Altered susceptibility to HIV-1 infection has been observed in multiple cohort studies around the world, with a small proportion of HIV-Exposed, Seronegative (HESN) individuals remaining uninfected despite repeated exposure. Previous work has shown association of three polymorphisms in interferon regulatory factor 1 (IRF1) with decreased susceptibility to HIV-1 infection and a reduced likelihood of seroconversion. Peripheral blood mononuclear cells (PBMCs) from patients with protective IRF1 haplotype exhibited significantly lower basal IRF1 expression, reduced responsiveness to IFNγ stimulation and significantly lower HIV-1 LTR transcription during the initial stages of infection. The goal of this thesis was to characterize the effect of three IRF1 polymorphisms associated with HIV-resistant phenotype on: (1) IRF1 gene transcription, alternative splicing of IRF1 mRNA and IRF1 mRNA/protein stability (2) plasma and cervical lavage (CVL) cytokine/chemokine expression and (3) HIV pathogenesis and disease progression. Furthermore since differences in hormone expression are known to regulate IRF1 function we set out to determine if differences in plasma hormone levels contribute to the natural resistance against HIV-1 infection in the Majengo HESN cohort. Polymorphisms in the IRF1 gene do not directly affect IRF1 transcription but instead act as intronic splicing regulators. PBMCs from individuals with protective IRF1 haplotype were associated with increased inclusion of exons 7/8 and decreased protein stability when compared to cells from individuals with nonprotective haplotype. Individuals with protective IRF1 haplotype also exhibited significantly higher plasma IL15, IFNγ and IL6 expression and significantly higher CVL IL15, IFNγ, IL2 and sIL2Rα expression (with univariate analysis only) compared to those with nonprotective IRF1 haplotype. Additionally, individuals with protective IRF1 haplotype expressed significantly lower levels of plasma prolactin when compared to individuals with nonprotective haplotype. IRF1 polymorphisms were found to not be associated with HIV disease progression, suggesting that the protective effect of IRF1 polymorphisms is limited to the early stages, prior to establishment of HIV-1 infection. Furthermore, independent of IRF1 genotypes, significantly lower plasma prolactin, estrogen, progesterone and cortisol levels were observed in HESN women suggesting that hormonal regulation may be one of the main factors regulating natural resistance to HIV-1 infection in the Majengo HESN cohort.

Heterosexual intercourse now accounts for the majority of HIV transmission within sub-Saharan Africa. The generation of microbicides and vaccines, therefore, requires a better understanding of the mucosal correlates of protection, including the role of HIV-specific IgA. It is now accepted that not all individuals are equally susceptible to HIV-1 infection, as exemplified by the HIV Exposed Seronegative (HESN) women of the Pumwani Cohort in Nairobi, Kenya. To assess whether mucosal IgA responses contribute to this protection, 3 novel IgA variable genes were cloned from HESN cervical B-cell cDNA. Nine monoclonal IgA Abs were produced, two of which were properly produced from cell culture. The HESN-derived A6/30L and A9/30L variants had a greater specificity for gp120IIIB than their A6/4L and A9/4L counterparts, while the A6 variant recognizes a distinct gp120 epitope compared to the broadly neutralizing antibody IgGb12. Further characterization of these IgA chains may suggest their suitability for use in microbicides or mucosal vaccines.

Heterosexual intercourse now accounts for the majority of HIV transmission within sub-Saharan Africa. The generation of microbicides and vaccines, therefore, requires a better understanding of the mucosal correlates of protection, including the role of HIV-specific IgA. It is now accepted that not all individuals are equally susceptible to HIV-1 infection, as exemplified by the HIV Exposed Seronegative (HESN) women of the Pumwani Cohort in Nairobi, Kenya. To assess whether mucosal IgA responses contribute to this protection, 3 novel IgA variable genes were cloned from HESN cervical B-cell cDNA. Nine monoclonal IgA Abs were produced, two of which were properly produced from cell culture. The HESN-derived A6/30L and A9/30L variants had a greater specificity for gp120IIIB than their A6/4L and A9/4L counterparts, while the A6 variant recognizes a distinct gp120 epitope compared to the broadly neutralizing antibody IgGb12. Further characterization of these IgA chains may suggest their suitability for use in microbicides or mucosal vaccines.

The innate immune system is at the interface between the host's immune system and the initial contact with HIV. Understanding the correlates of innate immune protection against Human Immunodeficiency Virus is an important goal for development of effective anti-HIV therapies or vaccines. Not all exposures to HIV end in infection. The innate immune system has been linked to the reduced susceptibility of HIV-exposed seronegative (HESN) female commercial sex workers in Kenya by a number of studies. This thesis is a comparison of Toll-like receptor (TLR) responses in different immune cells in peripheral blood mononuclear cells (PBMCs) from HESN and HIV negative (susceptible) female commercial sex workers (CSWs). This study tested the hypothesis that higher TLR8 responsiveness in PBMCs of HESN to ssRNA analogous to HIV's genetic material, would result in higher effector responses capable of making HIV target cells more refractory in vitro, compared to susceptible controls. The results showed that PBMCs of HESN were often hypo-responsive to TLR4 and TLR7 stimulations evidenced by often reduced cytokine responses to the corresponding ligands, but hyper-responsive to TLR8 following stimulation with ssRNA analogous to HIV's genetic material. The 'dichotomy' in TLR responsiveness of HESN PBMCs was associated with differential expression of cognate TLRs in PBMCs, and altered activation of TLR signalling pathways. The opposing pattern of TLR7 and TLR8 responsiveness corresponded to the ability of HIV to infect target cells in vitro; where pre-treatment of PBMCs with TLR7 enhanced HIV replication whereas TLR8 stimulation inhibited HIV replication. The differences in outcomes of the HIV infection assays were associated with distinct cytokine profiles, where TLR7 stimulation induced robust type I IFNs responses without proinflammatory TNF-α and IL-12 cytokine responses,while TLR8 stimulations produced type II IFN responses accompanied by robust proinflammatory responses in both groups. The cytokine milieu of HESN PBMCs prior to and following TLR4 and TLR8 stimulations was more tightly regulated, but was associated with higher activation of CD8+, NK cells, monocytes but not blood DCs. These results demonstrate that the lower activation or 'quiescent' state of HESN PBMCs did not limit the ability of their cells to recognize ssRNA analogous to HIV derived genetic material and mount potent responses capable of limiting HIV infection in vitro, supporting the overall hypothesis tested. This thesis contributes to the growing knowledge on the dichotomous outcomes between TLR7 and TLR8 treatments with respect to HIV infection that could be instrumental in the design of novel HIV inventions such as vaccines or microbicides. / May 2016

La majorité des infections par le VIH sont acquises hétérosexuellement surtout chez les femmes en Afrique subsaharienne. Le tractus génital féminin (TGF) est la principale porte d’entrée pour le VIH et joue un rôle important dans la défense de l’organisme. De concert avec les cellules épithéliales, les cellules dendritiques (DC) aident à maintenir une balance immunitaire entre tolérance et inflammation. Dans un groupe de travailleuses du sexe (CSW) à Cotonou, au Bénin, des femmes (CSW ≥ 8 ans) ont été identifiées comme hautement exposées séronégatives (HESN). La fréquence de populations cellulaires myéloïdes de type Monocytes-Derived Dendritic Cells (MoDC) présentant un potentiel antiviral et « tolérogénique/régulateur » est augmentée au niveau du TGF des HESNs et les monocytes pourraient être impliqués dans leur génération. Les résultats de RNA-seq sur les monocytes totaux permettent de constater une augmentation de gènes associés à des fonctions effectrices, de protection/contrôle de l’infection et de régulation chez les HESNs comparé aux contrôles (2,5-5 années CSWs HIV- « early HESN », CSWs HIV+ et des femmes de la population générale Non CSWs HIV-). Les résultats de cytométrie en flux (FACS) démontrent une proportion élevée de non-classiques comparé aux autres sous-populations de monocytes sanguins, exprimant davantage de molécules effectrices et régulatrices, suggérant un lien avec les MoDCs tolérogéniques observées. Cinq individus ont séroconverti et ont présenté des modifications bien avant la séroconversion, soit une diminution de β-chimiokines et des IgG anti-gp41 dans le compartiment sanguin et mucosal du TGF. Un bris du profil « tolérogénique/régulateur » pourrait donc favoriser la séroconversion. / Most HIV infection are acquired through heterosexual intercourse, mostly in women in subsaharian Africa. The female genital tract (FGT) is the principal portal of entry for HIV and plays a critical role in host defense. Together, epithelial cells and dendritic cells (DC) help maintain immunological balance between inflammation and tolerance. In a group of commercial sex worker (CSW) from Cotonou, in Benin, women (CSW 8 ≥ years) have been identified as HIV-1 highly exposed seronegative (HESN). The frequency of myeloid cell populations alike to Monocytes-Derived Dendritic Cells (MoDC) presenting an antiviral potential and a tolerogenic/regulating profile were increased in FGT of HESNs and their monocytes could be implied in their generation. The RNA-seq results on total blood monocytes show an increase expression of genes associated with effector, protection/control of HIV infection and regulation functions in HESNs compared with control groups (2,5-5 years CSWs HIV- « early HESN », CSWs HIV+ and women from general population Non CSWs HIV-). Our flow cytometry (FACS) results show an elevated frequency of non-classical compared with other sub-populations in blood monocytes, expressing more effector and regulator molecules, suggesting a link with observed tolerogenic MoDCs. Moreover, five individuals have seroconverted and presented modifications before seroconversion such as lower levels of β-chemokines and anti-gp41 IgG in blood and mucosal compartments in the FGT. A break of this “tolerogenic/regulating” profile could favor seroconversion.

No description available.

La plupart des infections par le VIH-1 sont acquises lors de rapports hétérosexuels. En Afrique subsaharienne on observe 71 % des infections mondiales et 60 % des nouvelles infections par le VIH-1 touchent les femmes. Le tractus génital féminin (TGF) constitue la principale porte d’entrée pour le VIH-1 et joue un rôle important dans la défense de l’organisme contre les microorganismes pathogènes tout en maintenant une tolérance de la flore commensale. On y trouve les cellules épithéliales qui participent à l’élaboration des réponses immunes en collaboration avec les cellules dendritiques (DCs), mais également d’autres types de cellules immunitaires qui confèrent une protection à la muqueuse vaginale, notamment à travers la production de cytokines et de chimiokines. Nous avons établi une cohorte de travailleuses du sexe (CSWs) au Bénin et nous avons identifié des femmes hautement exposées et séronégatives au VIH-1 (HESN), qui demeurent séronégatives après plus de sept années actives dans le travail du sexe. Les personnes HESN étant un excellent modèle d’immunité naturelle contre le VIH-1, le but de notre projet consiste donc à étudier les cellules immunitaires impliquées dans la protection de l’hôte face au VIH-1, au niveau du tractus génital féminin. Nous émettons l’hypothèse que le maintien de faibles conditions inflammatoires dans le TGF des femmes HESN préviendrait une activation immunitaire excessive en préservant l’intégrité de la barrière de la muqueuse vaginale et contribuerait ainsi à maintenir une protection contre l’infection par le VIH-1. Des études antérieures sur les HESN béninoises et kenyanes ont démontré que ces femmes présentent de faibles niveaux d’inflammation dans leur TGF inférieur. En accord avec cela, nous avons observé de faibles niveaux de BLyS/BAFF dans la muqueuse vaginale des HESN comparativement aux travailleuses du sexe séropositives (CSWs+ HIV+). BLyS/BAFF est une molécule importante pour la différenciation des cellules B et pour la sélection de cellules B de première ligne de la zone marginale (MZ). De ce fait, nous rapportons pour la première fois la présence de cellules B CD1c+ de type MZ qui sont capables de se lier naturellement à la gp120 glycosylée, au niveau de la muqueuse vaginale. Or, des cellules B CD1c+ exprimant IgG sont augmentées chez les CSWs+ HIV+ comparativement aux HESN, ce qui pourrait contribuer à l’hyperglobulinémie observée dans le TGF inférieur des CSWs+ HIV+. Les faibles niveaux de BLyS/BAFF retrouvés dans la muqueuse vaginale des HESN semblent donc préserver une homéostasie au sein du compartiment B et des cellules B CD1c+ du TGF. De plus, nous y détectons une réactivité des IgG1 avec la gp-41 de l’enveloppe virale, qui pourrait contribuer à leur immunité naturelle. Avec les cellules épithéliales, les DCs sont l’une des premières à être en contact avec le virus dans le TGF. Elles jouent un rôle essentiel dans l’orchestration des réponses immunitaires. Nous pensons que les DCs contribuent au maintien de faibles conditions inflammatoires dans le TGF des HESN, prévenant ainsi l’activation immunitaire excessive et préservant l’intégrité de la barrière muqueuse de façon à maintenir une protection/contrôle contre le virus. Nous avons caractérisé une population myéloïde endocervicale « tolérogénique » HLA-DR+CD14+CD11c+ exprimant HLA-G, ILT4, CD103 et de forts taux d’IFN-α et d’IL-10 dont la fréquence relative était augmentée au niveau du col de l’utérus des HESN comparativement aux CSWs+ HIV+. De plus, des populations Tregs/Tr1 étaient aussi augmentées chez les HESN. Ces données reflètent à la fois une réponse antivirale et une contribution au contrôle des conditions inflammatoires dans le TGF des HESN. Afin de mieux comprendre la nature des cellules myéloïdes tolérogéniques, nous avons voulu dériver des monocytes en cellules dendritiques (MoDCs). Toutefois, nous avons remarqué que la différenciation des MoDCs des HESN était altérée. Suite à cela, nous avons caractérisé le profil transcriptomique des monocytes. Les résultats préliminaires mettent en lumière l’éventuel rôle des récepteurs nucléaires NR4A dans la modulation des MoDCs et, possiblement, sur le plan des cellules myéloïdes tolérogéniques chez les HESN. Dans l’ensemble, ces résultats nous ont permis d’acquérir de nouvelles connaissances sur les mécanismes mis en place chez les HESN dans l’immunité naturelle contre le VIH-1. / Most HIV-1 infections are acquired through heterosexual intercourse. In sub-Saharan Africa, 71% of global infections are observed and 60% of new HIV-1 infections affect women. The female genital tract (FGT) constitutes a main portal of entry for HIV-1 and plays an important role in protecting the host against pathogens while maintaining a tolerance to a commensal flora. FGT immunity involves genital epithelial cells as well as dendritic cells (DCs) and many other types of immune cells which confer protection, through the production of chemokines and cytokines. We established a cohort of commercial sex workers (CSWs) in Benin and identified HIV-1 highly exposed seronegative (HESN) individuals, who remain uninfected after more than seven years of active prostitution. These HESN individuals being an exceptional model of natural immunity against HIV-1, the aim of our project is to characterize immune cells involved in protection from HIV-1 infection, in the female genital tract. We hypothesize that maintenance of low inflammatory conditions in the FGT of HESN women helps to prevent excessive immune activation likely preserving the mucosal barrier integrity and would help to maintain a protection against HIV infection. Previous studies of Beninese and Kenyan HESN have shown that these women have a low inflammatory profile in their lower FGT. Accordingly, we found that vaginal mucosa of HESN had lower soluble BLyS/BAFF levels when compared to HIV-infected CSWs (CSWs+ HIV+). BLyS/BAFF is highly recognized for its role in B-cell ontogenesis, as well as cell fate decision towards the innate marginal zone (MZ) B-cell pool. For the first time, we report the presence of genital MZ-like CD1c+ B-cells that naturally bind to fully glycosylated gp120 in the vaginal mucosa. However, CD1c+ B-cells expressing IgG are increased in the lower FGT of CSWs+ HIV+ when compared to HESN, suggesting that these cells could contribute to the hyperglobulinemia observed in the lower FGT of CSWs+ HIV+. The low levels of BLyS/BAFF found in the vaginal mucosa of HESN thus appear to preserve homeostasis of the FGT B cell compartment and CD1c+ B-cells. In addition, we detect a reactivity of IgG1 to HIV-gp41 in cervico-vaginal lavages (CVL) supernatants of HESN, which could contribute to their natural immunity. Epithelial cells and DCs are one of the earliest cell types to sense the virus in the FGT. They play a key role in the orchestration of immune responses. We characterized a "tolerogenic" endocervical myeloid HLA-DR+CD14+CD11c+ population expressing HLA-G, ILT4, CD103 and high levels of IFN-α and IL-10, that was increased in the cervix of HESN when compared to CSWs+ HIV+. In addition, frequencies of Tregs/Tr1 cells were also increased in HESN. We believe that DCs contribute to maintaining low inflammatory conditions in the FGT of HESN, preventing excessive immune activation and preserving the integrity of the mucosal barrier to maintain a protection/control against the virus. These data reflect both an antiviral response and a contribution to the control of inflammatory conditions in the FGT of HESN. To better understand the nature of tolerogenic myeloid cells, we wanted to derive monocyte-derived dendritic cells (MoDCs). However, derivation of blood MoDCs was impaired in HESN. As a result, we decided to characterize the transcriptomic profile of total blood monocytes. Preliminary results appear to demonstrate the possible role of NR4A nuclear receptors in MoDCs modulation, and possibly in tolerogenic myeloid cells in HESN. Overall, our results contribute to a better understanding of the mechanisms established by HESN in natural immunity to HIV-1.