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To establish a method for measurement of the neutralizing activity of a monoclonal antibody against the rabies virus. Twenty-four rabies street virus-positive samples were isolated by the mouse inoculation test. Isolated rabies street viruses were cultured and the virus titer tested in N2A cells. We established a rabies street virus bank with viruses that could adapt to the growth of N2A cells with a high titer. Then repeatability was evaluated after three detections of the TRN006 monoclonal antibody. Of the 24 positive samples,15 strains of the virus could adapt to N2A cells and form fluorescent foci in cells.Finally,10 strains with a high titer were selected for the rabies street virus bank, which covered nine Provinces of China and four Chinese lineages. Three lineages were used for the neutralization test for the monoclonal antibody, and the Student’s t-test showed that it had good repeatability.

China has the second highest number of rabies cases worldwide. There are millions of category III rabies exposure cases in China every year, which are treated with rabies immunoglobulin(RIG)and the rabies vaccine. The price of RIG is relatively expensive, and the application of RIG is relatively complicated. The rate of RIG use in post exposure prophylaxis(PEP)for rabies exposure category Ⅲ cases has remained low for a significant amount of time. Reducing the dosage of RIG could reduce the cost of PEP, while simplifying the use of RIG could make PEP easier. Together, these steps could improve the rate of RIG utilization in PEP. There are conflicting conclusions in studies of RIG on the immune effects of the rabies vaccine.Exploring the mechanism of action of RIG in the immune response to the rabies vaccine would help to explain the role of RIG in the immune effects mediated by the rabies vaccine. In this paper, the progress of research on the application of RIG is systematically reviewed in order to provide a reference for the formulation of new and more practical guidelines for the application of RIG.

To evaluate the neutralizing potency and spectrum of three recombinant human mAbs CR57(Ⅰ), RV08(Ⅱ), RV3A5 (Ⅲ) and the triple combination cocktail against antigenic site I, II and III on rabies virus glycoprotein, a standard fluorescent antibody virus neutralization test(FAVN)on several RV vaccine strains, fixed strains, and street strains of total 11 trains was performed by incubation of RV with varying concentrations of antibody followed by incubation with BHK-21 cells. To investigate whether the antibodies display neutralizing activity against a lethal RV infection in vivo, we performed a Syrian hamster study by infecting with 50LD(50)/100μl of CVS-11 strain intramuscularly (i. m.) in the gastrocnemius muscle. Three recombinant human mAbs CR57 (I), RV08 (II), RV3A5 (III) and the compatibility triple cocktail showed broad cross-neutralizing reactivity to all 11 RV strains. The cocktail composed of three mAbs CR57 (Ⅰ) RV08 (Ⅱ), RV3A5 (Ⅲ) by neutralizing titers of 1 : 1 : 1 has no less in neutralizing ability against these strains, indicating that no mutual interference between the three antibodies. The cocktail exhibited neutralizing synergistic activity against individual strains(JX08-45,Flury,SRV9).The treatment with CR57,RV08,RV3A5 or the triple combination cocktail alone respectively provided better protection with a survival range of 100%against the lethal RV infection compared HRIG immunized alone. Combined immunization with the vaccine, recombinant mAbs protected hamsters with a survival rate of 100%equally as well as HRIG after exposure to a lethal RV infection. Our results provide more candidates eligible for use in a mAb cocktail aimed at replacing RIG for rabies post-exposure prophylaxis.

To obtain neutralizing high affinity human recombinant antibodies for antigenic site Ⅲ of rabies virus(RV)glycoprotein, we chose scFv phage display technology to optimize CR4098 with chain shuffling. Using pHAL14-CR4098 as vector, the combinatorial shuffling scFv antibody phage libraries were constructed to replace CR4098 light or heavy chain genes respectively by antibody genes derived from the blood of RV-vaccinated donors. After package by hyperphage, the chain shuffling scFv phage library was panned and selected by ELISA with purified rabies virus aG strain. The specific antibody was converted to full human IgG antibody with the VH/VK Express cassettes. Affinity and neutralizing test were performed to verify the function of the IgG molecules. Fourteen unique human ScFv antibodies specific for the glycoprotein of rabies virus were obtained by ELISA,IFA and DNA sequencing. Further tested showed that RV3A5 has a high affinity of 2.8×10(-9) M and high neutralizing activity to rabies virus both aG strains and CVS strains. Competitive ELISA showed that RV3A5 and CR4098for antigenic Site III competed with each other, indicating that they had overlapping or shared the epitope. Our results provide more candidates eligible for use in a mAb cocktail aimed at replacing RIG for rabies post-exposure prophylaxis.

To study the B cell linear epitopes of rabies virus CVS-11 nucleoprotein, peptides were synthesized according to the amino acid sequences of B cell linear epitopes. Linear epitopes predicted by bioinformatics analysis were evaluated with immunological techniques. Indirect enzyme-linked immunosorbent assay showed that titers of antibodies to peptides (355-369 and 385-400 residues of rabies virus CVS-11 nucleoprotein) were above 1:12 800 in mouse sera. The antibodies recognized denatured rabies virus CVS-11 nucleoprotein in Western blot analysis. Purified anti-peptide antibodies recognized natural rabies virus CVS-11 nucleoprotein in BHK-21 cells in indirect fluorescent antibody test. The 355-369 and 385-400 residues of rabies virus CVS-11 nucleoprotein were validated as B cell linear epitopes.

To sequence and analyze the full-length gene sequence of rabies vaccine virus aG strain. The full-length gene sequence of aG strain was amplified by RT-PCR by 8 fragments,each PCR product was cloned into vector pGEM-T respectively, sequenced and assemblied; The 5' leader sequence was sequenced with method of 5' RACE. The homology between aG and other rabies vaccine virus was analyzed by using DNAstar and Mega4. 0 software. aG strain was 11 925nt(GenBank accession number: JN234411) in length and belonged to the genotype I . The Bioinformatics revealed that the homology showed disparation form different rabies vaccine virus. the full-length gene sequence of rabies vaccine virus aG strain provided a support for perfecting the standard for quality control of virus strains for production of rabies vaccine for human use in China.

To construct a recombinant baculovirus expressing glycoprotein (GP) of RV SRV9 strain and test the immunological efficacy in mice, open reading frame of rabies virus GP gene of SRV9 strain was cloned into the shuttle vector Bacmid to construct the recombinant shuttle plasmid Bacmid-G and transfection was performed into S f9 cells with the recombinant shuttle plasmid. CPE appeared in cell cultures was identified by electronmicroscopy. Western-blot, IFA and immunity tests in mice were performed to identify the immunoreactivity and immunogenicity of the expression products. Our results showed a recombinant baculovirus expressing GP protein of rabies virus SRV9 was obtained. The expression products possessed a favorable immunogenicity and fall immunized mice could develop 100% protective level of anti-rabies neutralizing antibody. In conclusion, The SRV9 glycoprotein expressed by the recombinant baculovirus in this study had good immunogenicity and could induce anti-rabies neutralizing antibody, which laid the foundation of further development of rabies subunit vaccine.

To construct a recombinant human adenovirus type 5 expressing glycoprotein (GP) of attenuated rabies virus SRV9 and testing immunological efficacy on the immunized mice. Open reading frame of rabies virus GP gene of SRV9 strain was cloned into the shuttle vector of adenovirus expression system in multiple cloning sites to construct the recombinant shuttle plasmid pacAd5 CMV-Gs9, cotransfection was performed into 293AD cells mediated by FuGENE Transfection Reagent with linearized backbone plasmid and recombinant shuttle plasmid, cell cultures were collected after CPE appearance and were identified by PCR and electronmicroscopy, virus titer was measured in 293AD cells. Kunming mice were intraperitoneally injected with 10(6) TCID50 adenovirus, blood for serum preparation was collected through caudal vein pre-immune and post-immune and tested for VNA appearance by fluorescent antibody virus neutralization test (FAVN) detection. Recombinant shuttle plasmid pacAd5 CMV-Gs9 was constructed correctly. A recombinant human adenovirus type 5 was obtained expressing GP protein of rabies virus SRV9. The virus titer reached 10(6) CFU/mL at the least. All mice developed a certain amount of the anti-rabies neutralizing antibody 14 days after intraperitoneal inoculation, while the effective protection rates were 90%. In conclusion, Recombinant adenovirus expressing the rabies virus GP was constructed successfully and a certain amount of neutralizing antibodies were induced in mice, which laid the material foundation for further development of new rabies vaccine.

Objective: The purpose of this study is to produce new antibody molecules to neutralize the rabies virus specifically by the way of constructing and expressing the human anti-rabies virus scdsFv (disulfide stabilized single chain antibody) gene, and characterizing its bioactivity., Methods: We obtained the sequence of variable region of heavy chain (VH) and variable region of light chain (VL) of RV monoclonal antibody SO57 from GenBank, and it respectively mutated into cysteine in the gene loci VH44 and VL100. The scdsFv gene was synthesized and inserted into a prokaryotic expression vector pET22b(+). Purified inclusion body scdsFv proteins were obtained by Ni-NTA His Bind Resin affinity chromatography and identified by SDS-PAGE gel and Western blot assay. The binding activity of scdsFv was identified by ELISA and mouse brain tissues infected with rabies virus CVS strain. The relative affinity of scdsFv was measured by ELISA using thiocyanate elution. The capacity of the scdsFv to neutralize the rabies virus CVS strains was determined by fluorescent antibody virus neutralisation test (FAVN). The fusion proteins neutralized the CVS strain in a standard in vivo neutralized assay where the virus was incubated with the scdsFv molecules before intracranial inoculation in mice., Results: The fragment genes of scdsFv to rabies virus were constructed successfully. ScdsFv protein was expressed in E. coli with approximate molecular weight of 30.0 kDa, which could be recognized by anti-His mAb. ELISA results demonstrated that scdsFv could bind antigen specificity. It was found that the strong reactivity of scdsFv to the smear of RV infected mouse-brain was demonstrated by IFA. As determined by FAVN with a reference serum, the titer of scdsFv was 41 IU/mL. In addition, scdsFv could be 55.6% protection of mice against lethal challenge with rabies virus CVS., Conclusion: The scdsFv can bind antigen specificity and has neutralization capacity to the virus in vitro and in vivo. The anti-rabies scdsFv is potential for application in rabies post-exposure prophylaxis.

A combinatorial human Fab library to the rabies virus was constructed using antibody genes derived from the blood of vaccinated donors. The library were panned and selected on purified rabies virus particles of aG or CTN strain with phage display. Eleven unique human Fab antibodies specific for the rabies virus glycoprotein were obtained by ELISA, IFA and DNA sequences analysis of these antibodies. Among these Fab antibodies, five human Fab antibodies were converted to full-length human IgG antibodies with recombinant baculovirus system. The five full-length human IgG antibodies were tested in vitro for rabies virus neutralization, resulting in all specificities to neutralize the virus. The obtained human anti-rabies antibodies lay the basis for the production of cocktail of anti-rabies monoclonal antibody with chinese intellectual property.

Objective: Compare the difference of the results referred to the WHO standard rabies immunoglobulin and the national standard human rabies immunoglobulin used in the rapid fluorescent focus inhibition test (RFFIT)., Methods: Setting the WHO standard immunoglobulin and the national standard immunoglobulin in the same system and testing 12 human serum at the same time. Compare the fluorescence percentage of the two different standard immunoglobulin; compare the 12 serum results calculated from the two different standard immunoglobulin used the calculation formula of neutralization antibody titer., Results: The Results display that the 50% percent of the two standard immunoglobulin are all between the fifth and the sixth well, but the percentage of the national standard immunoglobulin is lower than the WHO one. The same testserum result calculated from the WHO standard immunoglobulin is little higher than the national one., Conclusion: There is difference in the WHO standard immunoglobulin and the national one, but there is no influence in the results.

Objective: To evaluate the safety and immunogenicity of Rabipur produced in India., Methods: A random and single-blind study was conducted to compare the safety, effect and sero conversion rates. Rabipur produced in Germany used as control group., Results: The results showed that they were no severe systemic and local reaction occurred in trial group (Rabipur produced in India). The difference between trial group and control group was not significant. 14 day and 45 days after the first dose, the seroconversion rates of the two groups were 100%. 14 day and 45 days after the first dose, GMC of the two groups increased obviously and highly beyond 0.5TU/ml., Conclusion: Rabipur produced in India has excellent safety and immunogenicity. Trial Registration National Food Drugs Surveillance Administrative Bureau, "Medicine Clinical Experiment Written directive from a superior" 2005L04184.

To obtain a bivalence vaccine against canine rabies virus and canine parvovirus, a chimeric rabies virus expressing canine parvovirus VP2 protein was generated by the technique of reverse genetics. It was shown that the chimeric virus designated as HEP-Flury (VP2) grew well on BHK-21 cells and the VP2 gene could still be stably expressed after ten passages on BHK-21 cells. Experiments on the mice immunized with the chimeric virus HEP-Flury (VP2) demonstrated that specific antibodies against rabies virus and canine parvovirus were induced in immunized mice after vaccination with the live chimeric virus.

Objective: To establish a reverse genetic system of rabies virus for producing safe and efficient rabies vaccine., Method: By reverse genetic and molecular cloning technique, we established two rabies virus rescue systems including (1) help plasmids expressing N, P and L protein, and (2) CMV/T7 and T7 promoter., Results: Wild-type rERA-VC was rescued by both systems, and had the same growth kinetics as parental virus. The third generation virus could grow to high titer., Conclusion: The established reverse genetic system for rescuing wild-type rERA-VC provides the possibility of producing safe and efficient rabies virus vaccines.

Objective: To get the high specific and sensitive mononclonal antibodies against RV., Methods: The myeloma cell line SP2/0 fused with the spleen cell of 6 - 8 weeks old BALB/c mice immunized with the CVS-11 virus antigen. The hybridized fusing cells were chosen by indirect ELISA detection and the positive hybridizing cells were amplified through mouse abdomen injection and the mouse McAbs ascites was purified by Protein A Sepharose 4 Fast Flow (Pharmacia Company). The specificity and sensitivity of the McAbs was identified by indirect ELISA and indirect DFA test., Results: The cell fusion rate reachs 100% and the indirect ELISA results showed that the McAbs ascites titer were 1 x 10(4), 1 x 10(5), 1 x 10(4) and 1 x 10(5); The immunoglobulin G type McAbs show no cross reaction with other related viruses., Conclusion: The high specific and sensitive mononclonal antibodies of RV can be used for rapid RV diagnosis.

Objective: To study the biological features of the adapted rabies virus 3aG-V in Vero cell., Methods: The virus structure, culture condition, pathogenicity, immunocompetence, toxicity and protective efficiency were studied., Results: The rabies virus 3aG-V strain could well adapt to Vero cell, the pathogenicity was very weak and the immunogenicity was very good., Conclusion: The Vero cell-adapted rabies virus strain may possibly be used as a vaccine strain.

Objective: To construct and characterize recombinant adenoviruses containing glycoprotein (GP) gene from rabies virus CVS-N2C strain., Methods: To obtain the recombinant adenovirus by pAdEasy system, identify recombinant virus with cDNA sequencing, Northern blot, Dot blot, Western blot and challenge-protection experiment of mice., Results: Recombinant adenovirus showed typical adenovirus morphological characteristics; the viral genome was stable; GP specific mRNA and presence of glycoprotein were determined in rAdGPcvs and rAdGPcvs' infected cells. The glycoprotein produced by recombinant adenovirus had a molecular mass of 66,000, which was similar to that of natural glycoprotein. In the group of rAdGPcvs immunized mice, 87.5%-100% of mice survived from a 35.8LD50/38.0LD50 lethal rabies intracerebral challenge. Finally 73.3%-83.3% of the mice that had received eAdGPcvs survived, and all the Ad5 immunized mice succumbed to rabies., Conclusion: Recombinant adenovirus rAdGPcvs and rAdGPcvs' hold great potential to be developed as recombinant rabies vaccines, and at the same time, it is actually the first study that on high neuropathogenicity rabies CVS-N2C glycoprotein based adenoviral recombinants.

Objective: To evaluate the immune response, of mice immunized with E1, E3 deleted replicative deficient recombinant adenovirus which can express glycoprotein of Chinese 3aG rabies virus., Methods: Rapid fluorescent focus inhibition test (RFFIT) was used to determine the titer of neutralizing antibodies; specific antigen induced spleen lymphocyte proliferation was determined by in vitro 3H-TdR in-corporation assay, and lethal intracerebral challenge with CVS rabies virus was used to evaluate the protective effectiveness of the recombinant virus as candidate vaccine., Results: Mice immunized by 1 x 10(7) recombinant virus developed high levels of rabies virus neutralizing antibodies (VNA). The spleen lymphocyte of the immunized mice also demonstrated remarkable increased proliferation after stimulation with inactivated rabies virus; 70%-90% of the mice immunized by the recombinant virus were protected from lethal intracerebral challenge with CVS rabies virus., Conclusion: E1, E3 deleted replication defective recombinant adenovirus containing 3aG glycoprotein gene may be developed as effective rabies vaccine to control rabies in China.

Objective: Using Vero cell as basic cultural material to improve the quality of rabies vaccines and to produce rabies vaccines for humans., Methods: CTN-1V10 strain were used for production. Vero cell of 150th generation were used for cultivation. Rotating cultivatal method with rotating bottle was used. Fluids with virus were collected at different time. Puritied rabies vaccine was produced on Vero cell after clarification, condensation, purification and extermination. A batch of vaccines made by this techniques were used for immunological observation. Sixty-three people were injected with this rabies vaccine according to the procedure of time of exposure. Thirty of them were injected with vaccines made in France (Verorab) while the others were injected with vaccines to be tested. Side effect and neutralizing antibody were recorded., Results: The quality of this newly developed rabies vaccines has met the quality set by WHO. After all dosages of injection, the rates of positive antibody were both 100% in two groups. The neutralizing antibody among testing group was 11.94 IU/ml comparing with control as 11.69 IU/ml., Conclusion: Purified rabies vaccines on Vero cell for humans had reasonable manufacture technique and less little effect with good technological imnunogenicity.

The distribution of rabies virus antigen (RVAg) in human tongue and salivary glands from three cases was studied with ABC immunohistochemical method. Of the 5 tissue blocks from 2 tongues, large amounts of RVAg were found in 4 tissue blocks, located in the serous glands, terminal nerves, some striated muscle fibers and some covering epithelium including taste cells of circumvallate papillae. Only small amounts of RVAg were present in 4 of the 9 salivary gland blocks, either within the acini or in nerves. These results differ from that reported previously in dog, fox and skunk salivary glands, which contained abundant RVAg. The amount of RVAg in human tongue is apparently greater than that in the salivary glands. It is probable that the serous glands of the human tongue are sites of predilection for RVAg and may serve as a source of infection.