To be able to induce antibody (Ab) responses against the HIV-1 envelope (Env) spike vulnerable sites defined by broadly neutralizing antibodies (bnAbs) will likely lead to a protective vaccine. Many HIV bnAbs have been discovered and their epitopes have been carefully mapped on the surface of Env, and these epitopes have been categorized into a number of well characterized classes. However, a consistently induction of bnAb responses targeting any of those currently well characterized vulnerable sites is still extremely difficult because of the unusual features of these bnAbs and their associated vulnerable sites, such as the requirement of a long CDR H3 to penetrate the glycan shield of the Env spike. Thus, identification of additional vulnerable sites with more favorable characteristics for rational targeting may help to increase our capability of induction of bnAb responses. We have identified recently a new vulnerable site by mapping the epitope of bnAb M4008_N1 and characterizing its antibody-antigen interaction. M4008_N1 is a bnAb that can neutralize about 40% of the primary isolates tested and its lineage class-switched to both IgG and IgA. We showed that by cryo-EM the epitope of M4008_N1 is centered at the crown region of the third variable loop (V3) of gp120 in the prefusion Env trimer. The unique characteristics of the M4008_N1 epitope and its mode of interaction with the V3 crown suggest a strategy to induce M4008_N1-like Ab responses by a V3 priming and heterologous boosting with stabilized Env trimer. As we have demonstrated previously, the V3 crown Ab responses are relatively straightforward to elicit by V3 immunogens. We therefore hypothesize that, since the V3 crown -hairpin is a common structure and M4008_N1 can bind V3 loop itself, antibodies that engage the N-terminal strand by a β- sheet interaction, like that of M4008_N1, can be induced by V3 immunogens, and such M4008_N1-like antibody responses can then be boosted with a subsequent immunization with a V3-stabilized Env trimer, which harbors the whole epitope of bnAb M4008_N1. Early versions of the SOSIP immunogens were known to induce non-neutralizing V3 antibodies, but some V3-stabilized versions seemed to be able to purge non- neutralizing V3 antibody responses. These V3-stabilized SOSIP trimers can therefore be used for the heterologous boost to induce M4008_N1-like antibody responses. We will test our hypothesis in a rabbit model using a set of carefully selected to be optimal in the heterologous prime/boost strategy, and we have two Aims. (1) Selection of the best immunogen combination, (2): Rabbit immunization and characterization of the antibody responses. The expected outcome of this project is a proof of principle demonstration for the immunogenicity targeting the newly identified V3 crown vulnerable site and to lay a foundation for future extensive immunogenicity studies including potentially non-human primate studies.