ABSTRACT HIV-1 envelope glycoproteins (Envs) mediate viral entry into host cells and are the sole target of neutralizing antibodies. HIV-1 Envs of most primary HIV-1 strains typically exist in a closed conformation (State 1) and occasionally transit to downstream, more open conformations (State 2 and State 3). Thus, current knowledge guides immunogen design towards mimicking the Env closed conformation as the preferred target for eliciting broadly neutralizing antibodies (bnAbs) to block HIV-1 entry. However, Env preferred conformations of more than 37 million different circulating HIV-1 strains are unlikely to adopt exactly the same closed conformation. And we still do not know how different State 1 conformations of diverse natural HIV-1 variants are. Our hypothesis: State 1 Envs form a heterogenous group of Envs that exist in similar but not identical conformations. Accordingly, improved ability of bnAbs to recognize multiple Env conformations will broaden their neutralization activity. To test our hypothesis, we will assess the conformational heterogeneity of Envs from different strains and build mechanistic models for 1) Envs transitions, 2) generation and transmission of Envs that exist in specific conformations, and 3) broad recognition of different Env states. In Specific Aim 1 we will study the conformational diversity of transmitted/founder (T/F) HIV-1 strains, which can establish HIV-1 in vivo and are the only target of a preventive vaccine. We will study the mechanisms by which T/F Envs expose internal elements and how they transit to downstream conformations. In Specific Aim 2 we will investigate the generation and evolution of incompletely closed Env conformation. We will reconstruct the evolution pathway of T/Fs in patients by using available viral sequences to build evolved viruses at different time point followed by investigating changes in their conformational states over time. In parallel, we will study Env conformational diversity among HIV-1 strains that are archived in the latent HIV-1 reservoir of people living with HIV-1 and identify potential targets for therapeutic interventions. These approaches will allow us to build a mechanistic model for how incompletely closed Envs are generated, evolved, and transmitted. In Specific Aim 3 we will define important parameters for broad recognition of CD4-binding site (CD4-bs) on different Env conformations of diverse primary strains. We will study how the exceptionally broad N6 (CD4-bs) bnAb recognizes different Env conformations, will identify critical interactions with residues on open and closed Env conformations, and will solve cryo-EM structures of HIV-1 Envs in complex with N6 to define the mechanism of broad recognition. Overall, our study will provide high-resolution and comprehensive view on the biology of HIV-1 entry pathway, will refine the knowledge on HIV-1 Env conformations, and will form a strong basis for the development of new strategies for HIV-1 cure and vaccine de...