Several antivirals that target discrete steps in the HIV entry process are under pre clinical and clinical development, and T-20 (Enfuvirtide) has recently received FDA approval for use in combination therapy of treatment-experienced patients failing HAART. Entry inhibitors will complement and diversify current treatment regimens, increasing the prospect for durable therapy. However, these inhibitors target the highly variable envelope (Env) protein and primary viruses can exhibit marked differences in sensitivity to entry inhibitors, including T-20. Thus, the efficacy of entry inhibitors is likely to vary considerably within patients depending upon both host and viral factors. Additionally, HIV will clearly evolve resistance to entry inhibitors, just as it has to RT and protease inhibitors. Indeed, T-20 treatment selects for mutations in the HR1 region of gp41 that affect viral sensitivity to T-20. The long-term objectives of this application are to identify Env determinants that influence sensitivity to different entry inhibitors and to elucidate the consequences of escape from T-20, as this may be of prognostic value and suggest effective entry inhibitor combinations. The hypothesis of this Research Plan is that various Env determinants, in gp120 and gp41, will impact entry inhibitor sensitivity and that these determinants may have a significant impact on entry and viral fitness. Additionally, T-20 resistance mutations are likely to impact entry efficiency, viral fitness as well as sensitivity to other entry inhibitors. The consequence of coreceptor binding affinity on fusion, replication and entry inhibitor sensitivity will be investigated in Aim 1, using a panel of Env mutants constructed to contain single amino acid changes that modulate coreceptor affinity. The impact of T-20 resistance mutations on fusion kinetics, viral fitness and sensitivity to other entry inhibitors will be investigated in Aim 2, in the context of Envs with high or low coreceptor affinity. Correlates of primary virus entry inhibitor sensitivity will be sought in Aim 3, using a panel of Envs, including some that are genetically related, that exhibit differential sensitivity to one or more entry inhibitors. Assays that will be used to achieve these goals include mutagenesis, Env:receptor binding, cell:cell fusion and inhibition, fusion kinetic, pseudotype and replication competent virus infection and inhibition and viral fitness determinations. Together, these studies will identify envelope determinants that influence sensitivity to entry inhibitors and the consequences of T-20 'escape' mutations. ? ?
