Orla Crosbie

Innate immune genes synergize to predict increased risk of chronic disease in hepatitis C virus infection.

Hepatitis C is a common infection with significant morbidity and mortality, and only a minority of patients successfully clear the infection. Identification of factors that influence disease progression in HCV infection is difficult owing to the lack of well-defined patient cohorts. However, recent evidence supports a role for the innate immune system in virus clearance. In this study, we investigated innate immune genes for their contribution to disease progression in a unique cohort of well-controlled HCV-infected patients. The Irish cohort of HCV patients is uniquely homogenous; patients were infected with a single genotype of HCV from contaminated anti-D Ig. We genotyped 543 infected patients, including 247 patients who spontaneously resolved infection, for natural killer (NK) cell-associated killer cell Ig-like receptors (KIR) genes and the recently reported IL28B (IFNλ3) SNP. The NK cell gene KIR2DS3 was significantly increased in patients with chronic infection [odds ratio (OR) 1.90, 95% confidence interval (CI) 1.25–2.90, P < 0.002]. The IL28B “T” allele was also significantly increased in chronically infected patients (OR 7.38, 95% CI 4.93–11.07, P < 10−8). The presence of both markers synergized to significantly increase the risk of chronic infection over either factor alone (OR 20.11, 95% CI 9.05–44.68, P < 10−7). In functional experiments, we found that IL28A significantly inhibited IFN-γ production by NK cells. Thus, we demonstrate a functional link between NK cells and type 3 IFN. Our findings may contribute to the development of a prognostic test for HCV and identify therapeutic strategies for the clinical management of HCV-infected patients.

Orthotopic liver transplantation for veno-occlusive disease complicating autologous bone marrow transplantation.

BACKGROUND Veno-occlusive disease of the liver is a serious and often life-threatening complication after bone marrow transplantation. Although risk factors for the development of veno-occlusive disease have been postulated, there is no precise method for accurately identifying those patients who are at risk and for whom early intervention and treatment would have maximum potential benefit. Liver transplantation has been advocated as a treatment for veno-occlusive disease in selected patients. METHODS In this report, we describe a patient who underwent liver transplantation for life-threatening veno-occlusive disease after autologous bone marrow transplantation for acute lymphoblastic leukemia. RESULTS Liver engraftment was achieved, but the patient developed Pneumocystis pneumonia, which failed to respond to pentamidine. The patient died 6 months after liver transplantation. CONCLUSIONS While acknowledging the limited experience of orthotropic liver transplantation in this patient population, we suggest its consideration as a feasible, potentially beneficial treatment option in patients with severe veno-occlusive disease after bone marrow transplantation.

Correlation between pre-treatment quasispecies complexity and treatment outcome in chronic HCV genotype 3a.

Pre-treatment HCV quasispecies complexity and diversity may predict response to interferon based anti-viral therapy. The objective of this study was to retrospectively (1) examine temporal changes in quasispecies prior to the start of therapy and (2) investigate extensively quasispecies evolution in a group of 10 chronically infected patients with genotype 3a, treated with pegylated α2a-Interferon and ribavirin.The degree of sequence heterogeneity within the hypervariable region 1 was assessed by analyzing 20–30 individual clones in serial serum samples. Genetic parameters, including amino acid Shannon entropy, Hamming distance and genetic distance were calculated for each sample. Treatment outcome was divided into (1) sustained virological responders (SVR) and (2) treatment failure (TF).Our results indicate, (1) quasispecies complexity and diversity are lower in the SVR group, (2) quasispecies vary temporally and (3) genetic heterogeneity at baseline can be use to predict treatment outcome.We discuss the results from the perspective of replicative homeostasis.

Insertion and recombination events at hypervariable region 1 over 9.6 years of hepatitis C virus chronic infection.

Hepatitis C virus (HCV) exists as a quasispecies within an infected individual. We have previously reported an in-frame 3 bp insertion event at the N-terminal region of the E2 glycoprotein from a genotype 4a HCV isolate giving rise to an atypical 28 aa hypervariable region (HVR) 1. To further explore quasispecies evolution at the HVR1, serum samples collected over 9.6 years from the same chronically infected, treatment naïve individual were subjected to retrospective clonal analysis. Uniquely, we observed that isolates containing this atypical HVR1 not only persisted for 7.6 years, but dominated the quasispecies swarm. Just as striking was the collapse of this population of variants towards the end of the sampling period in synchrony with variants containing a classical HVR1 from the same lineage. The replication space was subsequently occupied by a second minor lineage, which itself was only intermittently detectable at earlier sampling points. In conjunction with the observed genetic shift, the coexistence of two distinct HVR1 populations facilitated the detection of putative intra-subtype recombinants, which included the identification of the likely ancestral parental donors. Juxtaposed to the considerable plasticity of the HVR1, we also document a degree of mutational inflexibility as each of the HVR1 subpopulations within our dataset exhibited overall genetic conservation and convergence. Finally, we raise the issue of genetic analysis in the context of mixed lineage infections.

Analysis of the Evolution and Structure of a Complex Intrahost Viral Population in Chronic Hepatitis C Virus Mapped by Ultradeep Pyrosequencing

ABSTRACT Hepatitis C virus (HCV) causes chronic infection in up to 50% to 80% of infected individuals. Hypervariable region 1 (HVR1) variability is frequently studied to gain an insight into the mechanisms of HCV adaptation during chronic infection, but the changes to and persistence of HCV subpopulations during intrahost evolution are poorly understood. In this study, we used ultradeep pyrosequencing (UDPS) to map the viral heterogeneity of a single patient over 9.6 years of chronic HCV genotype 4a infection. Informed error correction of the raw UDPS data was performed using a temporally matched clonal data set. The resultant data set reported the detection of low-frequency recombinants throughout the study period, implying that recombination is an active mechanism through which HCV can explore novel sequence space. The data indicate that polyvirus infection of hepatocytes has occurred but that the fitness quotients of recombinant daughter virions are too low for the daughter virions to compete against the parental genomes. The subpopulations of parental genomes contributing to the recombination events highlighted a dynamic virome where subpopulations of variants are in competition. In addition, we provide direct evidence that demonstrates the growth of subdominant populations to dominance in the absence of a detectable humoral response. IMPORTANCE Analysis of ultradeep pyrosequencing data sets derived from virus amplicons frequently relies on software tools that are not optimized for amplicon analysis, assume random incorporation of sequencing errors, and are focused on achieving higher specificity at the expense of sensitivity. Such analysis is further complicated by the presence of hypervariable regions. In this study, we made use of a temporally matched reference sequence data set to inform error correction algorithms. Using this methodology, we were able to (i) detect multiple instances of hepatitis C virus intrasubtype recombination at the E1/E2 junction (a phenomenon rarely reported in the literature) and (ii) interrogate the longitudinal quasispecies complexity of the virome. Parallel to the UDPS, isolation of IgG-bound virions was found to coincide with the collapse of specific viral subpopulations.

High mortality during direct acting antiviral therapy for hepatitis C patients with Child’s C cirrhosis: Results of the Irish Early Access Programme

Please cite this article as: Gray, E., O’Leary, A., Stewart, S., Bergin, C., Cannon, M., Courtney, G., Crosbie, O., De Gascun, C.F., Feeney, E., Houlihan, D.D., Kelleher, B., Lambert, J.S., Lee, J., Mallon, P., McConkey, S., McCormick, A., McKiernan, S., McNally, C., Murray, F., Sheehan, G., Norris, S., Fanning, L.J., on behalf of the Irish Hepatitis C Outcomes and Research Network (ICORN), High mortality during direct acting antiviral therapy for hepatitis C patients with Child’s C cirrhosis: Results of the Irish Early Access Programme, Journal of Hepatology (2016), doi: http://dx.doi.org/10.1016/j.jhep.2016.03.022

Autoimmune hepatitis in association with lymphocytic colitis.

Autoimmune hepatitis is a rare, chronic inflammatory disorder which has been associated with a number of other auto-immune conditions. However, there are no reports in the medical literature of an association with microscopic (lymphocytic) colitis. We report the case of a 53-year-old woman with several autoimmune conditions, including lymphocytic colitis, who presented with an acute hepatitis. On the basis of the clinical features, serology, and histopathology, we diagnosed autoimmune hepatitis. To our knowledge, this is the first report of autoimmune hepatitis in association with lymphocytic colitis, and lends support to the theory of an autoimmune etiology for lymphocytic colitis.

T Lymphocyte Subsets and Activation Status in Patients Following Liver Transplantation

Changes in T-lymphocyte subsets have previously been shown to relate to clinical events following liver transplantation and be of prognostic significance following renal transplantation. The aim of this study was to examine T lymphocyte subsets, their activation status and the mean fluorescence intensity of cell surface markers by flow cytometric analysis, in peripheral blood of patients following liver transplantation. Stable transplant patients (n=11) had a significantly higher level of activation (HLA-DR expression ) of all T cell subsets: CD3, CD4 and CD8 compared to healthy controls: 17.5% +/- 14.0 (mean +/- SD) vs 4.7 +/- 1.8 (p=0.04), 13.7% +/- 10.3 vs 4.3 +/- 1.7 (p=0.03) and 23.8% +/- 19.9 vs 3.6 +/- 2.4 (p=0.02) respectively. A further increase in activation status occurred in all T cell subsets in association with acute cellular rejection, reaching significance for the CD4+ population: 13.7% +/- 10.2 vs 23.3% +/- 20.6 (p=0.04). The mean fluorescence intensity of the CD3+DR- and CD3+ DR+ populations were increased to 1397 +/- 869 and 1282 +/- 810 following liver transplantation compared to values of 425 +/- 204 and 376 +/- 166 respectively for controls (p<0.05). T-lymphocytes maintain a high level of activation following liver transplantation and continue to express high levels of the surface marker CD3, which may account for the occurrence of acute cellular rejection despite immunosuppression in these patients.

Separation of Hepatitis C genotype 4a into IgG-depleted and IgG-enriched fractions reveals a unique quasispecies profile

BackgroundHepatitis C virus (HCV) circulates in an infected individual as a heterogeneous mixture of closely related viruses called quasispecies. The E1/E2 region of the HCV genome is hypervariable (HVR1) and is targeted by the humoral immune system. Hepatitis C virions are found in two forms: antibody associated or antibody free.The objective of this study was to investigate if separation of Hepatitis C virions into antibody enriched and antibody depleted fractions segregates quasispecies populations into distinctive swarms.ResultsA HCV genotype 4a specimen was fractionated into IgG-depleted and IgG-enriched fractions by use of Albumin/IgG depletion spin column. Clonal analysis of these two fractions was performed and then compared to an unfractionated sample. Following sequence analysis it was evident that the antibody depleted fraction was significantly more heterogeneous than the antibody enriched fraction, revealing a unique quasispecies profile. An in-frame 3 nt insertion was observed in 26% of clones in the unfractionated population and in 64% of clones in the IgG-depleted fraction. In addition, an in-frame 3 nt indel event was observed in 10% of clones in the unfractionated population and in 9% of clones in the IgG-depleted fraction. Neither of these latter events, which are rare occurrences in genotype 4a, was identified in the IgG-enriched fraction.ConclusionIn conclusion, the homogeneity of the IgG-enriched species is postulated to represent a sequence that was strongly recognised by the humoral immune system at the time the sample was obtained. The heterogeneous nature of the IgG-depleted fraction is discussed in the context of humoral escape.

Network Analysis of the Chronic Hepatitis C Virome Defines Hypervariable Region 1 Evolutionary Phenotypes in the Context of Humoral Immune Responses

ABSTRACT Hypervariable region 1 (HVR1) of hepatitis C virus (HCV) comprises the first 27 N-terminal amino acid residues of E2. It is classically seen as the most heterogeneous region of the HCV genome. In this study, we assessed HVR1 evolution by using ultradeep pyrosequencing for a cohort of treatment-naive, chronically infected patients over a short, 16-week period. Organization of the sequence set into connected components that represented single nucleotide substitution events revealed a network dominated by highly connected, centrally positioned master sequences. HVR1 phenotypes were observed to be under strong purifying (stationary) and strong positive (antigenic drift) selection pressures, which were coincident with advancing patient age and cirrhosis of the liver. It followed that stationary viromes were dominated by a single HVR1 variant surrounded by minor variants comprised from conservative single amino acid substitution events. We present evidence to suggest that neutralization antibody efficacy was diminished for stationary-virome HVR1 variants. Our results identify the HVR1 network structure during chronic infection as the preferential dominance of a single variant within a narrow sequence space. IMPORTANCE HCV infection is often asymptomatic, and chronic infection is generally well established in advance of initial diagnosis and subsequent treatment. HVR1 can undergo rapid sequence evolution during acute infection, and the variant pool is typically seen to diverge away from ancestral sequences as infection progresses from the acute to the chronic phase. In this report, we describe HVR1 viromes in chronically infected patients that are defined by a dominant epitope located centrally within a narrow variant pool. Our findings suggest that weakened humoral immune activity, as a consequence of persistent chronic infection, allows for the acquisition and maintenance of host-specific adaptive mutations at HVR1 that reflect virus fitness.