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

FEDERICO BOLCIC; NATALIA LAUFER; JORGE QUARLERI

Lisboa, Portugal

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

Background: HIV/HCV coinfected patients respond worse to peg-interferon and ribavirin (pegIFN+RBV) treatment than those HCV monoinfected ones. The HCV genotype 1 sustained virological response rate is lower than in genotypes 2 or 3. Non-structural 5A (NS5A) protein of HCV has the potential to block interferon (IFN)-induced RNA-dependent protein kinase (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-alfa. 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 coinfected patients were studied. All patients were under antiretroviral therapy with a CD4 T value of 456+/-248 (mean+/-SD) when initiate the peg-INF+RBV treatment. Given that patients had detectable HCV RNA after 12 weeks, the anti-HCV therapy was discontinued. Blood samples were taken at different time points as follows: basal, previous to initiate the pegIFN+RBV scheduled therapy, and 24 h and 4 wk during the therapy. From each sample by using Ficoll density gradient, serum and peripheral blood mononuclear cells (PBMC) were separated. 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). The genotype was determined by phylogenetic relatedness of NS5A as well as by using LiPA and RFLP of the 5`UTR. Results: The HCV genotype 1 NS5A sequences showed a clear clustering at the interpatient 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 during time, independently 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 coinfected patients, the genetic variability of HCV-1a NS5A was low pre- 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-SVR.