Le VHC est une cause majeure de cancer du foie. Le traitement actuel est caractérisé par à un cout élevé, la présence de toxicité et l’émergence de résistance virale. Dans la 1ère partie de ma thèse, je me suis intéressé à l’entrée virale. L’entrée est nécessaire pour l’initiation ; la dissémination et le maintien de l’infection et représente ainsi une cible intéressante dans le développement de thérapies antivirales : CD81 et SRBI sont les 1ers facteurs décrits comme importants pour l’entrée : Nous avons confirmé leur rôle clé dans l’entrée et les étapes suivant l’entrée. De plus, nous avons montré leur rôle crucial dans la transmission cellule/cellule. Le VHC infecte principalement les hépatocytes, nous avons étudié en seconde partie de ma thèse le tropisme restreint du VHC aux hépatocytes. En définissant les facteurs essentiels à l’infection de cellules non hépatiques et en développant un modèle cellulaire afin d’identifier de nouveaux facteurs d’assemblage et de réplication du VHC. / HCV infection is the leading cause of chronic liver disease. The current SOC is still limited by high costs, toxicity and emergence of viral resistance. In the first part of my thesis we focused our workon viral entry. Viral entry is required for initiation, spread, and maintenance of infection, and thus is a promising target for the development of new antiviral therapies. CD81 and SR-BI are the first entry factors identified as important for HCV entry. In our work we confirmed their crucial role in entry, especially at the post-binding step. In addition we proved their key role in viral dissemination through the cell-cell transmission. As HCV mainly infects hepatocytes, we studied in the second part of my thesis, the restricted cellular tropism of HCV to hepatocytes and we defined the minimal host factors rendering non hepatic cell lines susceptible to HCV infection by the establishment of a powerful tool to identify new assembly and replication factors.

Introdução: Pacientes com infecção crônica pelo VHC e superinfecção pelo vírus da hepatite A (VHA) ou o vírus da hepatite B (VHB), têm maior morbi-mortalidade quando comparados com pacientes que apresentam infecção aguda somente pelo VHA ou VHB. A mortalidade associada à hepatite A aguda pode estar particularmente elevada em pacientes com pré-existência de hepatite crônica causada pelo VHC. Por esta razão, a imunização ativa com vacinas contra o VHA e o VHB vem a ser obrigatória nesta população, e consequentemente esta sorologia deve ser determinada. Objetivos: O objetivo deste trabalho foi avaliar a prevalência de marcadores sorológicos da hepatite A e hepatite B em 1.000 pacientes com infecção crônica pelo VHC atendidos no Ambulatório de Hepatites da Divisão de Gastroenterologia e Hepatologia Clínica do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo. Resultados: O anti-VHA IgG foi positivo em 923 de 1000 pacientes (92,3%). Quando estratificados por idade, o anti-VHA IgG foi encontrado em 61% dos pacientes entre 20 e 29 anos, 70% entre 30 e 39 anos, 85% entre 40 e 49 anos, 94% entre 50 e 59 anos e 99% nos pacientes com mais de 60 anos . O anti-HBc total foi positivo em 244 pacientes (24%). Estratificados por idade, em 4,3% dos pacientes entre 20 e 29 anos, 17% entre 30e 39 anos, 21% entre 40 e 49 anos, 24% entre 50 e 59 anos, e 28% dos pacientes com mais de 60 anos. Dos 244 pacientes anti-HBc IgG positivos, 0,8% são HBsAg positivo, 8,5% anti-HBc IgG isolado e 16% anti-HBs positivo. Conclusões: A prevalência de anti-VHA IgG nod nossos pacientes com hepatite C crônica foi semelhante à da população geral no município de São Paulo. No entanto, o anti-HBc totaI foi maior em nossos pacientes, quando comparada historicamente à população geral dos países ocidentais, sugerindo fatores de risco semelhantes para as hepatites B e C, o que enfatiza a importância dos programas de imunização nesta população / Background and Aims: Patients with chronic HCV and superinfection by hepatitis A virus (HAV) or hepatitis B virus (HBV) have higher morbidity and mortality when compared with those without HCV. For this reason, HAV and HBV active immunization has become mandatory in this population and hence their serological markers must be determined. The aim of this study was to evaluate the prevalence of serological markers of HAV and HBV infection in patients with chronic HCV. Methods: 1.000 chronic HCV infected patients at the University of Sao Paulo School of Medicine outpatient Liver Clinic were evaluated for the prevalence of serological markers of HAV and HBV infection. Results: Anti-HAV IgG was positive in 923 of 1000 patients (92.3%). When stratified by age, the anti-HAV IgG was found in 61% of patients between 20-29 years, 70% between 30-39 years, 85% between 40-49 years, 94% between 50-59 years, and 99% over 60 years of age. Anti-HBc IgG was positive in 244 patients (24%). Stratified by age, anti-HBc IgG was found in 4.3% of patients between 20-29 years, 17% between 30-39 years, 21% between 40 -49 years, 24% between 50-59 years, and 28% of patients over 60 years of age. Of the 244 anti-HBc IgG positive patients, 0.8% were also HBsAg positive, 8.5% were anti-HBc IgG isolated and 16% were also anti-HBs positive. Conclusions: The prevalence of anti-HAV IgG was similar to the general population in the city of São Paulo. However, anti-HBc IgG was higher in our chronic HCV patients, when compared historically to the general population of western countries, suggesting similar risk factors for HBV and HCV acquisition, so emphasizing the importance of immunization programs in this population. Keywords: Hepatitis C, Chronic; Hepatitis C; Hepacivirus, Prevalence; Hepatitis A; Hepatitis B Título: Prevalência de Marcadores Sorológicos das Hepatites A e B em Pacientes com Hepatite C Crônica atendidos no Ambulatório de Hepatites do Serviço de Gastroenterologia Clínica do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo - HCFMUSP Background and Aims: Patients with chronic HCV and superinfection by hepatitis A virus (HAV) or hepatitis B virus (HBV) have higher morbidity and mortality when compared with those without HCV. For this reason, HAV and HBV active immunization has become mandatory in this population and hence their serological markers must be determined. The aim of this study was to evaluate the prevalence of serological markers of HAV and HBV infection in patients with chronic HCV. Methods: 1.000 chronic HCV infected patients at the University of Sao Paulo School of Medicine outpatient Liver Clinic were evaluated for the prevalence of serological markers of HAV and HBV infection. Results: Anti-HAV IgG was positive in 923 of 1000 patients (92.3%). When stratified by age, the anti-HAV IgG was found in 61% of patients between 20-29 years, 70% between 30-39 years, 85% between 40-49 years, 94% between 50-59 years, and 99% over 60 years of age. Anti-HBc IgG was positive in 244 patients (24%). Stratified by age, anti-HBc IgG was found in 4.3% of patients between 20-29 years, 17% between 30-39 years, 21% between 40 -49 years, 24% between 50-59 years, and 28% of patients over 60 years of age. Of the 244 anti-HBc IgG positive patients, 0.8% were also HBsAg positive, 8.5% were anti-HBc IgG isolated and 16% were also anti-HBs positive. Conclusions: The prevalence of anti-HAV IgG was similar to the general population in the city of São Paulo. However, anti-HBc IgG was higher in our chronic HCV patients, when compared historically to the general population of western countries, suggesting similar risk factors for HBV and HCV acquisition, so emphasizing the importance of immunization programs in this population

Na prática hemoterápica em todo mundo, a triagem e o diagnóstico da infecção pelo HCV continua sendo um desafio. Desta forma, considerando as variáveis genéticas do vírus da Hepatite C e as variáveis populacionais de cada região, o conhecimento mais profundo da realidade da epidemiologia molecular do vírus da Hepatite C na população de doadores de sangue em regiões específicas se torna cada vez mais imprescindível. Desta forma, este estudo visou realizar a caracterização sorológica e molecular da infecção pelo Vírus da Hepatite C (HCV) em doadores de sangue do Estado do Amazonas a fim de subsidiar conhecimentos para interpretação dos diferentes perfis laboratoriais e clínicos, além de contribuir para o conhecimento das rotas de transmissão e da epidemiologia da infecção do Amazonas. Foram estudados 154 doadores de sangue do Estado do Amazonas com sorologia repetidamente reativa para anti-HCV. Foram realizados testes sorológicos por ELISA e Imunoblot, testes de detecção de RNA viral plasmático por Nested PCR, determinação da carga viral e genotipagem do vírus por sequenciamento. O estudo foi realizado no período de setembro de 2005 a abril de 2007 quando o índice de descarte por anti-HCV reativo foi de 0,37%. Foi observado que 50% dos casos de Imunoblot indeterminado apresentaram positividade no Nested PCR indicando a presença de RNA plasmático. A carga viral baixa foi um fator limitante para a determinação do genótipo viral pelo método de sequenciamento de algumas amostras. A freqüência de casos presumidos de clearance viral plasmático na amostra estudada foi de 18,8%. O genótipo mais prevalente do HCV nos doadores do Estado do Amazonas foi o genótipo 1 (87,5%) seguido do genótipo 3 (12,5%). Considerando as nuances da história natural da Hepatite C e os diversos momentos clínicos que os doadores possam se encontrar devem ser adotadas mudanças de condutas do acompanhamento dos doadores sororeativos para anti-HCV no banco de sangue do Amazonas e no fluxograma de diagnóstico da infecção. / The screening and diagnostic of HCV infection continue to be a challenge on the hemotherapic practice because the unique characteristics of each population and the molecular variability of the virus. The aim of this study was to characterize the serologic and molecular profile of 154 anti-HCV+ Amazon blood donors from the Amazon Hematology and Hemoterapy Foundation. Screening for anti HCV antibody was performed using ELISA and recombinant Imunoblot assay. Seropositive samples were assessed further with Nested PCR, viral load and genotyping techniques. In the study period, 2005-2007 the anti-HCV discard rate was 0,37%. We observed that 50% of the indeterminate Immunoblot cases showed HCV RNA presence in plasma by Nested PCR. The most prevalent genotype between subjects of this study was genotype 1 (87,5%), followed by genotype 3 (12,5%). The presumed plasma clearance frequency was 18,8%. The low viral load was a determinant critical factor to the genotype determination in some samples. Considering the different stages of the HCV natural infection different conducts may be adopted with inclusion of molecular testes as the first option for confirmation to the follow up of the positive anti-HCV blood donors in the Amazon blood bank and in the algorithm of the infection diagnostic, saving resources and providing a better counseling for the reactive donors.

A doença hepática crônica causada pelo vírus da hepatite C (HCV) tornou-se, nos últimos anos, uma das principais comorbidades dos pacientes portadores do vírus da imunodeficiência humana (HIV) nos países desenvolvidos. Os pacientes coinfectados com HIV/HCV apresentam uma progressão mais rápida para a cirrose e as suas complicações que os pacientes monoinfectados com HCV. Embora os mecanismos responsáveis por esta evolução não estejam totalmente esclarecidos, a expressão da molécula de HLA-G, um HLA de classe Ib não clássico, que tem propriedades bem reconhecidas na regulação negativa da resposta imune, pode estar relacionada à progressão da doença hepática. Os objetivos deste trabalho foram analisar o perfil de expressão de HLA-G em tecido hepático de pacientes coinfectados HIV/HCV e identificar possíveis variáveis do hospedeiro, do HCV e do HIV que possam estar relacionadas com a expressão de HLA-G na biópsia hepática. Para isso, 57 amostras de biópsia hepática de pacientes coinfectados com HIV/HCV, nas quais a imuno-histoquímica para HLA-G foi realizada, foram analisadas retrospectivamente quanto à expressão desta molécula no tecido hepático. Avaliaram-se também outras características histopatológicas da biópsia como grau de fibrose, atividade inflamatória, deposição de ferro e gordura. Determinou-se o polimorfismo de inserção ou deleção de 14 pares de bases da região 3` não traduzida do exon 8 do gene do HLA-G, que está relacionada com a produção de RNA-mensageiro, em 43 destes pacientes, além do polimorfismo de IL-28B, relacionado com a resposta ao tratamento do HCV, em 44 deles. Características bioquímicas e virológicas, tanto do HIV quanto do HCV também foram avaliadas. O genótipo 1 do HCV foi o mais prevalente (87,75%), especialmente o subgenótipo 1a (60%). A expressão do HLA-G foi observada em 38 (66,7%) amostras de fígado, e foi mais frequente em estágios moderados e severos de fibrose do que em estágios mais leves (94,1% x 55%, P < 0,01). Não houve relação entre a expressão do HLA-G e os outros parâmetros estudados. Embora a progressão para a cirrose no contexto da coinfecção por HIV/ HCV seja um processo complexo, modulado por muitos factores, a associação da intensidade de fibrose com a expressão do HLA-G pode indicar que a expressão desta proteína desempenha um importante papel nos mecanismos que contribuem para a progressão da doença, por meio da regulação negativa da resposta imune contra o HCV na coinfecção pelo HIV. / Chronic liver disease induced by hepatitis C virus (HCV) infection has recently become one of the most common comorbidities in patients who are infected with the human immunodeficiency virus (HIV) in developed countries. HIV/HCV coinfected patients show faster progression to cirrhosis and its complications than the HCV monoinfected patients. Even though the responsible mechanisms for this evolution have not been entirely clarified yet, the expression of the HLA-G molecule, a HLA from the non-classic Ib class, with well-known properties of negatively regulating the immune response, may be related to the liver disease progression. The aims of the present work were to analyze the HLA-G expression profile in the liver micro ambience of HIV/HCV coinfected patients and to identify possible host factors, HIV or HCV, that may be related to the HLA-G expression on the liver biopsy. For this purpose, 57 liver biopsies of HIV/HCV coinfect patients, in which immunohistochemistry for HLA-G had been performed, were retrospectively analyzed according the HLA-G expression on the hepatic tissue. Other histopathological features in the liver biopsies, such as fibrosis degree, inflammatory activity, iron deposition and fat were also evaluated. The polymorphism of insertion or deletion in 14-base pairs of the 3`non-translated region of exon 8 of the HLA-G gene, which is related to the production of HLA-G messenger RNA, was evaluated in 43 of the patients. Also, the polymorphism of IL-28B, related to the response to HCV treatment, was evaluated in 44 of them. Biochemical and virological features of HIV and HCV were also evaluated. The HCV genotype 1 was the most prevalent (87.75%), especially the subgenotype 1a (60%). The expression of HLA-G was observed in 38 (66.7%) samples of the liver biopsies, and it was most frequent in moderate and severe stages of fibrosis than in the mild stages (94.1% x 55%, P < 0.01). There was no established relationship between HLA-G and other parameters studied. Although the progression to cirrhosis in the context of HIV/HCV coinfection is a complex process modulated by many factors, the association of HLA-G expression with the intensity of the liver fibrosis may indicate the protein expression play an important role in the mechanisms that contribute to the progression of the disease, through the negative regulation of the immune response against HCV setting of a coinfection with HIV.

Introdução e objetivos: A infecção crônica pelo VHC é considerada um grave problema de saúde pública mundial. O perfil epidemiológico vem mudando desde 1992 com a obrigatoriedade da pesquisa sorológica em doadores de sangue. Atualmente o uso de drogas ilícitas injetáveis é o fator de risco mais importante. Em muitos casos o mecanismo de contaminação não é identificado sendo definido como forma esporádica. O presente trabalho avaliou aspectos demográficos e epidemiológicos de pacientes com infecção crônica pelo VHC, em acompanhamento no ambulatório de Hepatologia do Serviço de Gastroenterologia da Divisão de Clínica Médica II do Hospital das Clínicas da FMUSP. Pacientes e métodos: Foram entrevistados 700 de um total de 1.112 pacientes adultos, no período de outubro de 2001 a novembro de 2003 (49% homens, 51% mulheres). Após a assinatura do termo de consentimento esclarecido, todos os pacientes foram submetidos à entrevista com questionário elaborado para este estudo, abrangendo aspectos demográficos, fatores de risco e uso de bebida alcoólica. O diagnóstico da infecção pelo VHC foi realizado através de teste sorológico Elisa de terceira geração e pesquisa do RNA viral através da reação em cadeia da polimerase. A determinação do genótipo do VHC foi realizada em 540 amostras de soro através do sequenciamento da região 5\' UTR. A biópsia hepática foi analisada em 470 pacientes e estadiada segundo critérios das Sociedades Brasileiras de Patologia e Hepatologia. Resultados: Não houve diferença significante entre a média de idade de homens e mulheres (49 ± 12,2 anos e 51 ± 12,5, respectivamente). 60% cursaram até o ensino fundamental, 19,7% o ensino médio e 12,4% o superior. 1% dos pacientes tem ocupações ligadas ao setor primário da economia, 8% ao setor secundário e 52% ao setor terciário. 62% foram classificados como brancos e 67% como católicos. 60% referiram relacionamento estável monogâmico. Em 68,5% dos casos o diagnóstico foi estabelecido através de exames de rotina e em 19,4% durante doação de sangue. O principal fator de risco para infecção foi a realização de hemotransfusão antes de 1993 (46,4%). 10% dos pacientes referiram uso de drogas injetáveis e 3%, apresentavam ambos os fatores. Em 42% dos casos o mecanismo de infecção foi considerado como forma esporádica. O genótipo 1 foi responsável por 70% das infecções seguidas pelo genótipo 3 (25%). Grau leve de fibrose classificado como 0 ou 1 foi encontrado em 48,7% dos pacientes, estádio 2 em 18,5%, estádio 3 em 12,3% e estádio 4 (cirrose) em 20,4% dos casos. A análise multivariada mostrou que os fatores de risco para desenvolvimento de cirrose foram: uso de álcool(> 60g/dia, odds ratio 2.01), raça branca (odds ratio 2,2) e idade acima de 55,8 anos (odds ratio 2,2). Conclusões: A infecção crônica pelo VHC foi caracterizada por alta freqüência de formas esporádicas e predominância dos genótipos 1 e 3. Na maioria dos casos o diagnóstico da infecção foi realizado através de exames de rotina. Consumo de álcool acima de 60 g/dia, raça branca e idade acima de 55,8 anos foram fatores de risco para a progressão para a cirrose / Background and aims: Hepatitis C virus infection is considered a world-wide serious public health problem. Since 1992, the epidemiological profile of the infection has changed with the systematic serological testing in blood donors. Nowadays the use of illicit intravenous drugs remains one of the most important risk factor. Nevertheless, in a variable percentage of cases, the mechanisms of contamination can not be identified and those cases are referred as sporadic forms. The current work was aimed at evaluating the demographic and epidemiological profile of HCV infection in outpatients attending the Hepatology branch of the Division of Gastroenterology of University of São Paulo School of Medicine teaching hospital. Patients and methods: From October 2001 and November 2003, 700 out of 1.112 adult patients were enrolled in this study (49% men, 51% women). After the written informed consent was obtained, all patients were interviewed, using standardized questionnaire for collecting data about demographic data, risk factors and alcohol use. Routine serological testing for HCV was performed using third-generation ELISA assays and circulating HCV-RNA was detected by polymerase chain reaction. HCV genotyping was performed in 540 subjects by sequencing of the 5\' UTR region. Liver biopsy slides from 470 patients were available for the assessment of the degree of fibrosis, which was performed according to the criteria of the Brazilian Societies of Pathology and Hepatology Results: There was no significant difference between the mean age of men and women (49 ± 12.2 years and 51 ± 12.5, respectively). 60% had completed no more than basic education, 19.7% had finished high school and 12.4% had graduated from university. 1% worked in activities of the primary sector of economy, 8% in the secondary and 52% in the tertiary one. 62% were caucasian descendants. 67% were catholic. 70% were born in the Southeastern region of Brazil and 97% lived in the State of São Paulo. 60% declared to have a monogamic relationship in the last 6 months. In 68.5% the diagnosis of HCV infection was established by routine check-up tests and in 19.4% during blood donation. The major risk factor for HCV infection was blood transfusion before 1993 (46.4%). 10% of the patients were intravenous drug users, and 3% had both risk factors. In 42% of the cases, the mechanism of infection was considered sporadic. Genotype 1 was found in 70% of the cases, followed by genotype 3 (25%) and genotype 2 (2.7%). Liver fibrosis stage 0 or 1 was found in 48.7%, stage 2 in 18.5%, stage 3 in 12.3% and stage 4 (liver cirrhosis) in 20.4% of cases. Linear regression multivariate analysis showed that risk factors for developing liver cirrhosis were: alcohol abuse (> 60g/day, odds ratio 2.01), caucasian origin (OR 2.2) and age > 55.8 year old (OR 2.2). Conclusions: HCV infection profile in this cohort was characterized by a high frequency of sporadic forms and predominance of genotypes 1 and 3. The infection was diagnosed in most of the cases by routine check-up tests. Heavy alcohol use, caucasian origin and older age were risk factors for progression to cirrhosis

Accès limité / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

Le virus de l'hépatite C (HCV) représente un problème de santé publique à l'échelle mondiale. Les thérapies actuelles ne permettent pas de guérir tous les patients infectés par le HCV et certains antiviraux ont des effets secondaires importants. Dans la première partie de ma thèse, nous avons identifié des combinaisons d'antiviraux à action directe (DAA) et d'inhibiteurs d'entrée caractérisés par un effet synergique dans la prévention et le traitement du HCV dans des modèles de culture cellulaire et les souris uPA-SCID avec un foie chimérique. Ceci représente une nouvelle stratégies de lutte contre l'infection par le HCV. Dans la seconde partie de ma thèse, nous avons démontré que le mode de transmission du HCV de cellule à cellule est la voie de transmission dominante dans les modèles de culture cellulaire. De plus, les virus résistant aux DAA se propagent efficacement grâce à la transmission de cellule à cellule. L'inhibition de la transmission de cellule à cellule en utilisant des inhibiteurs d'entrée est un moyen efficace pour empêcher l'émergence de virus résistant aux DAA et pour potentialiser l'efficacité antivirale des DAA pour éradiquer l'infection par le HCV. / Hepatitis C virus (HCV) poses a threat to global health with infecting about 170 million people. Current therapies cannot cure all the patients infected with HCV and have obvious side effects. In the first part of my thesis, we uncovered combinations of direct-acting antivirals (DAAs) and entry inhibitors caracterized by a synergistic effect in prevention and treatment of HCV infection using HCV cell culture models and human liver chimeric uPA-SCID mice, thereby providing a new strategy to control HCV infection. In the second part of my thesis, we demonstrated that HCV cell-cell transmission is the dominant transmission route in cell culture models and that DAA-resistant HCV spread efficiently through cell-cell transmission to develop viral resistance. Blocking cell-cell transmission using entry inhibitors allows to prevent the emergence of DAA-resistant virus and potentiates the antiviral efficacy of DAAs to clear HCV infection. In summary, we provide novel strategies to enhance antiviral efficacy by combining entry inhibitors and DAAs and to prevent viral resistance by blocking viral cell-cell transmission.

L’infection par le virus de l’hépatite C (HCV) est une cause majeure d’hépatite chronique, de cirrhose hépatique et ce carcinome hépatocellulaire dans le monde. La compréhension des relations entre le virus et sa cellule hôte, l’hépatocyte, est indispensable pour le développement de nouvelles stratégies thérapeutiques et d’un vaccin préventif. La particularité de ce virus est son lien étroit avec le métabolisme lipidique. Le virus circule dans le sang associé aux lipoprotéines, formant une lipo-viro-particule (LVP) infectieuse. L’apolipoprotéine E (apoE) est une protéine cellulaire faisant intégralement partie de la LVP. Elle joue un rôle majeur dans l’infection et à la production des particules virales. Durant ce travail de thèse, j’ai pu approfondir les connaissances sur le rôle d’apoE dans le cycle viral du HCV sous deux aspects très différents. D’une part, j’ai pu démontré que la forme libre d’apoE, non liée aux lipoprotéines, inhibe la réplication du HCV grâce à la régulation du métabolisme lipidique hépatique, en induisant un efflux de cholestérol ABCG1-dépendant. D’autre part, j’ai participé à une étude démontrant que l’apoE liée à la LVP contribue à l’échappement du virus au système immunitaire, en masquant les épitopes de la protéine virale E2 aux anticorps neutralisants. Ces études ont mis en évidence deux nouveaux rôles d’apoE dans la pathogenèse du HCV. / Hepatitis C virus (HCV) infection is a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma worldwide. Understanding virus-host interactions in hepatocytes will contribute towards the development of new therapeutic strategies and a protective vaccine. HCV life cycle and the lipid metabolism are inextricably intertwined. A particular feature of this virus is that, in the blood, HCV virions are associated with lipoproteins, forming an infectious lipoviroparticle (LVP). Apolipoprotein E (apoE) is a key component of LVPs that plays an essential role in HCV entry and virions production. My PhD project entailed a more detailed dissection of apoE’s role in the HCV life cycle. I demonstrated that lipid-free apoE inhibits HCV replication by regulating the hepatic lipid metabolism via an ABCG1-dependent cholesterol efflux. Furthermore, I contributed to a study demonstrating that LVP-associated apoE helps the virus escape host immunity by blocking access of neutralizing antibodies to the viral glycoprotein E2. The work presented highlights two new roles of apoE in HCV pathogenesis

Les vascularites cryoglobulinémiques associées au virus de l'hépatite C (VC-VHC) sont caractérisées par une expansion clonale de lymphocytes B mémoires anergiques CD27+IgM+CD21-/low (Bm21-), un défaut quantitatif en lymphocytes T régulateurs (Tregs) et une polarisation Th1. Les antiviraux d'action directe sans interferon (DAAs) sont très efficaces chez les patients VC-VHC mais leur mécanisme d'action sur l'immunité cellulaire reste inconnu. Nous montrons que les DAAs normalisent la plupart des perturbations de l'homéostasie lymphocytaire B et T, en diminuant l'expansion des Bm21- et T folliculaires, et en augmentant les T régulateurs (Tregs). Nous avons étudié l'effet des Bm21- sur les sous-populations lymphocytaires T, et les réactivités de leur récepteur B. Nous montrons que les Bm21- stimulés par le CpG favorisent la sécrétion d'IFNγ par les T effecteurs et induisent leur prolifération. Inversement, les Bm21- stimulés par CpG diminuent la capacité proliférative des Tregs. Nous montrons la diversité intraclonale des IgM mutés des Bm21-, conduite par maturation d'affinité antigène dépendante. Les anticorps (Ac) des Bm21- ont une activité facteur rhumatoïde (FR) mais ne sont ni polyréactifs, ni autoréactifs contre les autoantigènes ubiquitaires. Les Ac des Bm21- ne présentent pas de réactivité croisée contre des antigènes viraux du VHC. Les Bm21- stimulés par CpG ont une signature transcriptomique révélant un phénotype non tolérogène. Ainsi, ces résultats suggèrent le rôle majeur des Bm21- dans le défaut tolérance des patients CV-VHC, à la fois par la stimulation CpG conduisant à une réactivation des Bm21- anergiques, et par l'expression clonale des IgM à activité FR. / Hepatitis C virus-associated cryoglobulinemia vasculitis (HCV-CV) is characterized by an abnormal clonal expansion of anergic CD27+IgM+CD21-/low memory B cell (Bm21-), a quantitative defect in regulatory T cells (Tregs) and Th1 profile. Interferon-free direct-acting antivirals (DAAs) proved to be very effective in patients with HCV-CV but their mechanisms of action and their effects on cellular immunity remain poorly defined. Our results indicate that DAAs effectively normalizes many of the disturbances in peripheral B- and T-lymphocyte homeostasis of HCV-CV patients, by reducting Bm21- and T follicular helper expansion and promoting Tregs. Then, we investigated the effects of Bm21- on T-cell subpopulations and study the reactivities of their B-cell receptors. We show that CpG-stimulated Bm21- promote the secretion of IFNγ by effector T cells and induce their proliferation. Conversely, stimulated Bm21- reduce the proliferative capacity of regulatory T cells. Bm21- B-cell expansions show intraclonal diversity of highly mutated IgM antibodies that were shape by an antigen-driven maturation process. Bm21- antibodies possess rheumatoid factor activity but are neither polyreactive nor recognize ubiquitous autoantigens. No crossreactivity of Bm21- antibodies against several HCV antigens was observed. We also identify a transcriptional signature in CpG-stimulated Bm21- revealing a phenotype sufficient to break the immune tolerance. Thus, these results strongly suggest a major role for Bm21- in defective tolerance of HCV-CV patients, both through CpG stimulation leading to reactivation of anergic Bm21-, and through the clonal expression of IgM antibodies with RF activity.

Natural Killer (NK) cells, members of the innate lymphoid cells (ILCs), are known to play an important role in the defense against foreign cells and abnormal host cells that have arisen due to viral infection or cancer inducing mutations. The typical immune response of NK cells involves the release of cytotoxic granules containing perforin and granzyme, and the secretion of immune-regulatory cytokines such as interferon gamma (IFN-γ). Unlike the adaptive lymphocytes such as T cells and B cells, NK cells do not require prior sensitization, enabling them to initiate an immune response much faster. This unique feature of NK cells is made possible by the utilization of an array of germline encoded receptors; but on the other hand, it limits NK cells ability to respond against rapidly evolving pathogens. NK cells overcome this shortcoming with an antibody-assisted process called antibody dependent cellular cytotoxicity (ADCC). A novel subset of human NK cells, which displays potent and broad antiviral responsiveness in concert with virus-specific antibodies, was recently discovered in cytomegalovirus positive (CMV+) individuals. This NK cell subset, called g-NK cell, was characterized by a deficiency in the expression of FcεRIγ, an adaptor protein that associates with CD16 which enables ADCC. Surprisingly, despite this deficiency, g-NK cells displayed an enhanced ADCC as compared to their conventional counterparts. Furthermore, having a long-lasting memory-like NK-cell phenotype suggests a role for g-NK cells in chronic infections. This study investigates the importance of g-NK-cells in clinical settings, first by investigating whether the presence of g-NK cells is associated with the magnitude of liver disease during chronic hepatitis C virus (HCV) infection. Analysis of g-NK cell proportions and function in the peripheral blood mononuclear cells (PBMCs) of healthy controls and chronic HCV subjects showed that chronic HCV subjects had slightly lower proportions of g-NK cells, while having similarly enhanced ADCC responses compared to conventional NK cells. Notably, among CMV+ chronic HCV patients, lower levels of liver enzymes and fibrosis were found in those possessing g-NK cells. g-NK cells were predominant among the CD56neg NK cell population often found in chronic HCV patients, suggesting their involvement in the immune response against HCV. Rituximab is a chimeric anti-CD20 antibody used to treat B cell lymphoma patients; and studies have suggested that its efficacy is associated with the ADCC potency and CD16 affinity. Since g-NK cells are characterized by their superior ADCC compared to their conventional counterpart, I decided to investigate whether the presence of g-NK cells can improve the effectiveness of rituximab against malignant B cells in the context of lymphoma and leukemia. The analysis of g-NK cells’ ADCC response against rituximab-coated lymphoma cell lines and B cells from a CLL patient indicated a superior ADCC by g-NK cells compared to their conventional NK cell counterparts. Taken together, for the first time, my findings indicate that the presence of g-NK cells in CMV+ individuals is associated with milder liver disease in chronic HCV infection. In addition, an enhanced ADCC response by g-NK cells upon encountering rituximab coated target cells suggests the beneficial roles of g-NK cells, and opens an avenue for novel therapeutic approaches where g-NK cells can be utilized to treat persistent diseases such as chronic viral infection and cancer.