Abha Chopra

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.

The immune responses which drive the earliest selection of viral adaptations in heterosexual transmission provide insights into the responses most important for acute natural control of founder viruses, which should be harnessed by preventative vaccines. Similarly, those viral adaptations which revert early after transmission will reflect sites of strong constraint against escape from natural and vaccine-induced responses. Here we characterize in detail the virological and immunological events in an individual who presented with HIV (subtype A) infection only days after epidemiologically proven sexual transmission, and pre-seroconversion (Fiebig stage II). Quantitative changes to intra-patient viral sequences were evaluated with deep pyrosequencing of full-length HIV-1 genomes in both donor and recipient. Longitudinal Sanger and FLX 454 sequencing of HIV-1 was performed on 8 plasma samples from day 13 to 467 post-transmission. HIV-specific CD8 T cell responses were evaluated in 9 PBMC samples (day 35-656) using HLA-class 1 matched peptides in the IFNg ELISpot assay. The donor and recipient shared an HLA-C allele (HLA-C*04) but were discordant at all other HLA class I loci. Evolution of escape in an immunodominant HLA-B*1503-restricted Nef WL9 epitope was detected on day 264 and was present in 41% of viral quasi-species at day 264. T cell responses were detected prior to sequence change and decreased thereafter. Sequence change and CD8 T cell responses were also detected in relation to other published HLA-restricted epitopes. However, early reversion and subsequent re-selection was observed at position 133 of Nef which is not within a known epitope but corresponded to a HLA-C*0401 associated polymorphism in population-based studies. Overall, IFNg responses were detected to 3 of 28 HIV-1 peptides tested on day 35 but broadened to 20 responses while infection became established and reached peak viral load on day 383. T cell responses declined after commencement of antiviral therapy. Adaptive viral changes and reversions in early HIV-1 infection occur at novel sites not captured in immunological studies of chronically infected individuals. This data suggests the presence of novel immune hierarchies and escape mutations in early infection, which may be applied to HIV vaccine design against founder viruses.