La formation du complexe de fusion qui précède la fusion est essentielle à l’infection de la cellule-cible par le VIH-1. L’inhibition de la formation de ce complexe de fusion peut empêcher l’infection et représenter un moyen de traitement antirétroviral. Plusieurs équipes ont essayé de produire des anticorps neutralisants par immunisation de souris ou de lapins avec des peptides linéaires non conformationnels, mais sans résultat. L’élaboration d’une lignée de cellules eucaryotes exprimant la structure HR1-PID-HR2 TM viral nous a permis de le faire. Cette lignée est un outil susceptible d’engendrer des anticorps monoclonaux (mAb). Trois mAbs contre le complexe de fusion de la région gp41 ont été produits chez la souris. L’activité neutralisante des anticorps produits a été évaluée sur des souches de laboratoires et des isolats primaires de VIH-1 de clades B et C. Ils induisent des réponses neutralisantes à large spectre. Les résultats obtenus montrent aussi que les trois anticorps monoclonaux pouvaient inhiber la formation de syncytia et bloquer l’interaction entre les structures HR1 et HR2 / Envelope glycoprotein is the primary target for human immunodeficiency virus (HIV) vaccine design. The N-terminal and the C-terminal regions of the gp41 interact with each other to form six helix bundle which is responsible for the fusion between the viral membrane and the target cell membrane. Monoclonal antibodies that disrupt the formation of the six helix bundle inhibit the HIV fusion. During the last decade, several human monoclonal antibodies of potent antiviral capacity for neutralizing primary isolates of different clades have been developed. The most broadly neutralizing monoclonal antibodies (mAbs) were screened from HIV-1 seropositive patients and recognized linear epitopes within the membrane proximal region of gp41. In this study, we developed a stable transfected eucaryot cell line expriming a well folded complex. Three mAbs against HIV-1 gp41 were prepared in mice. Our results show that the three mAbs were able to neutralize and inhibit the HIV-1 infection. Two of the mAbs bound to the recombinant folded gp140 and recognized HR1/HR2 regions while one of them recognized linear epitope within the HR2. Interestingly, the results showed that the three mAbs could inhibit the syncytium formation and block the interaction between the HR1 and the HR2 region

Made available in DSpace on 2015-07-22T20:50:17Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-10-28. Added 1 bitstream(s) on 2015-08-11T03:25:28Z : No. of bitstreams: 1 Publico-00284.pdf: 1210351 bytes, checksum: 9468c9b00f4f6d4853d0e556a03096c4 (MD5) / Introdução: a falha ao HAART torna importante o desenvolvimento de novos fármacos que alvejam diferentes passos do ciclo de vida do HIV-1. O Enfuvirtide (T20) é um peptídeo sintético que mimetiza a região HR2 da gp41 do HIV-1, impedindo sua fusão e entrada na célula hospedeira. A presença de mutações de resistência primária ao T20 pode levar a falta de resposta virológica sustentada (RVS) em indivíduos em falha terapêutica ao HAART. Objetivos: genotipar a gp41 do HIV-1 de indivíduos considerados respondedores e não respondedores ao T20; verificar se a presença de mutações de resistência primária poderia interferir na RVS; correlacionar o status RVS com aspectos virais, imunológicos e tropismo. Metodologia: DNA genômico do baseline (antes do tratamento), 6 e 12 meses após o tratamento com T20, foi purificado utilizando QIAamp DNA Mini kit (Qiagen®, Valencia, Califórnia, USA). Todos pacientes receberam terapia otimizada mais T20 no inicio. 506 pb referentes as regiões HR1 e HR2 da gp41 foram amplificados por PCR “nested”. A PCR “nested” da região V3 para estudo do tropismo viral amplificou 654 pb. A PCR foi purificada utilizando Montage® PCR Centrifugal Filter Devises (Millipore®). As PCRs purificadas das regiões V3 e gp41 foram sequenciadas no ABI Prism 3130 Genetic Analyser (Applied Biosystems, Ca, USA) com o kit comercial BigDye® Terminator Cycle Sequencing versão 3.1 (Applied Biossystems, Foster City. Califórnia, USA). Iniciadores da segunda etapa da PCR “nested” foram utilizados para sequenciar. Resultados: sete indivíduos apresentavam um perfil de resposta ao T20 por 12 meses, 4 por pelo menos 6 meses e dois não responderam entre 6 e 12 meses. A média de idade foi de 44,92 ± 5,39, sendo 46,16% do sexo feminino e 53,84% do masculino. Dos 13 pacientes analisados, 12 pertencem ao subtipo B e 1 ao F1. Oito pacientes apresentaram o correceptor R5 e cinco o X4. Não foram encontradas mutações nas posições 36 a 45 da HR1 em 12 meses. Tivemos a N42S, que é responsável pela diminuição da susceptibilidade ao T20. De um total de 59 aminoácidos analisados na HR1, observamos 18,64% de trocas e na região HR2, 38,88%. Na HR1 no período de 8 meses ou mais em relação ao baseline, não encontramos modificações nas posições 36 a 45. De um total de 59 aminoácidos analisados, observamos 15,25% de trocas e na HR2, 36,11%. Verificamos a E137K sem a presença da N43D, que causou resistência. Verificamos a S138T como mutação de primaria com persistência de 12 meses sem a N43D e a falta de resposta ao tratamento. Na contagem de T CD4+, não encontramos diferença estatística entre as médias dos diferentes grupos (ANOVA – p=0,1). As cargas virais após 6 meses foram indetectáveis (<50 copias/mL) em 69,23% dos pacientes. Houve queda de carga viral do baseline para 6 meses (p=0.034), assim como para 6 meses e 8 meses ou mais. O uso do medicamento teve interferência significativa quando comparados o baseline e 6 meses (p=0.004) e entre o baseline e 12 meses (p=0.022), mas entre 6 e 12 meses a interferência não foi significativa, mostrando que após 6 meses ela permanece sustentada. Entre as variáveis, indivíduos respondedores e genótipo R5 houve alto valor de similaridade (76.98). Ocorreu agrupamento entre as duas amostras dos respondedores com genótipo X4 com as mutações 306, 311 e 320, que o caracterizam. Discussão: em relação ao tropismo, 38,47% dos pacientes que estudamos apresentaram correceptor X4 após alguns anos de infecção e múltiplos esquemas HAART. Este resultado mostra concordância com a literatura, que descreve uma taxa média de 50%. A N42S, observada em nosso estudo é relacionada com diminuição da susceptibilidade da droga. Apesar disso, somente um dos pacientes não respondeu ao tratamento, não obtendo ganho imunológico. Conclusão: o tropismo dos vírus HIV-1 na maioria dos indivíduos que responderam ao T20 está fortemente associado com o R5, que não causa progressão rápida. A presença da S138T foi suficiente para a falta de resposta ao T20, sem associação com a N43D. Mostramos evidente RVS e recuperação imunológica com o T20, mas que não foi suficiente para retirar estes indivíduos do risco de doenças oportunistas. Nosso estudo evidenciou a importância de iniciar o resgate quando a contagem de linfócitos T CD4+ está acima de 200 células/mm3. / Introduction: the failure to HAART becomes important to development of new drugs that target different steps of the life cycle of HIV-1. The Enfuvirtide (T20) is a synthetic peptide that mimics the HR2 region of gp41 of HIV-1, preventing its fusion and entry into the host cell. The presence of primary resistance mutations to T20 can lead to lack of sustained virologic response (SVR) in people in the HAART failure. Objectives: to genotype the gp41 of HIV-1 subjects considered responders and nonresponders to T20; verify the presence of primary resistance mutations could influence the SVR, SVR status correlate with aspects of viral, immunological and tropism. Methodology: genomic DNA from baseline (before treatment), 6 and 12 months after treatment with T20, was purified using QIAamp DNA Mini kit (Qiagen ®, Valencia, California, USA). All patients received optimal therapy more T20 at the beginning. 506 bp referring to the HR1 and HR2 regions of gp41 were amplified by PCR nested. PCR nested the V3 region to study viral tropism amplified 654 bp. The PCR was purified using Montage ® PCR Centrifugal Filter Devises (Millipore ®). The PCRs of purified V3 and gp41 regions were sequenced in ABI Prism 3130 Genetic Analyzer (Applied Biosystems, CA, USA) with commercial kit BigDye ® Terminator Cycle Sequencing version 3.1 (Applied Biossystems, Foster City. California, USA). Initiators of the second stage of PCR nested were used for sequencing. Results: seven patients had a higher response to T20 for 12 months, 4 for at least 6 months and two did not respond between 6 and 12 months. The average age was 44.92 ± 5.39, and 46.16% female and 53.84% male. Of the 13 patients analyzed, 12 belong to subtype B and 1 to F1. Eight patients had the coreceptor R5 and five the X4. There were no mutations at positions 36 to 45 of HR1 in 12 months. We acquired the N42S, which is responsible for decreased susceptibility to T20. From a total of 59 amino acids analyzed in HR1, we observed 18.64% of change and in HR2 region, 38.88%. In HR1 within 8 months or more compared to the baseline, we found no changes in positions 36 to 45. From a total of 59 amino acids analyzed, we observed 15.25% of change and in HR2, 36.11%. We checked the E137K without the presence of N43D, which caused resistance. We checked the S138T mutation as the primary persistence of 12 months without the N43D and the lack of response to treatment. On the count of CD4 + T cells, we found no statistical difference between the means of different groups (ANOVA - p = 0.1). The viral loads after 6 months were undetectable (<50 copies / mL) in 69.23% of patients. There was a decrease in viral load baseline to 6 months (p = 0.034) and for 6 months and 8 months or more. The use of the drug had significant interference when compared to baseline and 6 months (p = 0.004) and between baseline and 12 months (p = 0.022), but between 6 and 12 months the interference was not significant, showing that after 6 months it remains sustainable. Among the variables, responders and R5 genotype, there were high value of similarity (76.98). There was a grouping between the two samples of responders with X4 genotype with mutations 306, 311 and 320 which characterize it. Discussion: regarding tropism, 38.47% of the patients studied had correceptor X4 after a few years of infection and multiple HAART regimens. This result shows agreement with the literature, which describes an average rate of 50%. The N42S, observed in our study is associated with decreased drug susceptibility. However, only one patient did not respond to treatment, without increase of his immune. Conclusion: the tropism of HIV-1 in the majority of people who responded to the T20 is strongly associated with the R5, which causes rapid progression. The presence of S138T was sufficient for the lack of response to T20, but no association with N43D. It was clearly shown SVR and immune recovery with the T20, but that was not enough to remove these people from the risk of contracting opportunistic diseases. Our study showed the importance of starting the rescue when the count of CD4 + T cells are above 200 cells/mm3. / TEDE / BV UNIFESP: Teses e dissertações

The mucosal antibody repertoire of the cervical mucosa in commercial sex workers from Nairobi, Kenya, who are highly sexually exposed to human immune deficiency virus type-1 (HIV-1) but remain persistently IgG seronegative (HEPS), may represent a novel source of broadly neutralizing monoclonal antibodies (mAbs) against HIV-1. Mucosal IgA specific for HIV-1 envelope (Env) subunit gp41 has been suggested as a correlate of protection in HEPS individuals. The in depth studies at both the gene and function level required to confirm their role in HIV-1 resistance are possible only using recombinant monoclonal IgAs. Human mAbs have traditionally been selected from libraries displayed on the surface of microorganisms (phage, yeast). However, due to inherent limitations, such techniques may not be optimal for isolating such rare mAbs from a pool of cervical B cells. We have developed an antibody selection system based on surface display on mammalian cells and used this technology to isolate four novel monoclonal antibodies, against linear epitopes on gp41, from the IgA repertoire of the cervical mucosa in Kenyan HEPS. Furthermore, three of the four mAbs were shown to bind with surface expressed consensus clade B and clade C Env on mammalian cells. Characterization of the variable region cDNA of the two strongest binding mAbs reveals extensive somatic mutations with a bias of replacement mutations clustering in the complementary determining regions (CDR) indicating antigen-driven affinity maturation had occurred. Affinity matured monoclonal IgAs, such as these, may play a role in the identification of new, vulnerable epitopes on HIV-1, or act as a component in a topical microbicide.

The mucosal antibody repertoire of the cervical mucosa in commercial sex workers from Nairobi, Kenya, who are highly sexually exposed to human immune deficiency virus type-1 (HIV-1) but remain persistently IgG seronegative (HEPS), may represent a novel source of broadly neutralizing monoclonal antibodies (mAbs) against HIV-1. Mucosal IgA specific for HIV-1 envelope (Env) subunit gp41 has been suggested as a correlate of protection in HEPS individuals. The in depth studies at both the gene and function level required to confirm their role in HIV-1 resistance are possible only using recombinant monoclonal IgAs. Human mAbs have traditionally been selected from libraries displayed on the surface of microorganisms (phage, yeast). However, due to inherent limitations, such techniques may not be optimal for isolating such rare mAbs from a pool of cervical B cells. We have developed an antibody selection system based on surface display on mammalian cells and used this technology to isolate four novel monoclonal antibodies, against linear epitopes on gp41, from the IgA repertoire of the cervical mucosa in Kenyan HEPS. Furthermore, three of the four mAbs were shown to bind with surface expressed consensus clade B and clade C Env on mammalian cells. Characterization of the variable region cDNA of the two strongest binding mAbs reveals extensive somatic mutations with a bias of replacement mutations clustering in the complementary determining regions (CDR) indicating antigen-driven affinity maturation had occurred. Affinity matured monoclonal IgAs, such as these, may play a role in the identification of new, vulnerable epitopes on HIV-1, or act as a component in a topical microbicide.

One potential approach to vaccine development against HIV involves generating an immunogen that can elicit the production of broadly neutralizing monoclonal antibodies (bnmAbs), which target specific sites on the HIV-1 envelope. Using site-directed mutagenesis and ELISA assays, this thesis investigates the idea of a secondary binding site of one of the bnmAbs, 2F5, as suggested by previous studies that identified residues Asp64, Thr65, and Arg82B on 2F5 that are recognized by its anti-idiotypic antibody 3H6. Results show that 2F5 binds only very weakly to the gp41 ectodomain in its post-fusion conformation. However, a small but significant difference was observed between the binding of the mutants and the T-20 peptide, a fusion inhibiting drug. Due to the limited effect, the results need to be confirmed using more quantitative techniques and more optimal conformations of the antigen, but raise the prospect that design of immunogens to elicit HIV-specific antibodies might have to incorporate this novel interaction site.

One potential approach to vaccine development against HIV involves generating an immunogen that can elicit the production of broadly neutralizing monoclonal antibodies (bnmAbs), which target specific sites on the HIV-1 envelope. Using site-directed mutagenesis and ELISA assays, this thesis investigates the idea of a secondary binding site of one of the bnmAbs, 2F5, as suggested by previous studies that identified residues Asp64, Thr65, and Arg82B on 2F5 that are recognized by its anti-idiotypic antibody 3H6. Results show that 2F5 binds only very weakly to the gp41 ectodomain in its post-fusion conformation. However, a small but significant difference was observed between the binding of the mutants and the T-20 peptide, a fusion inhibiting drug. Due to the limited effect, the results need to be confirmed using more quantitative techniques and more optimal conformations of the antigen, but raise the prospect that design of immunogens to elicit HIV-specific antibodies might have to incorporate this novel interaction site.

MEDEIROS, Melissa Soares. Polimorfismo do RRE e resistência aos antirretrovirais do vírus da imunodeficiência humana e efeito citopático e replicativo in vitro da enfuvirtida no códon 36 do vírus modificado pNL4-3. 2011. 255 f. Tese (Doutorado em Farmacologia) – Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, 2011. / Submitted by denise santos (denise.santos@ufc.br) on 2016-03-28T11:42:34Z No. of bitstreams: 1 2011_tese_msmedeiros.pdf: 18233541 bytes, checksum: 79da766383cb7344f8326013c6aa304c (MD5) / Approved for entry into archive by denise santos(denise.santos@ufc.br) on 2016-03-28T11:45:13Z (GMT) No. of bitstreams: 1 2011_tese_msmedeiros.pdf: 18233541 bytes, checksum: 79da766383cb7344f8326013c6aa304c (MD5) / Made available in DSpace on 2016-03-28T11:45:13Z (GMT). No. of bitstreams: 1 2011_tese_msmedeiros.pdf: 18233541 bytes, checksum: 79da766383cb7344f8326013c6aa304c (MD5) Previous issue date: 2011 / 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, naïve 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 (naïve 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. / Introdução: Rev Responsive Element (RRE) é uma molécula RNA responsável pelo transporte do mRNA viral do HIV-1 do núcleo para o citoplasma da célula CD4, através da via RRE-Rev, essencial para a replicação viral. As mutações de resistência a Enfuvirtida são primariamente localizadas no perímetro de 10 aminoácidos do HR1, região correspondente no RRE. Caracterizar o RRE poderá fornecer uma nova abordagem terapêutica para a terapia do HIV. Objetivos: Sequenciar e caracterizar o RRE da gp41 para avaliar sua variabilidade e correlação com parâmetros laboratoriais em sequências de pacientes infectados pelo HIV-1 que receberam terapia antirretroviral ou virgens. Em estudo in vitro avaliar a mutação 36D na presença de Enfuvirtida. Metodologia: 62 amostras de pacientes com HIV-1 do Ceará foram coletadas e 35 sequências de RRE foram obtidas e distribuídas em três grupos para fins de análise comparativa: N (virgens de terapia), T (uso de antirretroviral sem inibidor de fusão) e F (uso de antirretroviral associados a Enfuvirtida). Sequências obtidas foram alinhadas com o banco de dados de Los Alamos para HIV usando HIV BLAST Search. Estudo in vitro utilizou dois vírus de laboratório pNLA-3 (36D e 36G) observando citopatogenicidade e proliferação na presença de doses crescentes de Enfuvirtida. Resultados: A análise filogenética demonstrou alta prevalência do HIV-1 subtipo B (97,2%). Observou-se aumento da resposta imunológica no grupo T (71,5%) comparado ao F (2,98%). Mutações 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%). Três amostras demonstraram mutações de resistência significativas para os inibidores de fusão. Análise dos sítios primários de ligação do RRE observou presença de mutação 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 exposição à Enfuvirtida. Não houve diferença estatística significativa nas curvas de p24 do vírus 36D comparado com 36G, independente de concentrações de T20 (p>0.05). Observou-se menor formação de sincício, com diminuição da capacidade fusogênica, sem impacto na infectividade. Conclusão: O estudo definiu as mutações e polimorfismos mais prevalentes no Ceará, sugerindo alta preservação nas regiões de sítio primário de ligação do Rev-RRE. Evidenciou baixo perfil de resistência a Enfuvirtida em regimes com falha utilizando esta medicação. Detectou-se pressão seletiva no HR1 do HIV-1 de pacientes em uso de Antirretroviral, independente de exposição à Enfuvirtida. Evidenciado in vitro menor formação sincicial no vírus 36D, com diminuição na atividade fusogênica, mantendo infectividade.

Fusion inhibitors are a class of antiretroviral drugs used to prevent entry of HIV into host cells. Many of the fusion inhibitors being developed, including the drug enfuvirtide (ENF), are peptides designed to mimic, and thereby competitively inhibit, the viral fusion protein gp41. An exception to this is a class of cyclic, cationic, antimicrobial peptides known as θ-defensins, which are produced by many non-human primates and exhibit broad-spectrum antiviral and antibacterial activity. Currently, the θ-defensin analog RC-101 is being developed as a microbicide to prevent sexual transmission of HIV-1. Understanding potential RC-101 resistance, and how resistance to other fusion inhibitors affects RC-101 susceptibility, is critical for future development. Partial drug resistance due to genetic variability within HIV-1 presents a major hurdle in microbicide development. Drug-resistance mutations, whether naturally occurring or resulting from selection during treatment, often apply to many drugs in a particular class. Combining different drug classes into a single microbicide should provide greater protection against the growing variability observed in HIV. Our work has identified the beneficial effects of combining the fusion inhibitor RC-101 and the RT inhibitor CSIC to prevent transmission of clinically isolated and drug-resistant HIV-1. Several aspects of HIV-1 virulence and pathogenesis are mediated by the envelope protein gp41. Additionally, peptides derived from the gp41 ectodomain have been shown to induce chemotaxis in monocytes and neutrophils. While this chemotactic activity has been characterized, it is not known how these peptides could be produced under biological conditions. Our findings demonstrate that the epithelial serine protease matriptase efficiently cleaves the gp41 HR1 region at conserved residues into a chemotactic peptide. Here, we present evidence that advances our understanding of resistance to peptide entry inhibitors, reveals a potential benefit to combining specific drugs in an antiviral microbicide, and identifies a pathway by which HIV-1 may generate peptides to exploit host immunity. This work thereby facilitates improved methods in countering drug resistance and the development of new antiviral approaches to prevent HIV-1 transmission. Additionally, we have revealed basic mechanistic evidence that shed light on our current understanding of HIV-1 infection. Specifically, our focus on gp41 provides much needed insight into its role in membrane fusion, drug susceptibility, and modification of host responses.

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 / Sexual transmission of the Human Immunodeficiency Virus is the major mode of contamination (80%) for this pathogen (UNAIDS). This mode of transmission involves a passage of the virus though the mucosa and an interaction with epithelial cells and immune cells present in the mucosa (dendritic cells, macrophages and lymphocytes). Mucosa represents the major site of exposure for the organism to environmental antigens. The IgA expressed in the lumen of mucosa are the first line of immune defence against infection and colonization of mucosa. IgA are able to interact with glycoproteins (gp) expressed on the surface of HIV and prevent infection and/or block epithelial transcytosis. In this study we have investigated the prevalence of SIgA anti-MPER present in the parotid saliva of Exposed to HIV but Seronegative individuals (ESN). This study has allowed us to validate gp41 as an immunogen of interest for the generation of neutralizing IgA. IgA1 were generated in a chimeric mice model α1Kl that produced humanized IgA1 type antibodies. Immunizations of these mice has led to the elicitation of six monoclonal antibodies specific to the MPER region able to recognize extended conformational neutralizing epitopes of 2F5 and 4E10, two broadly neutralizing monoclonal antibodies specific to MPER. Elicited IgA1 have potent neutralizing properties for both laboratory and primary HIV strains. Characterization studies of the antiviral functions of these antibodies will further define the mode of action of these antibodies. To our knowledge, these anti-MPER humanized monoclonal neutralizing IgA1 antibodies are the first of this type described to date in the literature. By their low immunogenicity and autoreactivity, these antibodies can be easily integrated into local therapeutic approaches or passive serotherapy for protection in animal models such as the macaque challenged with SHIV. All the results of my PhD work confirm the great interest of gp41-specific SIgA as therapeutic agents against HIV

In den vergangenen Jahren sind Lipide, Membranen und deren Organisationsformen mehr und mehr in den Fokus der biologischen Forschung gerückt. Es wurde vorgeschlagen, dass in zellulären Membranen selbstassemblierende, submikroskopische Aggregate aus Sphingolipiden, Cholesterol und bestimmten Proteinen existieren und man vermutet, dass insbesondere Viren diese “Lipid Rafts” für ihren Zusammenbau nutzen und auf diese Art ihre Proliferationseffizienz erhöhen. Gleichwohl sind die genaue biologische Funktion und auch die molekulare Basis der Assoziation bestimmter Protein mit Lipid Rafts auch weiterhin unbekannt. In der vorliegenden Arbeit wurde Fluoreszenz-Lebenszeit-Mikroskopie genutzt, um die Lipid-Raft-Anreicherung des HIV-1 Glycoproteins gp41 zu untersuchen. Förster-Resonanz-Energietransfer zwischen fluoreszenzmarkierten viralen und Raft-Marker-Proteinen wurde gemessen, um deren gemeinsame, lokale Aufkonzentrierung in Lipid Rafts nachzuweisen. Durch Verwendung verschiedener Deletions- und Mutationsvarianten des Proteins konnte nicht nur seine Lipid-Raft-Präferenz demonstriert, sondern auch das Cholesterol-Bindemotiv (CRAC) als entscheidender Faktor der lateralen Sortierung identifiziert werden. Wir haben in diesem Kontext auch eine systematische Zell-zu-Zell-Variabilität in unseren Daten bemerkt, die einen zugrundeliegenden zellbiologischen Mechanismus der Membranorganisation nahelegt. Mithilfe von Fluoreszenz-Polarisations-Mikroskopie konnte zudem eine klare CRAC-Abhängigkeit der gp41-Oligomerisierung aufgezeigt werden. Die von uns gewonnenen Daten erlauben einen tieferen Einblick in die molekulare Basis und die biologischen Folgen der cholesterol-abhängigen lateralen Proteinorganisation für Virusassemblierungsprozesse an biologischen Membranen. / In recent years, there has been a considerable interest in the molecular organization of biological membranes. It has been hypothesized that self-assembling, freely diffusing, submicroscopic domains consisting of sphingolipids, cholesterol and certain proteins exist and the prevailing view is that those lipid rafts serve as platforms for specific molecular interactions by the preferential exclusion and inclusion of proteins. It was presumed, that in particular viruses make use of plasma membrane lipid rafts to augment the infection process and spread efficiently. However, the exact biological function and physical basis of protein partitioning into microdomains remains an outstanding question in virus biology. In the present study, fluorescence lifetime imaging microscopy was used to study lipid raft partitioning of the HIV-1 glycoprotein gp41 by detecting Foerster Resonance Energy Transfer between fluorescently labeled viral and raft marker proteins in living cells. Plasma membrane microdomain association of gp41 was demonstrated and by introducing systematic mutations and truncations in different gp41 motifs, the cholesterol recognition amino acid consensus (CRAC) was identified as the crucial determinant of the lateral sorting. Interestingly, we observed a systematic cell-to-cell variability in our raft related data that suggests underlying cell-biological mechanisms of membrane organization. Moreover, fluorescence polarization microscopy revealed a distinct CRAC requirement for gp41 oligomerization whereas other properties, such as intracellular distribution and expression efficiency were clearly demonstrated to be CRAC independent. Our data provide further insight into the molecular basis and biological implications of the cholesterol dependent lateral protein sorting for virus assembly processes at cellular plasma membranes.