Summary Structural insights from our recent work provide a strong scientific premise for exploring the membrane-related components of HIV-1 envelope glycoprotein (Env), including the membrane proximal external region (MPER), the transmembrane domain (TMD) and the cytoplasmic tail (CT), for vaccine development. Our data indicate that all these regions influence the stability and antigenicity of the Env ectodomain. Major broadly neutralizing antibody (bnAb) epitopes on HIV-1 Env include CD4 binding site, V1V2-glycan, V3-glycan, the fusion peptide/gp120-gp41 interface and the MPER. The optimal presentation of these epitopes, critical for their antigenicity and immunogenicity, depends on the Env trimer organization and conformation. Recently, the fusion peptide-based immunogens have induced robust cross-clade neutralizing responses in animal models, suggesting that bnAbs may be elicited by vaccination. In this research project, our central hypothesis is that rationally designed HIV-1 Env immunogens in different conformations with various degrees of bnAb epitope exposure induce different B cell responses, which may lead to production of diverse bnAbs in animal models and to new strategies for HIV-1 vaccine immunogen design. The research team is formed by a group of outstanding investigators with diverse yet complementary expertise to carry out the proposed studies. This group has extensive experience in protein engineering, production and characterization, in B cell biology and vaccinology in animal models, and in detailed analysis of vaccine-elicited antibody responses. The group members have an extensive history of working together on HIV-1 and SARS-CoV-2 related projects. The team will capitalize on the newly determined structures of the membrane-related components of HIV-1 Env to develop two innovative immunogen-design strategies: (1) soluble Env trimer immunogens and (2) membrane-bound intact Env trimer immunogens. We propose two Specific Aims to test the hypothesis: Aim 1. We will design, characterize and produce Env-based immunogens in both the protein and mRNA forms and perform structural studies of Env-based immunogens and their complexes with antibodies. Aim 2. We will evaluate immunogenicity of novel Env-based protein immunogens and mRNA vaccines in VelocImmune human antibody mice.