Two broadly neutralizing HIV monoclonal antibodies (mAB; 2F5 and 4E10) that bind to the membrane external proximal region (MPR) of gp41 were isolated from patient material but scientist have been unable to identify immunogens that induce broadly neutralizing antibodies to MPR in mice, rabbits or primates. We think they failed because they used the wrong epitopes as immunogens. We hypothesize that post-translational modifications such as phosphorylation of MPR are critical to the induction of neutralizing antibodies against MPR. Post-translational modifications (e.g. phosphorylation and nitration) can arise in the inflammatory milieu of an HIV infection. Based on this hypothesis I predict that the 2F5 and 4E10 monoclonals will have a greater affinity for the appropriate chemically-modified epitopes than for the non-modified epitopes. I will prepare a peptide library containing the extended MPR (epitopes) with phosphorylated residues within the sequences. I will also synthesize the epitopes as trimers to mimic the native gp41/gp120 trimer bundle found in the HIV capsid. The epitopes will be attached to an ELISA plate for the measurement of 2F5 and 4E10 titers against the epitopes. This data will provide a map of the chemical modifications recognized by 2F5 and 4E10 and where the modifications reside in the sequence. Recognized epitopes will be attached to a surface plasmon resonance chip and the on-rate/off-rate and affinity of 2F5 and 4E10 to the epitopes will be measured. The data will generate a structure-activity relationship of the mAb binding site. The first phase of the 12-month study will include synthesis and characterization of the peptides. The second phase will analyze the affinity of 2F5/4E10 to the epitopes. In future studies we would determine if chemical modifications with the greatest affinity to 2F5/4E10 induce HIV neutralizing antibodies in immunized rabbits and form the basis of an HIV vaccine.