A Structural Model for the Ligand Binding of Pneumococcal Serotype 3 Capsular Polysaccharide-Specific Protective Antibodies

Abstract

Capsular polysaccharides (CPSs) are major virulence factors that decorate the surfaces of many human bacterial pathogens. In their pure form or as glycoconjugate vaccines, CPSs are extensively used in vaccines deployed in clinical practice worldwide. However, our understanding of the structural requirements for interactions between CPSs and antibodies is limited. A longstanding model based on comprehensive observations of antibody repertoires binding to CPSs is that antibodies expressing heavy chain variable gene family 3 (VH3) predominate in these binding interactions in humans and VH3 homologs in mice. Toward understanding this highly conserved interaction, we generated a panel of mouse monoclonal antibodies (MAb) against Streptococcus pneumoniae serotype 3 CPS, determined an X-ray crystal structure of a protective MAb in complex with a hexasaccharide derived from enzymatic hydrolysis of the polysaccharide, and elucidated the structural requirements for this binding interaction. The crystal structure revealed a binding pocket containing aromatic side chains, suggesting the importance of hydrophobicity in the interaction. Through mutational analysis, we determined the amino acids that are critical in carbohydrate binding. Through elucidating the structural and functional properties of a panel of murine MAbs, we offer an explanation for the predominant use of the human VH3 gene family in antibodies against CPSs with implications in knowledge-based vaccine design. IMPORTANCE Infectious diseases caused by pathogenic bacteria are a major threat to human health. Capsular polysaccharides (CPSs) of many pathogenic bacteria have been used as the main components of glycoconjugate vaccines against bacterial diseases in clinical practice worldwide, with various degrees of success. Immunization with a glycoconjugate vaccine elicits T cell help for B cells that produce IgG antibodies to the CPS. Thus, it is important to develop an in-depth understanding of the interactions of carbohydrate epitopes with the antibodies. Structural characterization of the ligand binding of polysaccharide-specific antibodies laid out in this study may have fundamental biological implications for our comprehension of how the humoral immune system recognizes polysaccharide antigens, and in future knowledge-based vaccine design.

Keywords: Streptococcus pneumoniae; VH3 gene family; capsular polysaccharide; carbohydrate antigens; conjugate vaccines; glycoconjugate vaccine; monoclonal antibodies; monoclonal antibody.

FIG 1

(A) Pn3P-specific monoclonal antibodies can bind to Pn3 hexasaccharide and tetrasaccharide. ELISA plates were coated with Pn3P-HSA and MAbs were premixed with Pn3P, Pn3 hexasaccharide, Pn3 tetrasaccharide, or PBS and then added to the wells. Both oligosaccharides inhibited antibody binding to polysaccharide, with the inhibition by Pn3 hexasaccharide significantly stronger. The PBS control group was considered to have 100% binding, against which the experimental groups were normalized. Using a two-tailed Student’s t test, statistical significance was determined compared to no- inhibitor wells; **, P < 0.01; *, P < 0.05; ns, not significant. (B and C) Pn3 hexasaccharide can dissociate Pn3P-bound antibodies, whereas tetrasaccharide cannot. In a BLI experiment, avidin sensors loaded with biotinylated Pn3P were incubated with MAb 75.3 (B) or MAb 5.6 (C) for 300 s (I). After incubation in BLI assay buffer with no competitor for 60 s (II), sensors were incubated in 50 μg/ml of either tetrasaccharide or hexasaccharide to compete with the polysaccharide for 600 s (III).

FIG 2

Crystal structure of Pn3 trisaccharide bound to MAb 5.6 Fab. (A) The crystal structure of MAb 5.6 Fab and the trisaccharide portion of Pn3 hexasaccharide was solved at 2.3 Å resolution. The trisaccharide Glc-GlcA-Glc fits to the electron density observed at the antigen binding site of the MAb 5.6 Fab and is depicted with sticks. Heavy and light chains are colored as orange and gray, respectively. (B) Fab is depicted with surfaces and the trisaccharide with sticks. Heavy and light chains are colored as orange and gray, respectively. (C) Antigen-binding site of the Fab is depicted with surfaces and the trisaccharide with sticks. Hydrophobic residues on the binding groove are colored as cyan, asparagine and serine residues involved in electrostatic and hydrogen binding interactions with the trisaccharide are colored as blue, and other residues as yellow. (D) There are seven aromatic residues around the ligand. The GlcA residue of the trisaccharide forms an aromatic stacking with Y66 of the heavy chain and a probable aromatic stacking with Y110 of the light chain. (E) The trisaccharide forms a hydrogen bond and electrostatic interactions with the side chains of S55 and R107. Three water bridges are formed between the trisaccharide and backbone atoms of G113, T109, and I58. The side chain of R107 is also involved in the water bridge formed between the trisaccharide and G113.

FIG 3

Hydrophobic amino acids of the VH3 gene family contribute to carbohydrate binding. (A) Hydrophobic amino acids of MAb 5.6 heavy chain variable gene segment located around the trisaccharide are indicated by arrows. Sequence logos for seven human VH gene families were generated to show conserved amino acids within the families. Amino acids indicated with stars were mutated to assess their potential roles in ligand binding. (B) In an ELISA, ligand binding of wild type and MAb 5.6 single site mutants M39W, V53I, G62S, Y66N, W52G, S55A, and R107A were evaluated; “>” indicates no signal was observed below 2,222 ng/ml. (C) Mutations on V53 (cyan) and G62 (orange) did not alter the ligand binding. (D) Mutations on M39 (green), Y66 (purple), W52 (red), R107 (light blue), and S55 (blue) reduced the ligand binding of 5.6 MAb. (E) R107 (light blue) in HCDR3 is essential for ligand binding. (F) M39 (green), Y66 (purple), W52 (red), and S55 (blue) are in framework regions and essential for ligand binding.

FIG 4

Pn3P-specific MAbs are protective. (A) MAbs were tested in a standard OPKA using differentiated HL-60 cells. The Spn3 WU2 strain was opsonophagocytosed with antibodies and HL-60 cells and plated onto blood agar plates to count CFU. Data are averages of duplicate samples. Statistical significance was determined with the two-tailed Student’s t test; **, P < 0.01. (B and C) The Spn3 B2 strain was incubated with increasing concentrations of various MAbs and nonspecific mouse IgG1 (control) and analyzed by flow cytometry. (B) Representative fluorescence-activated cell sorting (FACS) dot plots show the percentage of agglutination for all MAbs and isotype control (IgG1) at various concentrations. (C) Graph shows the percentage of agglutination for all samples with increasing concentrations of MAbs (1 to 20 μg/ml). Results represent 2 independent experiments. Statistical significance was determined with the two-tailed Student’s t test; ***, P < 0.001; ****, P < 0.0001. (D) Following i.p. injection of MAb 5.6 or its isotype control, two groups of mice were challenged with lethal dose of Spn3 WU2 strain. Graph shows the percentage of surviving mice in each group in a 10-day period. Statistical significance was determined with the logrank (Mantel-Cox) test; ***, P < 0.001.