February 29, 2012

Hepatitis C vaccine signals landmark University of Alberta discovery

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(Photo by Julianna Damer/The Gateway)

Last updated: February 29, 2012 5:23 pm

Vaccine could protect against all forms of virus

Andrew Jeffrey — The Gateway (University of Alberta)

EDMONTON (CUP) — A University of Alberta team has made a breakthrough in hepatitis C research, creating a vaccine that could potentially combat all forms of the liver-destroying virus.

The vaccine was developed by Michael Houghton, a U of A researcher who first discovered the hepatitis C virus (HCV) in 1989. The vaccine exposes the human body to a non-infectious sub-unit of the HCV so that it can begin developing antibodies to protect against the virus. These antibodies are able to cross-neutralize against the seven genotypes of the virus.

“Previously, many people believed that the virus was impossible to neutralize with just one type of vaccine because there are so many different genotypes,” explained John Law, a member of the U of A research team.

“This is a proof of principle finding — showing that we can make a vaccine that can allow cross protections of many different varieties of the hepatitis C virus.”

Houghton began developing a vaccine more than a decade ago, and was recruited by the U of A’s Li Ka Shing Institute of Virology three years ago to continue his research. Law attributes much of the success of their team to Houghton’s dedication.

“We didn’t really make anything special. Mostly it’s been the efforts of Dr. Houghton. We’ve persisted and been able to go through the hurdles of finally getting the samples, getting the trial, and eventually testing the results and being able to find out the answer,” Law said.

“He stayed with his idea and eventually carried it out to a point where now we can see there is a very good potential for things that are going to happen.”

Law said an obstacle in creating the vaccine is the HCV’s ability to mutate quickly and exists in a variety of genotypes, similar to AIDS.

Law predicts that it will still take another five to seven years before the vaccine is ready to be released. The research has only completed the first of three phases needed for the FDA to approve the vaccine. Although its safety has already been tested, the vaccine will require further testing in a clinical setting.

The vaccine has already been presented by the research team to various other members of the virology community.

Last weekend, the team travelled to Montreal to present their findings at a Canadian symposium for hepatitis C. Law is hopeful that the team can improve upon their vaccine further before releasing it to the public.

“We’re basically trying to understand those antibody responses, and trying to find out which part of the sub-unit the antibody recognizes. There might be some common area between the genotypes that the antibody can see that is therefore blocking infections of HCV,” Law explained.

“We can maybe learn the mechanism to increase the efficacy of the vaccine and be able to design a better vaccine and move forward.”

Source

Also See: The Scientist Who Discovered Hepatitis C Says He’s Now Discovered the Vaccine

Hepatitis C Virus Persistence After Sustained Virological Response to Antiviral Therapy in Patients With or Without Past Exposure to Hepatitis B Virus

From Journal of Viral Hepatitis

T. N. Q. Pham; C. S. Coffin; N. D. Churchill; S. J. Urbanski; S. S. Lee; T. I. Michalak

Posted: 02/28/2012; J Viral Hepat. 2012;19(2):103-111. © 2012 Blackwell Publishing

Abstract and Introduction
Abstract

Hepatitis C virus (HCV) and hepatitis B virus (HBV) frequently coinfect and persist long after clinical resolution. We assessed the incidence of low-level (occult) HCV infection (OCI) after sustained virological response (SVR) to standard anti-HCV therapy in individuals with or without past exposure to HBV to recognize whether HBV could influence the prevalence of OCI, HCV level and hepatic histology. Plasma and peripheral blood mononuclear cells (PBMC) were collected from 24 individuals at 6- to 12-month intervals for up to 72 months after SVR. Liver histology was available for nine patients. HCV and HBV genomes were detected with sensitivity <10 genome copies/mL. In individuals without HBV exposure (n = 15), comprehensive analyses of sequential plasma and PBMC samples revealed HCV RNA in all 15 cases (75% plasma and 61% PBMC). In the group with HBV exposure (n = 9), evidenced by circulating anti-HBc and/or HBV DNA detection by a highly sensitive assay, HCV RNA was identified in all cases (83% plasma and 59% PBMC), at levels similar to those in HBV nonexposed individuals. In both groups of patients, most liver biopsies included those reactive for viral genomes displayed low-grade inflammation (8 of 9) and fibrosis (7 of 9). Sequence polymorphisms at the 5`-UTR between PBMC and liver or plasma, as well as circulating HCV virion-like particles, were observed in patients with or without HBV exposure. In conclusion, the prevalence of OCI after SVR is comparable in individuals with or without past exposure to HBV. HCV loads and liver alterations in OCI appear to be unaffected by low-level HBV DNA carriage.

Introduction

There are at least 370 million people chronically infected with hepatitis B virus (HBV) and 170 million of those with chronic hepatitis C virus (HCV) infection worldwide. The chronic hepatitis induced by the viruses can lead to fibrosis, cirrhosis and hepatocellular carcinoma (HCC).[1,2] However, while 90–95% of adults infected with HBV spontaneously resolve acute hepatitis, up to 85% of those infected with HCV develop chronic hepatitis C (CHC).[3,4]

Although hepatocytes are major targets of HBV and HCV, both pathogens can also propagate in the cells of the immune system, as evidenced by the presence of their genomes and respective replicative intermediates and proteins in the lymphatic organs and peripheral blood mononuclear cells (PBMC) of patients with chronic hepatitis B (CHB) and C.[5–10] The existence of small amounts of HBV and HCV genomes in plasma, PBMC or liver after clinical resolution of hepatitis highlighted the natural propensity for these two viruses to induce a persistent infection.[11–15] Occult HBV infection (OBI) is defined as detection of HBV DNA in liver, plasma and/or PBMC in the absence of hepatitis B surface antigen (HBsAg) in serum. Occult HCV infection (OCI) is characterized by low-level HCV RNA persisting in plasma, liver or PBMC in anti-HCV antibody-reactive individuals who resolved hepatitis C either spontaneously or after antiviral treatment or seemingly anti-HCV antibody-negative individuals with infection of unknown aetiology.[14–17] Although the existence of OBI has been generally accepted, that of OCI remains controversial in that HCV persisting in plasma, liver and/or immune cells after resolution of hepatitis C has not been observed by all investigators. This inconsistency is likely related to variations in the sensitivity of HCV RNA detection assays employed, processing of clinical samples, the number of samples tested and the amount of material used for analysis.[18] Because of their shared modes of transmission, coinfection with HCV and HBV is common, particularly in high-risk populations and in areas considered to be endemic for both viruses. Molecular and pathological consequences of interactions between HCV and HBV in coinfected patients are not fully elucidated given the relatively contradicting data that are available. On one hand, acute HCV infection in the context of CHB and vice versa acute HBV infection or OBI in CHC have been associated with a more active liver disease and a greater likelihood of developing cirrhosis and HCC.[19–24] Along this line, HBV superinfection has been correlated with an assumed complete clearance of serum HCV RNA, or even both HCV RNA and HBV DNA, in patients with CHC.[24–26] The reverse was also true for HCV superinfection.[22,24] In cell culture studies, HCV core or nonstructural proteins (e.g. NS5A) were found to repress HBV DNA synthesis.[27–29] On the other hand, in arguing against the negative influence of one virus over the other, other investigations demonstrated not only a coexistence of hepatic HBV and HCV in coinfected patients,[30,31] but also a simultaneous replication of HCV and HBV within the same hepatoma cell.[32,33]

Nevertheless, given the lymphotropic propensity of HCV and HBV, what remained undefined was whether HCV and HBV could affect each other's ability to propagate in immune cells, especially in the context of occult infection. Such recognition would be important in furthering our understanding of the full extent of viral persistence. With this in mind, the current study examined whether HBV exposure would influence the prevalence of HCV occult infection, the level of persisting HCV and liver pathology in patients with clinical resolution of CHC.

Patients, Clinical Samples and Methods
Patients and Clinical Samples

Twenty-four patients determined to have resolved CHC according to clinical and laboratory assay criteria were randomly selected for the study. They were followed at the University of Calgary Liver Unit, Alberta, Canada. These individuals had achieved a sustained virological response (SVR) for duration of up to 6 years following treatment with interferon alpha (IFN) or pegylated IFN S1). None of them was subjected toand ribavirin (p-IFN/R) (Supplementary Table immunosuppressive or anticancerous therapy during follow-up. Liver function tests were repeatedly normal and serum HCV RNA negative by Roche Amplicor HCV v2.0 assay (sensitivity 500 IU/mL or 1000 virus genome equivalent (vge)/mL; Roche Molecular Diagnostics, Pleasanton, CA, USA). All patients were reactive for antibodies to HCV (anti-HCV) by immunoassays (Abbott Diagnostics, Mississauga, Canada) at the time of the first blood sample collection. Clinical charts revealed that eight of them were also positive for antibodies to HBV core S1), indicative of a past(HBV-C) antigen (anti-HBc) (Supplementary Table exposure to HBV, by the Abbot Corzyme assay (Abbott Laboratories, North Chicago, IL, USA). One individual was negative for anti-HBc while positive for antibodies to HBsAg (anti-HBs); however, plasma and PBMC from this person were HBV DNA reactive when tested by research polymerase chain reaction (PCR) assays employed in this study.

Plasma and corresponding PBMC were available from all 24 subjects (Supplementary Table S1). Up to three plasma and PBMC pairs were collected from 21 individuals every 6–12 months, while a single sample pair from the remaining three was obtained at 18 months post-SVR. Liver biopsies were available before and after therapy (up to 60 months post-SVR) for eight individuals and only after SVR for an additional patient. The study protocol was approved by local Human Investigation Committees. All patients provided written informed consent to participate in the study.

Preparation of Peripheral Blood Mononuclear Cells and Cell Cultures

Peripheral blood mononuclear cells were isolated by Ficoll-HyPaque (Pharmacia Biotech, Quebec, Canada) gradient fractionation and cultured, if required, for 72 h in the presence of phytohemagglutinin (PHA; 5 μg/mL; Sigma, Mississauga, Canada) and interleukin-2 (IL-2; 20 U/mL; Roche) prior to analysis for HCV RNA or HBV DNA.[14]

Nucleic Acid Extraction

RNA was usually extracted from 250 μL of plasma using Trizol LS (Invitrogen Life Technologies, Burlington, Ontario, Canada) or 1 × 107 naive (uncultured) PBMC using Trizol (Invitrogen).[14] DNA was extracted[34] from the same volume of plasma or number of PBMC. When necessary, extraction of 1-mL sample of test plasma was carried out and the nucleic acid analysed. If the sample remained virus nonreactive, 4 mL of plasma was ultracentrifuged at 40 000 rpm for 20 h at 4 °C and nucleic acid extracted from the resulting pellet was tested for HCV RNA or HBV DNA.

Hepatitis C Virus RNA-positive and Hepatitis C Virus RNA-negative Strand Detection

RNA from PBMC (1–2 μg RNA; equivalent of 1–2 × 106 cells), plasma (equivalent of 250 μL, 1 mL or 4 mL) or liver tissue (2 μg RNA) was analysed by reverse transcriptase-polymerase chain reaction (RT-PCR) for HCV RNA-positive strand using HCV 5'-untranslated region (5'-UTR)-specific primers, cycling conditions and quantification standards reported earlier.[14] In all instances, specificity of amplicons was confirmed by nucleic acid hybridization (NAH) using 32P-labelled recombinant HCV 5'-UTR-E2 fragment as a probe.[14] Sensitivity of this assay is <10 vge/mL or <2.5 vge/reaction. HCV RNA-negative strand was detected by strand-specific RT-PCR/NAH using rTth DNA polymerase.[14] This assay detects ~100 copies of the correct (negative) strand, while nonspecifically identifying ≥106 vge of the positive strand.[14] Specificity of PCR amplicons and validity of the controls were confirmed by NAH. In every analysis, we included a number of negative and contamination controls, as reported.[14] Briefly, for each group of samples subjected to nucleic acid extraction, a mock sample containing no patient material was extracted and analysed with test samples. In the PCR step, a water sample and a mock (no test cDNA) were amplified as controls.

Hepatitis B Virus DNA Detection

Hepatitis B virus DNA in plasma, PBMC or liver was detected by PCR/NAH using primers specific for HBV-C and X (HBV-X) genes (GenBank accession number X72702). For first-round amplification, the primers were as follows: 1847-TGTTCATGTCCCACTGTTCAAGC (HBV-C outer sense), 2274-AAGATAGGGGCATTTGGTGG (HBV-C outer antisense), 1266-CCATACTGCGGAACTCCTAGC (HBV-X outer sense) and 1779-ACAGACCAATTTATGCCTACAGCC (HBV-X outer antisense). For second-round amplification, the primers were 1893-TTTGGGGCATGGACATTGACC (HBV-C inner sense), 2301-ATAAGCTGGAGGAGTGCGAATCC (HBV-C inner antisense), 1310-CTGGAGCAAACATTATCGGG (HBV-X inner sense) and 1748-CAAAGACCTTTAACCTGATCTCC (HBV-X inner antisense). In all cases, amplifications were carried out for 40 cycles under the following conditions: 95 °C for 60 s (denaturation), 52 °C for 60 s (annealing) and 72 °C for 60 s (extension). Specificity of amplicons was confirmed by NAH using 32P-labelled recombinant full-length HBV DNA as a probe.[11] This assay detects ≤10 vge/mL or ≤2.5 vge/reaction. Negative, positive and contamination controls were routinely included, as reported.[11,34]

Clonal Sequencing

To assess possible sequence variations and compartmentalization of HCV, 5'-UTR amplicons were cloned and at least 10 clones from each PCR product sequenced bidirectionally.[8] Polymorphisms were analysed by Sequencher software version 4.7 (Gene Codes Corp., Ann Arbor, MI, USA).

Immunoelectron Microscopy

To examine the presence of HCV virions, 500 μL of plasma was incubated with anti-HCV E2 monoclonal antibody (mAb; AP33; provided by Dr. Arvind Patel, University of Glasgow, UK) or mAb isotype control, as reported.[35,36] In the case of 16–45/M plasma, HCV RNA-positive fractions recovered after ultracentrifugation (4 mL plasma) over a 30% sucrose cushion[36] were similarly incubated with anti-E2 mAb. HCV particles were detected with anti-mouse IgG conjugated with 12-nm gold particles (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA) and, after counterstaining with 1% phosphotungstic acid, examined under a JEM 120 EX microscope (JEOL Ltd., Tokyo, Japan).

Liver Histology

After routine processing, liver biopsies were blindly evaluated by a hepatopathologist according to the METAVIR scoring system.[37]

Results
Occult Hepatitis C Virus Infection in Individuals With Clinical Sustained Virological Response Not Exposed to Hepatitis B Virus

Using the highly sensitive RT-PCR/NAH assays established earlier,[14] 15 of 28 plasma samples from 13 of 15 individuals were found positive for HCV RNA when 250-μL samples were tested (Table 1). Of those samples that were virus nonreactive at 250 μL, further testing with one or 4 mL of plasma increased HCV RNA detection[38] in an additional four and two samples, respectively. Overall, 21 of 28 (75%) of plasma samples were HCV RNA reactive (Table 1). HCV RNA levels varied considerably (2.5–400 vge/mL) at different time points within the same patient and also between different patients. For example, plasma HCV levels fluctuated from 400 vge/mL in the first sample to undetectable 6 months later (e.g. 5–45/F and 18–54/F), only to be detected again at 100 vge/mL 1 year thereafter (e.g. 18–54/F). In PBMC, 8 of 28 unstimulated samples were positive for HCV RNA, with ex vivo stimulation[39,40] increasing virus identification in another nine (Table 1). As in plasma, viral genome levels in untreated or mitogen-treated PBMC varied between individuals, ranging between 5 and 500 vge/106 cells, but not substantially in the same patient.

Overall, of the 11 cases with sequential samples, HCV RNA was detectable at least once in plasma in all cases and in unstimulated PBMC in six. Ex vivo stimulation of PBMC led to HCV RNA detection in the other five cases, making all cases ultimately positive for HCV RNA in PBMC. Hepatic HCV RNA was identified in one of three cases from this study group (Fig. 1).

757489-fig1

Figure 1. Detection of hepatitis C virus (HCV) in liver biopsies from individuals years after clinical diagnosis of sustained virological response following treatment of chronic hepatitis C. Total liver RNA (4 μg) was amplified by nested reverse transcriptase-polymerase chain reaction (RT-PCR) with 5'-UTR-specific primers and amplicon specificity verified by nucleic acid hybridization (NAH). (a) Identification of HCV RNA-positive strand. (b) Detection of HCV RNA-negative strand using strand-specific RT-PCR/NAH. Tenfold serial dilutions of HCV sRNA-positive or HCV sRNA-negative strands were used as quantitative standards, respectively. Dilutions of HCV sRNA-positive strands were included in the negative strand assay as a specificity control. Contamination controls included water added instead of cDNA and amplified by direct (DW) and nested (NW) reactions and mock (M) treated as test RNA. Positive signals showed the expected 244-bp 5'-UTR sequence fragments.

The detection of HCV RNA-positive strand was unlikely due to viral adsorption to the cells because of the concurrent detection of the viral negative strand in liver and PBMC (Table 1 and Fig. 1), of viral sequence polymorphisms in PBMC relative to plasma or liver (2–53/M; Supplementary Table S2) and of circulating HCV-like particles (3–46/F; Fig. 2). The particles usually occurred singly and, occasionally, in small aggregates with estimated diameters between 50 and 75 nm. Collectively, these findings clearly indicated the persistence of authentic HCV replication at low level in persons with clinical resolution of CHC.

757489-fig2

Figure 2. Immunoelectron microscopic identification of hepatitis C virus (HCV) particles in the plasma of individuals with clinical sustained virological response (SVR) as visualized by immunogold staining with anti-E2 mAb. (a–c) HCV particles in unfractionated plasma of 3–46/F obtained at 54 months after achieving SVR (d–f). HCV RNA-reactive particles recovered from fractions 3 and 4 after ultracentrifugation over a 30% sucrose cushion of 16–45/M plasma collected at 66 months after SVR (g–i). HCV virions in unfractionated plasma of 18–54/F obtained at 36 months following clinical SVR. (j) The unfractionated plasma from 3 to 46/F exposed to the isotype mAb control instead of anti-E2 mAb. Preparations were counterstained with 1% phosphotungstic acid. Bars indicate 50 nm.

Occult Hepatitis C Virus Infection in Patients With Clinical Sustained Virological Response and Past Exposure to Hepatitis B Virus

In the first instance, HBV DNA was identified, using our highly sensitive PCR/NAH assay,[11,34] in 17 of 22 (78%) plasma, 9 of 18 (50%) PBMC and 2 of 2 (100%) liver samples tested (Table 1 and Fig. 3), giving a total case positivity of 8 of 9 for HBV (Table 2). However, the lack of available material prevented us from determining the status of HBV replication in these cases.

757489-fig3

Figure 3. Detection of dual occult infection with hepatitis C virus (HCV) and hepatitis B virus (HBV) in individuals with clinically apparent sustained virological response (SVR) to anti-HCV therapy and past exposure to HBV. HCV RNA and HBV DNA were detected in plasma (Pl), peripheral blood mononuclear cells (P) and liver (L) by nested RT-PCR/NAH (for HCV RNA) or nested PCR/NAH (for HBV DNA) as described in Patients, Clinical Samples and Methods. Total RNA or DNA extracted from a patient with symptomatic chronic hepatitis C or serum hepatitis B surface antigen-positive chronic hepatitis B, respectively, was used as a positive control (PC). Contamination controls included water added instead of cDNA (HCV RNA analysis) or DNA (HBV DNA analysis) and amplified by direct (DW) and nested (NW) reactions and mock (M) treated as test RNA or DNA.

Using the same approach to virus detection as discussed in previous section, HCV RNA was ultimately identified in 83% plasma, 54.5% PBMC and 100% liver samples of patients with past exposure to HBV. As in the case of individuals without a past history with HBV, there was also evidence for the presence of HCV-negative strand (Table 1), circulating HCV-like particles (cases 16–45M and 18–54/F; Fig. 2) and single-nucleotide polymorphisms in PBMC (case 24–34F; Supplementary Table S2). Overall, the data as a whole lend support to the conclusion that past HBV exposure seems to have no identifiable influence on the frequency and virological properties of occult HCV persistence.

Liver Histology in Individuals With Occult Hepatitis C Virus Infection and With or Without Past Hepatitis B Virus Exposure

Histological examination of liver biopsies obtained before and after treatment (post-SVR) (Table 3; Supplementary Fig. S1) revealed (i) a decrease in necroinflammatory activity after SVR in five cases; (ii) no remarkable changes between pre- and post-treatment in two patients; and (iii) increased disease activity after SVR in two cases (1–61/F, 3–46/F). It was evident that despite histological improvement in the majority of cases investigated, most biopsies still displayed some degree (predominantly grades 1 and 2) of necroinflammation with piecemeal necrosis and lymphocytic infiltrations, as well as minimal to mild fibrosis (mainly stages 1 and 2) after SVR (Supplementary Fig. 1). But in the end, no remarkable differences were observed in liver histology between individuals with or without past exposure to HBV.

Discussion

In this study, we examined the prevalence of OCI in patients with clinically resolved hepatitis C who had or did not have past exposure to HBV. Our findings overall suggest that low-level HBV DNA carriage in plasma, PBMC or liver does not have a noticeable effect on the prevalence, virological characteristics and, most likely, features of liver histology encountered in the course of OCI. The data also indicate that regardless of the existence of past HBV exposure, the levels of HCV RNA, especially in plasma, could vary significantly over time in OCI. Given that both HCV and HBV genomes were detectable in the same compartments in many of the cases examined, it can be proposed that the reciprocal inhibition of HBV and HCV replication observed in highly viraemic hepatitis patients[22,24,26,41] is not evident in low-level coinfection with these viruses.

Since its discovery in recent years,[14] OCI continuing after clinical resolution of hepatitis C has been a subject of investigations by many groups. Although we and others have collectively documented the presence of low-level HCV persistence in plasma, PBMC and/or liver in individuals with spontaneous or treatment-induced recovery from CHC,[13–15] studies from other groups came to rather opposite conclusions.[42–44] As evidenced in this study, HCV RNA, in both plasma and PBMC, could fluctuate considerably over time in low-level HCV infection. This, in consequence, highlights the importance of testing multiple samples and, if required, using a larger amount of starting material for nucleic acid extraction. In addition, as ex vivo stimulation of lymphoid cells with mitogens could greatly enhance virus detection,[14,39] we routinely adopt this approach to our investigation of OCI in PBMC. Nevertheless, this methodology was not used by most, if not all, studies that had refuted the existence of occult HCV persistence.[42–44] Perhaps, a combination of the differences mentioned elsewhere, together with other factors discussed elsewhere,[17,18] could help reconcile the discrepancies between the data that argued for[13–15] and those that were against[42–44] the notion of OCI persisting after clinically diagnosed recovery. Further, it is important to note that in this study, as well in those published elsewhere [8,13–15,36], low-level occult HCV persistence was not just about mere detection of HCV RNA. Indeed, not only the persistent expression of HCV replicative strand in PBMC and liver tissue was documented, but also HCV sequence polymorphisms in PBMC compared with plasma or liver, circulating virion-like particles and their replication competence were demonstrated.[36]

In most cases evaluated, liver histology showed improvement after otherwise successful treatment. Nevertheless, there was also an indication of persistent low-grade liver inflammation and fibrosis (see Table 3), which was seemingly irrespective of whether the individuals were concurrently positive or not for HBV DNA. The question of whether OBI could contribute to such alterations after achieving SVR could not be conclusively addressed in this study and will require a similar investigation with a larger number of cases. At this point, our finding of hepatic changes after SVR is consistent with that reported by others.[13,15,45]

Taken together, the current study provides new insights into characteristics of occult HCV persistence in general and in individuals with past exposure to HBV in particular. It also offers a standardized approach with greater uniformity and sensitivity in the identification of OCI. Our data indicate that a past encounter with HBV may not negatively influence the prevalence and characteristics of low-level HCV persistence continuing after resolution of CHC achieved following antiviral therapy.

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New Antivirals Show Promise for an Interferon-free Hepatitis C Treatment

From Journal Watch > Journal Watch Gastroenterology

Atif Zaman, MD, MPH

Posted: 02/28/2012; Journal Watch © 2012 Massachusetts Medical Society

Abstract and Introduction
Abstract

Patients with HCV genotype 1 and previous null response achieved a The current generation of direct acting antivirals (DAAs), telaprevir and boceprevir, in combination with peginterferon and ribavirin (PEG/RBV) have demonstrated limited efficacy in patients with hepatitis C virus (HCV) genotype 1 infection who have experienced a previous null response to PEG/RBV. Sustained virologic response (SVR) has been approximately 30%, with associated high rates of resistance. Now, researchers have explored the efficacy of two next-generation DAAs with and without PEG/RBV in this patient population.

This phase IIa, industry-sponsored, randomized study involved 21 patients with previous null response to PEG/RBV (defined as <2 log HCV RNA drop from baseline at 12 weeks). Eleven patients received 60 mg daily of daclatasvir (an NS5A replication complex inhibitor) and 600 mg twice daily of asunaprevir (an NS3 protease inhibitor) for 24 weeks (DAA-only group). Ten patients received both DAAs and PEG/RBV for 24 weeks (DAA+PEG/RBV group). Of note, 90% of the study cohort had IL28B genotypes CT or TT, which respond poorly to PEG/RBV. The primary endpoint was SVR at 12 weeks after stopping therapy.

The SVR in the DAA-only group was 36% (2 of 9 with genotype 1a and 2 of 2 with genotype 1b). Six patients, all with genotype 1a, had viral breakthrough that was associated with viral resistance mutations to both DAAs. Alternately, the SVR in the DAA+PEG/RBV group was 100% (90% with genotype 1a) at 12 weeks after therapy and 90% at 24 weeks. Most adverse events were mild to moderate and included diarrhea, fatigue, headache, and nausea.

Comment

These findings provide early evidence that the next generation of DAAs is more potent than the current generation in treating HCV genotype 1 infection. In this cohort of the most difficult-to-treat HCV patients — previous null responders with genotype 1 infection — a 24-week SVR of 90% was achieved after 24 weeks of quadruple therapy (2 DAAs and PEG/RBV). Even an interferon-free regimen of the 2 DAAs resulted in an SVR of 36%. Results are eagerly anticipated from the phase III trial currently under way.

References

  • Lok AS et al. Preliminary study of two antiviral agents for hepatitis C genotype 1. N Engl J Med 2012 Jan 19; 366:216.
  • Chung RT. A watershed moment in the treatment of hepatitis C. N Engl J Med 2012 Jan 19; 366:273.

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Serum Levels of Alanine Aminotransferase Decrease With Age in Longitudinal Analysis



Clinical Gastroenterology and Hepatology
Volume 10, Issue 3 , Pages 285-290.e1, March 2012

Mamie H. Dong, Ricki Bettencour, David A. Brenner, Elizabeth Barrett–Connor, Rohit Loomba

published online 24 October 2011.

Abstract

Background & Aims

An increased level of alanine aminotransferase (ALT) is a marker of liver injury. The mean ALT level has been reported to decrease with age; we performed a longitudinal analysis to determine whether serum levels of ALT changes with age among community-dwelling, older adults in the US.

Methods

We analyzed clinical data from 2 cohorts of individuals who participated in the Rancho Bernardo Study, in Southern CA. The first cohort comprised 1073 community-dwelling participants (59% women); clinical data was collected from 1984–1987 and 1992–1997. The second cohort comprised 416 participants (64% women); data was collected from 1984–1987, 1992–1997, and 1997–1999. Demographic, metabolic covariates, ALT, bilirubin, and albumin were measured. Changes in individual ALT over time were examined in unadjusted and multivariable-adjusted linear and logistic regression analyses.

Results

At the baseline visit, the patients' mean age was 65.7 years and body mass index was 24.9 kg/m2. In cohort 1, the mean levels of ALT decreased with age by 10% (from 21 to 19 IU/L) between the time periods of 1984–1987 and 1992–1997 (P < .0001). In cohort 2, they decreased by 20% (from 20 to 16 IU/L) between the time periods of 1984–1987 and 1997–1999 (P < .0001). Categorically-defined increases in ALT also decreased with age (P < .0001). Results remained consistent in sex-specific analyses and after adjusting for metabolic syndrome components, alcohol use, bilirubin, and serum levels of albumin (P < .0001).

Conclusions

In a longitudinal analysis, we observed that levels of ALT decrease with age, independent of sex, metabolic factors, alcohol use, and results from commonly used liver function tests (bilirubin and albumin). When interpreting serum levels of ALT, physicians should consider patients’ age especially in the elderly.

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Hepatitis Viruses Activated By Stress In Cells

Article Date: 15 Feb 2012 - 1:00 PST

People who have received a donor organ need lifelong immunosuppressant drugs to keep their immune system from attacking the foreign tissue. However, with a suppressed immune system, many infectious agents turn into a threat. Infections such as with human cytomegalovirus and a certain type of human polyomavirus frequently cause complications in transplant recipients. For these patients it would therefore be particularly beneficial to have substances that suppress the immune system and exert an antiviral activity at the same time - thus killing two birds with one stone.

Jointly with colleges from Heidelberg University Hospital of Internal Medicine, researchers Professors Karin and Felix Hoppe-Seyler of the German Cancer Research Center (Deutsches Krebsforschungszentrum, DKFZ) have now tested a number of drugs with such an activity profile. They also tested the substances in liver cells infected with hepatitis B viruses (HBV) in the culture dish. The result: Liver cells that had been treated even produced considerably more viral offspring than untreated ones.

The substances under investigation inhibit the synthesis of nucleotides, which are the basic building blocks of DNA. This is how they exert their immunosuppressive effect: They slow down multiplication of immune cells because these lack building material for duplicating their genetic material. "The lack of DNA building blocks can cause a kind of stress in specific cells, which shows in the activation of a stress protein called p38", says Felix Hoppe-Seyler. "In liver cells, p38 very effectively activates the replication of hepatitis B viruses. "

The findings of the DKFZ researchers are a definite indication that administering these drugs in transplant recipients bears risks. Liver transplants, in particular, often have to be done because the body's own organ has been destroyed by hepatitis B viruses. In such cases, drug-induced activation of remaining HBV in the body may lead to the donor organ being attacked by hepatitis B viruses again.

Felix Hoppe-Seyler suspects that besides the three substances the group has investigated there are other substances which also cause an activation of p38. "In cancer patients being treated by chemotherapy, there is often a reactivation of chronic HBV infections. This has always been put down to their weakened immune system. We will now investigate whether this may also be due to activation of stress protein p38," said the virologist outlining the goals of his further research.

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Ultrasound technology proves accurate in diagnosing cirrhosis from recurrent hepatitis C

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February 29, 2012

Researchers from the Mayo Clinic confirm that ultrasound-based transient elastography (TE) provides excellent diagnostic accuracy for detecting cirrhosis due to recurrent infection with hepatitis C virus (HCV) infection following liver transplantation. Findings from the study published in the March issue of Liver Transplantation, a journal of the American Association for the Study of Liver Diseases, suggest that detection of significant fibrosis is more accurate when comparing patients with chronic HCV of the native liver

According to the World Health Organization (WHO), chronic HCV affects up to 170 million people worldwide and could lead to more severe liver diseases such as cirrhosis and liver cancer. Experts estimate that on average 6,000 liver transplants are performed in the U.S. each year. Medical evidence shows that following liver transplantation recipients who are HCV RNA-positive at the time of transplantation are at risk of reinfection with HCV. Moreover, studies have determined that fibrotic tissue can develop more quickly in the transplanted liver resulting in rapid progression of cirrhosis and graft failure.

"The current gold standard for determining liver disease severity and progression is liver biopsy," explains lead author Dr. Jayant Talwalkar with the Mayo Clinic in Rochester, Minnesota. "However, biopsy following liver transplantation may not accurately determine fibrosis severity and non-invasive imaging technology has advanced to more accurately assess the severity of liver injury which includes an indirect assessment of elevated portal pressure." A prior study reported liver biopsy can understage cirrhosis in up to 30% of cases.

For the present study researchers reviewed studies of the diagnostic accuracy of ultrasound-based TE, a non-invasive technology used to assess fibrosis by measuring liver stiffness. The team analyzed the performance of TE compared to liver biopsy in detecting sever hepatic fibrosis caused by recurrent HCV post-transplantation. Compared to liver biopsy, TE is a reproducible diagnostic technique that is quick and painless for patients.

Six studies were identified, with five studies that evaluated significant fibrosis and cirrhosis. Analysis of the pooled estimates showed TE had a sensitivity and specificity of 83%, respectively for detecting fibrosis. Of the five studies analyzing TE for detecting cirrhosis, sensitivity estimates were 98% and specificity at 84%. "Ultrasound-based TE provides excellent diagnostic accuracy for identifying cirrhosis caused by recurrent HCV following liver transplantation," concludes Dr. Talwalkar. "Further studies that confirm our results could highlight the importance of TE as a diagnostic tool for liver transplant recipients infected with HCV."

More information: "Ultrasound-based Transient Elastography for the Detection of Hepatic Fibrosis in Patients with Recurrent HCV after Liver Transplantation: Systematic Review and Meta-analysis." Corlan O. Adebajo, Jayant A. Talwalkar, John J. Poterucha, W. Ray Kim, and Michael R. Charlton. Liver Transplantation; (DOI: 10.1002/lt.22460) Published online: February 24, 2012; Print Issue Date: March 2012.

Provided by Wiley (news : web)

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