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 ∼1013 glycopeptides containing multiple Man9 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.

A vast number of biological processes are mediated by multivalent ligand-receptor interactions, including cell adhesion, host invasion by pathogens, pathogen neutralization by host and numerous cell regulatory signaling pathways. [1] Multivalency is especially important for carbohydrate-receptor interactions: whereas individual glycans [2] may bind with low affinity to a single binding site, the clustering of glycans creates a high-avidity interaction with clustered binding sites. This "carbohydrate cluster effect" [1b] has been demonstrated experimentally with synthetic multivalent carbohydrate ligands which bind well to protein targets. These ligands have included oligo-and polyvalent clusters of glycans on diverse scaffolds, including small molecules, dendrimers, polymers and even viral capsids.To date, most glycocluster ligands have been designed for synthetic convenience rather than control of tertiary structure. However, the biological activity of the natural glycocluster may be influenced by tertiary structure and other elements which are not usually addressed in synthetic glycocluster designs, such as: 1) Glycan spacing and orientation -glycans are normally attached to synthetic scaffolds through long flexible linkers, and the scaffolds themselves are often flexible. [3] 2) Glycan internal flexibility -in a natural glycocluster, As an alternative to rational design, we have been interested in directed evolution-based design of glycocluster ligands. Figure 1 outlines this concept: a library of scaffold molecules is glycosylated, generating a library of glycoclusters. The "best" glycoclusters are selected from the pool by binding to the target protein. These selection winners are then replicated to form a second-generation library and the process is repeated for several rounds until the pool is sufficiently enriched in high-affinity binders. We have chosen DNA as our glycocluster scaffolding material because DNA is easy to synthesize, easy to replicate by PCR, can fold into diverse sequence-dependent structures, and is amenable to sequence-specific "glycosylation" by glycan azides using CuAAC [5] ("click") attachment to alkyne-modified nucleobases. Iterative selection/amplification of DNA structures (SELEX) is often performed to obtain DNAs which bind to a target. [6] Our method, by contrast, would yield DNA scaffolds whose major function would be to position and support glycans optimally for target binding. However, these DNAs might also contain elements which would interact directly with the target, mimicking any non-carbohydrate components necessary in the natural ligand.We decided to test this concept in the design of glycoclusters which mimic the epitope of 2G12, an antibody which protects against HIV infection and binds to a cluster of highmannose glycans on the HIV envelope protein gp120. [7] Rationally-designed clusters of these glycans have been tested as vaccines to elicit 2G12-like antibodies, but without success. [8] Our evolution-based design would be the product of the procedure outlined i...

SELMA (SELection with Modified Aptamers) is a directed evolution method which can be used to develop DNA-supported clusters of carbohydrates in which the geometry of clustering is optimized for strong recognition by a lectin of interest. Herein, we report a modification of SELMA which results in glycoclusters which achieve dramatically stronger target recognition (100-fold) with dramatically fewer glycans (2–3-fold). Our first applications of SELMA yielded clusters of 5–10 oligomannose glycans which were recognized by broadly neutralizing HIV antibody 2G12 with moderate affinities (150–500 nM Kd’s). In the present manuscript, we report glycoclusters containing just 3–4 glycans, which are recognized by 2G12 with Kd’s as low as 1.7 nM. These glycoclusters are recognized by 2G12 as tightly as is the HIV envelope protein gp120, and they are the first constructs to achieve this tight recognition with the minimal number of Man9units (3–4) necessary to occupy the binding sites on 2G12. They are thus of great interest as immunogens which might elicit broadly neutralizing antibodies against HIV.

Up to ∼20% of HIV-infected individuals eventually develop broadly neutralizing antibodies (bnAbs), and many of these antibodies (∼40%) target a region of dense high-mannose glycosylation on gp120 of the HIV envelope protein, known as the “high-mannose patch” (HMP). Thus, there have been numerous attempts to develop glycoconjugate vaccine immunogens that structurally mimic the HMP and might elicit bnAbs targeting this conserved neutralization epitope. Herein, we report on the immunogenicity of glycopeptides, designed by in vitro selection, that bind tightly to anti-HMP antibody 2G12. By analyzing the fine carbohydrate specificity of rabbit antibodies elicited by these immunogens, we found that they differ from some natural human bnAbs, such as 2G12 and PGT128, in that they bind primarily to the core structures within the glycan, rather than to the Manα1 → 2Man termini (2G12) or to the whole glycan (PGT128). Antibody specificity for the glycan core may result from extensive serum mannosidase trimming of the immunogen in the vaccinated animals. This finding has broad implications for vaccine design aiming to target glycan-dependent HIV neutralizing antibodies.

Glycan density and linker composition significantly influence binding on glycan microarrays.