Major Hepatitis C Virus Genotypes Research Articles

Update on Hepatitis C Vaccine: Results and Challenges.

Therapy against the Hepatitis C virus (HCV) has significantly improved with the introduction of direct-acting antiviral drugs (DAAs), achieving over 95% sustained virological response (SVR). Despite this, the development of an effective anti-HCV vaccine remains a critical challenge due to the low number of patients treated with DAAs and the occurrence of HCV reinfections in high-risk groups. Current vaccine strategies aim to stimulate either B-cell or T-cell responses. Vaccines based on E1 and E2 proteins can elicit broad cross-neutralizing antibodies against all major HCV genotypes, though with varying efficiencies and without full protection against infection. In humans, the neutralizing antibodies induced by such vaccines mainly target the AR3 region, but their levels are generally insufficient for broad neutralization. Various HCV proteins expressed through different viral vectors have been utilized to elicit T cell immune responses, showing sustained expansion of HCV-specific effector memory T cells and improved proliferation and polyfunctionality of memory T cells over time. However, despite these advancements, the frequency and effectiveness of T-cell responses remain limited.

Biosensor-based multiple cross displacement amplification platform for visual and rapid identification of hepatitis C virus.

Hepatitis C remains a global health problem, especially in poverty-stricken areas. A rapid and sensitive point-of-care (POC) diagnostic tool is critical for the early detection and timely treatment of hepatitis C virus (HCV) infection. Here, for the first time, we reported a novel molecular diagnostic assay, termed reverse transcription multiple cross displacement amplification integrated with a gold-nanoparticle-based lateral flow biosensor (RT-MCDA-AuNPs-LFB), which was developed for rapid, sensitive, specific, and visual identification of HCV. HCV-RT-MCDA induced rapid isothermal amplification through a specific primer set targeting the 5'untranslated region gene from the major HCV genotypes 1b, 2a, 3b, 6a, and 3a that are prevalent in China. The optimal reaction temperature and time for RT-MCDA-AuNPs-LFB were 68°C and 25 min, respectively. The limit of detection of the assay was 10 copies per test, and the specificity was 100% for the experimental strains. The whole detection procedure, including crude nucleic acid isolation (~5 min), RT-MCDA (68°C, 25 min), and visual AuNPs-LFB result confirmation (less than 2 min), was performed within 35 min. The preliminary results indicated that the HCV-RT-MCDA-AuNPs-LFB assay could be a valuable tool for sensitive, specific, visual, cost-saving, and rapid detection of HCV and has potential as a POC diagnostic platform for field screening and early clinical detection of HCV infection.

Transmission networks of hepatitis C virus among HIV/HCV-coinfected patients in Guangdong, China

BackgroundCoinfection with hepatitis C virus (HCV) is common in human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) patients due to shared routes of transmission. We aimed to investigate the characteristics of HCV subgenotypes among HIV/HCV-coinfected patients in Guangdong and explore the molecular transmission networks and related risk factors for HCV strains.MethodsPlasma samples were obtained from 356 HIV/HCV-coinfected patients for HCV NS5B region sequencing. A neighbor-joining phylogenetic tree was constructed to affirm HCV subgenotypes. The transmission networks based on maximum likelihood phylogenetic tree were determined by Cluster Picker, and visualized using Cytoscape 3.2.1.ResultsA total of 302 HCV NS5B sequences were successfully amplified and sequenced from the 356 plasma samples. A neighbor-joining phylogenetic tree based on the 302 NS5B sequences revealed the profile of HCV subgenotypes circulating among HIV/HCV coinfection patients in Guangdong. Two predominant strains were found to be 6a (58.28%, 176/302) and 1b (18.54%, 56/302), followed by 3a (10.93%, 33/302), 3b (6.95%, 21/302), 1a (3.64%, 11/302), 2a (0.99%, 3/302) and 6n (0.66%, 2/302). A molecular transmission network of five major HCV genotypes was constructed, with a clustering rate of 44.04%. The clustering rates of subgenotypes 1a, 3a, 3b, 1b, and 6a were 18.18% (2/11), 42.42%, 52.38%, 48.21%, and 44.89%, respectively. Multivariate logistic regression analysis showed no significant effects from sex, age, transmission route, geographical region, baseline CD4 + T cell count or subgenotype (P > 0.05), except marital status. Married or cohabiting people (compared with unmarried people) had more difficulty forming transmission networks.ConclusionsIn summary, this study, based on HCV NS5B subgenotypes, revealed the HCV subtype diversity and distribution among HIV/HCV-coinfected patients in Guangdong. Marital status inclined to be the factor influencing HCV transmission networks formation.

Impact of direct-acting antiviral regimens on hepatic and extrahepatic manifestations of hepatitis C virus infection

Hepatitis C virus (HCV) is a common cause of liver disease and is associated with various extrahepatic manifestations (EHMs). This mini-review outlines the currently available treatments for HCV infection and their prognostic effect on hepatic manifestations and EHMs. Direct-acting antiviral (DAA) regimens are considered pan-genotypic as they achieve a sustained virological response (SVR) > 85% after 12 wk through all the major HCV genotypes, with high percentages of SVR even in advanced fibrosis and cirrhosis. The risk factors for DAA failure include old males, cirrhosis, and the presence of resistance-associated substitutions (RAS) in the region targeted by the received DAAs. The effectiveness of DAA regimens is reduced in HCV genotype 3 with baseline RAS like A30K, Y93H, and P53del. Moreover, the European Association for the Study of the Liver recommended the identification of baseline RAS for HCV genotype 1a. The higher rate of hepatocellular carcinoma (HCC) after DAA therapy may be related to the fact that DAA regimens are offered to patients with advanced liver fibrosis and cirrhosis, where interferon was contraindicated to those patients. The change in the growth of pre-existing subclinical, undetectable HCC upon DAA treatment might be also a cause. Furthermore, after DAA therapy, the T cell-dependent immune response is much weaker upon HCV clearance, and the down-regulation of TNF-α or the elevated neutrophil to lymphocyte ratio might increase the risk of HCC. DAAs can result in reactivation of hepatitis B virus (HBV) in HCV co-infected patients. DAAs are effective in treating HCV-associated mixed cryoglobulinemia, with clinical and immunological responses, and have rapid and high effectiveness in thrombocytopenia. DAAs improve insulin resistance in 90% of patients, increase glomerular filtration rate, and decrease proteinuria, hematuria and articular manifestations. HCV clearance by DAAs allows a significant improvement in atherosclerosis and metabolic and immunological conditions, with a reduction of major cardiovascular events. They also improve physical function, fatigue, cognitive impairment, and quality of life. Early therapeutic approach with DAAs is recommended as it cure many of the EHMs that are still in a reversible stage and can prevent others that can develop due to delayed treatment.

Retraction Note: Small interfering RNA targeted to stem-loop II of the 5\u2032 untranslated region effectively inhibits expression of six HCV genotypes

The antiviral action of interferon alpha targets the 5' untranslated region (UTR) used by hepatitis C virus (HCV) to translate protein by an internal ribosome entry site (IRES) mechanism. Although this sequence is highly conserved among different clinical strains, approximately half of chronically infected hepatitis C patients do not respond to interferon therapy. Therefore, development of small interfering RNA (siRNA) targeted to the 5'UTR to inhibit IRES mediated translation may represent an alternative approach that could circumvent the problem of interferon resistance. Four different plasmid constructs were prepared for intracellular delivery of siRNAs targeting the stem loop II-III of HCV 5' UTR. The effect of siRNA production on IRES mediated translation was investigated using chimeric clones between the gene for green fluorescence protein (GFP) and IRES sequences of six different HCV genotypes. The siRNA targeted to stem loop II effectively mediated degradation of HCV IRES mRNA and inhibited GFP expression in the case of six different HCV genotypes, where as siRNAs targeted to stem loop III did not. Furthermore, intracytoplasmic expression of siRNA into transfected Huh-7 cells efficiently degraded HCV genomic RNA and inhibited core protein expression from infectious full-length infectious clones HCV 1a and HCV 1b strains. These in vitro studies suggest that siRNA targeted to stem-loop II is highly effective inhibiting IRES mediated translation of the major genotypes of HCV. Stem-loop II siRNA may be a good target for developing an intracellular immunization strategy based antiviral therapy to inhibit hepatitis C virus strains that are not inhibited by interferon.

Hepatitis C Virus Subtypes Novel 6g-Related Subtype and 6w Could Be Indigenous in Southern Taiwan with Characteristic Geographic Distribution.

Hepatitis C virus (HCV) genotype (GT) 6 is the most genetically diverse GT and mainly distributed in Southeast Asia and south China but not Taiwan. Earlier studies showed the major HCV GTs in Taiwan were GT 1b and 2 with very rare GT 6 except in injection drug users (IDUs), and subtype 6a is the main GT 6 subtype among IDUs. Recently, we reported a much higher prevalence (18.3%) of GT 6 in Tainan City, southern Taiwan. This study was designed to clarify the subtypes of GT 6 in this endemic area. A total of 3022 (1343 men and 1679 women) HCV viremic patients were enrolled. Subtypes of GT 6 were determined by sequencing of core/E1 and nonstructural protein 5B in 322 of 518 GT 6 patients. The overall GT 6 prevalence rate was 17.1% (518/3022), with higher prevalence districts (>25%) located in northern Tainan. A novel 6g-related subtype is the most prevalent subtype (81.0%), followed by 6w (10.8%), 6a (7.5%), and 6n (0.7%). The high GT 6 prevalence in Tainan was mainly due to a novel 6g-related subtype and 6w. These two subtypes could be indigenous in Tainan with characteristic geographic distribution.

Insights into the unique characteristics of hepatitis C virus genotype 3 revealed by development of a robust sub-genomic DBN3a replicon.

Hepatitis C virus (HCV) is an important human pathogen causing 400 000 chronic liver disease-related deaths annually. Until recently, the majority of laboratory-based investigations into the biology of HCV have focused on the genotype 2 isolate, JFH-1, involving replicons and infectious cell culture systems. However, genotype 2 is one of eight major genotypes of HCV and there is great sequence variation among these genotypes (>30 % nucleotide divergence). In this regard, genotype 3 is the second most common genotype and accounts for 30 % of global HCV cases. Further, genotype 3 is associated with both high levels of inherent resistance to direct-acting antiviral (DAA) therapy, and a more rapid progression to chronic liver diseases. Neither of these two attributes are fully understood, thus robust genotype 3 culture systems to unravel viral replication are required. Here we describe the generation of robust genotype 3 sub-genomic replicons (SGRs) based on the adapted HCV NS3-NS5B replicase from the DBN3a cell culture infectious clone. Such infectious cell culture-adaptive mutations could potentially promote the development of robust SGRs for other HCV strains and genotypes. The novel genotype 3 SGRs have been used both transiently and to establish stable SGR-harbouring cell lines. We show that these resources can be used to investigate aspects of genotype 3 biology, including NS5A function and DAA resistance. They will be useful tools for these studies, circumventing the need to work under the biosafety level 3 (BSL3) containment required in many countries.

Viral vectored hepatitis C virus vaccines generate pan-genotypic T cell responses to conserved subdominant epitopes

BackgroundViral genetic variability presents a major challenge to the development of a prophylactic hepatitis C virus (HCV) vaccine. A promising HCV vaccine using chimpanzee adenoviral vectors (ChAd) encoding a genotype (gt) 1b non-structural protein (ChAd-Gt1b-NS) generated high magnitude T cell responses. However, these T cells showed reduced cross-recognition of dominant epitope variants and the vaccine has recently been shown to be ineffective at preventing chronic HCV. To address the challenge of viral diversity, we developed ChAd vaccines encoding HCV genomic sequences that are conserved between all major HCV genotypes and adjuvanted by truncated shark invariant chain (sIitr). MethodsAge-matched female mice were immunised intramuscularly with ChAd (108 infectious units) encoding gt-1 and -3 (ChAd-Gt1/3) or gt-1 to -6 (ChAd-Gt1-6) conserved segments spanning the HCV proteome, or gt-1b (ChAd-Gt1b-NS control), with immunogenicity assessed 14-days post-vaccination. ResultsConserved segment vaccines, ChAd-Gt1/3 and ChAd-Gt1-6, generated high-magnitude, broad, and functional CD4+ and CD8+ T cell responses. Compared to the ChAd-Gt1b-NS vaccine, these vaccines generated significantly greater responses against conserved non-gt-1 antigens, including conserved subdominant epitopes that were not targeted by ChAd-Gt1b-NS. Epitopes targeted by the conserved segment HCV vaccine induced T cells, displayed 96.6% mean sequence homology between all HCV subtypes (100% sequence homology for the majority of genotype-1, -2, -4 sequences and 94% sequence homology for gt-3, -6, -7, and -8) in contrast to 85.1% mean sequence homology for epitopes targeted by ChAd-Gt1b-NS induced T cells. The addition of truncated shark invariant chain (sIitr) increased the magnitude, breadth, and cross-reactivity of the T cell response. ConclusionsWe have demonstrated that genetically adjuvanted ChAd vectored HCV T cell vaccines encoding genetic sequences conserved between genotypes are immunogenic, activating T cells that target subdominant conserved HCV epitopes. These pre-clinical studies support the use of conserved segment HCV T cell vaccines in human clinical trials.

Enhancing the antigenicity and immunogenicity of monomeric forms of hepatitis C virus E2 for use as a preventive vaccine

The E2 glycoprotein of hepatitis C virus (HCV) is the major target of broadly neutralizing antibodies (bNAbs) that are critical for the efficacy of a prophylactic HCV vaccine. We previously showed that a cell culture–derived, disulfide-linked high-molecular-weight (HMW) form of the E2 receptor–binding domain lacking three variable regions, Δ123-HMW, elicits broad neutralizing activity against the seven major genotypes of HCV. A limitation to the use of this antigen is that it is produced only at low yields and does not have a homogeneous composition. Here, we employed a sequential reduction and oxidation strategy to efficiently refold two high-yielding monomeric E2 species, D123 and a disulfide-minimized version (D123A7), into disulfide-linked HMW-like species (Δ123r and Δ123A7r). These proteins exhibited normal reactivity to bNAbs with continuous epitopes on the neutralizing face of E2, but reduced reactivity to conformation-dependent bNAbs and nonneutralizing antibodies (non-NAbs) compared with the corresponding monomeric species. Δ123r and Δ123A7r recapitulated the immunogenic properties of cell culture–derived D123-HMW in guinea pigs. The refolded antigens elicited antibodies that neutralized homologous and heterologous HCV genotypes, blocked the interaction between E2 and its cellular receptor CD81, and targeted the AS412, AS434, and AR3 domains. Of note, antibodies directed to epitopes overlapping with those of non-NAbs were absent. The approach to E2 antigen engineering outlined here provides an avenue for the development of preventive HCV vaccine candidates that induce bNAbs at higher yield and lower cost.

Frequencies and Epidemiology of HCV Genotypes in Hepatic Patients in Khartoum State - Sudan

Infection by hepatitis C virus (HCV) is the leading cause of chronic liver disease worldwide, the patients unaware of their infection and at risk for developing cirrhosis and hepatocellular carcinoma. At least six major HCV genotypes comprising numerous, more closely related subtypes have been identified.

Tree shrew bone marrow-derived mesenchymal stem cells express CD81, OCLN, and miR-122, facilitating the entire hepatitis C virus life cycle.

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease and associated cirrhosis, and hepatocellular carcinoma worldwide. At present, there is no prophylactic vaccine against HCV due to the lack of in vivo and in vitro model systems. Although most recombinants of all major HCV genotypes replicate in Huh-7 cell line and derivatives, these cells are human hepatoma-derived cell line. Therefore, the development of un-tumor-derived cell systems facilitating the entire HCV life cycle is urgently needed. In this study, we aimed to establish a novel tree shrew-derived bone marrow-derived mesenchymal stem cell (BM-MSC) system to reconstruct the HCV life cycle. We transduction cluster of differentiation 81 (CD81), occludin (OCLN), and microRNA-122 (miR-122) into BM-MSCs, then used a well-established HCV, produced from the J6/JFH1-Huh7.5.1 culture system, to infect the cells. We observed that BM-MSCs transduction with CD81/OCLN or CD81/OCLN/miR-122 support HCV RNA replication and infectious virus production. We also found that the addition of exogenous vascular endothelial growth factor (VEGF) can enhance HCV infectivity in BM-MSCs, with HCV virus load up to 105 copies/mL. In conclusion, we identified the minimum essential factors required for HCV replication in tree shrew-derived nonhuman nonhepatic BM-MSCs. Further, we identified that exogenous addition of VEGF, and exogenous expression of CD81, OCLN, and miR-122, facilitates efficient viral replication and production of infectious particles. Our results describe a novel cell system capable of supporting the entire HCV life cycle, which may provide an essential tool for anti-HCV drug discovery, vaccine development, and study of pathogenesis.

Identification of Interferon Receptor IFNAR2 As a Novel HCV Entry Factor by Using Chemical Probes.

Upon sensing pathogen-associated patterns and secreting interferons (IFNs) into the environment, host cells perceive extracellular type I IFNs by the IFNα/β receptors IFNAR1 and IFNAR2 to stimulate downstream innate immune signaling cascades. Through the use of chemical probes, we demonstrated that IFNAR2 facilitates hepatitis C virus (HCV) entry. Silencing of IFNAR2 significantly attenuated HCV proliferation. IFNAR2 binds infectious HCV virions through a direct interaction of its D2 domain with the C-terminal end of apolipoprotein E (apoE) on the viral envelope and facilitates virus entry into host cells. The antibody against the IFNAR2 D2 domain attenuates IFNAR2-apoE interaction and impairs HCV infection. The recombinant IFNAR2 protein and the chemical probe potently inhibit major HCV genotypes in various human liver cells in vitro. Moreover, the impact of a chemical probe on HCV genotype 2a is also documented in immune-compromised humanized transgenic mice. Our results not only expand the understanding of the biology of HCV entry and the virus-host relationship but also reveal a new target for the development of anti-HCV entry inhibitors.

Consensus on management of hepatitis C virus infection in resource-limited Ukraine and Commonwealth of Independent States regions.

Globally, 69.6 million individuals were infected with hepatitis C virus (HCV) infection in 2016. Of the six major HCV genotypes (GT), the most predominant one is GT1, worldwide. The prevalence of HCV in Central Asia, which includes most of the Commonwealth of Independent States (CIS), has been estimated to be 5.8% of the total global burden. The predominant genotype in the CIS and Ukraine regions has been reported to be GT1, followed by GT3. Inadequate HCV epidemiological data, multiple socio-economic barriers, and the lack of region-specific guidelines have impeded the optimal management of HCV infection in this region. In this regard, a panel of regional experts in the field of hepatology convened to discuss and provide recommendations on the diagnosis, treatment, and pre-, on-, and posttreatment assessment of chronic HCV infection and to ensure the optimal use of cost-effective antiviral regimens in the region. A comprehensive evaluation of the literature along with expert recommendations for the management of GT1-GT6 HCV infection with the antiviral agents available in the region has been provided in this review. This consensus document will help guide clinical decision-making during the management of HCV infection, further optimizing treatment outcomes in these regions.

Evolutionary Pathways to Persistence of Highly Fit and Resistant Hepatitis C Virus Protease Inhibitor Escape Variants.

Protease inhibitors (PIs) are important components of treatment regimens for patients with chronic hepatitis C virus (HCV) infection. However, emergence and persistence of antiviral resistance could reduce their efficacy. Thus, defining resistance determinants is highly relevant for efforts to control HCV. Here, we investigated patterns of PI resistance–associated substitutions (RASs) for the major HCV genotypes and viral determinants for persistence of key RASs. We identified protease position 156 as a RAS hotspot for genotype 1‐4, but not 5 and 6, escape variants by resistance profiling using PIs grazoprevir and paritaprevir in infectious cell culture systems. However, except for genotype 3, engineered 156‐RASs were not maintained. For genotypes 1 and 2, persistence of 156‐RASs depended on genome‐wide substitution networks, co‐selected under continued PI treatment and identified by next‐generation sequencing with substitution linkage and haplotype reconstruction. Persistence of A156T for genotype 1 relied on compensatory substitutions increasing replication and assembly. For genotype 2, initial selection of A156V facilitated transition to 156L, persisting without compensatory substitutions. The developed genotype 1, 2, and 3 variants with persistent 156‐RASs had exceptionally high fitness and resistance to grazoprevir, paritaprevir, glecaprevir, and voxilaprevir. A156T dominated in genotype 1 glecaprevir and voxilaprevir escape variants, and pre‐existing A156T facilitated genotype 1 escape from clinically relevant combination treatments with grazoprevir/elbasvir and glecaprevir/pibrentasvir. In genotype 1 infected patients with treatment failure and 156‐RASs, we observed genome‐wide selection of substitutions under treatment. Conclusion: Comprehensive PI resistance profiling for HCV genotypes 1‐6 revealed 156‐RASs as key determinants of high‐level resistance across clinically relevant PIs. We obtained in vitro proof of concept for persistence of highly fit genotype 1‐3 156‐variants, which might pose a threat to clinically relevant combination treatments.

In Vitro Neutralization Assay Using Cultured Hepatitis C Virus.

The method outlined here enables evaluation of the neutralization potency of monoclonal and polyclonal antibodies against in vitro cultured hepatitis C virus (HCV). The high variation in envelope protein sequence among HCV isolates necessitates the inclusion of several isolates, spanning the major genotypes of HCV, in order to make strong conclusions concerning the cross-reactive neutralization potential of a given antibody. This would be particularly relevant for any neutralization experiments aimed at uncovering novel therapeutic- or vaccine-relevant antibodies. In addition, these assays can also be used to compare neutralization sensitivity of novel cultured HCV to that of previously characterized isolates.

A Real-Life Study of New Antiviral Therapies in a High Prevalence Geographical Area for Hepatitis C Virus Infection

Background: Hepatitis C virus (HCV) infection represents one of the most important causes of chronic liver damage. The development of new therapeutic approaches based on the use of direct-acting antivirals allowed reaching the high rates of sustained virological response and on the other hand, the low rates of drug side effects. Objectives: The study aimed to evaluate the efficacy and safety of multiple direct-acting antiviral (DAA) therapies against the major HCV genotypes in Campania. Methods: We enrolled, in this monocentric observational study, 518 adult patients (> 18-years-old) affected by HCV who received a DAA anti-HCV-based therapy in the routine clinical practice. We collected direct data registered by the Hepatogastroenterology Division of the University of Campania “L. Vanvitelli”, which covers a catchment area from the entire Campania region. Results: A great number (98.2 %) of the 518 enrolled patients was naive to the antiviral treatment and genotype distribution was 1a = 32 (6.2%), 1b = 252 (48.7%), 2 = 146 (28.2%), 3 = 52 (10.1%), and 4 = 36 (6.9%). 300 patients were cirrhotic (57.9%) and most of them had a Child-Pugh A5 score. 79.56 % of the patients belonging to the population of our study were classified as fibrosis Metavir F3 or F4 by Fibroscan. All the enrolled patients completed the treatment with the exception of five (n = 5; 0.96%) who interrupted it due to adverse events. We observed a relapse of infection in three patients treated with Sofosbuvir and Simeprevir for 12 weeks (0.57%). Intention to treat analysis showed an overall rate of 98.46% (n = 510/518) sustained virological response. Six of the eight failure patients had a second line anti-HCV treatment and four of them obtained SVR (two patients are waiting for resistance test results). Conclusions: New antiviral regimens of DAA-based for HCV represent one of the greatest innovations in the scientific context in the last few years. Our prospective observational study confirms the elevated efficacy in terms of SVR12, independently from HCV genotype and disease stage, when these treatments are used as the methods of a good clinical practice.

Hepatitis C Virus Infection in a Tertiary Care Hospital in Mumbai, India: Identification of a Mixed and Novel Genotype

Purpose: Hepatitis C virus (HCV) is a leading cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma (HCC). HCV being a ribonucleic acid virus has considerable sequence variability. Assessment of viral load and genotype is necessary for designing treatment strategies and monitoring for viral resistance among HCV-infected cases. HCC is the most common form of liver cancer, often occurring in people with chronic hepatitis B or C. We undertook this study to observe genotype distribution of the virus in HCV patients from Mumbai. Materials and Methods: Between January 2017 and December 2017, the study was conducted on 120 chronic hepatitis outpatients from a tertiary care hospital, Mumbai, after obtaining ethics approval. All these diagnosed cases of HCV were subjected to molecular diagnosis in a research institute, Mumbai, by real-time polymerase chain reaction-based techniques. Results: Males were more preponderant than females with HCV infection, and the highest number of HCV-infected cases was observed in the age group of 41–50 years. Genotype 3 (n = 70; 58.3%) accounted for the highest number of cases followed by genotypes 1b (n = 29; 24.2%) and then 1a (n = 14; 11.7%). Mixed genotypes 1b + 3 and individual genotype 4 were found in two cases each (1.7%). A total of three samples (2.5%) were found with untypeable genotype. Conclusion: The major HCV genotype observed was 3 which is difficult to treat with direct-acting antivirals, owing to the more rapid progression of liver disease, increased rates of steatosis (non-alcoholic fatty liver disease), a higher risk for cancer (HCC). We believe this study is the first one to address the prevalence of mixed genotypes and untypeable genotype from India.

Role of the E2 Hypervariable Region (HVR1) in the Immunogenicity of a Recombinant Hepatitis C Virus Vaccine.

ABSTRACTCurrent evidence supports a protective role for virus-neutralizing antibodies in immunity against hepatitis C virus (HCV) infection. Many cross-neutralizing monoclonal antibodies have been identified. These antibodies have been shown to provide protection or to clear infection in animal models. Previous clinical trials have shown that a gpE1/gpE2 vaccine can induce antibodies that neutralize the in vitro infectivity of all the major cell culture-derived HCV (HCVcc) genotypes around the world. However, cross-neutralization appeared to favor certain genotypes, with significant but lower neutralization against others. HCV may employ epitope masking to avoid antibody-mediated neutralization. Hypervariable region 1 (HVR1) at the amino terminus of glycoprotein E2 has been shown to restrict access to many neutralizing antibodies. Consistent with this, other groups have reported that recombinant viruses lacking HVR1 are hypersensitive to neutralization. It has been proposed that gpE1/gpE2 lacking this domain could be a better vaccine antigen to induce broadly neutralizing antibodies. In this study, we examined the immunogenicity of recombinant gpE1/gpE2 lacking HVR1 (ΔHVR1). Our results indicate that wild-type (WT) and ΔHVR1 gpE1/gpE2 antigens induced antibodies targeting many well-characterized cross-genotype-neutralizing epitopes. However, while the WT gpE1/gpE2 vaccine can induce cross-genotype protection against various genotypes of HCVcc and/or HCV-pseudotyped virus (HCVpp), antisera from ΔHVR1 gpE1/gpE2-immunized animals exhibited either reduced homologous neutralization activity compared to that of the WT or heterologous neutralization activity similar to that of the WT. These data suggest that ΔHVR1 gpE1/gpE2 is not a superior vaccine antigen. Based on previously reported chimpanzee protection data using WT gpE1/gpE2 and our current findings, we are preparing a combination vaccine including wild-type recombinant gpE1/gpE2 for clinical testing in the future.IMPORTANCE An HCV vaccine is an unmet medical need. Current evidence suggests that neutralizing antibodies play an important role in virus clearance, along with cellular immune responses. Previous clinical data showed that gpE1/gpE2 can effectively induce cross-neutralizing antibodies, although they favor certain genotypes. HCV employs HVR1 within gpE2 to evade host immune control. It has been hypothesized that the removal of this domain would improve the production of cross-neutralizing antibodies. In this study, we compared the immunogenicities of WT and ΔHVR1 gpE1/gpE2 antigens as vaccine candidates. Our results indicate that the ΔHVR1 gpE1/gpE2 antigen confers no advantages in the neutralization of HCV compared with the WT antigen. Previously, we showed that this WT antigen remains the only vaccine candidate to protect chimpanzees from chronic infection, contains multiple cross-neutralizing epitopes, and is well tolerated and immunogenic in humans. The current data support the further clinical development of this vaccine antigen component.

Evolutionary Analysis Provides Insight Into the Origin and Adaptation of HCV.

Hepatitis C virus (HCV) belongs to the Hepacivirus genus and is genetically heterogeneous, with seven major genotypes further divided into several recognized subtypes. HCV origin was previously dated in a range between ∼200 and 1000 years ago. Hepaciviruses have been identified in several domestic and wild mammals, the largest viral diversity being observed in bats and rodents. The closest relatives of HCV were found in horses/donkeys (equine hepaciviruses, EHV). However, the origin of HCV as a human pathogen is still an unsolved puzzle. Using a selection-informed evolutionary model, we show that the common ancestor of extant HCV genotypes existed at least 3000 years ago (CI: 3192–5221 years ago), with the oldest genotypes being endemic to Asia. EHV originated around 1100 CE (CI: 291–1640 CE). These time estimates exclude that EHV transmission was mainly sustained by widespread veterinary practices and suggest that HCV originated from a single zoonotic event with subsequent diversification in human populations. We also describe a number of biologically important sites in the major HCV genotypes that have been positively selected and indicate that drug resistance-associated variants are significantly enriched at positively selected sites. HCV exploits several cell-surface molecules for cell entry, but only two of these (CD81 and OCLN) determine the species-specificity of infection. Herein evolutionary analyses do not support a long-standing association between primates and hepaciviruses, and signals of positive selection at CD81 were only observed in Chiroptera. No evidence of selection was detected for OCLN in any mammalian order. These results shed light on the origin of HCV and provide a catalog of candidate genetic modulators of HCV phenotypic diversity.

Glycan Shielding and Modulation of Hepatitis C Virus Neutralizing Antibodies

Hepatitis C virus (HCV) envelope glycoprotein heterodimer, E1E2, plays an essential role in virus entry and assembly. Furthermore, due to their exposure at the surface of the virion, these proteins are the major targets of anti-HCV neutralizing antibodies. Their ectodomain are heavily glycosylated with up to 5 sites on E1 and up to 11 sites on E2 modified by N-linked glycans. Thus, one-third of the molecular mass of E1E2 heterodimer corresponds to glycans. Despite the high sequence variability of E1 and E2, N-glycosylation sites of these proteins are generally conserved among the seven major HCV genotypes. N-glycans have been shown to be involved in E1E2 folding and modulate different functions of the envelope glycoproteins. Indeed, site-directed mutagenesis studies have shown that specific glycans are needed for virion assembly and infectivity. They can notably affect envelope protein entry functions by modulating their affinity for HCV receptors and their fusion activity. Importantly, glycans have also been shown to play a key role in immune evasion by masking antigenic sites targeted by neutralizing antibodies. It is well known that the high mutational rate of HCV polymerase facilitates the appearance of neutralization resistant mutants, and occurrence of mutations leading to glycan shifting is one of the mechanisms used by this virus to escape host humoral immune response. As a consequence of the importance of the glycan shield for HCV immune evasion, the deletion of N-glycans also leads to an increase in E1E2 immunogenicity and can induce a more potent antibody response against HCV.