This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Our goal is to identify characteristics in the HIV-1 envelope glycoprotein (Env) that would provide rational targets for vaccination. HIV-1 has thwarted vaccination efforts largely through high levels of diversification. This diversification is most apparent in the HIV-1 Env, the protein responsible for mediating receptor interactions and fusion between virus and host. Because of its location on the virus exterior, Env is the only immunogen that can elicit humoral, neutralizing antibody responses. While a few broadly neutralizing antibodies have been identified, no strategy has proved successful in directing their generation. This may be due to a poor understanding of the trajectory of HIV-1 evolution within infected individuals and a failure to identify universal determinants of virus transmissibility. HIV-1 fitness, in the absence of drug treatment, is directly correlated with the rate of virus entry. Likewise, transmissibility or 'transmission fitness'will be determined by characteristics of the envelope glycoprotein. We hypothesize that determinants within HIV-1 Env make the virus more fit and therefore contribute to viral pathogenesis. Moreover, we predict that there exist determinants in Env that promote viral transmission from person to person. In each case, these determinants need to be identified in order to appropriately vaccinate to promote humoral anti-Env responses. On the one hand, the development of such responses would be anticipated to have therapeutic efficacy by preventing or limiting virus evolution to highly fit and highly pathogenic variants;whereas, the identification of determinants of 'transmission fitness'would provide targets for developing prophylactic vaccines. We will utilize HIV-1 infectious molecular clones containing fluorescent reporter genes in a dual-infection competition system to do the following 1.) identify molecular determinants of HIV-1 fitness using longitudinal viral isolates from infected individuals;2.) identify most fit variants in Env through the use of randomized hypervariable sequences;3.) compete Env variant viruses through relevant mucosal organotypic cultures to model virus transmission and identify the determinants of successful transmission. Through these efforts we anticipate being able to synergize with our ongoing vaccine development strategies.