Hepatitis C virus (HCV) has a high rate of replication and lacks RNA-proofreading capabilities, thereby leading to variant or mutant viruses circulating within the host as a quasispecies. Previous work in our laboratory identified viral variants that emerged in a class-II immunodominant epitope NS3 358-375 of the non-structural-3 (NS3) protein region of HCV, the sequence of which is based on genotype 1A, the most prevalent genotype in the United States. Further work suggested that positive immune selection pressure was driving viral variation. Paradoxically, viral variants account for only a small percentage of the circulating virus in human beings and in chimpanzees, suggesting that passive evasion is not the only means of escape by HCV. This observation suggests a unique pathogenesis for HCV as it persists in the host. In the current study, we hypothesize that viral variants are acting as altered peptide ligands (APLs). To test this hypothesis, we used cloned T cells specific for NS3 358-375 peptide, which demonstrated attenuated T-cell and interferon-γ (IFN-γ) responses to individual variant peptides, when compared with the NS3 358-375 stimulated T-cell clones. Furthermore, such variants could act as APLs, based on their ability to antagonize the IFN-γ proliferative responses of clones specific for NS3 358-375. In addition, major histocompatibility complex (MHC) class II tetramer staining demonstrated that variant peptide–MHC complexes were able to specifically bind to NS3 358-375 T-cell clones and that both the variant and NS3 358-375 tetramers were able to bind to the same CD4+ T cells. Taken together, the results suggest that viral variants may act as APL to effectively blunt the T-cell response to an important HCV epitope.
T-cell activation occurs when a T-cell receptor (TCR) binds to cognate or specific peptide-bound to major histocompatibility complex (pMHC) molecules on the surface of antigen presenting cells (APCs) [1]. The engagement of the TCR to pMHC is necessary for the activation of CD4+ and CD8+ T cells, thereby leading to an effective adaptive immune response against an invading pathogen [2]. Single amino acid substitution in the cognate peptide, termed altered peptide ligands (APLs), can elicit a gradient of effector function changes in a specific T cell [3].
The recognition of these segments of a pathogen or epitope(s) presented by the pMHC to T cells is critical for the clearance of viruses [4]. As in the case of hepatitis C virus (HCV) infection, clearance of the virus has been found to be dependent upon the quality of the CD4+ T-cell response in up to 30% of individuals infected [5], [6], [7], [8]. Mueller et al. [9] have detected virus-specific CD4+ T cells in patients with chronic HCV, suggesting a novel mechanism of viral persistence [10], [11], [12], [13], [14].
Previous work in our laboratory identified viral variants that emerged in an MHC class II immunodominant epitope NS3 358-375 of the non-structural-3 (NS3) protein region of HCV [15]. Further work suggested that positive immune selection pressure was driving viral variation [16]. In contradiction to viral escape models, HCV variants account for only a small percentage of the circulating virus in human beings and in chimpanzees [17], [18]. Also, HCV is able to modulate the immune response specifically toward viral persistence, indicating that HCV may be able to exploit a different mechanism in place of a more passive evasion strategy, leading to nondetection of the HCV pathogen by the immune system [16], [19].
One such mechanism that HCV may be able to exploit for persistence includes APLs, which have been observed within the hypervariable region 1 [20], [21], and in other pathogens, such as hepatitis B virus (HBV) [4], [22], human immunodeficiency virus (HIV) [23], [24], and Plasmodium falciparium [25]. Our previous studies identified and tracked viral variants arising in a single chronic HCV–infected individual and further determined, using polyclonal assays, the effect that these variants had on the in vitro immune response [15], [16], [18]. Although HCV circulates in the host as a quasispecies, it appears that such variation does not lead to viral escape but, rather, that some variants are able to suppress the immune response in an antigen-specific manner.
The importance of T cells in HCV clearance leads to the supposition that fully activated HCV specific T cells correlate with HCV clearance; however, the impact of viral variants on TCR activation in conjunction with the cognate peptide has not been fully elucidated in HCV pathogenesis. There are several non–mutually exclusive factors that lead to a fully activated T cell, such as the quantity of pMHC presented on the surface of APCs and TCR avidity. Previous work demonstrated an attenuated T-cell proliferative response to the cognate peptide in polyclonal assays when peripheral blood mononuclear cells (PBMC) from a chronic HCV–infected patient were preincubated with the variant peptides [16], [19]. A question that stems from the previous polyclonal work is whether the variants that arise in this important Th1-viral–clearing epitope (NS3 358-375) are antagonizing the same CD4+ T cell or inducing a subset of CD4+ T cells to suppress the cognate T-cell response. To test the hypothesis that viral variants could act as APLs leading to a partially activated T cell and not the induction of another subset of T cells that can suppress the immune response, in this study we used cloned T cells specific for the MHC class II epitope NS3 358-375 peptide. The APL peptides alone stimulated low T-cell and cytokine responses in comparison with cognate peptide in polyclonal assays, all of which has been documented elsewhere [16]. Prepulsing T-cell clones with variant peptides revealed that variants were able to antagonize proliferative and interferon-γ (IFN-γ) responses to cognate NS3 358-375 peptide. MHC class II tetramers, loaded with variant peptides, are able to bind specifically to NS3 358-375 T-cell clones. Furthermore, both variant S370P and NS3 358-375 are able to bind to the same NS3 358-375–specific CD4+ T cells. Thus, viral epitope variants are able to blunt the responses of the very T cells that should help with the elimination of virus. Our results also help to explain observations that CD4+ T-cell responses to HCV antigens seem to be attenuated or missing in chronically infected patients; these results also bear on the findings that CD8+ killer T cells are ineffective at eliminating HCV-infected target cells [26], [27].
These studies have been reviewed and approved by the University of Utah and the Medical College of Wisconsin Institutional Review Boards. Isolation of lymphocytes and subsequent HLA typing was performed as previously described [28].
In vitro PBMC and T-cell clones were stimulated with synthetic peptides representing one human leukocyte antigen DRB1*1501 restricted-epitope surrounding HCV NS3 amino acids 358–375 (amino acids 1384–1401 of the HCV polyprotein), as previously described [16], [28].
To determine whether naturally occurring epitope variants are acting as APL and thus antagonizing or anergizing potential helper T-cell responses, we used both PBMC polyclonal assays (Figs. 1A, 1B) and T-cell clones specific for NS3 358-375. The T-cell clones specific for NS3 358-375 (Figs. 1C, 1D) were derived from a patient with resolved HCV infection, PH1127 (HLA-DR15), who is viral load negative but antibody positive and recognizes a previously characterized immunodominant NS3 358-375 epitope (
We demonstrate, in this study, that variant epitopes modulate in vitro immune responses to the cognate peptide. Our results suggest that naturally occurring variants within a protective immunodominant epitope may act as APLs, leading to changes in the quality of T-cell responses, which could allow viral persistence.
This viral persistence seems relevant, considering that HCV is able to modulate the immune response in an antigen-specific manner. This was shown by the fact that the variants were
This work was supported by the National Institutes of Health ( 5R01AI047347-11 ). The authors thank Drs Matthew Williams, Robert Fujinami, Curt Hagedorn, and Matthew Mulvey for helpful discussions, along with Jane Libbey for critical feedback regarding this manuscript.
