Structure and Immune Recognition of the HIV Glycan Shield

Abstract

Vaccine design efforts against the human immunodeficiency virus (HIV) have been greatly stimulated by the observation that many infected patients eventually develop highly potent broadly neutralizing antibodies (bnAbs). Importantly, these bnAbs have evolved to recognize not only the two protein components of the viral envelope protein (Env) but also the numerous glycans that form a protective barrier on the Env protein. Because Env is heavily glycosylated compared to host glycoproteins, the glycans have become targets for the antibody response. Therefore, considerable efforts have been made in developing and validating biophysical methods to elucidate the complex structure of the Env-spike glycoprotein, with its combination of glycan and protein epitopes. We illustrate here how the application of robust biophysical methods has transformed our understanding of the structure and function of the HIV Env spike and stimulated innovation in vaccine design strategies that takes into account the essential glycan components.

Keywords: EM structure; X-ray structure; broadly neutralizing antibody; glycan composition; glycosylation; viral glycoprotein.

Figure 1

Model of the processing status of individual N-linked glycosylation sites on the BG505 SOSIP.664 trimer. The model is adapted from Behrens et al. (7, 8) and is constructed from the cryo-EM structure of glycosylated SOSIP (78) with terminal glycans modelled in that are not apparent in the cryo-EM maps. The protein surface is depicted in gray and the surface of the glycans are coored according to the percentage of high-mannose gycans.

Figure 2

Examples of bnAb recognition of glycans. A. The light chain (dark gray) of 3BNC117 (PDB 5V8L), a CD4bs-directed bnAb, interacts primarily with the core GlcNAc moieties (blue) at the base of the glycan and less with the branched Man glycans (green). Note that the glycans are only resolved to Man-6 with the terminal branching glycans (indicated by arrows) disordered in the cryoEM map. B. The heavy chain (dark gray) of PGT128 (PDB 5ACO), an N332 supersite bnAb, interacts with extended Man-8 (shown) or Man-9 glycans. Because these glycan moieties interact strongly with the bnAb heavy (dark gray) and light (light gray) chains, they are typically well resolved in crystallographic or cryoEM maps. In fact, PGT128 binds high mannose so well that the structure of the Fab and Man-9 alone was solved by crystallography (PDB 3TV3) (103). Man-8 is resolved in the figure shown. C. The heavy chain (dark gray) of PGT151 (PDB 5FUU), a gp120/gp41 interface bnAb interacts with minimally a tri-antennary complex glycan. Glycan array binding data suggest that this N611 glycan may be contain terminal sialic acid residues (indicated by magenta *), but these were not resolved in the cryo-EM map. D. The heavy chain (dark gray) of PG16 (PDB 4DQO) interacts with a terminal sialic acid of a hybrid-type glycan at N173.

Figure 3

Glycan flexibility and resolution. A. Glycans exhibit differing degrees of flexibility wherein the base is constrained and the two faces of the glycan remain, whereas the branched glycans have additional degrees of freedom. Shown are example conformations for Man-5 (purple, PG9 bound; PDB 3U4E) or Man-6 (green, PGT145 bound; PDB 5V8L) of N160 and Man-8 (brown, PGT128 bound; PDB 5ACO) and Man-8 (cyan, 10-1074 bound; PDB 5T3X) of N332. B. Ideal density (mesh) for a Man-9 (white sticks) glycan filtered to resolutions ranging from 2-20 Å. C. Cryo-EM density map (EMD-3308) filtered to different resolutions highlighting several gp120 glycans. At the lowest resolution (3–4 Å), only the first 2–3 glycan moieties are observed, due to conformational variability in the extended sugars. In the case of the glycan at N448, which interacts with the bnAb PGT151, extended branching is observed. As the cryo-EM density map is filtered to progressively higher resolutions, more of the glycan density is observed and the map captures some of the conformational heterogeneity. Glycosylation sites that contain a heterogeneous population of glycoforms (as confirmed by MS) can further blur the resolution. Even at the highest filtered resolution cryo-EM maps (10 Å), the full extent of glycans is not resolved to the same extent as the idealized density shown in Panel B. This supports a high degree of conformational flexibility of the branched portions of glycans.

Figure 4

Penetrating the glycan shield. Early structures of bnAbs bound to small portions of gp120 revealed how bnAbs deployed long heavy chain CDR3 loops to insert between glycans and access the underlying peptide surface of Env. A. In PGT128, an N332 supersite bnAb, CDRH3 is sandwiched between glycans at N301 and N332. B. In PG9, an apex bnAb, CDRH3 is sandwiched between N156 and N160. C. VRC38 also targets N156 and N160 Man-5 glycans (20). D. PGT145, another apex bnAb inserts its very long CDRH3 between three symmetrically related N160 glycans at the three-fold axis. In all cases, one glycan more intimately interacts with the bnAb and is more fully resolved in the structures.