Hepatitis C Virus (HCV) infects more than 170 million people worldwide but no vaccine is available yet. HCV entry may represent a promising target for therapies and is mediated by two envelope glycoproteins, E1 and E2, assembled as heterodimer onto the virus surface. However, how E1 and E2 dialog, structurally rearrange and act together during these steps remain poorly defined. In this work, we aimed to clarify the interrelation of E1E2 during virus entry, thus opening ways to potential new therapeutic strategies. We first investigated whether a strong genetic divergence between E1E2 heterodimers may highlight distinct functions. We observed that B-cell derived E1E2 were specialized for B-cell infection, suggesting that new functions can emerge from the E1E2 conformational plasticity. In a second approach, we identified a conserved dialog between E1 and the domain III of E2 that was critical for virus binding and fusion. Moreover, a computational model predicted a strong co-evolution between E1 and E2 as well as potential structural rearrangements, suggesting that HCV E2 is likely a fusion protein able to fold over via its domain III through the mediation of E1. Altogether, these different works highlight that E1 and E2 are involved in complex dialogs that regulate the heterodimer folding and functions, suggesting that E1E2 heterodimer is more likely a single functional protein entity than an association of two proteins with specific functions.

Thesis (Ph.D.)--University of Alberta, 2009. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor in Philosophy in Experimental Surgery, Department of Surgery. Title from pdf file main screen (viewed on October 13, 2009). Includes bibliographical references.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2014-10-29T13:42:36Z No. of bitstreams: 1 Maria Isabel Magalhães Santos Perfil das....pdf: 1133942 bytes, checksum: bcc264f7cc24ec022846c05825fd31cf (MD5) / Made available in DSpace on 2014-10-29T13:42:36Z (GMT). No. of bitstreams: 1 Maria Isabel Magalhães Santos Perfil das....pdf: 1133942 bytes, checksum: bcc264f7cc24ec022846c05825fd31cf (MD5) Previous issue date: 2014 / Fundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Salvador, BA, Brasil / Introdução: A doença causada pelo vírus da hepatite B (HBV) é um problema de saúde pública mundial. No Brasil, o sistema único de saúde (SUS) tem disponibilizado drogas antivirais para o tratamento de hepatite B crônica há mais de 10 anos, mas um sistema para o monitoramento e avaliação de resistência a estas drogas ainda não está disponível. Objetivo: Este estudo teve como objetivo determinar o perfil de mutações do HBV associadas com a resistência aos análogos de nucleos(t)ídeos entre 81 pacientes com infecção crônica pelo HBV: virgens de tratamento para hepatite B e tratados com diferentes análogos de nucleosídeos e nucleotídeos, no Hospital Professor Edgar Santos (HUPES-UFBA)- Salvador-BA. Metodologia: O HBV-DNA foi isolado de amostras de soro, amplificado por nested-PCR, utilizando-se primers deduzidos da região flaqueadora da domínio rt do gene P e sequenciados (ABI Prism 3730, Applied Biosystems, EUA). Duas a seis sequências de cada isolado foram alinhados e os sítios conflitantes foram resolvidos usando o software CLC Main Workbench v. 5.0 por inspeção visual dos eletroferogramas. As sequências consenso tinham um tamanho de 1032 pb (compreendendo os aminoácido 1-344 da rt). Estas sequências foram submetidas ao banco de dados HBVrt DB (Stanford University, EUA) para a análise de cada mutação de acordo com o genótipo e tratamento. Resultado: O genótipo A1 foi o mais prevalente (85,2%) seguido pelo genótipo A2 (4,9%) F (6,2%) e C1, D2 e D4 (1,2% cada). Seis pacientes (7 %) apresentaram mutações de resistência para LAM, ETV, TDF: dois com o padrão L180M + M204V e quatro com padrões diversos (L80I + L180M + M204I ;L80V + L180M + M204V; M204I; A194T). Todas estas mutações foram associadas ao genótipo A (quatro A1 e dois A2). Além disso, foi encontrado um paciente com HBV genótipo C típico do leste da Ásia. Destes pacientes, dois foram virgens de tratamento e quatro tinham histórico de tratamento para HIV ou HBV. Foram detectadas quatro mutações no gene S (três casos com a mutação sI195M e um a mutação sW196L) associadas às mutações do domínio rt do gene P, correspondendo à uma taxa de 6% de mutações de escape vacinal. A prevalência das mutações de resistência às drogas antivirais variou de acordo com a duração do tratamento e com o nível da barreira genética da droga utilizada. Neste estudo, foi encontrada uma forte associação entre a ocorrência de mutações de resistência do HBV e positividade para o AgHBe, co-infecção com o HIV e histórico de tratamento para HBV e/ou HIV. Conclusão: Antes da terapia ser iniciada é extremamente importante o monitoramento da carga viral e a identificação destas mutações para suportar decisões clinicas sobre o manejo dos pacientes e prevenir a emergência de vírus multi- resistentes. / Introduction: Hepatitis B virus (HBV) infection is a public health issue. The Brazilian public health system (SUS) has provided antiviral drugs for chronic hepatitis B treatment for over 10 years, but a system for monitoring for drug-related resistance mutations is not available. Objective: This study aims to determine the presence of HBV mutations associated with resistance to nucleos(t)ide analogs among 81 patients with chronic HBV infection-naïve and treated from University Hospital Professor Edgard Santos, Salvador-BA (HUPES-UFBA). Methods: Briefly, HBV-DNA was PCR amplified with primers deduced from the flanking of the rt domain at the HBV P gene and sequenced using ABI Prism 3730 (Applied Biosystems, USA). From two to six forward and reverse sequences of each isolate were assembled and conflicting sites were resolved using software CLC Main Workbench v. 5.0 by visual inspection of the electropherograms. Consensus sequence extended 1032 bp and encompassed the entire rt domain (from amino acid 1 to 344). Those sequences were submitted to the HBV drug resistance database (HBVrt DB, Stanford University, USA) to retrieve each mutation according to genotype and treatment. Results: HBV genotype A1 (85.2%) was the most prevalent followed by genotype A2 (4.9%), F (6.2%), and C1, D2 and D4 (1.2% each). Six patients (7%) exhibited resistance mutations to LAM, ETV and TDF: two with patterns L180M + M204V and four with other different patterns: L80I + L180M + M204I; L80V + L180M + M204V; M204I; A194T. All of these mutations were present in patients with genotype A (four A1 and two A2). Furthermore, this study found one patient with genotype C, common in East Asian. Of these patients, two were naïve and four had a history of treatment for HIV or HBV. In addition, four mutations in gene S (sI195M three cases with the mutation and one with the mutations W196L) associated with mutations in the rt domain of the P gene were detected, corresponding to a rate of 6% of vaccine escape mutations. Prevalence of drug-related resistance mutations varied according to treatment duration and the level of genetic barrier for the drugs used. Conclusion: In this study a strong association was found between the occurrence of HBV resistance mutations and HBeAg positivity, co-infection with HIV and a history of treatment for HBV and / or HIV. Once the drug therapy is initiated it is extremely important to monitor viral load and identify those mutations in order to support clinical decisions about patient management and also to prevent the emergence of multidrug-resistant viruses.

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Salvador, BA, Brasil / INTRODUÇÃO: A hepatite B (VHB) é uma infecção dinâmica crônica, que apesar de existir programas de imunização e tratamento antiviral disponível, existe o risco de emergência de mutações de resistência aos análogos de núcleos(t)ídeos (AN) que devem ser rastreadas, devido as suas implicações clínicas. O Brasil disponibiliza pelo SUS cinco drogas para o tratamento antiviral: IFN, LAM, ADF, ETV e TDF e um guia de conduta clínica para orientar o tratamento no território nacional, o Protocolo de Diretrizes Terapêuticas para Hepatite B e co-infecções. OBJETIVO: O objetivo do presente estudo foi avaliar as mutações de resistência aos AN, mutações de escape vacinal e genótipos circulantes em pacientes com hepatite B crônica em dois centros de referencia em Hepatites, na Bahia (região Nordeste) e no Acre (região Norte) do Brasil. MATERIAL E MÉTODOS: Foi utilizadas ferramentas de biologia molecular e bioinformática, através de nested PCR e sequenciamento direto das amostras, para rastrear as mutações de resistência, a região alvo foi a transcriptase reversa (RT) do gene P e as mutações de escape vacinal foi a região do gene S do VHB, como também os genótipos e subgenotipos do VHB. RESULTADOS: Foram incluídos 527 pacientes durante o período de 2011-2015, sendo 320 pacientes do HUPES/BA e 207 do FUNDHACRE/AC. Os pacientes que representam a região Nordeste foram 59,3 % do sexo masculino e uma média de idade de 44,75±12,4 DP, os pacientes da região Norte 42% foram do sexo masculino e a média de idade foi de 40,36±13,9 DP. Todos os pacientes incluídos apresentaram AgHBs persistente por mais de seis meses e 86,1% apresentaram AgHBe negativo. Foram sequenciadas 296 amostras dos pacientes com VHB crônica. Foram encontradas mutações de resistência aos AN na Região Norte 1,2% (2), Região Nordeste 7,4%(8) e no global 3,8%(20). Os padrões de mutações de resistência primária encontrados foram: rtA194T, (3) rtL180M+M204V, rtL180M+M204I, rtS202I, rtM204I, rtA181S, rtA181E e rtA184S. Em relação ao escape vacinal a frequencia para a Região Norte foi de 7,1% (11), Região Nordeste 8,4% (9) e no global 7,6% (20). Nos pacientes virgens de tratamento (n=189), a frequência de mutações de resistência foi de 6%, somente nas amostras da região Nordeste. Não houve diferença estatisticamente significante entre o grupo com ou sem mutação dos pacientes virgens de tratamento. Não foram encontradas mutações de resistência nas amostras da região Norte. Os genótipos circulantes nas duas regiões foram A, D e F, e a região Nordeste foi encontrada o genótipo C (C2). CONCLUSÃO: Os resultados demonstram a importância de rastrear e monitorar as mutações de resistência aos AN e de escape vacinal devido a importância epidemiológica e clínica na conduta terapêutica. / INTRODUTION: Hepatitis B virus (HBV) is a chronic dynamic infection, which although there immunization programs and antiviral therapy available, there is a risk of emergence of resistance mutations cores analogs (t) ide to be screened, because of their implications clinics. The Brazil offers the SUS five drugs for antiviral treatment: IFN, LAM, ADF, ETV and TDF and clinical guide of conduct to guide treatment in the country, the Therapeutic Guidelines Protocol for Hepatitis B and co-infections. AIM: The aim of this study was to evaluate the resistance mutations core analogues (t) ide, vaccine escape mutations and circulating genotypes in patients with chronic hepatitis B in two reference centers in Hepatitis, Bahia (Northeast) and Acre (Northern region) of Brazil. MATERIAL AND METHODS: Was used tools of molecular biology and bioinformatics by nested PCR and direct sequencing of samples to track resistance changes, the target region is the reverse transcriptase (RT) P gene and vaccine escape mutations was region of the gene S of HBV, as well as the HBV genotypes and subgenotipos. RESULTS: 527 patients were included during the period 2011-2015, with 320 patients HUPES / BA and 207 FUNDHACRE / AC. Patients representing the Northeast were 59.3% male and an average age of 44.75 ± 12.4 PD patients in the northern region 42% were male and the average age was 40, 36 ± 13.9 DP. All patients had persistent HBsAg for more than six months and 86.1% were HBeAg negative. We were sequenced 296 samples from patients with chronic HBV. the cores of similar resistance mutations were found (t) ide in the North 1.2% (2), Northeast 7.4% (8) and 3.8% overall (20). The patterns of primary resistance mutations were: rtA194T (3) rtL180M + M204V, M204I + rtL180M, rtS202I, rtM204I, rtA181S, and rtA181E rtA184S. Regarding vaccine escape the frequency for the Northern Region was 7.1% (11), Northeast 8.4% (9) and the global 7.6% (20). In treatment-naïve patients (n = 189), the frequency of resistance mutations was 6%, only the samples in the Northeast. There was no statistically significant difference between the groups with or without mutation of naive patients. There were no resistance mutations in samples from the North. Circulating genotypes in the two regions A, D and F, and the Northeast found the C genotype (C2). CONCLUSION: The results demonstrate the importance of tracking and monitoring the resistance mutations similar cores (t) ide and vaccine escape due to epidemiological and clinical importance in the therapeutic approach.

Chez un individu infecté, le VHC circule sous la forme d’une population de variants viraux appelés quasi-espèce. Lors d’un évènement de transmission, certains variants viraux sont préférentiellement transmis et un effondrement de la diversité virale chez l’individu nouvellement infecté est souvent observé. Les propriétés électives de ces variants ainsi que leur rôle dans l’évolution clinique sont méconnus. L’objectif de cette étude est d’identifier si des déterminants moléculaires situés au niveau des glycoprotéines d’enveloppe du VHC sont associés à une plus grande capacité de transmission. Les propriétés fonctionnelles des variants transmis et non transmis seront étudiées, en particulier la sensibilité à la neutralisation autologue. Les échantillons étudiés proviennent de couples mère-enfant infectés chroniquement par le VHC issus de d’un essai clinique réalisé en Thaïlande. La composition des populations virales au sein de 3 paires a été étudiée à l’aide d’une technique d’amplification après dilution limite (SGA) suivie d’un séquençage profond (Illumina). Le variant majoritaire chez la mère était retrouvé majoritaire chez l’enfant pour les paires 1 et 3. Pour 2 paires (2 et 3), une moindre diversité génétique a été observée chez l’enfant par rapport à la mère témoignant d’un goulot d’étranglement génétique lors de la transmission. Après clonage des gènes E1E2, des tests d’infectivité sur cellules hépatocytaires ainsi que des tests de neutralisation par le sérum maternel sont réalisés avec le modèle de rétrovirus pseudotypés (VHCpp). Pour la 1ère paire, le variant majoritaire chez la mère (variant transmis à l’enfant) est infectieux et résistant au sérum autologue. Pour la deuxième paire, le variant minoritaire (transmis) est légèrement résistant à la neutralisation autologue. Un variant majoritaire non transmis apparait sensible à la neutralisation autologue. Des études complémentaires en système de virus réplicatifs issus de la culture cellulaire (VHCcc) sont en cours. Au final, les résultats de cette étude contribuent à comprendre les étapes précoces de l’infection par le VHC, afin de mieux appréhender de futures approches immunoprophylactiques ou vaccinales. / In infected individuals, HCV circulates as a complex mixture of genetically different, but closely related viral variants named quasispecies. In a transmission event, some viral variants are preferentially transmitted. The genetic and functional properties of these variants are still unknown. The aim of our work was to identify molecular determinants of E1E2 associates with a greater capacity of transmission. We also intend to study the functional properties of transmitted and no transmitted variants, as for example sensibility to autologous neutralization. Studied sera samples were obtained from three women and their child infected by the HCV, who were participating in HIV prevention clinical trial for the prevention of perinatal transmission of HIV in Thailand. Quasispecies were studied with single genome amplification (SGA) followed by deep sequencing (Illumina). A decrease in intra-host diversity (genetic bottleneck) was observed in the viral population of child near birth (week 6) compared with that observed in the mother (just before delivery). For 2 pairs, the major variant observed in the mother was the same as the major one identified in the child. Retroviral pseudotypes (HCVpp), bearing each transmitted and non-transmitted envelope glycoproteins were produced. For each one, the level of infectivity on HuH7 cells was measured as well as the neutralizing activity of the autologous sera. For the first pair, the major variant (transmitted) appears resistant to autologous neutralization. For the second pair, the transmitted minor variant appears slightly resistant to autologous neutralization. A non-transmitted major variant is sensitive to autologous neutralization. Complementary studies with HCV derived from cell culture (HCVcc) are in progress We hope that the results of this study may be helpful to better understand early steps of HCV infection, which is of great interest for the development of immunoprophylaxis and vaccine strategies.

Le virus de l'hépatite B (HBV) infecte de manière chronique 240 millions de personnes dans le monde, et est la principale cause de carcinome hépatocellulaire. Actuellement, les traitements standards permettent une suppression virale à long terme, mais ne sont pas capables d'éliminer complètement le virus, en raison de la persistance de l'ADN circulaire et clos de façon covalente (ADNccc). Ce minichromosome viral réside dans le noyau des hépatocytes infectés, grâce à sa structure chromatinienne. En effet, lors de l'infection d'un hépatocyte, l'ADN viral partiellement double brin (ADN relâché circulaire (rc)) est libéré dans le noyau, où il est réparé et enveloppé par des protéines histones, afin de former une structure d'épisome chromatinisé. Les mécanismes conduisant à la formation ainsi qu'à la chromatinisation de l'ADNccc sont encore largement inconnus, et leur élucidation constituerait une première étape vers l'identification de nouvelles cibles thérapeutiques, susceptibles d'altérer la persistance de l'ADNccc. Dans ce but, nous avons étudié le rôle des facteurs hôtes de réparation de l'ADN, et des voies d'assemblage des nucléosomes, dans la formation de l'ADNccc, à des stades précoces (entre 30 minutes et 72 heures) de l'infection, dans des lignées cellulaires d'HepG2-NTCP, ainsi que dans des hépatocytes primaires humains. Nous nous sommes particulièrement concentrés sur la protéine chaperone d'histones, HIRA, qui est connue pour déposer le variant d'histone 3.3 (H3.3) sur l'ADN cellulaire d'une manière indépendante de la réplication et en association avec le remaniement des nucléosomes pendant la transcription et la réparation de l'ADN. Nous avons été capables de détecter l'ADNccc dans la fraction nucléaire des hépatocytes dès 30 minutes et 24 heures post-infection, par qPCR et Southern Blotting (SB), respectivement. L'extinction de HIRA par ARN interférent (siARN) avant l'inoculation du virus, a conduit à une forte diminution de l'accumulation de l'ADNccc (à la fois par qPCR et Southern Blot), qui était indépendante de la protéine HBx (en utilisant un virus HBx-défectueux). Les niveaux d'ADNrc sont restés stables, indiquant soit une éventuelle transition de l'ADNrc en ADNccc incomplète, ou retardée. L'analyse par immunoprécipitation de la chromatine a montré que HIRA était liée à l'ADNccc dès 30 minutes après infection, et que son recrutement était concomitant avec le dépôt de l'histone H3.3, ainsi que la liaison de la protéine de capside du HBV (HBc). Après 24 heures d'infection, une augmentation de la liaison de H3.3 et de l'ARN polymérase II sur l'ADNccc a été observée, en corrélation avec l'initiation de la transcription de l'ARN viral de 3.5 kb. Par des expériences de co-immunoprécipitation et de test de proximité entre protéines (PLA), nous avons montré que HIRA était capable d'interagir avec HBc dans des hépatocytes infectés et dans une lignée cellulaire HepaRG exprimant HBc de manière inductible. En conclusion, nos résultats suggèrent que la chromatinisation de l'ADN viral entrant est un événement très précoce, nécessitant l'histone chaperone HIRA. Bien que HBx ne soit pas requis pour ce processus, HBc pourrait jouer un rôle majeur, suggérant que l'interaction entre HIRA et HBc pourrait représenter une nouvelle cible thérapeutique à étudier / Hepatitis B virus (HBV) chronically infects 240 million people worldwide and is the major cause of hepatocellular carcinoma (HCC). Currently standard-of-care treatments can achieve longterm viral suppression, but are not able to completely eliminate the virus, due to the persistence of the covalently closed circular DNA (cccDNA). cccDNA, the viral minichromosome, resides in the nucleus of infected hepatocytes by virtue of its chromatin structure. Indeed, upon entry into hepatocytes, the partially double stranded viral DNA (relaxed circular (rc)DNA) is released into the nucleus, where it is repaired and wrapped by histones to form an episomal chromatinized structure. The mechanisms leading to cccDNA formation and chromatinization are still largely unknown and their elucidation would be a first step toward the identification of new therapeutic targets to impair cccDNA persistence. To this aim, we investigated the role of host factors belonging to DNA repair and nucleosome assembly pathways in cccDNA formation at early time points (i.e. between 30 minutes and 72 hours) post-infection in both HepG2-NTCP cell line and Primary Human Hepatocytes (PHH). We particularly focused on the histone chaperone Hira, which is known to deposit histone variant 3.3 (H3.3) onto cellular DNA in a replication-independent manner and in association to nucleosome reshuffling during transcription and DNA repair. We were able to detect cccDNA in the nuclear fraction of hepatocytes as early as 30 minutes and 24h post-infection, by qPCR and Southern Blotting (SB), respectively. Knock-down of Hira by RNA interference before virus inoculation led to a strong decrease in cccDNA accumulation (both in qPCR and SB) which was independent from HBx protein expression (using an HBx defective virus). rcDNA levels remained stable, indicating either a possible incomplete or delayed rcDNA to cccDNA transition. Chromatin Immunoprecipitation analysis showed that Hira was bound to cccDNA already at 2 hours post-infection and that its recruitment was concomitant with the deposition of histone H3.3 and the binding of HBV capsid protein (HBc). After 24 hours of infection, an increase of H3.3 and Pol2 binding on cccDNA was observed, correlating with the initiation of the transcription of the 3.5 kb RNA. By Co-Immunoprecipitation and Proximity Ligation Assay experiments, we showed that Hira was able to interact with HBc in infected hepatocytes and in a HepaRG cell line expressing HBc in an inducible manner. Altogether, our results suggest that chromatinization of incoming viral DNA is a very early event, requiring the histone chaperone Hira. While HBx is not required for this process, HBc could play a major role, suggesting that the interaction between Hira and HBc could represent a new therapeutic target to be investigated

To enhance the global control of encephalitis and hepatitis caused by rabies virus (RABV), Japanese encephalitis virus (JEV), enterovirus 71 (EV71) and hepatitis B virus (HBV), novel immunisation strategies are needed. All four diseases particularly affect low income countries with marginal health services – an affordable combined vaccine strategy could alleviate the large burden of disease. Therefore, we aimed to construct a multipathogen vaccine assessing the immunising activity of a recombinant modified vaccinia Ankara (MVA), expressing key antigens (RABV-glycoprotein, JEV pre-membrane & envelope protein, EV71-P1 protein and large hepatitis B surface antigen) from the various pathogens. Successful delivery of the pathogen sequences into non-essential sites (deletion site I, II, VI) of MVA via homologous recombination with a transfer plasmid, was demonstrated by transient color selection (LacZ-marker) in vitro. The stable insertion of the expression cassettes was validated over ten virus passages by PCR with specific primer sets, targeting the pathogen sequence. Two recombinants, one carrying the EV71 and JEV pathogen sequence and one carrying the RABV-HBV pathogen sequence were generated and validated by PCR.To ensure similar expression of the key antigens, a T7-promoter was linked to the expression cassettes of all pathogen sequences. Direct regulation of this promoter was achieved through co-infection with a second T7-polymerase expressing MVA under the control of a vaccinia p7.5 promoter. Protein expression from recombinant MVA using the co-infection model of expression in vitro, was further characterised by Western blot, dot blot and immunocytochemistry. All inserted transgenes were expressed using an avian (chicken embryo fibroblasts) or mammalian (human fetal lung fibroblasts) cell culture system. To investigate the co-infection model of antigen delivery in vivo, a pilot murine immunogenicity study was performed in six Balb/c mice using the MVA-RABV-HBV recombinant in a homologous prime-boost regimen two weeks apart. To detect antibodies against the expressed pathogen sequences in the mouse serum an antibody-capture assay was performed (Western blot, dot blot). The antigen (used to capture murine antibodies) was purified RABV-glycoprotein or large hepatitis B surface antigen expressed from a baculovirus. The murine antibodies were detected by a secondary anti-mouse antibody, conjugated with horseradish peroxidase for a chemiluminescent reporter assay. Although, serum antibodies against MVA were induced in all mice, no serum antibodies against RABV or HBV could be detected. In summary, we were able to demonstrate that two transgenes, when inserted into one or two different loci in the MVA genome, can be expressed in vitro when using the co-infection model of gene expression with a T7-expression system. This project has provided new insights into a novel group of vaccines, the multipathogen viral vector vaccines, employing MVA as a vector. Future studies will be needed to further explore this vaccine-group, as well as the co-infection model of expression.

L'infection par le virus de l'hépatite C implique des processus d'interaction complexe entre l'hôte et le virus. Plusieurs facteurs de l'hôte incluant des polymorphismes génétiques et les réponses immunitaires influent sur l'infection et les réponses au traitement. Aussi, il est important d'identifier en amont les facteurs pour prédire la réponse au traitement. L'objectif de la thèse est d'étudier l'influence de certains polymorphismes génétiques de l'hôte sur la réponse à la bithérapie et sur le statut immunitaire du foie dans l'infection chronique par le VHC. L'étude a porté sur les polymorphismes des gènes de l'interféron lambda 3 et 4, l'interféron gamma, l'interleukine 10, et l'interleukine 17, conjointement à la réponse au traitement avec le peg-IFNα et la RBV et aux réponses immunitaires du foie chez les patients. Nous avons établi une méthode PCR-RFLP simple et fiable pour le typage de deux polymorphismes de l'IFNL3. En utilisant les enzymes de restriction BstUI et BrsDI permet le génotypage de deux variantes de IFNL3 (rs12979860 C/T et rs8099917 T/G, respectivement). Les résultats indiquent que cette méthode PCR-RFLP donne des résultats similaires à ceux des méthodes standard et présente un intérêt pour des analyses de routine en laboratoire clinique car elle est peu coûteuse. Nous avons analysé l'association des polymorphismes avec la réponse au traitement antiviral sur une cohorte de 108 patients infectés par le VHC de génotype 1 traités par la bithérapie. Nous avons ainsi démontré que le génotype de l'IFNL4 TT/TT de ss469415590 et une réponse virologique rapide sont des facteurs prédictifs indépendants pour atteindre un taux de réponse virologique soutenue (OR = 3,93, p = 0,004 et OR = 6,74, p = 0,021). D'autre part, une charge virale initiale haute est associée à l'échec au traitement (OR = 0,34, p = 0,023). Ainsi, ces paramètres sont utiles pour la définition d'un traitement personnalisé. Pour expliquer l'influence de ces polymorphismes dans l'infection chronique par le VHC, nous avons étudié l'association des polymorphismes IFNL3 et 4 avec la réponse immunitaire du foie chez les patients atteints d'une infection chronique par le VHC. En utilisant l'expression de CD107a comme marqueur de l'activité sécrétoires des lymphocytes, nous avons observé une activité de dégranulation des lymphocytes du foie plus importante les patients porteurs des génotypes de IFNL4 favorables en comparaison avec les patients porteurs de l'allèle défavorable. En utilisant des analyses de régression, les taux d'ALT sont en corrélation avec la fréquence des cellules NKT CD107a+ dans le foie. Enfin, chez les patients traités par la bithérapie, une forte activité de dégranulation est observée chez les patients avec les génotypes favorables IFNL3 et 4. Nous suggérons que les polymorphismes des gènes de l'interféron lambda sont associés à l'activité de la dégranulation des lymphocytes intra-hépatiques et contribuent à un mécanisme de clairance du VHC sous la bithérapie. Nous avons également étudié l'impact de plusieurs polymorphismes génétiques sur la gravité de l'hépatite C chronique. Les résultats montrent une association significative observée entre le polymorphisme de l'IFN-γ et la gravité de l'hépatite C chronique. Pour l'analyse de régression logistique, l'allèle T et la présence d'une stéatose sont des facteurs prédictifs indépendants de la sévérité de la maladie hépatique chronique associée au VHC. L'utilisation du génotypage de l'IFN-γ pourrait être utile dans la prise en charge des patients. En conclusion, nous avons montré que les polymorphismes des gènes des IFNL3 et 4 et de l'IFN-γ de l'hôte jouent un rôle important dans l'efficacité du traitement et les réponses immunitaires hépatiques. Ces résultats aident à définir un traitement personnalisé pour le contrôle de l'infection chronique par le VHC, en particulier dans les régions aux ressources limitées où les nouveaux traitements ne sont pas accessibles. / Hepatitis C virus (HCV) infection is a complex interaction process between the host and viral factors. The host immune responses and genetic polymorphisms have been shown to be associated with the outcome of HCV infections and the responses to treatments. Thus, it is very important to identify pre-treament factors to predict treatment outcomes. The overall aim of the thesis study is to investigate the role of host genetic polymorphisms on response to combination therapy and immune response in the liver in chronic HCV infection. The study has focused on polymorphisms in the interferon lambda (IFNL) genes, interferon gamma, interleukin 10, and interleukin 17 in relation to response to therapy with peg-IFNα and Ribavirin (RBV) and liver immune responses in patients with chronic HCV infection.First, we have established a simple and reliable method for genotyping of the IFNL3 polymorphisms. We designed primers and selected restriction enzymes BstUI and BrsDI for genotyping 2 variants rs12979860 C/T and rs8099917 T/G, respectively. The results indicate that this PCR-RFLP method yields to identical data than standard sequencing method and commercial kit. We suggest that PCR-RFLP method could be used routinely in conventionally clinical laboratory for genotyping of IFNL3 polymorphisms. Next, we analysed the association of these variants with response in combination therapy of peg-IFNα and RBV. Among 108 treated patients infected with HCV genotype 1, by using logistic regression model analyses, we showed that patients who had favorable IFNL4 genotype (genotype TT/TT of ss469415590) and presented a rapid virological response (RVR) were independent predictors of achieving sustained virological response rate (OR = 3.93, CI = 1.53 -10.08, p = 0.004 and OR = 6.74, CI = 1.33 - 34.06, p= 0.021), whereas patients with high baseline viral load level were associated with failure to treatment (OR = 0.34, CI = 0.13 - 0.87, p = 0.023). We suggest that patients had favorable IFNL4 genotype and achieved RVR should benefit an individualized treatment of combination therapy of peg-IFNα and RBV. To explain the influence of these polymorphisms in chronic HCV infection, we investigated the association of IFNL4 polymorphisms with immune response in the liver in patients with chronic HCV infection. By using marker CD107a, a marker expressing degranulation activity of cytotoxic lymphocytes, we indicated that degranulation process was found in liver lymphocytes in patients carrying favourable IFNL4 genotypes compared with patients with unfavourable genotypes. By using multiple regression analyses, we demonstrated that ALT levels correlate with frequency of CD107a+ NKT cells in the liver. Finally, in patients treated by peg-IFNα and RBV, high degranulation activity observed in patients with favourable genotypes of IFNL3 and IFNL4 (CC of rs12979860 and TT/TT of ss469415590). We suggest that polymorphisms in the interferon lambda genes associated with intrahepatic lymphocyte degranulation activity and contribute to clearance mechanism of HCV under combination treatment of peg-IFNα and RBV.We investigated the impact of several genetic polymorphisms on the severity of chronic hepatitis C. We showed a significant association observed between polymorphism of IFN-γ and the severity of chronic hepatitis C. By using logistic regression analysis, T allele of IFN-γ and the presence of steatosis are independent predictive factors of severity of HCV-1 - related liver disease. This suggests we can use genetic variant of IFN-γ in classification and management of chronic hepatitis C. In conclusion, we indicated that host genetic polymorphisms play critical roles both in responses to treatment and in the immunopathogenesis of chronic HCV infection. This study can help to reach a closer step to individualized medicine for the control of chronic HCV infection in resource-limited regions when new treatment regimens are not available.

Made available in DSpace on 2014-06-11T19:27:20Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-04-14Bitstream added on 2014-06-13T18:31:21Z : No. of bitstreams: 1 yamasaki_lht_me_sjrp.pdf: 6194517 bytes, checksum: 350d37acd1e46ef2885eecc1354a317c (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / A infecção pelo vírus da Hepatite C (HCV) é considerada um grande problema de saúde pública, desde a sua descoberta em 1989. Entretanto a terapia mais utilizada atualmente, baseada no uso de Peginterferon, tem sucesso em aproximadamente 50% dos pacientes com o genótipo 1. Embora os mecanismos envolvidos nesta resistência viral ainda não sejam esclarecidos, sugere-se que fatores virais e do hospedeiro participam deste. A proteína não-estrutural 5A (NS5A) está envolvida em diversos processos celulares e é um componente essencial para o HCV. Entretanto, sua estrutura e função ainda não foram bem elucidadas. A partir destes fatos, os objetivos do presente estudo foram elaborar um modelo teórico da NS5A e investigar as propriedades estruturais e funcionais in silico. Foram analisadas 345 sequências da proteína NS5A do HCV de 23 pacientes infectados com o genótipo 1 ou 3. As composições de aminoácidos e de estrutura secundária demonstraram que há diferença entre os genótipos, podendo indicar que há diferenças nas interações proteína-proteína entre os genótipos, o que pode estar relacionado com a diferença da taxa de resistência ao tratamento. A análise funcional foi realizada com o ProtFun, que sugeriu que a NS5A estaria envolvida nas funções celulares de metabolismo intermediário central, tradução, crescimento, tranporte, ligação e hormônio. Estas funções variaram entre os domínios, suportando a hipótese de que a NS5A é uma proteína multifuncional. A análise pelo PROSITE indicou vários sítios de glicosilação, fosforilação e miristoilação, que são altamente conservados e podem ter função importante na estabilização da estrutura e função, sendo assim possíveis alvos de novos antivirais. Alguns deles estão em regiões relacionadas com a resposta ao tratamento. Outro... / Hepatitis C virus (HCV) infects almost 3% of people worldwide and it is considered the main cause of liver chronic diseases and transplants. Until today, there is no effective vaccine and the current most used therapy, based on Peginterferon, is successful only in 50% of patients infected by genotype 1. Although the outcomes of this treatment resistance are unclear, it is suggested host and virus factors may participate in this mechanism. Non-structural 5A (NS5A) protein is involved in several cellular and virus processes and it is a critical component of HCV. However, its structure and function are still uncertain. Regarding these facts, the present study attachments were to elaborate a model of the NS5A protein and to investigate NS5A structural and functional features, using in silico tools. It was analyzed 345 sequences of HCV NS5A protein from 23 patients infected by genotypes 1 or 3. Residues and secondary structure composition of all sequences demonstrated that there are differences between genotypes. It may indicate that there are differences in interactions between genotypes, which could be related with the distinct average of treatment resistance. In addition, among those that varied between genotypes, there were amino acids in regions that studies suggested as related with virus persistence. Functional analysis was performed with ProtFun. It suggested that NS5A is involved with central intermediary metabolism, translation, growth, transport, ligation and hormone functions in the cell. These functions vary between the domains, strengthening the hypothesis that NS5A is a multifunctional protein. Prosite motif search indicated that there are many glicosilation, fosforilation and myristoilation sites, which are highly conserved and may play an important role in structural stabilization and... (Complete abstract click electronic access below)

L’infection par le virus de l’hépatite C (HCV) est une cause majeure de maladies hépatites sévères et constitue un problème majeur de santé public. Le HCV est associé aux lipopoprotéines formant une lipoviroparticule (LVP) qui est la forme infectieuse du virus. L’apolipoprotéine E (apoE), associée aux lipoprotéines, est impliquée dans les étapes précoces et tardives de l’infection. Elle interagit avec de récepteurs impliqués dans le métabolisme lipidique tels les héparanes sulfates protéoglycanes (HSPG). Durant ma thèse, j’ai démontré que les acides aminés chargés positivement du domaine de liaison aux HSPG d’apoE sont impliqués dans l’entrée du HCV dans l’hépatocyte. J’ai également démontré que la production et/ou l’infectiosité des particules virales est corrélée au taux d’expression d’apoE dans les cellules sans avoir d’impact sur la traduction ou la réplication virales. Enfin, j’ai identifié syndecan-4, un membre de la famille des HSPG, comme l’HSPG principal impliqué dans l’entrée du HCV dans les lignées Huh7.5.1. L’ensemble de ces résultats démontre qu’HCV utilise l’interaction apoE-SDC4 pour établir une infection virale efficace. / Hepatitis C virus (HCV) infection is a major cause of liver disease worldwide and represents a major health problem. HCV associates with host lipoproteins forming host/viral hybrid complexes termed lipoviral particles. Apolipoprotein E (apoE) is a lipoprotein component that interacts with heparan sulfate proteoglycans (HSPG) to mediate hepatic lipoprotein uptake, and may likewise mediate HCV entry. I sought to define the functional regions of apoE with an aim to identify critical apoE binding partners involved in HCV infection. I demonstrated a direct correlation of apoE expression and HCV infectivity, whereas no correlation exists with viral protein translation or replication. Mutating the HSPG binding domain (HSPG-BD) of apoE revealed key residues that are critical for mediating HCV infection. Finally, I identified Syndecan-4 (SDC4), an HSPG family member, as the principal HSPG mediating HCV entry. Our data demonstrate that HCV uses apoE-SDC4 interactions to enter hepatocytes and establish efficient viral infection.