Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 4 uppsatser.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed on June 24, 2009). Includes bibliographical references.

Thesis (Ph.D.). / Written for the Dept. of Medicine, Division of Experimental Medicine. Title from title page of PDF (viewed2009/06/10 ). Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves 92-115).

Les macrophages sont une cible cellulaire importante du VIH-1 et sont impliqués dans la propagation virale et la constitution du réservoir. Les patients infectés par le VIH-2 présentent un contrôle naturel de l'infection qui est généralement absent chez les patients infectés par le VIH-1. Nous avons étudié ici la relation entre les macrophages et le VIH-2 afin d'évaluer leur contribution à la physiopathologie de l'infection. L'assemblage de particules virales dans des macrophages dérivés de monocytes (MDM) infectés par le VIH-2 se fait au niveau de la membrane de compartiments internes semblables aux VCC documentés dans les MDM infectés par le VIH-1. Les VCC des MDM infectés par le VIH-1 et le VIH-2 partagent la même composition protéique, et la même morphologie. Contrairement à Gag du VIH-1, la protéine Gag du VIH-2 est absente du cytosol et presque exclusivement localisée dans les VCC, ce qui suggère que Gag du VIH-2 est rapidement transportée vers le VCC une fois synthétisée dans le cytosol. Les particules de VIH-2 produites de novo par les MDM peuvent mûrir, mais sont faiblement infectieuses et se transmettent inefficacement aux cellules T activés. Cette faible infectiosité n'est pas associée avec l'expression du facteur de restriction BST-2 et n'est pas non plus améliorée par une baisse des niveaux d'expression de BST-2 induite par Vpu. Nos données suggèrent que les macrophages infectés par le VIH-2 ne contribuent probablement pas à la production et à la dissémination du virus in vivo. Cependant, les macrophages infectés par le VIH-2 peuvent représenter une source potentielle d'antigènes viraux qui pourraient stimuler les réponses des cellules T spécifiques du virus. / Macrophages are an important cellular target of HIV-1 and are potentially involved in viral spreading and constitution of the viral reservoir. HIV-2-infected patients exhibit a natural virological control of the infection that is generally absent from HIV-1-infected patients. Here, we studied the relationship between macrophages and HIV-2 to approach their potential contribution to the physiopathology of HIV-2 infection. Viral particles assembly in HIV-2-infected monocyte-derived macrophages (MDMs) occurred at the limiting membrane of internal compartments similar to virus-containing compartments (VCCs) documented in HIV-1-infected MDMs. Indeed, VCCs from HIV-1 and HIV-2-infected MDMs shared protein composition, as seen by confocal microcopy, and morphology, as seen by electron microscopy. Strikingly, HIV-2 Gag was mostly absent from the cytosol and almost exclusively localized to the VCCs, whereas HIV-1 Gag was distributed in both locations, suggesting that HIV-2 Gag is rapidly transported to the VCC membranes once synthesized in the cytosol. HIV-2 particles produced de novo by MDMs can mature, but are poorly infectious and inefficiently transmitted to activated T cells. This low infectivity neither correlate with expression of the restriction factor BST-2, nor was improved by Vpu-induced down-modulation of BST-2 levels. Our data suggest that, HIV-2-infected macrophages are unlikely to contribute to viral production and dissemination in vivo. However, HIV-2-infected macrophages accumulate large amounts of intracellular virus that may represent a potential source of viral antigens that could stimulate virus specific T cell responses.

Contexte : L’infection par le VIH-2 touche un à deux millions de personnes en Afrique de l’Ouest et sa prise en charge se heurte à de nombreuses difficultés. En plus des erreurs diagnostiques observées avec les tests VIH, la résistance intrinsèque du VIH-2 aux Inhibiteurs non nucléosidiques de la transcriptase inverse et sa moindre sensibilité à certains inhibiteurs de protéase, rendent complexe le traitement de l’infection. A cela vient s’ajouter l’absence de données sur la mortalité et la rétention dans les soins,dans un contexte d’émergence de résistances aux différentes classes d’ARV. Méthode : Le présent travail de thèse, s’appuie sur la cohorte ouest africaine IeDEA-VIH-2 et la biothèque qui lui est rattachée, de même que l’essai ANRS 12294 FIT-2, pour décrire les modalités diagnostiques, explorer la réponse thérapeutique et décrire la mortalité et les perdus de vue, parmi les patients vivant avec le VIH-2 en Afrique de l’Ouest.Résultats : Un quart des patients VIH-2 ou doublement infectés, testés de novo avec les algorithmes nationaux, étaient en réalité monoinfectés par le VIH-1. Les tests GenieIII et ImmunoCombII se sont avérés être de bons candidats pour un algorithme d’identification des patients VIH-2. Pour ce qui est du traitement, les patients vivant avec le VIH-2 initiaient principalement des régimes ARV à base d’IP boostés, avec une bonne réponse immunologique et virologique. Les régimes à base de trois INTI produisaient une réponse inférieure à celles des IP, tandis que les régimes à base d’IPnon boosté produisaient une moins bonne réponse. Il existe chez les patients VIH-2,une mortalité sous traitement ARV qui était précoce et élevée (5,2 /100 patient-année),associée à l’anémie et à des CD4 bas (<100/mm3) à l’initiation des ARV. L’essai FIT-2 actuellement en cours permettra d’identifier les meilleures séquences d’initiation des régimes ARV disponibles dans les pays ouest africains. Conclusion De nombreuses questions restent encore en suspens concernant le VIH-2, les études épidémiologiques, immunologiques et génotypiques permettront d’améliorer la prise en charge des patients VIH-2 vivant en Afrique de l’Ouest. / Background : The holistic care of the one to two million HIV-2 infected individuals in West Africa remain a concern. The frequent misdiagnosis with rapid HIV tests, in addition to the intrinsic resistance of HIV-2 to non-nucleoside reverse transcriptase inhibitors, and its low susceptibility to some protease inhibitors, make the treatment of people living with HIV-2 very challenging. The lack of data on mortality and retention in care among people living with HIV-2, as well as the emergence of resistance to most ART drugs is a great concern for the West African ART program. Method : We relied on the WADA-HIV-2 cohort, its associated biobank and the ANRS 12294 FIT-2 trial, to describe challenges in diagnosis and treatment, as well as mortality and lost to follow up, among people living with HIV-2 in West Africa. Results : One quarter of HIV-2 or dually reactive individuals, according to the national algorithms for HIV diagnosis, were found infected by HIV-1 only, and the tests GenieIII® and ImmunoCombII® showed good performances for a more accurate algorithm. Considering treatment, HIV-2 patients often initiate boosted PI based regimen, with good immunologic and virologic response. A suboptimal response was obtained with either three NRTI based regimens and unboosted PI based regimen, however worse with the first one. HIV-2 infected individuals experienced early and relatively high mortality (5.2/100 person-years), that was associated with anemia and low CD4 count (<100/mm3) at ART initiation. The results of the ongoing ANRS 12294 FIT-2 trial are expected to identify the best strategy for the optimal use of available ART regimens in West African countries. Conclusion : Many questions remain unanswered regarding HIV-2. Epidemiologic, immunologic and genotypic resistances surveys will help improving the care of people living with HIV-2 the West African region.

Les cellules de la lignée myéloïde jouent un rôle majeur dans la pathogénèse du VIH, servant à la fois de réservoir viral et permettant la transmission du virus aux cellules T. Cependant, ces cellules sont relativement résistantes à l’infection lentivirale par comparaison aux cellules provenant d’autres lignées. Des études provenant de divers laboratoires, dont le notre, ont montré que les étapes précoces de l’infection semblent se dérouler beaucoup moins efficacement dans ces cellules. Nous avons donc cherché à identifier des facteurs spécifiquement exprimés dans ces cellules pouvant être à l’origine de ce blocage. Ainsi, nous avons pu identifier APOBEC3A (A3A), un membre de la famille des APOBEC3s. A3A est spécifiquement exprimée dans les cellules de la lignée myéloïde mais n’était pas connue pour bloquer la réplication du VIH. Nous avons pu montrer que le pool d’A3A présent dans les cellules cibles est capable de cibler les particules entrantes d’une manière dépendante de son activité enzymatique. Sa déplétion, au moyen d’ARNs interférents, permet d’augmenter l’accumulation de l’ADN viral. Nos données suggèrent donc que A3A induit la dégradation des génomes viraux et que son activité antivirale est dirigée plus généralement contre les lentivirus de Primates. Cependant, la protéine Vpx des membres de la famille VIH-2/SIVSM permet de protéger contre l’action de A3A en induisant sa dégradation via le protéasome.Nous avons mis à jour un nouveau rôle de A3A lors de l’infection lentivirale des cellules myéloïdes. Ces données remettent en question le mode de fonctionnement des membres de la famille des APOBECS et ouvrent de nouvelles questions sur leurs modes d’action et leurs régulations dans les cellules primaires. / Myeloid cells are important for HIV pathogenesis both for viral transmission to T cells and as a viral reservoir. Nonetheless, these cells are quite restrictive to HIV infection compared to established cell lines or primary T cells. Studies from our group as well as other laboratories suggest thatthe early steps of infection are particularly inefficient in these cells . We tried to identify cellular factors specifically expressed in myeloid cells that could be responsible for this block. We identified APOBEC3A as one of such factors. A3A is a member of the APOBEC3 family and is the sole specifically expressed in myeloid cells. We showed that A3A blocks HIV incoming viral particles and more generally primates lentiviruses specifically in myeloid cells in a cytidine deaminase dependant manner. Our data suggest that A3A decreases viral DNA accumulation by inducing the degradation of newly synthesized genome most probably after deamination. Among the proteins coded by primate lentivirus, the HIV-2/SIVsm Vpx protein interacts and degrades A3A thus providing partial protection against A3A.Overall, our data reveal a novel role for A3A in the infection of myeloid cells and raises important questions about the regulation of the cell type specific antiviral role of A3A in primary myeloid cells.

HIV-2 differs from HIV-1 in that many infected people experience normal survival, whilst only 20&percnt; progress rapidly to AIDS. Understanding mechanisms of delayed HIV-2 disease progression could provide new insights into HIV control. The Caio Community Cohort was established in Guinea-Bissau in the setting of high HIV-2 prevalence. This thesis investigates the role of polymorphic host restriction factors of the TRIM family in HIV-2 outcome. TRIM proteins are a family of E3 ubiquitin-ligases, where closely-related TRIM5&alpha; and TRIM22 are thought to inhibit HIV-1 transcription, uncoating and budding. There was an association between TRIM5&alpha; amino acid substitution R136Q and reduced HIV-2 viral load/prolonged survival. Conversely, P479L was enriched among HIV-2 infected participants and progressors with CD4+ T cell decline. TRIM22 was highly polymorphic in this cohort, revealing three novel coding variants. Although most substitutions were located in the putative virus-interacting PRYSPRY domain, two in the coiled-coil, D155N and R242T, showed significant and divergent associations with survival. R242T was enriched in HIV-2 infected participants, who progressed to death at twice the rate of wild-type controls. In silico studies predicted D282, D360, and R321 of TRIM22 to be highly conserved, exposed residues, for which polymorphisms would be deleterious. When aligned with sequences from the potent HIV-1 restriction factor, rhesus macaque TRIM5&alpha;, TRIM22 substitutions R321K, T415I, and D360Y were spatially relevant to residues involved in HIV-1 restriction. The role of TRIM22 in HIV restriction was supported by in vitro pilot studies showing that TRIM22 was upregulated by HIV-1 infection in a lymphoid cell line and co-localised with the HIV-1 capsid protein p24. Overexpression of TRIM22 resulted in the restriction of VSV-G pseudotyped HIV-1 and SIVmac. The R242T substitution diminished TRIM22's restriction of HIV-1 and SIVmac: protein analysis suggested that this may be due to the inability of the R242T mutant to fully dimerise.

Submitted by Priscila Nascimento (pnascimento@icict.fiocruz.br) on 2012-11-09T13:57:11Z No. of bitstreams: 1 antonio-gomes-pinto-ferreira.pdf: 3200920 bytes, checksum: 45691c2f346703ac5db93dd4465b77f3 (MD5) / Made available in DSpace on 2012-11-09T13:57:11Z (GMT). No. of bitstreams: 1 antonio-gomes-pinto-ferreira.pdf: 3200920 bytes, checksum: 45691c2f346703ac5db93dd4465b77f3 (MD5) Previous issue date: 2005-03 / Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil. / Nesta dissertação apresentamos o modelo utilizado pelo Instituto de Tecnologia em Imunobiológicos, Bio-Manguinhos, Fiocruz, para absorver uma nova tecnologia de produção de reativos para diagnóstico, de alta complexidade (imunocromatografia e fluxo lateral) e estratégico para a área de saúde de nosso país. Inicialmente, porque permitirá a produção de um Kitde diagnóstico (“Teste Rápido HIV-1/2 – Bio-Manguinhos”), de alto impacto social, indispensável para ampliar as ações de saúde pública em DST/Aids e, emseguida porque será capaz de democratizar o diagnóstico da infecção pelo HIV no Brasil. A absorção desta nova tecnologia redundará em uma economia de divisas para o Brasil com a conseqüente interrupção de grandes compras de Kits importados, além de gerar um potencial a ser futuramente usado no desenvolvimento e produçãode Kits para o diagnóstico de outras importantes enfermidades que acometem a população brasileira, com baixo custo. Finalmente, devido às repercussões positivasem outros setores relacionados à produção, melhorando e fortalecendo a área de Reativos para Diagnóstico de Bio-Manguinhos. Estes são argumentos significativos para a implementação deste projeto baseado em transferência de tecnologia. Bio-Manguinhos estabeleceu as articulações necessárias junto ao Ministério da Saúde, especialmente junto ao PNDST/Aids, utilizando a “demanda do setor público brasileiro”, por um período de três anos, como contrapartida nas negociações com a empresa cedente da tecnologia, evitando, desta forma, qualquer gasto adicional de recursos públicos para absorver as referidas tecnologias. A Aids, ou SIDA, ainda é um dos maiores problemas de saúde pública no mundo, apresentando números absolutamente alarmantes – cerca de 40 milhões de pessoas infectadas pelo HIV, 5 milhões de novos casos por ano e 3,5 milhões de óbitos em 2004. No Brasil, apesar de ser adotada uma boa política de assistência e tratamento, que vem sendo citada como exemplo no mundo inteiro, ainda convivemos com uma epidemia que continua a se alastrar em ritmo preocupante, com estimativa de 600.000 brasileiros infectados. Atualmente, estamos presenciando uma revolução científica e tecnológica impar e, neste contexto, Bio-Manguinhos vem tendo como missão “contribuir para a melhoria dos padrões de saúde pública brasileira, através da pesquisa tecnológica e da produção de imunobiológicos necessários para atender à demanda gerada pelo quadro epidemiológico do país”. Assim, esta Instituição vem investindo, intensamente, em projetos de pesquisa e desenvolvimento (P&D), bem como na aquisição e incorporação de novas tecnologias para a produção, em escala industrial, de produtos capazes de suprir as demandas dos programas nacionais de saúde pública do Ministério da Saúde. Neste contexto, vem seguindo as tendências na área de Reativos para Diagnóstico laboratorial que estão voltadas, principalmente, para uma melhor orientação da conduta terapêutica com diagnósticos mais precisos, diferenciais e mais precoces na detecção das doenças, maior rapidez nos resultados e realização dos testes afins nos próprios locais de atendimento de pacientes. / This dissertation describes the model adopted by Bio-Manguinhos in the incorporation of a brand-new technology (immunochromatography and lateral flow) for the production of reagents for diagnosis. This technology is strategic and extremely valuable for the country, as it allows for the production of a low-cost diagnostic kit (Rapid Test HIV-1/2 – Bio-Manguinhos) that will, in turn, promote the dissemination of more extensive public health measures in the prevention and control of STD/Aids, including the socialization of HIV diagnosis in Brazil. Also, the substitution of large bulks of imported tests by their nationally produced equivalent will result in great, monetary savings by the Brazilian Ministry of Health and that the technological platform applied in such tests can also be used in the development of rapid tests designed for the diagnosis of other endemic diseases that afflict the Brazilian population. Finally, due to the positive repercussions it may have on other production sectors, this initiative will ameliorateand strengthen the area of Reagents for Diagnosis in Bio-Manguinhos. All of these arguments, represented strong incentives for the establishment of the partnership, being essential for the implementation of the technology transfer process. Bio-Manguinhos undertook the necessary negotiationswith MoH, especially with the National Program for STD/Aids, offering a share of the Brazilian public market for a period of three years in exchange for the technology to be transferred by the American company that detains it, thus avoidingany extra use of public funds by the Brazilian government. Aids is still one of the biggest public health problems in the world today, presenting impacting figures: approximately 40 million people are infected with HIV worldwide, with 5 million new cases diagnosed per year and 3.5 million deaths being reported in 2004. Despite Brazil´s policy for universal Aids treatment and assistance coverage – which is considered an example for the rest of the world – the country, with its estimated 600,000 HIV positive citizens, still deals with an epidemic that continues to spread at alarming rates. Certainly, much is yet expected to result from today´s scientific and technological revolution. Within this context, Bio-Manguinhos, whose mission is to “contribute to the improvement of the Brazilian public health standards through technological research and development and the production of immunobiologicals necessary for the fulfillment of the demands generated by the country´s epidemiological status”, has been investing heavily in R&D projects as well as in the acquisition and incorporation of new technologies for the large scale manufacture of products that will feed MoH´s national public health programs. One of the new tendencies in the field of reagents for laboratorial diagnosis is geared toward guiding a better therapeutic approach and calls for a more precise, differential and early disease diagnosis. The time required to obtain results also tends to be shorter and the tests, designed in much simpler formats, can beperformed faster, without need for extensive professional expertise or fully – equipped facilities.