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Characterisation of hepatitis C virus evolution in HIV/HCV coinfected individuals using deep sequencing technology: Relevance for new antiviral drugs and disease outcome

Tschochner, M., Rohrbach, J., Cooper, D., Merani, S., Chopra, A., Witteck, A., Battegay, M., Furrer, H., Günthard, H., Mallal, S., Rauch, A. and Gaudieri, S. (2010) Characterisation of hepatitis C virus evolution in HIV/HCV coinfected individuals using deep sequencing technology: Relevance for new antiviral drugs and disease outcome. In: 22nd Annual Conference of the Australasian Society for HIV Medicine, 20 - 22 October 2010, Sydney, Australia


HIV/HCV co-infection is associated with diminished HCV specific T-cell responses and with higher HCV RNA levels. During successful HAART, HCV specific T-cell responses increase and HCV RNA levels decrease. However, HCV can evade the host immune response through accumulation of escape variants. Because little is known about the development of escape mutations in co-infected individuals with onset of HAART, we investigated the influence of increased immune pressure during HAART on HCV sequence evolution, in particular within T-cell epitopes. Furthermore, we examined HCV evolution at known drug-resistance sites to the new anti-HCV drugs.

HCV genotype-1 bulk sequences covering the immunogenic HLA-class-I epitopes (HLA-B*0801, HLAA* 0101 in NS3 and HLA-B*2705 in NS5B) were analyzed in HLA-carriers and in HLA-non-carriers before and during HAART (n=69). To study sequence evolution within these epitopes and the occurrence of drug resistance mutations in quasispecies present at >=1%, we performed ultra-deep sequencing using the FLX-454 Roche technology on five subjects before and on HAART.

Bulk sequencing analyses indicated a significant accumulation of immune escape variants in HLA-carriers (16% of sites) compared to non-carriers (only 6%) before the initiation of HAART (p=0.009). These mutations were maintained during HAART (HLA-carriers: 8%; HLA-non-carriers: 6%). The emergence of additional escape mutations with the onset of HAART was rare (7% of sites) and reversions of escape mutations were not seen. However, by utilizing the deep sequencing technique, minor quasispecies could be detected in 88% of 225 analyzed amino acid sites. Within the HLA-B*2705 epitope only, mixtures were found in 24 of 105 sites at the nucleotide level that were not found in bulk sequencing. Furthermore, investigating 35 sites associated with drug resistance in the protease and polymerase genes, six drug resistance mutations could be detected with FLX as minority species but not with Sanger sequencing.

In HCV/HIV co-infected individuals, the increase in HCV specific T-cell pressure during HAART appears to have only a modest effect on HCV escape. However, more sensitive sequencing techniques do identify additional escape variants at lower levels in these individuals suggesting changes in the host’s immune pressure on HCV following the commencement of HAART. It remains to be determined if these low frequency viral escape species affect overall HCV-specific immune responses or disease outcome. Furthermore, deep sequencing identifies biologically relevant low-level drug resistant mutations in HCV treatment naïve subjects that are not detected by conventional population-based sequencing.

Item Type: Conference Paper
Murdoch Affiliation(s): Centre for Clinical Immunology and Biomedical Statistics
Institute for Immunology and Infectious Diseases
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