Study On The Construction And Immunogenicity Of Replication-deficient Recombinant Rabies Virus

Rabies,also known as hydrophobia,is a neurotropic zoonotic infectious disease caused by the Rabies virus(RABV).Rabies has a 100% lethality rate.RABV can infect humans and various warm-blooded animals,and dogs are currently the main source of rabies transmission.According to the World Health Organization(WHO),about 60,000 people have killed by rabies each year.Rabies poses a major threat to global human health and public health security.Currently,there is no effective treatment for rabies.Rabies can be controlled by vaccination.WHO studies have shown that vaccination of more than 70% of dogs in endemic areas is effective in preventing the spread of rabies.Currently,all commercially available rabies vaccines are inactivated,which are safe but have problems such as high costs and unsustainable immunization effects.Live-attenuated viruses have been banned from production and use in China because of the possibility of causing disease.Therefore,research on safe and effective new vaccines is important for the comprehensive prevention and control of rabies.Replication of replication-incompetent viruses is dependent on the supplementation of exogenous proteins,and only a single round of infection occurs in normal host cells,which cannot replicate and proliferate.The biosafety of replication-incompetent viruses has been greatly improved compared to wild-type viruses.Studies have shown that when used as a vaccine,replication-incompetent viruses can induce immune protection by expressing antigens in infected cells,just like the Live-attenuated virus vaccine,and have the same safety profile as inactivated vaccines.In this study,cell lines expressing rabies virus glycoprotein(RABV G)were constructed using the Piggy Bac transposon system.The G-gene-deficient recombinant rabies virus called r SRV9-ΔG-e GFP was successfully constructed using the rabies virus reverse genetics system.Its safety and immunogenicity were evaluated by animal experiments.The experiment lays the foundation for the research of the new rabies vaccine and vaccine vector.Specific studies include:(1)Establishment and characterization of stable cell lines expressing rabies virus glycoproteinBased on the Piggy Bac transposon system,the SRV9 G gene was amplified and the recombinant plasmid YHM-SRV9-G was constructed.The recombinant plasmid was cotransfected with Piggy Bac transposase plasmid into BSR/T7 cells,and the monoclonal cell line was obtained by serial drug screening with Blasticidin S hydrochloride(Blasticidin).The results showed that the appropriate Blasticidin drug sieve concentration was 20 μg/m L.A monoclonal cell line(BSR-G)stably expressing RABV G protein was obtained after twice consecutive monoclonal purifications according to the limited dilution method.The BSR-G cell line was identified by PCR,indirect immunofluorescence,Western Blot,and laser confocal techniques.The screened stable transfer cell line BSR-G cells could stably express the RABV G protein,and the RABV G protein was mainly expressed on the cell membrane.(2)Construction and characterization of the replication-deficient G-gene-deficient recombinant rabies virusBased on the reverse genetic system of the RABV SRV9 strain established in the laboratory,a full-length infectious clone of the recombinant rabies virus(p D-SRV9-ΔG-e GFP)was constructed.,which has a deletion of the G gene and expresses the green fluorescent protein(e GFP)gene.The plasmid called p D-SRV9-ΔG-e GFP was cotransfected with four helper plasmids expressing N,P,G,and L in BSR-G cells to rescue the recombinant virus.The recombinant virus was identified by DFA,RT-PCR,and transmission electron microscopy,and their biological properties were analyzed.DFA results indicate successful rescue to recombinant rabies virus called r SRV9-ΔG-e GFP.RT-PCR results showed that the recombinant virus genome did not contain the RABV G gene and was genetically stable.Transmission electron microscopy results showed that r SRV9-ΔG-e GFP still had the typical bullet-like morphological characteristics.Growth curves of r SRV9-ΔG-e GFP showed that the recombinant virus had similar growth kinetic trends to the parental virus SRV9.The propagation characteristics studies showed that r SRV9-ΔG-e GFP could not proliferate on BSR cells and only proliferated effectively on BSR-G cells.(3)Immunogenicity studies of replication-incompetent G-gene-deficient recombinant rabies virusTo evaluate the safety of r SRV9-ΔG-e GFP,the recombinant virus and the parental virus SRV9 were infected with 5-day-old ICR suckling rats by intracerebral injection.The mental status,weight change,and mortality of the suckling rats were observed and recorded for 14 consecutive days.The results showed that the mice in the SRV9 group began to show clinical signs of depression,paralysis,and death at 4 d after virus infection,and all died at 11 d after infection.The r SRV9-ΔG-e GFP infected mice did not show symptoms of disease,and all survived.To assess the immunogenicity of the recombinant virus called r SRV9-ΔG-e GFP,the r SRV9-ΔG-e GFP,the inactivated parental virus,and the parental virus were immunized by intramuscular injection into mice to detect the cellular and humoral immune responses.The results showed that the activation of DC cells,T cells and B cells were detected in inguinal lymph node cells of mice after the first immunization with the r SRV9-ΔG-e GFP.Moreover,the activation of T cells and B cells and the production of memory T cells were detected in the spleen 1 week after triple immunization,and the production of IFN-γ and IL-4secreting splenocytes was significantly increased.RABV-neutralizing antibodies were detected in the serum of mice after triple immunization.The antibody potency reached the international standard(0.5 IU/m L).In summary,a replication-incompetent G-gene-deficient recombinant rabies virus called r SRV9-ΔG-e GFP was constructed using the rabies virus reverse genetics system.r SRV9-ΔG-e GFP could only proliferate effectively in the stable cell line BSR-G expressing the glycoprotein of the rabies virus.It was not pathogenic to 5-day-old suckling mice.When compared to the parental virus it has a higher safety profile compared with the parental virus.Immunization of mice with r SRV9-ΔG-e GFP stimulates the production of rabies virus-neutralizing antibodies,and specific cellular immune responses.The recombinant rabies virus could be used as a novel genetically engineered vaccine candidate for Rabies.