Request to DAIDS to Manufacture a protein encoding CAP256.wk34.c80 OPT4 envelope for induction of V2 Apex bnAbs Induction of an effective HIV-1 vaccine is a global priority; yet, no HIV-1 vaccine regimen reproducibly elicits broadly neutralizing antibodies (bnAbs) in humans. The complex immunology underlying human bnAb development suggests that a successful vaccine will likely contain a series of immunogens that guide bnAb development 1-3. Such complex vaccines will require iterative design and evaluation in humans, necessitating rapid cGMP production of potent immunogens. We and others have found that multimerizing HIV-1 Env on nanoparticles (NPs) can augment its immunogenicity. HIV-1 Env trimer NP immunogens have been made previously by encoding HIV-1 Env fused to NP subunits as a single gene. These fusion protein NPs display a mixture of high-quality and low-quality, misfolded HIV-1 Env on their surfaces that can elicit immunodominant non-neutralizing antibodies. Thus, laborious, time-consuming development work must be performed to optimize the HIV-1 envelope production such that predominately well-folded envelope is fused to nanoparticle subunits. Additionally, we have found during cGMP cell line development that protein yields of Env trimer NP may be insufficient to enable cGMP manufacturing. The significance of this Project is that it will utilize an already successful cGMP process to rapidly manufacture higher quality HIV-1 Env trimer NPs than previous NPs encoded by single genes. We have already utilized this technology to overcome the pitfalls incurred with fusion NPs, by using our innovative conjugation method that generates NPs with only well-folded HIV-1 Env on their surface. This method utilizes the high specificity of sortase A conjugation to link highly purified, well-folded HIV-1 Env to ferritin NPs. These sortase A-conjugated NPs (scNPs) can be assembled rapidly and preserve the antigenic character of the HIV-1 Env, features critical for a vaccine regimen aiming to elicit durable, protective bnAbs. This innovative, rapid NP platform is universal in nature, having been used for HIV-1 Env trimers of various clades, SIVcpz Env trimers, and Spike proteins from other human pathogens such as SARS- CoV-2. We will utilize this platform in combination with CAP256w34.80 designed to guide affinity maturation of V2 Apex bnAbs in humans.