Prediction and identification of human leukocyte antigen-A2-restricted cytotoxic T lymphocyte epitope peptides from the human papillomavirus 58 E7 protein

Persistent infection with high-risk human papilloma virus (HPV) is the primary cause of cervical intraepithelial neoplasia (CIN) and cervical carcinoma. HPV58 is the third most common HPV genotype in China after HPV16 and HPV18. HPV E6 and E7 are oncoproteins and are constitutively expressed in HPV-associated cancer cells, therefore they are considered to he ideal target antigens for immunotherapy, including HPV therapeutic vaccine. In the present study, human leukocyte antigen (HLA)-A2-restricted cytotoxic T lymphocyte (CTL) epitope peptides were predicted and screened from HPV58 E7 antigen and their immunogenicity was subsequently determined. A total of 6 HLA-A2-binding peptides derived from HPV58 E7 were predicted and selected using 3 different prediction programs. A negative control peptide and PBS were used as two negative controls. Peripheral blood mononuclear cells (PBMCs) with HLA-A2(+) allele were used to detect the specific cellular immune response among the 6 predicted peptides by enzyme-linked immunospot assay (ELISOPT). Following preliminary screening for the predicted peptides, the antigenicity of the peptide HPV58 E7(72-80) was further assessed by an immunoassay to a vaccine contained HPV58 E7 antigen. Specific humoral and cellular immunity were detected using the peptide FIPV58E7(72-80) as the specific antigen. A total of 6 peptides from HPV58 E7 protein were predicted and subsequently named P1 (E7(7-15): TLREYILDL), P2 (E7(14-22): DLHPEPTDL), P3 (E7(69-77): CINSTTTDV), and P4 (E7(72-80): STTTDVRTL), P5 (E7(72- 87): TLQQLLMGT) and P6 (E7(83-91): LLMGTCTIV). In the ELISPOT assay on HLA-A2 (+) human PBMCs, interferon (IFN)-gamma-production was evident in the P2 and P4 groups. The average numbers of IFN-gamma associated spots in the P2 and P4 groups was 50.61 +/- 5.37 spot-forming cells (SFC)/1x10(5) and 266 +/- 34.42 SFC/ 1x10(5), respectively. The numbers of spots in the two peptides were significantly increased compared with the other 4 peptides and the control groups (P<0.05). In the further antigenicity verification of P4 (HPV58 E7(72-80)), the peptide only stimulated the humoral immune response of the AD-HPV16/18/58 mE6E7 vaccine containing HPV58 E7 antigen. Compared with the 2 negative control groups (1:400), the antibody titers of the vaccine group (1:25,600) were significantly increased (P<0.05). In cellular immunoassays the average number of IFN-gamma associated spots was 143.3 +/- 32.13 SEC/1x10(5) in the vaccine group, which was significantly enhanced compared with the PBS group (8 +/- 5.29 SEC/1x10(5); P<0.01) and the AD-NC group (28 +/- 5.13 SFC/1x10(5); P<0.01). The peptide HPV58 E7(72-80) (SITI1.)VRTL) displayed sufficient antigenicity to a vaccine contained HPV58 E7 antigen. Therefore, HPV58 E7(72-80) peptide may be considered as a candidate epitope peptide for the construction of HPV58 peptide vaccines.