Introdução: O desenvolvimento de antirretrovirais (AVRs) para o vírus HIV em pacientes multiexperimentados é prioridade em saúde pública e, além de supressão virológica máxima, recuperação e estabilização imunológica, os ensaios clínicos randomizados (ECRs) que envolvem esquemas de antirretrovirais utilizados em terapias de resgate devem ser capazes de demonstrar segurança na sua utilização, a curto e longo prazos. Objetivo: Realizar revisão sistemática e caracterizar os achados de segurança da terapia antirretroviral (TARV) para a infecção pelo vírus HIV-1 nos ECRs que utilizaram esquemas otimizados de base (EOB) em pacientes multiexperimentados através, principalmente, da avaliação da qualidade da informação sobre riscos baseada na extensão do CONSORT sobre riscos e da avaliação da apresentação de achados laboratoriais que são proxy de desfechos conhecidamente relevantes. Metodologia: Foram revisadas as bases de dados MEDLINE, EMBASE, LILACS, Colaboração Cochrane, SCOPUS e ISI Web of Science visando identificar publicações entre janeiro/2003 e agosto/2014. Também foram revisadas as bases de dados dos seguintes congressos científicos internacionais: International AIDS Conference; Conference on Retroviruses and Opportunistic Infections (CROI); Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC); e International Congress on Drug Therapy in HIV Infection. Não foram feitas restrições em relação à língua da publicação dos estudos. Os critérios para inclusão dos estudos foram: ECRs com pelo menos 16 semanas de duração e com pelo menos 100 indivíduos que avaliaram segurança da terapia antirretroviral (TARV) em pacientes com infecção pelo HIV- 1 caracterizados como multiexperimentados. Resultados: Vinte e quatro ECRs foram incluídos, sendo 16 estudos originais e 8 extensões. Vinte e três (95,8%) estudos foram exclusivamente financiados pela indústria farmacêutica. Apenas 15 (62,5%) estudos apresentaram eventos adversos (EAs) clínicos graves. Ocorrência de limiar para a apresentação de resultados foi observada em 13 (86,7%) estudos que apresentaram EAs graves, 22 (95,6 %) estudos que apresentaram EAs considerados comuns e em 8 (42,1%) estudos que apresentaram informações sobre desfechos laboratoriais. Seis (25%) ECRs não apresentaram informações sobre os desfechos laboratoriais investigados. Oito (33,3%) estudos não apresentaram informações sobre triglicerídeos, colesterol total, LDL (low density lipoprotein) e hiperglicemia, resultando em ausência de informação sobre os referidos desfechos para os medicamentos maraviroque (MVC) e vincriviroc (VIC). Nove estudos não apresentaram informações sobre marcadores hepáticos, resultando em ausência de informação destes desfechos para o medicamento enfuvirtida (ENF). Dezoito (75%) estudos não apresentaram informações sobre creatinina, resultando em ausência de informação sobre este desfecho para os medicamentos ENF, tipranavir (TPV) e darunavir (DRV). Conclusões: A informação sobre riscos em ECRs envolvendo TARV para pacientes multiexperimentados é altamente seletiva e insuficiente. Além disso, desfechos conhecidamente relevantes não tem sido incluídos a priori nestes estudos. Os ECRs que avaliam novos ARVs ainda apresentam as características dos estudos do início da era TARV, quando, diante de um cenário com grande mortalidade associada ao HIV, o principal objetivo era preservar a vida dos pacientes, em detrimento das questões de segurança da terapia. / Introduction: The development of antiretroviral drugs (ARVs) for treating the HIV virus in treatment-experienced patients is a priority in public health. In addition to virological suppression, recovery and stabilization of the immune system, randomized clinical trials (RCTs) involving antiretroviral regimens used in recovery therapies must be able to demonstrate short and long term safety in their use. Objective: To perform a systematic review and to characterize the findings regarding the safety of cART for HIV-1 virus infections in RCTs that used optimized background regimen in treatmentexperienced patients primarily through assessing the quality of information based on the extent of the CONSORT on risks and evaluating the overall presentation of laboratory findings that are known to proxy relevant outcomes. Methodology: The following databases were researched: MEDLINE, EMBASE, LILACS, The Cochrane Collaboration, SCOPUS and ISI Web of Science. We searched for works published between January/2003 and August/2014. The databases of the following international scientific conferences were also researched: International AIDS Conference; Conference on Retroviruses and Opportunistic Infections (CROI); Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC); and International Congress on Drug Therapy in HIV Infection. There were no restrictions regarding the language of the studies. The criteria for inclusion were the following: RCTs that lasted at least 16 weeks and with at least 100 participants who evaluated the safety of antiretroviral therapies (cART) in treatment-experienced patients with HIV-1. Results: Twenty-four RCTs were included, sixteen original studies and eight extensions. Twenty-three (95.8%) of the studies were funded exclusively by the pharmaceutical industry. Only fifteen (62.5%) of the studies showed serious clinical adverse events (AEs). The occurrence threshold for the presentation of results was observed in thirteen (86.7%) of the studies that showed serious AEs, twenty-two (95.6%) of the studies that showed AEs considered to be common, and eight (42.1%) of the studies which presented information on laboratory outcomes. Six (25%) RCTs have not provided information on laboratory outcomes investigated. Eight (33.3%) studies did not provide information on triglycerides, total cholesterol, LDL (low density lipoprotein) and hyperglycemia, resulting in lack of information on these outcomes for the drugs maraviroc (MVC) and vicriviroc (VIC). Nine studies did not provide information on hepatic markers, resulting in lack of information on these outcomes for the drug enfuvirtide (ENF). Eighteen (75%) studies did not provide information on creatinine, resulting in lack of information on this outcome for the drugs ENF, tipranavir (TPV) and darunavir (DRV). Conclusions: Information on the risks of RCTs involving cART for treatment-experienced patients is highly selective and insufficient. Moreover, known relevant outcomes have not been included a priori in these studies. RCTs evaluating new ARVs have the same characteristics of studies of early ARVs, when, faced with a scenario of high mortality associated with HIV, the main goal was to preserve the lives of patients, at the expense of the safety in therapy.

O deslocamento humano e o comportamento sexual são os principais fatores que impulsionaram a pandemia do Vírus da Imunodeficiência Humana Tipo 1 (HIV-1) para o perfil atual. Dentro da extensa variabilidade genética do HIV-1, o subtipo B (HIV-1 B) é a variante mais disseminada geograficamente, relacionando-se a aproximadamente 11% de todas as infecções no mundo. A estrutura intrínseca da transmissão do HIV-1B entre diferentes indivíduos tem valiosa importância para a compreensão da epidemia e para as intervenções em saúde pública. Assim, o presente estudo tem como objetivo caracterizar epidemiologicamente a dinâmica de transmissão e disseminação do HIV-1 subtipo B. Duas abordagens metodológicas foram empregadas: (1) Caracterização genética e temporal da diversidade molecular do HIV-1B em diferentes pontos no Brasil, através de coleta, amplificação e sequenciamento do material genético viral e, (2) Identificação das cadeias de transmissão existentes na epidemia do HIV-1B no Brasil através da seleção de sequências disponíveis em bancos de dados e posterior reconstrução filogenética. Nossos resultados mostram que a epidemia de HIV-1B é largamente influenciada por tendências regionais e sugerem uma maior probabilidade de transmissão do HIV entre indivíduos de mesma origem geográfica. A aparente diferença na prevalência da variante B”-GWGR em distintas regiões do Brasil e a relação de assinaturas genéticas virais com grupos específicos de exposição em determinados pontos do país corrobora a dinâmica epidemiológica localizada. Este isolamento na epidemia, aparentemente particular para o Brasil e demais países da América do Sul, pode ser um reflexo da discreta conexão nodal entre as diferentes cidades no continente, garantindo importante limitação local da epidemia de HIV-1B. Além disso, identificamos um curto prazo na dinâmica de transmissão do vírus entre indivíduos, envolvendo em média 29 meses. No grupo de indivíduos homossexuais/bissexuais (HSH), especificamente, o intervalo foi estimado em um ano. Estes resultados revelam uma dinâmica particular para o grupo HSH na epidemia do HIV-1B, em que o intervalo de tempo entre as novas infecções é muito estreito e possui ampla participação de indivíduos recém-infectados. A ampla coleta de dados e a utilização de métodos estatísticos robustos permitirão melhor entendimento da dinâmica epidemiológica do HIV e, através de programas ativos de saúde pública, podem influenciar no direcionamento de campanhas de intervenção para populações específicas. / The human displacement and sexual behavior are the main factors driving the human immunodeficiency virus type 1 (HIV-1) pandemic to the current profile. Within the extensive genetic variability of HIV-1, subtype B (HIV-1 B) is the most widespread variant being related to approximately 11% of all infections worldwide. The intrinsic structure of the HIV transmission among different individuals has valuable importance for the understanding of the epidemic and for the public health response. Thus, this study aims to characterize the epidemiological dynamics of HIV-1B transmission and dissemination. Two methodological approaches are required: (1) genetic and temporal characterization of molecular diversity of HIV-1B at different points in Brazil, through collection, amplification, and sequencing of viral genetic material, and (2) identification of transmission clusters within the HIV-1B epidemic in Brazil by reconstruction of phylogenetic trees using sequences selected from public databases. Our results show that the HIV-1B epidemic is largely influenced by regional trends and suggest a higher probability of HIV transmission between individuals from the same geographic origin. The apparent difference in the prevalence of the B”-GWGR variant in different regions of Brazil and the relationship of viral genetic signatures with specific exposure groups in certain parts of the country also supports the main influence of local factors in the HIV-1B epidemic. This epidemiological isolation apparently particular for Brazil and other South American countries may be a reflection of the discrete nodal connection between different cities within the continent. In addition, we identified a short-term dynamic spread within the transmission clusters in which the mean transmission time is approximately two years. In the group of homosexual/bisexual (MSM) specifically the intertransmissions interval has been estimated at about 1 year. These results show a specific dynamic in the HIV epidemic for the MSM group, and show a narrow interval between new infections with extensive involvement of newly infected individuals. These data highlight the need for better characterization of the HIV epidemic in Brazil and, in addition, show the need for specific prevention measures for concentrated epidemics. Public health services can be broadly benefited from this kind of information in order to guide intervention programs in public health.

A infecção pelo vírus da imunodeficiência humana (HIV) foi largamente disseminada pela população humana a partir da década de 80. Diversos estudos retrocedem o surgimento da epidemia do HIV a vírus infectantes de símios africanos de vida livre e ao conseqüente contato entre estas espécies e humanos. As rápidas taxas evolutivas e a associação às inter-relações humanas foram suficientes para a disseminação e a diversificação das formas virais. Desta forma, a expansão do HIV-1 pelo mundo estabeleceu uma pandemia formada por diferentes variantes virais. O subtipo B foi a primeira forma detectada do vírus e atualmente está presente em um grande número de países, sendo responsável por aproximadamente 10% das infecções por HIV no mundo todo. Fora da África, o subtipo B emergiu na região do Caribe, se disseminou pelos países vizinhos e atingiu os Estados Unidos, onde estabeleceu a pandemia. O traçado geográfico das rotas de disseminação nas Américas a partir do Haiti é, até o momento, difuso ou ausente. O presente trabalho visa investigar a história evolutiva do subtipo B nas Américas e, através de filogenias e análises estatísticas, examinar o papel da América do Sul no estabelecimento da epidemia. Os resultados obtidos sugerem uma participação primária das populações da América do Sul na disseminação do subtipo B. A estruturação populacional e as filogenias corroboram uma ligação epidemiológica importante entre os países do Caribe e os da América do Sul, visto que um clado agrupando isolados sul-americanos emerge diretamente do clado caribenho. Além deste, um grande grupo, formado por sequências das três Américas, denominado cluster pandêmico, compartilha um ancestral comum com o clado da América do Sul. Possíveis explicações para estes resultados envolvem a co-dispersão para as Américas a partir do Caribe e também a intermediação da América do Sul entre o Caribe e os Estados Unidos no estabelecimento da pandemia. Dados históricos de migrações populacionais a partir do Caribe reforçam estes cenários. Os dados apresentados revelam uma nova perspectiva a respeito da epidemia do HIV-1 subtipo B. Além disso, destacam a utilidade de uma abordagem populacional para o entendimento da dispersão da epidemia, contribuindo para a avaliação entre a diversidade viral e a movimentação de populações humanas. / Infections with human immunodeficiency virus (HIV) have been widely disseminated by the human population from the 1980's. Several studies point the onset of the epidemic to virus infecting free-living African apes through blood contact between these species and humans. The fast evolutionary rates and the human relationships were sufficient for the spreading and diversification of the epidemic. Thus, the spread oh HIV-1 established a worldwide pandemic formed by different viral forms. Among them, subtype B was the first form detected and is currently present in a large number of countries, accounting for approximately 10% of HIV infections worldwide. Out of Africa, subtype B likely emerged in Haiti in the Caribbean region, reached neighboring countries and the United States, where the pandemic was established. The geographical layout of the routes of spread within the Americas, so far, is diffuse or absent. This study aimed to investigate the evolutionary history of subtype B in the Americas and, through phylogenetic and statistical analyses sought to examine the role of South America in the pandemic. Our main findings suggest a primary involvement of the populations of South America in the spread of subtype B. The genetic structure of the viral population and the phylogenetic analyses supported an epidemiological link among the countries of the Caribbean and South American ones since a clade grouping isolates from South America emerges directly from the Caribbean. Besides this, a large group, formed by sequences of the three Americas, called pandemic cluster, share a common ancestor with the clade of South America. The co-dispersion to the Americas from the Caribbean and the intermediation of South America between the Caribbean and North America epidemics are explanations to support our results. The data presented here outline a new perspective on the HIV-1 subtype B epidemic. Furthermore, we highlight the usefulness of a population-based approach to understanding the spread of the epidemic, contributing to the assessment between the viral diversity and the movement of human populations.

Understanding the mechanisms used by HIV-1 to evade antibody neutralisation may contribute to the design of a high-coverage vaccine. This thesis explores the mechanism used by a Tier 3 virus leading to its high antibody neutralisation resistance phenotype. This thesis also describes how the glycans at the base of the V3 loop contribute to (i) breadth and potency in a cohort of unselected HIV-1-infected individuals and (ii) the selective pressures resulting from the V3/glycans shielding the virus from neutralisation and the glycans themselves being targets of broad antibody responses. HIV-1 isolates that are highly resistant to broadly neutralising antibodies could limit the efficacy of an antibody-based vaccine. For this reason, it is important to understand the mechanisms behind high HIV-1 resistance to neutralising antibodies. Chapter 2 and Chapter 3 of this thesis describe virus 253-11, a highly neutralisation resistant virus, which is particularly resistant to commonly-elicited, anti-membrane proximal external region (MPER) antibodies in sera. To further understand its resistance, mutations in the MPER were introduced that are known to delay fusion following CD4-binding and thus increase the time the virus spends in the open conformation. Interestingly, we found that these mutations affect the 253-11 Envelope (Env) spike before CD4-binding by destabilising the closed trimer structure. From these data, we hypothesized that the neutralisation resistance of 253-11 was due to an unusually tight, compact pre-fusion Env trimer that resists transient changes to the open conformation. The open conformation frequently exposes narrowly-neutralising antibody epitopes. Because the unliganded 253-11 Env presumably transitions infrequently into the open conformation, it would be able to evade these responses. 253-11 was sensitive to most but not all of the most potent broadly neutralising antibodies (bnAbs) tested, most likely because those broadly neutralising antibodies can access their epitopes in the pre-fusion Env conformation. To gain further information about the structure of the 253-11 Env, we designed a recombinant 253-11 SOSIP trimer and found it to be stable and predominantly adopt a closed conformation. The crystal structure of the SOSIP trimer revealed structural elements likely responsible for 253-11 Env compactness including the inward disposition of the heptad repeat helices and gp120 protomers towards the trimer axis. Taken together, the data from Chapter 2 and Chapter 3 highlight an underappreciated Env compactness mechanism of HIV-1 resistance to neutralising antibodies and these data may be useful in HIV-1 immunogen design research. Previous candidate HIV vaccines have failed to induce wide-coverage neutralising antibodies capable of substantially protecting vaccinees. A key approach in HIV immunogen development has been to define and model epitopes recognised by anti-HIV bnAbs. Candidate immunogen models identified by bnAbs include the V3/glycans, the V2/apex and the MPER epitopes. Autoreactivity and polyreactivity of anti-V3/glycan and anti-MPER antibodies are thought to pose both direct and indirect barriers to achieving neutralisation breadth. Chapter 4 of this thesis explored which of these bnAb epitopes were associated with breadth and potency in a South African cohort of chronically HIV-infected individuals. The study found that antibodies targeting the V3/glycans were associated with breadth and potency. In contrast, antibodies to the V2/apex were not associated with neutralisation breadth/potency. This suggests that auto/polyreactivity are not critical factors in the development of breadth and potency and that the V3/glycans should remain a high-priority vaccine candidate. Since targeting the V3/glycans was associated with breadth and potency in this cohort, the study continued to look at this epitope to investigate the role of these glycans in neutralisation resistance of Tier 2 viruses. The HIV-1 Env is surrounded by glycans that often prevent antibody neutralisation, leading to the term the "glycan shield", however some bnAbs have evolved to recognise these carbohydrates. Chapter 4 of this thesis describes how the N-linked glycan at position N301 is critical for maintaining neutralisation resistance of one subtype C virus (Du156.12), but not for another subtype-matched virus (CAP45.2.00.G3). Thus, the loss of the N301 glycan may have a substantial antibody-related fitness cost for some viruses but not others. The N301 glycan, as well as glycans at positions 332 and 334, are the primary targets of the anti-V3/glycan class of neutralising antibodies, which may select for loss of the targeted glycan. The evidence presented in Chapter 4 suggests that in some viruses, loss of the N301 glycan may result in evasion of anti-V3/glycan antibody responses while maintaining overall neutralisation resistance. This phenomenon may impair efficacy of passively-infused anti-V3/glycan bnAbs or a therapeutic vaccine.

Abstract The immaturity of the neonatal immune system places infants at an increased risk of infections and also affects the effective induction of protective immune responses by vaccines. In utero sensitisation to infectious pathogens results in immune activation and can establish immunological memory and may influence the immune response to unrelated antigens. In this study, we investigated in early life (birth to 6-10 weeks) the development of interferon-gamma (IFN-g) responses in uninfected infants born to HIV-1 infected mothers (exposed uninfected (EU) infants). Whole blood cell cultures stimulated with phytohaemagglutinin (PHA) or Mycobacterium bovis bacillus Calmette-Guérin (BCG) showed that EU infants have a greater ability to produce IFN-g in response to PHA and BCG at birth compared to control infants (born to HIV-1 uninfected mothers). However, by six weeks of age control infants produced significantly more IFN-g in response to PHA only. These results suggest that responses amongst EU infants establish and mature earlier than in control infants. In fact, over time a greater number of EU infants have a reduced ability or an inability to respond to stimuli such as PHA or BCG compared to control infants (when comparing responses at six weeks of age to responses of the matched birth samples). Full blood counts (FBC) counts were carried out using an automated AcT 5 diff haematology analyser which measures proportions and absolute counts of the five groups of white blood cells, namely, lymphocytes, monocytes, neutrophils, eosinophils and basophils. Importantly, there were no significant differences in the absolute lymphocyte counts of control infants and EU infants either at birth or at six weeks which could account for the IFN-g production differences noted between these infant groups, although the EU infants did exhibit a trend of higher absolute lymphocyte counts at six weeks than control infants. Age-dependent maturational changes in cell counts were observed in both control and EU infant groups, with neutrophils predominating at birth and lymphocytes predominating by six weeks, indicative of immune development with age in both infant groups. Short-course antiretroviral exposure increased basophil counts of infants at birth but did not affect counts by six weeks. Flow Cytometry studies using an Intracellular Cytokine (ICC) assay were conducted to establish which mononuclear cell types are predominantly responsible for producing IFN-g in infants. ICC assays done on whole blood revealed that natural killer cells (NK) were predominantly responsible for the IFN-g produced by 10 week old EU infants, and also at birth (control and EU infants). At birth whole blood cultures of 48% of EU infants already showed BCG-induced IFN-g responses (prior to BCG vaccination); this could be explained by a non-specific response (NK cells) to the antigen but could also involve T cell responses (CD4+ and/or CD8+ T cells) as supported by ICC data obtained from control and EU newborn infants. The infant immune system was clearly unique from that of their mothers. In particular, mothers’ demonstrated significant changes in blood counts from labour to six weeks postpartum indicative that immuno-modulation plays an essential role in a successful pregnancy. The single dose of nevirapine (sdNVP) taken by the mother at the onset of labour did not influence maternal blood counts significantly and maternal CD4+ and CD8+ T cells, and NK cells produced significantly more IFN-g than the CD14+ monocytes and CD19+ B cells, with CD8+ T cells producing the most. Our results have shown that EU infants are distinct from control infants with respect to immunological responses as measured by IFN-g production, and that maturational differences do exist between control and HIV-1 exposed infants. While the clinical importance of these results remains undetermined it is important to establish whether such immunological changes may result in altered susceptibility to infectious diseases in this already vulnerable population, and how this may impact on the induction of protective immune responses by vaccines. It remains important to identify neonatal immune system deficiencies and understand the consequences of exposure to maternal HIV-1 (in the absence of acquiring HIV-1 infection), the understanding of which could contribute to the development of more effective vaccines to HIV-1 and other infectious diseases.

A thesis submitted to the School of Pathology, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy. Johannesburg, 2016 / Human Immunodeficiency Virus Type 1 (HIV-1) infects approximately two million people annually, highlighting the need for a preventative vaccine. An effective HIV-1 vaccine will likely need to elicit broadly neutralizing antibodies (bNAbs), which arise naturally in some infected individuals and recognize the envelopes (Env) of multiple HIV-1 strains. Understanding the molecular events that contribute to bNAb development during infection may provide a blueprint for vaccine strategies. Here we investigated the development of a V1V2-directed bNAb lineage in the context of viral co-evolution in an HIV-1 superinfected participant (CAP256). For this, clonally-related monoclonal antibodies (mAbs), with a range of neutralization breadth, were isolated. We determined their developmental pathway from strain-specificity towards neutralization breadth and identified viral variants responsible for initiating and maturing this bNAb lineage. MAbs were isolated by memory B cell culture or trimer-specific single B cell sorting and extensively characterized by Env-pseudotyped neutralization, cell surface-expressed Env binding, electron microscopy and epitope-predictive algorithms. Antibody next-generation sequencing (NGS) at multiple time-points enabled the inference of the unmutated common ancestor (UCA) of this lineage. Viral co-evolution was investigated using Env single genome amplification and V1V2 NGS. A family of 33 clonally-related mAbs, CAP256-VRC26.01-33, was isolated from samples spanning four years of infection. The UCA of this lineage possessed an unusually long heavy chain complementarity determining region 3 (CDRH3), which resulted from a unique recombination event. Surprisingly, this UCA potently neutralized later viral variants that had evolved from the superinfecting virus, which we termed bNAb-initiating Envs. Viral diversification, which peaked prior to the development of neutralization breadth, created multiple immunotypes at key residues in the V1V2 epitope. Exposure to these immunotypes allowed adaptation of some mAbs to tolerate this variation and thus mature towards neutralization breadth. Based on these data, we proposed a four-step immunization strategy which includes priming with bNAb-initiating Envs to engage rare B cells with a long CDRH3; followed by three sequential boosts (including select V1V2 immunotypes) to drive antibody maturation. In conclusion, this study has generated a testable HIV-1 vaccine immunization strategy through the delineation of mAb-virus co-evolution during the development of neutralization breadth. / MT2017

No description available.

It is estimated that 38 million people currently live with HIV-1. Of the 38 million currently infected, 19.2 million women are infected compared to 17 million men, indicating a clear disproportion. As such, understanding mechanisms that result in increased susceptibly in women is critical to develop more efficacious prevention strategies. The epithelial cells that line the lower female reproductive tract are the first line of defense against invading pathogens. The current consensus in the field is that HIV-1 can cross the mucosal epithelium in two ways: paracellular passage and transcytosis. There are several endocytic pathway associated proteins within vaginal epithelial cells that may have a role in viral transcytosis, however, little is known about their role. One of these is Lysosomal Associated Membrane Protein 3 (LAMP3), which has been shown to be upregulated following viral exposure and involved in viral trafficking. The mechanisms regarding the early events of transmission of HIV-1 across vaginal epithelial cells remains unclear and warrant further investigation. This study was designed to examine the mechanism of how HIV1 crosses vaginal epithelial cells and potential interactions of LAMP3 and HIV-1. / Thesis / Master of Science (MSc)

Na gestação, a expressão das moléculas HLA-E ocorre principalmente nos trofoblastos extravilosos da placenta e estão associados com a inibição do sistema imune resultando na imunotolerância ao feto. Esta inibição da resposta imunológica pode ser benéfica em condições fisiológicas como a gestação, mas prejudicial na vigência de tumores e infecções. Nessa condição, tem sido descrito que o HIV infecta células trofoblásticas e utiliza a molécula HLA-E como mecanismo de escape, aumentando sua expressão para inibir a atuação de células citotóxicas. Entretanto, apesar da continua exposição viral, o fato de a maioria dos recém-nascidos não serem verticalmente infectados sugere a existência de barreiras naturalmente protetoras que previnem a transmissão materno-infantil (TMI) do HIV. Frente à escassez de estudos avaliando as moléculas HLA-E na interação imunológica materno-fetal, mais especificamente na infecção do HIV-1, e à importância que este tema tem perante a prevenção da TMI, este projeto teve o objetivo de avaliar a expressão de HLA-E em tecidos placentários de mulheres infectadas ou não pelo HIV-1. Trata-se de um estudo transversal, ao qual foram submetidos ao processamento imunohistoquímico tecido placentário parafinado de 106 mulheres infectadas pelo HIV-1 e de 100 mulheres não infectadas. A expressão da molécula HLA-E foi analisada por um patologista e classificada qualitativamente como leve, moderada ou intensa e quantitativamente como negativa (<5% de marcação), 1+ (6-25%), 2+ (26-50%), 3+ (51-75%) e 4+ (>75%). Os resultados sociodemográficos apontam que as gestantes infectadas pelo HIV-1 eram mulheres não vivendo em união conjugal (p<0,0001) e com baixa escolaridade (p=0,0004). Na análise imunohistoquímica, ficou evidente que a expressão do HLA-E ocorreu principalmente na região do trofoblasto extraviloso e em células endoteliais, mas não nas vilosidades da placenta. Os testes de estatísticos utilizados foram Qui-quadrado e exato de Fisher. Os resultados mostraram que em mulheres portadoras da infecção pelo HIV-1, a expressão das moléculas HLA-E estava significantemente menor entre as que apresentavam carga viral indetectável (p=0,03), e não houve associação entre a expressão das moléculas HLA-E com outras condições como presença ou não da infecção pelo HIV, ocorrência de aborto em gestações anteriores, número de células CD4+ e terapia antirretroviral utilizada. Estes resultados sugerem que o HIV-1 induz a expressão de HLA-E em células placentárias, podendo ser utilizado como mecanismo de escape do sistema imunológico. Entretanto outros trabalhos com polimorfismos genéticos e microRNAs são necessários para ampliar o conhecimento sobre a molécula, sua atuação na infecção pelo HIV- 1 e seu papel na TMI / During pregnancy, the expression of HLA -E molecules occurs mainly in extravillous trophoblasts of the placenta and are associated with the inhibition of the immune system resulting in immune tolerance to the fetus. This inhibition of the immune response may be beneficial in physiological conditions such as pregnancy, but harmful in the presence of tumors and infections. In this condition, it has been reported that HIV infects trophoblast cells and uses the HLA -E as an escape mechanism, increasing its expression to inhibit the activity of cytotoxic cells. However, despite the continued viral exposure, the fact that most newborns are not vertically infected suggests the existence of naturally protective barriers that prevent mother to child transmission (MTCT) of HIV. Due to the lack of studies evaluating HLA-E in the maternal-fetal immunological interaction, specifically in HIV-1 infection, and the importance that this topic has towards the prevention of MTCT, this project aimed to evaluate the expression of HLA-E in placental tissues of infected women or not by HIV-1. It is a cross sectional study, which were submitted to immunohistochemical processing the paraffin- embedded placental tissue of 106 women infected with HIV-1 and 100 uninfected women. The expression of HLA-E was analyzed by a pathologist and classified qualitatively as mild, moderate or severe and quantitatively as negative (<5% markup), 1+ (6-25 %), 2+ (26-50%), 3+ (51-75%) and 4+ (>75%). The sociodemographic results indicate that pregnant women infected with HIV-1 were not women living in marital union (p<0,0001) and have lower education (p=0,0004). In immunohistochemical analysis, it became evident that the expression of HLA-E occurred mainly in the extravillous trophoblast and endothelial cells, but not in the villi of the placenta. The statistical tests used were chi-square and Fisher exact tests. The results showed that in women with the HIV-1 infection, the expression of HLA-E was significantly lower among those who had undetectable viral load (p=0,03), and there was no association between the expression of HLA- E with other conditions such as the presence or absence of HIV infection, miscarriage in previous pregnancies, number of CD4+ cells and antiretroviral therapy used. These results suggest that HIV-1 induces the expression of HLA-E placental cells, using it as a mechanism to escape the immune system. However other studies with genetic polymorphisms and microRNAs are needed to increase knowledge about the molecule, its role in HIV-1 infeccion and its role in MTCT

A descoberta do tropismo do Vírus da Imunodeficiência Humana tipo 1 (HIV-1) pelos correceptores CCR5 e CXCR4 propiciou o desenvolvimento de novas estratégias terapêuticas com alvo nestes correceptores. Os antagonistas de CCR5 são fármacos que bloqueiam o correceptor CCR5, impedindo a entrada do (HIV) na célula, levando a uma diminuição significativa da viremia. Porém, a administração deste medicamento depende da determinação do tropismo dos vírus do indivíduo infectado antes e ao longo do uso desta estratégia terapêutica. A alça V3 da gp120 do envelope do HIV-1 é considerada o principal determinante do tropismo, mas outros fatores como diferenças intrínsecas entre as variantes do HIV-1, o número de sítios de N-glicosilação, variações no comprimento de determinadas regiões e a carga elétrica global da gp120 também podem afetar o tropismo viral. Este estudo apresenta a caracterização molecular da gp120 e a relação dos fatores moleculares com o tropismo pelos correceptores CCR5 e CXCR4. Foram incluídos no estudo 283 indivíduos infectados pelo HIV-1, dos quais foi possível obter 265 sequências da alça V3, resultados de tropismo fenotípico para 78 amostras e 20 sequências da gp120 completa. O tropismo fenotípico demonstrou a prevalência de vírus R5 (70,5%) e a presença de apenas uma amostra contendo exclusivamente vírus X4. O tropismo genotípico classificou 71,7% das sequências como vírus R5 e 28,2% como capazes de utilizar o correceptor CXCR4. A concordância entre os resultados de tropismo fenotípico e genotípico foi de 74,5%. Houve a prevalência do subtipo B (82%), sendo que a variante B\'(GWGR) esteve presente em 34,5% dos casos. Em média, as sequências da gp120 apresentaram 22 sítios de N-glicosilação, concentrados nas regiões C2 e C4. A carga elétrica líquida apresentou médias semelhantes entre vírus R5 e X4 tanto para a alça V3 quanto para a gp120. As características moleculares descritas neste estudo apresentaram distribuição semelhante entre vírus R5 e X4, não sendo possível estabelecer relações entre estes fatores a determinação do tropismo. / The tropism of Human Immunodeficiency Virus type 1 (HIV - 1) for CCR5 and CXCR4 coreceptors has provided the development of new therapeutic strategies targeting these coreceptors. CCR5 antagonists are able to prevent the HIV cell entry by blocking the CCR5 coreceptor, improving the viremia decreasing. However, this drug administration depends on viral tropism determination before and during the use of this therapeutic strategy. The V3 loop of the HIV-1 gp120 envelope is the major tropism determinant, but other factors such HIV-1 genetic variability, number of potential N-linked glycosylation sites, length variations in some protein domains and the gp120 global net charge may also affect the viral tropism. This study presents the molecular characterization of gp120 and the relationship between molecular factors and the tropism for CCR5 and CXCR4 coreceptors. A total of 283 HIV-1 infected subjects were included in this study. It was possible to obtain 265 V3 loop sequences, 78 results of phenotypic tropism and 20 sequences of the whole gp120. Phenotypic tropism showed the prevalence of R5 viruses (70.5%) while exclusively X4 viruses were found in only one sample. Genotypic tropism classified 71.7% of the sequences as R5 and 28.2% as virus able to use the CXCR4 correceptor. The agreement between phenotypic and genotypic tropism results was 74.5%. There was a prevalence of subtype B (82%), and the B\' (GWGR) variant was present in 34.5% of the cases. gp120 showed a mean of 22 N-linked glycosylation sites, which were more frequent in the C2 and C4 regions. The net charge showed similar means between R5 and X4 viruses for both V3 loop and gp120. The molecular characteristics described in this study showed similar distribution between R5 and X4 viruses and it was not possible to establish relationships between these factors the tropism determination.