Convergent evolution and targeting of diverse E2 epitopes by human broadly neutralizing antibodies are associated with HCV clearance.

The early appearance of broadly neutralizing antibodies (bNAbs) in serum is associated with spontaneous hepatitis C virus (HCV) clearance, but to date, the majority of bNAbs have been isolated from chronically infected donors. Most of these bNAbs use the VH1-69 gene segment and target the envelope glycoprotein E2 front layer. Here, we performed longitudinal B cell receptor (BCR) repertoire analysis on an elite neutralizer who spontaneously cleared multiple HCV infections. We isolated 10,680 E2-reactive B cells, performed BCR sequencing, characterized monoclonal B cell cultures, and isolated bNAbs. In contrast to what has been seen in chronically infected donors, the bNAbs used a variety of VH genes and targeted at least three distinct E2 antigenic sites, including sites previously thought to be non-neutralizing. Diverse front-layer-reactive bNAb lineages evolved convergently, acquiring breadth-enhancing somatic mutations. These findings demonstrate that HCV clearance-associated bNAbs are genetically diverse and bind distinct antigenic sites that should be the target of vaccine-induced bNAbs.

Neutralizing antibodies evolve to exploit vulnerable sites in the HCV envelope glycoprotein E2 and mediate spontaneous clearance of infection.

Individuals who clear primary hepatitis C virus (HCV) infections clear subsequent reinfections more than 80% of the time, but the mechanisms are poorly defined. Here, we used HCV variants and plasma from individuals with repeated clearance to characterize longitudinal changes in envelope glycoprotein E2 sequences, function, and neutralizing antibody (NAb) resistance. Clearance of infection was associated with early selection of viruses with NAb resistance substitutions that also reduced E2 binding to CD81, the primary HCV receptor. Later, peri-clearance plasma samples regained neutralizing capacity against these variants. We identified a subset of broadly NAbs (bNAbs) for which these loss-of-fitness substitutions conferred resistance to unmutated bNAb ancestors but increased sensitivity to mature bNAbs. These data demonstrate a mechanism by which neutralizing antibodies contribute to repeated immune-mediated HCV clearance, identifying specific bNAbs that exploit fundamental vulnerabilities in E2. The induction of bNAbs with these specificities should be a goal of HCV vaccine development.

Corrigendum: Convergent antibody responses are associated with broad neutralization of hepatitis C virus.

[This corrects the article DOI: 10.3389/fimmu.2023.1135841.].

Convergent antibody responses are associated with broad neutralization of hepatitis C virus.

Introduction: Early development of broadly neutralizing antibodies (bNAbs) targeting the hepatitis C virus (HCV) envelope glycoprotein E2 is associated with spontaneous clearance of infection, so induction of bNAbs is a major goal of HCV vaccine development. However, the molecular antibody features important for broad neutralization are not known. Methods: To identify B cell repertoire features associated with broad neutralization, we performed RNA sequencing of the B cell receptors (BCRs) of HCV E2-reactive B cells of HCV-infected individuals with either high or low plasma neutralizing breadth. We then produced a monoclonal antibody (mAb) expressed by pairing the most abundant heavy and light chains from public clonotypes identified among clearance, high neutralization subjects. Results: We found distinctive BCR features associated with broad neutralization of HCV, including long heavy chain complementarity determining region 3 (CDRH3) regions, specific VH gene usage, increased frequencies of somatic hypermutation, and particular VH gene mutations. Most intriguing, we identified many E2-reactive public BCR clonotypes (heavy and light chain clones with the same V and J-genes and identical CDR3 sequences) present only in subjects who produced highly neutralizing plasma. The majority of these public clonotypes were shared by two subjects who cleared infection. A mAb expressing the most abundant public heavy and light chains from these clearance, high neutralization subjects had features enriched in high neutralization clonotypes, such as increased somatic hypermutation frequency and usage of IGHV1-69, and was cross-neutralizing. Discussion: Together, these results demonstrate distinct BCR repertoires associated with high plasma neutralizing capacity. Further characterization of the molecular features and function of these antibodies can inform HCV vaccine development.

Functional HIV-1/HCV cross-reactive antibodies isolated from a chronically co-infected donor.

Despite prolific efforts to characterize the antibody response to human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) mono-infections, the response to chronic co-infection with these two ever-evolving viruses is poorly understood. Here, we investigate the antibody repertoire of a chronically HIV-1/HCV co-infected individual using linking B cell receptor to antigen specificity through sequencing (LIBRA-seq). We identify five HIV-1/HCV cross-reactive antibodies demonstrating binding and functional cross-reactivity between HIV-1 and HCV envelope glycoproteins. All five antibodies show exceptional HCV neutralization breadth and effector functions against both HIV-1 and HCV. One antibody, mAb688, also cross-reacts with influenza and coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We examine the development of these antibodies using next-generation sequencing analysis and lineage tracing and find that somatic hypermutation established and enhanced this reactivity. These antibodies provide a potential future direction for therapeutic and vaccine development against current and emerging infectious diseases. More broadly, chronic co-infection represents a complex immunological challenge that can provide insights into the fundamental rules that underly antibody-antigen specificity.

Serum neutralization activity declines but memory B cells persist after cure of chronic hepatitis C.

The increasing incidence of hepatitis C virus (HCV) infections underscores the need for an effective vaccine. Successful vaccines to other viruses generally depend on a long-lasting humoral response. However, data on the half-life of HCV-specific responses are lacking. Here we study archived sera and mononuclear cells that were prospectively collected up to 18 years after cure of chronic HCV infection to determine the role of HCV antigen in maintaining neutralizing antibody and B cell responses. We show that HCV-neutralizing activity decreases rapidly in potency and breadth after curative treatment. In contrast, HCV-specific memory B cells persist, and display a restored resting phenotype, normalized chemokine receptor expression and preserved ability to differentiate into antibody-secreting cells. The short half-life of HCV-neutralizing activity is consistent with a lack of long-lived plasma cells. The persistence of HCV-specific memory B cells and the reduced inflammation after cure provide an opportunity for vaccination to induce protective immunity against re-infection.

Repeated exposure to heterologous hepatitis C viruses associates with enhanced neutralizing antibody breadth and potency.

A prophylactic hepatitis C virus (HCV) vaccine that elicits neutralizing antibodies could be key to HCV eradication. However, the genetic and antigenic properties of HCV envelope (E1E2) proteins capable of inducing anti-HCV broadly neutralizing antibodies (bNAbs) in humans have not been defined. Here, we investigated the development of bNAbs in longitudinal plasma of HCV-infected persons with persistent infection or spontaneous clearance of multiple reinfections. By measuring plasma antibody neutralization of a heterologous virus panel, we found that the breadth and potency of the antibody response increased upon exposure to multiple genetically distinct infections and with longer duration of viremia. Greater genetic divergence between infecting strains was not associated with enhanced neutralizing breadth. Rather, repeated exposure to antigenically related, antibody-sensitive E1E2s was associated with potent bNAb induction. These data reveal that a prime-boost vaccine strategy with genetically distinct, antibody-sensitive viruses is a promising approach to inducing potent bNAbs in humans.

SARS-CoV-2-specific immune responses in boosted vaccine recipients with breakthrough infections during the Omicron variant surge.

BackgroundBreakthrough SARS-CoV-2 infections in vaccinated individuals have been previously associated with suboptimal humoral immunity. However, less is known about breakthrough infections with the Omicron variant.MethodsWe analyzed SARS-CoV-2-specific antibody and cellular responses in healthy vaccine recipients who experienced breakthrough infections a median of 50 days after receiving a booster mRNA vaccine with an ACE2 binding inhibition assay and an ELISpot assay, respectively.ResultsWe found that high levels of antibodies inhibited vaccine strain spike protein binding to ACE2 but that lower levels inhibited Omicron variant spike protein binding to ACE2 in 4 boosted vaccine recipients prior to infection. The levels of antibodies that inhibited vaccine strain and Omicron spike protein binding after breakthrough in 18 boosted vaccine recipients were similar to levels seen in COVID-19-negative boosted vaccine recipients. In contrast, boosted vaccine recipients had significantly stronger T cell responses to both vaccine strain and Omicron variant spike proteins at the time of breakthrough.ConclusionOur data suggest that breakthrough infections with the Omicron variant can occur despite robust immune responses to the vaccine strain spike protein.FundingThis work was supported by the Johns Hopkins COVID-19 Vaccine-related Research Fund and by funds from the National Institute of Allergy and Infectious Disease intramural program as well as awards from the National Cancer Institute (U54CA260491) and the National Institutes of Allergy and Infectious Disease (K08AI156021 and U01AI138897).

An Antigenically Diverse, Representative Panel of Envelope Glycoproteins for Hepatitis C Virus Vaccine Development.

BACKGROUND & AIMS: Development of a prophylactic hepatitis C virus (HCV) vaccine will require accurate and reproducible measurement of neutralizing breadth of vaccine-induced antibodies. Currently available HCV panels may not adequately represent the genetic and antigenic diversity of circulating HCV strains, and the lack of standardization of these panels makes it difficult to compare neutralization results obtained in different studies. Here, we describe the selection and validation of a genetically and antigenically diverse reference panel of 15 HCV pseudoparticles (HCVpps) for neutralization assays. METHODS: We chose 75 envelope (E1E2) clones to maximize representation of natural polymorphisms observed in circulating HCV isolates, and 65 of these clones generated functional HCVpps. Neutralization sensitivity of these HCVpps varied widely. HCVpps clustered into 15 distinct groups based on patterns of relative sensitivity to 7 broadly neutralizing monoclonal antibodies. We used these data to select a final panel of 15 antigenically representative HCVpps. RESULTS: Both the 65 and 15 HCVpp panels span 4 tiers of neutralization sensitivity, and neutralizing breadth measurements for 7 broadly neutralizing monoclonal antibodies were nearly equivalent using either panel. Differences in neutralization sensitivity between HCVpps were independent of genetic distances between E1E2 clones. CONCLUSIONS: Neutralizing breadth of HCV antibodies should be defined using viruses spanning multiple tiers of neutralization sensitivity rather than panels selected solely for genetic diversity. We propose that this multitier reference panel could be adopted as a standard for the measurement of neutralizing antibody potency and breadth, facilitating meaningful comparisons of neutralization results from vaccine studies in different laboratories.

Mechanisms of HCV resistance to broadly neutralizing antibodies.

Broadly neutralizing antibodies (bNAbs) block infection by genetically diverse hepatitis C virus (HCV) isolates by targeting relatively conserved epitopes on the HCV envelope glycoproteins, E1 and E2. Many amino acid substitutions conferring resistance to these bNAbs have been characterized, identifying multiple mechanisms of bNAb escape. Some resistance substitutions follow the expected mechanism of directly disrupting targeted epitopes. Interestingly, other resistance substitutions fall in E2 domains distant from bNAb-targeted epitopes. These substitutions, which can confer broad resistance to multiple bNAbs, act by less clearly defined mechanisms. Some modulate binding of HCV to cell surface receptors, while others may induce conformational changes in the E2 protein. In this review, we discuss mechanisms of HCV bNAb resistance and implications for HCV vaccine development.