Project Summary/Abstract. Advances in B cell biology and molecular virology have enabled the discovery, characterization, and commercial development of several classes of broadly neutralizing antibodies (bnAbs), with applications for prevention, treatment, and cure of HIV disease are under study. Yet, virus resistance remains the central vulnerability of effective bnAb use. This application proposes to address the problem of bNAb escape by rationally selecting combination bNAb therapy that limits virus escape. We propose to apply lessons from the successful development of combination antiretrovial therapy (cART), whereby bidirectional phenotypic antagonism was exploited. Our multidisciplinary team has secured plasma virus or virus sequences from recent or ongoing prevention and treatment studies of VRC01-class CD4 binding site (CD4bs)-targeting monotherapy, as well as combination therapy with V3 glycan-targeting (Table 1). We will leverage these unique samples to map the in vivo escape pathways of virus replicating in the presence of sub-suppressive levels of these clinically relevant bNAbs (Aim 1). Using the evolving escape variants, we will identify putative complementary bNAbs with maintained or inverse antibody sensitivities from rationally designed panels of candidate bNAbs (Aim 1). We will then characterize the autologous neutralizing antibody (anAb) response in the treatment cohorts, to determine the capacity of anAbs to impede virus escape from administered bNAbs (Aim 2). Finally, we will test the most promising complementary bNAbs to restrict virus escape in vivo in a validated barcoded TF SHIV/NHP model (Aim 3). Our scientific premise is that in vivo mapping of virus escape from bNAbs, identification of complementary bNAbs, defining the role of autologous antibodies, and rigorous in vivo testing in an authentic NHP model will elucidate basic mechanisms of virus resistance to bNAbs and inform more effective use of bNAbs across the HIV prevention, treatment and cure fields. If accomplished we will (i) have defined the sensitivities of escaped viruses from clinical trials to alternate bNAbs, (ii) identified bNAbs that cannot mutually escape using the same pathway, (iii) defined the role of anAbs in bnAb escape and (iv) tested the ability of complementary bnAbs to improve therapy in an authentic NHP model.