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To investigate whether exosomes derived from human breast milk or plasma confer protection against HIV-1 infection of monocyte-derived dendritic cells (MDDCs) and subsequent viral transfer to CD4+ T cells.MDDCs were generated and milk and plasma-derived exosomes were isolated from healthy donors. To determine the capacity of exosomes to inhibit HIV-1 infection, MDDCs were preincubated with exosomes before exposure to HIV-1BaL. To investigate transfer of HIV-1 from MDDCs to CD4+ T cells, MDDCs preincubated with exosomes and HIV-1BaL were cocultured with allogeneic CD4+ T cells. To explore receptors used by MDDCs for binding of exosomes, blocking experiments were performed.Productive HIV-1 infection was analysed in MDDCs and CD4+ T cells by determining p24 expression by flow cytometry. Confocal microscopy and flow cytometry was used to investigate uptake of fluorescently labelled exosomes by MDDCs.Milk exosomes, but not plasma exosomes, bind MDDCs via DC-SIGN inhibiting HIV-1 infection of MDDCs and subsequent viral transfer to CD4+ T cells.We propose that milk exosomes act as a novel protective factor against vertical transmission of HIV-1 by competing with HIV-1 for binding to DC-SIGN on MDDCs.