Prediction And Immune Effect Identify Of B And T Cell Epitopes In GB And Pp150 Proteins Of Human Cytomegalovirus

Objective:Human cytomegalovirus(HCMV)infection is one of the global public health problems.HCMV can lead to potential infection in healthy people and pose a threat to fetal growth and development,causing congenital infection in newborns.In order to design a novel synthetic HCMV vaccine,it is necessary to understand the important role of B and T cell antigen epitopes in adaptive immunity.By using bioinformatics methods to predict the antigenic epitopes with immune activation function,the ability of these antigen epitopes to help improve the immune response of the body was verified in vivo and in vitro,so as to provide a reference for the development of vaccines to find appropriate antigenic epitopes.Methods:In this study,the possible B and T cell antigen epitopes of gB and pp150proteins in HCMV were predicted by bioinformatics method,and the immune effect was evaluated by the presentation effect and immunogenicity of the predicted antigens detected by in vitro macrophage system and immunized mice.The nucleotide and amino acid sequences of AD169,Towne and Toledo strains were searched in Genebank database,and then the information of them were downloaded.The homology of the three strains was analyzed by online tools Mult Alin and MEGA 7 and Gene Doc software.The B cell antigen epitopes of the two proteins were comprehensively analyzed and predicted by online tools such as Prot Param,Prot Scale,TMHMM and Protean.The four online software,SYFPEITHI,IEDB,Net MHC II pan and Net MHC II,were used to predict the potential CD4~+T cell antigen epitopes of the two proteins.And the CD8~+T cell antigen epitopes of the two proteins were predicted by SYFPEITHI,IEDB and Net MHC.The effect of the predicted B and T cell antigen epitopes synthesized by chemical method were evaluated in vivo and in vitro.U937 cells were treated with different concentrations of Phorbol myristate acetate(PMA)at 50ng/ml,100 ng/ml,150 ng/ml and 200 ng/ml for 24h or 48h.The cell attachment which was observed under a microscope,the expression of CD11b on the cell surface which was detected by flow cytometry(FCM)and the toxic effect of PMA on cells which was detected by CCK-8 were three means to determine the best conditions for the construction of macrophages.The polypeptide of 50?g/ml was acted on macrophages separately,and the expression of CD86 was detected by flow cytometry to determine whether it could be recognized by macrophages and play the role of antigen presentation.In the animal experiment,the group containing only normal saline was set as the negative control group,the group containing saline and adjuvants was set as the adjuvant control group,and the group containing adjuvants,peptides or viruses was set as the experimental group,the group containing Ovalbumin(OVA)and adjuvants was set as the positive control group.50?l of B and T cell antigen epitopes of protein and 50?l of Freund's adjuvant were injected intramuscularly to immunize mice.After three immunizations,spleen cells of mice were taken.Finally,we detected the expression of IL-4 and IFN-?in CD3~+CD4~+T cells and the expression of IFN-?and TNF-?in CD3~+CD8~+T cells through using flow cytometry.Results:The identity of amino acid and nucleotide sequences among AD169,Towne and Toledo strains were more than 90%,confirming the truth that the homology was high.Therefore,the sequence of amino acid in AD169 strain was selected as the reference in this study for predicting cell antigen epitopes.The result of the B-cell antigen epitope regions of gB protein were 406?421 and 449?464,and the B cell antigen epitopes of pp150 protein were 535?550,633?648,673?688,880?895,659?674,290?305 and 821?836,respectively.The CD4~+T cell antigen epitopes of gB protein were located in the regions 124?138 and294?308,CD8~+T cell antigen epitopes were located in 69?77 regions.The CD4~+T cell antigen epitopes of pp150 protein were located in 2?16 and 311?325 regions,and the predicted CD8~+T cell antigen epitopes were located in 311-319 region.The predicted antigen epitopes were synthesized for subsequent experiments.When macrophage model was constructed in vitro,CD11b expression on the surface of U937 cells treated with 100ng/ml or above PMA was significantly increased.CCK-8assay showed that the cell activity was significantly enhanced after 48h of PMA treatment,and the cell activity reached the highest at 50 ng/ml and 100ng/ml PMA treatment for 48h.The expression of CD86 in macrophages stimulated by the synthetic peptides(B2?B7,B9,T1,T3?T6)at the final volume concentration of 50?g/ml for 24h was significantly different from that in the control group.In the animal experiment,gB or pp150 protein groups could effectively activate CD3~+CD4~+T cells to secrete IL-4 and IFN-?,and stimulate CD3~+CD8~+T cells to produce new subsets and secrete IFN-?and TNF-?,which compared with the group with only adjuvant.Conclusion:In this study,we successfully predicted B and T cell antigen epitopes of gB and pp150 proteins by using bioinformatics tools,and identified their immunogenicity at both cellular and animal levels,providing certain reference for the study of antigen epitopes of HMCV vaccine.