Orientador: Maria Marluce dos Santos Vilela / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-07T08:42:49Z (GMT). No. of bitstreams: 1 Melo_NadjaRodriguesde_D.pdf: 2013115 bytes, checksum: 52276d586f25b6b0ea07f41ad6a754df (MD5) Previous issue date: 2006 / Resumo: O presente estudo caracterizou a flora oral de Candida em 52 crianças infectadas pelo HIV-1 em dois períodos, definidos como período I antes da introdução de inibidores de protease no esquema de terapia antiretroviral para HIV-1 e período II após a introdução de inibidores de protease. Comparou-se as espécies de Candida identificadas nos períodos I e II. Isolados do períodos I foram identificados e as crianças em sua maioria (80%) estavam colonizadas por C. albicans. Redução no percentual de colonização por C. albicans de 80% para 52%, nos períodos I e II respectivamente, sugere mudança na colonização oral por Candida após a introdução da terapêutica com inibidores de protease HIV (IP). Destaca-se particularmente o aumento da incidência de isolados Não¿albicans (p=0.005) no período II. No período I haviam 8 crianças que estavam colonizadas por espécies Não-albicans e no período II haviam 20 crianças colonizadas com isolados Não-albicans. Investigou-se a prevalência de C. dubliniensis na família de uma das crianças que estava colonizada por esta levedura. Do total de 52 crianças 38.4% mostraram manifestação oral associada a colonização por Candida. Observou-se alta sensibilidade dos isolados aos agentes antifúngicos testados, mas 4% dos isolados exibiram resistência ao fluconazol. Documentou-se resistência cruzada entre agentes antifúngicos em isolado de Candida albicans em uma criança infectada pelo HIV-1 não previamente exposta a azoles. Um isolado de C. tropicalis mostrou baixa susceptibilidade ao fluconazol (MIC = 64 µg/ml) (1). O presente estudo revelou mudança significativa na colonização oral por Candida em crianças infectadas pelo HIV-1 sob terapia HAART (Highly Active Antiretroviral Therapy). Houve alta diversidade de espécies de Candida, com emergência de espécies Não-albicans após o uso de Inibidores de protease / Abstract: This study characterized the Candida oral flora from 52 Brazilian HIV 1-infected children, comparing the Candida species identified in two periods before (PI) and under (PII) the introduction of the HIV Protease Inhibitor therapy. The majority (80%) of the children from the PI group were colonized by C. albicans. Children in the PII (52%) were colonized by C. albicans and 28% of them carried on mixed colonization (C. albicans and Non¿albicans isolates). Therefore when we compared the periods I and II, after the inhibitor protease usage there was an important decrease in the percentile of colonization for C. albicans of 80% to 52%, suggesting an important change of the Candida oral colonization after the HIV protease inhibitors introduction. Particularly with increase of the Non-albicans isolates incidence (p=0.005) in the period II. In PI there were 8 children that were colonized by Non-albicans species and in the PII there were 20 children colonized with Non¿albicans isolates. Rare Candida species were identified, particularly we investigated the C. dubliniensis prevalence in a HIV-infected child¿s family. Of 52 children in this study 20 (38.4%) of them showed oral lesions associated to the Candida colonization. In spite of the high susceptibility of the isolates to the antifungal agents tested in this study, 4.4% (n=2) exhibited resistance to the fluconazole. One of the isolates was C. albicans from a HIV-infected child not prior exposure to azoles, which showed antifungal cross-resistance. One C. tropicalis isolate has shown low susceptibility to fluconazol (MIC = 64 µg/ml). This study was succeeded in showing the inhibitory effect of ritonavir on a single hyphae tip growth of C. albicans. The present investigation revealed a significative change of the Candida oral colonization in Brazilian HIV-infected children under HAART, high diversity of Candida species and Non-albicans species emergence after IP usage / Doutorado / Pediatria / Doutor em Saude da Criança e do Adolescente

Submitted by Cláudia Bueno (claudiamoura18@gmail.com) on 2016-03-21T17:16:47Z No. of bitstreams: 2 Tese - Ludimila Paula Vaz Cardoso - 2009.pdf: 2018391 bytes, checksum: a45731b4e5b612ada7fa15b35c75c521 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-03-22T14:04:38Z (GMT) No. of bitstreams: 2 Tese - Ludimila Paula Vaz Cardoso - 2009.pdf: 2018391 bytes, checksum: a45731b4e5b612ada7fa15b35c75c521 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2016-03-22T14:04:38Z (GMT). No. of bitstreams: 2 Tese - Ludimila Paula Vaz Cardoso - 2009.pdf: 2018391 bytes, checksum: a45731b4e5b612ada7fa15b35c75c521 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2009-08-04 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / HIV-1 resistance to antiretroviral (ARV): nucleoside reverse transcriptase inhibitors (NRTI), non-nucleoside reverse transcriptase inhibitors (NNRTI), protease inhibitors (PI) and new ARV classes, like enfurvitide (T-20), represents a challenge for sustainable virological and immunologic responses. This study describes the prevalence of primary and secondary HIV-1 drug resistance and subtypes circulating in Central West Brazil. HIV-1 phylogenetic diversity and ARV resistance mutations were assessed among 97 ARV naïve patients, 48 ARV experienced patients and 77 HIV-1 pregnant women from Goiânia/GO. Protease (PR), partial reverse transcriptase (RT) and gp41 genes were amplified and sequenced from plasma RNA. HIV-1 pol subtypes were assigned by phylogenetic analysis and env/gag subtypes were identified by heteroduplex mobility analysis (HMA). ARV resistance mutations were analyzed by Stanford University Database, Internacional AIDS Society, Los Alamos Database and other sources. Among ARV naïve patients, primary drug resistance ranged from 8-10%. IP (n=2), NRTI (n=3), NRTI (n=1), IP+NRTI (n=1) e NRTI+NNRTI (n=3). Subtype B represented 82.5%, subtype F1 6.2%, subtype C 3.1%, BPR/F1RT 7.2% and one sample was F1PR/CBRT mosaic. Among ARV experienced patients, secondary drug resistance was 79%: NRTI (n=1), NNRTI (n=1), PI+NRTI or NRTI+NNRTI (n=20), PI+NRTI+NNRTI (n=16, considered multidrug resistant-MDR). In the HIV-1 pol gene, subtype B represented 79.2%, subtype F1 4.2%, subtype C 2.1%, F1PR/BRT 8.3% and BPR/F1RT 6.3%. Among MDR patients, the mosaic F1PR/BRT/F1ENV (n=1) and subtype BPR/BRT/BENV (n=13) were identified. G36E, N42T and N43S T-20 resistance mutations were observed in 3 MDR patients. In the group of pregnant women, none had primary drug resistance mutations. Among 42 ARV experienced pregnant women, 16.7% presented HIV-1 isolates with drug resistance mutations: NRTI (n=2), NRTI + NNRTI (n=2), PI + NTRI (n=2) and PI+NRTI+NNRTI (n=1). Regarding HIV-1 subtypes in env gag/PR RT genes, 66.2% were subtype B, 3.9% subtype C and 6.5% subtype F1. Our data from Central West Brazil show extensive genetic diversity with a significant proportion of distinct BF1 recombinants and the co-circulation of subtypes B, F1 and C. Moreover our data show that ARV naïve and ARV experienced patients, including pregnant women, can benefit from genotyping for ARV drug resistance, to improve clinical management and salvage therapy, which are also important to control HIV-1 transmission. / A resistência do HIV-1 aos antirretrovirais (ARV): inibidores da transcriptase reversa análogos a nucleosídeos e nucleotídeos (NRTI), inibidores da transcriptase reversa não análogos a nucleosídeos (NNRTI), inibidores da protease (IP) e novas classes de drogas antirretrovirais, como o enfurvitida (T-20), representa um desafio para a resposta virológica e imunológica dos pacientes HIV+/Aids. Este estudo descreve a prevalência de resistência primária e secundária do HIV-1 aos ARVs e os subtipos circulantes na região Centro-Oeste do Brasil. A diversidade filogenética do HIV-1 e as mutações de resistência foram avaliadas entre 97 pacientes virgens de tratamento, 48 pacientes em terapia ARV e 77 gestantes HIV+/Aids de Goiânia/GO. Os genes da protease (PR), parte da transcriptase reversa (TR) e da gp41 foram amplificados e sequenciados a partir do RNA plasmático. Os subtipos no gene pol foram avaliados através da análise filogenética e no gene env/gag foram identificados através da análise da mobilidade do heteroduplex (HMA). As mutações de resistência aos ARVs foram analisadas através do banco de dados da Universidade de Stanford, da Sociedade Internacional de Aids, de Los Alamos e outras fontes. Entre os pacientes virgens de tratamento, a prevalência de resistência primária variou de 8-10%: IP (n=2), NRTI (n=3), NRTI (n=1), IP+NRTI (n=1) e NRTI+NNRTI (n=3). O subtipo B representou 82,5%, o subtipo F1 6,2%, o subtipo C 3,1%, o mosaico BPR/F1RT 7,2% e uma amostra apresentou o mosaico F1PR/CBRT. Entre os pacientes em terapia ARV, a prevalência de resistência secundária foi de 79%: NRTI (n=1), NNRTI (n=1), IP+NRTI or NRTI+NNRTI (n=20), IP+NRTI+NNRTI (n=16, considerados multiresistentes-MDR). No gene pol do HIV-1, o subtipo B representou 79,2%, o subtipo F1 4,2%, o subtipo C 2,1%, o mosaico F1PR/BRT 8,3% e o mosaico BPR/F1RT 6,3%. Entre os pacientes MDR, foram identificados o mosaico F1PR/BRT/F1ENV (n=1) e o subtipo BPR/BRT/BENV (n=13). As mutações de resistência ao T-20, G36E, N42T e N43S, foram observadas em 3 pacientes MDR. No grupo das gestantes, nenhuma apresentou mutação de resistência primária. Entre 42 gestantes em tratamento ARV, 16,7% apresentaram isolados do HIV-1 com mutações de resistência aos ARVs: NRTI (n=2), NRTI+NNRTI (n=2), IP+NTRI (n=2) e IP+NRTI+NNRTI (n=1). Em relação aos subtipos do HIV-1 nos genes env gag/PR TR, 66,2% foram do subtipo B, 3,9% subtipo C e 6,5% subtipo F1. Nossos resultados do Centro-Oeste do Brasil mostram grande diversidade genética com significantiva proporção de recombinantes BF1 e a co-circulação de subtipos B, F1 e C. Além disso, nossos dados mostram que pacientes virgens de tratamento e em terapia ARV, incluindo gestantes, podem se beneficiar da genotipagem do HIV-1 para resistência aos ARVs, para melhorar a conduta clínica e a terapia de resgate, que também são importantes para o controle da transmissão do HIV-1.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2015-03-24T12:49:00Z No. of bitstreams: 2 Tese - Maria Edileuza Soares Moura - 2014.pdf: 2617829 bytes, checksum: 8fe2eea4d03e82310f1d867e6bfbcf37 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-03-24T14:13:59Z (GMT) No. of bitstreams: 2 Tese - Maria Edileuza Soares Moura - 2014.pdf: 2617829 bytes, checksum: 8fe2eea4d03e82310f1d867e6bfbcf37 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-03-24T14:13:59Z (GMT). No. of bitstreams: 2 Tese - Maria Edileuza Soares Moura - 2014.pdf: 2617829 bytes, checksum: 8fe2eea4d03e82310f1d867e6bfbcf37 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2014-02-11 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / In a vast country, like Brazil surveillance of transmitted drug resistance to antiretroviral drugs/TDR should be continuous and extended to different locations and exposure groups. In the last decade significant geographic differences have been reported in the Brazilian AIDS epidemic: reduction in incidence of AIDS cases and related mortality in the southeast region (epicenter of the epidemic) contrasting with significant rise in both parameters in the northeast region. This study describes TDR and HIV-1 subtypes in protease-PR and reverse transcriptase-RT regions among antiretroviral (ARV) naïve patients from Piauí State (n=89) and Maranhão State (n=106), northeast, Brazil recruited between August 2011 and June 2012. HIV-1 pol gene (protease/PR and 2/3 of reverse transcriptase/RT) was sequenced from RNA. TDR was evaluated using the Calibrated Population Resistance (CPR) tool/Stanford HIV-1 Database, HIV-1 subtypes were defined by REGA software and phylogenetic analyses. Among patients from Piauí State (44 females, 45 males, 22 of them were men who have sex with men-MSM), overall TDR rate was 13.5%. TDR rate among MSM was 27.3% while among heterosexual men TDR rate was 10% and 9.1% among females. Single-class mutations to ARV (RT nucleoside and non nucleoside inhibitors NRTI/NNRTI or PR inhibitors/PI) predominated (10/12): M46L/V82F/L90M (PI), M41L/D67N (NRTI); K103NS/E138A (NNRTI). Two patients had dual class mutations: T215L (NRTI) and E138G/Y188L (NNRTI); T215N (NRTI) and F227L (NNRTI). Subtype B predominated (86.6%), non-subtype B isolates were rare: subtype F1 (n=1), subtype C (n=1). B/F1, F1/B and B/C intersubtype recombinants were observed in 11.2%. In Maranhão State females predominated (54.7%), median age was 31 years (range: 18-72); 78.3% reported heterosexual unprotected sex, 17.9% were MSM, two reported intravenous drug use/IDU. SDRM TDR rate was 3.8% (4/106). TDR was identified in adults (21-45 years), mostly males (3/4), 2 MSM, one IDU. Single-class mutations associated with NRTI (M184V; T215S) or NNRTI (K103S/N) were detected. No major PI mutation was identified; four isolates presented accessory mutations to PI (L10I/F, A71T/V). Subtype B represented 81.1% (86/106), subtype F1 1.9% (2/106), subtype C 2.8% (3/106) and 14.2% (15/106) were recombinants. The moderate frequency of BF recombinants in PI and MA where subtype F1 is rare suggests that recombinants generated in southeast/central-western were introduced and are circulating in northeast Brazil. Among patients from PI, high TDR rate observed among MSM ccontrasts with moderate rate among heterosexual patients. This result indicates that genotyping tests to detect drug resistance and TDR, mainly among MSM can contribute to define surveillance policies and to delineate prevention strategies to help control AIDS epidemic in northeast, Brazil. / Em um país extenso como o Brasil, a vigilância da resistência transmitida a antirretrovirais/TDR deve ser contínua e estendida a diferentes grupos populacionais e regiões geográficas. Na última década, diferenças regionais significativas foram relatadas na epidemia de HIV/aids no Brasil: redução na incidência de casos de aids e mortalidade na região sudeste (epicentro da epidemia) contrastando com aumento significativo de ambos parametros na região nordeste. O objetivo deste estudo foi descrever a prevalência de TDR e subtipos de HIV-1 na protease-PR e parte da transcriptase reversa-TR em isolados de pacientes virgens de tratamento que vivem nos estados do Piauí e do Maranhão, nordeste, Brasil. Pacientes virgens de tratamento antirretroviral/ARV recrutados entre agosto 2011- junho 2012 (Maranhão, n=106; Piauí, n=89) tiveram o gene pol (protease/PR e 2/3 da transcriptasecreversa/TR) sequenciado a partir do RNA. TDR foi avaliada mediante uso da Calibrated Population Resistance (CPR) tool/Stanford HIV-1 Database, subtipos de HIV-1 foram definidos pelo software REGA e por análise filogenética. Entre os pacientes do Piauí (44 mulheres, 45 homens, 22 deles declararam-se homens que fazem sexo com homens/HSH), a taxa de TDR foi 13,5%. TDR entre HSH foi 27,3%. Entre os homens heterossexuais, a taxa de TDR foi de 10% e entre as mulheres 9,1%. Mutações de resistência a uma classe de ARV (inibidores nucleosidicos ou não-nucleosidicos da TR/ITRN/ITRNN ou inibidores da PR/IP) predominaram (10/12): M46L/V82F/L90M (IP); M41L/D67N (ITRN); K103NS/E138A (ITRNN). Dois pacientes tiveram mutações de resistência a duas classes de ARV: T215L (ITRN) e E138G/Y188L (ITRNN); T215N (ITRN) e F227L (ITRNN). O subtipo B representou 86,6%, subtipos não B foram raros: subtipo F1 (n=1) e o subtipo C (n=1). Recombinantes intersubtipos B/F1, F1/B e B/C foram observados em 11,2 % dos isolados. No Maranhão predominou mulheres (54,7%), com mediana de idade de 31 anos (variação: 18-72); 78,3% relatou relação heterossexual desprotegida, 17,9% dos homens declararam-se HSH, dois referiram uso de drogas injetáveis. Entre os pacientes do MA 3,8% apresentou TDR (4/106). TDR foi identificada em adultos (21-45 anos), a maioria do sexo masculino (3/4), 2 HSH, um usuário de droga injetável. Mutações associadas a resistência aos ITRN (M184V; T215S) e aos ITRNN (K103S/N) foram detectadas. Mutações principais aos IP não foram identificadas, quatro isolados apresentaram mutações acessórias aos IP (L10I/F, A71T/V). O subtipo B representou 81,1% (86/106) dos isolados, o subtipo F1 1,9% (2/106), o subtipo C 2,8% (3/106) e 14,2% (15/106) eram recombinantes. A moderada prevalência de recombinantes BF detectada no PI e MA onde a frequência de subtipo F1 é rara, sugere que recombinantes gerados no sudeste/centro-oeste tenham sido introduzidos e estão circulando no nordeste. Nos pacientes do PI, alta taxa de TDR observada entre HSH contrastou com taxa moderada entre heterossexuais. Este resultado indica que a realização de testes de genotipagem para resistência para avaliar TDR, principalmente entre HSH pode contribuir para definir diretrizes de vigilância e delinear estratégias de prevenção para auxiliar o controle da epidemia de aids na região nordeste, Brasil.

Entre mulheres com HIV/AIDS há um maior número de casos de infecções persistentes pelo HPV contribuindo para um risco aumentado do desenvolvimento de lesões intraepiteliais escamosas cervicais. Ademais, a expressão anormal de moléculas HLA-E pode modular o sistema imunológico através da ligação com o receptor inibitório (CD94/NKG2A) ou estimulatório (CD94/NKG2C) de células NK e linfóticos T CD8+, diminuindo imunovigilância favorecendo a evasão de céulas infectadas por vírus. Diante da escassez de estudos avaliando a molécula HLA-E na interação com o HPV, mais especificamente na infecção pelo HIV-1, este estudo teve como objetivo avaliar a expressão de HLA-E em lesões intraepiteliais cervicais em mulheres portadoras do HPV, apresentando ou não a infecção pelo HIV-1. Trata-se de um estudo transversal, ao qual foram submetidos ao processo imunohistoquímico tecido do colo do útero parafinado de 67 mulheres infectadas pelo HIV-1 e 62 mulheres não infectadas, todas com lesão intraepitelial cervical com HPV, o qual foi tipificado. A expressão da molécula HLA-E foi analisada quantitativamente como sem expressão, de 1% a 30%, de 31% a 70% e de 71% a 100%. Os resultados mostraram que a infecção por herpes vírus foi maior entre as participantes HIV+ (P=0,005). Na análise imunohistoquímica, ficou evidente que as lesões intraepiteliais cervicais de mulheres infectadas pelo HIV-1 apresentaram redução na expressão da molécula HLA-E de 31% a 100% em comparação com mulheres sem a infecção pelo HIV- 1 (P=0,001), sugerindo que essa redução possa ser um mecanismo de escape viral, que acarreta a redução da apresentação de peptídeos virais para os linfócitos T CD8+. A expressão do HLA-E não foi associada aos graus de lesões intraepiteliais cervicais. Outros estudos são necessários para melhor compreensão do padrão e da função da expressão das moléculas HLA-E em lesões intraepiteliais cervicais de mulheres infectadas pelo HIV- 1 / Among women with HIV / AIDS there is a greater number of cases of persistent HPV infections contributing to an increased risk of developing squamous intraepithelial lesions of the cervix. In addition, abnormal expression of HLA-E molecules can modulate the immune system by binding to the inhibitory (CD94 / NKG2A) or stimulatory (CD94 / NKG2C) receptor NK cells and CD8+ T lymphocytes, decreasing immunovigilance and favoring the evasion of infected cells infected with virus. Due to the lack of studies evaluating the HLA-E molecule in the interaction with HPV, more specifically in HIV-1 infection, this project aimed to evaluate the expression of HLA-E in cervical intraepithelial lesions of infected women or not by HIV- 1, with the infection by HPV. It is a cross-sectional study, which was submitted to immunohistochemical processing the paraffin-embedded cervix tissue of 67 HIV-1 women infected with HIV-1 and 62 uninfected women, all of them with cervical intraepithelial lesion with HPV, which was typified. The expression of HLA-E was quantitatively analyzed as non- expressed, 1% to 30%, 31% to 70% and 71% to 100%. The results showed that the herpes virus infection was higher among HIV + participants (P = 0.005). In immunohistochemical analysis, it became evident that cervical intraepithelial lesions in HIV-1 infected women showed a reduction the expression of HLA-E molecule from 31% to 100% compared to women without HIV-1 infection (P = 0.001) Suggesting that this reduction may be a viral escape mechanism, which leads to a reduction in the presentation of viral peptides to CD8+ T lymphocytes. The expression of HLA-E was not associated with cervical intraepithelial lesions. More studies are need to better understand the pattern and function of HLA-E molecule expression in cervical intraepithelial lesions of infected women by HIV-1

Monócitos podem ser classificados em três subgrupos de acordo com o nível de expressão dos marcadores CD14 e CD16. Monócitos clássicos são os mais abundantes no sangue. Eles expressam um fenótipo CD14+CD16-CCR2+, conferindo a eles a habilidade de migrar rapidamente a sítios inflamatórios, através da resposta à quimiocina CCL2 (CC chemokine ligand 2). Aqui apresentamos a identificação e caracterização de uma expansão de um novo subgrupo de monócitos durante a infecção por SIV e HIV. Essas células são indistinguíveis dos monócitos clássicos quanto à expressão de CD14 e CD16; porém não expressam CCR2 de superfície. A análise do transcriptoma de células selecionadas confirmou que elas representam uma subpopulação distinta que expressa níveis mais baixos de citocinas inflamatórias e marcadores de ativação que as CCR2+. Elas exibem fagocitose alterada e quimiotaxia deficiente em resposta a CCL2 e CCL7, além de serem refratárias à infecção por SIV e apresentarem atividade antiproliferativa. Nós as denominamos monócitos clássicos atípicos CCR2- (ACC monocytes), e acreditamos que elas têm um papel importante na patogenia da AIDS, possivelmente refletindo uma resposta anti-inflamatória contra a excessiva imunoativação observada durante a infecção por SIV e HIV. Tratamento antirretroviral leva a um declínio dessa subpopulação tanto em macacos como seres humanos, sugerindo que a replicação viral é capaz de induzir esse fenótipo atípico / Monocytes have been categorized in three main subpopulations based on CD14 and CD16 surface expression. Classical monocytes are the most abundant subset in the blood. They express a CD14+CD16-CCR2+ phenotype, which confers on them the ability to migrate to inflammatory sites by quickly responding to CC chemokine ligand 2 (CCL2) signaling. Here we identified and characterized the surge and expansion of a novel monocyte subset during SIV and HIV infection. They were undistinguishable from classical monocytes regarding CD14 and CD16 expression, but did not express surface CCR2. Transcriptome analysis of sorted cells confirmed that they represent a distinct subpopulation that expresses lower levels of inflammatory cytokines and activation markers than their CCR2+ counterparts. They exhibited impaired phagocytosis and deficient chemotaxis in response to CCL2 and CCL7, besides being refractory to SIV infection and showing antiproliferative activity. We named these cells atypical CCR2- classical (ACC) monocytes, and believe they play an important role in AIDS pathogenesis, possibly reflecting an anti-inflammatory response against the extreme immune activation observed during SIV and HIV infection. Antiretroviral therapy caused this population to decline in both macaque and human subjects, suggesting that this atypical phenotype may be induced by viral replication

Le VIH, grâce à sa variabilité génétique, présente une grande capacité à échapper au système immunitaire et s’adapter à l’environnement. La recombinaison, source importante de cette variabilité, est illustrée par le grand nombre de formes recombinantes observées dans l’épidémie. Le laboratoire s’est intéressé à l’étude de ce mécanisme et des lois qui gouvernent l’apparition de ces formes et ce en se focalisant sur la recombinaison inter sous-types d’isolats primaires le long du gène de l’enveloppe. En plus de mettre en évidence les paramètres impliqués dans la génération et la sélection de ces formes dans la nature, le laboratoire a pu déterminer qu’une majorité des recombinants dans la région C2 du gène de l’enveloppe présente des défauts majeurs de fonctionnalité. Ces défauts étaient sans doute dus à une incompatibilité entre les deux portions de sous-types différents réassociées par la recombinaison. Afin d’identifier les régions impliquées dans cette perte de fonctionnalité, des chimères entre différents sous-types ont été construites et testées en entrée virale. Ces chimères présentent la région C2, V1V2 et V3 de sous-types différents du reste de la protéine. Les résultats obtenus ont permis de mettre en évidence que les régions V1V2 et C2 sont importantes pour le maintien de la fonctionnalité des protéines et que V1V2 est essentielle au maintien de l’interaction entre les sous-unités de l’enveloppe, la gp120 et la gp41. Cette étude a donc permis de déterminer que V1V2 et C2 font partie d’un réseau de coévolution important impliquant des interactions indispensables au maintien de la fonctionnalité de la protéine. / HIV presents a high genetic variability that is necessary for the virus to escape to the host immune response and to adapt to the environment. Recombination is an important source of this variability and contributes to the generation of recombinant forms in the epidemic. Using inter subtypes from primary isolates, the laboratory focused on the study of recombination along the envelope gene and the determination of the parameters involved in the generation and selection of these recombinant forms. Previous works in the laboratory showed that a majority of recombinants in C2 region of the envelope gene displayed an important loss of function. This loss was probably due to an incompatibility of the parts associated by recombination and that come from different origins. To identify the regions involved in the loss of functionality of these recombinants, chimeras where C2 and surrounding regions (V1V2 and V3) from a different subtype than the rest of the gene, were created and tested for their ability to mediate viral entry. The functionality tests showed that V1V2 and C2 are important to maintain the functionality of the protein. Indeed, V1V2 seem to be involved in the stability of the Env trimer by maintaining the interaction between the two subunits of the protein, gp120/gp41. This work shows that V1V2 and C2 regions are involved in a coevolution network and their interactions with other portions are essential to maintain the functionality of the protein.

Le VIH-1 est un rétrovirus dont le génome est constitué d'ARN (ARNg). La conversion de cet ARNg en ADN est réalisée lors de la rétrotranscription (RTion). Elle permet de convertir l'ARNg simple brin en une molécule d'ADN double brin, qui sera ensuite intégrée dans le génome de la cellule hôte pour assurer la réplication du virus. La RTion est réalisée dans les premières heures de l'infection, dès l'entrée du virus. La protéine de nucléocapside (NC) participe activement à cette conversion. La NC est une petite protéine avec deux motifs en doigt à zinc (ZF1 et ZF2) qui avec sa partie basique N-term sont responsables de son activité chaperonne des acides nucléiques. L'équipe a récemment découvert un nouveau rôle de la NC en observant que la mutation des doigts de zinc ou de la région basique N-term déclenche prématurément la RTion avant le relargage des nouveaux virus, c'est à dire pendant les phases tardives de réplication du VIH. Nous parlons alors de "RTion tardive". Celle-ci génère des virus à forte teneur en ADN viral. Ces résultats originaux indiquent que la NC joue un rôle dans le contrôle spatio-temporel de la RTion au cours de la réplication du VIH. Mon projet de thèse a consisté à identifier les partenaires potentiels de la NC dans cette RT tardive et à élucider les mécanismes moléculaires mis en jeu. L'approche expérimentale principalement utilisée au cours de ma thèse, consiste à la production de particules VIH-1 (pNL4-3) et à l'analyse de leur contenu en acides nucléiques par PCR quantitative en temps réel. En mesurant l'effet de la présence et de l'absence de la protéine virale Vif sur le déclenchement de la RTion tardive dans des cellules produisant le VIH-1, nous avons démontré l'implication de Vif dans la régulation de la RTion tardive. Nous avons également montré que l'ARNg, et plus particulièrement sa région 5'UTR, est un partenaire essentiel de la NC et de Vif dans le contrôle du timing de la RTion tardive. Notre étude comparative avec un rétrovirus plus ancien et plus simple (pas de Vif) comme le Murine Leukemia Virus (MuLV), montre que cette propriété de la NC du VIH-1 à contrôler la RTion tardive n'est pas commune à tous les rétrovirus. Pour mieux comprendre le rôle de la NC au cours des phases tardives de la réplication du VIH-1, nous avons utilisé la technique de microscopie TIRF (Total Internal Reflection Fluorescence microscopy) en cellules vivantes. Cette technique de microscopie permet de visualiser les étapes d'assemblage, de bourgeonnement et de relargage des particules virales à la membrane plasmique de la cellule hôte. Bien que la NC n'influe pas sur la cinétique d'assemblage du virus, elle est en revanche fortement impliquée dans le contrôle du bourgeonnement et du relargage des particules VIH-1. Des expériences de trans-complémentation du VIH-1 mutant (délétion du ZF2) montrent que la NC contribue au recrutement des protéines cellulaires ESCRT (Endosomal Sorting Complex Required for Transport) aux sites d'assemblage viral, notamment via la voie Tsg101. / HIV-1 is a retrovirus whose genome consists of RNA (gRNA). Conversion of this gRNA into DNA is made during reverse transcription (RTion). It can convert single-stranded gRNA in a double-stranded DNA molecule, which is then integrated into the genome of the host cell to ensure the virus replication. The RTion is carried out in the early hours of infection, soon after virus entry. The nucleocapsid protein (NC) is actively involved in this conversion. The NC protein chaperones this process via its nucleic acid annealing activities and its interactions with the reverse transcriptase enzyme. To function the NC needs its two conserved zinc fingers and flanking basic residues. The team recently discovered a new role for the NC. When the NC has a mutation of its zinc fingers or its N-terminal basic region, the RTion is made prematurely before the release of new viruses, i.e. during the later stages of HIV replication. We call it «late RTion". This RTion generates virus with a high level of viral DNA. These results indicate that the NC plays a role in the spatio-temporal control of the RTion during HIV replication. My thesis project was to identify potential partners of the NC in the late RT process and to elucidate the molecular mechanisms involved.The experimental approach mainly used in my project is the production of HIV-1 particles (pNL4-3) and the analysis of their nucleic acid content by Q-PCR in real-time. By measuring the effect of the presence and absence of the viral protein Vif on the initiation of late RTion in cells producing HIV-1, we demonstrated the role of Vif in late RTion regulation. We also showed that the gRNA, and particularly its 5'UTR, is a key partner of the NC and Vif in the control of the late RTion. Our comparative study between HIV-1 and an old and simple (no Vif) retrovirus such as the Murine Leukemia Virus (MuLV) showed that the ability of the HIV-1 NC to control late RTion is not common to all retroviruses.To better understand the role of NC during the late stages of the HIV-1 replication, we used the TIRF microscopy (Total Internal Reflection Fluorescence Microscopy) in live cells. The TIRF allows the visualization of the viral assembly, budding and release of virus particles at the plasma membrane of the host cell. Although the NC does not contribute to the kinetic of the virus assembly, it is however strongly involved in the control of the budding and the release of the HIV-1 particles. Experiences of trans-complementation of a HIV-1 mutant (deletion of ZF2) showed that NC contributes to the recruitment of the cellular ESCRT machinery (Endosomal Sorting Complex Required for Transport) at the assembly sites, particularly through the Tsg101 pathway.

Le VIH-1 présentent une menace pour la santé publique car aucune thérapie antivirale efficace n’existe encore ciblant les étapes tardives de leur réplication. Au cours de ma thèse, j’ai choisi de m’intéresser aux mécanismes d'assemblage de ce rétrovirus bourgeonnant de la membrane plasmique des cellules infectées. Dans un contexte cellulaire, j’étudierais les étapes tardives de la réplication du VIH-1, en particulier, à la régulation de la dynamique membranaire modulée au cours de l’assemblage du HIV-1 grâce à des techniques de pointe en microscopie de fluorescence à haute résolution. Ainsi, nous chercherons à comprendre les mécanismes moléculaires et les facteurs cellulaires, protéiques ou lipidiques, impliqués dans l’assemblage et le bourgeonnement du HIV-1 dans les cellules hôtes, le lymphocyte T CD4+ / HIV-1 presents a threat for the public health because either no vaccine or no effective antiviral therapy still exists targeting the late stages of their replication. During my thesis, I chose to be interested in the mechanisms of assembly of this burgeoning retrovirus of the plasmic membrane of the infected cells. In a cellular context, I would study the late stages of the replication of the HIV 1, in particular, in the regulation of the dynamics membranaire modulated during the assembly of the HIV-1 thanks to state-of-the-art techniques in microscopy of high-resolution fluorescence. So, we shall try to understand the molecular mechanisms and the cellular, protein or lipid factors, involved in the assembly and the budding of the HIV-1 in hosts cells, the lymphocyte T CD4 +

Despite the existence of a treatment, there is still not a cure for HIV-1 infection and there arearound 700 000 deaths per year from AIDS-related diseases. A major barrier for a cure is theestablishment of latent reservoirs that are impossible to distinguish from healthy cells and thuscan escape the immune system. One potential solution is called shock-and-kill strategy, whichaims to induce HIV-1 reactivation, exposing latently infected cells to the immune system andmaking them susceptible to cell death. In our lab, it was seen that when NK cells are stimulatedwith a pan-caspase inhibitor, they acquire the “shock” ability, but it is still unknown how. Inthis project, we observed that the supernatant from pan-caspase inhibitor-stimulated NK cellscan increase HIV-1 reactivation in two different latency models. Furthermore, the protein levelsof three HIV-1 suppressors were found to be increased in the same supernatant. For this reason,their effect in HIV-1 reactivation in latently infected cells was analysed. Although we did notobserve an increase in HIV-1 reactivation, the upregulation of these three proteins can be usefulin the clinical context. Since they are HIV-1 suppressors, their presence can prevent theinfection from spreading after latent cells are reactivated. Altogether, our results show that NKcells stimulated with a pan-caspase inhibitor are secreting a biological product that inducesHIV-1 reactivation. This indicates that there is a pathway in NK cells that can potentially beexploited in order for them to be able to induce HIV-1 reactivation.

Passive immunization with neutralizing antibodies (nAbs) may prevent mother-to-child transmission (MTCT) of HIV-1 and/or impact HIV-1 exposed infant outcomes. This study compared plasma neutralizing activity against heterologous HIV-1 variants and the quasispecies present in the infected mothers among exposed uninfected infants (HEU) to infants that eventually acquired infection and between transmitting versus non-transmitting mothers. HEU (n = 42), compared to those that eventually acquired infection (n = 21), did not possess higher nAb responses against heterologous envelopes (p = 0.46) or their mothers’ variants (p = 0.45). Transmitting as compared to non-transmitting mothers, however, had significantly higher plasma neutralizing activity against heterologous envelopes (p = 0.03), although these two groups did not have significant differences in their ability to neutralize autologous strains (p = 0.39). Furthermore, infants born to mothers with greater neutralizing breadth and potency were significantly more likely to have a serious adverse event (p = 0.03). These results imply that pre-existing anti-HIV-1 neutralizing activity does not prevent breast milk transmission. Additionally, high maternal neutralizing breadth and potency may adversely influence both frequency of breast milk transmission and subsequent infant morbidity. In addition to neutralization, passively acquired maternal antibodies that mediate antibody dependent cellular cytotoxicity (ADCC) may impact both breast milk transmission and infant outcomes. To date, no study has rigorously compared ADCC activity against a broad panel of heterologous strains or circulating maternal viruses among HEU versus infants that eventually acquire infection and among transmitting versus non-transmitting mothers. We developed a high-throughput assay that measures the lysis of infected reporter target cells in the presence and absence of antibodies. This assay yields similar results as other methods that use decreases in reporter signal from target cells or lysis of primary cells estimated by flow cytometry. In contrast to other ADCC methods, we show that our assay allows assessment of ADCC breadth and potency in a relatively high throughput manner because it uses novel target cells that are susceptible to infection by diverse HIV-1 variants. Estimating ADCC breadth and potency and activity against maternal strains will have important insights about the impact of passively acquired maternal antibodies on MTCT.