WO1993011795A1

WO1993011795A1 - Anti-idiotypic vaccine for protection against bovine respiratory syncytial virus

Info

Publication number: WO1993011795A1
Application number: PCT/US1992/010903
Authority: WO
Inventors: Paul J. Hall; Thomas B. Anderson
Original assignee: Integrated Biotechnology Corporation
Priority date: 1991-12-10
Filing date: 1992-12-10
Publication date: 1993-06-24

Abstract

The present invention provides a novel vaccine for protection of cattle against bovine respiratory syncytial virus (BRSV) infection. A vaccine composition includes an anti-idiotypic monoclonal antibody reactive against an idiotypic determinant of a primary antibody selected to interactably bind immune response evoking epitopes of BRSV, along with a pharmaceutically acceptable carrier for the anti-idiotypic monoclonal antibody. The vaccine composition is introduced into an animal to evoke an immune response in the animal that mimics the active immune response to BRSV proteins, effectively immunizing the animal.

Description

ANTI-IDIOTYPIC VACCINE FOR PROTECTION AGAINST BOVINE RESPIRATORY SYNCYTIAL VIRUS

Background and Summary of the Invention This invention relates to anti-idiotypic antibodies capable of binding to primary antibodies produced in response to bovine respiratory syncytial virus proteins. More particularly, the present invention is directed to monoclonal anti-idiotypic antibodies and their preparation and use for the production of vaccines promoting active immune response against bovine respiratory syncytial virus infection.

One of the major causes of lower respiratory tract infections in cattle is bovine respiratory syncytial virus (BRSV) , so named because of the characteristic formation of large syncytial masses in infected cell cultures. A related virus, Respiratory Syncytial Virus (RSV) was first identified in 1956 as the cause of an outbreak of upper respiratory disease in a chimpanzee colony (Morris et al., 1956, Recovery of

Cytopathogenic Agent from Chimpanzees with Coryza. Proc Soc Exp Biol Med 92:544-9.). and was initially called "Chimpanzee Coryza Agent". The following year antigenically identical viruses were isolated from two human infants, and the virus was named for its characteristic cytopathologic effects exerted on infected tissue (Channock et al., 1957, Recovery from Infants with Respiratory Illness of a Virus Related to Chimpanzee Coryza Agent (CCA). II. Epidemiologic Aspects of Infection in Infants and Young Children. AM J Hyg

66,.'291-300) . RSV is now recognized as the leading cause of hospitalization of children during the first year of life (Stott et al. , 1985, Respiratory Syncytial Virus. Brief Review. Archiv Virol 81:1-52). and also as the major viral cause of nosoco ial illness in children already hospitalized for other reasons (Arditin, M.S. (1988) Nosocomial Viral Infections in Neonatal Units. Part II. J Noso Infect 5(11:10-20.) . Half of all infants are estimated to become infected with RSV during -the first year of life, and virtually all infants have been infected by their second year.

The possibility of RSV infection in bovine populations was suggested by the discovery of antibodies to RSV in cattle serum (Dogget, 1965, Antibodies to Respiratory Syncytial Virus in Human Sera From Different Regions of the World. Bull World Health Org (32. :849- 853) . Bovine RSV (BRSV) was isolated two years later in Switzerland during an outbreak of respiratory disease in dairy cattle. Shortly after this report, a large epidemic of BRSV was reported in Japan (Inaba et al. , 1973, Physicochemical Properties of Bovine Respiratory Syncytial Virus, Jap J Micro 17:211-16; Inaba et al. , 1972, Bovine Respiratory Syncytial Virus. Studies on an Outbreak in Japan 1968-1969. Jap J Micro 16:373-83). BRSV was first isolated in the U.S. in 1974 from feedlot cattle in Iowa (Smith et al. , 1974, Isolation, Characterization, and Pathogenicity Studies of a Bovine Respiratory Syncytial Virus. Arch Virol 47:237-47.; and Missouri (Rosenquist, 1974, Isolation of Respiratory Syncytial Virus from Calves with Acute Respiratory

Disease. J Infect Pis 130(21:177-182.) , and BRSV is now known to infect all types of cattle, including nursing and weanling calves, feedlot steers, dairy calves and heifers, and lactating dairy cows (Ciszewski, D.K. (1990) Bovine Respiratory Syncytial Virus. Proc Iowa State Vet Assoc Ann Mtg:201-13.)

While BRSV produces clinical signs of infection in cattle of all ages, symptoms are most notable in young beef calves. Infection is evidenced by nasal and lacrimal discharge, increased r.espiratory rate, and elevated rectal temperature (104° to 108° F) . Other early clinical signs include mild depression, decreased feed intake and hypersalivation. In the later stages of disease manifestation, dyspnea with mouth breathing and frothing of saliva becomes pronounced. Subcutaneous emphysema may arise, and secondary bacterial pneumonia is a common complication. Serum antibodies to BRSV are now found in 60-80% of the cattle population of the United States and Canada (Baker, J.C. (1986) Bovine Respiratory Syncytial Virus: Pathogenesis, Clinical Signs, Diagnosis, Treatment, and Prevention. The Compendium (Food Animal 8(91:31-38.) . Morbidity during epidemic outbreaks is usually high, and mortality has been reported in up to 20% of affected cattle. Human, Bovine, Sheep, and Goat RSV, along with pneumonia virus of mice, have been classified into the genus pneumovirus within the paramyxoviridae family (Stott et al. (1985) Respiratory Syncytial virus. Brief Review. Archiv Virol 81:1-52: Baker et al. (1985) Bovine Respiratory Syncytial Virus. Vet Clinics of N Amer: Food Animal Practice 1(2) :259-275) . This family of viruses is comprised of pleomorphic enveloped virons containing a linear, single-stranded, negative RNA genome. Pneumoviruses are distinguished from other paramyxoviridae by the absence of neura inidase. RSV is further distinguished from other paramyxoviridae by its lack of hemagglutinin. The viruses of the paramyxoviridae family are known to be highly labile, and quickly lose viability outside host cells (Ciszewski (1990) Bovine Respiratory Syncytial Virus. Proc Iowa State Vet Assoc Ann Mtg:201-13) .

The genome of Human RSV has been studied in considerable detail, and codes for at least 10 distinct proteins. These proteins have been classified by as F, G, L, M, M2, N, NS, NS2, SH, and P (Stott et al (1985), Respiratory Syncytial Virus. Brief Review. Archiv Virol 81:1-52). Of these proteins, five have proposed or established functions: F (fusion glycoprotein) , G (attachment glycoprotein) , L (polymerase) , M (envelope matrix protein) , and N (nucleoplasmid protein) . The function of the remaining proteins M2 (also an envelope protein) , P (phosphoprotein) , NS (nonstructural) , and NS2, and SH (small hydrophile) , have yet to be determined. Although classification of Bovine RSV proteins has not been as extensive, molecular weights and functions of viral proteins have been identified in the bovine species (Cash et al. (1977) , A Comparison of Human and Bovine Respiratory Syncytial Viruses and Murine Pneumonia Virus. Virol 82:369-79.; Merz et al. (1980) , Importance of Antibodies to the Fusion Glycoprotein of Paramyxoviruses in the Prevention of Spread and Infection. J Exp Med 151:275-88.) . More recently, RNA analysis of Bovine RSV has revealed ten genes coding for ten proteins (Lerch et al., (1989) Characterization of

Bovine Respiratory Syncytial Virus Proteins and mRNAs and Generation of cDNA clones to the Viral mRNAs. J Virol 63(21:833-40.) . In this study, the Bovine RSV proteins N, P, M, F, and G were tentatively identified. Of these proteins, N, P, and M were found to be serologically related to human RSV using Western blot analysis and immunoprecipitation.

Of the 10 RSV proteins identified, protein F appears to be especially critical to the pathogenesis of infection, and is in fact the key protein mediating infection for all the paramyxoviridae (Merz et al. (1980) , Importance of Antibodies to the Fusion Glycoprotein of Paramyxoviruses in the Prevention of Spread and Infection. J Exp Med 151:275-88.) . The native fusion protein (usually designated FO) of this family of viruses is apparently a 70 kDa disulfide bonded glycoprotein that reduces to two fragments Fl (49 kDa) and F2 (20 kDa) (Lambert et al (1983) , Respiratory Syncytial Virus Glycoproteins. Virology 77:125-134.) . Protein F is involved in cell-fusing activity of the virus leading to syncytia formation. In addition, protein F mediates the hemolyzing activity of BRSV as well as viral penetration via fusion of viral and cell membranes. Bovine RSV vaccines effective to neutralize the F-protein have been developed. Killed virus vaccines are currently available from a variety of sources ( e.g.. Diamond Scientific, Inc., Norden, Grand Laboratories, and Boehringer Animal Health, Inc.). However, the use of such killed vaccines has currently fallen out of favor because of questionable efficacy and high associated costs. In addition, formalin-inactivation typically used to kill the virus is believed to alter the chemical and steric properties of the neutralizing epitopes of the F protein (Prince et al. (1986) , The Use of Purified

Immunoglobulin in the Therapy of Respiratory Syncytial Virus Infection. Ped Infect Pis 5(3):S201-3) .

The virus vaccines available from Diamond Scientific and Norden have been widely used by the cattle industry. The vaccines are administered intra-muscularly (IM) , and are recommended by the manufacturers for cattle of all ages. After three weeks, a booster injection is recommended, and repeated annual boosters are required. Though administration of these vaccines is considered beneficial, their efficacy in reducing morbidity and mortality in infected cattle remains controversial. While several studies have suggested substantial reductions in respiratory disease and mortality (Stott et al. (1987) Immune & Histopathological Responses In Animals Vaccinated With Recombinant Vaccinia Viruses That Express Individual Genes Of Human RSV. J Virol 61(121:3855-3861; Syvrud et al. (1988) Testing the Efficacy of BRSV vaccination in Herds with Variable Respiratory Problems. Vet Med April:429-30. ; Syvrud, (1989) Vaccination for Bovine Respiratory Syncytial Virus: Benefits for both Cow/Calf Herds and Feedlot Cattle. Bov Proceed 21:204-6.) , other workers have disputed these findings. For example, in a recent study involving five separate cattle herds, immunization with BRSV vaccine was carried out on 8,401 calves and yearlings. Variable effects were observed, and only two herds showed statistically significant benefit from vaccination. One herd actually recorded an increase in the treatment of respiratory infections. The authors of this study concluded that the cost of the present vaccines may not justify the reduction in BRSV infection attributable to vaccination (Donkersgoed et al. (1990) : Five Field Trials on the Efficacy of a Bovine Respiratory Syncytial Virus Vaccine. Can Vet J 31:93-100.) . In the Donkersgoed study, an initial vaccine administration was followed by administration of a vaccine booster injection. However, multiple injections for each animal are very costly in materials, time, and labor. Industry sources (Indiana Cattlemen's Association) indicate that the logistics of repeated vaccine injections precludes animal producers from using these vaccine products, or from using the products correctly for maximum protective effect.

Formalin-inactivation of BRSV is believed to account for need for multiple booster injections of the vaccine. Formalin treatment of live virus alters the neutralizing epitopes of F protein, and renders the vaccine incapable of inducing a highly effective active immunity. In fact, the vaccine itself may be responsible for severe disease observed in vaccine recipients (Prince et al. (1986) , The Use of Purified Immunoglobulin in the Therapy of Respiratory Syncytial Virus Infection. Ped Infect Pis 5(31:5201-3) . Prince and his colleagues showed that formalin-inactivated vaccine generated an unbalanced immune response in cotton rats, suggestive of an Arthus (Type HI) reaction, followed by Type IV delayed hypersensitivity. These authors suggest that the vaccine primes the host for an accelerated immune response to non-protective epitopes, resulting in the auto- immunopathology. Interestingly, formalin alteration of surface glycoproteins has also been associated with the failures of killed measles and killed mumps vaccines (Norrby et al (1976), Measles Vaccination. VII. The Occurrence of Antibodies of Antibodies Against Virus Envelope Components after Immunization with Inactivated Vaccine. Effects of Revaccination with Live Measles Vaccine. Acta Paediatr Scand 65:171-176.; Deinhardt et al. (1965) Immunization Against Mumps Prog Med Virol 11:126153.. Anti-idiotypic based vaccines present an alternative to conventional killed or live attenuated vaccines. An anti-idiotypic antibody is functionally defined as antibody that immunologically interacts with a primary antibody, binding idiotypes presented by that primary antibody. The idiotypes of the primary antibody are sets of antigenic determinants that interact with epitopes presented by a given antigen (e.g. , BRSV proteins) . Since the spatial and electronic characteristics of an idiotype are complementary to the antigen, an anti-idiotypic antibody is in effect a complementary image of a complementary image of the antigen, and presents steric and electronic characteristics corresponding to epitopes of the antigen. Anti-idiotypic antibody based bovine respiratory syncytial vaccines in accordance with the present invention have a number of advantages. First, because no viral material is used in the vaccine, the possibility of virulent infection from either the live virus or attenuated viral strains capable of reverting to a fully virulent state is non-existent. An anti-idiotypic antibody vaccine evokes an active immune response in an animal by mimicking the steric and electronic properties of viral protein epitopes, but the anti-idiotypic antibody is not otherwise functionally equivalent to the viral proteins. Second, the risk of contamination of the vaccine from viral proteins is reduced, again because viral proteins are not directly required to produce the anti-idiotypic antibody based vaccine. A third advantage is the stable steric configuration of anti-idiotypic antibody based vaccines. In contrast to killed virus vaccines, in which those viral proteins most suitable for stimulating immune response are often destroyed or altered by the killing agent (such as heat, acid, or alkali treatment) , an- anti-idiotypic antibody based vaccine does not require subjecting the immune stimulating anti-idiotypic antibodies to harsh deactivating conditions that may cause steric or electronic destabilization. Because of these advantages, as well as the low cost of production of anti-idiotypic antibodies from monoclonal hybridomas in accordance with the invention, vaccine production based upon anti-idiotypic monoclonal antibodies presenting epitopes mimicking BRSV protein immunogenic epitopes are superior agents for manufacture of a BRSV vaccine.

The present invention provides a novel vaccine for protection of cattle against bovine respiratory syncytial virus infection. A vaccine composition includes an anti-idiotypic monoclonal antibody reactive against an idiotypic determinant of a primary antibody selected to interactably bind immune response evoking epitopes of BRSV, along with a pharmaceutically acceptable carrier for the anti-idiotypic monoclonal antibody. The vaccine composition is introduced into an animal to evoke an immune response in the animal that mimics the active immune response to BRSV proteins, effectively immunizing the animal.

In accordance with the present invention, a hybridoma cell line (e.g., deposited cell line ATCC HB10901) is selected to produce an anti-idiotypic monoclonal antibody immunologically reactive against an idiotypic determinant of monoclonal antibody that specifically reacts with bovine respiratory syncytial virus proteins (e.g., monoclonal antibody 15C7, supplied by Dept. of Veterinary Science, Univ. of Nebraska,

Lincoln, Nebraska) . Antibodies secreted by cultures of this deposited cell line can be used to prepare BRSV vaccine compositions.

A vaccine protecting against bovine respiratory syncytial virus infection can be produced from a monoclonal antibody presenting an epitope having steric and electronic similarity to an epitope of bovine respiratory syncytial virus. The steric and electronic similarity must be sufficiently close so that antibodies produced in response to challenge of a bovine immune system by the monoclonal antibody will cross-react with any introduced bovine respiratory syncytial virus.

Additional features and advantages of the invention will become apparent to those skilled in the art upon consideration of the following detailed description of preferred embodiments exemplifying the best mode of carrying out the invention as presently perceived. Detailed Description of the Invention

Production and testing of an anti-idiotypic based vaccine in accordance with the present invention requires an antibody capable of immunologically reacting to specifically bind whole bovine respiratory syncytial virus (BRSV) , proteins derived by chemical modification or physical dissociation of BRSV virions, or proteins recombinantly produced to mimic BRSV proteins. The binding antibody may be either a monoclonal antibody secreted by a hybridoma, or an antibody selected from polyclonal antisera. Alternatively, chemically or genetically modified antibody binding fragments (e.g. , Fv, Fabs) , expression products of an antibody producing gene, other conventional antigen binding proteins capable of binding to BRSV fragments, or electronic and steric mimics of BRSV epitopes may be used. Such BRSV-binding proteins are identified as "primary" antibodies, a term also intended to embrace any protein that is an electronic and steric complement to BRSV proteins. The present invention includes production of proteins capable of binding primary antibodies that bind to BRSV. The binding protein can be an antibody or antibody fragment (e.g., Fv, Fabs), and includes a monoclonal antibody secreted by a hybridoma, or an antibody isolated and purified from polyclonal antisera. Such proteins are identified as "anti-idiotypic" antibodies, a term intended to embrace those antibodies capable of reacting to bind a primary antibody, a chemically modified primary antibody, or recombinantly produced electronic and steric primary antibody mimics.

Anti-idiotypic antibodies are commonly produced as a result of an animal's immune response, the response being triggered by injection of a primary antibody into the animal. Injection of the primary antibody is preferably into animal species phylogenetically distinct from bovine species, with mice, rabbits, chickens, or guinea pigs being preferred. When mice are used, laboratory strains including BALB/c, C57BL/b, DBA/2 or A strain mice are preferred, although other inbred strains may be used. Outbred or wild-type mice may also be used. Injection may be directly into the animal's popliteal lymph nodes, although more typically, intraperitoneal, intrasplenic, multiple intradermal (subcutaneous) sites, or a single intramuscular site are used. When multiple intradermal injections are used, the primary antibody is injected at 20-40 sites spread over the body of the animal. Because of variability of immune response, the use of multiple animals, booster injections (secondary injections, tertiary injections, etc.) of the primary antibody, or alternative conventional immunization methods may be required.

Immune response of injected animals can be enhanced by coupling (conjugating) the primary antibody to an exogenous carrier molecule. Proteins (both glycosylated and non-glycosylated) , lectins, polysaccharides, and water soluble vitamins may be chemically coupled to the primary antibody to augment immune response. Because of their high immunogenic potential and ease of chemical coupling, large carrier proteins such as keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA) are often chemically coupled to the primary antibody, although other conventional protein carriers may be used.

Co-injected adjuvants can also be used to enhance immune response of animals injected with a primary antibody. Preferred adjuvants include water and oil emulsions; inorganic compounds (metals, salts, etc.); synthetic polynucleotides; hormones; cyclic nucleotides; endotoxins; and lymphokines or monokines. Preferred water and oil emulsions include both Freund's incomplete adjuvant and Freund's complete adjuvant (composed of Freund's incomplete adjuvant plus heat killed and dried mycobacterium tuberculosis) , both which can be administered to enhance immune response. Preferred inorganic adjuvants include alum (potassium aluminum sul ate) , aluminum hydroxide, or calcium phosphate. Mineral oils, nitrocellulose filters, polyaerylamide, or even charcoal can also be used. Other preferred adjuvants can include muramyl dipeptide (MDP) , interleukins including recombinant bovine interleukin-2 (rBo-Il-2) , liposomes, metabolizable oils, and synthetic lipid A analogs.

Injection of an optimal amount of the primary antibody into an animal is critical, in part because injection of too little primary antibody can result in failure to evoke an immune response with consequent anti- idiotypic antibody production. However, injection of large amounts of the primary antibody is also undesirable since it can lead to the establishment of a tolerant state in the injected animal. To determine the optimal amount, batches of animals may be injected with different amounts of primary antibody. Those animals receiving too little of the primary antibody to elicit an immune response can later be given a larger dose of the primary antibody. Booster injections are often necessary, with the first of these booster injections being given when an animal's primary immune response (which includes production of anti-idiotypic antibodies) is past its peak (typically about 3-7 weeks after the primary injection of antigen in the case of rabbits) . About 7-10 days after the first booster injection test bleeds may be taken, and further booster injections given at regular intervals to those animals showing maximum response to the primary antibody. Since required boost interval for maximum immune response may vary from animal to animal, a boost is typically given when titer of circulating anti- idiotypic antibody begins to drop. The amount of primary antibody used for boosting injections is usually two- to three-fold less than that used for primary immunization. Booster injections can be made intramuscularly or subcutaneously.

Spleen lymphocytes of animals exhibiting desired anti-idiotypic antibody titer levels are selected for hybridization with immortalized myeloma-like cell lines. The resultant hybridomas secrete monoclonal antibodies, and mixed populations of the resulting hybrid cells are tested for their ability to secrete antibody of the desired specificity. Cell clone lines derived from individual cells are established. Ideally, the supernatant of the monoclonal hybridoma contains up to 50 μg/ml of the anti-idiotypic antibody. However, even greater amounts of antibody (5-20 mg/ml) can be obtained by growing the cloned hybridoma as an ascites tumor in the peritoneal cavity of mice. The anti-idiotypic antibody can be purified by affinity separation or conventional chromatographic separation techniques.

Whole anti-idiotypic antibodies are not required for vaccine preparation. As previously discussed, anti-idiotypic monoclonal antibodies can be chemically modified (e.g., with Fc fragment cleaved), provided that the immunogenicity of the anti-idiotypic antibody binding region is not degraded. Of course, such antibody fragments may be coupled to carrier molecules or co-administered with adjuvants as described in the following.

Vaccine preparation and administration is by conventional techniques. An appropriate anti-idiotypic antibody is diluted in a suitable excipient or pharmaceutical effective carrier solution (typically physiologic saline, although pure water or other conventional liquids may be used) , and the vaccine is administered to the animal. Various routes of immunization are possible, including intra-muscular, subcutaneous, oral, or intra-nasal administration. Exogenous carrier molecules such as those previously described can be coupled to the anti-idiotypic antibody to enhance immune response. Co-administered adjuvants, including oil emulsions, interleukins, bacterial MDP analogues, or liposomes that enhance not only humoral immune responses but local mucosal IgA immune responses may be used. Typical dose levels of anti-idiotypic antibody to a calf ranges from an initial injection of about 10 micrograms to about 1000 micrograms of anti- idiotypic antibody, although preferably from about 100 micrograms to about 200 micrograms is injected and most preferably about 150 micrograms is injected. Secondary or booster injections, if required, can be of either higher or lower dose levels.

In addition to use in a BRSV vaccine, anti- idiotypic antibodies produced in accordance with the present invention may be used for detection and quantitation of BRSV in bovines. For example, qualitative or quantitative assays using radioactively or enzymatically labelled anti-idiotypic antibodies can detect circulating antibodies to BRSV in cattle sera, enabling diagnosis of bovine populations at risk for BRSV infection.

The following Examples describing preparation and testing of an anti-idiotypic antibody and an anti- idiotypic antibody based vaccine to protect against bovine respiratory syncytial virus infection. These Examples are not intended to limit the scope of the invention, but are presented to aid in the understanding of the present invention. EXAMPLE 1

Production of anti-idiotypic antibody producing hybridomas

Myeloma cell lines obtained from the Transplant

Immunology Laboratory of the Department of Surgery at Indiana University School of Medicine were used as controls to check for contamination. Monoclonal antibodies 8G12 and 15C7, specific for bovine respiratory syncytial virus (BRSV) , were purchased from the Department of Veterinary Science, Institute of Agriculture and Natural Resources, University of Nebraska-Lincoln, Lincoln, Nebraska. Characterization of the antibodies showed that the antibodies neutralized BRSV and inhibited syncytia formation in vitro. These antibodies were shown by indirect fluorescent assay to stain an external envelope protein (Protein F) of living BRSV infected cells, and were further recognized by the presence of a 48K subunit of the viral fusion protein as indicated by Western blot analysis of bovine respiratory syncytial virus infected cell lysates.

The specificity of MAb's 8G12 and 15C7 for BRSV were verified by determining their ability to bind to a conventional BRSV vaccine in an enzyme linked immunoabsorbent assay (ELISA) . Attenuated live BRSV vaccine preparation (NORDEN Laboratories) was used as the target antigen. MAb's 8G12 and 15C7 were conjugated to horseradish peroxidase (HRP) using standard conjugation procedures. BRSV vaccine solution (50 μg) was incubated on a microtiter plate (Immunlon II) for two hours at room temperature. The plate was then washed with phosphate buffered saline (PBS) and 0.05% TWEEN-20. A solution of 2% fetal bovine serum and 10 mM lysine was added to each well to block non-specific binding sites. The plates were washed again with PBS/Tween-20 and a serial dilution of HRP-labeled 15C7 and 8G12 was added to each well and incubated at room temperature for one hour. The plate was again washed with PBS/Tween-20 and enzyme substrate indicator solution was added and incubated for approximately 15 minutes until a colorimetric reaction was observed. The optical density was measured at 492 nm on a Dynatech microtiter plate reader.

Monoclonal antibodies 8G12 and 15C7 were then chemically coupled (conjugated) to Keyhole Limpet Hemocyanin (KLH) , a carrier protein useful for imparting immunogenicity to the covalently coupled hapten molecule (the 8G12 and 15C7 antibodies in this example) . BALB/c mice were immunized with this conjugated preparation (the "primary" antibodies) by biweekly intraperitoneal injections, as well as with unconjugated 8G12 and 15C7 by direct intrasplenic injection.

Intra-peritoneal immunizations required 50-100 μg of the primary antibodies 8G12 and 15C7 emulsified in 200 μl complete Freund's adjuvant. After two weeks, a boost of 50 μg of the primary antibodies emulsified in 200 μl incomplete Freund's adjuvant was administered. This injection was repeated again after two more weeks. Following each boost, the animals were test bled, and the blood serum analyzed by enzyme linked immunoabsorbent assay (ELISA) for anti-idiotypic antibodies produced in response to injection of the primary antibodies.

Each intra-splenic injection (504 μg primary antibody in 200 μl AdjuPrime, Pierce Chemicals) required anesthetizing a BALB/c mouse with nembutal (300 μl of 4mg/ml solution) and making a 1-1.5 cm oblique incision in the skin just under the left rib cage. Blunt dissection of the skin layer was performed and the peritoneum incised. The spleen was visible just under the stomach, allowing the medial pole to be gently lifted, pulled through the incision to the exterior of the abdominal cavity and the primary antibody/AdjuPrime mixture injected directly into the spleen through a 1 ml tuberculin syringe with a 271/2 gauge needle. The spleen was then replaced and the muscle and skin layers were sutured with a 4-0 surgical silk. Serum was collected at routine intervals by retro-ocular orbital puncture. Pre-immunized mouse serum was used as negative control in ELISA assays to ensure that labeled 15C7 and 8G12 antibodies do not react with other antibodies present in mice sera. BRSV vaccine was used as a positive control. Three days following the final boost, the animals were bled by intra-cardiac puncture, and immediately sacrificed by cervical dislocation. The spleens were removed and splenocytes were cultured by the following procedures.

Hybridoma culture production was maintained in a base medium consisting of a 1/1 mixture of RPMI and Delbecco's Modified Eagle Medium supplemented with glutamine, pyruvate, MEM non-essential amino acids,

2-mercaptoethanol, and 1% Nutridoma. The murine myeloma cell line SP2/0 was selected because of its adaptability to serum free culture medium. A stock- of SP2/0 (100,000 - 500,000 cells/ml) was constantly maintained in culture flasks for continuous fusions. Myeloma cells were periodically exposed to 8-azaguanine to eliminate revertant mutants and maintained at an optimal concentration of 2-3 x 10 5 cells/ml to ensure a logarithmic growth phase. Splenocytes derived from primary antibody injected BALB/c mice and myeloma cells were mixed 1:1, and centrifuged at 400 g for five minutes to form a single pellet. The supernatant was aspirated, and the cells were resuspended in 6 mis of serum-free medium. Three mis of polyethylene glycol (PEG 1500) were slowly added with stirring over about one minute, and the cells were left at room temperature an additional seven minutes. The cells were centrifuged at 400 g for five, minutes, and allowed to sit an additional seven minutes. The cells were diluted to 50 mis, centrifuged, resuspended, and placed in culture media.

After fusion, the cells were resuspended, divided into 200 μl aliquots in 96 well plates, and incubated in 7% C0₂ at 37°C. Two to three days after fusion, media containing hypoxanthine-aminopterin- thymidine (HAT media) was introduced, and the cells were maintained in HAT for the next week. Feeding was accomplished by aspiration of approximately half of the culture medium, and replacement with fresh media. Culture in HAT was continued for one to two weeks until clones become visible. Cells were then moved to hypoxanthine-thymidine (HT) media. After five feedings with HT, the cells were maintained on the base media.

New hybridoma cultures producing anti-idiotypic antibodies were identified by ELISA using original primary antibodies as the antigen and following conventional procedures known in the art. Supernatants from a master plate containing clones were screened by ELISA for anti-idiotypic antibody production. Supernatants were sampled by placing 50 to 100 μl aliquots of supernatant in 96 well microtiter plates and storing overnight at 4°C. Subsequently, 200 μl of a 2% fetal bovine serum solution was added to each well to block any free binding sites. Horseradish peroxidase- labeled primary antibodies were incubated with the prepared plates to detect the presence of any potential anti-idiotypic antibodies present in the hybridoma supernatants.

Following the above procedure, a hybridoma cell line producing monoclonal anti-idiotypic immunologically reactive against idiotypic sites of monoclonal antibodies 15C7 was identified and deposited with American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland, USA, 20852 under Accession Number ATCC ΗB10901. Ascites production was used to increase the available amount of anti-idiotypic antibody. Mice were primed by intra-peritoneal injection of 0.5 ml pristane. Approximately 100 hybridoma cells were injected after priming. After ascites production was evident (7-21 days) , 3-5 mis of ascites fluid was removed every 2-3 days until death. Cells were removed from the ascites fluid by centrifugation at 400 g for five minutes, counted, and frozen in liquid nitrogen or reinjected into other pristane primed mice. In addition to ascites production, tissue culture based production of the anti-idiotypic antibody was also investigated. Although antibody production in cell culture was only about 1/1000 relative to ascites based production, up to 100 mis of culture can be obtained without special equipment. The cell culture supernatant was free of contamination by host immunoglobulins found in ascites fluid, and in serum-free media the monoclonal anti-idiotypic antibody is the dominant protein in solution. Monoclonal anti-idiotypic antibodies were partially purified by ammonium sulfate precipitation. Saturated ammonium sulfate was prepared and added dropwise to the sample while stirring, 50% concentration. Stirring was continued an additional 20-30 minutes, and followed by centrifugation at 10,000 g for five minutes. The resu. ant formed pellet was washed twice with 50% ammonium sulfate, and then dissolved in phosphate buffered saline (PBS) . Ammonium ion was subsequently removed by dialysis in PBS.

Affinity chromatography was also used for characterization and testing of the anti-idiotypic antibody. Cyanogen bromide (CNBr) activated agarose beads were first washed with distilled water followed by 2M NaHC0₃-Na₂C0₃. Activation was achieved by the addition of CNBr dissolved in acetonitrile with constant stirring to a concentration of 100 mg CNBr per gram of agarose gel. After 15-20 minutes, the gel was washed on a sintered glass funnel with 1 mM HCI, followed by distilled water. The activated agarose was weighed, and scooped into a solution of anti-idiotypic antibody (about 1 g wet agarose per 10 mg antibody) in 0.1 M NaHC0₃ at pH 8. This solution was mixed at room temperature for two hours. The remaining active sites on the beads were neutralized by the addition of 0.01 M glycine, and incubated at room temperature for an additional two hours. Beads were recovered by centrifugation, and washed three times with alternating solutions of 0.1M sodium acetate in 0.5M NaCl at pH 4, followed by 0.1M Tris-HCl in 0.5 M NaCl at pH 4. The beads were resuspended in PBS, pH 7.4, and packed into a column. The column was pre-cycled with elution buffer, followed by extensive washing with PBS. Approximately 2-2.5 mg anti-idiotypic antibody per mg of agarose-bound primary antibody were applied to the column in PBS, pH 7.4, at a flow rate around 1-2 ml/min. Elution of the column was done with glycine-HCl, pH 2.2-2.8. Eluate was dialyzed against phosphate buffered saline and concentrated by Amicon filtration to a working dilution.

EXAMPLE 2 Vaccine Preparation and Testing

The affinity isolated anti-idiotypic antibodies were prepared as vaccines by routine procedures. Two types of test vaccines have been made, one with 150 μg anti-idiotypic antibody emulsified in 0.5 ml complete Freund's adjuvant and a second with 150μg anti-idiotypic antibody emulsified in alum (Pierce Chemical Co.). For controls, these same emulsion types were made with a BALB/c myeloma, MOPC-21. One week old, random sex dairy calves obtained from local sources (Hoosier Stockyards, Lebanon, Indiana) were used to test the ability of anti- idiotype antibody vaccines to stimulate anti-BRSV immune responses in cattle. Only calves sero-negative for BRSV antibodies were used in initial immunization trials. The animals were injected with the vaccine and control solutions at multiple intramuscular and subcutaneous locations. Secondary injections of both vaccine and control were given two weeks after the initial injection, followed by third and fourth injections of antigens at two week intervals. The fifth administration of antigen was given intra-nasally and consisted of lOOμg antigen in sterile saline. On days 3, 5, 7, 11, and 14 following vaccine administration, calves were bled by jugular veni- puncture, and serial dilutions of the blood sera were screened in a BRSV specific ELISA system. Preliminary results indicate that only those calves given anti- idiotypic based vaccine have increased anti-BRSV antibody titers.

Claims

Claims:

1. Hybridoma cell line ATCC HB10901.

2. A hybridoma selected to produce an anti-idiotypic monoclonal antibody immunologically reactive against an idiotypic determinant of one of monoclonal antibody 15C7 and monoclonal antibody 8G12.

3. A hybridoma producing an anti-idiotypic monoclonal antibody immunologically reactive with an idiotypic determinant of a primary monoclonal antibody, the idiotypic determinant of said primary monoclonal antibody immunologically reactive with bovine respiratory syncytial virus.

4. A monoclonal antibody produced by hybridoma cell line ATCC HB10901.

5. An anti-idiotypic monoclonal antibody immunologically reactive with an idiotypic determinant of one of monoclonal antibody 15C7 and monoclonal antibody 8G12.

6. An immunologically reactive protein presenting an antigen binding region specific to bind an idiotypic determinant of a primary monoclonal antibody immunologically reactive against bovine respiratory syncytial virus.

7. A vaccine for protection against bovine respiratory syncytial virus infection, the vaccine comprising an anti-idiotypic antibody immunologically reactive with a primary antibody, the primary antibody being immunologically reactive with bovine respiratory syncytial virus proteins, and a pharmaceutically effective carrier therefor.

8. A vaccine for protection against bovine respiratory syncytial virus infection, the vaccine comprising an anti-idiotypic monoclonal antibody reactive against an idiotypic determinant of a primary antibody, the idiotypic determinant of said primary antibody being i munologically reactive to bind with bovine respiratory syncytial virus, and a pharmaceutically effective carrier therefor.

9. An immunodiagnostic agent comprising an anti-idiotypic monoclonal antibody reactive against an idiotypic determinant of a primary antibody, the idiotypic determinant of said primary antibody being immunologically reactive with bovine respiratory syncytial virus, and means for labelling the anti-idiotypic monoclonal antibody.