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Background: Use of direct-acting antiviral drugs (DAAs) that target the Hepatitis C virus may be hampered by the rapid selection of viral strains that harbour drug resistance-associated variants (RAVs). These RAVs are often associated with a fitness cost and tend to occur on low frequency strains within treatment naive subjects. To address the clinical relevance of low frequency RAVs in the setting of DAAs, this study utilised a Primer ID ultra-deep sequencing approach to mitigate PCR errors and bias to accurately quantify viral sequences in subjects that failed DAA treatment. Methods: Subjects were enrolled in the follow-up study P05063 of previous treatment with boceprevir and all had detectable RAVs at virological failure (VF) based on Sanger-based population sequencing. Twelve subjects had three time-points available: baseline, VF, and follow-up (median 830.5 days). Viral RNA was amplified using unique primer identifiers (primer IDs) and sequenced using 454 ultra-deep sequencing. Results: The sequencing strategy used in this study improved the detection of clinically relevant low frequency strains bearing RAVs compared to population sequencing and showed that these strains can persist for up to two years post-treatment failure. Strains carrying multiple RAVs were common in breakthrough viruses. Putative compensatory mutations were identified. Conclusions: The Primer ID ultra-deep sequencing approach identifies RAVs that can reduce drug sensitivity at levels below the detection threshold for population sequencing. The approach also removes PCR errors and biases, suggesting this sequencing strategy should become the standard approach by which to perform temporal quasispecies studies and resistance screening.

BackgroundThe immune response to hepatitis B virus (HBV) is important for both viral control and disease pathogenesis. A detailed understanding of the HBV-specific T-cell responses may potentially lead to novel therapeutic strategies for HBV.MethodsAll English language journal articles (including articles in press) up to October 2007 were retrieved using searches of MEDLINE, EMBASE and the Cochrane Controlled Trial Registry. An extensive database of HBV sequences (SeqHepB) and GenBank were used to assess the degree of sequence variation in each epitope. The new standardized nomenclature for HBV amino acid position number was applied to all previously defined epitopes.ResultsForty-four HBV-specific human leukocyte antigen (HLA) class I restricted and 32 HBV-specific HLA class II restricted epitopes have been defined and have been identified in all HBV genes. The majority of HLA class I restricted epitopes have been defined in HLA-A2-positive individuals in the setting of acute HBV infection. There is significant sequence variation of these epitopes within and between HBV genotypes. Newer HBV immunotherapeutics appear promising but are still in early phases of development.ConclusionsIdentification of HBV-specific epitopes in non-HLA-A2-positive individuals and recognition of genotypic variation across epitopes are important for the future development of novel immunotherapeutic strategies for the management of chronic HBV infection.

The aim of this study was to evaluate the effect of CD4+ T-cell counts and other characteristics of HIV-infected individuals on hepatitis C virus (HCV) RNA levels.All HIV-HCV-coinfected Swiss HIV Cohort Study participants with available HCV RNA levels and concurrent CD4+ T-cell counts before starting HCV therapy were included. Potential predictors of HCV RNA levels were assessed by multivariate censored linear regression models that adjust for censored values.The study included 1,031 individuals. Low current and nadir CD4+ T-cell counts were significantly associated with higher HCV RNA levels (P = 0.004 and 0.001, respectively). In individuals with current CD4+ T-cell counts200/microl, median HCV RNA levels (6.22 log10 IU/ml) were +0.14 and +0.24 log10 IU/ml higher than those with CD4+ T-cell counts of 200-500/microl and500/microl. Based on nadir CD4+ T-cell counts, median HCV RNA levels (6.12 log10 IU/ml) in individuals with200/microl CD4+ T-cells were +0.06 and +0.44 log10 IU/ml higher than those with nadir T-cell counts of 200-500/microl and500/microl. Median HCV RNA levels were also significantly associated with HCV genotype: lower values were associated with genotype 4 and higher values with genotype 2, as compared with genotype 1. Additional significant predictors of lower HCV RNA levels were female gender and HIV transmission through male homosexual contacts. In multivariate analyses, only CD4+ T-cell counts and HCV genotype remained significant predictors of HCV RNA levels.Higher HCV RNA levels were associated with CD4+ T-cell depletion. This finding is in line with the crucial role of CD4+ T-cells in the control of HCV infection.

The immune response to hepatitis B virus (HBV) is important for both viral control and disease pathogenesis. A detailed understanding of the HBV-specific T-cell responses may potentially lead to novel therapeutic strategies for HBV.All English language journal articles (including articles in press) up to October 2007 were retrieved using searches of MEDLINE, EMBASE and the Cochrane Controlled Trial Registry. An extensive database of HBV sequences (SeqHepB) and GenBank were used to assess the degree of sequence variation in each epitope. The new standardized nomenclature for HBV amino acid position number was applied to all previously defined epitopes.Forty-four HBV-specific human leukocyte antigen (HLA) class I restricted and 32 HBV-specific HLA class II restricted epitopes have been defined and have been identified in all HBV genes. The majority of HLA class I restricted epitopes have been defined in HLA-A2-positive individuals in the setting of acute HBV infection. There is significant sequence variation of these epitopes within and between HBV genotypes. Newer HBV immunotherapeutics appear promising but are still in early phases of development.Identification of HBV-specific epitopes in non-HLA-A2-positive individuals and recognition of genotypic variation across epitopes are important for the future development of novel immunotherapeutic strategies for the management of chronic HBV infection.