J. Dietz

Deep Sequencing Reveals Mutagenic Effects of Ribavirin during Monotherapy of Hepatitis C Virus Genotype 1-Infected Patients

ABSTRACT The preeminent mode of action of the broad-spectrum antiviral nucleoside ribavirin in the therapy of chronic hepatitis C is currently unresolved. Particularly under contest are possible mutagenic effects of ribavirin that may lead to viral extinction by lethal mutagenesis of the hepatitis C virus (HCV) genome. We applied ultradeep sequencing to determine ribavirin-induced sequence changes in the HCV coding region (nucleotides [nt] 330 to 9351) of patients treated with 6-week ribavirin monotherapy (n = 6) in comparison to placebo (n = 6). Baseline HCV RNA levels maximally declined on average by −0.8 or −0.1 log10 IU/ml in ribavirin- versus placebo-treated patients. No general increase in rates of nucleotide substitutions in ribavirin-treated patients was observed. However, more HCV genome positions with high G-to-A and C-to-U transition rates were detected between baseline and treatment week 6 in ribavirin-treated patients in comparison to placebo-treated patients (rate of 0.0041 transitions per base pair versus rate of 0.0022 transitions per base pair; P = 0.049). Similarly, the sensitive detection of low-frequency minority variants by statistical filtering indicated significantly more positions with G-to-A and C-to-U transitions in ribavirin-treated patients than in placebo-treated patients (rate of 0.0331 transitions versus rate of 0.0186 transitions per G/C-containing position at baseline; P = 0.018). In contrast, non-ribavirin-associated A-to-G and U-to-C transitions were not enriched in the ribavirin group (P = 0.152). We conclude that ribavirin exerts a mutagenic effect on the virus in patients with chronic hepatitis C by facilitating G-to-A and C-to-U nucleotide transitions.

Dual function of the NK cell receptor 2B4 (CD244) in the regulation of HCV-specific CD8+ T cells

The outcome of viral infections is dependent on the function of CD8+ T cells which are tightly regulated by costimulatory molecules. The NK cell receptor 2B4 (CD244) is a transmembrane protein belonging to the Ig superfamily which can also be expressed by CD8+ T cells. The aim of this study was to analyze the role of 2B4 as an additional costimulatory receptor regulating CD8+ T cell function and in particular to investigate its implication for exhaustion of hepatitis C virus (HCV)-specific CD8+ T cells during persistent infection. We demonstrate that (i) 2B4 is expressed on virus-specific CD8+ T cells during acute and chronic hepatitis C, (ii) that 2B4 cross-linking can lead to both inhibition and activation of HCV-specific CD8+ T cell function, depending on expression levels of 2B4 and the intracellular adaptor molecule SAP and (iii) that 2B4 stimulation may counteract enhanced proliferation of HCV-specific CD8+ T cells induced by PD1 blockade. We suggest that 2B4 is another important molecule within the network of costimulatory/inhibitory receptors regulating CD8+ T cell function in acute and chronic hepatitis C and that 2B4 expression levels could also be a marker of CD8+ T cell dysfunction. Understanding in more detail how 2B4 exerts its differential effects could have implications for the development of novel immunotherapies of HCV infection aiming to achieve immune control.

Consideration of Viral Resistance for Optimization of Direct Antiviral Therapy of Hepatitis C Virus Genotype 1-Infected Patients.

Different highly effective interferon-free treatment options for chronic hepatitis C virus (HCV) infection are currently available. Pre-existence of resistance associated variants (RAVs) to direct antiviral agents (DAAs) reduces sustained virologic response (SVR) rates by 3–53% in hepatitis C virus (HCV) genotype 1 infected patients depending on different predictors and the DAA regimen used. Frequencies of single and combined resistance to NS3, NS5A and NS5B inhibitors and consequences for the applicability of different treatment regimens are unknown. Parallel population based sequencing of HCV NS3, NS5A and NS5B genes in 312 treatment-naïve Caucasian HCV genotype 1 infected patients showed the presence of major resistant variants in 20.5% (NS3), 11.9% (NS5A), and 22.1% (NS5B) with important differences for HCV subtypes. In NS3, Q80K was observed in 34.7% and 2.1% of subtype 1a and 1b patients, respectively while other RAVs to second generation protease inhibitors were detected rarely (1.4%). Within NS5A RAVs were observed in 7.1% of subtype 1a and 17.6% in subtype 1b infected patients. RAVs to non-nucleoside NS5B inhibitors were observed in 3.5% and 44.4% of subtype 1a and 1b patients, respectively. Considering all three DAA targets all subtype 1a and 98.6% of subtype 1b infected patients were wildtype for at least one interferon free DAA regimen currently available. In conclusion, baseline resistance testing allows the selection of at least one RAVs-free treatment option for nearly all patients enabling a potentially cost- and efficacy-optimized treatment of chronic hepatitis C.

A heterogeneous hierarchy of co-regulatory receptors regulates exhaustion of HCV-specific CD8 T cells in patients with chronic hepatitis C

BACKGROUND & AIMS The functionality of virus-specific T cells is regulated by a sophisticated network of an expanding repertoire of co-regulatory receptors, which could be harnessed for immunotherapeutic applications. However, targeting particular pathways during persistent virus infections has resulted in variable outcomes. The extent to which T cell exhaustion can be reversed, by targeting multiple co-regulatory pathways, still remains not fully investigated. METHODS We analysed the phenotype and in vitro functionality of HCV-specific CD8(+) T cells expressing PD-1, CTLA-4, TIM-3 or 2B4 either alone or in various combinations and compared expression levels to those of cytomegalovirus (CMV) and Epstein-Barr virus (EBV) specific T cells in peripheral blood mononuclear cells (PBMCs) from the same cohort of patients with chronic hepatitis C (CHC) infection. RESULTS Blockade and/or crosslinking of distinct co-regulatory pathways in exhausted HCV-specific CD8(+) T cells resulted in rather diverse and individualized T cell responses, irrespective of the type and number of receptors targeted. Overall, in vitro manipulations of these pathways yielded three response possibilities: (i) total non-response (ii) good single blockade response and (iii) good dual/multiple blockade response, with each comprising approximately one-third of the patients tested. The diversity of the in vitro responsiveness of HCV-specific T cells was reflected by an enormous ex vivo phenotypic heterogeneity. Despite this broad heterogeneity, HCV-specific CD8(+) T cells differed from EBV- and CMV-specific T cells in particular by TIM-3 expression, which also correlated with liver disease activity and viral load. CONCLUSIONS HCV-specific CD8(+) T cell functionality, upon co-regulatory receptor manipulations, was characterized by an individual pattern of responses in patients with CHC, suggesting that treatment approaches, targeting these receptors, should consider inter-individual differences and be personalized.

Interferon lambda 4 genotypes and resistance‐associated variants in patients infected with hepatitis C virus genotypes 1 and 3

Single‐nucleotide polymorphisms (SNPs) in the interferon lambda 4 (IFNL4) gene are predictors for treatment success in patients with hepatitis C virus (HCV) infection. For direct‐acting antiviral combinations only weak association with IFNL4 SNPs was observed. Little is known about potential selections of resistance‐associated variants (RAVs) by the IFNL4 genotype. This study analyzed the prevalence of RAVs to currently approved direct‐acting antivirals in a large European population in correlation to SNPs in IFNL4. Samples of 633 patients chronically infected with HCV genotypes 1a (n = 259), 1b (n = 323), and 3 (n = 51) were genotyped for rs12979860 (formerly known as IL28B) and rs368234815. RAVs in NS3, NS5A, and NS5B were detected by population‐based sequencing. In addition, IFNL4 SNPs and NS5A RAVs were analyzed including deep sequencing (n = 109) in an independent replication cohort of HCV genotype 1‐infected patients (n = 201). No significant correlation was found between IFNL4 SNPs and rare and common RAVs within NS3 and NS5B. In contrast, the NS5A RAV Y93H was detected frequently in HCV genotype 1b (14%) and significantly associated with the beneficial IFNL4 SNPs (P < 0.001 and P = 0.002, respectively). Moreover, the presence of Y93H in HCV genotype 1b patients was significantly associated with the second site variant T83M (P < 0.001). Independent factors significantly associated with the presence of Y93H were IFNL4 genotype and high baseline viral load. Conclusion: The NS5A RAV Y93H is significantly associated with the presence of beneficial IFNL4 SNPs and a high baseline viral load in HCV genotype 1‐infected patients, which may explain a lack of correlation or even an inverse correlation of treatment response with IFNL4 genotype in some NS5A inhibitor containing IFN‐free regimens. (Hepatology 2016;63:63–73)

Variations in serum sphingolipid levels associate with liver fibrosis progression and poor treatment outcome in hepatitis C virus but not hepatitis B virus infection

Ablation of very‐long‐chain ceramides (Cers) with consecutive elevations in sphinganine levels has been shown to cause a severe hepatopathy in a knockout mouse model. We have recently shown that serum sphingolipids (SLs) are deregulated in patients with chronic liver disease. However, their role as possible biomarkers in liver fibrosis remains to date unexplored. We assessed, using liquid chromatography/tandem mass spectrometry, serum concentrations of various SL metabolites in 406 patients with chronic viral hepatitis, 203 infected with genotype 1 hepatitis C virus (HCV) and 203 with hepatitis B virus (HBV), respectively. We observed significant variations of serum SLs, with sphingosine and sphinganine being, both in univariate (P < 0.05) as well as in multivariate analysis, significantly associated to severity of liver fibrosis in HCV‐infected patients (odds ratio [OR]: 1.111; confidence interval [CI]: 1.028‐1.202; P = 0.007 and OR, 0.634; CI, 0.435‐0.925; P = 0.018, respectively). Serum SLs correlated significantly with serum triglyceride and cholesterol levels as well as with insulin resistance, defined by the homeostatic model assessment index, in HCV patients. Sustained viral response rates in HCV patients were independently predicted by serum C24Cer (OR, 0.998; CI, 0.997‐0.999; P = 0.001), its unsaturated derivative C24:1Cer (OR, 1.001; CI, 1.000‐1.002; P = 0.059), and C18:1Cer (OR, 0.973; CI, 0.947‐0.999; P = 0.048), together with ferritin (OR, 1.006; CI, 1.003‐1.010; P < 0.001), alkaline phosphatase (OR, 1.020; CI, 1.001‐1.039; P = 0.032), and interleukin‐28B genotype (OR, 9.483; CI, 3.139‐28.643; P < 0.001). Conclusion: Our study demonstrates a tight interaction between variations in serum SL levels and progression of liver fibrosis as well as responsiveness to antiviral therapy. Particularly, sphingosine, sphinganine, and C24Cer appear as promising novel biomarkers in chronic HCV infection and should be further evaluated within the noninvasive prediction of liver fibrosis. (Hepatology 2015;61:812–822)

IFN lambda 4 genotypes and resistance-associated variants in HCV genotype 1 and 3 infected patients.

Single‐nucleotide polymorphisms (SNPs) in the interferon lambda 4 (IFNL4) gene are predictors for treatment success in patients with hepatitis C virus (HCV) infection. For direct‐acting antiviral combinations only weak association with IFNL4 SNPs was observed. Little is known about potential selections of resistance‐associated variants (RAVs) by the IFNL4 genotype. This study analyzed the prevalence of RAVs to currently approved direct‐acting antivirals in a large European population in correlation to SNPs in IFNL4. Samples of 633 patients chronically infected with HCV genotypes 1a (n = 259), 1b (n = 323), and 3 (n = 51) were genotyped for rs12979860 (formerly known as IL28B) and rs368234815. RAVs in NS3, NS5A, and NS5B were detected by population‐based sequencing. In addition, IFNL4 SNPs and NS5A RAVs were analyzed including deep sequencing (n = 109) in an independent replication cohort of HCV genotype 1‐infected patients (n = 201). No significant correlation was found between IFNL4 SNPs and rare and common RAVs within NS3 and NS5B. In contrast, the NS5A RAV Y93H was detected frequently in HCV genotype 1b (14%) and significantly associated with the beneficial IFNL4 SNPs (P < 0.001 and P = 0.002, respectively). Moreover, the presence of Y93H in HCV genotype 1b patients was significantly associated with the second site variant T83M (P < 0.001). Independent factors significantly associated with the presence of Y93H were IFNL4 genotype and high baseline viral load. Conclusion: The NS5A RAV Y93H is significantly associated with the presence of beneficial IFNL4 SNPs and a high baseline viral load in HCV genotype 1‐infected patients, which may explain a lack of correlation or even an inverse correlation of treatment response with IFNL4 genotype in some NS5A inhibitor containing IFN‐free regimens. (Hepatology 2016;63:63–73)

Origin, prevalence and response to therapy of hepatitis C virus genotype 2k/1b chimeras

BACKGROUND & AIMS Little is known about the epidemiology and frequency of recombinant HCV genotype 2/1 strains, which may represent a challenge for direct antiviral therapy (DAA). This study aims to identify the epidemiology and phylogeny of HCV genotype 2/1 strains and encourages genotype screening, to select the DAA-regimen that achieves the optimal sustained virologic response. METHODS Consecutive samples from HCV genotype 2 infected patients, according to commercial genotyping, from Germany, Italy and Israel were re-genotyped by Sanger-based sequencing. Virologic, epidemiological, and phylogenetic analyses including other published chimeras were performed. RESULTS Sequence analysis of 442 supposed HCV genotype 2 isolates revealed 61 (genotype 2k/1b (n=59), 2a/1b (n=1) or 2b/1a (n=1)) chimeras. No chimeras were observed in Italy, but the frequency was 14% and 25% in Germany and Israel. Treatment of viral chimera with sofosbuvir/ribavirin led to virologic relapse in 25/27 patients (93%). Nearly all patients treated with genotype 1-based DAA-regimens initially (n=8/9), or after relapse (n=13/13), achieved a sustained virologic response. Most patients with 2k/1b chimeras (88%) were originally from eight different areas of the former Soviet Union. All known 2k/1b chimeras harbour the same recombination breakpoint and build one phylogenetic cluster, while all other chimeras have different phylogenies. CONCLUSIONS The HCV genotype 2k/1b variant derives from one single recombination event most likely in the former Soviet Union, while other chimeras are unique and develop independently. A relatively high frequency has been observed along the migration flows, in Germany and Israel. In countries with little migration from the former Soviet Union the prevalence of 2k/1b chimeras is expected to be low. Treatment with sofosbuvir plus ribavirin is insufficient, but genotype 1-based regimens seem to be effective. Lay summary: The frequency of recombinant HCV is higher than expected. A novel recombinant variant (HCV genotype 2a/1b) was identified. Screening for recombinant viruses would contribute to increased response rates to direct antiviral therapy.

Divergent preS Sequences in Virion-Associated Hepatitis B Virus Genomes and Subviral HBV Surface Antigen Particles From HBV e Antigen-Negative Patients

Background Hepatitis B virus (HBV) surface proteins (HBsAg) coat the viral particle and form subviral particles (SVPs). Loss of HBsAg represents a functional cure and is an important treatment goal. Methods We analyzed the impact of the HBV genotypes A-E and pre-S mutations on SVP expression in hepatitis B virus e antigen (HBeAg)-negative chronic HBV-infected patients. A HBV genome harboring a preS1-deletion was analyzed in hepatoma cells. Results We observed a genotype-specific ratio of the 3 surface proteins (SHBs/MHBs/LHBs), reflecting differences in the morphology and composition of SVPs. Deletions/mutations in the preS1/preS2 domain, detected in released viral genomes, did not affect the molecular weight of MHBs and LHBs in these patients. In contrast, LHB molecular weight was altered in vitro using an HBV genome harboring a preS1-deletion derived from one of these patients. Conclusion Differences in composition of SVPs may result in genotype-specific immunogenicity and pathogenesis. In the patients with preS-mutations, secreted HBsAg and released viral genomes cannot be derived from the same genetic source. As viral genomes are derived from covalently closed circular DNA (cccDNA), HBsAg is presumably derived from integrated DNA. This important HBsAg source should be considered for novel antiviral strategies in HBeAg-negative chronic HBV-infected patients.

P0818 : Analysis of NS3 protease resistance-associated variants and phenotypes for the prediction of treatment response to HCV triple therapy

Triple therapy of chronic hepatitis C virus (HCV) infection with boceprevir (BOC) or telaprevir (TVR) leads to virologic failure in many patients which is often associated with the selection of resistance-associated variants (RAVs). These resistance profiles are of importance for the selection of potential rescue treatment options. In this study, we sequenced baseline NS3 RAVs population-based and investigated the sensitivity of NS3 phenotypes in an HCV replicon assay together with clinical factors for a prediction of treatment response in a cohort of 165 German and Swiss patients treated with a BOC or TVR-based triple therapy. Overall, the prevalence of baseline RAVs was low, although the frequency of RAVs was higher in patients with virologic failure compared to those who achieved a sustained virologic response (SVR) (7% versus 1%, P = 0.06). The occurrence of RAVs was associated with a resistant NS3 quasispecies phenotype (P<0.001), but the sensitivity of phenotypes was not associated with treatment outcome (P = 0.2). The majority of single viral and host predictors of SVR was only weakly associated with treatment response. In multivariate analyses, low AST levels, female sex and an IFNL4 CC genotype were independently associated with SVR. However, a combined analysis of negative predictors revealed a significantly lower overall number of negative predictors in patients with SVR in comparison to individuals with virologic failure (P<0.0001) and the presence of 2 or less negative predictors was indicative for SVR. These results demonstrate that most single baseline viral and host parameters have a weak influence on the response to triple therapy, whereas the overall number of negative predictors has a high predictive value for SVR.