Submitted by Caroline Falcao (caroline.rfalcao@ufpe.br) on 2017-04-06T17:05:06Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) 2012-dissertação-EvônioBarrosCampeloJunior.pdf: 1895687 bytes, checksum: 45a651f79dfdcc2ce3328e3201f91911 (MD5) / Made available in DSpace on 2017-04-06T17:05:06Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) 2012-dissertação-EvônioBarrosCampeloJunior.pdf: 1895687 bytes, checksum: 45a651f79dfdcc2ce3328e3201f91911 (MD5) Previous issue date: 2012-12-27 / Desde 1967, momento do seu surgimento no mundo científico, o GB vírus C (GBV-C) permanece conhecido como vírus indolente, todavia não obstante,pesquisas têm reveladoadespeito da ausência de vinculação do GBV-C com alguma doença, que a associação entre o GBV-C e HIV promove uma progressão mais lenta na doença pelo HIV em indivíduos coinfectados. Essa interaçãoteriacomo mecanismo a competição com o vírus da imunodeficiência humana (HIV) na ligação aos correceptores CCR5 e CXCR4 edessa formatem sidoobjeto de análise com intuito de promover a compreensão da dinâmica imunológica do HIV. Portanto, admitindoaimportância do GBV-C na pandemia de HIV/aids, esse trabalho determinou a prevalência da infecção pelo GBV-Cem pessoas vivendo com HIV/aids acompanhadas noServiço de Doenças Infecciosas e Parasitárias do HC-UFPE, verificou o perfil sócio-demográficoda população, identificou fatores de riscoe fez a associação entre a contagem de linfócitos TCD4 e a infecção peloGBV-C.Representou um estudode corte transversal, analisado como caso-controle, no qual a presença do GBV-C RNA foi investigada em249 pessoas vivendo com HIV/aids, de acordo com a demanda espontânea do ambulatório de Doenças Infecciosas e Parasitárias HC-UFPE,no período de junho adezembro de 2011 e que preenchiam os critérios de inclusão. Foram coletadas amostras de sangue de cada participante, os quais também assinaram um termo de consentimento livre e esclarecido e responderam questionário elaborado especificamente para a pesquisa. Adetecção do RNA do GBV-C foi realizado por PCR (Reação em CadeiadaPolimerase).O resultado da pesquisa indicou uma prevalência de 24,3%, compatível com outros estudos nacionais e internacionais,indicou o perfil sócio-demográfico, apontoua via parenteral como a mais prevalente na infecção pelo GBV-Ceassociou a contagem de linfócitos T CD4 e a infecção peloGBV-C,em pessoas vivendo com HIV/aids na fase inicial da infecção. Também a discussão apontou a necessidade de realização de trabalhos de coorte para melhor compreensão de fatores implicados na transmissão do GBV-C. / Since 1967, the time of its emergence in the scientific world, the GB virus C (GBV-C) virus remains known as indolent, but nevertheless, research has revealed despite the absence of binding of GBV-C with some disease, the association between GBV-C and HIV promotes a slower progression in HIV disease in coinfected individuals. This interaction mechanism would compete with the human immunodeficiency virus (HIV) in binding to CCR5 and CXCR4coreceptorsand thus has been the subject of analysis in order to promote understanding of the dynamics of HIV immune.Therefore, admitting the importance of GBV-C in the HIV/AIDS pandemic, this study estimated the prevalence of GBV-C infection in people living with HIV/AIDS followed in the Service of Infectious and Parasitic HC-UFPE, there the socio-demographic profile of the population, identified risk factors and made the association between CD4 count and GBV-C infection.Represented a cross-sectional study, analyzed as case-control study in which the presence of GBV-C RNA was investigated in 249 people living with HIV/AIDSat the outpatient of Infectious and Parasitic Diseases HC-UFPE, between June and December 2011 and who met the inclusion criteria. Blood samples were collected from each participant, who also signed a consent form and a questionnaire developed specifically for research. Detection of GBV-C RNA was performed by PCR (Polymerase Chain Reaction).The result of the survey indicated a prevalence of 24.3%, consistent with other national and international studies, said the socio-demographic profile, pointed the parenteral route as the most prevalent in the GBV-C infection and associated with CD4 count and GBV-C infection in people living with HIV/AIDS in the early phase of infection. Also the discussion pointed to the need to perform cohort studiesto better understand the factorsinvolvedinthe transmission of GBV-C.

Human Pegivirus (HPgV; originally called GB virus C) is an RNA virus within the Pegivirus genus of the Flaviviridae that commonly causes persistent infection. Worldwide, approximately 750 million people are infected with HPgV. No causal association between HPgV and disease has been identified; however, several studies found an association between persistent HPgV infection and prolonged survival of HIV-infected individuals that appears to be related to a reduction in host immune activation. HPgV replicates well in vivo (>10 million genome copies/ml plasma) but grows poorly in vitro and systems to study this virus are limited. Consequently, mechanisms of viral persistence and host immune modulation remain poorly characterized, and the primary permissive cell type(s) has not yet been identified. The overall goals of my thesis were to characterize HPgV tropism, effects of HPgV infection on host immune response and mechanisms of viral persistence. Previous studies found HPgV RNA in T and B lymphocytes and ex vivo infected lymphocytes produce viral particles. To further characterize HPgV tropism, we quantified HPgV RNA in highly purified CD4+ and CD8+ T cells, including naïve, central memory, and effector memory populations, and in B cells (CD19+), NK cells (CD56+) cells and monocytes (CD14+) obtained from persistently infected humans using real time RT-PCR. Single genome sequencing was performed on virus within individual cell types to estimate genetic diversity among cell populations. HPgV RNA was present in CD4+ and CD8+ T lymphocytes (9 of 9 subjects), B lymphocytes (7 of 9), NK cells and monocytes (both 4 of 5). HPgV RNA levels were higher in naïve (CD45RA+) CD4+ cells than in central memory and effector memory cells (p<0.01). HPgV sequences were highly conserved between patients (0.117 ± 0.02 substitutions per site) and within subjects (0.006 ± 0.003 substitutions per site). The non-synonymous/synonymous substitution ratio was 0.07 suggesting low selective pressure. CFSE-labeled HPgV RNA-positive microvesicles (SEV) from serum delivered CFSE to uninfected monocytes, NK cells, T and B lymphocytes, and HPgV RNA was transferred to peripheral blood mononuclear cells (PBMCs) with evidence of subsequent viral replication. Thus, HPgV RNA-positive SEV may contribute to delivery of HPgV to PBMCs in vivo, explaining the apparent broad tropism of this persistent human RNA virus. Although HPgV infection reduces NK cell activation in HIV-infected individuals, the mechanism by which this occurs is not characterized. We studied HPgV effects on NK cell non-cytolytic function in HIV-infected people by measuring expression of IL-12 induced interferon gamma (IFNg) and cytolytic function by measuring K562 target-cell induced CD107a and granzyme B. IFNg expression was lower in HIV-HPgV co-infected subjects compared to HIV mono-infected subjects treated with combination antiretroviral therapy (p=0.02). In contrast, cytolytic NK cell functions were not affected by HPgV. Inhibition of IFNg was due to inhibition of tyrosine kinase (Tyk2) by HPgV envelope protein E2. HPgV positive human sera, extracellular vesicles containing E2 protein, recombinant E2 protein and synthetic E2 peptides containing a predicted Tyk2 interacting motif inhibited IL-12-mediated IFNg release by NK cells. Thus HPgV-E2 inhibits NK cell non-cytolytic functions. Inhibition of NK cell-induced proinflammatory/antiviral cytokines may contribute to both HPgV's ability to persist with high viral loads (>10 million genome copies/ml plasma) and reduce immune cell activation. Understanding mechanisms by which HPgV alters immune activation may contribute towards novel immunomodulatory therapies to treat HIV and inflammatory diseases.

GB virus C (GBV-C) is a common, apathogenic virus that can inhibit human immunodeficiency virus (HIV) replication in vitro. Persistent coinfection with GBV-C has been associated with improved survival among HIV-infected adults while loss of GBV-C viremia has been associated with poor survival. If GBV-C does inhibit HIV replication, it is possible that GBV-C infection may reduce mother-to-child-transmission (MTCT) of HIV. This study investigated whether maternal or infant GBV-C infection was associated with reduced MTCT of HIV infection. The study population consisted of 1,783 pregnant women from three Bangkok perinatal HIV transmission studies (1992-94, 1996-7, 1999-2004). We tested plasma collected at delivery for GBV-C RNA, GBV-C antibody, and GBV-C viral genotype. If maternal GBV-C RNA was detected, the four- or six-month infant specimen was tested for GBV-C RNA. Rates of MTCT of HIV in GBV-C-infected and GBV-C-uninfected women and infants were compared using multiple logistic regression as were associations with MTCT of GBV-C and prevalence of GBV-C infection. The prevalence of GBV-C infection (i.e. presence of RNA or antibody) was 33% among HIV-infected women and 15% among HIV-uninfected women. Forty-one percent of GBV-C-RNA-positive women transmitted GBV-C to their infants. Only two of 101 (2.0%) GBV-C-RNA-positive infants acquired HIV infection compared to 162 (13.2%) of 1,232 of GBV-C-RNA-negative infants (RR 0.15, p<0.0001). This association remained after adjustment for maternal HIV viral load, antiretroviral prophylaxis, CD4+ count and other covariates. MTCT of HIV was not associated with presence of maternal GBV-C RNA or maternal GBV-C antibody. Maternal receipt of antiretroviral therapy was associated with increased MTCT of GBV-C, as was high GBV-C viral load, vaginal delivery and absence of infant HIV infection. GBV-C infection among women was independently associated with more than one lifetime sexual partner, intravenous drug use and HIV-infection. We observed a higher prevalence of GBV-C infection among HIV-infected compared to HIV-uninfected pregnant women in Thailand, likely due to common risk factors. Antiretroviral therapy appears to increase MTCT of GBV-C. Infant GBV-C acquisition, but not maternal GBV-C infection, was significantly associated with reduced MTCT of HIV. Mechanisms for these later two associations are unknown. / Bhanich Supapol W, Remis RS, Raboud J, Millson M, Tappero J, Kaul R, Kulkarni P, McConnell MS, Mock PA, Culnane M, McNicholl J, Roongpisuthipong A, Chotpitayasunondh T, Shaffer N, Butera S. 2008. Reduced mother-to-child transmission of HIV associated with infant but not maternal GB virus C infection. J Infect Dis 197(10):1369-1377. Bhanich Supapol W, Remis RS, Raboud J, Millson M, Tappero JW, Kaul R, Kulkarni P, McConnell MS, Mock PA, McNicholl JM, Vanprarar N, Asavapiriayanont S, Shaffer N, Butera ST. 2009. Mother-to-child transmission of GB virus C in a cohort of women coinfected with GB virus C and HIV in Bangkok, Thailand. J Infect Dis 200:227-235.

GB virus C (GBV-C) is a common, apathogenic virus that can inhibit human immunodeficiency virus (HIV) replication in vitro. Persistent coinfection with GBV-C has been associated with improved survival among HIV-infected adults while loss of GBV-C viremia has been associated with poor survival. If GBV-C does inhibit HIV replication, it is possible that GBV-C infection may reduce mother-to-child-transmission (MTCT) of HIV. This study investigated whether maternal or infant GBV-C infection was associated with reduced MTCT of HIV infection. The study population consisted of 1,783 pregnant women from three Bangkok perinatal HIV transmission studies (1992-94, 1996-7, 1999-2004). We tested plasma collected at delivery for GBV-C RNA, GBV-C antibody, and GBV-C viral genotype. If maternal GBV-C RNA was detected, the four- or six-month infant specimen was tested for GBV-C RNA. Rates of MTCT of HIV in GBV-C-infected and GBV-C-uninfected women and infants were compared using multiple logistic regression as were associations with MTCT of GBV-C and prevalence of GBV-C infection. The prevalence of GBV-C infection (i.e. presence of RNA or antibody) was 33% among HIV-infected women and 15% among HIV-uninfected women. Forty-one percent of GBV-C-RNA-positive women transmitted GBV-C to their infants. Only two of 101 (2.0%) GBV-C-RNA-positive infants acquired HIV infection compared to 162 (13.2%) of 1,232 of GBV-C-RNA-negative infants (RR 0.15, p<0.0001). This association remained after adjustment for maternal HIV viral load, antiretroviral prophylaxis, CD4+ count and other covariates. MTCT of HIV was not associated with presence of maternal GBV-C RNA or maternal GBV-C antibody. Maternal receipt of antiretroviral therapy was associated with increased MTCT of GBV-C, as was high GBV-C viral load, vaginal delivery and absence of infant HIV infection. GBV-C infection among women was independently associated with more than one lifetime sexual partner, intravenous drug use and HIV-infection. We observed a higher prevalence of GBV-C infection among HIV-infected compared to HIV-uninfected pregnant women in Thailand, likely due to common risk factors. Antiretroviral therapy appears to increase MTCT of GBV-C. Infant GBV-C acquisition, but not maternal GBV-C infection, was significantly associated with reduced MTCT of HIV. Mechanisms for these later two associations are unknown. / Bhanich Supapol W, Remis RS, Raboud J, Millson M, Tappero J, Kaul R, Kulkarni P, McConnell MS, Mock PA, Culnane M, McNicholl J, Roongpisuthipong A, Chotpitayasunondh T, Shaffer N, Butera S. 2008. Reduced mother-to-child transmission of HIV associated with infant but not maternal GB virus C infection. J Infect Dis 197(10):1369-1377. Bhanich Supapol W, Remis RS, Raboud J, Millson M, Tappero JW, Kaul R, Kulkarni P, McConnell MS, Mock PA, McNicholl JM, Vanprarar N, Asavapiriayanont S, Shaffer N, Butera ST. 2009. Mother-to-child transmission of GB virus C in a cohort of women coinfected with GB virus C and HIV in Bangkok, Thailand. J Infect Dis 200:227-235.

Made available in DSpace on 2014-12-17T14:03:41Z (GMT). No. of bitstreams: 1 MagdaFDC.pdf: 1127289 bytes, checksum: e8f867febd165499040c2d067e137209 (MD5) Previous issue date: 2005-02-23 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / No Brasil, a Leishmania chagasi ? a esp?cie que causa a leishmaniose visceral (LV). A Leishmania tem a capacidade de causar um estado de parasitismo intracelular nos macr?fagos. A destrui??o dos parasitas, tanto no in?cio da infec??o, como na fase tardia, depende da ativa??o de macr?fagos por citocinas. Leishmania evade da resposta imunol?gica do hospedeiro e sobrevive sem o macr?fago durante a doen?a. A infec??o por Leishmania chagasi pode induzir hepatites, independente da co-infec??o com v?rus de hepatites (A-E). A infec??o por GBV-C est? sendo associada com prote??o ? progress?o da doen?a, e aumento da sobreviv?ncia em indiv?duos HIV-1 soro positivos. Neste estudo, objetivou-se avaliar se a infec??o subcl?nica por Leishmania Chagasi depende da modula??o de outros pat?genos dentre eles o v?rus da hepatite G. Um total de 322 indiv?duos, residentes em ?reas end?micas para LV e um total de 82 pacientes de banco de sangue participaram deste estudo. Usando a t?cnica de RT-PCR seguida do sequenciamento do fragmento da PCR da regi?o 5 NTR e submetendo-se os dados obtidos a um banco de genes (BLAST) para uma an?lise comparativa de seq??ncias do Genebank obteve-se uma identidade de 98% do clone GBV1 com o isolado 34,5 da hepatite G v?rus, 5 -UTR, parcial seq??ncia (AF489995. 1). Dentre os fen?tipos avaliados, DTH+, DTH-, e LV constatou-se uma maior preval?ncia do fen?tipo DTH+ nos indiv?duos GBV-C positivos se comparados com DTH- e LV( DTH+ 57%, DTH- 10%,LV 33%).Um subgrupo de 4 indiv?duos de fen?tipo LV e positivos para GBV-C no ano de 1996 foi submetido a novas coletas em 2004. An?lises bioqu?micas de enzimas,AST,ALT , YGT,LDH, Fosfatasse Alcalina e bilirrubinas foram realizadas, bem como, dosagens de marcadores de outras hepatites.Constatou-se que as dosagens bioqu?micas n?o sofreram altera??es significativas e que os pacientes positivos no ano de 1996 para GBV-C; no ano de 2004 apresentaram HVAG positivo, com marcadores negativos, para hepatite B e C. Sugerimos uma poss?vel intera??o do v?rus da hepatite G com pacientes do fen?tipo LV e DTH+, com poss?vel ativa??o da resposta imune celular

GB virus C (GBV-C) is a lymphotropic human virus which was recently assigned to a new genus Pegivirus within the Flaviviridae family. GBV-C infection is found worldwide, and viremia prevalence is about 1% to 4% in healthy blood donors and up to 42% in HIV-infected individuals. In clinical studies, GBV-C coinfection is associated with prolonged survival of HIV-infected individuals. GBV-C infection modestly alters T cell homeostasis in vivo through various mechanisms, including modulation of chemokine and cytokine release and receptor expression, and by diminution of T cell activation, proliferation and apoptosis, all of which may contribute to improved HIV clinical outcomes. This thesis explores the interrelationship between GBV-C infection and immunoactivation and identifies potential mechanisms by which GBV-C reduces immunoactivation. Chronic HIV infection is associated with persistent immunoactivation which contributes to the immune dysfunction. In particular, T cell activation supports HIV replication and correlates with HIV viral load (VL). Persistent immunoactivation also contributes to the depletion of uninfected bystander cells by mechanisms of activation induced cell death (AICD). Although treatment with combination antiretroviral therapy (cART) reduces HIV VL, T cell activation does not return to levels found in HIVuninfected individuals. Sustained immunoactivation is also associated with lower virological response to cART suggesting therapies to reduce immunoactivation in combination with cART may benefit HIV-infected individuals. Since GBV-C infection is associated with reduced immunoactivation, understanding mechanisms by which GBV-C modulates these signaling pathways may provide insights into novel approaches to treat HIV infection and chronic immunoactivation. The effect of GBV-C infection on T cell activation and IL-2 signaling pathways were studied in a cohort of HIV-positive individuals. GBV-C viremic HIV positive individuals on cART have reduced T cell activation which was significantly associated with higher percentage of immunomodulatory CD3 +CD4-CD8-T cells. Ex vivo GBV-C infection was associated with reduced lymphocyte proliferation in response to IL-2, lower frequency of reactivation of latent HIV and protection against AICD. In vitro expression of GBV-C envelope glycoprotein E2 in CD4+ T cell lines inhibited T cell receptor (TCR) induced IL-2 secretion and inhibited IL-2 signaling pathways. This effect was mediated at least in part by reducing activation of lymphocyte specific tyrosine kinase (Lck). Through deletion mutagenesis, the inhibitory motif within the viral protein was mapped to a region that contains a predicted Lck substrate, a highly conserved tyrosine at position 87 (Y87). Lck phosphorylated GBV-C E2 protein in vitro and mutation of Y87 residue abolished the inhibitory effects of E2 protein. Synthetic peptides containing this inhibitory motif competed for Lck phosphorylation and inhibited TCR signaling in primary human T cells. The number of GBV-C infected T cells was found to be low in vivo, yet GBV-C infection reduced global TCR signaling. GBV-C RNA and E2 protein were detected in extracellular microvesicles purified from GBV-C infected human serum or the culture supernatant of E2 expressing cells, and these microvesicles inhibited TCR signaling in uninfected bystander T cells. Together, these data identify a novel mechanism by which GBV-C infection leads to global reduction in T cell activation and IL-2 signaling in the infected host, and provide a working model in which the viral envelope glycoprotein serves as a substrate for Lck and competes for Lck phosphorylation in the infected T cells and in uninfected bystander T cells.

A epidemia HIV/AIDS é um grave problema de saúde enfrentado no Brasil e no mundo. Desde o surgimento do vírus, na década de 80, muitos esforços foram realizados para esclarecer o curso da infecção que resulta no comprometimento do sistema imune em indivíduos sem tratamento. A ativação imune crônica pode levar a um status de imunossenescência exacerbada, morte celular, alteração da resposta imune e uma imunodeficiência generalizada. Percebe-se que diversos fatores do hospedeiro interferem na progressão para Aids, como deleção de 32 pares de base do gene CCR5 (CCR5delta32), perfis de HLA desfavoráveis (*B35) e coinfecções, principalmente citomegalovírus, tuberculose e hepatites B e C. Estudos recentes com o GBV-C, pertencente à família Flaviviridae, gênero Pegivirus, possibilitaram uma nova perspectiva no entendimento do curso da infecção causada pelo HIV, uma vez que nenhuma doença foi relacionada à presença do vírus GB tipo C, além de promover um atraso na progressão para a Aids e aumento da sobrevida dos pacientes portadores do vírus. Assim, o objetivo desse estudo foi avaliar o perfil de ativação, senescência e exaustão celular em indivíduos recém-infectados pelo HIV-1 e coinfectados pelo GBVC. Foram investigados a contagem de linfócitos T CD4+ e CD8+, razão CD4/CD8, presença do GBV-C e HIV-1, além da análise da expressão de marcadores de ativação (CCR5, CD38 e HLA-DR), senescência e exaustão celular (PD-1, CD95, CD57 e CD28). Diante dos critérios de inclusão do estudo, foram selecionados nove pacientes com infecção persistente com o vírus GBV-C para o grupo 1 (HIV-1+/GBV-C+), e oito pacientes sem viremia para GBV-C foram incluídos no grupo 2 (HIV-1+/GBV-C-), sendo a média de idade dos pacientes selecionados de 31,6 e 31,7 anos, respectivamente, sexo masculino e homens que fazem sexo com homens (HSH). Na visita de inclusão no estudo (V1) nenhum dos dados analisados (células T CD4+ e CD8+, carga viral e razão CD4/CD8) apresentou diferença estatística, assim como os marcadores de ativação, senescência e exaustão celular. Na análise longitudinal da diferença (deltaVn-V1), percebeu-se uma diminuição dos marcadores de ativação e senescência no grupo HIV-1+/GBV-C +, sem significância estatística entre esses dados. Foi observado, contudo, que houve uma diminuição de células T CD4+ e CD8+ naïve no grupo HIV-1+/GBV-C+, também notou-se redução na subpopulação de células T CD8+ naïve e memória central expressando CD28, houve uma diminuição das subpopulações de memória intermediária e efetora terminal, assim como na subpopulação efetora terminal expressando HLA-DR+, no grupo HIV-1+/GBV-C+. Os resultados demonstraram que a infecção pelo GBV-C reflete na diminuição da estimulação imune, ativação celular e também na redução de marcadores de senescência e exaustão celular nas subpopulações de células T, sugerindo um envolvimento na modulação da progressão do HIV / The HIV/AIDS epidemic is a serious health problem in Brazil and in the world. Since its emergence in the 1980s, many efforts have been made to understand this infection, resulting in a compromised immune system if left untreated. Chronic immune activation may lead to exacerbated immunosenescence, cell death, altered immune response, and a generalized immunodeficiency. Several host factors play an important role in the progression to AIDS, such as the 32 base pairs deletion in the CCR5 gene (CCR5delta32), unfavorable HLA molecules (*B35), and coinfections, mainly cytomegalovirus, tuberculosis, and hepatitis B and C. Recent studies with the GBV-C (Flaviviridae family, genus Pegivirus) have provided a new perspective in the understanding of the HIV infection natural history. GBV-C coinfection delays progression to Aids and increases patient survival. In addition, no symptoms have been associated to its occurrence. The aim of this study was to evaluate the profile of cellular activation, senescence, and exhaustion in recently HIV-infected individuals coinfected with GBVC. Patients were selected from a prospective cohort diagnosed with recent HIV-1 infection with known results for levels of CD4+ and CD8+ T lymphocytes, CD4/CD8 ratio, GBV-C plasma levels, HIV-1 plasma viremia, and markers for cellular activation (CCR5, CD38, and HLA-DR) and senescence and exhaustion (PD-1, CD95, CD28, and CD57). Nine presented persistent GBV-C infection and were selected for group 1 (HIV- 1/GBV-C+), mean age of 31.6 years. Another set of eight patients without GBV-C viremia were selected as controls and included in group 2 (HIV-1/GBV-C-), mean age of 31.7 years. All participants were male, in most cases men who have sex with men (MSM). At baseline visit (V1), no variable (levels of CD4+ and CD8+ lymphocytes, viral load, CD4/CD8 ratio, and cellular activation, senescence, and exhaustion markers) presented no statistical significant differences, suggesting that all selected patients shared similar characteristics. Longitudinal analysis (delta, Vn-V1) revealed a nonsignificant decrease in activation and senescence markers for both groups. However, it was observed a decrease in naïve CD4+ and CD8+ T cells in group 1, and also a reduction in the subpopulations of naïve and central memory (CD28+) CD8+ T cells. The HIV+/GBV-C+ group also presented diminished intermediate memory and terminal effector subpopulations, as well a decrease in HLA-DR+ terminal effector cells. The data demonstrate that GBV-C infection results in reduced immune stimulation, cellular senescence, and cell exhaustion, suggesting an involvement in the modulation of HIV progression

El virus C (GBV-C) o virus de la hepatitis G (HGV) está relacionado con el virus de la hepatitis C (HCV) ya que tiene una organización genómica y una homología en la secuencia muy próxima pero, sin embargo, no parece causar hepatitis ni ningún otro tipo de patología. En los últimos años, se han publicado numerosos trabajos en los que se asocia la co-infección del GBV-C/HGV y el virus de la inmunodeficiencia humana (VIH) con una menor progresión de la enfermedad causada por el virus VIH así como con una mayor supervivencia de los pacientes una vez que el SIDA se ha desarrollado. Basándonos en un modelo de co-infección, existen diversas teorías de cómo el virus de la hepatitis G puede influir en la enfermedad del VIH, se cree que puede inducir las quemoquinas, regular negativamente los co-receptores del VIH, así como provocar otros efectos indefinidos en los linfocitos huésped. Pero el mecanismo responsable del efecto beneficioso que el GBV-C/HGV tiene sobre el curso de la infección causada por VIH no está todavía definido. Teniendo en cuenta el posible uso de los péptidos sintéticos, el objetivo de la actual tesis doctoral es llevar a cabo la selección de péptidos de la proteína de envoltura E1 del GBV-C/HGV con potencial capacidad de inhibición del péptido de fusión del VIH-1, mediante la síntesis múltiple en paralelo de secuencias peptídicas lineales que cubren la totalidad de la estructura primaria de esta proteína y su posterior evaluación mediante ensayos biofísicos. Con ello se pretende plantear la utilización de péptidos sintéticos del GBV-C/HGV en el diseño de inhibidores de la entrada del VIH en la célula huésped. Se pretende así tratar de comprender mejor el efecto de la presencia de anticuerpos anti-GBV-C/HGV en la progresión de la enfermedad causada por la infección del VIH y de profundizar en el conocimiento actual acerca de la interacción entre estos dos virus. Se realiza la síntesis en fase sólida de 58 péptidos correspondientes a la proteína de envoltura E1 del GBV-C/HGV y mediante el ensayo de liberación de contenidos vesiculares se seleccionan 5 péptidos capaces de inhibir la capacidad del péptido de fusión (PF) del VIH-1 de formar poros en las membranas. Se realizan estudios detallados de los péptidos y de la inhibición del proceso de fusión, inducido por el péptido de fusión del VIH-1, por parte de secuencias del GBV-C/HGV. Para ello se realizan estudios de citotoxicidad, ensayos de hemólisis, estudio de la estructura mediante dicroísmo circular, ensayos de agregación de liposomas inducida por los péptidos y ensayos de transferencia de energía por resonancia. Por otra parte se realizan ensayos utilizando monocapas de extensión como modelos de membrana mediante la técnica de Langmuir-Blodgett, y se calculan las concentraciones superficial de exceso y las de saturación, información que servirá para conocer la capacidad de los péptidos para formar monocapas estables en la interfase aire/agua. Los diferentes ensayos muestran una interacción de los péptidos de la proteína E1 con el péptido de fusión del VIH-1, dando lugar a una inhibición de la actividad que el péptido de fusión del VIH-1 tiene sobre las membranas lipídicas. Esto sugiere que estos péptidos pueden ser candidatos para ser utilizados en futuras terapias antivirales. / "BIOPHYSICAL STUDIES OF SYNTHETIC PEPTIDES OF THE ENVELOPE PROTEIN E1 OF GBV-C/HGV AND ITS RELATIONSHIP WITH HIV" The virus C (GBV-C) or hepatitis G virus (HGV) is related to hepatitis C virus (HCV) because it has similar genome organization and sequence homology; however, it does not seem to cause hepatitis or any other pathology. In recent years, there have been several studies in which the GBV-C/HGV and human immunodeficiency virus (HIV) co-infection is associated with less progression of the disease caused by HIV as well as improved survival of patients after AIDS has developed. Based on a model of co-infection, there are several theories of how the hepatitis G virus can affect HIV disease: it could induce chemokines, negatively regulate the HIV co-receptors and cause other undefined effects in the host cell. But the mechanism responsible for the beneficial effect that GBV-C/HGV has over the course of infection by HIV is not yet defined. Taking into account the possible use of synthetic peptides, the aim of the present thesis is to carry out the selection of peptides from the envelope protein E1 of GBV-C/HGV with potential ability to inhibit the HIV-1fusion peptide. The aim is to better understand the effect of the presence of antibodies anti-GBV-C/HGV in the progression of AIDS and to study the interaction between these two viruses. The solid phase synthesis of 58 peptides corresponding to the envelope protein E1 of GBV-C/HGV was made and 5 peptides capable of inhibiting the ability of the fusion peptide (FP) of HIV-1 to form pores in membranes were selected by means of the leakage of vesicular contents assay. Detailed studies of the E1 peptides and their ability to inhibit the fusion process induced by the fusion peptide of HIV-1 were done. Monolayers and liposomes were used as model membranes. The different tests show an interaction of the E1 peptides with the HIV-1fusion peptide, resulting in an inhibition of the activity that the fusion peptide has on lipid membranes. This suggests that these peptides could be good candidates for future antiviral therapies.

A epidemia HIV/AIDS é um grave problema de saúde enfrentado no Brasil e no mundo. Desde o surgimento do vírus, na década de 80, muitos esforços foram realizados para esclarecer o curso da infecção que resulta no comprometimento do sistema imune em indivíduos sem tratamento. A ativação imune crônica pode levar a um status de imunossenescência exacerbada, morte celular, alteração da resposta imune e uma imunodeficiência generalizada. Percebe-se que diversos fatores do hospedeiro interferem na progressão para Aids, como deleção de 32 pares de base do gene CCR5 (CCR5delta32), perfis de HLA desfavoráveis (*B35) e coinfecções, principalmente citomegalovírus, tuberculose e hepatites B e C. Estudos recentes com o GBV-C, pertencente à família Flaviviridae, gênero Pegivirus, possibilitaram uma nova perspectiva no entendimento do curso da infecção causada pelo HIV, uma vez que nenhuma doença foi relacionada à presença do vírus GB tipo C, além de promover um atraso na progressão para a Aids e aumento da sobrevida dos pacientes portadores do vírus. Assim, o objetivo desse estudo foi avaliar o perfil de ativação, senescência e exaustão celular em indivíduos recém-infectados pelo HIV-1 e coinfectados pelo GBVC. Foram investigados a contagem de linfócitos T CD4+ e CD8+, razão CD4/CD8, presença do GBV-C e HIV-1, além da análise da expressão de marcadores de ativação (CCR5, CD38 e HLA-DR), senescência e exaustão celular (PD-1, CD95, CD57 e CD28). Diante dos critérios de inclusão do estudo, foram selecionados nove pacientes com infecção persistente com o vírus GBV-C para o grupo 1 (HIV-1+/GBV-C+), e oito pacientes sem viremia para GBV-C foram incluídos no grupo 2 (HIV-1+/GBV-C-), sendo a média de idade dos pacientes selecionados de 31,6 e 31,7 anos, respectivamente, sexo masculino e homens que fazem sexo com homens (HSH). Na visita de inclusão no estudo (V1) nenhum dos dados analisados (células T CD4+ e CD8+, carga viral e razão CD4/CD8) apresentou diferença estatística, assim como os marcadores de ativação, senescência e exaustão celular. Na análise longitudinal da diferença (deltaVn-V1), percebeu-se uma diminuição dos marcadores de ativação e senescência no grupo HIV-1+/GBV-C +, sem significância estatística entre esses dados. Foi observado, contudo, que houve uma diminuição de células T CD4+ e CD8+ naïve no grupo HIV-1+/GBV-C+, também notou-se redução na subpopulação de células T CD8+ naïve e memória central expressando CD28, houve uma diminuição das subpopulações de memória intermediária e efetora terminal, assim como na subpopulação efetora terminal expressando HLA-DR+, no grupo HIV-1+/GBV-C+. Os resultados demonstraram que a infecção pelo GBV-C reflete na diminuição da estimulação imune, ativação celular e também na redução de marcadores de senescência e exaustão celular nas subpopulações de células T, sugerindo um envolvimento na modulação da progressão do HIV / The HIV/AIDS epidemic is a serious health problem in Brazil and in the world. Since its emergence in the 1980s, many efforts have been made to understand this infection, resulting in a compromised immune system if left untreated. Chronic immune activation may lead to exacerbated immunosenescence, cell death, altered immune response, and a generalized immunodeficiency. Several host factors play an important role in the progression to AIDS, such as the 32 base pairs deletion in the CCR5 gene (CCR5delta32), unfavorable HLA molecules (*B35), and coinfections, mainly cytomegalovirus, tuberculosis, and hepatitis B and C. Recent studies with the GBV-C (Flaviviridae family, genus Pegivirus) have provided a new perspective in the understanding of the HIV infection natural history. GBV-C coinfection delays progression to Aids and increases patient survival. In addition, no symptoms have been associated to its occurrence. The aim of this study was to evaluate the profile of cellular activation, senescence, and exhaustion in recently HIV-infected individuals coinfected with GBVC. Patients were selected from a prospective cohort diagnosed with recent HIV-1 infection with known results for levels of CD4+ and CD8+ T lymphocytes, CD4/CD8 ratio, GBV-C plasma levels, HIV-1 plasma viremia, and markers for cellular activation (CCR5, CD38, and HLA-DR) and senescence and exhaustion (PD-1, CD95, CD28, and CD57). Nine presented persistent GBV-C infection and were selected for group 1 (HIV- 1/GBV-C+), mean age of 31.6 years. Another set of eight patients without GBV-C viremia were selected as controls and included in group 2 (HIV-1/GBV-C-), mean age of 31.7 years. All participants were male, in most cases men who have sex with men (MSM). At baseline visit (V1), no variable (levels of CD4+ and CD8+ lymphocytes, viral load, CD4/CD8 ratio, and cellular activation, senescence, and exhaustion markers) presented no statistical significant differences, suggesting that all selected patients shared similar characteristics. Longitudinal analysis (delta, Vn-V1) revealed a nonsignificant decrease in activation and senescence markers for both groups. However, it was observed a decrease in naïve CD4+ and CD8+ T cells in group 1, and also a reduction in the subpopulations of naïve and central memory (CD28+) CD8+ T cells. The HIV+/GBV-C+ group also presented diminished intermediate memory and terminal effector subpopulations, as well a decrease in HLA-DR+ terminal effector cells. The data demonstrate that GBV-C infection results in reduced immune stimulation, cellular senescence, and cell exhaustion, suggesting an involvement in the modulation of HIV progression

Die vorliegende Habilitationsschrift befasst sich schwerpunktmäßig vor allem mit der Klinik und Therapie der Hepatitis C. Evaluiert wurden: 1. verschiedene therapeutische Strategien, 2. die Ursachen der "Non-Response" auf eine anti-virale Therapie sowie 3. die klinische Relevanz der neu entdeckten Hepatitis-assoziierten Viren und 4. ihre Bedeutung bei Patienten mit akuter bzw. chronischer Lebererkrankung unklarer Ätiologie sowie bei Patienten vor und nach Lebertransplantation. Ad 1. Aus dem Vergleich verschiedener Therapie-Konzepte wie der Kurzzeit- Kombinationstherapie, Triple-Therapie, Hochdosis-Interferon?-Therapie und der Anwendung antiviraler Substanzen wie Ribavirin und Amantadin ergaben sich neue Erkenntnisse hinsichtlich relevanter prognostischer Parameter für die Therapieresponse. Ad 2. Analysiert wurden die möglichen molekularen Mechanismen der Therapieresponse bzw. Non-Response sowie der Stellenwert von Interaktionen bestimmter HCV-Proteine (NS5A, E2, sogenannte PKR-eIF2a Phosphorylisations-Homologie-Domäne [PePHD]) mit den Interferon? induzierten Effektorproteinen. Es konnte gezeigt werden, daß die Anzahl der Mutationen innerhalb des NS5A Proteins einen prognostischen Parameter darstellen hinsichtlich der Response auf eine Interferon?-Therapie. Dagegen spielen Mutationen innerhalb der PePHD-Region keine Rolle. Ad 3. Aus den Untersuchungen zur klinischen Relevanz der neu entdeckten Hepatitis-assoziierten Viren GB Virus-C/Hepatitis G Virus (GBV-C/HGV) und TT-Virus (TTV) ergaben sich keine Hinweise bzgl. eines Einflusses von GBV-C/HGV bzw. TTV-Infektionen auf den Verlauf der chronischen Hepatitis C. Die durchgeführten Verlaufsuntersuchungen bei koinfizierten Patienten sprechen dafür, daß es sich um Interferon-sensitive Viren handelt; jedenfalls beeinflussen sie nicht die IFN?-induzierte Response. Ad 4. Untersucht wurden ferner die Prävalenz, Transmission und Relevanz der GBV-C/HGV und TTV-Infektion im Hinblick auf ihre Hepatitis-induzierenden Eigenschaften. Die Ergebnisse belegen, dass beide Viren parenteral übertragen werden, und dass sie eine hohe Prävalenz bei Patienten mit parenteralen Risikofaktoren besitzen. Eine Hepatitis-induzierende Potenz dieser Viren konnten wir nicht beobachten; bei der Mehrzahl aller chronisch infizierter Personen ließen sich keine Zeichen einer chronischen Hepatitis finden. / The major goal of this thesis is the analysis of the clinical outcome of patients with Hepatitis C virus (HCV) infection and the response to therapy. Analysed were 1. different types of therapeutic strategies 2. causes responsible for ineffective antiviral therapy (non-response) 3. clinical relevance of the newly discovered hepatitis-associated viruses and 4. the role of these viruses in patients with acute or chronic hepatitis of unknown causes and in those receiving liver grafts. Ad 1. Compared were different therapeutic concepts such as short-term combination therapy, triple-therapy, high dose IFN?-therapy and the use of antiviral substances such as ribavirin and amantadine. It emerged that relevant prognostic parameters can be deduced with respect to the therapeutic response rate. Ad 2. Analysed were possible molecular mechanisms, which may interfere with response or non-response to antiviral therapy. In this respect, we focussed on the interaction of certain HCV-proteins as NS5A, E2, so-called PKR-eIF2a phosphorylisation-homology-domain (PePHD). with the interferon-?-induced effector proteins. There is evidence, that number of mutations within the NS5A proteins are of prognostic relevance with respect to the response to interferon?-therapy. In contrast, mutations within the PePHD-region do not play any role in this respect. Ad 3. We also studied the clinical relevance of the newly discovered viruses GBV-C/HGV and TTV, and found, that they have no impact concerning the course of chronic hepatitis C. These viruses are interferon-sensitive and do not influence the IFNa-response as it could be documented by following the course of co-infected patients. Ad 4. Our studies also focused on the prevalence, transmission and relevance of GBV-C/HGV and TTV infections with respect to their role as hepatitis-inducing agents. We can show that both virus types are parenterally transmitted. There is a high prevalence for both types in patients confronted with risk factors for parenteral factors. From analysis of many patients being chronically infected with these viruses it became quite clear that they lack any important potency to provoke chronic liver disease.