Julian Schulze zur Wiesch

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.)

Expansion of HIV-specific T follicular helper cells in chronic HIV infection

HIV targets CD4 T cells, which are required for the induction of high-affinity antibody responses and the formation of long-lived B cell memory. The depletion of antigen-specific CD4 T cells during HIV infection is therefore believed to impede the development of protective B cell immunity. Although several different HIV-related B cell dysfunctions have been described, the role of CD4 T follicular helper (TFH) cells in HIV infection remains unknown. Here, we assessed HIV-specific TFH responses in the lymph nodes of treatment-naive and antiretroviral-treated HIV-infected individuals. Strikingly, both the bulk TFH and HIV-specific TFH cell populations were significantly expanded in chronic HIV infection and were highly associated with viremia. In particular, GAG-specific TFH cells were detected at significantly higher levels in the lymph nodes compared with those of GP120-specific TFH cells and showed preferential secretion of the helper cytokine IL-21. In addition, TFH cell expansion was associated with an increase of germinal center B cells and plasma cells as well as IgG1 hypersecretion. Thus, our study suggests that high levels of HIV viremia drive the expansion of TFH cells, which in turn leads to perturbations of B cell differentiation, resulting in dysregulated antibody production.

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.

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.

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.

Comprehensive Analysis of Frequency and Phenotype of T Regulatory Cells in HIV Infection: CD39 expression of FoxP3+ T regulatory cells correlates with progressive disease.

ABSTRACT There are conflicting data about the frequency and role of regulatory T cells (Tregs) during the course of HIV infection. Peripheral blood of a large cohort of HIV-infected patients (n = 131) at different stages of disease, including 15 long-term nonprogressors and 21 elite controllers, was analyzed to determine the frequency and phenotype of Tregs, defined as CD4+, CD25high, CD127low, FoxP3high cells. A significantly increased relative frequency of Tregs within the CD4+ compartment of HIV+ patients compared to that of healthy controls (P < 0.0001) was observed. Additionally, the relative frequency of Tregs directly correlated with HIV viral load and inversely with CD4+ counts. However, the absolute Treg number was reduced in HIV-infected patients versus healthy controls (P < 0.0001), with the exception of elite controllers (P > 0.05). The loss of absolute Treg numbers coincided with rising markers of immune activation (P < 0.0006). The initiation of antiviral therapy significantly increased absolute Treg numbers (P < 0.0031). We find that the expression of CD39, a newly defined ectonucleotidase with immunomodulatory functions on Tregs, correlated with progressive HIV disease, HIV viral load, and immune activation. Of note, when tested in peripheral blood mononuclear cells of healthy volunteers, the in vitro capacity to suppress T-cell proliferation was limited to CD4+, CD25high, CD39+ T cells. Interestingly, Tregs of elite controllers exhibited not only the highest expression of CCR5, CTLA-4, and ICOS but also the lowest level of CD39. The data presented here reconcile the seemingly contradictory results of previous studies looking at Tregs in HIV and highlight the complexity of Treg-mediated immunoregulation during human viral infections.

Broadly directed virus-specific CD4+ T cell responses are primed during acute hepatitis C infection, but rapidly disappear from human blood with viral persistence

Early after symptom onset, HCV-specific CD4+ T cell responses are primed and detectable in patients regardless of clinical outcome, but without early antiviral therapy these T cells become exhausted or deleted in chronically infected patients.

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.)