Effectiveness of interferon-free therapy for the treatment of HCV-patients with compensated cirrhosis treated through the Irish early access program.

BACKGROUND: We investigated the real-world effectiveness of interferon-free regimens for the treatment of patients with compensated cirrhosis infected with hepatitis C virus (HCV). METHOD: Using the Irish national HCV treatment registry, the effectiveness and safety of interferon-free regimens for HCV-infected patients treated between April 2015 and August 2016, was determined. RESULTS: A SVR12 was achieved in 86% of subjects treated with sofosbuvir/ledipasvir ± ribavirin (SOF/LDV±RBV), 93% treated with paritaprevir, ombitasvir and ritonavir combined with dasabuvir ± ribavirin (3D±RBV) and 89% treated with sofosbuvir/daclatasvir ± ribavirin (SOF/DCV±RBV). The discontinuation rate was 5% and the on-treatment mortality rate was 1%. CONCLUSION: The availability of interferon-free regimens represents a significant breakthrough for the treatment of HCV infection. Treatments options, with high SVR12 rates, are now available for patients with compensated cirrhosis who were unsuitable for treatment with interferon-based regimens. Data obtained from studies conducted in real world practice provide robust information fundamental for input into future economic evaluations for agents used for the treatment of HCV infection.

Hepatitis E Virus (HEV) Infection in Ireland.

Hepatitis E virus (HEV) is a single stranded RNA virus causing infection worldwide. In developing countries HEV genotypes 1 and 2 spread faeco-orally via water. Recently, infections with HEV have been detected in Europe and North America in patients with no travel history. These are food-borne HEV genotypes 3 and 4, a pig-associated zoonosis. Most infections are asymptomatic but morbidity and chronic infection may occur with prior liver disease or immunosuppression. International seroprevalence rates vary and with improved diagnostics have increased. To determine the current prevalence in this region we studied anonymised serum samples submitted in 2015 for routine testing. We detected anti-HEV IgG in 16/198 (8%) individuals, highest rate in 40-59 year olds (43.8%). This is higher than reported for the same region in 1995 (0.4%) using a previous generation assay. This study provides evidence of HEV circulation in Ireland and reinforces the need for ongoing surveillance.

In vitro replication models for the hepatitis C virus.

Soon after the discovery of the hepatitis C virus (HCV), attention turned to the development of models whereby replication of the virus could be investigated. Among the HCV replication models developed, the HCV RNA replicon model and the newly discovered infectious cell culture systems have had an immediate impact on the study of HCV replication, and will continue to lead to important advances in our understanding of HCV replication. The aim of this study is to deal with developments in HCV replication models in a chronological order from the early 1990s to the recent infectious HCV cell culture systems.

Viral load change and sequential evolution of entire hepatitis C virus genome in Irish recipients of single source-contaminated anti-D immunoglobulin*.

In hepatitis C virus (HCV) infection, serum viral load is important in the prediction of therapeutic efficacy. However, factors that affect the viral load remain poorly understood. To identify viral genomic elements responsible for the viral load, we investigated samples from a population of Irish women who were iatrogenically infected from a single HCV source by administration of HCV 1b-contaminated anti-D immune globulin between 1977 and 1978 (Kenny-Walsh, N Engl J Med 1999; 340: 1228). About 15 patients were divided into two groups, viral load increasing group (11 patients) and decreasing group (4 patients). Pairs of sera were collected from each patient at interval between 1.1 and 5.8 years. Full-length sequences of HCV genome were determined, and analyzed for changes in each patient. Sliding window analysis showed that the decreasing group had significantly higher mutation rates in a short segment of NS5B region that may affect the activity of RNA-dependent RNA polymerase. By comparing each coding regions, significantly higher mutation numbers were accumulated in NS5A region in the increasing group than the decreasing group (0.92 vs 0.16 nucleotides/site/year, P = 0.021). The mutation in certain positions of the HCV genome may be determinant factors of the viral load in a relatively homogeneous patient population.

A strategy for obtaining near full-length HCV cDNA clones (assemblicons) by assembly PCR.

Hepatitis C virus (HCV) genotype is a predictor of response, and guides the duration of antiviral therapy. However, with the exception of HCV genotype 1a, 1b and 2a, a limited number of clones from other genotypes exist. Here we report the optimization of long RT-PCR to generate three overlapping amplicons that span the near full length HCV genome from a panel of HCV genotypes (1a, 1b, 2a, 2b, 3a, 4a, 5a). Assembly-PCR (As-PCR) was used to construct near full-length cDNA clones (assemblicons) for each genotype. The optimization of the long RT-PCR on genotype 1a and 1b indicated that QIAamp Viral RNA kit (Qiagen, UK), Expand RT and Expand Long Template PCR system (Roche, UK), were the most efficient in producing the requisite three overlapping amplicons and assemblicons for each genotype. The genotype of each assemblicon was confirmed. Assemblicon generation was only possible when the overlapping amplicons were biotinylated. As-PCR obviated the need for time consuming ligations and cloning. The use of three overlapping amplicons in the construction of HCV assemblicons minimised the chimeric nature of the resultant clone. As-PCR may prove a methodological avenue through which a larger panel of consensus HCV clones could be made available for HCV in vitro investigation.

Inferred hepatitis C virus quasispecies diversity is influenced by choice of DNA polymerase in reverse transcriptase-polymerase chain reactions.

The hepatitis C virus (HCV) is known to circulate in vivo as a quasispecies, a population of closely related, but genetically nonidentical virions. HCV reverse transcriptase (RT)-(nested) polymerase chain reaction (PCR) strategies are used to study quasispecies diversity at certain important viral genetic loci, predominantly at hypervariable region 1 (HVR 1) of the E2 envelope gene, and the interferon sensitivity determining region (ISDR) of the nonstructural 5a (NS5a) gene. We have found that the choice of DNA polymerase employed in viral PCR has effects on the inferred viral diversity at two distinct loci on the HCV genome. Nested HVR 1 and ISDR PCR was performed with both proofreading (Pwo) and nonproofreading (Taq) DNA polymerases on identical cDNA derived from three separate HCV-positive sera. Amplicons were cloned and sequences determined for 18-20 individual clones per sample. Quasispecies diversity determined from HVR 1 and ISDR PCR products showed that there was a marked effect on the inferred diversity depending on which DNA polymerase was employed in the PCR. The deduced amino acid sequences of the major variants within each specimen were identical for both Taq and Pwo DNA polymerase-mediated PCRs. However, a greater number of minor variants were observed in the Taq-generated amplicons, 80% of which were not observed in the Pwo-generated amplicons. Primer editing in the Pwo-generated amplicons was observed in 19% (20/104) of clones examined. Single-strand conformational polymorphism analysis of multiple replicates of each amplicon revealed good intra-PCR reproducibility in terms of genetic heterogeneity, and that as such the observations were not due to poor PCR reproducibility. The use of nonproofreading DNA polymerases to assess viral diversity can yield an incorrect quasispecies spectrum and affect RT-PCR assay performance. The contribution of Taq-induced errors and lack of adaptability of primers to potentially heterologous template-binding sites indicate that proofreading DNA polymerases should be the enzyme of choice in these systems.

HLA class II genes determine the natural variance of hepatitis C viral load.

The aim of this study was to investigate the relationship between human leukocyte antigen (HLA) class II genes and the natural fluctuations in hepatitis C viral load in a homogeneous patient population. The study group consisted of 57 viremic (hepatitis C virus [HCV] 1b) women for whom HLA class II DRB1 and DQB1 haplotyping, virologic, histologic, and biochemical markers of disease activity were available. All patients were infected with HCV 1b from the same source of hepatitis C-contaminated anti-D immunoglobulin during the period from May 1977 to November 1978. The mean slope of change of viral load was 0.34 (SD +/- 0.73) log(10) viral copies/mL/year, which is significantly different from zero, P < 10(-9). Analysis of the relationship between the slope of change of viral load and HLA class II haplotype indicated a significantly different slope of change of viral load between the alleles of (1) DRB1(*)15 and DRB1(*)0701, and (2) DQB1(*)0602 and DQB1(*)0201, P(c) =.036 and P(c) =.026 after Bonferroni correction for multiple comparisons, respectively. Significant differences for grade and stage of disease at liver biopsy were observed for DQB1(*)0501 and DQB1(*)0201 alleles; P =.019, r(s) =.64, and P =.047, r(s) =.57, respectively. In addition, significant differences in stage of disease were found to exist between DRB1(*)13 and DRB1(*)0701, P =.031, r(s) = -.71. Our results define an association between the slope of change of viral load and HLA class II haplotype in patients infected with genotype 1b of HCV. This suggests a role for host immunogenetic factors in HCV infection in this homogeneous group.

Viral clearance in hepatitis C (1b) infection: relationship with human leukocyte antigen class II in a homogeneous population.

The aim of this study was to investigate the possibility of a significant relationship between human leukocyte antigen (HLA) class II and the clearance of hepatitis C virus (HCV). The study group consisted of 156 Irish women who iatrogenically received HCV 1b-contaminated Anti-D immunoglobulin between May 1977 and November 1978. Thus, the study population was homogeneous in terms of gender, source of infection, and ethnicity. On Screening in 1994, all individuals were anti-HCV antibody positive by recombinant immunoblot assay, while 46% (n = 72) of the group were HCV-positive by reverse transcriptase-polymerase chain reaction (RT-PCR). HLA DRB1 and DQB1 status was molecularly defined by high resolution reverse line probe hybridization methodology. Clearance of HCV 1b was found to be associated with DRB1*01. However, this association was lost after Bonferroni correction for multiple comparisons. Extended haplotype analysis between specific DRB1 and DQB1 allelic combinations identified a significant reduction in the frequency of DQB1*0501 in the presence of DRB1*0701 in the persistently infected individuals in the study group (P <.05). No associations with either viral clearance or persistence were found at the DQB1 locus. Our results suggest that HLA DRB1*01 appears to contribute to the spontaneous resolution of a primary HCV infection in the Irish population. The presence of DRB1*0701 in the absence of DQB1*0501 possibly reflects an influence of this allele in persistence of HCV infection. Defined and homogeneous patient populations offer the best opportunity to illuminate previously disguised immunogenetic factors important in the clearance of HCV 1b.

Development of the immunoglobulin repertoire.

In this Review we will include studies on the development immunoglobulin repertoire. We will discuss the pattern of V, (D), and J rearrangement in both normal B cells and autoimmune disorders. We will define the role of the recombination signal sequences and the importance of the nucleotide sequence of these highly conserved motifs. Whether deviations from the consensus recombination signal sequence will be tolerated by the recombination mechanism and the importance of the recombination-activating genes are also discussed. We will address the issue of whether pathogenic autoantibodies are generated as part of the normal immune repertoire and the importance of receptor editing as a means by which the immune system deletes autoreactive B cells.

Mouse VH7183 recombination signal sequences mediate recombination more frequently than those of VHJ558.

Mouse VH gene segments are conventionally classified into 13 families on the basis of sequence similarity. The 7183 family lies close to the 3' end of the locus and is preferentially used in BALB/c mice; J558, the largest family, lies close to the 5' end of the VH stretch and is preferentially used in C57BL/6 mice. To investigate whether differential effectiveness of the RSSs in the two families might contribute to the overusage of 7183 in the primary repertoire of BALB/c, we constructed recombination substrates in which the recombination signal sequences (RSSs) of VH segments 7183 and J558 compete with each other for a single RSS, DFL16.1, after transfection into two transformed cell lines derived from C57BL/6 and two cell lines from BALB/c mice. In both strains, the 7183 RSS was found to be preferentially used (83%). Thus, the 7183 RSS mediates recombination more frequently than does that of J558, and this preference must thereby influence the primary repertoire, but the strain difference cannot be accounted for by a difference in the RSSs.