HCV螺旋酶N端HLA-A2限制性CTL表位的变异和CTL免疫应答

A HLA-A2-restricted-CTL epitope variation at the N-terminal of HCV helicase and the immune response of CTLs

目的 :研究丙型肝炎病毒 (HCV)螺旋酶N端HLA A2限制性细胞毒性T细胞 (CTL)表位的变异及其增殖反应。方法 :对两例慢性HCV感染者随访 7年 ,用第 1年和第 5年的外周血提取病毒RNA ,再以RT PCR扩增HCV螺旋酶N端基因 ,并亚克隆及测序。根据测序结果 ,合成CTL抗原表位肽段。用血清学方法进行HLA分型。从感染者第 7年外周血中分离淋巴细胞 ,检测CTL对抗原肽的增殖反应。结果 :在具有HLA A2表型的感染者中 ,第 1年病毒抗原表位的起始氨基酸均存在琥珀突变 ,5年后该突变消失。在无HLA A2表型的感染者中 ,其感染后 5年病毒抗原表位肽段既无共有序列的变异 ,也无琥珀突变。HCV感染后 7年 ,两例感染者的淋巴细胞对该抗原表位肽段均无增殖反应。结论 :螺旋酶N端抗原表位出现的无义突变 ,可能与病毒的免疫逃避有关。在慢性感染的后期 ,病毒感染者对螺旋酶N端的CTL表位肽不出现免疫应答。

AIM: To investigate a HLA A2 restricted CTL epitope variation at the N terminal of hepatitis C virus (HCV) helicase and the proliferative response of cytotoxic T lymphocytes (CTLs) to this epitope. METHODS: Two patients infected with HCV were followed up for 7 years. Blood samples taken at the first year and the fifth year were used for viral RNA extraction. Using RT PCR, the N terminal gene of helicase region was amplified from the extracted RNA. The amplified segments were subcloned and sequenced to assess the epitope variation. A HLA A2 restricted CTL epitope peptide was synthesized according to the sequencing result. HLA types of the two patients were determined by serological method. The peripheral blood lymphocytes were separated from the two patients at 7th year after infection and cultured with the synthetic CTL epitope peptide to assess the CTL’s proliferative response. RESULTS: In the HLA A2 + individual, there was an amber mutation at the first amino acid of the epitope in the first year after infection, but the amber mutation disappeared after 5 years. However, in the HLA A2 - individual, the epitope had neither variation nor amber mutation in 5 years after infection. 7 years after HCV infection, T lymphocytes from both individuals had no proliferative response to the epitope peptide. CONCLUSION: The amber mutation of the epitope at N terminal of helicase may be associated with viral immune escape. CTLs have no response to the epitope peptide in the N terminal of HCV helicase in the later stage of chronic HCV infection.