A key goal of HIV-1 vaccine development is to induce long-lasting broadly neutralizing antibodies (bnAbs) that can inhibit HIV-1 infection. Messenger (m) RNA has emerged as a promising new vaccine modality that can elicit potent immune responses, while avoiding the safety risks and anti-vector immunity associated with some live virus vaccines. Important targets for bnAb induction are N301, N332 glycans at the base of the gp120 V3 loop. Our overall goals in this grant are 1) To design an mRNA that encodes a V3-glycan mimetope that, when expressed, will bind a V3 glycan UCA; 2) To select and produce mRNA formulations non-GMP that encode HIV-1 Envs for immunization in humanized mice and RMs; and 3) To produce the sequential V3-glycan mRNA vaccine under CGMP conditions, perform toxicity studies, and prepare an IND for testing in a Phase I trial in man. Overall Specific Aim 1. Develop mRNA delivery constructs for sequential Env trimers for V3-glycan bnAb B cell lineage vaccinations. Hypotheses: Messenger RNA vaccination of humanized mice, Rhesus macaques and humans will induce long-lasting anti-V3 glycan bnAb epitope antibodies, and mRNA vaccination will promote sequential somatic hypermutations and affinity maturation in V3-glycan targeted B cell lineages. Overall Specific Aim 2. Produce CGMP mRNA immunogens. Hypotheses: Messenger RNAs can be produced and encapsulated in potent nanoparticle formulations under CGMP for use in human Phase I trials, and will be safe and immunogenic. Moreover, the mRNA immunogens selected for CGMP production will produce stable Env trimers upon transfection of cell lines in vitro and after immunization in vivo in humanized mice. Expectations and Impact on the Field. Messenger RNAs are the current most promising vaccine strategy for inducing high-titered and long-lasting antibody responses. A successful first in man Phase I clinical trial with clinical trials materials produced in this IPCAVD will change the field by showing the plausibility of initiation of V3-glycan bnAb B cell lineages.