Disease outcomes in a cohort of women in Ireland infected by hepatitis C-contaminated anti-D immunoglobulin during 1970s.

BACKGROUND & AIM: In the mid-1990s, a group of Rh negative women was diagnosed with hepatitis C virus (HCV) genotype 1b infection, following administration of contaminated anti-D immunoglobulin in 1977-79. We aimed to describe their disease history and estimate the effect of selected host and treatment factors on disease progression. METHODS: We conducted a cohort study on the women infected with HCV. Information was collected from records at seven HCV treatment centres on demographics, treatment and health outcomes up to the 31st December 2013. We calculated cumulative incidence, case fatality, and sub hazard ratios (SHR) for disease progression using competing risks regression. RESULTS: Six hundred and eighty-two patients were included in the study. Among the chronically infected patients (n=374), 35% completed interferon-based antiviral treatment; 42% of whom had a sustained virological response. At the end of 2013, 19%, 1.9%, and 4.9% of chronically infected patients had developed cirrhosis, hepatocellular carcinoma, and liver-related death, respectively, compared with 10%, 0.8%, and 2.4% at the end of 2008. At the end of 2013, 321 (86%) of the chronically infected patients remained alive, 247 (77%) of whom were still chronically infected. Factors associated with increased cirrhosis rates included high alcohol intake (aSHR=4.9 [2.5-9.5]) and diabetes mellitus (aSHR=5.0 [2.9-8.8]). CONCLUSIONS: Development of liver-related outcomes accelerated with time, with the risk of cirrhosis, hepatocellular carcinoma, and liver-related death doubling in the last five years of follow-up, particularly in women with high alcohol consumption and diabetes mellitus. We recommend that patients with chronic HCV infection be advised of the additive harmful effect of alcohol, and that data be collected on this cohort after a further five years to analyse the effect of subsequent antiviral treatment during this rapidly evolving period in HCV treatment history. LAY SUMMARY: In the mid-1990s, a group of women were diagnosed with chronic hepatitis C virus (HCV) infection following receipt of contaminated anti-D immunoglobulin between 1977 and 1979 in Ireland. Seventy-two (19%) developed cirrhosis and 18 had died from liver-related causes (5%) after 36years of infection. Disease progression accelerated in the last five years of follow-up, particularly in women with diabetes mellitus and high alcohol consumption. We recommend that patients with chronic HCV infection be advised of the additive harmful effect of high alcohol consumption.

Humoral immune system targets clonotypic antibody-associated hepatitis C virus.

Hypervariable region 1 (HVR1) is one of the potential neutralization domains in the E2 glycoprotein of hepatitis C virus (HCV). Point mutations of the HVR1 can lead to humoral immune escape in HCV-infected patients. In this study, we segregated the chronically infected viraemic sera from HCV-infected patients into populations of antibody-free virus and antibody-associated virus (AAV) and mapped potential epitopes within the E1E2 gene junction of AAV sequences (residues 364-430). Furthermore, we generated HCV pseudoparticles (HCVpp) derived from AAV sequences to assess their infectivity. We studied the neutralization potential of virus-free Fab obtained from antibody-virus complexes, in the HCVpp system. We observed selective targeting of clonotypic HCV variants from the quasispecies pool. Moreover, we identified potential neutralizing epitopes within the HVR1 and an additional epitope that overlapped with a broadly neutralizing AP33 epitope (amino acid 412-423 in E2). We observed a marked difference in the infectivity of HCVpp generated using E1E2 sequences isolated from AAV. We document reduction in the infectivity of HCVpp-H77 and HCVpp derived from AAV sequences when challenged with virus-free Fab. Our results provide novel insights into the complexities of engagement between HCV and the humoral immune system.

A single amino acid change in the hypervariable region 1 of hepatitis C virus genotype 4a aids humoral immune escape.

Longitudinal analysis of chronic hepatitis C virus (HCV) infection has shown that the virus has several adaptive strategies that maintain persistence and infectivity over time. We examined four serum samples from the same chronically infected HCV genotype 4a patient for the presence of IgG antibody-associated virus. RNA was isolated from antibody-associated and antibody-free virions. Subsequent to sequence analysis, 27 aa hypervariable region 1 (HVR1) peptides were used to test the humoral immune escape. We demonstrated that differential peptide binding of Fab was associated with a single amino acid change. We provide direct evidence of natural humoral immune escape by HCV within HVR1.

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

UNLABELLED: 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.

Intensive temporal mapping of hepatitis C hypervariable region 1 quasispecies provides novel insights into hepatitis C virus evolution in chronic infection.

Hepatitis C virus (HCV) is an RNA virus which exists as swarms of closely related viruses known as quasispecies (QS). A number of studies have demonstrated associations between QS hypervariable region 1 (HVR1) characteristics (diversity and complexity) and treatment success. We investigated HCV QS change in chronic infection over intervals of 2-4 weeks in 23 chronically infected individuals to describe the natural history of virus evolution and establish whether HCV QS characteristics could be used to individualize treatment regimens at a molecular level. HVR1 QS diversity, complexity and divergence continue to change in an unpredictable fashion in chronic infection even where there is little phylogenetic change, which is likely to preclude the use of these features in treatment individualization. Our phylogenetic analysis identified no change in the HVR1 QS in 12 subjects, minor change in four subjects and we describe a time-ordered phylogeny for the first time over a period as short as 16 weeks in seven subjects. We identified the existence of multiple subpopulation infections using partitioned analysis of QS and illustrated how subpopulations were sequentially replaced in a number of subjects. We illustrated marked variation in the nucleotide substitution per codon position between patients with sequence change and those without change in the phylogenetic tree. Analysis of codon-specific selection pressures identified a number of codons under purifying selection, suggesting that these code for structurally conserved amino acids. We also identified sections of the HVR1 under positive selection with marked sequence heterogeneity, suggesting that these may be potential epitope-binding sites.

Analysis of the evolution and structure of a complex intrahost viral population in chronic hepatitis C virus mapped by ultradeep pyrosequencing.

UNLABELLED: 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.

Hepatitis C community prevalence is over-estimated: a prospective, birth cohort study.

BACKGROUND: Hepatitis C virus infection is often asymptomatic, and many patients may be unaware they are infected. Community-based, birth cohort screening has been advocated to identify these patients. It has been estimated that 0.7-1% of individuals born between 1965 and 1985 in Ireland are infected. The cost-effectiveness of screening is critically dependent on the population prevalence. AIMS: The aim is to determine the community prevalence of hepatitis C virus infection in the birth cohort 1965-1985. METHODS: Residual serum samples from blood tests ordered by community general practitioners were anonymised and analysed for the presence of hepatitis C antibody ± antigen. Twelve large general hospitals throughout the country participated. RESULTS: A total of 14,320 samples were tested, 9347 of which were from the birth cohort 1965-1985. Seventy-two samples were positive for hepatitis C antibody of which 12 were positive for hepatitis C antigen (17%). The overall prevalence of hepatitis C antigen in the birth cohort was 0.09%. A higher prevalence (0.39%) was identified in males in two urban areas of Dublin. CONCLUSIONS: Hepatitis C virus seroprevalence was much lower than previously estimated. The proportion of antibody positive patients with hepatitis C antigen was also lower than expected suggesting the effects of treatment and/or high spontaneous viral clearance. Universal birth cohort screening is unlikely to be cost-effective. Targeted birth cohort screening in high prevalence areas could be considered.

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.

The pan-genotype specificity of the hepatitis C virus anti-core monoclonal antibody C7-50 is contingent on the quasispecies profile of a population.

The inter/intra-genotype quasispecies makeup of hepatitis C virus (HCV) has retarded the development of antibodies capable of pan-genotype reactivity. Mutations, even in conserved domains, are tolerated to a degree. In this report, we characterise the pan-genotype specificity of the commercially available monoclonal anti-HCV core antibody C7-50. We demonstrate the antibody's ability to detect HCV core protein following infection of Huh7 cells with serum-derived HCV of genotypes 2-5 and that a single-site polymorphism in a genotype 3a core amino acid sequence is sufficient to disrupt antibody recognition of the epitope. This same polymorphism is a feature of genotype 3 viruses.

Small intestinal bacterial overgrowth in nonalcoholic steatohepatitis: association with toll-like receptor 4 expression and plasma levels of interleukin 8.

BACKGROUND: Experimental and clinical studies suggest an association between small intestinal bacterial overgrowth (SIBO) and nonalcoholic steatohepatitis (NASH). Liver injury and fibrosis could be related to exposure to bacterial products of intestinal origin and, most notably, endotoxin, including lipopolysaccharide (LPS). AIM: To compare the prevalence of SIBO and its relationships to LPS receptor levels and systemic cytokines in NASH patients and healthy control subjects. METHODS: Eighteen NASH patients (eight males) and 16 age-matched and gender-matched healthy volunteers were studied. SIBO was assessed by the lactulose breath hydrogen test (LHBT), plasma lipopolysaccharide binding protein (LBP) levels by ELISA, and expression (as a percentage) of TLR-2 and 4 on CD14-positive cells by flow cytometry. Pro-inflammatory cytokines (IL-1ß, IL-6, IL-8, and TNF-α) were measured in plasma. RESULTS: SIBO was more common in NASH patients than control subjects (77.78% vs. 31.25%; P < 0.0001). LBP levels and TLR-2 expression were similar in both groups, TLR-4/MD-2 expression on CD14 positive cells was higher among NASH patients: expression, mean ± SEM, NASH vs. control: 20.95 ± 2.91% vs. 12.73 ± 2.29%, P < 0.05. Among the examined cytokines, only IL-8 levels were significantly higher in patients than control (P = 0.04) and correlated positively with TLR-4 expression (r = 0.5123, P = 0.036). CONCLUSION: NASH patients have a higher prevalence of small intestinal bacterial overgrowth which is associated with enhanced expression of TLR-4 and release of IL-8. SIBO may have an important role in NASH through interactions with TLR-4 and induction of the pro-inflammatory cytokine, IL-8.

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 alpha2a-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.

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

BACKGROUND: Hepatitis 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. RESULTS: A 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. CONCLUSION: In 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.

Ultradeep Pyrosequencing of Hepatitis C Virus to Define Evolutionary Phenotypes.

Analysis of hypervariable regions (HVR) using pyrosequencing techniques is hampered by the ability of error correction algorithms to account for the heterogeneity of the variants present. Analysis of between-sample fluctuations to virome sub-populations, and detection of low frequency variants, are unreliable through the application of arbitrary frequency cut offs. Cumulatively this leads to an underestimation of genetic diversity. In the following technique we describe the analysis of Hepatitis C virus (HCV) HVR1 which includes the E1/E2 glycoprotein gene junction. This procedure describes the evolution of HCV in a treatment naïve environment, from 10 samples collected over 10 years, using ultradeep pyrosequencing (UDPS) performed on the Roche GS FLX titanium platform ( Palmer et al., 2014 ). Initial clonal analysis of serum samples was used to inform downstream error correction algorithms that allowed for a greater sequence depth to be reached. PCR amplification of this region has been tested for HCV genotypes 1, 2, 3 and 4.

Reverse epitope mapping of the E2 glycoprotein in antibody associated hepatitis C virus.

The humoral immune system responds to chronic hepatitis C virus (HCV) infection by producing neutralising antibodies (nAb). In this study we generated three HCV pseudoparticles in which E1E2 glycoprotein sequence was targeted by the host humoral immune system. We used patient derived virus free Fabs (VF-Fabs) obtained from HCV genotype 1a (n = 3), genotype 1b (n = 7) and genotype 3a (n = 1) for neutralisation of HCVpp produced in this study both individually and in combination. Based on the available anti-HCV monoclonal nAb mapping information we selected amino acid region 384-619 for conformational epitope mapping. Amongst our notable findings, we observed significant reduction in HCVpp infectivity (p<0.05) when challenged with a combination of inter genotype and subtype VF-Fabs. We also identified five binding motifs targeted by patient derived VF-Fab upon peptide mapping, of which two shared the residues with previously reported epitopes. One epitope lies within an immunodominant HVR1 and two were novel. In summary, we used a reverse epitope mapping strategy to identify preferred epitopes by the host humoral immune system. Additionally, we have combined different VF-Fabs to further reduce the HCVpp infectivity. Our data indicates that combining the antigen specificity of antibodies may be a useful strategy to reduce (in-vitro) infectivity.

Serendipitous identification of natural intergenotypic recombinants of hepatitis C in Ireland.

BACKGROUND: Recombination between hepatitis C single stranded RNA viruses is a rare event. Natural viable intragenotypic and intergenotypic recombinants between 1b-1a, 1a-1c and 2k-1b, 2i-6p, respectively, have been reported. Diagnostically recombinants represent an intriguing challenge. Hepatitis C genotype is defined by interrogation of the sequence composition of the 5' untranslated region [5'UTR]. Occasionally, ambiguous specimens require further investigation of the genome, usually by interrogation of the NS5B region. The original purpose of this study was to confirm the existence of a suspected mixed genotype infection of genotypes 2 and 4 by clonal analysis at the NS5B region of the genome in two specimens from two separate individuals. This initial identification of genotype was based on analysis of the 5'UTR of the genome by reverse line probe hybridisation [RLPH]. RESULTS: The original diagnosis of a mixed genotype infection was not confirmed by clonal analysis of the NS5B region of the genome. The phylogenetic analysis indicated that both specimens were natural intergenotypic recombinant forms of HCV. The recombination was between genotypes 2k and 1b for both specimens. The recombination break point was identified as occurring within the NS2 region of the genome. CONCLUSION: The viral recombinants identified here resemble the recombinant form originally identified in Russia. The RLPH pattern observed in this study may be a signature indicative of this particular type of intergenotype recombinant of hepatitis C meriting clonal analysis of NS2.

Comparison of three stool antigen assays with the 13C- urea breath test for the primary diagnosis of Helicobacter pylori infection and monitoring treatment outcome.

BACKGROUND: The urea breath test (UBT) is the gold-standard non-invasive test for the detection of Helicobacter pylori infection, however, the lack of availability of the UBT due to the high cost of the test, and in particular the need for expensive analytical instrumentation, limits the usefulness of this method. Stool antigen assays may offer an alternative non-invasive method for the diagnosis of infection. OBJECTIVE: To compare the accuracy of three stool antigen assays (HpSA, IDEIA HpStAR, and ImmunoCard STAT) against the UBT for the primary diagnosis of H. pylori infection and for monitoring treatment outcome. METHODS: A total of 102 patients attending two gastroenterology day-case clinics for the investigation of dyspepsia were included. Each patient provided breath and stool samples for analysis. Patients who tested positive for H. pylori by the validated UBT were prescribed triple therapy and invited to return for repeat breath and stool sample analysis 6 weeks post-treatment. RESULTS: Of the 102 patients tested, 48 were diagnosed with H. pylori infection by the UBT. The HpSA assay interpreted 38 of these as positive (79% sensitive). Of the 54 UBT-negative patients the HpSA assay interpreted all 54 as negative (100% specific). The IDEIA HpStAR assay correctly identified 44 patients as positive (92% sensitive) and 50 as negative (92.5% specific). The ImmunoCard STAT assay interpreted 38 patients as positive (79% sensitive) and 52 as negative (96.3% specific). CONCLUSION: The findings indicate that the IDEIA HpStAR stool antigen kit is the most accurate assay of the three assays evaluated, and possibly represents a viable alternative to the UBT for the primary diagnosis of H. pylori infection and for monitoring treatment outcome.