Peptide P5 (residues 628–683), comprising the entire membrane proximal region of HIV-1 gp41 and its calcium-binding site, is a potent inhibitor of HIV-1 infection

The membrane proximal region (MPR) of the transmembrane subunit, gp41, of the HIV envelope glycoprotein plays a critical role in HIV-1 infection of CD4+ target cells and CD4-independent mucosal entry. It contains continuous epitopes recognized by neutralizing IgG antibodies 2F5, 4E10 and Z13, and is therefore considered to be a promising target for vaccine design. Moreover, some MPR-derived peptides, such as T20 (enfuvirtide), are in clinical use as HIV-1 inhibitors. We have shown that an extended MPR peptide, P5, harbouring the lectin-like domain of gp41 and a calcium-binding site, is implicated in the interaction of HIV with its mucosal receptor. We now investigate the potential antiviral activities of P5 and other such long MPR-derived peptides. Structural studies of gp41 MPR-derived peptides using circular dichroism showed that the peptides P5 (a.a.628–683), P1 (a.a.648–683), P5L (a.a.613–683) and P7 (a.a.613–746) displayed a well-defined α-helical structure. Peptides P5 inhibited HIV-1 envelope mediated cell-cell fusion and infection of peripheral blood mononuclear cells by both X4- and R5-tropic HIV-1 strains, whereas peptides P5 mutated in the calcium binding site or P1 lacked antiviral activity, when P5L blocked cell fusion in contrast to P7. Strikingly, P5 inhibited CD4-dependent infection by T20-resistant R5-tropic HIV-1 variants. Cell-cell fusion studies indicated that the anti-HIV-1 activity of P5, unlike T20, could not be abrogated in the presence of the N-terminal leucine zipper domain (LZ). These results suggested that P5 could serve as a potent fusion inhibitor

Galactosyl ceramide expressed on dendritic cells can mediate HIV-1 transfer from monocyte derived dendritic cells to autologous T cells

AbstractMucosa, comprising epithelial and dendritic cells, are the major sites for Human Immunodeficiency Virus type 1 (HIV-1) transmission. There, DCs can capture incoming HIV-1 and in turn transfer virus to CD4+ T lymphocytes in a two-phase process, thereby initiating HIV-1 dissemination. We show that the glycosphingolipid Galactosyl Ceramide (GalCer), acting as mucosal epithelial receptor for HIV-1, was expressed by human monocyte derived immature DCs (iDCs), human primary DCs isolated from blood and mucosal tissue and in situ on mucosal tissue and acts as HIV-1-gp41 receptor. Blocking both GalCer and CD4 with specific mAbs results in a >95% transfer inhibition of HIV-1 from human monocyte-derived iDCs to autologous resting T cells. GalCer interaction with HIV-1 controls the early infection-independent phase of HIV-1 transfer to T cells. Thus, GalCer appears as an initial receptor for HIV-1, common to both mucosal epithelial cells and iDCs

Calcitonin gene-related peptide inhibits Langerhans cell-mediated HIV-1 transmission.

International audienceUpon its mucosal entry, human immunodeficiency virus type 1 (HIV-1) is internalized by Langerhans cells (LCs) in stratified epithelia and transferred locally to T cells. In such epithelia, LCs are in direct contact with peripheral neurons secreting calcitonin gene-related peptide (CGRP). Although CGRP has immunomodulatory effects on LC functions, its potential influence on the interactions between LCs and HIV-1 is unknown. We show that CGRP acts via its receptor expressed by LCs and interferes with multiple steps of LC-mediated HIV-1 transmission. CGRP increases langerin expression, decreases selected integrins, and activates NF-κB, resulting in decreased HIV-1 intracellular content, limited formation of LC-T cell conjugates, and elevated secretion of the CCR5-binding chemokine CCL3/MIP-1α. These mechanisms cooperate to efficiently inhibit HIV-1 transfer from LCs to T cells and T cell infection. In vivo, HIV-1 infection decreases CGRP plasma levels in both vaginally SHIV-challenged macaques and HIV-1-infected individuals. CGRP plasma levels return to baseline after highly active antiretroviral therapy. Our results reveal a novel path by which a peripheral neuropeptide acts at the molecular and cellular levels to limit mucosal HIV-1 transmission and suggest that CGRP receptor agonists might be used therapeutically against HIV-1

HIV-1 Efficient Entry in Inner Foreskin Is Mediated by Elevated CCL5/RANTES that Recruits T Cells and Fuels Conjugate Formation with Langerhans Cells

Male circumcision reduces acquisition of HIV-1 by 60%. Hence, the foreskin is an HIV-1 entry portal during sexual transmission. We recently reported that efficient HIV-1 transmission occurs following 1 h of polarized exposure of the inner, but not outer, foreskin to HIV-1-infected cells, but not to cell-free virus. At this early time point, Langerhans cells (LCs) and T-cells within the inner foreskin epidermis are the first cells targeted by the virus. To gain in-depth insight into the molecular mechanisms governing inner foreskin HIV-1 entry, foreskin explants were inoculated with HIV-1-infeceted cells for 4 h. The chemokine/cytokine milieu secreted by the foreskin tissue, and resulting modifications in density and spatial distribution of T-cells and LCs, were then investigated. Our studies show that in the inner foreskin, inoculation with HIV-1-infected cells induces increased CCL5/RANTES (1.63-fold) and decreased CCL20/MIP-3-alpha (0.62-fold) secretion. Elevated CCL5/RANTES mediates recruitment of T-cells from the dermis into the epidermis, which is blocked by a neutralizing CCL5/RANTES Ab. In parallel, HIV-1-infected cells mediate a bi-phasic modification in the spatial distribution of epidermal LCs: attraction to the apical surface at 1 h, followed by migration back towards the basement membrane later on at 4 h, in correlation with reduced CCL20/MIP-3-alpha at this time point. T-cell recruitment fuels the continuous formation of LC-T-cell conjugates, permitting the transfer of HIV-1 captured by LCs. Together, these results reveal that HIV-1 induces a dynamic process of immune cells relocation in the inner foreskin that is associated with specific chemokines secretion, which favors efficient HIV-1 entry at this site

Visualisation en temps réel de l’infection de la muqueuse génitale par le VIH

International audienc

Transmission vaginale du VIH-1 (développement d'un modèle expérimental humain pour l'étude des événements précoces)

Lors de rapports hétérosexuels, la muqueuse génitale féminine, plus vulnérable à l'infection que son homologue masculin, est la principale porte d'accès à l'entrée du virus. Le développement de stratégies visant à prévenir la transmission du VIH au niveau de ce site nécessite une meilleure compréhension des événements précoces de l infection. Au niveau de ce tissu, le VIH rencontres ses premières cellules cibles, s amplifie localement, se diffuse et établit une infection systémique. Malgré de nombreux efforts, les mécanismes détaillés de la transmission du VIH au niveau des voies génitales féminines restent largement inconnus. Avec toutes les restrictions évidentes de l'étude chez des sujets humains, la compréhension de ces mécanismes dépend du développement de modèles expérimentaux adéquats. Les modèles de culture ex vivo d explants de tissus servent de pont entre les études sur cultures cellulaire et celles in vivo. Nous avons développé un tel modèle d infection ex vivo d explants humain cervico-vaginaux. Récemment, il a été établi que 80% des cas de transmission sexuelles au VIH sont établies par un seul virion/génome viral. Afin de déterminer si les virus transmis ont des caractéristiques spécifiques - et en particulier l enveloppe - favorisant la transmission, nous avons infecté ces explants de tissus avec des virus isogéniques comportant l enveloppe des virus transmit ou celle des variants adaptées aux conditions de laboratoire; et comparé l'efficacité de réplication virale, ainsi que de l état d'activation des cellules cibles. Nous n'avons trouvé aucune différence entre des virus exprimant les enveloppes des souches de laboratoire et celles des virus transmisIn heterosexual transmission, the genital mucosa is the main gateway for viral entry, in particular the female lower genital tract, which is more susceptible to viral infection than its male counterpart. The development of strategies to prevent HIV transmission at this vulnerable site requires a better understanding of the early events leading to a viral infection. In this tissue, HIV-1 encounters its first target cells and, after an initial local amplification, eventually disseminates and establishes a systemic infection. In spite of many efforts, the detailed mechanisms of HIV-1 transmission in the female lower genital tract remain largely unknown. With all the obvious restriction on studying these mechanisms in humans, their understanding depends on the development of adequate experimental models. Ex vivo explants tissues model serve as a bridge between cell cultures and tissues in vivo. We have developed such an ex vivo model of infection of human cervico-vaginal explants. Recently, it was established that 80% of sexual HIV-1 transmissions are established by a single virion/viral genome. To investigate whether transmitted/founder (TF) viruses have specific trait - and specifically the envelope - favoring transmission, we infected the tissue explants with isogenic HIV-1 viruses encoding Env sequences from TF or laboratory-adapted HIV-1 variants and compared viral replication efficiencies, the viruses cellular targets and their activation status. We found no differences between viruses expressing envs from laboratory strains and a small panel of T/F virusesPARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF

Un passager clandestin: Un VIH infectieux caché dans les plaquettes alimente les macrophages tissulaires

International audienc

The Protective HIV-1 Envelope gp41 Antigen P1 Acts as a Mucosal Adjuvant Stimulating the Innate Immunity

International audienceMucosal nasal vaccine development, although ideal to protect from pathogens invading mucosally, is limited by the lack of specific adjuvant. We recently used P1, a conserved region of HIV-1 gp41-envelope glycoprotein, as efficient antigen in a prophylactic HIV-1 mucosal vaccine applied nasally. Herein, P1 immunomodulation properties were assessed on human nasal mucosal models by measuring induction of cytokine and chemokine production, intracellular signaling pathways, mucosal dendritic cell (DC) activation, and T cell proliferation. P1 adjuvant properties were evaluated by quantification of antigen-specific B cell responses against a model antigen in an in vitro immunization model. We now demonstrated that P1 has additional immunological properties. P1 initiates immune responses by inducing nasal epithelial cells to secrete the Th2-cytokine thymic stromal lymphopoietin (TSLP), a described mucosal adjuvant. Secreted TSLP activates, in turn, intracellular calcium flux and PAR-2-associated NFAT signaling pathway regulated by microRNA-4485. Thereafter, P1 induces mucosal dendritic cell maturation, secretion of TSLP in a TSLP-receptor (R)-dependent autocrine loop, but also IL-6, IL-10, IL-8, CCL20, CCL22, and MMP-9, and proliferation of CD4+ T cells. Finally, P1 acts as an adjuvant to stimulate antigen-specific B cell responses in vitro. Overall, P1 is a multi-functional domain with various immuno-modulatory properties. In addition to being a protective vaccine antigen for HIV prevention, P1 acts as adjuvant for other mucosal vaccines able to stimulate humoral and cellular antigen-specific responses