Immunogenicity Of Hcmv GH And PP65 Protein Peptide Vaccines

Objective Bioinformatics techniques were used to analyze envelope glycoproteins g H and phosphoprotein 65(PP65)encoded by human cytomegalovirus(HCMV).Linear B cell dominant epitopes,CD8~+T cell epitopes and CD4~+T cell epitopes of the two proteins were screened out,and the obtained epitopes were synthesized into polypeptides.The immunogenicity of the polypeptides was verified at the cellular and animal levels,which laid the foundation for the research of HCMV subunit vaccine.Methods 1.Eleven bioinformatics softwares,such as Prot Param,Prot Scale and SOPMA,were used to predict the physicochemical properties,structure and function,linear B cell epitopes,CD8~+T cell epitopes and CD4~+T cell epitopes of HCMV g H and pp65 proteins.A total of 13 peptides were synthesized by biosynthesis technology.According to the biological characteristics,they were dissolved in appropriate solvents.In vitro and in vivo experiments were used to detect the immune activation effect of the synthetic peptides.2.In vitro,human monocyte line U937 was induced into macrophages by phorbol myristate acetate(PMA)to evaluate the activation of synthetic peptides on antigen-presenting cells.U937 cells were induced by different concentrations of PMA and different treatment time.The morphological changes of cells were observed under microscope,and the expression of marker CD11b on macrophages was detected by flow cytometry,so as to determine the best induction conditions.The cytotoxicity of U937 cells under different induction conditions was detected by CCK-8 test.50?g/ml polypeptide was added into macrophages,and the non-polypeptide was the control group,after 24h of culture,the expression of marker CD86 on M1 macrophages was detected by flow cytometry to explore the immune activation effect of each polypeptide on macrophages.3.In vivo experiments,the vaccine prepared by mixing OVA with Freund's adjuvant was set as the positive group,the vaccine prepared by mixing g H protein polypeptide,PP65 protein polypeptide,g H protein polypeptide+PP65 protein polypeptide and inactivated HCMV with Freund's adjuvant was set as the experimental group,the normal saline group was set as the negative group,and the normal saline and Freund's adjuvant mixed group was set as the adjuvant control.The mice were immunized with Freund's complete adjuvant for the first time,Freund's incomplete adjuvant for the second time and the third time by intramuscular injection for three times.One week after the third immunization,the spleen of mice was taken and the splenocyte suspension was obtained.The expression of IFN-?,TNF-?and IL-4 by T cells was analyzed by flow cytometry.Results The amino acid sites of the dominant epitopes of g H protein and pp65 protein were predicted by bioinformatics software as follows:the linear B cell epitopes of g H protein were 179-194,383-398 and 526-541,and the linear B cell epitopes of PP65protein were 382-397 and 463-478;the CD8~+T cell epitopes of g H protein were 719-727,and the CD8~+T cell epitopes of PP65 protein were 495-503;the CD4~+T cell epitopes of g H protein were 236-250,340-354 and 707-721,while those of pp65 protein were 45-59,110-124 and 287-301.The results of cell induction experiment showed that U937 cells could be induced into macrophages well when the concentration of PMA was 100ng/ml and the treatment time was 48h.CCK-8 experiment showed that there was no cytotoxicity under this induction condition.The expression of CD86 in macrophages was activated by the peptide with the final concentration of 50?g/ml.The synthesis of peptide of g H protein or pp65 protein combined with adjuvant can effectively enhance the response of CD4~+T cells and CD8~+T cells,stimulate the expression of cytokines IFN-?,IL-4 and TNF-?.Conclusions The predominant epitopes of B and T cells were predicted by bioinformatics,and the macrophage model was constructed in vitro to prove that peptides can stimulate the expression of the macrophage surface marker CD86 to activate the innate immune response.In vivo,it can stimulate the activation of CD4~+T cells,express related cytokines,and activate CD8~+T cells to produce cytotoxicity.It has good immunogenicity and provides superior epitopes for the research of HCMV subunit vaccines.