Identification Of Effective Adjuvants For Zika Virus EDⅢ Subunit Vaccine And Exploration Of Non-Neutralizing Epitopes

Background:Zika virus(ZIKV)was first isolated from a rhesus macaque in Uganda in 1947and first detected in humans in Nigeria in 1953.To date,ZIKV infection has been reported in more than 60 countries worldwide.ZIKV causes neurological diseases such as Guillain-Barre syndrome and congenital Zika syndrome(symptoms include microcephaly,brain abnormalities and other congenital malformations).The virus is transmitted by Aedes Aegypti and belongs to the flavivirus genus together with yellow fever virus,dengue virus,Japanese encephalitis virus and West Nile virus.ZIKV is an enveloped virus with an approximately 10.7-kb positive-sense RNA genome and has three structural proteins such as capsid(C),precursor of membrane/membrane(pr M/M)and envelope(E),in which the protein E has important roles in infection and pathogenesis.The E protein is a transmembrane protein and consists of three domains termed DⅠ,DⅡ,and EDⅢ,a fusion loop(FL)and a stalk region(S).The DⅠ–DⅡ region of ZIKV E protein is a target for cross-reactive antibodies with other flaviviruses,such as dengue virus(DENV)and West Nile virus(WNV),whereas the EDⅢ domain only induces ZIKV-specific antibodies,and therefore,is a key target for the development of ZIKV vaccine and therapeutic antibodies.Currently,ZIKV vaccine candidates under development include inactivated virus,live attenuated virus,nucleic acid vaccines,viral vectors,virus-like particles(VLPs),and viral proteins(or subunit vaccines).However,these vaccines are still under clinical trials and none is approved for use in humans.Our research group has designed a fusion protein subunit vaccine based on ZIKV EEDⅢ domain and human Ig G Fc segment in the early stage,and verified that it can induce specific neutralizing antibodies in mice.Purposes:The purpose of this study is to compare the adjuvanticity of MF59,Alum,MPL,or Alum+MPL combination(refer to AS04),in a hope to identify an adjuvant or adjuvant combination that is able to promote the EEDⅢ-based ZIKV vaccine to induce a balanced immune response and potent protection against ZIKV infection.This study also aims to find the non-neutralizing dominant epitopes of EEDⅢ subunit vaccine and further optimize the design of subunit vaccine.Methods:(1)ZIKV-M375N/E377T-Fc protein was expressed successfully in eukaryotic expression system and purified by Protein A affinity chromatography.Then,mice were immunized with ZIKV protein and different adjuvants,and were boosted three weeks later.The sera were collected at 7 days post-last dose.ZIKV EEDⅢ protein-specific antibody titers,E protein-specific antibody titers,and h Fc antibody titers were analyzed by ELISA.Neutralizing antibodies against three Zika virus strains R103451,PAN2016,and PRVABC59were measured by plaque reduction neutralization test(PRNT).Flow cytometry analysis was performed to detect the frequency of CD4~+T cells and CD8~+T cells that activate and secrete cytokines in peripheral blood.The PRNT and q PCR were used to analyse the virus titers and ZIKV RNA copies in sera and tissues of challenged mice.(2)Looking for non-neutralizing dominant epitopes of Zika virus EEDⅢ subunit vaccine.First,seven distinct epitopes were selected on ZIKV EEDⅢ based on its crystal structure and functional characteristics.Glycan probe was introduced to each of 7 epitopes by site-directed mutation PCR.Mutant proteins were then expressed and purified in 293T cells and confirmed by Western Blot.The antigenicity of ZIKV mutant proteins was analyzed by ELISA.After that,the above EEDⅢ proteins were used to inject BALB/c mice and A129 mice,followed by the analysis of ZIKV specific antibodies,neutralize immunogenicity,protective immune response induced by the mutant proteins,and passive immune protection.Results:(1)The effects of different adjuvants on the efficacy of the protein vaccine were analyzed by mouse models.ELISA test shows that,the combination of Alum with MPL adjuvant induced significantly higher levels of EEDⅢ-specific antibodies and induced a more balanced antibody response,indicated by a low Ig G1/Ig G2a ratio;Plaque reduction neutralization test shows that for all three ZIKV strains,the neutralizing antibody titers induced by EEDⅢ when Alum was used in combination with MPL were significantly higher than other groups;Compared to the use of individual adjuvants,the combination of Alum with MPL enhanced the EEDⅢ-specific cellular immune response(Th1,Th2,Th17)and the frequency of IFN-γ~+/CD8~+T cells and IL-4~+/CD8~+T cells.(2)The greatest level of protection against a high-dose ZIKV challenge was achieved when EEDⅢ vaccine is formulated with both Alum and MPL adjuvants.(3)To explore the non-neutralizing dominant epitopes of EEDⅢ subunit vaccines,the effects of glycosylation modification of 7 epitopes of ZIKV EEDⅢ protein on the immune protection of vaccines were analyzed.Protein antigenicity analysis showed that the glycan probe located at residue 366 bound to all neutralizing antibodies tested,but did not bind to ZV-2 which has no neutralizing activity in vitro;the glycan probe located at residue 393 abolished the binding of EEDⅢ protein to 3 neutralizing antibodies;and the glycan probe located at residue 333 also abolished the binding of EEDⅢ protein to 3 neutralizing antibodies;Evaluation of neutralizing immunogenicity of these EEDⅢ epitopes demonstrated that EEDⅢ proteins containing a glycan probe located at residue333 or 366 induced significantly higher neutralizing antibodies titers.In contrast,a glycan probe located at residue 309 or 393 of EEDⅢ protein can reduce the neutralizing antibody titers.The EEDⅢ proteins with a glycan probe located at residue 315,351,or 369 induced insignificant changes in neutralizing antibody titers,as compared with wild type EEDⅢ vaccine.(4)Live virus protective experiments shows that EEDⅢ proteins containing a glycan probe located at residue 333 or 366 could significantly improve the active immune protection and passive immune protection of the vaccine,while the EEDⅢ mutant proteins containing a glycan probe located at residue 309 or 393 effectively reduced the immune protection of the vaccine.Conclusion:The immunogenicity and the ability to protect against ZIKV infection are significantly enhanced when both Alum and MPL are used in the same vaccine formulation.This study has identified the combination of Alum with MPL as an effective adjuvant formulation for ZIKV EEDⅢ-based subunit vaccines.Glycosylation at four epitopes(333,366,393,and 309)can effectively change the immune protection of the EEDⅢ subunit vaccine.Modification of epitopes 333 or 366 of the ZIKA EEDⅢ subunit by glycosylation can successfully block non-neutralizing dominant epitopes and improve the efficacy to induce neutralizing antibodies,immunogenicity and immune protection of the vaccines.