Construction And Immunological Study Of VLPs And Recombinant RABV Expressing Glycoprotein Of MARV

Marburg haemorrhagic fever?MHF?is an acute and severe zoonotic disease caused by Marburg virus?MARV?.Currently,there aren't effective drugs or licensed vaccines to control the outbreak and spread of this disease.As MARV is a Biosafety Level 4?BSL-4?pathogen,it further hinders the development of its traditional vaccine.Therefore,development of safe and effective novel vaccine has important practical applications for reacting to MHF outbreaks and possible bioterrorism attacks.Virus-like particles?VLPs?mimic the morphology of the natural virus and there was non-replicative and non-infective.VLPs stimulate innate immunity and further elicit adaptive immune.Therefore,VLPs represent a major advancement in the development of subunit vaccines with enhanced immunogenicity.Human papilloma virus?HPV?VLPs and hepatitis?HBV?VLPs have been approved to commercial production.In recent years,with the deepening of research,viral vector vaccines have played an important role in the prevention and control of severe infectious diseases.RABV has many advantages as a vaccine vector,such as simple genome,stable expression of foreign proteins,insertion of multiple genes and large fragments,decreased pathogenicity of attenuated strains,stable immunogenicity and so on.The study used the baculovirus/insect cell expression system?Bac-to-Bac/IC?to construct MARV VLPs and constructed a recombinant rabies virus expressing MARV GP using rabies virus SRV9 strain as a vector,and respectively evaluated their safety and immunogenicity in mice,horses,and non-human primates,which lays the foundation for the development of safe and efficient MARV vaccines.Thus,the present study chose two proteins to construct and rescue one recombinant baculoviruses that co-express GP and VP40 by Bac-to-Bac/IC.The results of genomic PCR,indirect immunofluorescence and Western Blot showed that rescued recombinant baculoviruses successful expressed MARV GP and VP40 protein.The recombinant baculovirus infected Sf9 cells and successfully obtained MARV VLPs.Transmission electron microscopy showed that VLPs are similar to native MARV and have a typical filamentous structure with the diameters of about 600-1000 nm.MARV VLPs was purified by ultracentrifugation and sucrose density gradient centrifugation.The immunogenicity of MARV VLPs alone or mixed with three adjuvants(Poria cocos polysaccharide[PCP-II],Polyinosinic Acid-Polycytidylic Acid[PolyI/C]and aluminium hydroxide[Alum]were evaluated after intramuscular vaccination in BALB/c mice and rhesus macaques.The results of mice study showed that vaccination with the MARV VLPs induced neutralizing antibodies and cellar immune responses.MARV VLPs mixed with PCP-II adjuvant group resulted in high titres of MARV-specific antibodies,activated relatively higher numbers of B cells and T cells in peripheral blood mononuclear cells?PBMCs?,and induced greater cytokine secretion from splenocytes than the other adjuvants.The results of rhesus macaques showed that vaccinated with MARV VLPs mixed with PCP-II adjuvant produced a GP-specific IgG titer of up to 1:1280 and virus-neutralizing antibody titers that reached 1:320.MARV VLPs also elicited interferon-??IFN-??and interleukin-4?IL-4?secretion associated with T-helper 1 cell?Th1?-and T-helper 2 cell?Th2?-mediated immunity,as detected using ELISpot assays.These data indicated that MARV VLPs mixed with a PCP-II djuvant have excellent immunogenicity and could induced specific humoral and cellular immune responses in mice and rhesus macaques.Passive immunotherapy with anti-serum was an effective and economic approach to the prevention and treatment of viruses and bacteria infectious.Healthy horses were inoculated with purified MARV VLPs to produce hyperimmune serum and F?ab'?₂.Titers of serum IgG reached 1:20480 and virus-neutralizing antibody titers reached1:640.The median effect concentrations?EC₅0?of horse anti-MARV IgG and F?ab'?₂were 0.316 mg/mL and 10 mg/mL,respectively.Based on the reverse genetic manipulation platform of rabies virus SRV9 strain established,the G gene of MARV was introduced between the P and M genes,rescued recombinant rabies virus expressing Glycoprotein of MARV.The results of direct immunofluorescence and Western Blot showed that the recombinant virus r SRV9-MGP could successfully expressed MARV GP.rSRV9-MGP was stablely duplicated in BSR cell and the titers of rSRV9-MGP were up to 10⁸ FFU/mL.Compared with the parent strain rSRV9,the G gene of MARV didn't affected growth property and pathogenicity of rSRV9-MGP.To evaluate the immunogenicity of recombinant viruses,rSRV9 and r SRV9-MGP were inactivated and then alone or mixed with PCP-II adjuvant to vaccinated mice via the intramuscular route.The results revealed that mice inoculated with rSRV9-MGP induced virus-neutralizing antibody titers against RABV and MARV.rSRV9-MGP mixed with PCP-II could significantly enhance cellular immune responses and virus-neutralizing responses than other groups.In summary,MARV VLPs and recombinant virus rSRV9-MGP could successfully induce specific humoral and cellular immune responses,and provided technical support and material reserves for the further development of novel vaccines against MARV.