Katja Nitschke

Human leukocyte antigen B27 selects for rare escape mutations that significantly impair hepatitis C virus replication and require compensatory mutations

Human leukocyte antigen B27 is associated with spontaneous viral clearance in hepatitis C virus (HCV) infection. Viral escape within the immunodominant, HLA‐B27‐restricted, HCV‐specific, cluster of differentiation (CD)8+ T‐cell epitope, nonstructural protein (NS)5B2841‐2849 (ARMILMTHF), has been shown to be limited by viral fitness costs as well as broad T‐cell cross‐recognition, suggesting a potential mechanism of protection by HLA‐B27. Here, we studied the subdominant HLA‐B27‐restricted epitope, NS5B2936‐2944 (GRAAICGKY), to further define the mechanisms of protection by HLA‐B27. We identified a unique pattern of escape mutations within this epitope in a large cohort of HCV genotype 1a–infected patients. The predominant escape mutations represented conservative substitutions at the main HLA‐B27 anchor residue or a T‐cell receptor contact site, neither of which impaired viral replication capacity, as assessed in a subgenomic HCV replicon system. In contrast, however, in a subset of HLA‐B27+ subjects, rare escape mutations arose at the HLA‐B27 anchor residue, R2937, which nearly abolished viral replication. Notably, these rare mutations only occurred in conjunction with the selection of two equally rare, and structurally proximal, upstream mutations. Coexpression of these upstream mutations with the rare escape mutations dramatically restored viral replication capacity from <5% to ≥70% of wild‐type levels. Conclusion: The selection of rare CTL escape mutations in this HLA‐B27‐restricted epitope dramatically impairs viral replicative fitness, unless properly compensated. These data support a role for the targeting of highly constrained regions by HLA‐B27 in its ability to assert immune control of HCV and other highly variable pathogens. (HEPATOLOGY 2011;)

HLA-B*27 subtype specificity determines targeting and viral evolution of a hepatitis C virus-specific CD8+ T cell epitope

BACKGROUND & AIMS HLA-B*27 is associated with spontaneous HCV genotype 1 clearance. HLA-B*27-restricted CD8+ T cells target three NS5B epitopes. Two of these epitopes are dominantly targeted in the majority of HLA-B*27+ patients. In chronic infection, viral escape occurs consistently in these two epitopes. The third epitope (NS5B2820) was dominantly targeted in an acutely infected patient. This was in contrast, however, to the lack of recognition and viral escape in the large majority of HLA-B*27+ patients. Here, we set out to determine the host factors contributing to selective targeting of this epitope. METHODS Four-digit HLA class I typing and viral sequence analyses were performed in 78 HLA-B*27+ patients with chronic HCV genotype 1 infection. CD8+ T cell analyses were performed in a subset of patients. In addition, HLA/peptide affinity was compared for HLA-B*27:02 and 05. RESULTS The NS5B2820 epitope is only restricted by the HLA-B*27 subtype HLA-B*27:02 (that is frequent in Mediterranean populations), but not by the prototype HLA-B*27 subtype B*27:05. Indeed, the epitope is very dominant in HLA-B*27:02+ patients and is associated with viral escape mutations at the anchor position for HLA-binding in 12 out of 13 HLA-B*27:02+ chronically infected patients. CONCLUSIONS The NS5B2820 epitope is immunodominant in the context of HLA-B*27:02, but is not restricted by other HLA-B*27 subtypes. This finding suggests an important role of HLA subtypes in the restriction of HCV-specific CD8+ responses. With minor HLA subtypes covering up to 39% of specific populations, these findings may have important implications for the selection of epitopes for global vaccines.

Synergism of tapasin and human leukocyte antigens in resolving hepatitis C virus infection

CD8+ T‐cell responses to hepatitis C virus (HCV) are important in generating a successful immune response and spontaneously clearing infection. Human leukocyte antigen (HLA) class I presents viral peptides to CD8+ T cells to permit detection of infected cells, and tapasin is an important component of the peptide loading complex for HLA class I. We sought to determine if tapasin polymorphisms affected the outcome of HCV infection. Patients with resolved or chronic HCV infection were genotyped for the known G/C coding polymorphism in exon 4 of the tapasin gene. In a European, but not a US, Caucasian population, the tapasin G allele was significantly associated with the outcome of HCV infection, being found in 82.5% of resolvers versus 71.3% of persistently infected individuals (P = 0.02, odds ratio [OR] = 1.90 95% confidence interval [CI] = 1.11‐3.23). This was more marked at the HLA‐B locus at which heterozygosity of both tapasin and HLA‐B was protective (P < 0.03). Individuals with an HLA‐B allele with an aspartate at residue 114 and the tapasin G allele were more likely to spontaneously resolve HCV infection (P < 0.00003, OR = 3.2 95% CI = 1.6‐6.6). Additionally, individuals with chronic HCV and the combination of an HLA‐B allele with an aspartate at residue 114 and the tapasin G allele also had stronger CD8+ T‐cell responses (P = 0.02, OR = 2.58, 95% CI‐1.05‐6.5). Conclusion: Tapasin alleles contribute to the outcome of HCV infection by synergizing with polymorphisms at HLA‐B in a population‐specific manner. This polymorphism may be relevant for peptide vaccination strategies against HCV infection. (Hepatology 2013;53:881–889)

CD8+ T-Cell Responses in Hepatitis B and C: The (HLA-) A, B, and C of Hepatitis B and C.

Approximately 500 million people are chronically infected with the hepatitis B virus (HBV) or hepatitis C virus (HCV) worldwide and are thus at high risk of progressive liver disease, leading to liver fibrosis, cirrhosis and ultimately hepatocellular cancer. Virus-specific CD8+ T-cells play a major role in viral clearance in >90% of adult patients who clear HBV and in approximately 30% of patients who clear HCV in acute infection. However, several mechanisms contribute to the failure of the adaptive CD8+ T-cell response in those patients who progress to chronic infection. These include viral mutations leading to escape from the CD8+ T-cell response as well as exhaustion and dysfunction of virus-specific CD8+ T-cells. Antiviral efficacy of the virus-specific CD8+ T-cell response also strongly depends on its restriction by specific human leukocyte antigens (HLA) class I alleles. Our review will summarize the role of HLA-A, B and C-restricted CD8+ T-cells in HBV and HCV infection. Due to the current lack of a comprehensive database of HBV- and HCV-specific CD8+ T-cell epitopes, we also provide a summary of the repertoire of currently well-described HBV- and HCV-specific CD8+ T-cell epitopes. A better understanding of the factors that contribute to the success or failure of virus-specific CD8+ T-cells may help to develop new therapeutic options for HBV eradication in patients with chronic HBV infection (therapeutic vaccination and/or immunomodulation) as well as a prophylactic vaccine against HCV infection.

Tetramer Enrichment Reveals the Presence of Phenotypically Diverse Hepatitis C Virus-Specific CD8+ T Cells in Chronic Infection

ABSTRACT Virus-specific CD8+ T cells are rarely detectable ex vivo by conventional methods during chronic hepatitis C virus (HCV) infection. In this study, however, we were able to detect and characterize HCV-specific CD8+ T cells in all chronically HCV genotype 1a-infected, HLA-A*02:01-positive patients analyzed by performing major histocompatibility complex (MHC) class I tetramer enrichment. Two-thirds of these enriched HCV-specific CD8+ T-cell populations displayed an effector memory phenotype, whereas, surprisingly, one-third displayed a naive-like phenotype despite ongoing viral replication. CD8+ T cells with an effector memory phenotype could not expand in vitro, suggesting exhaustion of these cells. Interestingly, some of the naive-like CD8+ T cells proliferated vigorously upon in vitro priming, whereas others did not. These differences were linked to the corresponding viral sequences in the respective patients. Indeed, naive-like CD8+ T cells from patients with the consensus sequence in the corresponding T-cell epitope did not expand in vitro. In contrast, in patients displaying sequence variations, we were able to induce HCV-specific CD8+ T-cell proliferation, which may indicate infection with a variant virus. Collectively, these data reveal the presence of phenotypically and functionally diverse HCV-specific CD8+ T cells at very low frequencies that are detectable in all chronically infected patients despite viral persistence. IMPORTANCE In this study, we analyzed CD8+ T-cell responses specific for HLA-A*02:01-restricted epitopes in chronically HCV-infected patients, using MHC class I tetramer enrichment. Importantly, we could detect HCV-specific CD8+ T-cell populations in all patients. To further characterize these HCV-specific CD8+ T-cell populations that are not detectable using conventional techniques, we performed phenotypic, functional, and viral sequence analyses. These data revealed different mechanisms for CD8+ T-cell failure in HCV infection, including T-cell exhaustion, viral escape, and functional impairment of naive-like HCV-specific CD8+ T cells.

The cyclophilin-inhibitor alisporivir stimulates antigen presentation thereby promoting antigen-specific CD8+ T cell activation

Background & Aims Cyclophilin-inhibitors have potent antiviral activity against Hepatitis C virus (HCV) and are promising candidates for broad-spectrum antiviral therapy. Cyclosporine A (CsA) acts immunosuppressive by blocking T cell activation and antigen presentation. Alisporivir, a non-immunosuppressive CsA analog in clinical development, does not inhibit T cell activation. In this study we explored the impact of alisporivir on antigen presentation. Methods Hepatoma cells endogenously expressing the epitope-restricting major histocompatibility complex-class I (MHC-I) allele HLA-A2 and constitutively expressing a viral antigen were established to study the impact of cyclophilin-inhibitors on antigen presentation. Antigen-specific CD8+ T cell activation and MHC-I surface expression were measured to quantify antigen presentation. Results Our work establishes a novel cell culture model to study antigen presentation in liver-derived cells. Authentic regulation of antigen presentation was ensured by the action of pro- and anti-inflammatory cytokines. Alisporivir pretreatment stimulated antigen presentation by hepatoma target cells, leading to enhancement of antigen-specific CD8+ T cell activation by 40%. Alisporivir, as well as a panel of other cyclophilin-inhibitors, induced an increase of MHC-I and beta-2 microglobulin on the surface of several cell lines. The drug neither enhanced MHC-I transcript or protein levels nor affected surface expression of other proteins or protein trafficking in general. Proteasome-inhibitors completely blocked the alisporivir-directed enhancement of surface MHC-I, suggesting an influence of the drug on peptide-availability. Conclusions Alisporivir stimulates antigen presentation by inducing enhanced MHC-I surface expression, thereby promoting antigen-specific CD8+ T cell activation. This immunostimulatory function might further contribute to the antiviral activity of non-immunosuppressive cyclophilin-inhibitors.

Synergism of tapasin and human leukocyte antigens in resolving hepatitis C virus infection: Hepatology

CD8+ T‐cell responses to hepatitis C virus (HCV) are important in generating a successful immune response and spontaneously clearing infection. Human leukocyte antigen (HLA) class I presents viral peptides to CD8+ T cells to permit detection of infected cells, and tapasin is an important component of the peptide loading complex for HLA class I. We sought to determine if tapasin polymorphisms affected the outcome of HCV infection. Patients with resolved or chronic HCV infection were genotyped for the known G/C coding polymorphism in exon 4 of the tapasin gene. In a European, but not a US, Caucasian population, the tapasin G allele was significantly associated with the outcome of HCV infection, being found in 82.5% of resolvers versus 71.3% of persistently infected individuals (P = 0.02, odds ratio [OR] = 1.90 95% confidence interval [CI] = 1.11‐3.23). This was more marked at the HLA‐B locus at which heterozygosity of both tapasin and HLA‐B was protective (P < 0.03). Individuals with an HLA‐B allele with an aspartate at residue 114 and the tapasin G allele were more likely to spontaneously resolve HCV infection (P < 0.00003, OR = 3.2 95% CI = 1.6‐6.6). Additionally, individuals with chronic HCV and the combination of an HLA‐B allele with an aspartate at residue 114 and the tapasin G allele also had stronger CD8+ T‐cell responses (P = 0.02, OR = 2.58, 95% CI‐1.05‐6.5). Conclusion: Tapasin alleles contribute to the outcome of HCV infection by synergizing with polymorphisms at HLA‐B in a population‐specific manner. This polymorphism may be relevant for peptide vaccination strategies against HCV infection. (Hepatology 2013;53:881–889)

SYNERGISM OF TAPASIN AND HLA IN RESOLVING HCV INFECTION

CD8+ T‐cell responses to hepatitis C virus (HCV) are important in generating a successful immune response and spontaneously clearing infection. Human leukocyte antigen (HLA) class I presents viral peptides to CD8+ T cells to permit detection of infected cells, and tapasin is an important component of the peptide loading complex for HLA class I. We sought to determine if tapasin polymorphisms affected the outcome of HCV infection. Patients with resolved or chronic HCV infection were genotyped for the known G/C coding polymorphism in exon 4 of the tapasin gene. In a European, but not a US, Caucasian population, the tapasin G allele was significantly associated with the outcome of HCV infection, being found in 82.5% of resolvers versus 71.3% of persistently infected individuals (P = 0.02, odds ratio [OR] = 1.90 95% confidence interval [CI] = 1.11‐3.23). This was more marked at the HLA‐B locus at which heterozygosity of both tapasin and HLA‐B was protective (P < 0.03). Individuals with an HLA‐B allele with an aspartate at residue 114 and the tapasin G allele were more likely to spontaneously resolve HCV infection (P < 0.00003, OR = 3.2 95% CI = 1.6‐6.6). Additionally, individuals with chronic HCV and the combination of an HLA‐B allele with an aspartate at residue 114 and the tapasin G allele also had stronger CD8+ T‐cell responses (P = 0.02, OR = 2.58, 95% CI‐1.05‐6.5). Conclusion: Tapasin alleles contribute to the outcome of HCV infection by synergizing with polymorphisms at HLA‐B in a population‐specific manner. This polymorphism may be relevant for peptide vaccination strategies against HCV infection. (Hepatology 2013;53:881–889)

Differential virus-specific CD8+ T-cell epitope repertoire in hepatitis C virus genotype 1 versus 4

Virus‐specific CD8+ T‐cell responses play an important role in the outcome of hepatitis C virus (HCV) infection. To date, most HCV‐specific CD8+ T‐cell epitopes have been defined in HCV genotype 1 infection. In contrast, the HCV genotype 4‐specific CD8+ T‐cell response is poorly defined. Here, we analysed whether known HCV‐specific CD8+ T‐cell epitopes are also recognized in HCV genotype 4‐infected patients and set out to identify the first HCV genotype 4‐specific CD8+ T‐cell epitopes. We studied patients chronically infected with HCV genotype 1 (n = 20) or 4 (n = 21) using 91 well‐described HCV‐specific epitope peptides. In addition, we analysed 24 genotype 4‐infected patients using 40 epitope candidates predicted using an in silico approach. HCV‐specific CD8+ T‐cell responses targeting previously described epitopes were detectable in the majority of genotype 1‐infected patients (11 of 20). In contrast, patients infected with HCV genotype 4 rarely targeted these epitopes (4 of 21; P = .0247). Importantly, we were able to identify eight novel HCV genotype 4‐specific CD8+ T‐cell epitopes. Only one of these epitopes was shared between genotype 1 and genotype 4. These results indicate that there is little overlap between CD8+ T‐cell repertoires targeting HCV genotype 1 and 4. Prophylactic vaccination studies based on HCV genotype 1 are currently underway. However, in countries with the highest prevalence of HCV infection, such as Egypt, most patients are infected with HCV genotype 4. Thus, prophylactic vaccination strategies need to be adapted to HCV genotype 4 before their application to regions where HCV genotype 4 is endemic.