Screening Of New Antigen Based On Zaire Ebola Virus Glycoprotein And Construction Of RVSV-based Ebola Vaccine

Ebola virus is the pathogen of severe Ebola hemorrhagic fever and belongs toClass A biological warfare agents that can be used as biological weapons and forbioterrorism. Because severe disease symptoms emerge very fast, human body cannot produce effective immune response. No effective treatments and licensed vaccinesare now available for EBOV infection. Up till now, viral vector-based Ebola vaccinesexpressing GP, such as recombinant adenovirus vector vaccine (rAD5-GP) andrecombinant vesicular stomatitis virus vector vaccine (rVSV-GP), have been provedto give effective protection against the lethal challenge of Ebola virus in no humanprimates (NHP). However, rAD5-GP vaccine has the potential pre-existing immunityand needs high immunizing dose and the long immunization time. In contrast,rVSV-GP vaccine has no pre-existing immunity and induces effective immuneresponse quickly, and provides therapeutical effects. Its combined vaccines, which arecomposed of monovalent rVSV-based vaccines, could provide effective protectionagainst the lethal challenge of multiple Ebola viruses in NHP. It is known that thecombined vaccines are costly and need to be evaluated seperately. While, rVSV vectorcan only express the shortly exogenous gene bellow4.5kb, so it is difficult toconstruct polyvalent vaccine which expressing several GP based on one rVSV vector.Since the advantages of rVSV vaccines, we constructed an Ebola GP’sfragment-based VSV multiple vaccine. Three antigen fragments (GP1ΔM，MLD，L)from ZEBOV-GP were designed to construct pVAX1-based recombinant plasmidsaccording to the epitope distributions, the structure and function of ZEBOV-GP.Through analysis of antibody levels of the sera of the immunized BALB/c mice, thefragment L from the link region of GP (aa393-556) was selected as the candidateantigen. The immunogenicity and the critical immunogenic region of fragment L wasthen determined by using the immunization with its recombinant protein or pVAX1plasmid individually. Because the fusion proteins linked by FMDV-2A could beeffectively cleaved in the absence of protease, FMDV-2A was used to constructmultiple vaccines by using reverse genetic technology. The major results are as follow:1. Fragment L possesses high immunogenicity and produces effective neutralizingantibody(1) Fragment L from the link region of ZEBOV-GP(aa393-556) induced stronghumoral immune response and effective cellular immune responseThe glycoprotein of the most virulent Zaire Ebola virus was selected as thetemplate for the screening of antigen fragments. According to the distribution ofepitopes in GP and structure of GP, we designed three antigen fragments(GP1ΔM,MLD, L), constructed recombinant plasmids based on pVAX1vector and immunizedBALB/c mice. Through analysis of antibody levels in immune serum, we found thatafter3times immunization, the levels of antibody in the sera from mice immunizedwith pVAX1-L or pVAX1-GP were significantly higher than those from mice thatreceived pVAX1-GP1ΔM and pVAX1-MLD. More importantly, the antibody levelsinduced by pVAX1-L were as high as those induced by pVAX1-GP. Furtherly, thepVAX1-GP and pVAX1-L immunization groups exhibited similar IgG2a/IgG1ratios.These results indicated that the ability of L to induce humoral immune response isconsistent with GP, and significantly stronger than GP1ΔM and MLD. On the otherhand, fragment L induced a weaker cellular immune response than GP, the SI ofsplenocytes proliferation and the levels of IFN-γ and IL-2of the L immunizationgroup were all significantly higher than those of the control group, indicating L couldinduce moderate cellular immune response.(2) Fragment L induced the secretion of neutralizing antibody effectivelyThe analysis of neutralizing antibody levels was carried out usingGP-pseudovirus. pVAX1-L immunization serum exhibited an inhibitory rate as highas that of pVAX1-GP immunization serum, indicating that the levels of neutralizingantibody were similar between the GP immunization group and the L immunizationgroup. The result further demonstrated that immunogenicity of L is similar to GP.(3) The critical immunogenic region of L located at the area containing furin site andinternal fusion loopAccording to the structure and function of L, three smaller fragmentsLA(aa393-479), LM(aa436-519) and LB(aa480-556) were designed to constructpVAX1-based recombinant plasmids and immunize BALB/c mice. No significant differences were observed in the antibody levels of sera among pVAX1-L,pVAX1-LM, and pVAX1-LB immunization groups except pVAX1-LA immunizationgroup after3times immunization. The inhibitory rates of pVAX1-LM and pVAX1-LBimmune serum to GP-pseudovirus infection were similar to that of pVAX1-L immuneserum, although the pVAX1-LA immune serum did not inhibit GP-pseudovirusinfection as effectively as the pVAX1-L immune serum. These results indicated thatthe high immunogenicity and high induced neutralizing activity of fragment L may beattributed to the structure and function of the furin site and the internal fusionloop(aa479-556of GP).2. The rVSVΔG-based bivalent vaccine containing Sudan-GP-2A-L sequenceinduced cross-immunity against Zaire-GP and Sudan-GP effectivelyIn order to demonstrate the possibility of the bivalent vaccines based onFMDV-2A and rVSVΔG vector, we constructed the bivalent vaccinerVSVΔG-SGP-2A-L that contained Sudan-GP and L. Afte the SGP and L gene werelinked by FMDV-2A and inserted into rVSV vector to construct a recombinantbivalent vaccine vector, rVSVΔG-SGP-2A-L vaccine and two monovalent vaccinesrVSVΔG-SGP and rVSVΔG-ZGP were rescued using reverse genetic technology,respectively. Fresh BHK21cells were then infected with the recombinant vaccinesand the cells lysate was applied to the detection of antigens expression. The resultsshowed that the recombinant vaccines induced CPE and expressed the SGP and Lidentified by western blot. After immunized Balb/c mouse, IgG and neutralizingantibody titers showed that rVSVΔG-SGP-2A-L produced significant cross-immunityagainst Zaire-GP and Sudan-GP. These results indicated that it is efficient for theconstruction of rVSV-based polyvalent vaccines using FMDV-2A sequence andfragment L.In conclusion, antigen L induces strong humoral immune response and elicitsefficient neutralizing antibodies, similar to the full-length GP. The criticalimmunogenicity region of L locates at the area including sequence around furin siteand internal fusion loop (aa479-556of GP). The bivalent vaccines based on rVSVΔGvector and FMDV-2A expresses the two antigens Sudan-GP and L and inducescross-immunity against Zaire-GP and Sudan-GP effectively. The fragment L could beused to replace GP to construct monovalent vaccine or polyvalent vaccine based on rVSVΔG vector and FMDV-2A.