Construction And Evaluation Of Zika Virus DNA Vaccine

Since 2015,Zika virus spreads from Brazil and other South American countries,to cause a large outbreak throughout more than 60 countries,rising extensive concern world widely.In taxonomy,Zika virus belongs to the flavivirus family and is a plus-stranded RNA virus with a genome size of approximately 11 KB.The viral genome has only one open reading frame encoding a polyprotein of 3423 amino acids,which is processed into three structural proteins and seven non-structural proteins by the cleavage of the host and viral proteases.Among these proteins,viral membrane precursor protein（PRM）and envelope protein（E）,as well as non-structural protein（NS1）have been widely reported as vaccine targets.As an RNA virus transmitted by Aedes aegypti,Zika virus can infect both Aedes aegypti and human beings,and is able to overflow repeatedly from Aedes aegypti that serves as a reservoir of the virus,threating severely to lives and health of the people who live in the tropical and subtropical areas,such as South America.In this study,we developed DNA vaccines for Zika virus which provide safe and effective protection against Zika virus,by inducing the body to elicit powerful immune responses thereby preventing the intracellular replication of the virus.Furthermore,we characterized the immunological features of the mice given Zika vaccine,by applying transcriptome sequencing and single cell sequencing,to find the potential TCR and BCR sequences specially targeting Zika virus.We also applied a novel strategy in Zika DNA vaccine design,that is,targeting multiple antigens from Zika virus,on one hand,to enhance the immune effort of the vaccine and,on the other hand,to reduce the possibility of virus escaping from immune-mediated clearance resulting from rapid mutation-induced antigen drift.In addition,viruses from flavivirus family adapt to similar natural environments,coexist in the same time and location,therefore it is easy to cause side effects such as antibody-dependent enhancement（ADE）.Current Zika virus DNA vaccines almost target pr ME,some of which have gone to clinical trial.In our study,we selected pc DNA3.1（+）vector as the backbone,drove the expression of target protein by using CMV promoter,and put poly A tail following vaccine DNA sequence.We designed four DNA vaccines,expressing Zika pr ME（VPC-pr ME）,Zika NS1（VPC-NS1）,Zika pr ME and NS1 fusion protein（VPC-pr ME-NS1）and co-expressed motif III of the Zika E protein and NS1,separated by IRES2 and appended by a signal peptide at the end（VPC-EIII-NS1）.Among them,VPC-pr ME is previously reported to successfully induce immune response and selected as a control in our study.The other three vaccines are characterized by containing a non-structural protein,NS1.We use different strategy to express NS1 in designed vaccines and want to evaluate which one would have the best potential to induce spatial immune defense and to avoid the ADE effect.The aim of this study is to generate a safe and effective vaccine to protect individual against Zika reencounter through establishing a strong immune memory to Zika virus antigens.We chose the DNA vaccine as the main theme.To evaluate the effects of the DNA vaccines we developed,we first tested the intracellular expression of the antigens.Each of four vaccine candidates expressed corresponding virus protein,respectively.BALB/c mice were intramuscular administered with 100 micrograms of DNA vaccine using a two-inoculation method at day 0 and 14,and then the blood samples were harvested from the tail at day 7,21and 35 after the first immunization.In humoral immune response assays,the level of total Ig G increased consistently and markedly,and reached the peak at the 35 days after the first vaccination.In contrast,Ig G from the control groups,PBS group and mock vector group,only exhibited mildly increase.We then assessed Zika virus-specific Ig G levels by using ELISA assays with inactivated Zika virus as coating antibody.At day 7 after the first vaccination,Zika virus-specific neutralizing Ig G was detected in VPC-pr ME group,whereas the Ig G started to be detected at day 21 in the other three vaccination groups.At day 35,Zika virus-specific Ig G from all groups markedly increased and reached peak.Because a relatively high concentration of Zika virus-specific Ig G at day 35,we performed virus neutralization assays by using serum collected at day 35.The results showed that,serum from PBS group hardly protected Vero cells from Zika virus;serum from VPC-pr ME-NS1,VPS-pr ME,VPC-NS1 and VPC-EIII-NS1exhibited significant protective ability against Zika virus.The PD₅₀ values,which indicates the protective ability of serum,from the four vaccine groups,were 78 times dilution,93 times dilution,37 times dilution and 90 times dilution,respectively.Among them,neutralizing effect of the serum from the VPC-EIII-NS1 was considered better and more stable due to the smallest value variation within group.In cell-mediated immune response assays,at day 35 of the first immunization,mice were sacrificed and spleens were collected for single splenocyte preparation.After getting the splenocyte suspension,cells were stained with fluorescence-labeled surface and intracellular marker antibodies followed by flow cytometry analysis for the different cell subsets like B cell,T cell and myeloid cell,and the antigen-specifically T cell subsets by detecting IFN-?secretion.Our data showed that in the spleen,the main cell subsets,B cell,T cell and myeloid cell,were not changed upon vaccination,in comparison with PBS,indicating that the DNA vaccines has significant safety.In a Zika virus-specific IFN-?assay,upon corresponding Zika antigen stimulation,antigen-specific CD4+T cell expanded more than ten times in VPC-NS1,VPC-pr ME and VPC-pr ME-NS1 groups,in comparison with control.Similarly,antigen-specific CD8+T cell also increased significantly,with VPC-NS1 inducing the most increase,fourteen times than control.At last,we characterized the immunological features of the mice spleen from each group by using single cell sequencing for transcriptome and TCR and BCR reptoire.Collectively,we established a novel Zika virus DNA vaccine targeting both conventional and non-structural epitope,potentially enhancing cell-mediated immune response and avoiding ADE effects.