Anthony Brown

Global distribution and prevalence of hepatitis C virus genotypes

Hepatitis C virus (HCV) exhibits high genetic diversity, characterized by regional variations in genotype prevalence. This poses a challenge to the improved development of vaccines and pan‐genotypic treatments, which require the consideration of global trends in HCV genotype prevalence. Here we provide the first comprehensive survey of these trends. To approximate national HCV genotype prevalence, studies published between 1989 and 2013 reporting HCV genotypes are reviewed and combined with overall HCV prevalence estimates from the Global Burden of Disease (GBD) project. We also generate regional and global genotype prevalence estimates, inferring data for countries lacking genotype information. We include 1,217 studies in our analysis, representing 117 countries and 90% of the global population. We calculate that HCV genotype 1 is the most prevalent worldwide, comprising 83.4 million cases (46.2% of all HCV cases), approximately one‐third of which are in East Asia. Genotype 3 is the next most prevalent globally (54.3 million, 30.1%); genotypes 2, 4, and 6 are responsible for a total 22.8% of all cases; genotype 5 comprises the remaining <1%. While genotypes 1 and 3 dominate in most countries irrespective of economic status, the largest proportions of genotypes 4 and 5 are in lower‐income countries. Conclusion: Although genotype 1 is most common worldwide, nongenotype 1 HCV cases—which are less well served by advances in vaccine and drug development—still comprise over half of all HCV cases. Relative genotype proportions are needed to inform healthcare models, which must be geographically tailored to specific countries or regions in order to improve access to new treatments. Genotype surveillance data are needed from many countries to improve estimates of unmet need. (Hepatology 2015;61:77–87)

Novel Adenovirus-Based Vaccines Induce Broad and Sustained T Cell Responses to HCV in Man

An adenoviral HCV vaccine induces antiviral T cell responses in human volunteers. Hepatitis Hide and Seek Like venture capitalists and Wall Street bankers, patients receiving results of their blood work don’t like surprises, and more than money is at stake. Because infections caused by the hepatitis C virus (HCV) frequently are asymptomatic, patients might not know they’ve been infected: Symptoms don’t usually appear until irreversible liver scarring has occurred, which may cause cirrhosis, liver failure, or cancer. Even if infection is caught early, current therapies to combat this stealth virus have serious side effects, and there is no vaccine to prevent or treat HCV infection. Now, Barnes et al. demonstrate that vaccines developed with adenoviral vectors can induce broad and sustained immune responses to HCV in humans. Adenoviral vectors have shown promise in vaccine trials in animal models; however, preexisting immunity to common serotypes in humans has limited their use. In a phase 1 clinical trial, Barnes et al. vaccinated healthy subjects with two rare serotype adenoviral vectors that expressed an HCV protein. Both the human and the chimp adenoviral vaccinations elicited HCV-specific immune responses in the recipients that responded to multiple HCV antigens, were sustained for at least a year with boost, and elicited memory responses. And the researchers got a surprise they liked: Vaccination primed T cells to respond to multiple HCV strains at a level consistent with protective immunity. Further trials will be needed to confirm protective or therapeutic roles in HCV-infected individuals. Currently, no vaccine exists for hepatitis C virus (HCV), a major pathogen thought to infect 170 million people globally. Many studies suggest that host T cell responses are critical for spontaneous resolution of disease, and preclinical studies have indicated a requirement for T cells in protection against challenge. We aimed to elicit HCV-specific T cells with the potential for protection using a recombinant adenoviral vector strategy in a phase 1 study of healthy human volunteers. Two adenoviral vectors expressing NS proteins from HCV genotype 1B were constructed based on rare serotypes [human adenovirus 6 (Ad6) and chimpanzee adenovirus 3 (ChAd3)]. Both vectors primed T cell responses against HCV proteins; these T cell responses targeted multiple proteins and were capable of recognizing heterologous strains (genotypes 1A and 3A). HCV-specific T cells consisted of both CD4+ and CD8+ T cell subsets; secreted interleukin-2, interferon-γ, and tumor necrosis factor–α; and could be sustained for at least a year after boosting with the heterologous adenoviral vector. Studies using major histocompatibility complex peptide tetramers revealed long-lived central and effector memory pools that retained polyfunctionality and proliferative capacity. These data indicate that an adenoviral vector strategy can induce sustained T cell responses of a magnitude and quality associated with protective immunity and open the way for studies of prophylactic and therapeutic vaccines for HCV.

A Human Vaccine Strategy Based On Chimpanzee Adenoviral and MVA Vectors That Primes, Boosts and Sustains Functional HCV Specific T-Cell Memory

A prime-boost HCV vaccine strategy induces durable and broad T cell responses, characteristic of those associated with viral control. An Ounce of HCV Prevention Chronic hepatitis C virus (HCV) infection causes liver inflammation that can lead to diminished liver function or liver failure. Recent approval of antiviral drugs for HCV affords health care providers with treatment options; however, these new therapies are expensive with limited availability, leaving the door open for preventative approaches such as vaccines. Swadling et al. report a first-in-human trial of a prime-boost vaccine strategy for HCV. They prime with a simian adenoviral vector followed by a modified vaccinia Ankara vector encoding HCV proteins, which induces a T cell response similar to that found in HCV control in natural infection. If this strategy can show efficacy in later-stage studies, this approach could be used in a preventative HCV vaccine. A protective vaccine against hepatitis C virus (HCV) remains an unmet clinical need. HCV infects millions of people worldwide and is a leading cause of liver cirrhosis and hepatocellular cancer. Animal challenge experiments, immunogenetics studies, and assessment of host immunity during acute infection highlight the critical role that effective T cell immunity plays in viral control. In this first-in-man study, we have induced antiviral immunity with functional characteristics analogous to those associated with viral control in natural infection, and improved upon a vaccine based on adenoviral vectors alone. We assessed a heterologous prime-boost vaccination strategy based on a replicative defective simian adenoviral vector (ChAd3) and modified vaccinia Ankara (MVA) vector encoding the NS3, NS4, NS5A, and NS5B proteins of HCV genotype 1b. Analysis used single-cell mass cytometry and human leukocyte antigen class I peptide tetramer technology in healthy human volunteers. We show that HCV-specific T cells induced by ChAd3 are optimally boosted with MVA, and generate very high levels of both CD8+ and CD4+ HCV-specific T cells targeting multiple HCV antigens. Sustained memory and effector T cell populations are generated, and T cell memory evolved over time with improvement of quality (proliferation and polyfunctionality) after heterologous MVA boost. We have developed an HCV vaccine strategy, with durable, broad, sustained, and balanced T cell responses, characteristic of those associated with viral control, paving the way for the first efficacy studies of a prophylactic HCV vaccine.

A Drosophila ortholog of the human cylindromatosis tumor suppressor gene regulates triglyceride content and antibacterial defense

The cylindromatosis (CYLD) gene is mutated in human tumors of skin appendages. It encodes a deubiquitylating enzyme (CYLD) that is a negative regulator of the NF-κB and JNK signaling pathways, in vitro. However, the tissue-specific function and regulation of CYLD in vivo are poorly understood. We established a genetically tractable animal model to initiate a systematic investigation of these issues by characterizing an ortholog of CYLD in Drosophila. Drosophila CYLD is broadly expressed during development and, in adult animals, is localized in the fat body, ovaries, testes, digestive tract and specific areas of the nervous system. We demonstrate that the protein product of Drosophila CYLD (CYLD), like its mammalian counterpart, is a deubiquitylating enzyme. Impairment of CYLD expression is associated with altered fat body morphology in adult flies, increased triglyceride levels and increased survival under starvation conditions. Furthermore, flies with compromised CYLD expression exhibited reduced resistance to bacterial infections. All mutant phenotypes described were reversible upon conditional expression of CYLD transgenes. Our results implicate CYLD in a broad range of functions associated with fat homeostasis and host defence in Drosophila.

Failure to Detect Xenotropic Murine Leukemia Virus-Related Virus in Blood of Individuals at High Risk of Blood-Borne Viral Infections

A xenotropic murine leukemia virus-related virus (XMRV) has recently been reported in association with prostate cancer and chronic fatigue syndrome, with a prevalence of up to 3.7% in the healthy population. We looked for XMRV in 230 patients with human immunodeficiency virus type 1 or hepatitis C infection. XMRV was undetectable in plasma or peripheral blood mononuclear cells by polymerase chain reaction targeting XMRV gag or env. T cell responses to XMRV Gag were undetectable in peripheral blood mononuclear cells by ex vivo gamma interferon enzyme-linked immunospot assay. In our cohorts, XMRV was not enriched in patients with blood-borne or sexually transmitted infections from the United Kingdom and Western Europe.

Short-term starvation of immune deficient Drosophila improves survival to gram-negative bacterial infections.

Background Primary immunodeficiencies are inborn errors of immunity that lead to life threatening conditions. These predispositions describe human immunity in natura and highlight the important function of components of the Toll-IL-1- receptor-nuclear factor kappa B (TIR-NF-κB) pathway. Since the TIR-NF-κB circuit is a conserved component of the host defence in higher animals, genetically tractable models may contribute ideas for clinical interventions. Methodology/Principal Findings We used immunodeficient fruit flies (Drosophila melanogaster) to address questions pertaining to survival following bacterial infection. We describe here that flies lacking the NF-κB protein Relish, indispensable for countering Gram-negative bacteria, had a greatly improved survival to such infections when subject to dietary short-term starvation (STS) prior to immune challenge. STS induced the release of Nitric Oxide (NO), a potent molecule against pathogens in flies, mice and humans. Administering the NO Synthase-inhibitory arginine analog N-Nitro-L-Arginine-Methyl-Ester (L-NAME) but not its inactive enantiomer D-NAME increased once again sensitivity to infection to levels expected for relish mutants. Surprisingly, NO signalling required the NF-κB protein Dif, usually needed for responses against Gram-positive bacteria. Conclusions/Significance Our results show that NO release through STS may reflect an evolutionary conserved process. Moreover, STS could be explored to address immune phenotypes related to infection and may offer ways to boost natural immunity.

Comparison of Next-Generation Sequencing Technologies for Comprehensive Assessment of Full-Length Hepatitis C Viral Genomes.

ABSTRACT Affordable next-generation sequencing (NGS) technologies for hepatitis C virus (HCV) may potentially identify both viral genotype and resistance genetic motifs in the era of directly acting antiviral (DAA) therapies. This study compared the ability of high-throughput NGS methods to generate full-length, deep, HCV sequence data sets and evaluated their utility for diagnostics and clinical assessment. NGS methods using (i) unselected HCV RNA (metagenomics), (ii) preenrichment of HCV RNA by probe capture, and (iii) HCV preamplification by PCR implemented in four United Kingdom centers were compared. Metrics of sequence coverage and depth, quasispecies diversity, and detection of DAA resistance-associated variants (RAVs), mixed HCV genotypes, and other coinfections were compared using a panel of samples with different viral loads, genotypes, and mixed HCV genotypes/subtypes [geno(sub)types]. Each NGS method generated near-complete genome sequences from more than 90% of samples. Enrichment methods and PCR preamplification generated greater sequence depth and were more effective for samples with low viral loads. All NGS methodologies accurately identified mixed HCV genotype infections. Consensus sequences generated by different NGS methods were generally concordant, and majority RAVs were consistently detected. However, methods differed in their ability to detect minor populations of RAVs. Metagenomic methods identified human pegivirus coinfections. NGS provided a rapid, inexpensive method for generating whole HCV genomes to define infecting genotypes, RAVs, comprehensive viral strain analysis, and quasispecies diversity. Enrichment methods are particularly suited for high-throughput analysis while providing the genotype and information on potential DAA resistance.

Full-Length Characterization of Hepatitis C Virus Subtype 3a Reveals Novel Hypervariable Regions under Positive Selection during Acute Infection

ABSTRACT Hepatitis C virus subtype 3a is a highly prevalent and globally distributed strain that is often associated with infection via injection drug use. This subtype exhibits particular phenotypic characteristics. In spite of this, detailed genetic analysis of this subtype has rarely been performed. We performed full-length viral sequence analysis in 18 patients with chronic HCV subtype 3a infection and assessed genomic viral variability in comparison to other HCV subtypes. Two novel regions of intragenotypic hypervariability within the envelope protein E2, of HCV genotype 3a, were identified. We named these regions HVR495 and HVR575. They consisted of flanking conserved hydrophobic amino acids and central variable residues. A 5-amino-acid insertion found only in genotype 3a and a putative glycosylation site is contained within HVR575. Evolutionary analysis of E2 showed that positively selected sites within genotype 3a infection were largely restricted to HVR1, HVR495, and HVR575. Further analysis of clonal viral populations within single hosts showed that viral variation within HVR495 and HVR575 were subject to intrahost positive selecting forces. Longitudinal analysis of four patients with acute HCV subtype 3a infection sampled at multiple time points showed that positively selected mutations within HVR495 and HVR575 arose early during primary infection. HVR495 and HVR575 were not present in HCV subtypes 1a, 1b, 2a, or 6a. Some variability that was not subject to positive selection was present in subtype 4a HVR575. Further defining the functional significance of these regions may have important implications for genotype 3a E2 virus-receptor interactions and for vaccine studies that aim to induce cross-reactive anti-E2 antibodies.

Chronic hepatitis C viral infection subverts vaccine‐induced T‐cell immunity in humans

Adenoviral vectors encoding hepatitis C virus (HCV) nonstructural (NS) proteins induce multispecific, high‐magnitude, durable CD4+ and CD8+ T‐cell responses in healthy volunteers. We assessed the capacity of these vaccines to induce functional HCV‐specific immune responses and determine T‐cell cross‐reactivity to endogenous virus in patients with chronic HCV infection. HCV genotype 1‐infected patients were vaccinated using heterologous adenoviral vectors (ChAd3‐NSmut and Ad6‐NSmut) encoding HCV NS proteins in a dose escalation, prime‐boost regimen, with and without concomitant pegylated interferon‐α/ribavirin therapy. Analysis of immune responses ex vivo used human leukocyte antigen class I pentamers, intracellular cytokine staining, and fine mapping in interferon‐γ enzyme‐linked immunospot assays. Cross‐reactivity of T cells with population and endogenous viral variants was determined following viral sequence analysis. Compared to healthy volunteers, the magnitude of HCV‐specific T‐cell responses following vaccination was markedly reduced. CD8+ HCV‐specific T‐cell responses were detected in 15/24 patients at the highest dose, whereas CD4+ T‐cell responses were rarely detectable. Analysis of the host circulating viral sequence showed that T‐cell responses were rarely elicited when there was sequence homology between vaccine immunogen and endogenous virus. In contrast, T cells were induced in the context of genetic mismatch between vaccine immunogen and endogenous virus; however, these commonly failed to recognize circulating epitope variants and had a distinct partially functional phenotype. Vaccination was well tolerated but had no significant effect on HCV viral load. Conclusion: Vaccination with potent HCV adenoviral vectored vaccines fails to restore T‐cell immunity except where there is genetic mismatch between vaccine immunogen and endogenous virus; this highlights the major challenge of overcoming T‐cell exhaustion in the context of persistent antigen exposure with implications for cancer and other persistent infections. (Hepatology 2016;63:1455‐1470)

HCV genotype-3a T cell immunity: specificity, function and impact of therapy

Background Hepatitis C virus (HCV) genotype-3a infection is now the dominant strain in South Asia and the UK. Characteristic features include a favourable response to therapy; the reasons for this are unknown but may include distinct genotype-3a-specific T cell immunity. In contrast to genotype-1 infection, T cell immunity to this subtype is poorly defined. Objectives The aims of the study were to (1) define the frequency, specificity and cross-reactivity of T cell immunity across the whole viral genome in genotype-3a infection and (2) assess the impact of interferon (IFN)-α/ribavirin on T cell immunity. Design T cell responses in chronic and resolved HCV genotype-3a were analysed in comparison with genotype-1 infection (total n=85) using specific peptide panels in IFN-γ ELISpot assays. T cell responses were followed longitudinally in a subset of genotype-3a infected patients receiving therapy. Responses were further defined by CD4 and CD8 subset analysis, sequencing of autologous virus and cross-reactivity of genotype-3a with genotype-1a/-1b antigens. Results CD8 T cell responses commonly targeted the non-structural (NS) proteins in chronic genotype-3a infection whereas in genotype-1 infection CD4 responses targeting HCV core predominated (p=0.0183). Resolved infection was associated with CD4 T cells targeting NS proteins. Paradoxically, a sustained response to therapy was associated with a brisk decline in virus-specific and total lymphocyte counts that recovered after treatment. Conclusion HCV genotype-3a exhibits a distinct T cell specificity with implications for vaccine design. However, our data do not support the theory that genotype-3a viral clearance with therapy is associated with an enhanced antiviral T cell response. Paradoxically, a reduction in these responses may serve as a biomarker of IFN responsiveness.