In Vitro Selection of Cyclized, Glycosylated Peptide Antigens That Tightly Bind HIV High Mannose Patch Antibodies

In vitro selection is typically limited to discovery of peptides, proteins, and nucleic acids. Given the importance of carbohydrate-protein interactions in diverse areas of biology including cell adhesion/recognition, immunoregulation and host-pathogen interactions, directed-evolution-based methods for discovery of potent glycoligands are greatly needed. We have previously reported a method for in vitro selection of glycopeptides that combines mRNA display, alkynyl amino acid incorporation, and CuAAC "click" glycosylation. Herein, we describe extensions of this method that incorporate chemical cyclization, removal of N-terminal glycosylation sites, and next-generation sequencing; as an approach to HIV immunogen design, we have used this method to develop mimics of the High Mannose Patch (HMP), the region on HIV envelope protein gp120 most commonly targeted by HIV broadly neutralizing antibodies (bnAbs). We prepared libraries of 10^12-14 glycopeptides about 50 amino acids in length, containing variable placement of high mannose (Man₉GlcNAc₂) glycans and cyclization sites. From selection, we obtained binders to HIV bnAbs PGT128, PGT122, and gl-PGT121, a germline precursor of PGT122, and chemically synthesized numerous glycopeptide hits. Several glycopeptides bound very tightly to their target HIV bnAb, e.g., with a K _D as low as 0.5 nM for PGT128. These glycopeptides are of interest as immunogens and tools for HIV vaccine design.