Joerg Timm

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.)

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.

Broad repertoire of the CD4+ Th cell response in spontaneously controlled hepatitis C virus infection includes dominant and highly promiscuous epitopes.

A vigorous hepatitis C virus (HCV)-specific Th cell response is regarded as essential to the immunological control of HCV viremia. The aim of this study was to comprehensively define the breadth and specificity of dominant HCV-specific CD4+ T cell epitopes in large cohorts of subjects with chronic and spontaneously resolved HCV viremia. Following in vitro stimulation of PBMC, HCV-specific cell cultures from each subject were screened with an overlapping panel of synthetic 20-mer peptides spanning the entire HCV polyprotein. Of 22 subjects who spontaneously controlled HCV viremia, all recognized at least one of a group of six epitopes situated within the nonstructural (NS) proteins NS3, NS4, and NS5, each of which was detected by >30% of subjects, but most subjects recognized additional, more heterogeneous specificities. In contrast, none of the most frequently targeted epitopes was detected by >5% of persons with chronic infection. The most frequently recognized peptides showed promiscuous binding to multiple HLA-DR molecules in in vitro binding assays and were restricted by different HLA-DR molecules in functional assays in different persons. These data demonstrate that predominant CD4+ T cell epitopes in persons with resolved HCV infection are preferentially located in the nonstructural proteins and are immunogenic in the context of multiple class II molecules. This comprehensive characterization of CD4+ T cell epitopes in resolved HCV infection provides important information to facilitate studies of immunopathogenesis and HCV vaccine design and evaluation.

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.

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.

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.

Increased cytotoxic T-lymphocyte epitope variant cross-recognition and functional avidity are associated with hepatitis C virus clearance.

ABSTRACT Hepatitis C virus (HCV) clearance has been associated with reduced viral evolution in targeted cytotoxic T-lymphocyte (CTL) epitopes, suggesting that HCV clearers may mount CTL responses with a superior ability to recognize epitope variants and prevent viral immune escape. Here, 40 HCV-infected subjects were tested with 406 10-mer peptides covering the vast majority of the sequence diversity spanning a 197-residue region of the NS3 protein. HCV clearers mounted significantly broader CTL responses of higher functional avidity and with wider variant cross-recognition capacity than nonclearers. These observations have important implications for vaccine approaches that may need to induce high-avidity responses in vivo.

Acute Hepatitis C Virus Infection in Incarcerated Injection Drug Users

BACKGROUND The Centers for Disease Control and Prevention has emphasized the need for interventional programs regarding hepatitis C virus (HCV) infection for injection drug users, the group of persons who are at highest risk of acquiring acute infection. METHODS We designed a pilot study to assess the feasibility of identifying injection drug users with acute HCV infection in correctional and detoxification facilities. On-site medical providers were educated regarding risk factors and signs and symptoms of infection and were instructed to refer all patients with hepatitis to our specialty clinic. RESULTS Over a 30-month period, 21 patients received a diagnosis of acute hepatitis C, 3 received a diagnosis of hepatitis B, and 1 received a diagnosis of hepatitis A. Of the 21 patients with acute hepatitis C, 19 were identified in the prison setting shortly after incarceration. Of the 17 patients who were observed serially (mean duration of observation, 6.3 months), 8 had spontaneous virologic clearance. Early therapy with pegylated interferon was initiated for 5 patients with persistent viremia and led to a sustained virologic response in 2 individuals. All patients agreed to undergo human immunodeficiency virus counseling and testing, as well as to receive immunization for hepatitis A and B. CONCLUSIONS Incarceration presents a unique opportunity to identify injection drug users with acute HCV infection, to initiate counseling regarding other bloodborne pathogens, and to facilitate immunizations and HCV treatment.