Developing a vaccine that can elicit broadly neutralizing antibodies (bnAbs) against HIV remains a significant challenge in the fight against HIV/AIDS. One major vulnerable site on the envelope protein that bnAbs recognize is the fusion peptide (FP) on the Env. Although considerable efforts and progress have been made to induce bnAb responses targeting this site by chemically conjugating FP with carrier proteins such as KLH, which is difficult to use in human trials, broad and potent neutralization has not yet been achieved. In a recent rabbit pilot study, we demonstrated that a recombinant FP-cholera toxin subunit B (FP-CTB) fusion immunogen, in combination with a heterologous Env trimer boost, elicited tier 2 neutralizing antibody responses against a standard global panel of viruses with unprecedented breadth and potency. CTB is the B subunit of the AB toxin from the pathogen Vibrio cholerae and belongs to the well-known AB toxins with pentameric B subunits. There are four main families of AB toxins: Cholera Toxin (CT), Pertussis Toxin (PT), Shiga toxin (ST), and Subtilase cytotoxin (SU), each with different immunomodulatory properties. CTB is particularly known for its mucosal adjuvant properties. We have previously used the pentameric B subunits to carry gp120 V3 and V1V2 domains and demonstrated their strong capacity for immunofocusing and induction of durable epitope-specific Ab responses. Furthermore, recombinant CTB has already been used in human vaccines, making FP-CTB a promising vaccine candidate. Our central hypothesis is that fusing the flexible FP onto a highly immunogenic toxin B subunit can help focus immune responses on the FP epitope, and these responses can be guided to develop neutralization breadth by heterologous boosting using native Env trimers. Building upon our exciting and promising preliminary results, we propose to advance the development of the FP and toxin B subunit-based immunogens to improve the potency and breadth of bnAb responses. Our team has the necessary expertise and resources to achieve this goal, and we have three aims, including Aim 1: Lay the groundwork for a novel AB toxin B subunit-based FP vaccine, Aim 2: Improve the Breadth and Potency of Neutralizing Antibody Responses, and Aim 3: Test the immunogenicity of refined FP immunogens in NHPs. Upon completion of this project, our panel of novel FP immunogens will be fully developed and characterized for structure, antigenicity, and immunogenicity, and selected immunogens can then be moved forward in the pipeline for NHP challenge studies.