Proteasome-independent degradation of HIV-1 in naturally non-permissive human placental trophoblast cells

<p>Abstract</p> <p>Background</p> <p>The human placenta-derived cell line BeWo has been demonstrated to be restrictive to cell-free HIV-1 infection. BeWo cells are however permissive to infection by VSV-G pseudotyped HIV-1, which enters cells by a receptor-independent mechanism, and to infection by HIV-1 via a cell-to-cell route.</p> <p>Results</p> <p>Here we analysed viral entry in wild type BeWo (CCR5⁺, CXCR4⁺) and BeWo-CD4⁺(CD4⁺, CCR5⁺, CXCR4⁺) cells. We report that HIV-1 internalisation is not restricted in either cell line. Levels of internalised p24 antigen between VSV-G HIV-1 pseudotypes and R5 or X4 virions were comparable. We next analysed the fate of internalised virions; X4 and R5 HIV-1 virions were less stable over time in BeWo cells than VSV-G HIV-1 pseudotypes. We then investigated the role of the proteasome in restricting cell-free HIV-1 infection in BeWo cells using proteasome inhibitors. We observed an increase in the levels of VSV-G pseudotyped HIV-1 infection in proteasome-inhibitor treated cells, but the infection by R5-Env or X4-Env pseudotyped virions remains restricted.</p> <p>Conclusion</p> <p>Collectively these results suggest that cell-free HIV-1 infection encounters a surface block leading to a non-productive entry route, which either actively targets incoming virions for non-proteasomal degradation, and impedes their release into the cytoplasm, or causes the inactivation of mechanisms essential for viral replication.</p

Distinct efficacy of HIV-1 entry inhibitors to prevent cell-to-cell transfer of R5 and X4 viruses across a human placental trophoblast barrier in a reconstitution model in vitro

<p>Abstract</p> <p>Background and methods</p> <p>HIV-1 cell-to-cell transmission is more efficient than infection of permissive cells with cell-free particles. The potency of HIV-1 entry inhibitors to inhibit such transmission is not well known. Herein, we evaluated the efficacy of this new class of antiretrovirals to block cell-to-cell transmission of HIV-1 in a model of reconstitution of the human placental trophoblast barrier <it>in vitro</it>.</p> <p>Results</p> <p>Our data show that CCR5 antagonists and T20 inhibit the passage of the virus across the BeWo cell monolayer in contact with PBMCs infected with an R5 (Ba-L) and a dualtropic (A204) HIV-1 with IC50s in the range of 100 – 5,000 nM for TAK779; 90 to 15,000 nM for SCH-350581 and 3,000 to 20,000 nM for T20. The CXCR4 antagonist AMD3100 is also effective against X4 HIV-1 infected PBMCs in our model with IC50 comprised between 4 nM and 640 nM. HIV-1 entry inhibitors are less efficient to block cell-to-cell virus transmission than cell-free HIV-1 infection of PBMCs and CCR5 antagonists do not prevent PBMC infection by dual tropic HIV-1 in contrast to cell-to-cell infection in our model.</p> <p>Surprisingly, T20 (and C34) do not block cell-to-cell transmission of X4 HIV-1 but, rather, increase 80 to 140 fold, compared to control without drug, the passage of the virus across the trophoblast barrier. Additional experiments suggest that the effect of T20 on BeWo/PBMC-X4 HIV-1 is due to an increase of effector-target cells fusion.</p> <p>Conclusion</p> <p>Our results support further evaluation of HIV-1 coreceptor antagonists, alone or combined to other antiretrovirals, in a perspective of prevention but warn on the use of T20 in patients bearing X4 HIV-1 at risk of transmission.</p

Seminal Plasma Exposures Strengthen Vaccine Responses in the Female Reproductive Tract Mucosae

HIV-1 sexual transmission occurs mainly via mucosal semen exposures. In the female reproductive tract (FRT), seminal plasma (SP) induces physiological modifications, including inflammation. An effective HIV-1 vaccine should elicit mucosal immunity, however, modifications of vaccine responses by the local environment remain to be characterized. Using a modified vaccinia virus Ankara (MVA) as a vaccine model, we characterized the impact of HIV-1+ SP intravaginal exposure on the local immune responses of non-human primates. Multiple HIV-1+ SP exposures did not impact the anti-MVA antibody responses. However, SP exposures revealed an anti-MVA responses mediated by CD4+ T cells, which was not observed in the control group. Furthermore, the frequency and the quality of specific anti-MVA CD8+ T cell responses increased in the FRT exposed to SP. Multi-parameter approaches clearly identified the cervix as the most impacted compartment in the FRT. SP exposures induced a local cell recruitment of antigen presenting cells, especially CD11c+ cells, and CD8+ T cell recruitment in the FRT draining lymph nodes. CD11c+ cell recruitment was associated with upregulation of inflammation-related gene expression after SP exposures in the cervix. We thus highlight the fact that physiological conditions, such as SP exposures, should be taken into consideration to test and to improve vaccine efficacy against HIV-1 and other sexually transmitted infections

Decidual soluble factors participate in the control of HIV-1 infection at the maternofetal interface

International audienceBackgroundMaternofetal transmission (MFT) of HIV-1 is relatively rare during the first trimester of pregnancy despite the permissivity of placental cells for cell-to-cell HIV-1 infection. Invasive placental cells interact directly with decidual cells of the uterine mucosa during the first months of pregnancy, but the role of the decidua in the control of HIV-1 transmission is unknown.ResultsWe found that decidual mononuclear cells naturally produce low levels of IL-10, IL-12, IL-15, TNF-α, IFN-α, IFN-γ and CXCL-12 (SDF-1), and large amounts of CCL-2 (MCP1), CCL-3 (MIP-1α), CCL-4 (MIP-1β), CCL-5 (Rantes), CXCL-10 (IP-10), IL-6 and IL-8. CCL-3 and CCL-4 levels were significantly upregulated by in vitro infection with R5 HIV-1 but not X4. Decidual CD14+ antigen presenting cells were the main CCL-3 and CCL-4 producers among decidual leukocytes. R5 and X4 HIV-1 infection was inhibited by decidual cell culture supernatants in vitro. Using HIV-1 pseudotypes, we found that inhibition of the HIV-1 entry step was inhibited by decidual soluble factors.ConclusionOur findings show that decidual innate immunity (soluble factors) is involved in the control of HIV-1 infection at the maternofetal interface. The decidua could thus serve as a mucosal model for identifying correlates of protection against HIV-1 infection

Dynamics of Vaginal and Rectal Microbiota Over Several Menstrual Cycles in Female Cynomolgus Macaques

International audienceThe composition of the microbiota in cynomolgus macaques is only partially characterized, although this animal model is often used to study pathogenesis and preventive strategies against infections. We thus performed, for the first time, a longitudinal characterization of the vaginal and rectal microbiota of five cycling female cynomolgus macaques. Samples were collected weekly for 15 weeks and the V3/V4 regions of the16S rRNA gene sequenced. Sequences were analyzed with QIIME for OTU detection and taxonomic assignment. Progesterone levels were also determined to evaluate hormonal influence on bacteria relative abundance. The rectal and vaginal bacterial composition in cynomolgus macaques is polymicrobial and clearly distinct, with larger individual variability in the vagina. Rectal microbiota profiles were consistent between animals, whereas they were highly variable and animal-specific in the vagina. In the rectum, the most abundant taxa were Ruminococcaceae, Prevotella, and Clostridiales. In the vagina, the most abundant genera were Sneathia, Porphyromonas, Prevotella, and Fusobacterium. Lactobacillus were found at relative abundances higher than 1% in only one animal and were not predominant. Comparison of the vaginal cynomolgus macaque microbiota with that of humans showed similarity to community state type IV-A usually associated with dysbiosis. In the vagina, the relative abundance of 12 bacterial genera was found to be associated with progesterone levels. Our study provides a detailed characterization of the rectal and vaginal microbiota in female cynomolgus macaques and opens new perspectives of this animal model

Activation of Toll-Like Receptors Differentially Modulates Inflammation in the Human Reproductive Tract: Preliminary Findings

International audienceThe female reproductive tract (FRT) is the main site of entry of sexually transmitted infections (STIs). Toll-like receptors (TLRs) that recognize pathogenic motifs are widely expressed in the FRT. TLR stimulation induces immune activation and local production of inflammatory mediators. In the FRT, this response should also be compatible with reproductive functions and symbiosis with host microbiota. With a view to develop efficient mucosal vaccines to prevent STI acquisition, the role of TLR ligands in the FRT needs to be explored. We have therefore investigated the cytokine profiles of the different compartments of the FRT (vagina, endocervix, ectocervix, and uterus) before and after stimulation of mononuclear cells from human tissue specimens. The comparison with PBMCs allowed us to highlight the FRT specificities. We first characterized the main immune cell populations in each compartment and observed that their distribution was different through the compartments. The CD45+ cells represented a maximum of 11% in the FRT in contrast to 96% in PBMCs. We identified two main populations among the CD45+ cells in the four compartments of the FRT: CD3+ T cells (CD4+ and CD8+) and CD14+ APCs. B cell populations (CD19+) were much less frequent than T cells in all the FRT regions and were equally distributed. NK CD56+ cells were detected in all compartments and were more abundant in the uterus. Stimulation of the mononuclear cells was then performed with TLR agonists: R848 for TLR7/8, Poly I:C for TLR3, LPS for TLR4 and ODN CpG for TLR9. Cytokine levels in unstimulated cultures of cells isolated from all FRT compartments were higher than in cultures of unstimulated PBMCs. In contrast, after stimulation with TLR agonists, cytokine responses induced by TLR agonists were moderate in the FRT and significantly lower than in PBMCs. These responses were varied with different TLR ligands and FRT compartments. The cytokine profile induced by TLR activation in the FRT supports the role of these tissues in genital anti-microbial immunity and in the control of inflammation while allowing maintenance of its reproductive function