Thomas Kuntzen

Naturally Occurring Dominant Resistance Mutations to Hepatitis C Virus Protease and Polymerase Inhibitors in Treatment-Naïve Patients

Resistance mutations to hepatitis C virus (HCV) nonstructural protein 3 (NS3) protease inhibitors in <1% of the viral quasispecies may still allow >1000‐fold viral load reductions upon treatment, consistent with their reported reduced replicative fitness in vitro. Recently, however, an R155K protease mutation was reported as the dominant quasispecies in a treatment‐naïve individual, raising concerns about possible full drug resistance. To investigate the prevalence of dominant resistance mutations against specifically targeted antiviral therapy for HCV (STAT‐C) in the population, we analyzed HCV genome sequences from 507 treatment‐naïve patients infected with HCV genotype 1 from the United States, Germany, and Switzerland. Phylogenetic sequence analysis and viral load data were used to identify the possible spread of replication‐competent, drug‐resistant viral strains in the population and to infer the consequences of these mutations upon viral replication in vivo. Mutations described to confer resistance to the protease inhibitors Telaprevir, BILN2061, ITMN‐191, SCH6 and Boceprevir; the NS5B polymerase inhibitor AG‐021541; and to the NS4A antagonist ACH‐806 were observed mostly as sporadic, unrelated cases, at frequencies between 0.3% and 2.8% in the population, including two patients with possible multidrug resistance. Collectively, however, 8.6% of the patients infected with genotype 1a and 1.4% of those infected with genotype 1b carried at least one dominant resistance mutation. Viral loads were high in the majority of these patients, suggesting that drug‐resistant viral strains might achieve replication levels comparable to nonresistant viruses in vivo. Conclusion: Naturally occurring dominant STAT‐C resistance mutations are common in treatment‐naïve patients infected with HCV genotype 1. Their influence on treatment outcome should further be characterized to evaluate possible benefits of drug resistance testing for individual tailoring of drug combinations when treatment options are limited due to previous nonresponse to peginterferon and ribavirin. (HEPATOLOGY 2008;48:1769–1778.)

Escape from the Dominant HLA-B27-Restricted Cytotoxic T-Lymphocyte Response in Gag Is Associated with a Dramatic Reduction in Human Immunodeficiency Virus Type 1 Replication

ABSTRACT Human leukocyte antigen (HLA)-B27-positive subjects are uncommon in their ability to control infection with human immunodeficiency virus type 1 (HIV-1). However, late viral escape from a narrowly directed immunodominant Gag-specific CD8+ T-lymphocyte (CTL) response has been linked to AIDS progression in these individuals. Identifying the mechanism of the immune-mediated control may provide critical insights into HIV-1 vaccine development. Here, we illustrate that the CTL escape mutation R264K in the HLA-B27-restricted KK10 epitope in the capsid resulted in a significant defect in viral replication in vitro. The R264K variant was impaired in generating late reverse transcription products, indicating that replication was blocked at a postentry step. Notably, the R264K mutation was associated in vivo with the development of a rare secondary mutation, S173A, which restored viral replication in vitro. Furthermore, infectivity of the R264K variant was rescued by the addition of cyclosporine A or infection of a cyclophilin A-deficient cell line. These data demonstrate a severe functional defect imposed by the R264K mutation during an early step in viral replication that is likely due to the inability of this variant to replicate efficiently in the presence of normal levels of cyclophilin A. We conclude that the impact of the R264K substitution on capsid structure constrains viral escape and enables long-term maintenance of the dominant CTL response against B27-KK10, providing an explanation for the protective effect of HLA-B27 during HIV infection.

High Level of PD-1 Expression on Hepatitis C Virus (HCV)-Specific CD8+ and CD4+ T Cells during Acute HCV Infection, Irrespective of Clinical Outcome

ABSTRACT We monitored expression of PD-1 (a mediator of T-cell exhaustion and viral persistence) on hepatitis C virus (HCV)-specific CD8+ and CD4+ T cells from blood and liver during acute and chronic infections and after the resolved infection stage. PD-1 expression on HCV-specific T cells was high early in acute infection irrespective of clinical outcome, and most cells continued to express PD-1 in resolved and chronic stages of infection; intrahepatic expression levels were especially high. Our results suggest that an analysis of PD-1 expression alone is not sufficient to predict infection outcome or to determine T-cell functionality in HCV infection.

Structural and Functional Constraints Limit Options for Cytotoxic T-Lymphocyte Escape in the Immunodominant HLA-B27-Restricted Epitope in Human Immunodeficiency Virus Type 1 Capsid

ABSTRACT Control of human immunodeficiency virus type 1 (HIV-1) by HLA-B27-positive subjects has been linked to an immunodominant CD8+ cytotoxic T-lymphocyte (CTL) response targeting the conserved KK10 epitope (KRWIILGLNK263-272) in p24/Gag. Viral escape in KK10 typically occurs through development of an R264K substitution in conjunction with the upstream compensatory mutation S173A, and the difficulty of the virus to escape from the immune response against the KK10 epitope until late in infection has been associated with slower clinical progression. Rare alternative escape mutations at R264 have been observed, but factors dictating the preferential selection of R264K remain unclear. Here we illustrate that while all observed R264 mutations (K, G, Q, and T) reduced peptide binding to HLA-B27 and impaired viral replication, the replicative defects of the alternative mutants were actually less pronounced than those for R264K. Importantly, however, none of these mutants replicated as well as an R264K variant containing the compensatory mutation S173A. In assessing the combined effects of viral replication and CTL escape using an in vitro coculture assay, we further observed that the compensated R264K mutant also displayed the highest replication capacity in the presence of KK10-specific CTLs. Comparisons of codon usage for the respective variants indicated that generation of the R264K mutation may also be favored due to a G-to-A bias in nucleotide substitutions during HIV-1 replication. Together, these data suggest that the preference for R264K is due primarily to the ability of the S173A-compensated virus to replicate better than alternative variants in the presence of CTLs, suggesting that viral fitness is a key contributor for the selection of immune escape variants.

Spontaneous Control of HCV Is Associated With Expression of HLA-B*57 and Preservation of Targeted Epitopes

BACKGROUND & AIMS HLA class I alleles are linked to spontaneous control of hepatitis C virus (HCV) and human immunodeficiency virus-1, but for HCV the roles of particular alleles and corresponding CD8(+) T-cell responses remain incompletely defined. We aimed to determine the correlations between these alleles and natural outcomes of HCV and determine associated key T-cell responses. METHODS In a cohort of HCV individuals, we determined HLA class I alleles, HCV outcomes, T-cell responses, and examined sequence data for mutational changes within key epitopes. RESULTS Carriage of HLA-B 57 was associated with a higher rate of viral clearance (risk ratio = 2.0; 95% confidence interval: 1.2-3.4), while HLA-B 08 was associated with a lower rate (risk ratio = 0.34; 95% confidence interval: 0.1-0.9]. Two HLA-B 57-restricted T-cell epitopes were targeted in spontaneous clearance; subjects with chronic viremia expressing HLA-B 57 harbored HCV strains with a high frequency of mutations in key residues. HLA-B 57-mediated escape was supported by diminished immune recognition of these variants and acute HCV infection revealing viral evolution toward less recognized variants. Analysis of a genotype 1b strain from a single-source HCV outbreak in which HLA-B 57 was not protective revealed sequence variations that interfere with immunogenicity, thereby preventing HLA-B 57-mediated immune pressure. CONCLUSIONS Our data indicate a role of HLA-B 57-restricted CD8(+) T-cell responses in mediating spontaneous clearance and evolution in HCV infection, and viral strains containing epitope variants that are less recognized abrogate the protective effects of HLA-B 57. The finding that HLA-B 57-mediated antiviral immunity is associated with control of both human immunodeficiency virus-1 and HCV suggests a common shared mechanism of a successful immune response against persistent viruses.

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

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.)

Viral Sequence Evolution in Acute Hepatitis C Virus Infection

ABSTRACT CD8+-T-cell responses play an important role in the containment and clearance of hepatitis C virus (HCV) infection, and an association between viral persistence and development of viral escape mutations has been postulated. While escape from CD8+-T-cell responses has been identified as a major driving force for the evolution of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV), a broader characterization of this relationship is needed in HCV infection. To determine the extent, kinetics, and driving forces of HCV sequence evolution, we sequenced the entire HCV genome longitudinally in four subjects monitored for up to 30 months after acute infection. For two subjects the transmission sources were also available. Of 53 total nonenvelope amino acid substitutions detected, a majority represented forward mutations away from the consensus sequence. In contrast to studies in HIV and SIV, however, only 11% of these were associated with detectable CD8+ T-cell responses. Interestingly, 19% of nonenvelope mutations represented changes toward the consensus sequence, suggesting reversion in the absence of immune pressure upon transmission. Notably, the rate of evolution of forward and reverse mutations correlated with the conservation of each residue, which is indicative of structural constraints influencing the kinetics of viral evolution. Finally, the rate of sequence evolution was observed to decline over the course of infection, possibly reflective of diminishing selection pressure by dysfunctional CD8+ T cells. Taken together, these data provide insight into the extent to which HCV is capable of evading early CD8+ T-cell responses and support the hypothesis that dysfunction of CD8+ T cells may be associated with failure to resolve HCV infections.

Impaired Hepatitis C Virus-Specific T Cell Responses and Recurrent Hepatitis C Virus in HIV Coinfection

Background Hepatitis C virus (HCV)-specific T cell responses are critical for spontaneous resolution of HCV viremia. Here we examined the effect of a lymphotropic virus, HIV-1, on the ability of coinfected patients to maintain spontaneous control of HCV infection. Methods and Findings We measured T cell responsiveness by lymphoproliferation and interferon-γ ELISPOT in a large cohort of HCV-infected individuals with and without HIV infection. Among 47 HCV/HIV-1-coinfected individuals, spontaneous control of HCV was associated with more frequent HCV-specific lymphoproliferative (LP) responses (35%) compared to coinfected persons who exhibited chronic HCV viremia (7%, p = 0.016), but less frequent compared to HCV controllers who were not HIV infected (86%, p = 0.003). Preservation of HCV-specific LP responses in coinfected individuals was associated with a higher nadir CD4 count (r 2 = 0.45, p < 0.001) and the presence and magnitude of the HCV-specific CD8+ T cell interferon-γ response (p = 0.0014). During long-term follow-up, recurrence of HCV viremia occurred in six of 25 coinfected individuals with prior control of HCV, but in 0 of 16 HIV-1-negative HCV controllers (p = 0.03, log rank test). In these six individuals with recurrent HCV viremia, the magnitude of HCV viremia following recurrence inversely correlated with the CD4 count at time of breakthrough (r = −0.94, p = 0.017). Conclusions These results indicate that HIV infection impairs the immune response to HCV—including in persons who have cleared HCV infection—and that HIV-1-infected individuals with spontaneous control of HCV remain at significant risk for a second episode of HCV viremia. These findings highlight the need for repeat viral RNA testing of apparent controllers of HCV infection in the setting of HIV-1 coinfection and provide a possible explanation for the higher rate of HCV persistence observed in this population.

Transmission and Long-Term Stability of Compensated CD8 Escape Mutations

ABSTRACT Human immunodeficiency virus effectively evades CD8+ T-cell responses through the development of CD8 escape mutations. Recent reports documenting reversion of transmitted mutations and the impact of specific escape mutations upon viral replication suggest that complex forces limit the accumulation of CD8 escape mutations at the population level. However, the presence of compensatory mutations capable of alleviating the impact of CD8 escape mutations on replication capacity may enable their persistence in an HLA-mismatched host. Herein, we illustrate the long-term stability of stereotypic escape mutations in the immunodominant HLA-B27-restricted epitope KK10 in p24/Gag following transmission when accompanied by a specific compensatory mutation.

Hepatitis C virus (HCV) sequence variation induces an HCV-specific T-cell phenotype analogous to spontaneous resolution

ABSTRACT Hepatitis C virus (HCV)-specific CD8+ T cells in persistent HCV infection are low in frequency and paradoxically show a phenotype associated with controlled infections, expressing the memory marker CD127. We addressed to what extent this phenotype is dependent on the presence of cognate antigen. We analyzed virus-specific responses in acute and chronic HCV infections and sequenced autologous virus. We show that CD127 expression is associated with decreased antigenic stimulation after either viral clearance or viral variation. Our data indicate that most CD8 T-cell responses in chronic HCV infection do not target the circulating virus and that the appearance of HCV-specific CD127+ T cells is driven by viral variation.