Chiara Taibi

Autophagy plays an important role in the containment of HIV-1 in nonprogressor-infected patients

Recent in vitro studies have suggested that autophagy may play a role in both HIV-1 replication and disease progression. In this study we investigated whether autophagy protects the small proportion of HIV-1 infected individuals who remain clinically stable for years in the absence of antiretroviral therapy, these named long-term nonprogressors (LTNP) and elite controllers (EC). We found that peripheral blood mononuclear cells (PBMC) of the HIV-1 controllers present a significantly higher amount of autophagic vesicles associated with an increased expression of autophagic markers with respect to normal progressors. Of note, ex vivo treatment of PBMC from the HIV-1 controllers with the MTOR inhibitor rapamycin results in a more efficient autophagic response, leading to a reduced viral production. These data lead us to propose that autophagy contributes to limiting viral pathogenesis in HIV-1 controllers by targeting viral components for degradation.

Dynamics of HCV genotype 4 resistance-associated variants during virologic escape with pIFN/RBV + daclatasvir: A case study using ultra deep pyrosequencing

BACKGROUND Daclatasvir (DCV) is an approved NS5A inhibitor with potent anti-HCV activity and broad genotype coverage. DCV resistance-associated variants (RAVs) have been described for patients infected with genotype (GT) 1, but increased GT4 prevalence in European countries as a result of immigration has boosted interest in this genotype. OBJECTIVES Establishment of NS5A variability in treatment-naive patients with HCV genotype 4 infection and a case study of the dynamics of resistance-associated variants in a virologic failure receiving pIFN/RBV+DCV, as assessed by ultra-deep sequencing. STUDY DESIGN Five treatment-naïve GT4 patients (GT4a [n = 1], GT4d [n = 3], GT4o [n = 1]) were evaluated for inclusion in the COMMAND-4 study and treatment with pIFN/RBV±DCV. RESULTS Patient (Pt) 1 received pIFN/RBV; Pts2-4 received pIFN/RBV + DCV; Pt5 was a screening failure. Pt1 relapsed; Pt2 experienced breakthrough at Wk4; Pts3 and 4 achieved a sustained virologic response. No substitutions associated with DCV-resistance were detected at baseline. In terms of viremic time points for Pts1 and 2, the extent of NS5A diversity pre-treatment was not significantly related to viral load (r = -0568; p = 0.035). In Pt2, multiple substitutions associated with DCV-resistance were observed after breakthrough at NS5A amino acid positions 28, 31 and 93. These substitutions were frequently observed on the same haplotype (L28S + M31I = 55.52, 82.50, and 99.36% at Wk4, 8 and 9; L28S + M31I + Y93H = 11.77, 5.01 and <0.6% at Wk4, 8 and 9). CONCLUSIONS This is the first report to describe DCV-resistance in patients infected with GT4d, supporting a possible role for a recently described RAV (L28S), and presenting the dynamics of HCV quasispecies during therapy failure, with indications of changes of diversity and association of mutations.

IFNL4 and IFNL3 Associated Polymorphisms Strongly Influence the Spontaneous IFN-Alpha Receptor-1 Expression in HCV-Infected Patients

Single-nucleotide polymorphism in IFNL3 gene (rs12979860) predicts spontaneous and therapy-induced HCV clearance. In a previous study from our group PBMC from patients with favourable rs12979860 genotype showed higher levels of IFNAR-1 mRNA. Recently, a dinucleotide polymorphism, ss469415590 (TT or ΔG), has been discovered in the region upstream IFNL3 gene, which is in high linkage disequilibrium with rs12979860. ss469415590[ΔG] is a frameshift variant that creates a novel gene, designed IFNL4, encoding the interferon-lambda 4 protein (IFNL4). The aim of the present study was to extend the analysis of IFNAR-1 mRNA levels to the ss469415590 variants. Our results highlight that the difference of IFNAR-1 mRNA levels between favourable and unfavourable genotype combinations, at both rs12979860 and ss469415590 loci, is stronger than that observed for single polymorphisms at each locus. These findings suggest may represent the biological basis for the observed association between IFNL3 CC and IFNL4 TT/TT genotypes and favourable outcome of either natural HCV infection (clearance vs chronic evolution) or IFN-based therapy.

HCV NS3 quasispecies in liver and plasma and dynamics of telaprevir-resistant variants in breakthrough patients assessed by UDPS: A case study

BACKGROUND The impact of pre-existing variants in hepatitis C virus (HCV) quasispecies, carrying resistance-associated mutations (RAMs), on the outcome of treatment with direct acting antiviral agents (DAA) is debated and it is complicated by the lack of knowledge of quasispecies distribution between the viral reservoir (liver) and the circulating compartment. OBJECTIVE To evaluate NS3 protease heterogeneity and presence of RAMs on baseline plasma and liver biopsy samples. Plasma dynamics were also analyzed during therapy and after its suspension. Study design Ultra-deep pyrosequencing (UDPS) was performed in two HCV genotype 1a patients who received telaprevir (TVR)-based therapy and developed treatment failure due to TVR-resistance. RESULTS In both patients the baseline diversity of NS3 quasispecies in plasma was higher than in liver (183.6×10(-4) vs 47.8×10(-4) and 246.0×10(-4) vs 55.0×10(-4) nt substitution/site, respectively, p<0.0001), but phylogenetic trees did not evidence compartmentalization between the two compartments. At baseline RAMs (i.e. V36A, T54A) were detected very low levels (range: 0.31-0.52%) in both specimen types. However, phylogenetic analyses revealed that the viral variants carrying these mutations at baseline were different from those that became fixed at breakthrough, when combined V36M+R155K, conferring high-level resistance to TVR, were observed. The frequency of resistance-associated variants declined after withdrawal of drug selective pressure. CONCLUSIONS UDPS allowed extensive evaluation of quasispecies compartmentalization and of their dynamics after withdrawal of TVR. Plasma and liver NS3 quasispecies, including low level RAMs, do not show significant difference.

Near full length hepatitis C virus genome reconstruction by next generation sequencing based on genotype-independent amplification

BACKGROUND Deep sequencing has a deep impact on the study of rapidly mutating RNA viruses, such as hepatitis C virus, proving to be an invaluable tool for analyzing virus diversity and evolution. AIM Genotype-independent high-throughput pyrosequencing was used to obtain near full length hepatitis C virus genome sequence reconstruction directly from clinical samples. METHODS Samples from hepatitis C virus infected subjects harbouring different subtypes (1a, 1b, 2c) were analyzed (viral load range: 1.2-20.8 × 10(6)IU/ml). Data were generated with a modified sequence-independent single primer amplification method followed by 454 sequencing. RESULTS the extent of reconstructed hepatitis C virus genome varied from 79.95% to 99.64%. No correlation between extent of genome reconstruction and either viral load (r=0.4857, p=0.3556) or number of HCV reads (r=0.08571, p=0.9194) was observed. CONCLUSION This study describes a protocol for obtaining whole genome sequences from different hepatitis C virus patients with different genotypes in a single sequencing run.

The clinical significance of HCV core antigen detection during Telaprevir/Peg-Interferon/Ribavirin therapy in patients with HCV 1 genotype infection.

BACKGROUND Direct-acting antiviral drugs (DAA) regimen improve the SVR rate. However, adverse effects often lead to therapy interruption. This underlines the importance to find some predictive parameters of response in order to consider the possibility of a shorter time of antiviral treatment in the appearance of adverse effects without affecting the success of the therapy. OBJECTIVES We aimed to examine the HCVAg kinetics in the early phase of treatment and its predictive value of SVR in patients undergoing TPV/Peg-IFN/RBV treatment. STUDY DESIGN Twenty-three patients infected by HCV genotype 1 (1a n=11; 1b n=12) were included in this prospective study. RESULTS At baseline the median Log of HCVAg concentration in RVR and EVR patients were 3.15 fmol/L and 3.45 fmol/L, respectively with no significant differences. The baseline median HCV-RNA to HCVAg ratio was 233.77, this ratio was significantly lower when measured on day 1 (27.52) and on day 6 (24.84) (p<0.001). The two-tailed Fishers exact test indicated that the SVR response is statistically significantly different in patients with detected HCVAg at week1 compared to patients with no detectable HCVAg (p=0.05). The sensitivity, specificity, and negative and positive predictive values (NPV, PPV) were 53.8, 87.5, 53.8 and 87.5%, respectively. The area under the ROC curve was 0.71 at day T6, the best cut-off of 3 fmol/L when evaluated with the HCVAg plasma concentration at day T6. CONCLUSION Undetectable HCVAg in the early phase of TPV/Peg-IFN/RBV treatment could represent an important parameter for predicting SVR.

IFN-Alpha Receptor-1 Upregulation in PBMC from HCV Naïve Patients Carrying CC Genotype. Possible Role of IFN-Lambda

Background and Aims IL-28B gene polymorphisms predict better therapeutic response and spontaneous clearance of HCV. Moreover, higher expression of IFN-lambda has been reported in patients with the rs12979860 CC favourable genotype. The study aim was to establish possible relationships between IL-28B rs12979860 genotypes and expression of IFN-alpha receptor-1 (IFNAR-1) in naïve HCV patients, and to explore the possible role of IFN-lambda. Methods IFNAR-1 mRNA levels were measured in PBMC from naïve patients with chronic hepatitis C with different IL-28 genotypes. The ability of IFN-lambda to up-regulate the expression of IFNAR-1 was established in PBMC from healthy donors carrying different IL-28B genotypes. Results Lower IFNAR-1 mRNA levels were observed in PBMC from HCV-infected naïve patients as compared to healthy donors. In healthy donors, IFNAR-1 mRNA levels were independent from IL-28B genotype, while in HCV patients, an increasing gradient was observed in TT vs CT vs CC carriers. In the latter group, a direct correlation between IFNAR-1 and endogenous IL-28B expression was observed. Moreover, IFN-lambda up-regulated IFNAR-1 expression in normal PBMC in a time-and dose-dependent manner, with a more effective response in CC vs TT carriers. Conclusion Endogenous levels of IFN-lambda may be responsible for partial restoration of IFNAR-1 expression in HCV patients with favourable IL-28 genotype. This, in turn, may confer to CC carriers a response advantage to either endogenous or exogenous IFN-alpha, representing the biological basis for the observed association between CC genotype and favourable outcome of either natural infection (clearance vs chronicization) or IFN therapy.

Characterization of Naturally Occurring NS5A and NS5B Polymorphisms in Patients Infected with HCV Genotype 3a Treated with Direct-Acting Antiviral Agents

Hepatitis C virus (HCV) genotype (GT)3 is associated with increased risk of steatosis, development of cirrhosis and hepatocellular carcinoma. Limited data are available regarding genetic variability and use of direct-acting antiviral agents in these patients. non-structural protein 5A (NS5A) and non-structural protein 5B (NS5B) sequencing was performed on 45 HCV GT3-infected Italian patients subsequently treated with sofosbuvir ± daclatasvir (SOF ± DCV). Novel GT3a polymorphisms were observed by Sanger sequencing in three NS5A (T79S, T107K, and T107S) and three NS5B (G166R, Q180K, and C274W) baseline sequences in patients who achieved sustained virological response (SVR). Baseline NS5A resistance-associated substitutions (RASs) A30K and Y93H were detected in 9.5% of patients; one patient with A30K did not achieve SVR. Phylogenetic analyses of sequences showed no distinct clustering. Genetic heterogeneity of NS5A and NS5B was evaluated using ultra-deep pyrosequencing (UDPS) in samples longitudinally collected in patients not achieving SVR. Some novel NS5A and NS5B polymorphisms detected at baseline may not impact treatment outcome, as they were not enriched in post-failure samples. In contrast, the clinically novel GT3 NS5A-L31F RAS emerged in one treatment failure, and I184T, G188D and N310S, located on the same NS5B haplotype, became predominant after failure. These findings suggest a potential impact of these novel substitutions on the treatment outcome; however, their significance requires further investigation.

Ultrasensitive HCV RNA Quantification in Antiviral Triple Therapy: New Insight on Viral Clearance Dynamics and Treatment Outcome Predictors.

Objectives Identifying the predictive factors of Sustained Virological Response (SVR) represents an important challenge in new interferon-based DAA therapies. Here, we analyzed the kinetics of antiviral response associated with a triple drug regimen, and the association between negative residual viral load at different time points during treatment. Methods Twenty-three HCV genotype 1 (GT 1a n = 11; GT1b n = 12) infected patients were included in the study. Linear Discriminant Analysis (LDA) was used to establish possible association between HCV RNA values at days 1 and 4 from start of therapy and SVR. Principal component analysis (PCA) was applied to analyze the correlation between HCV RNA slope and SVR. A ultrasensitive (US) method was established to measure the residual HCV viral load in those samples which resulted “detected <12IU/ml” or undetectable with ABBOTT standard assay, and was retrospectively used on samples collected at different time points to establish its predictive power for SVR. Results According to LDA, there was no association between SVR and viral kinetics neither at time points earlier than 1 week (days 1 and 4) after therapy initiation nor later. The slopes were not relevant for classifying patients as SVR or no-SVR. No significant differences were observed in the median HCV RNA values at T0 among SVR and no-SVR patients. HCV RNA values with US protocol (LOD 1.2 IU/ml) after 1 month of therapy were considered; the area under the ROC curve was 0.70. Overall, PPV and NPV of undetectable HCV RNA with the US method for SVR was 100% and 46.7%, respectively; sensitivity and specificity were 38.4% and 100% respectively. Conclusion HCV RNA “not detected” by the US method after 1 month of treatment is predictive of SVR in first generation Protease inhibitor (PI)-based triple therapy. The US method could have clinical utility for advanced monitoring of virological response in new interferon based DAA combination regimens.

Lack of reduction in serum alpha-fetoprotein during treatment with direct antiviral agents predicts hepatocellular carcinoma development in a large cohort of patients with hepatitis C virus-related cirrhosis

Risk of hepatocellular carcinoma (HCC) in hepatitis C virus cirrhotic patients treated with direct‐acting antiviral agents (DAA) is still debating. We investigated it in a large cohort. The cohort comprised 1045 cirrhotic patients who completed treatment with DAA, with a median follow‐up of 17.3 months after end of treatment (EOT), including 943 patients without history of HCC and 102 previously treated for HCC. The majority were men (59.9%), with compensated cirrhosis (88.8%), genotype 1b (44.7%). Univariate, multivariate analysis and Kaplan‐Meier curves were performed to detect predictors of HCC in patients with and without reduction in alpha‐fetoprotein (AFP) during treatment. SVR12 was 95.6%. HCC developed in 95 (9.9%), including 54 of 943 (5.7%) occurrent and 41 of 102 (39%) recurrent tumours. De novo were more often unifocal (P = 0.01) and curable (P = 0.03). AFP decreased from 16.1 ± 36.2 mg/dL (baseline) to 11.4 ± 55 mg/dL (EOT). At univariate analysis, predictors were a previous HCC, older age, higher model for end‐stage liver disease, prolonged INR, lower platelets, baseline and EOT AFP, virological failure and no reduction in AFP during treatment. Kaplan‐Meier curves showed lower incidence of HCC in patients showing any reduction in AFP (P = 0.001). Those with AFP <6 ng/mL had the lowest risk (P = 0.0002). At logistic regression, platelets (P = 0.009, OR 0.99 CI: 0.99‐1.00), previous HCC (P < 0.000 01, OR: 10.76, 95% CI: 5.89‐19.34) and no reduction in AFP during treatment (P = 0.0005, OR: 2.98, CI: 1.60‐5.54) were independent predictors of HCC. In conclusion, risk of HCC after DAA treatment remains substantial. It is higher among patients with previous HCC, low platelets and without reduction in AFP during treatment.