A vaccine is needed to stop the HIV-1/AIDS pandemic, however, the development of a protective vaccine remains a great challenge and requires novel strategies. Various approaches to stabilize the whole extracellular domain of the HIV-1 envelope (Env) has led to greatly improved vaccine designs, e.g., the development of the SOSIP trimer, which, in addition to having provided a refined structural understanding of the Env trimer, is considered as a promising immunogen because it harbors all the known vulnerable sites on the Env trimer targeted by bnAbs. However, SOSIP-based immunogens have achieved only limited success due to unwanted distracting epitope sites that divert Ab responses to strain-specific ones. Unlike whole Env extracellular domain approaches, we have been using a divide-and-conquer strategy by starting with individual domains of the Env trimer to develop domain-specific immunogens which have the advantage of immune- focusing Ab responses to selected Env domains and epitopes and of avoiding induction of Abs to unwanted epitope regions. We started with the V1V2 domain, not only for its domain structure spatially located at the Env apex but also its Abs inversely correlate with the risk of infection in the RV144 trial, have developed a panel of trimeric V1V2 domain immunogens by scaffolding V1V2 of gp120 on trimeric non-HIV proteins, and demonstrated that they can induce V1V2-focused Ab responses. Moreover, further design efforts have resulted in a single chain tandem V1V2 trimer showing antigenic reactivity with trimer-specific bnAbs. We hypothesize that such V1V2-domain immunogens can be improved to present the native Env apex configuration by further structural characterization and engineering. To expand from the Env apex, we have then designed a ‘re-cored’ Env trimer immunogen by replacing the gp120 inner domain and gp41 in the prefusion trimer structure with a stable trimeric non-HIV scaffold protein; antigenicity tests demonstrated that this molecule harbors all key bnAb binding sites of the trimeric Env apex and EM visualization showed it to have a well-formed trimeric configuration. This novel construct provides a starting point to develop a trimeric immunogen that carries key vulnerable sites of the Env trimer which could become an attractive alternative to the SOSIP trimers, offering greater focus on the most promising bnAb epitopes. The goal of this R01 project is to structurally characterize these rationally-designed immunogens and further apply our structure-based platform to develop new generations of them so that they serve as effective immunogens targeting native trimeric configurations. We have three Aims. 1) Characterize and refine the trimeric V1V2-scaffold constructs mimicking the native prefusion conformation of the Env trimer apex. 2) Develop the re-cored Env trimer immunogen harboring key bnAb sites. 3) Test the immunogenicity of these refined immunogens in animals. At the completion of this project, our novel trim...