T cell responses from blood donors infected with different HCV genotypes against HCV 1a proteins

Hepatitis C virus (HCV) infection, which can cause chronic liver diseases, cirrhosis and hepatocellular carcinoma, is still a major public health problem worldwide. Upon comparing the sequences of variants from different geographical areas, at least six major genotypes have been identified and classified. Several methods such as competitive PCR, PCR-RFLP, INNO-LiPA and sequencing have been used for HCV genotyping. The objective of this study were to investigate the distribution of HCV genotypes found in Thai blood donors and study the immune response against HCV 1a proteins to gain information for further vaccine development. In order to investigate a reliable method for genotyping HCV commonly found in Southeast Asia, 4 methods (2 RFLPs, INNO-LiPA, direct sequencing) were compared and performed in 35 samples. According to our data, direct sequencing still seemed to be the most reliable method for genotyping and was therefore used in HCV genotyping for further study. In order to investigate the distribution of HCV genotypes presented in Thai blood donors, 100 anti-HCV positive blood donors were enrolled in this study. RT-PCR for HCV RNA was performed and ninety samples were HCV RNA positive. Seventy-seven samples were selected for further molecular characterization of the core region of HCV, genotype 1 (39%), genotype 3 (44.2%) and genotype 6a (16.8 %) viruses were identified. HCV specific response of PBMCs from 41 samples against HCV 1a proteins was studied using proliferation and IFN-γ production assays. There was no significant difference in HCV specific response among PBMCs from donors infected with different HCV genotypes and among all of the HCV proteins used (core, NS3/4, NS5), HCV NS3/4 was the most immunogenic protein in a large portion of the individuals tested. Since proteins from HCV genotype 1a were used in the study and there were responses to these proteins detected in blood donors infected with other genotypes, this suggested that there might be genotype cross reactivity of immune response against HCV proteins. In addition to soluble HCV 1a proteins, BLCL transfected with plasmid expressing NS3/4 protein was also tested for the ability to stimulate HCV specific response in HCV infected individuals. IFN-γ production against HCV protein was detected when liver-infiltrating lymphocytes were used as responder cells but not when PBMCs were used. One explanation may be that there is a very low frequency of HCV specific T cells in PBMCs. In conclusion, this study provides the information on HCV genotypes presented in Thai blood donors and HCV specific response in blood donors infected with different HCV genotypes, which cross reactivity against HCV 1a proteins, were also demonstrated. The information obtained may be useful for further vaccine development especially for infected individuals in Southeast Asian.