Epitope-targeting strategy for a vaccine against human immunodeficiency virus type-1; peptide ligands for the broadly-neutralizing antibodies b12, 2F5 and 2G12

The research presented here is part of an ongoing effort to develop an epitopetargeted vaccine against the Human Immunodeficiency Virus type 1. We have used phage-displayed peptide libraries, in conjunction with other biochemical and structural techniques, to generate and characterize peptides specific for the broadly-neutralizing, human monoclonal antibodies (Mab) b12,2F5 and 2G12. First, peptide ligands for Mab b12 were isolated. One peptide, B2.1, was characterized in detail. The crystal structure of B2.1 in complex with b12 Fab was solved and compared with a model of the antibody docked to gp120. B2.1 was used to immunize mice, seeking to generate a b12-like, neutralizing antibody response. Minimal structural resemblance between the putative b12 epitope and B2.1 peptide was observed, indicating different mechanisms of binding to b12. Moreover, the anti B2.1 response from the immunized mice did not cross-react with gp120, further indicating this difference. Second, we demonstrate that Mab 2F5, which binds to the sequence ELDKWA on gp41, selects peptides with multiple, different amino acid sequences flanking its threeresidues DKW native core epitope. High-affinity binding was achieved by peptides with unrelated flanking sequences C-terminal to the core epitope, with the nature and location of critical binding residues differing between peptides. Our results implicate multiple mechanisms of binding, and suggest the existence of two distinctive functional regions in the 2F5 paratope, one that is DKW-specific and one that is multi-specific. Finally, a peptide ligand specific for the carbohydrate-binding Mab 2G12 was isolated, characterized and optimized. The peptide competes with carbohydrate ligands for binding to 2G12. The crystal structure of the 2G12-peptide complex, and its comparison with the 2G 1 2-Man9GlcNAc2 complex, shows that the peptide is not a mimic of Man9GlcNAc2. They occupy separate sites in the antibody's paratope, with minimal overlaping, therefore the peptide and Man,GlcNAc, make a different set of contacts with the Mab. In summary, we have observed that peptide mimicry of the b12,2F5 and 2G12 epitopes is essentially functional, not structural. Our results indicate that these ligands can be used as antibody-specific markers, and that most likely, conventional immunizations with so-called peptide mimics will not elicit these antibodies.