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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.

Resistance mutations to hepatitis C virus (HCV) nonstructural protein 3 (NS3) protease inhibitors in1% of the viral quasispecies may still allow1000-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.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.

Hepatitis C virus (HCV) is an enveloped, positive-strand RNA virus belonging to the Hepacivirus genus in the Flaviviridae family. Seven confirmed genotypes (1–7) are generally distinguished by phylogenetic methods and pair-wise distance calculations [1, 2]. On the basis of full genome nucleotide sequences, HCV genotypes diverged from each other by a pair-wise distance of >30%. Individual genotypes can be further divided into more closely related subtypes that diverged by a pair-wise distance of 15%–30%. All viral genotypes retain their repertoire of colinear structural and nonstructural genes, thereby facilitating preliminary genotype classification on the basis of partial genome sequences of short fragments (approximately 300–400 nucleotides) in the structural core/E1 region and the nonstructural NS5B region [3]. However, full genome sequences remain indispensible for the detection of genome recombination events. While only recombinant HCV genotype 2 k/1b viruses have been found to actively circulate in the population so far [4–7], recombinants of genotypes 2/5, 2b/1b, 2b/1a, and 2i/6p have been detected in single isolates from humans [8–11]. Furthermore, the frequency of HCV intergenotype and intragenotype recombination may be underestimated because of the lack of robust detection methods [12, 13]. Although genotyping based on core/E1 or NS5B sequences has resulted in the provisional classification of a large number of subtype variants within each genotype, at least 1 but preferably ≥2 full genome sequences are required to confirm subtype designation [2, 12]. Accurate genotype and subtype classification is clinically important because major genotypes differ considerably in their response rates to treatment with pegylated interferon and ribavirin and with directly acting antiviral drugs (DAAs) that are designed mainly against genotype 1 isolates. The limited efficacy of DAAs against other genotypes has been shown for genotype 3 [14], and treatment response rates for drug regimens containing boceprevir (an NS3 protease inhibitor) or BMS-790052 (an NS5A inhibitor) were found to vary even between subtypes 1a and 1b [15, 16]. Different baseline frequencies of drug resistance mutations may account for these differences [17], and such variation might become more significant if interferon-free regimens containing 1 or more DAAs could indeed displace the current standard of care [18]. Until clinically tolerable DAAs with high barriers to resistance against the complete spectrum of HCV genotypes become available [18, 19], accurate subtype determination and, possibly, drug resistance profiling of the individual's viral population may guide the optimal choice of drugs. Full genome deep sequencing, as performed in this study, allows the identification of drug resistance mutations across the genome that may exist as dominant or minor variants in treatment-naive patients, thereby informing the design of therapeutic regimens.

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 (NS5B 2629–2637 ) (KSKKTPMGF) and E2 541–549 (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.

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., Author Summary The ability of HIV-1 and other highly variable pathogens to rapidly mutate to escape vaccine-induced immune responses represents a major hurdle to the development of effective vaccines to these highly persistent pathogens. Application of next-generation or deep sequencing technologies to the study of host pathogens could significantly improve our understanding of the mechanisms by which these pathogens subvert host immunity, and aid in the development of novel vaccines and therapeutics. Here, we developed a 454 deep sequencing approach to enable the sensitive detection of low-frequency viral variants across the entire HIV-1 genome. When applied to the acute phase of HIV-1 infection we observed that the majority of early, low frequency mutations represented viral adaptations to host cellular immune responses, evidence of strong host immunity developing during the early decline of peak viral load. Rapid viral escape from the most dominant immune responses however correlated with loss of this initial viral control, suggestive of the importance of mounting immune responses against more conserved regions of the virus. These data provide a greater understanding of the early evolutionary events subverting the ability of host immune responses to control early HIV-1 replication, yielding important insight into the design of more effective vaccine strategies.

Open Access Poster presentation P09-20 LB. Ultra-deep sequencing of full-length HIV-1 genomes identifies rapid viral evolution during acute infection MR Henn1, C Boutwell3, N Lennon1, K Power3, C Malboeuf1, P Charlebois1, A Gladden3, J Levin1, M Casali1, L Philips3, A Berlin1, A Berical3, R Erlich1, S Anderson1, H Streeck3, M Kemper3, E Ryan1, Y Wang3, L Green1, K Axten3, Z Brumme3, C Brumme3, C Russ1, E Rosenberg3, H Jessen2, M Altfeld3, C Nusbaum1, B Walker3, B Birren1 and TM Allen*3

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.

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.

Chronic hepatitis C virus (HCV) infection is typically characterized by a lack of virus-specific CD4+ T-cell–proliferative responses, but strong responses have been described in a subset of persons with persistent viremia. One possible explanation for these responses is that they were primed by an earlier resolved infection and do not recognize the current circulating virus. We defined all targeted epitopes using overlapping peptides corresponding to a genotype 1a strain in 44 patients chronically infected with different HCV genotypes (GT). Surprisingly, more HCV-specific CD4+ T-cell responses were detected in patients with chronic non-GT1 infection compared with patients with chronic GT1 infection (P = .017). Notably, we found serologic evidence of a previous exposure to GT1 in 4 patients with non-GT1 infection, and these persons also demonstrated significantly more responses than non-GT1 patients in whom genotype and HCV serotype were identical (P < .001). Comparison of recognition of GT1-specific peptides to peptides representing autologous virus revealed the absence of cross-recognition of the autologous circulating virus. These data indicate that persisent HCV infection can occur in the presence of an HCV-specific T-cell response primed against a heterologous HCV strain, and suggest that clearance of 1 GT does not necessarily protect against subsequent exposure to a second GT.