Evolution of the hepatitis C virus second envelope protein hypervariable region in chronically infected patients receiving alpha interferon therapy

Abstract

Sustained hepatitis C virus (HCV) RNA clearance is achieved in 8 to 12% of patients with chronic HCV infection treated with alpha interferon (IFN-alpha) at the approved dose of 3 MU three times a week for 6 months and in about 25% of those receiving this treatment for 12 months. We used single-strand conformation polymorphism analysis combined with cloning and sequencing strategies to characterize the genetic evolution of HCV second envelope gene hypervariable region 1 (HVR1) quasispecies during and after IFN therapy in patients who failed to clear HCV RNA. Sustained HCV RNA clearance was achieved in 6% of patients. Profound changes in HVR1 quasispecies major variants were estimated to occur in 70% of the patients during and after therapy. These changes were evolutionary and were characterized by shifts in the virus population, related to selection and subsequent diversification of minor pretreatment variants. The quasispecies changes appeared to be induced by changes in the host environment likely resulting from the IFN-induced enhancement and post-IFN attenuation of neutralizing and possibly cytotoxic responses against HVR1. The remaining patients had no apparent changes in HVR1 quasispecies major variants, suggesting selection of major pretreatment variants, but some changes were observed in other genomic regions. We conclude that IFN-alpha administration and withdrawal profoundly alters the nature of circulating HCV quasispecies, owing to profound changes in virus-host interactions, in patients in whom sustained HCV RNA clearance fails to occur. These changes are associated with profound alterations of the natural outcome of HCV-related liver disease, raising the hypothesis of a causal relationship.

FIG. 1

Selection of the study group. tiw, three times a week.

FIG. 2

SSCP analysis of 351-bp HCV E1/E2 PCR products containing HVR1 in specimens obtained before, during, and after treatment from three patients receiving 3 × 10⁶ U of IFN-α2a subcutaneously three times a week for 6 months. (A) Nonresponder followed up until month 12 (M12). This is an example of a patient without HVR1 quasispecies major variant changes during follow-up: the SSCP patterns were identical at the various times before (M0), during (M1 to M6), and after (M8 to M12) IFN treatment. (B) Nonresponder followed up until month 12 (M12). This is an example of a patient with HVR1 quasispecies major variant changes: HVR1 quasispecies changed from one time point to the next during treatment (M1 to M6) and after IFN withdrawal (M7 to M12) until the end of follow-up. (C) Virological responder-relapser re-treated with the same dose of IFN for 6 months (M9 to M15) and followed up for a further 6 months. This is an example of a patient with HVR1 quasispecies major variant changes: HVR1 quasispecies changed after the first course of IFN (M9 versus M0) and from one time point to the next during re-treatment (M10 to M15) and after IFN withdrawal until the end of follow-up (M16 to M21).

FIG. 3

Phylogenetic trees of HVR1 in the five patients studied before IFN treatment and at various times during and after therapy. The phylogenetic reconstructions are neighbor-joining trees with bootstrap proportions of more than 500 of 1,000 bootstrap replicates shown (in percentages) at appropriate branch points. Letters stand for amino acid sequences, and numbers following “M” stand for the months when the sequences were isolated (e.g., M3-B, amino acid sequence B isolated at month 3).