ATTENUATION OF VACCINES WITH MONOCLONAL ANTIBODY

Abstract: Live vaccines having attenuated virulence are made by incubating live infectious organisms with excess neutralizing monoclonal antibody and growing surviving variants.

Description: ATTENUATION OF VACCINES WITH MONOCLONAL ANTIBODY The invention described herein was made in the course of .work under a grant from the Department of Health and Human Services. This invention relates to the attenuation of live vaccines of infectious organisms and pertains more specifically to a process for iτι vitro attenuation of live vaccines which eliminates the need for blind passages of the vaccine through animals or tissue culture materials. The classical method for attenuating live vaccines involves repeated blind passages of the disease causing virus or other infectious orgamism through successive animals or tissue culture materials, assaying its virulence after each such passage, until its virulence has been attenuated to the desired degree. This is a random process which operates by chance, so that the results cannot be predicted; in the case of some diseases, e.g., yellow fever, a very large number of successive passages through animals have been required to achieve an acceptable attenuation, making the production of such vaccines very time-consuming and expensive. Moreover, attenuation of viruses by such methods alters the product from the original virus in ways which are not well understood. It has now been found that live vaccines can be attenuated jLn vitro without the need for passage through an animal or tissue culture. The method of attenuating virus of the present invention comprises incubating a live infectious organism with excess neutralizing monoclonal antibody specific to the organism to cause the antibody to bind to the organism, and separating surviving unbound organism variants from said bound organism to provide vaccine of attenuated virulence. While the method of the present invention can be employed with any live infectious organism such as bacteria, fungi, parasites or the like, it is of particular value when the organism is a virus, especially a reovirus or a rotavirus, as well as any of those organisms which have been reliably attenuated only with difficulty, such as respiratory syncitial virus. The desired neutralizing monoclonal antibody can be prepared by immunizing an animal such as a mouse by injection with the infectious organism, removing the spleen cells of the animal and fusing them with myeloma cells to form hybridoma cells, cloning the hybridoma cells in a serum-containing medium, separating the monoclonal antibodies produced from the medium, and screening them by neutralization assay for those monoclonal antibodies capable of neutralizing the infectivity of the organism. The organism is then grown in a suitable medium containing excess neutralizing monoclonal antibody. The neutralizing antibody binds specifically to the site on the orgamism which is involved in binding to the host cell receptors and kills almost all of the infectious organisms, only a very few variants surviving. However, the small number of variants that do survive have lost all or essentially all of their capacity to produce lethal infection. These essentially non-virulent variant organisms can be grown normally in tissue culture cells and are effective to induce protective neutralization antibody formation upon injection into mammals. They are consequently useful as vaccines and can be administered by any form of percutaneous injection in the usual manner. The following example will serve to illustrate more fully the nature of the present invention without acting as a limitation upon its scope. - Example Immunization of mice Adult mice were immunized with mammalian reovirus type 3 (Dearing) , an in ectious organism which results in a highly fatal encephalitis in suckling mice. BALB/σ mice were obtained from the National Institutes- of Health. The mice did not ' have neutralizing antibody against reovirus at the time of the initial immunization. The mice were immunized by 7 intraperitoneal injection of 5 x 10 PFU of gradient purified type 3 reovirus (Dearing) in gelatin-saline (0.8% NaCl, 0.003% CaCl 2 , 0.07% MgCl 2 , 6H 2 0, 0.12% H 3 B0 3 , 0.005% a 2 B 4 0 ? , 10H 2 0, 0.3% gelatin) . Injection occurred initially four months and again one month prior to the myeloma cells fusions. Three, days prior to the fusion, the animals were inoculated intravenously with 3 x 10 PFU of virus in gelatin-saline. The spleens of three immunized animals were aseptically removed and the spleen cells were dispersed by repeated flushing with Dulbecco's modified minimal essential medium (DMEM) at 4°. Myeloma Cells The mouse myeloma cell line BALB/c P-3-NSl/l-Ag -l (NS1) (Kohler and Milstein, 1976) was maintained in suspension culture in DMEM supplemented with 7.5% heat inactivated in suspension culture in DMEM supplemented with 7.5% heat inactivated (56°, 30 min.) fetal calf serum (FCS) and 7.5% heat inactivated agamma newborn calf serum (DMEM-15%) . Construction of Hybridomas The spleen cells were washed with DMEM and mixed with NS1 myeloma cells at a ratio of 4:1. The cells were pelleted by centrifugation and fused with 50% polyethylene glycol (PEG 1500) (Nowinski et al., 1979, Lostrom et al., 1979). After fusion, the cells were pelleted and resuspended in DMEM containing 20% fetal calf serum (DMEM-20%) and were dispersed into 96 well icrotiter plates (Linbro) . On day one, a half 4 volume of.DMEM-20% containing 10 M hypoxanthine, 4 x -7 —5 10 M aminopterin, 1.6 x 10 M thymidine and 3 x 10 —6 M glycine (HAT media) was added. .On day 2, 3, 6, 9, 12 and 15 half the media was removed and replaced with HAT media. After day 15, media was replaced with DMEM-20% containing 10 -4M hypoxanthine and 1.6 x 10~ 5 M thymidine (Nowinski et.al., 1979). Colonies became visible between the second and third week after initiation of the culture. Culture supernatants were screened for anti-viral antibodies by a radioimmunoassay procedure. The reovirus was added to polyvinyl chloride plates (Cooke Engineering Co.) at q 10 pfu/well in 25 μl PBS and allowed to air dry. The plates were then incubated with 50 μ 1 of methanol per well until dry; and then with 5% bovine serum albumin (BSA) for 2-3 hours. Culture fluids (25 μl) from the hybridoma cells were added to each well and kept at 37° for one hour. The wells were then washed 4 times with PBS-1% BSA ( ostrom et al., 1979). Rabbit antimouse IgG antisera (Cappel) that had been iodinated with chlora ine T (Greenwood et al., 1963) was added ς (10 cpm/well) and the plates were incubated overnight at 4°. The specific activity of the iodinated antiseru was 1.8 x 10 cpm/mg of protein. Wells were washed five times with PBS, cut out, and counted in a gamma counter. Hybridoma Cell Cloning Hybrid cell lines producing monoclonal antibodies, as determined by the foregoing radioimmunoassay, were cloned by dilution and growth in 0.3% agar (Cotton et al., 1973). Colonies were allowed to grow until readily visible by eye. They were then transferred with a pasteur pipette to one well of a 96-well plate containing 0.1 ml of IMEMZO (improved minimal essential media supplemented with zinc, insulin, and H ' epes buffer) media (International Biological Laboratories, Inc.) containing 15% FCS. Cells were grown up to mass culture and subsequently maintained in IMEMZO containing 5% heat inactivated fetal calf serum and 5% agamma newborn calf serum. Isolation of Monoclonal IgG Antibody Cloned hybridoma cell cultures, grown up to a 400 ml volume by daily feedings with fresh media were maintained at this volume for 48 hours and the cells were removed by centrifugation at 400 g for 20 min. The supernatants were removed and phenylmethylsulfonyl fluoride (PMSF) was added to a concentration of 100 yg/ml. The supernatant was applied to a 3 ml bed volume column of protein A-Sepharose CL-4B (Sigma) at a flow rate of approximately 25 ml/hr (Lee et al., 1981) . The column was washed with PBS until no protein was detectable in the eluent and the IgG was eluted with 0.1M glycine-HCL (pH 2.5). The fractions containing IgG were pooled (4 ml) and dialyzed against PBS (4 liters). Aliquots were stored at -20°. All antibodies were used at a concentration of -1-2 mg/ml. Neutralization Assays Virus preparation were diluted to 2 x 10 PFϋ/ l in gelatin-saline. Purified and isolated hybridoma IgGs prepared as described were diluted serially in 5-fold steps in gelatin-saline and 0.2 ml of the IgG dilutions was mixed with 0.2 ml of the virus. The neutralization was carried out at 34° for 1 hr (Weiner and Fields, 1977). Duplicate 0.1 ml .samples were then plated in 6 well cluster dishes (Costar) and allowed to adsorb for 1 hr. The cells were then overlaid with agar and the plaque assays were performed as reported previously (Fields ' and Joklik, 1969) . The neutralization titer is expressed as the highest dilution of antibody which neutralized 80% of the input virus (Weiner and Fields, 1977). The desired monoclonal antibody, designated as G5, exhibited no detectable neutralization of reoviruses of serotype 1 (Lang) or 2 (Jones) at a 1:10 dilution of antibody, but exhibited effective neutralization titer of 12,500 against the reovirus serotype 3 (Dearing) which had been used to immunize the mice, as well as neutralization of additional type 3 isolates from natural sources (Hrdy et al., 1979). The antibody subclass was determined by ouchterlony analysis to be IgG- . It was determined to be specific to the viral σ -1 protein, a viral outer capsid protein of serotype 3 by immunoprecipitation of S-methionine labelled infected cell lysates, and thus was specific to the site which has been well known to be involved in binding the virus to host cell receptors. ~ . Incubation of Virus With Neutralizing Monoclonal Antibody Reovirus serotype 3 was incubated with an excess of the G5 neutralizing monoclonal antibody. Viral plaques that appeared on the plates were isolated and passaged a second time in the presence of G5 antibody to eliminate clones not actually resistant to this antibody. Resistant viruses (variants) were then grown into virus stocks in mouse L cells. Three variant viruses (A, F. and K) isolated in this manner were tested to determine their relative capacity to resist neutralization by the G5 monoclonal antibody. All three variants were altered in their response toG5 antibody: the F and K variants were totally resistant to the antibody while the A variant was neutralized to a level intermediate between the Dearing parent and the F and K variants. For example, the A variant was neutralized at the 1:20 and 1:100 dilutions of G5, but the plaque reduction at 1:500 was of borderline significance. In contrast to the results with the G5 monoclonal antibody, the three variants were indistinguishable from the parental reovirus type 3 Dearing virus in neutralization tests using a type-specific hyperimmune antiserum. These data indicate that all three variants encode an antigenically altered he agglutinin with antigeniσ alterations that are readily detected with monoclonal antibodies but not with a standard hyperimmune antiserum. Determination of Virulence To determine if the variants were altered in terms of virulence, they were examined as to their relative capacity to cause fatal neurological disease in suckling mice. The type 3 Dearing parent is highly neurovirulent; as few as 10 PFU inoculated intracerebrally (i.e.) into suckling mice kills 50% of mice by 14 days while higher does kill 100% of mice. To compare the neurovirulence of type 3 Dearing and the variant viruses, we injected varying doses of virus into suckling mice and determined the survival pattern for each infected group of animals. The F and K 7 variants had LD-. n values of 3 x 10 and > 3 x 10 7 PFU respectively while the A variant had an intermediate LD 5Q of 1.8 x 10 PFU. These results indicated that all of the variants were at least 10,000 times less neurovirulent than the Dearing strain from which they were derived. Interestingly, the antigenic variant that was intermediate in antigenic reactivity, the A variant, also had an intermediate LD CΛ (1.8 x 5 5 10 ) while, in contrast, the F and K variants that had totally lost the capacity to be neutralized by G5 were most dramatically altered in virulence (LD,- n 3 x 7 7 10 and >3 x 10 ) . These data suggest that the loss of antigenic reactivity with the G5 antibody is directly related to the neurovirulence. The A, F and K variants all were found to grow to high titer in mouse L cells, and were not temperature sensitive for such growth. When injected into mice, these non-virulent variants induced the development of protective neutralizing antibodies to an extent comparable to that of the original virulent Dearing type 3 reovirus, as shown by the results set forth in the following table: Table I Neutralization of Reovirus Type 3 (Dearing) and Variants Using Hyperimmune Mouse Antisera Percent of Inoculum Resistant to Antibody Vi rus es Dilution of - Var ants Ant body Antibody Dearing A_ F K Anti- Dearing 1:10 0% 0% 0% 0% 1:100 4% 9% 4% 0.72 1:1000 . 6% 20% 8% 4% Aπti-A 1:10 Q% % 0% 0% 1:100 15% 18% 4% 5% 1:1000 19% 32% 17% 6% Anti-F mo 0% 0% 0% 0% 1:100 5% 17% 3% 1% 1:1000 24% 26% 4% 1% Aπti-K 1:10 ' Q% _% 0% 0% 1:100 4% 11% 3% 2% 1:1000 19% 13% 4% 2% The variants are consequently effective vaccines against reovirus type 3 infection when mammals are inoculated with effective amounts of the variants. Similar results may be obtained with other viruses and infectious organisms. An approach to enrich the selection of mutants makes use of the affinity of the G5 monoclonal to the HA3 bearing virus. In this case G5 is plated on polystyrene plates (1 mg/ml) . To this immobilized G5 monoclonal is now added 10 12 PFU of reovirus. The reovirus will bind to the G5 monoclonal. The reovirus has a mutation rate of 10 —6, which indicates that one in a million reoviruses will not bind to the G5. The non adherent viruses are collected after one hour at 4°C and passed again on a second plate to which G5 is immobilized. After this second cycle the non adherent virus is collected and is grown up in the standard manner. The only virus which escapes binding (i.e. is incapable of binding G5) will be mutant or variant in G5. In this system, second mutants at other sites can be selected quickly. Anti Vic (a second viral protein) monoclonal antibodies can be plated on polystyrene dishes, and the G5 variants subjected to negative selection by this approach as well. By this technology mutants at any one or multiple sites can be selected.

Claims: What is claimed is: . - 11 - 1. The method of attenuating live vaccines which comprises incubating a live infectious organism with excess monoclonal antibody specific to the organism to cause the antibody to bind to the organism, and separating surviving unbound organism variants from the bound organism to provide vaccine of attenuated virulence. 2. The method as claimed in claim 1 in which said organism is a virus. 3. The method as claimed in claim 1 in which said organism is a reovirus. 4. The method as claimed in claims 1, 2 or 3 in which said antibody binds specifically to the site of the organism involved in binding to host cell receptors. OMPI \r . VlfO

Inventors: FIELDS BERNARD N, FIELDS, BERNARD, N.
Applicants: HARVARD COLLEGE, PRESIDENT AND FELLOWS OF HARVARD COLLEGE
Application year: 1983
Main classification: A61K39
IPC codes: A61K39, C07K16