Experimental Study On Identification And Molecular Modification Of HLA-A2-restricted Cytotoxic T Lymphocyte Epitopes Derived From E7 Antigen Of HPV-16

The human papillomavirus(HPV)6, 11, 16and 18 are the common HPV types associated with various infections in humans. Among them, HPV-6 and HPV-11 belongs to benign types, and HPV-16 and HPV18, which some times become causative agents in the pathogenesis of cervical cancer or its precursor lesions, such as cervical intraepithelial neoplasia (CIN) besides condyloma acuminatum (CA), are considered as "high risk" or malignant HPV types. Some therapies such as interferon, and antivirus drugs, are used to treat the various infections of HPV with little effect. It is urgently needed that specific immunotherapy is developed to effectively clear HPV infection, and prevent the infection from recurrent CA and canceration. HPV E gene, one domain of HPV genomes, contains 8 open reading frames (ORFs), which are involved in DNA replication, transcription, translation, etc. E7 protein is in close connection with cell transformation and cancerization. Many researchers have paid more attention to the protein. It is a very important step to predict, synthesize, identify and modify the epitopes derived from E7 protein sequeuce for the development of specific immunotherapy.Cytotoxic T Lymphocytes (CTL) epitopes derived from E7 protein can be presented by HLA-A2 and HLA-A3. About 53% of Chinese people are HLA-A2 positive, so it is very important to predict HLA-A2-restricted CTL epitopes derived from E7 antigen for wide application in future. HLA-A2-binding peptide supermotif prediction method, polynomial method and quantitative motif method were used to predict epitopes and determine the peptides E7₇-15, E7₁1-19, E7₄9-57, E7₆6-74 and E7₈2-90 to be synthesized. The peptides were synthesized with solid phase strategies, purified with reverse-phase HPLC and identified with mass spectrometry. The purity of the peptides were ＞95% and the measured values of their molecular weight were identical with their theoretical values, which was theprerequisite and basis for identifying epitopes.The CTL epitope candidates were assayed by a LDH release assay to determine their abilities in inducing the generation of specific CTLs. Among these, peptide HPV16 E7₁1-19 and HPV E7₄9-57 were confirmed to be CTL epitopes presented by HLA-A2.This study provided experimental basis for design and development of therapeutic peptide vaccines in HPV infection.Although some peptides, such as HPV E7₇-15, E7₆6-74 and E7₈2-90, have high affinity theoretically for HLA-A2, they have little antigenicity. 51Cr release assay was used to determine the relationship between epitopes and other peptides predicted. The results demonstrated that there was competitive inhibition between peptides (E7₇-15, E7₆6-74, E7₈2-90) and epitopes (E7₁1-19, E7₄9-57), and specific CTLs induction was influenced by the peptides distinctively. It was due to the lower affinity of the epitopes for HLA-A2. Competitive inhibition of the peptides may be one of the important reasons of recurrence and cancerization, and the modification of amino acid substitution was needed as well.Based of the lower affinity of the epitope E7₄9-57 for HLA-A2, modification of amino acid substitution was used to identify novel HLA-A2-restricted CTL epitopes. Quantitative method was used to evaluate the affinity of those peptides substituted, and determine the CTL epitope candidates to be synthesized and identified. The molecular models of CTL epitope candidates bound to the HLA-A2 molecule and of the HLA-A2-peptide complex were established by computer molecular modeling. It was suggested that the two peptides substituted may be the CTL epitopes restricted by HLA-A2 molecule. This became the basis for novel epitope identification.Cytotoxic experiment (LDH release assay) was used to identify the two peptides substituted and synthesized. The results showed that modified peptide Pâ‘ (RLHYNIVTF) had the antigenicity similar to HPV17E7₄9-57,and the other modified peptide Pâ‘¥(RLHYNIVTV) had no antigenicity. So Pâ‘ (RLHYNIVTF) was the novel HLA-A2-restricted epitope.To observe if the CT...