The overall goal of this proposal is to develop a vaccination strategy that induces strong and durable humoral and cellular immunity against HIV, providing long-term protection even with low neutralizing antibody (Ab) responses using DNA-LNP technology. Developing an effective HIV-1 vaccine has been an elusive goal for over four decades. An ideal HIV vaccine should generate a potent and broadly cross-reactive neutralizing antibody response (bnAb) that remains at high titers for many years. Native-like trimeric HIV envelope (Env) gp140 immunogens, such as SOSIP, NFL, and UFO, have significantly advanced the field by eliciting autologous nAbs; however, they do not induce an antibody response with neutralization breadth. We and others have shown that a serum neutralization titer of around 300 or higher is necessary for nearly complete protection in non-human primates (NHPs). These findings underscore the importance of developing vaccination methods that maintain persistent nAb levels at or above this threshold. Besides nAbs, CD8 T cells—especially tissue-resident memory T cells—are crucial for protection against HIV. Our recent studies indicate that adding a T cell-inducing vaccine to an nAb-inducing vaccine can significantly lower the neutralization titer threshold needed for long-term protection against intravaginal SHIV challenges, bringing it below detectable levels. Therefore, we hypothesize that vaccine approaches that induce strong, lasting CD8 T cell responses, along with neutralizing antibodies, will improve protection against HIV—even when serum neutralizing antibody levels are low. In ongoing studies, we compared the immunogenicity of mRNA-LNPs with DNA-LNPs expressing SIV Gag in rhesus macaques (RMs). We observed that DNA-LNPs elicit 25-fold higher levels of Gag-specific CD8 T cell responses compared to mRNA-LNPs. Moreover, the CD8 T cells induced by the DNA-LNP vaccine were durable, showing only about 2- fold contraction over four months post-boost. The DN