Resistance to hepatitis C virus:
potential genetic and immunological determinants
Michael Mokhlis Mina
A thesis submitted in fulfilment of the requirement for the degree of
Doctor of Philosophy
The Kirby Institute
The University of New South Wales
September 2020 GRIS 23/2/21, 6:22 pm
Thesis Title and Abstract Declarations Inclusion of Publications Corrected Thesis and Statement Responses
Thesis Title
Resistance to hepatitis C virus: potential genetic and immunological determinants
Thesis Abstract
Studies of highly exposed individuals who remain seronegative (HESN) for HIV infection led to the discovery that homozygosity for the d32 mutation in the CCR5 chemokine receptor gene abrogated viral entry into target cells, and was associated with resistance to infection. In addition, evidence for protective immunity has been found in some HESN groups, such as sex workers in the Gambia.
Population studies of those at high risk for hepatitis C virus (HCV) infection suggest that a HESN phenotype exists. There is a growing body of evidence for protective immunity, which allows clearance of HCV without seroconversion, and proof-of- principle evidence from in vitro studies that genetic polymorphisms may confer resistance to establishment of infection.
This doctoral research project explores evidence for protective immunity, including via genetically programmed variations in host responses and provides evidence that genetic mutations confer resistance against HCV. The data generally strengthens the notion that investigations of naturally occurring polymorphisms within the HCV interactome, and genetic association studies of well-characterised HESN individuals, may identify potential targets for vaccine design and inform novel therapies.
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Thesis Title and Abstract Declarations Inclusion of Publications Corrected Thesis and Statement Responses
ORIGINALITY STATEMENT
" I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged.
COPYRIGHT STATEMENT
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For any substantial portions of copyright material used in this thesis, written permission for use has been obtained, or the copyright material is removed from the final public version of the thesis.
AUTHENTICITY STATEMENT
" I certify that the Library deposit digital copy is a direct equivalent of the final officially approved version of my thesis.
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Thesis Title and Abstract Declarations Inclusion of Publications Corrected Thesis and Statement Responses
UNSW is supportive of candidates publishing their research results during their candidature as detailed in the UNSW Thesis Examination Procedure.
Publications can be used in your thesis in lieu of a Chapter provided:
You contributed greater than 50% of the content in the publication and are the "primary author", i.e. you were responsible primarily for the planning, execution and preparation of the work for publication. You have approval to include the publication in their thesis in lieu of a Chapter from your Supervisor and Postgraduate Coordinator. The publication is not subject to any obligations or contractual agreements with a third party that would constrain its inclusion in the thesis.
" My thesis has publications - either published or submitted for publication - incorporated into it in lieu of a Chapter/s. Details of these publications are provided below.
Publication Details #1
Full Title: Resistance to hepatitis C virus: potential genetic and immunological determinants
Authors: Michael M Mina, Fabio Luciani, Barbara Cameron, Rowena A Bull, Michael R Beard, David Booth, Andrew R Lloyd
Journal or Book Name: Lancet Infectious Diseases
Volume/Page Numbers: 2015;15: 451–60
Date Accepted/Published: Published Online February 19, 2015
Status: published
The Candidate's Contribution to Review fully written by Michael Mina the Work:
Location of the work in the thesis This paper is chapter 1 of the thesis. It sets the scene for the whole PhD. The paper and/or how the work is provides evidence that an HESN phenotype exists and it introduces the hypothesis of incorporated in the thesis: the PhD and illustrates that studies of individuals who were highly exposed but seronegative (HESN) for HIV infection led to the discovery that homozygosity for the Δ32 deletion mutation in the CCR5 gene prevents viral entry into target cells, and is associated with resistance to infection.
Additionally, evidence for protective immunity has been noted in some HESN groups, such as sex workers in The Gambia. Population studies of individuals at high risk for hepatitis C virus infection suggest that an HESN phenotype exists.
This Review discusses the possibility that genetic mutations confer resistance against hepatitis C virus, and also explores evidence for protective immunity, including via genetically programmed variations in host responses.
Publication Details #2
Full Title: Natural killer cells in highly exposed hepatitis C-seronegative injecting drug users
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Authors: M. M. Mina, B. Cameron, F. Luciani, U. Vollmer-Conna, A. R. Lloyd
Journal or Book Name: Journal of Viral Hepatitis
Volume/Page Numbers: 2016 Jun;23(6):464-72
Date Accepted/Published: Accepted for publication December 2015
Status: published
The Candidate's Contribution to First author and performed experiments the Work: 80% contribution
Location of the work in the thesis This is chapter 2 and follows on nicely from the literature review showing evidence that and/or how the work is there is protective immunity in the HESN phenotype. incorporated in the thesis: I describe a very robust algorithm to identify HESN and then use this cohort for immunity analysis.
This is the first of 2 chapters that discuss immunity in the HESN phenotype. This chapter discusses innate immunity and chapter 3 provides evidence of adaptive immunity.
Publication Details #3
Full Title: Anti-envelope antibody responses in highly exposed seronegative individuals may be associated with protection from HCV infection
Authors: Michael Mina, Alexander Underwood, Auda Eltahla, Bing-Ru Wu, Melanie R. Walker, Rowena A. Bull, Andrew R. Lloyd
Journal or Book Name: Journal of Viral Hepatitis
Volume/Page Numbers: 2020;00:1–10
Date Accepted/Published: 11 May 2020
Status: published
The Candidate's Contribution to First author and performed experiments the Work: Write manuscript, identification subjects, perform majority experimental work, data analysis, completing revisions
Location of the work in the thesis This is chapter 3 in the thesis and is the second paper to discuss protective immunity. and/or how the work is Chapter 2 focuses on innate immunity whilst chapter 3 studies adaptive immunity. incorporated in the thesis: The chapter describes anti-HCV Envelope (E) antibody responses in the HESN Phenotype.
The chapter also provides evidence of neutralization activity and HCV E–specific memory B cells.
These findings suggest that HESN subjects may be resistant to HCV infection through humoral immune-mediated mechanisms which flows on nicely from chapters 1 and 2.
Publication Details #4
Full Title: Polymorphisms in DOCK2 are associated with protection against hepatitis C infection
Authors: Michael M. Mina, Hui Li, Auda Eltahla, Fabio Luciani, Louisa Degenhardt, William D. Rawlinson, Nick Martin, Elliot C. Nelson, David Booth, Andrew R. Lloyd
Journal or Book Name: Journal of Infectious Diseases
Volume/Page Numbers:
Date Accepted/Published:
Status: submitted
The Candidate's Contribution to First author and performed experiments. >80% contribution. the Work:
Location of the work in the thesis This is chapter 4 of the thesis and flows on from the concept introduced in chapter 1 and/or how the work is regarding genetic resistance in the HESN phenotype. incorporated in the thesis: The chapter provides evidence linking polymorphisms in the immunoregulatory https://gris.unsw.edu.au/examinations/thesub/view/92987/responses#incl_of_pubs_stmt_view Page 2 of 3 GRIS 23/2/21, 6:24 pm
proteins encoded by DOCK-2 to resistance against HCV infection.
CANDIDATE’S DECLARATION
" I declare that I have complied with the Thesis Examination Procedure.
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Acknowledgements vi
List of Tables vii
List of Figures viii
List of Publications related to this PhD ix
Abbreviations x
Introduction 1
Chapter 1. Literature Review - Resistance to hepatitis C virus: potential genetic and 3 immunological determinants
Abstract 4
1.1 Introduction 4
1.2 Evidence for resistance and protection against hepatitis C virus infection 4
1.3 Protective immunity against hepatitis C 7
1.3.1 Primary Infection 7
1.3.2 Reinfection 7
1.3.3 HESN phenotype 8
1.3.4 Occult infection 8
1.4 Genetically determined resistance against hepatitis C virus infection 9
1.5 Conclusion 10
1.6 References 11
1.7 Supplementary 14
1.7.1 Supplementary Table 1 References 24
1.7.1 Supplementary Table 2 References 34
Chapter 2. Natural killer cells in highly exposed hepatitis C-seronegative injecting 37 drug users
Summary 38
2.1 Introduction 38
2.2 Materials and Methods 39
2.2.1 Study Cohort 39
ii
2.2.2 Analysis of risk behaviour and composite risk scores 39
2.2.3 NK cell phenotyping 40
2.2.4 In vitro stimulation with TLR ligands 41
2.2.5 Flow cytometry analysis 41
2.2.6 Statistical analysis 41
2.3 Results 41
2.3.1 Subjects 41
2.3.2 NK subsets 41
2.3.3 Principal components analysis identified phenotypic and 42
functional patterns associated with the HESN incident
phenotype
2.3.4 Baseline vs follow-up within the incident group 42
2.3.5 Baseline HESN vs baseline incident 42
2.4 Discussion 42
2.5 Acknowledgements and Disclosures 44
2.6 Funding & Conflicts of Interest 44
2.7 References 44
Chapter 3. Anti-envelope antibody responses in highly exposed seronegative 47 individuals may be associated with protection from HCV infection
Abstract 48
3.1 Introduction 48
3.2 Materials and Methods 49
3.2.1 Study cohort 49
3.2.2 Ethics 49
3.2.3 Identification of HESN subjects 49
3.2.4 Control subjects 49
3.2.5 HCVpp production and collection of E1/E2 lysate 50
3.2.6 Capture E1/E2 enzyme-linked immunosorbent assays (ELISAs) 50
3.2.7 Recombinant genotype 1a E2 (rE2) binding ELISA 50
iii
3.2.8 HCVpp neutralization assay 50
3.2.9 Analysis of HCV-specific B cells 51
3.2.10 Statistical analysis 51
3.3 Results 51
3.3.1 Subjects 51
3.3.2 Reactivity of HESN and incident plasma to anti-E ELISAs 51
3.3.3 Isotyping IgG-positive HESN subjects 52
3.3.4 Neutralization of HCVpp 53
3.3.5 E2-specific B cells in HESN subjects 53
3.4 Discussion 53
3.5 Acknowledgements 56
3.6 Conflict of Interests 56
3.7 References 56
3.8 Supplementary Tables 58
Chapter 4. Polymorphisms in DOCK2 are associated with protection against hepatitis C 61 infection
Abstract 64
4.1 Introduction 66
4.2 Materials and methods 69
4.2.1 Study cohorts 69
4.2.2 Selection of HESN and control subjects 69
4.2.3 SNP genotyping 70
4.2.4 Statistical analysis 71
4.3 Results 72
4.3.1 HESN cases and Incident control subjects 72
4.3.2 Associations in the HITS-p cohort 72
4.3.3 Replication analysis in CATS 73
4.3.4 Haplotype analyses in the DOCK-2 region 74
4.4 Discussion 75
iv 4.5 Acknowledgements and Disclosures 79
4.6 Funding & Conflicts of interest 79
4.7 Contributors 79
4.8 References 86
Chapter 5. Discussion and conclusion 90
5.1 Discussion 91
5.2 Conclusion 96
References to Introduction and Discussion 98
v Acknowledgements
I am most indebted to my supervisor Professor Andrew Lloyd for his invaluable support and guidance.
I would also like to thank Dr. Barbara Cameron, Dr. Auda Eltahla, Mrs Elizabeth
Keoshkerian and Mrs Hui Li for their valuable technical advice and input in my PhD project.
Dedicated to:
Monica, Gabrielle, Juliette, Christie and Luke. I love you more than you will ever know.
To those who gave me special words of encouragement.
vi
List of Tables
Page
1.1 Supplementary Table 1 15
Hepatitis C virus interacting host proteins
1.2 Supplementary Table 2 32
Genetic associations with HCV clearance
2.1 Demographics of the three subject groups at baseline, 40
Selected by composite risk score
2.1 Logistic regression analysis of principal components describing the 42
phenotype and function of NK subsets
3.1 Supplementary Table 1 58
Demographic characteristics of HESN and Incident subject groups at
baseline
3.2 Supplementary Table 2 59
Anti-HCV Envelope antibody positivity in HESN subjects
3.3 Supplementary Table 3 60
IgG subclasses in HESN subjects positive for IgG against HCV Envelope
4.1 Demographic and risk behaviour characteristics of HESN cases and Incident 80
control subjects in the prospective HITS-p cohort and the replication CATS
cohort
4.2 SNPs associated with the HESN phenotype in the HITS-p and CATS cohorts 81
4.3 DOCK-2 haplotype distribution in HESN cases and Incident control groups in 83
the HITS-p and CATS cohorts
vii List of Figures
Page
1.1 The hepatitis C virus life cycle 5
1.2 Network diagram showing connections between HCV proteins, HCV- 6
interacting human proteins, and candidate proteins from the HCVcc siRNA
screen
1.3 Hepatitis C virus prevalence in relations to time at risk 7
1.4 Identification of highly exposed seronegative individuals 8
2.1 Schematic overview of the subject groups, samples and statistical analyses 40
2.2 Analyses of NK subpopulations 43
3.1 IgG binding against rE2 and E1/E2 in the plasma of HESN cases at three 52
separate time points, and in pre-infection and post-infection plasma from
Incident control subjects.
3.2 IgM binding against rE2 and E1/E2 in the plasma of HESN cases at three 53
separate time points, and in pre-infection and post-infection plasma from
Incident control subjects.
3.3 IgA binding against rE2 and E1/E2 in the plasma of HESN cases at three 54
separate time points, and in pre-infection and post-infection plasma from
Incident control subjects.
3.4 IgG1, IgG2, IgG3 and IgG4 isotyping against rE2 and E1/E2 in the plasma 55
of HESN cases.
3.5 Neutralizing activity (%) of plasma from HESN cases positive for IgG and 55
pre-infection time point of Incident control subjects against HCVpp containing
Envelopes of H77 and UKN3A13.6.
3.6 Frequency of HCV-specific B cells (% of total B-cell population; CD 19+CD20+ 56
CD10-IgD-) in the PBMCs of HESN and the pre-infection time point of Incident
control subjects.
4.1 Schematic overview of HESN subject groups in HITS-p and CATS, with 84
associated and replicated SNPs
4.2 Locus zoom plot for association signals in regions containing DOCK-2 85
viii List of Publications related to this PhD
Included in thesis as chapters for examination
1. Mina M, Luciani F, Cameron B, Bull R, Beard MR, Booth D and Lloyd AR. Resistance to
hepatitis C infection: potential genetic and immunological determinants. Lancet Infectious
Diseases 2015; 15(4):451-460.
2. Mina M, Cameron B, Luciani F, Vollmer-Conna U and Lloyd AR. Natural killer cells in highly
exposed hepatitis C-seronegative injecting drug users. Journal of Viral Hepatitis 2016;
23(6):464-472.
3. Mina M, Underwood A, Eltahla A, Wu BR, Walker MR, Bull RA and Lloyd AR. Anti-envelope
antibody responses in highly exposed seronegative individuals may be associated with
protection from HCV infection. Journal of Viral Hepatitis 2020 Jun 4. doi:
10.1111/jvh.13339. Online ahead of print.
Chapter 4 submitted for publication
4. Polymorphisms in DOCK2 are associated with protection against hepatitis C infection. Mina M,
Li H, Eltahla A, Eltahla F, Degenhardt L, Rawlinson WD, Martin NG, Jeffries P, Nelson EC,
Booth D, Lloyd AR.
ix Abbreviations
Ab Antibody
ADCC Antibody-dependent cellular cytotoxicity
ATSI Aboriginal or Torres Strait Islander
CATS Comorbidity and Trauma Study
CCL Chemokine (C-C motif) ligand
CCR Chemokine (C-C motif) receptor
CXCL CXC- chemokine ligand
CXCR CXC- chemokine receptor
DAA Directly acting antivirals
DOCK2 Dedicator of cytokinesis 2
ELISA Enzyme-linked immunosorbent assay
FMO Fluorescence-minus-one
GNA Galanthus nivalis
GT Genotype
GWAS Genome-wide association study
HCl Hydrochloric acid
HCV Hepatitis C virus
HCVpp HCV pseudoparticle
HESN Highly exposed seronegative
HITS-p Hepatitis C Incidence and Transmission
Study in prison
HIV Human immunodeficiency virus
HLA Human leucocyte antigen
IDU Injecting drug use
IDUs Injecting drug users
IFN Interferon
IFNg Interferon gamma
ISGs Interferon-stimulated genes
KIR Killer inhibitory receptor
x NCRs Natural cytotoxicity receptors
KIR Killer cell immunoglobulin-like receptor
NCR Natural cytotoxicity receptor
NK Natural killer
NKG2D Natural killer group 2 member D
NSP Non-structural proteins
OST Opioid substitution therapy
PBMC Peripheral blood mononuclear cells
PC Principal component
PCA Principal components analysis
PI4K-IIIa Phosphatidylinositol 4-kinase-III alpha
PWID People who inject drugs
RIPA Radioimmunoprecipitation assay
RNA Ribonucleic Acid
RNAi RNA interference
SNP Single nucleotide polymorphisms
SVR Sustained viral response
TLR Toll-like receptor
TNF Tumour necrosis factor
TNFa Tumour necrosis factor alpha
UNSW University of New South Wales
VZV Vesicular stomatitis virus
χ² Chi-square tests
xi Introduction
This doctoral research project addresses the hypotheses that genetic mutations confer resistance against hepatitis C (HCV), and that resistance is mediated immunologically, including via genetically programmed variations in host responses that protect against establishment of chronic HCV infection.
Injecting drug use (IDU) is the major risk factor for HCV transmission with the prevalence of infection rising progressively in relation to accumulated years of IDU. A small minority people who inject drugs
(PWID) who have been undertaking IDU over many years remain seronegative and aviraemic, despite prolonged probable exposure to HCV - termed highly exposed, seronegative (HESN) subjects. The first chapter in this thesis is a literature review published in Lancet: Infectious Diseases entitled “Resistance to hepatitis C virus: potential genetic and immunological determinants” and introduces the hypotheses of this doctoral research project. This chapter has been written fully by myself and I have also composed a list of host genes that play a role in HCV infection (Supplementary Table). It sets the scene for the whole thesis and provides evidence that an HESN phenotype exists.
The research studies in this thesis were underpinned by a framework for reliable identification of the
HESN phenotype utilising a risk-behaviour algorithm predicting incident infection from longitudinally- collected risk and infection data similar to the procedure used to model coronary heart disease risks,
(1) and HIV infection risks. (2) Each of the experimental chapters utilised a case-control study design in which long-standing, high risk, HCV-uninfected HESN cases were compared to controls who became
HCV infected (i.e ‘Incident control’ subjects who were non-protected) during longitudinal follow-up. The aims of the second and third chapters in the thesis, entitled “Natural killer cells in highly exposed hepatitis C-seronegative injecting drug users,” and “Anti-envelope antibody responses in highly exposed seronegative individuals may be associated with protection from HCV infection,” both published in the Journal of Viral Hepatitis, were to establish whether innate immunity (natural killer cells) and adaptive humoral immunity (anti-Envelope [E] antibodies, neutralisation of HCV pseudoparticles, and E2-specific B cells), were associated with protection against HCV infection in HESN cases versus
Incident controls. Natural killer cells in highly exposed hepatitis C-seronegative injecting drug users, provides evidence that there is protective immunity in the HESN phenotype. I describe a very robust algorithm to identify HESN and then use this cohort for immunity analysis. I am first author of this paper
1 and performed all experiments. Statistical advice was given by Roy Wilson from the Australian Centre for Commercial Mathematics and I performed the analysis. Anti-envelope antibody responses in highly exposed seronegative individuals may be associated with protection from HCV infection, describes anti-
HCV Envelope (E) antibody responses in the HESN Phenotype. The chapter also provides evidence of neutralization activity and HCV E–specific memory B cells. I wrote the manuscript, identified the subjects, and performed the majority of the experimental work and data analysis. The HCVpp neutralization assay was carried out by Dr Alexander Underwood and the analysis of HCV-specific B cells was performed by Dr Bing-Ru Wu.
The aim of chapter 4 which is described in the submitted manuscript entitled “Polymorphisms in DOCK2 are associated with protection against hepatitis C infection,” was to explore the hypothesis that host genetic polymorphisms may confer resistance to HCV infection. The chapter provides evidence linking polymorphisms in the immunoregulatory proteins encoded by DOCK-2 to resistance against HCV infection. I identified the cohort genotyped using the multiple sclerosis (MS) Chip, an Illumina Infinium
HD custom array that was developed by the International MS Genetics Consortium and wrote the paper.
I was assisted by Hui Li with the statistical analysis.
2 Chapter 1
Literature Review - Resistance to hepatitis C virus: potential genetic and
immunological determinants
3 Review
Resistance to hepatitis C virus: potential genetic and immunological determinants
Michael M Mina, Fabio Luciani, Barbara Cameron, Rowena A Bull, Michael R Beard, David Booth, Andrew R Lloyd
Studies of individuals who were highly exposed but seronegative (HESN) for HIV infection led to the discovery that Lancet Infect Dis 2015; homozygosity for the Δ32 deletion mutation in the CCR5 gene prevents viral entry into target cells, and is associated 15: 451–60 with resistance to infection. Additionally, evidence for protective immunity has been noted in some HESN groups, Published Online such as sex workers in The Gambia. Population studies of individuals at high risk for hepatitis C virus infection February 19, 2015 http://dx.doi.org/10.1016/ suggest that an HESN phenotype exists. The body of evidence, which suggests that protective immunity allows S1473-3099(14)70965-X clearance of hepatitis C virus without seroconversion is growing. Furthermore, proof-of-principle evidence from in- Infl ammation and Infection vitro studies shows that genetic polymorphisms can confer resistance to establishment of infection. This Review Research Centre, School of discusses the possibility that genetic mutations confer resistance against hepatitis C virus, and also explores evidence Medical Sciences, University of for protective immunity, including via genetically programmed variations in host responses. The data generally New South Wales, Sydney, NSW, Australia (M M Mina MRCP, strengthens the notion that investigations of naturally arising polymorphisms within the hepatitis C virus interactome, F Luciani PhD, B Cameron PhD, and genetic association studies of well characterised HESN individuals, could identify potential targets for vaccine R A Bull PhD, Prof A R Lloyd MD); design and inform novel therapies. School of Molecular and Biomedical Science, University of Adelaide, Adelaide, SA, Introduction blood transfusion (before the introduction of serological Australia (M R Beard PhD); Infectious diseases are a leading cause of human morbidity screening globally in the early 1990s).8 Centre for Cancer Biology, SA and mortality, and therefore act as a major selective Hepatitis C virus encodes very few proteins and Pathology, Adelaide, SA, pressure on the evolution of the human genome.1 therefore depends heavily upon host factors for pro- Australia (M R Beard); and Westmead Millennium 9 Accordingly, clear evidence suggests that genetic variation pagation (fi gure 1). Viruses enter hepatocytes via inter- Institute, University of Sydney, in human populations contributes to a population’s actions between viral envelope proteins E1 and E2, and Sydney, NSW, Australia susceptibility to infectious diseases, and in some individuals four known host receptors, CD81 (a widely expressed (D Booth PhD) confers resistance to establishment of infection. This tetraspanin), scavenger receptor class B type-1, and the Correspondence to: resistance is best exemplifi ed by the recognition that tight junction proteins claudin-1 and occludin.10–12 Dr Michael M Mina, Infl ammation and Infection individuals who are homozygous for a common loss of Additionally, other molecules including heparan sulfate, Research Centre, School of function variant of the FUT2 gene do not express the both dendritic cell and liver specifi c intracellular Medical Sciences, University of H type-1 oligosaccharide ligand needed for Norwalk virus adhesion molecule-3-grabbing non-integrins,13 low- New South Wales, Sydney binding, and cannot be infected with this pathogen.2 density lipoprotein-receptor,14 and the Niemann–Pick C1- NSW 2052, Australia [email protected] Once infection is established, interactions between like 1 cholesterol absorption receptor15 have all been determinants of microbial virulence and host immune implicated in hepatitis C virus cell attachment and entry. defence mechanisms underpin the pathogenesis of the Investigators using infectious cell culture systems with infectious disease. The desired outcome for the host is both hepatitis C virus replicons (which do not contain the effi cient elimination of the pathogen, with restricted full genome) and the full-length-genome Japanese tissue injury, and long lasting immunological protection fulminant hepatitis-1 strain, have identifi ed hundreds of against reinfection.3 Conversely, the ideal outcome for the host-viral protein interactions, many potentially relevant evolutionary survival of the pathogen is to ensure to resistance and protective immunity (fi gure 2).16 For transmission to another host—ie, by induction of example, RNA interference (RNAi) screens have restricted tissue injury and infection of suffi cient duration uncovered many host cofactors for hepatitis C virus to ensure transmission to a new host. The generation of replication, with phosphatidylinositol 4-kinase-IIIα protective immunity against pathogens is clearly, at least (PI4K-IIIα) the most consistently identifi ed factor.17,18 This in part, genetically defi ned.4 enzyme phosphorylates phosphatidylinositol in the Hepatitis C virus infection is a major problem for 4 position of the inositol ring to generate phosph- public health programmes worldwide, with an estimated atidylinositol 4-phosphate. PI4K-IIIα kinase activity is 180 million individuals infected.5 Acute hepatitis C virus needed for membranous web formation, and silencing of infection is usually asymptomatic and results in clearance PI4K-IIIα results in an aggregation of double-membrane in 25% of individuals infected.6 For chronically infected vesicles and hepatitis C virus replication complexes.19 individuals, the virus drives sustained hepatic necro- AL-9, a member of the 4-anilinoquinazoline-containing infl ammation and fi brosis, and persists throughout life kinase inhibitor family, inhibits hepatitis C virus unless cured by antiviral treatment.7 Transmission of replication in vitro by directly inhibiting PI4K-IIIα.20 hepatitis C virus is predominantly associated with Another RNAi screen targeting about 4000 human genes sharing and re-use of injecting apparatus among identifi ed nine cellular genes that regulate viral replication. injecting drug users (IDUs), but other parenteral means Investigators silencing these genes reported an inhibition can cause transmission too, including tattooing and of viral replication by more than 60%.21
4 www.thelancet.com/infection Vol 15 April 2015 451 Review
Endoplasmic 2 Fusion and uncoating reticulum
3 Translation
RNA
1 Entry Structural proteins
Non-structural proteins
Nucleus
4 RNA replication
Lipid droplet Lipoviroparticle
6 Maturation Golgi
5 Assembly 7 Release
Figure 1: The hepatitis C virus life cycle Reproduced with permission from Herker and Ott.9 ER=endoplasmic reticulum.
The expression of many host cellular proteins, which Evidence for resistance and protection against might modulate hepatitis C virus replication, such as hepatitis C virus infection heat shock protein 27,22 α-actinin,23 nucleolin,24 Identifi cation of individuals who are highly exposed eukaryotic initiation factor 4A-I,25 and Rho GDP- but seronegative dissociation inhibitor 226 can be increased in the Injecting drug use is the major risk factor for hepatitis C presence of replicating hepatitis C virus. Prominent in virus transmission in developed countries, with this list too are host immune response proteins, notably seroprevalence in cross-sectional studies of IDUs including the interferon-stimulated genes such as ranging from 50% to more than 90%.32 A few long-term viperin, which interacts with both hepatitis C virus IDUs remain seronegative and aviraemic, despite NS5A, core proteins, and vesicle-associated membrane extended and probably repeated exposure to hepatitis C protein-associated protein subtype A, which results in virus through sharing of drug-injection equipment.33 the disruption of viral replication.27 Additionally, strong This highly exposed but seronegative (HESN) group evidence exists for a role of apolipoprotein E as a have been termed exposed uninfected, which suggests hepatitis C virus infectivity factor because viral the absence of demonstrable infection, as defi ned by replication is closely associated with cellular lipids.28,29 conventional antibody or RNA testing.34 Several Small interfering RNAs or inhibitors that target prospective studies of high-risk IDUs have likewise components of very low density lipoprotein synthesis, reported subgroups that have remained seronegative inhibit infectious hepatitis C virus secretion.28,30,31 despite longstanding high-risk behaviour,35 who might Much remains to be discovered in relation to the represent a phenotype that are resistant, or became hepatitis C virus-host protein interactome and the host infected but had effi cient viral clearance before immune response characteristics against this pathogen. seroconversion.36 A quantitative meta-analysis of the Nevertheless, evidence suggests that hepatitis C virus prevalence of hepatitis C virus infection in relation to replication is dependent on many host factors and time since onset of injecting-drug use, showed both investigation of genetically defi ned variations in these linear and quadratic eff ects (fi gure 3), with a 95% CI of host proteins could uncover mechanisms of resistance hepatitis C virus prevalence ranging between 93% and and protection against hepatitis C virus infection. 99% after 15 years of use.37 In view of the apparent
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ADAMTS4 HCV-encoded proteins Interactions between HCV-encoded FCGRT HCV-interacting proteins (human) (HHCV) proteins and HCV-interacting proteins STMN2 HCVcc siRNA screen hits Interactions between HCVcc siRNA SRI HCV-interacting proteins that also scored screen hits and HCV-interacting proteins in the HCVcc siRNA screen TRRAP B2M ELAVL1 BCAN NMT1 EEF1A1 UBA52 C20orf20 PRKRIP1 UBQLN4 FN1 CALCOCO2 p7 GNB2 EFEMP1 STIM2 CYBA IKBKE KPNA1 KHDRBS1 MAPK7 LCK TRIO SRC HCK XPNPEP1 MORF4L1 PRPF40A PTMA EIF2AK2 PIK3R1 PRKACA JUN FES RBM4 ZNF467 ZNF148 FMNL1 RINT1 APLP2 SIAH1 ARFIP2 TAF1 NS3 STAT3 AKT1 TBK1 RAC1 ITGB1 TGFB1I1 FYN TICAM1 GNB2L1 USHBP1 YY1AP1 CDC6 CCNB2 SORBS3 F WDR37 CDKN1A MBP AGT EWSR1 GRB2 FLT4 ERCC5 C1QBP MYO3A MAPK1 FRS3 TGFBR1 CORE BCAR1 E2F2 YY1 CREBBP BCL6 NUAK2 CHUK YWHAZ CDCA3 TRAF2 MARK3 CNOT3 YWHAE CNOT2 WWTR1 CNOT1 IRF3 YWHAB PPIB SRRM2 NPAT VIM TP73 KCNK3 PROX1 EP300 TP53 PSME1 TFAP2A FADD TNFRSF1A TWIST1 FAS TBP TOP1 ZEB1 STAT1 RIPK2 HIPK3 BRF1 SMAD3 MCM2 RAF1 JAK1 LIMS2 PML RXRA JAK2 SMURF1 KRT8 DDX3X PLSCR1 PPP2R1A PKM2 KRT18 NR4A1 SMURF2 CDK6 DNAJA3 NS5A PPIA FHL2 NS4A NOC4L E1 NS5B RAB14 GSK3B NUP62 HSPA1A SMAD6 LYN NS2 ARHGEF10L E2 SORBS2 SMAD5 ITGA7 VAPA CDH1 VAPB TUBB2C CANX UBQLN1 DLGAP4 DNAJB1 NCL OSBP VAMP1
Figure 2: Network diagram showing connections between HCV proteins, HCV-interacting human proteins, and candidate proteins from the HCVcc siRNA screen The appendix shows a summary of the HCV interactome. Reproduced from Li and colleagues,16 by permission from National Academy of Sciences. See Online for appendix HCV (cc)=hepatitis C virus (cell culture system). plateau in the prevalence datasets, some or all of the cohort of Australian IDUs in prison38 to allow investigation remaining 1–7% of the group are plausibly HESN of genetic determinants of resistance and protective individuals. immunity.39 A risk–behaviour algorithm predicting In February, 2014, we reported a framework for reliable incident infection was developed, similar to the procedure identifi cation of the HESN phenotype in a prospective used to model coronary heart disease risk40 and HIV
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interferons α and β) and thereafter many interferon- 100 stimulated genes with antiviral properties. Increased interferon-stimulated gene expression in the liver during acute hepatitis C virus has been associated with 47 80 spontaneous clearance. Natural killer (NK) cell activation has likewise been shown to predict clearance,48,49 although activation has been reported irrespective of outcome.50 Early, vigorous, and sustained CD4-positive T-cell 60 proliferative responses against many hepatitis C virus proteins predict disease resolution.51 In-vivo depletion studies in chimpanzees have shown that virus-specifi c CD8-positive T cells and CD4-positive T cells are key 40 eff ector cells for the host when controlling hepatitis C 52,53
Hepatitis C virus prevalence (%) Hepatitis virus replication. During primary infection, CD8- positive T cells often show an exhausted phenotype with 54 20 impaired ability to secrete interferon γ. Additionally, a close reciprocal association exists between CD8-positive T-cell exhaustion and viral escape.55 Neutralising antibodies that block hepatitis C virus cell entry are 0 directed against epitopes in the E2 region of the envelope, 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 including the hypervariable regions. Studies that used Time since onset of drug injection (years) hepatitis C virus pseudoparticles to model systems Figure 3: Hepatitis C virus prevalence in relation to time at risk enabled the investigation of virus entry and showed that 72 studies and 293 categories of time at risk, hepatitis C Virus Synthesis Project, 1989–2006. Blue line shows neutralising antibodies are sometimes induced in the model-predicted prevalence in relation to time since onset of drug injecting for studies in developed countries after 1995. Reproduced with permission from Hagan and colleagues.37 early phase of infection in patients who subsequently clear the virus.56 Conversely, in the chronic progressors infection risks.41 The index includes many recognised (analogous to HIV infection), these antibodies typically behavioural risk factors for transmission (eg, injecting appear late and have neutralisation activity against drug use, sharing of injecting apparatus, and injecting variants no longer present in the quasispecies.57 heroin) weighted on the basis of the individual hazard ratio for prediction of incident infection. Application of Reinfection this index at each timepoint to longitudinal data about Early studies in the chimpanzee model showed that risk behaviours in the cohort showed that a small subset repeated exposure to homologous and heterologous of individuals in the upper risk tertile had consistently strains of hepatitis C virus could result in repeated raised risk indices, comparable with, or higher than, the infection, although reinfection was generally associated incident cases, but remained uninfected (fi gure 4). with reduced periods of viraemia and a heightened Although this algorithm and the associations identifi ed probability of clearance.58 Reinfected chimpanzees had need to be replicated in other cohorts, this approach rapid acquisition of specifi c cytolytic activity by liver off ers a framework for improved reliability in the resident CD8-positive T cells and expansion of memory defi nition of HESN for future studies. CD4-positive and CD8-positive T cells in the blood,53 featured reduced peak amounts of alanine amino- Protective immunity against hepatitis C virus transferase, and produced interferon γ and tumour Primary infection necrosis factor α (TNFα) earlier and at higher amounts If researchers are able to understand the diff erences in than normal.59 This phenotype has been confi rmed in immune response characteristics between individuals who human beings.35 For instance, individuals with primary clear chronic infection and those who develop it, they will infection were reported to be 12-times more likely to gain insights into potentially protective immunity43,44 develop chronic infection, and have average hepatitis C because acute hepatitis C virus infection results in clearance viral loads 2 logs higher than did those who became in 25% of cases,6 and is associated with innate and adaptive infected after having previously cleared hepatitis C immune responses in most individuals. However, virus.60 However, a study in 2013 of hepatitis C virus pre- reinfection in high-risk individuals is common, including exposed chimpanzees did not reproduce this fi nding.61 rapidly after an initial episode,45,46 suggesting that the The chimpanzees were repeatedly exposed to human immunity induced by primary infection generally has plasma with trace amounts of hepatitis C virus, and had restricted cross-protective capacity.35 induction of hepatitis C virus-specifi c T cells without Soon after hepatitis C virus infection, an innate seroconversion and systemic viraemia, but were not immune response is evident in the liver and in the blood, protected upon subsequent hepatitis C virus challenge.61 featuring induction of antiviral proteins (notably type 1 Conversely, the investigators reported suppression of the
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AB 0·8 Incident Uninfected T1 T 3 T 3 Uninfected T2 0·6 Uninfected T3
T 2 T 2 0·4 Risk score
0·2 T 1 T 1
0·0 Baseline Final Baseline Mid Final
Figure 4: Identifi cation of highly exposed seronegative individuals Composite risk scores shown as means with standard deviation bars. Uninfected participants were split into risk tertiles T1–T3 on the basis of the fi nal timepoint of subsequently infected participants.38 Reproduced from Sugden and colleagues,38 by permission from John Wiley and Sons. Data from the Hepatitis C Incidence and Transmission Study in prisons.42 chimpanzees’ immune responses, and two of three pre- show established infection, leading to processing and exposed chimpanzees developed chronic infection after presentation of viral peptides through class I MHC. rechallenge with the virus, with concomitant regulatory Hence, detection of hepatitis C virus-specifi c cytotoxic T-cell expansion.61 An important point to emphasise is T-cell responses in HESN individuals potentially suggests that the inoculum of hepatitis C virus infection is often that these individuals have been previously infected with not known in studies on human cohorts. Another study62 hepatitis C virus.72 Previous infection might also be reported that only fi ve of nine patients who had associated with rapid seroreversion. Follow-up studies successfully eliminated a previous hepatitis C virus after spontaneous clearance suggest that 20–50% of infection were able to spontaneously resolve a subsequent individuals serorevert, to become hepatitis C virus infection. Spontaneous resolution of reinfection was antibody negative more than a decade after spontaneous associated with a rise in both the magnitude and breadth viral clearance.73,74 Alternatively, T-cell responses to not of the total hepatitis C virus-specifi c T-cell response, previously encountered antigens can occur, and have suggesting generation of de-novo T-cell responses.62 been associated with enhanced or diminished immunity and changed immunopathological eff ects.75 In hepatitis C HESN phenotype virus, a cross-reactive epitope is shared between the NS3 Cross-sectional studies have documented hepatitis C protein and the infl uenza virus,76 and CD8-positive T-cell virus-specifi c CD4-positive and CD8-positive T cell responses, specifi c for this epitope, have been reported in responses in the absence of viral antibodies or viraemia, acute hepatitis C virus and associated with severe illness.77 in a range of high risk groups who remain uninfected. These studies include those of family members of Occult infection patients with chronic hepatitis C virus,63 sexual partners Another plausible explanation for the HESN phenotype is of people with chronic hepatitis C virus,64 health-care that these individuals are infected with a replication- workers with needlestick injuries,65 children born to defective viral variant, or one with a non-hepatocyte chronically infected mothers,66 children living with cellular tropism, allowing generation of cellular immunity hepatitis C virus-infected siblings,67 and high-risk without seroconversion or evidence of viraemia in the uninfected IDUs.34 NK cell activity might also have a role circulation.78 Such so-called occult hepatitis C virus in abrogating of established infection in high-risk infection has been reported in B and T lymphocytes, uninfected IDUs,68 and in infants born to chronically dendritic cells, and monocytes from patients who have infected mothers.69 Additionally, one report70 has shown cleared the virus, albeit at very low proportions.79,80 This hepatitis C virus-specifi c cytotoxic T-cell responses in occult hepatitis C virus infection might be analogous to conjunction with established, but ultimately transient, so-called elite controllers of HIV infection, in which some viraemia without seroconversion, in prisoners who individuals harbour HIV proviral DNA in resting CD4- acknowledged use of injecting drugs and sharing of the positive T cells at proportions 10⁴–10⁶ times lower than injecting apparatus. Similar fi ndings were reported in those in most infected individuals, which is much lower chimpanzees inoculated with very low doses of than the detection levels of conventional assays.81 This low hepatitis C virus.71 degree of infection might plausibly be benefi cial to the Demonstration of CD8-positive cytotoxic T-cell host by providing persisting antigenic stimulation to responses against viral antigens is usually assumed to sustain or enhance both innate and either HIV or
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hepatitis C virus specifi c cellular immunity, thereby receptor (KIR) genotype (2DL2 or 2DL3), in combination reducing the possibility of reinfection.82,83 with the absence of the HLA-C1 ligand for this KIR, has been reported to be associated with HESN in African sex Genetically determined resistance against hepatitis C workers.91 virus infection Furthermore, in HIV the characteristics of potentially Substantial genetically determined resistance to HIV protective immunity, particularly in HESN sex workers infection with the macrophage-tropic HIV variants, in Nairobi, Kenya, have been a focus of investigation.92 which predominate in transmission, was proven to be Rowland-Jones and colleagues93 postulated that these conferred via homozygosity for the Δ32 truncation women were initially exposed to viral inocula that mutation in the CCR5 gene, which acts as a major co- effi ciently primed a cell-mediated immune response in receptor for HIV entry.84 This discovery underpins the the absence of antibody synthesis. Consistent with this notion of a similar scenario in relation to hepatitis C notion, HESN individuals have been reported to have virus. However, resistance to hepatitis C virus infection polarised Th1 cytokine responses;94 cytotoxic T-cell in HESN individuals is largely unexplored. The proof-of responses to both HIV envelope and gag epitopes;95 principle for such genetic resistance is evidenced by in- enhanced NK cell production of interferon γ, TNFα, and vitro studies examining host proteins, which interact the chemokine ligands for the CCR5 receptor, CCL3, with hepatitis C virus. Cyclophilin A, a member of a CCL4, and CCL5;96 and diminished cell surface family of cellular peptidyl-prolyl-isomerases, is a host- expression of the major chemokine coreceptors for viral encoded factor that is essential for hepatitis C virus entry CCR5 and CXCR4.97 Notably, several of these replication and possibly particle assembly too.85 women subsequently became infected after a period of Peptidyl-prolyl-isomerase catalyses the isomerisation of reduction in the frequency of, or an interruption to, sex peptide bonds from the trans to the cis form at proline work, suggesting that the initial protective mechanisms residues and helps with protein folding. Several were maintained by repeated exposure to HIV.98 hepatitis C virus non-structural proteins (NS2, NS5A, In hepatitis C, a well characterised combination of and NS5B) have been reported to interact with genetic and environmental factors account for much of cyclophilin A,86 and cyclophilin A mutants without the varied probability of clearance from primary isomerase activity do not sustain viral replication.85 A infection. The key genetic factors are single nucleotide report87 in 2012, showed that homozygosity at any of the polymorphisms in the IFNL3 gene locus, which have three naturally arising single nucleotide polymorphisms been associated with both spontaneous clearance99–101 and in the region of the peptidyl-prolyl-isomerase gene, clearance via interferon-based antiviral treatment.102 The resulted in an unstable cyclophilin A protein, IFNL3 locus encodes interferon λ 3, which shares a intracellular cyclophilin A depletion, and a hepatitis C common signalling pathway with type I interferons.103 virus refractory phenotype in vitro. However, our 2014 Genome-wide association studies101 have shown that the study38 of the potential association between these single three times diff erences in the rates of spontaneous nucleotide polymorphisms and the HESN phenotype in hepatitis C virus clearance in diverse ethnic groups, such 210 Australian prisoners who injected drugs reported as African–Americans and Japanese individuals, are that no participants were homozygous for the minor probably caused by the frequency of these IFNL3 single allele. nucleotide polymorphisms. In 2013 a new transcript Claudin-1 is a coreceptor needed for late stage binding encoding an interferon γ 4 protein was identifi ed of hepatitis C virus to hepatocytes.88 A study89 of 68 IDUs upstream of the IFNL3 locus, and clearance was strongly who were not infected with hepatitis C virus reported associated with the interferon γ 4-TT variant, which is in that these individuals carried single nucleotide strong linkage disequilibrium with the other clearance- polymorphisms –15312C, –7153A, and –5414C in the associated single nucleotide polymorphisms.104,105 claudin-1 promoter region more often than individuals A report106 in 2011 identifi ed HESN individuals as with chronic hepatitis C virus infection (n=658) and having a signifi cantly lower frequency of the protective hepatitis C virus clearers (n=199). IFNL3 genotype than anti-hepatitis C virus-positive Resistance in HESN individuals might also be spontaneous resolvers, but a similar frequency to patients attributable to genetic variations in immunological who were chronically infected. Another report107 in HESN proteins, which might contribute to very effi cient individuals suggested the same association, but did not clearance of established infection, thus representing include infected participants as a comparator. Our study38 genetically determined protection. In HIV, HESN in well characterised HESN individuals, uninfected low individuals are more likely to carry specifi c MHC class I risk individuals and incident cases in the Australian IDU and II alleles than are seropositive individuals,90 notably a prisoner cohort reported no association. cluster of closely related HLA alleles (the A2 or 6802 The association between HLA alleles with spontaneous supertype).90 These alleles are known to present the same hepatitis C virus clearance has been studied extensively, peptide epitopes for T-cell recognition. Additionally, in view of the evidence for CD4-positive and CD8-positive heterozygosity for the NK cell expressed killer inhibitory T-cell responses in clearance of primary infection108
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(see appendix for a summary of the HLA associations of 12 p40 (IL12B) is polymorphic and located on 5q31–33, spontaneous hepatitis C virus clearance). The class I and a functional single nucleotide polymorphism (A→C) alleles HLA-A*1101, HLA-A*03, HLA-B*57, HLA-B*27, of the 3' untranslated region at position 1188 has been and HLA-Cw*0102 have all been associated with identifi ed.117 The homozygous CC genotype, which clearance. HLA-B*57 and HLA-B*27 are of particular confers high interleukin 12 production, has been interest in view of their protective eff ect in HIV, with over identifi ed as a marker of resistance in a cohort of HESN representation of HLA-B*57 in HIV-elite controllers, and IDUs compared with healthy control participants and a consistent association of HLA-B*27 with a more individuals with chronic hepatitis C virus infection.107 favourable course of untreated HIV infection.109 However, Hepatitis C virus-specifi c T cells secrete the antiviral and the most widely reported associations with spontaneous immunomodulatory cytokine interferon γ.65 A −764G→C hepatitis C virus clearance are with HLA class II alleles, single nucleotide polymorphism in the interferon γ such as HLA-DRB1*03 and HLA-DQB1*0301.110 No promoter region, which confers increased gene association between HESN and either class I or II alleles transcripts, has been associated with viral clearance in was reported in the Australian IDU prisoner cohort, one study.118 The anti-infl ammatory cytokine interleukin although the small sample size of 210 individuals, 10 downregulates the Th1 response and suppresses the including eight HESN individuals, did not exclude a secretion of TNFα and interferon γ. Several studies119 possible association.38 suggest polymorphisms in this region of the genome are Associations between KIR loci and the outcome of important in hepatitis C virus clearance. Particularly, a acute hepatitis C virus infection have also been reported. promoter region haplotype (−1117A, −854T, −627A), The KIR locus shows substantial population diversity,111 which is associated with reduced interleukin 10 with diff erent individuals harbouring varying numbers expression, was proven to be more frequent in patients of KIR genes, and wide-ranging allelic polymorphisms.112 who clear primary infection.120 Transforming growth KIR proteins react with one of two epitopes at aminoacid factor β suppresses NK cell activity and inhibits position 80 on HLA-Cw (and positions on a few rare interferon γ and interleukin 12 production. The C allele HLA-B) molecules. An association between individuals at −509 in the promoter, which leads to reduced homozygous for KIR2DL3 (an inhibitory receptor gene) expression of transforming growth factor β1, has been and HLA-C1 (KIR2DL3’s ligand gene) and spontaneous associated with hepatitis C virus clearance in a study of clearance of acute hepatitis C virus infection has been Japanese patients.121 reported.113 This receptor–ligand combination could provide weaker inhibitory signals than could other Conclusion inhibitory KIR–HLA-C receptor–ligand pairings, and Clear precedents exist for genetically determined thus prime a more responsive NK cell phenotype.114,115 resistance and protection against human infectious KIR gene activation is estimated to account for only 20% diseases. Some epidemiological evidence exists to of the variance in clearance rates. suggest that a small subset of individuals remain Knapp and colleagues116 studied 29 HESN IDUs from apparently uninfected with hepatitis C virus, despite very needle exchange or community drug services and longlasting high-risk behaviours and probable repeated 19 from a correctional centre, and compared these exposure. Some individuals repeatedly clear established individuals with 257 patients with chronic hepatitis C infection via both innate and adaptive immune virus infection. The HESN IDUs group included mechanisms. In combination these data suggest individuals who had a broad range of injecting duration plausibility for both hepatitis C virus resistance and (0·5–24 years) and lifetime injection episodes (36–17 520). immunological protection, and that genetics probably Of the putative HESN individuals, only 27% reported contribute to these phenotypes. present intravenous drug use and 100% reported sharing A framework for consistent defi nition of the HESN of needles or other drug injection equipment. No other phenotype using longitudinal data among high risk selection criteria were applied to the group. A greater individuals has been proposed because plausible frequency of homozygosity for the KIR2DL3/HLA-C mechanistic subpopulations within the HESN label allotypes was noted among the HESN group (25·0% vs cannot yet be delineated. With a consistent working 9·7%, odds ratio 3·1).116 By contrast, no association was defi nition of the HESN phenotype, genetic and reported in the Australian IDU prisoner cohort; again, the small sample size did not exclude a possible association.38 Search strategy and selection criteria Many associations between polymorphisms in cytokine We searched PubMed between Jan 1, 1979, and Sept 30, genes and spontaneous hepatitis C virus clearance have 2014 with the search terms “hepatitis C virus”, “protective been reported; however, none has been consistently immunity”, “genetics of resistance”, “intravenous drug use”, reproduced. Interleukin 12 is a heterodimer of p35 and “highly exposed seronegative”, and “natural killer cells”. We p40 subunits and is a key cytokine in promotion of only included articles published in English. antiviral Th1 responses. The gene encoding interleukin
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immunological associations of the phenotype can be 20 Bianco A, Reghellin V, Donnici L, et al. Metabolism of explored, and compared with other traditional hepatitis C phosphatidylinositol 4-kinase IIIα-dependent PI4P Is subverted by HCV and is targeted by a 4-anilino quinazoline with antiviral virus outcomes, including those with chronic infection activity. PLoS Pathog 2012; 8: e1002576. and spontaneous clearers who seroconvert, some of 21 Ng TI, Mo H, Pilot-Matias T, et al. Identifi cation of host genes whom might become protected from reinfection. These involved in hepatitis C virus replication by small interfering RNA technology. Hepatology 2007; 45: 1413–21. investigations of well characterised HESN individuals 22 Choi YW, Tan YJ, Lim SG, Hong W, Goh PY. Proteomic approach could identify the genetic correlates and host immune identifi es HSP27 as an interacting partner of the hepatitis C virus response characteristics of the HESN phenotype, which NS5A protein. 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13 460 www.thelancet.com/infection Vol 15 April 2015 Supplementary webappendix This webappendix formed part of the original submission and has been peer reviewed. We post it as supplied by the authors. Supplement to: Mina MM, Luciani F, Cameron B, et al. Resistance to hepatitis C virus: potential genetic and immunological determinants. Lancet Infect Dis 2015; published online Feb 19. http://dx.doi.org/10.1016/S1473-3099(14)70965-X.
14 Supplementary Table 1
Hepatitis C virus interacting host proteins
Host gene Human protein name Interacting HCV factor Reference Host proteins interacting with HCV viral entry 1. CANX Calnexin E1/E2 Li1 2. CD81 CD81 molecule E2 Pileri2 3. CLDN1 Claudin-1 E1/E2 Liu3 4. CLEC4M C-type lectin domain family 4, member M E1/E2 Pohlmann4 5. EGFR Epidermal growth factor receptor Promote CD81-CLDN1 Lupberger5 association and membrane fusion 6. EIF2AK2 Eukaryotic translation initiation factor 2- E2 Li1 alpha kinase 2 7. EphA2 Ephrin receptor A2 Promote CD81-CLDN1 Lupberger5 association and membrane fusion 8. HS Heparin sulphate E1/E2 Barth6 9. ITGB1 Integrin, beta 1 E2 Li1 10. LDL-R Low-density lipoprotein -receptor E1/E2 Agnello7 11. LTF Lactotransferrin E1/E2 Yi8 12. NPC1L1 Niemann-Pick C1-like 1 cholesterol virion-associated cholesterol Sainz9 absorption receptor 13. NR4A1 Nuclear receptor subfamily 4, group A, E1/E2 Li1 member 1 14. OCLN Occludin E1/E2 Liu3 15. PFN1 Profilin 1 E1 de Chassey10 16. SCARB1 Scavenger receptor class B, member 1 E2 Scarselli11 17. SDC2 Syndecan 2 E2 Barth6 18. SIGN DC-specific intracellular adhesion E1/E2 Cormier12 molecule-3-grabbing non-integrin 19. L-SIGN Liver-specific (L)-SIGN E1/E2 Cormier12 20. SRB-1 Scavenger receptor class B type-1 E1/E2 Scarselli11 21. TF Transferrin E2 Nozaki13 Host proteins interacting with HCV viral replication 22. ACP1 Acid phosphatase 1, soluble Core Kang14 23. ACTN1 Alpha-Actinin 1 NS3/NS5B de Chassey10 24. ACTN2 Alpha-Actinin 2 NS3 Lan15 25. ACLY ATP citrate lyase NS5A de Chassey10 26. ACY3 Aspartoacylase (aminocyclase) 3 Core Chen16 27. AEBP1 AE binding protein 1 NS3 de Chassey10 28. AGRN Agrin Core de Chassey10 29. AGT Angiotensinogen (serpin peptidase F Huang17 inhibitor, clade A, member 8) 30. AHNAK AHNAK nucleoprotein NS5A Ahn18 31. AKT1 v-akt murine thymoma viral oncogene NS5A Randall19 homolog 1 32. ANKRD12 Ankyrin repeat domain 12 NS3 de Chassey10 33. ANKRD28 Ankyrin repeat domain 28 NS3 de Chassey10 34. ARAP1 ArfGAP with RhoGAP domain, ankyrin NS5A Ahn18 repeat and PH domain 1 35. ARFIP1 ADP-ribosylation factor interacting NS5A de Chassey10 protein 1 (arfaptin 1) 36. ARFIP2 ADP-ribosylation factor interacting NS3 de Chassey10 protein 2 (arfaptin 2) 37. ARHGEF6 Rac/Cdc42 guanine nucleotide exchange NS3 de Chassey10 factor (GEF) 6 38. ARNT Aryl hydrocarbon receptor nuclear NS3 de Chassey10 translocator 39. ARS2 Arsenate resistance protein 2 NS3 de Chassey10 40. ATF6A Activating transcription factor 6 NS4B Tong20 41. ATF6B Activating transcription factor 6 beta NS4B Tong20 42. ATM Ataxia telangiectasia mutated NS3/NS4A/ NS5B Ariumi21 43. AXIN1 Axin 1 NS5A de Chassey10 44. AZGP1 Alpha-2-glycoprotein 1, zinc-binding F Huang17 45. hB-ind1 Human butyrate-induced transcript 1 NS5A Taguwa22
46. B2M Beta-2-microglobulin NS3 de Chassey10 47. BAX BCL2-associated X protein NS5A Chung23 48. BCAN Brevican NS3 de Chassey10 49. BCAR1 Breast cancer anti-estrogen resistance 1 Core de Chassey10 50. BCKDK Branched chain ketoacid dehydrogenase NS3 de Chassey10
15 kinase 51. BCL6 B-cell CLL/lymphoma 6 (zinc finger NS3 de Chassey10 protein 51) 52. BCL2A1 BCL2-related protein A1 NS3 de Chassey10 53. BEND5 BEN domain containing 5 NS3 de Chassey10 54. BEND7 BEN domain containing 7 NS5A de Chassey10 55. BIN1 Bridging integrator 1 NS5A Zech24 56. BZRAP1 Benzodiazapine receptor (peripheral) NS3 de Chassey10 associated protein 1 57. C1orf94 Chromosome 1 open reading frame 94 NS3 de Chassey10 58. C9orf6 Chromosome 9 open reading frame 6 NS5A Ahn18 59. C9orf30 Chromosome 9 open reading frame 30 NS3 de Chassey10 60. C10orf6 Chromosome 10 open reading frame 6 NS3 de Chassey10 61. C10orf18 Chromosome 10 open reading frame 18 NS3 de Chassey10 62. C12orf41 Chromosome 12 open reading frame 41 NS3 de Chassey10 63. C14orf135 Chromosome 14 open reading frame 135 F Huang17 64. C16orf7 Chromosome 16 open reading frame 7 NS3 de Chassey10 65. C19orf66 Chromosome 19 open reading frame 66 NS3 de Chassey10 66. C1QBP Complement component 1, q Core Kittlesen25 subcomponent binding protein 67. C7 Complement component 7 NS2 de Chassey10 68. CADPS Ca2+-dependent secretion activator NS5A de Chassey10 69. CADPS2 Ca2+-dependent activator protein for NS5A de Chassey10 secretion 2 70. CALCOCO Calcium binding and coiled-coil domain 2 NS3 de Chassey10 2 71. CALR Calreticulin E1/E2 Choukhi26 72. CAMLG Calcium modulating ligand NS4A Chen27 73. CASP8 Caspase 8, apoptosis-related cysteine NS3 Prikhod`ko28 peptidase 74. CCDC21 Coiled-coil domain containing 21 NS3 de Chassey10 75. CCDC37 Coiled-coil domain containing 37 NS3 de Chassey10 76. CCDC52 Coiled-coil domain containing 52 NS3 de Chassey10 77. CCDC66 Coiled-coil domain containing 66 NS3 de Chassey10 78. CCDC86 Coiled-coil domain containing 86 NS5A Ahn18 79. CCHCR1 Coiled-coil alpha-helical rod protein 1 NS3 de Chassey10 80. CCNH Cyclin H Core Ohkawa29 81. CD5L CD5 molecule-like NS3 de Chassey10 82. CD68 CD68 molecule Core de Chassey10 83. CDC2 Cell division cycle 2, G1 to S and G2 to NS5A Arima30 M 84. CDK2 Cyclin-dependent kinase 2 NS5A Arima30 85. CDK6 Cyclin-dependent kinase 6 NS5A Randall19 86. CDKN1A Cyclin-dependent kinase inhibitor 1A Core Wang31 87. CENPC1 Centromere protein C 1 NS5A de Chassey10 88. CEP57 Centrosomal protein 57kDa NS5A Ahn18 89. CEP63 Centrosomal protein 63kDa NS5A de Chassey10 90. CEP68 Centrosomal protein 68kDa NS5B de Chassey10 91. CEP120 Centrosomal protein 120kDa NS5A de Chassey10 92. CEP152 Centrosomal protein 152kDa NS3 de Chassey10 93. CEP192 Centrosomal protein 192kDa NS3 de Chassey10 94. Centrosomal protein 250kDa NS5A/NS5B de Chassey10 CEP250 95. CFL1 Cofilin 1 (non-muscle) Core Kang14 96. CFP Complement factor properdin NS3 de Chassey10 97. ChK2 Checkpoint kinase 2 NS5B Ariumi21 98. CHPF Chondroitin polymerizing factor NS3 de Chassey10 99. CHUK Conserved helix-loop-helix ubiquitous NS5B Choi32 kinase 100. CIDEB Cell death-inducing DFFA-like effector b NS2 Erdtmann33 101. CINP Cyclin-dependent kinase 2 interacting NS5B Lan15 protein 102. COL4A2 Collagen, type IV, alpha 2 Core de Chassey10 103. CORO1B Coronin, actin binding protein, 1B NS3 de Chassey10 104. CRABP1 Cellular retinoic acid binding protein 1 NS5A Ahn18 105. Creatine Creatine kinase B NS4A Hara34 kinase B
106. CREB3 CAMP responsive element binding Core/NS4A/p7 Jin35 protein 3
107. CREBB CREB binding protein Core Gomez-Gonzalo36
16 108. CSK C-src tyrosine kinase NS5A Masumi37
109. CSNK2A1 Casein kinase 2, alpha 1 polypeptide NS5A Kim38 110. CSNK2B Casein kinase 2, beta polypeptide NS3 de Chassey10 111. CTGF Connective tissue growth factor NS3 de Chassey10 112. CTSB Cathepsin B F Huang17 113. Cyclophilin Peptidylprolyl isomerase A NS5A/NS5B Liu3 A 114. DDX3 DEAD (Asp-Glu-Ala-Asp) box helicase 3 Core Ariumi21
115. DDX5 DEAD (Asp-Glu-Ala-Asp) box Core/NS5B Kang14 polypeptide 5 116. DES Desmin NS3 de Chassey10 117. DLAT Dihydrolipoamide S-acetyltransferase (E2 NS3 de Chassey10 component of pyruvate dehydrogenase complex) 118. DNAJA3 DnaJ (Hsp40) homolog, subfamily A, NS5A de Chassey10 member 3 119. DOCK7 Dedicator of cytokinesis 7 NS3 de Chassey10 120. DPF1 D4, zinc and double PHD fingers family 1 NS3 de Chassey10 121. DPP7 Dipeptidyl-peptidase 7 NS3 de Chassey10 122. EEF1A1 Eukaryotic translation elongation factor 1 NS3/NS4A de Chassey10 alpha 1 123. EFEMP1 EGF-containing fibulin-like extracellular NS3/NS5A de Chassey10 matrix protein 1 124. EFEMP2 EGF-containing fibulin-like extracellular NS3 de Chassey10 matrix protein 2 125. EGFL7 EGF-like-domain, multiple 7 Core de Chassey10 126. EIF1 Eukaryotic translation initiation factor 1 NS3 de Chassey10 127. EIF2AK2 Eukaryotic translation initiation factor 2- Core/E2/NS5A Yan39 alpha kinase 2 128. EIF2AK3 Eukaryotic translation initiation factor 2- E2 Pavio40 alpha kinase 3 129. EIF4A2 Human eukaryotic initiation factor 4AII NS5B Kyono41 (heIF4AII) 130. EIF4ENIF1 Eukaryotic translation initiation factor 4E NS3 de Chassey10 nuclear import factor 1 131. EP300 E1A binding protein p300 Core Gomez-Gonzalo36 132. ERC1 ELKS/RAB6-interacting/CAST family NS3 Hidajat42 member 1 133. FAM135A Family with sequence similarity 135, E2 de Chassey10 member A 134. FAS Fas cell surface death receptor Core Moorman42 135. FADD Fas (TNFRSF6)-associated via death Core Li1 domain 136. FAM65A Family with sequence similarity 65, NS3 de Chassey10 member A 137. FBF1 Fas (TNFRSF6) binding factor 1 NS3 de Chassey10 138. FBL2 F-box and leucine-rich repeat protein 2 NS5A Wang31
139. FBLN1 Fibulin 1 NS3 de Chassey10 140. FBLN2 Fibulin 2 Core/NS3/p7 de Chassey10 141. FBLN5 Fibulin 5 Core/NS2/NS3 de Chassey10 142. FBN1 Fibrillin 1 NS3 de Chassey10 143. FBN3 Fibrillin 3 NS3 de Chassey10 144. FBXL2 F-box and leucine-rich repeat protein 2 NS5A/NS5B Wang43 145. FBXL20 F-box and leucine-rich repeat protein 20 NS5A Wang43 146. FES Feline sarcoma oncogene NS3 de Chassey10 147. FKBP7 FK506 binding protein 7 Core Kang14 148. FKBP8 FK506 binding protein 8 NS5A Wang44 149. FGG Fibrinogen gamma chain NS4B Liu3 150. FHL2 Four and a half LIM domains 2 NS5A de Chassey10 151. FIGNL1 Fidgetin-like 1 NS3 de Chassey10 152. FMNL1 Formin-like 1 p7 Huang45 153. FN1 Fibronectin 1 NS3 de Chassey10 154. FERM and PDZ domain containing 4 NS3 de Chassey10 FRMPD4 155. FRS3 Fibroblast growth factor receptor NS3 de Chassey10 substrate 3 156. FTH1 Ferritin, heavy polypeptide 1 NS3/NS5A de Chassey10 157. FUCA2 Fucosidase, alpha-L- 2, plasma NS3 de Chassey10 158. FUNDC2 FUN14 domain containing 2 Core Li46 159. FUSE Polykaryocytosis promoter NS5A Zhang47
17 binding protein 160. FXYD6 FXYD domain containing ion transport p7 de Chassey10 regulator 6 161. FYN FYN oncogene related to SRC, FGR, NS5A Macdonald48 YES 162. G3BP1 Ras-GTPase-activating protein binding Yi49 protein-1 163. GAA Glucosidase, alpha; acid (Pompe disease, NS3 de Chassey10 glycogen storage disease type II) 164. GAB1 GRB2-associated binding protein 1 NS5A He50 165. GAPDH Glyceraldehyde-3-phosphate Core de Chassey10 dehydrogenase 166. GBP2 Guanylate binding protein 2, interferon- NS3 de Chassey10 inducible
167. GFAP Glial fibrillary acidic protein NS3 de Chassey10.
168. GIN1 Gypsy retrotransposon integrase 1 NS5A de Chassey10 169. GLRX3 Glutaredoxin 3 Core Kang14 170. GOLGA2 Golgi autoantigen, golgin subfamily a, 2 NS5A de Chassey10 171. GNB2 Guanine nucleotide binding protein (G NS3 de Chassey10 protein), beta polypeptide 2 172. GPS2 G protein pathway suppressor 2 NS5A de Chassey10 173. GRB2 Growth factor receptor-bound protein 2 NS5A Tan51 174. GRN Granulin Core de Chassey10 175. GSK3A Glycogen synthase kinase 3 alpha NS5A Randall19 176. GSK3B Glycogen synthase kinase 3 beta NS5A Randall19 177. H19 H19, imprinted maternally expressed p7 Huang45 untranslated mRNA 178. HAO1 Hydroxyacid oxidase (glycolate oxidase) NS5B Lan15 1 179. HCK Hemopoietic cell kinase NS5A Macdonald48 180. HLA-A Major histocompatibility complex, class I, Core Nattermann52 A 181. HLA-E Major histocompatibility complex, class I, Core Nattermann52 E 182. HIST3H2B Histone cluster 3, H2bb NS3 Borowski53 B 183. HIST4H4 Histone cluster 4, H4 NS3 Borowski53 184. HIVEP2 Human immunodeficiency virus type I Core/NS3 de Chassey10 enhancer binding protein 2 185. HNRNPA1 Heterogeneous nuclear NS5B Kim54 ribonucleoprotein A1 186. HNRNPK Heterogeneous nuclear ribonucleoprotein Core/NS3 de Chassey10 K 187. HOXD8 Homeobox D8 Core/E2/NS2/NS4A/NS5B de Chassey10 188. HSPA5 Heat shock 70kDa protein 5 (glucose- E1/E2 Choukhi26 regulated protein, 78kDa) 189. HSPD1 Heat shock 60kDa protein 1 (chaperonin) Core Kang14 190. Hsp27 Heat shock protein 27 NS5A Choi55
191. HSP90AA1 Heat shock protein 90 NS5A/NS3 Okamoto56 192. IGLL1 Immunoglobulin lambda-like polypeptide NS5A de Chassey10 1 193. IKBKE Inhibitor of kappa light polypeptide gene NS3 Otsuka57 enhancer in B-cells, kinase epsilon 194. INF2 Inverted formin, FH2 and WH2 domain NS3 de Chassey10 containing 195. INO80E INO80 complex subunit E NS3 de Chassey10 196. IPO4 Importin 4 NS5A Randall19 197. IPO5 Importin 5 NS5A Chung58 198. IQWD1 IQ motif and WD repeats 1 NS3 de Chassey10 199. IRF3 Interferon regulatory factor 3 NS3 Foy58 200. ISLR Immunoglobulin superfamily containing p7 Huang17 leucine-rich repeat
18 201. ITGB1 Integrin, beta 1 (fibronectin receptor, beta E2 de Chassey10 polypeptide, antigen CD29 includes MDF2, MSK12) 202. ITGB4 Integrin, beta 4 NS3 de Chassey10 203. ITGAL Integrin, alpha L (antigen CD11A (p180), NS5A de Chassey10 lymphocyte function-associated antigen 1; alpha polypeptide) 204. JAG2 Jagged 2 NS3 de Chassey10 205. JAK1 Janus kinase 1 Core/NS5A Hosui59 206. JAK2 Janus kinase 2 Core Hosui59 207. JUN Jun proto-oncogene E1/NS3 de Chassey10 208. KIAA1549 KIAA1549 protein NS3 de Chassey10 209. KIAA2022 KIAA2022 NS3 de Chassey10 210. KIF7 Kinesin family member 7 NS3 de Chassey10 211. KIF17 Kinesin family member 17 NS3 de Chassey10 212. KPNA1 Karyopherin alpha 1 (importin alpha 5) Core/NS3 de Chassey10 213. KRT8 Keratin 8 Core Kang14 214. KRT18 Keratin 18 Core Kang14 215. KRT19 Keratin 19 Core Kang14 216. LAMA5 Laminin, alpha 5 NS3 de Chassey10 217. LAMB2 Laminin, beta 2 (laminin S) NS3 de Chassey10 218. LAMC3 Laminin, gamma 3 NS3 de Chassey10 219. LCK Lymphocyte-specific protein tyrosine NS5A Macdonald48 kinase 220. LDB1 LIM domain binding 1 NS3 de Chassey10 221. LIMS2 LIM and senescent cell antigen-like NS5A de Chassey10 domains 2 222. LMNB1 Lamin B1 p7 de Chassey10 223. LPXN Leupaxin Core de Chassey10 224. LSM1 LSM1 homolog, U6 small nuclear RNA Untranslated region (UTR) RNA de Chassey10 associated 225. LRRC7 Leucine rich repeat containing 7 NS3 de Chassey10 226. LRRTM1 Leucine rich repeat transmembrane Core de Chassey10 neuronal 1 227. LTBP4 Latent transforming growth factor beta Core/NS3 de Chassey10 binding protein 4 228. LTBR Lymphotoxin beta receptor (TNFR Core Matsumoto60 superfamily, member 3) 229. LYN V-yes-1 Yamaguchi sarcoma viral related NS5A Macdonald48 oncogene homolog 230. LZTS2 Leucine zipper, putative tumor suppressor NS3 de Chassey10 2 231. M2PK Pyruvate kinase isoenzyme type M2 NS5B Wu61
232. MAGED1 Melanoma antigen family D, 1 Core/NS3 de Chassey10 233. MAPK12 Mitogen-activated protein kinase 12 NS5A Randall19 234. MBP Myelin basic protein NS3 Borowski53 235. MCL1 Myeloid cell leukemia sequence 1 (BCL2- Core Mohd-Ismail62 related) 236. MEGF6 Multiple EGF-like-domains 6 Core de Chassey10 237. MEGF8 Multiple EGF-like-domains 8 NS3 de Chassey10 238. MGC2752 Hypothetical LOC65996 NS5B de Chassey10 239. MGP Matrix Gla protein NS5A Ahn18 240. MLXIP MLX interacting protein NS3 de Chassey10 241. MMRN2 Multimerin 2 Core de Chassey10 242. MPDU1 Mannose-P-dolichol utilization defect 1 F Huang17 243. MORC4 MORC family CW-type zinc finger 4 NS3 de Chassey10 244. MORF4L1 Mortality factor 4 like 1 NS3 de Chassey10 245. MS4A6A Membrane-spanning 4-domains, p7 Huang17 subfamily A, member 6A 246. MT2A Metallothionein 2A NS4A Liu3 247. MT-CO2 Mitochondrially encoded cytochrome c NS4A Liu3 oxidase II 248. MT-CO3 Mitochondrially encoded cytochrome c NS4B Liu3 oxidase III 249. MVP Major vault protein NS3 de Chassey10 250. MYD88 Myeloid differentiation primary response NS5A Abe63 gene (88) 251. NAP1L1 Nucleosome assembly protein 1-like 1 NS3/NS5A de Chassey10 252. NAP1L2 Nucleosome assembly protein 1-like 2 NS3/NS5A de Chassey10 253. Nuclear Nuclear factors NF90, NF110, NF45 UTR RNA Isken64 factors
19 NF90, NF110, NF45
254. NCAN Neurocan NS3 de Chassey10 255. NCL Nucleolin NS5B Hirano65
256. NDRG1 N-myc downstream regulated gene 1 NS5A Ahn18 257. NDUFV3 NADH dehydrogenase (ubiquinone) NS4B Liu66 flavoprotein 3, 10kDa 258. NEFL Neurofilament, light polypeptide 68kDa NS3 de Chassey10 259. NEFM Neurofilament, medium polypeptide NS3 de Chassey10 150kDa 260. NFE2 Nuclear factor (erythroid-derived 2), NS5A de Chassey10 45kDa 261. NID1 Nidogen 1 NS3 de Chassey10 262. NID2 Nidogen 2 (osteonidogen) NS3 de Chassey10 263. N-PAC Cytokine-like nuclear factor n-pac NS3 de Chassey10 264. NPM1 Nucleophosmin (nucleolar Core Mai67 phosphoprotein B23, numatrin) 265. NR4A1 Nuclear receptor subfamily 4, group A, Core/E1/E2/NS2/NS4A/NS5B de Chassey10 member 1 266. NUCB1 Nucleobindin 1 NS5A de Chassey10 267. NUP62 Nucleoporin 62kDa NS3 de Chassey10 268. NUP214 Nucleoporin 214kDa p7 Huang17 269. OAS1 2`,5`-oligoadenylate synthetase 1, NS5A Taguchi68 40/46kDa 270. OBSCN Obscurin, cytoskeletal calmodulin and NS3 de Chassey10 titin-interacting RhoGEF 271. OS9 Amplified in osteosarcoma NS5B de Chassey10 272. PABPN1 Poly(A) binding protein, nuclear 1 Core de Chassey10 273. PAK4 P21(CDKN1A)-activated kinase 4 Core de Chassey10 274. PARP4 Poly (ADP-ribose) polymerase family, NS3 de Chassey10 member 4 275. PARVG Parvin, gamma NS5A de Chassey10 276. PCYT2 Phosphate cytidylyltransferase 2, NS3 de Chassey10 ethanolamine 277. PDE4DIP Phosphodiesterase 4D interacting protein NS3 de Chassey10 (myomegalin) 278. PDLIM5 PDZ and LIM domain 5 NS3 de Chassey10 279. PDPK1 3-phosphoinositide dependent protein NS5A Randall19 kinase-1 280. PFDN2 Prefoldin subunit 2 F Tsao69 281. PFDN5 Prefoldin subunit 5 F Ma70 282. PI4KA Phosphatidylinositol 4-kinase, catalytic, NS5A Shulla71 alpha 283. PICK1 Protein interacting with PRKCA 1 NS3 de Chassey10 284. PIK3CB Phosphoinositide-3-kinase, catalytic, beta NS5A Street72 polypeptide 285. PIK3R1 Phosphoinositide-3-kinase, regulatory NS5A He50 subunit 1 (p85 alpha) 286. PITX1 Paired-like homeodomain 1 NS5A Ghosh73 287. PKM2 Pyruvate kinase, muscle NS5B de Chassey10 288. PKN2 Protein kinase N2 NS5B Kim74 289. PKNOX1 PBX/knotted 1 homeobox 1 NS3 de Chassey10 290. PLEKHG4 Pleckstrin homology domain containing, NS3 de Chassey10 family G (with RhoGef domain) member 4 291. PLSCR1 Phospholipid scramblase 1 Core Li1 292. PML Promyelocytic leukemia Core Herzer75 293. PMVK Phosphomevalonate kinase NS5A de Chassey10 294. PNPLA8 Patatin-like phospholipase domain NS3 de Chassey10 containing 8 295. PSMA6 Proteasome (prosome, macropain) E2 de Chassey10 subunit, alpha type, 6 296. POU3F2 POU class 3 homeobox 2 NS2 de Chassey10 297. PPARA Peroxisome proliferator-activated receptor Core Tanaka76 alpha 298. PPIB Peptidylprolyl isomerase B (cyclophilin NS5A/NS5B Liu77 B) 299. PPP1R13L Protein phosphatase 1, regulatory NS5A de Chassey10 (inhibitor) subunit 13 like 300. PPP2R4 Protein phosphatase 2A activator, NS5A Georgopoulou78
20 regulatory subunit 4 301. PRKACA Protein kinase, cAMP-dependent, NS3 Borowski53 catalytic, alpha 302. PRM1 Protamine 1 NS3 Borowski53 303. PRMT1 Protein arginine methyltransferase 1 NS3 Rho79 304. PRMT5 Protein arginine methyltransferase 5 NS3 Rho79 305. PRRC1 Proline-rich coiled-coil 1 NS3 de Chassey10 306. PSMB4 Proteasome (prosome, macropain) NS5B Lan15 subunit, beta type, 4 307. PSMB8 Proteasome (prosome, macropain) NS3 Khu80 subunit, beta type, 8 (large multifunctional peptidase 7) 308. PSMB9 Proteasome (prosome, macropain) NS3/NS5A/NS5B de Chassey10 subunit, beta type, 9 (large multifunctional peptidase 2) 309. PSME3 Proteasome (prosome, macropain) Core/NS3 Saito81 activator subunit 3 (PA28 gamma; Ki) 310. PTB Polypyrimidine tract binding protein UTR RNA Aizaki82
311. PTBP2 Polypyrimidine tract binding protein 2 NS3/NS5B Domitrovich83 312. PTMA Prothymosin, alpha (gene sequence 28) NS5A Ahn18 313. PTPLAD1 Protein tyrosine phosphatase-like A NS5A Taguwa22 domain containing 1 314. PTPRN2 Protein tyrosine phosphatase, receptor NS3 de Chassey10 type, N polypeptide 2 315. RAB14 RAB14, member RAS oncogene family F Huang17 316. RAB5A RAB5A, member RAS oncogene family NS4B Stone84 317. RABEP1 Rabaptin, RAB GTPase binding effector NS3 de Chassey10 protein 1 318. RAF1 V-raf-1 murine leukemia viral oncogene NS5A Burckstummer85 homolog 1 319. RAI14 Retinoic acid induced 14 NS3 de Chassey10 320. RASAL2 RAS protein activator like 2 NS3 de Chassey10 321. RBM4 RNA binding motif protein 4 NS3 de Chassey10 322. RBP4 Retinol binding protein 4, plasma NS4B Liu66 323. RCN3 Reticulocalbin 3, EF-hand calcium NS3 de Chassey10 binding domain 324. RGNEF Rho-guanine nucleotide exchange factor NS3 de Chassey10 325. RICS Rho GTPase-activating protein NS3 de Chassey10 326. RINT1 RAD50 interactor 1 NS3 de Chassey10 327. RNA RNA-dependent helicase p68 (DEAD-box NS5B Goh86 helicase protein p68) p68 328. RNF31 Ring finger protein 31 Core/NS3 de Chassey10 329. RPL18A Ribosomal protein L18a NS5A de Chassey10 330. RSF1 Remodeling and spacing factor 1 Core Chen87 331. RSAD2 Viperin NS5A, Core Helbig88 332. RTN3 Reticulon 3 NS4B Liu66 333. RUSC2 RUN and SH3 domain containing 2 NS3 de Chassey10 334. RXRA retinoid X receptor, alpha Core Tsutsumi87 335. SBF1 SET binding factor 1 NS3 de Chassey10 336. SDCCAG8 Serologically defined colon cancer NS3 de Chassey10 antigen 8 337. SECISBP2 SECIS binding protein 2 NS3 de Chassey10 338. SEPT6 Septin 6 NS5B Kim54
339. SEPT10 Septin 10 NS3 de Chassey10 340. SERPINC1 Serpin peptidase inhibitor, clade C F Huang17 (antithrombin), member 1 341. SERPINF2 Serpin peptidase inhibitor, clade F (alpha- NS3 Drouet89 2 antiplasmin, pigment epithelium derived factor), member 2 342. SERPING1 Serpin peptidase inhibitor, clade G (C1 NS3 Drouet89 inhibitor), member 1, (angioedema, hereditary) 343. SERTAD1 SERTA domain containing 1 NS3 de Chassey10 344. SESTD1 SEC14 and spectrin domains 1 NS3 de Chassey10 345. SETD2 SET domain containing 2 Core/E1/E2/NS2/NS5B de Chassey10 346. SF3B2 Splicing factor 3b, subunit 2, 145kDa NS3 de Chassey10 347. SFRP4 Secreted frizzled-related protein 4 NS5A Ahn18 348. SGM Sphingomyelin NS5B Sakamoto90 349. SHARPIN SHANK-associated RH domain interactor NS5A/NS5B de Chassey10 350. SLC22A7 Solute carrier family 22 (organic anion Core Kang14
21 transporter), member 7 351. SLC31A2 Solute carrier family 31 (copper Core de Chassey10 transporters), member 2 352. SMAD3 SMAD family member 3 Core/NS3 Cheng91 353. SMURF2 SMAD specific E3 ubiquitin protein NS3 de Chassey10 ligase 2 354. SMYD3 SET and MYND domain containing 3 NS5A de Chassey10 355. SNRPD1 Small nuclear ribonucleoprotein D1 NS3 Iwai92 polypeptide 16kDa 356. SNX4 Sorting nexin 4 NS3 de Chassey10 357. SORBS2 Sorbin and SH3 domain containing 2 NS5A de Chassey10 358. SORBS3 Sorbin and SH3 domain containing 3 NS5A de Chassey10 359. SP110 SP110 nuclear body protein Core Watashi93 360. SPOCK3 Sparc/osteonectin, cwcv and kazal-like NS3 de Chassey10 domains proteoglycan (testican) 3 361. SPON1 Spondin 1, extracellular matrix protein NS3 de Chassey10 362. SRC V-src sarcoma (Schmidt-Ruppin A-2) NS5A Masumi37 viral oncogene homolog (avian) 363. SRCAP Snf2-related CBP activator protein NS5A Ghosh73 364. SRPX2 Sushi-repeat-containing protein, X-linked NS3 de Chassey10 2 365. SSB Sjogren syndrome antigen B (autoantigen NS5A Houshmand94 La) 366. SSR4 Signal sequence receptor, delta p7 Huang17 (translocon-associated protein delta) 367. SSX2IP Synovial sarcoma, X breakpoint 2 NS3 de Chassey10 interacting protein 368. ST3GAL1 ST3 beta-galactoside alpha-2,3- F Huang17 sialyltransferase 1 369. STAB1 Stabilin 1 NS3 de Chassey10 370. STAT1 Signal transducer and activator of Core/NS5A de Chassey10 transcription 1, 91kDa 371. STAT3 Signal transducer and activator of Core/NS3 de Chassey10 transcription 3 372. STRBP Spermatid perinuclear RNA binding p7 Huang17 protein 373. SVEP1 Sushi, von Willebrand factor type A, EGF NS3 de Chassey10 and pentraxin domain containing 1 374. SYNCRIP Synaptotagmin binding, cytoplasmic UTR RNA Liu95 RNA interacting protein 375. SYNE1 Spectrin repeat containing, nuclear NS3 de Chassey10 envelope 1 376. SYNPO2 Synaptopodin 2 NS3 de Chassey10 377. TACSTD2 Tumor-associated calcium signal NS5A Ahn18 transducer 2 378. TAF1 TAF1 RNA polymerase II, TATA box NS3 de Chassey10 binding protein (TBP)-associated factor, 250kDa 379. TAF9 TAF9 RNA polymerase II, TATA box NS5A Lan96 binding protein (TBP)-associated factor, 32kDa 380. TAF11 TAF11 RNA polymerase II, TATA box Core Otsuka57 binding protein (TBP)-associated factor, 28kDa 381. TATDN1 TatD DNase domain containing 1 Core Kang14 382. TBC1D20 TBC1 domain family, member 20 NS5A Sklan97
383. TBC1D2B TBC1 domain family, member 2B NS3 de Chassey10 384. TBK1 TANK-binding kinase 1 NS3 Otsuka57 385. TBP TATA box binding protein Core/NS5A Li1 386. TBXAS1 Thromboxane A synthase 1 (platelet, NS3 de Chassey10 cytochrome P450, family 5, subfamily A) 387. TGFBR1 Transforming growth factor, beta receptor NS5A Choi98 I (activin A receptor type II-like kinase, 53kDa) 388. THAP1 THAP domain containing, apoptosis NS3 de Chassey10 associated protein 1 389. THBS1 Thrombospondin 1 NS5A de Chassey10 390. TICAM1 Toll-like receptor adaptor molecule 1 NS3 Ferreon99 391. TMEM63B Transmembrane protein 63B NS3 de Chassey10 392. TMF1 TATA element modulatory factor 1 NS5A de Chassey10 393. TMSB4X Thymosin, beta 4, X-linked E1 de Chassey10 394. TNF Tumor necrosis factor (TNF superfamily, Core Chung100
22 member 2) 395. TNFRSF1 Tumor necrosis factor receptor Core Li1 A superfamily, member 1A 396. TNXB Tenascin XB NS4B Tong20 397. TLR2 Toll-like receptor 2 Core/NS3 Dolganiuc101 398. TP53 Tumor protein p53 Core/NS3/NS5A Li1 399. TP73 Tumor protein p73 Core Alisi102 400. TRADD TNFRSF1A-associated via death domain Core/NS5A Chung100 401. TRAF2 TNF receptor-associated factor 2 Core/NS5A Chung100
402. TRAF3IP3 TRAF3 interacting protein 3 NS4A de Chassey10 403. TRIM23 Tripartite motif-containing 23 NS3 de Chassey10 404. TRIM27 Tripartite motif-containing 27 NS2/NS3 de Chassey10 405. TRIO Triple functional domain (PTPRF NS3 de Chassey10 interacting) 406. TRIOBP TRIO and F-actin binding protein NS5A de Chassey10 407. TRIP11 Thyroid hormone receptor interactor 11 NS3 de Chassey10 408. TSN Translin Core Li103 409. TTC4 Tetratricopeptide repeat domain 4 NS5B Lan15 410. TXNDC11 Thioredoxin domain containing 11 NS3/NS5A de Chassey10 411. TUBB2C Tubulin, beta 2C NS5B de Chassey10 412. TUT1 Terminal uridylyl transferase 1, U6 NS4A Liu104 snRNA-specific 413. UBA3 Ubiquitin-like modifier activating enzyme NS3 de Chassey10 3 414. UBASH3A Ubiquitin associated and SH3 domain NS5A de Chassey10 containing, A 415. UBQLN1 Ubiquilin 1 p7/NS4A/NS5B Li1 416. UBQLN4 Ubiquilin 4 p7 Li1 417. USHBP1 Usher syndrome 1C binding protein 1 NS3 de Chassey10 418. USP19 Ubiquitin specific peptidase 19 NS5A de Chassey10 419. UXT Ubiquitously-expressed transcript NS3 de Chassey10 420. VAPA VAMP (vesicle-associated membrane NS5A/NS5B Gao105 protein)-associated protein A, 33kDa 421. VAPB VAMP (vesicle-associated membrane NS5A/NS5B Hamamoto106 protein)-associated protein B and C 422. VAP-33 Vesicle-associated membrane protein NS5A, NS5B Lu107 (VAMP)-associated protein of 33 kDa 423. VCAN Versican NS3 de Chassey10 424. VIM Vimentin Core/NS3 Kang14 425. VISA Virus-induced signaling adapter NS3 Johnson108 426. VTN Vitronectin F Huang17 427. VWF Von Willebrand factor Core/NS3 de Chassey10 428. XAB2 XPA binding protein 2 NS3 de Chassey10 429. XRN2 5`-3` exoribonuclease 2 NS3 de Chassey10 430. YWHAB tyrosine 3-monooxygenase/tryptophan 5- Core Aoki109 monooxygenase activation protein, beta 431. YWHAE Tyrosine 3-monooxygenase/tryptophan 5- Core Aoki109 monooxygenase activation protein, epsilon 432. YWHAZ Tyrosine 3-monooxygenase/tryptophan 5- Core Aoki109 monooxygenase activation protein, zeta 433. YY1 YY1 transcription factor Core Mai67 434. YY1AP1 YY1 associated protein 1 NS3 de Chassey10 435. ZBTB1 Zinc finger and BTB domain containing 1 NS3 de Chassey10 436. ZCCHC7 Zinc finger, CCHC domain containing 7 NS3 de Chassey10 437. ZHX3 Zinc fingers and homeoboxes 3 NS3 de Chassey10 438. ZMYM2 Zinc finger, MYM-type 2 NS3 de Chassey10 439. ZNF83 Zinc finger protein 83 F Huang17 440. ZNF271 Zinc finger protein 271 Core de Chassey10 441. ZNF281 Zinc finger protein 281 NS3 de Chassey10 442. ZNF410 Zinc finger protein 410 NS3 de Chassey10 443. ZNF646 Zinc finger protein 646 NS5A de Chassey10 444. ZZZ3 Zinc finger, ZZ-type containing 3 NS3 de Chassey10 Host proteins interacting with HCV particle assembly and envelopment 445. AP2M1 Adaptor-related protein complex 2, mu 1 Core Neveu110 subunit 446. PLA2G4A Group IVA phospholipase A2 Core Menzel111 447. MTTP Microsomal triglyceride transfer protein NS5A Huang112 448. APOA1 Apolipoprotein A-I NS5A Shi113 449. APOA2 Apolipoprotein AII Core Barba114 450. APOB Apolipoprotein B (including Ag(x) NS5A Icard115
23 antigen) 451. APOE Apolipoprotein E NS5A Jiang116
Supplemental references
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31 Supplementary Table 2
Genetic associations with HCV clearance
IL28B Clearance rate n HR / OR Population studied Reference (%) rs12979860 CC 98 (37) HR 4·56 International collaboration of Grebley1 CT 46 (22) HR 2·66 prospective cohorts, 82% TT 15 (24) HR 2·66 Caucasian (n = 632 patients, 9 studies)
CC OR 3·77 Meta-analysis (42 studies) Chayama2 (overall) North Africans OR 1·67 Hispanics OR 7·17 Meta-analysis (n=2,340 patients, CC OR 3·2 7 studies) Jimenez-Sousa3 (overall) Meta-analysis (7 studies) mainly Zheng4 OR 3·75 Caucasian rs8099917 TT 77 (98) OR 17·27 Chinese of Han ancestry (n = Rao5 864)
TT OR 3·86 Meta-anlysis (39 studies) Chayama2 (overall) TT OR 4·82 Asians OR 2·71 Caucasians
TT OR 3·6 Meta-analysis 1,783 patients, 4 Jimenez-Sousa3 (overall) studies Zheng4 TT OR 2·665 Meta-analysis 4 studies mainly Caucasian rs10853728 CC 67 (83) OR 2·32 Chinese of Han ancestry (n = Rao5 864) rs12980275 AA 77 (97) OR 7·92 Chinese of Han ancestry (n = Rao5 864) rs8105790 TT 77 (98) OR 14·88 Chinese of Han ancestry (n = Rao5 864) rs12980275, rs8105790, rs8099917, rs10853728 AA,TT,TT,CC 64 (82) OR 2·12 Chinese of Han ancestry (n = Rao5 864) Interferon lambda 4 Effect size (RNA - OR Population studied Reference gene ve vs. RNA+ve; % unless stated) 31.3 vs. 40.9 0·95 890 women (555 African Aka6 TT/TT 34.9 vs. 12.7 3·59 American, 185 Hispanic, 150 TT /ΔG white)
HLA alleles or haplotype Alleles or haploptype Effect size (RNA – OR / RR Population studied Reference ve vs. +ve % unless stated) DQB1*0301 53 vs. 18 OR 5·09 Caucasian Cramp7 DQA1*03 51 vs. 18 OR 4·69 Clinic pts vs HCW DRB1*04 47 vs. 16 OR 4·52 49 RNA +ve One or more of above 3 69 vs. 27 NS 55 RNA –ve
DRB1*1101 40 vs. 10 NS Caucasian Alric8 DQB1*0301 84 vs. 31 NS 103 RNA +ve 25 RNA –ve DRB1*11 31 vs. 8 RR 0·19 Caucasian Minton9 DQB1*0301 51 vs. 24 RR 0·75 3% asian 137 RNA +ve
32 35 RNA -ve DRB1*1104 18 vs. 5 OR 4·51 Caucasian Mangia10 DQB1*0301 53 vs. 29 OR 4·52 149 RNA +ve DRB1*1104- 18 vs. 5 OR 7·38 35 RNA –ve DQB1*0301 DRB1*01 27 vs. 7 OR 4·9 Caucasian (Irish female anti-D Barrett11 immunogloblin recipients) 73 RNA –ve 84 RNA +ve DRB1*0101 32 vs. 9 NS Caucasian (Irish female anti-D McKiernan12 DQB1*0501 37 vs. 14 immunogloblin recipients) (in linkage) 95 RNA –ve DRB1*03011 17 vs. 42 148 RNA +ve DQB1*0201 16 vs. 43 (in linkage) DRB1*15011 44 vs. 6 RR 13·6 Caucasian Lechmann13 9 RNA –ve 18 RNA +ve DQB1*0301 25 vs. 19 OR 0·72 Caucasian IDU and haemophiliac Thio14 DRB1*0101 10 vs. 5 OR 0·45 cohorts DQB1*0501- 10 vs. 5 OR 0·48 200 RNA –ve DRB1*0101 374 RNA +ve (The latter associations not found in African American subjects) HLA-A*1101 5 vs. 3 OR 0·49 Caucasian IDU and haemophiliac Thio15 HLA-B*57 7 vs. 5 OR 0·62 cohorts HLA-Cw*0102 4 vs. 2 OR 0·43 231 RNA –ve 444 RNA +ve DRB1*1101 34 vs. 15 Pc=0·012 Caucasian Alric16 DQB1*0301 64 vs. 29 Pc=0·003 63 RNA –ve 282 RNA +ve HLA-C1C1 38 vs. 30 OR 1·4 Caucasian and African American Khakoo17 685 RNA +ve 352 RNA -ve KIR Effect size (RNA – OR / RR Population studied Reference ve vs. +ve % unless stated) KIR2DL3 - HLA-C1 19 vs. 12 OR 1·71 Caucasian and African American Khakoo17 ligand 685 RNA +ve 352 RNA -ve Chemokine gene Effect size (RNA – OR / χ² Population studied Reference ve vs. + ve % unless stated) IL12B 1188A/C 50 vs. 66 χ² 4·12 European Caucasian Houldsworth18 123 RNA +ve 72 RNA -ve IL12B 11 HESN vs. 1 OR 12 Caucasian Hegazy19 1188C/C controls 76 HESN IDU 105 healthy controls IFNG 764G (promoter NS OR 3·51 92% African American, 8% Huang20 mutation) Caucasian American IDU − 166 RNA +ve 85 RNA -ve
33 IL10 rs6703630 19 vs. 32 OR 0.52 African Americans Oleksyk21 183 RNA +ve 91 RNA -ve rs6693899 27 vs. 39 OR 0.58 African Americans Oleksyk21 183 RNA +ve 91 RNA -ve rs1800896 32 vs. 36 OR 0.84 African Americans Oleksyk21 183 RNA +ve 91 RNA -ve rs1800872 43 vs. 38 OR 1.22 African Americans Oleksyk21 183 RNA +ve 91 RNA -ve rs3024498 9 vs. 17 OR 0.53 African Americans Oleksyk21 183 RNA +ve 91 RNA -ve TGF-beta 1 18 vs. 39 OR 2.4 Japanese Kimura22 -509CC 184 RNA +ve 46 RNA -ve Superoxide dismutase Effect size (RNA – χ² Population studied Reference gene vs. + % unless stated) TT 27 vs. 7 11.64 European Caucasian Houldsworth23 123 RNA +ve 72 RNA -ve
OR odds ratio NS not stated RR relative risk HR hazard ratio Pc P value corrected for multiple comparisons KIR killer inhibitory receptor HESN highly exposed seronegative χ² chi-square
Supplemental references
1. Grebely J, Page K, Sacks-Davis R, van der Loeff MS, Rice TM, Bruneau J, et al. The effects of female sex, viral genotype, and IL28B genotype on spontaneous clearance of acute hepatitis C virus infection. Hepatology. 2014; 59(1): 109-20. 2. Chayama K, Hayes CN. Interleukin-28B polymorphisms and hepatitis C virus clearance. Genome medicine. 2013; 5(1): 6. 3. Jimenez-Sousa MA, Fernandez-Rodriguez A, Guzman-Fulgencio M, Garcia- Alvarez M, Resino S. Meta-analysis: implications of interleukin-28B polymorphisms in spontaneous and treatment-related clearance for patients with hepatitis C. BMC medicine. 2013; 11: 6. 4. Zheng MH, Li Y, Xiao DD, Shi KQ, Fan YC, Chen LL, et al. Interleukin-28B rs12979860C/T and rs8099917T/G contribute to spontaneous clearance of hepatitis C virus in Caucasians. Gene. 2013; 518(2): 479-82. 5. Rao HY, Sun DG, Jiang D, Yang RF, Guo F, Wang JH, et al. IL28B genetic variants and gender are associated with spontaneous clearance of hepatitis C virus infection. Journal of viral hepatitis. 2012; 19(3): 173-81. 6. Aka PV, Kuniholm MH, Pfeiffer RM, Wang AS, Tang W, Chen S, et al. Association of the IFNL4-DeltaG Allele With Impaired Spontaneous Clearance of Hepatitis C Virus. J Infect Dis. 2013. 7. Cramp ME, Carucci P, Underhill J, Naoumov NV, Williams R, Donaldson PT. Association between HLA class II genotype and spontaneous clearance of hepatitis C viraemia. J Hepatol. 1998; 29(2): 207-13.
34 8. Alric L, Fort M, Izopet J, Vinel JP, Charlet JP, Selves J, et al. Genes of the major histocompatibility complex class II influence the outcome of hepatitis C virus infection. Gastroenterology. 1997; 113(5): 1675-81. 9. Minton EJ, Smillie D, Neal KR, Irving WL, Underwood JC, James V. Association between MHC class II alleles and clearance of circulating hepatitis C virus. Members of the Trent Hepatitis C Virus Study Group. Journal of Infectious Diseases. 1998; 178(1): 39-44. 10. Mangia A, Gentile R, Cascavilla I, Margaglione M, Villani MR, Stella F, et al. HLA class II favors clearance of HCV infection and progression of the chronic liver damage. Journal of Hepatology. 1999; 30(6): 984-9. 11. Barrett S, Ryan E, Crowe J. Association of the HLA-DRB1*01 allele with spontaneous viral clearance in an Irish cohort infected with hepatitis C virus via contaminated anti-D immunoglobulin. Journal of Hepatology. 1999; 30(6): 979-83. 12. McKiernan SM, Hagan R, Curry M, McDonald GS, Nolan N, Crowley J, et al. The MHC is a major determinant of viral status, but not fibrotic stage, in individuals infected with hepatitis C. Gastroenterology. 2000; 118(6): 1124-30. 13. Lechmann M, Schneider EM, Giers G, Kaiser R, Dumoulin FL, Sauerbruch T, et al. Increased frequency of the HLA-DR15 (B1*15011) allele in German patients with self-limited hepatitis C virus infection. European Journal of Clinical Investigation. 1999; 29(4): 337-43. 14. Thio CL, Thomas DL, Goedert JJ, Vlahov D, Nelson KE, Hilgartner MW, et al. Racial differences in HLA class II associations with hepatitis C virus outcomes. Journal of Infectious Diseases. 2001; 184(1): 16-21. 15. Thio CL, Gao X, Goedert JJ, Vlahov D, Nelson KE, Hilgartner MW, et al. HLA-Cw*04 and hepatitis C virus persistence. Journal of Virology. 2002; 76(10): 4792-7. 16. Alric L, Fort M, Izopet J, Vinel JP, Bureau C, Sandre K, et al. Study of host- and virus-related factors associated with spontaneous hepatitis C virus clearance. Tissue Antigens. 2000; 56(2): 154-8. 17. Khakoo SI, Thio CL, Martin MP, Brooks CR, Gao X, Astemborski J, et al. HLA and NK cell inhibitory receptor genes in resolving hepatitis C virus infection. Science. 2004; 305(5685): 872-4. 18. Houldsworth A, Metzner M, Rossol S, Shaw S, Kaminski E, Demaine AG, et al. Polymorphisms in the IL-12B gene and outcome of HCV infection. J Interferon Cytokine Res. 2005; 25(5): 271-6. 19. Hegazy D, Thurairajah P, Metzner M, Houldsworth A, Shaw S, Kaminski E, et al. Interleukin 12B gene polymorphism and apparent resistance to hepatitis C virus infection. Clin Exp Immunol. 2008; 152(3): 538-41. 20. Huang Y, Yang H, Borg BB, Su X, Rhodes SL, Yang K, et al. A functional SNP of interferon-gamma gene is important for interferon-alpha-induced and spontaneous recovery from hepatitis C virus infection. Proc Natl Acad Sci U S A. 2007; 104(3): 985-90. 21. Oleksyk TK, Thio CL, Truelove AL, Goedert JJ, Donfield SM, Kirk GD, et al. Single nucleotide polymorphisms and haplotypes in the IL10 region associated with HCV clearance. Genes Immun. 2005; 6(4): 347-57. 22. Kimura T, Saito T, Yoshimura M, Yixuan S, Baba M, Ji G, et al. Association of transforming growth factor-beta 1 functional polymorphisms with natural clearance of hepatitis C virus. J Infect Dis. 2006; 193(10): 1371-4.
35 23. Houldsworth A, Metzner M, Shaw S, Kaminski E, Demaine AG, Cramp ME. Polymorphic differences in SOD-2 may influence HCV viral clearance. Journal of medical virology. 2014.
36 Chapter 2
Natural killer cells in highly exposed hepatitis C-seronegative injecting
drug users
37 Journal of Viral Hepatitis, 2016 doi:10.1111/jvh.12511
Natural killer cells in highly exposed hepatitis C-seronegative injecting drug users M. M. Mina,1 B. Cameron,1 F. Luciani,1 U. Vollmer-Conna,2 A. R. Lloyd,1 on behalf of the † HITS-p investigators 1Inflammation and Infection Research Centre, School of Medical Sciences, University of New South Wales, Sydney, Australia; and 2School of Psychiatry, University of New South Wales, Sydney, Australia
Received October 2015; accepted for publication December 2015
SUMMARY. Injecting drug use remains the major risk factor of the major NK subpopulations (based on CD56 and CD16 for hepatitis C (HCV) transmission. A minority of long-term co-expression), before logistic regression analysis to identify injecting drug users remain seronegative and aviraemic, associations with exposed, seronegative individuals. The despite prolonged exposure to HCV – termed highly CD56dimCD16+ (P = 0.05, OR 6.92) and CD56dimCD16À exposed seronegative subjects. Natural killer (NK) cells (P = 0.05, OR 6.07) principal components differed between have been implicated in this apparent protection. A longi- exposed, seronegative individuals and pre-infection samples tudinal nested, three group case–control series of subjects of the other two groups. These included CD56dimCD16+ was selected from a prospective cohort of seronegative and CD56dimCD16À subsets with CD56dimCD16+ IFN-c and injecting drug users who became incident cases (n = 11), TNF-a on unstimulated cells, and CD56dimCD16À CD69+, remained seronegative (n = 11) or reported transient high- CD107a+, IFN-c and TNF-a following TLR stimulation. The risk behaviour and remained uninfected (n = 11). The cytotoxic CD56dim NK subset thus distinguished highly groups were matched by age, sex and initial risk behaviour exposed, seronegative subjects, suggesting NK cytotoxicity characteristics. Stored peripheral blood mononuclear cells may contribute to protection from HCV acquisition. Fur- were assayed in multicolour flow cytometry to enumerate ther investigation of the determinants of this association natural killer cell subpopulations and to assess functional and prospective assessment of protection against HCV activity using Toll-like receptor ligands before measure- infection are warranted. ment of activation, cytokine production and natural cyto- toxicity receptor expression. Principal components were Keywords: hepatitis C, highly exposed seronegative, natural derived to describe the detailed phenotypic characteristics killer cells, principal component analysis, protective immunity.
INTRODUCTION highly exposed seronegative (HESN) group have also been termed ‘exposed uninfected’ indicating the absence of Injecting drug use (IDU) remains the major risk factor for demonstrable infection as defined by conventional antibody hepatitis C virus (HCV) transmission in developed coun- or RNA testing [3]. Several prospective studies of high-risk tries, with seroprevalence rates in cross-sectional studies of IDUs have reported subgroups that remain seronegative injecting drug users (IDUs) ranging from 50% to 90% [1]. despite long-standing risk behaviour [4], who may repre- A minority of long-term IDUs remain seronegative and avi- sent a phenotype that are resistant to infection, or who raemic, despite prolonged and likely repeated exposure to may become infected but have efficient viral clearance HCV through sharing of drug injection equipment [2]. This prior to seroconversion [5].
Abbreviations: ADCC, antibody-dependent cellular cytotoxicity; ATSI, aboriginal or torres strait islander; v², chi-square tests; FMO, fluores- cence-minus-one; HCV, hepatitis C Virus; HESN, highly exposed seronegative; HITS-p, hepatitis C incidence and transmission study; IDU, injecting drug use; IDUs, injecting drug users; IFN-c, interferon gamma; ISGs, interferon-stimulated genes; KIR, killer cell immunoglobulin- like receptor; NCRs, natural cytotoxicity receptors; NKG2D, natural killer group 2 member D; NK, natural killer; PBMC, peripheral blood mononuclear cells; PCA, principal components analysis; PC, principal component; TLR, toll-like receptor; TNF-a, tumour necrosis factor alpha. Correspondence: Dr Michael Mina, Inflammation and Infection Research Centre, School of Medical Sciences, University of New South Wales, Sydney NSW 2052, Australia. E-mail: [email protected] †The Hepatitis C Incidence and Transmission Study in prisons (HITS-p) investigators are listed in Acknowledgements.
© 2016 John Wiley & Sons Ltd
38 2 M. M. Mina et al.
Multiple factors, many immunological, have been impli- In relation to HESN, there is a paucity of studies of the cated in determining the varied outcomes from HCV infec- role of NK cells as rapid effectors. We recently reported tion [6,7]. Individuals may be exposed and remain higher numbers of activated (CD69+) NK cells in the seronegative and individuals may have naturally clear CD56dimCD16+ subset, and cytotoxic (NKp30+) cells in the infection and remain seropositive; or the majority develop CD56brightCD16+ subset amongst HESN subjects, with chronic infection. The latter group have progressive hep- higher frequencies of IFN-c and lower frequencies of atic fibrosis with the ultimate risk of cirrhosis, liver failure CD107a expression [19]. Other studies have reported and hepatocellular carcinoma. Innate antiviral mecha- higher proportions of the CD56dim subset amongst IDUs nisms constitute the first line of defence in acute HCV, who are HESN, with increased expression of NKp30 [20]. whilst informing and directing the adaptive immune The influence of TLR activation on protection from HCV response [8]. The interplay between the host cells and infection has not been previously investigated. virus at this early stage may set the stage for efficient HCV Using a highly selected group of HESN individuals and clearance or chronic infection. matched comparison subjects, this study aimed to charac- Natural killer cells are regarded as sentinels of the innate terize the phenotype and function of NK cells in HESN, and immune system, playing a key role in the initial host defence elucidate whether they have a role in protective immunity against many viruses [9]. They are identified as CD3-CD56+ against HCV infection. lymphocytes that are regulated by the integration of signals from both activating and inhibitory cell surface receptors MATERIALS AND METHODS [10]. The two major human NK subsets are designated via CD56 bright or dim phenotypes, with evidence for associated Study cohort differences in effector functions [11]. The CD56dim subset expresses more perforin and predominantly acts by cytotoxi- Longitudinally collected demographic and risk behaviour city through cell-to-cell contact. The CD56bright subset, data as well as cryopreserved peripheral blood mononuclear found in higher frequencies in tissues compared to periph- cells (PBMC) were available from IDUs (n = 500) enrolled eral blood, acts mainly through secretion of immunomodu- in the Hepatitis C Incidence and Transmission Study in pris- latory cytokines such as IFN-c and TNF-a [12]. ons (HITS-p), based in correctional centres in New South The large number of activating and inhibitory cell surface Wales, Australia [21]. Subjects who had HCV antibody- receptors on NK cells include killer cell immunoglobulin-like negative test results (Abbott Architect HCV assay; Abbott receptors (KIRs), lectin-like receptors (NKG2A-F), natural Laboratories, Abbot Park, Illinois, OH, USA) in the preced- cytotoxicity receptors (NCRs: NKp30, NKp44 and NKp46) ing 12 months prior were recruited, completed enrolment and the natural killer group 2 member D (NKG2D) receptor screening for HCV antibodies and RNA (Cobas AmpliPrep [13]. The inhibitory receptors and their corresponding Cobas TaqMan HCV assay; Roche Molecular Diagnostics, ligands include KIR2DL1:HLA-C group 2, KIR2DL2/3:HLA- Pleasanton, CA, USA) and were followed at six- to 12- C group 1, KIR3DL1:HLA-B alleles, KIR3DL2:HLA-A alleles month intervals, to screen for HCV seroconversion and vir- and NKG2A:HLA-E [11]. A large number of activation aemia. Subjects completed a detailed interview recording receptors have been characterized, most of which are risk behaviours at each time point [22]. PBMC were stored expressed by all NK cells [14]. In addition to KIRs and between 2005 and 2014 using a well-established and NCRs, NK cells also express several Toll-like receptors quality-controlled methodology [23]. (TLRs), and evidence from hepatitis B suggests important functional roles in antiviral immunity [15]. Analysis of risk behaviour and composite risk scores Early after HCV infection, an innate immune response is evident in the liver and in the blood, featuring induction of Using our recently described phenotypic definition for antiviral proteins, notably the type 1 IFNs and thereafter HESN [24], which is similar to a procedure used to model numerous interferon-stimulated genes (ISGs) with antiviral risk factors for coronary heart disease [25] and HIV infec- properties [16]. In the chimpanzee model, greater ISG tion risk [26], a composite risk score based on both logistic expression in the liver during acute HCV was correlated and Cox regression multivariate analyses of predictors of with spontaneous clearance [16]. In humans with acute incident HCV infection in the cohort was applied to identify HCV infection, both CD56bright and CD56dim populations HESN as very high-risk individuals who remained consis- have been shown to have increased NK2GD expression tently uninfected, despite high-risk behaviours [21]. Signifi- and enhanced IFN-c production and cytotoxicity – regard- cant risk factors from univariate analysis (P ≤ 0.1) and all less of the subsequent clearance or chronic infection out- risk factors previously associated with incident infection come [17]. NK cells are believed to become activated were included in the multivariate analysis. Time-dependent during acute HCV approximately 8–14 weeks after infec- Cox regression for predictors of incident HCV infection dur- tion, when liver enzymes and viraemia reach high levels ing follow-up using hazard ratios was used to calculate [18]. composite risk scores for the follow-up period. Odds and
© 2016 John Wiley & Sons Ltd
39 NK Cells In HESN Injecting Drug Users 3 hazard ratios were summed and then normalized to a NK cell phenotyping range of 0–1.0 to create a composite risk score for each individual at multiple time points. Scores were divided To define NK subpopulations, thawed PBMC were stained into tertiles across the cohort to create ‘high-risk’, ‘med- with LIVE/DEAD Fixable Dead Cell Stain (Life Technologies, ium-risk’ and ‘low-risk’ scoring bands (Supplementary Carlsbad, CA, USA) and then fluorochrome-conjugated Table 1). antibodies for cell surface markers: anti-CD3-APC-H7 Three subject groups matched by age and sex with lon- (Clone SK7), anti-CD56-BV421 (Clone NCAM16.2) and gitudinally collected PBMC samples were then selected for anti-CD16-AF700 (Clone 3G8). In addition, cells were co- analysis: (i) HESN cases, defined as having high composite stained with anti-CD69-PerCP-Cy5.5 (Clone FN50) and risk indices of ‘lifetime’, ‘pre-imprisonment’ and/or ‘since anti-CD107a-FITC (Clone H4A3) to examine activation sta- imprisonment’ risk behaviour. These cases also had high- tus and functional activity, respectively, and after perme- risk scores for at least two follow-up time points during abilization (Cytofix/Cytoperm solution, BD Biosciences, San which they remained uninfected, and were excluded if they Jose, CA, USA), with intracellular IFN-c (BV605; Clone had a low-risk score at any time point; (ii) matched unin- B27) and TNF-a (PE; Clone MAb11), all from BD Bio- fected, but high-risk subjects who become infected during sciences, San Jose, CA, USA. follow-up (termed ‘Incident’); and (iii) matched uninfected Cells were also stained for the natural cytotoxicity recep- subjects with an initial high-risk index, but who subse- tors, anti-NKp44-PE (Clone p44-8.1), anti-NKp46-PE-Cy7 quently ceased high-risk behaviour and remained unin- (clone 9E2), anti-NKp30-AF647 (Clone p30-15) and anti- fected (termed here ‘Hi_Lo’). The latter group were NKG2D-PE-CF594 (Clone 1d11), all from BD Biosciences. included to examine associations between NK parameters Finally, the following Toll-like receptors were stained: anti- and risk behaviour. Figure 1 has a schematic overview of TLR4-PE-Cy7 (Clone HTA125, LifeSpan BioSciences, Seat- the subject groups and the statistical analyses. PBMC from tle, WA, USA) on the cell surface, before permeabilization the earliest available postinfection time point of those who and staining with anti-TLR3-FITC (Novus Biologicals, Lit- became infected (Incident) were studied – generally approx- tleton, CO, USA); anti-TLR7-PE (Clone IMG4G6, LifeSpan imately 6 months after infection and before antiviral treat- BioSciences, Seattle, WA, USA); and anti-TLR9-BV421 ment was offered. (Clone eB72-1665, BD Biosciences, San Jose, CA, USA).
Table 1 Demographics of the three HESN* Incident* Hi_Lo* subject groups at baseline, selected by (n = 11) (n = 11) (n = 11) composite risk score Mean age in years (sd) 33.2 (5.9) 32.1 (4.2) 34.6 (5.6) Male n (%) 9 (81) 9 (81) 7 (64) ATSI n (%) 2 (18) 4 (36) 7 (64) Mean no. times in prison (sd) 4.64 (1.7) 2.15 (1.5) 5.27 (1.4) Mean time inside prison (months) (sd) 7 (7.8) 12.5 (18.0) 6.4 (5.0) Previous juvenile detention n (%) 6 (55) 6 (55) 7 (74) Mean number of tattoos (sd) 3.8 (6.1) 3.1 (3.1) 2.8 (4.1) Ever inject heroin n (%) 8 (73) 9 (81) 9 (81) Ever inject buprenorphine/methadone 6 (55) 6 (55) 7 (64) n (%) Duration of injecting (years) (sd) 8.6 (3.7) 7.1 (5.4) 9.1 (8.0)
sd, standard deviation; ATSI, Aboriginal or Torres Strait Islander. *No significant differences between groups.
Key Time point 1 Time point 2 High risk uninfected
High risk infected HESN Low risk uninfected
Analysis 1 Incident Analysis 2 Fig. 1 Schematic overview of the Analysis 3 subject groups, samples and statistical Hi_Lo analyses. Analysis 4
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40 4 M. M. Mina et al.
of <0.05 was considered significant unless otherwise sta- In vitro stimulation with TLR ligands ted. Peripheral blood mononuclear cells were cultured at Analyses were conducted using SPSS or Prism (SPSS 7.5 9 105 cells/well for 20 h at 37 °C in complete med- statistics v20, IBM, Armonk, NY, USA; GraphPad Prism v. ium comprised of RPMI-1640 (Gibco BRL, Grand Island, 6.0c GraphPad Software, La Jolla, CA). NY, USA) supplemented with 10% (v/v) heat-inactivated human AB serum (HI-ABS; Sigma Aldrich, Australia), RESULTS 2mML-glutamine, 100 U/mL penicillin and 100 lg/mL streptomycin in sterile 96-well U-bottom plates (Greiner Subjects Bio-One, Monroe, NC, USA) with TLR ligands or media alone controls, including poly-I-C (PIC) [25 lg/mL] (TLR3; Two time points from 11 HESN, 11 Infected (including an Invivogen, San Diego, CA, USA), lipopolysaccharide (LPS) uninfected and infected time point) and 11 ‘Hi_Lo’ subjects [100 ng/mL] (TLR4; Invivogen), R848 [5 lg/mL] (TLR7/ (high risk and low risk, uninfected) were included in this 8; Invivogen) and CpG (TLR9; Invivogen). The concentra- study. There were no significant differences in demographic tions of these TLR ligands and culture conditions were as or risk behaviour characteristics between the 11 subjects previously described [27]. The Golgi transport inhibitor in each of the three study groups (Table 1). There were 9 Brefeldin A (Golgiplug, Becton Dickinson, Franklin Lakes, males in the HESN group (81%), 9 in the Incident group NJ, USA) was added for the final 4 h of culture. The cells (81%) and 7 (64%) in the Hi_Lo group. Of the HESN cases, were then stained for flow cytometry as above. 2 (18%) were of Aboriginal or Torres Strait Islander des- cent, in comparison with 4 (36%) in the Incident group and 7 (64%) ‘Hi_Lo’ subjects. There were 8 (73%) HESN Flow cytometry analysis subjects who reported injecting heroin, and 6 (55%) had Cells were acquired on a LSRFortessaTM flow cytometer (BD injected buprenorphine or methadone. The mean number Biosciences) and fluorescence analysed in 14 colours using of tattoos was 3.8, 3.1 and 2.8 for HESN, Incident and Flowjo software (Flowjo 10.0.6, Treestar, Ashland, OR, ‘Hi_Lo’ groups, respectively. The mean period of this USA). Gating was performed as previously described [20] imprisonment at baseline was 7, 12.5 and 6.4 months for with fluorescence-minus-one (FMO) controls used to set the three groups, and the numbers who had previously gates (Figure S1). been in juvenile detention were 6 (55%), 6 (55%) and 7 (74%) for HESN, Incident and ‘Hi_Lo’ groups, respectively. Statistical analysis NK subsets Variables from the NK cell phenotyping assays and the in vitro stimulation assay were combined and then divided Several analyses were performed to assess statistical differences into data sets describing four NK subsets: CD56brightCD16+, in the frequency and functional phenotypes of circulating NK CD56brightCD16À, CD56dimCD16+ and CD56dimCD16À.A cells (CD3À CD56+) longitudinally in each group (analyses principal components analysis (PCA) was carried out on 1–3, Fig. 1), and between groups (analysis 4, Fig. 1). the data set for each of these four NK subsets to identify No differences were evident in the frequencies of the the two most significant principal components (PCs) for total NK cell population between baseline and follow-up each. A preliminary analysis by paired t-test was under- time points within the HESN subject group (7.9% vs 6.9%, taken to verify that there was no significant difference in P = 0.07). There were also no differences between baseline the PCs between the two uninfected time points of the (uninfected) and follow-up (infected) time points in the HESN group (analysis 1, Fig. 1). Incident group (9.3% vs 10.3%, P = 0.45), or between Logistic regression was then performed on the 8 PCs to baseline (high risk) and follow-up (low risk) in the ‘Hi_Lo’ identify differences between baseline (when all subjects group (8.26% vs 9.17%, P = 0.97; all paired t-tests). Simi- were uninfected) and the HCV infected time point of the larly, there was no significant difference between HESN Incident group (analysis 2, Fig. 1). Baseline and follow-up and baseline data of the Incident group (7.6% vs 9.3%, comparison was also made within the ‘Hi_Lo’ group (anal- P = 0.38 unpaired t-test). ysis 3, Fig. 1). Similarly, the HESN data set was contrasted There were no significant differences in the percentages with the baseline data set of the Incident subject group of CD56bright and CD56dim subpopulations as a proportion (analysis 4, Fig. 1). of total NK cells between baseline and follow-up time Demographic and risk behaviour data were analysed points in the (i) HESN group (8.1% vs 9.1%, P = 0.45; using two-tailed paired and unpaired t-tests for continuous 91.9% vs 90.9%, P = 0.45), (ii) Incident group (8.5% vs variables and chi-square tests (v²) for categorical variables. 10.1%, P = 0.09; 91.5 vs 89.7, P = 0.09, respectively) Elimination logistic regression analysis was used to identify and (iii) ‘Hi_Lo’ group (9.1% vs 7.2%, P = 0.13; 90.9% vs associations with HCV infection status. A two-sided P-value 92.8%, P = 0.13, respectively).
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41 NK Cells In HESN Injecting Drug Users 5
There were also no significant differences at baseline in IFN-c+-producing cells after TLR 7/8 stimulation increased CD56bright and CD56dim subpopulations between HESN and with incident infection. Incident groups (8.1% vs 8.5%, P = 0.85; 91.9% vs 91.5%, P = 0.85, respectively). Similarly, there were no Baseline HESN vs baseline incident differences in the NK subsets CD56dimCD16+ cytotoxic NK cells, or the potential antibody-dependent cellular cytotoxi- Logistic regression analysis of the same eight PCs compar- city (ADCC) effector NK cells (CD56brightCD16+) between ing the baseline HESN and baseline Incident group data any of the groups (Table S2). sets identified differences in the CD56dimCD16+ population in the second PC (P = 0.05, OR 6.92) and CD56dimCD16À population in the first PC (P = 0.05, OR 6.07, Table 2). Principal components analysis identified phenotypic and The variables constituting these PCs are illustrated in functional patterns associated with the HESN and Fig. 2(c,d). This difference was associated with increased incident phenotype CD56dimCD16+ NK cell population frequencies before and The analysis of phenotypic and functional properties of NK after TLR stimulation, as well as by increased cytokine pro- cells included a total of 227 unique NK cell parameters for duction in the unstimulated samples. The logistic regres- each subject’s time point. For data reduction, a PC for of sion also revealed differences in the frequencies of each the four major NK subsets was derived: CD56dimCD16À cells expressing IFN-c, TNF-a, CD107a and CD56brightCD16+, CD56brightCD16À, CD56dimCD16+ and CD69, following the stimulation of TLR 3, 4, 7/8 and 9. CD56dimCD16À. From these PCs, the top two solutions within each NK subset, accounting for 30–40% of the vari- DISCUSSION ance in the data set were selected for further comparative analyses (Table 2 and Supplementary Table S3). This study is the first comprehensive characterization of NK subpopulations by phenotype and function in carefully characterized HESN individuals who are likely to have Baseline vs follow-up within the incident group been repeatedly exposed to HCV through high-risk drug Logistic regression analysis including the eight PCs, identi- injecting over many years, in comparison with matched fied differences between baseline and follow-up time points subjects who became infected or reduced their injecting of the Incident group in the CD56brightCD16+ in the second risk behaviours. The findings presented here reveal that PC (P = 0.01, OR 29.07) and CD56dimCD16+ in the second the cytotoxic NK subset, CD56dim, differed between HESN PC (P = 0.01, OR 21.7). The variables constituting these and comparison subjects, within both CD16+ and CD16À two PCs are illustrated in Fig. 2(a,b). The proportions of subpopulations. This gives further credence to the hypothe- CD56brightCD16+ and CD56brightCD16+TNF-a-producing sis that NK cells contribute to anti-HCV defence in the cells following stimulation with TLR 3, 4, 7/8 and 9 ligands earliest stages of infection, potentially providing protection were most associated with new infection status. By con- from HCV acquisition. The data also demonstrate that sub- trast, the CD56brightCD16+ CD107a+ and CD56brightCD16+ jects who become infected with HCV have an altered NK CD69+ subpopulations following TLR 7/8 stimulation profile following infection within both CD56brightCD16+ decreased with incident infection, and the CD56brightCD16+ and CD56dimCD16+ subsets, affirming the role of these cells
Table 2 Logistic regression analysis of Baseline vs Baseline vs Baseline principal components describing the follow-up follow-up within HESN vs phenotype and function of NK subsets within incident ‘Hi_Lo’ incident
Principal component P OR P OR P OR
CD56bright16+, PC1 0.84 0.84 0.40 0.05 0.55 1.68 CD56bright16+, PC2 0.01 29.07 0.80 0.62 0.76 0.74 CD56dim16+, PC1 0.12 0.17 0.26 998.6 0.98 1.03 CD56dim16+, PC2 0.01 21.70 0.64 6.26 0.05 6.92 CD56bright16À, PC1 0.08 0.21 0.12 45.86 0.97 1.03 CD56bright16À, PC2 0.39 0.58 0.54 0.17 0.30 0.54 CD56dim16À, PC1 0.06 7.44 0.32 0.07 0.05 6.07 CD56dim16À, PC2 0.07 0.12 0.48 0.37 0.42 2.14
Significant P values bolded. PC1, first principal component; PC2, second princi- pal component; OR, odds ratio.
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42 6 M. M. Mina et al.
4 (a) 100 (b) 80 Baseline Incident 3 60 Follow-up Incident 2 40 1 20 Mean % positive (SD) 0 0
PIC_total PIC_ total PIC_TNF- R848_totalLPS_ total PIC_CD69CPG_ total CPG_ total LPS_ total LPS_TNF- CPG_TNF- R848_CD69 R848_ IFN- R848_ total R848_ TNF- LPS_CD107a Unstim_ total R848_CD107a Unstim_IFN-Unstim_TNF-
100 (c) 60 (d) Baseline HESN 80 40 Baseline Incident 60
40 20 20 Mean % positive (SD) 0 0
PIC_ total LPS_totalPIC_total PIC_CD69 CPG_ total LPS_ total LPS_CD69 CPG_CD69 CPG_totalCPG_IFN- PIC_ PIC_TNF-IFN- R848_ total R848_ total PIC_CD107a R848_IFN- CPG_TNF- Unstim_ total CPG_CD107a Unstim_ total LPS_CD107a Unstim_IFN-Unstim_TNF- Unstim_CD69 R848_CD107a
Fig. 2 Analysis of NK subpopulations. Significant differences in NK subpopulations were observed between: (a) Baseline and follow-up time points of the Incident group within CD56brightCD16+. PIC: TLR3, LPS: TLR4, R848: TLR7/8, CPG: TLR9, total indicates % of CD56 of live cells. Data shown are mean+SD. (b) Baseline and follow-up time points of the Incident group within CD56dimCD16+. PIC: TLR3, LPS: TLR4, R848: TLR7/8, CPG: TLR9, total indicates % of CD56 of live cells. Data shown are mean+SD. (c) Baseline time point comparisons of HESN and Incident subjects within CD56dimCD16+. PIC: TLR3, LPS: TLR4, R848: TLR7/8, CPG: TLR9, total indicates % of CD56 of live cells. Data shown are mean+SD. (d) Baseline time point comparisons of HESN and Incident subjects within CD56dimCD16À. PIC: TLR3, LPS: TLR4, R848: TLR7/8, CPG: TLR9, total indicates % of CD56 of live cells. Data shown are mean+SD. in the response to established infection. There was no asso- ing NK cells in apparent protection from HIV infection in ciation between risk status and alteration of NK phenotype high-risk exposed individuals [34,35]. Similarly, NK cells or function. were found to be activated early in healthcare workers fol- NK cells secrete cytokines that inhibit replication, pro- lowing accidental percutaneous exposure to HCV and may mote dendritic cell maturation and induce production of have contributed to protection in those who remained avi- chemokines that recruit lymphoid and inflammatory cells raemic [36]. Similar observations have been made in [28]. NK cells can also modulate adaptive immune spouses of HCV-infected subjects [37,38]. Total NK cell responses by killing T cells and antigen-presenting cells populations amongst uninfected IDUs have previously been (APCs) [29]. NK cells from individuals acutely infected reported to be enriched for CD56dim effector NK cells dis- with HCV and, during chronic infection, have previously playing enhanced IL-2-induced cytolytic activity and been shown to display a bias towards cytotoxicity, rather higher levels of NKp30-activating NCRs [20], although no than cytokine production [30,31]. Expression of CD107a, detailed description of IDU or risk behaviour was included which marks degranulating NK cells as a surrogate marker in this study. Another study of acute, chronic and resolved of cytotoxicity, has been correlated with the magnitude of HCV infections observed lower frequencies of NKp30+, the HCV-specific T-cell response, measured by IFN-c ELI- NKp46+ and NKG2D+ NK cells in patients who were sub- SPOT, in a cohort of in patients with HCV chronic evolu- sequently able to clear HCV infection than in those becom- tion and HCV spontaneous resolution during the acute ing chronically infected, suggesting that these surface phase of HCV and after 1-month follow-up [30]. The pre- markers may play a role in the control of the virus [39]. sent study is consistent with these and other recent reports Increased expression of NKG2D has also been detected in that have identified subjects acutely infected with HCV as patients with acute HCV, irrespective of the outcome, com- having altered ratios of cytotoxic and IFN-c-producing NK pared with healthy controls [17,30], whilst increased IFN-c subsets [32,33]. production was enriched in KIR2DL3-expressing NK cells, Growing evidence of a role for innate immunity in pro- suggesting that this allele is associated with spontaneous tection from viral infection is provided by studies implicat- resolution and protection from HCV potentially due to less
© 2016 John Wiley & Sons Ltd
43 NK Cells In HESN Injecting Drug Users 7
NK cell inhibition [17]. In this regard, the finding in the CD56dimCD16À, accounting for up to 40% of the variance present study that the cytotoxic NK subset (CD56dim) dif- in the data set – which may be regarded as relatively mod- fered between HESN and Incident subject groups at base- est (Table S3). Nevertheless, this approach offers the line is interesting. Co-expression of CD16 (the low-affinity advantage that it is systematic and comprehensive, thereby FccRIII, which is almost exclusively found on the CD56dim accounting for the large, exploratory data set. Although subset) has been associated with ADCC. The evidence for the subjects and samples included here feature both careful increased expression of the activation marker, CD69, and case characterization and longitudinal sample collection of the IgG receptor, FccRIII, which mediates ADCC, raises allowing within-subject analyses, a larger study size will be the possibility of undetected anti-HCV antibodies sustaining required to confirm and extend the findings. NK cell activation and cytotoxicity in HESN. This finding Efforts to develop a prophylactic vaccine are in their supports the hypothesis that cytotoxic antiviral mecha- infancy. The observation that some IDUs remain unin- nisms and HCV-specific antibodies via ADCC may have a fected despite continued exposure to HCV suggests a role role in protection from HCV. This notion requires further for innate immunity in natural protection. Better under- evaluation, as prior HCV exposure may have resulted in standing of the innate immune correlates of protection incomplete seroconversion or rapid seroreversion. against HCV acquisition in studies of HESN has the poten- A previous study by Knapp and colleagues studied 48 tial to inform vaccine development, particularly in relation HCV HESN IDUs from both needle exchange and commu- to adjuvant selection. nity drug services (n = 29) and a correctional centre (n = 19) and compared these with individuals with chronic ACKNOWLEDGEMENTS AND DISCLOSURES HCV (n = 257). The HESN IDUs group included individuals who had a broad range of injecting duration (0.5– The HITS-p investigators include Andrew Lloyd, Kate 24 years) and lifetime injection episodes (36-17,520). Dolan, Michael Levy, Peter White, Bill Rawlinson, Carla There were only 27% who reported current intravenous Treloar, Paul Haber, Greg Dore and Lisa Maher. The statis- drug use. No other selection criteria were applied to the tical advice of Roy Wilson, Australian Centre for Commer- group. This limited characterization of HESN may obscure cial Mathematics, is gratefully acknowledged. key findings. For instance, a greater frequency of homozy- gosity for KIR2DL3/HLA-C allotypes was found amongst FUNDING & CONFLICTS OF INTEREST the HESN group (25.0% vs 9.7%, OR 3.1) [40]. By con- trast, no association was found in the HITS-p cohort from The HITS-p cohort has been supported by a Program Grant which the current study sample was drawn [24]. Further from National Health and Medical Research Council studies of larger HESN cohorts are required to explore the (NHMRC) of Australia (No. 510448), a NHMRC Partner- relationship between NK function and genetic diversity. ship Grant (No. 1016351), a research contract with New The limitations of the current study warrant considera- South Wales Health (10/1392) and Strategic Priorities tion. Principal component analysis (PCA) is a popular data Funding from the University of New South Wales. ARL is processing and dimension reduction technique, which supported by a Practitioner Fellowship from the NHMRC seeks linear combinations of the original variables such (No. 1043067).The authors have no conflict of interests to that the derived variables capture maximal variance. It has declare. an obvious drawback, that is, each PC is a linear combina- tion of all variables and the loadings are typically nonzero. CONTRIBUTORS It should be noted that the PCAs in the current study iden- tified two PCs within each of the four major NK subsets, All authors contributed to the writing of this review and CD56brightCD16+, CD56brightCD16À, CD56dimCD16+ and agree with its content and conclusions.
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BMC Public Health 32 Golden-Mason L, Rosen HR. Natural 11 Bryceson YT, March ME, Ljunggren 2010; 10: 633. killer cells: multifaceted players with HG, Long EO. Activation, coactiva- 23 Dyer WB, Pett SL, Sullivan JS et al. key roles in hepatitis C immunity. tion, and costimulation of resting Substantial improvements in perfor- Immunol Rev 2013; 255(1): 68–81. human natural killer cells. Immunol mance indicators achieved in a 33 Edlich B, Ahlenstiel G, Zabaleta Rev 2006; 214: 73–91. peripheral blood mononuclear cell Azpiroz A et al. Early changes in 12 Vivier E, Tomasello E, Baratin M, cryopreservation quality assurance interferon signaling define natural Walzer T, Ugolini S. Functions of program using single donor sam- killer cell response and refractori- natural killer cells. Nat Immunol ples. Clin Vaccine Immunol 2007; 14 ness to interferon-based therapy of 2008; 9(5): 503–510. (1): 52–59. hepatitis C patients. Hepatology 13 Kanto T, Hayashi N. Innate immu- 24 Sugden PB, Cameron B, Luciani F, 2012; 55(1): 39–48. nity in hepatitis C virus infection: Lloyd AR, Investigators H. Explo- 34 Scott-Algara D, Truong LX, Vers- Interplay among dendritic cells, nat- ration of genetically determined misse P et al. Cutting edge: ural killer cells and natural killer T resistance against hepatitis C infec- increased NK cell activity in HIV-1- cells. Hepatol Res 2007; 37(Suppl tion in high-risk injecting drug exposed but uninfected Vietnamese 3): S319–S326. users. J Viral Hepatitis 2014; 21(8): intravascular drug users. J Immunol 14 Lanier LL. NK cell recognition. Annu e65–e73. 2003; 171(11): 5663–5667. Rev Immunol 2005; 23: 225–274. 25 D’Agostino RB, Russell MW, Huse 35 Tomescu C, Abdulhaqq S, Montaner 15 Luangsay S, Ait-Goughoulte M, DM et al. Primary and subsequent LJ. Evidence for the innate immune Michelet M et al. 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Hepatology 2013; 58(5): Activation of natural killer cells 2005; 39(4): 489–495. 1621–1631. during acute infection with hepatitis 27 Howell J, Sawhney R, Testro A 37 Bronowicki JP, Vetter D, Uhl G et al. C virus. Gastroenterology 2010; 138 et al. Cyclosporine and tacrolimus Lymphocyte reactivity to hepatitis C (4): 1536–1545. have inhibitory effects on toll-like virus (HCV) antigens shows evidence 18 Ahlenstiel G. The Natural Killer Cell receptor signaling after liver trans- for exposure to HCV in HCV-serone- Response to HCV Infection. Immune plantation. Liver Transpl 2013; 19 gative spouses of HCV-infected Network 2013; 13(5): 168–176. (10): 1099–1107. patients. JInfectDis1997; 176(2): 19 Sugden PB, Cameron B, Mina M, 28 Dustin LB, Rice CM. Flying under 518–522. Lloyd AR on behalf of the Hi . Pro- the radar: the immunobiology of 38 Koziel MJ, Wong DK, Dudley D, tection against hepatitis C infection hepatitis C. Annu Rev Immunol Houghton M, Walker BD. 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45 NK Cells In HESN Injecting Drug Users 9
and T helper cell responses in acute HCV infection may predict 2DL3 and group 1 human leukocyte seronegative persons. J Infect Dis viral clearance. J Hepatol 2011; 55 antigen-C following exposure to hep- 1997; 176(4): 859–866. (2): 278–288. atitis C virus. Hepatology 2010; 51(4): 39 Alter G, Jost S, Rihn S et al. Reduced 40 Knapp S, Warshow U, Hegazy D et al. 1168–1175. frequencies of NKp30+NKp46+, Consistent beneficial effects of killer CD161+, and NKG2D+ NK cells in cell immunoglobulin-like receptor
SUPPORTING INFORMATION Additional Supporting Information behavioural predictors of incident Incident group; the ‘Hi_Lo’ group and may be found in the online version of HCV infection from data regarding baseline comparison of HESN and this article: ‘lifetime’, ‘3 months prior imprison- Incident groups. Table S3. Principal Figure S1. Representative staining ment’, ‘since imprisonment’ (n = components analyses of NK sub-popu- of CD56dim and CD56bright CD3À NK 500) and follow-up periods (n = lations from the NK assays enumerat- cells with an NKG2D fluorescence- 210). Table S2. Mean percentages of ing ex vivo expression of NCRs and minus-one (FMO) control illus- subsets of NK cells from baseline and TLRs as well as TLR stimulation trated. Table S1. Demographic and follow-up within the HESN group; the assays.
© 2016 John Wiley & Sons Ltd
46 Chapter 3
Anti-envelope antibody responses in highly exposed seronegative individuals
may be associated with protection from HCV infection
47 Received: 25 March 2020 | Accepted: 11 May 2020 DOI: 10.1111/jvh.13339
ORIGINAL ARTICLE
Anti-envelope antibody responses in highly exposed seronegative individuals may be associated with protection from HCV infection
Michael Mina1,2 | Alexander Underwood1,2 | Auda Eltahla1,2 | Bing-Ru Wu1,2 | Melanie R. Walker1,2 | Rowena A. Bull1,2 | Andrew R. Lloyd1
1Viral Immunology Systems Program, The Kirby Institute, The University of New South Abstract Wales, Sydney, New South Wales, Australia In rare cases, individuals with a history of long-term injecting drug use remain seron- 2 School of Medical Sciences, Faculty of egative and aviraemic, despite prolonged and likely repeated exposure to Hepatitis Medicine, The University of New South Wales, Sydney, New South Wales, Australia C virus (HCV) through high-risk behaviour. We describe anti-HCV Envelope (E) an- tibody responses in a prospective cohort of carefully defined highly exposed but Correspondence Michael Mina, Viral Immunology Systems uninfected subjects (HESN) and comparison subjects who were also high risk and Program, The Kirby Institute, The University uninfected, but rapidly became HCV infected (Incident). Longitudinally collected of New South Wales, Sydney, NSW 2052, Australia. samples from HESN cases (n = 22) were compared to Incident controls (n = 22). IgG, Email: [email protected] IgM and IgA from sera were tested by ELISA to genotype 1a and 3a E glycoproteins,
Funding information and recombinant genotype 1a E2 antigen. IgG subclass isotyping was performed for National Health and Medical Research those positive for IgG. Virus-neutralizing activity was assessed on HCV pseudoparti- Council of Australia; NHMRC Research Fellowships, Grant/Award Number: cles, and HCV E–specific B cells analysed using flow cytometry. A significant minority 1130128, 1080001 and 1084706 of HESN cases (n = 10; 45%) had anti-E, predominantly in the IgG2 subclass, which was not found in the pre-infection time point of the Incident cases (n = 1; 5%). A sub- set of the HESN subjects also had neutralizing activity and HCV-specific B cells de- tected significantly more than Incident cases pre-infection. In conclusion, the HESN phenotype is associated with IgG2 anti-E antibodies, neutralization activity and HCV E–specific memory B cells. These findings suggest that HESN subjects may be resist- ant to HCV infection through humoral immune-mediated mechanisms.
KEYWORDS antibodies, hepatitis C virus, highly exposed seronegative, humoral immunity
1 | INTRODUCTION inject drugs (PWID) ranging from 50% to 90%.2 Whilst effective antiviral medications are now available,3 eradication will only be Hepatitis C virus (HCV) infection is a major problem for public possible if robust preventative strategies are also implemented.4 health programmes, with an estimated 71 million individuals in- As HCV replication is dependent on many host factors,5 study of fected worldwide.1 Injecting drug use (IDU) remains the major individuals who consistently resist infection could uncover mech- risk factor for HCV transmission in developed countries, with anisms of resistance and protection against HCV, and inform vac- seroprevalence rates in cross-sectional studies of people who cine design.
Abbreviations: ELISA, enzyme-linked immunosorbent assays; HCl, hydrochloric acid; HCV, hepatitis C virus; HESN, highly-exposed but uninfected subjects; HRP, horseradish peroxidase; IDU, injecting drug use; NK, natural killer; PBMC, peripheral blood mononuclear cells; PWID, people who inject drugs; TMB, 3', 5, 5'-tetramethylbenzidine; VSV, vesicular stomatitis virus.
J Viral Hepat. 2020;00:1–10. wileyonlinelibrary.com/journal/jvh © 2020 John Wiley & Sons Ltd | 1 2 | MINA et al.
In rare cases, individuals with a history of long-term injecting drug (Abbott Architect HCV assay; Abbott Laboratories, Abbot Park) use (IDU) remain seronegative and aviraemic, despite prolonged and in the preceding 12 months were recruited, completed enrolment likely repeated exposure to HCV through sharing of drug injection screening for anti-HCV and RNA (Cobas AmpliPrep Cobas TaqMan equipment.6 These individuals are termed ‘highly exposed seroneg- HCV assay; Roche Molecular Diagnostics), and then were followed at ative’ (HESN) or ‘exposed uninfected’ due to the absence of demon- approximately 6-month intervals, to screen for HCV seroconversion strable infection tested through sensitive diagnostics, which include and viraemia. Subjects completed a detailed interview recording risk commercially available enzyme-linked immunosorbent assays (ELISAs) behaviours at each time point.22,24 Plasma and PBMC were sepa- and HCV RNA testing.7,8 It should be noted that none of the currently rated and maintained in long-term storage in vapour phase nitrogen available commercial ELISAs test for antibodies against the HCV using quality-assured methodology, as described previously.25 Envelope proteins, E1 and E2, which are key for attachment and entry of HCV virions into target cells.9 In most primary infection cases, an- ti-E is not detected until approximately 50 days or longer after estab- 2.2 | Ethics lished infection.10,11 In the case of HESN individuals, it is plausible that seroconversion against E proteins occurs without subsequent devel- Ethical approvals were obtained from the Human Research Ethics opment of antibodies against the other viral antigens included in the Committees of Justice Health (reference number GEN 31/05), the assays. This theory is supported by a recent study that demonstrated New South Wales Department of Corrective Services (reference a significant increase of HCV antibody response to E1/E2 in a group number 05/0884) and the University of New South Wales (reference of exposed uninfected individuals as compared to healthy controls, numbers 05094 and 08081), all located in Sydney, Australia. Written with some of these individuals demonstrating neutralizing activity.12 informed consent was obtained from the participants. Such responses may be virus-neutralizing and hence protective.13-15 In addition, previous cross-sectional studies have documented HCV-specific CD4 and CD8 T cell responses in the absence of anti-HCV 2.3 | Identification of HESN subjects or viraemia, in a range of high-risk groups who remain uninfected,7,16-19 potentially indicating that they were either previously infected with The phenotypic definition for HESN was based on an algorithm used to HCV, or had cross-reactive cellular immunity. HESN individuals have model risk factors for coronary artery disease,26 and HIV infection.27 We also been shown to display raised levels of natural killer (NK) cell activ- have previously described the calculation of composite risk scores based ity, as well as enhanced cytokine production with IL-6, IL-8 and TNF-α, on both logistic and Cox regression multivariate analyses of predictors of compared to individuals infected with HCV.20,21 These HESN individu- incident HCV infection in high-risk IDU prisoners enrolled in the HITS-p als may thus represent a phenotype with immune-mediated resistance cohort.28 In brief, significant risk factors from univariate analyses (P ≤ .1), to establishment of infection, and so may guide vaccine design. and all risk factors previously associated with incident infection, were In this study, an improved framework for reliable identification included in a multivariate analysis. Hazard ratios derived from time-de- of HESN individuals was used with risk behaviour and incident pendent Cox regression for predictors of incident HCV infection during infection data sets from a prospective cohort of uninfected high- follow-up were used to calculate the composite risk scores. Odds and risk PWID. An extreme phenotype of individuals, who were likely hazard ratios were summed and then normalized to a range of 0-1.0 to be highly exposed over a prolonged period via risk behaviour to create a composite risk score for each individual at each time point. but remained uninfected (ie HESN cases), and a matched compar- Scores were then divided into tertiles across the cohort to create ‘high- ison group of incident cases were identified. Stored samples were risk’, ‘medium-risk’ and ‘low-risk’ scoring bands.29 This enabled the iden- assayed for anti-HCV E activity in ELISAs, virus neutralization, and tification of HESN as high-risk individuals who remained consistently HCV E–specific B-cell responses. This study aimed to determine the uninfected, despite ongoing high-risk behaviours. HESN subjects were presence of functional anti-HCV E responses as a correlate of poten- defined as having high composite risk indices for the observation periods tially protective immunity against HCV infection. of: ‘lifetime’, ‘pre-imprisonment’ and/or ‘since imprisonment’ risk behav- iour, along with high-risk scores for at least two follow-up time points during which they remained uninfected. Subjects were excluded if they 2 | MATERIALS AND METHODS had a low-risk score at any time point. Stored samples from baseline and two follow-up time points (at least 12 months later) were analysed. 2.1 | Study cohort
Longitudinally collected demographic and risk behaviour data as well 2.4 | Control subjects as cryopreserved plasma and peripheral blood mononuclear cells (PBMC) were available from PWID (n = 590) enrolled in the Hepatitis Comparison subjects who became infected during follow-up (termed C Incidence and Transmission Study in prisons (HITS-p), based in here ‘Incident’) were matched as a group by age, sex and risk be- correctional centres in New South Wales, Australia.22,23 Recently haviours to HESN subjects. Stored samples from the latest available imprisoned subjects who had HCV antibody-negative test results pre-infection time point of those who became infected (termed here
49 MINA et al. | 3
‘pre-infection’) and a follow-up time point approximately six months IgA (Sigma-Alrich) conjugated with HRP was added. For purified after incident infection (‘post-infection’) and before antiviral treat- IgG, secondary antibodies including mouse anti-human IgG1 (Clone ment was offered were studied. HP6001; SouthernBiotech), IgG2 (Clone 31-7-4; SouthernBiotech), In addition, negative control samples for assay standardization IgG3 (Clone HP6050; SouthernBiotech) and IgG4 (Clone HP6025; were obtained from healthy subjects (n = 8) who were HCV negative, SouthernBiotech) all conjugated with horseradish peroxidase (HRP) had no history of IDU, or any other risk factors associated with HCV were added. The detection of antibodies bound to E1/E2 was done infection. through the addition of 3, 3', 5, 5'-tetramethylbenzidine (TMB, Thermo Fisher Scientific) for 15 minutes. The reaction was then stopped by the addition of 1 mol/L hydrochloric acid (HCl) and 2.5 | HCVpp production and collection of E1/ measured at 450 nm using a CLARIOstar microplate reader (BMG E2 lysate Labtech). Positive binding was determined by an optical density value greater than the mean plus three standard deviations of the Two HCV E1/E2 expression plasmids representing HCV gen- values obtained in the eight healthy control samples. Binding values otypes (gt)-1 (H77, GenBank accession AF011751) and gt3 are represented as the optical density/ background cut-off, where a (UKN3A13.6, AY894683) were used to generate HCV pseudo- value above 1.0 was considered positive. particles (HCVpps) and were kind gifts from Professor Jonathan Ball (University of Nottingham, UK). The luciferase-encoding re- porter plasmid (pTG126) and the murine leukaemia virus (MLV) 2.7 | Recombinant genotype 1a E2 (rE2) gag/pol-encoding packaging construct (phCMV-5349) were binding ELISA kindly provided by Prof. Francois-Loic Cosset (University of Lyon, France). Recombinant E2 (rE2) was prepared as described previously.33 In Retroviral HCVpp was prepared as described elsewhere30,31 brief, constructs containing the H77 rE2 (gt1a) were expressed in based on the protocols developed by Bartosch et al.9 In brief, HCVpp Freestyle-HEK293 cells (Thermo Fisher Scientific). The culture su- was generated by co-transfecting pTG126, phCMV-5349 and an pernatant was harvested 48 hours later and rE2 was captured and HCV E1/E2 clone into HEK293T cells seeded the night before using concentrated using His-Trap (GE Healthcare). a mammalian Calphos transfection kit (Macherey-Nagel). Following a For rE2 binding ELISAs, 96-well Maxisorp microtitre plates were 72 hours incubation, the supernatant containing the HCVpp was col- directly coated with purified rE2 from H77 (gt1). The plates were lected, the infectivity tested and titrated to standardize the HCVpp then blocked with a solution of 5% nonfat dry milk diluted in TBS- to be 10- to 20-fold more infectious than mock pseudoparticles lack- T. Purified IgG was then analysed for binding to rE2 in the same ing HCV E1/E2. The HEK293T cells were collected and lysed using manner as the assay using GNA-lectin captured E1/E2, as described a radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher above. Similarly, positive binding was determined by an optical den- Scientific) containing a cocktail protease inhibitor (Sigma-Alrich) di- sity value greater than the mean plus 3 standard deviations of the luted at a ratio of 1:100. After a 30 minutes incubation at 4°C, the values obtained in the 8 healthy control samples. Binding values cells were spun at 13 000 rpm for 10 minutes and the supernatant are represented as the optical density/ background cut-off, where a containing the E1/E2 collected. value above 1.0 was considered positive.
2.6 | Capture E1/E2 enzyme-linked immunosorbent 2.8 | HCVpp neutralization assay assays (ELISAs) Neutralization assays were performed as previously described.34 Galanthus nivalis (GNA; Sigma-Alrich) capture ELISAs were per- In brief, titrated HCV pseudoparticles (HCVpp) were incubated formed as described previously.32 In brief, 96-well Nunc Maxisorp for 1 hour with heat-inactivated plasma at a 1:20 dilution before microtitre plates (Thermo Fisher Scientific) were prepared by being added to Huh7.5 cells (Apath) seeded the night before. After coating each well with 500 ng of GNA lectin (Sigma-Alrich), fol- 72 hours, the cells were lysed with a lysis buffer (Promega), Bright- lowed by blocking of the wells with a solution of 5% nonfat dry Glo reagent (Promega) was added and luminescence was measured milk in Tris-buffered saline with Tween (TBS-T; 20 mmol/L Tris- on a CLARIOstar microplate reader (BMG Labtech). Percentage
HCl, pH 7.5 150 mmol/L NaCl, 0.1% Tween 20). E1/E2 lysates neutralization was calculated as (1- RLU test plasma/ RLU healthy control) from H77 (Gt1) and UKN3A13.6 (3A) were then captured onto x 100. A human-derived monoclonal Ab, HC84.27, kindly provided the lectin-coated plates. Plasma or IgG, purified from HESN indi- by Professor Steven Foung (Stanford University, USA) was used as a viduals, Incident and healthy control samples using Protein G Mag positive control for neutralization experiments. Neutralization was Sepharose Xtra (GE Healthcare), was then added to lectin bound also tested against vesicular stomatitis virus (VSV)pp to demonstrate E1/E2. For plasma, goat anti-human IgG (Sigma), IgM (Sigma) and neutralization specificity.
50 4 | MINA et al.
2.9 | Analysis of HCV-specific B cells 3.2 | Reactivity of HESN and incident plasma to anti-E ELISAs Analysis of HCV-specific B cells in HESN and Incident subjects was undertaken as previously described.33 In brief, cryopreserved In order to determine whether individuals in the HESN and Incident PBMCs were thawed for flow cytometric staining with HCV E2–spe- groups had anti-E antibodies (Abs), anti-IgG, anti-IgM and anti-IgA cific B cells detected using a rE2 cocktail tetramer with both gt1a ELISAs were performed on all plasma using gt1a and gt3a GNA- (H77) and gt3a (UKN3A13.6) proteins, as well as anti-CD10, anti- captured E1/E2, as well as purified gt1a rE2. ELISAs were initially CD20, anti-CD38, anti-IgD, anti-CD19, anti-CD27 and anti-IgG anti- performed on plasma from 8 healthy individuals to determine a bodies. Flow cytometry data were analysed using FlowJo version 10 threshold for positivity (mean + 3 SD). For IgG, the OD450 thresh- software (Tree Star Inc). The threshold for detection of HCV E2–spe- olds were 0.134, 0.175 and 0.129, for IgM the thresholds were 0.445, cific B cells was established from the mean plus three standard de- 0.256 and 0.608, and for IgA the thresholds were 0.229, 0.908 and viations of the frequencies observed in samples from the 8 healthy 1.25 for rE2, gt1a E1/E2 and gt3a E1/E2, respectively. Of the 22 control subjects. HESN subjects, 10 (45%) were found to have significant anti-E IgG binding to at least one of the E proteins, including in the first analysis time point, and typically with positive responses in at least two of 2.10 | Statistical analysis the three time points tested (Table S2 for details of the response pat- terns over time in these 10 subjects). By contrast, in the pre-infec- Statistical analysis was conducted using GraphPad Prism 8 tion samples of the Incident comparison group only one subject had Software (GraphPad Software) and SPSS v. 19.09 (IBM). Fisher's a positive binding signal for anti-rE2 IgG (P = .0039, Fisher's exact exact test was used to compare categorical data. Wilcoxon's rank- test). There was no significant difference in the median OD/cut-off sum test and Mann-Whitney tests were used to evaluate statisti- values for the positive responses in the two groups at this time point cally significant differences between paired and unpaired groups, (Figure 2). As expected, after incident infection, samples from sub- respectively. Statistical significance was defined as a P-value less jects in the Incident group had significantly higher IgG binding levels than .05. for all three E proteins tested, when compared to both their pre- infection time point (P < .001 Wilcoxon's t tests) and values obtained in HESN subjects (P < .01, Mann-Whitney t tests Figure 1). 3 | RESULTS For anti-E IgM, 15/22 HESN subjects (68%) and 17 (77%) of pre-infection samples of the Incident group had positive responses 3.1 | Subjects for at least one of the E proteins tested (P = .736, Fisher's exact test). No significant differences were observed in the frequency of Of the 590 subjects enrolled in the HITS-p cohort, 22 were identi- positive binding against one or more of the three antigens between fied as HESN individuals, as well as 22 Incident comparison sub- HESN time points (P > .05, Fisher's exact test). Similarly, no signifi- jects who were initially uninfected but developed incident HCV cant differences were observed in the frequency of positive binding infection, who were matched as a group on demographic and between pre- and post-infection time points in Incident subjects risk behaviour characteristics (Table S1). There were no signifi- (P > .05, Fisher's exact test). The OD/cut-off values were comparable cant differences in the groupwise comparisons for the key vari- between HESN time points (P > .05, Wilcoxon's t tests), and between ables. There were 15 males in the HESN group (68%) and 18 in each HESN time point and the pre-infection time point of Incident the Incident group (81%). Of the HESN cases, 5 (23%) were of subjects (P > .05, Mann-Whitney t tests) no significant differences Aboriginal or Torres Strait Islander descent, in comparison with were observed for binding to any E protein (Figure 2). Incident sub- 7 (32%) in the Incident group. The overall risk behaviour status jects at the post-infection time point had significantly higher anti-E was comparable with 17 (77%) HESN cases and 15 (68%) Incident IgM titres in gt1a and gt3a E1/E2 when compared to HESN subjects cases who reported recent injecting of heroin, and 11 (50%) and (P < .05, Mann-Whitney t tests Figure 2B,C). However, this was not 10 (45%) who had injected diverted methadone or buprenorphine significant for the rE2 (P > .05, Mann-Whitney t tests, Figure 2A). in the HESN and Incident groups, respectively. The mean period Further, Incident subjects at the post-infection time point had sig- of imprisonment at the first sampling point for this analysis was nificantly higher anti-E IgM titres in rE2 and gt1a E1/E2 when com- 19.8 and 38.8 months in the HESN and Incident groups respec- pared to pre-infection (P < .01, Wilcoxon's t test, Figure 2A,B). There tively. The interval to the next follow-up sampling point was 6.3 was no significant difference in the gt3a E1/E2. and 4.8 months after the initial sampling time point the HESN and For anti-E IgA, 7/22 HESN subjects (27%) and 3/22 (14%) pre-in- Incident groups, respectively (representing the ‘post-incident’ in- fection samples of the Incident group had positive responses for fection time point in the Incident group). The genotype of the inci- at least one of the E proteins tested (P = .280, Fisher's exact test). dent infections, included 8 (36%) with gt1a, 9 (41%) with gt3a and For the final time point in HESN subjects, the frequency of positive 5 (23%) had an unknown genotype due to low viral load (<100 IU/ binding was significantly higher in gt1a E1/E2 (P = .0211, Fisher's mL). exact test) and gt3a E1/E2 (P = .0459, Fisher's exact test). No other
51 MINA et al. | 5
rE2 IgG FIGURE 1 IgG binding against rE2 (gt1a, A) and E1/E2 (gt1a, B; gt3a, C) in the plasma of HESN cases (grey) at three separate time (A) **** points, and in pre-infection and post-infection plasma from Incident 25 control subjects (white). The dotted line represents the threshold for positivity as determined by the mean plus 3 standard deviations 20 of binding in healthy control samples. Comparisons between HESN 15 cases and Incident control subjects were done using a Mann- Whitney t test. Comparisons between time points were done 10 using the Wilcoxon t test. (*P < .05, **P < .01, *** P < .001 and 5 ****P < .0001) 2.0
1.5 differences were observed in the frequency of positive binding be- 1.0 tween HESN subjects, between Incident time points or between
0.5 HESN subjects and Incident subjects (P > .05, Fisher's exact tests). When the OD/cut-off values were compared between each HESN 0.0 time point, the second and third time points had significantly higher anti-E IgA titres when compared to the first time point when com- Gt1a E1/E2 pared in binding to the gt3a E1/E2 (P < .0001, Wilcoxon's t tests, (B) **** Figure 3). No other differences were observed between HESN time 25 points for the other antigens. When each HESN time point was com- ) 20 pared to the pre-infection time point in Incident subjects, the third HESN time point had greater IgA binding to the gt1a E1/E2 and gt3a E1/ 15 Incident E2 (P < .05, Mann-Whitney t tests, Figure 3B,C). No significant dif-
/cut-off 10
0 ferences were observed in IgA binding against rE2 (Figure 3A). Post-
45 5 infection, Incident subjects had significantly higher anti-E IgA titres 2.0
OD only in the rE2 antigen (P < .01, Mann-Whitney t tests Figure 3A), 1.5 but not for the other antigens (P > .05, Mann-Whitney t tests, g(
1.0 Figure 3B,3C). When compared to the pre-infection time point, the post-infection time point in Incident subjects had significantly higher 0.5 Bindin anti-E IgA responses against rE2 and gt3a E1/E2 (P < .05, Wilcoxon's 0.0 t tests, Figure 3A,C) but not the gt1a E1/E2 (P > .05, Wilcoxon's t test Figure 3B). Gt3a E1/E2 ** 3.3 | Isotyping IgG-positive HESN subjects (C) *** 10 For each of the sampling time points from the 10 HESN subjects where positive anti-E IgG binding to one or more E proteins was 8 demonstrated (n = 14), IgG was purified from the plasma, and IgG 6 subclass isotyping of the response was performed on a single time
4 point. This time point was selected based on the best titre across all time points and likely cross-reactivity (positive for more than one
2.0 ELISA). The threshold for positivity (mean + 3 SD) for each subclass was established on plasma from eight healthy individuals. For IgG1, 1.5 the thresholds were 0.051, 0.240 and 0.417; for IgG2, the thresholds 1.0 were 0.054, 0.107 and 0.126, for IgG3 the thresholds were 0.066, 0.5 0.286 and 0.505; and for IgG4, the thresholds were 0.100, 0.226 and
0.0 0.260 for rE2, gt1a E1/E2 and gt3a E1/E2, respectively. n 1 2 3 Interestingly, for all HESN samples which were IgG positive for on io ti either gt1a or gt3a E1/E2 (n = 7), only IgG2 subclass binding was de- ec Timepoints tected above threshold (Figure 4). Similarly, in samples IgG positive nf -i e-infect
st for rE2 binding, 8 of 10 were positive for IgG2. The binding levels Pr
Po (OD/cut-off) were only modestly above the healthy control thresh- old. In addition, four subjects had one or two samples which had
52 6 | MINA et al.
rE2 FIGURE 2 IgM binding against rE2 (gt1a, A) and E1/E2 (gt1a, (A) B; gt3a, C) in the plasma of HESN cases (grey) at three separate 6 ** time points, and in pre-infection and post-infection plasma from Incident control subjects (white). The dotted line represents the 5 threshold for positivity as determined by the mean plus 3 standard deviations of binding in healthy control samples. Comparisons between HESN cases and Incident control subjects was done using 4 a Mann-Whitney t test. Comparisons between time points were done using the Wilcoxon t test. (*P < .05, **P < .01, *** P < .001 and 3 ****P < .0001)
2 binding above threshold against rE2 utilizing IgG3, and five subjects had binding above threshold for IgG4. These IgG subclass anti-E 1 binding patterns are summarized in Table S3.
0 3.4 | Neutralization of HCVpp Gt1a E1/E2 (B) 8 The samples from the 10 IgG-positive HESN subjects were also tested HESN Incident for neutralization against the HCVpp containing E1/E2 of H77 (gt1a)
) 7 **
ff or UKN3A13.6 (gt3a). These samples were compared to the neutrali-
-o 6 zation capacity of plasma from Incident subjects at the pre-infection ut time point (n = 22)—of which only one sample demonstrated IgG bind- /c 0 5 ing against rE2 (see above). As shown in Figure 5, varied but generally 45
D low-level neutralization to both HCVpps was observed. The neutrali- 4 (O zation activity was significantly higher in the HESN samples for both
ng 3 HCVpp tested (P < .001, Mann-Whitney t tests). ndi
Bi 2
1 3.5 | E2-specific B cells in HESN subjects
0 To further characterize the HESN phenotype, subjects that tested positive for anti-Envelope IgG and had stored PBMCs available Gt3a E1/E2 (C) (n = 8) were analysed for E2-specific B cells in flow cytometry in * comparison with samples from the pre-infection time point of the 2.5 Incident control group (n = 7).33 The number of subjects with detect- able cells and the mean frequencies of HCV E2-specific memory B cells (events/106 PBMCs) were compared. The prevalence of detect- 2.0 able HCV E2–specific B cells 5/8 (63%) was higher than in the pre- infection time point of Incident group subjects (2/7, 29%) (Figure 6). 1.5 There was no significant difference in the mean frequencies be- tween these two groups (P = .1206).
1.0
4 | DISCUSSION 0.5 Previous studies of the immunological correlates of the HCV HESN status have been cross-sectional in design with retrospective recall 0.0 1 2 3 n of risk behaviour to characterize the probability of exposure and on io t ti hence the phenotype.7,20,21,35,36 These studies have identified HCV-
Timepoints ec specific T-cell responses, natural killer (NK) cell activation and anti-E nf -i
e responses in a significant minority of the cases. This study extends st-i nfec Pr Po these previous findings with an improved designation of the HESN
53 MINA et al. | 7
(A) rE2 FIGURE 3 IgA binding against rE2 (gt1a, A) and E1/E2 (gt1a, B; and gt3a, C) in the plasma of HESN cases (grey) at three separate 10 ** time points, and in pre-infection and post-infection plasma from Incident control subjects (white). The dotted line represents the 8 threshold for positivity as determined by the mean plus 3 standard 6 deviations of binding in healthy control samples. Comparisons between HESN cases and Incident control subjects was done using 4 a Mann-Whitney t test. Comparisons between time points were done using the Wilcoxon t test. (*P < .05, **P < .01, *** P < .001 and 2.0 ****P < .0001)
1.5 case and incident control phenotypes from analysis of prospectively 1.0 collected risk behaviour and infection status, along with further de- 0.5 lineation of anti-E as being in the IgG2 subclass, as well as neutrali- 0.0 zation activity and HCV E–specific memory B cells. These findings suggest that HESN subjects are resistant to HCV infection through Gt1a E1/E2 humoral immune-mediated mechanisms. (B) Over recent years mounting evidence has shown that early ** induction of HCV E–specific antibodies, particularly neutral- 2.0 HESN Incident izing antibodies, plays an important role in controlling primary 14,15,34,37,38
) HCV infection. However, neutralizing antibodies 1.5 are also commonly found in chronic HCV infections, and even when found in those who clear primary infection they do not 14,39 /cut-off necessarily protect from reinfection. In our recent study of
45 0 1.0 subjects with early primary HCV infection, all subjects had de- tectable E-specific IgG responses, but clearance of the infection OD was associated with the early development of IgG1 and IgG3 g( 0.5 isotype responses,40 along with autologous transmitted-founder virus-specific neutralization,38 highlighting the link between
Bindin the timing and IgG subclass response elicited and infection out- 0.0 come. In the present study, a significant minority of the carefully identified HESN cases had repeatedly detectable IgG responses Gt3a E1/E2 against gt1 or gt3 E (which constitute approximately 90% of inci- **** dent and prevalent genotypes in Australia).41,42 These responses (C) were almost exclusively in the IgG2 subclass. This finding is note- * worthy as IgG2 responses are rare in primary HCV infection,40 2.0 * but do have unique functional characteristics of potential rele- vance to protective immunity. IgG2 antibodies are recognized to bind glycans independent of T-cell help (typically targeting bac- 1.5 terial polysaccharides, but also viral glycans).43,44 In addition, this isotype binds to the neonatal Fc receptor (FcRn) which is widely expressed on epithelial cells in adult life, including hepatocytes, 1.0 and is recognized in this context to facilitate mucosal transport of immune complexes for antigen sampling45 and phagocytosis.46
0.5 A negative in vitro investigation of the role of FcRn neutraliza- tion of HCV in hepatocytes has been reported, but this inves- tigation only included an IgG1 monoclonal antibody (AR3) and 47 0.0 serum from a patient with chronic HCV. Further exploration of
1 2 3 the potential role IgG2 anti-HCV E antibodies in neutralization on of hepatocyte uptake and survival of HCV virions is warranted. Timepoints ecti nfection nf Given the cross-reactivity commonly associated with IgM Abs, it -i
re is perhaps unsurprising that a large number of subjects were positive P
Post-i for anti-E IgM in both HESN and pre-infection Incident individuals.
54 8 | MINA et al.
(A) IgG1 (B) IgG2 FIGURE 4 IgG1 (A), IgG2 (B), IgG3 (C) and IgG4 (D) isotyping against rE2 (gt1a, 1.0 1.5 black) and E1/E2 (gt1a, grey; gt3a, white) 0.8 in the plasma of HESN cases (n = 10) 1.0 positive for IgG binding. The dotted line 0.6 represents the threshold for positivity as 0.4 determined by the mean plus 3 standard 0.5 deviations of binding for the isotype in 0.2 healthy control samples ) 0.0 0.0
009 078 356 362 382 432 452 481 4016 4017 /cut-off 009 078 356 362 382 432 452 481 4016 4017
45 0 (C) IgG3 (D) IgG4
OD 2.5 1.5 g( 2.0 1.0
Bindin 1.5
1.0 0.5
0.5
0.0 0.0
009 078 356 362 382 432 452 481 009 078 356 362 382 432 452 481 4016 4017 4016 4017 SubjectID
rE2 Gt1aE1/E2 Gt3a E1/E2
Surprisingly, however, anti-E IgA was significantly associated with during primary HCV infection, very few subjects had detectable HESN individuals when compared to the pre-infection time point of anti-HCV E IgA responses and there was no association with clear- incident individuals. In our previous report of serological responses ance.40 Given the recognized role of polymeric IgA (pIgA) in muco- sal protection, and the previous analysis of incident case samples H77 from this cohort showing elevated serum levels of pIgA,48 it may be 100 HESN of interest to further characterize these IgA responses. Consistent with the previous report,12 virus neutralization activ- 75 Pre-infection ity was found in the plasma of the HESN cases and (with a single exception) absent from the pre-infection samples of the Incident 50 *** control group. Given that transmitted-founder virus-specific neu- tralizing antibodies are recognized to be an important contributor to 38 n 25 clearance in primary infection, it is possible that HESN cases have io broader neutralization activity that offers the potential for repeated at clearance of diverse viral strains. Further assessment of the breadth
is 0 of the neutralization activity in a wider panel of HCVpp, and in the UKN3A13.6 HCVcc system, is warranted.
eutral 100 Finally, the finding of HCV E–specific memory B cells in a sub- set of HESN cases is both novel and significant, as this further sup- %N 75 **** ports the likelihood of prior HCV exposure and the potential for antibody-mediated protection. This finding opens the potential for 50 further characterization of the antibody repertoire, specificities and functional activities of these HCV-specific B cells in HESN via single 25 cell sorting and monoclonal antibody synthesis.
0 FIGURE 5 Neutralizing activity (%) of plasma from HESN cases
n (grey) positive for IgG and pre-infection time point of Incident control subjects (white) against HCVpp containing Envelopes of ctio HESN H77 (gt1a, A) and UKN3A13.6 (gt3a, B). Comparisons between time points were done using the Wilcoxon t test. (*P < .05, **P < .01, *** e-infe
Pr P < .001 and ****P < .0001)
55 MINA et al. | 9
2. Mehta SH, Astemborski J, Kirk GD, et al. Changes in blood- 0.10 borne infection risk among injection drug users. J Infect Dis. 2011;203:587-594. 3. Kim DY, Ahn SH, Han KH. Emerging therapies for hepatitis C. Gut 0.08 Liv. 2014;8:471-479. 4. Hagan LM, Schinazi RF. Best strategies for global HCV eradication. Liver Int. 2013;33(Suppl 1):68-79. 5. Mina MM, Luciani F, Cameron B, et al. Resistance to hepatitis C 0.06 virus: potential genetic and immunological determinants. Lancet Infect Dis. 2015;15:451-460. 6. Thomas DL, Vlahov D, Solomon L, et al. Correlates of hepatitis C 0.04 virus infections among injection drug users. Medicine (Baltimore). 1995;74:212-220. 7. Thurairajah PH, Hegazy D, Chokshi S, et al. Hepatitis C virus (HCV)– 0.02 specific T cell responses in injection drug users with apparent resis- tance to HCV infection. J Infect Dis. 2008;198:1749-1755. 8. Grebely J, Prins M, Hellard M, et al. International Collaboration of Incident HIV, Hepatitis CiIC. Hepatitis C virus clearance, reinfec- 0.00 tion, and persistence, with insights from studies of injecting drug AB HCV-specific of Bcells (% CD19+ CD20+ CD10-IgD-) users: towards a vaccine. Lancet Infect Dis. 2012;12:408-414. 9. Bartosch B, Dubuisson J, Cosset FL. Infectious hepatitis C virus FIGURE 6 Frequency of HCV-specific B cells (% of total B-cell pseudo-particles containing functional E1–E2 envelope protein population; CD19+CD20 + CD10-IgD-) in the PBMCs of HESN complexes. J Exp Med. 2003;197:633-642. (A) and the pre-infection time point of Incident control subjects 10. Major ME, Dahari H, Mihalik K, et al. Hepatitis C virus kinetics and (B). The dotted line represents the threshold of detection as host responses associated with disease and outcome of infection in determined by the mean plus 3 standard deviations of detection in chimpanzees. Hepatology. 2004;39:1709-1720. healthy control samples 11. Thimme R, Oldach D, Chang KM, Steiger C, Ray SC, Chisari FV. Determinants of viral clearance and persistence during acute hepa- titis C virus infection. J Exp Med. 2001;194:1395-1406. In combination, these further studies provide new insights into 12. Swann RE, Mandalou P, Robinson MW, et al. Anti-envelope anti- the humoral immune correlates of potentially protective immunity body responses in individuals at high risk of hepatitis C virus who against HCV. resist infection. J Viral Hepat. 2016;23:873-880. 13. Bartosch B, Cosset FL. Cell entry of hepatitis C virus. Virology. 2006;348:1-12. ACKNOWLEDGEMENTS 14. Osburn WO, Fisher BE, Dowd KA, et al. Spontaneous control of We would like to thank the HITS-p investigators including Kate primary hepatitis C virus infection and immunity against persistent Dolan, Paul Haber, William Rawlinson, Carla Treloar, Greg Dore, reinfection. Gastroenterology. 2010;138:315-324. Lisa Maher, and Andrew Lloyd and Fabio Luciani. We would like 15. Raghuraman S, Park H, Osburn WO, Winkelstein E, Edlin BR, Rehermann B. Spontaneous clearance of chronic hepatitis C virus to thank Francois-Loic Cosset for the MLV plasmids, Jonathan infection is associated with appearance of neutralizing antibodies Ball and Alexander Tarr for HCV envelope encoding plasmids, and reversal of T-cell exhaustion. J Infect Dis. 2012;205:763-771. and Steven Foung, Mansun Law and Richard Urbanowicz for as- 16. Al-Sherbiny M, Osman A, Mohamed N, et al. Exposure to hepatitis sistance with the HCVpp assay and provision of monoclonal an- C virus induces cellular immune responses without detectable vire- mia or seroconversion. Am J Trop Med Hyg. 2005;73:44-49. tibodies. Research support for the HITS-p cohort included grants 17. Della Bella S, Riva A, Tanzi E, et al. Hepatitis C virus-specific re- from National Health and Medical Research Council of Australia activity of CD4+-lymphocytes in children born from HCV-infected (NHMRC)—Project Nos. 222887 and 1146082, Partnership No. women. J Hepatol. 2005;43:394-402. (eng). 1016351, and Program Nos. 510488 and 1053206. AAE, RAB 18. Kamal SM, Amin A, Madwar M, et al. Cellular immune responses in seronegative sexual contacts of acute hepatitis C patients. J Virol. and ARL are supported by NHMRC Research Fellowships (Nos: 2004;78:12252-12258. 1130128, 1080001 and 1084706). 19. Koziel MJ, Wong DK, Dudley D, Houghton M, Walker BD. Hepatitis C virus-specific cytolytic T lymphocyte and T helper cell responses CONFLICT OF INTERESTS in seronegative persons. J Infect Dis. 1997;176:859-866. 20. Golden-Mason L, Cox AL, Randall JA, Cheng L, Rosen HR. Increased The authors declare that there is no conflict of interest regarding the natural killer cell cytotoxicity and NKp30 expression protects publication of this article. against hepatitis C virus infection in high-risk individuals and inhib- its replication in vitro. Hepatology. 2010;52:1581-1589. ORCID 21. Warshow UM, Riva A, Hegazy D, et al. Cytokine profiles in high Michael Mina https://orcid.org/0000-0001-8644-5702 risk injection drug users suggests innate as opposed to adaptive im- munity in apparent resistance to hepatitis C virus infection. J Viral Hepat. 2012;19:501-508. REFERENCES 22. Cunningham EB, Hajarizadeh B, Amin J, et al. Investigators HI-p. 1. Polaris Observatory HCVC. Global prevalence and genotype dis- Longitudinal injecting risk behaviours among people with a history tribution of hepatitis C virus infection in 2015: a modelling study. of injecting drug use in an Australian prison setting: The HITS-p Lancet Gastroenterol Hepatol. 2017;2:161-176. study. Int J Drug Policy. 2018;54:18-25.
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Freeman AJ, Ffrench RA, Post JJ, et al. Prevalence of production How to cite this article: Mina M, Underwood A, Eltahla A, of virus-specific interferon-gamma among seronegative hepati- et al. Anti-envelope antibody responses in highly exposed tis C-resistant subjects reporting injection drug use. J Infect Dis. seronegative individuals may be associated with protection 2004;190:1093-1097. (eng). 37. Pestka JM, Zeisel MB, Blaser E, et al. Rapid induction of virus-neu- from HCV infection. J Viral Hepat. 2020;00:1–10. https://doi. tralizing antibodies and viral clearance in a single-source outbreak org/10.1111/jvh.13339 of hepatitis C. Proc Natl Acad Sci U S A. 2007;104:6025-6030.
57 Supplementary Table 1: Demographic characteristics of HESN and Incident subject groups at baseline
HESNa Incident Variable (n=22) (n=22) P value Mean age in years (SDb) 26.7 (4.8) 24.1 (4.2) 0.57 Male n (%) 15 (68) 18 (81) 0.16 ATSIc n (%) 5 (23) 7 (32) 0.50 Mean no. times in prison (SD) 5 (1.7) 3.4 (3.5) 0.48 Mean time inside prison months (SD) 19.8 (16.6) 38.8 (37.1) 0.19 Previous juvenile detention n (%) 9 (41) 12 (55) 0.38 Mean number of tattoos (SD) 3.8 (5.1) 2.7 (2.7) 0.18 Ever inject heroin n (%) 17 (77) 15 (68) 0.48 Ever inject buprenorphine/methadone n (%) 11 (50) 10 (45) 0.49 Duration of injecting years (SD) 8.2 (3.4) 6.8 (4.7) 0.26 a HESN: highly-exposed seronegative b SD: standard deviation c ATSI: Aboriginal and Torres Strait Islander
58 Supplementary Table 2. Anti-HCV Envelope antibody positivity in HESN subjects
IgG IgM IgA Subject b Time Gt1a Gt3a Gt1a Gt3a Gt1a Gt3a a ID point rE2 E1/E2 E1/E2 rE2 E1/E2 E1/E2 rE2 E1/E2 E1/E2 1 + - + ------2 - - - + + - - + + 300009 3 - - + ------1 + ------2 - - + - - + - - - 300078 3 ------1 - - - - + - - - - 2 - - - + + - - - - 300356 3 + - + - + - - - - 1 ------+ + 2 ------300362 3 + ------1 - - - - + - - - - 2 + - - + + + - + + 300382 3 - - - + + - - + + 1 + + + - + - - - - 2 + + + ------300432 3 + - + - + - - - - 1 ------2 + ------300452 3 - - - - + - - - - 1 + ------2 - - - + - - - - - 300481 3 ------1 + - - - - - + + + 2 ------+ - - 400016 3 ------+ + + 1 ------+ - - 2 + - - + - - + - - 400017 3 ------a ID: Identifier b Gt: Genotype
59 Supplementary Table 3. IgG subclasses in HESN subjects positive for IgG against HCV
Envelope
Gt1a rE2 Subject Time ID point IgG1 IgG2 IgG3 IgG4 300009 1 - + - - 300078 1 - - - - 300356 3 - + - - 300362 3 - - + - 300382 2 - + - - 300432 2 - + - - 300452 2 - + - - 300481 1 - - + - 400016 1 - + + - 400017 2 - + + - Gt1a E1/E2 Subject Time ID point IgG1 IgG2 IgG3 IgG4 300432 2 - + - -
Gt3a E1/E2 Subject Time ID point IgG1 IgG2 IgG3 IgG4 300009 1 - + - - 300078 2 - + - - 300356 3 - + - - 300362 3 - + - - 300382 2 - + - - 300432 2 - + - - 300452 2 - + - - 300481 1 - + - - 400016 1 - + - -
60 Chapter 4
Polymorphisms in DOCK2 are associated with protection against hepatitis C
infection
61 Polymorphisms in DOCK2 are associated with protection against hepatitis C infection
Michael M. Mina1, Hui Li1, Auda Eltahla1, Fabio Luciani1, Louisa Degenhardt2, William D.
Rawlinson3, Nicholas G Martin4, Paul Jeffries5, Elliot C. Nelson5, David Booth6, Andrew R.
Lloyd1
1 The Kirby Institute, University of New South Wales, Sydney, Australia, 2052
2 National Drug and Alcohol Research Centre, University of New South Wales, Sydney,
Australia, 2052
3 Serology, Virology and OTDS Laboratories, NSW Health Pathology Randwick, Australia
2031
4 QIMR Berghofer Medical Research Institute, Herston, Australia, 4006
5 Department of Psychiatry, Washington University School of Medicine, Missouri, United
States, 63110
6 The Westmead Institute for Medical Research, Westmead, Australia, 2145
Correspondence to:
Dr Michael M. Mina,
Viral Immunology Systems Program, The Kirby Institute, University of New South Wales,
Sydney NSW 2052
Australia
Email: [email protected]
Ph: +612 9385 2534
Fax: +612 9385 1389
Abbreviations:
CATS - Comorbidity and Trauma Study
1 62 DAA – directly acting antivirals
DOCK2 - dedicator of cytokinesis 2
GWAS - Genome-wide association study
HCV – hepatitis C virus
HESN – highly exposed seronegative
IDU – injecting drug use
IDUs – injecting drug users
HITS-p – Hepatitis C Incidence and Transmission Study
OST - opioid substitution therapy
SNP - single nucleotide polymorphisms
χ² – Chi-square tests
2 63 Abstract
Background: Injecting drug use (IDU) is the major risk factor for hepatitis C (HCV) transmission with the prevalence of infection rising progressively in relation to accumulated years of IDU. A small minority of long-term people who inject drugs (PWID) remain seronegative and aviraemic, despite prolonged probable exposure to HCV - termed highly exposed, seronegative (HESN) subjects. Host genetic polymorphisms may confer such resistance.
Methods: Two independent, carefully-selected longitudinal case-control series of subjects who either remained HESN (n=11 +11), or became incident infection cases (n=22+22), were selected from a prospective cohort of high risk, seronegative, prisoners (the HITS-p cohort; n=590) as an initial test and replication dataset. A further replication case-control group (n=28) was then selected from a cross-sectional cohort of PWID with known HCV status (the CATS cohort; n=1459), using retrospective data to designate HESN (or prevalent infection). The case- control groups for each comparison were matched by age, sex, and high-risk behaviour characteristics. DNA was isolated from the HITS-p subjects and analysed using a customised genome-wide single nucleotide polymorphism (SNP) chip. Existing GWAS data was available for the CATS cohort.
Results: Almost 15,000 SNPs were associated at p<0.05 in the HITS-p test dataset in either autosomal recessive or dominant models (logistic regression), of which only two were directly replicated (in the RP11-463J7.2 gene and in the zinc finger protein 827 (ZNF827) gene. A total of six additional SNPs within or near to the human dedicator of cytokinesis-2 (DOCK-2) gene were also associated but not directly replicated. Of these associations, three of the DOCK-2
SNPs were also replicated in the CATS cohort in the same additive, dominant and recessive
3 64 test models. Two common DOCK-2 haplotypes (minor haplotype frequency, MHF >5%), which consisted of replicated SNPs, were associated with HESN status in both HITS-p and
CATS cohorts.
Conclusion: This study provides replicated evidence linking polymorphisms in the immunoregulatory proteins encoded by DOCK-2 to resistance against HCV infection. Further investigation of the biological basis of the DOCK-2 association in protection against HCV infection is warranted.
Key words: hepatitis C, highly exposed seronegative, DOCK2, genetic resistance.
4 65 Introduction
Genetic variation in human populations contributes to the susceptibility to infectious diseases, which is exemplified by the recognition that individuals who are homozygous for a common loss of function variant of the FUT2 gene do not express the H type-1 oligosaccharide ligand needed for Norwalk virus binding, and cannot be infected with this particular Norovirus. (1) In other individuals, genetically-determined relative resistance to the establishment of chronic infection may be conferred, exemplified the CCR5 d-32 mutation and HIV resistance. (2-4)
Despite the advent of highly effective directly acting antivirals (DAAs) for hepatitis C (HCV) infection, (5, 6) this infectious disease remains a major problem for public health programmes worldwide, with an estimated 71 million individuals chronically infected. (7) Primary HCV infection is usually asymptomatic and results in clearance in 25% of individuals. (8) For chronically infected individuals, the persistent viral infection drives sustained hepatic necro- inflammation and progressive fibrosis, culminating in cirrhosis, liver failure and an increased risk of hepatocellular carcinoma. (9) These therapies are very expensive. A better understanding of HCV immune response may indicate new therapeutic approaches.
Injecting drug use (IDU) is the dominant risk factor for HCV transmission in developed countries, with seroprevalence rates in cross-sectional studies of people who inject drugs
(PWID) ranging from 50% to 90%. (10) In these surveys, a small minority of high risk, long- term PWID are seronegative and aviraemic, despite likely repeated exposure to HCV through prolonged periods of sharing of drug injection equipment. (11) Further, several prospective studies of high-risk PWID have reported subgroups that remain seronegative despite documented long-standing risk behaviour, who may represent a phenotype that are resistant to infection. (8, 12) This highly exposed seronegative (HESN) group have also been termed
5 66 ‘exposed uninfected’, indicating the absence of demonstrable infection as defined by highly sensitive diagnostic tests for HCV antibodies and RNA. (13) The basis for resistance to infection in the HESN is unknown. It is likely to be genetic, and the protective genetic variants should indicate immunological processes which may indicate therapeutically useful targets. In this study we sought genetic variants that were over-represented in this group.
HCV encodes very few proteins and therefore depends heavily upon host factors for propagation. (14) RNA interference (RNAi) screens and targeted gene silencing studies have identified multiple cellular genes that reduce viral replication, (15-19) and several studies have examined associations between genetic polymorphisms in critical host proteins and HCV replication in vitro. For instance, three naturally occurring single nucleotide polymorphisms
(SNPs) in the region of the peptidyl-prolyl isomerase A (PPIA) gene encoding cyclophilin A, were shown to abrogate HCV replication in vitro, (20) however, the minor allele frequencies for these SNPs are very low in the population, and have not been identified in HESN. (21) In addition, multiple studies have examined polymorphisms in candidate genes encoding known
HCV entry co-receptors, including Tetraspanin CD81, Claudin-1 (CLDN1), Claudin-6
(CLDN6), Claudin-9 (CLDN9), Occludin (OCLN), Scavenger receptor-B1 (SCARB1), Low density lipoprotein receptor (LDL-R), and Niemann-Pick C1-like 1 (NPC1L1) - with a small number of putative associations with the HESN phenotype found, each awaiting replication.
(22-29) In addition, a study of HIV-infected men-who-have sex-with men (MSM) or PWID who were at high risk of HCV infection but remained uninfected, implicated three promoter region SNPs in DC-SIGN (dendritic cell specific ICAM-grabbing non-integrin, CD209) which has also been found to support viral entry into dendritic cells. (28, 30) The association was found only in MSM, and not in PWID.
6 67 It is also clear that genetic variations in key innate response pathways can facilitate efficient clearance of primary infection, particularly including variations in the interferon lambda (IFN)- l -3 and -4 genes, but also in natural killer (NK) cell receptors. (31-33) Spontaneous clearance has also been linked to human leucocyte antigens (HLA) influencing the characteristics of the
CD4 and CD8 adaptive T cell responses against the virus. (34-37) Several studies of HESN have identified anti-Envelope antibody responses in a minority of subjects (HCV Envelope proteins are not included in diagnostic anti-HCV antibody assays), as well as more prevalent natural killer (NK), and HCV-specific CD4 and CD8 T cell responses – all suggestive of efficient viral clearance without traditional seroconversion as the biological basis of the HESN phenotype. (38, 39) Indeed, an early report from the high risk PWID cohort studied here described four cases of sustained HCV viraemia with ultimate clearance, without seroconversion, but with cellular immune responses against HCV recorded. (40)
Using a repeated test and replication cohort design, highly-selected groups of HESN individuals and matched high risk PWID comparison subjects who became HCV infected were identified, and genetic polymorphisms associated with immune-mediated resistance to HCV infection were initially sought via a custom immunological SNP array in the two case-control groups from the first prospective cohort, before the associations were confirmed in the second replication cohort using an existing genome wide association study (GWAS) dataset.
7 68 Materials and methods
Study cohorts
HESN and matched Infected subjects were identified from HITS-p, and the confirmatory
CATS cohort.
The HITS-p Cohort
Longitudinally collected demographic and risk behaviour data, as well as DNA were available from HCV-uninfected PWID (n =590) enrolled in the Hepatitis C Incidence and Transmission Study in prisons (HITS-p), which was based in correctional centres in New
South Wales, Australia. (41) Subjects were followed at three- to 12-month intervals, to screen for HCV seroconversion and viraemia, and to complete a detailed interview recording risk behaviours at each time point. (42) This study ran for a decade and recorded high rates of ongoing IDU, sharing of injection devices, as well as incident HCV infections. (12, 43)
The CATS Study
The Comorbidity and Trauma (CATS) Study (n=1459) was a cross-sectional study of opiate- dependent PWID subjects recruited from opioid substitution therapy clinics in New South
Wales, Australia investigating genetic association of opiate dependency. (44-46) Genome wide association study (GWAS) data, as well as HCV antibody and viraemic status, were available for all subjects in this study, which was used as a confirmatory dataset for the associations identified in the HITS-p cohort. (47, 48)
Selection of HESN and control subjects
The phenotypic definition for HESN subjects in HITS-p was based on an algorithms previously used to model risk factors for coronary heart disease (49) and HIV infection, (50) as previously described. (51) In brief, the algorithm utilised composite risk scores derived from both logistic
8 69 and Cox regression multivariate analyses of predictors of incident HCV infection in the HITS- p cohort. The hazard ratios for each associated risk factor were used to calculate the composite risk scores, which were summed and then normalized to a range of 0–1.0 for each individual at each follow-uptime point. Scores were divided into tertiles across the cohort to create ‘high’,
‘medium’ and ‘low’ scoring bands. (21) HESN subjects within HITS-p were thus defined as having high composite risk indices for each of the following time windows in the longitudinal dataset: ‘lifetime’, ‘pre-imprisonment’ and/or ‘since imprisonment’, as well as having at least two follow-up time points during which they remained uninfected (Supplementary Table 1).
Potential HESN were excluded if they had a low-risk score at any time point, or were non-
Caucasian. Two separate groups of HESN subjects (n=11 for each) were matched groupwise to Incident control subjects (n=22 for each) by age and gender (all Caucasian).
The HESN subjects from the CATS cohort (n=14) were also selected based on Caucasian ethnicity, and the risk status from retrospectively recorded dataset from within the group who were HCV antibody and RNA negative, but with high risk status designated based on the combination of ‘lifetime’ and ‘recent’ risk behaviors recorded in the cross-sectional survey interview (Supplementary Table 1). ‘Incident’ cases (i.e those confirmed to be HCV infected) from the CATS cohort (n=24) were selected as HCV antibody positive Caucasian subjects, with either chronic or cleared infection, who were matched as a group by age and gender to the
HESN.
SNP genotyping
The HITS-p cohort was genotyped using the multiple sclerosis (MS) Chip, an Illumina
Infinium HD custom array, which was developed by the International MS Genetics
Consortium. The MS Chip content included the entire content of the Illumina exome chip
9 70 which includes ~300,00 SNPs that fine map the exons of known genes in human (as described http://genome.sph.umich.edu/wiki/Exome_Chip_Design#Illumina_Exome_Arrays), with additional custom content including a further ~85,000 genetic variants associated with auto- immune disease, as described previously. (52) Notably also, the major histocompatibility complex (MHC) is densely covered with ~15,000 SNPs and allows for imputation of classical
HLA alleles with all available current methods. The CATS subjects had previously been genotyped at the Johns Hopkins Center for Inherited Disease Research (CIDR) using the
Illumina Human660W-Quad Bead Chip which identifies ~660K genetic variations per sample.
(47)
Statistical analysis
Demographic and risk behaviour data for the HESN cases and Incident control subjects were analysed using two-tailed t tests for continuous variables and Chi-square tests (χ²) for categorical variables. Logistic regression was used to assess the genetic associations of each
SNP considering HESN and Incident as binary phenotypes with age and gender as co-variates.
Definition of linkage disequilibrium block structures and estimation of haplotypes were performed using Haploview (Broad Institute, https://www.broadinstitute.org/haploview/haploview). Analyses were conducted using SPSS,
Prism (SPSS statistics v20, IBM, Armonk, NY, USA; GraphPad Prism v. 6.0c GraphPad
Software, La Jolla, CA) and PLINK (https://zzz.bwh.harvard.edu/plink/). Odd ratios (ORs) and
95% confidence intervals (CI) were determined. A conventional threshold of p value <0.05 was considered to indicate statistical significance.
10 71 Results
HESN cases and Incident control subjects
There were two sets of n=11 HESN and n=22 matched Incident control subjects identified from
HITS-p (total n=66), and n=14 HESN and n=28 Incident control (i.e infected) subjects from the confirmatory CATS cohort, respectively. There were no significant differences in demographic characteristics between the subject groups within each study (Table 1). There were a total of 15 males in the HESN group (68%) and 18 in the Incident group (81%) from
HITS-p, and 11 males in the HESN (79%) and 21 males in the Incident group (75%) from
CATS. Of the HESN cases, 17 (77%) from HITS-p and 12 (86%) from CATS reported recent injecting of heroin; and 11 (50%) and 8 (57%) reported recent injecting of buprenorphine or methadone respectively. The mean duration of IDU was 8.2 years in HESN compared to 6.8 years in the Incident group from HITS-p, and 1.4 years in HESN from CATS, compared to 1.6 years in the Incident group.
Associations in the HITS-p cohort
In the first test HITS-p case-control group, there were 15,000 SNPs associated with the HESN phenotype with a significance threshold of p<0.05. Of these two SNPs were directly replicated with one SNP (rs814346) in the RP11-463J7.2 gene consistently associated with HESN in a recessive model, and one SNP (rs6537384) in the zinc finger protein 827 (ZNF827) gene with a dominant positive association with Incident infection. In addition, a total of six SNPs within or near to the human dedicator of cytokinesis-2 (DOCK-2) gene on chromosome 5 (rs7735341, rs7733443, rs10067968, rs6873253, rs17562153, and rs13190445) were associated with HESN in a recessive model in one case-control group or the other suggesting a consistent association by gene (rather than an association by SNP). To resolve whether the latter finding was the result of limited sample size and hence power, these candidate SNPs associations were re-examined
11 72 with the two case-control groups from HITS-p combined. This analysis revealed 18 SNPs in
DOCK-2, that were significantly different in frequency distribution between HESN and
Incident groups (all p<0.05) (Figure 1). The most significant DOCK-2 SNP (rs12055169) was detected under both recessive and dominant model tests (Table 2, Figure 2). The single RP11-
463J7.2 and ZNF827 (rs6537384) SNPs remained associated in an unchanged pattern.
Replication analysis in CATS
Confirmation of each of these genetic associations was sought in the case-control series from the cross-sectional CATS cohort using the previously completed GWAS dataset. The RP11-
463J7.2 and ZNF827 SNPs were not present on the Illumina Human660W-Quad Bead
Chiparray, but had proxies identified based on evidence of linkage disequilibrium between the
SNPs in data from the 1000 Genomes Project (r2>0.85). There was no association found with the proxy SNPs in the RP11-463J7.2 or ZNF827 genes in the CATS cohort.
By contrast, replication at the 0.05 level was evident for 3 SNPs in the DOCK-2 gene, which were obtained using the same model used in the primary analysis. (Figure 1) In a dominant model, the rs7735431 allelic frequency, which differed significantly between HESN and
Incident subjects in the HITS-p sample (OR 3.51, p = 0.027), also differed between HESN and
Incident subjects in CATS (OR 5.59, p = 0.040). Further, in an additive model, the rs1477316 allelic frequency, which differed significantly between HESN and Incident subjects in HITS- p, (OR 2.03, p = 0.045), was also associated in CATS (OR 3.58, p = 0.042). Furthermore, the rs749965 allelic frequency, which differed significantly between HESN and Incident subjects in HITS-p (OR 0.27, p = 0.023), was also associated in CATS (OR 0.07, p = 0.036) in a recessive model.
12 73 Haplotype analyses in the DOCK-2 region
The genetic effects of the DOCK-2 allelic combinations on the risk of HCV infection were evaluated by haplotype-based analyses in HITS-p. First, the rs7735431 and rs1477316 SNPs for the associations were replicated in both cohorts were assembled into a haplotype to assess potential additive effects of these alleles. Four haplotypes accounting for 99.9% of subjects analysed in the HITS-p cohort were resolved. The most common GA haplotype was protective against HCV infection (OR 0.41, p=0.015). The second analysis included all 3 replicated SNPs
(rs7735431, rs1477316 and rs749965) in a larger haplotype block, which consisted of seven combinations with frequencies >1%. A strong association with protection from HCV infection was found in the GAA haplotype carrying minor alleles for rs749965 (OR 0.37, p = 0.008).
The direction of the observed (protective) association was consistent with those observed for the individual SNPs (Table 3). These protective haplotypes in HITS-p were also validated in
CATS cohort with the GA haplotype and GAA haplotype being over-represented in HESN above the Incident subject groups (OR 0.13, p = 0.011; OR 0.23; p = 0.046 respectively).
13 74 Discussion
This study provides novel, replicated evidence linking several variations in the DOCK-2 gene with resistance against HCV infection, potentially indicative of an immunoregulatory effect underpinning the HESN phenotype. There are rare, biallelic inherited mutations of DOCK-2 in humans, which cause markedly reduced protein expression, and have been shown to result in impaired induction of IFN-a and IFN-l, chemokine-induced lymphocyte migration, NK cell activation, and T cell proliferation. (53, 54) Accordingly, it is reasonable to hypothesise that enhanced expression of DOCK-2 may be protective against HCV via these same functions resulting in highly efficient clearance.
The risk-behaviour algorithm based on prediction of incident infection used to identify the
HESN phenotype in the prospective cohort provides a state of the art, probabilistic approach to characterise likely repeated prior exposures to HCV via IDU. This approach is comparable to that developed for risk assessment of sexual transmission of HCV among MSM. (55) The algorithm used here included many recognised behavioural risk factors for transmission, including injecting drug use, sharing of injecting apparatus, and injecting of heroin, applied to each longitudinal time point in HITS-p, in order to identify a small subset of individuals in the upper risk tertile who remained uninfected despite consistently raised risk indices. These risk scores were comparable to, or higher than those of the actual incident cases. This provides a more robust approach to identifying the HESN phenotype compared to previous studies in
PWID. (24, 35, 39, 56) It should be noted that the risk phenotyping in the replication CATS cohort was intrinsically less robust as a result of the cross-sectional nature of the dataset.
Previous studies exploring resistance to HCV infection have adopted candidate gene approaches informed by understanding of the host factors for viral entry and replication. (22-
29) The positive findings in these studies all await replication. Further corroborative in vitro
14 75 investigations of the effect of the variants on HCV entry have not verified protection (i.e reduced infectivity). (23) The DC-SIGN promoter SNPs associated with a reduced risk of sexual acquisition of HCV were shown to reduce transcription. (28) An initial report associating the high-producing C allele of the antiviral interleukin (IL)-12B gene with resistance to HCV infection in PWID, was not confirmed in a subsequent study from the same group. (35, 57) Interestingly, none of these previous candidate gene associations were confirmed in the present study. This may reflect differences in the HESN phenotyping and the fact that the majority of the previous studies were amongst HIV-infected subjects, including a
GWAS which had essentially negative findings in this group. (26)
Several investigations using GWAS have confirmed that the differences in the rates of spontaneous HCV clearance amongst ethnic groups, such as African-Americans and Japanese, are strongly associated with SNPs in the IFNL3 gene locus, which encodes IFN-λ3, which shares a common signalling pathway with type I interferons. (33) A novel transcript encoding the IFN-l4 protein was identified upstream of the IFNl3 locus, and clearance was also strongly associated with the IFNl4-TT variant, which is in strong linkage disequilibrium with the other clearance-associated SNPs. (58, 59) Perhaps surprisingly, Knapp et al, previously identified
HESN individuals as having a significantly lower frequency of the protective IFNl3 genotype when compared to HCV antibody positive spontaneous clearers, but a similar frequency to patients who were chronically infected. Another report in HESN individuals suggested the same association, but did not include HCV-infected participants as a comparator. (57) By contrast, a previous report from the HITS-p cohort reported no association with IFNl3 genotype (21). Again, the analysis reported here did not identify an association with IFNl3 genotype.
15 76 Using a more open-ended immunogenetic discovery approach, the present study has identified
18 SNPs in the DOCK-2 gene that were significantly different in frequency distribution between longitudinally-identified HESN and Incident groups in HITS-p, of which 3 SNP associations were replicated in the cross-sectional CATS cohort. Further, haplotype-based analysis using these replicated SNPs found a substantial over-representation in HESN above
Incident subject groups. The DOCK family of proteins are guanine nucleotide exchange factors and include four proteins (A-D), which are required for the activation of GTPases to catalyse the hydrolysis of GTP to GDP and thereby release ATP in a response to a variety of stimuli. (53) The DOCK-A sub-family includes DOCK-1, DOCK-2, and DOCK-5, all of which activate the G protein, Rac. (60) Among these subfamily members, DOCK-2 is specifically expressed on immune cells and has been shown to regulate the migration, proliferation and activation of T and B lymphocytes as well as NK cells, by affecting membrane polarization and cytoskeletal dynamics through Rac activation. (53) DOCK-2 is also essential for Toll-like receptor (TLR)-7 and -9-mediated interferon (IFN)-α induction in plasmacytoid dendritic cells
(pDCs). (54) Deficiency of DOCK-2 in mice and in humans leads to impaired Rac activation and thereby to defects in actin polymerization, chemokine-induced lymphocyte migration, NK- cell activation and associated IFN-a and IFN-l production, as well as T-cell proliferation.
These immune defects lead to uncontrolled and often fatal viral and bacterial infections. (54,
61) The precise mechanism of how DOCK-2 is activated and exerts its effects remains unclear, however, its inhibition has shown to reduce Rac activity in vitro. (53) The key question arising from the association found here is what are the biological effects of over-expression or enhanced activation of DOCK-2. In this regard the association between enhanced colonic cancer tissue expression of DOCK-2 and greater tumour infiltration with CD8 T lymphocytes may be noteworthy. (62) Paradoxically, a recent report of studies in the DOCK-2 knockout mouse has also revealed that the mice have a pronounced expansion of the CD8 memory
16 77 T cell compartment associated with a selective increase in T cell receptor sensitivity to antigen presentation. (63)
Although this study used a repeated test and replication case-control study design, the overall sample size was small, hence there is significant residual potential for Type 1 error. Further replication of the DOCK-2 association in larger prospective PWID cohorts followed for HCV infection is warranted to confirm the finding and to underpin further mechanistic investigations.
The choice of using the exome chip rather than whole genome sequencing of a highly selected polar HESN phenotype with a high probability of a single common variant was driven by cost, and by the intrinsic uncertainty associated with the probabilistic definition of the HESN phenotype and its genetic basis.
Taken together the repeatedly replicated DOCK-2 associations with the HESN phenotype point to immunogenetic factors in determining this protective effect. Interestingly, along with the absence of associations with SNPs in the IFNl3 locus, these findings imply that efficient clearance without seroconversion but followed by protective immunity, may occur using unique (DOCK-2-dependent) mechanisms, in a highly selected minority of high-risk individuals. Further studies to better understand the biological basis of the DOCK-2 genotype- phenotype relationship are warranted and the circumstances under which such apparent immune protection may arise.
17 78 Acknowledgements and Disclosures
The HITS-p investigators include: Andrew Lloyd, Kate Dolan, Michael Levy, Peter White, Bill
Rawlinson, Carla Treloar, Paul Haber, Greg Dore, and Lisa Maher.
Funding & Conflicts of interest:
The HITS-p cohort was supported by a Program Grant from National Health and Medical
Research Council (NHMRC) of Australia (No. 510448), and a NHMRC Partnership Grant (No.
1016351).
ARL is supported by a NHMRC Practitioner Fellowship (No. 1137587).
The authors have no conflicts of interest to declare.
Contributors
All authors contributed to the writing of this manuscript and agree with its content and conclusions.
18 79 Table 1: Demographic and risk behaviour characteristics of HESN cases and Incident control
subjects in the prospective HITS-p cohort and the replication CATS cohort
HITS-p CATS
HESN Incident HESN Incident
(n = 22) (n = 44) (n = 14) (n = 28)
Mean age, years (SD) 26.7 (4.8) 24.1 (4.2) 35.9 (6.6) 38.5 (9.3)
Male, n (%) 15 (68) 18 (81) 11 (79) 21 (75)
Mean no. times imprisoned (SD) 4.64 (1.7) 2.15 (1.5) 3.9 (1.6) 3.1 (2.5)
Mean duration imprisonment, months (SD) 7 (7.8) 12.5 (18.0) 8 (6.7) 9 (8.2)
Previous juvenile detention, n (%) 12 (55) 12 (55) 7 (50) 11 (40)
Mean no. tattoos (SD) 3.8 (6.1) 3.1 (3.1) 3.9 (5.1) 3.5 (3.2)
Ever injected heroin, n (%) 17 (77) 15 (68) 12 (86) 23 (82)
Ever injected buprenorphine/methadone, n (%) 11 (50) 10 (45) 8 (57) 16 (57)
Duration of IDU in years (SD) 8.2 (3.4) 6.8 (4.7) 1.4 (0.5) 1.6 (0.7)
SD standard deviation
19 80 Table 2. SNPs associated with the HESN phenotype in the HITS-p and CATS cohorts
HITS-p CATS SNP for Nearest Gene SNP Position A1/A2 OR (95% CI) p value replication r2 Position A1/A2 OR (95% CI) p value Model RP11-463J7.2 rs814346 190764733 A/G 0.08 (0.02-0.31) 0.0002 rs650913 0.99 190764092 G/A 3.63 (0.57-23.09) 0.172 Recessive ZNF827 rs6537384 146870765 A/C 9.07 (2.80-29.32) 0.0002 rs6537382 0.87 146867376 A/C 1.40 (0.29-6.68) 0.676 Dominant DOCK2 rs10044453 169451743 G/A 2.46 (1.10-5.48) 0.028 rs10044453 1 169451743 G/A 1.19 (0.46-3.09) 0.726 Additive DOCK2 rs10067968 169391558 G/A 2.21 (1.02-4.81) 0.045 rs10067968 1 169391558 G/A 0.66 (0.22-2.00) 0.461 Additive DOCK2 rs12055169 169466391 A/T 4.11 (1.63-10.40) 0.003 rs12055169 1 169466391 A/T 1.06 (0.45-2.50) 0.890 Additive DOCK2 rs13161756 169281567 G/A 0.21 (0.05-0.84) 0.027 rs13161756 1 169281567 G/A 0.73 (0.11-4.94) 0.751 Additive DOCK2 rs1477316 169439728 G/A 2.03 (1.02-4.06) 0.045 rs1477316 1 169439728 G/A 3.58 (1.05-12.27) 0.042 Additive DOCK2 rs1875478 169450045 G/A 2.54 (1.19-5.40) 0.016 rs1875478 1 169450045 G/A 1.34 (0.53-3.39) 0.532 Additive DOCK2 rs6873253 169410622 A/G 0.39 (0.18-0.87) 0.022 rs6873253 1 169410622 A/G 2.30 (0.62-8.55) 0.214 Additive DOCK2 rs10044453 169451743 G/A 2.62 (1.18-5.83) 0.017 rs10044453 1 169451743 G/A 1.16 (0.46-2.91) 0.753 Allelic DOCK2 rs10067968 169391558 G/A 2.24 (1.05-4.79) 0.035 rs10067968 1 169391558 G/A 0.87 (0.35-2.15) 0.758 Allelic DOCK2 rs11134602 169440299 A/G 4.67 (1.02-21.3) 0.031 rs11134602 1 169440299 A/G 2.33 (0.73-7.49) 0.147 Allelic DOCK2 rs12055169 169466391 A/T 3.44 (1.54-7.66) 0.002 rs12055169 1 169466391 T/A 1.00 (0.40-2.48) 1.000 Allelic DOCK2 rs12653315 169471291 A/G 4.67 (1.02-21.30) 0.031 rs12653315 1 169471291 A/G 1.39 (0.36-5.39) 0.634 Allelic DOCK2 rs13161756 169281567 G/A 0.25 (0.07-0.91) 0.026 rs13161756 1 169281567 G/A 0.78 (0.14-4.32) 0.780 Allelic DOCK2 rs13190445 169520897 A/C 2.28 (1.05-4.93) 0.034 rs13190445 1 169520897 A/C 1.44 (0.58-3.57) 0.437 Allelic DOCK2 rs1477316 169439728 G/A 2.50 (1.16-5.39) 0.018 rs1477316 1 169439728 G/A 1.95 (0.77-4.91) 0.155 Allelic DOCK2 rs1875478 169450045 G/A 2.86 (1.32-6.19) 0.007 rs1875478 1 169450045 G/A 1.24 (0.50-3.08) 0.643 Allelic DOCK2 rs6873253 169410622 A/G 0.31 (0.13-0.73) 0.006 rs6873253 1 169410622 A/G 1.84 (0.63-5.35) 0.259 Allelic DOCK2 rs749965 169441749 A/G 0.43 (0.21-0.91) 0.025 rs749965 1 169441749 A/G 0.56 (0.22-1.41) 0.215 Allelic DOCK2 rs956303 169476723 A/G 2.60 (1.03-6.53) 0.038 rs956303 1 169476723 A/G 1.17 (0.45-3.05) 0.743 Allelic DOCK2 rs10447205 169438392 A/T 0.41 (0.17-0.99) 0.044 rs10447205 1 169438392 A/T 1.26 (0.40-3.89) 0.694 Allelic DOCK2 rs261068 169320689 C/A 2.66 (1.00-7.04) 0.044 rs261068 1 169320689 C/A 1.17 (0.45-3.05) 0.743 Allelic DOCK2 rs17562153 169526837 G/A 0.23 (0.07-0.76) 0.017 rs17562153 1 169526837 G/A 0.26 (0.02-2.69) 0.255 Recessive
20 81 Table 2. Continued
DOCK2 rs749965 169441749 A/G 0.27 (0.09-0.83) 0.023 rs749965 1 169441749 A/G 0.07 (0.01-0.85) 0.036 Recessive DOCK2 rs10044453 169451743 G/A 3.44 (1.18-10.03) 0.023 rs10044453 1 169451743 G/A 2.38 (0.50-11.47) 0.278 Dominant DOCK2 rs10067968 169391558 G/A 4.67 (1.53-14.22) 0.007 rs10067968 1 169391558 G/A 0.41 (0.07-2.29) 0.310 Dominant DOCK2 rs10447205 169438392 A/T 0.29 (0.10-0.88) 0.028 rs10447205 1 169438392 A/T 1.76 (0.37-8.34) 0.474 Dominant DOCK2 rs12055169 169466391 A/T 5.40 (1.73-16.82) 0.004 rs12055169 1 169466391 T/A 0.89 (0.21-3.73) 0.868 Dominant DOCK2 rs13161756 169281567 G/A 0.21 (0.05-0.84) 0.027 rs13161756 1 169281567 G/A 0.73 (0.11-4.94) 0.751 Dominant DOCK2 rs17738692 169483172 G/A 3.00 (1.02-8.83) 0.046 rs17738692 1 169483172 G/A 0.98 (0.20-4.76) 0.984 Dominant DOCK2 rs1875478 169450045 G/A 5.62 (1.83-17.25) 0.003 rs1875478 1 169450045 G/A 1.91 (0.37-9.87) 0.440 Dominant DOCK2 rs6873253 169410622 A/G 0.29 (0.10-0.88) 0.028 rs6873253 1 169410622 A/G 3.25 (0.75-14.11) 0.116 Dominant DOCK2 rs7700885 169480559 A/G 4.14 (1.39-12.35) 0.011 rs7700885 1 169480559 A/G 2.16 (0.43-10.92) 0.353 Dominant DOCK2 rs7735431 169201727 A/G 3.51 (1.15-10.67) 0.027 rs7735431 1 169201727 A/G 5.59 (1.08-28.97) 0.040 Dominant A1 refers to the minor allele and A2 refers to the major allele.
Odds ratios (OR) are presented as the effect of minor allele. An OR <1 indicates the SNP is associated with protection against HCV infection.
p values in bold are for SNP associations replicated in the two cohorts
SD standard deviation
21 82 Table 3. DOCK-2 haplotype distribution in HESN cases and Incident control groups in the
HITS-p and CATS cohorts
Haplotype Frequency HESN Incident OR STAT P HITS-p Haplotypes GGA 0.066 0.024 0.094 3.78 1.360 0.244 AAA 0.046 0.045 0.051 1.00 <0.001 0.997 GAA 0.319 0.499 0.219 0.37 7.010 0.008 AGG 0.195 0.159 0.206 1.35 0.446 0.504 GGG 0.178 0.112 0.212 2.20 1.900 0.168 AAG 0.054 0.023 0.073 5.00 1.460 0.227 GAG 0.142 0.138 0.146 1.04 0.004 0.949 Haplotypes AG 0.202 0.159 0.215 1.44 0.638 0.425 GG 0.237 0.136 0.296 2.54 3.270 0.071 AA 0.093 0.068 0.115 1.83 0.608 0.435 GA 0.467 0.636 0.374 0.41 5.970 0.015
CATS Haplotypes AAA 0.218 0.240 0.116 1.55 0.321 0.571 GAA 0.234 0.260 0.241 0.23 3.970 0.046 AGG 0.112 0.046 0.283 224.00 5.940 0.015 GGG 0.281 0.293 0.217 1.16 0.042 0.838 AAG 0.039 0.018 0.101 10.70 1.920 0.165 GAG 0.116 0.143 0.042 0.36 1.150 0.284
Haplotypes AG 0.120 0.052 0.250 187.00 6.460 0.011 GG 0.272 0.287 0.250 1.11 0.022 0.883 AA 0.249 0.252 0.250 2.39 1.340 0.247 GA 0.358 0.409 0.250 0.13 6.440 0.011 Two SNP haplotype consisting of rs7735431 and rs1477316 with the haplotype GA was protective against HCV infection.
Three SNP haplotype consisting of rs7735431, rs1477316 and rs749965 with the haplotype
GAA was also protective against HCV infection.
22 83 Figure 1. Schematic overview of HESN subject groups in HITS-p and CATS, with associated and replicated SNPs
HITS-p Test cohort Illumina MS Chip n=11 HESN cases / n=22 Incident controls
~15,000 associated SNPs P<0.05
HITS-p Replication cohort Illumina MS Chip n=11 HESN cases / n=22 Incident controls 2 directly replicated SNPs and 6 gene-associated SNPs P<0.05 Combined HITS-p case-control cohorts n=22 HESN cases / n=44 Incident controls 20 candidate SNPs p<0.05 Illumina Human 660W CATS Replication cohort Quad Bead Chip n=14 HESN cases / n=28 Infected controls 3 replicated DOCK-2 SNPs P<0.05
23 84 Figure 2. Locus zoom plot for association signals in regions containing DOCK-2
Position on chr5 (Mb)
24 85 References
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28 89 Chapter 5
Discussion and Conclusion
90 Discussion
The studies in this thesis utilised a framework for reliable identification of the HESN phenotype originally developed using longitudinally collected demographic and risk behaviour data from HCV-uninfected
PWID (n =590) enrolled in the Hepatitis C Incidence and Transmission Study in prisons (HITS-p). (3)
The algorithm for the HESN phenotype provided a probabilistic approach to characterise likely repeated prior exposures to HCV via IDU. This approach is comparable to that which was subsequently developed for identification of HESN subjects via longitudinal datasets based on risk behaviours associated with sexual transmission of HCV among men who have sex with men (MSM). (4) The algorithm used here included many recognised behavioural risk factors for HCV transmission amongst
PWID, including injecting drug use, sharing of injecting apparatus, and injecting of heroin, in order to identify a small subset of individuals in the upper risk tertile who remained uninfected despite consistently raised risk indices. These risk scores were comparable to, or higher than those of the actual incident cases. This approach provides the best available identification strategy for the HESN phenotype compared to previous studies in PWID. (5-8).
Clinicians and researchers frequently use patient risk characteristics to stage, (9-11) prognosticate, (12,
13) and sometimes to diagnose diseases. (14) There is a substantial body of literature indicating that some people who have been repeatedly exposed to Human Immunodeficiency Virus (HIV) infection may remain uninfected. (2) Indeed, the discovery of the d32 mutation in the HIV-coreceptor, CCR5 in two HIV HESN individuals, led to recognition that this relatively common mutation conferred near complete resistance to HIV infection in homozygotes and partial protection in heterozygotes. (15) The studies in this thesis add to the body of work that can be used to generate risk scores amongst PWID with regard to their risk of contracting HCV, (16) and contributes further to studies that have utilised risk scores to provide a basis for inclusion into immunological or genetic studies. (2, 17-19)
Using the HESN phenotype described in this thesis, the manuscript described in Chapter 2 adds to the body of evidence that describes multiple factors contributing to protection from HCV infection. The chapter describes a comprehensive characterisation of NK subpopulations by phenotype and function in the carefully characterised HESN individuals who are likely to have been repeatedly exposed to HCV
91 through high-risk IDU over many years, in comparison with matched subjects who became infected or reduced their injecting risk behaviours. This study is the first comprehensive characterization of NK subpopulations by phenotype and function in carefully characterized HESN individuals, who are likely to have been repeatedly exposed to HCV through high-risk drug injecting over many years, in comparison with matched subjects who became infected or reduced their injecting risk behaviours. The findings reveal that the frequencies, and functional activity, of the CD56dim, cytotoxic NK subset, differed between HESN and comparison subjects, within both CD16+ and CD16- subpopulations, giving credence to the hypothesis that NK cells contribute to anti-HCV defence in the earliest stages of infection, potentially providing protection from HCV acquisition, and supporting previous reports that
NK cells secrete cytokines that inhibit replication, promote dendritic cell maturation and induce production of chemokines that recruit lymphoid and inflammatory cells. (20) Increased frequencies of
NK cells expressing CD56bright have been observed in patients chronically infected with HCV, but not those that cleared the acute infection, suggesting an overall more effector NK cell repertoire during chronic infection. (21) It is also worth noting that NK cells can also modulate adaptive immune responses by killing T cells and antigen-presenting cells (APCs). (22) Co-expression of CD16 (the low- affinity IgG receptor, FcγRIII, which is almost exclusively found on the CD56dim subset) has been associated with ADCC. (23) The observed evidence for increased expression of the activation marker,
CD69, and of FcγRIII, raises the possibility of undetected anti-HCV antibodies - particularly anti-
Envelope antibodies as identified in Chapter 3 - sustaining NK cell activation and cytotoxicity via ADCC in HESN. This finding supports the hypothesis that cytotoxic antiviral mechanisms and HCV-specific antibodies via ADCC may have a role in protection from HCV. This notion requires further investigation, as prior HCV exposure may have resulted in incomplete seroconversion with anti-Envelope antibodies only or rapid seroreversion.
The limitations of this NK study warrant consideration. Principal component analysis (PCA) is a popular data processing and dimension reduction technique, which seeks linear combinations of the original variables such that the derived variables capture maximal variance. It has an obvious drawback, that is, each PC is a linear combination of all variables and the loadings are typically non-zero. It should be noted that the PCAs identified two PCs within each of the four major NK subsets, CD56brightCD16+,
CD56brightCD16−, CD56dimCD16+ and CD56dimCD16−, accounting for up to 40% of the variance in the
92 dataset – which may be regarded as relatively modest. Nevertheless, this approach offers the advantage that it is systematic and comprehensive, thereby accounting for the large, exploratory data set. Although the subjects and samples included here feature both careful case characterization and longitudinal sample collection allowing within-subject analyses, a larger study size will be required to confirm and extend the findings.
These data are consistent with evidence that early inhibition of innate immunity contributes to the development of persistence in HCV infection. (24) Conversely, placenta and cord blood studies of HCV infected mothers have demonstrated increased NK cells in maternal decidua compared to maternal peripheral blood, (25) representing a potential mechanism by which the placenta prevents vertical transmission during pregnancy. With regard to the HESN phenotype in HIV, the characteristics of potentially protective immunity, particularly in HESN sex workers in Nairobi have been a particular focus of investigation. (26) It has been hypothesised that these women were initially exposed to viral inocula that efficiently primed a cell-mediated immune response in the absence of antibody production. (27)
Consistent with this notion, HESN subjects have been reported to demonstrate a variety of pathogen- sepcific cellular immune responses, namely polarised Th1 cytokine responses (28) and cytotoxic T cell
(CTL) responses to both HIV Envelope and Gag epitopes. (29) It is worth highlighting that a number of these women subsequently became infected following a period of reduction in the frequency of, or an interruption to, sex work, suggesting that the initial protective mechanisms were maintained by repeated exposure to HIV. (30)
Furthermore, HIV HESN subjects have been reported to carry specific MHC class I and II alleles, (31) notably a cluster of closely related HLA alleles – the A2/6802 supertype. (31) These alleles are known to present the same peptide epitopes for T cell recognition. In addition, heterozygosity for the NK cell expressed Killer Inhibitory Receptor (KIR) genotype - 2DL2/2DL3, in combination with the absence of the HLA-C1 ligand for this KIR, has been reported to be associated with HESN in African sex workers.
(32) A similar association has been reported with KIR3DL1 homozygosity in the absence of its cognate
HLA-Bw4 ligand, (32) and co-expression of KIR3DL1 and HLA-B57 has been associated with slow progression to AIDS. (33)
93 In HCV, a well characterised combination of genetic and environmental factors account for much of the varied probability of clearance from primary infection. The association between HLA alleles with spontaneous HCV clearance has also been much studied, given the evidence for CD4+ and CD8+ T cell responses in clearance of primary infection. (34) The most widely reported associations with spontaneous HCV clearance are with HLA Class II alleles, such as HLA-DRB1*03 and –DQB1*0301,
(35) and associations between KIR loci with the outcome of acute HCV infection have also been reported. Individuals homozygous for KIR2DL3 (an inhibitory receptor gene) and HLA-C1 (its ligand gene) and spontaneous clearance of acute HCV infection has previously been reported. (36) This receptor-ligand combination could provide weaker inhibitory signals than other inhibitory KIR:HLA-C receptor-ligand pairings and thus prime a more responsive NK cell phenotype. (37, 38) KIR gene activation accounts approximately for only 20% of the variance in clearance rates and therefore suggests a number of potential mechanisms of protection and resistance against HCV.
Support for the hypothesis of an adaptive component of protective immunity amongst HESN in HCV was also provided by the studies in Chapter 3 which indicated that IgG anti-E responses, as well as neutralisation activity and HCV E-specific memory B cells, were significantly greater in HESN when compared with subjects who subsequently became infected. Further, these IgG responses were generally repeatedly detectable, and were almost exclusively in the IgG2 subclass, which is noteworthy as IgG2 responses are rare in primary HCV infection, (39) but do have unique functional characteristics of potential relevance to protective immunity. This finding is novel and supports the likelihood of prior
HCV exposure and the potential for efficient Envelope-targeted responses mediating protection. A significant minority of the carefully identified HESN cases had repeatedly detectable IgG responses against gt1 or gt3 E (which constitute approximately 90% of incident and prevalent genotypes in
Australia). IgG2 antibodies are recognized to bind glycans independent of T-cell help (typically targeting bacterial polysaccharides, but also viral glycans). In addition, this isotype binds to the neonatal Fc receptor (FcRn) which is widely expressed on epithelial cells in adult life, including hepatocytes, and is recognized in this context to facilitate mucosal transport of immune complexes for antigen sampling
(40) and phagocytosis. (41) Further exploration of the potential role IgG2 anti-HCV E antibodies in neutralization of hepatocyte uptake and survival of HCV virions is warranted, as well as investigation of the Fc-dependent functions such as ADCC. The findings in this chapter also open the potential for
94 further characterisation of the antibody repertoire, specificities and functional activities of these HCV- specific B cells in HESN via single cell sorting and monoclonal antibody synthesis.
Similar findings have been reported in HIV HESN studies, (42-44) and as in the study described in
Chapter 3, have identified antibodies against epitopes that are not detectable by routine diagnostic assays. These observations point to the presence of humoral responses with potentially novel specificities in both HCV and HIV HESN, and suggest that further characterisation of these specificities may help to identify novel protective epitopes targeted by these individuals which would be of interest for vaccine design.
The final paper (chapter 4) provides novel evidence underpinning the hypothesis that polymorphisms in host immune response genes may confer resistance to HCV infection. This chapter presents replicated evidence linking several variations in DOCK-2 genes to resistance against HCV infection – indicative of immunoregulatory effects underpinning the HESN phenotype. Eighteen SNPs in the
DOCK-2 gene were found to be significantly different in frequency distribution between longitudinally- identified HESN and Incident groups in HITS-p, of which 3 SNP associations were replicated in the cross-sectional CATS cohort. Further, haplotype-based analysis using these replicated SNPs found a substantial over-representation in HESN above Incident subject groups. The findings in this chapter suggest that enhanced expression of DOCK-2 may be protective against HCV resulting in highly efficient clearance. The key question arising from the association found here is what are the biological effects of over-expression or enhanced activation of DOCK-2. In this regard, the association between enhanced colonic cancer tissue expression of DOCK-2 and greater tumour infiltration with CD8 T lymphocytes may be noteworthy. (45)
Although this study used a repeated test and replication case-control study design, the overall sample size was small, hence there is significant residual potential for Type 1 error. Further replication of the
DOCK-2 association in larger prospective PWID cohorts followed for HCV infection is warranted to confirm the finding and to underpin further mechanistic investigations. The choice of the exome chip rather than whole genome sequencing of a highly selected polar HESN phenotype with a high probability of a single common variant was driven by cost, and by the intrinsic uncertainty associated
95 with the probabilistic definition of the HESN phenotype and its genetic basis. With all this said, the evidence for the role of DOCK-2 as a protective phenotype against HCV infection generated in this chapter warrants further investigation.
Taken together, the repeatedly replicated DOCK-2 associations with the HESN phenotype point to immunogenetic factors in determining this protective effect. Interestingly, along with the absence of associations with SNPs in the IFNl3 locus, these findings imply that efficient clearance without seroconversion but followed by protective immunity, may occur using unique (DOCK-2-dependent) mechanisms, in a highly selected minority of high-risk individuals. Further studies to better understand the biological basis of the DOCK-2 genotype-phenotype relationship are warranted. These may include investigation of the HCV-specific cellular and humoral immune responses in selected HESN subjects and matched controls from the HITS-p cohort who cleared HCV infection and who carry the DOCK-2 protective haplotype. Transcriptional analysis of peripheral blood mononuclear cells (PBMC) from
HESN and matched subjects who became infected, may help to further elucidate this immune protection. (46) Sequencing of highly selected HESN could help identify a potential unique mutation within the DOCK-2 locus, (47) with a view to over-expression and knockdown experiments, including hepatocytes to examine examine the infectivity and replication efficiency of HCV (JFH-1), and the associated interferon-stimulated gene expression. (48, 49)
Conclusion
Taken together, the studies presented in this thesis provide evidence for a protective phenotype associated with HESN individuals. These include the replicated DOCK-2 association, alongside a robust innate immunity in the form of CD56dim, cytotoxic NK subset, and IgG antibodies of anti-E responses, as well as neutralisation activity and HCV E-specific memory B cells within the HESN phenotype point to both immunological and genetic factors in determining this protective effect.
Epidemiological evidence exists to suggest that a small subset of individuals remain apparently uninfected with hepatitis C virus, despite very long-lasting high-risk behaviours and probable repeated
96 exposure. Some individuals repeatedly clear established infection via both innate and adaptive immune mechanisms. In combination the studies in this thesis clearly support the existence of an HCV resistant phenotype and argue for further studies to elucidate the mechanisms.
97 References
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