Background:  Hepatitis C Virus (HCV) evades HLA-restricted immune pressure through accumulation of viral escape variants. We have shown previously that successful HAART increases HCV specific T cell immune responses. Here, we investigated the influence of an increased immune pressure during HAART on HCV escape mutations.

Methods:  HCV sequences covering the immunogenic epitopes HLA-B*0801 (NS3-1395), -A*0101 (NS3-1436), and -B*2705 (NS5B-284) were analyzed in individuals with the restricting HLA-allele (HLA-carriers) and in those without these HLA-types (HLA-non-carriers) before and on successful HAART (median follow-up time 33 months). Analyses were restricted to known escape positions within HCV epitopes. Bulk sequencing was performed in all study participants (N = 31). To study sequence evolution in minor quasispecies within these epitopes, we performed high resolution ultra-deep sequencing using the FLX 454 Roche technology before and on HAART in 5 individuals. Threshold for mixtures was set as 20% for bulk sequencing and 1% for ultra-deep sequencing.

Results:  Bulk sequencing analyses indicated a significant accumulation of immune escape variants in HLA-carriers compared to non-carriers pre-HAART: In HLA-carriers, 50% of sites contained the escape variant compared to only 17% of sites in HLA-non-carriers (P =0.009). After starting HAART, there was no significant increase in the proportion of escape variants (+3% for HLA-carriers and +2% for non-carriers). Emergence of escape mutations post-HAART was rare (7% of sites), and we observed no reversion from escape variants to wildtype amino acids. Ultra-deep sequencing revealed mixtures in 24 of 105 (23%) sites at the nucleotide level within the HLA-B*2705 epitope that were not found in bulk sequencing. At one site conferring resistance to HCV-polymerase inhibitors, the escape variant was detected in ultra-deep, but not in bulk-sequence in HLA-carriers.

Conclusions:  We show that HCV escapes from HLA-restricted immune pressure in HCV/HIV co-infected individuals. Emergence of new escape mutations and reversions on successful HAART were rare. This suggests that most viral escape mutations occur early in HCV infection and are maintained long-term, and that the increase in HCV specific T cell pressure on HAART has only a modest effect on HCV escape and on the emergence of HCV drug resistance mutations. However, ultra-deep sequencing may provide additional insight into biologically relevant low-level frequency mutations.