Construction And Immunological Evaluation Of Ebola Virus DNA Vaccine

BackgroundEbola virus（EBOV）is a member of the Filoviridae family.It can cause sever and fatal hemorrhagic fever in humans and non-human primates（NHPs）,with a fatality rate of up to 90%.EBOV is highly contagious and can be spread through the body fluids from patiens.The virus can be used as a biological weapon to threat mankind.At the same time,natural outbreak of EBOV epidemic also leads to live threat and property damage.Epidemillogical studies have shown that licensed drugs or vaccines are needed to prevent EBOV outbreak.And vaccination is considered to be the main strategy to control the spread of human infectious diseases.EBOV envelope glycoprotein（GP）mediates the entire process of virus entering host cells,including adhesion,cell entry and fusion.At present,most of the target antigen of EBOV vaccines were GP.Thus,GP related immunological research is particularly important.With the development of bioinformatics technology,the predictive analysis methods of antigen epitopes have gradually become familiar to researchers.It provides new ideas and methods for the study of antigen characteristics and vaccine design.DNA vaccines have attracted increasing attention in recent years because of their advanteges like good stability,simple construction,and easy to stroge.Researchers have constructed various DNA vaccines for GP to explore its immunological properties.Traditional DNA vaccines mainly induce cellular immune responses,which activate CD8⁺T cells through the endogenous antigen presentation pathway.However,many studies have shown that a strong humoral immune response to EBOV GP plays an important role in the prevention of EBOV.Therefore,it is necessary to construct a DNA vaccine that can elicit a strong cellular and humoral immune response.Lysosome-associated membrane protein（LAMP）is a lysosomal-specific membrane protein,which can transform the antigen presentation pathway and activate CD4⁺T cells to induce humoral immune responses.ObjectivesIn order to screen major histocompatibility complex（MHC）I/II restricted epitopes of Ebola virus glycoprotein and analyze their characteristics for vaccine development,bioinformatic prediction and downstream analyses were used as well as experimental validation.The Zaire Ebola virus glycoprotein plasmid containing LAMP molecule gene was constructed by using molecular biology methods.After three consecutive immunization,mice were detected for the cellular and humoral immune response.MethodsThe dominant epitopes of Zaire Ebola virus glycoprotein were predicted by using IEDB and other prediction tool.Cluster analysis,conservative analysis and molecular docking of the dominant epitopes were performed.Meanwhile,the plasmid p VAX-GP_EBOencoding glycoprotein were used to immunize mice.And ELISpot experiment was performed to verify the predictive epitopes.The mice were simultaneously immunized by plasmids p VAX-LAMP/GP_EBO and p VAX-GP_EBO.The cellular and humoral immune response of mice to Ebola virus glycoprotein was verified by experiments such as ELISA,ELISpot and flow cytometry.ResultsFor EBOV GP,for MHC I restricted epitopes,there were 148 immunogenicity dominant epitopes in the human leukocyte antigen（HLA）I superfamily with population coverage rate over 97%,and 25 immunogenicity dominant epitopes in the H-2 subtype.In the prediction of MHC II restricted epitopes,19 HLA II superfamily which constitutes a population coverage rate of 99%restrictive 15-mer epitopes were screened and 13epitopes were screened by H-2.ELISpot experiment results showed that p VAX-GP_EBOelicited mouse spleen cells to produce cellular immune responses against synthetic dominant epitopes,among which LFLRATTELRTFSIL and GYYSTTIRYQATGFG significantly activated cellular immune response in C3H mice.In the results of conservative analysis,the HLA I restricted dominant epitopes were more conservative than the H-2 restricted ones in the MHC I restricted dominant epitopes.There were dominant epitopes of both interspecific and intraspecies conservatism,but no epitopes of only interspecific conservatism either intraspecies non-conservatism.Molecular docking showed that dominant epitopes can obtain good docking scores in human HLA I/II superfamily molecules,which further confirmed the feasibility of its application in human immunity.The results of ELISA experiments showed that the presence of LAMP molecule induced a stronger humoral immune response in mice,and the ELISpot experiment showed that the cellular immune response in mice were enhanced.Flow cytometry observed that more CD4⁺T cells were activated.And the proportion of effector and effector memory T cells increased,suggesting that it probably helped to maintain long-term immune responses.ConclusionsIn the epitope prediction analysis,the 9-mer epitopes（GPCAGDFAF,GAFFLYDRL and TVIYRGTTF）and the 15-mer epitopes（LFLRATTELRTFSIL and GYYSTTIRYQATGFG）can be used as candidate epitopes for the Ebola virus epitope vaccine.The construction of DNA plasmids combining LAMP molecules gene realized the transformation of the antigen presentation pathway and improved the mice cellular and humoral immune responses against EBOV.