Construction And Immunological Study Of Recombinant RABV Expressing Rift Valley Fever Virus Egn Protein And Rift Valley Fever Virus Bacterial-Like Particles

Rift Valley Fever is a zoonotic disease caused by Rift Valley Fever virus,which is a significant threat to domestic ruminants and humans.RVFV can be transmitted by mosquito bites,direct contact with sick animals and their products,and aerosols.RVFV was first discovered in 1931 in Kenya's Great Rift Valley.First outbreak outside of Africa was in Arabian Peninsula in 2000.Both the United Kingdom's Anti-terrorism,Crime and Security Act in 2001 and the United States Patriot Act in 2002 listed RVFV as a potential bioterrorism agent.The World Health Organization(WHO)'s first Workshop on Prioritization of Pathogens assigned RVF to the list of"severe emerging diseases with potential to generate a public health emergency" in 2015(8+3)and 2018(9+X).RVF remains on the World Organization for Animal Health(OIE)'s list of notifiable animal diseases of concern.As global warming leads to an expanded distribution of insect vectors and an increased international trade,the risk of RVFV introduction into countries where RVFV is not endemic is increasing.China identified the first RVF imported case in July 2016.The outbreak of the RVF epidemic not only threatens human health but the resulting trade barriers also seriously affect the country's economic and trade development.Traditional inactivated vaccines and attenuated vaccines have the disadvantages of increased virulence or virus recombinant,which make their use is severely restricted in RVF non-endemic areas and no approved RVF vaccine is available for use in non-endemic areas.The development of vaccines and therapeutic antibodies for the prevention and treatment of RVF is a great significance for preventing and controlling the spread of RVF,protecting patients,and reducing economic losses.Preventing and controlling RVF from animal sources can effectively control the spread of RVFV,and it is imminent to develop a safe and immunogenic veterinary RVF vaccine.Therefore,the construction and immunological study of recombinant RABV expressing RVFV eGn protein and RVFV bacterial-like particle were carried out.1.Construction and identification of recombinant rabies virus rSRV9-eGn expressing RVFV eGn proteinBased on the infectious clone of the attenuated SRV9 RABV strain in our laboratory,this study inserted the gene of RVFV major antigen epitope Gn ectodomain into the SRV9 genome by molecular biology technology to construct a recombinant plasmid rSRV9-eGn.BSR cells were co-transfected with recombinant plasmid rSRV9-eGn and helper plasmids pD-N,pD-P,pD-G,pD-L,which successfully rescued the recombinant virus rSRV9-eGn.Direct immunofluorescence results showed that the recombinant virus rSRV9-eGn was successful rescued;RT-PCR results demonstrated that the RVFV eGn gene has been successfully inserted into SRV9 genome and can be stably inherited;Indirect immunofluorescence,SDS-PAGE and Western Blot results showed that RVFV eGn protein was successfully expressed and co-chimeric with RABV G protein on the surface of RABV virions,laying the foundation for inactivation of rSRV9-eGn;observed under the electron microscope,the recombinant virus rSRV9-eGn has typical elastic morphological characteristics;the growth curve showed that the recombinant virus rSRV9-eGn had similar growth kinetics with rSRV9,and cultured for 72 h was the optimal harvest time of the viruses;intracranial injection of recombinant virus rSRV9-eGn and rSRV9 at different viral titers in ICR suckling mice and adult mice showed that insertion of the RVFV eGn gene reduced the neurotoxicity of the rSRV9 strain.In summary,this part of the study successfully rescued the RVF eGn gene chimeric recombinant virus rSRV9-eGn.2.Immunogenicity evaluation of recombinant rabies virus rSRV9-eGn expressing RVFV eGn proteinIn order to assess the immunogenicity of the recombinant virus rSRV9-eGn,mice were immunized with live rSRV9 and live recombinant virus rSRV9-eGn,and inactivated recombinant virus rSRV9-eGn was immunized alone or with three different adjuvants.The results showed that only inactivated recombinant virus rSRV9-eGn combined with Poly I:C and ISA201VG compound adjuvants group could induce mice to produce anti-RVFV eGn-specific IgG antibodies,with IgG1 as the dominant subtype and IgG1/IgG2a>1,this indicated that it could trigger a Th2-type humoral immune response.The RVFV pseudovirus packaged with the VSV-based system was used to verify neutralizing antibodies in immunized mouse sera,and it was found that there was no anti-RVFV neutralizing antibody in mouse serum containing high level RVFV eGn-specific IgG antibody.Comprehensive analysis of cytokine secretion in sera and splenocytes of immunized mice revealed that inactivated recombinant virus rSRV9-eGn combined with Poly I:C and ISA201VG compound adjuvants could induce mice to develop a humoral immune response produced by Th2 cells.In addition,inactivated recombinant virus rSRV9-eGn combined with Poly I:C and ISA201VG compound adjuvants can also induce the increase of central and effector memory T cells in mice,in which effector memory T cells are dominant.Inactivated recombinant virus rSRV9-eGn combined with Poly I:C and ISA201VG compound adjuvants immunized mice,which could induce mice to produce high level RVFV-specific IgG antibodies and anti-RABV neutralizing antibodies.RABV in vivo and in vitro neutralization experiments showed that one week after first immunization was 30%,two weeks after first immunization was 70%and one week after first booster immunization was 100%positive for anti-RABV neutralizing antibodies.CVS-11 street strain challenged immunized mice one week after the first immunization and one week after the second immunization,with survival rates of 30%and 100%,respectively.The above results show that the rSRV9 strain has good immunogenicity and is a potential viral vector.3.Construction and identification of RVFV bacterial-like particles displayed by GEMUsed insect cell-baculovirus expression system in our laboratory,RVFV bacterial-like particles displayed by GEM was prepared.The first is to fusc the RVFV Gn head gene with the Lactococcus lactis MG 1363 PA gene by fusion PCR to achieve the expression of the RVFV Gn head-PA fusion protein.The food-grade Lactococcus lactis MG 13 63 was acidified to prepare GEM particles.The RVFV Gn head-PA fusion protein interacts with the GEM particles,and the RVFV Gn head-PA fusion protein is displayed on the surface of the GEM particles through non-covalent binding.PCR amplification results showed that the HBM-His-RVFV Gn head-PA fusion gene has been successfully inserted into the recombinant baculovirus genome;indirect immunofluorescence,SDS-PAGE,and Western Blot results indicated that the RVFV Gn head-PA fusion protein has been successfully expressed and secreted into supernatant,but this recombinant baculovirus could only be passed to the fourth generation,and no fusion protein was detected in the fifth generation cell culture.The results of indirect immunofluorescence,flow cytometry and immunoelectron microscopy showed that RVFV Gn head-PA fusion protein has been successfully displayed on the surface of GEM particles.The ultra-thin section results determined that the untreated Lactococcus lactis MG1363 cell wall was clearly visible and the internal texture was uniform;GEM particles retain the smooth morphology of Lactococcus lactis,removed its own genetic material and proteins,and interior was hollow;after the RVFV Gn head-PA fusion protein was displayed on the GEM surface,a layer of floc was attached to the GEM surface.To sum up,the RVFV Gn head-PA fusion protein,which expressed by insect cell-baculovirus expression system,was successfully displayed on the surface of GEM particles,and were prepared as RVF bacterial-like particle,RVFV-BLPs.4.Immunogenicity evalution of GEM displayed RVFV baterial-like particlesIn order to evaluate the immunogenicity of RVFV-BLPs and the effect of different antigen amounts on their immunogenicity,20 ?g,50 ?g and 100 ?g doses of RVFV-BLPs were used to immunize mice.The results showed that RVFV Gn head specific IgG antibodies could be detected in all three groups,but the level of specific IgG antibodies was not directly proportional to the dose of immunogen.Among them,RVFV-BLPs(50 ?g)group had the best immune effect and showed early antibody appearance,long duration,slow decay.By examining the IgG subtypes,it was found that the IgG subtypes produced by three different doses of RVFV-BLPs stimulated mice also showed different characteristics.Only IgG1 was detected in the RVFV-BLPs(20 ?g)group;the more potent IgG1 was detected in the RVFV-BLPs(50 ?g)group,and IgG2a and IgG3 were detected at different time points after immunization;the RVFV-BLPs(100?g)group was only detected IgGl and IgG2a,of which IgG1 was dominant.The IgG antibodies produced by immunizing mice with three different amounts of RVFV-BLPs were maily IgG1 subtypes,and IgGl/IgG2a were all greater than I,indicating that RVFV-BLPs immunized mice tended to Th2 type humoral immune response.Three groups of mouse serum were taken for cytokine analysis.It was found that mice immunized with different antigen doses of RVFV-BLPs could induce mice to produce different types and amounts of cytokines,indicating that the antigen dose may strongly affect the type of functional effector cells induced during the immunization process type.The RVFV-BLPs(20?g)group and RVFV-BLPs(50 ?g)group mice could produce Th1 type cytokines and Th2 type cytokines;the RVFV-BLPs(100?a)group were predominantly Th2 type cytokines.Based on the analysis of specific IgG subtypes and cytokine types in serum of immunized mice,the mice were induced to produce Th2 type humoral immune responses after immunized with RVFV-BLPs,and selected RVFV-BLPs(50 ?g)group for further research.The RVFV pseudovirus packaged with the VSV-based system was used to verify neutralizing antibodies in mouse serum harvested two weeks after third immunization,it was found that serum diluted 40 times can neutralize 50%RVFV pseudovirus.Spleen cells harvested from two weeks after third immunization of RVFV-BLPs(50 ?g)group mice were tested for cellular immunity.It was found that RVFV-BLPs(50 ?g)could promote the proliferation of mouse lymphocytes,and also promoted the production of IFN-y and IL-4.Compared with the PBS group,the number of central memory T cells of CD4+and CD8+T lymphocytes increased,and the number of dominant effector memory T cells decreased.This indicated that after immunized mice with RVFV-BLPs(50 ?g),central memory T cells produced by the mice were induced to settle in the T cell area of peripheral immune organs.When stimulated by the antigen,they differentiated into effector cells and effector molecules,and did not directly exert effects.When stimulated again,it is redifferentiating into effector cells and quickly exerting an immune effect.To sum up,the two RVFV-preventing immunogens prepared in this study,namely RVFV eGn protein recombinant rabies virus rSRV9-eGn and GEM display RVFV bacterial-like particles RVFV-BLPs,both are the first applications of RABV vector and GEM-displayed bacterial-like particle methods to the exploration of new RVF vaccines.The characteristics of the two are different:both can stimulate the body to produce specific cellular and humoral immune responses;the use of inactivated recombinant virus rSRV9-eGn combinated with adjuvant was expected to develop a dual vaccine that could prevent both RABV infection and RVFV infection;GEM displays RVFV bacterial-like particles RVFV-BLPs were easy to produce,store and transport.Both two have the potential to develop into RVF candidate vaccine,laying the foundation for the research of new RVF vaccines.