Rare HIV-1 infected individuals display a significant level of broadly neutralizing antibodies that reflect the pattern of neutralization (i.e. breadth) and potency (i.e. relatively high titer). We hypothesize that the definition of the neutralization specificities present in broadly neutralizing patient sera will tell us the best targets upon which to focus immunogen design efforts to elicit broadly neutralizing responses. Because it is not known the regions of Env that are the targets for sera displaying breadth, this is important information to guide immunogen design. Like the 4 known broadly neutralizing antibodies, such sera demonstrate that given the proper immunogenic stimulus, it is possible to elicit broadly neutralizing HIV antibodies. Using a multi prong approach we intend to identify the neutralization specificity in the sera by a combination of selective protein-peptide adsorptions, chimeric viruses and by the direct isolation of novel B cell clones. In parallel, we will analyze candidate Env-based immunogens in small animals and will map the specificity of the elicited neutralizing activity. a) Mapping the Neutralizing Specificity in Sera by Selective Protein Adsorption We identified a subset of HIV-1 patients that display broad neutralizing activity in their sera. We are actively pursuing the analysis of the neutralizing specificity in the sera to elucidate how breadth is accomplished by the natural infection process. We have demonstrated that for the two most potent sera, V3 and V1/2 peptides do not affect the neutralizing capacity of the sera, whereas full-length gp120 coupled to solid phase beads can adsorb most of the HIV neutralizing activity. We will add selected point mutant gp120 glycoproteins to create a protein panel with different antigenic properties to selectively remove neutralizing activity. We have already coupled BaL CD4 binding site point mutant gp120 proteins to the beads (368D/R) and have observed that for the two most potent sera, the mutant proteins do not remove the neutralizing activity directed against 4 diverse viral strains. The mutant proteins can remove the neutralizing and binding activity of monoclonals of with specificities distal to the CD4 binding region. These data indicate that much of the broad neutralizing activity in these two potent neutralizing sera is directed against the CD4 binding site. We will add additional controls, for example, a denatured gp120 bead and a BSA coupled bead , and will perform the neutralization/adsorptions over a range of serum dilutions. We will remove or purify IgG from the sera to confirm that the neutralization is antibody meditated. For detection of gp41 membrane-proximal-directed neutralization (MPR), we will generate MPR miniprotein beads and an alanine-mutated version as a negative control. We will continue the analysis of the patient B cell repertoire by EBV-induced immortalization by new methods developed by Antonio Lanzavecchia and perform direct neutralization of pseudo-typed virus with the supernatants from the immortalized B cells in collaboration with Mark Connors. We plan to collaborate with Dennis Burton to generate antibody phage display libraries from the bone marrow of selected patients. And we will continue to develop a panel of reciprocal HIV/SIV chimeric viruses as another means to confirm the neutralization specificity in the patient sera. In the future, with the appropriate validation, these tools will be extremely valuable to map the specificity in vaccine-elicited neutralizing sera.