Early mutations in HCV genotype 1-NS5A sequence after peginterferon-ribavirin treatment in non-responders HIV coinfected patients

BOLCIC F, LAUFER N., QUARLERI J.

Lisboa, Portugal

Workshop; 5th International HIV and Hepatitis Co-infection workshop; 2009

Background: HCV/HIV co-infected patients respond worse to peginterferon and ribavirin (pegIFN+RBV) treatment than those HCV mono-infected ones. The HCV genotype 1 sustained virological response rate is lower than genotypes 2 or 3. Non-structural 5A (NS5A) protein of HCV has the potential to block interferon (IFN)-induced RNA-dependent protein kinase R (PKR) and may therefore interfere with the response to IFN therapy. Genetic variability within the NS5A dsRNA-dependent protein kinase binding domain (PKR-BD) of HCV has been associated with responsiveness to IFN-α. The aim of this study was to analyze the HCV genotype 1 NS5A genomic heterogeneity at the PKR-BD region, including the interferon sensitivity determining region (ISDR) from HIV/HCV pegIFN+RBV non-responder patients.  Methods: Five non-responders HIV/HCV co-infected patients were studied. All patients were under antiretroviral therapy with CD4 T value of 456±248 (mean±SD) when initiated the peg-INF+RBV treatment. Given that patients did not reach at least a 2 log decay in HCV RNA at week 12, therapy was discontinued. Blood samples were drawn at different time points as follows: baseline -previous to initiate pegIFN+RBV therapy-24 hs, week 4 and 12 during therapy. From each sample, serum and peripheral blood mononuclear cells (PBMC) were separated by Ficoll density gradient. HCV RNA was isolated from both compartments. HCV viremia was quantitated at days 0, 1, and weeks 4 and 12 of treatment. The HCV NS5A region, PKR-BD, including ISDR, was amplified by RT-nested-PCR and then sequenced (aa 2209-2274). Genotypes were determined by phylogenetic relatedness of NS5A as well as by LiPA and RFLP of the 5´UTR. Results: The HCV genotype 1 NS5A sequences showed a clear clustering at the inter-patient level when the phylogenetic relatedness was established. Each patient group comprised the NS5A sequences from both compartments –serum and PBMC- and from the three different studied time points. At the intra-host level, the NS5A sequences exhibited a high degree of conservation, independently of the compartment analyzed, serum and PBMC, which was also sustained over time, regardless of pegIFN+RBV therapy pressure. All sequences were assigned to genotype 1a. When comparing with a prototype HCV genotype 1a sequence, the number of mutations at the PKRBD, was less than five mutations, and the mean number of mutations was 2.8 (2-4). Comparisons between baseline, 24 h and week-4 NS5A sequences did not show significant increases in mutations. Conclusions: In HIV co-infected patients, the genetic variability of HCV-1a NS5A was low previous to and early during pegIFN+RBV therapy, in both compartments serum and PBMC, showing a patient-stamp. No selection of IFN-resistant HCV strains was observed and a low diverse (< or =5) ISDR or/and PKR-BD sequence appears to contribute to non-sustained virologic response.