Hepatitis C subtyping assay failure in UK patients born in sub-Saharan Africa: Implications for global treatment and elimination.

BACKGROUND AND AIMS: The newly developed direct-acting antivirals have revolutionized the treatment of chronic hepatitis C virus (HCV), with cure rates as high as 98% in some cohorts. Although genome sequencing has demonstrated that some subtypes of HCV naturally harbor drug resistance associated substitutions (RAS), these are often overlooked as "rarities." Furthermore, commercial subtyping assays and associated epidemiological findings are skewed towards Western cohorts and whole-genome sequencing can be problematic to deploy without significant infrastructure and training support. We thus aimed to develop a simple, robust and accurate HCV subtyping pipeline, to optimize and streamline molecular detection and sequence-based typing of diverse RAS-containing subtypes. METHODS: HCV serum derived from 146 individuals, whose likely source of infection was from sub-Saharan Africa (SSA) was investigated with a novel panel of single round polymerase chain reaction (PCR) assays targeting NS5B and NS5A genomic regions. Virus subtype assignments were determined by pairwise-distance analysis and compared to both diagnostic laboratory assignments and free-to-use online typing tools. RESULTS: Partial NS5A and NS5B sequences were respectively obtained from 131 to 135 HCV-positive patients born in 19 different countries from SSA but attending clinics in the UK. We determined that routine clinical diagnostic methods incorrectly subtyped 59.0% of samples, with a further 6.8% incorrectly genotyped. Of five commonly used online tools, Geno2Pheno performed most effectively in determining a subtype in agreement with pairwise distance analysis. CONCLUSION: This study provides a simple low-cost pathway to accurately subtype in SSA, guide regional therapeutic choice and assist global surveillance and elimination initiatives.

Interferon-Induced Transmembrane Proteins Mediate Viral Evasion in Acute and Chronic Hepatitis C Virus Infection.

Although adaptive immune responses against hepatitis C virus (HCV) infection have been studied in great detail, the role of innate immunity in protection against HCV infection and immune evasion is only partially understood. Interferon-induced transmembrane proteins (IFITMs) are innate effector proteins restricting host cell entry of many enveloped viruses, including HCV. However, the clinical impact of IFITMs on HCV immune escape remains to be determined. Here, we show that IFITMs promote viral escape from the neutralizing antibody (nAb) response in clinical cohorts of HCV-infected patients. Using pseudoparticles bearing HCV envelope proteins from acutely infected patients, we show that HCV variants isolated preseroconversion are more sensitive to the antiviral activity of IFITMs than variants from patients isolated during chronic infection postseroconversion. Furthermore, HCV variants escaping nAb responses during liver transplantation exhibited a significantly higher resistance to IFITMs than variants that were eliminated posttransplantation. Gain-of-function and mechanistic studies revealed that IFITMs markedly enhance the antiviral activity of nAbs and suggest a cooperative effect of human monoclonal antibodies and IFITMs for antibody-mediated neutralization driving the selection pressure in viral evasion. Perturbation studies with the IFITM antagonist amphotericin B revealed that modulation of membrane properties by IFITM proteins is responsible for the IFITM-mediated blockade of viral entry and enhancement of antibody-mediated neutralization. Conclusion: Our results indicate IFITM proteins as drivers of viral immune escape and antibody-mediated HCV neutralization in acute and chronic HCV infection. These findings are of clinical relevance for the design of urgently needed HCV B-cell vaccines and might help to increase the efficacy of future vaccine candidates.

Broad neutralization of hepatitis C virus-resistant variants by Civacir hepatitis C immunoglobulin.

Hepatitis C virus (HCV)-induced end-stage liver disease is the major indication for liver transplantation (LT). However, reinfection of the liver graft is still common, especially in patients with detectable viral load at the time of LT. Limited data are available on direct-acting antivirals in the transplant setting for prevention of graft infection. The human hepatitis C immunoglobulin (HCIG) Civacir is an investigational drug that is currently being developed in an ongoing phase 3 clinical trial assessing its safety and efficacy at preventing HCV recurrence after liver transplantation (LT) in the United States. Using well-characterized patient-derived HCV variants selected during LT, we studied the molecular mechanism of action of Civacir. Inhibition of HCV infection was studied using infectious HCV models including HCV pseudoparticles (HCVpp) and cell culture-derived HCV (HCVcc) containing patient-derived viral envelope glycoproteins from 22 HCV variants isolated from patients before and after LT. The human hepatitis C immune globulin Civacir is an investigational drug that is currently being developed in an ongoing phase 3 clinical trial assessing safety and efficacy to prevent HCV recurrence after LT in the United States. Using well-characterized patient-derived HCV variants selected during LT, we studied the molecular mechanism of action of Civacir. Inhibition of HCV infection was studied using infectious HCV models including HCV pseudoparticles and cell culture-derived HCV containing patient-derived viral envelope glycoproteins from 22 HCV variants isolated from patients before and after liver transplantation. Additionally, we studied neutralization of different HCV genotypes and of direct-acting antiviral-resistant viruses. Our results indicate that Civacir potently, broadly, and dose-dependently neutralizes all tested patient variants in HCV pseudoparticles and cell culture-derived HCV assays including variants displaying resistance to host neutralizing antibodies and antiviral monoclonal antibodies. The half-maximal inhibitory concentrations were independent of the phenotype of the viral variant, indicating that virus neutralization by Civacir is not affected by viral selection. Furthermore, Civacir is equally active against tested direct-acting antiviral-resistant HCV isolates in cell culture. CONCLUSION: Collectively, these results demonstrate broad neutralizing activity of Civacir against resistant viruses, likely due to synergy between anti-HCV antibodies derived from different plasma donors, and support its further clinical development for prevention of liver graft infection. (Hepatology 2016;64:1495-1506).

Flexible and rapid construction of viral chimeras applied to hepatitis C virus.

A novel and broadly applicable strategy combining site-directed mutagenesis and DNA assembly for constructing seamless viral chimeras is described using hepatitis C virus (HCV) as an exemplar. Full-length HCV genomic cloning cassettes, which contained flexibly situated restriction endonuclease sites, were prepared via a single, site-directed mutagenesis reaction and digested to receive PCR-amplified virus envelope genes by In-Fusion cloning. Using this method, we were able to construct gene-shuttle cassettes for generation of cell culture-infectious JFH-1-based chimeras containing genotype 1-3 E1E2 genes. Importantly, using this method we also show that E1E2 clones that were not able to support cell entry in the HCV pseudoparticle assay did confer entry when shuttled into the chimeric cell culture chimera system. This method can be easily applied to other genes of study and other viruses and, as such, will greatly simplify reverse genetics studies of variable viruses.

Simultaneous determination of HCV genotype and NS5B resistance associated substitutions using dried serum spots from São Paulo state, Brazil.

Hepatitis C virus (HCV) is responsible for more than 180 million infections worldwide, and about 80 % of infections are reported in Low and Middle-income countries (LMICs). Therapy is based on the administration of interferon (INF), ribavirin (RBV) or more recently Direct-Acting Antivirals (DAAs). However, amino acid substitutions associated with resistance (RAS) have been extensively described and can contribute to treatment failure, and diagnosis of RAS requires considerable infrastructure, not always locally available. Dried serum spots (DSS) sampling is an alternative specimen collection method, which embeds drops of serum onto filter paper to be transported by posting to a centralized laboratory. Here, we assessed feasibility of genotypic analysis of HCV from DSS in a cohort of 80 patients from São Paulo state Brazil. HCV RNA was detected on DSS specimens in 83 % of samples of HCV infected patients. HCV genotypes 1a, 1b, 2a, 2c and 3a were determined using the sequence of the palm domain of NS5B region, and RAS C316N/Y, Q309R and V321I were identified in HCV 1b samples. Concerning therapy outcome, 75 % of the patients who used INF +RBV as a previous protocol of treatment did not respond to DAAs, and 25 % were end-of-treatment responders. It suggests that therapy with INF plus RBV may contribute for non-response to a second therapeutic protocol with DAAs. One patient that presented RAS (V321I) was classified as non-responder, and combination of RAS C316N and Q309R does not necessarily imply in resistance to treatment in this cohort of patients. Data presented herein highlights the relevance of studying circulating variants for a better understanding of HCV variability and resistance to the therapy. Furthermore, the feasibility of carrying out genotyping and RAS phenotyping analysis by using DSS card for the potential of informing future treatment interventions could be relevant to overcome the limitations of processing samples in several location worldwide, especially in LMICs.

Challenges on the development of a pseudotyping assay for Zika glycoproteins.

Introduction. Zika virus (ZIKV) emerged as a public health concern on the American continent during late 2015. As the number of infected grew so did the concerns about its capability to cause long-term damage especially with the appearance of the congenital Zika syndrome (CZS). Proteins from the TAM family of receptor tyrosine kinases (RTKs) were proposed as the cellular receptors, however, due to the ability of the virus to infect a variety of cell lines different strategies to elucidate the tropism of the virus should be investigated.Hypothesis. Pseudotyping is a powerful tool to interrogate the ability of the glycoprotein (GP) to permit entry of viruses.Aim. We aimed to establish a highly tractable pseudotype model using lenti- and retro-viral backbones to investigate the entry pathway of ZIKV.Methodology. We used different glycoprotein constructs and different lenti- or retro-viral backbones, in a matrix of ratios to investigate production of proteins and functional pseudotypes.Results. Varying the ratio of backbone and glycoprotein plasmids did not yield infectious pseudotypes. Moreover, the supplementation of the ZIKV protease or the substitution of the backbone had no positive impact on the infectivity. We showed production of the proteins in producer cells implying the lack of infectious pseudotypes is due to a lack of successful glycoprotein incorporation, rather than lack of protein production.Conclusion. In line with other reports, we were unable to successfully produce infectious pseudotypes using the variety of methods described. Other strategies may be more suitable in the development of an efficient pseudotype model for ZIKV and other flaviviruses.

Rationally derived inhibitors of hepatitis C virus (HCV) p7 channel activity reveal prospect for bimodal antiviral therapy.

Since the 1960s, a single class of agent has been licensed targeting virus-encoded ion channels, or 'viroporins', contrasting the success of channel blocking drugs in other areas of medicine. Although resistance arose to these prototypic adamantane inhibitors of the influenza A virus (IAV) M2 proton channel, a growing number of clinically and economically important viruses are now recognised to encode essential viroporins providing potential targets for modern drug discovery. We describe the first rationally designed viroporin inhibitor with a comprehensive structure-activity relationship (SAR). This step-change in understanding not only revealed a second biological function for the p7 viroporin from hepatitis C virus (HCV) during virus entry, but also enabled the synthesis of a labelled tool compound that retained biological activity. Hence, p7 inhibitors (p7i) represent a unique class of HCV antiviral targeting both the spread and establishment of infection, as well as a precedent for future viroporin-targeted drug discovery.

Elevated serum activity of MBL and ficolin-2 as biomarkers for progression to hepatocellular carcinoma in chronic HCV infection.

Hepatocellular carcinoma (HCC) is an uncommon but significant outcome of chronic hepatitis C virus (HCV) infection. A serum biomarker for predicting progression to HCC would have a major impact on patient monitoring and clinical management. We explored circulating liver-expressed lectins, ficolin-2, ficolin-3 and mannose binding lectin (MBL), as potential biomarkers for the development of HCC. The activity of these three lectins were analysed in HCV positive patients who developed HCC (n = 31) with comparable HCV-positive HCC-negative patients (n = 106) and healthy controls (n = 79). Serum binding activity of ficolin-2 and MBL were elevated compared to controls. Analysis of pre-HCC onset samples revealed that MBL levels were significantly elevated up to 3 years, and ficolin-2 was elevated up to 1 year, prior to diagnosis of HCC over controls. This preliminary study identifies MBL and ficolin-2 as potential biomarkers for the development of HCC in chronic HCV infection.

Expression of human ficolin-2 in hepatocytes confers resistance to infection by diverse hepatotropic viruses.

The liver-expressed pattern recognition receptors mannose-binding lectin (MBL), ficolin-2 and ficolin-3 contribute to the innate immune response by activating complement. Binding of soluble ficolin-2 to viral pathogens can directly neutralize virus entry. We observed that the human hepatoma cell line HuH7.5, which is routinely used for the study of hepatotropic viruses, is deficient in expression of MBL, ficolin-2 and ficolin-3. We generated a cell line that expressed and secreted ficolin-2. This cell line (HuH7.5 [FCN2]) was more resistant to infection with hepatitis C virus (HCV), ebolavirus and vesicular stomatitis virus, but surprisingly was more susceptible to infection with rabies virus. Cell-to-cell spread of HCV was also inhibited in ficolin-2 expressing cells. This illustrates that ficolin-2 expression in hepatocytes contributes to innate resistance to virus infection, but some viruses might utilize ficolin-2 to facilitate entry.

Characterization of hepatitis C RNA-containing particles from human liver by density and size.

Hepatitis C virus (HCV) particles found in vivo are heterogeneous in density and size, but their detailed characterization has been restricted by the low titre of HCV in human serum. Previously, our group has found that HCV circulates in blood in association with very-low-density lipoprotein (VLDL). Our aim in this study was to characterize HCV RNA-containing membranes and particles in human liver by both density and size and to identify the subcellular compartment(s) where the association with VLDL occurs. HCV was purified by density using iodixanol gradients and by size using gel filtration. Both positive-strand HCV RNA (present in virus particles) and negative-strand HCV RNA (an intermediate in virus replication) were found with densities below 1.08 g ml(-1). Viral structural and non-structural proteins, host proteins ApoB, ApoE and caveolin-2, as well as cholesterol, triglyceride and phospholipids were also detected in these low density fractions. After fractionation by size with Superose gel filtration, HCV RNA and viral proteins co-fractionated with endoplasmic reticulum proteins and VLDL. Fractionation on Toyopearl, which separates particles with diameters up to 200 nm, showed that 78 % of HCV RNA from liver was >100 nm in size, with a positive-/negative-strand ratio of 6 : 1. Also, 8 % of HCV RNA was found in particles with diameters between 40 nm and 70 nm and a positive-/negative-strand ratio of 45 : 1. This HCV was associated with ApoB, ApoE and viral glycoprotein E2, similar to viral particles circulating in serum. Our results indicate that the association between HCV and VLDL occurs in the liver.