PROJECT SUMMARY With the advent of highly successful antiretroviral therapy, mortality and morbidity associated with human immunodeficiency virus (HIV) infection has been significantly reduced. Despite this, hundreds of thousands of new HIV infections occur each year, demonstrating the need for a safe and effective vaccine. HIV vaccines based on the elicitation of broadly neutralizing antibodies (bNAbs) are at the forefront of HIV research. Stabilized HIV immunogens have now shown the ability to elicit tier 2 neutralizing antibodies in rhesus macaques, and numerous bNAbs have been isolated from human subjects. Furthermore, germline targeting immunogens have demonstrated initial success in humans in eliciting bNAb germline precursors. A key aspect in the effectiveness of HIV immunogens is the delivery method. Somatic hypermutation is a critical event for optimal and efficient bNAb elicitation. Recombinant HIV Env proteins are poorly immunogenic and fail to follow typical lymph node (LN) trafficking pathways due to high glycosylation and poor recognition by natural IgM. Instead, recombinant HIV Env proteins are captured by interfollicular macrophages rather than by macrophages in the subcapsular sinus, leading to poor antigen availability and limited repeated somatic hypermutation. The scientific premise for this proposal is that since recombinant HIV Env proteins have altered LN trafficking, biodegradable LN targeting nanoparticles that facilitate transfer of soluble HIV Env trimers directly to LNs are a promising tool to circumvent current barriers. Our objective is to test the hypothesis that a lymph node (LN) targeting nanoparticle (NP) technology will improve immune responses generated by human immunodeficiency virus (HIV) envelope (Env) protein immunogens. This proposal is high risk, as the elicitation of broadly neutralizing antibodies (bNAbs) at a high titer and long duration is challenging; nonetheless, if successful, it offers high reward by substantially improving HIV Env vaccine responses. In Specific Aim 1, we will determine if PLGA-b-PEG NPs more efficiently deliver germline targeting immunogens to LN follicles and increase somatic hypermutation compared to unencapsulated immunogens. Mice will be immunized with germline targeting immunogen trimers encapsulated within the NP platform, and serological and cellular analysis will be completed, including monitoring of somatic hypermutation using single cell sequencing approaches. LN trafficking and accumulation in follicles will also be assessed. In Specific Aim 2, we will determine if co-encapsulation of diverse HIV Envs in NPs enhances elicitation of tier 2 neutralization breadth. HIV Env SOSIP trimers from two viral clades will be combined in a single NP, and serological responses will be assessed. This proposal is highly collaborative, combining Dr. Jarrod Mousa, a structural immunologist and HIV/AIDS Vaccine Scholars Program recipient, and Dr. Hai-Quan Mao, a biomaterials and nanomedi...