Modeling of pneumococcal serogroup 10 capsular polysaccharide molecular conformations provides insight into epitopes and observed cross-reactivity

Abstract

Streptococcus pneumoniae is an encapsulated gram-negative bacterium and a significant human pathogen. The capsular polysaccharide (CPS) is essential for virulence and a target antigen for vaccines. Although widespread introduction of pneumococcal conjugate vaccines (PCVs) has significantly reduced disease, the prevalence of non-vaccine serotypes has increased. On the basis of the CPS, S. pneumoniae serogroup 10 comprises four main serotypes 10A, 10B, 10C, and 10F; as well as the recently identified 10D. As it is the most prevalent, serotype 10A CPS has been included as a vaccine antigen in the next generation PCVs. Here we use molecular modeling to provide conformational rationales for the complex cross-reactivity reported between serotypes 10A, 10B, 10C, and 10F anti-sera. Although the highly mobile phosphodiester linkages produce very flexible CPS, shorter segments are conformationally defined, with exposed $\beta$ -D-galactofuranose ( $\beta$ DGalf) side chains that are potential antibody binding sites. We identify four distinct conformational epitopes for the immunodominant $\beta$ DGalf that assist in rationalizing the complex asymmetric cross-reactivity relationships. In particular, we find that strongly cross-reactive serotypes share common epitopes. Further, we show that human intelectin-1 has the potential to bind the exposed exocyclic 1,2-diol of the terminal $\beta$ DGalf in each serotype; the relative accessibility of three- or six-linked $\beta$ DGalf may play a role in the strength of the innate immune response and hence serotype disease prevalence. In conclusion, our modeling study and relevant serological studies support the inclusion of serotype 10A in a vaccine to best protect against serogroup 10 disease.

Keywords: S. pneumoniae; capsular polysaccharide; carbohydrate epitopes; cross-protection; molecular modeling; serogroup 10; vaccine antigen.

FIGURE 1

S. pneumoniae serogroup 10 rabbit antisera cross-reactivity trends showing the cross-reactivity of antisera ( $\mathit{\alpha }$ ) raised against serogroup 10 CPSs ( $\mathit{\alpha }$ Pn10A, $\mathit{\alpha }$ Pn10B, $\mathit{\alpha }$ Pn10C, and $\mathit{\alpha }$ Pn10F) (Henrichsen, 1995). Self-cross-reactivity titer was set as 100% with 50% self-titer, 25% self-titer, and 3%–7% self-titer represented by solid arrows, dashed arrows, and dotted arrows, respectively.

FIGURE 2

S. pneumoniae serogroup 10 CPS repeating unit structures for (A) Pn10A, (B) Pn10B, (C) Pn10C, and (D) Pn10F represented with the SNFG (Symbol Nomenclature for Glycans) system (Varki et al., 2015). Similarities and differences in repeating units are indicated by background color shading.

FIGURE 3

(A) The end-to-end distance, r, is indicated on the 6 RU Pn10A molecule. Time series graphs (left column) of r and corresponding histograms (right column) for the 3,000 ns simulation trajectories are shown for: (B) Pn10A, (C) Pn10B, (D) Pn10C, and (E) Pn10F. Conformational snapshots at 50 ns intervals are shown above the time series plots. The histograms are labeled with the standard deviations ( $\mathit{\sigma }$ ) and modal peak r value(s).

FIGURE 4

Main conformational families identified for the 6 RU S. pneumoniae serotype CPS, with the terminal repeat units (RU 1 and RU 6) excluded from analysis: (A) Pn10A, (B) Pn10B, (C) Pn10C, and (D) Pn10F. Backbone galactose residues are shown in yellow, ribitol residues shown in grey, phosphate in purple, side group galactofuranose residues in orange, and side group galactopyranose residues in light green. The primary clusters occurred regularly throughout the trajectory. The secondary cluster of Pn10B occurred regularly in the second half of the simulation along with the primary cluster; for Pn10C the secondary cluster occurred only for ∼400 ns around the 1,500 ns timestep.

FIGURE 5

Main conformational families identified for RU 4 in the backbone of 6 RU S. pneumoniae serotype CPS: (A) Pn10A, (B) Pn10B, (C) Pn10C, and (D) Pn10F. Backbone galactose residues shown in yellow, Rib-ol-5P residues shown in grey, phosphate in purple, side group $\mathit{\beta }$ Galf residues in orange, side group $\mathit{\beta }$ Gal residues in light green, ETD portion of $\mathit{\beta }$ Galf in green, and ETD portion of Rib-ol-5P residues in cyan.

FIGURE 6

Conformational epitopes of the $\mathit{\beta }$ DGalNAc— $\mathit{\beta }$ DGalf disaccharide in S. pneumoniae serogroup 10, RU 4. (A) Super-imposed conformational families (>5%) of the $\mathit{\beta }$ DGalNAc— $\mathit{\beta }$ DGalf disaccharide with associated percentages for the four CPS molecules. (B) Representative conformational epitopes (EP1-4) of the $\mathit{\beta }$ DGalNAc— $\mathit{\beta }$ DGalf disaccharide for the four CPS molecules, with associated percentage occupancies, grouped according to conformational similarity.

FIGURE 7

Human intelectin-1 (hIntL-1) binding. (A) Monomer of the crystal structure of hIntL-1 bound with allyl-beta-galactofuranose (PDB ID: 4WMY). Example conformations of the 3 RU CPSs with the $\mathit{\beta }$ DGalf side chain positioned in the hIntL-1 binding site are shown for (B) Pn10A, (C) Pn10B, (D) Pn10C, and (E) Pn10F. The end-to-end distance (r) of these conformations are 33 Å, 41 Å, 16 Å, and 33 Å for Pn10A, Pn10B, Pn10C, and Pn10F, respectively. Overlay of our CPS molecules with the hIntL-1 protein binding site were created by aligning the O4, C5, O5, C6, and O6 atoms of the central side group (RU 2) $\mathit{\beta }$ DGalf residue from each CPS molecule with that of the allyl $\mathit{\beta }$ DGalf in the hIntL-1 binding site. We then identified frames where the molecular conformation aligns with the hIntL-1 binding site free from protein-CPS intersection with good alignment of the galactofuranose rings and exocyclic terminal-1,2-diol (ETD) moieties.