsion. Emulsification to a twenty percent tissue concentration in distilled water was done in a Waring Blendor. The emulsions were dispensed into glass ampoules, flame sealed, the contents shell frozen and stored at ~55°C. The mouse brain virus (NIH-CVS) was obtained from the National Institutes of Health. White Swiss mice of eighteen to twenty grams were inoculated intracerebrally with 0.03 ml of a 1:100 dilution of the mouse brain virus. Four days after inoculation they exhibited symptoms of fixed virus rabies and twentybfour to thirty-six hours later were sacrificed under ether. The brains were removed, pooled in a common container, weighed and stored at -55°C until the time of emulsion. Emulsification was performed in the same manner as for the rabbit brain. B. INOCULATION OF RABBITS Healthy young white rabbits weighing approximately six pounds were selected for this study. Age was considered important, since it was necessary to carry the study over a period of eight months. The backs of the rabbits were closely clipped. The intracutaneous route was selected because the work of Habel (15) indicated that it yielded the highest antibody titers. In general, inoculations were started with 0.25 ml of the centrifuged supernatant liquid of a ten percent brain emulsion. Inoculations were made twice weekly for a period of three weeks, and the dosage increased by 0.25 ml every three weeks until a maximum of 1.5 ml was reached. Inoculations were then made only weekly. No more than 0.5 to 0.75 ml was inoculated into any one site. Twelve rabbits were hyperimmunized with the MDH virus and eighteen with the NIH—CV8 virus. Eight rabbits inoculated with the NIH virus developed fixed virus rabies, data which are in agreement with the findings of Habel (15). None of the rabbits that received the MDH virus exhibited symptoms. 0. BLEEDING OF RABBITS The rabbits were bled from the heart four times at monthly inter- vals, beginning two months after the start of the experiment. They were then bled twice monthly for the last two months of the experiment, sacrificing the animals at the final bleeding. The animals received no immunizing injection one week prior to bleeding. Serum was separated from.the clotted blood the day after it was drawn. Approximately 1.5 ml from each rabbit was set aside for complement fixation tests. For 6. virus neutralization testing, pooled serums were used. The rabbits were divided into groups of five or six each, two groups for MDH virus immu- nization (groups 1 and 2) and two groups for NIH-CV3 virus immunization (groups 3 and A). At each bleeding a serum pool was prepared for each group, using 0.5 ml of serum from each rabbit. The remainder of the serum.was pooled in two bottles according to the strain of virus used in the immunizing injections. All serums were shell frozen and stored at -55°C. D. IN VITRO NEUTRALIZATION All tests were performed with sixteen to eighteen gram white Swiss mice of random sex and six weeks of age. The first tests were carried out with the raw serum.mixed with mouse brain fixed virus, but later tests were done with both the mouse and the rabbit brain fixed viruses. The following dilutions of fixed virus were prepared: 1:5, 1:50, etc., through l:500,000,000. Serum dilutions of 1:2, 1:h, 1:8 were also pre- pared. Serum and fixed virus mixtures were prepared by adding equal volumes of the diluted serum to diluted virus. Thus, the final dilution of virus would be 1:10, 1:100, 1:1,000 and 1:10,000, and the final serum dilutions 1:2, 1:4, 1:8 and 1:16. The higher dilutions of virus were come bined with equal volumes of normal rabbit serum for inoculation into the control mice. The mixtures with fixed virus were incubated in a 37°C water bath for one hour, immediately chilled, and inoculated intracere- brally into mice in 0.03 ml amounts. Mice were observed for fourteen days. In the experiments with street virus, cow or dog brain suspensions were used. They were first treated with 1,000 units of penicillin and 2,000 units of streptomycin per ml to destroy or inhibit any bacteria that may have been present. Saline was used as the diluent for making the virus suspensions. The virus-serum mixtures were made as described in the preceding paragraph except that the final serum dilutions were made as high as 1:16,000. Only purified serum was used in these tests. The mixtures were incubated at 37°C for one hour prior to inoculation of the mice. The animals were observed for symptoms of street virus rabies for twenty-eight days. The final dilutions of both the fixed and street viruses for inoculation of the control mice were made with undiluted normal rabbit serum. E. COMPLEMENT FIXATION The method of Kolmer (17) was used in the performance of these tests. The reagents were prepared in the laboratory except for the hemolysin, which was purchased from Difco Laboratories.* The normal mouse brain and infected mouse brain antigens were prepared according to the procedure outlined by Espana and Hammon (18). F. PREPARATION OF REFINED SERUM The serums were removed from storage at -55°C and kept overnight at +5°C for thawing. Serum.obtained from rabbits immunized with MDH antigen and that obtained from.rabbits immunized with NIH antigen were purified separately. The method used in purification was that of Heidelberger_gt_al. (19). The protocols are as follows: Concentration and Refinement of Rabies Antiserum 1. The volume of raw serum was MDH 1280 m1 and NIH 1100 m1. 2. A temperature of 25°C was used for the concentration of the serum. * Difco Laboratories, Detroit, Michigan 3. h. 5. 13. 8. The pH was adjusted to 7.3 with 1:5 glacial acetic acid. Anhydrous sodium sulfate was added to a 16% concentration and the solution was stored overnight at 25°C to permit the precipitate to reach equilibrium.with the supernatant solution. The precipitate was separated by centrifugation and then washed four times with 16% sodium sulfate by resuspending and recentrifuging. The precipitate was dialyzed through two layers of #300 cellophane* (.001 inch thick) for eighteen hours at 15°C in running tap water and for forty-eight hours at 5°C in running distilled water. The volume of concentrated serum.was MDH 100 m1 and NIH 108 ml. Phenol was added to 0.5% concentration. NaCl crystals were added to 0.85% concentration. Super-ce1** was added to 1.0% concentration to assist in the removal of suspended particles. The solution was then centrifuged for one hour at 3000 R.P.M. and the precipitate discarded. Analysis of the final solution for total solids indicated the follow- ing: MDH 14.1% and NIH 13.7%. The solution was sterilized by filtration through one ST-3 pad,*** 6 cm diameter. * E. I. Dupont De Nemours Company, Cellophane Division, Wilmington, Delaware. %* Johns Manville Company, 832 Fisher Building, Detroit, Michigan. *** Hercules Filter Company, 20h Twenty-First Avenue, Patterson, New Jersey. 15. l6. 17. 18. In both lots the sterility test on the bulk products was performed by planting two tubes consisting of 15 ml of thioglycollate medium with 0.5 ml each and observed for seven days. No evidence of growth was observed. The final solutions of purified antiserums were dispensed into bottles at a volume of 10 ml each and immediately frozen. The frozen serum was then dried from the frozen state by sublimation. The sterility test on the reconstituted dried serum was performed in the same manner as for the bulk product. No evidence of growth was observed. The pyrogen test was performed in rabbits. They were injected intravenously with 3.0 ml of serum.for each kilogram of body weight. Two animals were used for each serum. No significant changes in body temperature were noted during the required three-hour observa- tion period. A safety test was performed on the reconstituted dried serum and consisted of the intraperitoneal inoculation of 0.5 ml into each of two 20 gram mice and 5 m1 into each of two 300 to 350 gram guinea pigs. They showed no reaction or loss in weight over the seven-day observation period. 10. IV. EXPERIMENTAL RESULTS A. IN VITRO NEUTRALIZATION TESTS The method of Reed and Muench (20) for calculating fifty percent endpoints was followed for determination of the neutralization index. To obtain an index, the fifty percent endpoint dilution of the protected mice was subtracted from the fifty percent endpoint dilution of the un- protected control mice, with the difference being the neutralization index. Animals can be a source of considerable variation when they are used for biological titration purposes. It is generally accepted that it is necessary to obtain nearly a ten-fold difference in the results from one test to another in order for them to be significant. 1. Eixed virus. The indices obtained for the unpurified serum pools from each bleeding are given in Table I. The serums of all the pools showed a satisfactory index at the first bleeding, but only in pool 1 when tested with NIH-CV8 virus was there a significant rise in titer from the first to the last bleeding. The titer of the serum pools showed considerable variation from one bleeding to another. The raw serums from the rabbits immunized with MDH and NIH antigens contained less antibody against MDH challenge virus at the conclusion of the experiment than was present in the serum at the time of the initial bleeding. 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