Mechanisms of Hepatitis C Virus Infection and Pathogenesis

Lead Research Organisation: MRC Virology Unit

Abstract

We are investigating the properties of the protein components of the Hepatitis C virus (HCV) particles and their interactions with host proteins. Our aim is to understand exactly how the virus initiates infection of its target cell and how it interfers with the workings of the host cell to cause disease. HCV is a major cause of serious liver disease. It is estimated that approximately 200 million people globally are infected with this virus, a significant number of whom will go on to develop serious liver disease, including cirrhosis and hepatocellular carcinoma. Current worldwide projections estimate that HCV in particular will be a major source of morbidity and mortality before the end of this decade. Although new promising treatments are now becoming available, there are many unmet needs and there is as yet no vaccine available. Therefore, basic research on viral proteins to enhance our understanding of the mechanisms of virus infection is needed to develop better anti-viral therapies. Our investigations of the functions and structures of the relevant virus and host proteins will allow better understanding of the processes involved in virus life cycle and disease. The information gained will be crucial in developing novel antiviral drugs and vaccines.

Technical Summary

Hepatitis C virus (HCV) is a major cause of chronic liver disease that often leads to cirrhosis and hepatocellular carcinoma. Promising new treatments are now becoming available, but they are focused on one viral genotype. There are many unmet needs and therefore HCV will remain a serious threat for the foreseeable future. There is as yet no vaccine available. To develop new targets for a broad and more effective therapy, and preventative vaccines, it is necessary to gain greater insights into the processes involved in virus life cycle and their effects on cellular metabolism.

Our research is focused on investigating the mechanisms of HCV entry into target cells and the interactions between viral and cellular components involved in virus life cycle and pathogenesis. Specifically, we have been studying virus neutralizing antibodies and their mode of action both at structural and functional level, modes of virus transmission in cultured cells, and the functional significance of the interaction between the HCV core protein and host factors. Closely aligned to the above work, we have (1) developed several robust cell-based high-throughput screening assays that have enabled us to identify novel anti-viral compounds targeting different stages of the virus life-cycle and (2) an on-going translational project on the development of antibody-based vaccine against HCV.
My group is a member of the Glasgow HCV Network (a consortium of clinicians and scientists) and a national consortium called HCV Research UK focused on fundamental research, clinical studies and translational approaches on HCV. Our aim is to investigate molecular mechanisms that contribute to HCV-associated pathogenesis. The work performed under this programme includes whole genome transcriptome analysis in diseased liver, and investigating host factors that determine viral clearance or progression to chronicity and extrahepatic disorders.
In addition, we are studying the interaction between HCV core and the cellular DEAD-box protein, DDX3. Our data show that this interaction is dispensable for virus life cycle, but may be involved in apoptosis, oncogenesis and aspects of innate immunity. We also find that DDX3 interacts with a variety of cellular factors involved in host RNA metabolism thus implicating the core-DDX3 interaction in these processes, which may in turn contribute to viral pathogenesis. We are investigating the mechanisms for the sequestration of the host factors and their functions in infected cells. Identification of functional pathways is likely to provide novel targets for pharmacological intervention and treatment of HCV-related diseases.

Organisations

 
Description Marie Curie Research Training Network
Amount £330,000 (GBP)
Organisation Marie Sklodowska-Curie Actions 
Sector Academic/University
Country Global
Start 09/2006 
End 08/2010
 
Title Antibodies to HCV proteins 
Description The tools are a large collection of rabbit polyclonal and mouse monoclonal antibodies to hepatitis C virus core, E1 and E2 proteins. 
Type Of Material Antibody 
Provided To Others? Yes  
Impact These are highly valuable reagents. They have had substantial impact in research on structural and functional analysis of the relevant proteins. Indeed, we recently showed that one of our antibodies, MAb AP33, has potent and broad virus neutralizing property. MRCT has filed patents for therapeutic development of AP33 and its humanised version, MRCT10. 
 
Title Antiviral compounds 
Description Novel compounds inhibiting various stages of HCV infection have been generated following high-throughput screening 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact Very useful tools to disect pathways and processes affecting virus life cycle. 
 
Title DDX3 antibodies 
Description Rabbit polyclonal and mouse monoclonal antibodies to the human DEAD-box protein, DDX3 
Type Of Material Antibody 
Provided To Others? Yes  
Impact This antibodies have been useful in elucidating the properties and functions of DDX3 in normal and malignant cells and tissue. 
 
Title DDX3 plasmids 
Description The tools are plasmid constructs carrying the gene (or derivatives of it) encoding the human DEAD-box protein, DDX3. The nucelotide sequence of DDX3 has been deposited in Gene-Bank (Accession no. AF061337) 
Type Of Material Technology assay or reagent 
Provided To Others? Yes  
Impact This constructs have been useful in elucidating the properties and functions of DDX3 in normal and malignant cells and tissue 
 
Title Generation of recombinant antibodies to HCV proteins 
Description Genes encoding antibodies of interest have been cloned and stable cell lines expressing them have been generated. 
Type Of Material Antibody 
Provided To Others? No  
Impact The antibody expressing clones and cell lines are useful tools in studies to perform sturcture-function studies on proteins of interest. Furthermore, the clones are amenable to genetic manipulation with a view to to generating higher/better affinity antibodies and to aiding above studies. 
 
Title HHL cell lines 
Description A panel of human hepatocyte cell lines. Commercially available primary hepatocytes derived from a normal human liver were immortalized by retrovirus mediated transduction of human papillomavirus E6 and E7 antigens. A total of 15 cell lines were established that were able to grow in normal, serum containing medium and in a defined, serum free medium. 
Type Of Material Cell line 
Year Produced 2006 
Provided To Others? Yes  
Impact The cell lines have been used by a number of groups to look at liver-specific gene expression and function, and immunological response. 
 
Title Huh7-J20 SEAP reporter cell line 
Description This is a novel cell line (Huh7-20) that is capable of secreting a reporter (Secreted Alkaline Phosphatase or SEAP) into the culture medium upon infection with hepatitis C virus (HCV). Current methods to quantitate virus infectivity in cell culture are time-consuming and labour-intensive. We developed the Huh7-J20 reporter cell line to facilitate in vitro studies of HCV infection and replication. This cell line enables rapid and sensitive quantification of HCV infection and viral replication in cell culture, and is ideal for hihg-throughput screening of antiviral compounds 
Type Of Material Cell line 
Year Produced 2009 
Provided To Others? Yes  
Impact Huh7-J20 has several distinct advantages: (i) it offers a rapid and convenient infectivity assay in which SEAP activity is directly measured in a sample of the culture medium without compromising cell integrity; (ii) it provides a tool to indirectly measure infectivity, translation and viral RNA replication; (iii) it can be used in virus neutralization assays and for high throughput screening of viral entry inhibitors; and (iv) it can be used with any inter- or intra-genotypic derivative of HCV strain JFH1, and possibly other cell culture-infectious viral isolates that may be isolated in future. This work was published in 2009. 
 
Title Method to synchronize HCV infection 
Description We have very recently developed a new magnetic adsoption method to rapidly synchronize hepatitis C virus infection. It involves adsorbing the virus to magnetic nanoparticles and then applying magnetic field for rapid attachment (within 2 minutes at room-temperature) of the virus to cells. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact Hepatitis C Virus entry into target cells is a multistep process involving several cellular factors and receptors. However, their precise role during virus entry is unclear. Investigation of the mechanisms of HCV entry, such as the order of intervention by the cellular receptors, requires synchronizing infections. Our new method allows greater efficiency of virus adsorption to cells, and increased virus infectivity, as compared to the standard protocol involving incubation of the virus with cells at 4°C for 1 hr, or to a standard infection at 37°C. It alleviates the potential anamolies associated with conditions used in standard protocols. Furthermore, we found that magnetic adsorption respects the natural entry route of the virus, making this system suitable to study the early stages of HCV infection. This work has just been submitted for possible publication. The method will become available to researchers once published. 
 
Title Plasmids expressing HCV proteins 
Description Plasmids expressing different HCV-encoded proteins 
Type Of Material Technology assay or reagent 
Provided To Others? Yes  
Impact These reagents have been useful in many functional studies of viral proteins which have resulted in sevreal publications 
 
Title Plasmids expressing cellular proteins 
Description Genes encoding cellular proteins of interest were generated for use in studies investigating virus-host interactions. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact Extremely useful tools in studies investigating functions of host proteins in virus life cycle. 
 
Description AP33 and DDX3 
Organisation University of Texas
Country United States 
Sector Academic/University 
PI Contribution Provision of reagents for the study involving in vitro selection of neutralisation-escape mutants of hepatitis C virus In a separate project my team had used reagents supplied by the collborator for our studies on dissection of the interaction of hepatitis C virus core with the cellular DEAD-box proten DDX3
Collaborator Contribution Experimental data, provision of reagents
Impact Generated HCV mutants that escape neutralization by the broadly neutralizing antibody AP33. Publication: PMID - 19052239 The reagents supplied allowed us to show that the HCV core-DDX3 interaction is not required for virus replication. Publication: 19793905; 20237087
Start Year 2006
 
Description Anti-E2 human fabs 
Organisation San Raffaele Hospital
Department San Raffaele Scientific Institute (SRSI)
Country Italy 
Sector Academic/University 
PI Contribution Sigificant intellectual input, provision of training of a clinical research fellow from the collaborator's lab, and supplying reagents
Collaborator Contribution Allowed us to further define the role of a broadly neutralizing epitope of HCV E2 and map epitopes recognized by several novel anti-E2 human fabs
Impact One publication: PMID - 17989176; 20011511
Start Year 2006
 
Description Application of MAb AP33 in histochemistry 
Organisation University of Texas
Department Health Science Center at Houston
Country United States 
Sector Academic/University 
PI Contribution Provision of antibody AP33 in large quantities, intellectual input
Collaborator Contribution The histopathological distinction between acute cellular rejection (ACR) and recurrent hepatitis C virus (HCV) infection in liver biopsy specimens after liver transplantation (LT) can be difficult. The aim of our study was to determine whether immunohistochemical (IHC) staining for HCV is useful in LT setting. Having first established the methods for the detection of HCV E2 glycoprotein by immuno-histochemical (IHC) staining of patient liver biopsy specimen using MAb AP33, 85 formalin fixed, paraffin embedded liver needle biopsy specimens from HCV-infected LT recipients were stained for the viral glycoprotein. The results showed that the expression of the HCV E2 glycoprotein in liver grafts is associated with positive response to anti-viral therapy, and that positive HCV-IHC using the AP33 antibody correlates with increased serum viral load.
Impact Our work was presented at the United States & Canadian Academy of Pathology Meeting in San Diego, CA, in March 2014. Also, a manusript describing this work has been submited for publication.
Start Year 2012
 
Description Broadly Neutralizing Antibodies - Mechanism for Escape 
Organisation Genentech, Inc
Country United States 
Sector Private 
PI Contribution Generation and provision of humanized derivative of our broadly neutralizing antibody AP33.
Collaborator Contribution This particular work led to the development of an affinity matured version of the humanized antibody. The study provided an experimental evidence of glycan shift as a mechanism of escape from virus neutralization by this antibody.
Impact PMID: 23458406
Start Year 2007
 
Description Broadly Neutralizing Antibodies - Mechanism for Escape 
Organisation MRC-Technology
Department MRCT Centre for Therapeutics Discovery (CTD)
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Generation and provision of humanized derivative of our broadly neutralizing antibody AP33.
Collaborator Contribution This particular work led to the development of an affinity matured version of the humanized antibody. The study provided an experimental evidence of glycan shift as a mechanism of escape from virus neutralization by this antibody.
Impact PMID: 23458406
Start Year 2007
 
Description Characterisation of neutralising antibody responses to HCV in a large cohort of acutely or chronically infected patients 
Organisation University of Glasgow
Department MRC - University of Glasgow Centre for Virus Research
Country United Kingdom 
Sector Academic/University 
PI Contribution Identification and functional characterization of HCV E1/E2 antibody responses during evolving spontaneous clearance and progression to chronicity in a cohort of HCV-infected or HIV-HCV co-infected individuals. Provided our expertise in this area of work and training to PhD student, a clinical research fellow and a post-doc.
Collaborator Contribution Characterizing diversification within the HCV E1 and E2 envelope genes over time in HIV patients co-infected with HCV that have spontaneously cleared HCV or have fluctuating viremia. Provision of patient samples
Impact Two patients with fluctuating viramia were studied using samples collected at multiple time points. In the first patient (pt100), samples from before and after the negative viral load, purifying selection was evident at multiple positions with HCV glycoproteins E1 and E2. Amino acid changes were observed outwitwith the known receptor-binding sites. An unexpected observation was that no positive selection was present within the previously well-described E2 hypervariable region 1 (HVR-1) - this region is the most extensively described target for neutralising antibodies in patients with acute HCV. It seems highly likely that there are neutralising antibodies targeting regions outwith of HVR1 and this forms the basis of further study. In the second patient (p56), the viral load never became undetectable but did drop by >1log10. The level of selection estimated across E1 and E2 appeared to be higher than that in the pt100. An amino acid change V313I was present within the same fairly conserved region of E1 as R317H in p100. This may well represent evidence of a previously undescribed B cell epitope. Again there was very little evidence of selection occurring within E2 HVR1. We are also investigating neutralizing antibody responses in spontaneous clearers and chronically-infected patients Multi-disciplinary: Fundamental and clinical science
Start Year 2013
 
Description Characterization of HCV structural proteins 
Organisation Pasteur Institute, Lille
Department Center for Infection & Immunity of Lille
Country France 
Sector Academic/University 
PI Contribution Provision of reagents; characterization of antibody-mediated neutralization directed against the hypervariable region 1 of HCV E2 glycoprotein
Collaborator Contribution Define the proeprties of HCV glycoproteins in virus-infected cultured cells
Impact Publication: PMID - 19100780; 16501092; 20668082; 21084495
Start Year 2006
 
Description Characterization of a broadly neutralizing antibody 
Organisation University of Nottingham
Department School of Biomedical Sciences Nottingham
Country United Kingdom 
Sector Academic/University 
PI Contribution Analysed host neutralizing responses to the hepatitis C virus E2 E2 glycoproten epitope recognized by the broadly neutralizing monoclonal antibody, AP33. Performed detailed characterisation of AP33 epitope
Collaborator Contribution Experimental data
Impact This collaboration resulted in three publications: PMID: 17502679 PMID: 17947521 PMID: 16496330 PMID: 16912317
 
Description Chimeric HCV isolates 
Organisation University of Glasgow
Department MRC - University of Glasgow Centre for Virus Research
Country United Kingdom 
Sector Academic/University 
PI Contribution Generated chimeric HCV incorporating the viral glycoproteins sequences from diverse genotypes which were supplied to us by our collaborator.
Collaborator Contribution Plasmids carrying sequences of interest supplied by our collaborator to facilitate this study
Impact The chimeric viruses are currently being characterised.
 
Description Encapsidation of HCV subgenomic RNA 
Organisation University of Southampton
Department School of Medicine Southampton
Country United Kingdom 
Sector Academic/University 
PI Contribution Provided significant intellectual input and supplied relevant reagents
Collaborator Contribution Development of transencapsidation system provided insight into HCV RNA packaging functions
Impact Publication: PMID - 19223490
 
Description Functional studies on HCV p7 
Organisation Hannover Medical School
Department Twincore
Country Germany 
Sector Academic/University 
PI Contribution Provision of reagents by us for studies on HCV p7. The CD81 low cell lijes provided by the collaborator to us were very useful in our studies on HCV cell-to-cell transmission in cultured cells.
Collaborator Contribution Further define the role of p7 in HCV morphogenesis. Provision of CD81 low lines for our studies on HCV cell-to-cell transmission
Impact Publications: 17658949; 19088272; 19726506
Start Year 2006
 
Description HCV NS4B and cellular DDX3 
Organisation University of Glasgow
Department MRC - University of Glasgow Centre for Virus Research
Country United Kingdom 
Sector Academic/University 
PI Contribution Provided reagents for studies on NS4B. Collaborators antibodies were very useful for our studies on the dissection of the HCV core-DDX3 interaction Proteomics studies of cells infected with wild-type HCV or a viral mutant carrying a substitution in the core protein that abrogates core-DDX3 interaction. Identified a set of novel differentially expressed genes potentially implicated in processes/pathways contributing to virus-associate pathogenesis.
Collaborator Contribution Defined the role of NS4B in HCV replication and morphogenesis. Separately, provided reagents for our studies on delineating the interaction between HCV core and cellular DDX3.
Impact Publications: PMID - 19073716: 19793905 From our proteomics studies we have identified a set of novel differentially expressed genes potentially implicated in processes/pathways contributing to virus-associate pathogenesis. Experiments involving further validation of selected genes and elucidation of their functional significance remain to be performed.
Start Year 2007
 
Description HCV cell-to-cell transmission 
Organisation Rockefeller University
Country United States 
Sector Academic/University 
PI Contribution We demonstrated that CD81 is dispensable for hepatitis C virus cell-to-cell transmission in cultured cells. Recent data show that the tight junction proteins claudin 1 and occludin 1 play a central role in HCV cell-to-cell transmission.
Collaborator Contribution Provision of CD-81 low cell lines
Impact One publication: PMID - 19088272
Start Year 2007
 
Description HCV core 
Organisation Institute of Biology and Chemistry of Proteins (IBCP)
Country France 
Sector Academic/University 
PI Contribution Our group identified crucial residues in the domain I of HCV core that are improtant for virus assembly and spread. Recently, we reported a single mutation (Y35A) within domain I of JFH1 core that disrupted its interaction with the cellular RNA helicase DDX3 and that this virus-host interaction is dispensable for HCV replication. Here, further mutagenesis of domain I of core identified two JFH1 mutants (G33A and F24A) with unique phenotypes that were unrelated to the core-DDX3 interaction. Following transfection, the G33A RNA replicated efficiently yet the extracellular and intracellular infectious virus titers were reduced by approximately 2 logs, indicating that this residue is important for infectious virus assembly. During serial passaging of cells replicating this mutant RNA, we reproducibly observed the reversion of virus infectivity to WT levels. Moreover, this increase in infectivity coincided with compensatory mutations arising in close proximity to the original G33A substitution. In contrast to the G33A phenotype, the F24A mutant achieved peak infectious virus titers similar to WT following transfection. However, the infection kinetics of the released virus particles was substantially reduced, indicating a defect in virus spread.
Collaborator Contribution Structural analysis of the helix-loop-helix motif of HCV core in the context of G33A and F24A mutations.
Impact Examination of core protein [residues 2-45] structure (PDB entry 1CWX) indicates that the residues G33 and F24 are in contact with each other, and that the G33A mutation induces a steric clash with F24 which is reversed by the observed compensatory mutations bringing them spatially close to F24. Molecular simulations revealed that these mutations disturb the distribution of core conformational preferences, which is likely related to the modulation of infection kinetics. Together, these data highlight the plasticity of core domain I conformation and illustrate the relationship between its structural tolerance to mutations and virus assembly and virus spread. PMID: 22072760 Multi-disciplinary: Virology, molecular biology, protein chemistry/structure
Start Year 2008
 
Description HCV entry and neutralizing antibodies 
Organisation National Institute of Health and Medical Research (INSERM)
Department INSERM U748 (Virus Host Interactions and Liver Diseases)
Country France 
Sector Public 
PI Contribution From our part, provision of training to a clinical scientist, reagents and significant intellectual input. Collaborator's reagents were very useful in our studies on synchronization of virus infection, virus neutralization assays, and functional characterization of mutant viruses
Collaborator Contribution Provided insights into host neutralizing responses to HCV infection, studied receptor functions and the role of apoE in virus infection, and supplied reagents. Identified novel entry factors. Performed in vivo analysis of the neutralizing capability of our antibody in a chimeric human liver mouse model.
Impact Publications: PMID - 16928753; 17392433; 18718838; 19100780; 20237087; 20713596; 21516087; 22414763; 24830295; 26710081; 26404951. Passive infusion of our neutralizing MAb AP33 into chimeric human liver mice afforded complete protection upon challenge with a clinical isolate of HCV. Demonstrated a role of ApoE in HCV evasion from neutralizing antibodies.
Start Year 2006
 
Description HCV entry and neutralizing antibodies 
Organisation University of Ghent
Department Center for Vaccinology
Country Belgium 
Sector Academic/University 
PI Contribution From our part, provision of training to a clinical scientist, reagents and significant intellectual input. Collaborator's reagents were very useful in our studies on synchronization of virus infection, virus neutralization assays, and functional characterization of mutant viruses
Collaborator Contribution Provided insights into host neutralizing responses to HCV infection, studied receptor functions and the role of apoE in virus infection, and supplied reagents. Identified novel entry factors. Performed in vivo analysis of the neutralizing capability of our antibody in a chimeric human liver mouse model.
Impact Publications: PMID - 16928753; 17392433; 18718838; 19100780; 20237087; 20713596; 21516087; 22414763; 24830295; 26710081; 26404951. Passive infusion of our neutralizing MAb AP33 into chimeric human liver mice afforded complete protection upon challenge with a clinical isolate of HCV. Demonstrated a role of ApoE in HCV evasion from neutralizing antibodies.
Start Year 2006
 
Description HCV-like particles 
Organisation Albert Ludwig University of Freiburg
Country Germany 
Sector Hospitals 
PI Contribution Provision of training of relevant assay systems and and reagents
Collaborator Contribution Functional studies of HCV-like particles
Impact Following two publications have arisen from this collaboration: PMID - 16360642; 17553880; 15308699; 18718838; 16928753
 
Description HCV-like particles 
Organisation Osaka University
Country Japan 
Sector Academic/University 
PI Contribution Provision of training of relevant assay systems and and reagents
Collaborator Contribution Functional studies of HCV-like particles
Impact Following two publications have arisen from this collaboration: PMID - 16360642; 17553880; 15308699; 18718838; 16928753
 
Description Host factors influencing HCV pathogenesis 
Organisation NHS Health Scotland
Country United Kingdom 
Sector Public 
PI Contribution We are looking at the factors influencing infection and pathogenesis of HCV infections in humans at the molecular level. To date we have looked at host responses to HCV genotypes and studied the effects of chronic HCV on the liver transcriptome. We have established a pipeline for the analysis of RNA-Seq data from archived liver biopsies. Our key findings are: (a) significant enrichment of interferon-regulated and immune pathways, (b) changes that occur during disease progression (i.e. fibrosis/cirrhosis) are not present at the early stages of chronic infection, (c) genotype 1 HCV induces a higher level of pro-inflammatory gene expression compared to genotype 3 HCV, (d) up-regulation of type I IFN inducible genes is seen in early stage chronic HCV, (e) pro-apoptotic IFN genes are not strongly induced, and (f) antiviral pathways that are able to down-regulate IFN signalling are up-regulated.
Collaborator Contribution Provided valuable patient samples; made important contributions in terms of ideas and input from clinical perspective
Impact Five studies published (PMID:23931242, PMID: 25200131, PMID: 25800823, PMID: 26912610, PMID: 27405885). Further studies on hightroughput sequencing of data patient biopsies have revealed interesting information. Briefly, HCV gentoype (gt) 1-infected biopsies displayed elevated levels of transcripts regulated by Type I and Type III interferon (IFN), including genes that predict response to IFNa therapy. In contrast, genes controlled by IFN? were induced in gt3-infected biopsies. Moreover, IFN? levels were higher in gt3 samples compared to gt1 and control biopsies. Analysis of hepatocyte-derived cell lines confirmed that the genes up-regulated in gt3 infection were predominantly induced by IFN?. The distinct interactions of the two viral genotypes with hepatic cells may explain differences in the progression of disease pathology and treatment response under both IFN-based treatments and novel direct acting antivirals. A separate study demonstrated the presence of anti-HCV envelope antibody responses in individuals at high risk of HCV who resist infection. These antibodies may be a factor that helps these individuals resist HCV infection. We are also obtaining data on antibody responses in a small cohort of patients and at-risk individuals as follows: • Studied broad cross-genotypic antibody responses in clinical cohorts, their epitope targets and clinical associations. Showed patients with broad neutralizing responses usually target multiple epitopes and they are associated with lower rates of fibrosis and may predict slower disease progression. • In collaboration with Dr Paul Kellam (University of Cambridge) we are investigating the diversity of immunoglobulin repertoire in acute and chronic infections using next generation sequencing methods. In parallel, we plan to set up assays to measure B-cell repertoire (from patients at different disease stages and infected with different genotypes) at the functional level (manuscript in prepatration). • Studied neutralising anti-envelope antibodies in unifected individuals that are repeatedly exposed to HCV (in collaboration with Matthew Cramp, University of Plymouth). This is a unique cohort of individuals who resist HCV infection despite repeated exposure. We showed that a subset of individuals produce neutralizing anti-envelope antibodies which may contribute to host immunity. Further exploring regions targeted by these antibodies may yield novel epitopes for vaccine design (manuscript published). Multi-disciplinary: Clinical and fundamental science
Start Year 2010
 
Description Host factors influencing HCV pathogenesis 
Organisation Plymouth Hospitals NHS Trust
Country United Kingdom 
Sector Public 
PI Contribution We are looking at the factors influencing infection and pathogenesis of HCV infections in humans at the molecular level. To date we have looked at host responses to HCV genotypes and studied the effects of chronic HCV on the liver transcriptome. We have established a pipeline for the analysis of RNA-Seq data from archived liver biopsies. Our key findings are: (a) significant enrichment of interferon-regulated and immune pathways, (b) changes that occur during disease progression (i.e. fibrosis/cirrhosis) are not present at the early stages of chronic infection, (c) genotype 1 HCV induces a higher level of pro-inflammatory gene expression compared to genotype 3 HCV, (d) up-regulation of type I IFN inducible genes is seen in early stage chronic HCV, (e) pro-apoptotic IFN genes are not strongly induced, and (f) antiviral pathways that are able to down-regulate IFN signalling are up-regulated.
Collaborator Contribution Provided valuable patient samples; made important contributions in terms of ideas and input from clinical perspective
Impact Five studies published (PMID:23931242, PMID: 25200131, PMID: 25800823, PMID: 26912610, PMID: 27405885). Further studies on hightroughput sequencing of data patient biopsies have revealed interesting information. Briefly, HCV gentoype (gt) 1-infected biopsies displayed elevated levels of transcripts regulated by Type I and Type III interferon (IFN), including genes that predict response to IFNa therapy. In contrast, genes controlled by IFN? were induced in gt3-infected biopsies. Moreover, IFN? levels were higher in gt3 samples compared to gt1 and control biopsies. Analysis of hepatocyte-derived cell lines confirmed that the genes up-regulated in gt3 infection were predominantly induced by IFN?. The distinct interactions of the two viral genotypes with hepatic cells may explain differences in the progression of disease pathology and treatment response under both IFN-based treatments and novel direct acting antivirals. A separate study demonstrated the presence of anti-HCV envelope antibody responses in individuals at high risk of HCV who resist infection. These antibodies may be a factor that helps these individuals resist HCV infection. We are also obtaining data on antibody responses in a small cohort of patients and at-risk individuals as follows: • Studied broad cross-genotypic antibody responses in clinical cohorts, their epitope targets and clinical associations. Showed patients with broad neutralizing responses usually target multiple epitopes and they are associated with lower rates of fibrosis and may predict slower disease progression. • In collaboration with Dr Paul Kellam (University of Cambridge) we are investigating the diversity of immunoglobulin repertoire in acute and chronic infections using next generation sequencing methods. In parallel, we plan to set up assays to measure B-cell repertoire (from patients at different disease stages and infected with different genotypes) at the functional level (manuscript in prepatration). • Studied neutralising anti-envelope antibodies in unifected individuals that are repeatedly exposed to HCV (in collaboration with Matthew Cramp, University of Plymouth). This is a unique cohort of individuals who resist HCV infection despite repeated exposure. We showed that a subset of individuals produce neutralizing anti-envelope antibodies which may contribute to host immunity. Further exploring regions targeted by these antibodies may yield novel epitopes for vaccine design (manuscript published). Multi-disciplinary: Clinical and fundamental science
Start Year 2010
 
Description Host factors influencing HCV pathogenesis 
Organisation The Wellcome Trust Sanger Institute
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution We are looking at the factors influencing infection and pathogenesis of HCV infections in humans at the molecular level. To date we have looked at host responses to HCV genotypes and studied the effects of chronic HCV on the liver transcriptome. We have established a pipeline for the analysis of RNA-Seq data from archived liver biopsies. Our key findings are: (a) significant enrichment of interferon-regulated and immune pathways, (b) changes that occur during disease progression (i.e. fibrosis/cirrhosis) are not present at the early stages of chronic infection, (c) genotype 1 HCV induces a higher level of pro-inflammatory gene expression compared to genotype 3 HCV, (d) up-regulation of type I IFN inducible genes is seen in early stage chronic HCV, (e) pro-apoptotic IFN genes are not strongly induced, and (f) antiviral pathways that are able to down-regulate IFN signalling are up-regulated.
Collaborator Contribution Provided valuable patient samples; made important contributions in terms of ideas and input from clinical perspective
Impact Five studies published (PMID:23931242, PMID: 25200131, PMID: 25800823, PMID: 26912610, PMID: 27405885). Further studies on hightroughput sequencing of data patient biopsies have revealed interesting information. Briefly, HCV gentoype (gt) 1-infected biopsies displayed elevated levels of transcripts regulated by Type I and Type III interferon (IFN), including genes that predict response to IFNa therapy. In contrast, genes controlled by IFN? were induced in gt3-infected biopsies. Moreover, IFN? levels were higher in gt3 samples compared to gt1 and control biopsies. Analysis of hepatocyte-derived cell lines confirmed that the genes up-regulated in gt3 infection were predominantly induced by IFN?. The distinct interactions of the two viral genotypes with hepatic cells may explain differences in the progression of disease pathology and treatment response under both IFN-based treatments and novel direct acting antivirals. A separate study demonstrated the presence of anti-HCV envelope antibody responses in individuals at high risk of HCV who resist infection. These antibodies may be a factor that helps these individuals resist HCV infection. We are also obtaining data on antibody responses in a small cohort of patients and at-risk individuals as follows: • Studied broad cross-genotypic antibody responses in clinical cohorts, their epitope targets and clinical associations. Showed patients with broad neutralizing responses usually target multiple epitopes and they are associated with lower rates of fibrosis and may predict slower disease progression. • In collaboration with Dr Paul Kellam (University of Cambridge) we are investigating the diversity of immunoglobulin repertoire in acute and chronic infections using next generation sequencing methods. In parallel, we plan to set up assays to measure B-cell repertoire (from patients at different disease stages and infected with different genotypes) at the functional level (manuscript in prepatration). • Studied neutralising anti-envelope antibodies in unifected individuals that are repeatedly exposed to HCV (in collaboration with Matthew Cramp, University of Plymouth). This is a unique cohort of individuals who resist HCV infection despite repeated exposure. We showed that a subset of individuals produce neutralizing anti-envelope antibodies which may contribute to host immunity. Further exploring regions targeted by these antibodies may yield novel epitopes for vaccine design (manuscript published). Multi-disciplinary: Clinical and fundamental science
Start Year 2010
 
Description Host factors influencing HCV pathogenesis 
Organisation University of Glasgow
Department Institute of Health and Wellbeing
Country United Kingdom 
Sector Academic/University 
PI Contribution We are looking at the factors influencing infection and pathogenesis of HCV infections in humans at the molecular level. To date we have looked at host responses to HCV genotypes and studied the effects of chronic HCV on the liver transcriptome. We have established a pipeline for the analysis of RNA-Seq data from archived liver biopsies. Our key findings are: (a) significant enrichment of interferon-regulated and immune pathways, (b) changes that occur during disease progression (i.e. fibrosis/cirrhosis) are not present at the early stages of chronic infection, (c) genotype 1 HCV induces a higher level of pro-inflammatory gene expression compared to genotype 3 HCV, (d) up-regulation of type I IFN inducible genes is seen in early stage chronic HCV, (e) pro-apoptotic IFN genes are not strongly induced, and (f) antiviral pathways that are able to down-regulate IFN signalling are up-regulated.
Collaborator Contribution Provided valuable patient samples; made important contributions in terms of ideas and input from clinical perspective
Impact Five studies published (PMID:23931242, PMID: 25200131, PMID: 25800823, PMID: 26912610, PMID: 27405885). Further studies on hightroughput sequencing of data patient biopsies have revealed interesting information. Briefly, HCV gentoype (gt) 1-infected biopsies displayed elevated levels of transcripts regulated by Type I and Type III interferon (IFN), including genes that predict response to IFNa therapy. In contrast, genes controlled by IFN? were induced in gt3-infected biopsies. Moreover, IFN? levels were higher in gt3 samples compared to gt1 and control biopsies. Analysis of hepatocyte-derived cell lines confirmed that the genes up-regulated in gt3 infection were predominantly induced by IFN?. The distinct interactions of the two viral genotypes with hepatic cells may explain differences in the progression of disease pathology and treatment response under both IFN-based treatments and novel direct acting antivirals. A separate study demonstrated the presence of anti-HCV envelope antibody responses in individuals at high risk of HCV who resist infection. These antibodies may be a factor that helps these individuals resist HCV infection. We are also obtaining data on antibody responses in a small cohort of patients and at-risk individuals as follows: • Studied broad cross-genotypic antibody responses in clinical cohorts, their epitope targets and clinical associations. Showed patients with broad neutralizing responses usually target multiple epitopes and they are associated with lower rates of fibrosis and may predict slower disease progression. • In collaboration with Dr Paul Kellam (University of Cambridge) we are investigating the diversity of immunoglobulin repertoire in acute and chronic infections using next generation sequencing methods. In parallel, we plan to set up assays to measure B-cell repertoire (from patients at different disease stages and infected with different genotypes) at the functional level (manuscript in prepatration). • Studied neutralising anti-envelope antibodies in unifected individuals that are repeatedly exposed to HCV (in collaboration with Matthew Cramp, University of Plymouth). This is a unique cohort of individuals who resist HCV infection despite repeated exposure. We showed that a subset of individuals produce neutralizing anti-envelope antibodies which may contribute to host immunity. Further exploring regions targeted by these antibodies may yield novel epitopes for vaccine design (manuscript published). Multi-disciplinary: Clinical and fundamental science
Start Year 2010
 
Description Host factors influencing HCV pathogenesis 
Organisation University of Glasgow
Department MRC - University of Glasgow Centre for Virus Research
Country United Kingdom 
Sector Academic/University 
PI Contribution We are looking at the factors influencing infection and pathogenesis of HCV infections in humans at the molecular level. To date we have looked at host responses to HCV genotypes and studied the effects of chronic HCV on the liver transcriptome. We have established a pipeline for the analysis of RNA-Seq data from archived liver biopsies. Our key findings are: (a) significant enrichment of interferon-regulated and immune pathways, (b) changes that occur during disease progression (i.e. fibrosis/cirrhosis) are not present at the early stages of chronic infection, (c) genotype 1 HCV induces a higher level of pro-inflammatory gene expression compared to genotype 3 HCV, (d) up-regulation of type I IFN inducible genes is seen in early stage chronic HCV, (e) pro-apoptotic IFN genes are not strongly induced, and (f) antiviral pathways that are able to down-regulate IFN signalling are up-regulated.
Collaborator Contribution Provided valuable patient samples; made important contributions in terms of ideas and input from clinical perspective
Impact Five studies published (PMID:23931242, PMID: 25200131, PMID: 25800823, PMID: 26912610, PMID: 27405885). Further studies on hightroughput sequencing of data patient biopsies have revealed interesting information. Briefly, HCV gentoype (gt) 1-infected biopsies displayed elevated levels of transcripts regulated by Type I and Type III interferon (IFN), including genes that predict response to IFNa therapy. In contrast, genes controlled by IFN? were induced in gt3-infected biopsies. Moreover, IFN? levels were higher in gt3 samples compared to gt1 and control biopsies. Analysis of hepatocyte-derived cell lines confirmed that the genes up-regulated in gt3 infection were predominantly induced by IFN?. The distinct interactions of the two viral genotypes with hepatic cells may explain differences in the progression of disease pathology and treatment response under both IFN-based treatments and novel direct acting antivirals. A separate study demonstrated the presence of anti-HCV envelope antibody responses in individuals at high risk of HCV who resist infection. These antibodies may be a factor that helps these individuals resist HCV infection. We are also obtaining data on antibody responses in a small cohort of patients and at-risk individuals as follows: • Studied broad cross-genotypic antibody responses in clinical cohorts, their epitope targets and clinical associations. Showed patients with broad neutralizing responses usually target multiple epitopes and they are associated with lower rates of fibrosis and may predict slower disease progression. • In collaboration with Dr Paul Kellam (University of Cambridge) we are investigating the diversity of immunoglobulin repertoire in acute and chronic infections using next generation sequencing methods. In parallel, we plan to set up assays to measure B-cell repertoire (from patients at different disease stages and infected with different genotypes) at the functional level (manuscript in prepatration). • Studied neutralising anti-envelope antibodies in unifected individuals that are repeatedly exposed to HCV (in collaboration with Matthew Cramp, University of Plymouth). This is a unique cohort of individuals who resist HCV infection despite repeated exposure. We showed that a subset of individuals produce neutralizing anti-envelope antibodies which may contribute to host immunity. Further exploring regions targeted by these antibodies may yield novel epitopes for vaccine design (manuscript published). Multi-disciplinary: Clinical and fundamental science
Start Year 2010
 
Description Host factors influencing HCV pathogenesis 
Organisation University of Oxford
Department Nuffield Department of Experimental Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution We are looking at the factors influencing infection and pathogenesis of HCV infections in humans at the molecular level. To date we have looked at host responses to HCV genotypes and studied the effects of chronic HCV on the liver transcriptome. We have established a pipeline for the analysis of RNA-Seq data from archived liver biopsies. Our key findings are: (a) significant enrichment of interferon-regulated and immune pathways, (b) changes that occur during disease progression (i.e. fibrosis/cirrhosis) are not present at the early stages of chronic infection, (c) genotype 1 HCV induces a higher level of pro-inflammatory gene expression compared to genotype 3 HCV, (d) up-regulation of type I IFN inducible genes is seen in early stage chronic HCV, (e) pro-apoptotic IFN genes are not strongly induced, and (f) antiviral pathways that are able to down-regulate IFN signalling are up-regulated.
Collaborator Contribution Provided valuable patient samples; made important contributions in terms of ideas and input from clinical perspective
Impact Five studies published (PMID:23931242, PMID: 25200131, PMID: 25800823, PMID: 26912610, PMID: 27405885). Further studies on hightroughput sequencing of data patient biopsies have revealed interesting information. Briefly, HCV gentoype (gt) 1-infected biopsies displayed elevated levels of transcripts regulated by Type I and Type III interferon (IFN), including genes that predict response to IFNa therapy. In contrast, genes controlled by IFN? were induced in gt3-infected biopsies. Moreover, IFN? levels were higher in gt3 samples compared to gt1 and control biopsies. Analysis of hepatocyte-derived cell lines confirmed that the genes up-regulated in gt3 infection were predominantly induced by IFN?. The distinct interactions of the two viral genotypes with hepatic cells may explain differences in the progression of disease pathology and treatment response under both IFN-based treatments and novel direct acting antivirals. A separate study demonstrated the presence of anti-HCV envelope antibody responses in individuals at high risk of HCV who resist infection. These antibodies may be a factor that helps these individuals resist HCV infection. We are also obtaining data on antibody responses in a small cohort of patients and at-risk individuals as follows: • Studied broad cross-genotypic antibody responses in clinical cohorts, their epitope targets and clinical associations. Showed patients with broad neutralizing responses usually target multiple epitopes and they are associated with lower rates of fibrosis and may predict slower disease progression. • In collaboration with Dr Paul Kellam (University of Cambridge) we are investigating the diversity of immunoglobulin repertoire in acute and chronic infections using next generation sequencing methods. In parallel, we plan to set up assays to measure B-cell repertoire (from patients at different disease stages and infected with different genotypes) at the functional level (manuscript in prepatration). • Studied neutralising anti-envelope antibodies in unifected individuals that are repeatedly exposed to HCV (in collaboration with Matthew Cramp, University of Plymouth). This is a unique cohort of individuals who resist HCV infection despite repeated exposure. We showed that a subset of individuals produce neutralizing anti-envelope antibodies which may contribute to host immunity. Further exploring regions targeted by these antibodies may yield novel epitopes for vaccine design (manuscript published). Multi-disciplinary: Clinical and fundamental science
Start Year 2010
 
Description Human monoclonal antibodies to HCV E2 
Organisation Stanford University
Country United States 
Sector Academic/University 
PI Contribution Continuing characterization of the broadly neutralizing properties of several several human human monoclonal antibodies to hepatitis C virus E2 glycoprotein. Provided reagents and made intellectual contributions. A detailed functional analyses of different HCV mutants carrying substitution mutations in a region of the viral E2 glycprotein that is a target of our broadly neutralizing antibody AP33
Collaborator Contribution Provision of antibodies and experimental data, including performing characterization of mutant viruses.
Impact Several publications: PMID: 17079294; 17990788; 18272755; 18385242; 18400849; 19321602; 22511875; 23097455; 20237087; 21697343; 22993159; 25122476
Start Year 2006
 
Description Immune functions of hepatocytes 
Organisation University of Oxford
Department Nuffield Department of Clinical Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution Provision of novel human hepatocyte cell lines
Collaborator Contribution Provided insights into the immune functions of novel human hepatocyte cell lines generated in our lab
Impact Publication: PMID - 17726521; 15780065
 
Description In vivo analysis of HCV neutralizing antibodies 
Organisation University of Ghent
Department Center for Vaccinology
Country Belgium 
Sector Academic/University 
PI Contribution Assessment of antibodies of interest in in vitro assays and in cultured cells infected with HCV.
Collaborator Contribution Antibodies of interest have been tested and analysed for their effects on HCV infection in the chimeric human liver uPA-SCID mouse model. Specifically, we show that our broadly neutralizing antibody AP33 is capable of protecting mice upon challenge with a clinical isolate of hepatitis C virus. Our humanised antibody AP33 is currently being tested the above uPA-SCID mouse model.
Impact Data presented preseneted at the HCV2012 and AASLD 2012 meeting. A paper published (PMID: 26710081). Two moe papers published recently (PMID: 29074219, 29128754)
Start Year 2011
 
Description In vivo analysis of HCV neutralizing antibodies 
Organisation University of Strasbourg
Country France 
Sector Academic/University 
PI Contribution Assessment of antibodies of interest in in vitro assays and in cultured cells infected with HCV.
Collaborator Contribution Antibodies of interest have been tested and analysed for their effects on HCV infection in the chimeric human liver uPA-SCID mouse model. Specifically, we show that our broadly neutralizing antibody AP33 is capable of protecting mice upon challenge with a clinical isolate of hepatitis C virus. Our humanised antibody AP33 is currently being tested the above uPA-SCID mouse model.
Impact Data presented preseneted at the HCV2012 and AASLD 2012 meeting. A paper published (PMID: 26710081). Two moe papers published recently (PMID: 29074219, 29128754)
Start Year 2011
 
Description Investigating functions of DDX3 
Organisation Martin Luther University of Halle-Wittenberg
Department Institute of Biochemistry and Biotechnology
Country Germany 
Sector Academic/University 
PI Contribution Parallel investigation of the role of DDX3 in HCV life cycle
Collaborator Contribution Investigated the role of DDX3 in the assembly of functional 80S ribosomes
Impact Data presented at HCV2011 meeting.
Start Year 2010
 
Description Investigating the structural basis HCV entry and neutralization by cryo-EM 
Organisation University of Glasgow
Department MRC - University of Glasgow Centre for Virus Research
Country United Kingdom 
Sector Academic/University 
PI Contribution Generation and optimization of virus particles and neutralization assays for electron tomography. Provided reagents and advice to generate recombinant hepatitis B virus (HBV) capsid carrying an hepatitis C virus (HCV) E2 glycoprotein fragment representing an epitope of our braodly neutralizing mouse monoclonal antibody (MAb) AP33.
Collaborator Contribution Developing imaging and image processing protocols, performing electron tomography protocols. Cryo-electron micrography/image reconstruction of HBV capsid carrying the MAb AP33 epitope.
Impact Work in progress. The HBV capsid particles displaying the MAb AP33 epitope will be used for vaccine development. Multi-disciplinary: Virology, molecular biology, electron microscopy, structural
Start Year 2011
 
Description Novel anti-HCV antibodies and their analyses 
Organisation Hannover Medical School
Department Institute for Virology
Country Germany 
Sector Academic/University 
PI Contribution Generated novel mouse monoclonal antibodies targeting different regions of HCV envelope glycoprotein E2 and performed their detailed functional characterization. Two of these (MAbs DAO1 and DAO3) were found to target novel conformation-sensitive epitopes on E2. We also identified one antibody (MAb DAO5) that bound to a major virus receptor binding site (aa 523-540) which forms part of the central immunoglobulin (Ig)-like domain within HCV E2. A detailed epitope mapping experiments identified aa D535 to be crucial for DAO5 recognition of E2. Interestingly, D535 is also critical for the interation between HCV E2 and CD81, a host factor essential for virus entry. Curiously though, MAb DAO5 did not neutralize virus infection. We have now obtained a crystal structure of the epitope peptide in compelx with MAb DAO5 in collaboration with scientists at Pasteur Inst, Paris. Together, our results suggest that the E2 segment spanning aa 529-540 is structurally flexible.
Collaborator Contribution Determined the crystal structure of analysis of MAb DAO5 in complex with a peptide representing its epitope (E2 aa 529-540). These data showed that the peptide in complex with DAO5 adopted a helical conformation that is in stark contrast to the extended conformation observed in the E2 core structure. It was also observed that MAb DAO5 binds to soluble E2 (sE2) in a temperature-dependent manner and despite the lack of neutralizing activity it directly cross-competes with the virus receptor CD81 and other conformational neutralizing mAbs recognizing a critical epitope residue (D535). These results suggest that mAb DAO5 recognizes natively folded sE2 locally displaying a high degree of flexibility in this crucial CD81 binding region.
Impact A manuscript describing the structural basis of the conformational flexibility in the central Ig-like domain of E2 is published in mBio (PMID: 28512091). Multidisciplinary: Virology, molecular biology, immunology, protein structure and function
Start Year 2010
 
Description Novel anti-HCV antibodies and their analyses 
Organisation Pasteur Institute, Paris
Country France 
Sector Academic/University 
PI Contribution Generated novel mouse monoclonal antibodies targeting different regions of HCV envelope glycoprotein E2 and performed their detailed functional characterization. Two of these (MAbs DAO1 and DAO3) were found to target novel conformation-sensitive epitopes on E2. We also identified one antibody (MAb DAO5) that bound to a major virus receptor binding site (aa 523-540) which forms part of the central immunoglobulin (Ig)-like domain within HCV E2. A detailed epitope mapping experiments identified aa D535 to be crucial for DAO5 recognition of E2. Interestingly, D535 is also critical for the interation between HCV E2 and CD81, a host factor essential for virus entry. Curiously though, MAb DAO5 did not neutralize virus infection. We have now obtained a crystal structure of the epitope peptide in compelx with MAb DAO5 in collaboration with scientists at Pasteur Inst, Paris. Together, our results suggest that the E2 segment spanning aa 529-540 is structurally flexible.
Collaborator Contribution Determined the crystal structure of analysis of MAb DAO5 in complex with a peptide representing its epitope (E2 aa 529-540). These data showed that the peptide in complex with DAO5 adopted a helical conformation that is in stark contrast to the extended conformation observed in the E2 core structure. It was also observed that MAb DAO5 binds to soluble E2 (sE2) in a temperature-dependent manner and despite the lack of neutralizing activity it directly cross-competes with the virus receptor CD81 and other conformational neutralizing mAbs recognizing a critical epitope residue (D535). These results suggest that mAb DAO5 recognizes natively folded sE2 locally displaying a high degree of flexibility in this crucial CD81 binding region.
Impact A manuscript describing the structural basis of the conformational flexibility in the central Ig-like domain of E2 is published in mBio (PMID: 28512091). Multidisciplinary: Virology, molecular biology, immunology, protein structure and function
Start Year 2010
 
Description Novel in vitro model for HCV infection and replication 
Organisation Shantou University Medical College, China
Country China 
Sector Academic/University 
PI Contribution This study led to the establishment of a novel human embryonic stem cell-derived hepatocyte system for propagation of hepatitis C virus in my lab. We also showed that this system is phenotypically closer to human liver hepatocytes and as such has allowed us to identify novel virus-host interactions that are not seen in the existing widely used hepatoma-based cell culture system for studying HCV life cycle. This work was funded by the MRC UK-China Stem Cell partnership Development Initiative (PI - Edinburgh partner; Co-PI - myself).
Collaborator Contribution University of Edinburgh partner provided the stem cell-derived hepatocytes and related functional characterization. Shantou partners performed the work stem cell-related work at Edinburgh (differentiation into human hepatocytes and phenotypic characterization) and virus-related study (in vitro infection, virus spread assays, virus-host interaction) in my lab.
Impact PMID: 24201130 PMID: 25068132 Multidisciplinary: Regenerative medicine, stem cell biology, virology, molecular biology
Start Year 2011
 
Description Novel in vitro model for HCV infection and replication 
Organisation University of Edinburgh
Department MRC Centre for Regenerative Medicine
Country United Kingdom 
Sector Public 
PI Contribution This study led to the establishment of a novel human embryonic stem cell-derived hepatocyte system for propagation of hepatitis C virus in my lab. We also showed that this system is phenotypically closer to human liver hepatocytes and as such has allowed us to identify novel virus-host interactions that are not seen in the existing widely used hepatoma-based cell culture system for studying HCV life cycle. This work was funded by the MRC UK-China Stem Cell partnership Development Initiative (PI - Edinburgh partner; Co-PI - myself).
Collaborator Contribution University of Edinburgh partner provided the stem cell-derived hepatocytes and related functional characterization. Shantou partners performed the work stem cell-related work at Edinburgh (differentiation into human hepatocytes and phenotypic characterization) and virus-related study (in vitro infection, virus spread assays, virus-host interaction) in my lab.
Impact PMID: 24201130 PMID: 25068132 Multidisciplinary: Regenerative medicine, stem cell biology, virology, molecular biology
Start Year 2011
 
Description Reporter cell line 
Organisation Heidelberg University
Country Germany 
Sector Academic/University 
PI Contribution Our lab generated and characterized a reporter cell line for rapid and sensitive evaluation of HCV infection and replication. This cell line, first of its kind, has application for high throughput screening of anti-viral compounds
Collaborator Contribution Provision of a chimeric HCV virus.
Impact Publication: PMID - 19397930
Start Year 2008
 
Description Role of DDX3 in mRNA translation 
Organisation Harvard University
Department Department of Molecular and Cellular Biology
Country United States 
Sector Academic/University 
PI Contribution Provided significant intellectual input and supplied relevant reagents
Collaborator Contribution Further defined the multifunctional role of the DEAD-box protein DDX3 in normal cells
Impact Publication: PMID - 18628297
 
Description Screening of antiviral compounds 
Organisation King's College School
PI Contribution Acquired state-of-the art robotic systems to establish high-throughput screening facility within the CVR. Developed and validated HTS assays to investigate the different stages of HCV life cycle (requiring work under containment levels 2 and 3). Compound library screens identified several novel hits inhibiting different stages of HCV entry and post-entry events. These compounds are proving very useful in our studies to dissect viral processes at the mechanistic level, and their antiviral potential is being further developed for treatment to control and eliminate infection.
Collaborator Contribution Provision of novel DNA minor groove compounds, nucleoside analogues and estrogens. A large part of the study involving the latter was performed at Università del Piemonte Orientale, Novara, Italy. More recently, compounds developed by our King's College partners were evaluated.
Impact Identified novel inhibitors acting at different stages of HCV life cycle (e.g. virus entry, genome translation & amp; replication, virus assembly and secretion). 4 papers published to date (PMID: 26428870, 27406141, 27885811, 30661824).
Start Year 2011
 
Description Screening of antiviral compounds 
Organisation Northumbria University
Country United Kingdom 
Sector Academic/University 
PI Contribution Acquired state-of-the art robotic systems to establish high-throughput screening facility within the CVR. Developed and validated HTS assays to investigate the different stages of HCV life cycle (requiring work under containment levels 2 and 3). Compound library screens identified several novel hits inhibiting different stages of HCV entry and post-entry events. These compounds are proving very useful in our studies to dissect viral processes at the mechanistic level, and their antiviral potential is being further developed for treatment to control and eliminate infection.
Collaborator Contribution Provision of novel DNA minor groove compounds, nucleoside analogues and estrogens. A large part of the study involving the latter was performed at Università del Piemonte Orientale, Novara, Italy. More recently, compounds developed by our King's College partners were evaluated.
Impact Identified novel inhibitors acting at different stages of HCV life cycle (e.g. virus entry, genome translation & amp; replication, virus assembly and secretion). 4 papers published to date (PMID: 26428870, 27406141, 27885811, 30661824).
Start Year 2011
 
Description Screening of antiviral compounds 
Organisation Russian Academy of Sciences
Department Engelhardt Institute of Molecular Biology (EIMB)
Country Russian Federation 
Sector Public 
PI Contribution Acquired state-of-the art robotic systems to establish high-throughput screening facility within the CVR. Developed and validated HTS assays to investigate the different stages of HCV life cycle (requiring work under containment levels 2 and 3). Compound library screens identified several novel hits inhibiting different stages of HCV entry and post-entry events. These compounds are proving very useful in our studies to dissect viral processes at the mechanistic level, and their antiviral potential is being further developed for treatment to control and eliminate infection.
Collaborator Contribution Provision of novel DNA minor groove compounds, nucleoside analogues and estrogens. A large part of the study involving the latter was performed at Università del Piemonte Orientale, Novara, Italy. More recently, compounds developed by our King's College partners were evaluated.
Impact Identified novel inhibitors acting at different stages of HCV life cycle (e.g. virus entry, genome translation & amp; replication, virus assembly and secretion). 4 papers published to date (PMID: 26428870, 27406141, 27885811, 30661824).
Start Year 2011
 
Description Screening of antiviral compounds 
Organisation University of Eastern Piedmont
Country Italy 
Sector Academic/University 
PI Contribution Acquired state-of-the art robotic systems to establish high-throughput screening facility within the CVR. Developed and validated HTS assays to investigate the different stages of HCV life cycle (requiring work under containment levels 2 and 3). Compound library screens identified several novel hits inhibiting different stages of HCV entry and post-entry events. These compounds are proving very useful in our studies to dissect viral processes at the mechanistic level, and their antiviral potential is being further developed for treatment to control and eliminate infection.
Collaborator Contribution Provision of novel DNA minor groove compounds, nucleoside analogues and estrogens. A large part of the study involving the latter was performed at Università del Piemonte Orientale, Novara, Italy. More recently, compounds developed by our King's College partners were evaluated.
Impact Identified novel inhibitors acting at different stages of HCV life cycle (e.g. virus entry, genome translation & amp; replication, virus assembly and secretion). 4 papers published to date (PMID: 26428870, 27406141, 27885811, 30661824).
Start Year 2011
 
Description Screening of antiviral compounds 
Organisation University of Strathclyde
Department Strathclyde Institute of Pharmacy & Biomedical Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Acquired state-of-the art robotic systems to establish high-throughput screening facility within the CVR. Developed and validated HTS assays to investigate the different stages of HCV life cycle (requiring work under containment levels 2 and 3). Compound library screens identified several novel hits inhibiting different stages of HCV entry and post-entry events. These compounds are proving very useful in our studies to dissect viral processes at the mechanistic level, and their antiviral potential is being further developed for treatment to control and eliminate infection.
Collaborator Contribution Provision of novel DNA minor groove compounds, nucleoside analogues and estrogens. A large part of the study involving the latter was performed at Università del Piemonte Orientale, Novara, Italy. More recently, compounds developed by our King's College partners were evaluated.
Impact Identified novel inhibitors acting at different stages of HCV life cycle (e.g. virus entry, genome translation & amp; replication, virus assembly and secretion). 4 papers published to date (PMID: 26428870, 27406141, 27885811, 30661824).
Start Year 2011
 
Description Screening of antiviral compounds 
Organisation Volgograd State Medical University
Country Russian Federation 
Sector Academic/University 
PI Contribution Acquired state-of-the art robotic systems to establish high-throughput screening facility within the CVR. Developed and validated HTS assays to investigate the different stages of HCV life cycle (requiring work under containment levels 2 and 3). Compound library screens identified several novel hits inhibiting different stages of HCV entry and post-entry events. These compounds are proving very useful in our studies to dissect viral processes at the mechanistic level, and their antiviral potential is being further developed for treatment to control and eliminate infection.
Collaborator Contribution Provision of novel DNA minor groove compounds, nucleoside analogues and estrogens. A large part of the study involving the latter was performed at Università del Piemonte Orientale, Novara, Italy. More recently, compounds developed by our King's College partners were evaluated.
Impact Identified novel inhibitors acting at different stages of HCV life cycle (e.g. virus entry, genome translation & amp; replication, virus assembly and secretion). 4 papers published to date (PMID: 26428870, 27406141, 27885811, 30661824).
Start Year 2011
 
Description Structure of AP33 and related vaccine studies 
Organisation Imperial College London
Country United Kingdom 
Sector Academic/University 
PI Contribution Generated and purified large quantities of the hepatitis C virus (HCV) monoclonal antibody (MAb) AP33 Fab fragment (a pan-genotypic virus neutralizing antibody) and supplied appropriate peptides to University of St Andrews for co-crystallisation studies. We used MAb AP33 as a template to reverse engineer an immunogen that induces similar antibodies upon vaccination. To our knowledge, this is the first time in the HCV vaccine field, the success of such a focused, structure-based approach has been demonstrated. We have supplied this candidate vaccine to our University of St Andrews collaborator for structural studies. We recently established collaoartion with ICL to validate this vaccine in vivo in an HCV infection small animal model In collaboration with the group at University of Naples Federico II, Naples we characterised monoclonal antibodies generated in animals following immunisation with a cyclic peptide representing the AP33 epitope for binding to the HCV glycoprotein E2 and for virus neutralising activity. In collaboration with the Intercollegiate Faculty of Biotechnology , Gdansk, analysed sera from animals immunised with the hepatitis B virus surface antigen-based virus-like particles displaying the AP33 epitope.
Collaborator Contribution Determined the crystal structure of the antibody AP33 bound to its epitope peptide and just recently recently, that of our vaccine candidate in complex with AP33. Further structural analysis of the latter is currently in progress. The University of Naples Federico II group generated antibodies to the cyclic variant of the peptide corresponding to the AP33 epitope and performed their structural analyses when bound to the peptide. Intercollegiate Faculty of Biotechnology , Gdansk, developed a hepatitis B virus surface antigen-based virus-like particles displaying the AP33 epitope for vaccine research Our collaborators at the ICL have shown protection from HCV infection in a chimeric human live mouse model following passive immunization with serum IgGs derived from mice vaccinated with our anti-idiotype antibody vaccine.
Impact We have recently crystallized MAb AP33 bound to its cognate epitope sequence which identified antibody residues essential for this interaction. Based on this we are generated mutated derivatives of this antibody which are proving very useful in our on-going studies on understanding the antigen-antibody interactions at both structural and biochemical levels (Paper published: PMID 22993159). As an extension to this work, we have a novel vaccine candidate capable of eliciting MAb AP33-like anitbodies in immunized animals. Furthermore, a crystal structure of this antibody-based vaccine candidate in complex with its ligand has just been generated. Further work (including in vivo evaluation of the vaccine) is in progress. Indeed, more recently our collaborators at the ICL have shown protection from HCV infection in a chimeric human live mouse model following passive immunization with serum IgGs derived from mice vaccinated with our anti-idiotype antibody vaccine. We explored the potential of cyclic peptides mimicking the AP33 epitope structure to elicit anti-HCV antibodies. Antibodiess that specifically recognize a cyclic variant of the epitope bound to soluble E2 with a lower affinity than other blocking antibodies and do not neutralize virus. The structure of the complex between one such antibody and the cyclic epitope, together with new structural data showing the linear peptide bound to neutralizing antibodies in extended conformations, suggests that the epitope displays a conformational flexibility that contributes to neutralization escape. Such features can be of major importance for the design of epitope-based anti-HCV vaccines (published PMID:26819303). Collaboration is multidisciplinary involving Protein biochemistry, molecular biology, structural biology and virology.
Start Year 2009
 
Description Structure of AP33 and related vaccine studies 
Organisation Medical University of Gdansk
Department Intercollegiate Faculty of Biotechnology
Country Poland 
Sector Academic/University 
PI Contribution Generated and purified large quantities of the hepatitis C virus (HCV) monoclonal antibody (MAb) AP33 Fab fragment (a pan-genotypic virus neutralizing antibody) and supplied appropriate peptides to University of St Andrews for co-crystallisation studies. We used MAb AP33 as a template to reverse engineer an immunogen that induces similar antibodies upon vaccination. To our knowledge, this is the first time in the HCV vaccine field, the success of such a focused, structure-based approach has been demonstrated. We have supplied this candidate vaccine to our University of St Andrews collaborator for structural studies. We recently established collaoartion with ICL to validate this vaccine in vivo in an HCV infection small animal model In collaboration with the group at University of Naples Federico II, Naples we characterised monoclonal antibodies generated in animals following immunisation with a cyclic peptide representing the AP33 epitope for binding to the HCV glycoprotein E2 and for virus neutralising activity. In collaboration with the Intercollegiate Faculty of Biotechnology , Gdansk, analysed sera from animals immunised with the hepatitis B virus surface antigen-based virus-like particles displaying the AP33 epitope.
Collaborator Contribution Determined the crystal structure of the antibody AP33 bound to its epitope peptide and just recently recently, that of our vaccine candidate in complex with AP33. Further structural analysis of the latter is currently in progress. The University of Naples Federico II group generated antibodies to the cyclic variant of the peptide corresponding to the AP33 epitope and performed their structural analyses when bound to the peptide. Intercollegiate Faculty of Biotechnology , Gdansk, developed a hepatitis B virus surface antigen-based virus-like particles displaying the AP33 epitope for vaccine research Our collaborators at the ICL have shown protection from HCV infection in a chimeric human live mouse model following passive immunization with serum IgGs derived from mice vaccinated with our anti-idiotype antibody vaccine.
Impact We have recently crystallized MAb AP33 bound to its cognate epitope sequence which identified antibody residues essential for this interaction. Based on this we are generated mutated derivatives of this antibody which are proving very useful in our on-going studies on understanding the antigen-antibody interactions at both structural and biochemical levels (Paper published: PMID 22993159). As an extension to this work, we have a novel vaccine candidate capable of eliciting MAb AP33-like anitbodies in immunized animals. Furthermore, a crystal structure of this antibody-based vaccine candidate in complex with its ligand has just been generated. Further work (including in vivo evaluation of the vaccine) is in progress. Indeed, more recently our collaborators at the ICL have shown protection from HCV infection in a chimeric human live mouse model following passive immunization with serum IgGs derived from mice vaccinated with our anti-idiotype antibody vaccine. We explored the potential of cyclic peptides mimicking the AP33 epitope structure to elicit anti-HCV antibodies. Antibodiess that specifically recognize a cyclic variant of the epitope bound to soluble E2 with a lower affinity than other blocking antibodies and do not neutralize virus. The structure of the complex between one such antibody and the cyclic epitope, together with new structural data showing the linear peptide bound to neutralizing antibodies in extended conformations, suggests that the epitope displays a conformational flexibility that contributes to neutralization escape. Such features can be of major importance for the design of epitope-based anti-HCV vaccines (published PMID:26819303). Collaboration is multidisciplinary involving Protein biochemistry, molecular biology, structural biology and virology.
Start Year 2009
 
Description Structure of AP33 and related vaccine studies 
Organisation University of Naples
Department Institute of Biostructures and Bioimaging
Country Italy 
Sector Academic/University 
PI Contribution Generated and purified large quantities of the hepatitis C virus (HCV) monoclonal antibody (MAb) AP33 Fab fragment (a pan-genotypic virus neutralizing antibody) and supplied appropriate peptides to University of St Andrews for co-crystallisation studies. We used MAb AP33 as a template to reverse engineer an immunogen that induces similar antibodies upon vaccination. To our knowledge, this is the first time in the HCV vaccine field, the success of such a focused, structure-based approach has been demonstrated. We have supplied this candidate vaccine to our University of St Andrews collaborator for structural studies. We recently established collaoartion with ICL to validate this vaccine in vivo in an HCV infection small animal model In collaboration with the group at University of Naples Federico II, Naples we characterised monoclonal antibodies generated in animals following immunisation with a cyclic peptide representing the AP33 epitope for binding to the HCV glycoprotein E2 and for virus neutralising activity. In collaboration with the Intercollegiate Faculty of Biotechnology , Gdansk, analysed sera from animals immunised with the hepatitis B virus surface antigen-based virus-like particles displaying the AP33 epitope.
Collaborator Contribution Determined the crystal structure of the antibody AP33 bound to its epitope peptide and just recently recently, that of our vaccine candidate in complex with AP33. Further structural analysis of the latter is currently in progress. The University of Naples Federico II group generated antibodies to the cyclic variant of the peptide corresponding to the AP33 epitope and performed their structural analyses when bound to the peptide. Intercollegiate Faculty of Biotechnology , Gdansk, developed a hepatitis B virus surface antigen-based virus-like particles displaying the AP33 epitope for vaccine research Our collaborators at the ICL have shown protection from HCV infection in a chimeric human live mouse model following passive immunization with serum IgGs derived from mice vaccinated with our anti-idiotype antibody vaccine.
Impact We have recently crystallized MAb AP33 bound to its cognate epitope sequence which identified antibody residues essential for this interaction. Based on this we are generated mutated derivatives of this antibody which are proving very useful in our on-going studies on understanding the antigen-antibody interactions at both structural and biochemical levels (Paper published: PMID 22993159). As an extension to this work, we have a novel vaccine candidate capable of eliciting MAb AP33-like anitbodies in immunized animals. Furthermore, a crystal structure of this antibody-based vaccine candidate in complex with its ligand has just been generated. Further work (including in vivo evaluation of the vaccine) is in progress. Indeed, more recently our collaborators at the ICL have shown protection from HCV infection in a chimeric human live mouse model following passive immunization with serum IgGs derived from mice vaccinated with our anti-idiotype antibody vaccine. We explored the potential of cyclic peptides mimicking the AP33 epitope structure to elicit anti-HCV antibodies. Antibodiess that specifically recognize a cyclic variant of the epitope bound to soluble E2 with a lower affinity than other blocking antibodies and do not neutralize virus. The structure of the complex between one such antibody and the cyclic epitope, together with new structural data showing the linear peptide bound to neutralizing antibodies in extended conformations, suggests that the epitope displays a conformational flexibility that contributes to neutralization escape. Such features can be of major importance for the design of epitope-based anti-HCV vaccines (published PMID:26819303). Collaboration is multidisciplinary involving Protein biochemistry, molecular biology, structural biology and virology.
Start Year 2009
 
Description Structure of AP33 and related vaccine studies 
Organisation University of St Andrews
Department Centre for Biomolecular Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Generated and purified large quantities of the hepatitis C virus (HCV) monoclonal antibody (MAb) AP33 Fab fragment (a pan-genotypic virus neutralizing antibody) and supplied appropriate peptides to University of St Andrews for co-crystallisation studies. We used MAb AP33 as a template to reverse engineer an immunogen that induces similar antibodies upon vaccination. To our knowledge, this is the first time in the HCV vaccine field, the success of such a focused, structure-based approach has been demonstrated. We have supplied this candidate vaccine to our University of St Andrews collaborator for structural studies. We recently established collaoartion with ICL to validate this vaccine in vivo in an HCV infection small animal model In collaboration with the group at University of Naples Federico II, Naples we characterised monoclonal antibodies generated in animals following immunisation with a cyclic peptide representing the AP33 epitope for binding to the HCV glycoprotein E2 and for virus neutralising activity. In collaboration with the Intercollegiate Faculty of Biotechnology , Gdansk, analysed sera from animals immunised with the hepatitis B virus surface antigen-based virus-like particles displaying the AP33 epitope.
Collaborator Contribution Determined the crystal structure of the antibody AP33 bound to its epitope peptide and just recently recently, that of our vaccine candidate in complex with AP33. Further structural analysis of the latter is currently in progress. The University of Naples Federico II group generated antibodies to the cyclic variant of the peptide corresponding to the AP33 epitope and performed their structural analyses when bound to the peptide. Intercollegiate Faculty of Biotechnology , Gdansk, developed a hepatitis B virus surface antigen-based virus-like particles displaying the AP33 epitope for vaccine research Our collaborators at the ICL have shown protection from HCV infection in a chimeric human live mouse model following passive immunization with serum IgGs derived from mice vaccinated with our anti-idiotype antibody vaccine.
Impact We have recently crystallized MAb AP33 bound to its cognate epitope sequence which identified antibody residues essential for this interaction. Based on this we are generated mutated derivatives of this antibody which are proving very useful in our on-going studies on understanding the antigen-antibody interactions at both structural and biochemical levels (Paper published: PMID 22993159). As an extension to this work, we have a novel vaccine candidate capable of eliciting MAb AP33-like anitbodies in immunized animals. Furthermore, a crystal structure of this antibody-based vaccine candidate in complex with its ligand has just been generated. Further work (including in vivo evaluation of the vaccine) is in progress. Indeed, more recently our collaborators at the ICL have shown protection from HCV infection in a chimeric human live mouse model following passive immunization with serum IgGs derived from mice vaccinated with our anti-idiotype antibody vaccine. We explored the potential of cyclic peptides mimicking the AP33 epitope structure to elicit anti-HCV antibodies. Antibodiess that specifically recognize a cyclic variant of the epitope bound to soluble E2 with a lower affinity than other blocking antibodies and do not neutralize virus. The structure of the complex between one such antibody and the cyclic epitope, together with new structural data showing the linear peptide bound to neutralizing antibodies in extended conformations, suggests that the epitope displays a conformational flexibility that contributes to neutralization escape. Such features can be of major importance for the design of epitope-based anti-HCV vaccines (published PMID:26819303). Collaboration is multidisciplinary involving Protein biochemistry, molecular biology, structural biology and virology.
Start Year 2009
 
Description Studies on HCV p7 and NS5A 
Organisation University of Leeds
Department School of Molecular & Cellular Biology
Country United Kingdom 
Sector Academic/University 
PI Contribution Provision of reagents, contribution of experimental data, and provison of training to a PhD student
Collaborator Contribution Further enhanced our understanding of the functions of HCV p7 and NS5A
Impact Publications: PMID - 18828153; 19656877
Start Year 2008
 
Title Broadly Specific Anti-HCV Antibody (AP33) 
Description The invention relates to a surprising discovery that one well known anti-Hepatitis virus (HCV) antibody AP33 recognises and neutralises infection of all 6 known HCV genotypes. The use of the antibody in therapeutics and use of its epitope as a vaccine are covered in the claims. 
IP Reference WO2006100449 
Protection Patent granted
Year Protection Granted 2006
Licensed Yes
Impact AP33 antibody continues to be of great value to many HCV researchers around the world. Due to its unique properties, it has proven to be an excellent tool in functional studies on HCV glycoprotein.
 
Title Humanised AP33 - MRCT10 
Description This is the humanized version of AP33 which was developed in collaboration with MRC Technology's Therapeutic Antibody Group 
IP Reference WO2009081285 
Protection Patent application published
Year Protection Granted 2009
Licensed Yes
Impact This antibody has therapeutic potential against HCV. Indeed, it has been recently licenced by the MRCT to a pharma in China.
 
Title Mouse monoclonal antibody (MAb) AP33 and its humanized derivative, MRCT10 
Description MAb AP33 recognizes a highly conserved linear region of the hepatitis C virus E2 glycoprotein which is essential for virus entry into target cells. This antibody blocks the interaction of E2 with CD81, a cellular receptor essential for virus entry. It also neutralizes infection of cells with diverse viral genotypes and subtypes. Given its broadly neutralizing properties, MAb AP33 was humanized in collaboration with the MRCT Therapeutic Antibody Group. The latter has recently been licenced to a pharma for therapeutic development. 
Type Therapeutic Intervention - Drug
Current Stage Of Development Initial development
Year Development Stage Completed 2007
Development Status Actively seeking support
Impact The humanised MAb AP33, called MRCT10, is expected to be of direct clinical benefit for passive immunotherapy to reduce the risk of HCV infection after needlestick or other accidental exposure, and in the liver-transplant setting to reduce the incidence of graft re-infection. Furthermore, given its broadly neutralising properties, the epitope recognised by MAb AP33/MRCT10 can be considered a valid lead for vaccine design. 
 
Description Glasgow Science Festival - 'Outbreak' 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Type Of Presentation Workshop Facilitator
Geographic Reach Regional
Primary Audience Schools
Results and Impact Since 2012, approximately 80 high school students have attended a workshop where they investigate a fictional virus outbreak using molecular biology and diagnostic methods.

The workshop received excellent evaluation feedback from participants.
Year(s) Of Engagement Activity 2012,2013