Rabies virus G protein epitope, and rabies virus neutralising binding molecule that binds specifically thereto

Abstract

This invention relates to a rabies virus G protein epitope and a rabies-virus-neutralizing binding molecule that binds specifically thereto, wherein different epitope sites of rabies virus G protein are identified and binding molecules that bind thereto and a cocktail thereof can be found to retain neutralizing activity against various rabies viruses.

Claims

1. A rabies-virus-neutralizing antibody which binds to an epitope located between amino acid residues 33 and 215 or an epitope located between amino acid residues 331 and 333 of the wild-type rabies virus G protein, the sequence of which protein is set forth in SEQ ID NO:2, wherein the antibody comprises: a) a heavy chain variable domain comprising the CDR1 of SEQ ID NO:3, the CDR2 of SEQ ID NO:4, and the CDR3 of SEQ ID NO:5; and b) a light chain variable domain comprising the CDR1 of SEQ ID NO:6, the CDR2 of SEQ ID NO:7, and the CDR3 of SEQ ID NO:8; or a) a heavy chain variable domain comprising the CDR1 of SEQ ID NO:9, the CDR2 of SEQ ID NO:10, and the CDR3 of SEQ ID NO:11; and b) a light chain variable domain comprising the CDR1 of SEQ ID NO:12, the CDR2 of SEQ ID NO:13, and the CDR3 of SEQ ID NO:14; or a rabies-virus-neutralizing fragment of such an antibody.

2. The antibody of claim 1, wherein the binding molecule has a binding affinity (K.sub.D) less than 110.sup.8 M.

3. The antibody of claim 1, wherein if the antibody comprises a) a heavy chain variable domain comprising the CDR1 of SEQ ID NO:3, the CDR2 of SEQ ID NO:4, and the CDR3 of SEQ ID NO:5; and b) a light chain variable domain comprising the CDR1 of SEQ ID NO:6, the CDR2 of SEQ ID NO:7, and the CDR3 of SEQ ID NO:8, the heavy chain variable region comprises the sequence set forth in SEQ ID NO:15.

4. The antibody of claim 1, wherein if the antibody comprises a) a heavy chain variable domain comprising the CDR1 of SEQ ID NO:3, the CDR2 of SEQ ID NO:4, and the CDR3 of SEQ ID NO:5; and b) a light chain variable domain comprising the CDR1 of SEQ ID NO:6, the CDR2 of SEQ ID NO:7, and the CDR3 of SEQ ID NO:8, the light chain variable region comprises the sequence set forth in SEQ ID NO:16.

5. The antibody of claim 1, wherein if the antibody comprises a) a heavy chain variable domain comprising the CDR1 of SEQ ID NO:9, the CDR2 of SEQ ID NO:10, and the CDR3 of SEQ ID NO:11; and b) a light chain variable domain comprising the CDR1 of SEQ ID NO:12, the CDR2 of SEQ ID NO:13, and the CDR3 of SEQ ID NO:14, the heavy chain variable region comprises the sequence set forth in SEQ ID NO:17.

6. The antibody of claim 1, wherein if the antibody comprises a) a heavy chain variable domain comprising the CDR1 of SEQ ID NO:9, the CDR2 of SEQ ID NO:10, and the CDR3 of SEQ ID NO:11; and b) a light chain variable domain comprising the CDR1 of SEQ ID NO:12, the CDR2 of SEQ ID NO:13, and the CDR3 of SEQ ID NO:14, the light chain variable region comprises the sequence set forth in SEQ ID NO:18.

7. The antibody of claim 3, wherein the sequence of the heavy chain is set forth in SEQ ID NO:19.

8. The antibody of claim 4, wherein the sequence of the light chain is set forth in SEQ ID NO:20.

9. The antibody of claim 5, wherein the sequence of the heavy chain is set forth in SEQ ID NO:21.

10. The antibody of claim 6, wherein the sequence of the light chain is set forth in SEQ ID NO:22.

11. A composition comprising the antibody of claim 1 and a suitable carrier.

12. The composition of claim 11, further comprising a second antibody.

13. A method of preventing or treating rabies in a subject which comprises administering to the subject a prophylactically or therapeutically effective amount of the antibody of claim 1.

14. A method of diagnosing rabies in a subject which comprises contacting a sample from the subject with the antibody of claim 1 under conditions permitting formation of a complex between the antibody and any rabies virus present in the sample and detecting any complex so formed.

15. A package comprising: 1) the composition of claim 11; and (2) a package insert providing instructions for use of the composition.

Description

BRIEF DESCRIPTION OF DRAWING

(1) FIG. 1 shows the survival rates of mice against SV2 virus in an animal test.

MODE FOR INVENTION

(2) A better understanding of the present invention may be obtained via the following examples, which are set forth to illustrate, but are not to be construed as limiting the scope of the present invention. The documents cited herein are incorporated by reference into this application.

Example 1: Epitope Identification of Antibody

(3) In order to identify epitopes of two antibodies (hereinafter, referred to as antibody 1 or antibody 2) among four antibodies finally selected in Example 4 disclosed in Korean Patent Application No. 10-2014-0178030, a single mutation in the rabies virus G protein (SEQ ID NO:1) was induced, and the binding activity of antibody 1 and antibody 2 to each mutant was evaluated. The preparation of each mutant and the analysis of the binding activity of antibody 1 and antibody 2 thereto were performed using a shotgun mutagenesis method (J Am Chem Soc. 2009; 131(20): 6952-6954) of Integral Molecular, USA.

(4) A shotgun mutagenesis method enables the expression and analysis of a library of mutated target proteins in eukaryotic cells. Particularly, all residues of the target protein may be mutated with different amino acids and may be expressed in a mammalian cell line to analyze changes in monoclonal antibody (MAb) binding or function.

(5) A shotgun mutagenesis method was performed in two steps, in which the G protein expression plasmid of the wild-type rabies virus CVS-11 strain was prepared and expressed in a mammalian cell line, after which the antibody concentration for binding activity analysis of antibody 1 and antibody 2 was optimized.

(6) Thereafter, a mutant library (hereinafter, referred to as single mutant library), in which respective amino acid residues in the G protein of the rabies virus CVS-11 strain were substituted with other amino acids, was prepared using a shotgun mutagenesis method, and the binding activity thereof to the antibody was analyzed, and thus the epitopes of antibody 1 and antibody 2 were confirmed.

Example 1-1: Preparation of Plasmid Expressing Rabies Virus G Protein

(7) A plasmid was prepared using a gene (Genbank Ref # AAC34683.1) expressing the G protein of the rabies virus CVS-11 strain, and the C-terminus thereof was tagged with V5/HIS6 in order to verify the expression thereof. Next, the plasmid was expressed in HEK293 cells. The corresponding plasmid was temporarily transfected to the HEK293 cells, followed by incubating the plasmid containing only a vector as a negative control group and a positive control group, that is, a structure (WT RABV) expressing the rabies virus G proteome, in a 384-well plate for one day.

Example 1-2: Preparation of Single Mutant Library of Rabies Virus G Protein

(8) A single mutant library, in which each of amino acid residues at positions 1505 of rabies virus G protein (SEQ ID NO:2), except for a signal peptide, was substituted with a different amino acid, was prepared using a shotgun mutagenesis method. The plasmid prepared in Example 1-1 was used as a parent plasmid. Alanine scanning mutagenesis was performed as a mutagenesis strategy to produce 505 mutant clones (hereinafter referred to as alanine scanning mutants). The alanine scanning mutagenesis is a method of substituting the protein amino acid with alanine to evaluate the contribution of the specific amino acid site to the function, stability or external response of the whole protein. Also, using an escape mutant virus testing method (Korean Patent Application No. 10-2014-0178030), each of the amino acids at positions 34, 210, 331, 336 and 413 of the rabies virus G protein was substituted not with alanine but with the other amino acid residue to produce single mutant clones (hereinafter referred to as customized mutants). Therefore, the total library size consisted of 512 mutant clones, as shown in Table 1 below.

(9) TABLE-US-00001 TABLE 1 Properties of library used for epitope identification No. of Clone name Clone clones Alanine Substitution of each of rabies G protein amino 505 scanning acids 1-505 .fwdarw. Ala(A) mutants Substitution of Ala(A) in rabies G protein amino acids 1-505 .fwdarw. Ser(S) Customized Substitution of rabies G protein amino acid 34 7 mutants Gly(G) .fwdarw. Val(V) Substitution of rabies G protein amino acid 34 Gly(G) .fwdarw. Glu(E) Substitution of rabies G protein amino acid 34 Gly(G) .fwdarw. Arg(R) Substitution of rabies G protein amino acid 210 Val(V) .fwdarw. Glu(E) Substitution of rabies G protein amino acid 331 Ser(S) .fwdarw. Leu(L) Substitution of rabies G protein amino acid 336 Asn(N) .fwdarw. Lys(K) Substitution of rabies G protein amino acid 413 Glu(E) .fwdarw. Asp(D) Total number 512 *Amino acid position was numbered except for the signal peptide of rabies virus G protein

Example 1-3: Epitope Identification of Antibody

Example 1-3-1: Epitope Identification of Antibody Using Alanine Scanning Mutant

(10) In order to identify epitopes of antibody 1 and antibody 2, the binding activity to antibody 1 and antibody 2 was analyzed using the alanine scanning mutant library prepared in Example 1-2.

(11) In order to evaluate the binding of the alanine scanning mutant library of Example 1-2 and antibody 1 and antibody 2, immunofluorescent FACS was performed two times, and thus mutants were selected.

(12) When the whole antibody body of antibody 1 and antibody 2 was used as it is for the epitope analysis test, the corresponding analysis discrimination was decreased. Hence, antibody 1 and antibody 2 were treated with papain, whereby the Fc portion was removed from the antibody and only the Fab portion was left behind, thus determining the use of Fab of antibody 1 and Fab of antibody 2. Through the test using Fc-free Fab, a secondary antibody for analytical detection was a Fab-specific Alexa Fluor 488-conjugated secondary antibody (Jackson Immunoresearch Alexa fluor488 Goat anti-human F(ab)2 fragment).

(13) Also, the epitope analysis test was performed under the condition that the optimal concentration of antibody 1 Fab was 0.33 g/ml and the optimal concentration of antibody 2 Fab was 0.33 g/ml. As the control antibodies, Abcam 1C5 (Abcam Inc.) and QED 20501 (QED Bioscience Inc.) antibodies were used, and analysis was performed under the condition that the optimal concentration of the control antibody Abcam 1C5 was 0.25 g/ml and the optimal concentration of the control antibody QED 20501 was 0.5 g/ml.

(14) In the library selection, selected were alanine scanning mutants expressed over 80% of the control antibody Abcam 1C5 or QED 20501 and simultaneously less than 15% of antibody 1 Fab or antibody 2 Fab, compared to the binding reactivity to the positive control group of Example 1-1.

(15) TABLE-US-00002 TABLE 2 Binding reactivity (% WT) Antibody Antibody Abcam QED 20501 Mutant 1 Fab 2 Fab 105 Mab MAb E33A 1.3 59.3 81.2 93.4 G34A 0.3 95.9 98.2 91.7 C35A 3.9 83.7 101.6 94.9 L38A 5.4 130.2 127.6 113.8 A200S 10.5 102.9 109.1 87.6 K202A 4.2 132 113 105.5 L215A 6.6 100.1 101.7 100.0 R333A 68.6 5.4 103.1 109.1

(16) As is apparent from the results of Table 2, in the case of antibody 1, a total of seven alanine scanning mutants were selected, and the epitope sites of antibody 1 were identified as being located at amino acid positions 33, 34, 35, 38, 200, 202 and 215 of the rabies virus G protein through the selected mutants.

(17) In the case of antibody 2, a total of one alanine scanning mutant was selected, and the epitope site of antibody 2 was identified as being located at amino acid position 333 of the rabies virus G protein through the selected mutant.

Example 1-3-2: Epitope Identification of Antibody Using Customized Mutant

(18) In order to identify epitopes of antibody 1 and antibody 2, the binding activity to antibody 1 and antibody 2 was analyzed using the customized mutant library prepared in Example 1-2.

(19) In order to evaluate the binding of the customized mutant library of Example 1-2 and antibody 1 and antibody 2, immunofluorescent FACS was performed as in Example 1-3-1, and mutants were selected. The selection results are shown in Table 3 below. Here, Anti-V5 was used as an analytical control group for confirming the expression of the rabies virus G protein with the expression vector.

(20) TABLE-US-00003 TABLE 3 Binding reactivity (% WT) Antibody Antibody Abeam 105 QED 20501 Mutant 1 Fab 2 Fab MAb MAb Anti-V5 G34V 2.1 108 101 74.6 91.9 G34E 1.4 138 167 124 75.2 G34R 1.4 76.6 120 56.7 72.3 V210E 23.9 8.3 1.6 1 85.9 S331L 60.5 0.1 82.3 50.4 111 N336K 85.3 101 84.4 59 96.1 E413D 78.6 77.7 115 40.7 80.4

(21) As is apparent from the results of able 3, in the case of antibody 1, three customized mutants were selected, and the epitope site of antibody 1 was identified as being located at amino acid position 34 of the rabies virus G protein through the selected mutants.

(22) In the case of antibody 2, one customized mutant was selected, and the epitope site of antibody 2 was identified as being located at amino acid position 331 of the rabies virus G protein through the selected mutant.

Example 1-3-3: Results of Epitope Identification of Antibody

(23) Based on the results of Examples 1-3-1 and 1-3-2, the epitope of antibody 1 was found to be located at amino acid positions 33, 34, 35, 38, 200, 202 and 215 of the rabies virus G protein (SEQ ID NO:2), and the epitope of antibody 2 was found to be located at amino acid positions 331 and 333 of the rabies virus G protein, as shown in Table 4 below.

(24) The rabies virus binding sites are represented as the antigenic sites of surface glycoprotein of the rabies virus, and the antigenic structure of rabies glycoprotein for each site was first defined by Lafon et al. (J. Gen. Virol. 64:843-8451 1983). The currently identified rabies virus binding sites were classified into antigenic sites I, II, III, IV, and a, and were defined by Marissen et al. (J Virol. 2005 April; 79(8):4672-8.).

(25) TABLE-US-00004 TABLE 4 Amino acid residue Antibody Amino acid Amino acid Rabies virus ID No. mutant position* binding site Antibody 1 1 Glu(E)-->Ala(A) 33 Not known 2 Gly(G)-->Ala(A) 34 Antigenic site II 3 Cys(C)-->Ala(A) 35 Antigenic site II 4 Leu(L)-->Ala(A) 38 Antigenic site II 5 Ala(A)-->Ser(S) 200 Antigenic site II 6 Lys(K)-->Ala(A) 202 Not known 7 Leu(L)-->Ala(A) 215 Not known Antibody 2 1 Ser(S)-->Leu(L) 331 Antigenic site III 2 Arg(R)-->A1a(A) 333 Antigenic site III *Amino acid position was numbered except for the signal peptide of the rabies virus G protein

Example 2: Determination of Antigen-Antibody Binding Affinity Using Surface Plasmon Resonance Technique

(26) A surface plasmon resonance assay (Biacore Inc.) was used to determine the binding affinity of the antibody by epidemiological measurement of the forward and reverse reaction rate constants. Therefore, the antigen-antibody binding affinity of antibody 1 and antibody 2 was determined using a surface plasmon resonance technique.

(27) Particularly, the binding affinity of purified rabies virus G protein and antibody 1 and antibody 2 was determined through a surface plasmon resonance assay by means of a Biacore T200 (GE Healthcare) using an analytical buffer HBS-EP (10 mM HEPES [pH 7.4], 150 mM NaCl, 3 mM EDTA and 0.005% surfactant P20) at 25 C. 50 g/ml rabies virus G protein diluted in 10 mM sodium acetate (pH 5.0) was directly immobilized by about 500 RU on a CM5 biosensor chip using an amine coupling kit in accordance with the manufacturer's guidelines and procedures. The unreacted portion of the biosensor surface was blocked with ethanolamine. For the reaction analysis, Biacore T200 control software and Biacore T200 evaluation software were used. Antibody 1 and antibody 2 were diluted in a HBS-EP buffer. During the assay, all measurements were performed using, as a control group, the biosensor surface having no immobilized rabies virus G protein. The association and dissociation rate constants Ka (M.sup.1s.sup.1) and Kd (s.sup.1) were determined at a flow rate of 30 l/min. The rate constant was obtained by measuring the binding in the antibody concentration range of 2.46 to 200 nM through a three-fold serial dilution and using the buffer as a control group. Subsequently, the equilibrium dissociation constant KD (M) for the reaction between the antibody and the target antigen was calculated using the following equation from the reaction rate constants: KD=Kd/Ka. The binding was recorded by calculating the function of time and reaction rate constant.

(28) Based on the results thereof, as shown in Table 5 below, the binding affinity of antibody 1 and antibody 2 to the purified rabies virus G protein was determined. Both antibody 1 and antibody 2 exhibited high affinity to the rabies virus G protein.

(29) TABLE-US-00005 TABLE 5 Measurement of binding affinity using rabies virus G protein Sample ka1 (1/Ms) kd1 (1/s) KD AVR KD Antibody 1 2.52E+05 9.39E04 3.73E09 4.54E09 2.14E+05 1.15E03 5.36E09 Antibody 2 2.11E+06 0.0005743 2.72E10 2.24E10 1.54E+06 2.70E04 1.75E10

Example 3: Evaluation of Neutralizing Activity of Antibody Against Escape Virus Produced by CR4098 Antibody

Example 3-1: Production of Escape Virus for CR4098 Antibody

(30) The DNA sequence for the CR4098 antibody (Crucell) was verified with reference to U.S. Pat. No. 7,579,446 and the NCBI (National Center for Biotechnology Information) database, and was synthesized in Enzynomics Co. Ltd. (located in Yuseong-gu, Daejeon), cloned to a pCT146 expression vector (according to Korean Patent Application No. 10-2014-0178030), and then delivered to Celltrion Inc. The sequence of the synthesized portion was confirmed through restriction enzyme cleavage analysis and DNA sequencing. The CR4098 antibody was produced in the F2N78 cell line through a transient transduction method.

(31) For intracellular transient transduction a formulation for transfecting plasmid DNA into eukaryotic cells, FREESTYLE Max (Invitrogen, 16447-100), was used, and transduction was performed in accordance with the manufacturer's manual. The day before transduction, F2N78 cells (Korean Patent No. 10-1005967, Patentee: Celltrion) growing on an EX-CELL 293 serum-free medium (Sigma, 14571C: hereinafter referred to as EX-CELL 293 medium) were centrifuged, the medium was replaced with a FreeStyle293 serum-free medium (Gibco, 12338), and the cells were inoculated in an amount of 50 ml each (a total of 100 ml) at a cell concentration of 0.810.sup.6 per ml using two 250 ml shaker flasks. On the day of transduction, each of 125 g of pCT178 DNA containing an antibody gene and 125 l of the FREESTYLE Max reagent formulation for transfecting plasmid DNA into eukaryotic cells was diluted to a volume of 2 ml using an OptiPRO SFM II medium (Invitrogen, 12309), and gently mixed. The immediately diluted FreeStyle Max reagent formulation solution was mixed with a DNA-diluted solution and then reacted at room temperature for 17 min. During the reaction at room temperature for 17 min, the number of inoculated F2N cells to be used for transduction was counted, and the cell concentration was diluted to 1.010.sup.6 using the FreeStyle293 medium. After 17 min, transduction was carried out by treating a mixed solution of DNA and the formulation for transfecting plasmid DNA into eukaryotic cells, FREESTYLE Max reagent, with F2N cells. The day after transduction, the same amount of EX-CELL 293 medium was added to the transduced cells and incubated for 7 days, thereby producing the CR4098 antibody.

(32) Thereafter, CR4098 was used to perform the test for producing the escape mutant virus for the corresponding antibody.

(33) A rabies virus CVS-1 strain having an infectivity of 10.sup.6 per ml was subjected to a 1.5-fold serial dilution in a 96-well cell culture plate and then reacted with 1040 IU/ml CR4098 antibody at 37 C. for 1 hr. After reaction for 1 hr, 210.sup.5 cells/ml BHK cells were added and incubated for 3 days. After 3 days, the virus was obtained, the cells were immobilized, and the expression of rabies nucleocapsid protein was measured through staining in order to evaluate whether the cells were infected with the CVS-11 strain (JENO Biotech, 9061). In the second passage, an increased amount of CR4098 antibody was added to the virus obtained in the first passage, and the same test as above was repeated. In the first or second passage, virus 45 clones, in which the virus, obtained from the wells that had not been infected or had been little infected with the rabies virus CVS-11 strain, was confirmed to cause infection or to increase the extent of infection despite the addition of the CR4098 antibody in a gradually increasing amount, were amplified, after which RNA was isolated using a QIAamp Viral RNA Mini kit (QIAGEN, 52904).

(34) Using RNA as a template, cDNA was synthesized by means of a SuperScriptIII First-strand Synthesis system for RT-PCR (Invitrogen, 18080-051), and then amplified by Takara ExTaq (Takara, RR001A), followed by sequencing. Based on the results of sequencing, the changed sequence of the escape mutant virus for the CR4098 antibody was showed in N336K.

Example 3-2: Evaluation of Neutralizing Activity of Antibody Against Escape Virus Produced by CR4098 Antibody

(35) Whether antibody 1 and antibody 2 had neutralizing activity against the escape virus (N336K) produced by the CR4098 antibody in Example 3-1 was evaluated.

(36) The test for the neutralizing activity was performed using a FAVNT method. The Fluorescent Antibody Virus-Neutralizing Test (FAVNT) is a test method for determining the level of rabies virus antibody in human or animal serum through a cell culture method.

(37) In the test method of FAVNT, the dilution testing antibody (or HRIG) and a predetermined amount of rabies virus [100 TCID50/0.1 ml] were placed in a 96-well plate and reacted for 60 min, and baby hamster kidney cells were incubated for 3648 hr together with a culture medium containing 10% bovine serum (Eagle's minimum essential medium). After the incubation, the cells were immobilized, and then the anti-rabies antibody, ABC kit (VECTASTAIN) and DAB kit (VECTASTAIN) were used to stain the cells infected with the rabies virus. Finally, the number of stained cells was counted and the neutralizing activity of the test antibody relative to HRIG, the neutralizing activity of which is precisely known, was calculated.

(38) As results thereof, as shown in Table 6 below, both antibody 1 and antibody 2 exhibited neutralizing activity against the CR4098 antibody escape virus (N336K), and only the CR4098 antibody had no neutralizing activity.

(39) TABLE-US-00006 TABLE 6 Results of evaluation of neutralizing activity of antibody against escape virus produced by CR4098 antibody Virus Antibody IU/mg CR4098 antibody escape virus (N336K) CR4098 All infected Antibody 1 1352 Antibody 2 3081

Example 4: Evaluation of Neutralizing Activity of Antibody Against Various Kinds of Rabies Virus

(40) Approximately 50 varieties of rabies virus from around the world were tested for in-vitro neutralizing activity of antibody 1 and antibody 2 through RFFIT. The results are shown in Table 7 below.

(41) Upon the neutralizing activity testing, a Rapid Fluorescent Focus Inhibition Test (RFFIT) was performed to measure the activity of the antibody exposed by the rabies virus antigen. The neutralizing antibody was induced by glycoprotein on the outer surface of the virus. The Disease Control Center adopted the RFFIT method, which is rapid, economic, sensitive and reproducible.

(42) Like FAVNT, RFFIT is a test method for determining the level of rabies virus antibody in human or animal serum through the cell culture method. The immunofluorescent staining was used to evaluate virus amplification and required about 20 hr.

(43) RFFIT was performed in a multi-well slide using the mixed dilution test serum and the predetermined amount of rabies virus [50-50% Fluorescing Foci Doses (50 FFD50)/0.1 ml]. The mixed slide containing neuroblastoma and culture medium (Eagle's minimum essential medium) and 10% bovine serum was incubated in a 37 C. carbon dioxide incubator for 90 min. The form of serum-virus-cell was incubated for 20 hr in a 37 C. carbon dioxide incubator. Thereafter, incubation, washing, immobilization, and staining of the rabies virus conjugate with a label were performed, and observation was conducted using a fluorescence microscope.

(44) TABLE-US-00007 TABLE 7 Results of evaluation of neutralizing activity of antibody against various kinds of rabies virus Antibody 1 Antibody 2 Working conc.(g/ml) No. Virus ID 0.20 1 Mongoose RSAMongoose, South Titer 270 320 Africa IU/mL 7.7 9.1 IU/mg 7714 9143 2 Dog TunDog, Tunisia Titer 230 270 IU/mL 2.3 2.7 IU/mg 2300 2700 3 Dog thaiDog, Thailand Titer 320 1300 IU/mL 2.3 9.3 IU/mg 2286 9286 4 Dog sonDog, Mexico Titer 340 1100 IU/mL 2.7 8.8 IU/mg 2720 8800 5 Phi 002 (231)Human/dog, Titer 250 250 Philippines IU/mL 2.5 2.5 IU/mg 2500 2500 6 DR MXBat, Mexico Titer 250 180 IU/mL 2.4 1.7 IU/Ing 2381 1714 7 DR BrazilBat, Brazil Titer 56 45 IU/mL 2.2 1.8 IU/mg 2240 1800 8 WA BatBat, Washington, USA Titer 270 250 IU/mL 2.2 2.0 IU/mg 2160 2000 9 rv61Human (ex, dog),UK(ex, Titer 200 280 India) IU/mL 1.7 2.4 IU/mg 1739 2435 10 AL BatBat, California, USA Titer 1100 1100 IU/mL 7.6 7.6 IU/mg 7586 7586 11 AZ BatBat , Arizona Titer 125 1300 IU/mL 1.1 11.3 IU/mg 1087 11304 12 phi dogHuman/dog, Philippines Titer 75 280 IU/mL 0.28 1.04 IU/mg 1389 5185 13 Rv342 ChinaCow/dog, China Titer 280 1300 IU/mL 0.5 2.4 IU/mg 2545 11818 14 TX SK 4384Skunk, Texas, USA Titer 75 250 IU/mL 0.21 0.71 IU/mg 1071 3571 15 AK FXArctic Fox, Alaska, USA Titer 60 270 Failed IU/mL 0.44 2.00 IU/mg 2222 10000 16 Gray FX-AZ (AZ fox)Gray Fox, Titer 16 170 Arizona, USA IU/mL 0.53 5.67 IU/mg 2667 28333 17 323RDog/Coyote, Texas, USA Titer 60 625 IU/mL 0.33 3.47 IU/mg 1667 17361 18 RVHNHuman (ex, wolf), Russia, Titer 54 42 Arctic IU/mL 0.40 0.31 IU/mg 2000 1556 19 TN-269Bat, Tennessee, USA Titer 145 390 IU/mL 1.16 3.12 IU/mg 5800 15600 20 China dog 2005Dog, China Titer 12 13 IU/mL 0.43 0.46 IU/mg 2143 2321 21 TN410Bat, Tennessee, USA Titer 19 200 IU/mL 0.51 5.33 IU/mg 2533 26667 22 Dog ArgDog, Argentina Titer 145 170 IU/mL 0.36 0.43 IU/mg 1813 2125 23 TX SK 4380Skunk, Texas, USA Titer 50 70 IU/mL 0.40 0.56 IU/mg 2000 2800 24 RACRaccoon, Georgia, USA Titer 95 145 IU/mL 0.70 1.07 IU/mg 3519 5370 25 TX CoyoteCoyote, Texas, USA Titer 56 170 IU/mL 0.33 1.00 IU/mg 1647 5000 26 Mongoose PRMongoose, Titer 54 56 Puerto-Rico IU/mL 0.39 0.40 IU/mg 1929 2000 27 I-151Dog, India Titer 125 320 IU/mL 0.74 1.88 IU/mg 3676 9412 28 Sri LankaCow, Sri Lanka Titer 54 50 IU/mL 0.43 0.40 IU/mg 2160 2000 29 Wu ABLVAustralian bat lyssa, Titer 250 440 Genotype 7 IU/mL 0.38 0.68 IU/mg 1923 3385 30 ABV (SM 4476) Australian bat Titer 54 210 lyssa, Genotype 7 IU/mL 0.40 1.56 IU/mg 2000 7778 31 3860 CA BatBat, California, Titer 56 210 USA IU/mL 0.40 1.50 IU/mg 2000 7500 32 Gabon dogDog, Gabon Titer 65 170 IU/mL 0.41 1.06 IU/mg 2031 5313 33 CVS-11 Titer 70 250 IU/mL 0.41 1.47 IU/mg 2059 7353 34 EBLV 1 A09-3484Genotype 5 Titer 56 540 IU/mL 0.56 5.40 IU/mg 2800 27000 35 EBLV 2 A03-4659Genotype 6 Titer 54 280 IU/mL 0.60 3.11 IU/mg 3000 15556 36 DuvenhageGenotype 4 Titer 13 180 IU/mL 0.44 7.20 IU/mg 2200 36000 37 EBLV 1 A09-3485Genotype 5 Titer 50 180 IU/mL 1.85 6.67 IU/mg 9259 33333 38 EBLV 2 A09-3483Genotype 6 Titer 50 125 IU/mL 2.22 5.56 IU/mg 11111 27778 39 CASKSkunk, California, USA Titer 50 54 IU/mL 0.40 0.43 IU/mg 2000 2160 40 Bat EfBat, Pennsylvania, USA Titer 50 50 IU/mL 1.05 1.05 IU/mg 5263 5263 41 C1434Bat, Alabama, USA Titer 19 36 IU/mL 0.51 0.96 IU/mg 2533 4800 42 VA 399Bat, Virginia, USA Titer 10 50 IU/mL 0.56 2.78 IU/mg 2778 13889 43 TN 132Bat, Tennessee, USA Titer 56 280 IU/mL 2.00 10.00 IU/mg 10000 50000 44 TXFXGray fox, TX Titer 56 250 IU/mL 0.41 1.85 IU/mg 2074 9259 45 I-148Dog, India Titer 13 10 IU/mL 0.52 0.40 IU/mg 2600 2000 46 LC NYBat, New York, USA Titer 11 45 IU/mL 0.52 2.14 IU/mg 2619 10714 47 857rRaccoon dog, Russia, Far Titer 56 54 East IU/mL 2.80 2.70 IU/mg 2800 2700 48 NC SKSkunk, Wisconsin, USA Titer 280 270 IU/mL 2.24 2.16 IU/mg 2240 2160 49 MI1625Bat, Tennessee, USA Titer 11 70 IU/mL 0.39 2.50 IU/mg 1964 12500 50 MyotisBat, Washington, USA Titer 230 280 IU/mL 1.64 2.00 IU/mg 8214 10000 51 ERA Titer 50 250 IU/mL 0.36 1.79 IU/mg 1786 8929

Example 5: Evaluation of Neutralizing Activity of Antibody Against Rabies Virus Isolated in India

Example 5-1: In-Vitro Test

(45) Using antibody 1, antibody 2 and an antibody cocktail (antibody 1+antibody 2), a wild-type virus of Table 8 below, flourishing in India, was subjected to RFFIT as in Example 4. The test was conducted in the National Institute of Mental Health and Neuro-Sciences (NIMHANS), India. Each antibody was at a concentration of about 1 g, and testing was carried out in Neuro-2a cells with 100 FFD.sub.50 of each virus of Table 8 below. The results of RFFIT exhibited neutralizing activity against each virus in all of three antibodies.

(46) TABLE-US-00008 TABLE 8 Rabies virus isolated in India Virus abbreviation Virus-isolated animal Virus-isolated area SV1 Dog Kerala, India SV2 Dog Kerala, India SV3 Human Karnataka, India SV4 Human Karnataka, India SV5 Dog Chennai, Tamilnadu ,India SV6 Dog Chennai, Tamilnadu, India

Example 5-2: In-Vivo Test

(47) In-vivo testing was performed in mice following the in-vitro testing of Example 5-1. As shown in Table 9 below, all antibodies in animal test groups exhibited high neutralizing activity. 10 mice were used in each animal test group, and the virus used was 100 LD50 (in 0.1 mL). 3 hr after virus inoculation (muscle injection), each antibody (about 1 g) was inoculated at the same position (muscle injection), after which the survival rate was observed for 30 days. FIG. 1 is a graph showing the mouse survival rate against SV2 virus among a total of six viruses (SV1SV6) in an animal test.

(48) In the present animal test, the survival rate against each of SV1 to SV6 viruses is shown in Table 9 below.

(49) TABLE-US-00009 TABLE 9 Results of evaluation of neutralizing activity of antibody against rabies virus isolated in India through animal testing SV1 SV2 SV3 SV4 SV5 SV6 Antibody 1 100 100 100 100 100 100 Antibody 2 80 90 90 80 90 90 Antibody 1 + Antibody 2 100 100 100 100 100 100 Control group 0 0 0 0 0 0

Example 6: Evaluation of Interference Effect of Cocktail of Antibody 1 and Antibody 2

Example 6-1: Evaluation of Interference Effect of Cocktail of Antibody 1 and Antibody 2 Using Test for Measuring Neutralizing Activity Against Wild-Type Rabies Virus and Rabies Virus CVS-11 Strain

(50) Using antibody 1, antibody 2 and an antibody cocktail (antibody 1+antibody 2), twelve wild-type rabies viruses and the rabies virus CVS-11 strain were subjected to RFFIT as in Example 4.

(51) As set forth in Table 10 below, all of antibody 1, antibody 2 and the cocktail comprising antibody 1 and antibody 2 mixed at the same ratio exhibited superior neutralizing activity against twelve wild-type rabies viruses and the rabies virus CVS-11 strain, whereby antibody 1 and antibody 2 in the cocktail were confirmed to have no interference effect upon biological neutralizing activity evaluation.

(52) TABLE-US-00010 TABLE 10 Results of evaluation of neutralizing activity of antibody cocktail against various kinds of rabies virus Antibody Antibody Cocktail 1 2 (1:1) No. Virus (IU/mg) (IU/mg) (IU/mg) 1 LC NY Bat, New York, USA 2619 10714 8571 2 Bat Ef Bat, Pennsylvania, USA 5263 5263 4421 3 TX Coyote Coyote, Texas, USA 1647 5000 2500 4 1-151 Dog, India 3676 9412 5000 5 Gabon dog Dog, Gabon 2031 5313 4531 6 Sri Lanka Cow, Sri Lanka 2160 2000 2000 7 NC SK Skunk, Wisconsin, USA 2240 2160 2240 8 China dog 2005 Dog, China 2143 2321 1964 9 Dog thai Dog, Thailand 2286 9286 7857 10 phi dog Human/dog, Philippines 1389 5185 4630 11 Mongoose RSA Mongoose, South 7714 9143 9143 Africa 12 Myotis Bat, Washington, USA 8214 10000 8929 13 CVS-11 2059 7353 6765

Example 6-2: Evaluation of Interference Effect of Cocktail of Antibody 1 and Antibody 2 Using Molar Excess Testing

(53) Whether antibody 1 and antibody 2 exhibited the interference effect was also evaluated through molar excess testing. To this end, the concentration ratio of two antibodies was set in the range of 1/9 to 9/1 for the rabies virus CVS-11 strain tested in Example 6-1, and REFIT was performed three times as in Example 4. The results are shown in Tables 11 and 12 below.

(54) Through the testing at different concentration ratios of antibody 1 and antibody 2, even when the ratio of antibody 1 and antibody 2 was adjusted to 1:9 to 9:1, all of antibody 1, antibody 2, and the cocktail of antibody 1 and antibody 2 exhibited the rabies-virus-neutralizing activity shown in Tables 11 and 12 below.

(55) TABLE-US-00011 TABLE 11 Evaluation of neutralizing activity of antibody cocktail against rabies virus CVS-11 strain Test Results (IU/mg) (Measurement using antibody 1) Anti- Anti- body body Standard 1 (%) 2 (%) 1 2 3 Mean deviation A 0 100 N/A N/A N/A N/A N/A B 10 90 45000 34185 102556 60581 36752 C 25 75 41023 18000 23688 27570 11992 D 50 50 6837 3948 11844 7543 3995 E 75 25 2000 1519 3464 2328 1013 F 90 10 962 731 1667 1120 487 G 100 0 380 380 658 473 161

(56) TABLE-US-00012 TABLE 12 Evaluation of neutralizing activity of antibody cocktail against rabies virus CVS-11 strain Test Results (IU/mg) (Measurement using antibody 2) Anti- Anti- body body Standard 1 (%) 2 (%) 1 2 3 Mean deviation A 0 100 7793 4500 10256 7516 2888 B 10 90 5000 3798 11395 6731 4084 C 25 75 13674 6000 7896 9190 3997 D 50 50 6837 3948 11844 7543 3995 E 75 25 6000 4558 10391 6983 3038 F 90 10 8659 6580 15000 10080 4386 G 100 0 N/A N/A N/A N/A N/A

(57) Dunnett's test, one of the multiple comparison test methods, was used as a statistical analysis method to evaluate the significance of the neutralizing activity difference between the test group and the control group to confirm the interference effect. Particularly, if the adjusted p-value in the Dunnett analysis is greater than 0.05, which is a significance threshold, it is not significant, indicating no difference between groups being compared. On the other hand, if the adjusted p-value is less than 0.05, it indicates that there is a significant difference between the groups being compared. The results of Dunnett's analysis based on Tables 11 and 12 are respectively shown in Tables 13 and 14 below.

(58) As set forth in Tables 13 and 14 below, in Table 13, the adjusted p-value was 0.05 or less only when the proportion of antibody 2 was 90% or more, indicating that there was no interference effect except for the case of 90% content of antibody 2. However, in the case where the proportion of antibody 2 is 90% or more, the interference effect may exist statistically, but it is considered to be due to the very strong neutralizing activity of antibody 2 and cannot be regarded as the interference effect. In Table 14, even when the proportion of antibody 1 was increased, the adjusted p-value was 0.05 or more, confirming that there was no interference effect. Therefore, it can be concluded that there is no interference effect between two antibodies at all concentrations.

(59) TABLE-US-00013 TABLE 13 Multiple comparison test results for rabies virus CVS-11 strain at antibody 1 concentration Least-squares mean difference Multiple Standard Degrees of Adjusted comparison test Estimate error freedom t value Probability > |t| p-value Result B vs G 60108 12961 12 4.64 0.0006 0.0024 Significant C vs G 27098 12961 12 2.09 0.0585 0.1958 Not significant D vs G 7070.4 12961 12 0.55 0.5954 0.9718 Not significant E vs G 1855.1 12961 12 0.14 0.8886 0.9999 Not significant F vs G 647.41 12961 12 0.05 0.961 1 Not significant

(60) TABLE-US-00014 TABLE 14 Multiple comparison test results for rabies virus CVS-11 strain at antibody 2 concentration Least-squares mean difference Multiple Standard Degrees of Adjusted comparison test Estimate error freedom t value Probability > |t| p-value Result B vs A 785.08 3080.8 12 0.25 0.8032 0.9991 Not significant C vs A 1673.77 3080.8 12 0.54 0.5969 0.9723 Not significant D vs A 26.7002 3080.8 12 0.01 0.9932 1 Not significant E vs A 533.29 3080.8 12 0.17 0.8655 0.9999 Not significant F vs A 2563.39 3080.8 12 0.83 0.4216 0.8691 Not significant

(61) Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that a variety of modifications and equivalents able to substitute therefor may be provided, without departing from the scope and spirit of the invention as disclosed in the accompanying claims. Accordingly, the present invention is not intended to be limited to particular embodiments, which are merely disclosed as the best form for performing the present invention, and should be construed to include all embodiments falling within the accompanying claims.