Spontaneous clearance in Patients acutely infected with HCV - Immune profiling, Novel biomarkers and X-omics approaches | SPHINX | Project | News & Multimedia | FP7 | CORDIS

Final Report Summary - SPHINX (Spontaneous clearance in Patients acutely infected with HCV - Immune profiling, Novel biomarkers and X-omics approaches)

Executive Summary:
The SPHINX Consortium was a North / South partnership aimed at identifying novel biomarkers, and providing new insight into the mechanisms of spontaneous clearance of hepatitis C virus (HCV) during acute infection. Hepatitis C is a major public health problem with high incidence in regions such as Egypt and other Mediterranean Partner Countries (MPCs).
In line with the EC request, we applied “-omics” or hypothesis-generating approaches to perform: (i) multi-analyte profiling of plasma analytes; (ii) characterization of HCV-specific cellular immune responses during the clearance phase; and (iii) assessment of genetic and epigenetic variations that predict viral clearance. Our Consortium has been able to study the pathogenesis of acute HCV infection in regions where disease incidence is high and the recruitment of acute symptomatic HCV individuals permitted the establishment of relevant patient cohorts. We exploited and continue to exploit this unique position by assessing which host factors are critical for spontaneous clearance, by preventing HCV entry into uninfected cells or allowing for rapid targeting of HCV infected hepatocytes. Relevant host factors being analyzed by the Consortium include metabolic analytes, innate immune molecules and components of the cellular and humoral immune system.
Together, this has helped provide a framework for understanding the host response to acute HCV. In addition, we established a cooperative training program under the umbrella of the EU/MPC Research Collaboration – Fighting Hepatitis in Egypt, which enhanced training of the next generation of scientists in Clinical Epidemiology and Biomarker Discovery. Cross-Consortia collaborations with the EU funded HepaCute Project further enhanced the impact of our work. Additional impact from dissemination of the project relates to our consortiums efforts to have HCV genotype 4 recognised as a neglected infectious disease. We worked in collaboration with the G-Finder group which helped result in the official recognition of HCV genotype4 as a neglected infectious disease. Finally biomarker discoveries, both genetic and protein, from Sphinx are currently being applied to novel point of care based technologies as part of a follow up FP7 project PoC-HCV. This project will result in the implementation of biomarker based solutions for improved HCV patient management in Europe and resource limited settings.
In summary results obtained from this project will impact the management of acute hepatitis C in Egypt; provide critical tools for studying the host response to HCVg4; and though the delivery of greater insight into the mechanisms of spontaneous clearance, we have identified potential correlates of vaccine protection.

Project Context and Objectives:
The SPHINX Consortium was a North / South partnership aimed at identifying novel biomarkers, and providing new insight into the mechanisms of spontaneous clearance of hepatitis C virus (HCV) during acute infection. Hepatitis C is a major public health problem with high incidence in regions such as Egypt and other Mediterranean Partner Countries (MPCs).
In line with the EC request, we applied “-omics” or hypothesis-generating approaches to perform: (i) multi-analyte profiling of plasma analytes; (ii) characterization of HCV-specific cellular immune responses during the clearance phase; and (iii) assessment of genetic and epigenetic variations that predict viral clearance. Our Consortium has been able to study the pathogenesis of acute HCV infection in regions where disease incidence is high and the recruitment of acute symptomatic HCV individuals permitted the establishment of relevant patient cohorts. We exploited and continue to exploit this unique position by assessing which host factors are critical for spontaneous clearance, by preventing HCV entry into uninfected cells or allowing for rapid targeting of HCV infected hepatocytes. Relevant host factors being analyzed by the Consortium include metabolic analytes, innate immune molecules and components of the cellular and humoral immune system. The components of the Sphinx project were divided across 7 complementary work packages, described as follows:
WP1 Acute HCV Cohorts in Egypt
The main objective of WP1 was to maintain a well-characterized cohort of patients with acute hepatitis C in Egypt and identify patients with and without spontaneous viral clearance. This resulted in the creation of a large biobank of patient samples (plasma, DNA and RNA) collected from acute HCV patients followed from the time of presentation with symptomatic disease which were made available for work proposed in the other work packages.
WP2 Inflammatory and antibody signatures
The main objective of WP2 was to construct a signature of the inflammatory response for acute viral hepatitis using multi-analyte profiling (MAP) and validate plasma biomarkers that are predictive for spontaneous viral clearance. This was achieved through two complementary strategies, the first focused on multiplexed host protein biomarkers, and the second through the development of a novel patient classification strategy using antibody detection assays.
WP3 Analysis of cellular activation during spontaneous clearance of HCV
The main objective of WP3 was to define cellular signatures predictive of spontaneous clearance of acute HCV and provide mechanistic insight into the determinants of anti-viral immunity. This was achieved through High-throughput HLA-based assays and the identification of novel HCVg4 epitopes, testing of novel tools on patient samples, and through the definition of the cytokine production profile of HCV-specific CD4 and CD8 T cells.
WP4 Genetic analysis of polymorphisms correlating with spontaneous resolution of HCV
The main objective of WP4 was to identify the genetic polymorphisms pre-disposing HCV clearance vs. chronicity in an Egyptian population, which was performed by Candidate gene approach with replication studies, and in addition through an miRNA screen for evaluation of correlations with spontaneous clearance.
WP5 Training opportunities in Clinical Epidemiology and Biomarker Discovery
The main objective of WP5 was to provide training in clinical epidemiology and biomarker discovery. This was achieved through a Biomarker discovery workshop held for 2 weeks at the Institut Pasteur, Tunis and thoughout the course of the project numerous exchange programme for spreading excellence throughout the Consortium.
WP6 Project Management
The main objective of WP6 was to establish a Consortium that collaborates efficiently and works toward the stated objectives, while protecting intellectual property and fulfilling EC requirements. This was achieved through coordinated and regular communication and dissemination activities that included peer reviewed publications, presentations, and conference workshops.
WP7 Collaboration with HepaCute and STDF projects
The main objective of WP7 was to exploit synergies between EU and Egyptian hepatitis research programs during the proposed work. Furthermore, we propose to coordinate efforts for disseminating information about the public health priorities in Egypt and work towards a sustained cooperation between North and South research partners. This was achieved through joint meetings with the HepaCute consortium, as well as shared workshops at the EASL meetings.

Project Results:
WP1 Acute HCV Cohorts in Egypt

Objective: to maintain a well-characterized cohort of patients with acute hepatitis C in Egypt and identify patients with and without spontaneous viral clearance.

1.1 Dissemination of banked materials to members of the Consortium

Banked samples (plasma, DNA and RNA) collected from acute HCV patients followed from the time of presentation with symptomatic disease were made available for work proposed in WP2 and WP4. This shipment was made in anticipation of the initiation of the SPHINX project, and used to support initial biomarker studies. A second shipment was made in August 2011 to Institut Pasteur for dissemination to the relevant partners for continued biomarker studies.

A third shipment was made in April 2012. This shipment included the following:
− Extracted DNA for IL28B genotyping of patients to complete the genetic studies on IL28 and other SNP polymorphisms and their association to viral clearance in the acute infectious phase.
− Serum intake samples for the algorithm validation (predictive algorithm of early viral clearance in acute C hepatitis infected patients). These samples have been used to measure serums proteins using Luminex assays as defined in WP2.
− HCVg4 antibody studies using newly developed multiplex immunoassays on acute HCV serum samples and chronic patients (NMI WP2).

An analysis on acute hepatitis C patients with fluctuating viraemia was performed. This was not initially planned, but was identified to be of epidemiological importance. 596 samples have been retested by quantitative PCR.

A fourth shipment was sent in June 2012 and consisted of extracted DNA from
− HCV negative Blood donors for genetics studies (WP4)
− HCV EIA & NAT positive Blood donors (WP4)
− HCV EIA positive & NAT negative Blood donors (WP4)
This DNA collection was completed with other extracted DNA shipments sent in September 2013 and May 2014.

A plasma shipment was also made in November 2012.

Unfortunately we could not receive cells processed from HLA typed HCV EIA positive blood bags originally scheduled to be sent in May 2012. Official authorization for sending them abroad was too difficult to obtain in the recent political climate.

1.2 New recruitment of acute HCV patients

From March 2010 to March 2013, patients were included according to the following inclusion criteria:
− Presentation with fever and jaundice of less than 21 days in duration.
− ALT ≥3 x ULN.

We added 2 new fever hospitals (Assouan and Alexandria) to the initial two hospitals of Cairo to increase our recruitment capacity. Taken together, 1 637 patients were enrolled in the 4 centres, as summarized in the following table:

Abbassia Imbaba Assiut Alexandria Total
No. of Patients (%) 256 (16) 272 (17) 544 (33) 565 (34) 1 637
Male (%) 162 (63) 165 (61) 409 (75) 402 (71) 1 138 (67)
Age
min-max mean
[18-65] 30
[18-60] 41
[18-71] 29
[18-59] 34
[18-71] 32
Table 1. Numbers of new patients recruited in the 4 fever hospitals between March 2010 to March 2013.

The frequency over time of the recruitment is shown in Fig 1.1 While recruitment was impacted by the political events during winter 2010-11, we have managed to achieve the majority of the objectives proposed in the initial application. Among those 1,637 enrolled patients with acute hepatitis, 382 were diagnosed as HCV PCR positive (Fig 1.2). The recruitment of acute C infected patients followed the same scheme of inclusion over time as the whole population, with a decrease during the winter 2010-11 due to the political events happening at that time in Egypt (Fig 1.3).

1.3 Follow-up and management of patients

The project had a standard procedure for following-up patients who present with symptomatic acute HCV infection. This definition was reviewed by leaders of the SPHINX and HepAcute Consortia at the beginning of the project and a consensus definition was applied to both projects. All patients with definite or probable acute hepatitis C were referred to the LDRU for clinical and biochemical follow-up. Patients were offered free medical treatment during the follow-up, as well as during 8 visits (week 4, 8, 12, 26, 52, 78, and 104, and after symptoms). At each time point, HCV viral load was assessed in order to determine the disease course and if needed treatment recommendations were made. Among the 382 recruited HCV PCR+ patients, 200 (52%) were diagnosed with acute hepatitis C, and followed as shown in Fig 1.4.

The main objective of WP1 was the recruitment of 150 patients with acute hepatitis C over the three-year period of the project (i.e. corresponding approximately to 4 patients per month), with 50% having a follow-up of at least 6 months. We achieved 200 patients with acute hepatitis C of whom 50% were followed-up for at least 3 months and 38% for least 6 months. Follow-up rates were lower than those expected but 73 patients with complete follow-up is very close to the 75 expected and this is in fact a great success considering the difficult political context.

1.4 Data management

Clinical and biological data were compiled for the acute HCV cohort by the team of Mohamed Abdel-Hamid. Maha Gafay was recruited as data manager for the acute hepatitis C cohort at Ain Shams University. She is responsible for gathering biological, clinical and epidemiological data for the main database.

1.5 Processing of blood and HLA typing (Rafik)
As part of routine work for the cohort, whole blood analysis and separation is performed according to standard operating procedures for isolation of PBMCs and plasma. An aliquot of the patient PBMCs is stored in Trizol for RNA extraction; an aliquot (from one time point) is stored for genomic DNA extraction and molecular haplotyping; and the remainder of the cells are frozen and stored in liquid nitrogen for cellular profiling and functional studies. We began with samples from the Cairo fever hospital (TMRI) after which recruitment began from Alexandria and Assiut. Finally in February recruitment began also in Minia and Tanta.

• HLA Typing
Due to the lack of information on the normal haplotypes of healthy Egyptians 126 normal healthy Egyptians were haplotypes for both HLA Class 1 A and B by the SSOP technique (INNO-LiPA by Innogenetics). This was completed in May 2011. The rationale behind this was to provide insight into HLA alleles with a high frequency within the Egyptian population which is necessary for the development MHC tetramer-based assays for WP3. The frequency of HLA-A and HLA-B alleles in healthy Egyptians is represented in Fig 1.5.

1.6 Oversight of epidemiological studies and analysis
Clinical and sample databases, and documentation of patient recruitment were followed twice a year by Arnaud Fontanet’s team (L Le Fouler, A Bernier IP Paris). It included data cleaning, queries to the epidemiological team in Egypt to clarify the data when needed, selection of patients and samples for the consortium to request the necessary samples for analyses, and bi-annual reports to follow patient recruitment.

A case control study has been performed to identify current risk factors for hepatitis C infection. Multivariate models were built to identify risk factors associated with HCV transmission among non-drug users and drug users separately. Among non-drug users, hospital admission was independently associated with acute HCV infection. Among drug users, risk factors were: multiple sexual relations, intravenous drug use and shaving at the barber. Illiteracy and marriage were significant risk factors in both groups. This study showed that invasive medical procedures are still a major risk for acquiring new HCV infections in Egypt. An analysis on acute HCV patients with fluctuating viremia has been performed to characterize fluctuating (or intermittent) viremia and to assess the threshold for defining chronicity among PCR+ patients over time. Viral load dynamics according to viral clearance patterns are shown in Fig 1.6.
Changes in lipids and glucose during acute phase according to viral clearance patterns support findings in the scientific literature, as shown in Fig 1.7.

WP2 Inflammatory and antibody signatures

Objective: To construct a signature of the inflammatory response for acute viral hepatitis using multi-analyte profiling (MAP) and validate plasma biomarkers that are predictive for spontaneous viral clearance.

2.1 Assay development and extension of biomarker screen (Schneiderhan-Marra)
At EDI miniaturized multiplexed sandwich immunoassays were implemented to quantitate a number of serum analytes, requiring only limited amounts of patient samples. Prior using these assays for precious sample screening, assays were analyzed to demonstrate the performance and the reliability of the assays. Assay validation was performed according to guidelines edited by regulatory bodies (e.g European Medicine Agency, 2009; 2001 and Food and Drug Administration 2013). Calibration curve validation ensures that the curve fitting model used for the calculation of the standard curve is adapted to the assay and provides a robust description of the concentration-response of the assay.

Recovery was assayed using spiked sample studies using an appropriate matrix to visualize potential sample matrix effects. Recovery evaluations were made by using human serum and plasma samples with no or low analyte concentrations. These samples were spiked using a minimum of three different concentrations of the standard curve reagent (low, mid, high). These three types of reference samples were always included as duplicates within every microtiter plate of samples during the screening procedures. The recovery values for these samples were typically within 80-120% over the working range of the assay. We assessed Linearity/Reportable ranges by using plasma or serum with a measureable concentration and dilute with matrix or our sample diluent. The assays revealed a linear range over the range of dilutions necessary to drive the measured concentration in a patient sample into the quantification range. Precision was analyzed using intra- and inter-assay experiments. We compared replicates of samples with a measurable concentration within one run on the same day, and on independent runs on 3 different days. Lower limit of detection (LOD) was defined as the concentration of an analyte that is significantly different from a blank sample + three standard deviations (SD). The functional sensitivity LLOQ- ULOQ was reported accurately determined based on spike in experiments. We considered an LLOQ for these assays, which reveal a recovery within 75-125%, combined with the back-calculated concentration of the lowest calibrator revealing a CV below 20%. The ULOQ was reported accurately based on the reproducibility of spike in results where recovery was within 75-125% and the back calculated concentration of the highest calibrator revealed a CV below 20%. These values for 47 analytes are shown in Table 2. These assays were applied to HCV samples and analyzed as outlined below.

Apolioproteins as biomarkers of HCV clearance
Based on previous findings identifying the importance of lipoproteins in the response to HCV we examined in detail a panel of 11 apolipoproteins in a cohort of aHCV-infected individuals over the time course of infection. We identified three apolipoproteins; apolipoprotein H (apoH), D (apoD) and CIII (apoCIII) that were significantly associated with viral clearance. Increased levels of these apolipoproteins in the plasma of patients was correlated with spontaneous viral clearance through the acute phase of HCV infection (Figure 2.1). Interestingly, neither apoH, nor apoD have previously been identified as co-factors or biomarkers involved in HCV replication or clearance. ApoCIII has been previously identified as a possible serum biomarker for the response to therapy in the context of chronic HCV infection, both validating our analytical approach and suggesting that there may be similar immune mechanisms involved in both acute and chronic infection. Strikingly, plasma apoH levels remained consistently higher throughout follow-up among patients who cleared the virus, as compared to those who remained persistently infected. These results introduce a new apolipoprotein, apoH, as possible co-factor of HCV pathogenesis and biomarker of spontaneous viral clearance. These results have recently been published in J Hepatol. 2014 Jun 3. pii: S0168-8278(14)00388-2.

2.2 Focusing of multiplexed assays and validation studies on the newly recruited cohort (Joos)

We employed certified multiplexed protein immune assays to identify biomarker signatures of viral hepatitis in order to define unique and common responses for three different acute viral infections of the liver. We performed multi-analyte profiling, measuring the concentrations of 182 serum proteins obtained from acute Hepatitis A (18), B (18), and C (28) infected patients. Virus specific biomarker signatures were identified and validation was performed using a second independently recruited patient population. A core signature of 46 plasma proteins were commonly modulated in all 3 infections, as compared to healthy controls. Principle Component Analysis (PCA) revealed a host response based upon 34 proteins, which could distinguish HCV patients from HAV and HBV-infected individuals or healthy controls (Figure 2.2 left). When HAV and HBV groups were compared directly, 34 differentially expressed serum proteins allowed the separation of these two patient groups. A validation study was performed on an additional 111 patients, to confirm the relevance of our initial findings and helped define the 17 most robust analytes that reproducibly segregated the patient populations (Figure 2.2 right). These analytes with their associated p and q values as measured by ANOVA are listed in Table 3. This combined discovery and biomarker validation approach revealed a previously unrecognized virus-specific induction of host proteins. The identification of hepatitis virus specific signatures provides a foundation for functional studies and the identification of potential correlates of viral clearance.

These results were published in Hepatology 2014 Apr;59 (4):1273-82.
2.3 Development of a novel patient classification strategy using antibody detection assays (Joos)

Antigens for assay setup – proteins and peptides
Different HCV proteins were required to set up the serological assay. The target HCV proteins were selected by the Institut Pasteur based on the consensus HCVg4 outlined in WP3.1. As these proteins are not commercially available, plasmids vectors were created and the recombinant form of the proteins (CORE, NS3, NS4a, NS4b, NS5a, NS5b; HCV genotype 4ga) were expressed at the NMI.

Firstly the protein sequences selected for expression were optimized based on hydrophobicity and immunogenicity plots, thus increasing the chance of expression in bacteria. Additionally, all gene sequences were codon-optimization for expression in E.coli to improve expression yield. The selected DNA sequences were synthesized and cloned into pET-28 expression vectors containing a 6x-His Tag for affinity purification. E. coli hosts were transformed with the plasmids for vector amplification. Purified plasmids were analyzed by restriction digest to verify the identity of the insert by its size using gel-electrophoresis.

Recombinant expression of His-tagged HCV genotype 4a proteins (CORE, NS3, NS5A and NS5B) in E. coli and purification by affinity chromatography using Ni-NTA columns on the ÄktaExpress system was performed at the NMI. Although different E. coli strains and different plasmids were tested, recombinant expressions of NS4A and NS4B proteins were not satisfying. For compensation of these proteins two commercially available NS4 mosaic proteins were purchased and the small NS4A protein was also synthesized as three overlapping peptides. Additionally other peptides (c22, 5-1-1 and c100), which are used in the CHIRON RIBA HCV 3.0 SIA, were produced. For the 5-1-1- peptide differences in the sequence of genotype 1a and 4a were observed and both sequences were used.

Furthermore commercial available HCV antigens from different HCV genotypes and antigens from other pathogens (HAV, HBV, HDV, HEV, EBV, CMV, T. gondii and H. pylori), which also cause unspecific symptoms like fever and/or hepatitis, were included in the assay setup.

Development of a novel HCV serological test and a classification system to discriminate between acutely and chronically infected patients
A multiplex assay for the classification of acutely or chronically HCV infected patients was developed on the Luminex platform. Therefore 25 antigens (different genotypes, different manufacturer) of the Hepatitis C virus were immobilized on color-coded beads. All beads were incubated with human serum or plasma samples obtained from HCV infected specimens, allowing anti-HCV antibodies to bind their antigen. Parallel runs were performed for the detection of human IgG or human IgM antibodies directed against the immobilized antigens.

The development of the assays included optimization of sample dilution, buffer and matrix conditions, and testing of suitable detection antibodies. Both assays were technically validated before the first screening. In the first sample screenings detection of both, IgG and IgM antibodies directed against the pathogen antigens, were performed. In the context of HCV infection, the IgM assay seems to be not meaningful and not of sufficient sensitivity, therefore no further work has been done on this assay.
With the IgG assay a screening of 227 samples (Table 4) was performed to generate a classification system for the discrimination of healthy donors, acutely and chronically infected HCV patients.

After first screening, the initial set of 25 HCV antigens was reduced to 12 antigens (3x CORE region, 3x NS3 region, 3x NS4 region, 3x NS5 region), which were identified to be essential for the classification system. The classification was designed as a two-step system. The first step leads to the primary test result (see confusion matrix 1), which is a classification into negative, indeterminate or positive result according to the ‘CHIRON RIBA HCV 3.0 SIA’ and ‘INNO-LIA HCV Score’ tests. For the classification some of the criteria as described for the above tests were adopted, some were slightly changed and new assessment criteria were implemented.
Overall 93.4% of the instances were correctly classified, 3.5% were indeterminate and 3.1% were falsely classified. Regarding the sensitivity and the specificity good results were achieved: 96.6% sensitivity and 97.3% specificity. Numbers of the indeterminate classified samples cannot be integrated in these calculations.

The most difficult part of the classification is the discrimination of healthy donors and early acutely infected HCV patients. No samples from the chronic HCV patients were classified as negative. However, it should be mentioned that the clinical status is based on HCV PCR data, but negative PCR does not necessarily mean negative antibody testing and positive PCR samples need not be positive in antibody testing. Due to the fact, that there was no according ELISA or SIA data for the samples available, a comparison of antibody testing results could not be performed. The secondary test result provides information whether a HCV patient is acutely or chronically infected. Therefore only the positively classified patients from the primary test results were used. The achieved results are presented in the confusion matrix 2. Overall 82.1% of the instances were correctly classified. These early results are considered as a favorable result as the discrimination of acutely and chronically HCV infection can only be based on the antibody profile of a sample.

Development of a multiplex serological test for 9 infectious pathogens
As mentioned in the section “antigens for assay setup” other pathogenic antigens were included in the multiplex assay setup. The aim of this assay is to have just one test that requires to be performed, to identify which pathogen is causing acute symptoms (fever and/or hepatitis) of a patient, in Egyptian fever hospitals. In total, the assay was set up with antigens of 9 common pathogens (Hepatitis A/B/C/D/E virus, Epstein Barr virus, Cytomegalovirus, Toxoplasma gondii and Helicobacter pylori) so that the serological profiles of these pathogens could be monitored simultaneously.

As an example for the multiplex serological test for 9 pathogens a single patient kinetic (out of 15 currently tested) is shown in the Figure 2.3. An increase of antibodies directed against some HCV proteins was observed over the time, whereas almost no changes were seen for the other pathogens. The seroconversion is dependent on the individual immune response and often can only be observed, if the first time point is dated correctly. In general, this concept has shown promise and the technical validation of the assay showed stable results (mean of intra-assay variation on all antigens and tested samples (n=3) + Blank in 20 replicates: 5.7% CV, mean of inter-assay variation on all antigens and tested samples n=6 + Blank in 4 runs measured in triplicates: 5.4% CV). For multiplexed assays the clinical validation is often difficult and requires an extensive set of samples, which were not completely available before the end of the project reporting. Such validation tests will be completed after the project duration, and we are seeking additional financial support to continue the development. Multiplexed pathogen detection tests such as described herein have a high potential application and are especially interesting for countries like Egypt. Therefore a performance verification of this serological test for 9 pathogens with suitable samples is still planned and currently ongoing.

WP3 Analysis of cellular activation during spontaneous clearance of HCV

Objective: To define cellular signatures predictive of spontaneous clearance of acute HCV and provide mechanistic insight into the determinants of anti-viral immunity.

3.1 High-throughput HLA-based assays & identification of HCVg4 epitopes (Schumacher)

MHC-based technology to described HCV-specific CD8 T cell immunity
Results of the Rafik team (Ain Shams University, Cairo) have demonstrated that the Egyptian population expresses a great variety of HLA types. Within these HLA types, there is substantial further variability, in the form of HLA micropolymorphisms. This makes it necessary to understand to what extent such micropolymorphisms influence the interaction of peptide-HLA complexes with T cell receptors and – when this is significant – develop technology to allow T cell monitoring for a large series of HLA class I micropolymorphic variants.

Confronted with the above mentioned extensive HLA subtype variability we developed HLA peptide exchange technology for a large series of HLA A*02 subtypes (together with our collaborator G. Grotenbreg, Singapore). Specifically, we developed MHC peptide exchange technology for a series of eight different HLA A*02 subtypes. To subsequently assess whether HLA micropolymorphisms influence T cell interactions, we have created HLA multimers for each of these HLA A*02 subtypes with 10 different peptide antigens for which we have generated HLA A*02:01 restricted T cell clones.
Importantly, whereas this set of T cell clones was reliably stained with the matched HLA A*02:01 multimers, staining with multimers for other HLA A*02 subtypes was highly variable. An example of such differential staining is shown in Figure 3.1A in which a HLA A*02:01 restricted EBVGLC T cell clone was stained with the different HLA A*02 subtype multimers. All other analyses are summarized within Fig 3.1B. Furthermore, we have also documented that this micropolymorphic variation affects T cell detection in large scale screening efforts (van Buuren et al, 2013).

To evaluate whether the observed differential staining is a result of altered peptide MHC binding or of altered binding of the T-cell receptor to the pMHC complex, we have performed a large series of pHLA ELISAs, to reveal which peptides are able to stabilize the different subtype HLA complexes. This analysis reveals that in most cases, impaired T cell detection is not due to altered peptide binding, but rather by alteration of the TCR exposed surface (Figure 3.1). Collectively, these data reveal that HLA micropolymorphisms strongly affect pHLA based T cell monitoring but also provide the tools to solve this issue for HLA A*02 subtypes that are common within the Egyptian population.
Having generated the biochemical tools to allow T cell monitoring for HLA A*02 subtypes that are common within the Egyptian population, we have in parallel invested in establishing the optimization of algorithms that can be used to predict which peptides from HCV genomes are most likely to represent T cell epitopes. To this end, we assessed the added value different optimization strategies, using a set of well-established HIV epitopes for benchmarking. The results of this benchmarking study (van Buuren et al., unpublished) very clearly demonstrate that epitope predictions on the basis of only the predicted HLA binding capacity still have a relatively low specificity. Importantly, specificity can be shown to increase substantially through the simultaneous use of algorithms that describe the likelihood of proteasomal processing, and that describe the dissimilarity of the peptide to the endogenous peptide repertoire. This optimized prediction algorithm facilitates the identification of relevant HCV epitopes, but will also be of value to predict T cell epitopes in other human diseases.

3.2 Evaluation of HCVg4-specific T cell responses across the patient cohort (Albert)
Due to political and logistic challenges in shipping HCV patient cells out of Egypt, we were unable to evaluate the newly developed HCVg4 specific T cell tools on the acute HCV patient cohort. Nevertheless a backup strategy was implemented, which was to collect and analyse patient samples from Parisian HCV cohorts. This has allowed us to validate our tools, which we plan to ultimately test on acute patient samples.

First, a HCV g4a consensus sequence was defined based on a 9 amino acid full length isolate collected in Egypt (source : Los alamos National Laboratory HCV database). It was compared with the HCV g1a reference sequence (H77 strain), and results from epitope discovery prediction tools (Artificial Neural Network, NetMHCpan). Accordingly, three groups of potential epitopes were identified. Group 1 consists of 17 potential epitopes whose sequences are 100% identical in both genotypes. Most of them have been already tested in previous studies on g1 patients, and are in general only occasionally and weakly immunogenic. Group 2 consists of 12 epitopes sometimes identified in HCV genotype 1 patients, and whose sequences are very close in the genotype 4 as compared to g1. Group 3 are 144 neo-epitopes present uniquely in genotype 4, as compared to the genotype 1 sequence. To this list, 11 peptides recently described as immunogenic in the hepatocellular carcinoma were added.

These in silico predicted epitopes have been synthetized in Ton Schumacher’s laboratory in Amsterdam, and tested for their in vitro binding capability by using the peptide exchange technology as a screening tool (Figure 3.4). Elisa plates are coated with unlabeled streptavidin, then incubated with the A2 photocleavable monomer. Peptides to be tested are dispensed in each well, as well as peptides for calibration (high, medium and low affinity peptides). The plate is then put one hour under the UV lamp to disintegrate the stock complex. Monomers will be rescue only if the tested peptide has a sufficient affinity. Detection of refolded molecules is performed by using an HRP-labelled anti-hb2m antibody which is specific to the folded conformation of the hβ2m. In that way, only refolded multimer will be detected, and the intensity of the signal, calibrated based on the known peptides, will give a semi-quantitative appreciation of the in vitro binding capability of a given epitope. With this screen, 31 predicted epitopes were excluded, all from group 3. Altogether, we now have with a list of 173 potential epitopes to be screened on HCV patients (Figure 3.4).
Tetramer assay standardization
In parallel, and in an attempt to evaluate and improve our practice using multimer assays, we participated in testing proficiency panels organized by the Cancer Immunotherapy (CIMT) Association (http://cimt.eu). The principle behind this project is to evaluate and standardize multimer staining practices across European laboratories. In doing so we have improved conditions for our assays while contributing to standardisation of this important practice. For example, in the first proficiency panel (CIP_ID12) known amounts of TCR-transgenic T cells were spiked into constant amount of healthy PBMCs, thus generating null- (0 spiked cells), mid- (1:14 ratio), or high- (1:7 ratio) frequency artificial antigen-specific populations. These samples were evaluated for intra (in two separate experiments) and inter laboratory variation using standard staining protocols. In the second proficiency panel (CIP_ID13) the staining capacities of four distinct fluorochromes used in the same sample (PE, APC, QD605, QD705) were evaluated. Also examined was inter laboratory variability for the detection of 4 antigen-specific T cell populations using the same multimers with calibration beads as internal controls. Altogether, participation to these external quality controls was very useful for optimizing our knowledge and practice on antigen-specific CD8 T cell detection.

3.3 Define the cytokine production profile of HCV-specific CD4 and CD8 T cells (Barnaba)

Tuning of regulatory T cells by tumor or non-tumor signals in HCV infection.
In this study, we provided the first comprehensive analysis of Treg phenotypic and functional complexity within diverse human tissue microenvironments, characterized by chronic inflammation, fibrosis or oncogenic transformation, within the same organ (i.e. the liver). We found that cirrhosis and tumor microenvironments favour the expansion of CD4+CD127lowFoxp3+ Tregs, as compared with those infiltrating non-cirrhotic tissues or derived from peripheral blood (Fig. 3.5). In addition, tumor or cirrhotic Tregs express significantly higher levels of membrane OX40 (Fig. 3.6) and produced significantly lower levels of IFN-γ (Fig. 3.7) OX40 thus representing a marker of Treg stability and specialized Th1-suppression. Indeed, OX40+ Tregs T-bethighIFN-γ– performed higher suppression function than the OX40– T-bet+IFN-γ+ ones and correlated with a molecular signature of Treg stability, including higher expression of Helios (a transcription factor associated with Treg molecular and functional stability) and CD39 (Fig. 3.8). On the contrary, chronic HCV-associated liver inflammation in the absence of cirrhosis provides a series of pro-inflammatory signals that rather promoted the preferential diversion of Tregs into Th1-like cells (OX40–, Helioslow, CD39–,low suppressive function) (Fig. 3.5-3.8). In addition, the finding that the percentage of OX40-expressing Tregs is positively correlated with Treg frequency and proliferation ex vivo supports the idea that OX40 can provide signals contributing to Treg survival and expansion in cirrhotic or tumor microenvironments in humans. Taken together, these data suggest that the cirrhotic microenvironment favours the recruitment and expansion of committed Tregs, thus establishing a state of immune tolerance, ultimately contributing to the evolution of cirrhosis into cancer. By contrast, recruitment and expansion of unstable Th1-like Tregs in the inflammatory liver microenvironment would contribute to amplify inflammation rather than suppression.
The natural ligand of OX40 (OX40L) can be expressed by a variety of cell types including macrophages. We showed that M2-like mono/MF express high OX40L levels and expand highly suppressive OX40+ Tregs, directly by the OX40/OX40L interaction. These mono/MF spontaneously produced TNF-α and IL-10, a phenotype resembling IL-10-producing M2-like tumor-associated macrophages and reminiscent of chronic, low-dose, TNF-α exposure that plays critical roles in tumor-promoting inflammation. However, such “alternatively activated” macrophages appear equally distributed in NT-LIVnc and NT-LIVc/TUM districts, suggesting that other signals likely drive the differential accumulation of Th1-like versus Th1-suppressing Tregs in distinct contexts. Contrary to OX40L, the expression of its receptor OX40 in Tregs is indeed differently modulated in distinct microenvironments, being expressed at higher frequency in cirrhosis and tumor. Not only OX40L itself, but also other signals may be involved in OX40+ Treg accumulation at those sites. Among others, we have underscored a negative role for IFN-γ in OX40 up-regulation in vitro, an observation suggesting that, in NT-LIVnc, OX40+ Treg frequency could be maintained at lower levels by the higher IFN-γ amounts produced by Th1 cells in that context, which are sustained by IL-12. Conversely, in cirrhosis and cancer, OX40+ Tregs are allowed to accumulate and to interact with OX40L+ mono/MF. In turn, OX40L may stabilize their phenotype, further increasing their proliferation and OX40 expression and counteracting Th1-like plasticity. Therefore, the dichotomy in the distribution of Th1-suppressing and Th1-like Tregs in different districts may be, at least partly, attributable to the interplay between OX40/OX40L axis and type-1 cytokines, especially to IL-12 that is available in NT-LIVnc, but not in NT-LIVc/TUM districts (probably depending on both quality and quantity of the inflammatory response in each tissue), and can achieve a significant polarization of human Th1-like Tregs in vitro. This observation recommends the possible usage of compounds stimulating IL-12 production in tumors in order to favor the generation of anti-tumor effector cells rather than immunosuppressive committed Tregs. In tumor/pre-tumor contexts, the paucity of type-1 cytokines may not only prevent an extensive Th1-like Treg polarization, but also allow OX40 expression (which is inhibited by IFN-γ) and the OX40/OX40L interaction with OX40L+ mono/MF that can contribute to the expansion of OX40+ Tregs only in those districts. In turn, OX40L signal can help amplifying and fixing this Treg phenotype by fostering Treg proliferation, inhibiting Treg plasticity, and up-regulating its own receptor OX40.

OX40L expression by mono/MF directly correlated with HCV viral load. We are tempted to speculate that high HCV load may support tumor progression also through OX40L-mediated local Treg expansion and the resulting immune suppression. This hypothesis can account for the observation that both OX40+ Treg expansion and high mono/MF-OX40L expression in cirrhosis or tumor was peculiar of CHC, but not of hepatitis B or alcoholic fatty liver disease pts. Further studies are in progress to verify if Treg expansion in tumor and cirrhosis is dependent on the synergistic effects by OX40 and antigen-specific (HCV-related? Tumor-associated?) signals.

Our results support the notion that Helios may label truly committed Tregs with stable FOXP3 expression and demethylated TSDR, which are less susceptible to diversion into Th1-like cells, but are more prone to up-regulate OX40, to proliferate and to become specialized Th1-suppressors. In particular, while Helios appears to mark committed Tregs as developmentally determined, OX40 may be viewed as an activation marker, expressed preferentially by committed Tregs infiltrating tumor or pre-tumor microenvironments (i.e. HCC and cirrhosis). Therefore, the predominant effect of OX40 triggering in vivo would be the expansion of highly suppressive OX40+ Tregs, which in turn would favor tumor progression, an issue that should be carefully taken into account when designing OX40-targeted anti-cancer immunotherapies. In this context, targeting, rather than triggering, the OX40 receptor may represent a suitable strategy to block Tregs in cancer immunotherapy in humans (at least in HCC), by eliminating/paralyzing tumor-associated OX40+ Tregs, comprising specialized Th1-suppressing and committed (Helioshigh) cells, while sparing systemic immune homeostasis, maintained by peripheral Helios+ Tregs not expressing OX40. In conclusion, our data point out that human Tregs phenotypically and functionally adapt to microenvironmental signals in liver tissues affected by peculiar pathological conditions, and suggest that a global comprehension of Treg heterogeneity and plasticity is mandatory to well-designed therapies for chronic inflammatory diseases and cancer.

3.4 Evaluate determinants of immune exhaustion (Barnaba)

Chronic Immune activation, CD8 T cells specific to apoptotic epitopes, and HCV infection
We hypothesised that in chronic inflammatory conditions, such as chronic infections and systemic autoimmune diseases, a large number of T lymphocytes are activated and undergo apoptosis after performing their effector functions. We demonstrated that only apoptotic activated T cells retaining CD40L can induce dendritic cell (DC) maturation. Therefore, when activated apoptotic T cells expressing CD40L are engulfed by DC, the latter are able to cross-prime CD8+ T cells specific for the antigens derived by apoptotic cells. We have demonstrated by two-dimentional electrophoresis (2DE) that the proteome of apoptotic T lymphocytes comprises caspase-cleaved proteins and the majority of them are cross-presented by DCs to a wide repertoire of autoreactive CD8+ T cells (Rawson, Nat Med 2007). Live T cells as well as apoptotic T cells proteome includes proteins derived from cytoskeleton, cytoplasmic, or nuclear structures (e.g. vimentin, non-muscle myosin, Rho-GDP-DI-2, human nuclear ribonucleoprotein K, lamin B1, actin cytoplasmic 1), as identified by mass-spectometry-based proteomics approach (Rawson, Nat Med 2007). However, while in live cells we found only entire proteins, in apoptotic cells we observed fragments of those proteins derived from caspase cleavage. The crosspriming of CD8+ T cells specific for the antigens derived by the caspase cleavage generates a pool of CTLs that participates to the maintenance of a low-level grade of chronic inflammation and induces a vicious cycle when they undergo apoptosis as well. This mechanism has been already demonstrated by our group in HIV (Rawson, Nat Med 2007) and acute HCV infections (Franceschini D, PLoS Pathog 2012). The results on HIV patients showed increased apoptotic epitope-specific responses that were directly correlated with the number of circulating apoptotic CD4+ T cells, inversely correlated with the absolute number of circulating live CD4+ T cells, and ultimately correlated with the disease progression (Rawson, Nat Med 2007). The results in acute HCV subjects showed that polyfunctional (Th1, Th2, Th17) CD8+ T cell responses were robust in acute HCV patients and were significantly higher in those that experienced a chronic evolution (Franceschini D, PLoS Pathog 2012).

Objectives
To evaluate the role of apoptotic-epitope (AE)-specific CD8+ T cells in subjects with chronic HCV in treatment with Peg-Interferon-alpha (Peg-IFN) and Ribavirin (RBV) in order to demonstrate:
• Any possible role in liver disease progression (inflammation/fibrosis)
• Any possible role as predictive marker of favourable response to treatment

To characterise the phenotype, the state of activation/exhaustion and the effector functions of AE-specific CD8+ T cells. Blood samples were collected at baseline and after 1 and 6 months of therapy from HLA-A2+ subjects with HCV. IFN-g responses against 12 pools containing a total of 90 synthetic HLA-A2-binding apoptotic peptides and 10 pools containing 60 HLA-A2-binding HCV-specific peptides were analysed by ELISPOT assay at each time point. 
The frequency of AE- and HCV- specific CD8+ T cells were evaluated by using dextramers of HLA- A*0201 molecules complexed to 5 apoptotic peptides (non-muscle myosin, vimentin, actin) and 4 viral peptides (NS3 1406-1415, NS4b, NS5b, core) and were analysed by flow cytometry. AE- and HCV- specific CD8+ T cells function was analysed evaluating the cytokine production (IFN- gamma, IL-17) and degranulation (CD107a) in response to specific stimulation, by intracellular staining and flow cytometry analysis. Then, AE and HCV- specific CD8+ T cells were characterised looking at the expression of CCR7 and CD45RA, dividing them in four subsets: naïve (CCR7+CD45RA+), central memory (CCR7+CD45RA-), effector memory (CCR7-CD45RA-) and effector memory CD45RA+ (CCR7-CD45RA+). We evaluated also the proliferation (Ki67) and the expression of markers such as PD1, Tbet, Eomes, HLA-DR, CD69, CD95 in order to better characterise the activation state of apoptotic epitope specific CD8+ T cells.
Patients had higher AE- (Fig. 3.9) and HCV-specific (not shown) CD8+ T cell frequencies than controls at all times. Moreover, AE- (Fig. 3.9) but not HCV-specific (not shown), CD8+ T cell frequencies significantly increased after only 1 month from the starting of therapy with pegIFN and ribavirin. Patients were divided into Relapsers (R) and Sustained Virological Responders (SVR) based on treatment response (Fig. 3.10). At baseline, no difference between NR and SVR was observed in HCV- and AE-specific CD8+ T cell frequencies. However, while HCV-specific CD8+ T cells increased but not significantly (P=0,05) only after 6 months of therapy in R, AE-specific responses significantly increased only in R starting from the 4th week of treatment (Fig. 3.10 A,B).

At baseline, PD1 expression in autoreactive CD8+ T cells was higher in NR patients compared to SVR suggesting an increased IFN-I signature only in this group. Both AE- and HCV- specific CD8+ T cells produce small amount of cytokines in chronic HCV infection but degranulate (CD107a) and proliferate (Ki67) sensitively.

Our data suggest a possible involvement of AE-specific CD8+ T cells in chronic immune activation and disease progression in HCV. Moreover, patients that fail to respond to treatment may have a heightened IFN-I signature that increase with Peg-IFNα/Ribavirin (Sarasin-Filipowicz M, PNAS 2008) and autoreactive CD8+ T cells could represent a marker of this phenomenon. IFN-I signature is characterized by immunosuppression and hyperimmune activation (Sarasin-Filipowicz M, PNAS 2008). As AE-specific CD8+ T cells express more PD1 in NR patients, this may signify that these cells are more sensitive to the IFN-I signalling pathway and participate to the hyperimmune activation. The increase of AE-specific CD8+ T cell frequencies may predict an unfavourable response to therapy in HCV infection. Moreover, they may reveal patients with increased IFN-I signature that could benefit from interferon-free therapies.
WP4 Genetic analysis of polymorphisms correlating with spontaneous resolution of HCV

Objective: To identify the genetic polymorphisms pre-disposing HCV clearance vs. chronicity in an Egyptian population.

4.1 Candidate gene approach with replication studies (Abel)

The objective of this work package was to identify the human genetic variants controlling the HCV clearance / chronic infection phenotype in an Egyptian population. It was based on a two-step strategy:

(i) A hypothesis-based candidate gene approach providing a detailed exploration of some candidate genes, in particular IL28B.
(ii) A hypothesis-generating genome-wide exploration that should be conducted at the end of the grant.

During the first part of the project, we conducted the hypothesis-based approach. We selected a total of 42 candidate genes involved in anti-viral (in particular anti-HCV) responses (list in table 5). Single nucleotide polymorphism (SNP) selection within those candidate genes was based on tag SNP information publicly available from the International HapMap project (http://www.hapmap.org/). Tag SNPs were selected in order to ensure a high coverage for SNPs with minor allele frequency (MAF) >0.05. While no Egyptian population is included in the HapMap database, it is interesting to note that our previous GWAS performed in a Moroccan population have shown that the patterns of LD observed in Morocco were similar to those observed in the European HapMap population (unpublished data). We selected the tag SNPs in order to capture >80% of reported SNPs with MAF > 0.05 at a r2 of 0.8 in the European population, respectively. In addition, we performed a specific SNP selection within the IL28B region (see next paragraph). SNPs were genotyped in a first sample consisting of 261 HCV infected subjects (130 with spontaneous clearance and 131 with chronic infection), and 126 HCV negative Egyptian subjects. Except for IL28B variants (see next paragraph), analyses of the SNPs in other candidate genes did not identify any additional significant signals associated with spontaneous HCV clearance in this first sample.

Therefore, we focused our analyses in the IL28B region. We genotyped nine SNPs within the IL28B genomic region which covered the linkage disequilibrium (LD) block known to be associated with HCV clearance in European populations. The SNPs the most significantly associated with spontaneous clearance were rs12979860 (P=1.6x10-7) and the non-synonymous IL28B SNP, rs8103142 (P=1.6x10-7) (Figure 4.1). The magnitude of the association with rs12979860 observed is similar to that reported in many studies conducted on HCV genotype 1 infection 1,2. SNP rs8103142 replaces a lysine residue by an arginine residue at position 70 (K70R) in IFN-λ3 (encoded by IL28B), a substitution predicted to be benign by Polyphen. Interestingly, three SNPs at the two bounds of the region were monomorphic, reducing the size of the LD block in which the causal variants are potentially located to ~20 kilobases, excluding the IL28A genomic region. Therefore, we could show that HCV clearance in Egypt was associated with a region of IL28B smaller than that identified in European populations, and involved the non-synonymous IL28B SNP, rs8103142, and these results led to a publication3. More recently, a study identified a new dinucleotide variant ss469415590 (TT vs ΔG) in strong LD with rs12979860, and the ΔG allele is a frameshift variant that created a novel gene denoted as IFNL4, encoding the IFN-λ4 protein4. The ΔG allele was found to be superior to the rs12979860 T allele in predicting poorer response to HCV treatment4,5. We are currently genotyping this novel variant in our newly collected Egyptian samples to investigate its effect in an Egyptian population.

The second phase of the project was to conduct a hypothesis-generating genome-wide (GW) exploration of variants associated with clearance. The initial idea was to use a strategy based on GW association studies (GWAS) which are designed to search for the role of common variants (frequency > 0.02) and need large sample size. We decided to change this strategy for two main reasons: 1) due to the general political situation in Egypt, the data collection was delayed and sample sizes were not large enough to have substantial power to conduct a GWAS; 2) a recent GWAS on spontaneous clearance was published in 2013 in a large samples of different ethnic origin, and only found a weak signal in the HLA region in addition to the strong known association with IL28B variants6. Therefore, it was very unlikely that we could identify the role of new common variants through a GWAS conducted in an Egyptian sample of limited size. Our new strategy was to search for the role of more rare variants (frequency < 0.02) which have not been investigated so far taking advantage of the new next-generation sequencing technologies, in particular whole exome sequencing (WES). In recent months, WES has led to the identification of the molecular basis of single-gene inborn errors in small numbers of unrelated affected individuals, and our laboratory has pioneered the use of WES in the genetic investigation of rare and severe infectious diseases to identify monogenic defects7-11.

It has been strongly suggested that WES could also be a powerful approach for investigations of the role of rare variants in more common diseases12. This strategy was recently validated by the successful identification of a modifier gene for a Mendelian trait: DCTN4, which influences Pseudomonas aeruginosa infection in patients with cystic fibrosis13. This discovery was based on the investigation of 91 patients with “extreme phenotypes”, which showed that 12 of the 43 patients with early infections were carriers of missense variants of DCTN4, whereas none of the 48 patients with late-onset infections carried these variants. In a first step to investigate the role of rare variants involved in spontaneous clearance, we decided to perform WES in a sample of 60 subjects with “extreme phenotypes” to increase the likelihood of finding additional variants that may be involved in spontaneous clearance. From our initial sample we selected 30 subjects who presented a spontaneous clearance while they had the IL28B genotype at risk for chronic infection, and 30 subjects who developed a chronic HCV infection while they had the IL28B genotype predisposing to clearance. WES has been completed for these subjects and the analysis of these data is ongoing. We are searching for genes (genetic homogeneity approach) and pathways (physiological homogeneity) displaying differences in frequencies of rare variants in subjects with clearance as compared to subjects with chronic infection using appropriate methods12,14. The most promising genes will be further investigated for replication studies in the remaining Egyptian subjects.

As already mentioned above, analyses of the other candidate genes did not identify any additional significant signals associated with spontaneous HCV clearance in our first Egyptian sample. However, when analyzing the HCV infection phenotype by itself, also denoted as HCV infection per se (ie comparing HCV negative subjects to all HCV positive subjects either cleared or chronic), preliminary analyses identified several potential interesting signals, in particular in the gene DDX58 encoding RIG-I that needed to be confirmed in replication samples. Our initial plan was to collect additional samples of both HCV infected and HCV negative samples, through both fever hospitals and blood banks in order to perform replication studies. Because of the general political Egyptian situation, this collection was substantially delayed, and we decided to pursue this investigation in samples of European origin that we had available at that time.

For this study of HCV infection per se, we used European cohorts who had previous high-throughput genotyping in the context of GWAS. Two cohorts from France and Switzerland were recently used in a GWAS investigating the genetic variants predisposing to HCV-related liver fibrosis, and consisted of 1390 patients chronically infected by HCV15. We also used two cohorts of controls: one consisting of 1542 subjects who were included in a GWAS investigation on venous thrombosis16, and the second consisting of 2837 individuals who are the publicly available controls provided by the Illumina company. While the HCV status of the two cohorts of controls is unknown, it is reasonable to assume that the proportion of HCV-infected subjects should be at most the overall HCV seroprevalence in this kind of populations (<1%). Therefore, the lack of power resulting for the misclassification of some control subjects could be considered as negligible.

As these cohorts have been genotyped with different GWAS arrays, we first compared the overall sample of 1390 HCV infected subjects to the first control cohort of 1542 subjects genotyped with the array which was the closest to those used for infected subjects. These cohorts have been genotyped with Illumina arrays including from 370,000 to 1 million SNPs. To homogeneize the SNPs to be analyzed and to increase their number, we first conducted an imputation procedure. Genotypes were imputed in these cohorts using the 1000 genome database17 as a template and by means of the IMPUTE2 software18. A total of 7,865,932 SNPs with high imputation quality were retained for the association analyses. We first tested the SNPs that were found interesting in our initial Egyptian study (especially in the gene DDX58), and none of them were replicated.

We then conducted the GWAS analysis of the whole set of available SNPs (Figure 4.2). Only one significant signal (p<5x10-8) was found on chromosome 19, with several SNPs associated with HCV infection. All these SNPs are located within the IL28B region and the signal corresponds to the known association of IL28B variants with clearance. As our HCV-infected sample consists only of patients who have chronic infection, the frequency of their IL28B variants significantly differ from a non-infected population. In addition, we observed a total of 81 SNPs who presented a suggestive association (p <10-5) with HCV infection (figure 2). These 81 SNPs were grouped into 29 independent regions. To further investigate these associations we included the second control group of 2837 in the analyses. Only three SNPs showed an improved association with the additional control group, one on chromosome 3 (going from 6x10-7 to 5x10-10), one on chromosome 6 within the HLA region (going from 8x10-6 to 4x10-8), and one on chromosome 13 going from 4x10-6 to 3x10-6). As expected, the IL28B SNPs also showed an improved association with combined p-values < 10-9.

Overall, this GWAS analysis identified 3 SNPs (in addition to IL28B variants) that may be associated with HCV infection. As explained for the IL28B SNPs (and because we only have chronically infected subjects), it is not possible to determine at this step if these SNPs could influence HCV infection per se, or HCV spontaneous clearance. We are presently conducting analyses in additional independent samples of HCV-infected and control subjects to 1) confirm or not that our results for these three SNPs are true associations, 2) determine if these associations influence HCV infection per se or HCV clearance by genotyping samples of subjects with spontaneous clearance. These analyses are ongoing in samples of European origin, and we could soon perform the same kind of analyses in Egyptian samples. Our Egyptian partners had now available samples of 400 subjects with chronic HCV infection, 400 subjects with spontaneous clearance, and 1200 HCV-negative controls. These samples will also be used to genotype the new ss469415590 mentioned previously.

4.3 miRNA screen and evaluation of correlations with spontaneous clearance (Albert)
We performed stimulation of whole blood and PBMCs from multiple healthy donors and hepatic cell lines (Huh7 cells) with IFNα, IFNβ, IFNγ, IFNλ, and TNFα to identify Interferon induced microRNAs. 12hours after cytokine stimulation total RNA was extracted and analyzed for quantity and quality of micro RNAs using Agilent BioAnalyzer. This revealed that RNA purification by the RNAEasy kit (Qiagen) lead to the loss of microRNAs. Stimulations were therefore repeated and total RNA extracted using Trizol precipitation extraction. MicroRNA quality was confirmed by the Agilent BioAnalyzer and samples were sent to Fasteris (CH) for full sequencing of known microRNAs. Results from sequencing indicated good quality of microRNAs in all samples, the raw data aligned and analyzed for quality control using bow tie, before alignment with by Katie Siddle (Quintana lab)
Base qualities and nucleotide distributions were good with the exception of one sample, and the results were compared with the mir database to identify induced microRNAs. ANOVA was applied to identify differentially induced mirRNAs specific to each stimulation condition, and these identified miRNAs were employed in hierarchical clustering (Fig 4.3 upper) and principle component analysis (Fig 4.3 lower). This enabled the selection of microRNA sequences for inclusion in the candidate SNP analysis.

WP5 Training opportunities in Clinical Epidemiology and Biomarker Discovery

Objective: To provide training in clinical epidemiology and biomarker discovery.

5.1 Biomarker discovery workshop
The major focus of the Sphinx training opportunities was a 2 week workshop held at Institute Pasteur Tunis from June 11-22, which consisted of:

(1) Didatic lectures (June 11-13)
• Epidemiology: Epidemiology of viral diseases, Biostatistical Approaches, Database Management.
• Virology & Molecular Biology : Liver tropic viruses, Molecular biology techniques, Genome Sequencing.
• Immunology : Immune cell interactions, Metabolic and Immune interactions, Chemokines and Cytokines.

(2) Guest Research Lectures (June 14-15)
Occurrence of the SPHINX mid term meeting in parallel with the workshop enabled participation in the workshop by the majority of the Sphinx partners through guest lectures. Students learned about ethical and commercial aspects of biomarker discovery with real life examples.

(3) Practical Training (June 18-22)
Divided into small working groups, the candidates took part in an investigative study on viral and Leishmania associated biomarkers using Luminex technology and flow cytometric analyzing real biomarker data. Biomarker candidates identified as part of WP2.1 were assessed at different stages of hepatitis disease progression. The results generated were analyzed using statistical software and approaches tailored for biomarker discovery and validation.

A call was made for candidates through the SPHINX project website, with advertisements also made through the Pasteur International Network, ANRS, Nature Events and Nature Middle East. This lead to numerous applications from which the best qualified 25 students were selected by the workshop committee with preference given to applicants from Mediterranean Partner Countries (including Hepacute and STDF projects).

5.2 Exchange programme for spreading excellence throughout the Consortium (Fontanet)
Unfortunately due to the local political disturbances in Egypt, the exchange of researchers within the project during the second 18 months of the project remained challenging. However a number of successful visits did occur as listed below in Table 6.
An additional successful outcome of the workshop held in Tunis was the development of a new collaborative project between Institut Pasteur Tunis and Paris. This lead to a PhD student Wafa Kammoun from IP Tunis successfully applying for a Pasteur International fellowship which allowed her to visit the laboratory of Matthew Albert for a duration of 4 months to learn and apply biomarker discovery techniques such as Luminex and flow cytometry to her patient samples. She was also able to attend the high level course in Immunology held annually at Institut Pasteur, Paris. The results from her collaborative project are currently being prepared for publication.

WP6 Project Management

Objective: To establish a Consortium that collaborates efficiently and works toward the stated objectives, while protecting intellectual property and fulfilling EC requirements

6.1 Operational management of the project (Albert)

Despite political challenges that have arisen within the Consortium we have succeeded in meeting all of our management objectives;
(1) Successful Kick Off meeting was held in Cairo with all partners.
(2) A productive 12 month meeting was held October 4th 2011 via WebEx, with the participation of all partners (except MOH). It helped to identify priorities required for the continued success of the project goals.
(3) Successful Mid term meeting was held at IP Tunis 16-17th 2012 with representatives from the majority of partners, despite local political disturbances preventing the travel of a number of partners.
(4) A 2nd WebEx was held online including all partners on Jan 15th 2014. This allowed for a group meeting to discuss progress and identify opportunities and priorities during the remainder of the project, without the need for travel and the associated expenses.
(5) Successful Final meeting was held in London 9th April 2014 with all partners.
(6) All deliverables and milestones have been successfully met.

Here we would like to highlight the passing of Dr Mostafa Mohammed, which was both a personal loss to many within the project as well as a great loss for the epidemiology and viral hepatitis scientific communities. Dr Mohammed was a driving force behind the project and an inspirational leader for the Egyptian partners and the Consortium as a whole. While his loss is still felt, the SPHINX partners are even more committed to the success of the project as a tribute to his lasting vision and commitment to public health concerns. A meeting was held in Cairo with Matthew Albert, Arnaud Fontanet, Darragh Duffy and all Egyptian partners in late October 2011 to identify the best solutions for ensuring the successful continuation of the project.

A second major managerial challenge has been the unexpected political disruptions in Egypt during the course of the project. The impact of these events on patient recruitment has been reported in WP1 review. With respect to management issues, the continued changes at the Ministry of Health prevented their official signing of the Consortium Agreement and entry into the project. As a result of the close working relationship between Gothi officials and Fever Hospital staff, all project tasks continued in the absence of these official signatures. Renewed efforts were made to obtain the official signature by the new Ministry of Health which were obtained and sent to the EC.

6.2 Communication & Dissemination (Albert)

Communication was maintained throughout the consortium through regular emails and conference calls between small working groups. Biweekly conference calls were held between Institut Pasteur and Ain Shams University/Gothi. Institut Pasteur held regular conference calls with NMI/EDI, and NKI on specific work package tasks.

The main focus of all dissemination activities is the project website www.sphinx-hcv.eu which gathers presentations and publications from the project, which are also listed below.

• Presentation of the project results at conferences were made at the following venues:
Measuring Antigen-Specific Immune Responses (MASIR) conference Dubrovnik Croatia May 2013

Defining the influence of chronic HCV infection on the T cell preimmune repertoire: implications for vaccination and hepatocellular carcinoma.
European Association for the Study of the Liver (EASL) Amsterdam April 2013

EASL-EPEMED Biomarker Development Challenges in Viral Hepatitis

Dechema Conference on Functional Genomics and Proteomics - Applications, Molecular Diagnostics & Personalized Medicine
Frankfurt January 2013
Multiplexed Antigen Arrays for the Analysis of the Human Serological Response for HCV genotype 4

C-HEP 2nd World Congress "Controversies in the Management of Viral Hepatitis"
Berlin 18-20 October 2012
PLENARY SESSION
DO WE NEED TO KNOW GENETIC FACTORS SUCH AS IL28B FOR TREATMENT DECISION MAKING? Matthew Albert/Vincent Soriano

European Congress of Immunology (ECI) Glasgow September 2012

The role of dipeptidylpeptidase IV in lymphocyte migration and HCV disease pathogenesis
Poster abstract : ABC: Exploring the signature of viral hepatitis

• Scientific publications from this period of the project included the following (additional publications are currently being prepared for submission):

Journal of Hepatology 2014 Jun 3. S0168-8278(14)00388-2
Apolipoprotein H expression is associated with IL28B genotype and viral clearance in Hepatitis C virus infection.
Laird, Melissa E., Mohsen, Amira, Duffy, Darragh, Mamdouh, Rasha, LeFouler, Lenaig, Casrouge, Armanda, El-Daly, Mai, Rafik, Mona , Abdel-Hamid, Mohamed, Soulier, Alexandre, Pawlotsky, Jean-Michel, Hézode, Christophe , Rosa, Isabelle, Renard, Philippe , Mohamed, Mostafa K. , Bonnard, Philippe, Izopet, Jacques, Mallet, Vincent, Pol, Stanislas, Albert, Matthew L., Fontanet, Arnaud

Hepatology 2014 Apr;59(4):1273-82
The ABCs of viral hepatitis that define biomarker signatures of acute viral hepatitis.
Duffy D, Mamdouh R, Laird M, Soneson C, Le Fouler L, El-Daly M, Casrouge A, Decalf J, Abbas A, Eldin NS, Fontes M, Abdel-Hamid M, Mohamed MK, Rafik M, Fontanet A, Albert ML.

J Immunol. 2014 Jan 15;192(2):641-8
HLA Micropolymorphisms Strongly Affect Peptide–MHC Multimer–Based Monitoring of Antigen-Specific CD8+ T Cell Responses.
van Buuren MM1, Dijkgraaf FE, Linnemann C, Toebes M, Chang CX, Mok JY, Nguyen M, van Esch WJ, Kvistborg P, Grotenbreg GM, Schumacher TN.

Cytokine 2013 Aug;63(2):105-12
CXCL10 antagonism and plasma sDPPIV correlate with increasing liver disease in chronic HCV genotype 4 infected patients.
Ragab D, Laird M, Duffy D, Casrouge A, Mamdouh R, Abass A, Shenawy DE, Shebl AM, Elkashef WF, Zalata KR, Kamal M, Esmat G, Bonnard P, Fontanet A, Rafik M, Albert ML.

PLOS Pathogens 2012 Jun;8(6):e1002759
Polyfunctional type-1, -2, and -17 CD8(+) T cell responses to apoptotic self-antigens correlate with the chronic evolution of hepatitis C virus infection.
Franceschini D, Del Porto P, Piconese S, Trella E, Accapezzato D, Paroli M, Morrone S, Piccolella E, Spada E, Mele A, Sidney J, Sette A, Barnaba V

• Additional impact from dissemination of the project relates to our consortiums efforts to have HCV genotype 4 recognised as a neglected infectious disease. This effort was driven by results from our epidemiological studies (WP1) that highlighted the neglected clinical need, and the viral genotype analysis for tetramer design (WP3) which highlighted how different genotype 4 is compared to the more studied genotype 1 and 2 variants. We worked in collaboration with the G-Finder group to define the following 3 categories which helped result in the official recognition of HCV genotype4 as a neglected infectious disease:

1. The disease disproportionately affects people in developing countries

The World Health Organization has declared hepatitis C a global health problem, based on an estimate of 2-3% of the world population (around 150 million people) infected with HCV. Notably, the global distribution of infection is variable, with developing countries in Africa having 3-14% prevalence, often with viral genotypes different from those present in developed countries (Lavanchy, 2011).

Since the discovery of the hepatitis C virus in the 1990s, collaborations between academia and industry have translated into new treatments, including the recently-approved anti-protease therapies for HCV genotype 1 (HCVg1) – the most common genotype in the US and western Europe. While the new drug regimens represent an exciting breakthrough, there has been a troubling consequence: success in treating HCV in Europe and the USA has created a two-tiered system, with the specific needs of HCV infected persons in Africa and the Middle East being largely overlooked. Specifically, there are two issues: first, the HCV strains circulating in resource poor countries are not identical to the developed countries. For example, HCV genotype 4 (HCVg4) represents 80% of HCV infections in Africa and the Middle East; it is not sensitive to the recently approved anti-protease inhibitors; and it will not be covered by current vaccination efforts that focus exclusively on HCVg1. Indeed, several HCV genotypes have been identified with at least 6 major genotypes and over 76 subtypes described. Second, there are insufficient financial resources to treat those in need of therapy, not to mention the increased cost that will be associated with new drug regiments. For example in Egypt, the National treatment centers have succeeded in curing 120K persons over the past 4yrs (of the nearly 2 million in need of treatment); and meanwhile ~500K new people were infected. New avenues of support must be identified in order to ensure access of pan-genotype specific treatments in developing countries; and to establish viable plans for prevention (e.g. public health campaigns, vaccine development).

The special case of HCVg4 in Africa
The prevalence of HCVg4 is highest in Africa, reaching 5.3% of adults in Central Africa (representing ~50% of HCV infections), and 15% of adults in Egypt (with HCVg4 representing >95% of HCV infections). The origin of the epidemic in Egypt has been attributed to the mass campaigns of parenteral anti-schistosomiasis treatment in rural areas in the 1960s–70s (Frank). Since then, the virus has continued to spread, mainly through intravenous injections and other medical procedures. The magnitude of the problem translates to >8 million people infected with HCVg4 in Egypt alone, and the incidence of new infections being the highest, worldwide (Kamal). Compounding the burden of disease prevalence, there is a single nucleotide polymorphism (SNP) that is disproportionately high in persons of African-decent (i.e. IL28B risk allele is present in ~90% of African populations as compared to 40-65% in Europeans and 10-15% in Asians), conferring higher failure rates of HCV treatment (Thomas).

HCV as a public health burden
In approximately 80% of infections, HCV becomes a chronic disease, with devastating long-term sequella. These include: (i) cirrhosis of the liver, which develops in about 10–20% of infected individuals; (ii) liver failure which develops in about 20–25% of cirrhotic individuals; and (iii) liver cancer, which carries a 1-2% risk per year of infection with chronic HCV. Notably, HCV is the leading cause of hepatocellular carcinoma (HCC) and in Egypt, HCC is the leading cause of cancer-related death.

2. There is no existing product to treat/ prevent the disease OR a product exists but improved or additional products are needed (e.g. due to resistance, unsuitable formulations, missing DC strains);

HCV genotypes differ from each other by 31-33% at the nucleotide level. The 6 genotypes are further divided into epidemiologically distinct subtypes (over 76 subtypes) differing by 20-25% in nucleotide sequence from one another (Timm). The sequence diversity of HCV presents a significant hurdle for the development of pan-genotype vaccines. Current vaccine strategies are designed to induce adaptive immune responses and principally target conserved regions of the genome; however, it will not be possible to utilize HCVg1 vaccine candidates (the few that are still in development) for protecting individuals from HCVg4. The designation of HCVg4 as a neglected disease will support its prioritization as a target for vaccine development.

Regarding the treatment of already infected individuals, patients with HCVg4 have been considered difficult to treat, with sustained virological response rates (SVR) being achieved in ~50% of those who receive pegylated IFN in combination therapy with ribavirin (Aljumah). The recently approved direct acting antiviral agents (Boceprevir and Teleprevir) have been developed to target the non-structural protein 3 (NS3) of HCV genotype 1, and as such are not recommended for usage in the management of HCVg4 patients recent EASL guidelines. Even if the possibility existed to use these drugs off-label, the price of these new therapies makes them prohibitively expensive for resource poor countries (~50,000$ per patient). New direct anti-viral drugs are being developed that are anticipated to cover HCVg4, but again, the pharmaceutical and public health communities have failed to establish a program that will enable access to HCV patients in Africa or the Middle East.

3. There is no commercial market to attract R&D from private industry

Hepatitis C virus genotype 4 disproportionately infects persons living in developing countries where limited access to healthcare facilities results in significant numbers of undiagnosed and untreated persons. With respect to the commercial market for HCV, the USA, Japan and 5 EU countries (France, Germany, Italy, Spain, and UK) represent 67% of the total capital expenditures (SCRIP). Therefore, it is not surprising that new therapeutic regiments have been, and are being developed to target the viral genotypes predominant in these countries: HCVg1 in the US and Europe; and HCVg2 in Japan (SCRIP).

The first hurdle in combatting HCVg4 lies in the fact that current diagnostic tools were developed for detection of HCVg1. This makes accurate epidemiological studies in countries with potentially heavy HCVg4 burdens challenging. Second, there is a need for improved development and better access to direct anti-virals that inhibit HCVg4. Finally, there is an urgent need to establish and execute a plan for vaccine development targeting HCVg4. Currently, there is no vaccine for HCV. The size of the potential therapeutic market for HCVg1/g2 in the top 7 countries, combined with the differing demographics compared to developing countries, makes the development of HCVg4 vaccines uninteresting from a purely commercial perspective. Moreover, current efforts focus on therapeutic vaccines, whereas preventative vaccines are required for limiting HCVg4 infections – of note, the market strategy and developmental hurdles are different for a preventative vaccine. In Egypt, it is very unlikely that the generalized epidemic can be controlled through infection control measures only. These have been in place for the past ten years, are very expensive and difficult to implement logistically, and have had little impact on the course of the epidemic so far. Only a vaccine would have the potential to stop the spread of this deadly virus.

In conclusion, the general hepatitis C field has benefitted recently from novel drug developments, stimulated by the incredible commercial market that exists in the USA, Japan and EU (estimated to be 15B Euro expenditure per year). The paradoxical side effect of this successful focus on the western HCV subtypes, however, has resulted in HCVg4 becoming a neglected infectious disease.

According to the criteria suggested by the G-Finder document, classification of HCVg4 as a neglected disease is justified; such a classification would help scientists and epidemiologists establish programs to combat the 30 million HCVg4 infections worldwide, most of which impact persons living in resource poor countries. Designation of HCVg4 as a neglected disease will aid the development of specific diagnostic, therapeutic and preventative approaches for combating this chronic infectious disease. Failure to grant such a designation will leave Western market forces as the driver of HCVg4 management –likely resulting in an estimated 10yr delay in access to drugs, with no viable plan for vaccine development.

WP7 Collaboration with HepaCute and STDF projects

Objective: To exploit synergies between EU and Egyptian hepatitis research programs during the proposed work. Furthermore, we propose to coordinate efforts for disseminating information about the public health priorities in Egypt and work towards a sustained cooperation between North and South research partners.

7.1 Joint meetings of the EU-funded SPHINX and HepaCute, together with Egyptian STDF projects.

Joint meetings between SPHINX and HepaCute consortia were held in parallel with the EASL 2013 meeting in Amsterdam, and EASL 2014 meeting in London. The objectives of these meetings were to develop links and collaborations between the two EC-funded projects.

7.2 2nd Joint Meetings on the subject of Acute HCV in EU and MPC.
As part of the EASL 2012 International Liver Congress on April 18th in Barcelona, SPHINX and HepaCute organized a joint satellite workshop under the title of “Fighting Viral Hepatitis C”. Researchers from both projects representing Northern and Southern perspectives gave presentations covering epidemiology, health impact, and management of hepatitis C. This was followed by a panel discussion including SPHINX, HepaCute, STDF and EC representatives focusing on future EU-MPC interaction in the context of Hepatitis C research.

As part of the EASL 2014 International Liver Congress on April 10th in London, SPHINX and HepaCute organized a joint satellite workshop under the title of “Hepatitis C genotype 4: hope for a neglected infectious disease”. Invited speakers gave presentations on the epidemiology, viral diversity of HCV genotype 4, and speakers from the MSF and WHO discussed the future for new treatments for HCV genotype 4. Researchers from both projects gave presentations demonstrating the new technological tools developed for HCV genotype 4 covering epidemiology, health impact, and management of hepatitis C. This was followed by a panel discussion focused on what is required to combat HCV genotype 4. In addition to this workshop Sphinx researchers Matthew Albert and Arnaud Fontanet chaired workshops on personalized medicine approaches, and access to new therapies in research limited settings respectively, both highlighting relevant findings from the Sphinx consortium.

7.3 Open Training Courses.
The HepaCute workshop that was held at Institut Pasteur, Morocco from 25-27th November 2011 was attended by four SPHINX researchers; Dr Amal Abbas and Dr Dina Elshinawy from Ain Shams University, Cairo, Dr Abo Bakr Mostafa from GOTHI, Cairo and Yousr Ben Ayed from Institut Pasteur, Tunis.

The SPHINX Clinical Epidemiology and Biomarker Discovery Workshop was held from June 11-23, 2012 at the Institut Pasteur, Tunis and was attended by two Hepacute researchers; Dr Ezzikouir Sayeh Institut Pasteur, Morocco and Dr Mohamed Aboghrip VACSERA, Cairo and an STDF researcher Tawfeek Abdelhafez, National Research Centre, Cairo.

7.4 Coordination between EU-funded SPHINX and HepaCute Consortia and with the

Egyptian National Committe for Prevention and Control of Viral Hepatitis.
Regular conference calls have been held between Dr Matthew Albert (Institut Pasteur) and Dr Helmut Diepolder (KUM) throughout the course of the projects. The project management teams lead by Dr Darragh Duffy (Institut Pasteur) and Dr Jerome Weinback (Inserm Transfert) for each project have kept in regular email and personal contact.

Opportunities for collaboration with two STDF projects were identified within the context of the Sphinx project, but these were not realised due to the removal of funding for STDF projects by the Egyptian government.

Potential Impact:
Infection with HCV can cause acute or chronic liver damage and ultimately leads to liver failure, cirrhosis or liver cancer. Major management decisions include questions of when, whether and how to treat patients with chronic HCV. Our Consortium has been able to study the pathogenesis of acute HCV infection in regions where disease incidence is high and the recruitment of acute symptomatic HCV individuals permits the establishment of relevant patient cohorts. We exploited and continue to exploit this unique position by assessing which host factors are critical for spontaneous clearance, by preventing HCV entry into uninfected cells or allowing for rapid targeting of HCV infected hepatocytes. Relevant host factors being analyzed by the Consortium include metabolic analytes, innate immune molecules and components of the cellular and humoral immune system. These biomarkers are currently being transferred onto point of care based platforms for easy implementation in resource limited settings. This recently initiated FP7 project, PoC-HCV is building upon the discoveries and findings of Sphinx through collaborations with 3 leading European biotech SMEs (www.poc-hcv.eu). In addition, to aid their uptake and utilisation our consortium has worked with the Policy Cures G finder group to have HCV genotype 4 recognised as a neglected infectious disease.

Major management decisions for the treatment of HCV patients include questions of when, whether and how to treat. Clinicians and pharmaceutical companies are beginning to explore the concept of personalised medicine, and notably this concept has been partly realised. For example, Fibrotest® and FibroMax® are two fibrosis biomarker panels that are employed for helping make the decision of whether or not to treat (BioPredictive); and HCV genotype is used for determining duration of treatment (Roche Diagnostics and others). Our consortium and others have identified several useful biomarkers, but they have yet to be widely applied to clinical practice. These include haplotype differences in IL28B, which is predictive of response to pegylated interferon / ribavirin (Peg-IFN/RBV) therapy. Moreover, our work has contributed to the identification of immune (IP-10) and metabolic proteins (Apolioprotein H) that can be measured from plasma or serum, and used to predict treatment response in several patient populations. In addition our discoveries around Apo H in the acute Sphinx cohort has lead to a fundamental understanding of how host protein-pathogen interactions can differentially result in infection (unpublished results).

There is a specific and urgent need in Egypt for validated baseline predictors of Peg-IFN/RBV therapy, where 6 million individuals are chronically infected with HCV. Regarding European and USA practices, new treatment regiments have become available with the approval of two direct acting antivirals (NS3 protease inhibitors) with other agents in late-stage clinical development. This will increase the complexity of patient management and will demand the introduction of predictive tests capable of guiding therapy, limiting adverse effects and managing costs. The value of personalised approaches for managing patients with HCV infection is rooted in positive outcomes for the patient: saving lives and improving the quality of life. Other stakeholders will also benefit: physicians will realise easier decision-making, and better prediction of treatment outcome. Payers will benefit from optimised use of resources: initiating early treatment for those who are likely to respond; discontinuing treatment when risk of failure or adverse reactions is high; and permitting rapid modifications of treatment plans. Regulators and policymakers will find a stronger basis for the development of cost-effective and/or cost-saving treatment guidelines. Simply stated, point-of-care testing for the management of HCV patients makes sense.

Additional impact from dissemination of the project relates to our consortiums efforts to have HCV genotype 4 recognised as a neglected infectious disease. This effort was driven by results from our epidemiological studies (WP1) that highlighted the neglected clinical need of HCVg4, and the viral genotype analysis for tetramer design (WP3) which highlighted how different genotype 4 is compared to the more studied genotype 1 and 2 variants. We worked in collaboration with the G-Finder group to define the 3 categories which helped result in the official recognition of HCV genotype4 as a neglected infectious disease.

1. The disease disproportionately affects people in developing countries
The World Health Organization has declared hepatitis C a global health problem, based on an estimate of 2-3% of the world population (around 150 million people) infected with HCV. Notably, the global distribution of infection is variable, with developing countries in Africa having 3-14% prevalence, often with viral genotypes different from those present in developed countries (Lavanchy, 2011). Since the discovery of the hepatitis C virus in the 1990s, collaborations between academia and industry have translated into new treatments, including the recently-approved anti-protease therapies for HCV genotype 1 (HCVg1) – the most common genotype in the US and western Europe. While the new drug regimens represent an exciting breakthrough, there has been a troubling consequence: success in treating HCV in Europe and the USA has created a two-tiered system, with the specific needs of HCV infected persons in Africa and the Middle East being largely overlooked. Specifically, there are two issues: first, the HCV strains circulating in resource poor countries are not identical to the developed countries. For example, HCV genotype 4 (HCVg4) represents 80% of HCV infections in Africa and the Middle East; it is not sensitive to the recently approved anti-protease inhibitors; and it will not be covered by current vaccination efforts that focus exclusively on HCVg1. Indeed, several HCV genotypes have been identified with at least 6 major genotypes and over 76 subtypes described. Second, there are insufficient financial resources to treat those in need of therapy, not to mention the increased cost that will be associated with new drug regiments. For example in Egypt, the National treatment centers have succeeded in curing 120K persons over the past 4yrs (of the nearly 2 million in need of treatment); and meanwhile ~500K new people were infected. New avenues of support must be identified in order to ensure access of pan-genotype specific treatments in developing countries; and to establish viable plans for prevention (e.g. public health campaigns, vaccine development).

The special case of HCVg4 in Africa
The prevalence of HCVg4 is highest in Africa, reaching 5.3% of adults in Central Africa (representing ~50% of HCV infections), and 15% of adults in Egypt (with HCVg4 representing >95% of HCV infections). The origin of the epidemic in Egypt has been attributed to the mass campaigns of parenteral anti-schistosomiasis treatment in rural areas in the 1960s–70s (Frank). Since then, the virus has continued to spread, mainly through intravenous injections and other medical procedures. The magnitude of the problem translates to >8 million people infected with HCVg4 in Egypt alone, and the incidence of new infections being the highest, worldwide (Kamal). Compounding the burden of disease prevalence, there is a single nucleotide polymorphism (SNP) that is disproportionately high in persons of African-decent (i.e. IL28B risk allele is present in ~90% of African populations as compared to 40-65% in Europeans and 10-15% in Asians), conferring higher failure rates of HCV treatment (Thomas).

HCV as a public health burden
In approximately 80% of infections, HCV becomes a chronic disease, with devastating long-term sequella. These include: (i) cirrhosis of the liver, which develops in about 10–20% of infected individuals; (ii) liver failure which develops in about 20–25% of cirrhotic individuals; and (iii) liver cancer, which carries a 1-2% risk per year of infection with chronic HCV. Notably, HCV is the leading cause of hepatocellular carcinoma (HCC) and in Egypt, HCC is the leading cause of cancer-related death.

2. There is no existing product to treat/ prevent the disease OR a product exists but improved or additional products are needed
HCV genotypes differ from each other by 31-33% at the nucleotide level. The 6 genotypes are further divided into epidemiologically distinct subtypes (over 76 subtypes) differing by 20-25% in nucleotide sequence from one another (Timm). The sequence diversity of HCV presents a significant hurdle for the development of pan-genotype vaccines. Current vaccine strategies are designed to induce adaptive immune responses and principally target conserved regions of the genome; however, it will not be possible to utilize HCVg1 vaccine candidates (the few that are still in development) for protecting individuals from HCVg4. The designation of HCVg4 as a neglected disease will support its prioritization as a target for vaccine development. Regarding the treatment of already infected individuals, patients with HCVg4 have been considered difficult to treat, with sustained virological response rates (SVR) being achieved in ~50% of those who receive pegylated IFN in combination therapy with ribavirin (Aljumah). The recently approved direct acting antiviral agents (Boceprevir and Teleprevir) have been developed to target the non-structural protein 3 (NS3) of HCV genotype 1, and as such are not recommended for usage in the management of HCVg4 patients recent EASL guidelines. Even if the possibility existed to use these drugs off-label, the price of these new therapies makes them prohibitively expensive for resource poor countries (~50,000$ per patient). New direct anti-viral drugs are being developed that are anticipated to cover HCVg4, but again, the pharmaceutical and public health communities have failed to establish a program that will enable access to HCV patients in Africa or the Middle East.

3. There is no commercial market to attract R&D from private industry
Hepatitis C virus genotype 4 disproportionately infects persons living in developing countries where limited access to healthcare facilities results in significant numbers of undiagnosed and untreated persons. With respect to the commercial market for HCV, the USA, Japan and 5 EU countries (France, Germany, Italy, Spain, and UK) represent 67% of the total capital expenditures (SCRIP). Therefore, it is not surprising that new therapeutic regiments have been, and are being developed to target the viral genotypes predominant in these countries: HCVg1 in the US and Europe; and HCVg2 in Japan (SCRIP). The first hurdle in combatting HCVg4 lies in the fact that current diagnostic tools were developed for detection of HCVg1. This makes accurate epidemiological studies in countries with potentially heavy HCVg4 burdens challenging. Second, there is a need for improved development and better access to direct anti-virals that inhibit HCVg4. Finally, there is an urgent need to establish and execute a plan for vaccine development targeting HCVg4. Currently, there is no vaccine for HCV. The size of the potential therapeutic market for HCVg1/g2 in the top 7 countries, combined with the differing demographics compared to developing countries, makes the development of HCVg4 vaccines uninteresting from a purely commercial perspective. Moreover, current efforts focus on therapeutic vaccines, whereas preventative vaccines are required for limiting HCVg4 infections – of note, the market strategy and developmental hurdles are different for a preventative vaccine. In Egypt, it is very unlikely that the generalized epidemic can be controlled through infection control measures only. These have been in place for the past ten years, are very expensive and difficult to implement logistically, and have had little impact on the course of the epidemic so far. Only a vaccine would have the potential to stop the spread of this deadly virus. In conclusion, the general hepatitis C field has benefitted recently from novel drug developments, stimulated by the incredible commercial market that exists in the USA, Japan and EU (estimated to be 15B Euro expenditure per year). The paradoxical side effect of this successful focus on the western HCV subtypes, however, has resulted in HCVg4 becoming a neglected infectious disease.

According to the criteria suggested by the G-Finder document, classification of HCVg4 as a neglected disease is justified; and such a classification would help scientists and epidemiologists establish programs to combat the 30 million HCVg4 infections worldwide, most of which impact persons living in resource poor countries. Designation of HCVg4 as a neglected disease will aid the development of specific diagnostic, therapeutic and preventative approaches for combating this chronic infectious disease. Failure to grant such a designation will leave Western market forces as the driver of HCVg4 management –likely resulting in an estimated 10yr delay in access to drugs, with no viable plan for vaccine development.

Sphinx has also resulted in the development of new tools for the study, and potentially for the diagnosis of HCV patients. These include new Major Histocompatibility Complex class I tetramers and multiplexed HCV antibody and pathogen Luminex assays. Indeed work conduced in Sphinx has highlighted the complexity of the MHC in particular in diverse north African populations such as Egypt and the need for specific research tools designed and tailored to the genetics of the local population to be studied. In addition results from the antibody assay development have highlighted the need for tools to be also tailored to the genetics of the local infectious agents, as diagnostic tools developed for western viral genotypes can give very different results to those adapted for local viral genotypes. Work from both of these research areas is still ongoing and we believe will results in additional findings as well as potentially useful diagnostic tools, in particular for HCVg4 screening.

Finally additional impact from Sphinx has been through our dissemination activities related to publications, conferences, and training. Sphinx partners have published X papers in leading immunology, hepatology, and infection specific journals, with an additional 5 manuscripts in preparation. We have also succeeded in disseminating our work and the public health care problem of HCV though engagement with general public media. As well as numerous conference presentations two highly successful workshops were held in collaboration with HepaCute at the EASL international liver conference, focused on building upon the fighting hepatitis alliance and the concept of HCVg4 as a neglected infectious disease. For training a 2 week biomarker discovery workshop was held at the Institut Pasteur, Tunis were 24 junior scientists from the North African and Mediterranean region were trained by leading experts in the field. Practical and bioinformatics sessions were also held on multi analyte protein analysis (Luminex) and multidimensional flow cytometry and statistical analysis. This high level training will hopefully inspire and equip the next generation of researchers for additional biomarker discovery research in hepatitis and beyond.

In summary results obtained from this project will impact the management of hepatitis C in Egypt; provide critical tools for studying the host response to HCVg4; and through the delivery of greater insight into the mechanisms of spontaneous clearance, we have identified potential correlates of vaccine protection. Direct results are currently being translated onto point of care technologies for implementation of biomarker guided patient management in resource limited settings.

List of Websites:
www.sphinx-hcv.eu

Institut Pasteur
matthew.albert@pasteur.fr
darragh.duffy@pasteur.fr

Spontaneous clearance in Patients acutely infected with HCV - Immune profiling, Novel biomarkers and X-omics approaches

Final Report Summary - SPHINX (Spontaneous clearance in Patients acutely infected with HCV - Immune profiling, Novel biomarkers and X-omics approaches)

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