HCV: Virology and Pathogenesis

Sun, Nov 02 - 8:00 AM

 

Extrahepatic Manifestations

 

993. Much of the HCV RNA in Infected Human Cells Is in Double-stranded RNA (dsRNA) Replication Complexes and Is Invisible to Standard Detection Methods. 

A. L. Klepper; J. Garber; J. L. Walewski; J. A. Gutierrez; T. D. Schiano; A. D. Branch.

Background:

HCV replication has been reported in extrahepatic cells, however, ability to detect HCV RNA in these cells often fluctuates. We tested the hypothesis that denaturation prior to RT/PCR is required to liberate HCV from replication complexes to allow detection.

 

Methods:

Mononuclear cells from ascitic fluid (AMCs;n=5) and PMBCs (n=1) were isolated, cultured for 1-8 wks, and RNA was Trizol extracted. Prior to RT/PCR, dsRNA was denatured by heating to 105 C. HCV RNA content was measured by PCR/gel electrophoresis using one of two assays: 1) amplifying nucleotides 234-992 using Superscript III System for a 40-cycle one-step PCR, followed by 35-cycle PCR using the HotStar Kit, or 2) A 5’UTR assay using Superscript III and random hexamers for reverse transcription followed by a 35-cycle amplification of nucleotides 77-324 using the HotStar Kit. Relative band intensities were determined (Scion Image). A TaqMan assay was performed using the LC RNA Master Kit, targeting nucleotides 62-309, with a FAM/BHQ probe (nucleotides 94-111).

 

Results:

4/5 of the AMC cultures tested using the sensitive PCR assay had detectable HCV RNA. All positive cultures showed increased HCV RNA after heating to 105 C. HCV RNA was detectable before and after heating in 3 samples, with a 4.5x increase (±3.2) in the heated samples. In the 4th, HCV RNA was detectable only after heating. To compare the reported Tm for dsRNA (≈99 C) to the temperature at which HCV RNA became detectable in this sample, unheated (UH) RNA was compared to RNA heated at 90, 100, and 105 C. HCV RNA was detectable only when heated at 100 and 105 C. We also compared heated vs. UH RNA from AMCs, liver, and PBMCs isolated from 3 separate HCV patients. Heating the RNA to 105 C produced a 3.2x boost in HCV RNA from AMCs, a 1.9x boost in liver, and a 6.2x boost in PBMCs. TaqMan PCR was used to compare the impact of heating RNA from liver and H77 full-length replicon cells, to H77 T7 genomic transcripts. Heating RNA to 105 C yielded 1.9x more detectable HCV RNA in heated vs. UH liver, and 2.8x more detectable HCV in heated vs. UH H77 full-length replicon cells. In contrast, heating single-stranded H77 T7 transcripts resulted in a 3.4x decrease in detectable HCV RNA.

 

Discussion:

These studies suggest that much of the HCV RNA human cells is in dsRNA complexes, answering a persistently vexing question in the investigation of (extrahepatic) HCV replication: Why it is so difficult to consistently detect HCV RNA in infected cells and human specimens? The answer: RNA is there, it is just present in structures that evade detection by conventional RT/PCR methods. (NIH DA016156/DK066939)

 


HCV Drug Pipeline – Drug Resistance

 

994. Dynamical Archiving - a Novel Mechanism for Post-Treatment Long Term Persistence of Resistant HCV Strains to Direct Anti-viral Drugs. 

A. U. Neumann.

Background:

Current paradigm assumes that HCV has no virological mechanism to archive mutations and that resistant strains (RES) have lower relative-fitness than wild-type strain (WT). Thus, once a direct anti-HCV drug is discontinued, any RES that evolved will decline back to baseline level and WT takes over. However, it was recently shown that RES to HCV protease-inhibitors persist in frequencies larger than baseline level up to 6 months post treatment. Here we present a novel mechanism that can allow HCV RES to persist in relatively high frequencies even after cessation of drug pressure.

 

Methods:

A new mathematical model combining intra-cellular HCV replication dynamics with cell infection dynamics is used. Intra-cellular RNA (ICR) forms replication units (RU), which in turn synthesize more ICR that is partially packaged and exported as virions. RES, both in term of RU and ICR, which have lower drug-sensitivity but also lower relative-replication-fitness compared to WT, evolve intra-cellularly. The model is generalized to allow a distribution of infected cells with different proportions of RES ICR and RU.

 

Results:

Simulation of the ODE model shows that if RES has developed during treatment then most infected cells harbor mostly RES RU and ICR. Once specific drug pressure is removed, WT starts to take over intra-cellularly due its higher relative-replication-fitness in the absence of the drug. However, replacement of RES RU will not be immediate and thus resistant virus will transiently continue to be exported. Since the envelope of RES virus did not change, RES has the same relative-de-novo-infection-fitness as WT. Therefore, RES still has high probability to infect new cells. Furthermore, when a cell is newly infected with RES, its lower relative-replication-fitness is not relevant since there is no WT competition. Thus, RES RU will be created and will synthesize resistant virus until reverse mutation produces enough WT to replace the RES RU. During that time, enough RES can be exported to infect new cells and repeat the cycle.

 

This dynamical archiving process, within a large range of parameters, allows for establishment of relatively high level of RES in equilibrium. Conditions that allow for dynamical archiving as function of relative fitness, drug sensitivity, reverse mutation rate and half-life of RU, were calculated. This was also confirmed by stochastic simulations.

 

Conclusions:

In contradiction to the current paradigm, resistant HCV strains can persist after the end of treatment with direct anti-virals by a process of dynamical archiving, which is first described here allowing to calculate the conditions necessary to prevent it.

 


HCV Drug Pipeline – Nitazoxanide

 

1000. Nitazoxanide Inhibits Hepatitis C Virus Replication In Vitro. 

T. Schaninger; J. Hong; G. G. Luo.

Background.

Nitazoxanide is a thiazolide anti-infective with activity against a number of protozoa, bacteria, and viruses. It is FDA approved for treatment of Cryptosporidium and Giardia. Nitazoxanide inhibits replication of hepatitis C virus, hepatitis B virus, and rotavirus in vitro. Based on its broad antiviral activity, the mechanism of action is likely through cellular processes rather than specific antiviral targets. Rossignol and colleagues recently reported that the use of nitazoxanide in combination with peginterferon alfa-2a with or without ribavirin among treatment-naive hepatitis C patients infected with genotype 4 significantly improved viral response compared to the standard of care (peginterferon alfa-2a plus ribavirin). The sustained virologic response was 79%, 61%, and 50% respectively.

 

Method.

We tested nitazoxanide and its active metabolite, tizoxanide, in HCV cell culture. Time of addition experiments were carried out in the infectious HCV genotype 2a (JFH1) cull culture model to investigate which steps of the replicative cycle are inhibited by nitazoxanide.

 

Result.

Nitazoxanide and tizoxanide exhibited potent antiviral activity against subgenomic HCV 1b replicon as well as infectious HCV genotype 2a (JFH1). Nitazoxanide and tizoxanide significantly inhibited HCV infection when they were added during the infection period. However, they showed reduced activity when added post infection.

 

Conclusion.

These findings indicate thiazolide affects the early stage of the HCV infection cycle. A proposed mechanism of action of nitazoxanide against HCV replication will be described and discussed.

 


HIV/HBV Coinfection

 

1002. HBV-HCV Coinfection: Insights from a Novel Model System. 

P. Bellecave; J. Gouttenoire; M. Gajer; V. Brass; G. Koutsoudakis; H. E. Blum; M. Nassal; R. Bartenschlager; D. Moradpour.

Background:

Coinfection with hepatitis B virus (HBV) and hepatitis C virus (HCV) has been associated with severe liver disease and frequent progression to liver cirrhosis and hepatocellular carcinoma. Clinical evidence suggests reciprocal replicative suppression of the two viruses ('viral interference'). However, virtually nothing is known about molecular interactions between HBV and HCV due to the lack of appropriate model systems.

 

Methods:

A tetracycline-regulated gene expression system was used to generate a panel of stable Huh-7 cell lines inducibly replicating HBV. These cell lines and control Huh-7 cell lines inducibly expressing the green fluorescent protein (GFP) were transfected with selectable HCV replicons or infected with cell culture-derived HCV (HCVcc).

 

Results:

Three successive transfection and selection steps allowed the establishment of Huh-7 cells inducibly replicating HBV and constitutively replicating subgenomic HCV RNA. In these cell lines, it is possible to regulate the expression of HBV proteins, HBV genome replication, and infectious viral particle formation by the concentration of tetracycline in the culture medium while HCV proteins are expressed and HCV RNA replicated constitutively. The presence or absence of replicating HBV did not influence the antiviral effect of interferon-alpha against HCV. In addition, specific inhibition of one virus did not significantly affect gene expression, replication and release of the other. Experiments using HCVcc did not reveal any significant superinfection exclusion of HCV by HBV. Finally, cell culture-adapted HCVcc isolated from long-term cultures did not display any HBV-specific adaptation.

 

Conclusion:

HBV and HCV can replicate in the same cell without any significant direct interaction. Therefore, the viral interference observed in vivo in coinfected patients is likely due to innate and/or adaptive host immune responses. These findings provide new insights into the pathogenesis of HBV-HCV coinfection and may contribute in the future to its clinical management.

 


Epidemiology/Transmission – General

 

1004. Investigating the Vertical Transmission and Evolution of Hepatitis C Virus between Mother and Infant Pairs. 

K. Goto; Y. Tanaka; T. Endo; K. Ito; A. A. Khan; M. Mizokami.

Background/Aim:

The incidence of mother-to-infant transmission is approximately 5–10% in infants born to HCV-RNA-positive mothers. This is an important route of HCV infection in childhood because HCV infection through blood transfusion has been effectively managed by blood donor screening. Many data are available for HCV evolution in adults but there is little information regarding the HCV evolutionary mechanism in mothers and their infants who got infection by vertical transmission. Our aims are to investigate the vertical transmission and characteristics of HCV evolution of mother–to-infant pairs.

 

Methods:

Seven Japanese mother-to-infant pairs with vertical transmission of HCV were studied. The 336-bp sequences in the NS5b gene, which is an essential viral replicating enzyme encoding the viral RNA-dependent RNA polymerase, were determined from serum samples at 2-6 points (~12.0 years intervals) in each patient. The sequences were phylogenetically analyzed and the evolution of HCV was compared between mothers and their infants.

 

Results:

The distribution of HCV subtypes in pairs was as follows: 3 pairs with 1b; 3 pairs with 2a; and 1 pair with 2b. The sequences obtained from each pair were clustering together in a phylogenetic tree, providing evidence of vertical transmission with a 97.3-99.7% identity of sequences of infants with their respective mothers. Examining the evolution of HCV in each patient, nucleotide substitutions within the NS5b region were identified in 4 of 7 mothers (0.26-4.46 x 10^-3 substitutions/site/year). Except for one infant who showed a nucleotide substitution rate of 0.37 x 10^-3 /site/year, substitutions were not found in other 5 infants during a follow-up of ~12.0 years.

 

Conclusions:

The study provides evidence of vertical transmission of HCV from HCV-RNA-positive mothers to their infants. Less evolution of HCV observed in infants may be due to low selective immunological pressure generated by the infant host, as compared to HCV evolution in adults.

 


HCV Drug Pipeline – Telaprevir

 

1011. Long term follow up of patients previously treated with telaprevir. 

N. Forestier; S. Susser; M. W. Welker; U. Karey; S. Zeuzem; C. Sarrazin.

Background:

Telaprevir (TVR) is a highly selective inhibitor of the hepatitis C virus NS3/4A protease with blocking of HCV replication in patients with hepatitis C. In this study, we describe the follow up status of patients who were initially treated with telaprevir. So far, little is known, about what happens after stopping treatment with TVR.

 

Methods:

We followed up patients with HCV who were treated in a phase 1b study for 14 days with TVR (monotherapy study, n=34) and in addition patients who were treated in another study for 14 days with either TVR alone, TVR+peginterferon or with placebo+peginterferon (combination study, n=20). Most of the patients from the monotherapy study received no further treatment after the 14 days treatment with TVR, however nearly all of the patients from the combination study received after the initial 14 days treatment with TVR standard of care treatment (SOC) with peginterferon (Peg-IFN) and ribavirin (RBV). Long term follow up of the patients was performed outside the studies and some patients were lost to follow up. We performed HCV-RNA testing as well as amplification and clonal sequencing of the NS3 protease gene in patients showing TVR resistant variants during treatment with TVR.

 

Results:

From the monotherapy study 5 patients were enrolled and all patients showed TVR resistant variants during the initial treatment with TVR. Patient 1 received no further treatment afterwards and 9 months after EOT HCV-RNA is 83000 IU/mL. Patient 2 received also no further treatment and HCV-RNA 1 year after EOT is 263000 IU/mL. Patient 3 received a 48 week course of Peg-IFN and RBV after the study and 24 weeks after the end of SOC treatment HCV-RNA is 590000 IU/mL. Patient 4 received no further treatment and 8 months after EOT HCV-RNA is 96000 IU/mL. Patient 5 also received no further therapy and 3 years after EOT HCV-RNA is 310000 IU/mL. From the combination study 16 from 20 patients were initially treated with TVR/TVR+Peg-IFN and 15 patients continued with SOC treatment. 10 patients remained HCV-RNA undectable after EOT with a SVR. From the relapsed patients we followed up 3 patients, all 3 patients showed TVR resistant variants during the initial TVR treatment as well as at the timepoint of relapse. In patient 1 HCV-RNA is 468000 IU/mL 24 weeks after EOT, in patient 2 HCV-RNA is 376000 IU/mL and in the last patient 253 000 IU/mL.

 

Conclusions:

Variable outcomes have been detected in patients treated with telaprevir alone or in combination with Peg-IFN for 14 days in phase 1 studies. A detailed virologic status of the patients as well as sequencing analyses results in these patients will be presented at the meeting.

 


HCV Treatment – Predictors of Treatment Response

 

1019. Hepatitis C Viral Genotype Age is a Predictor of the Clinical Response to Interferon Therapy. 

P. S. Pang; P. J. Planet; J. S. Glenn.

Purpose:

To determine why patients infected with the various hepatitis C viral genotypes require different durations of therapy and achieve different sustained viral response rates to interferon plus ribavirin therapy.

 

Methods:

The first phylogenetic analysis to include all available HCV genomic sequences was performed. The resultant tree was then used, in conjunction with a review of all prospective clinical trials that studied interferon and ribavirin therapy, to determine if a correlation existed between genomic age and clinical outcome. The viral loci potentially responsible for interferon treatment sensitivity differences were then screened for using ancestral protein sequence reconstruction.

 

Results:

A new cladogram of HCV was constructed, which, for the first time reveals the relative evolutionary ages of all the major HCV genotypes. We determined that genotype-specific clinical outcomes directly correlate with genotype age. This relationship allowed us to predict that genotypes 5 and 6, for which there currently are no published prospective trials, will likely have intermediate response rates, similar to genotype 3. Two viral loci that likely play a direct role in determining genotype-specific outcomes were also identified. Prior studies have independently identified these exact two loci as having a role in inhibiting the innate immune factor PKR.

 

Conclusion:

A primary determinant of genotype-specific response rates to interferon therapy is a set of viral factors that evolved to progressively acquire an increasing ability to inhibit the innate immune response.

 


HCV Disease Progression – Race

 

1022. Severity of Liver Disease in Chronic Hepatitis C Patients: Latino versus Caucasian. 

M. Y. Sheikh; M. H. Bashir; A. A. Afaq; H. Sadiq; J. Choudhury.

Background

Ethinicity can affect the natural history of chronic hepatitis C (CHC) and treatment response. Recently, Latino patients were noted to have a lower response to HCV treatment than non-Latino whites. Latinos were also noted to have more rapid estimated rate of fibrosis than Caucasians in the past. A recent Veterans study showed no difference in fibrosis rates in Latinos compared to Caucasians. The aim of this study was to evaluate the stages of fibrosis at the time of initial liver biopsy in chronic hepatitis C in Latino as compared to Caucasian.

 

Methods

The clinical data of 481 Caucasians and 472 Latinos with CHC were retrospectively evaluated between years 2000-06. METAVIR scoring system (stage0-4) was used to assess fibrosis. Scores of 0 was taken as no fibrosis, 1 and 2 as low-grade fibrosis and 3 and 4 were considered as high-grade fibrosis and cirrhosis respectively. Steatosis was assessed with Brunt Scores. Patients with co infections or other liver diseases were excluded.

 

Results

Of 481 Caucasians, 58.1% were males and 41.9% females with a mean age of 46.8 years and mean BMI of 28.6. Of 472 Latinos, 51.5% were males and 48.5% were females with a mean age of 45.6 years and mean BMI of 31.1. Of 481 Caucasian HCV patients, 80(16.6%) had stage 0, 183(38%) stage 1, 115(23.9%) stage 2, 22(12.9%) stage 3 and 41(8.5%) stage 4 fibrosis. Whereas, among 472 Latino HCV patients, 85(18%), 137(29%), 103(21.8%), 82(17.4%) and 65(13.8%) had stage 0,1,2,3 and 4 fibrosis respectively. Higher stages of fibrosis were noted in Latinos as compared to Caucasians (p=0.001)(Table). 90% of HCV patients were Genotype 1. Brunt scores for steatosis were significantly higher in Latinos as compared to Caucasians (p=0.001). There was no significant difference noted between the two ethnic groups based upon age, sex and BMI.

 

Conclusions

At the time of HCV diagnosis, Latinos have advanced stages of hepatic fibrosis as compared to Caucasians, indicating higher disease progression rates. There is a high prevalence of steatosis in Latinos as compared to Caucasians. Further studies are needed to evaluate various factors affecting the natural history in Latino patients with CHC

 

Ethnicity

0

1-2

3-4

Total

Caucasian #(%)

80(16.6%)

298(62%)

103(21.4%)

481(100%)

Latino # (%)

85(18%)

240(50.8)

147(31.1%)

472(100%)

Total # (%)

165(17.3%)

538(56.5%)

250(26.2%)

953(100%)

 


Disease Progression – Race

 

1031. Differential Gene Expression between Caucasian and African American Patients with Chronic Hepatitis C (CH-C). 

A. Afendy; M. Stepanova; A. Baranova; N. Hossain; I. Younossi; A. M. Wheeler; Z. M. Younossi.

Background:

African American (AA) with CH-C have lower response rates to pegylated interferon and Ribavirin (PEG-IFN/RBV).

 

Aims:

To compare gene expression of Caucasians (CA) with CH-C to those of AA with CH-C.

 

Methods:

CH-C patients who were scheduled to receive PEG-IFN/RBV (Standard doses and duration) were included in this analysis. Blood samples were collected prior to treatment as well as 24 hours, 7 days, 28 days and 56 days after the initiation of PEG-IFN/RBV treatment. From the peripheral blood mononuclear cells, total RNA was extracted, quantified and used for one step RT- PCR to profile 317 mRNAs (160 genes consisting of interferon-inducible, interferon pathway, immune response, and housekeeping genes). Expression levels of mRNAs were normalized with 6 “housekeeping” genes and a reference RNA. Differential gene expression between AA and CA cohorts was calculated for each gene using Wilcoxon non-parametric test (p-values not exceeding 0.05 after Benjamini-Hochberg correction were considered significant) and reported as CA/AA expression ratios.

 

Results:

A total of 53 CH-C patients were included in this analysis. Of these, 42 patients were CA and 11 were AA with CH-C (Age: 47.8±5.9, 58% male, 74% genotype 1). Overall sustained virologic response was 45% in CA and 18% in AA. Prior to treatment, AA patients had lower expression of the following genes: MMP9 (CA/AA: 1.373), STAT3 (CA/AA:1.563), PLAUR (CA/AA:1.503) and IRF-1 (CA/AA: 1.222). After 24 hrs of treatment, CA showed a higher expression of ILI16, IFITM1, GBP2, ADAR, MNDA, TLR2, PSMB8IKBKG, GTPBP2 AND PTEN genes. After 7 days of treatment, CA showed a higher expression of MMP9 gene. After 28 and 56 days of treatment, AA patients showed a higher expression of B2M and IFITM3; respectively.

 

Conclusions:

AA CH-C patients seem to have significantly lower expression of a number of genes involved in interferon signaling pathway, prior to and shortly after initiation of PEG-IFN and RBV. This data can provide important information to explain the lower responsiveness of AA to PEG-IFN and RBV.

 


HIV/HCV Coinfection

 

1047. Hepatitis C Virus (HCV) Specific CD4+ and CD8+ T-Cell Responses in Patients with Chronic Hepatitis C and the Impact of HIV Coinfection. 

N. I. Rallón; M. López; V. Soriano; J. García-Samaniego; M. Romero; P. Labarga; J. González-Lahoz; J. M. Benito.

Background:

Cellular responses against HCV seem to be low in most patients with chronic hepatitis C. They may be even further impaired in HIV patients. Herein, we examine the functional profile of HCV-specific T-cells and the impact of HIV coinfection in patients with chronic hepatitis C.

 

Methods:

HCV-specific CD4+ and CD8+ T-cell responses were examined in 30 interferon-naive patients, 10 HCV-monoinfected and 20 HIV/HCV-coinfected. The profile of cytokine production (IFN-γ, IL-2 and TNF-α) in response to 324 genotype-matched overlapping peptides spanning five HCV proteins (E2, p7, NS3, NS5a, NS5b) was measured using 5-colour flow cytometry. Differences between groups for the different immune parameters were tested using non-parametric tests.

 

Results:

CD4 counts and plasma HIV-RNA in coinfected patients were 390+213 cells/μl and 2.6+1.3 log copies/ml (65% of them were on HAART). Serum HCV-RNA in monoinfected and coinfected patients was 5.7+0.8 and 6.4+0.7 log IU/ml, respectively (p=0.03). Overall, 90% of monoinfected patients had genotype 1 whereas in coinfected patients 45% had HCV-1 and 55% had HCV-3.

 

The proportion of patients having a detectable CD4 or CD8 response against the five HCV proteins was high (>80%) and similar in both mono- and coinfected patients. Levels of total CD4 and CD8 responses against the five HCV proteins were also similar in both groups. The cytokine profile of HCV-specific CD4 and CD8 T-cells was dominated, in both groups, by cells producing only TNF-α. However, the contribution of single TNF-α+ cells to CD4 responses against NS3 and NS5a was significantly higher in coinfected than in monoinfected patients. In contrast, the contribution of cells producing only IL-2 into the CD4 response against the different proteins was higher in monoinfected than in coinfected patients, although differences did not reach statistical significance.

 

In HCV-1 patients there was a positive and significant correlation between the contribution of single TNF-α+ cells to CD4 responses against NS5a and serum HCV-RNA, after adjusting for HIV load (r=0.86, p=0.01).

 

Conclusions:

The functional profile of HCV-specific T-cells is limited to a single function and dominated by TNF-α. The ability of CD4 T-cells to produce IL-2 against HCV is impaired in HIV-coinfected patients. The greater level of single TNF-α+ cells contributing to the CD4 response in coinfected patients might explain at least in part a lower control of HCV replication in this population.

 


Disease Progression – HCC

 

1048. High Incidence of Hepatocellular Carcinoma associated with Hepatitis C Virus Genotype 3a in Pakistan. 

A. A. Khan; Y. Tanaka; Z. Azam; Z. Abbas; F. Kurbanov; S. S. Hamid; S. Jafri; M. Mizokami.

Background:

Infection with hepatitis C virus (HCV) genotype 1b is reportedly associated with higher incidence of hepatocellular carcinoma (HCC) than infection with other genotypes however, differences among HCV genotypes in pathogenicity are not clear and have not been proven. Aims: The study was conducted to investigate the etiologies of chronic liver diseases (CLDs) in Pakistan, where HCC incidence is reportedly rising.

 

Methods:

A total of 189 CLDs patients including 82 with HCC were enrolled in this study. HCV genotypes were determined by phylogenetic analysis in the NS5B region, and the epidemic history of HCV genotype 3a (HCV-3a) was examined using methods based on coalescent theory.

 

Results:

HCV-3a was the predominant genotype (81.4%) in the studied cohort, followed by 3b (9.3%), 3k (2.3%), 1a (1.5%), 1c (1.5%), 1b (0.8%) and 2a (0.8%). The mean HCV RNA levels were significantly higher in HCC and chronic hepatitis (CH) than in liver cirrhosis (LC) (p<0.00001) patients. The main predictors of HCC were high mean age (SD) 55.8 (+ 9.9) (p<0.0001), and male gender (p<0.001). Molecular evolutionary analysis disclosed a distinct phylogenetic cluster of HCV-3a in Pakistan {Fig.(a)}and estimation of the effective number of HCV infections dated HCV-3a in this region around 1920s and a rapid exponential growth in 1950s {Fig.(b)}. This indicates that the epidemic spread of HCV-3a occurred earlier in Pakistan than in other countries from which this genotype has been reported.

 

Conclusions:

HCV-3a having earlier epidemic spread in Pakistan might be associated with rising incidence of HCC. The duration of infection rather than HCV genotype is associated in development of HCC.

 

 


HIV/HCV Coinfection

 

1053. Immune restoration and HCV quasispecies evolution in HIV coinfected patients.  

P. Vaghefi; G. Maurin; D. Lavillette; C. Feray; E. Dussaix; A. Roque-Afonso.

Introduction:

HCV genetic variability is due to the high error rate of the viral RNA polymerase and to negative and positive selection pressures. We investigated the contribution of immune selection pressure on HCV quasispecies evolution by studying the hypervariable region (HVR) in HIV co-infected subjects upon immune restoration.

 

Patients and methods:

Twenty eight patients were studied at 2 time points: 15 patients before and after antiretroviral treatment initiation with a mean interval of 24.3 +/- 10.6 months and 13 non-treated patients with a mean interval of 18.1 +/- 12 months. Quasispecies complexity and its qualitative modifications were assessed by SSCP and by cloning and sequencing. Analysis was performed according to CD4 cell count, HIV and HCV viral load, HCV genotype, aminotransferases levels (ALT) and neutralizing activity of sera measured by HCV pseudoparticles (HCVpp) experiments.

 

Results:

In the treated group, CD4 cell count increased and HIV viral load decreased significantly. A trend towards increased neutralizing activity of sera was also observed. No significant change in HCV viremia and quasispecies complexity was noted in both groups. Complexity was higher in the 14 patients infected by HCV genotype 1, particularly at the 2nd time point (5.57 versus 3.5), independently of the viral load. Emergence of variants and global modification of quasispecies (emergence + disappearing) was correlated to the initial level of ALT (r=0.53). Disappearing of variants was correlated to the raise of CD4 counts (r=0.48) and the raise of neutralizing activity (r=0.58). Phylogenetic analysis in 4 patients was consistent with SSCP results and showed a profound shift of quasispecies in 3 cases, associated in 1 case with a ratio of non-synonymous /synonymous mutations (dN/dS) >1.

 

Conclusion:

We had previously shown that qualitative variation of HVR was not related to CD4 cell count and depended on the initial complexity (Roque-Afonso, J Infect Dis 2002). Immune restoration, reflected by CD4 counts increase, appears to be associated with an increase of neutralizing antibodies titer, allowing the elimination of part of circulating variants, without any impact on HCV viremia. In association with humoral response, the cellular immunity also contributes to the quasispecies evolution as suggested by the link between liver cytolysis and variants emergence.

 


Disease Progression

 

1057. Natural history of HCV superinfection and reinfection. 

P. A. White; J. Grebely; J. K. Flynn; G. Matthews; M. K. Renkin; B. Yeung; W. Rawlinson; J. Kaldor; M. Hellard; A. R. Lloyd; R. A. Ffrench; G. J. Dore.

Purpose:

To characterise the natural history of HCV superinfection and reinfection.

 

Methods:

ATAHC is a prospective study of the natural history and treatment of acute/early chronic HCV. Treated subjects received PEG-IFN alfa-2a (24 weeks). Secondary infection was defined as new HCV infection among treated subjects with persistent HCV viremia (superinfection) and treatment-induced HCV virologic suppression (2 RNA < 10 IU/ml) or spontaneous HCV clearance (reinfection). Envelope glycoprotein 1 and hypervariable region 1 were amplified using RT-PCR. Amplicons were sequenced and nucleotide divergence calculated to confirm secondary infection. HCV-specific T cell responses were tested in PBMCs collected 12 weekly during follow-up and assessed by HCV peptide ELISpot and multiplex in vitro cytokine production assays.

 

Results:

167 subjects were enrolled, 107 received treatment. Nine cases of superinfection (n=5) and reinfection (n=4) were identified, including 7 during (n=3, all superinfection) or following treatment (n=4, all reinfection). 8 cases had ongoing injecting reported, 5 had temporal ALT flares >400 U/L and 8 were infected with genotypes different from the primary strain (1 with divergent G3a strain, 10% sequence diversity). Broad and strong T cell responses to HCV G1a proteins were observed in 1 subject with initial G3a infection with spontaneous clearance upon reinfection with G1a (Figure), while weak or non-existent proliferative responses were observed in the 3 reinfection subjects without clearance. Clearance of HCV superinfection was observed in 2 subjects. One subject demonstrated three genotype switches (2b - 1a - 3a - 1) during and following treatment.

 

Conclusion:

These findings describe a heterogeneous natural history of HCV superinfection and reinfection, consistent with chimpanzee studies. Protection from viral persistence upon reinfection can occur, but is not universal.

 

 


Disease Progression

 

1062. Hepatitis C as a risk factor for primary renal cell carcinoma. 

S. C. Gordon; D. Moonka; K. a. Brown; M. Y. Huang; S. E. Anteau; L. Lamerato.

Background:

Chronic HCV mediates the development of HCC and various lymphoproliferative diseases. We observed a higher than expected prevalence of HCV infection among patients with primary renal cell carcinoma (RCC) by examining our health system cancer registry. We found that 42/724 (5.8%) consecutive RCC cases tested for anti-HCV were antibody positive (HCV+). We therefore sought to determine whether HCV confers an increased risk for the development of RCC.

 

Methods:

We used administrative data from a large integrated health care system to explore the incidence of RCC among HCV infected persons. Adults (over 18 yrs) testing HCV+ between 1997 and 2006 were compared to a control cohort testing anti-HCV negative (HCV-) during the same time period; HBsAg and HIV positive patients were excluded. Data from the system’s registry identified all RCC pts diagnosed between 1997 and April 2008. HCC, prostate and colorectal neoplasms were examined as controls. We used logistic regression to calculate the unadjusted Odds Ratio (OR) with 95% confidence intervals (CI) for HCV status and cancer, and multivariate models adjusting for age, gender and race.

 

Results:

The cohort consisted of 74,570 pts with 12.6% HCV+ (n=9,401) and 65,169 HCV negative. Males and African Americans (AA) had a higher prevalence of HCV (15.4% males vs. 9.8% females, p<0.001 and 16.4% AA vs. 9.8% non-AA, p<0.001). The mean age of HCV+ patients was older than the HCV- (52 vs. 48, p<0.001). There were 163 RCC patients (0.25/100) in the unaffected vs. 35 (0.37/100) in the HCV+ group. The unadjusted OR for RCC was 1.49 (CI: 1.03, 1.83, p=0.03). The mean age at RCC diagnosis was much younger in HCV+ individuals (52 vs. 63, p<0.001). When adjusting for age, race and gender the overall OR for HCV among RCC patients was 1.2 (CI: 0.83, 1.74, p=0.34). However, at particular risk were males less than age of 50 at HCV diagnosis, where the adjusted OR for RCC was 4.8 (CI: 2.48, 9.17, p<0.001). As expected, the risk of HCC among HCV+ patients was greatly increased (adjusted OR = 12.52, CI: 9.52, 16.48, p<0.001), without increased risk for colorectal or prostate cancer.

 

Conclusions:

Chronic HCV is associated with a higher incidence of primary renal cell carcinoma than non-infected persons, and this risk is particularly high, with nearly 5-fold increase, among younger males. Potential limitations include unmeasured risk factors and a referral bias at a tertiary medical center; larger studies are required to confirm these findings.

 


Extrahepatic Manifestations

 

1066. Is HOMA-IR the best marker of insulin resistance in chronic hepatitis due to HCV infection? 

R. Narita; M. Hiura; S. Abe; Y. Kihara; A. Tabaru; M. Otsuki.

Background and Aims:

Recent epidemiological studies have suggested that hepatitis C (HCV) infection is associated with an increased risk of development of type 2 diabetes mellitus (DM). Elucidation of the relationship between chronic HCV infection (CHC) and insulin resistance (IR) is of great clinical relevance, because IR promotes liver fibrosis and exerts some influences on the response to antiviral therapy. The hyperinsulinemic-euglycemic clamp is the gold standard for measuring whole-body IR. However, the insulin clamp is time consuming and difficult to perform, and cannot be applied in ordinary clinical practice or in large-scale epidemiological studies. The index of IR is usually calculated basis on the fasting serum glucose and insulin leveals, according to the homeostasis model assessment (HOMA-IR). In this study, we determined plasma glucose and insulin concentrations during the oral glucose tolerance test (OGTT) to evaluate whether the HOMA-IR is the best marker of IR in CHC.

 

Methods:

We enrolled a total of 202 CHC patients in this study, and performed liver biopsy and 75g OGTT in all of these patients. We calculated the total area under curve (AUC) of serum glucose and insulin for 30 and 180 min after glucose loading. The degree of necroinflammatory activity and fibrosis of the biopsy specimen were scored semiquantitatively, as described by Scheuer. Liver fibrosis was quantified by the image analysis. Pearson’s correlation coefficient was used to study the relationship between HOMA-IR and other data.

 

Result:

There was a significant correlation between [AUC]0-180 min of glucose or insulin and HOMA-IR. Pearson’s correlations coefficient (r) and the P values for these correlations were r = 0.334 and 0.562, and P<0.0001 and <0.001 respectively. Matsuda index [104/√(mean insulin after OGTT x mean glucose after OGTT x fasting glucose x fasting insulin)] strongly correlated with HOMA-IR (r = 0.855, P<0.0001). Glucose 0-30[AUC] x insulin 0-30[AUC], which reflects an index of hepatic resistance to insulin, had a significant but much weaker correlation with HOMA-IR (r = 0.534, P<0.0001).

 

Conclusion:

Although HOMA-IR is an useful index of IR, determination of hepatic insulin resistance using of HOMA-IR is not feasible in CHC patients.

 


Disease Progression – HCC

 

1073. Ethanol and acetaldehyde enhance hepatitis C virus replication in human hepatoma cells supporting infectious virus production. 

S. Seronello; C. Ito; T. Wakita; J. Choi.

Introduction:

Hepatitis C virus (HCV) causes severe alteration of the host redox status, and ethanol is a well-known cofactor in the hepatopathogenesis induced by HCV. Ethanol metabolism generates reactive oxygen species (ROS) and other metabolites, including acetaldehyde. The mechanism of how oxidative stress and ethanol interact with HCV to induce liver diseases remains unclear. However, studies have shown that ethanol can modulate HCV titer in patients as well as the HCV RNA level in vitro, in Huh7 human hepatoma cells that continuously support the subgenomic HCV RNA replication without virus production, suggesting that ethanol enhances HCV replication both in the presence and absence of the complete viral lifecycle.

 

Methods:

In this study, we use JFH1 strain that produces infectious virus particles in Huh7 cells to demonstrate that ethanol, at subtoxic and physiologically relevant concentrations, elevates HCV RNA level in the context of the complete lifecycle of HCV. Ethanol also increases HCV RNA in the subgenomic JFH1-Luc (genotype 2a) and Con1-Neo (genotype 1b) RNA-transfected cells, suggesting that the HCV RNA genome replication is affected, independent of the genotype. Acetaldehyde likewise elevates HCV RNA at physiological concentrations. In contrast, decreasing intracellular glutathione (GSH), a major endogenous antioxidant defense, with L-buthionine S,R-sulfoximine (BSO) decreases HCV RNA's, suggesting that endogenous oxidative stress suppresses HCV replication, and this suppression is attenuated by GSH ethyl ester, but not exogenous GSH. H2O2, generated with bolus H2O2 or glucose oxidase plus glucose also decreases HCV replication, and BSO enhances the suppression by H2O2.

 

Conclusion:

Therefore, ethanol is likely to enhance HCV replication through acetaldehyde rather than ROS. As HCV titer is associated with liver diseases and adversely affects patients' responses to antiviral therapy, increased viral replication may constitute a key mechanism of pathological interactions between ethanol and HCV.

 


Extrahepatic Manifestations

 

1075. Hepatitis C virus infection enhances insulin resistance induced by visceral fat accumulation. 

Y. Eguchi; T. Mizuta; E. Ishibashi; T. Eguchi; A. Matsunobu; N. Oza; S. Nakashita; Y. Kitajima; H. Takahashi; Y. Kawaguchi; R. Iwakiri; I. Ozaki; N. Ono; K. Fujimoto.

Background:

Recently, an association between hepatitis C virus (HCV) infection and insulin resistance (IR) has been noted. Although visceral fat accumulation is closely related to IR in subjects with metabolic syndrome and nonalcoholic fatty liver disease (NAFLD), the possible effect of HCV on visceral fat accumulation remains unclear.

 

Aim:

To evaluate the relationship between IR and visceral fat accumulation in HCV-infected patients and then compared the incidence of IR between HCV-infected patients and NAFLD patients with or without visceral fat accumulation.

 

Methods:

A total of 87 HCV-infected patients in comparison with 125 sex- and age-matched patients with NAFLD were recruited. In order to exclude a confounding effect of advanced hepatic fibrosis on IR, the patients were limited to HCV patients with fibrosis stages 1 and 2 (n=87; 39 males and 48 females) and platelet counts of >15 x 104 /mm3. The patients were included if they fulfilled the following criteria: absence of diabetes mellitus; absence of markers of hepatitis B virus; no evidence of autoimmune liver disease or alcoholic liver disease (>20 g of alcohol per day). We evaluated the degree of visceral fat area (VFA; cm2) at the umbilical level was measured by computed tomography and divided into two grades: no visceral obesity, VFA<100; visceral obesity, VFA≥100. IR was evaluated by homeostasis model assessment of IR (HOMA-IR) and the quantitative insulin sensitivity check index (QUICKI). Pancreatic beta-cell function was evaluated by homeostasis model assessment of beta-cell function (HOMA-beta). Serum soluble TNF-receptors 1 and 2 and adiponectin were measured.

 

Results:

Body mass index, waist circumference, VFA, total cholesterol, triglyceride and fasting plasma glucose were significantly higher in NAFLD patients than in HCV-infected patients, whereas aminotransferase and HOMA-beta were significantly higher in HCV-infected patients than in NAFLD patients. HOMA-IR and QUICKI was correlated with visceral fat accumulation, and higher in HCV patients than in NAFLD patients with visceral obesity. HOMA-beta was higher in HCV patients than in NAFLD patients for each VFA grade. There was no difference between the levels of adiponectin in HCV-infected patients and NAFLD patients with visceral obesity. Serum soluble TNF-receptor 1 and 2 were higher in HCV patients than in NAFLD patients with visceral obesity.

 

Conclusions:

These results indicate that HCV infection is a risk factor for development of IR, particularly in patients with visceral obesity. The progression of IR may be enhanced by an increase in TNF activity in HCV-infected patients with visceral obesity.

 


Extrahepatic Manifestations

 

1084. Microsomal triglyceride transfer protein (MTP) - 493 G/T polymorphism is associated with insulin resistance in chronic hepatitis C (CHC) genotype 1 infection. 

É. F. Siqueira; C. P. Oliveira; M. Corrêa-Giannella; J. Stefano; A. Cavaleiro; M. Z. Fortes; M. C. Muniz; K. A. Silva; F. S. Silva; L. B. Pereira; F. J. Carrilho.

Background & Aims:

Chronic hepatitis C (CHC) infection has been shown to promote insulin resistance and hepatic steatosis independent of host metabolic factors. Hepatic steatosis affects 40–86% of patients with CHC and leads to increased disease progression and decreased response to antiviral therapy. Besides, reduced hepatic MTP activity resulting in fatty liver could contribute to the severity of hepatic steatosis in CHC. The G allele of a common functional polymorphism in the MTP gene promoter, −493 G/T has been associated with lower gene transcription than the T allele. We therefore investigated this polymorphism in the MTP gene and its relation with metabolic and histological variables in patients with CHC.

 

Methods:

Eight six untreated patients with HCV RNA and liver biopsy proven CHC were genotyped for the − 493G/T MTP polymorphism. Genomic DNA was extracted from peripheral blood cells and the promoter region of the MTP gene was amplified by PCR. The – 493 G/T polymorphism was determined by the PCR-based restriction fragment length polymorphism. All patients were negative for markers of Wilson’s disease, hemochromatosis and autoimmune diseases and had current and past daily alcohol intake less than 100g/week. The patients were divided according to HOMA-IR (Homeostasis model assessment-insulin resistance) into two groups: HOMA >2.1 and HOMA<2.1. A set of metabolic and serum lipid markers were also measured at the time of liver biopsies.

 

Results:

MTP genotype GG/GT is associated with insulin resistance (HOMA > 2.1) in HCV genotype 1- infected patients (OR 5.2; CI (1.20 – 25.44) whereas this association was not observed in patients with HCV genotype 3-infected patients. No association was found between the polymorphism and the degree of steatosis and fibrosis in both HCV genotype.

 

Conclusion:

Our results indicate that the presence of G allele of MTP − 493G/T polymorphism is associated with insulin resistance in patients infected with HCV genotype 1 and this genetic aspect, which is possibly associated with a lower MTP hepatic expression, could be important to increased disease progression and decreased response to antiviral therapy.

 


Disease Progression – HCC

 

1087. Hepatitis C virus proteins can modulate microRNA expression and chemotherapeutic responses in Hepatocellular Carcinoma (HCC). 

C. Braconi; F. Meng; N. Huang; T. Suzuki; C. Taccioli; C. Croce; T. Patel.

Background:

HCC is a highly chemoresistant tumor. In order to develop more effective therapies, knowledge of the determinants of chemoresistance is essential. We have recently shown that alterations in selected miRNA can modulate tumor cell responses to chemotherapy. Hepatitis C (HCV) is the major cause of HCC in the USA. The role of HCV viral proteins in tumor behavior are unknown and we postulated that modulation of host cell miRNA expression by HCV viral proteins could contribute to chemoresistance in HCV associated HCC.

 

Methods:

HepG2 human HCC cells were stably transfected to express the entire open reading frame of HCV genotype 1b genome (Hep-394) or empty vector controls (SWX). Chemosensitivity to sorafenib and doxorubicin was assessed using a viable cell assay. microRNA expression profiling was performed using a microarray and transfection with pre-miRs was performed by nuclear transfection. Western blotting for PARP and Mcl-1 was performed in total cell lysate and for cytochrome C in cytosolic fractions obtained by ultracentrifugation. Caspase 3/7 activation was assessed using a luminometric assay.

 

Results:

Compared to controls, HCV-positive Hep-394 cells had a lower IC50 to sorafenib (1.0 +/- 0.1 vs 3.5+/- 0.8 μΜ) or doxorubicin (0.8 +/- 0.1 vs 1.7 +/- 0.1 μΜ). Compared to controls, HCV positive cells had significant alterations in expression of selected miRNA, with 10 miRNA (3.1% of total) > 2-fold down-regulated and 27 miRNA (8.4% of total) > 2-fold up-regulated (p<0.01). Of these, miR-193b and miR-29b were prominently increased in expression (by 3.6 and 1.8-fold respectively), whereas miR-206 and miR-627 were decreased (by 5.0 and 3.0-fold respectively) in HCV-positive HCC cells. Interrogation of target prediction databases identified the anti-apoptotic protein Mcl-1 as a putative target of both miR-193b and miR-29b. The expression of Mcl-1 was decreased in HCV-positive cells, compared to controls. Moreover, there was an increase in caspase-3/7 activity, PARP cleavage, and cytosolic cytochrome C release in HCV-positive cells compared to controls under basal conditions and during incubation with 10 μΜ sorafenib. Both Mcl-1 expression and the IC50 to sorafenib was decreased in HCV positive cells transfected with precursors to miR-193b compared to control precursor miRNAs (2.1 +/- 0.1 vs 5.5 +/- 1.0 μΜ, p< 0.01).

 

Summary and Conclusions:

Cellular expression of HCV proteins increases sensitivity to sorafenib by miRNA-dependent modulation of Mcl-1 and cellular apoptosis. We conclude that enhancing the miRNA responses involved in response to chemotherapy may be useful for individualized therapy in HCC based on etiology.