Objective:To apply mass spectrometry as well as bioinformatics techniques to analyze HBV peptides derived from Pol and X proteins presented by human immortalized B lymphocytes(BLCLs),and to screen for effective T-cell epitopes,which lays the foundation for the development of therapeutic vaccines.Methods:The group has constructed a genome-wide expression plasmid containing 1.2-fold HBVC2 isoforms and transfected it into BLCLs by electro-transfection,isolated and identified HBV peptides by LC-MS/MS,bioinformatically predicted peptide sequences in terms of sensitization,antigenicity,toxicity,HLA molecular affinity,and the ability of peptide HLA-class Ⅰ complex to bind to specific T cells,and retrieved reported sequences.Results:HBV peptides from lysates of five immortalized B cells(BLCLs-1 to BLCLs-5)carrying HBVC2 subtype-expressing recombinants were analyzed,and 141 peptides with sequence lengths of no less than 8 amino acid residues(≥8 aa)were successfully isolated and identified,of which 133 were derived from Pol,and 8 from X proteins.The 141 HBV peptides were analyzed for sensitization,antigenicity and toxicity,and 50 antigenic,non-toxic and sensitizing HBV peptides(47 from Pol and 3 from X protein)were screened for affinity analysis with HLA-class Ⅰ and Ⅱ molecules and for prediction of the binding ability of the peptide HLA-class Ⅰ complexes to specific T cells.Among these peptides,37 had affinity to the corresponding genotypes of HLA-class Ⅰ and Ⅱ molecules.Finally,we obtained 37 peptides with antigenic,nontoxic,and sensitizing properties with IC50<500 nmol/L to HLA-class Ⅰ and Ⅱ molecules,which have affinitied to the corresponding genotypes but have not been reported,and can be continued to be explored as potential epitopes.Finally,15 HBV peptide hotspot core regions were formed by intra-and inter-strain common,contained,overlapping or neighboring sequence analysis of 141 peptides,with 7 core region sequences with antigenic,nontoxic,and sensitizing properties restricted to the corresponding geno-types.Peptides with affinity IC50<500 nmol/L had an affinity core sequence overlap of no less than 8 amino acids,and can be priori-tized for candidate T cell epitopes for subsequent studies.Conclusion:Peptides derived from Pol and X proteins have been successfully isolated and identified,and potential T cell epitopes have been screened.

Objective:To apply mass spectrometry as well as bioinformatics techniques to analyze HBV peptides derived from Pol and X proteins presented by human immortalized B lymphocytes(BLCLs),and to screen for effective T-cell epitopes,which lays the foundation for the development of therapeutic vaccines.Methods:The group has constructed a genome-wide expression plasmid containing 1.2-fold HBVC2 isoforms and transfected it into BLCLs by electro-transfection,isolated and identified HBV peptides by LC-MS/MS,bioinformatically predicted peptide sequences in terms of sensitization,antigenicity,toxicity,HLA molecular affinity,and the ability of peptide HLA-class Ⅰ complex to bind to specific T cells,and retrieved reported sequences.Results:HBV peptides from lysates of five immortalized B cells(BLCLs-1 to BLCLs-5)carrying HBVC2 subtype-expressing recombinants were analyzed,and 141 peptides with sequence lengths of no less than 8 amino acid residues(≥8 aa)were successfully isolated and identified,of which 133 were derived from Pol,and 8 from X proteins.The 141 HBV peptides were analyzed for sensitization,antigenicity and toxicity,and 50 antigenic,non-toxic and sensitizing HBV peptides(47 from Pol and 3 from X protein)were screened for affinity analysis with HLA-class Ⅰ and Ⅱ molecules and for prediction of the binding ability of the peptide HLA-class Ⅰ complexes to specific T cells.Among these peptides,37 had affinity to the corresponding genotypes of HLA-class Ⅰ and Ⅱ molecules.Finally,we obtained 37 peptides with antigenic,nontoxic,and sensitizing properties with IC50<500 nmol/L to HLA-class Ⅰ and Ⅱ molecules,which have affinitied to the corresponding genotypes but have not been reported,and can be continued to be explored as potential epitopes.Finally,15 HBV peptide hotspot core regions were formed by intra-and inter-strain common,contained,overlapping or neighboring sequence analysis of 141 peptides,with 7 core region sequences with antigenic,nontoxic,and sensitizing properties restricted to the corresponding geno-types.Peptides with affinity IC50<500 nmol/L had an affinity core sequence overlap of no less than 8 amino acids,and can be priori-tized for candidate T cell epitopes for subsequent studies.Conclusion:Peptides derived from Pol and X proteins have been successfully isolated and identified,and potential T cell epitopes have been screened.