ABSTRACT
BACKGROUND & AIMS: The adaptive immune response against hepatitis C virus (HCV) is significantly shaped by the host's composition of HLA-alleles with the consequence that the HLA phenotype is a critical determinant of viral evolution during adaptive immune pressure. In the present study, we aimed to identify associations of HLA class I alleles with HCV subtypes 1a and 1b genetic variants. METHODS: The association between HCV genetic variants and specific HLA-alleles was investigated in a cohort of 159 patients with chronic HCV genotypes 1a- and 1b-infection who were treated with pegylated interferon-alfa 2b and ribavirin in a prospective controlled trial for 48 weeks by direct sequencing of the genes encoding the HCV proteins E2, NS3, and NS5B and by HLA class I-genotyping of patients. HCV genetic variants were associated with specific HLA-alleles and the binding strength of accordant amino acid sequences to the corresponding HLA-allele was assessed by using the SYFPEITHI-algorithm. RESULTS: Overall, associations between HLA class I alleles and HCV sequence variation were rare. Five unknown HLA class I-associated viral genetic variations were identified, which in part affected the binding of predicted HCV CD8+ T cell epitopes to the respective HLA-allele. In addition, different patterns of HLA class I-allele/HCV sequence associations between the two subtypes were observed. CONCLUSIONS: We identified several unknown HLA class I-restricted HCV variants which in part impair binding to predicted HCV CD8+ T cell epitopes with remarkable differences between HCV subtypes 1a and 1b quasispecies.
Subject(s)
Genes, MHC Class I , Hepacivirus/genetics , Hepatitis C, Chronic/immunology , Hepatitis C, Chronic/virology , Adolescent , Adult , Aged , Alleles , Base Sequence , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/virology , Cohort Studies , DNA Primers/genetics , Female , Genetic Association Studies , Genetic Variation , Genotype , Hepacivirus/classification , Hepacivirus/immunology , Hepatitis C, Chronic/genetics , Host-Pathogen Interactions/genetics , Host-Pathogen Interactions/immunology , Humans , Male , Middle Aged , Phylogeny , Prospective Studies , RNA, Viral/blood , RNA, Viral/genetics , Viral Envelope Proteins/genetics , Viral Nonstructural Proteins/genetics , Young AdultABSTRACT
BACKGROUND: Diagnosis of hepatitis C virus (HCV) infection and its therapy is based on qualitative and quantitative measurement of HCV RNA. OBJECTIVES: A new assay that employs automated specimen extraction and real-time RT-PCR (COBAS Ampliprep/COBAS TaqMan, "CAP/CTM", Roche Diagnostics, Pleasanton, USA) was designed for linear quantification and highly sensitive detection of HCV RNA. STUDY DESIGN: The performance characteristics of CAP/CTM were compared to standard RT-PCR-based COBAS Amplicor Monitor 2.0 (CAM) assay in a multicenter study. RESULTS: The limit of detection of CAP/CTM was 7.4 IU/ml (95% CI 6.2-10.6) and clinical specificity was 99%. The linear range of HCV RNA quantification by CAP/CTM was between 28 and 1.4 x 10(7) IU/ml, with a correlation coefficient between expected and observed results of >0.99. A fivefold dilution of serum- or plasma-samples showed a linear correlation of HCV RNA levels in undiluted and diluted samples. Analyses of the mean intra- and inter-assay imprecision within the linear range of quantification showed a coefficient of variation of 3% and 3%, respectively. HCV genotypes 1a/b, 2b, 3a, 4, 5 and 6 were equally quantified by the CAP/CTM and CAM assay with mean deviations ranging from -0.29log(10) to 0.32log(10) IU/ml. HCV RNA quantification by CAP/CTM and CAM was highly concordant (correlation coefficient of 0.96). CONCLUSIONS: The CAP/CTM assay is a reliable and robust assay for highly sensitive detection and quantification of HCV RNA within a broad linear range.
