Construction And Immunogenicity Of Inactivated Recombinant Rabies Virus Vector Vaccines And Bacteria-like Particle Vaccines Against Nipah Virus Encephalitis

Nipah virus encephalitis is a zoonotic infectious disease caused by Nipa virus（NiV）.Since it was first emerged from Malaysia in 1998,NiV outbreaks have also spread to sub-tropical areas such as the Malaysia,Bangladesh,Philippines,Singapore and India.NiV spreads through the main transmission routes of fruit bats-pigs-humans transmission,with humans presenting with fever,headache,vomiting,encephalitis and respiratory diseases following NiV infection,with a mortality rate of40%-80%.Infection in pigs presents with neurological and respiratory symptoms and can also lead to mispregnancy and death.Because of its high lethality and destructiveness,Nipah virus encephalitis has been included in the World Health Organization（WHO）listed it in the blueprint list of priority research diseases^[1].In June 2022,the Ministry of Agriculture and Rural Affairs of China issued a notice listing NiV as a Class I animal epidemic disease.The National Institute of Allergy and Infectious Diseases and the Centers for Disease Control and Prevention（CDC）list NiV as the pathogen of biological defense concern,and the CDC also classifies it as Class C biological weapon according to the level of danger.To reduce the risk of transmission of Nipah virus encephalitis in our country and to provide technical assistance and supplies for the prevention and control of Nipah virus encephalitis in our country,this study used rabies virus（RABV）and gram-positive enhancer matrix as carriers,constructed the recombinant RABV vaccines and bacterium-like particles（BLPs）vaccines,respectively,carrying the major protective antigen F or G protein of NiV on the surface,and evaluated the immunogenicity of the two candidate vaccines.I have mainly done the following research:1.Construction and identification of RABV with recombinant NiV-F/G geneIn this study,the two recombinant viruses with surface chimeric NiV glycoprotein F or G was rescued by using RABV/SRV9 strain as reverse genetic vector.The F or G gene of NiV was inserted between the P and M genes of RABV vector,respectively,to construct RABV infectious clones.Then,BSR cells were then co-transfected with two full-length plasmids and four auxiliary plasmids,pc DNA3.1-SRV9-N,pc DNA3.1-SRV9-P,pc DNA3.1-SRV9-L and pc DNA3.1-SRV9-G,to obtain two recombinant viruses,r SRV9-NiV-F and r SRV9-NiV-G.The Western blot result showed that the purified two recombinant viruses could express the F or G protein of NiV;Under immuno-transmission electron microscopy,the surface of the two recombinant viruses was identified and labeled with specific donkey anti-mouse and donkey anti-rabbit gold labeled antibodies,respectively.The results showed that the F or G protein of NiV were embedded on the surface of RABV/SRV9 respectively.The growth kinetics curves showed that there was no significant difference between the two recombinant viruses and the vector viruses.the F or G gene of NiV were still positive by PCR,indicating that the two recombinant viruses could pass stably in BSR cells.2.Construction of BLPs displaying NiV-F/G proteinThe two BLPs were constructed using the GEM-PA system to display NiV-F_etcand NiV-G_etc proteins.A baculovirus insect cell expression system was used to express fusion proteins including the extracellular domains of the F or G protein of NiV and anchor hook protein PA,respectively.The two fusion proteins were combined with GEM to obtain two BLPs,BLP-NiV-F_etc and BLP-NiV-G_etc.Western blot and indirect immunofluorescence assay showed that the two fusion proteins were expressed in Sf9 cells;Under immune-transmission electron microscopy,the specific donkey anti-mouse gold labeled antibodies could effectively recognize and bind to the BLP-NiV-F_etc and BLP-NiV-G_etc surfaces.This indicated that the F or G protein of NiV,was displayed on the surface of BLP,respectively.3.Immunogenicity of RABV with recombinant NiV-F/G geneMice were immunized with inactivated recombinant virus to detect their cellular and humoral immune levels.The recombinant viruses r SRV9-NiV-F and r SRV9-NiV-G were inactivated withβ-propionolactone,and the inactivated viruses were added to ISA 201 VG and poly（I:C）complex adjuvants to immunize SPF grade BALB/c mice aged 4-6 weeks to detect their immune response parameters.The results showed that there was no significant change in the body weight of the mice after immunization compared to the control group.After the third immunization,spleen lymphocytes were isolated from the mice.The experimental results showed that after stimulation with NiV glycoprotein,the proliferation ability of spleen cells in the immunized group were significantly higher than that in the control group（p<0.001）,and the IFN-γand IL-4 cytokines secreted by spleen lymphocytes were significantly higher than those in the control group（p<0.01～0.001）.Using a commercially available ELISA cytokine detection kit to test Th1（IFN-γ,TNF-αand IL-2）and Th2（IL-4,IL-6 and IL-10）cytokines,the results showed that there were different levels of improvement compared to the control group.Both recombinant viruses can also stimulate the production of specific Ig G in mice.After the third immunization,the r SRV9-NiV-F and r SRV9-NiV-G immunization groups alone,and their co-immunization groups reached specific Ig G titers of 1:106,496,1:155,648 and 1:40,960,respectively.The results of antibody subtype identification showed that the Ig G2a/Ig G1 induced by the three immune groups was greater than 1,indicating that the immune response was dominated by Th1-biased immune responses.The results of IEM identification of the immunogenicity of the recombinant virus immune serum showed that the recombinant virus had good reactogenicity and could specifically bind to the BLP antigen.4.Immunogenicity of BLPs displaying NiV-F/G proteinMice were immunized with BLPs to detect their cellular and humoral immune levels.The prepared BLP-NiV-F_etc and BLP-NiV-G_etc were added to ISA 201 VG and poly（I:C）complex adjuvants,respectively,to immunize SPF grade BALB/c mice aged 4-6 weeks,and their immune response parameters were measured.The results showed that there was no significant effect on body weight of mice after immunized with the BLPs compared with the control group.After the third immunization,spleen lymphocytes were isolated from the mice and their proliferative capacity and cytokine secretion levels were measured.The results showed that after stimulation with the F or G protein of NiV,CCK-8 was added to stimulate spleen cell proliferation,and the stimulation index was higher than that of the control group（p<0.001）.The number of spleen lymphocytes secreting IFN-γand IL-4 factors was also significantly higher than that of the control group（p<0.05～0.001）.The cytokines（IFN-γ,TNF-α,IL-2,IL-4,IL-6 and IL-10）secreted by splenic lymph into the cell supernatant also increased to varying degrees.BLPs can stimulate the production of specific Ig G in mice after immunization.After the third immunization,the BLP-NiV-F_etc and BLP-NiV-G_etcimmunization groups alone,and their co-immunization groups reached specific Ig G titers of 1:8,192,1:16,384 and 1:45,056,respectively.The results of antibody subtype identification showed that the Ig G2a/Ig G1 induced by the three immune groups after the third immunization were all less than 1,indicating that the immune response tended to be dominated by the Th2-biased immune response.In conclusion,this study has successfully prepared the inactivated recombinant RABV vector vaccines and BLPs vaccine expressing NiV-F or G protein.Both vaccine candidates showed good immunogenicity and can induce cellular and humoral immune responses.After three immunizations with r SRV9-NiV-G,the specific antibody titer reached 1:155,648;After three times of combined immunization with BLP-NiV-F_etc and BLP-NiV-G_etc,the specific antibody titer reached 1:45,056.This study lays the foundation for the development of a novel type of vaccine with strong immunogenicity,safety and efficacy against Nipah virus encephalitis,and also provides technical support and material reserves for the prevention and control of Nipah virus encephalitis in our country.