Michaela Lucas

CD8 Epitope Escape and Reversion in Acute HCV Infection

In the setting of acute hepatitis C virus (HCV) infection, robust HCV-specific CD8+ cytotoxic T lymphocyte (CTL) responses are associated with initial control of viremia. Despite these responses, 70–80% of individuals develop persistent infection. Although viral escape from CD8 responses has been illustrated in the chimpanzee model of HCV infection, the effect of CD8 selection pressure on viral evolution and containment in acute HCV infection in humans remains unclear. Here, we examined viral evolution in an immunodominant human histocompatibility leukocyte antigen (HLA)-B8–restricted NS3 epitope in subjects with acute HCV infection. Development of mutations within the epitope coincided with loss of strong ex vivo tetramer and interferon γ enzyme-linked immunospot responses, and endogenous expression of variant NS3 sequences suggested that the selected mutations altered processing and presentation of the variant epitope. Analysis of NS3 sequences from 30 additional chronic HCV-infected subjects revealed a strong association between sequence variation within this region and expression of HLA-B8, supporting reproducible allele-specific selection pressures at the population level. Interestingly, transmission of an HLA-B8–associated escape mutation to an HLA-B8 negative subject resulted in rapid reversion of the mutation. Together, these data indicate that viral escape from CD8+ T cell responses occurs during human HCV infection and that acute immune selection pressure is of sufficient magnitude to influence HCV evolution.

Regulatory T cells suppress in vitro proliferation of virus-specific CD8+ T cells during persistent hepatitis C virus infection.

ABSTRACT The basis of chronic infection following exposure to hepatitis C virus (HCV) infection is unexplained. One factor may be the low frequency and immature phenotype of virus-specific CD8+ T cells. The role of CD4+CD25+ T regulatory (Treg) cells in priming and expanding virus-specific CD8+ T cells was investigated. Twenty HLA-A2-positive patients with persistent HCV infection and 46 healthy controls were studied. Virus-specific CD8+ T-cell proliferation and gamma interferon (IFN-γ) frequency were analyzed with/without depletion of Treg cells, using peptides derived from HCV, Epstein-Barr virus (EBV), and cytomegalovirus (CMV). CD4+CD25+ Treg cells inhibited anti-CD3/CD28 CD8+ T-cell proliferation and perforin expression. Depletion of CD4+CD25+ Treg cells from chronic HCV patients in vitro increased HCV and EBV peptide-driven expansion (P = 0.0005 and P = 0.002, respectively) and also the number of HCV- and EBV-specific IFN-γ-expressing CD8+ T cells. Although stimulated CD8+ T cells expressed receptors for transforming growth factor beta and interleukin-10, the presence of antibody to transforming growth factor beta and interleukin-10 had no effect on the suppressive effect of CD4+CD25+ regulatory T cells on CD8+ T-cell proliferation. In conclusion, marked CD4+CD25+ regulatory T-cell activity is present in patients with chronic HCV infection, which may contribute to weak HCV-specific CD8+ T-cell responses and viral persistence.

Ex vivo analysis of human memory CD4 T cells specific for hepatitis C virus using MHC class II tetramers

Containment of hepatitis C virus (HCV) and other chronic human viral infections is associated with persistence of virus-specific CD4 T cells, but ex vivo characterization of circulating CD4 T cells has not been achieved. To further define the phenotype and function of these cells, we developed a novel approach for the generation of tetrameric forms of MHC class II/peptide complexes that is based on the cellular peptide-exchange mechanism. HLA-DR molecules were expressed as precursors with a covalently linked CLIP peptide, which could be efficiently exchanged with viral peptides following linker cleavage. In subjects who spontaneously resolved HCV viremia, but not in those with chronic progressive infection, HCV tetramer-labeled cells could be isolated by magnetic bead capture despite very low frequencies (1:1,200 to 1:111,000) among circulating CD4 T cells. These T cells expressed a set of surface receptors (CCR7+CD45RA-CD27+) indicative of a surveillance function for secondary lymphoid structures and had undergone significant in vivo selection since they utilized a restricted Vbeta repertoire. These studies demonstrate a relationship between clinical outcome and the presence of circulating CD4 T cells directed against this virus. Moreover, they show that rare populations of memory CD4 T cells can be studied ex vivo in human diseases.

Comprehensive Analysis of CD8+-T-Cell Responses against Hepatitis C Virus Reveals Multiple Unpredicted Specificities

ABSTRACT The hepatitis C virus (HCV)-specific CD8+-T-cell response is thought to play a critical role in HCV infection. Studies of these responses have largely relied on the analysis of a small number of previously described or predicted HCV epitopes, mostly restricted by HLA A2. In order to determine the actual breadth and magnitude of CD8+-T-cell responses in the context of diverse HLA class I alleles, we performed a comprehensive analysis of responses to all expressed HCV proteins. By using a panel of 301 overlapping peptides, we analyzed peripheral blood mononuclear cells (PBMC) from a cohort of 14 anti-HCV-positive, HLA A2-positive individuals in an enzyme-linked immunospot assay. Only four subjects had detectable HLA A2-restricted responses in PBMC, and only 3 of 19 predicted A2 epitopes were targeted, all of which were confirmed by tetramer analysis. In contrast, 9 of 14 persons showed responses with more comprehensive analyses, with many responses directed against previously unreported epitopes. These results indicate that circulating HCV-specific CD8+-T-cell responses can be detected in PBMC in the majority of infected persons and that these responses are heterogeneous with no immunodominant epitopes consistently recognized. Since responses to epitopes restricted by single HLA alleles such as HLA A2 do not predict the overall response in an individual, more comprehensive approaches, as shown here, should facilitate definition of the role of the CD8+-T-cell response in HCV infection. Moreover, the low level or absence of responses to many predicted epitopes provides a rationale for immunotherapeutic interventions to broaden cytotoxic-T-lymphocyte recognition.

Preferential loss of IL-2–secreting CD4+ T helper cells in chronic HCV infection†

Hepatitis C virus (HCV) becomes persistent in the majority of infected individuals. In doing so, the virus evades host adaptive immune responses, although the mechanisms responsible in this evasion are not clear. Several groups have demonstrated weak or absent HCV‐specific CD4+ T cell responses during chronic HCV infection using proliferation assays and, more recently, class II tetramers. However, the functional status of HCV‐specific CD4+ T cells in resolved and persistent infection is poorly understood. Using interferon γ (IFN‐γ) and interleukin 2 (IL‐2) enzyme‐linked immunospot assays, we analyzed cytokine secretion patterns in chronically infected patients and compared them with those with resolved infection. In the spontaneous resolver group, strong IL‐2 secretion in relation to IFN‐γ secretion was observed. However, in the persistently infected group, a consistent and significant loss of IL‐2–secreting cells, compared with IFN‐γ–secreting cells, was identified. In vitro addition of IL‐2 had a substantial effect in restoring CD4+ T cell activity. In conclusion, failure of IL‐2 secretion, as opposed to physical deletion or complete functional unresponsiveness, appears to be an important determinant of the status of CD4+ T cell populations in chronic HCV infection. Loss of IL‐2 secretory capacity may lead to disruption of IFN‐γ and proliferative function in vivo—a status that characterizes the cellular immune response in both CD4+ and CD8+ compartments in chronic disease. (HEPATOLOGY 2005;41:1019–1028.)

Hepatitis C virus drug resistance and immune-driven adaptations: Relevance to new antiviral therapy†

The efficacy of specifically targeted anti‐viral therapy for hepatitis C virus (HCV) (STAT‐C), including HCV protease and polymerase inhibitors, is limited by the presence of drug‐specific viral resistance mutations within the targeted proteins. Genetic diversity within these viral proteins also evolves under selective pressures provided by host human leukocyte antigen (HLA)‐restricted immune responses, which may therefore influence STAT‐C treatment response. Here, the prevalence of drug resistance mutations relevant to 27 developmental STAT‐C drugs, and the potential for drug and immune selective pressures to intersect at sites along the HCV genome, is explored. HCV nonstructural (NS) 3 protease or NS5B polymerase sequences and HLA assignment were obtained from study populations from Australia, Switzerland, and the United Kingdom. Four hundred five treatment‐naïve individuals with chronic HCV infection were considered (259 genotype 1, 146 genotype 3), of which 38.5% were coinfected with human immunodeficiency virus (HIV). We identified preexisting STAT‐C drug resistance mutations in sequences from this large cohort. The frequency of the variations varied according to individual STAT‐C drug and HCV genotype/subtype. Of individuals infected with subtype 1a, 21.5% exhibited genetic variation at a known drug resistance site. Furthermore, we identified areas in HCV protease and polymerase that are under both potential HLA‐driven pressure and therapy selection and identified six HLA‐associated polymorphisms (P ≤ 0.05) at known drug resistance sites. Conclusion: Drug and host immune responses are likely to provide powerful selection forces that shape HCV genetic diversity and replication dynamics. Consideration of HCV viral adaptation in terms of drug resistance as well as host “immune resistance” in the STAT‐C treatment era could provide important information toward an optimized and individualized therapy for chronic hepatitis C. (HEPATOLOGY 2009.)

Human Immunodeficiency Virus Type 1-Hepatitis C Virus Coinfection: Intraindividual Comparison of Cellular Immune Responses against Two Persistent Viruses

ABSTRACT Both human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) lead to chronic infection in a high percentage of persons, and an expanding epidemic of HIV-1-HCV coinfection has recently been identified. These individuals provide an opportunity for simultaneous assessment of immune responses to two viral infections associated with chronic plasma viremia. In this study we analyzed the breadth and magnitude of the CD8+- and CD4+-T-lymphocyte responses in 22 individuals infected with both HIV-1 and HCV. A CD8+-T-lymphocyte response against HIV-1 was readily detected in all subjects over a broad range of viral loads. In marked contrast, HCV-specific CD8+-T-lymphocyte responses were rarely detected, despite viral loads in plasma that were on average 1,000-fold higher. The few HCV-specific responses that were observed were relatively weak and limited in breadth. CD4-proliferative responses against HIV-1 were detected in about half of the coinfected subjects tested, but no proliferative response against any HCV protein was found in these coinfected persons. These data demonstrate a major discordance in immune responses to two persistent RNA viruses. In addition, they show a consistent and profound impairment in cellular immune responses to HCV compared to HIV-1 in HIV-1-HCV-coinfected persons.

Full-Breadth Analysis of CD8+ T-Cell Responses in Acute Hepatitis C Virus Infection and Early Therapy

ABSTRACT Multispecific CD8+ T-cell responses are thought to be important for the control of acute hepatitis C virus (HCV) infection, but to date little information is actually available on the breadth of responses at early time points. Additionally, the influence of early therapy on these responses and their relationships to outcome are controversial. To investigate this issue, we performed comprehensive analysis of the breadth and frequencies of virus-specific CD8+ T-cell responses on the single epitope level in eight acutely infected individuals who were all started on early therapy. During the acute phase, responses against up to five peptides were identified. During therapy, CD8+ T-cell responses decreased rather than increased as virus was controlled, and no new specificities emerged. A sustained virological response following completion of treatment was independent of CD8+ T-cell responses, as well as CD4+ T-cell responses. Rapid recrudescence also occurred despite broad CD8+ T-cell responses. Importantly, in vivo suppression of CD3+ T cells using OKT3 in one subject did not result in recurrence of viremia. These data suggest that broad CD8+ T-cell responses alone may be insufficient to contain HCV replication, and also that early therapy is effective independent of such responses.

Pervasive Influence of Hepatitis C Virus on the Phenotype of Antiviral CD8 + T Cells

Recent studies using MHC class I tetramers have shown that CD8+ T cell responses against different persistent viruses vary considerably in magnitude and phenotype. At one extreme, hepatitis C virus (HCV)-specific CD8+ T cell responses in blood are generally weak and have a phenotype that is perforin low and CCR7 high (early memory). At the other, specific responses to CMV are strong, perforin high, and CCR7 low (mature or effector memory). To examine the potential mechanisms behind this diversity, we compared CMV-specific responses in HCV-infected and healthy individuals. We find a striking difference in the phenotype of CMV-specific CD8+ T cells between these groups. In the HCV-infected cohort, CMV-specific CD8+ T cells lost markers associated with maturity; they had increased expression of CCR7 and reduced expression of Fas and perforin. They nevertheless responded to Ag in vitro in a manner similar to controls, with strong proliferation and appropriate acquisition of effector memory markers. The reduction in mature CD8 T cells in HCV-infected individuals may arise through either impairment or regulation of T cell stimulation, or through the early loss of mature T cells. Whatever the mechanism, HCV has a pervasive influence on the circulating CD8+ T cell population, a novel feature that may be a hallmark of this infection.

Evidence of Viral Adaptation to HLA Class I-Restricted Immune Pressure in Chronic Hepatitis C Virus Infection

ABSTRACT Cellular immune responses are an important correlate of hepatitis C virus (HCV) infection outcome. These responses are governed by the hosts human leukocyte antigen (HLA) type, and HLA-restricted viral escape mutants are a critical aspect of this host-virus interaction. We examined the driving forces of HCV evolution by characterizing the in vivo selective pressure(s) exerted on single amino acid residues within nonstructural protein 3 (NS3) by the HLA types present in two host populations. Associations between polymorphisms within NS3 and HLA class I alleles were assessed in 118 individuals from Western Australia and Switzerland with chronic hepatitis C infection, of whom 82 (69%) were coinfected with human immunodeficiency virus. The levels and locations of amino acid polymorphisms exhibited within NS3 were remarkably similar between the two cohorts and revealed regions under functional constraint and selective pressures. We identified specific HCV mutations within and flanking published epitopes with the correct HLA restriction and predicted escaped amino acid. Additional HLA-restricted mutations were identified that mark putative epitopes targeted by cell-mediated immune responses. This analysis of host-virus interaction reveals evidence of HCV adaptation to HLA class I-restricted immune pressure and identifies in vivo targets of cellular immune responses at the population level.