Andrew Berical

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.)

Whole genome deep sequencing of HIV-1 reveals the impact of early minor variants upon immune recognition during acute infection

Deep sequencing technologies have the potential to transform the study of highly variable viral pathogens by providing a rapid and cost-effective approach to sensitively characterize rapidly evolving viral quasispecies. Here, we report on a high-throughput whole HIV-1 genome deep sequencing platform that combines 454 pyrosequencing with novel assembly and variant detection algorithms. In one subject we combined these genetic data with detailed immunological analyses to comprehensively evaluate viral evolution and immune escape during the acute phase of HIV-1 infection. The majority of early, low frequency mutations represented viral adaptation to host CD8+ T cell responses, evidence of strong immune selection pressure occurring during the early decline from peak viremia. CD8+ T cell responses capable of recognizing these low frequency escape variants coincided with the selection and evolution of more effective secondary HLA-anchor escape mutations. Frequent, and in some cases rapid, reversion of transmitted mutations was also observed across the viral genome. When located within restricted CD8 epitopes these low frequency reverting mutations were sufficient to prime de novo responses to these epitopes, again illustrating the capacity of the immune response to recognize and respond to low frequency variants. More importantly, rapid viral escape from the most immunodominant CD8+ T cell responses coincided with plateauing of the initial viral load decline in this subject, suggestive of a potential link between maintenance of effective, dominant CD8 responses and the degree of early viremia reduction. We conclude that the early control of HIV-1 replication by immunodominant CD8+ T cell responses may be substantially influenced by rapid, low frequency viral adaptations not detected by conventional sequencing approaches, which warrants further investigation. These data support the critical need for vaccine-induced CD8+ T cell responses to target more highly constrained regions of the virus in order to ensure the maintenance of immunodominant CD8 responses and the sustained decline of early viremia.

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.

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.

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.

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.

Whole Genome Pyrosequencing of Rare Hepatitis C Virus Genotypes Enhances Subtype Classification and Identification of Naturally Occurring Drug Resistance Variants

BACKGROUND  Infection with hepatitis C virus (HCV) is a burgeoning worldwide public health problem, with 170 million infected individuals and an estimated 20 million deaths in the coming decades. While 6 main genotypes generally distinguish the global geographic diversity of HCV, a multitude of closely related subtypes within these genotypes are poorly defined and may influence clinical outcome and treatment options. Unfortunately, the paucity of genetic data from many of these subtypes makes time-consuming primer walking the limiting step for sequencing understudied subtypes. METHODS  Here we combined long-range polymerase chain reaction amplification with pyrosequencing for a rapid approach to generate the complete viral coding region of 31 samples representing poorly defined HCV subtypes. RESULTS  Phylogenetic classification based on full genome sequences validated previously identified HCV subtypes, identified a recombinant sequence, and identified a new distinct subtype of genotype 4. Unlike conventional sequencing methods, use of deep sequencing also facilitated characterization of minor drug resistance variants within these uncommon or, in some cases, previously uncharacterized HCV subtypes. CONCLUSIONS  These data aid in the classification of uncommon HCV subtypes while also providing a high-resolution view of viral diversity within infected patients, which may be relevant to the development of therapeutic regimens to minimize drug resistance.

Temporal Dynamics of a Predominant Protease Inhibitor–Resistance Mutation in a Treatment-Naive, Hepatitis C Virus–Infected Individual

The dramatic antiviral activities of drugs that specifically inhibit hepatitis C virus replication can be tempered by baseline mutations that confer resistance. We describe the kinetics of an R155K mutation in hepatitis C virus (HCV) NS3 protease known to confer resistance to specific protease inhibitors in an individual coinfected with human immunodeficiency virus-1 and HCV. Longitudinal sequences revealed changes in the relative frequency with which this variant was observed independent of HCV replication levels, illustrating that this mutation coexists with wild-type strains in vivo in the absence of drugs. The persistence of drug-resistance mutations argues for baseline resistance genotyping at the time therapy is initiated to accurately predict the efficacy of treatment.

Compensatory Mutations Restore the Replication Defects Caused by Cytotoxic T Lymphocyte Escape Mutations in Hepatitis C Virus Polymerase

ABSTRACT While human leukocyte antigen B57 (HLA-B57) is associated with the spontaneous clearance of hepatitis C virus (HCV), the mechanisms behind this control remain unclear. Immunodominant CD8+ T cell responses against the B57-restricted epitopes comprised of residues 2629 to 2637 of nonstructural protein 5B (NS5B2629–2637) (KSKKTPMGF) and E2541–549 (NTRPPLGNW) were recently shown to be crucial in the control of HCV infection. Here, we investigated whether the selection of deleterious cytotoxic T lymphocyte (CTL) escape mutations in the NS5B KSKKTPMGF epitope might impair viral replication and contribute to the B57-mediated control of HCV. Common CTL escape mutations in this epitope were identified from a cohort of 374 HCV genotype 1a-infected subjects, and their impact on HCV replication assessed using a transient HCV replicon system. We demonstrate that while escape mutations at residue 2633 (position 5) of the epitope had little or no impact on HCV replication in vitro, mutations at residue 2629 (position 1) substantially impaired replication. Notably, the deleterious mutations at position 2629 were tightly linked in vivo to upstream mutations at residue 2626, which functioned to restore the replicative defects imparted by the deleterious escape mutations. These data suggest that the selection of costly escape mutations within the immunodominant NS5B KSKKTPMGF epitope may contribute in part to the control of HCV replication in B57-positive individuals and that persistence of HCV in B57-positive individuals may involve the development of specific secondary compensatory mutations. These findings are reminiscent of the selection of deleterious CTL escape and compensatory mutations by HLA-B57 in HIV-1 infection and, thus, may suggest a common mechanism by which alleles like HLA-B57 mediate protection against these highly variable pathogens.

A set of reference sequences for the Hepatitis C genotypes 4d, 4f and 4k covering the full open reading frame

Infection with genotype 4 of the Hepatitis C virus is common in Africa and the Mediterranean area, but has also been found at increasing frequencies in injection drug users in Europe and North America. Full length viral sequences to characterize viral diversity and structure have recently become available mostly for subtype 4a, and studies in Egypt and Saudi Arabia, where high proportions of subtype 4a infected patients exist, have begun to establish optimized treatment regimens. However knowledge about other subtype variants of genotype 4 present in less developed African states is lacking. In this study the full coding region from so far poorly characterized variants of HCV genotype 4 was amplified and sequenced using a long range PCR technique. Sequences were analyzed with respect to phylogenetic relationship, possible recombination and prominent sequence characteristics compared to other known HCV strains. We present for the first time two full‐length sequences from the HCV genotype 4k, in addition to five strains from HCV genotypes 4d and 4f. Reference sequences for accurate HCV genotyping are required for optimized treatment, and a better knowledge of the global viral sequence diversity is needed to guide vaccines or new drugs effective in the world wide epidemic. J. Med. Virol. 80:1370–1378, 2008.