Quasispecies heterogeneity within the E1/E2 region as a pretreatment variable during pegylated interferon therapy of chronic hepatitis C virus infection

Abstract

A series of 29 patients undergoing treatment for chronic hepatitis C virus (HCV) genotype 1 infection with pegylated alpha-2a interferon plus ribavirin were studied for patterns of response to antiviral therapy and viral quasispecies evolution. All patients were treatment naive and had chronic inflammation and fibrosis on biopsy. As part of an analysis of pretreatment variables that might affect the outcome of treatment, genetic heterogeneity within the viral E1-E2 glycoprotein region (nucleotides 851 to 2280) was assessed by sequencing 10 to 15 quasispecies clones per patient from serum-derived PCR products. Genetic parameters were examined with respect to response to therapy based on serum viral RNA loads at 12 weeks (early viral response) and at 24 weeks posttreatment (sustained viral response). Nucleotide and amino acid quasispecies complexities of the hypervariable region 1 (HVR-1) were less in the responder group in comparison to the nonresponder group at 12 weeks, and genetic diversity was also less both within and outside of the HVR-1, with the difference being most pronounced for the non-HVR-1 region of E2. However, these genetic parameters did not distinguish responders from nonresponders for sustained viral responses. Follow-up studies of genetic heterogeneity based on the HVR-1 in selected responders and nonresponders while on therapy revealed greater evolutionary drift in the responder subgroup. The pretreatment population sequences for the NS5A interferon sensitivity determinant region were also analyzed for all patients, but no correlations were found between treatment response and any distinct genetic markers. These findings support previous studies indicating a high level of genetic heterogeneity among chronically infected HCV patients. One interpretation of these data is that early viral responses are governed to some extent by viral factors, whereas sustained responses may be more influenced by host factors, in addition to effects of viral complexity and diversity.

FIG. 1.

Phylogenetic analysis of pretreatment viral clones. A neighbor-joining tree was constructed as described in Materials and Methods. The tree depicts evolutionary relationships for responders (blue) and nonresponders (red) or partial responders (black) based on the 12-week endpoint.

FIG. 2.

Phylogenetic trees for baseline and follow-up sequences of the HVR-1 for representative patients within the early viral responder and nonresponder subgroups. Trees were constructed with the neighbor-joining method, and the reliability of tree topology confirmed with 100 bootstrap replicates at the major branch points. The left panel shows the tree for responder patient 12 (baseline samples are indicated by 12-3, 12-6, 12-9, etc.; follow-up samples are indicated by 12-1-4, 12-1-9, etc., and 12-2-6, 12-2-10f, etc. [see Table 5]). The right panel shows the tree for nonresponder patient 15 (baseline and follow-up samples are indicated as described for patient 12).

FIG. 3.

Analysis of NS5A sequences and relationship to treatment response. (A) Phylogenetic relationship of population sequences for the 206 nucleotide sequence from baseline serum samples of all 29 patients in the study, together with HCV01 (HCV genotype 1a prototype [M62321]). (B) Genetic comparison of NS5A sequences for nonresponders (top) and responders (bottom) with respect to the 1a prototype sequence. The reference sequence is indicated on the top line. The P value refers to the comparison of genetic distances of responders and HCV01 versus nonresponders and HCV01.