Catherine Fauvelle

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.

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.

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.

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.

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

Significance Hepatitis C virus is a major public health concern, infecting approximately 3% of the world’s population, with no vaccine currently available. To enable rational vaccine design for this highly diverse and dynamic virus, we performed alanine scanning of nearly all positions of the E2 envelope protein, which is the primary target of the antibody response, using a panel of 16 human monoclonal antibodies that target a broad range of epitopes. This approach provided an unprecedented global view of the determinants of E2 stability, residue connectivity, and neutralizing antibody recognition. These insights and mapping data provide a framework to engineer E2 to modulate antibody recognition and optimize its capacity to induce broadly neutralizing antibodies in the context of a vaccine. 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.

Hepatitis C virus vaccine candidates inducing protective neutralizing antibodies

ABSTRACT 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.

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).

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.

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

&NA; 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. Graphical Abstract Figure. No caption available. HighlightsDisease outcome and viral escape are linked to transcriptional differences in T cellsT cells from different outcomes share a core of co‐regulated T cell identity genesMetabolic, nucleosome, and immune genes are dysregulated early in chronic infectionDysregulation correlates with sex, age, and presence of HCV‐specific CD4 T cells &NA; Wolski et. al show that transcriptional dysregulation of metabolic, nucleosomal, and immune processes in virus‐specific CD8+ T cells during early persistent HCV infection is both under tight transcriptional control and associated with differences in predictors of disease outcome, like patient sex, age, and the presence of HCV‐specific CD4+ T cells.

1150 HEPATITIS C VIRUS LIVER TRANSPLANTATION ESCAPE VARIANT IS CHARACTERIZED BY BOTH ENHANCED TRIGLYCERIDE-RICH LIPOPROTEIN ASSOCIATION AND SENSITIVITY TO apoE ANTIBODIES

cEVR (p =0.003). We found no relationship with the expression of mir-122 for total cholesterol, triglycerids and HCV viral load. Mir122 expression showed a 50% reduction in HCV infected patients and is not modified at the different stages of fibrosis. Conclusions: Patients IL28B CT/TT who failed to PEG-IFN plus ribavirin presented a reduction of mir-122 expression. Whereas, in vitro mir-122 stimulates HCV replication, there was no correlation between viral load and hepatic mir-122 expression. The stage of the disease was not associated with a modification of mir122 expression. Altogether, these results suggest that the level of expression of mir-122 needs to be maintained to regulate its different activity.