Adaptation Of Chinese Rabies Isolate To Growth In Cells And Development Of Highly Effective Rabies Vaccine With Adjuvants

Rabies, a devastating and preventable viral disease, poses a serious threat tohumans and animals (especially canid species) in China. More than95%of the2,000to3,000annual human rabies cases in China are caused by rabid dog bites. Thissituation is largely the result of the low coverage of rabies immunization in dogs inthe country. In China, domestic live vaccines (including ERA and Flury-LEP) that areof low potency and with poor safety records in humans and dogs are still used widely.Although the use of four domestically inactivated rabies dog vaccines has beenlicensed in the past three years, none has been marketed for several reasons (e.g.,potency, cost, etc). Two methods for enhancing the potency were commonly used ininactivated rabies vaccine in China, including adaptation of rabies virus to high titergrowth in cell lines and addition of immunopotentiators/adjuvants.Based on above problems, our studies are as follows:1. Adaptation of a Chinese ferret badger strain of rabies to growth to high titer inBHK-21cells1.1. Adaptation of JX08-45to growth in BHK-21cellsJX08-45was added to a BHK-21cell monolayer. The inoculated cells weresuccessively passaged until an acceptable virus titer for vaccine production wasreached and were harvested for TCID50assay. The cell culture-adapted JX08-45wasrenamed JX08-45CC.The results showed that the titer of JX08-45virus gradually increased after serialpassage in BHK-21cells, reaching a plateau at passage level120, and maintainingthis at about108.0TCID50/mL.1.2. Pathogenicity of JX08-45CCSerial10-fold dilutions of JX08-45CC were injected respectively I.C. and I.M.into adult mice. Unvaccinated beagle dogs were respectively injected I.M.(rightmasseter muscle) with of JX08-45CC and JX08-45.All adult mice died of intracerebral injection of JX08-45CC at doses of>106TCID50/mL. At lower titers, some animals survived. Following I.M. injection, someanimals survived even doses of108TCID50/mL. Rabies symptoms, when presented,were observed7-9days post-challenge with deaths occurring within24-48h later.Ninety days post-challenge, none of the challenged dogs with JX08-45CC developedrabies and died, however,3dogs challenged with JX08-45died of rabies respectivelyat14thday,18thday and20thday.1.3. Genomic analysis of JX08-45CC Nucleotide sequences of the complete genomes of JX08-45and JX08-45CC werecompared with some vaccine and street strains.Each genome of both JX08-45and JX08-45CC contained11,922nt, with17ntchanges in the cell culture-adapted virus. One nucleotide substitution was located inthe untranslated region (UTR) between the P-and M-coding regions with7substitutions in the G-, and8in the L-coding regions, respectively. The nucleotidesubstitutions resulted in a total of7amino acid changes (genome positions:3523nt,3761nt,3925nt,4110nt,4414nt,4646nt and5484nt) with one in the L protein(genome position:5484nt) and the others in the G protein. The amino acid at position333of G protein remained Arg. Amino acid comparisons with other rabies vaccinestrains revealed that4the7amino acid substitutions in the JX08-45(genome position:3761nt,3925nt,4110nt and4646nt) were mostly the same as those at the samegenome position in the other vaccine strains examined. The6amino acid changes(genome positions:3523nt,3761nt,3925nt,4110nt,4414nt and4646nt) wererespectively located at the G protein amino acid positions51,130,191,247,348and425, none of which is in either of the major antigenic sites (site II and site III) of theglycoprotein.1.4. Immunization of miceAdult mice were randomized and injected in the upper hind legs with JX08-45,CVS-11, ERA, SRV9or Flury-LEP. Serum samples were collected from theretro-orbital plexus on the14thd after treatment for FAVN test.The differences of antibody titers between the mean values were not statisticallysignificant. From a numerical viewpoint, the mean value of JX08-45was almost thesame as that of Flury-LEP and slightly higher than those of the other strains.2. Preparation of adjuvants2.1. Oil-in-water emulsion and botanical polysaccharidesOil-in-water emulsion and botanical polysaccharides (Astragalus, Echinacea,wolfberry, and kelp polysaccharides) were individually tested to screen for highlyeffective immunoenhancers/adjuvants for inactivated rabies vaccines throughimmunization of mice.The results showed that average titers of oil-in-water emulsion and Astragaluspolysaccharides groups were significantly higher than that of the Alum group.2.2. Mechanisms of stimulation of antibody response by adjuvantsPeripheral blood lymphocyte proliferation assay in vitro, identification of Th/Tcsubsets and Cytokine measurement were respectively performed to study themechanism of stimulation of antibody response by adjuvants.Lymphocyte proliferation in vaccinated mice was observed to be significantlyenhanced in the presence of oil-in-water emulsion and Astragalus polysaccharides. Asignificant enhancement in IFN-γ by all polysaccharides was observed, which is indicative of Th1and Tc1responses, whereas Th2and Tc2responses were notenhanced. The adjuvants enhanced the release of a range of cytokines within1d afterinoculation.2.3. Toxicity analysis in miceMice were injected subcutaneously with10doses of oil-in-water emulsion.Fertility and body weight of inoculated mice were measured. Fragments of spleen,lung, kidney, liver, heart and gut were processed by the routine techniques of paraffinembedding and hematoxylin-eosin (HE) staining.The results showed that the djuvants used in vaccines carried no toxicity risk tofertility, body weight and viscera of the inoculated mice.3. A highly effective vaccine with adjuvants3.1. Preparation of the inactivated rabies vaccine (JX08-45strain)The vaccine production process was determined by immunization in dogs. Threedifferent batches of the inactivated vaccine were produced and tested in ourlaboratory.The potency of three different batches of the inactivated vaccine is respectively3.15IU/mL,3.10IU/mL and3.05IU/mL.3.2. Serological potency assay based on the FAVN testOn the14thday after vaccination, the median (20.58IU/ml) of rabies-neutralizingantibody titers of group Vac1exceeded reference3(15.64IU/ml), and the medians ofgroup Vac2, Vac3and Vac4were semiquantitated as being between reference2(3.01IU/ml) and reference3(15.64IU/ml). As a result, the potencies of the four testvaccines were in accord with their required qualities.To evaluate the accuracy and stability of the serological potency assay-basedFAVN, repeat tests (3times) and single-dose mouse protection test were performed.All results of repeat tests by the serological potency assay and of single-dose mouseprotection test accorded with the required qualities of test vaccines.The potency of test vaccines was evaluated by comparison of the protectiveefficacy of the test and reference vaccines in dogs. Median titer (6.15IU/ml) of therabies-neutralizing antibody of the group Vac1was equal to that of the groupreference3; however, the IQR (7.34IU/ml) of Vac1was less than that (8.20IU/ml)of reference3; i.e., the protective efficacy of vac1in dogs was higher than that ofreference3(6.15IU/ml). The protective efficacy of vac2, vac3and vac4in dogswas estimated to be between reference2(1.32IU/ml) and reference3(6.15IU/ml);i.e., the protective efficacy of the test vaccines in dogs was in line with their requiredpotency.3.3. Comparison of inactivated vaccine efficacy of JX08-45-related vaccine andcommercial vaccine in ChinaUnvaccinated dogs were injected with1mL JX08-45CC, or Nobivac Rabies in the triceps brachii muscle. Serum samples were collected from the brachial veinbefore vaccination and every1month for6months.The antibody titers of JX08-45CC and Nobivac Rabies both reached a peak inthe1stmonth with both slowly declining to about half of these titers by6monthspost-vaccination. Ninety days post-challenge, all dogs vaccinated with Nobivac andJX08-45CC inactivated rabies vaccines survived, and all negative control dogsdeveloped rabies from8d to24d after inoculation.As per above results, our conclusions are as fllows:1. The cell culture-adapted rabies virus derived from a Chinese isolate the viruscan be used as a vaccine candidate with clear biological background and highimmunogenicity in China.2. The oil-in-water emulsion and Astragalus polysaccharides can enhance rabiesantibody response by cell immune approach and have potential for use asimmunopotentiators/adjuvants in inactivated rabies vaccines for veterinary use.3. The inactivated rabies vaccine (JX08-45CC strain) could been used to protectdogs from rabies safely and effectively.