Infection with hepatitis C virus (HCV) can cause liver damage known as fibrosis, which often leads to liver disease and hepatocellular carcinoma. The impairment of circulating, bulk (non-specific and specific) CD8+ T cells within HCV-infection, characterized by an altered phenotype and the increased expression of pro-apoptotic genes, is observed when compared to uninfected controls. The relationship between bulk CD8+ T cell function and the extent of liver damage has not been demonstrated. In this study, widespread immune alterations were observed in untreated HCV infection with advanced liver fibrosis. Untreated HCV-infected individuals with advanced fibrosis possessed a significantly decreased proportion of naïve CD8+ T cells and an increased proportion of late effector memory CD8+ T cells compared to uninfected controls. Upon T cell receptor (TCR) stimulation, these individuals also had an increased intracellular IFN-γ expression for four CD8+ T cell subsets, a decreased CD107a expression for central memory CD8+ T cells, and a decreased perforin induction for naïve and central memory CD8+ T cells. These immune alterations did not reverse 24 weeks after viral cure. This study indicates there is a relationship between the differentiation and function of bulk CD8+ T cells and the extent of liver damage within HCV infection.

Recentemente estudos demonstraram que os SNPs (polimorfismos de base única) rs8099917 e rs12979860 localizados próximos ao gene da IL28B explicam a variação de resposta à infecção e tratamento do paciente contra o genótipo 1 do HCV, porém não para o genótipo 3 deste vírus. Este trabalho encontrou associação significativa entre resposta à infecção devida ao genótipo 3 pelo tratamento (PEG-INF e RBV) e o polimorfismo rs8099917 em uma amostra da população de Santos - SP. Para o polimorfismo rs12979860, esta associação somente foi encontrada ao se parear indivíduos para sexo, idade e grau de fibrose hepática, demonstrando a importância da retirada de efeitos de estratificação neste tipo de análise. Estes resultados se confirmam ao se agregar dados de uma população proveniente da Bahia em uma meta-análise. Além disso, fez-se um estudo GWAS a fim de se conhecer outras variações genéticas envolvidas nessa resposta. Esta análise indicou a existência de alguns SNPs candidatos com sugestão de associação, dentre eles a tiroglobulina, relacionada aos hormônios da tireóide. / Recently, studies have shown that SNPs (single nucleotide polymorphisms) rs8099917 and rs12979860, located near the gene IL28B explain the changes in the response to infection and treatment of a patient against the HCV genotype 1, but not for the genotype 3 of the virus. This study found a significant association between response to infection due to treatment by genotype 3 (PEG-INF and RBV) and the rs8099917 polymorphism in a population sample from Santos - SP. To the rs12979860 polymorphism, this association was only found when individuals are paired for sex, age and degree of hepatic fibrosis, demonstrating the importance of the withdrawal effects of stratification in this type of analysis. These results confirm the aggregate data from a population of Bahia in a meta-analysis. In addition, a GWAS was made in order to search other genetic variations involved in this response. This analysis indicated the existence of some candidate SNPs with suggestion of association, including thyroglobulin, thyroid hormones related to.

A infecção pelo vírus da hepatite C acomete cerca de 180 milhões de pessoas em todo mundo. Trata-se de doença com pouca manifestação clinica, onde cerca de 75% a 85% dos casos evolui para cronificação e aproximadamente 15% para hepatocarcinoma. Entre os fatores de risco mais conhecidos estão a realização de transfusões de sangue e hemoderivados anterior a 1993, uso de drogas endovenosas e relações sexuais desprotegidas. Este trabalho tem por objetivo estimar a prevalência de sorologia positiva para hepatite C e seus possíveis fatores de risco no distrito de Botafogo, município de Bebedouro, SP, onde a elevada freqüência de casos de hepatite chamou a atenção dos seus próprios moradores. Da população de 1318 habitantes, 353 foram sorteados para participar da pesquisa, sendo submetidos a questionário padronizado e coleta de sangue. Infecção pelo vírus da hepatite C foi pesquisada através de exames imunoenzimáticos e de PCR, no Instituto Oswaldo Cruz (Fiocruz), e por teste imunocromatográfico, no Laboratório de Sorologia do Hospital da Clínicas de Ribeirão Preto-USP . A prevalência encontrada foi de 8,8% (IC95%: 5,8 11,7). As variáveis que mostraram associação na análise univariada foram submetidas a um procedimento multivariado por aplicação do modelo de log binomial. As variáveis preditoras independentes de infecção pela hepatite C foram sexo masculino, tempo de residência acima de trinta anos e uso de medicações parenterais com material esterilizado por técnica de fervura. Uma possível explicação para a elevada prevalência nessa população reside na possibilidade de disseminação do vírus a partir de um antigo morador, que exercia informalmente atividades ligadas ao atendimento à saúde, especialmente aplicação de injeções, numa época anterior ao uso de seringas descartáveis. / Approximately 180 million people worldwide are infected by the hepatitis C virus. It is an illness with little clinical manifestation where about 75% - 85% of the cases evolve to chronification and about 15% to hepatocarcinoma. Among the bestknown risk factors are blood and blood by-product transfusions prior to 1993, use of intravenous drugs and unprotected sexual relations. This study has the objective of estimating the prevalence of positive serology for hepatitis C and its possible risk factors in the district of Botafogo, municipality of Bebedouro, São Paulo, where the high frequency of hepatitis cases caught the attention of the population itself. Out of a population of 1318 inhabitants, 353 were selected to participate in the research, being submitted to a standard questionnaire and blood collection. Hepatitis C infection was researched through immunoenzimatic and PCR exams at the Oswaldo Cruz Institute (FIOCRUZ), and by immunochromatographic tests at the Serology Laboratory of the Hospital das Clinicas in Ribeirão Preto USP. The prevalence found was of 8,8% (CI95%: 5,8 11,7). The variables that demonstrated an association in the univariate analysis were submitted to a multivariate procedure through the application of the binomial log model. The independent predictors for hepatitis C infection were male sex, local residence time over thirty years and use of parenteral medication with material sterilized through boiling technique. A possible explanation for the high prevalence in this population lies in the possibility of dissemination of the virus from an older inhabitant who informally exercised medical activities, especially the application of injections in a period before there was use of dischargeable syringes.

Orientador: Domingues Alves Meira / Banca: Alexandrina Sartori / Banca: Ricardo Sobhie Diaz / Banca: Fernando Lopes Gonçalves Júnior / Resumo: A aids e a hepatite C crônica são infecções caracterizadas por importante processo inflamatório contínuo, regulado por uma complexa interação entre citocinas. A persistência da atividade inflamatória crônica está intimamente relacionada com a progressão da patogênese da aids, bem como na indução de fibrose na hepatite C. Com o objetivo de avaliar o padrão de citocinas na infecção pelo HIV e na hepatite C crônica, as citocinas IL-2, IL-4, IL-10, TNF-α, INF-γ, TGF-β foram dosadas por Elisa e RT-PCR em cinco grupos: pacientes coinfectados pelo HIV/VHC (n=22), monoinfectados pelo HIV com supressão virológica pelo tratamento, e sem supressão virológica (n=17), monoinfectados pelo VHC (n=22) e um grupo controle composto por indivíduos doadores de sangue (n=10). IL-4 e IL-10 estiveram aumentadas consistentemente nos quatro grupos de estudo, determinando predomínio do perfil Th-2. INF-γ, TNF-α e TGF-β estiveram aumentados apenas nos grupos com infecção pelo VHC, com ou sem coinfecção pelo HIV. No grupo de monoinfectados pelo HIV com supressão virológica, a IL-2 dosada por RT-RCR esteve aumentada, porém os níveis séricos dosados por Elisa estavam normais. A alta produção de citocinas pró-inflamatórias INF-γ, TNF-α e TGF-β nos dois grupos de pacientes com infecção pelo VHC refletem o processo progressivo de acúmulo de inflamação e fibrose hepática. Já o predomínio de IL-4 e IL-10 em todos os grupos, citocinas ligadas ao perfil Th-2, demonstram a incapacidade de produção de uma resposta citotóxica Th-1, perpetuando a infecção e a inflamação crônica, mesmo naqueles indivíduos com supressão virológica pelo tratamento. Além de drogas antivirais, novos tratamentos imunomoduladores têm sido propostos para a erradicação viral, ou a interrupção das lesões causadas pelo estado inflamatório crônico... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Both AIDS and chronic hepatitis C (HCV) are characterized by continuous inflammatory process, regulated by a complex interaction between cytokines. The persistence of chronic inflammatory activity is closely related to the progression of the pathogenesis of AIDS, as well as the induction of fibrosis in HCV. In order to analyze the role of cytokines in HIV/HCV coinfection and the fibrosis progression, IL-2, IL-4, IL-10, TNF- α, INF-γ, TGF-β were measured by ELISA and RT -PCR in five groups: HIV/HCV coinfected patients (n = 22), HCV monoinfected patients (n = 22), HIV monoinfected patients with and without virological suppression (n = 17) and a control group composed by blood donors (n = 10). Hepatic biopsy and METAVIR classification were performed in all HCV patients (n=44). The baseline characteristics (sex, age and race) of all groups were similar. No correlations were found between cytokines and hepatic fibrosis. IL-4 and IL-10 were consistently increased in the four study groups, findings associated to a Th-2 profile. INF- γ, TNF-α and TGF-β were increased only in groups with HCV infection. In the group of HIV monoinfected patients with virological suppression, IL-2 measured by RT-RCR was increased, but serum levels measured by ELISA were normal. The high production of proinflammatory cytokines INF-γ, TNF-α and TGF-β in two groups of patients with HCV infection reflect the gradual process of inflammation and liver fibrosis. The predominance of IL-4 and IL-10 in all study groups demonstrates an inability to promote a cytotoxic Th-1 response. Even in HIV monoinfected patients with virological suppression with increased IL-2 expression, Th-2 cytokines were the predominant, perpetuating the chronic inflammation. In addition to antiviral drugs, new immunomodulatory treatments have been proposed... (Complete abstract click electronic access below) / Doutor

Chronic infection with the hepatitis C virus infection (HCV) affects over 170 million individuals worldwide. In this thesis the natural history and management of hepatitis C in North- East London was investigated. The prevalence of cirrhosis in patients with chronic hepatitis C rises with increasing duration of infection. In Asian patients infected at birth, infection over 60 years causes cirrhosis in 71 % of infected individuals. Since the rate of fibrosis progression in Asian patients is the same as that seen in Caucasian patients, it is likely that similar rates of cirrhosis will be seen in all patients who are infected with HCV for over 60 years. Factors found to be associated with fibrosis progression were:- age and alcohol excess. Insulin resistance was associated with fibrosis progression. However, elevated serum ferritin or hepatiC iron were not. Knowledge of hepatitis C in the East of London was examined and found to be poor despite the Department of Health information campaign. Educational meetings and postal surveys improved the level of knowledge of HCV. However as our group only assessed knowledge immediately after completion of the sessions, such a testing regime does not address long-term knowledge retention. We examined current and novel management strategies for patients with chronic HCV. Current therapy involves pegylated interferon and ribavirin. We found that insulin resistance was a poor predictor of sustained virological response. Chinese herbal treatments for hepatitis C are widely used but poorly studied. Our group designed a randomised controlled double blind study to assess whether Chinese herbal treatment is effective and results from this study show that recruitment and retention in trials of alternative therapies are problematic and that the herbal remedy had little effect on viraemia and quality of life, although liver function tests did improve a little.

人類免疫缺陷病毒 (HIV) 是人獲得性免疫缺陷綜合征 (愛滋病，AIDS) 的致病原，2010年全球有180萬人死於愛滋病，HIV/AIDS已成為全球健康的嚴重挑戰。人類免疫缺陷病毒與乙型肝炎病毒 (HBV) ，丙型肝炎病毒 (HCV) 的合併感染非常普遍，已演變成具有嚴重臨床後果的新健康問題。儘管對於人類免疫缺陷病毒的研究已有很大的進展，但由於受研究模型的限制，人體免疫系統對人類免疫缺陷病毒感染的應答，特別是對乙型肝炎病毒，丙型肝炎病毒與人類免疫缺陷病毒合併感染的免疫應答，仍值得進一步的闡明。 / 在本研究中，我們首先對深圳人類免疫缺陷病毒，乙型肝炎病毒，丙型肝炎病毒合併感染的流行情況進行研究。共選取914份人類免疫缺陷病毒感染者的血漿，經過對乙型肝炎病毒表面抗原 (HBsAg) 和抗丙型肝炎病毒抗體 (anti-HCV) 的檢測，發現10.9% (100/914) 的被檢測者是人類免疫缺陷病毒/乙型肝炎病毒合併感染，14.6% (133/914) 為人類免疫缺陷病毒/丙型肝炎病毒合併感染，3.7% (34/914) 為人類免疫缺陷病毒/乙型肝炎病毒/丙型肝炎病毒三重感染。多元邏輯回歸分析證明人類免疫缺陷病毒傳染的危險行為與合併感染顯著相關聯。大多數的人類免疫缺陷病毒/乙型肝炎病毒合併感染者都是通過性接觸感染人類免疫缺陷病毒，包括異性傳播與同性傳播 (95/100, 95%); 大多數的人類免疫缺陷病毒/丙型肝炎病毒合併感染者是靜脈注射吸毒者 (89/133, 66.9%); 人類免疫缺陷病毒/乙型肝炎病毒/丙型肝炎病毒三重感染者中，大多數是靜脈注射吸毒者 (28/34, 82.4%)。靜脈注射吸毒人群中，大部分是男性 (108/122, 88.5%)，約半數人的年齡介乎27至32歲 (56/122, 45.9%) 。有接近一半的經過血液和血液製品傳播人類免疫缺陷病毒的人是人類免疫缺陷病毒/丙型肝炎病毒合併感染者 (10/23, 43.5%) 。性別與人類免疫缺陷病毒感染的危險行為有顯著關係，大部份的靜脈注射吸毒者是男性。 / 進一步，我們利用酶聯免疫吸附測定法 (ELISA) 檢測深圳愛滋病陽性樣本血漿中白細胞介素27 (IL-27) 的濃度。結果顯示，對比健康參照者，人類免疫缺陷病毒單獨感染者，人類免疫缺陷病毒/乙型肝炎病毒合併感染者，人類免疫缺陷病毒/丙型肝炎病毒合併感染者的血漿IL-27濃度顯著升高。隨後我們進一步發現，人類免疫缺陷病毒單獨感染組，人類免疫缺陷病毒/乙型肝炎病毒，人類免疫缺陷病毒/乙型肝炎病毒/丙型肝炎病毒合併感染組之間的血漿IL-27濃度沒有顯著差異，而人類免疫缺陷病毒/丙型肝炎病毒合併感染組與人類免疫缺陷病毒/乙型肝炎病毒/丙型肝炎病毒三重感染組的血漿IL-27濃度差異顯著。我們還發現人類免疫缺陷病毒單獨感染組中，血漿IL-27濃度與CD4⁺ T 淋巴細胞數量顯著正相關 (r = 0.177, P = 0.034)。 / 我們進一步分析了人類免疫缺陷病毒和丙型肝炎病毒的病毒載量對血漿IL-27濃度的影響，發現HIV單獨感染組中人類免疫缺陷病毒載量與血漿IL-27濃度沒有顯著相關 (r = - 0.063, P = 0.679)，而人類免疫缺陷病毒/丙型肝炎病毒合併感染組中，人類免疫缺陷病毒載量與血漿IL-27濃度顯著正相關 (r = 0.362, P = 0.049)。人類免疫缺陷病毒/丙型肝炎病毒合併感染組中，人類免疫缺陷病毒載量與丙型肝炎病毒載量缺少顯著線性關聯 (r = - 0.072, P = 0.704)，而人類免疫缺陷病毒/丙型肝炎病毒合併感染組可根據人類免疫缺陷病毒與丙型肝炎病毒的病毒載量再細分成血漿IL-27濃度差異顯著的三組 (P = 0.014) ， 丙型肝炎病毒載量與血漿IL-27濃度缺少顯著關聯 (r = - 0.119, P = 0.530) 。 / 我們利用TaqMan®等位基因分型技術測定深圳男同性戀人群中IL-27 p28基因的單核苷酸多態性 (SNP)。結果顯示，人類免疫缺陷病毒感染組IL-27 p28 -964A/G 和4603G/A的基因型與健康男同性戀參照組的基因型沒有顯著差異， IL-27 p28 -964A/G 和4603G/A的等位基因比率也沒有顯著差異。結果也顯示，IL-27 p28 2905T/G的TG基因型可減少2.77倍的人類免疫缺陷病毒感染風險，等位基因G可減少2.72倍的人類免疫缺陷病毒感染風險。連鎖不平衡在IL-27 p28 -964A/G 和2905T/G 中存在 ( / 綜上所述， 在本研究中，我們首次調查了深圳人類免疫缺陷病毒，乙型肝炎病毒，丙型肝炎病毒合併感染的流行情況，並分析了合併感染的風險因素。 發現人類免疫缺陷病毒單獨感染者，人類免疫缺陷病毒/乙型肝炎病毒合併感染者， 及人類免疫缺陷病毒/丙型肝炎病毒合併感染者的血漿IL-27濃度比健康參照組顯著地升高；人類免疫缺陷病毒單獨感染組中，血漿IL-27濃度與CD4⁺ T淋巴細胞數量顯著正相關。人類免疫缺陷病毒/丙型肝炎病毒合併感染組中，人類免疫缺陷病毒載量與血漿IL-27濃度顯著正相關 (r = 0.362, P = 0.049)。分析深圳男同性戀人群IL-27 p28基因的單核苷酸多態性，發現IL-27 p28 2905T/G 與人類免疫缺陷病毒感染相關，GGG單型可降低男同性戀人群人類免疫缺陷病毒感染的風險。 / Human Immunodeficiency Virus (HIV) is the causative agent of Acquired Immunodeficiency Syndrome (AIDS); HIV/AIDS caused 1.8 million deaths world-widely in 2010 and became a major global health challenge. HIV co-infections with Hepatitis B virus (HBV), Hepatitis C virus (HCV) are common and have emerged into new health problems with severe clinical consequences. Since the discovery of HIV, massive progress in understanding of the pathogen has been achieved. Due to the restriction of research model, how human immune system responds to HIV infection, particularly, to HBV or HCV co-infections is still worthy further elucidation. / A cohort study was first conducted in Shenzhen regarding the seroprevalence of HBV, HCV infections among HIV-infected population. Totally 914 HIV positive individuals were recruited in the study and tested for HBsAg and anti-HCV antibodies. The results showed a 10.9% (100/914) HIV/HBV co-infection rate, 14.6% (133/914) HIV/HCV co-infection prevalence and 3.7% (34/914) HIV/HBV/HCV triple-infection prevalence. Multivariate logistic regression revealed that HIV transmission risk behavior was significantly associated with HIV, HBV, HCV co-infections. Most HIV/HBV co-infection cases got HIV through sexual contact including heterosexual and homosexual behaviors (95/100, 95%); while most HIV/HCV co-infection subjects were injection drug users (IDUs) (89/133, 66.9%). In the case of HIV/HBV/HCV triple-infection, IDUs accounted for a large ratio (28/34, 82.4%). Among IDUs, most of them were male (108/122, 88.5%) and nearly half were aged 27 to 32 years old (56/122, 45.9%). Near half people who got HIV through blood and blood products were HIV/HCV co-infected (10/23, 43.5%). Gender has a significant correlation with HIV risk behavior and most IDUs were male. / Next, we applied ELISA to test HIV positive clinical samples and proved that plasma interleukin-27 (IL-27) level was significantly elevated in HIV mono-infected, HIV/HBV co-infected and HIV/HCV co-infected subjects when compared with healthy controls. Later, we further revealed that plasma IL-27 titer was not significantly varied among HIV, HBV and HCV co-infections except between HIV/HCV co-infections and HIV/HBV/HCV triple-infections. We also observed a significant positive correlation between CD4⁺ T cell counts and plasma IL-27 titer within HIV mono-infected group (r = 0.177, P = 0.034). / We further analyzed the impact HIV and HCV viral loads on plasma IL-27 titer. We found there was no significant correlation between HIV viral load and IL-27 titer among HIV mono-infected individuals (r = - 0.063, P = 0.679); while a significant positive correlation was observed between HIV viral load and IL-27 titer in HIV/HCV co-infected individuals (r = 0.362, P = 0.049). In the case of HIV/HCV co-infection, there was no significant linear correlation between HIV and HCV viral loads (r = - 0.072, P = 0.704) but exist obvious subdivision of samples in terms of HIV and HCV viral loads with significant IL-27 titer variance (P = 0.014). No correlation was observed between HCV viral load and IL-27 titer (r = - 0.119, P = 0.530). / IL-27 p28 polymorphisms were genotyped with TaqMan® Allelic Discrimination Assay in Chinese men who have sex with men (MSM) population in Shenzhen and the results revealed that proportions of IL-27 p28 -964A/G and 4603G/A genotypes were not significantly different from the healthy controls; IL-27 p28 -964A/G and 4603G/A allele frequencies were similar between HIV positive MSM group and healthy control MSM group. Results also showed that for IL-27 p28 2905T/G polymorphism, TG genotype has a 2.77-fold decreased risk of HIV susceptibility and subjects with G allele has a 2.72-fold decreased risk of HIV susceptibility. Linkage disequilibrium (LD) coefficients were observed between IL-27 p28 -964A/G and 2905T/G ( / In conclusion, the seroprevalences of HBV and HCV infection among HIV positive population in Shenzhen were surveyed and risk factors associated with co-infections were analyzed. Plasma IL-27 titer was significantly elevated in HIV mono-infected, HIV/HBV co-infected and HIV/HCV co-infected individuals. IL-27 level was correlated with CD4⁺ T cell counts within HIV mono-infected people. A significant positive correlation was found between HIV viral load and IL-27 titer in HIV/HCV co-infected individuals (r = 0.362, P = 0.049). IL-27 p28 2905T/G was associated with individual susceptibility to HIV infection and haplotype GGG showed a protective role in restricting HIV infection in MSM population. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / He, Lai. / "October 2012." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 135-155). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract (English) --- p.iii / Abstract (Chinese) --- p.vi / Acknowledgements --- p.ix / Contents --- p.x / List of Tables --- p.xv / List of Figures --- p.xvi / Abbreviations --- p.xvii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Human Immunodeficiency Virus --- p.1 / Chapter 1.1.1 --- HIV virology --- p.1 / Chapter 1.1.1.1 --- HIV structure and genome organization --- p.1 / Chapter 1.1.1.2 --- HIV life cycle --- p.3 / Chapter 1.1.1.3 --- HIV genotypes --- p.5 / Chapter 1.1.2 --- HIV epidemiology --- p.6 / Chapter 1.1.2.1 --- Global HIV epidemiology --- p.6 / Chapter 1.1.2.2 --- HIV epidemiology in China --- p.9 / Chapter 1.1.3 --- HIV pathogenesis --- p.13 / Chapter 1.1.3.1 --- Natural history of HIV infection --- p.13 / Chapter 1.1.3.2 --- HIV transmission --- p.15 / Chapter 1.1.3.3 --- HIV tropism --- p.17 / Chapter 1.1.4 --- Immune responses to HIV infection --- p.19 / Chapter 1.1.4.1 --- Innate immune response --- p.19 / Chapter 1.1.4.2 --- Adaptive immune response --- p.21 / Chapter 1.1.5 --- Diagnosis --- p.24 / Chapter 1.1.6 --- HIV prevention --- p.25 / Chapter 1.1.7 --- Anti-HIV therapy --- p.25 / Chapter 1.1.8 --- Hepatitis B virus, Hepatitis C virus infection --- p.26 / Chapter 1.1.8.1 --- HBV infection natural history, diagnosis, disease progression and epidemiology --- p.26 / Chapter 1.1.8.2 --- HCV infection natural history, diagnosis, disease progression and epidemiology --- p.30 / Chapter 1.1.9 --- HIV, HBV, HCV co-infections --- p.32 / Chapter 1.2 --- Interleukin-27 --- p.36 / Chapter 1.2.1 --- Biology of IL-27 --- p.36 / Chapter 1.2.2 --- IL-27 on immune system --- p.37 / Chapter 1.2.3 --- IL-27 anti-tumor properties --- p.38 / Chapter 1.2.4 --- IL-27 antiviral features --- p.40 / Chapter 1.2.5 --- IL-27 with hepatitis --- p.41 / Chapter 1.3 --- Single-nucleotide polymorphisms (SNPs) --- p.42 / Chapter 1.3.1 --- Types of SNPs --- p.43 / Chapter 1.3.2 --- Functions of SNPs --- p.43 / Chapter 1.4 --- Objectives of the study --- p.45 / Chapter Chapter 2 --- Seroprevalence of HBV, HCV infection among HIV positive individuals in Shenzhen --- p.52 / Chapter 2.1 --- Introduction --- p.52 / Chapter 2.2 --- Materials and methods --- p.54 / Chapter 2.2.1 --- Study participants --- p.54 / Chapter 2.2.2 --- Measure of HBV, HCV seroprevalence --- p.55 / Chapter 2.2.3 --- Statistical analysis --- p.60 / Chapter 2.3 --- Results --- p.61 / Chapter 2.3.1 --- HIV infection in Shenzhen --- p.61 / Chapter 2.3.2 --- Seroprevalence of HBV, HCV infection among HIV positive individuals in Shenzhen --- p.61 / Chapter 2.4 --- Discussion --- p.65 / Chapter 2.4.1 --- HIV infection in Shenzhen --- p.65 / Chapter 2.4.2 --- HIV, HBV, HCV co-infections in Shenzhen --- p.68 / Chapter 2.4.3 --- Limitations of the study --- p.71 / Chapter Chapter 3 --- Upregulation of Interleukin-27 titer in HIV infected persons --- p.78 / Chapter 3.1 --- Introduction --- p.78 / Chapter 3.2 --- Materials and methods --- p.80 / Chapter 3.2.1 --- Study participants --- p.80 / Chapter 3.2.2 --- Measure of HIV, HBV, HCV infection --- p.80 / Chapter 3.2.3 --- Detection of IL-27 in plasma --- p.81 / Chapter 3.2.4 --- CD4 counting --- p.84 / Chapter 3.2.5 --- Statistical analysis --- p.84 / Chapter 3.3 --- Results --- p.84 / Chapter 3.3.1 --- Demographics of study participants --- p.84 / Chapter 3.3.2 --- Upregulation of IL-27 levels in HIV infected persons --- p.85 / Chapter 3.3.3 --- Correlation of plasma IL-27 titer with CD4⁺ T cell count --- p.86 / Chapter 3.4 --- Discussion --- p.86 / Chapter Chapter 4 --- Impact of HIV, HCV viral loads on Interleukin-27 titer among Antiretroviral Therapy- Naïve HIV positive Chinese --- p.95 / Chapter 4.1 --- Introduction --- p.95 / Chapter 4.2 --- Materials and methods --- p.96 / Chapter 4.2.1 --- Study participants --- p.97 / Chapter 4.2.2 --- HIV, HBV and HCV Serological assays --- p.97 / Chapter 4.2.3 --- CD4 counting --- p.97 / Chapter 4.2.4 --- Detection of plasma IL-27 --- p.98 / Chapter 4.2.5 --- Quantification of HIV, HCV viral loads --- p.98 / Chapter 4.2.6 --- Statistical analysis --- p.102 / Chapter 4.3 --- Results --- p.102 / Chapter 4.3.1 --- Demographics of study participants --- p.102 / Chapter 4.3.2 --- Plasma IL-27 was elevated in HIV-positive persons --- p.103 / Chapter 4.3.3 --- Correlation of IL-27 titer and CD4⁺ T cell count --- p.103 / Chapter 4.3.4 --- Correlation of HIV viral load and IL-27 titer --- p.104 / Chapter 4.3.5 --- Correlation of HCV viral load and IL-27 titer --- p.104 / Chapter 4.4 --- Discussion --- p.105 / Chapter Chapter 5 --- Association of Interleukin-27 polymorphisms with the susceptibility to HIV infection in a Chinese men who have sex with men population --- p.116 / Chapter 5.1 --- Introduction --- p.116 / Chapter 5.2 --- Materials and methods --- p.118 / Chapter 5.2.1 --- Study participants --- p.118 / Chapter 5.2.2 --- HIV screening --- p.118 / Chapter 5.2.3 --- Genomic DNA extraction --- p.119 / Chapter 5.2.4 --- IL-27 p28 -964A/G, 2905T/G and 4603G/A genotyping --- p.120 / Chapter 5.2.5 --- Statistical analysis --- p.121 / Chapter 5.3 --- Results --- p.122 / Chapter 5.3.1 --- Demographics of study participants --- p.122 / Chapter 5.3.2 --- IL-27 genotypes and allele frequencies in HIV MSM and healthy MSM controls --- p.122 / Chapter 5.3.3 --- LD analysis and haplotype analysis --- p.123 / Chapter 5.4 --- Discussion --- p.124 / Chapter Chapter 6 --- Summary and perspectives --- p.130 / Chapter 6.1 --- Summary --- p.130 / Chapter 6.2 --- Perspectives --- p.132 / Bibliography --- p.135

Hepatitis C virus (HCV) infection has become a global public health concern with over 130 million people chronically infected and over 350,000 deaths every year from HCV-related liver diseases. GB virus-B (GBV-B) infection in tamarins is a surrogate model for acute HCV infection. Whilst HCV infection commonly leads to chronicity, GBV-B is naturally cleared. To better understand this natural clearance, this project aimed to study the associated humoral immune response to GBV-B. Additionally, GBV-B-specific antibodies were produced with the aim of characterising the pathology of the virus. Previously, there was no available GBV-B neutralisation assay to identify antibodies in this animal model. Therefore, a GBV-B neutralisation assay, based on a method that is known to be successful for the closely-related HCV, was developed. This method involved producing pseudotyped retroviral particles (PV) expressing the GBV-B envelope that could infect a human hepatocarcinoma cell line. GBV-B PV production was confirmed by western blotting. Future studies can now test archived tamarin sera in this assay for the presence of neutralising antibodies. Neutralising antibodies found through this model could be epitope mapped, and incorporated into HCV vaccine design strategies. To study the pathology of GBV-B infection, GBV-B-specific antibodies were also produced using two techniques in parallel- classical hybridoma technology and ribosome display. Antibodies targeting the nucleocapsid core protein of GBV-B have been previously detected in tamarins and served as the target for production of GBV-B antibodies using both aforementioned technologies. GBV-B core-specific antibodies were successfully isolated using both technologies and can now be used in downstream techniques, such as immunohistochemistry, to characterise the pathology of GBV-B infection thereby further validating the use of the animal model.

Sofosbuvir is a uridine based nucleotide inhibitor of the hepatitis C viral (HCV) polymerase that is the backbone of many treatment regimens. In combination with drugs targeting other viral enzymes (including the poorly potent guanosine analogue ribavirin or highly potent inhibitors of viral NS5A or protease) most patients clear virus and resistance to sofosbuvir is rare, allowing effective retreatment with sofosbuvir. Patients with Genotype 3 HCV respond less well than other genotypes and response is reduced in those previously exposed to interferon. Here we show that patientderived virus from patients with Genotype 3 HCV who relapse to sofosbuvir-based therapies have a reduced sensitivity to SOF in an in-vitro phenotyping assay. Analysis of viral sequencing data revealed two distinct polymorphisms (A150V and K206E) in the HCV polymerase that are associated with treatment failure and in-vitro; they reduce sofosbuvir sensitivity against genotype 3 hepatitis C virions. However both polymorphisms modify the cellular response to type I interferon and in cells lacking response to interferon the impact on sofosbuvir sensitivity is minimal. The A150V polymorphism reduces the response to interferon 70 fold whereas the K206E substitution has minimal effects on interferon in isolation but in combination with A150V reduces the response 100 fold. Preliminary data indicates that the A150V polymorphism interferes with the late response to type I interferons enabling the virus to overcome the induction of interferon-stimulated genes. These data indicate a complex interaction between direct acting antiviral drugs and the innate antiviral response.

Introdução: A Organização Mundial da Saúde estima que cerca de 71 milhões de pessoas estejam vivendo infectadas pelo vírus da hepatite C (HCV), o que corresponde a 1% da população mundial. A infecção crônica pelo HCV é uma das principais causas de cirrose hepática e carcinoma hepatocelular. O tratamento farmacológico visa a erradicação do vírus e melhora da atividade inflamatória. Em 2015, três fármacos de ação direta (DAA), simeprevir (SMV), sofosbuvir (SOF) e daclatasvir (DCV), foram incluídos no protocolo brasileiro de tratamento da hepatite C crônica. Estudos demonstram que o uso destes medicamentos está associado a elevadas taxas de resposta ao tratamento e a menor incidência de reações adversas em relação às terapias baseadas no uso do interferon e de inibidores de protease de primeira geração. Outros trabalhos indicam que pode haver associação entre a presença de determinadas mutações de resistência viral com a resposta ao tratamento farmacológico. Objetivos: Verificar a efetividade e a segurança dos esquemas de tratamento da hepatite C crônica que envolvem os DAA e analisar se a presença das mutações de resistência impacta negativamente na resposta ao tratamento. Casuística e Métodos: Trata-se de um estudo quase experimental de braço único realizado no Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto. Foram incluídos indivíduos maiores de 18 anos, infectados pelo genótipo 1 do HCV, que iniciaram tratamento com esquema que continha SOF em associação com DCV ou SMV. Foram coletados dados sociodemográficos, antropométricos, clínicos e realizou-se levantamento da ocorrência de reações adversas durante o tratamento, por meio do sistema informatizado do hospital. A coleta de sangue, para verificação da presença de mutações de resistência viral, foi realizada em até 15 dias antes do início do tratamento. O principal desfecho considerado foi a RVS12, definida como a ausência de carga viral detectável na 12ª semana após o fim do tratamento. Resultados: 262 indivíduos foram incluídos no estudo, dos quais 58,0% eram do sexo masculino e 79,4% foram classificados como brancos. A média de idade calculada para a amostra foi 55 anos, com desvio padrão de 10 anos. Quanto aos esquemas de tratamento propostos, 49,6% dos pacientes receberam tratamento com SOF e DCV e 50,4% fizeram uso de SOF e SMV. A taxa global de RVS12 foi de 92,7% (93,9% para SOF + DCV e 91,7% para SOF + SMV). Não foram identificados fatores associados a RVS, segundo a análise estatística. As reações adversas mais comuns foram anemia, náuseas, cefaleia e fadiga. Não foi encontrada evidência de associação entre a presença de mutações associadas a resistência ao tratamento e a falha no tratamento. Conclusão: O uso de esquemas de tratamento compostos por SOF e DCV ou SOF e SMV apresentou alta taxa de RVS e bom perfil de tolerabilidade em pacientes com genótipo 1 do HCV. / Background: The World Health Organization estimates that about 71 million people are living with hepatitis C virus (HCV), which accounts for 1% of the world population. Chronic HCV infection is one of the major causes of liver cirrhosis and hepatocellular carcinoma. The pharmacological treatment aims to eradicate the virus and improve inflammatory activity. In 2015, three direct-acting antivirals (DAA), simeprevir (SMV), sofosbuvir (SOF) and daclatasvir (DCV) were included in the brazilian protocol for the treatment of chronic hepatitis C. Studies have shown that the use of these drugs is associated with higher rates of response to treatment and to lower incidence of adverse reactions, compared to therapies based on the use of interferon and first-generation protease inhibitors. Other studies indicate that there may be an association between the presence of certain viral resistance mutations and the response to pharmacological treatment. Aims: To verify the effectiveness and safety of chronic hepatitis C treatment regimens involving DAA and to analyze whether the presence of resistance mutations negatively affects SVR rates. Methods: It is an almost experimental single-arm study performed at the Clinics Hospital of the Ribeirão Preto Medical School. We included individuals older than 18 years old, infected with HCV genotype 1, who started treatment with a regimen containing SOF in combination with DCV or SMV. Sociodemographic, anthropometric and clinical data were collected and the occurrence of adverse reactions during the treatment was investigated through the computerized system of the hospital. Blood collection for search for viral resistance mutations was performed within 15 days prior to initiation of treatment. The main outcome was SVR12, defined as the absence of detectable viral load at the 12th week after the end of treatment. Results: 262 subjects were included in the study, of which 58.0% were male and 79.4% were white. The mean age calculated for the sample was 55 years, with standard deviation of 10 years. Regarding the treatment regimens, 49.6% of the patients received SOF and DCV and 50.4% used SOF and SMV. The SVR12 rate was 92.7% (93.9% for SOF + DCV and 91.7% for SOF + SMV). No factors associated with SVR were identified according to the statistical analysis. The most common adverse reactions were anemia, nausea, headache and fatigue. No evidence of association was found between the presence of mutations associated with treatment resistance and treatment failure. Conclusion: The use of treatment regimens composed of SOF and DCV or SOF and SMV showed a high rate of SVR and a good tolerability profile in patients with HCV genotype 1.

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