Quadrivalent humanized anti-rabies virus monoclonal antibody and application thereof

Abstract

The invention discloses a humanized tetravalent anti-rabies virus monoclonal antibody, the light chain amino acid sequence of the antibody is as shown in SEQ ID No.5, and the heavy chain amino acid sequence of the antibody is as shown in SEQ ID No.3. Compared with a bivalent antibody, the antibody disclosed by the invention can enhance the neutralizing effect on the rabies virus at the current clinical common titer concentration (100IU / mL); meanwhile, the local diffusion speed of molecules is reduced, and the local residence rate of the tetravalent antibody is increased; the comprehensive biological efficacy of local application of the tetravalent antibody can reach 1.9 times of that of a bivalent antibody, and the dosage of the antibody in prevention measures adopted after clinical exposure of the III-level rabies virus can be reduced.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a novel tetravalent human anti-rabies virus monoclonal antibody and application thereof. Background Art

[0002] Rabies is an ancient animal epidemic disease, a fatal infectious disease caused by the bite of a dog infected with the rabies virus, with a mortality rate of nearly 100%. Until the 21st century, the disease still exists widely, causing serious harm to human health; especially in developing countries, due to the lack of appropriate epidemiological control and related resources, about 59,000 people die from rabies every year worldwide. Therefore, rabies is still one of the serious problems facing global health care.

[0003] Although rabies cannot be cured, it can be prevented. Pre-exposure and post-exposure prevention is the only way to deal with it. Pre-exposure prevention is the vaccination of rabies vaccine, which is given to high-risk people such as dog trainers, pet doctors and some scientific researchers; post-exposure prevention is the treatment of people who have contact with suspected rabid animals. The WHO vaccination program (2010) and my country's "Rabies Exposure Prevention and Treatment Work Specifications (2023 Edition)" regulations and recommendations are the standard documents currently used for clinical diagnosis and treatment.

[0004] In current clinical practice, the preventive measures adopted after exposure to grade III rabies virus include the use of purified anti-rabies virus polyclonal antibodies in human serum, local infiltration injection plus intramuscular injection away from the wound, but there are problems such as difficulty in obtaining, unstable supply, large dosage and high cost; currently, human anti-rabies virus monoclonal antibodies produced by bioengineering technology are on the market, which are bivalent antibodies, but still have the characteristics of large dosage and high price, which imposes a certain economic burden on patients. According to the "Third Edition Report of the World Health Organization Expert Consultation on Rabies" and the "Expert Consensus on Rabies Exposure Prevention and Treatment" (2019), it is feasible for grade III exposed people to only infiltrate immunoglobulin in and around the wound, and it is no longer recommended to inject excess immunoglobulin away from the wound. Local application is the trend, but studies have shown that only 24.4% of the activity remains after local injection of immunoglobulin in the wound for 24 hours. Therefore, further improving the local neutralization effect of anti-rabies immunoglobulin on rabies virus and prolonging the local retention rate are the future development directions. Summary of the invention

[0005] On the one hand, the present invention provides a human tetravalent anti-rabies virus antibody, the light chain amino acid sequence of the antibody is shown in SEQ ID No.5, and the heavy chain amino acid sequence of the antibody is shown in SEQ ID No.3.

[0006] In one embodiment, the antibody is a monoclonal antibody, a recombinant antibody or a polyclonal antibody, preferably a monoclonal antibody.

[0007] On the other hand, the present invention also provides a DNA encoding the above antibody.

[0008] In one embodiment, the encoding DNA of the light chain is as shown in SEQ ID No.6.

[0009] In one embodiment, the encoding DNA of the heavy chain is as shown in SEQ ID No.4.

[0010] On the other hand, the present invention also provides an expression vector, which comprises the DNA encoding the above antibody.

[0011] As used herein, the term "vector" refers to a nucleic acid construct designed for transfer between different host cells. An "expression vector" refers to a vector that has the ability to incorporate and express heterologous DNA fragments in exogenous cells. Many prokaryotic and eukaryotic expression vectors are commercially available. The selection of an appropriate expression vector is well known to those skilled in the art.

[0012] In one embodiment, the expression vector is pcDNA3.4.

[0013] On the other hand, the present invention also provides a host cell, wherein the host cell comprises the above antibody or the above expression vector.

[0014] In one embodiment, the host cell includes prokaryotic cells and eukaryotic cells, preferably mammalian cells, for example, 293F cells.

[0015] In another aspect, the present invention provides a pharmaceutical composition comprising the above antibody and a pharmaceutically acceptable carrier.

[0016] On the other hand, the present invention also provides the use of the above-mentioned antibody in preparing an immune reagent for rabies virus.

[0017] The present invention obtains a tetravalent humanized anti-rabies virus monoclonal antibody by modifying the antibody sequence. At the currently commonly used clinical titer concentration (100 IU / mL), the neutralization effect on rabies virus can be enhanced compared with the bivalent antibody; at the same time, the local diffusion rate of the molecule is reduced, and the local retention rate of the tetravalent antibody is improved; the comprehensive biological efficacy of the local application of the tetravalent antibody can reach 1.9 times that of the bivalent antibody, which can reduce the amount of antibody used in the preventive measures adopted after clinical exposure to grade III rabies virus, thereby alleviating the problems of difficulty in obtaining, unstable supply, large clinical dosage and high cost, and can also reduce the suffering of patients and reduce the burden on society and patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1.Antibody configuration diagrams of SO57 and 4RSO57-08Lds; the left diagram shows the IgG configuration of the original bivalent antibody SO57, and the right diagram shows the IgG configuration of the tetravalent antibody 4RSO57-08Lds.

[0019] Figure 2 . Gel electrophoresis of anti-rabies virus monoclonal antibody protein. The left band in the right picture is divalent SO57; the right band is tetravalent 4RSO57-08Lds. The position of the bands is consistent with the molecular weight.

[0020] Figure 3 .Comparison results of SO57 and 4RSO57-08Lds antibody titers.

[0021] Figure 4 : The results of the local retention experiment of two antibodies in mice. The figure shows the cy7-labeled fluorescence signal at the skin lesion on the right thigh of the mouse. DETAILED DESCRIPTION

[0022] The present invention is further described below in conjunction with the examples. The following description is only a preferred embodiment of the present invention and is not intended to limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change it into an equivalent embodiment with equivalent changes. The materials, reagents, instruments and methods used in the following examples are conventional materials, reagents, instruments and methods in the art unless otherwise specified, and can be obtained through commercial channels.

[0023] Example 1. Design and expression of tetravalent human anti-rabies virus monoclonal antibody:

[0024] In this embodiment, the design and modification are based on the bivalent human anti-rabies virus monoclonal antibody SO57.

[0025] The light chain amino acid sequence of monoclonal antibody SO57 is shown in SEQ ID No.1 (the light chain variable region is underlined), and the light chain DNA sequence is shown in SEQ ID No.2; the heavy chain amino acid sequence is shown in SEQ ID No.3 (the heavy chain variable region is underlined), and the heavy chain DNA sequence is shown in SEQ ID No.4.

[0026] qsaltqprsvsgspgqsvtisctgtssdigynfvswyqqhpgkapklmiydatkrpsgvpdrfsgsk sgntasltisglqaedea dyyccsyagdytpgvvfgggtkltvl gqpkaapsvtlfppsseelqankatlvclisdfypgavtvawkadsspvkagvetttpskqsnn kyaassylsltpeqwkshrsyscqvthegstvektvaptecs(SEQ ID No. 1).

[0027] cagagcgccctgacccagcccaggagcgtgagcggcagccccggccagagcgtgaccatcagctgcaccggcaccagcagcgacatcggcggctacaacttcgtgagctggtaccagcagcaccccggcaaggcccccaagctgatgatctacgacgccaccaagaggcccagcggcgtgcccgacaggttcagcggcagcaagagcggcaacaccgccagcctgaccatcagcggcctgcaggccgaggacgaggccgactactactgctgcagctacgccggcgactacacccccggcgtggtgttcggcggcggcaccaagctgaccgtgctgggccagcccaaggccgcccccagcgtgaccctgttcccccccagcagcgaggagctgcaggccaacaaggccaccctggtgtgcctgatcagcgacttctaccccggcgccgtgaccgtggcctggaaggccgacagcagccccgtgaaggccggcgtggagaccaccacccccagcaagcagagcaacaacaagtacgccgccagcagctacctgagcctgacccccgagcagtggaagagccacaggagctacagctgccaggtgacccacgagggcagcaccgtggagaagaccgtggcccccaccgagtgcagc(SEQ ID No.2)。

[0028] qvqlvqsgaevkkpgssvkvsckasggtfnrytvnwvrqapgqglewmggiipifgtanyaqrfqgrl titadeststaymelsslrsddtavyfcarenldnsgtyyyfsgwfdpwgqgtlvtvssastkgpsvfplatsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpsslgtqtyicnvnhkpsntkvdkrvepkscdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk (SEQ ID No. 3).

[0029]

[0030] In this embodiment, the light chain of monoclonal antibody SO57 is modified. Specifically, antibody NC08 that binds to different epitopes from SO57 antibody is selected, and its scFv sequence is analyzed. The glycine at position 44 in its VH sequence and the tryptophan at position 100 in its VL sequence are mutated to cysteine ​​to form a disulfide bond, thereby obtaining a dsFv sequence. The dsFv is connected to the C-terminus of the light chain of the original bivalent antibody SO57 through a (G4S)3 sequence. The amino acid sequence of the modified light chain is shown in SEQ ID No.5, and its encoding DNA sequence is shown in SEQ ID No.6. The heavy chain of the modified monoclonal antibody SO57 still uses the sequence shown in SEQ ID No.3. The modified tetravalent human anti-rabies virus monoclonal antibody is named: 4RSO57-08Lds.

[0031] qsaltqprsvsgspgqsvtisctgtsssdiggynfvswyqqhpgkapklmiydatkrpsgvpdrfsgsksgntasltisglqaedeadyyccsyagdytpgvvfgggtkltvlgqpkaaps vtlfppsseelqankatlvclisdfypgavtvawkadsspvkagvetttpskqsnnkyaassylsltpeqwkshrsyscqvthegstvektvaptecsggggsggggsggggsqvqlqesg pglvkpsetlsltctvsggsissvnsywgwirqppckglewignfyysgnthynpslksrvtisvgtsknqfslklnsvtaadtavyycarqstiggffdywgqgtlvtvssggggsgggg sggggsqsaltqpasvsgslgqsitisctgtssdignynlvswyqqhpgkapkliiyevtkrpsgvsnrfsgsksgntasltisglqaedeanyycssytatknycifgggtkltvr(SEQ ID No.5).

[0032]

[0033] The antibody configurations of SO57 and 4RSO57-08Lds are as follows Figure 1 shown.

[0034] Example 2: Construction and expression of antibody expression plasmid:

[0035] Antibody expression plasmid construction:

[0036] The light chain and heavy chain DNA sequences of the original bivalent antibody SO57 and the light chain DNA sequence fragment of the tetravalent antibody 4RSO57-08Lds were synthesized respectively, and inserted into the vector plasmid Pcdna3.4 to obtain three expression plasmids: Pcdna3.4-SO57LC, Pcdna3.4-SO57HC, and Pcdna3.4-4RSO57-08Lds;

[0037] The bacterial solution containing the target expression plasmid was cultured and amplified, and the plasmid was extracted in large quantities using the Tiangen endotoxin-free plasmid extraction kit. The specific operations were carried out according to the instructions.

[0038] Antibody expression and purification:

[0039] Mammalian 293F cell culture, using FreeStyle TM 293 Expression Medium culture medium was used and the culture was cultured in a 37°C, 5% CO2 incubator with a shaking flask at 125 rpm.

[0040] Cell transfection (taking 50ml culture medium as an example):

[0041] Cell preparation: One day before transfection, place 6-7x10 5 Cells were seeded at 1x10 cells / ml. The cell density should be 1x10 6 cells / ml; prepare transfection complex: take 50 μg of target plasmid (Pcdna3.4-SO57LC plus Pcdna3.4-SO57HC plasmid for expressing divalent antibody SO57, and Pcdna3.4-4RSO57-08Lds plus Pcdna3.4-SO57HC plasmid for expressing tetravalent antibody 4RSO57-08Lds); dilute with 2 ml of OptiPROTM SFM (Invitrogen) serum-free culture medium, mix thoroughly to prepare DNA dilution solution, take 250 μl of transfection reagent PEI and add it to the DNA dilution solution to prepare transfection complex, mix thoroughly, and leave at room temperature for 15 minutes; add the transfection complex to 293F cell culture medium, and continue to shake the flask and culture at 37°C, 5% CO2 incubator at 125 rpm for 96 hours.

[0042] Antibody protein expression and purification: After transfecting 293F cells with a plasmid containing the target gene, continue to culture on a shaker for 96 hours, centrifuge at 200g for 3 minutes to discard the cell pellet, collect the supernatant culture medium, and then centrifuge at 10000g for 15 minutes again to remove impurities in the culture medium, then filter with a 0.45μm filter membrane, and then use a 30kd concentrator tube at 3800rmp, 4℃ centrifuge to concentrate the volume 20 times, and continue to purify as required.

[0043] Purification of antibody protein:

[0044] Using Protein A SefinoseTM-5ml (Pre-Packed Gravity Column) chromatography column,

[0045] Pre-prepared: Bingding buffer: 0.1M Na3PO4, 0.15M NaCl, PH 7.2; elution buffer: 0.1M citric acid, PH 2.7; neutralizing buffer: 1M Tris-HCl PH 9.0; wash and balance: first rinse with 50ml ddH2O, then wash the column with 50ml elution buffer, and then balance with 50ml bingding buffer; load: drop the supernatant containing the target protein into the column, and load 2-3 times; elution: after loading, wash with 50ml bingding buffer to remove non-specific binding; prepare 10 15ml centrifuge tubes, add 800μl of pre-prepared neutralizing buffer to each 15ml centrifuge tube; elute the protein with 30ml elution buffer, and wash with 50ml bingding buffer to remove non-specific binding; prepare 10 15ml centrifuge tubes, add 800μl of pre-prepared neutralizing buffer to each 15ml centrifuge tube; elute the protein with 30ml elution buffer, and wash with 50ml neutralizing buffer Buffer prepared 15ml centrifuge tube connected to the eluent, every 3ml tube change; Segment recovery: collect the eluent, use nanodrop to determine the protein concentration of each tube, discard those below 0.05mg / ml, select ultrafiltration tubes of different specifications according to the molecular weight of the target protein, and concentrate the eluent with ultrafiltration tubes; Protein preservation: the final protein is packaged, quick-frozen in liquid nitrogen, and stored at -80℃. Wash the chromatography column and store it: wash the chromatography column with 50ml of bingding buffer, then wash it with 50ml of ddH2O, and finally wash it with 50ml of 20% ethanol and seal it.

[0046] Protein gel electrophoresis results Figure 2 shown.

[0047] Example 3, antibody neutralization titer detection and comparison:

[0048] The rapid fluorescent focus inhibition test (RFFIT) method was used to compare the neutralizing titers of antibodies by binding to rabies virus (conducted at Henan Zhongkang Medical Testing Co., Ltd.);

[0049] Experimental methods:

[0050] The viruses, cells and standards used in the experiment were prepared as follows: the international standard attack strain of rabies virus CVS-11 is a fixed virus; BSR cells are clones of hamster kidney cells BHK-21; the standard serum recommended by WHO is human immunoglobulin with a titer of 30 IU / ml.

[0051] (1) BSR cell culture: Trypsinize the BSR monolayer cells, perfuse the cells with DMEM high-glucose culture medium containing 10% fetal bovine serum, count the cells with a cell counting plate, and inoculate 2×10 cells per flask. 6 The cells were supplemented with culture medium and incubated in a 37°C 5% CO2 incubator.

[0052] (2) Virus titer determination: After the virus was thawed, two adjacent rows were selected on a 96-well plate and the virus was serially diluted 5-fold in DMEM culture medium containing 10% fetal bovine serum (30 μl of virus was added to 120 μl of DMEM culture medium). 50 μl of a 1×10 6 cells / ml of BSR cell suspension, incubate in a 37℃ 5% CO2 incubator for 24h; discard the culture supernatant, wash once with PBS; fix with 80% cold acetone at 4℃ for 30min; discard the acetone, add 50μl anti-rabies virus nucleoprotein fluorescent antibody staining solution (the fluorescent antibody is diluted 1:50 with PBS), incubate at 37℃ for 30min, and then wash three times with PBS; add 60% glycerol, count the number of fluorescent foci in each well under a fluorescence microscope, and calculate the titer.

[0053] (3) Virus preparation: Prepare cell suspension normally and count cells, add CVS-11 virus seed at MOI = 0.1 to the cell suspension, incubate in a 37°C 5% CO2 incubator for 1 hour, shake once every 15 minutes, transfer the virus and cell suspension to a cell culture bottle, incubate in a 37°C 5% CO2 incubator for 72 hours, collect the culture supernatant and dispense into small tubes, mark the batch (this is the stock virus), and store at -70°C. Prepare stock virus in large quantities using the same method.

[0054] (4) Titration of the optimal attack volume of the virus: The optimal attack volume of the stored virus was titrated. After the virus was thawed, a row was selected on a 96-well plate and the virus was serially diluted 2-fold with DMEM culture medium containing 10% fetal bovine serum (70 μl of virus was added to 70 μl of DMEM culture medium); 50 μl of the virus of each dilution was taken out and added to each well of another row (containing 50 μl of DMEM culture medium); 50 μl of a concentration of 1×10 6 The cell suspension was prepared at 50 cells / ml and cultured in a 37°C 5% CO2 incubator for 24 h. The highest dilution that allowed 80% of the cells to be infected (producing fluorescent foci) was detected by RFFIT.

[0055] (5) Setting of control system: The control system of this experiment includes standard serum control, negative serum control and virus control. The standard serum control adopts the international standard anti-rabies virus serum with a titer of 30 IU / ml recommended by WHO; the negative serum is the serum of people who have not been vaccinated against rabies.

[0056] (6) RFFIT method: The antibody protein to be tested was inactivated at 56°C for 30 min, and then diluted three-fold in series in a 96-well plate using DMEM culture medium containing 10% fetal bovine serum (50 μl of the serum to be tested was added to 100 μl of DMEM culture medium); the standard serum and negative serum were diluted in series using the same method; the virus control wells were diluted two-fold in series (the method was the same as the optimal attack volume titration); 50 μl of the optimal attack volume virus was added to each well except the virus control well, and after incubation at 37°C 5% CO2 for 1 h, 50 μl of a 1×10 6 cells / ml of cell suspension; incubated in a 37℃ 5% CO2 incubator for 24h; determined by RFFIT.

[0057] (7) Calculation of neutralizing antibody titer: Based on the application software Microsoft Excel, according to the dilution gradient determined in the operating procedure and in combination with the Reed & Muench method formula, a formula for calculating the neutralizing antibody titer was designed with the international standard serum titer as the reference coefficient and the dilution multiple before and after 50% infection wells of the tested sample as the variable.

[0058] The results are shown in Table 1 and Figure 3 shown.

[0059] Table 1. Results of the two antibodies neutralizing rabies virus using the RFFIT method

[0060]

[0061] The results showed that compared with the original bivalent human anti-rabies virus antibody SO57, the neutralization titer of the tetravalent human anti-rabies virus antibody 4RSO57-08Lds was improved at a molar concentration of ≤2.0nmol / ml.

[0062] It is worth noting that the titer concentration of the currently commonly used bivalent human anti-rabies virus antibody in clinical practice is 100 IU / mL. As can be seen from the figure, the titer concentration of the tetravalent antibody 4RSO57-08Lds at the corresponding equimolar concentration is about 130 IU / mL, which is 1.3 times the titer of the bivalent human antibody SO57.

[0063] Example 4: Antibody protein fluorescent labeling and local retention experiment in small animals:

[0064] 4.1 Use anthocyanin dye Cy7 to label the original bivalent antibody SO57 and the tetravalent human anti-rabies virus 4RSO57-08Lds antibody;

[0065] 4.2 Local residence experiment in small animals:

[0066] Using small animal in vivo imaging methods,

[0067] Balb / c albino mice, 8 experimental mice and one blank control, all weighing about 20g each, randomly numbered and prepared:

[0068] 24 h before the experiment, eight experimental mice were used to establish a mouse thigh skin injury model: the outer skin of the right thigh of the mouse was depilated, the skin was scratched with a surgical knife tip for about 0.5 cm, and then disinfected with 0.5% iodine tincture;

[0069] Before the formal experiment, the fluorescence signal of the Cy7-labeled protein sample was detected using a small animal live imaging instrument;

[0070] SO57 group, 4 rats, 100 μl of the prepared Cy7-labeled bivalent antibody SO57 (0.4 mg / ml) was injected subcutaneously at the skin lesion of the right thigh of each rat;

[0071] 4RSO57-08Lds group, 4 mice, 100 μl of the prepared Cy7-labeled tetravalent antibody, 4RSO57-08Lds (0.56 mg / ml), was subcutaneously injected into the skin lesion of the right thigh of each mouse;

[0072] Each group of mice was tested with a small animal live imaging instrument. The intensity of the Cy7-labeled fluorescence signal at the skin lesion on the right thigh of the mice was detected at 0h, 2h, 4h, 6h, and 24h after injection. A blank control mouse was added when taking pictures of each group;

[0073] The results are as follows Figure 4 And as shown in Table 2.

[0074] Table 2. Results of the local retention rate experiment of two antibodies in mice

[0075]

[0076] Note: Fluorescence value unit (10E5*p / s / cm 2 / sr);

[0077] As shown in Table 2, the average local retention rate of antibody 4RSO57-08Lds at 2h, 4h, 6h, and 24h was 1.48 times that of antibody SO57, and had a higher local retention rate.

[0078] In current clinical practice, the preventive measures adopted after exposure to grade III rabies virus require the use of anti-rabies virus polyclonal antibodies purified from human serum, which are injected locally by infiltration and injected intramuscularly away from the wound. However, there are problems such as difficulty in obtaining, unstable supply, large dosage and high cost.

[0079] The present invention obtains a tetravalent humanized anti-rabies virus monoclonal antibody, which can enhance the neutralization effect on rabies virus compared with a bivalent antibody at the currently commonly used clinical titer concentration (100 IU / mL); at the same time, the local diffusion rate of molecules is reduced, and the local retention rate of the tetravalent antibody is improved; the comprehensive biological efficacy of the local application of the tetravalent antibody can reach 1.9 times that of the bivalent antibody, which can reduce the amount of antibodies used in the preventive measures adopted after clinical exposure to grade III rabies virus, thereby alleviating the problems of difficulty in obtaining, unstable supply, large clinical dosage and high cost, and can also reduce the suffering of patients and reduce the burden on society and patients.

[0080] The preferred embodiments of the present invention are described in detail above. The detailed description of the technical scheme of the present invention with the help of the optimized embodiment is illustrative rather than restrictive. It cannot be determined that the specific embodiments of the present invention are limited to this. For ordinary technicians in the technical field to which the present invention belongs, without departing from the concept of the present invention, modifications to the technical schemes recorded in the embodiments or equivalent replacement of some of the technical features therein should be deemed to fall within the scope of patent protection determined by the claims submitted by the present invention.

Claims

1. A human tetravalent anti-rabies virus antibody, the light chain amino acid sequence of the antibody is shown in SEQ ID No.5, and the heavy chain amino acid sequence of the antibody is shown in SEQ ID No.

3.

2. The antibody according to claim 1, characterized in that The antibody is a monoclonal antibody, a recombinant antibody or a polyclonal antibody, preferably a monoclonal antibody.

3. A DNA encoding the antibody according to claim 1 or 2.

4. The DNA according to claim 3, characterized in that The light chain encoding DNA of the antibody is shown as SEQ ID No.6, and the heavy chain encoding DNA of the antibody is shown as SEQ ID No.

4.

5. An expression vector comprising the DNA according to claim 3 or 4. A host cell comprising the antibody according to claim 1 or 2, or the expression vector according to claim 5.

7. A pharmaceutical composition comprising the antibody according to claim 1 or 2 and a pharmaceutically acceptable carrier.

8. Use of the antibody according to claim 1 or 2 in preparing an immune reagent for rabies virus.

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