Where to Next? Research Directions after the First Hepatitis C Vaccine Efficacy Trial.

Thirty years after its discovery, the hepatitis C virus (HCV) remains a leading cause of liver disease worldwide. Given that many countries continue to experience high rates of transmission despite the availability of potent antiviral therapies, an effective vaccine is seen as critical for the elimination of HCV. The recent failure of the first vaccine efficacy trial for the prevention of chronic HCV confirmed suspicions that this virus will be a challenging vaccine target. Here, we examine the published data from this first efficacy trial along with the earlier clinical and pre-clinical studies of the vaccine candidate and then discuss three key research directions expected to be important in ongoing and future HCV vaccine development. These include the following: 1. design of novel immunogens that generate immune responses to genetically diverse HCV genotypes and subtypes, 2. strategies to elicit broadly neutralizing antibodies against envelope glycoproteins in addition to cytotoxic and helper T cell responses, and 3. consideration of the unique immunological status of individuals most at risk for HCV infection, including those who inject drugs, in vaccine platform development and early immunogenicity trials.

Hepatitis C Virus Vaccine Research: Time to Put Up or Shut Up.

Unless urgently needed to prevent a pandemic, the development of a viral vaccine should follow a rigorous scientific approach. Each vaccine candidate should be designed considering the in-depth knowledge of protective immunity, followed by preclinical studies to assess immunogenicity and safety, and lastly, the evaluation of selected vaccines in human clinical trials. The recently concluded first phase II clinical trial of a human hepatitis C virus (HCV) vaccine followed this approach. Still, despite promising preclinical results, it failed to protect against chronic infection, raising grave concerns about our understanding of protective immunity. This setback, combined with the lack of HCV animal models and availability of new highly effective antivirals, has fueled ongoing discussions of using a controlled human infection model (CHIM) to test new HCV vaccine candidates. Before taking on such an approach, however, we must carefully weigh all the ethical and health consequences of human infection in the absence of a complete understanding of HCV immunity and pathogenesis. We know that there are significant gaps in our knowledge of adaptive immunity necessary to prevent chronic HCV infection. This review discusses our current understanding of HCV immunity and the critical gaps that should be filled before embarking upon new HCV vaccine trials. We discuss the importance of T cells, neutralizing antibodies, and HCV genetic diversity. We address if and how the animal HCV-like viruses can be used for conceptualizing effective HCV vaccines and what we have learned so far from these HCV surrogates. Finally, we propose a logical but narrow path forward for HCV vaccine development.

A study based on four immunoassays: Hepatitis C virus antibody against different antigens may have unequal contributions to detection.

BACKGROUND: All commercial Hepatitis C virus antibody (anti-HCV) assays use a combination of recombinant antigens to detect antibody response. Antibody responses to individual antigenic regions (core, NS3/4 and NS5) used in assays have not been investigated. METHODS: In this study, we quantified HCV viral load, tested anti-HCV with four commercial assays (Ortho-ELISA, Murex-ELISA, Architect-CMIA and Elecsys-ECLIA) in 682 plasma specimens. In antigenic region ELISA platform, microwells were coated with three antigens: core (c22-3), NS3/4 (c200) and NS5 individually. The signal-to-cutoff (S/Co) values of different assays, and antibody responses to individual antigens were compared. The specimens were divided into HCV RNA positive group, anti-HCV consistent group, and anti-HCV discrepant group. RESULTS: Anti-core and anti-NS3/4 were simultaneously detected in 99.2% of HCV RNA positive specimens and showed great consistency with total anti-HCV signals. Responses to the core region were more robust than those to the NS3/4 region in anti-HCV consistent group (p < 0.001). Anti-NS5 only occurred in companying with responses to the core and NS3/4 antigens, and failed to affect the final anti-HCV positive signals. In anti-HCV discrepant group, 39.0% of positive signals could not be traced back to any single antigenic region. CONCLUSION: Antibody responses to the core and NS3/4 antigens were stronger, whereas responses to the NS5 antigen were the weakest, indicating that individual antigenic regions played different roles in total anti-HCV signals. This study provides an impetus for optimizing commercial anti-HCV assays.

From Structural Studies to HCV Vaccine Design.

Hepatitis C virus (HCV) is a serious and growing public health problem despite recent developments of antiviral therapeutics. To achieve global elimination of HCV, an effective cross-genotype vaccine is needed. The failure of previous vaccination trials to elicit an effective cross-reactive immune response demands better vaccine antigens to induce a potent cross-neutralizing response to improve vaccine efficacy. HCV E1 and E2 envelope (Env) glycoproteins are the main targets for neutralizing antibodies (nAbs), which aid in HCV clearance and protection. Therefore, a molecular-level understanding of the nAb responses against HCV is imperative for the rational design of cross-genotype vaccine antigens. Here we summarize the recent advances in structural studies of HCV Env and Env-nAb complexes and how they improve our understanding of immune recognition of HCV. We review the structural data defining HCV neutralization epitopes and conformational plasticity of the Env proteins, and the knowledge applicable to rational vaccine design.

Structure-Based and Rational Design of a Hepatitis C Virus Vaccine.

A hepatitis C virus (HCV) vaccine is a critical yet unfulfilled step in addressing the global disease burden of HCV. While decades of research have led to numerous clinical and pre-clinical vaccine candidates, these efforts have been hindered by factors including HCV antigenic variability and immune evasion. Structure-based and rational vaccine design approaches have capitalized on insights regarding the immune response to HCV and the structures of antibody-bound envelope glycoproteins. Despite successes with other viruses, designing an immunogen based on HCV glycoproteins that can elicit broadly protective immunity against HCV infection is an ongoing challenge. Here, we describe HCV vaccine design approaches where immunogens were selected and optimized through analysis of available structures, identification of conserved epitopes targeted by neutralizing antibodies, or both. Several designs have elicited immune responses against HCV in vivo, revealing correlates of HCV antigen immunogenicity and breadth of induced responses. Recent studies have elucidated the functional, dynamic and immunological features of key regions of the viral envelope glycoproteins, which can inform next-generation immunogen design efforts. These insights and design strategies represent promising pathways to HCV vaccine development, which can be further informed by successful immunogen designs generated for other viruses.

Clinical utility of hepatitis C virus core antigen (HCVcAg) assay to identify active HCV infection in hemodialysis and renal transplant patients.

BACKGROUND: The prevalence of HCV infection is high and it is a major cause of liver-related morbidity and mortality in hemodialysis and renal transplant patients. Diagnosis of hepatitis C virus (HCV) infection requires both HCV antibody screening and confirmatory nucleic acid testing (NAT). Hepatitis C virus core antigen (HCVcAg) is a reliable direct viral marker to identify active HCV infection. AIM: To assess the clinical utility of HCV core antigen to identify active HCV infection in hemodialysis and renal transplant patients. METHODS: A representative total of 231 plasma samples with a predominance of low viral load were included for HCVcAg testing and its performance characteristics were compared with the gold standard HCV RNA. RESULTS: Comparison of HCVcAg with HCV RNA showed an excellent specificity of 99% (95% CI: 94.7 to 100%) and sensitivity of 80.62% (95% CI: 73.59 to 87.7%). Likewise, the PPV and NPV of HCVcAg were 99.1% (95% CI: 93.7% to 99.9%) and 80.2% (95% CI: 74% to 85.2%) respectively. The correlation between HCVcAg and HCV RNA was found to be good (R2 = 0.86, p<0.0001). Among common Indian HCV genotypes (1, 3 & 4), good correlation was observed between HCV RNA and HCVcAg (R2 = 0.81, p <0.0001). CONCLUSIONS: It is the first Indian study to show that HCVcAg is a reliable, cost-effective direct marker to identify active HCV infection in hemodialysis and renal transplant patients. Implementation of HCVcAg testing could improve the accessibility to efficacious and affordable disease management in hemodialysis and renal transplant patients. In HCVcAg negative cases, sequential testing with anti-HCV antibody followed by HCV RNA could be a reliable and cost-effective approach.

In Vivo and In Vitro Potency of Polyphosphazene Immunoadjuvants with Hepatitis C Virus Antigen and the Role of Their Supramolecular Assembly.

Two well-defined synthetic polyphosphazene immunoadjuvants, PCPP and PCEP, were studied for their ability to potentiate the immune response to the hepatitis C virus (HCV) E2 glycoprotein antigen in vivo. We report that PCEP induced significantly higher serum neutralization and HCV-specific IgG titers in mice compared to other adjuvants used in the study: PCPP, Alum, and Addavax. PCEP also shifted the response toward the desirable balanced Th1/Th2 immunity, as evaluated by the antibody isotype ratio (IgG2a/IgG1). The in vivo results were analyzed in the context of antigen-adjuvant molecular interactions in the system and in vitro immunostimulatory activity of formulations. Asymmetric flow field flow fractionation (AF4) and dynamic light scattering (DLS) analysis showed that both PCPP and PCEP spontaneously self-assemble with the E2 glycoprotein with the formation of multimeric water-soluble complexes, which demonstrates the role of polyphosphazene macromolecules as vaccine delivery vehicles. Intrinsic in vitro immunostimulatory activity of polyphosphazene adjuvants, which was assessed using a mouse macrophage cell line, revealed comparable activities of both polymers and did not provide an explanation of their in vivo performance. However, PCEP complexes with E2 displayed greater stability against agglomeration and improved in vitro immunostimulatory activity compared to those of PCPP, which is in line with superior in vivo performance of PCEP. The results emphasize the importance of often neglected antigen-polyphosphazene self-assembly mechanisms in formulations, which can provide important insights on their in vivo behavior and facilitate the establishment of a structure-activity relationship for this important class of immunoadjuvants.

Antibody Responses in Hepatitis C Infection.

Antibody responses in hepatitis C virus (HCV) have been a rather mysterious research topic for many investigators working in the field. Chronic HCV infection is often associated with dysregulation of immune functions particularly in B cells, leading to abnormal lymphoproliferation or the production of autoantibodies that exacerbate inflammation and extrahepatic diseases. When considering the antiviral function of antibody, it was difficult to endorse its role in HCV protection, whereas T-cell response has been shown unequivocally critical for natural recovery. Recent breakthroughs in the study of HCV and antigen-specific antibody responses provide important insights into viral vulnerability to antibodies and the immunogenetic and structural properties of the neutralizing antibodies. The new knowledge reinvigorates HCV vaccine research by illuminating a new path for the rational design of vaccine antigens to elicit broadly neutralizing antibodies for protection.

Hepatitis C virus antigen detection is an appropriate test for screening and early diagnosis of hepatitis C virus infection in at-risk populations and immunocompromised hosts.

Early diagnosis remains key for effective prevention and treatment. Unfortunately, current screening with anti-hepatitis C virus antibody (anti-HCV Ab) test may have limited utility in the diagnosis of HCV infection and reinfection. This is of special concern to at-risk population, such as immunocompromised hosts and end-stage renal failure patients on hemodialysis. HCV antigen (Ag) could be useful in identifying the ongoing infection in such clinical scenarios. Hence, we aimed to study the utility of HCV Ag testing for the diagnosis of acute and chronic hepatitis C. Of 89 samples studied, 19 were from acute hepatitis C patients who were immunocompromised or were on hemodialysis, 43 were from active chronic hepatitis C patients and 27 were from patients treated for chronic hepatitis C. All samples were tested for HCV Ag using the Abbott ARCHITECT HCV Ag assay. HCV Ag was reactive in 19/19 samples from acute hepatitis C patients and 42/43 samples from active chronic hepatitis C patients. It was nonreactive in all samples from treated patients. The test showed a sensitivity and specificity of 98.4% and 100.0%, respectively. The positive and negative predictive values were 100.0% and 96.4%, respectively. The HCV antigen test has high clinical sensitivity and specificity and is useful for the diagnosis of acute and chronic hepatitis C infection in at-risk and immunocompromised patients. Its short turnaround time and relatively low cost are advantageous for use in patients on hemodialysis and other at-risk patients who require monitoring of HCV infection and reinfection.

Optimized cell systems for the investigation of hepatitis C virus E1E2 glycoproteins.

Great strides have been made in understanding and treating hepatitis C virus (HCV) thanks to the development of various experimental systems including cell-culture-proficient HCV, the HCV pseudoparticle system and soluble envelope glycoproteins. The HCV pseudoparticle (HCVpp) system is a platform used extensively in studies of cell entry, screening of novel entry inhibitors, assessing the phenotypes of clinically observed E1 and E2 glycoproteins and, most pertinently, in characterizing neutralizing antibody breadth induced upon vaccination and natural infection in patients. Nonetheless, some patient-derived clones produce pseudoparticles that are either non-infectious or exhibit infectivity too low for meaningful phenotyping. The mechanisms governing whether any particular clone produces infectious pseudoparticles are poorly understood. Here we show that endogenous expression of CD81, an HCV receptor and a cognate-binding partner of E2, in producer HEK 293T cells is detrimental to the infectivity of recovered HCVpp for most strains. Many HCVpp clones exhibited increased infectivity or had their infectivity rescued when they were produced in 293T cells CRISPR/Cas9 engineered to ablate CD81 expression (293TCD81KO). Clones made in 293TCD81KO cells were antigenically very similar to their matched counterparts made parental cells and appear to honour the accepted HCV entry pathway. Deletion of CD81 did not appreciably increase the recovered titres of soluble E2 (sE2). However, we did, unexpectedly, find that monomeric sE2 made in 293T cells and Freestyle 293-F (293-F) cells exhibit important differences. We found that 293-F-produced sE2 harbours mostly complex-type glycans whilst 293T-produced sE2 displays a heterogeneous mixture of both complex-type glycans and high-mannose or hybrid-type glycans. Moreover, sE2 produced in 293T cells is antigenically superior; exhibiting increased binding to conformational antibodies and the large extracellular loop of CD81. In summary, this work describes an optimal cell line for the production of HCVpp and reveals that sE2 made in 293T and 293-F cells are not antigenic equals. Our findings have implications for functional studies of E1E2 and the production of candidate immunogens.

Increased early acute cellular rejection events in hepatitis C-positive heart transplantation.

BACKGROUND: Increased utilization of hepatitis C virus (HCV)-positive donors has increased transplantation rates. However, high levels of viremia have been documented in recipients of viremic donors. There is a knowledge gap in how transient viremia may impact acute cellular rejections (ACRs). METHODS: In this study, 50 subjects received hearts from either viremic or non-viremic donors. The recipients of viremic donors were classified as nucleic acid amplification testing (NAT)+ group, and the remaining were classified as NAT-. All patients were monitored for viremia levels. Endomyocardial biopsies were performed through 180 days, evaluating the incidence of ACRs. RESULTS: A total of 50 HCV-naive recipients received hearts between 2018 and 2019. A total of 22 patients (44%) who received transplants from viremic donors developed viremia at a mean period of 7.2 ± 0.2 days. At that time, glecaprevir/pibrentasvir was initiated. In the viremia period (<56 days), 14 of 22 NAT+ recipients (64%) had ACR vs 5 of 28 NAT- group (18%) (p = 0.001). Through 180 days, 17 of 22 NAT+ recipients (77%) had a repeat rejection biopsy vs 12 of 28 NAT- recipients (43%) (p = 0.02). NAT+ biopsies demonstrated disparity of ACR distribution: negative, low-grade, and high-grade ACR in 84%, 12%, and 4%, respectively, vs 96%, 3%, and 1%, respectively, in the NAT- group (p = 0.03). The median time to first event was 26 (interquartile range [IQR]: 8-45) in the NAT+ group vs 65 (IQR: 44-84) days in the NAT-. Time to first event risk model revealed that NAT+ recipients had a significantly higher rate of ACR occurrences, adjusting for demographics (p = 0.004). CONCLUSIONS: Transient levels of viremia contributed to higher rates and severity of ACRs. Further investigation into the mechanisms of early immune activation in NAT+ recipients is required.

Protective cellular immune response against hepatitis C virus elicited by chimeric protein formulations in BALB/c mice.

The eradication of hepatitis C virus (HCV) infection is a public health priority. Despite the efficiency of treatment with direct-acting antivirals, the high cost of the therapy and the lack of accurate data about the HCV-infected population worldwide constitute important factors hampering this task. Hence, an affordable preventive vaccine is still necessary for reducing transmission and the future disease burden globally. In this work, chimeric proteins (EnvCNS3 and NS3EnvCo) encompassing conserved and immunogenic epitopes from the HCV core, E1, E2 and NS3 proteins were produced in Escherichia coli, and their immunogenicity was evaluated in BALB/c mice. The impact of recombinant HCV E2.680 protein and oligodeoxynucleotide 39M (ODN39M) on the immune response to chimeric proteins was also assessed. Immunization with chimeric proteins mixed with E2.680 enhanced the antibody and cellular response against HCV antigens and chimeric proteins. Interestingly, the combination of NS3EnvCo with E2.680 and ODN39M as adjuvant elicited a potent antibody response characterized by an increase in antibodies of the IgG2a subclass against E2.680, NS3 and chimeric proteins, suggesting the induction of a Th1-type response. Moreover, a cytotoxic T lymphocyte response and a broad response of IFN-γ-secreting cells against HCV antigens were induced with this formulation as well. This T cell response was able to protect vaccinated mice against challenge with a surrogate model based on HCV recombinant vaccinia virus. Overall, the vaccine candidate NS3EnvCo/E2.680/ODN39M might constitute an effective immunogen against HCV with potential for reducing the likelihood of viral persistence.

Domain-Scan: Combinatorial Sero-Diagnosis of Infectious Diseases Using Machine Learning.

The presence of pathogen-specific antibodies in an individual's blood-sample is used as an indication of previous exposure and infection to that specific pathogen (e.g., virus or bacterium). Measurement of the diagnostic antibodies is routinely achieved using solid phase immuno-assays such as ELISA tests and western blots. Here, we describe a sero-diagnostic approach based on phage-display of epitope arrays we term "Domain-Scan". We harness Next-generation sequencing (NGS) to measure the serum binding to dozens of epitopes derived from HIV-1 and HCV simultaneously. The distinction of healthy individuals from those infected with either HIV-1 or HCV, is modeled as a machine-learning classification problem, in which each determinant ("domain") is considered as a feature, and its NGS read-out provides values that correspond to the level of determinant-specific antibodies in the sample. We show that following training of a machine-learning model on labeled examples, we can very accurately classify unlabeled samples and pinpoint the domains that contribute most to the classification. Our experimental/computational Domain-Scan approach is general and can be adapted to other pathogens as long as sufficient training samples are provided.

Antibody Responses to Immunization With HCV Envelope Glycoproteins as a Baseline for B-Cell-Based Vaccine Development.

BACKGROUND & AIMS: We investigated antibody responses to hepatitis C virus (HCV) antigens E1 and E2 and the relevance of animal models for vaccine development. We compared antibody responses to vaccination with recombinant E1E2 complex in healthy volunteers, non-human primates (NHPs), and mice. METHODS: We analyzed 519 serum samples from participants in a phase 1 vaccine trial (ClinicalTrials.gov identifier NCT00500747) and compared them with serum or plasma samples from C57BL/6J mice (n = 28) and rhesus macaques (n = 4) immunized with the same HCV E1E2 antigen. Blood samples were collected at different time points and analyzed for antibody binding, neutralizing activity, and epitope specificity. Monoclonal antibodies from the immunized NHPs were isolated from single plasmablasts and memory B cells, and their immunogenetic properties were characterized. RESULTS: Antibody responses of the volunteers, NHPs, and mice to the non-neutralizing epitopes on the E1 N-terminus and E2 hypervariable region 1 did not differ significantly. Antibodies from volunteers and NHPs that neutralized heterologous strains of HCV primarily interacted with epitopes in the antigen region 3. However, the neutralizing antibodies were not produced in sufficient levels for broad neutralization of diverse HCV isolates. Broadly neutralizing antibodies similar to the human VH1-69 class antibody specific for antigen region 3 were produced in the immunized NHPs. CONCLUSIONS: In an analysis of vaccinated volunteers, NHPs, and mice, we found that recombinant E1E2 vaccine antigen induces high-antibody titers that are insufficient to neutralize diverse HCV isolates. Antibodies from volunteers and NHPs bind to the same neutralizing epitopes for virus neutralization. NHPs can therefore be used as a preclinical model to develop HCV vaccines. These findings also provide useful baseline values for development of vaccines designed to induce production of neutralizing antibodies.

Progress and challenges of a pioneering hepatitis C elimination program in the country of Georgia.

BACKGROUND & AIMS: Georgia, with a high prevalence of HCV infection, launched the world's first national hepatitis C elimination program in April 2015. A key strategy is the identification, treatment, and cure of the estimated 150,000 HCV-infected people living in the country. We report on progress and key challenges from Georgia's experience. METHODS: We constructed a care cascade by analyzing linked data from the national hepatitis C screening registry and treatment databases during 2015-2018. We assessed the impact of reflex hepatitis C core antigen (HCVcAg) testing on rates of viremia testing and treatment initiation (i.e. linkage to care). RESULTS: As of December 31, 2018, 1,101,530 adults (39.6% of the adult population) were screened for HCV antibody, of whom 98,430 (8.9%) tested positive. Of the individuals who tested positive, 78,484 (79.7%) received viremia testing, of whom 66,916 (85.3%) tested positive for active HCV infection. A total of 52,576 people with active HCV infection initiated treatment and 48,879 completed their course of treatment. Of the 35,035 who were tested for cure (i.e., sustained virologic response [SVR]), 34,513 (98.5%) achieved SVR. Reflex HCVcAg testing, implemented in March 2018, increased rates of monthly viremia testing by 97.5% among those who screened positive for anti-HCV, however, rates of treatment initiation decreased by 60.7% among diagnosed viremic patients. CONCLUSIONS: Over one-third of people living with HCV in Georgia have been detected and linked to care and treatment, however, identification and linkage to care of the remaining individuals with HCV infection is challenging. Novel interventions, such as reflex testing with HCVcAg, can improve rates of viremia testing, but may result in unintended consequences, such as decreased rates of treatment initiation. Linked data systems allow for regular review of the care cascade, allowing for identification of deficiencies and development of corrective actions. LAY SUMMARY: This report describes progress in Georgia's hepatitis C elimination program and highlights efforts to promote hepatitis C virus screening and treatment initiation on a national scale. Georgia has made progress towards eliminating hepatitis C, treating over 50,000 people, approximately one-third of the number infected, and achieving cure for 98.5% of those tested. However, identifying infected individuals and linking them to care remains challenging. Novel approaches to increase diagnostic testing can have unintended consequences further down the care cascade.

Hepatitis C Core-Antigen Testing from Dried Blood Spots.

In order to expand hepatitis C virus (HCV) screening, a change in the diagnostic paradigm is warranted to improve accessibility and decrease costs, such as utilizing dried blood spot (DBS) collection. In our study, blood from 68 patients with chronic HCV infection was spotted onto DBS cards and stored at the following temperatures for one week: -80 °C, 4 °C, 21 °C, 37 °C, and alternating 37 °C and 4 °C; to assess whether temperature change during transportation would affect sensitivity. Sample was eluted from the DBS cards and tested for HCV antibodies (HCV-Ab) and HCV core antigen (core-Ag). HCV-Abs were detected from 68/68 DBS samples at -80 °C, 4 °C, 21 °C, and 67/68 at 37 °C and alternating 37 °C and 4 °C. Sensitivity of core-Ag was as follows: 94% (-80 °C), 94% (4 °C), 91% (21 °C), 93% (37 °C), and 93% (37 °C/4 °C). Not only did temperature not greatly affect sensitivity, but sensitivities are higher than previously reported, and support the use of this assay as an alternative to HCV RNA. We then completed a head-to-head comparison (n = 49) of venous versus capillary samples, and one versus two DBS. No difference in core-Ag sensitivity was observed by sample type, but there was an improvement when using two spots. We conclude that HCV-Abs and core-Ag testing from DBS cards has high diagnostic accuracy and could be considered as an alternative to HCV RNA in certain settings.

Evaluation of a hepatitis C virus core antigen assay from venepuncture and dried blood spot collected samples: A cohort study.

The global scale-up of hepatitis C virus (HCV) diagnosis requires simplified and affordable HCV diagnostic pathways. This study evaluated the sensitivity and specificity of the HCV Architect core antigen (HCVcAg) assay for detection of active HCV infection in plasma and capillary whole blood dried blood spots (DBS) compared with HCV RNA testing in plasma (Abbott RealTime HCV Viral Load). Samples were collected from participants in an observational cohort enrolled at three sites in Australia (two-drug treatment and alcohol clinics and one homelessness service). Of 205 participants, 200 had results across all samples and assay types and 186 were included in this analysis (14 participants receiving HCV therapy were excluded). HCV RNA was detected in 29% of participants ([95% CI: 22.6-36.1], 54 of 186). The sensitivity of HCVcAg for detection of active HCV infection in plasma was 98.1% (95% CI: 90-100) and 100% (95% CI: 93-100) when compared to HCV RNA thresholds of ≥12 and ≥1000 IU/mL, respectively. The sensitivity of the HCVcAg assay for detection of active HCV infection in DBS was 90.7% (95% CI: 80-97) and 92.5% (95% CI: 82-98) when compared to HCV RNA thresholds of ≥12 and ≥1000 IU/mL, respectively. The specificity of HCV core antigen for detection of active infection was 100% (95% CI: 97-100) for all samples and RNA thresholds. These data indicate that the detection of HCVcAg is a useful tool for determining active HCV infection; to facilitate enhanced testing, linkage to care and treatment particularly when testing plasma samples are collected by venepuncture.

A Recombinant Hepatitis C Virus Genotype 1a E1/E2 Envelope Glycoprotein Vaccine Elicits Antibodies That Differentially Neutralize Closely Related 2a Strains through Interactions of the N-Terminal Hypervariable Region 1 of E2 with Scavenger Receptor B1.

The global health burden for hepatitis C virus (HCV) remains high, despite available effective treatments. To eliminate HCV, a prophylactic vaccine is needed. One major challenge in the development of a vaccine is the genetic diversity of the virus, with 7 major genotypes and many subtypes. A global vaccine must be effective against all HCV genotypes. Our previous data showed that the 1a E1/E2 glycoprotein vaccine component elicits broad cross-neutralizing antibodies in humans and animals. However, some variation is seen in the effectiveness of these antibodies to neutralize different HCV genotypes and isolates. Of interest was the differences in neutralizing activity against two closely related isolates of HCV genotype 2a, the J6 and JFH-1 strains. Using site-directed mutagenesis to generate chimeric viruses between the J6 and JFH-1 strains, we found that variant amino acids within the core E2 glycoprotein domain of these two HCV genotype 2a viruses do not influence isolate-specific neutralization. Further analysis revealed that the N-terminal hypervariable region 1 (HVR1) of the E2 protein determines the sensitivity of isolate-specific neutralization, and the HVR1 of the resistant J6 strain binds scavenger receptor class-B type-1 (SR-B1), while the sensitive JFH-1 strain does not. Our data provide new information on mechanisms of isolate-specific neutralization to facilitate the optimization of a much-needed HCV vaccine.IMPORTANCE A vaccine is still urgently needed to overcome the hepatitis C virus (HCV) epidemic. It is estimated that 1.75 million new HCV infections occur each year, many of which will go undiagnosed and untreated. Untreated HCV can lead to continued spread of the disease, progressive liver fibrosis, cirrhosis, and eventually, end-stage liver disease and/or hepatocellular carcinoma (HCC). Previously, our 1a E1/E2 glycoprotein vaccine was shown to elicit broadly cross-neutralizing antibodies; however, there remains variation in the effectiveness of these antibodies against different HCV genotypes. In this study, we investigated determinants of differential neutralization sensitivity between two highly related genotype 2a isolates, J6 and JFH-1. Our data indicate that the HVR1 region determines neutralization sensitivity to vaccine antisera through modulation of sensitivity to antibodies and interactions with SR-B1. Our results provide additional insight into optimizing a broadly neutralizing HCV vaccine.

Broadly Neutralizing Antibodies Targeting New Sites of Vulnerability in Hepatitis C Virus E1E2.

Increasing evidence indicates that broadly neutralizing antibodies (bNAbs) play an important role in immune-mediated control of hepatitis C virus (HCV) infection, but the relative contribution of neutralizing antibodies targeting antigenic sites across the HCV envelope (E1 and E2) proteins is unclear. Here, we isolated thirteen E1E2-specific monoclonal antibodies (MAbs) from B cells of a single HCV-infected individual who cleared one genotype 1a infection and then became persistently infected with a second genotype 1a strain. These MAbs bound six distinct discontinuous antigenic sites on the E1 protein, the E2 protein, or the E1E2 heterodimer. Three antigenic sites, designated AS108, AS112 (an N-terminal E1 site), and AS146, were distinct from previously described antigenic regions (ARs) 1 to 5 and E1 sites. Antibodies targeting four sites (AR3, AR4-5, AS108, and AS146) were broadly neutralizing. These MAbs also displayed distinct patterns of relative neutralizing potency (i.e., neutralization profiles) across a panel of diverse HCV strains, which led to complementary neutralizing breadth when they were tested in combination. Overall, this study demonstrates that HCV bNAb epitopes are not restricted to previously described antigenic sites, expanding the number of sites that could be targeted for vaccine development.IMPORTANCE Worldwide, more than 70 million people are infected with hepatitis C virus (HCV), which is a leading cause of hepatocellular carcinoma and liver transplantation. Despite the development of potent direct acting antivirals (DAAs) for HCV treatment, a vaccine is urgently needed due to the high cost of treatment and the possibility of reinfection after cure. Induction of multiple broadly neutralizing antibodies (bNAbs) that target distinct epitopes on the HCV envelope proteins is one approach to vaccine development. However, antigenic sites targeted by bNAbs in individuals with spontaneous control of HCV have not been fully defined. In this study, we characterize 13 monoclonal antibodies (MAbs) from a single person who cleared an HCV infection without treatment, and we identify 3 new sites targeted by neutralizing antibodies. The sites targeted by these MAbs could inform HCV vaccine development.