The goal of this proposal is to determine how antigen exposure shapes subsequent Natural Killer cell responses to HIV. We propose to identify NK functional subsets in naïve and antigen-primed human NK cells using single-cell sequencing and multi-parametric flow cytometry. We propose to verify the relevance of such functional subsets to NK cell-mediated host protection from HIV disease using our established in vitro and in vivo functional assays. NK cells are innate lymphocytes that live up to their name by their ability to kill infected or tumor cells within minutes of exposure. However, the targeting of NK cell effector functions has not been a significant focus in vaccine development, which has mainly focused on their T and B cell counterparts in the adaptive immune system. Recent findings from the PI of this application indicate that NK cells deserve more attention. We recently published exciting new data that human NK cells remember prior antigen- encounters and mediate enhanced recall responses to HIV-Envelope in humanized mice. Here, we present unpublished new data that HIV-Env-primed memory NK cells suppress HIV viral titers upon experimental viral challenge. These findings have opened the opportunity to harness NK memory functions for vaccine design. However, high NK cell receptor repertoire diversity is associated with an increased risk of HIV acquisition, and NK cell receptor repertoires diversify throughout life, presumably in response to antigen exposure. These data present a challenge for vaccine design, as both protective NK memory responses and potentially risky NK repertoire diversifications are consequences of antigen exposure. The identification of specific functional subsets of HIV-responsive, host-protective NK cells and their mechanisms of host protection is therefore critically needed. Their discovery will open the door for revised vaccine designs that endure the incorporation of NK memory, rather than harmful receptor repertoire diversity, as a host protective outcome. Our data will enable the pre-screening of vaccines for the induction of protective NK functional subsets in pre- clinical models and allow for improved vaccine efficacy evaluations in humans. Thereby, our studies will provide the rationale to develop novel vaccines that exploit the antiviral activity of NK cells to protect humans from HIV infection while avoiding harmful activity.