IFN-free therapy is associated with restoration of type I IFN response in HIV-1 patients with acute HCV infection who achieve SVR.

Interferon (IFN)-free direct-acting antiviral agents (DAAs) have revolutionized chronic hepatitis C virus (HCV) treatment; early studies suggest excellent efficacy in acute HCV. However, changes in innate immune responses during DAA therapy for acute HCV are unknown. We studied interferon-stimulated gene (ISG) expression and related cytokines/chemokines in HIV-infected patients with acute HCV receiving sofosbuvir plus ribavirin (SOF+RBV) as part of the A5327 clinical trial. ISG expression was determined from PBMCs, and circulating cytokines/chemokines were quantified from serum from study participants. The overall sustained virologic response (SVR) was 57%; all treatment failures were due to virologic relapse. Apart from NOS2a, baseline ISG/chemokine/cytokine levels were similar irrespective of treatment outcome. Downregulation of ISGs was observed at treatment week four and end of treatment (EOT), implicating HCV in establishing elevated ISGs early during HCV infection. Levels of many of these ISGs increased at post-treatment week 12 (PTW12) in relapsers only, coinciding with recurrent HCV RNA. Eleven ISGs were differentially expressed in responders vs relapsers. On-treatment viral suppression was also associated with a reduction in IP-10, CXCL11 and MIP-1ß levels. In contrast, circulating IFN-α levels were significantly higher at EOT and PTW12 in responders vs relapsers. Upregulation of peripheral ISG expression is established early in the course of HCV infection during acute HCV infection, but did not predict subsequent treatment outcome with SOF+RBV. ISGs were downregulated during therapy and increased post-therapy in relapsers. IFN-α levels were higher in responders at EOT/PTW12, suggesting that impaired type I IFN production/secretion may contribute to relapse.

Analysis of resistance-associated substitutions in acute hepatitis C virus infection by deep sequencing across six genotypes and three continents.

Several direct-acting antivirals (DAAs) have been approved for the treatment of chronic hepatitis C virus (HCV) infections, opening the door to highly effective interferon-free treatment regimens. Resistance-associated substitutions (RASs) have been reported both in treatment-naïve patients and following treatment with protease (NS3), phosphoprotein (NS5A) and polymerase (NS5B) inhibitors. The prevalence of naturally occurring RASs in untreated HCV-infected individuals has mostly been analysed in those infected with genotype 1 (GT1), in the late phase of infection, and only within limited regions of the genome. Furthermore, the geographic distribution of RASs remains poorly characterized. In this study, we used next-generation sequencing to analyse full-length HCV genomes for the prevalence of RASs in acute HCV infections identified in nine international prospective cohorts. RASs were analysed in 179 participants infected with all six major HCV genotypes (GT1-GT6), and the geographic distribution of RASs was assessed in 107 GT1a and GT3a samples. While RASs were detected at varied frequencies across the three genomic regions, and between genotypes, RASs relevant to multiple DAAs in the leading IFN-free regimens were rarely detected in combination. Low-frequency RASs (<10% of the viral population) were also shown to have a GT-specific distribution. The main RASs with geographic associations were NS3 Q80K in GT1a samples and NS5B N142T in GT3a. These data provide the backdrop for prospective surveillance of RASs during DAA treatment scale-up.

Phylogenetic analysis of full-length, early infection, hepatitis C virus genomes among people with intravenous drug use: the InC3 Study.

Cross-continental phylogenetic analysis is important to understand subtle molecular differences of currently circulating hepatitis C virus (HCV) subtypes. Existence of such differences can be crucial in pursuing a universal hepatitis C vaccine. We characterized molecular epidemiology of early HCV infections identified across nine cohorts [North America (n=4), Australia (n=4) and Europe (n=1)] in the International Collaborative of Incident HIV and Hepatitis C in Injecting Cohorts (InC3 ). One hundred and ninety-two full-length HCV genomes were amplified from plasma of incident infections and subjected to next generation sequencing to establish the largest cross-continental, full-length acute HCV genomic data set available to date. Genomes from the most common subtypes (1a: n=94, 2b: n=15 and 3a: n=68) were used in phylogenetic analysis. Using full genome trees, 78 sequences (44%) were found to lie within 29 phylogenetic clusters/pairs defined on the basis of molecular similarity of consensus sequences. Of these, 26 each had exclusively Australian or North American sequences indicating a strong geographical bias for molecular similarity. On further analysis of behavioural and demographic associations, binary logistic regression analysis showed that older age and non-Caucasian ethnicity were significantly associated with clustering. HCV probably evolves in micro-epidemics within geographically isolated communities.

Genome-wide association study of hepatitis C virus- and cryoglobulin-related vasculitis.

The host genetic basis of mixed cryoglobulin vasculitis is not well understood and has not been studied in large cohorts. A genome-wide association study was conducted among 356 hepatitis C virus (HCV) RNA-positive individuals with cryoglobulin-related vasculitis and 447 ethnically matched, HCV RNA-positive controls. All cases had both serum cryoglobulins and a vasculitis syndrome. A total of 899 641 markers from the Illumina HumanOmni1-Quad chip were analyzed using logistic regression adjusted for sex, as well as genetically determined ancestry. Replication of select single-nucleotide polymorphisms (SNPs) was conducted using 91 cases and 180 controls, adjusting for sex and country of origin. The most significant associations were identified on chromosome 6 near the NOTCH4 and MHC class II genes. A genome-wide significant association was detected on chromosome 6 at SNP rs9461776 (odds ratio=2.16, P=1.16E-07) between HLA-DRB1 and DQA1: this association was further replicated in additional independent samples (meta-analysis P=7.1 × 10(-9)). A genome-wide significant association with cryoglobulin-related vasculitis was identified with SNPs near NOTCH4 and MHC Class II genes. The two regions are correlated and it is difficult to disentangle which gene is responsible for the association with mixed cryoglobulinemia vasculitis in this extended major histocompatibility complex region.

Acute hepatitis C infection lowers serum lipid levels.

Chronic hepatitis C infection is associated with hypolipidaemia that resolves with viral clearance. Lipid levels in a subgroup of patients rebound to levels that may increase the risk of coronary heart disease. The impact of acute hepatitis C infection and its clearance on lipid levels is unknown. We undertook a retrospective evaluation of subjects with acute hepatitis C infection evaluating lipid levels before, during and following acute infection. Thirty-eight subjects with acute hepatitis C infection had lipid levels available. Twelve patients had pre-infection and intra-infection lipid levels available. Cholesterol (197.8-152.4 mg/dL, P = 0.025), low-density lipoprotein (LDL) (116.1-76.3 mg/dL, P = 0.001) and non-high-density lipoprotein (non-HDL) cholesterol (164.0-122.7 mg/dL, P = 0.007) decreased dramatically during acute hepatitis C virus infection. Nineteen patients who achieved viral clearance had lipid levels available during infection and following resolution of infection. In these patients, cholesterol (145.0-176.0 mg/dL, P = 0.01), LDL (87.0-110.1 P = 0.0046) and non-HDL cholesterol (108.6-133.6 mg/dL, P = 0.008) increased significantly. No change was seen in patients who developed chronic infection. Four patients had lipid levels before, during and following resolution of infections and had increased postinfection LDL, cholesterol and non-HDL cholesterol from pre-infection levels, indicating acute infection may be associated with an increase in postinfection lipid levels and may confer an increased risk of coronary heart disease. Acute hepatitis C infection results in hypolipidaemia with decreased LDL, cholesterol and non-HDL cholesterol levels that increase following infection resolution. Levels may increase above pre-infection baseline lipid levels and should be monitored.

Characterization of full-length hepatitis C virus genotype 4 sequences.

Over 85% of the world's nearly 170 million hepatitis C virus (HCV)-infected subjects exist in regions of Africa, Southeast Asia and Middle Eastern countries where genotypes 4-6 are very common. In particular, HCV genotype 4 is highly prevalent in Egypt with more than 19% of the population infected and chronic HCV representing one of the top five leading causes of death, due in part to ineffective interferon alpha treatment against this genotype. Despite this, very little work has been carried out to characterize the sequence diversity of genotype 4, which will be critical to the development of effective vaccines and antiviral therapies against this genotype. As a result of the paucity of sequence data available for HCV genotype 4, for which only one full genome sequence is currently available, we were interested in characterizing additional genotype 4 sequences and to provide reagents for amplification of this genotype. Here we describe seven unique HCV genotype 4a full genomes, in addition to a single genotype 4d genome, and characterize their sequence diversity in relation to other more closely characterized HCV genotypes.

Detection of diverse hepatitis C virus (HCV)-specific cytotoxic T lymphocytes in peripheral blood of infected persons by screening for responses to all translated proteins of HCV.

Broadly directed hepatitis C virus (HCV)-specific cytotoxic T lymphocytes (CTL) have been identified from liver-infiltrating lymphocytes but have been more difficult to assess in peripheral blood of infected persons. To enhance the detection of CTL from peripheral blood mononuclear cells (PBMC), we cocultured PBMC with autologous Epstein-Barr virus-transformed B-lymphoblastoid cell lines that had been infected with recombinant vaccinia virus constructs so that they expressed the entire translated polyprotein of HCV-H, a type 1a strain. These stimulated cells from HCV-infected as well as exposed seronegative persons were then cloned at limiting dilution and tested for HCV-specific CTL activity using a standard (51)Cr release assay. HCV-specific CTL were detected in PBMC from seven of nine persons with chronic hepatitis, including five of seven in whom CTL had previously been detected from liver biopsy specimens but not PBMC. In a single person with chronic HCV infection, CTL directed against as many as five different epitopes were detected in peripheral blood and were similar in specificity to those detected in liver tissue. This technique was used to evaluate eight subjects identified to be at high risk for HCV exposure due to continued injection drug abuse; no evidence of CTL in PBMC was found. We conclude that CTL can be detected in PBMC from the majority of persons with chronic HCV infection but are present at lower levels or absent in exposed but persistently seronegative persons. The high degree of concordance of HCV epitopes identified from liver and PBMC suggests that this strategy is a reasonable alternative to liver biopsy for characterizing the CTL response to HCV in chronically infected persons.