nÃo hà / IntroduÃÃo: Rev Responsive Element (RRE) à uma molÃcula RNA responsÃvel pelo transporte do mRNA viral do HIV-1 do nÃcleo para o citoplasma da cÃlula CD4, atravÃs da via RRE-Rev, essencial para a replicaÃÃo viral. As mutaÃÃes de resistÃncia a Enfuvirtida sÃo primariamente localizadas no perÃmetro de 10 aminoÃcidos do HR1, regiÃo correspondente no RRE. Caracterizar o RRE poderà fornecer uma nova abordagem terapÃutica para a terapia do HIV. Objetivos: Sequenciar e caracterizar o RRE da gp41 para avaliar sua variabilidade e correlaÃÃo com parÃmetros laboratoriais em sequÃncias de pacientes infectados pelo HIV-1 que receberam terapia antirretroviral ou virgens. Em estudo in vitro avaliar a mutaÃÃo 36D na presenÃa de Enfuvirtida. Metodologia: 62 amostras de pacientes com HIV-1 do Cearà foram coletadas e 35 sequÃncias de RRE foram obtidas e distribuÃdas em trÃs grupos para fins de anÃlise comparativa: N (virgens de terapia), T (uso de antirretroviral sem inibidor de fusÃo) e F (uso de antirretroviral associados a Enfuvirtida). SequÃncias obtidas foram alinhadas com o banco de dados de Los Alamos para HIV usando HIV BLAST Search. Estudo in vitro utilizou dois vÃrus de laboratÃrio pNLA-3 (36D e 36G) observando citopatogenicidade e proliferaÃÃo na presenÃa de doses crescentes de Enfuvirtida. Resultados: A anÃlise filogenÃtica demonstrou alta prevalÃncia do HIV-1 subtipo B (97,2%). Observou-se aumento da resposta imunolÃgica no grupo T (71,5%) comparado ao F (2,98%). MutaÃÃes mais comuns e polimorfismos do Grupo N foram Q32L (41,6%), N42S (8,3%), R46K (33,3%), L54M (41,6%); no grupo T: Q32R (8,3%), R46K (25%), L54M (33,3%); e no grupo F: Q32L (18,2%), G36D (9,1%), V38A (9,1%), N42S (27,3%), N42T (9,1%), R46K (27,3%), L54M (45,4%), K77R (54,5%). TrÃs amostras demonstraram mutaÃÃes de resistÃncia significativas para os inibidores de fusÃo. AnÃlise dos sÃtios primÃrios de ligaÃÃo do RRE observou presenÃa de mutaÃÃo 28A em 27,2% e 8,3% nos grupos F e N respectivamente, e 27S em 8,3% no grupo T. Houve pressÃo seletiva da regiÃo HR1 do HIV-1 de pacientes usando antirretroviral, independente da exposiÃÃo à Enfuvirtida. NÃo houve diferenÃa estatÃstica significativa nas curvas de p24 do vÃrus 36D comparado com 36G, independente de concentraÃÃes de T20 (p>0.05). Observou-se menor formaÃÃo de sincÃcio, com diminuiÃÃo da capacidade fusogÃnica, sem impacto na infectividade. ConclusÃo: O estudo definiu as mutaÃÃes e polimorfismos mais prevalentes no CearÃ, sugerindo alta preservaÃÃo nas regiÃes de sÃtio primÃrio de ligaÃÃo do Rev-RRE. Evidenciou baixo perfil de resistÃncia a Enfuvirtida em regimes com falha utilizando esta medicaÃÃo. Detectou-se pressÃo seletiva no HR1 do HIV-1 de pacientes em uso de Antirretroviral, independente de exposiÃÃo à Enfuvirtida. Evidenciado in vitro menor formaÃÃo sincicial no vÃrus 36D, com diminuiÃÃo na atividade fusogÃnica, mantendo infectividade. / Introduction: Rev Responsive Element (RRE) is a RNA molecule responsible to mRNA from HIV-1 virus nuclear transportation to cytoplasm through RRE-Rev pathway, essential to virus replication. Enfuvirtide resistance mutations are primary located in a perimeter of 10 amino acids of HR1, a corresponded region of RRE. Characterize RRE should provide a new approach for HIV therapy. Objectives: Sequence and characterize RRE from gp41 to evaluate variability and correlate with laboratory parameters in sequences from HIV-1-infected patients, which were receiving regimens including Enfuvirtide, naÃve or rescue therapy. Also evaluated mutation G to D at codon 36 in the presence of fusion inhibitor (enfuvirtide). Methods: Sixty-two samples from HIV patients in Ceara/Brazil were collected and Thirty-five RRE sequences and clinical follow-up were analyzed, distributed into three groups: N (naÃve therapy), T (treated patients with rescue regimens) and F (rescue regimens containing Enfuvirtide). Sequences obtained were aligned with Los Alamos HIV sequence database by using the HIV BLAST Search. Culture Study was performed using two different pNLA-3 (36D and 36G) with increasing amounts of enfuvirtide. Results: A phylogenetic analyses demonstrated higher prevalence of HIV-1 subtypes B (97.2%). An increased immunology response was observed in CD4 count higher on group T (71.5%) compared with F (2.98%). Group N most common mutations and polymorphisms were Q32L (41.6%), N42S (8.3%), R46K (33.3%), L54M (41.6%); group T: Q32R (8.3%), R46K (25%), L54M (33.3%); and group F: Q32L (18.2%), G36D (9.1%), V38A (9.1%), N42S (27.3%), N42T (9.1%), R46K (27.3%), L54M (45.4%), K77R (54.5%). Three samples demonstrated significant resistance mutations to fusion inhibitors. Analysis of RRE nucleotide primary sites observed mutation 28A in 27.2% and 8.3% on groups F and N respectively, and 27S in 8.3% on group T. There was selective pressure on HR1 region from HIV-1 patients using antiretroviral, independent of enfuvirtide exposure. There was no statistical difference between p24 curves of virus 36D compared with 36G, independent of T20 concentrations (p>0.05). It was observed less syncytial formation in 36D virus, with diminished fusogenic activity besides keeping infectivity. Conclusions: This study defined most prevalent RRE polymorphisms in Ceara/Brazil and suggests highly preserved regions primary sites to Rev connection. Observed a low resistance profile to enfuvirtide in failing regimens with this drug. Selective pressure on HR1 region in failed regimens with out fusion inhibitors was detected. A less syncytial formation in 36D virus with diminished fusogenic activity was detected.

With the explosion of protein tools as popular platforms for discovery and therapeutics, we see greater need for regulator systems that work congruently within these frameworks, especially safe and effective tools that can be implemented in humans. To this end, we endeavor to create orthogonal, precise and flexible protein modulators that can be easily employed to control protein tools with little need to iterate design for novel contexts. Hepatitis C NS3 protease is employed as a stabilizable linker between protein domains, enabling control over protein localization with FDA approved anti-viral drugs. The power of this tool is demonstrated by controlling gene expression through the controlled tether and release of a transcription factor. Inteins have already been employed to modulate proteins in synthetic contexts, however we observe that natural systems lack the avenues of control necessary to make them indispensable. We employ existing protein tools to construct a system of modular protein association, as well as drug and light inducible schema that reveal gaps in our knowledge of how to repurpose inteins in vivo. Despite this, we use inteins in the construction of a novel cargo delivery platform based on the fusogenic properties of the viral envelope glycoprotein from Vesicular stomatitis virus (VSV-G). We confirm reduced tropism of cargo delivery based on an intein lock-and-key mechanism that has implications for both biosafety as well as targeted delivery in vivo of natively folded proteins to target cells. / 2024-05-23T00:00:00Z

No description available.

<p>Majority of new HIV-1 infections world-wide occur via the genital tract. Therefore, achieving effective mucosal immunity will be a critical component of vaccine strategies in the prevention and control of infection during early stages of transmission itself. Rigorous efforts have been made to identify conserved epitopes and develop rational design of immunogens, in order to elicit broadly-reactive protective Abs against HIV. Newly emerging data have highlighted the significance of Ab effector functions other than classical IgG-mediated neutralization in HIV infection. In the studies contributing towards this thesis, an optimized vaccine model is described that successfully elicits potent systemic and mucosal Abs against the highly conserved epitopes of the membrane-proximal external region and the coiled coil region of gp41. Intriguing observations are reported on the IgA-inducing capacity of the coiled coil epitope, QARVLAVERY, which highlight the potential of this epitope as an attractive candidate for mucosal vaccines. Most importantly, the epitope-specific Abs proved to be functional in neutralizing HIV in a standardized assay. With particular relevance to mucosal protection, epitope-specific IgA also effectively inhibited the transcytosis of HIV in an optimized transwell assay. The effect of gp41-specific Abs was also assessed against a novel panel of HIV-1 viral clones, and exhibited significant protection. These clones selectively express envelopes from viruses that would be desired targets of prophylactic immune responses, the earliest founder population in the mucosa after virus transmission.</p> / Doctor of Philosophy (PhD)

Le virus de l'immunodéficience humaine (VIH) est responsable du syndrome d'immunodéficience humaine acquise (SIDA). Le virus est très variable et est classé en divers sous-types dont le sous-type B qui est le plus étudié et le C qui est le plus répandu. L'enveloppe (Env) à la surface du virus lui permet d'infecter des cellules du système immunitaire, parmi lesquelles les lymphocytes T CD4 (LT CD4) et les macrophages sont des cibles privilégiées. Nous avons étudié le rôle de variations de séquences dans la partie de Env qui n'est pas exposée à la surface du virus (la queue intravirale (gp41CT)). Nos résultats montrent que ce domaine contribue à la réplication du virus dans les LT CD4 et que des variations dans ce domaine entravent l'assemblage des protéines de structure du virus, diminuant la production de nouveaux virus. Ce défaut n'est pas observé dans les macrophages, suggérant qu'un facteur cellulaire est impliqué. Identifier ce facteur pourrait fournir de nouvelles cibles antivirales / The human immunodeficiency virus (HIV) is responsible for the pandemic of acquired immunodeficiency syndrome (AIDS). Being highly variable, the virus has been subdivided into viral subtypes. Subtype B is the most studied, while subtype C is the most spread. The envelope (Env) expressed at the surface of the virion enables infection of cells involved in the immune system, like CD4 cells (CD4 TL) and macrophages. We studied the Env region not exposed at the viral surface (intraviral tail, gp41CT), which also harbors sequence characteristics linked to viral subtype. Viruses with subtype C gp41CT had lower replication capacities in CD4 TL. Microscopy analysis showed a defect in clustering of the viral structural protein Gag, revealing that changes in gp41CT affect assembly of all viral components. This defect was seen in CD4 TL but not in macrophages, suggesting the involvement of a cellular factor. Identifying this factor could open new therapeutic leads

Introdução: estudos com Enfuvirtida (ENF) mostraram que mutações na HR1 da gp41 levam à resistência primária de cepas em pacientes sem tratamento prévio com inibidores de fusão (Derdeyn et al., 2000; Rimsky et. al., 1998; Sista et. al., 2002). Outros, que o uso contínuo de HAART leva à falha virológica (Shafer et. al., 1998; Shafer & Vuitton, 1999). Para a melhor escolha terapêutica recomenda-se usar a Genotipagem do HIV1 (Ministério da Saúde, 2008b; Perez-Elias et. al., 2003; Shafer et. al., 2001). Objetivos: avaliar o perfil de resistência do HIV1 ao ENF pelo sequenciamento da HR1 da gp41 em pacientes sob HAART, sem uso de inibidor de fusão. Realizar fenotipagem virtual da pol do HIV1 em trinta e duas cepas com resistência aos NRTI, NtRTI , NNRTI e PI para verificar indicação do ENF. Métodos: investigamos 877 prontuários de pacientes do CRT-DST/AIDS entre 5/2002 a 7/2005 e verificamos que 92 que haviam realizado o Teste de Genotipagem do HIV1 em nosso laboratório, sem tratamento prévio com inibidores de entrada poderiam participar do nosso estudo. O primeiro grupo, com 60 pacientes apresentou cepas sensíveis à pelo menos um antirretroviral (ARV) e o segundo com 32 cujas cepas apresentaram resistência parcial e completa às drogas. A região HR1/HR2 da gp41 foi seqüenciada com o kit Big Dye Terminator vs. 3.1. Alinhamos as seqüências e as comparamos com HXB2 através do programa BioEdit vs.7.0.9.0. As mutações de resistência ao ENF foram determinadas pelo algoritmo Francês (ANRS) e pelo da Universidade de Stanford. Submetemos as sequências ao Virtual Phenotype Assay (Virco, Bélgica) para obtermos a Fenotipagem Virtual. Os subtipos da HR1/HR2 da gp41 e do pol foram determinados por análises filogenéticas e os recombinantes, pelo Simplot v 3.5.1. Resultados: 89(97%) amostras foram seqüenciadas, 2 (2%) não amplificaram e 1 (1%) não tinha material suficiente. Destas, 82 (92%) eram do subtipo B, 6(7%) do F1 e 1(1%), do BF na gp41. No pol, 80(87%) eram B, 4(4%) eram F e 7(8%), eram FB. Três (3%) apresentaram resistência primária ao ENF, determinada pela N42D (1%), N43H (1%) e L44M (1%). Dez amostras tinham os polimorfismos Q39R(10%), N42H(10%), N42R(10%), N42N/S(10%) e N42S(60%). Na HR2, as mutações N126N/K, N126K/Q/E, E137E/K, E137K e S138S/A estavam presentes em 26% das cepas. Apesar das cepas apresentarem resistência aos ARV pelo teste de genotipagem, a fenotipagem virtual mostrou que 10% dos ARV ainda atuavam sobre elas, enquanto que 75% delas apresentaram resistência parcial e 15%, completa às drogas. Conclusão: nosso estudo sugere a introdução do teste de genotipagem da HR1/HR2 da gp41 antes de indicar do ENF em pacientes com falha terapêutica e que a Fenotipagem Virtual pode ser usada para a escolha do melhor esquema antirretroviral que permita uma supressão viral sustentada. / Background: studies using Enfuvirtide (ENF) have showed that mutations in the HR1 (gp41) have been associated with primary resistance in strains of the patients without previous treatment with fusion inhibitors. (Rimsky et. al.,1998; Derdeyn et al.,2000, Sista et. al.,2002). The widespread use of HAART may achieve maximal suppression of the viral load, but the viral rebound may occur, leading to virologic failure (Shafer et. al.,1998; Shafer & Vuitton,1999). To optimize therapeutic choices it is recommended to use the HIV1 Genotypic Assay. Objective: to evaluate the resistance profile in the HR1 in patients submitted to HAART, but naïve entry inhibitors. To make the Virtual Phenotypic Assay in the pol of the HIV1 strains isolated of the 32 patients with resistance to the antiretroviral (ARV) and to verify the possibility of using the ENF. Methods: we investigated 877 handbook patients of the CRT-DST/AIDS from May/2002 to July/2005. We identified 92 naïve patients to entry inhibitors included in our cohort to do genotypic assay; they were included in our research. The first group was composed by 60 persons with sensible strains to only one ARV. The second, with 32 presented strains with resistance to all ARV. HR1/HR2 was sequenced (ABIPrism BigDye Terminator vs 3.1). Bioedit Software vs.7.0.9.0 was used to compare sequences with the HIVHXB2. The resistance mutations to ENF were analyzed using French ANRS and Stanford HIV Drug Resistance Algorithm. The results by Virtual Phenotype Assay of 32 patients that presented regimen failure by Genotypic Assay predicted HIV phenotypic resistance. Subtype analysis were did by Phylogeny and the recombinants strains results, confirmed by Simplot v.3.5.1. Results: 89(97%) out of 92 samples were sequenced. Two of them (2%) could not be amplified and one (1%) we did not have enough material to use. In the HR1/HR2 82(92%) of the samples were typed as B, 6(7%), as F and 1(1%) as FB. In the pol 80(87%) were classified as B, 4(4%), as F and 7(8%) as FB. According to algorithms used, 3(3%) out of 89 ENF naïve patients presented complete resistance to this drug. One virus harbored the N42D mutation (1%), another, the N43H(1%) and the other one the L44M(1%). In 10 samples we observed the polymorphisms Q39R(1%), N42H(1%), N42R(1%), N42N/S(1%) and N42S(7%). In the HR2 domain the substitutions N126N/K, N126K/Q/E, E137E/K, E137K and S138S/A were present in 26% of the samples. Although strains presented resistance to the ARV by Genotypic Assay, in the Virtual Phenotypic Assay we verified that 10% of the drugs had some effect upon them. 75% of the strains presented partial and 15% completed resistance to ARV. Conclusion: it is important to introduce the HR1/HR2 genotyping test to verify resistance mutations to ENF before indicating this drug to patients with virologic failure. The virtual phenotypic assay can be used to select the better combination therapy to obtain viral suppression sustained response.

Les protéines membranaires sont particulièrement riches en acides aminés aromatiques, tels que le tryptophane (W). On retrouve cette originalité dans beaucoup de peptides antimicrobiens et dans les protéines de fusion virales. La glycoprotéine de l'enveloppe de HIV-1, gp41, en est un exemple. Manifestement, les résidus W sont impliqués dans la perturbation des membranes et la formation des pores. L'objectif de ce travail est d'étudier le rôle des résidus W dans de telles activités en utilisant l'optique non linéaire. Pour cela, nous avons préalablement déterminé l'hyperpolarisabilité (le potentiel non linéaire) du W par la diffusion Hyper Raleigh (HRS). Puis nous avons montré une évolution de la réponse non linéaire de petits peptides synthétiques en fonction du nombre croissant de leurs résidus W. Ces résultats ont permis de suivre l'implication des tryptophanes de deux peptides K3W et gp41W, lors de leurs interactions avec des monocouches lipidiques à l'interface air-eau par la génération de second harmonique (SHG). D'autre part, l'influence de telles interactions sur la structure secondaire et l'orientation des peptides a été déterminée par le PM-IRRAS. Nous avons ainsi montré la cohérence entre les modifications du signal SHG, liées à des changements d'orientation des tryptophanes et celles des spectres de PM-IRRAS, dues à des changements d'orientation de la structure secondaire de gp41W / Membrane proteins are extremely rich in aromatic amino acids, like tryptophan (W). This particularity is found in many antimicrobial peptides and in several virus fusion proteins. An example of these fusion proteins is the HIV-1 envelop glycoprotein, the gp41. It is clear that the W residues are implicated in membrane perturbation and pore formation. The aim of this work was the investigation of the W residue role in such activities, using the nonlinear optic. First, we determined the W hyperpolarizabilité (nonlinear potential) by the Hyper Rayleigh Scattering (HRS). Then, the evolution of the nonlinear signal of small synthetic peptides, as function of the increasing number of their W residues, was demonstrated. These results allowed us to follow the W residue involvement of two peptides, K3W4 and gp41W, in the interaction with lipids monolayer at the air-water interface, using the second harmonic generation (SHG). The influence of such interaction in the peptide structure and orientation was determined using the PM-IRRAS. In conclusion, we showed the coherence between the SHG signal variation, due to the W orientation changes, and the PMIRRAS spectra modification, due to the gp41W helix orientation changes

Introdução: estudos com Enfuvirtida (ENF) mostraram que mutações na HR1 da gp41 levam à resistência primária de cepas em pacientes sem tratamento prévio com inibidores de fusão (Derdeyn et al., 2000; Rimsky et. al., 1998; Sista et. al., 2002). Outros, que o uso contínuo de HAART leva à falha virológica (Shafer et. al., 1998; Shafer & Vuitton, 1999). Para a melhor escolha terapêutica recomenda-se usar a Genotipagem do HIV1 (Ministério da Saúde, 2008b; Perez-Elias et. al., 2003; Shafer et. al., 2001). Objetivos: avaliar o perfil de resistência do HIV1 ao ENF pelo sequenciamento da HR1 da gp41 em pacientes sob HAART, sem uso de inibidor de fusão. Realizar fenotipagem virtual da pol do HIV1 em trinta e duas cepas com resistência aos NRTI, NtRTI , NNRTI e PI para verificar indicação do ENF. Métodos: investigamos 877 prontuários de pacientes do CRT-DST/AIDS entre 5/2002 a 7/2005 e verificamos que 92 que haviam realizado o Teste de Genotipagem do HIV1 em nosso laboratório, sem tratamento prévio com inibidores de entrada poderiam participar do nosso estudo. O primeiro grupo, com 60 pacientes apresentou cepas sensíveis à pelo menos um antirretroviral (ARV) e o segundo com 32 cujas cepas apresentaram resistência parcial e completa às drogas. A região HR1/HR2 da gp41 foi seqüenciada com o kit Big Dye Terminator vs. 3.1. Alinhamos as seqüências e as comparamos com HXB2 através do programa BioEdit vs.7.0.9.0. As mutações de resistência ao ENF foram determinadas pelo algoritmo Francês (ANRS) e pelo da Universidade de Stanford. Submetemos as sequências ao Virtual Phenotype Assay (Virco, Bélgica) para obtermos a Fenotipagem Virtual. Os subtipos da HR1/HR2 da gp41 e do pol foram determinados por análises filogenéticas e os recombinantes, pelo Simplot v 3.5.1. Resultados: 89(97%) amostras foram seqüenciadas, 2 (2%) não amplificaram e 1 (1%) não tinha material suficiente. Destas, 82 (92%) eram do subtipo B, 6(7%) do F1 e 1(1%), do BF na gp41. No pol, 80(87%) eram B, 4(4%) eram F e 7(8%), eram FB. Três (3%) apresentaram resistência primária ao ENF, determinada pela N42D (1%), N43H (1%) e L44M (1%). Dez amostras tinham os polimorfismos Q39R(10%), N42H(10%), N42R(10%), N42N/S(10%) e N42S(60%). Na HR2, as mutações N126N/K, N126K/Q/E, E137E/K, E137K e S138S/A estavam presentes em 26% das cepas. Apesar das cepas apresentarem resistência aos ARV pelo teste de genotipagem, a fenotipagem virtual mostrou que 10% dos ARV ainda atuavam sobre elas, enquanto que 75% delas apresentaram resistência parcial e 15%, completa às drogas. Conclusão: nosso estudo sugere a introdução do teste de genotipagem da HR1/HR2 da gp41 antes de indicar do ENF em pacientes com falha terapêutica e que a Fenotipagem Virtual pode ser usada para a escolha do melhor esquema antirretroviral que permita uma supressão viral sustentada. / Background: studies using Enfuvirtide (ENF) have showed that mutations in the HR1 (gp41) have been associated with primary resistance in strains of the patients without previous treatment with fusion inhibitors. (Rimsky et. al.,1998; Derdeyn et al.,2000, Sista et. al.,2002). The widespread use of HAART may achieve maximal suppression of the viral load, but the viral rebound may occur, leading to virologic failure (Shafer et. al.,1998; Shafer & Vuitton,1999). To optimize therapeutic choices it is recommended to use the HIV1 Genotypic Assay. Objective: to evaluate the resistance profile in the HR1 in patients submitted to HAART, but naïve entry inhibitors. To make the Virtual Phenotypic Assay in the pol of the HIV1 strains isolated of the 32 patients with resistance to the antiretroviral (ARV) and to verify the possibility of using the ENF. Methods: we investigated 877 handbook patients of the CRT-DST/AIDS from May/2002 to July/2005. We identified 92 naïve patients to entry inhibitors included in our cohort to do genotypic assay; they were included in our research. The first group was composed by 60 persons with sensible strains to only one ARV. The second, with 32 presented strains with resistance to all ARV. HR1/HR2 was sequenced (ABIPrism BigDye Terminator vs 3.1). Bioedit Software vs.7.0.9.0 was used to compare sequences with the HIVHXB2. The resistance mutations to ENF were analyzed using French ANRS and Stanford HIV Drug Resistance Algorithm. The results by Virtual Phenotype Assay of 32 patients that presented regimen failure by Genotypic Assay predicted HIV phenotypic resistance. Subtype analysis were did by Phylogeny and the recombinants strains results, confirmed by Simplot v.3.5.1. Results: 89(97%) out of 92 samples were sequenced. Two of them (2%) could not be amplified and one (1%) we did not have enough material to use. In the HR1/HR2 82(92%) of the samples were typed as B, 6(7%), as F and 1(1%) as FB. In the pol 80(87%) were classified as B, 4(4%), as F and 7(8%) as FB. According to algorithms used, 3(3%) out of 89 ENF naïve patients presented complete resistance to this drug. One virus harbored the N42D mutation (1%), another, the N43H(1%) and the other one the L44M(1%). In 10 samples we observed the polymorphisms Q39R(1%), N42H(1%), N42R(1%), N42N/S(1%) and N42S(7%). In the HR2 domain the substitutions N126N/K, N126K/Q/E, E137E/K, E137K and S138S/A were present in 26% of the samples. Although strains presented resistance to the ARV by Genotypic Assay, in the Virtual Phenotypic Assay we verified that 10% of the drugs had some effect upon them. 75% of the strains presented partial and 15% completed resistance to ARV. Conclusion: it is important to introduce the HR1/HR2 genotyping test to verify resistance mutations to ENF before indicating this drug to patients with virologic failure. The virtual phenotypic assay can be used to select the better combination therapy to obtain viral suppression sustained response.

Les réponses des cellules T CD4 (Thelper, TH) jouent un rôle clé dans l'immunité antivirale. Cependant, celles générées par l'infection au VIH et les vaccins candidats sont variables. Des données chez la souris et l'humain suggèrent que des réponses TH antivirales peuvent être générées avant l'exposition à l'antigène spécifique par réaction croisée avec d'autres microorganismes et influencer les réponses TH ultérieures. Les réponses TH au VIH chez des individus séronégatifs seront investiguées et comparées à celles de sujets infectés. Une haute prévalence de réponses TH prolifératives au VIH a été observée chez des sujets VIH-. Gag montre une légère prédominance sur les autres protéines du VIH Env, Nef et Pol (33% des donneurs VIH- ont une réponse contre Gag >1% par test CFSE), mais qui diffère de l’immunodominance observée chez les donneurs VIH+. Malgré les réponses prolifératives plus petites chez les donneurs VIH-, des lignées cellulaires de TH spécifiques pour Gag ou Env ont pu être générées. Un marquage intracellulaire a validé leur spécificité et leurs fonctions montrant des réponses dominées par l'expression de TNF et CD40L comparativement à celles dérivées de donneurs VIH+ produisant beaucoup d’IFN-γ. L’affinité antigénique varie chez les sujets VIH-, mais peut être améliorée chez certains donneurs en optimisant la présentation antigénique. Une cartographie d’épitopes pour Env gp41 à identifier des épitopes reconnus par les TH. Les résultats montrent la présence de TH spécifiques au VIH chez une proportion de donneurs séronégatifs. Ces cellules pourraient influencer le développement de réponses vaccinales et spécifique au VIH durant l’infection aiguë. / CD4+ T cell (Thelper, TH) responses play a key role in antiviral immunity. However, HIV-specific TH responses generated either by infection or by vaccine candidates are highly variable. Studies in mice and humans suggest that antiviral TH responses can be generated before exposure to the specific viral pathogen through cross-reactivity with other microorganisms These pre-existing responses may influence development of TH responses upon pathogen or immunogen exposure. We investigated HIV-specific TH responses in HIV-uninfected individuals (UD) and compared them to those of HIV-infected donors (HI). The prevalence of HIV-specific proliferative TH responses in UD was surprisingly high: 33% of UD had a robust Gag response >1% by CFSE assay. While Gag was more frequently targeted than the alternative HIV proteins Env, Nef and Pol, we did not observe the strong Gag immunodominance pattern seen in HI. Proliferative responses were overall lower in UD than HI, but strong expansion was occasionally observed. We derived Gag- and Env-specific short-term TH cell lines from UD and used intracellular staining to confirm their specificity and functions. TNF-α and CD40L dominated TH responses in UD lines, contrasting with HI lines that were robust IFN- producers. Functional affinity in UD was variable and could be improved in some subjects by optimization of antigen presentation. Gp41 epitope mapping identified peptides recognized by TH from UD. The results show that functional HIV-specific CD4 T cells exist in a substantial proportion of UD. Such pre-existing CD4 T cell could impact development of virus-specific TH responses at the time of acute HIV infection and influence responses to vaccine candidates.

La transmission du Virus de l'Immunodéficience Humaine (VIH) par voie sexuelle représente le mode majoritaire de contamination (80%) (UNAIDS). Ce mode de contamination implique le passage du virus à travers les muqueuses et une interaction avec les cellules épithéliales et les cellules immunitaires présentes au sein de ces muqueuses (cellules dendritiques, macrophages ou lymphocytes). Les muqueuses représentent le principal site d'exposition de l'organisme aux antigènes de l'environnement. Les SIgA (IgA sécrétoires) présentes dans la lumière de ces muqueuses représentent la première ligne de défense immunitaire contre l'infection et la colonisation des muqueuses. Les IgA sont capables d'interagir avec les glycoprotéines (gp) exprimées à la surface du VIH et de bloquer l'infection et/ou la transcytose à travers l'épithélium muqueux. Nous avons pu étudier la prévalence des SIgA anti-gp41 et plus précisément anti-MPER présentes dans la salive parotidienne de personnes Exposées au VIH Séronégatives (ESN) et leur rôle dans l'inhibition de l'infection par le virus in vitro. Nous avons pu démontrer que ces sujets présentaient un taux plus important de SIgA anti-MPER neutralisantes. Ce premier travail nous a permis de valider la gp41 comme immunogène d'intérêt pour la génération de SIgA neutralisantes. Nous avons pu générer des IgA1 dans un modèle murin α1Kl chimérique capable de produire des anticorps IgA1 humanisés. L'immunisation de ces souris a permis la production de 6 anticorps monoclonaux spécifiques de la région MPER capables de reconnaître des épitopes conformationnels élargis, correspondant aux épitopes reconnus par le 2F5 et le 4E10. Les IgA1 présentaient de fortes capacités neutralisantes pour différentes souches de laboratoire et de souches primaires du VIH. Les études de caractérisation des fonctions antivirales de ces anticorps permettront de mieux définir le mode d'action de ces anticorps. A notre connaissance, ces IgA1 neutralisantes anti-MPER sont les premières décrites à ce jour dans la littérature. De par leur faible immunogénicité et leur faible autoréactivité, ces anticorps peuvent facilement être intégrés dans des approches thérapeutiques locales ou par sérothérapie passive pour la protection après administration de SHIV dans des modèles animaux comme le macaque. L'ensemble de mes travaux de thèse ont confirmé l'intérêt thérapeutique potentiel des SIgA dans la lutte contre le VIH et notamment celles dirigées contre la partie gp41 de l'enveloppe