Development Of A Novel ZIKV T Cell Vaccine Based On Comprehensive Epitope Mapping

Zika virus(ZIKV)is a flavivirus transmitted by mosquitoes.It belongs to the flavivirus genus of the Flaviviridae family and is widespread in tropical and subtropical regions.Since it was first discovered in Zika Forest in Uganda in 1947,no epidemic has erupted globally for more than 60 years.In 2007,after the first outbreak of ZIKV on Yap Island in the western Pacific Ocean,it caused some concern;but it was not until the outbreak of ZIKV in South America in 2015 that it received widespread attention from the international community.Based on the major threat that the virus poses to global public health security,ZIKV infection was officially listed as a global public health emergency in 2016,and research on it has also continued.The surface of ZIKV is covered with an envelope.The inner nucleocapsid forms an icosahedral structure.Its genome is a single-stranded RNA of about 11 kb in size.After being infected with ZIKV,the patient's symptoms are generally mild,including fever,rash,arthralgia,and conjunctivitis.Studies have also found that it is necessary to be associated with neurological diseases(such as Guillain-Barré syndrome);more serious,Congenital ZIKV infection may cause serious clinical diseases,including skeletal abnormalities,eye vision lesions,microcephaly,hydrocephalus,etc.ZIKV's possible vertical transmission,sexual transmission,blood transmission and many other transmission routes also make the prevention and treatment of ZIKV more difficult and more likely to cause a serious public health crisis.After ZIKV attracted wide attention from the international community,its vaccine research and development also entered the "fast lane".At present,the types of ZIKV vaccines under development mainly include live attenuated vaccines,inactivated vaccines,nucleic acid vaccines,and recombinant vaccines.These vaccines under development are focused on inducing the production of neutralizing antibodies.Antibodies can inhibit the replication of viruses by neutralizing virus particles,but although the antibodies produced may be protective,they may also cause virus infection.The occurrence of antibody dependent enhancement(ADE)response.There is experimental evidence that ZIKV antibodies can help dengue virus to enhance infection of the body or cells(ADE)in an in vitro experimental model,and vice versa.It has been reported that the T cell response during ZIKV infection plays an important role.The memory CD4 + and CD8 + T cell responses of ZIKV have been shown to have protective effects against ZIKV in animal models,and the T cell response itself has no possibility of ADE.In this project,through the development of a protective and non-ADE risk T cell vaccine,the feasibility and effectiveness of T cell vaccine to prevent viral infections are studied,and then the role of T cell response in the process of ZIKV infection and T cell vaccine Mechanisms that provide protection during ZIKV infection.The determination of ZIKV T cell epitopes has an important role in the research of T cell vaccines and T cell immunity.Therefore,this topic first focuses on the determination of T cell epitopes in mice infected with ZIKV.Based on the protein sequence of ZIKV,we designed and synthesized 427 overlapping peptides,each of which is 18 amino acids long,with 10 amino acids overlapping between adjacent peptides,covering the full length of the viral protein sequence.Using the IFN-? ELISPOT kit,we detected 63 positive peptides.The FACS further analyzed the secretion of cytokines stimulated by ZIKV peptides in different T cell subsets,and finally identified 11 peptides containing CD4 + T cell epitopes and 19 peptides containing CD8 + T cell epitopes.Seven of these peptides are located on structural proteins,and 21 are located on non-structural proteins.In order to focus on the T cell vaccine and avoid the interference of antibody responses,we constructed three ZIKV T cell vaccines using peptides selected from non-structural proteins containing T cell epitopes,including the peptide containing only CD4 + T cell epitopes NS2A(95-112),NS2B(13-30),NS2B(37-54),NS2B(93-110),NS3(43-60).Only peptides containing CD8 + T cell epitopes NS1(263-280),NS4A(66-83),NS4B(36-53),NS4B(76-93),NS4B(84-101),and the CD4 + T cells epitope peptide NS2A(95-112),NS2B(13-30),NS2B(37-54),NS2B(93-110),NS3(43-60)are mixed with the CD8 + T cell epitope peptide NS1(263-280),NS4A(66-83),NS4B(36-53),NS4B(76-93),NS4B(84-101).We then infected the immunized mice with ZIKV to verify the protective effects of the three ZIKV T cell vaccines,and found that all three ZIKV T cell vaccines can provide protection to infected mice and contain both CD4 + and CD8 + T cell epitopes Peptide vaccine can provide the best protection.We also found that the ZIKV T cell vaccine has a good effect on removing viruses in the eyes,spleen,and liver tissue.Through neutralization experiments and ADE experiments,we confirmed that after multiple immunizations using these T cell epitope peptides,neither neutralizing antibodies nor ADE were produced during the acute infection period.In a further in vivo killing test,it was found that mice vaccinated with these three peptide vaccines could kill target cells co-incubated with the corresponding peptides.Mice vaccinated simultaneously with CD4 + and CD8 + T cell epitope peptide vaccines showed the best in vivo killing rate,reaching a specific killing rate of 40.6%.Those mice immunized with CD4+ or CD8+ T cell epitope vaccine alone had a specific T cell killing rate of 24.5% or 19.1%,respectively.These data indicate that the protective effect produced by the T cell epitope vaccine is partly due to the cytotoxic effect mediated by T cells.And this protective effect does not depend on neutralizing antibodies,so there is no risk of inducing ADE.Therefore,T cell epitopes have development prospects.We also tried the combination of T cell vaccine and traditional antibody vaccine to provide better vaccine efficacy.Especially in the field of flavivirus vaccines,the risk of ADE in vivo can be suppressed by enhancing protective T cell immunity,which is a question worth discussing.Here,we designed a combined immunization protocol for the ZIKV T cell peptide vaccine and E80 protein vaccine.We found that on the third day after infection,the viremia level of all vaccine groups was significantly reduced Detection threshold(25 PFU / ml).However,in mice immunized with E80 only,the viral RNA(including viral RNA in cells in the blood)detected in the blood was not significantly reduced.In contrast,the viral RNA in the blood of mice immunized with CD4+ or CD8+ T cell vaccines was significantly reduced.This difference was also observed on day 7 after infection.These data suggest that the addition of CD4+ or CD8+ T cell vaccine can improve the efficacy of E80 protein vaccine.In summary,this study first confirmed that ZIKV infection can cause a strong and extensive T cell response in mice,and identified a series of novel CD4 + and CD8 + T cell epitopes,and found that some of these epitopes can activate cytotoxicity.T cell function.Subsequently,using these newly identified peptides with immunodominant epitopes,we developed a novel ZIKV T-cell alternative vaccine,demonstrating that the vaccine can confer the ability of vaccinated mice to resist ZIKV infection.We also found that the combination of the T cell epitope vaccine and the E80 recombinant protein vaccine can provide better protection than any vaccine alone.These results will provide a theoretical basis and pilot product for the development of ZIKV T cell vaccines based on CD4 + and CD8 + T cell epitopes.And the discovery also helps to break the inherent thinking of T virus vaccine development of other viruses,and has important reference significance.