Human leukocyte antigen–associated sequence polymorphisms in hepatitis C virus reveal reproducible immune responses and constraints on viral evolution†

Abstract

CD8 ؉ T cell responses play a key role in governing the outcome of hepatitis C virus (HCV) infection, and viral evolution enabling escape from these responses may contribute to the inability to resolve infection. To more comprehensively examine the extent of CD8 escape and adaptation of HCV to human leukocyte antigen (HLA) class I restricted immune pressures on a population level, we sequenced all non-structural proteins in a cohort of 70 chronic HCV genotype 1a-infected subjects (28 subjects with HCV monoinfection and 42 with HCV/human immunodeficiency virus [HIV] coinfection). Linking of sequence polymorphisms with HLA allele expression revealed numerous HLA-associated polymorphisms across the HCV proteome. Multiple associations resided within relatively conserved regions, highlighting attractive targets for vaccination. Additional mutations provided evidence of HLA-driven fixation of sequence polymorphisms, suggesting potential loss of some CD8 targets from the population. In a subgroup analysis of mono-and co-infected subjects some associations lost significance partly due to reduced power of the utilized statistics. A phylogenetic analysis of the data revealed the substantial influence of founder effects upon viral evolution and HLA associations, cautioning against simple statistical approaches to examine the influence of host genetics upon sequence evolution of highly variable pathogens. Conclusion: These data provide insight into the frequency and reproducibility of viral escape from CD8 ؉ T cell responses in human HCV infection, and clarify the combined influence of multiple forces shaping the sequence diversity of HCV and other highly variable pathogens. (HEPATOLOGY 2007;46:339-349.) R ecent studies suggest that immune control of hepatitis C virus (HCV) is possible 1-3 and the role of CD8 T cells is supported by studies linking particular human leukocyte antigen (HLA) class I alleles with control of HCV. 4 How viral infection persists in the face of an activated host immune response is poorly understood. Several mechanisms have been suggested that may contribute to the failure to contain HCV: these include impairment of cellular effector functions, 3,5 suppression of antigen-specific cells by regulatory T cells, 6-8 dendritic cell dysfunction, 9 T cell exhaustion, 10,11 or deletion of antigen-specific T cells in the liver. 12 However, persistence of HCV may also be facilitated by viral evolution that enables evasion of host immune responses occurring over the course of an individual infection. In the chimpanzee model, a strong association has been demonstrated between viral persistence and the development of CD8 escape mutations. 13,14 Moreover, recent studies have begun to clarify the propensity for viral escape from CD8 ϩ

Divergent Adaptation of Hepatitis C Virus Genotypes 1 and 3 to Human Leukocyte Antigen–Restricted Immune Pressure†

“…The HLA associations observed in that study are reproduced in the analysis performed here (using the Fisher's exact test for the entire dataset) but are not included as they fall above the stringent cutoff set in this study. Three sites shown in Tables 2 and 3 overlapped with HLA-associated viral polymorphisms identified by Timm et al 5 (NS3-1398, NS3-1444, NS5B-2841). In addition, three sites identified in the report by Timm et al were of borderline significance in our study (NS2-881, P ϭ 0.04; NS3-1019, P ϭ 0.07; NS5A-2153, P ϭ 0.02).…”

“…Viral polymorphisms within or flanking these epitopes can result in escape from HLA-restricted CTL responses, providing an effective mechanism for the virus to subvert host immune control (reviewed by Bowen and Walker 3 ). We and others 4,5 have shown that viral polymorphisms in the HCV genotype 1 genome are associated with specific HLA class I alleles, indicating adaptation of HCV to HLA-restricted immune pressure.…”

“…Others have previously used phylogenetic methods to adjust for these potential confounding factors. 5,12 In this study, we addressed this issue by identifying clusters of possibly related sequence and assessing the potential impact of such relatedness by performing analyses stratified by clusters. This approach is based on the notion that within relatively homogeneous clusters of possibly related sequences, the distributions of HLA types should be random.…”

“…Associations with an odds ratio Ͻ1 indicate overrepresentation of consensus in individuals that carry the respective HLA allele, indicating sites where the consensus viral sequence appears best adapted to T cell responses acting at these sites across the host population. 4,5,18,19 In order to minimize confounding through founder effects, 12 the analysis was adjusted for viral phylogenetic relatedness (see Statistical Methods).…”

Genetic Variation of Host Immune Response Genes and Their Effect on Hepatitis C Infection and Treatment Outcome

“…Studies utilizing population-based genetic approaches, which examine the association between specific viral polymorphisms and the HLA types of individuals within a host population, have demonstrated viral escape (adaptation) to HLA-restricted immune pressure evident at the population level for both HIV and HCV (Gaudieri, Rauch et al 2006;Timm, Li et al 2007) . The approach is based on the premise that the host's HLA molecules regulate immune responses by presenting specific viral epitopes to T lymphocytes and viral polymorphisms within or flanking these epitopes that compromise the efficacy of these T lymphocyte responses (i.e.…”