ABSTRACT HIV-1 envelope glycoproteins (Envs) mediate viral entry into host cells and are the sole target of neutralizing antibodies. Broadly neutralizing antibodies (bnAbs) target highly conserved sites on HIV-1 Envs and neutralize a wide range of diverse strains from different clades. Nevertheless, bnAb immunotherapy aiming to suppress HIV-1 replication sometimes leads to development of bnAb-resistant HIV-1 strains, and HIV-1 strains with pre- existing bnAb resistance can be identified by prescreening before treatment. Thus, understanding the underlying mechanisms of bnAb resistance are critical for the future application of bnAbs for immunotherapy and prevention. Mechanisms that lead to multi-bnAbs resistance and indirect mechanisms that facilitate escape of bnAbs from different groups are of particular public health concern. Our study is structured to provide important insights into bnAb resistance at different levels. In Specific Aim 1 we will study direct resistance mechanisms of rebounded HIV-1 strains that are resistant to multiple bnAbs. We will screen samples from clinical studies of bnAb therapy, identify Envs of HIV-1 strains that exhibit the highest degree of resistance to several bnAbs, study Env sequence, function, glycosylation patterns and determine the structures of resistant Envs at atomic level resolution. Our comprehensive approach will provide unique profiles of selected multi-bnAb resistant Envs that integrate all potential mechanisms contributing to bnAb resistance. In a parallel direction, we will study the ability of rebounded HIV-1 strains to spread through cell-cell transmission, which allows efficient viral replication in the presence of different groups of bnAbs. We will test the hypothesis that bnAb-sensitive HIV-1 strains that replicate despite high levels of bnAb in the serum of participants from the RV397 trial can efficiently spread by cell-cell transmission. Additionally, we will investigate the molecular mechanisms of strains that exhibit increased cell-cell transmission efficiency and bnAb resistance. In Specific Aim 2 we will define optimal bnAb combinations to overcome bnAb resistance and use antibody yeast display technology to bioengineer recombinant bnAbs with improved affinity against bnAb-sensitive and resistant HIV-1 strains. This approach will allow us to confirm mechanisms of HIV-1 resistance to bnAbs and to test the hypothesis that specific changes in bnAbs can improve bnAb breadth and allow targeting of a subset of resistant HIV-1 strains. Overall, our study will provide high-resolution and comprehensive view on multi-bnAb resistant HIV-1 Envs, on alternative pathways of HIV-1 resistance in vivo, and on potential approaches to overcome bnAb resistance. Our results will form a strong basis for the development of new strategies for HIV-1 immunotherapy and prevention efforts.