Abstract Findings that certain infections induce immunity not only against the causing agent, but also against an unrelated pathogen have intrigued investigators for many years. During recent years, underlying mechanisms of this phenomenon have started to come to light. It was found that the key cells responsible for heterologous protection are innate immune cells such as natural killer cells (NKs), dendritic cells, and monocytes/macrophages. These cells are ‘primed’ by initial infection, allowing them to provide enhanced response to subsequent infection by the same or unrelated agent. This phenomenon of innate immune memory was termed trained immunity. The proposed mechanism for trained immunity involves activation by the first stimulus of metabolic pathways that lead to epigenetic changes, which maintain the cell in a "trained" state allowing enhanced responses to a subsequent stimulus. Innate immune memory can lead either to enhanced responses or to suppression of subsequent responses (‘tolerance’), depending on the strength and length of the initial stimulation of the immune cells. In the context of HIV infection, it remains unknown whether innate memory is induced by infection, although limited evidence suggests a lasting activation of NK cells following HIV exposure. In this application, we present the first evidence that extracellular vesicles carrying the HIV-1 protein Nef (exNef) induce trained immunity in human monocytes, characterized by increased response to stimulation. The mechanism of this training appears to depend on exNef-mediated effect on cholesterol homeostasis. Given that exNef are released into the blood even after HIV replication had been suppressed by ART, trained monocytes/macrophages can be maintained for a long time after virus suppression. We propose to investigate the mechanism of exNef-induced innate immune memory training and its effect on susceptibility of macrophages to HIV infection. The proposed aims address the two scientific questions in the FOA: ‘Does HIV exposure or infection induce innate immune memory?’ and ‘What mechanisms regulate innate immune memory which impact HIV acquisition?’. The study also involves a specific approach mentioned in the FOA: ‘Metabolic changes leading to reprogramming of innate immune cells’.