Stefan Oscarson

The innate immune protein human surfactant protein D (SP-D) recognises pathogens in the lungs via binding to carbohydrate surface structures. SP-D has been shown to target gram-negative bacterial lipopolysaccharide via calcium-dependent binding, preferentially to the inner core heptose (HepI). To further investigate this recognition, we have determined the high-resolution crystal structures of a trimeric recombinant fragment of human SP-D complexed with synthetic di- and trisaccharides, HepI-Kdo, HepIII-HepII-HepI, and HepII-HepI phosphorylated at either HepI or HepII, inner core motifs common to the lipopolysaccharide of many gram-negative bacteria. In contrast to acid-hydrolysed lipopolysaccharide used in several previous studies, these synthetic saccharides allow presentation of both the innermost Kdo in its natural pyranose form and heptose phosphorylation. The structures confirm the flexibility of SP-D to adopt an alternative binding mode when the preferred epitope is not available, reveal a preference for recognition of the reducing terminal heptose (HepI) via the glyceryl group, indicate that a single Kdo attached to HepI does not have a significant role in ligand recognition, and provide evidence that recognition of phosphorylated inner core diheptosyl ligands varies dependent on whether HepI or HepII is phosphorylated. The disaccharide with HepII O4’ phosphorylation binds via the preferred HepI glyceryl-hydroxyls, while HepI O4’ phosphorylation reveals HepII binding via the pyranose ring O3’ and O4’ hydroxyls. The ability of the HepII O4’ phosphate to prevent preferred HepI recognition suggests a role for heptose phosphorylation in shielding the bacterial LPS inner core from immune recognition.

Cryptococcus neoformans is a fungus classified by the World Health Organization as a critically important pathogen, which poses a significant threat to immunocompromised individuals. In this study, we present the chemical synthesis and evaluation of two semisynthetic vaccine candidates targeting the capsular polysaccharide glucuronoxylomannan (GXM) of C. neoformans. These semisynthetic glycoconjugate vaccines contain an identical synthetic decasaccharide (M2 motif) antigen. This antigen is present in serotype A strains, which constitute 95% of the clinical cryptococcosis cases. This synthetic oligosaccharide was conjugated to two proteins (CRM197 and Anthrax 63 kDa PA) and tested for immunogenicity in mice. The conjugates elicited a specific antibody response that bound to the M2 motif but also exhibited additional cross-reactivity toward M1 and M4 GXM motifs. Both glycoconjugates produced antibodies that bound to GXM in ELISA assays and to live fungal cells. Mice immunized with the CRM197 glycoconjugate produced weakly opsonic antibodies and displayed trends toward increased median survival relative to mice given a mock PBS injection (18 vs 15 days, p = 0.06). These findings indicate promise, achieving a successful vaccine demands further optimization of the glycoconjugate. This antigen could serve as a component in a multivalent GXM motif vaccine.

Cryptococcus neoformans is a fungal pathogen responsible for cryptococcosis and cryptococcal meningitis. The C. neoformans ’ capsular polysaccharide and its shed exopolysaccharide function both as key virulence factors and to protect the fungal cell from phagocytosis. Currently, a glycoconjugate of these polysaccharides is being explored as a vaccine to protect against C. neoformans infection. In this study, NOE and J -coupling values from NMR experiments were consistent with a converged structure of the synthetic decasaccharide, GXM10-Ac ₃ , calculated from MD simulations. GXM10-Ac ₃ was designed as an extension of glucuronoxylomannan (GXM) polysaccharide motif (M2) which is common in the clinically predominant serotype A strains and is recognized by protective forms of GXM-specific monoclonal antibodies. The M2 motif is a hexasaccharide with a three-residue α-mannan backbone, modified by β-(1→2)-xyloses (Xyl) on the first two mannoses (Man) and a β-(1→2)-glucuronic acid (GlcA) on the third Man. Combined NMR and MD analyses reveal that GXM10-Ac ₃ adopts an extended structure, with Xyl/GlcA branches alternating sides along the α-mannan backbone. O -acetyl esters also alternate sides and are grouped in pairs. MD analysis of a twelve M2-repeating unit polymer supports the notion that the GXM10-Ac ₃ structure is uniformly represented throughout the polysaccharide. This derived GXM model displays high flexibility while maintaining a structural identity, yielding insights to further explore intermolecular interactions between polysaccharides, interactions with anti-GXM mAbs, and the cryptococcal polysaccharide architecture.

Cryptococcus neoformans is a fungus classified by the World Health Organization as a critically important pathogen, posing a significant threat to immunocompromised individuals. In this study, we present the chemical synthesis and evaluation of two semi-synthetic vaccine candidates targeting the capsular polysaccharide glucuronoxylomannan (GXM) of C. neoformans. These semi-synthetic glycoconjugate vaccines contain the identical synthetic decasaccharide (M2 motif) antigen. This motif is present in serotype A strains, which constitute 95% of clinical cryptococcosis cases. This synthetic oligosaccharide was conjugated to two proteins (CRM197 and Anthrax 63 kDa PA) and tested for immunogenicity in mice. The conjugates elicited a specific antibody response that bound to the M2 motif but also exhibited additional cross-reactivity towards M1 and M4 GXM motifs. Both glycoconjugates produced antibodies that bound to GXM in ELISA assays and to live fungal cells. Mice immunized with the CRM197 glycoconjugate produced opsonic antibodies and displayed trends toward increased median survival relative to mice given a mock PBS injection (18 vs 15 days, p = 0.06). While these findings indicate promise, achieving a successful vaccine demands further optimization of the glycoconjugate. It could serve as a component in a multi-valent GXM motif vaccine, enhancing both strength and breadth of immune responses.

The synthesis of gram quantities of the TF antigen (β-ᴅ-Gal-(1→3)-α-ᴅ-GalNAc) and its 3’-sulfated analogue with a TEG-N₃ spacer attached is described. The synthesis of the TF antigen comprises seven steps, from a known N-Troc-protected galactosamine donor, with an overall yield of 31%. Both the spacer (85%) and the galactose moiety (79%) were introduced using thioglycoside donors in NIS/AgOTf-promoted glycosylation reactions. The 3’-sulfate was finally introduced through tin activation in benzene/DMF followed by treatment with a sulfur trioxide–trimethylamine complex in a 66% yield.

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".

Cryptococcus neoformans is a fungal pathogen responsible for cryptococcosis and cryptococcal meningitis. The C. neoformans capsular polysaccharide and shed exopolysaccharide functions both as a key virulence factor and to protect the fungal cell from phagocytosis. Currently, a glycoconjugate of these polysaccharides is being explored as a vaccine to protect against C. neoformans infection. In this combined NMR and MD study, experimentally determined NOEs and J -couplings support a structure of the synthetic decasaccharide, GXM10-Ac ₃ , obtained by MD. GXM10-Ac ₃ was designed as an extension of glucuronoxylomannan (GXM) polysaccharide motif (M2) which is common in the clinically predominant serotype A strains and is recognized by protective forms of GXM-specific monoclonal antibodies. The M2 motif is characterized by a 6-residue α-mannan backbone repeating unit, consisting of a triad of α-(1→3)-mannoses, modified by β-(1→2)-xyloses on the first two mannoses and a β-(1→2)-glucuronic acid on the third mannose. The combined NMR and MD analyses reveal that GXM10-Ac ₃ adopts an extended structure, with xylose/glucuronic acid branches alternating sides along the α-mannan backbone. O -acetyl esters also alternate sides and are grouped in pairs. MD analysis of a twelve M2-repeating unit polymer supports the notion that the GXM10-Ac ₃ structure is uniformly represented throughout the polysaccharide. This experimentally consistent GXM model displays high flexibility while maintaining a structural identity, yielding new insights to further explore intermolecular interactions between polysaccharides, interactions with anti-GXM mAbs, and the cryptococcal polysaccharide architecture. Significance Statement: This study utilized a combined NMR and MD approach to elucidate the structure of a Cryptococcus neoformans GXM synthetic decasaccharide (GXM10-Ac ₃ ), recognized by protective anti-GXM mAbs. The data revealed an extended structure in which the xylose/glucuronic acid branches and pairs of 6- O -acetyl esters predominantly alternate sides along the α-mannan backbone. MD analysis of a GXM polysaccharide predicts that the decasaccharide structure is uniformly represented in the polysaccharide. Additionally, the GXM exhibits high flexibility while maintaining structural identity. These findings lay the foundation for future studies aimed at understanding anti-GXM antibody-polysaccharide interactions.

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 fungal critical priority pathogen, infection results in antibodies targeting capsular glucuronoxylomannan (GXM). These antibodies produce diverse outcomes, however the molecular basis for antigen recognition remain unknown. To address this, we constructed a microarray containing twenty-six glycans representative of those found in highly virulent cryptococcal strains and utilised it to study sixteen monoclonal antibodies with distinct in vivo functions. Notably, protective and non-protective antibodies shared conserved reactivity to the M2 motif of GXM, irrespective of their origin from infection or vaccination. Interestingly, only two antibodies (12A1 and 18B7) exhibited diverse tri-valent GXM motif reactivity. Additionally, IgG subclass antibodies associated with protective responses showed cross-reactivity to at least two GXM motifs. The specificity and affinity of glycan binding were modulated by somatic mutation (12A1) and isotype switching (3E5 and 4H3), subsequently influencing their functional effects. This enhanced understanding of antibody binding epitopes was used to map the antigenic diversity of two Cryptococcus neoformans strains. Our results highlight the exceptional complexity of fungal capsular polysaccharides and underscore the imperative for developing multi-GXM motif vaccines. Such a vaccine holds the potential to elicit a comprehensive and effective immune response capable of neutralising the vast antigenic diversity presented by cryptococcal capsules.