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Le virus de l'hépatite C (HCV) est une cause majeure de maladie chronique du foie et de carcinome hépatocellulaire. Les options thérapeutiques pour traiter l'hépatite chronique sont limitées par des coûts élevés, des effets secondaires et une résistance virale. L'entrée du HCV est la première étape d'interaction entre le virus et la cellule hôte. Elle est requise pour l'initiation, la propagation et le maintien de l'infection, ce qui en fait une cible prometteuse pour les traitements antiviraux. L’entrée du HCV nécessite l'interaction coopérative de plusieurs facteurs cellulaires, y compris CD81 et claudine-1 (CLDN1). Nous avons récemment identifié un rôle pour le récepteur à l’EGF (EGFR) et le récepteur à l’ephrine A2 (EphA2) dans l'entrée du HCV par la régulation de la formation du complexe de co-récepteurs CD81-CLDN1, ce qui suggère que la signalisation de ces récepteurs joue un rôle dans l'entrée du virus. Nous avons voulu identifier les mécanismes moléculaires de signalisation de l’EGFR requis pour l'entrée du HCV et avons identifié HRas comme un transducteur de signalisation clé de l'hôte. Des études d'imagerie ont révélées que la signalisation de HRas peut moduler la diffusion et le trafic membranaire de CD81, ce qui permet l’assemblage du complexe de récepteurs. De plus, HRas s’associe avec les récepteurs de l'hôte CD81 et CLDN1 et des facteurs d’entrée du HCV inconnus jusque là: l’intégrine beta1 et Rap2B. Le HCV profite donc de la signalisation de HRas pour l'entrée cellulaire. Ces données améliorent notre compréhension des mécanismes moléculaires de l'entrée du HCV induite par l’EGFR et ouvrent de nouvelles perspectives pour le développement d'antiviraux. / Hepatitis C virus (HCV) is a major cause of chronic liver disease and hepatocellular carcinoma worldwide. Therapeutic options to treat chronic viral hepatitis are limited by high costs, side effects and viral resistance in most patients. HCV entry is the first step of interaction between the virus and the host cell. It is required for the initiation, propagation and maintenance of infection, making it a promising target for antiviral therapy. HCV entry requires the cooperative interaction of several cellular factors, including CD81 and claudin-1 (CLDN1). We have recently identified a role for EGF receptor (EGFR) and ephrin receptor A2 (EphA2) in HCV entry by regulating the formation of the co-receptor complex CD81-CLDN1, suggesting that the signaling of these receptors might play a role in viral entry. However, the precise mechanisms of regulation are unknown. We wanted to identify the molecular mechanisms of EGFR signaling required for the HCV entry process. We identify HRas as key host signaling transducer for HCV entry. Advanced imaging studies have revealed that HRas signaling can modulate the lateral diffusion and membrane trafficking of CD81. A modified diffusion of CD81 allows the assembly of the receptors complex. In addition, HRas associates with tetraspanin microdomains containing the host receptors CD81 and CLDN1 and HCV entry factors previously unknown: the integrin beta1 and Rap2B. HCV therefore exploits HRas signaling for cellular entry. These data improve our understanding of the molecular mechanisms of HCV entry induced by EGFR and open new perspectives for the development of antivirals targeting signaling pathways.

Hepatitis C virus (HCV), a blood-borne pathogen, is a small enveloped RNA virus belonging to the Hepacivirus genus of the Flaviviridae family. HCV infection represents one of the major health concerns affecting approximately 170 million people globally. Patients with chronic HCV infection are at risk of developing hepatic fibrosis, cirrhosis and hepatocellular carcinoma. No protective anti-HCV vaccine is available yet. Until recently, standard therapy based on pegylated interferon plus ribavirin, was inadequate in treating all the patients as it results in a sustained virological response in only 40 to 50 percent of patients infected with the most common genotype (gt 1). Advances in understanding host-HCV interactions have helped developing newer anti-HCV agents such as telaprevir and boceprevir. However, treatment success is still limited due to different factors including genotype specificity, high cost, potential drug-drug interactions, substantial side effects etc. The positive-sense single-stranded RNA genome of HCV is approximately 9.6kb long which is flanked by highly structured and conserved 5’ and 3’ untranslated regions (UTRs) at both ends. Unlike cap-dependent translation of host cell mRNAs, HCV translation is mediated by an internal ribosomal entry site (IRES) present majorly within the 5’UTR. Several reports have demonstrated the interaction of different cellular proteins with HCV-5’UTR and/or 3’UTR, which include human La protein, polypyrimidine tract binding protein (PTB), poly (rC)-binding protein 2 (PCBP2) etc. These interactions of trans-acting factors with the UTRs may be important for HCV translation and/or replication. Earlier study from our laboratory revealed the importance of interaction of human La protein, by its central RNA recognition motif (RRM), with the HCV IRES around a tetranucleotide sequence GCAC near initiator AUG in influencing HCV translation. However, the role of this interaction, if any, in HCV RNA replication was not known. In the first part of the thesis, we characterized the interaction between human La protein and the GCAC to understand its role in HCV replication. We incorporated mutation, which altered the binding of La, in the GCAC motif in HCV monocistronic replicon and checked HCV RNA replication by reverse transcriptase polymerase chain reaction (RT-PCR). The mutation drastically inhibited HCV replication. Interestingly, overexpression of La could reverse the effect of this mutation and significantly enhanced HCV RNA levels. Using a bicistronic replicon, we observed that decrease in replication was independent of translation inhibition. Furthermore, mutation at the GCAC motif reduced the association between La and viral polymerase, NS5B as seen in co-immunoprecipitation assays. Moreover, this mutation affected translation to replication switch regulated by the interplay between HCV-NS3 protease and human La protein. Our analyses of point mutations, based on RT-PCR and luciferase assays, revealed distinct roles of each nucleotide of the GCAC motif in HCV replication and translation. Finally, 5’-3’ crosslink assays revealed that specific interaction of the GCAC motif with human La protein is important for linking 5’ and 3’ends of HCV genome. Results clearly demonstrate the mechanism of regulation of HCV replication by interaction of cis-acting element GCAC within the HCV IRES with human La protein. HCV is highly species-specific. Under natural conditions, HCV infects only humans and chimpanzees. This restricted host-tropism has prevented the development of a small animal model to study HCV infection in vivo. Although several human-specific entry factors have been identified to be responsible for this species selectivity, full multiplication of the HCV in animals (other than humans and chimpanzees) is still not possible. In the second part of the thesis, we showed that a post-entry host factor –‘La protein’ may also contribute in determining HCV host tropism. We aligned La protein sequences from different species and interestingly we found that HCV RNA interacting beta-turn sequence (KYKETDL) in central RRM (residues 112-184) is conserved only in human and chimpanzee. Earlier, it was shown from our laboratory that a heptameric peptide comprising of this sequence (derived from human La) could inhibit HCV translation by competing with La interaction with the IRES element. However, in the current study, another peptide corresponding to the mouse La sequence (KYKDTNL) was unable to inhibit HCV RNA translation. Similarly, wild-type mouse La (mLa) failed to stimulate HCV IRES function, but addition of chimeric mouse La protein bearing human beta-turn sequence (mLahN7) significantly increased HCV IRES mediated translation in vitro. Also, exogenous supplementation of mLahN7 enhanced HCV translation in cell culture system. Moreover, quantitative as well as tagged RT-PCR analyses showed an enhanced HCV replication upon overexpression of mLahN7. The findings obtained in this part raise a possibility of creating HCV mouse model using human specific cellular entry factors and a humanized form of La protein. Hepatitis C has emerged as a major challenge to the medical community. Developing more potent and safe anti-HCV regimens is need of the hour. As described above, a linear hepatapeptide (KYKETDL) was synthesized and shown to reduce HCV translation. However, this linear peptide was stable only for a shorter time scale. Therefore, in the third part of the thesis, effect of a more stable cyclic form of this peptide has been described. NMR spectroscopy suggested that the beta turn conformation is preserved in cyclic peptide as well. Also, using in vitro bicistronic reporter assay, we demonstrated that cyclic peptide inhibits HCV translation in a dose dependent manner. In fact, due to its higher stability, cyclic peptide reduced HCV translation and replication more efficiently than the corresponding linear peptide at longer post-treatment time point. Additionally, we observed that cyclic peptide is non-toxic in cell culture system. Our results suggest that cyclic peptide might emerge as a promising lead compound against hepatitis C. Due to availability of only partially effective liver protective drugs in modem medicine, complementary and alternative medicine approach, based on plant derived compounds, is also being utilised against HCV. Plant derived compounds have advantages of having high chemical diversity, drug-likeliness properties and ability of being metabolized by the body with little or no toxicity than synthetic ones. Different studies have shown that phytochemicals may exert anti-HCV activities by acting as direct-acting antivirals and play a potential therapeutic role in treating HCV infection. Also, from our laboratory, it was shown that methanolic extract of Phyllanthus amarus (P. amarus) plant inhibited HCV replication. The fourth part of the thesis describes the study on the anti-HCV properties of several bioactive components from P. amarus extract. Using a fluorimetric assay, we demonstrated that two principal components of this extract, phyllanthin and corilagin reduced the HCV NS3 protease activity significantly in vitro. We also observed a sharp reduction in HCV negative sense RNA levels in cell culture system. Structural knowledge-based molecular docking studies showed interactions of phyllanthin and corilagin with the amino acid residues of the catalytic triad of NS3 protease. Further, these compounds were found to be non-toxic in cell culture. Also, phyllanthin and corilagin displayed antioxidant properties by blocking HCV induced oxidative stress generated by reactive oxygen species suggesting their hepatoprotective nature. More importantly, our in vivo toxicity analyses and pharmacokinetics studies proved their safety, tolerability, metabolic stability, and systemic oral bioavailability and support their potential as novel anti-HCV therapeutic candidates. Altogether, the study deciphers mechanistic details of translation and replication of HCV RNA and demonstrates novel antiviral agents targeting these important viral processes.

L'hépatite C (VHC) est l'une des causes principales de maladies du foie dans le monde, qui représentent un risque élevé d'évoluer vers la cirrhose et le carcinome hépatocellulaire. Actuellement, le traitement standard de l’infection par le VHC est l'interféron pégylé-(peg-IFN) et la ribavirine. Bien que le taux de la réponse virale soutenue (RVS) au traitement se soit améliorée au cours de ces années, cette thérapie n'est pas efficace chez tous les patients. En outre, plusieurs effets secondaires toxiques, de complications et le coût élevé limitent la compliance du patient et l'efficacité du traitement. Il n'existe pas de modèle simple d'infection par le VHC et il est nécessaire de développer des modèles in vitro et in vivo utiles pour étudier la physiopathologie de l'infection par le VHC, y compris les événements précoces de l'infection aiguë (l'entrée du virus, des mécanismes immunologiques et génétiques prédictifs) ainsi que l'évaluation de la puissance des médicaments antiviraux contre le VHC. Nous rapportons ici, nos efforts visant à développer des modèles appropriés de l'infection par le VHC. Dans un premier temps, nous avons établi un modèle de petit animal pour étudier l'infection par le VHC. Tupaia est un petit animal, apparenté aux primates et peu couteux. Dans notre travail, nous avons étudié la susceptibilité du tupaia à l'infection par VHC. Douze tupaias adultes ont été inoculés avec le VHC provenant de sérum de patient et d'ARN du VHC (génotype 1a). Trois jeunes tupaias ont été artificiellement nourris pendant un mois et ensuite inoculés par le VHC provenant de sérum du patient. L'ARN du VHC, les anticorps anti-VHC et l’évolution des quasi-espèces du VHC ont été déterminées chez l'animal avant et après l'inoculation. L'infection transitoire et intermittente s'est produite chez deux des 3 jeunes tupaias et l’infection chronique par le VHC s’est produite chez quatre tupaias sur 12 tupaias adultes. Le tupaia devrait représenter un modèle utile pour l'étude de l’infection chronique par le VHC. Dans une deuxième étape, un système de culture in vitro d'hépatocytes primaires de Tupaia a été établi, dans lequel l'infection par le VHC ne pouvait être bloquée ni par le CD81 soluble ni par des anticorps dirigés contre le CD81. Pour comprendre ces résultats, nous avons cloné, séquencé la grande boucle extracellulaire (LEL) du CD81 chez le Tupaia et analysé l'interaction de la protéine d’enveloppe E2 du VHC avec la LEL du CD81 chez le Tupaia par un test « enzyme-linked immunosorbent assay » (EIA). Nous avons constaté que chez le Tupaia, la séquence d'acides aminés du LEL de CD81 qui se lie au VHC présentait en 6 résidus d'acides aminés différents par rapport à la séquence humaine et la capacité de LEL de CD81 à se lier à la proteine d’enveloppe E2 du VHC a également diminuée. La structure différente de CD81 chez l’homme et chez le tupaia pourrait expliquer l'altération de l'interaction entre CD81 et la proteine E2 du VHC. Ce résultat démontre un rôle important de LEL du CD81 pour l'entrée du VHC. Dans une troisième étape, nous avons développé un modèle ex vivo de culture de tranches de foie humain et leur infection par le VHC. Le développement de lignées cellulaires provenant d’hepatocarcinome, permissives à la réplication du VHC, a fourni d'importants nouveaux outils virologiques pour étudier les mécanismes de l'infection par le VHC, mais ce modèle expérimental reste relativement éloigné des conditions physiologiques et pathologiques. Nous rapportons ici le développement d'un nouveau modèle ex vivo utilisant la culture de tranches de foie humain adulte, démontrant, pour la première fois, la capacité d’isolats primaires ainsi que JFH -1, H77/C3, Con1/C3 (HCVcc), de répliquer et de produire de novo des particules virales infectieuses ayant un titre viral élevé… / Hepatitis C virus (HCV) is one of the major causes of liver disease all over the world which has a high risk to progress to cirrhosis and hepatocellular carcinoma. Currently, the licensed standard treatment of HCV infection is Pegylated-interferon (peg-IFN) and ribavirin. Although the sustained viral response (SVR) rate of treatment has improved during these years, this therapy is not effective in all patients. In addition, several toxic side effects, complication and high cost limit the patient compliance and the efficacy of the treatment. There is no easy model of HCV infection and it is necessary to develop useful in vitro and in vivo models to study the pathobiology of HCV infection, including early events of acute infection (viral entry, immunological mechanisms, and genetic predictors) as well as the evaluation of the potency of the HCV antiviral drugs. We report here in our efforts in developing suitable models of HCV infection. In a first step, we preliminary established a small animal model to study HCV infection. Tupaia is a small, closed related to primate and cost-effective animal. In our work, we investigated the susceptibly of tupaia to HCV infection. Twelve adult tupaias were inoculated with native HCV from patient serum and full-length HCV RNA (Genotype 1a). Three young tupaias were artificially breeded for a month and then inoculated by native HCV from patient serum. HCV RNA, anti-HCV and HCV quasi species evolution were determined in the animal before and after inoculation. Transient and intermittent infection occurred in two among 3 young tupaias and HCV chronic infection occurred in four among 12 adult tupaias. Tupaia should represent a useful model for study HCV chronic infection. In a second step, an in vitro culture system of primary tupaia hepatocytes has been established in which HCV infection could be blocked neither by the soluble CD81 nor by antibodies against CD81. To understand these results, we cloned, sequenced the large extracellular loop (LEL) of tupaia CD81 and analyzed the interaction of HCV E2 with the tupaia CD81 LEL by enzyme-linked immunosorbent assay (EIA). We found that in the tupaia the amino acids sequence of HCV CD81 LEL presented in 6 different amino acid residues compared with human CD81 LEL sequence and the CD81 LEL ability to bind to HCV E2 was also decreased. The different structure of CD81 between human and tupaia could explain the alteration of the interaction between HCV E2 and CD81. This result demonstrated an important role of CD81 LEL for HCV entry. In a third step, we developed an ex vivo model of human liver slices culture and their infection with HCV. The development of human cultured HCV-replication-permissive hepatocarcinoma cell lines has provided important new virological tools to study the mechanisms of HCV infection; however this experimental model remains distantly related to physiological and pathological conditions. Here, we report the development of a new ex vivo model using human adult liver slices culture, demonstrating, for the first time, the ability of primary isolates to undergo de novo viral replication with the production of high titer infectious virus, as well as JFH-1, H77/C3, Con1/C3 (HCVcc). This experimental model was validated by demonstrating the HCV neutralization or HCV inhibition, in a dose-dependent manner, either by CD81 or E2 specific antibodies or convalescent serum from a recovered HCV patient, or by anti-viral drugs. This new ex vivo model represents a powerful tool for studying the viral life cycle, dynamics of virus spread in the liver and also for evaluating the efficacy of the new antiviral drugs. In the last step, we evaluated the efficacy of the new antiviral drugs with our ex vivo model of human adult liver slices. HCV NS3/4A protease is essential for viral replication and has been one of the most important target for developing specific antiviral drug

Unravelling the regulators of translation and replication of Hepatitis C virus Hepatitis C virus (HCV) is a positive sense, single stranded RNA virus belonging to the genus Hepacivirus and the family Flaviviridae. It infects human liver cells predominantly. Although, the treatment with α interferon and ribavirin can control HCV in some cases, they fail to achieve sustained virological response in others, thus emphasizing the need of novel therapeutic targets. The viral genome is 9.6 kb long consisting of a 5’ untranslated region (5’UTR), a long open reading frame (ORF) that encodes the viral proteins and the 3’ untranslated region (3’UTR). The 5’UTR contains a cis acting element, the internal ribosome entry site (IRES) that mediates the internal initiation of translation. The HCV 5’UTR is highly structured and consists of four major stem-loops (SL) and a pseudoknot structure. HCV proteins are synthesized by the IRES mediated translation of the viral RNA, which is the initial obligatory step after infection. The viral proteins are synthesized in the form of a long continuous chain of proteins, the polyprotein, which is then processed by the host cell and the viral proteases. Once viral proteins are synthesized sufficiently, the viral RNA is replicated. However the mechanism of switch from translation to viral RNA replication is not well understood. Several host proteins as well as the viral proteins help in the completion of various steps in the HCV life cycle. In this thesis, the role of two such factors in HCV RNA translation and replication has been characterized and exploited to develop anti-HCV peptides. The HCV proteins are categorized into two major classes based on the functions broadly: the non structural and the structural proteins. HCV NS3 protein (one of the viral non structural proteins) plays a central role in viral polyprotein processing and RNA replication. In the first part of the thesis, it has been demonstrated that the NS3 protease (NS3pro) domain alone can specifically bind to HCV-IRES RNA, predominantly in the SLIV region. The cleavage activity of the NS3 protease domain is reduced upon HCV-RNA binding owing to the participation of the catalytic triad residue (Ser 139) in this RNA protein interaction. More importantly, NS3pro binding to the SLIV region hinders the interaction of La protein, a cellular IRES-trans acting factor required for HCV IRES-mediated translation, thus resulting in the inhibition of HCV-IRES activity. Moreover excess La protein could rescue the inhibition caused by the NS3 protease. Additionally it was observed that the NS3 protease and human La protein could out-compete each other for binding to the HCV SL IV region indicating that these two proteins share the binding region near the initiator AUG which was further confirmed using RNase T1 foot printing assay. Although an over expression of NS3pro as well as the full length NS3 protein decreased the level of HCV IRES mediated translation in the cells, replication of HCV RNA was enhanced significantly. These observations suggested that the NS3pro binding to HCV IRES reduces translation in favour of RNA replication. The competition between the host factor (La) and the viral protein (NS3) for binding to HCV IRES might contribute in the regulation of the molecular switch from translation to replication of HCV. In the second part the interaction of NS3 protease and HCV IRES has been elucidated in detail and the insights obtained were used to target HCV RNA function. Computational approach was used to predict the putative amino acid residues within the protease that might be involved in the interaction with the HCV IRES. Based on the predictions a 30-mer peptide (NS3proC-30) was designed from the RNA binding region. This peptide retained the RNA binding ability and also inhibited IRES mediated translation. The NS3proC-30 peptide was further shortened to 15-mer length (NS3proC-C15) and demonstrated ex vivo its ability to inhibit translation as well as replication. Additionally, its activity was tested in vivo in a mice model by encapsulating the peptide in Sendai virus based virosome followed by preferential delivery in mice liver. This virosome derived from Sendai virus F protein has terminal galactose moiety that interacts with the asialoglycoprotein receptor on the hepatocytes leading to membrane fusion and release of contents inside the cell. Results suggested that this peptide can be used as a potent anti-HCV agent. It has been shown earlier from our laboratory, that La protein interacts with HCVIRES near initiator AUG at GCAC motif by its central RNA recognition motif, the RRM2 (residues 112-184). A 24 mer peptide derived from this RRM2 of La (LaR2C) retained RNA binding ability and inhibited HCV RNA translation. NMR spectroscopy of the HCV-IRES bound peptide complex revealed putative contact points, mutations at which showed reduced RNA binding and translation inhibitory activity. The residues responsible for RNA recognition were found to form a turn in the RRM2 structure. A 7-mer peptide (LaR2C-N7) comprising this turn showed significant translation inhibitory activity. The bound structure of the peptide inferred from transferred NOE (Nuclear Overhauser Effect) experiments suggested it to be a βturn. Interestingly, addition of hexa-arginine tag enabled the peptide to enter Huh7 cells and showed inhibition HCV-IRES function. More importantly, the peptide significantly inhibited replication of HCVRNA. Smaller forms of this peptide however failed to show significant inhibition of HCV RNA functions suggesting that the 7-mer peptide as the smallest but efficient anti-HCV peptide from the second RNA recognition motif of the human La protein. Further, combinations of the LaR2C-N7 and NS3proC-C15 peptide showed better inhibitory activity. Both the peptides were found to be interacting at similar regions of SLIV around the initiator AUG. The two approaches have the potential to block the HCV RNA-directed translation by targeting the host factor and a viral protein, and thus can be tried in combination as a multi drug approach to combat HCV infection. Taken together, the study reveals important insights about the complex regulation of the HCV RNA translation and replication by the host protein La and viral NS3 protein. The interaction of the NS3 protein with the SLIV of HCV IRES leads to dislodging of the human La protein to inhibit the translation in favour of the RNA replication. These two proteins thus act as the regulators of the translation and the replication of viral RNA. The peptides derived from these regulators in turn regulate the functions of these proteins and inhibit the HCV RNA functions.

Introduction International guidelines endorse the screening of patients for human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV) and Chlamydia trachomatis before assisted reproductive techniques (ART). At present no such guidelines exists in South Africa. At the Reproductive and Endocrine Unit (referred to as “the Unit”) of Steve Biko Academic Hospital, all patients with unknown HIV status are counselled and a blood sample is collected during the initial visit for automated laboratory based HIV screening. These HIV results are not available before semen samples are processed. Furthermore, patients are not screened for HBV, HCV and Chlamydia trachomatis. Couples attending the Unit are of a low to middle socio-economic status and experience financial constraints. Moreover, automated laboratory based assays are expensive to perform. Rapid testing is a cost effective and practical method from screening patients, with a 20–30 minute result turnover time. Until screening at the Unit is improved, the possible identification of semen characteristics that could indicate HIV infection would be a useful tool. Materials and Methods The following rapid point-of-care assays were evaluated: Determine® HIV-1/2 combo test (n=100), Determine® HBsAg test (n=100), DIAQUICK HCV kit (n=74), and the DIAQUICK Chlamydia trachomatis kit (n=30). For profiling, parameters from a basic semen analysis of HIV-positive males (n=60) were compared with HIV-negative males (n=60). Information pertaining to CD4 count, antiretroviral treatment and plasma viral load of HIV-positive males were analysed. Results From all patients included in the study, 8% tested positive for HIV. The risk of a female being HIV-positive was 3.73 times higher than for males. In the pilot study to explore rapid testing for HBV and HCV, 1% and 1.4% of patients tested positive respectively. When testing for Chlamydia trachomatis 31.3% of females, but no males tested positive. Comparing semen profiles, no significant differences were found between samples from HIV positive and negative males or between HIV positive males categorised by CD4 cell count (p>0.05). For the HIV-positive group with a detectable plasma HIV viral load (>40 copies/ml), a significant difference was observed in the semen viscosity (p=0.0460). Significant differences were noted in the sperm motility (immotile sperm p=0.0456, progressive sperm p=0.0192) of patients receiving antiretroviral (ARV) therapy. Discussion and Conclusion The use of rapid testing is an acceptable and feasible option for improving current screening protocols at the Unit. The absence of definite alterations in the semen characteristics of HIV-positive men further motivates the need for a simpler, point-of-care screening protocol. The prevalence of HBV was lower than that reported in the general population of South Africa and further investigation is needed. Although the sample size was small, HCV prevalence was similar to that of the general population. One third of females tested positive for Chlamydia trachomatis. The methodology used was possibly not appropriate for males. This study highlighted the need for guidelines that address the specialised needs of ART clinics in resource-limited and developing countries with a high HIV prevalence. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Obstetrics and Gynaecology / unrestricted

This research project aimed to conduct a strategic analysis of the implementation of a supervised injecting facility (SIF) in Montérégie. Using a mixed design, we first completed a portrait of the injection drug user (IDU) population. We then explored the perceptions of IDU and stakeholders with regard to the relevance of implementing a SIF in the region. Although some similarities were found with the IDU populations of Montreal and the province of Quebec, this population in Montérégie is characterized by a lower frequency of injections in public, less homeless people and lower rates of HIV and HCV infections. Despite these differences, the IDU population in Montérégie was found to have important physical and psychosocial needs. Although the relevance of a SIF in Montérégie is undeniable, improvements regarding the accessibility, continuity and appreciation of the actual services dedicated to IDU remain a priority. / Ce projet de recherche visait à réaliser une analyse stratégique de l’implantation d’un service d’injection supervisée (SIS) en Montérégie. Utilisant un devis mixte, son premier volet consistait à tracer un portrait de la population usagère de drogues par injection (UDI) montérégienne, alors que le second explorait les perceptions des UDI et des acteurs stratégiques œuvrant auprès d’eux quant à l’implantation d’un SIS dans la région. Bien que similaire aux populations UDI montréalaise et du Québec, celle de la Montérégie s’en distingue par le fait qu’elle s’injecte moins souvent dans des lieux publics, qu’elle soit sans domicile fixe à moindre proportion et par ses taux inférieurs d’infection au VIH et au VHC. Elle présente toutefois des besoins physiques et psychosociaux importants. Bien qu’un SIS soit jugé pertinent en Montérégie, une amélioration de l’accessibilité, de la continuité et de l’appréciation de l’offre de services actuelle dédiés aux UDI est considérée comme prioritaire.

La majorité des individus exposés au virus de l’hépatite C (VHC) développent une infection chronique. Une réponse immunitaire adaptative forte et soutenue est associée avec la guérison spontanée du VHC, mais les mécanismes sous-jacents demeurent mal définis. Le rôle des cellules NK et des cellules dendritiques (DC) dans la guérison spontanée du VHC est encore méconnu. Les cellules NK sont la population effectrice la plus importante de l’immunité innée car elles tuent les cellules infectées et sécrètent diverses cytokines. Les DC reconnaissent des agents infectieux et elles sont les premières à initier et réguler l’immunité adaptative. Les cellules NK et les DC interagissent également entre elles afin de réguler l’immunité innée et adaptative. L’hypothèse du projet de doctorat est que l'activité des cellules NK pendant la phase aiguë de l'infection par le VHC module la fonction des DC afin que ces dernières puissent générer une réponse immunitaire adaptative capable d'éliminer le VHC. Le premier objectif était d’établir une corrélation entre l'activité des cellules NK et l'évolution de l'infection au VHC. Nous avons observé une augmentation de la cytotoxicité, mais une diminution de la sécrétion de cytokines par les cellules NK chez les patients chroniques et qui ont résolu spontanément pendant la phase aiguë en comparaison aux contrôles non infectés, démontrant alors une dissociation entre ces deux fonctions. Nos résultats suggèrent que les cellules NK sont activées pendant la phase aiguë indépendamment de l’évolution de l’infection. Le deuxième objectif était d’établir une corrélation entre le phénotype et la fonction des DC, et l'évolution de l'infection. Nous avons d’abord observé que les DC plasmacytoïdes de tous les patients infectés ont un phénotype plus immature que les contrôles, et que ce phénotype est plus prononcé chez les patients ayant résolu spontanément. De plus, en réponse à des stimulations, nous avons observé que pendant la phase aiguë précoce, les DC myéloïdes (mDC) de tous les patients infectés indépendamment de l’évolution de l’infection produisent davantage de cytokines en comparaison aux contrôles. Cependant, cette hyperréactivité n’est pas soutenue au cours de l’évolution chronique. Le troisième objectif était d’établir une corrélation entre les interactions NK/DC et l’évolution de l’infection. Nous avons étudié la capacité des cellules NK à lyser les DC potentiellement tolérogéniques, ainsi que la capacité des DC matures à activer les cellules NK, et nous avons observé aucune différence entre les patients infectés et les contrôles. Finalement, nous avons démontré pour la première fois la capacité des DC immatures à inhiber la fonction des cellules NK. En conclusion, nous avons démontré que les cellules NK sont activées pendant la phase aiguë de l’infection par le VHC indépendamment de l’évolution de l’infection. De plus, la capacité des cellules NK à éliminer les DC potentiellement tolérogéniques est intacte. Finalement, les mDC sont hyperréactives pendant la phase aiguë de l’infection, mais cette hyperréactivité n’est pas soutenue avec la persistance de l’infection. Cette perte d’hyperréactivité des mDC ne semble pas affecter la capacité des DC à activer les cellules NK, mais elle pourrait jouer un rôle dans l’inefficacité de l’immunité adaptative à éliminer le VHC. / The majority of individuals exposed to the hepatitis C virus (HCV) develop a chronic infection. It is known that a strong and sustained adaptive immune response is associated with the spontaneous clearance of HCV, however the underlying mechanisms are not well defined. The role of natural killer (NK) cells and dendritic cells (DCs) during the spontaneous resolution of HCV remains unknown. NK cells are the primary effector population of the innate immune response which are able to kill infected cells and secrete various cytokines. On the other hand, DCs are the first cell type to initiate and regulate adaptive immunity after recognizing infectious pathogens. NK cells and DCs can also interact reciprocally to further regulate innate and adaptive immunity. Our hypothesis is that NK cell activity during acute HCV will modulate DC function to prime a highly efficient adaptive immune response resulting in viral clearance. The first aim of my project was to establish a correlation between NK cell activity and the outcome of HCV infection. We observed increased NK cell cytotoxicity, but decreased cytokine secretion during acute HCV in patients with chronic evolution as well as spontaneous resolution, further demonstrating a dissociation between these two NK cell functions. Our results suggest that NK cells are activated during acute HCV infection regardless of infection outcome. The second aim was to establish a correlation between DC phenotype, function and the outcome of infection. We observed that plasmacytoid DCs (pDCs) from all HCV-infected patients have a more immature phenotype as compared to negative controls, yet this is more pronounced in spontaneous resolvers. Furthermore, we observed that during the early acute phase, myeloid DCs (mDCs) from all HCV-infected patients, regardless of outcome, have increased production of cytokines as compared to un-infected controls in response to stimulation. However, this hyperresponsiveness of mDCs is not sustained with chronic evolution. The third aim was to establish a correlation between the NK/DC cross-talk and infection outcome. We studied the capacity of NK cells to kill potentially tolerogenic DCs, as well as the capacity of mature DCs to activate NK cells, and we observed no major differences between different stages of HCV infection and un-infected controls. However, we obtained unprecedented data which suggests that immature DCs have the capacity to inhibit NK cell function. In conclusion, our results demonstrate that NK cells are activated during acute HCV infection regardless of its outcome. Furthermore, the capacity of NK cells to kill potentially tolerogenic DCs is intact for all groups of patients. Finally, mDCs are hyperresponsive during acute HCV, but this hyperresponsiveness is not sustained with persistence of viremia. The loss of mDC hyperresponsiveness does not seem to affect the capacity of DCs to activate NK cells, but might play a role in the capacity of DCs to prime a highly efficient adaptive immune response resulting in viral clearance.