Directed Evolution of Multivalent Glycopeptides Tightly Recognized by HIV Antibody 2G12

[Image: see text] Herein, we report a method for in vitro selection of multivalent glycopeptides, combining mRNA display with incorporation of unnatural amino acids and “click” chemistry. We have demonstrated the use of this method to design potential glycopeptide vaccines against HIV. From libraries of ∼10(13) glycopeptides containing multiple Man(9) glycan(s), we selected variants that bind to HIV broadly neutralizing antibody 2G12 with picomolar to low nanomolar affinity. This is comparable to the strength of the natural 2G12–gp120 interaction, and is the strongest affinity achieved to date with constructs containing 3–5 glycans. These glycopeptides are therefore of great interest in HIV vaccine design.