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It was proposed to develop a structure-based approach for synthetic vaccine development designed to recapitulate the activities of potent neutralizing monoclonal antibodies (MAbs) using constrained peptides as immunogens. We focused on two antibodies, a MAb 58.2 that binds a sequential V3 epitope and IgG bl2 that binds a discontinuous epitope, both on HIV-1 gp120. We validated the structure-based approach with a V3 mimetic using MAb 58.2 and demonstrated the enormous effect that peptide conformation can have on affinity and immunogenicity. We were unable to mimic the more challenging discontinuous IgG bl2 epitope despite considerable effort. The enormous affinity enhancements we observed for the constrained V3 peptide and dearth of potent HIV-1 neutralizing antibodies, particularly those specific for sequential epitopes, led us to an important discovery. It suggested that new antibodies might be identified with constrained peptides that go undetected with peptides. It was found that several helix-stabilized gpl20 peptides detect new antibodies that go undetected with linear peptides implying a general phenomenon. Constrained peptides, tailored to mimic short, sequential regions of proposed neutralization sites might be used to widen the selection of template antibodies best suited for synthetic vaccine development.

Our proposal was directed to the identification of synthetic peptide mimetics corresponding to neutralizing epitopes recognized by IgG1b12 and MAb 58.2. Herein, we report recent results on MAb 58.2 which provides important new information regarding methods and outcomes for synthetic vaccines as well as specific information about the role that conformation plays in neutralizing immune responses to the V3 epitope. MAb 58.2 displays broad reactivity against Clade B gp120s from primary isolates. It weakly neutralizes a cloned macrophage-tropic primary isolate, JR-CSF. We identified a constrained V-3 loop mimetic that binds >1,000 times better to MAb 58.2 than the corresponding linear peptide. Rabbit polyclonal antibodies to the constrained peptide but not the linear peptide bind rgp120 (MN). Furthermore, the polyclonal immune response to a constrained V3 peptide mimics the conformational preference and neutralizing activity of MAb 58.2. HIV-1 neutralization correlates with conformational preference and affinity for gp120. These results and others presented in the report establish beyond doubt the remarkable improvements in antigenicity and immunogenicity of constrained peptides compared to linear peptides.

HIV-1 initiates infection by attaching itself to CD4 on immune system cells. Two reagents, IgG1b12 and tetrameric CD4-IgG2, bind the envelop glycoprotein, gp12O, on HIV-1, block this reaction and prevent infection. We have focused our research on the identification and mimicry of epitopes recognized- by these reagents for use as a vaccine. We present 4 reports. A constrained DPE peptide loop based on Asp368ProGlu37O from the gp12O sequence has been used to isolate a neutralizing MAb similar to IgGlbl2. A 3-D structure prediction for the Vl/V2 domain on gp12O which plays a role in the neutralization of HIV-1 by 1gG1b12 is presented and provides a basis for the design of constrained peptide vaccines. Both phage-peptide and synthetic peptide libraries have been extensively screened with Fab 12 and sCD4. Synthetic peptides characterized by Asp, Glu and rich in trptophans have been identified that block sCD4/gp120 and Fab 12/gp12O binding with IC5O = 10-100 uN. Finally, the DPE loop has been ligated to a 'universal' T-cell epitope to yield a pure, well characterized four-branched loop-NAPS that is immunogenic in outbred mice and stimulates antibodies that bind recombinant gp120 IlIB.