DC-SIGN expression was monitored by flow cytometry analysis using FITC-labelled anti-DC-SIGN antibodies clones DCN46 (BD Biosciences, Missisaugua, Canada) and 5D7 (Santa Cruz Biotechnology, Santa Cruz, USA). The cells were also incubated with isotype-matched control antibodies. Flow cytometry was performed using a BD FACS-Scan (BD Biosciences). Full-term placentas were obtained following non-complicated pregnancies and deliveries at Hôpital St-Luc and Hôpital Bethesda in Cotonou, Benin. All infants were delivered vaginally except for two who were delivered by caesarean section and none of the mothers presented with signs of sexually transmitted infections or placental malaria infection. Placentas with signs of inflammation (chorioamnionitis) were excluded. These HIV-1-infected mothers received a combination of three antiviral drugs (full regimen) during pregnancy and delivery. Infants received a full regimen or a single dose nevirapine at delivery and none of them were HIVinfected. A small piece of each placenta was collected and processed within 3 hours following the delivery and washed extensively with PBS to remove blood and maternal cells. Mononuclear cells were mechanically isolated from placental tissue using a Medimachine (BD Biosciences) and purified on Histopaque gradient (Sigma-Aldrich, Oakville, Canada). Placental mononuclear cells were cryopreserved until flow cytometry analysis. Cells from 4 wild-type (WT) p-336T/p-201C and 11 homozygote or heterozygote p-336C/p-201A infants for the promoter variants born to HIV-1-negative mothers were analysed. Placental cells from 3 WT and 6 homozygote or heterozygote infants born to HIV-1-positive mothers were also analysed. Placental cells were analysed by flow cytometry using CD3-, CD19-(eBioscineces, San Diego, USA), and CD56-PerCPCy5.5 (BD Biosciences), CD14-Alexa700 (BioLegend, San Diego, USA), CD163-APC (R&D System, Minneapolis, USA), DC-SIGN-FITC, CD68-PE and HLA-DR-PECy7 (BD Biosciences) antibodies and isotype-matched controls. Dead cells stained with Live/ Death Aqua (Invitrogen) and lineage cells stained with CD3-, CD19-and CD56-PerCPCy5.5 antibodies were excluded. Placental macrophages were initially gated for CD14 expression and high granularity. Geometric mean fluorescence intensity (MFI) was calculated in DC-SIGN + CD163 + and DC-SIGN + CD163 2 subsets to assess the level of DC-SIGN expression and their level of maturation. Change geometric MFI represents the difference between specific marker expression and its FMO (fluorescence minus one). Flow cytometry was performed using a BD LSR-Fortessa (BD Biosciences).