Early Transcriptional Divergence Marks Virus-Specific Primary Human CD8+ T Cells in Chronic versus Acute Infection.

Distinct molecular pathways govern the differentiation of CD8+ effector T cells into memory or exhausted T cells during acute and chronic viral infection, but these are not well studied in humans. Here, we employed an integrative systems immunology approach to identify transcriptional commonalities and differences between virus-specific CD8+ T cells from patients with persistent and spontaneously resolving hepatitis C virus (HCV) infection during the acute phase. We observed dysregulation of metabolic processes during early persistent infection that was linked to changes in expression of genes related to nucleosomal regulation of transcription, T cell differentiation, and the inflammatory response and correlated with subject age, sex, and the presence of HCV-specific CD4+ T cell populations. These early changes in HCV-specific CD8+ T cell transcription preceded the overt establishment of T cell exhaustion, making this signature a prime target in the search for the regulatory origins of T cell dysfunction in chronic viral infection.

Broadly neutralizing antibodies from an individual that naturally cleared multiple hepatitis C virus infections uncover molecular determinants for E2 targeting and vaccine design.

Cumulative evidence supports a role for neutralizing antibodies contributing to spontaneous viral clearance during acute hepatitis C virus (HCV) infection. Information on the timing and specificity of the B cell response associated with clearance is crucial to inform vaccine design. From an individual who cleared three sequential HCV infections with genotypes 1b, 1a and 3a strains, respectively, we employed peripheral B cells to isolate and characterize neutralizing human monoclonal antibodies (HMAbs) to HCV after the genotype 1 infections. The majority of isolated antibodies, designated as HMAbs 212, target conformational epitopes on the envelope glycoprotein E2 and bound broadly to genotype 1-6 E1E2 proteins. Further, some of these antibodies showed neutralization potential against cultured genotype 1-6 viruses. Competition studies with defined broadly neutralizing HCV HMAbs to epitopes in distinct clusters, designated antigenic domains B, C, D and E, revealed that the selected HMAbs compete with B, C and D HMAbs, previously isolated from subjects with chronic HCV infections. Epitope mapping studies revealed domain B and C specificity of these HMAbs 212. Sequential serum samples from the studied subject inhibited the binding of HMAbs 212 to autologous E2 and blocked a representative domain D HMAb. The specificity of this antibody response appears similar to that observed during chronic infection, suggesting that the timing and affinity maturation of the antibody response are the critical determinants in successful and repeated viral clearance. While additional studies should be performed for individuals with clearance or persistence of HCV, our results define epitope determinants for antibody E2 targeting with important implications for the development of a B cell vaccine.

Functional microRNA screen uncovers O-linked N-acetylglucosamine transferase as a host factor modulating hepatitis C virus morphogenesis and infectivity.

OBJECTIVE: Infection of human hepatocytes by the hepatitis C virus (HCV) is a multistep process involving both viral and host factors. microRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene expression. Given that miRNAs were indicated to regulate between 30% and 75% of all human genes, we aimed to investigate the functional and regulatory role of miRNAs for the HCV life cycle. DESIGN: To systematically reveal human miRNAs affecting the HCV life cycle, we performed a two-step functional high-throughput miRNA mimic screen in Huh7.5.1 cells infected with recombinant cell culture-derived HCV. miRNA targeting was then assessed using a combination of computational and functional approaches. RESULTS: We uncovered miR-501-3p and miR-619-3p as novel modulators of HCV assembly/release. We discovered that these miRNAs regulate O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) protein expression and identified OGT and O-GlcNAcylation as regulators of HCV morphogenesis and infectivity. Furthermore, increased OGT expression in patient-derived liver tissue was associated with HCV-induced liver disease and cancer. CONCLUSION: miR-501-3p and miR-619-3p and their target OGT are previously undiscovered regulatory host factors for HCV assembly and infectivity. In addition to its effect on HCV morphogenesis, OGT may play a role in HCV-induced liver disease and hepatocarcinogenesis.

Global mapping of antibody recognition of the hepatitis C virus E2 glycoprotein: Implications for vaccine design.

The E2 envelope glycoprotein is the primary target of human neutralizing antibody response against hepatitis C virus (HCV), and is thus a major focus of vaccine and immunotherapeutics efforts. There is emerging evidence that E2 is a highly complex, dynamic protein with residues across the protein that are modulating antibody recognition, local and global E2 stability, and viral escape. To comprehensively map these determinants, we performed global E2 alanine scanning with a panel of 16 human monoclonal antibodies (hmAbs), resulting in an unprecedented dataset of the effects of individual alanine substitutions across the E2 protein (355 positions) on antibody recognition. Analysis of shared energetic effects across the antibody panel identified networks of E2 residues involved in antibody recognition and local and global E2 stability, as well as predicted contacts between residues across the entire E2 protein. Further analysis of antibody binding hotspot residues defined groups of residues essential for E2 conformation and recognition for all 14 conformationally dependent E2 antibodies and subsets thereof, as well as residues that enhance antibody recognition when mutated to alanine, providing a potential route to engineer E2 vaccine immunogens. By incorporating E2 sequence variability, we found a number of E2 polymorphic sites that are responsible for loss of neutralizing antibody binding. These data and analyses provide fundamental insights into antibody recognition of E2, highlighting the dynamic and complex nature of this viral envelope glycoprotein, and can serve as a reference for development and rational design of E2-targeting vaccines and immunotherapeutics.

Apolipoprotein E Mediates Evasion From Hepatitis C Virus Neutralizing Antibodies.

BACKGROUND & AIMS: Efforts to develop an effective vaccine against hepatitis C virus (HCV) have been hindered by the propensity of the virus to evade host immune responses. HCV particles in serum and in cell culture associate with lipoproteins, which contribute to viral entry. Lipoprotein association has also been proposed to mediate viral evasion of the humoral immune response, though the mechanisms are poorly defined. METHODS: We used small interfering RNAs to reduce levels of apolipoprotein E (apoE) in cell culture-derived HCV-producing Huh7.5-derived hepatoma cells and confirmed its depletion by immunoblot analyses of purified viral particles. Before infection of naïve hepatoma cells, we exposed cell culture-derived HCV strains of different genotypes, subtypes, and variants to serum and polyclonal and monoclonal antibodies isolated from patients with chronic HCV infection. We analyzed the interaction of apoE with viral envelope glycoprotein E2 and HCV virions by immunoprecipitation. RESULTS: Through loss-of-function studies on patient-derived HCV variants of several genotypes and subtypes, we found that the HCV particle apoE allows the virus to avoid neutralization by patient-derived antibodies. Functional studies with human monoclonal antiviral antibodies showed that conformational epitopes of envelope glycoprotein E2 domains B and C were exposed after depletion of apoE. The level and conformation of virion-associated apoE affected the ability of the virus to escape neutralization by antibodies. CONCLUSIONS: In cell-infection studies, we found that HCV-associated apoE helps the virus avoid neutralization by antibodies against HCV isolated from chronically infected patients. This method of immune evasion poses a challenge for the development of HCV vaccines.

Affinity maturation of a broadly neutralizing human monoclonal antibody that prevents acute hepatitis C virus infection in mice.

Direct-acting antivirals (DAAs) have led to a high cure rate in treated patients with chronic hepatitis C virus (HCV) infection, but this still leaves a large number of treatment failures secondary to the emergence of resistance-associated variants (RAVs). To increase the barrier to resistance, a complementary strategy is to use neutralizing human monoclonal antibodies (HMAbs) to prevent acute infection. However, earlier efforts with the selected antibodies led to RAVs in animal and clinical studies. Therefore, we identified an HMAb that is less likely to elicit RAVs for affinity maturation to increase potency and, more important, breadth of protection. Selected matured antibodies show improved affinity and neutralization against a panel of diverse HCV isolates. Structural and modeling studies reveal that the affinity-matured HMAb mediates virus neutralization, in part, by inducing conformational change to the targeted epitope, and that the maturated light chain is responsible for the improved affinity and breadth of protection. A matured HMAb protected humanized mice when challenged with an infectious HCV human serum inoculum for a prolonged period. However, a single mouse experienced breakthrough infection after 63 days when the serum HMAb concentration dropped by several logs; sequence analysis revealed no viral escape mutation. CONCLUSION: The findings suggest that a single broadly neutralizing antibody can prevent acute HCV infection without inducing RAVs and may complement DAAs to reduce the emergence of RAVs. (Hepatology 2016;64:1922-1933).

A prophylactic hepatitis C virus vaccine: a distant peak still worth climbing.

Hepatitis C virus (HCV) infects an estimated more than 150 million people and is a leading cause of liver disease worldwide. The development of direct-acting antivirals (DAAs) will markedly improve the outcome of antiviral treatment with cure of the majority of treated patients. However, several hurdles remain before HCV infection can be considered a menace of the past: High treatment costs will most likely result in absent or limited access in middle and low resource countries and will lead to selective use even in wealthier countries. The limited efficacy of current HCV screening programs leads to a majority of cases being undiagnosed or diagnosed at a late stage and DAAs will not cure virus-induced end-stage liver disease such as hepatocellular carcinoma. Certain patient subgroups may not respond or not be eligible for DAA-based treatment strategies. Finally, reinfection remains possible, making control of HCV infection in people with ongoing infection risk difficult. The unmet medical needs justify continued efforts to develop an effective vaccine, protecting from chronic HCV infection as a mean to impact the epidemic on a global scale. Recent progress in the understanding of virus-host interactions provides new perspectives for vaccine development, but many critical questions remain unanswered. In this review, we focus on what is known about the immune correlates of HCV control, highlight key mechanisms of viral evasion that pose challenges for vaccine development and suggest areas of further investigation that could enable a rational approach to vaccine design. Within this context we also discuss insights from recent HCV vaccination studies and what they suggest about the best way to go forward.

Role of seipin in lipid droplet morphology and hepatitis C virus life cycle.

Infectious hepatitis C virus (HCV) particle assembly starts at the surface of lipid droplets, cytoplasmic organelles responsible for neutral fat storage. We analysed the relationship between HCV and seipin, a protein involved in lipid droplet maturation. Although seipin overexpression did not affect the total mean volume occupied by lipid droplets nor the total triglyceride and cholesterol ester levels per cell, it caused an increase in the mean diameter of lipid droplets by 60 %, while decreasing their total number per cell. The latter two effects combined resulted in a 34 % reduction of the total outer surface area of lipid droplets per cell, with a proportional decrease in infectious viral particle production, probably due to a defect in particle assembly. These results suggest that the available outer surface of lipid droplets is a critical factor for HCV release, independent of the neutral lipid content of the cell.

Mutations that alter use of hepatitis C virus cell entry factors mediate escape from neutralizing antibodies.

BACKGROUND & AIMS: The development of vaccines and other strategies to prevent hepatitis C virus (HCV) infection is limited by rapid viral evasion. HCV entry is the first step of infection; this process involves several viral and host factors and is targeted by host-neutralizing responses. Although the roles of host factors in HCV entry have been well characterized, their involvement in evasion of immune responses is poorly understood. We used acute infection of liver graft as a model to investigate the molecular mechanisms of viral evasion. METHODS: We studied factors that contribute to evasion of host immune responses using patient-derived antibodies, HCV pseudoparticles, and cell culture-derived HCV that express viral envelopes from patients who have undergone liver transplantation. These viruses were used to infect hepatoma cell lines that express different levels of HCV entry factors. RESULTS: By using reverse genetic analyses, we identified altered use of host-cell entry factors as a mechanism by which HCV evades host immune responses. Mutations that alter use of the CD81 receptor also allowed the virus to escape neutralizing antibodies. Kinetic studies showed that these mutations affect virus-antibody interactions during postbinding steps of the HCV entry process. Functional studies with a large panel of patient-derived antibodies showed that this mechanism mediates viral escape, leading to persistent infection in general. CONCLUSIONS: We identified a mechanism by which HCV evades host immune responses, in which use of cell entry factors evolves with escape from neutralizing antibodies. These findings advance our understanding of the pathogenesis of HCV infection and might be used to develop antiviral strategies and vaccines.

Hepatitis C virus vaccines--progress and perspectives.

Approximately 170 million individuals, representing 3% of the global population, are infected with hepatitis C virus (HCV). Whereas strategies for antiviral therapies have markedly improved resulting in clinical licensing of direct-acting antivirals, the development of vaccines has been hampered by the high genetic variability of the virus as well as by the lack of suitable animal models for proof-of-concept studies. Nevertheless, there are several promising vaccine candidates in preclinical and clinical development. After a brief summary of the molecular virology and immunology relevant to vaccine development, this review explains the model systems used for preclinical vaccine development, and highlights examples for most recently developed HCV vaccine candidates.

[Hepatitis C virus and neutralizing antibodies]. / Virus de l'hépatite C et anticorps neutralisants.

Neutralizing antibodies represent a key component of immune control in many viral infections. For a long time, these antibodies were thought to play a marginal role in hepatitis C virus (HCV) infection, and that cellular immune responses are more critical in immune control. The development of model systems to study HCV entry and significant advances in our understanding of viral entry and escape, have reenergized interest in the potential of protective neutralizing antibodies against HCV. Yet further investigations are needed to completely elucidate the role of neutralizing antibodies, recent data on their clinical impact during HCV infection and recent development of monoclonal antibodies capable of neutralizing autologous and heterologousHCVstrains, represent our best hope for the development of novel therapeutic or preventive vaccine.

Follicular T helper cells shape the HCV-specific CD4+ T cell repertoire after virus elimination.

BACKGROUNDChronic hepatitis C virus (HCV) infection is characterized by a severe impairment of HCV-specific CD4+ T cell help that is driven by chronic antigen stimulation. We aimed to study the fate of HCV-specific CD4+ T cells after virus elimination.METHODSHCV-specific CD4+ T cell responses were longitudinally analyzed using MHC class II tetramer technology, multicolor flow cytometry, and RNA sequencing in a cohort of patients chronically infected with HCV undergoing therapy with direct-acting antivirals. In addition, HCV-specific neutralizing antibodies and CXCL13 levels were analyzed.RESULTSWe observed that the frequency of HCV-specific CD4+ T cells increased within 2 weeks after initiating direct-acting antiviral therapy. Multicolor flow cytometry revealed a downregulation of exhaustion and activation markers and an upregulation of memory-associated markers. Although cells with a Th1 phenotype were the predominant subset at baseline, cells with phenotypic and transcriptional characteristics of follicular T helper cells increasingly shaped the circulating HCV-specific CD4+ T cell repertoire, suggesting antigen-independent survival of this subset. These changes were accompanied by a decline of HCV-specific neutralizing antibodies and the germinal center activity.CONCLUSIONWe identified a population of HCV-specific CD4+ T cells with a follicular T helper cell signature that is maintained after therapy-induced elimination of persistent infection and may constitute an important target population for vaccination efforts to prevent reinfection and immunotherapeutic approaches for persistent viral infections.FUNDINGDeutsche Forschungsgemeinschaft (DFG, German Research Foundation), the National Institute of Allergy and Infectious Diseases (NIAID), the European Union, the Berta-Ottenstein-Programme for Advanced Clinician Scientists, and the ANRS.

In vivo combination of human anti-envelope glycoprotein E2 and -Claudin-1 monoclonal antibodies for prevention of hepatitis C virus infection.

Despite the development of direct-acting antivirals (DAAs), hepatitis C virus (HCV) infection remains a major cause for liver disease and cancer worldwide. Entry inhibitors block virus host cell entry and, therefore, prevent establishment of chronic infection and liver disease. Due to their unique mechanism of action, entry inhibitors provide an attractive antiviral strategy in organ transplantation. In this study, we developed an innovative approach in preventing HCV infection using a synergistic combination of a broadly neutralizing human monoclonal antibody (HMAb) targeting the HCV E2 protein and a host-targeting anti-claudin 1 (CLDN1) humanized monoclonal antibody. An in vivo proof-of-concept study in human liver-chimeric FRG-NOD mice proved the efficacy of the combination therapy at preventing infection by an HCV genotype 1b infectious serum. While administration of individual antibodies at lower doses only showed a delay in HCV infection, the combination therapy was highly protective. Furthermore, the combination proved to be effective in preventing infection of primary human hepatocytes by neutralization-resistant HCV escape variants selected during liver transplantation, suggesting that a combination therapy is suited for the neutralization of difficult-to-treat variants. In conclusion, our findings suggest that the combination of two HMAbs targeting different steps of virus entry improves treatment efficacy while simultaneously reducing treatment duration and costs. Our approach not only provides a clinical perspective to employ HMAb combination therapies to prevent graft re-infection and its associated liver disease but may also help to alleviate the urgent demand for organ transplants by allowing the transplantation of organs from HCV-positive donors.

A meta-analysis of home range studies in the context of trophic levels: Implications for policy-based conservation.

Home ranges have been widely-used as ecological tools, though using home range estimates in decision-support for conservation biology is a relatively new idea. However, trophic levels are rarely taken into consideration when estimating home range. This lapse could present issues when interpreting past studies, especially in policy-based conservation. The objectives of this study were to survey the current literature, to critically analyse published articles with home range analyses, and to compare home range size by species' trophic level. We predicted that animals residing in higher trophic levels would have significantly larger home ranges than animals occupying lower trophic levels. We found that terrestrial carnivores had larger home ranges than terrestrial herbivores, though terrestrial mesocarnivores had the largest home ranges. We also found that aquatic herbivores had larger home ranges than both aquatic carnivores and aquatic mesocarnivores. Our results are important to consider for planning and management sectors, to avoid the implementation of ineffective conservation policies.

A cinnamon-derived procyanidin type A compound inhibits hepatitis C virus cell entry.

BACKGROUND AND AIMS: Chronic hepatitis C virus (HCV) infection is a major cause of liver disease worldwide. Although direct-acting antivirals can cure the large majority of treated patients, important limitations remain, including treatment failure and high costs precluding access to therapy in resource-limited settings. We report herein the anti-HCV effects of IND02, a procyanidin type A molecule, isolated and characterized from cinnamon. METHODS AND RESULTS: Using cellculture-derived HCV (HCVcc), HCV pseudoparticles (HCVpp), and subgenomic replicons, we demonstrated that IND02 markedly and dose-dependently inhibited HCV cell entry. Kinetic assays demonstrated that IND02 inhibits HCV entry most likely at a postbinding step. Experiments performed using primary human hepatocytes confirmed inhibition of HCV entry by IND02, demonstrating the functional impact in the most physiological cell-based system for studying HCV-host interactions. CONCLUSIONS: The natural compound IND02 exhibits potent HCV cell entry inhibition and provides a novel perspective for development of a low-cost antiviral for treatment of HCV infection.

Hepatitis C virus vaccine candidates inducing protective neutralizing antibodies.

INTRODUCTION: With more than 150 million chronically infected people, hepatitis C virus (HCV) remains a substantial global health burden. Direct-acting antivirals have dramatically improved viral cure. However, limited access to therapy, late stage detection of infection and re-infection following cure illustrate the need for a vaccine for global control of infection. Vaccines with induction of neutralizing antibodies (nAbs) have been shown to protect successfully against infections by multiple viruses and are currently developed for HCV. Areas covered: Here we review the progress towards the development of vaccines aiming to confer protection against chronic HCV infection by inducing broadly nAbs. The understanding or viral immune evasion in infected patients, the development of novel model systems and the recent structural characterization of viral envelope glycoprotein E2 has markedly advanced our understanding of the molecular mechanisms of virus neutralization with the concomitant development of several vaccine candidates. Expert commentary: While HCV vaccine development remains challenged by the high viral diversity and immune evasion, marked progress in HCV research has advanced vaccine design. Several vaccine candidates have shown robust induction of nAbs in animal models and humans. Randomized clinical trials are the next step to assess their clinical efficacy for protection against chronic infection.

Unraveling hepatitis C virus structure.

The high variability and the limited knowledge of the structure of the hepatitis C virus (HCV) envelope glycoproteins (GP) are challenging hurdles for vaccine design. Recently, Kong et al. published a new model of HCV E2 GP structure in Science, revealing a globular structure, starkly contrasting from the extended model of class II fusion proteins from other Flaviviridae viruses.

Neutralizing antibodies and pathogenesis of hepatitis C virus infection.

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. The interplay between the virus and host innate and adaptive immune responses determines the outcome of infection. There is increasing evidence that host neutralizing responses play a relevant role in the resulting pathogenesis. Furthermore, viral evasion from host neutralizing antibodies has been revealed to be an important contributor in leading both to viral persistence in acute liver graft infection following liver transplantation, and to chronic viral infection. The development of novel model systems to study HCV entry and neutralization has allowed a detailed understanding of the molecular mechanisms of virus-host interactions during antibody-mediated neutralization. The understanding of these mechanisms will ultimately contribute to the development of novel antiviral preventive strategies for liver graft infection and an urgently needed vaccine. This review summarizes recent concepts of the role of neutralizing antibodies in viral clearance and protection, and highlights consequences of viral escape from neutralizing antibodies in the pathogenesis of HCV infection.