Synthetic Glycans Reveal Determinants of Antibody Functional Efficacy against a Fungal Pathogen

Abstract

Antibodies play a vital role in the immune response to infectious diseases and can be administered passively to protect patients. In the case of Cryptococcus neoformans, a WHO critical priority fungal pathogen, infection results in antibodies targeting capsular glucuronoxylomannan (GXM). These antibodies yield protective, non-protective, and disease-enhancing outcomes when administered passively. However, it was unknown how these distinct antibodies recognized their antigens at the molecular level, leading to the hypothesis that they may target different GXM epitopes. To test this hypothesis, we constructed a microarray containing 26 glycans representative of those found in highly virulent cryptococcal strains and utilized it to study 16 well-characterized monoclonal antibodies. Notably, we found that protective and non-protective antibodies shared conserved reactivity to the M2 motif of GXM, irrespective of the strain used in infection or GXM-isolated to produce a conjugate vaccine. Here, only two antibodies, 12A1 and 18B7, exhibited diverse trivalent GXM motif reactivity. IgG antibodies associated with protective responses showed cross-reactivity to at least two GXM motifs. This molecular understanding of antibody binding epitopes was used to map the antigenic diversity of two Cryptococcus neoformans strains, which revealed the exceptional complexity of fungal capsular polysaccharides. A multi-GXM motif vaccine holds the potential to effectively address this antigenic diversity. Collectively, these findings underscore the context-dependent nature of antibody function and challenge the classification of anti-GXM epitopes as either "protective" or "non-protective".

Keywords: Cryptococcus neoformans; antibodies; epitopes; glycans; immunology; vaccines.

Figure 1

Common structural motifs found in glucuronoxylomannan (GXM).

Figure 2

Library of synthetic glucuronoxylomannan glycans.

Figure 3

Microarray profile of several mAbs against synthetic GXM microarray. (a) Binding profile of seven mAbs. (b) Heatmap of IgGs binding to microarray. (c) Heatmap of IgMs binding to microarray. X-axes are glycan numbers defined in Figure 2.

Figure 4

Isotype switching alters GXM antibody specificity and affinity. (a) Microarray binding data for mAbs 3E5 IgG₁, 3E5 IgG_2a, 3E5 IgG_2b, 3E5 IgG₃, 3E5 IgE, and 3E5 IgA. (b) Heatmap of mAb 3E5 isotype variants binding to microarray. (c) Heatmap of mAb 4H3 isotype (IgG₁, IgG_2b, and IgG₃) variants binding to microarray. X-axis are glycan numbers defined in Figure 2.

Figure 5

(a) Multiple sequence alignment analysis of complementarity-determining regions (CDRs) of GXM-specific mAbs. CDRs are numbered according to the Chothia numbering scheme. Positions highlighted with orange boxes make contacts with the GXM antigen in the putative model of the 2H1:GXM (M2 motif) 10-mer 15. Positions highlighted in cyan boxes also make contact with the GXM antigen and are basic residues that contribute to the electropositive region of the paratope. (b) Representative model of the 2H1:GXM complex from Vina-Carb molecular docking, with the 2H1 Fab represented as an electrostatic surface. The GXM antigen is displayed with the mannan backbone and 6-O-acetylation as green and red spheres, and the xylose and glucuronic acid side chain glycans are displayed as orange and cyan surfaces. (c) The same model of the complex, with the 2H1 Fab displayed in cartoon representation and light- and heavy-chain CDRs highlighted in blue and red, respectively. (d) Expanded view of the interacting residues in this model, where contacting residues of mAb 2H1 are displayed as sticks and colored according to their CDR, as in panel c. The basic residues that form the electropositive pocket are highlighted with blue boxes. (e) Expanded view of the modeling results Motif 2 glycan docking the with mAb 2H1. Glycan side chains that are paratope- or solvent-facing are indicated with yellow and blue labels, respectively. (f) GXM 15 is extracted from the docking complex with 2H1 and rotated slightly to clearly demonstrate the solvent-exposed and paratope-interacting faces of the glycan, which are formed by alternating glycan branches along the mannan backbone. (g) Symbol Nomenclature for Glycans (SNFG) representation of 15.

Figure 6

Composite epitopes recognized by mAbs 12A1 and 13F1 and antibody localization on capsules of single-motif C. neoformans strains. (a) Full reactivity profiles of mAbs 12A1 and 13F1. Each individual glycan in the array for which binding was observed is displayed with transparency levels that reflect the relative degree of binding, and the glycan structures are grouped into boxes with respect to their motif composition: M1 (red), M2 (green), and M4 (yellow). Energy-minimized models of the composite epitopes-based reactivity with glycan structures in the array. The mannan backbone and 6-O-acetyl groups are displayed as green or red spheres, respectively. Xylose or glucuronic acid branches are displayed as space-filling surfaces and are colored orange and blue, respectively. β-1,4-Xylose groups are displayed with a degree of transparency that reflects their relative binding to M4 structures compared to M2. The minimal glycan length was defined as the structure that was bound with the highest affinity in the array for a given mAb. For 12A1, xylose groups on mannose-b^n and n-1 are colored a slightly lighter shade of orange to reflect the tolerance to the absence of these groups for these mAbs. (b) Immunofluorescence staining with mAbs 12A1 and 13F1 on a single motif expressing C. neoformans cells. Left panels: Immunofluorescence staining of Mu-1 (M2) and ATCC 24067 (M1) C. neoformans with mAb 12A1. Staining is annular for both strains. Right panels: Immunofluorescence staining of Mu-1 and ATCC 24067 was performed with mAb 13F1. Mu-1 staining is annular, and ATCC 24067 staining is punctate.