Synthetic Glycans to Improve Current Glycoconjugate Vaccines and Fight Antimicrobial Resistance

Abstract

Antimicrobial resistance (AMR) is emerging as the next potential pandemic. Different microorganisms, including the bacteria Acinetobacter baumannii, Clostridioides difficile, Escherichia coli, Enterococcus faecium, Klebsiella pneumoniae, Neisseria gonorrhoeae, Pseudomonas aeruginosa, non-typhoidal Salmonella, and Staphylococcus aureus, and the fungus Candida auris, have been identified by the WHO and CDC as urgent or serious AMR threats. Others, such as group A and B Streptococci, are classified as concerning threats. Glycoconjugate vaccines have been demonstrated to be an efficacious and cost-effective measure to combat infections against Haemophilus influenzae, Neisseria meningitis, Streptococcus pneumoniae, and, more recently, Salmonella typhi. Recent times have seen enormous progress in methodologies for the assembly of complex glycans and glycoconjugates, with developments in synthetic, chemoenzymatic, and glycoengineering methodologies. This review analyzes the advancement of glycoconjugate vaccines based on synthetic carbohydrates to improve existing vaccines and identify novel candidates to combat AMR. Through this literature survey we built an overview of structure-immunogenicity relationships from available data and identify gaps and areas for further research to better exploit the peculiar role of carbohydrates as vaccine targets and create the next generation of synthetic carbohydrate-based vaccines.

Figure 1

Surface glycans commonly found on Gram-positive and Gram-negative bacteria. Gram-positives lack an outer membrane and present a thick peptidoglycan (PG) layer, to which lipoteichoic (LTA) and wall teichoic acids (WTA) are connected. The Gram-negative bacterial cell wall features two membranes and a thin PG layer. Both can be surrounded by capsular polysaccharide (CPS). The Gram-negative outer membrane is decorated with lipopolysaccharide (LPS) or a truncated form lacking of the O-antigen, termed lipooligosaccharide (LOS).

Figure 2

Approaches for manufacturing of glycans for vaccine development based on (A) bacterial fermentation, including classic polysaccharide extraction from pathogens and Protein Glycan Coupling Technology (PGCT) and (B) total synthesis of oligosaccharides via solution phase chemical/chemoenzymatic approaches or solid phase automated synthesis.

Figure 3

Synthetic polyribosyl-ribitol-phosphate (PRP) antigen of Quimi-Hib, the first synthetic glycan-based vaccine for prevention of Haemophilus influenzae type b, is produced by a controlled polycondensation of a H-phosphonate building block.

Figure 4

Meningococcal capsular polysaccharide structures and related synthetic conjugates. On the right the structure of the repeating unit of the natural CPS is described. Synthetic structures include copies of the natural polysaccharide (4–6) and analogues (7 and 8). In conjugate (6) the 11-mer molecule was obtained in a one-pot fashion using the enzymatic elongation of a synthetic acceptor.

Figure 5

Pneumococcal vaccine candidates based on synthetic glycans. Synthetic oligosaccharides mimicking several Streptococcus pneumoniae serotypes (ST) have been synthesized and tested at the preclinical level to identify key glycan epitopes.

Figure 6

Pseudaminic acid (marked in red) containing octasaccharide conjugated to CRM₁₉₇ as vaccine candidate against A. baumannii. Raised antibodies recognized the exopolysaccharide (EPS) and induced bacterial killing.

Figure 7

(A) Chemical structures of C. difficile PSI, PSII, and PSIII. (B) Synthetic routes for PSI and related fragments have been developed enabling identification of the disaccharide epitope (26).

Figure 8

(A) Synthetic fragments of PSII from C. difficile. (B) Recognition of PSII on ELISA by the conjugated fragments showed the importance of the phosphate group for cross-reactivity with the natural PS. (C) Recognition of PSII on the bacterial surface assessed by confocal microscopy with anti-PSII (left part) and anti-(27) antibodies (right part). Adapted with permission from ref (180). Copyright 2012 American Chemical Society.

Figure 9

Synthetic routes for conjugates of the E. coli O-antigen core pentasaccharide (A) and repeating unit of O1 (B). A conjugate of the core pentasaccharide was shown to induce functional antibodies. The conjugate of the O1 fragment was recognized by anti-O1 chicken serum. The β-Rha-(1–3)-GlcNAc linkage was successfully generated by boron mediated insertion of the GlcNAc building block in the rhamnose-1,2-epoxide intermediate. The assembled structure was deprotected and conjugated to BSA (40). This conjugate was shown to recognize chicken anti-O1 serum, using an ELISA assay.

Figure 10

Enterococcus faecalis and faecium synthetic antigens, including capsular polysaccharides (DHG), as well as lipoteichoic acids (LTA) and wall teichoic acids (WTA). Glycoconjugates of synthetic DHG (45 and 46) and LTA (51) fragments elicited antibodies in rabbit, inducing bacterial opsonophagocytic killing (OPK) in vitro and providing protection from bacterial colonization in vivo.

Figure 11

Synthetic strategies for group A Streptococcus antigens and collected immune data. (A) Immunization with a GAS cell wall hexasaccharide conjugated to BSA through squarate chemistry induced antibodies identical to a natural CPS-TT conjugate. In silico conformational analysis of the immunogenic fragment suggested that the polyrhamnose backbone forms a helix with the GlcNAc moieties on the periphery. Adapted with permission from ref (230). Copyright 2004 Elsevier). (B) The GAS dodecasaccharide conjugated to CRM₁₉₇ elicited a high titer of anti-GAS opsonic antibodies. (C) Chemoenzymatically assembled nonasaccharide conjugated to the GAS protein ScpA (GAS C5a peptidase) induced antibodies against both antigens. (D) Site-selective conjugation of natural GAS polysaccharide to the streptococcal protein SpyAD induced antibodies able to provide in vivo protection in mice. (E) GAS trisaccharide conjugated to a synthetic peptidolipid, containing the T-cell peptide epitope PADRE.

Figure 12

(A) X-ray crystallography of semisynthetic GBS III-DP2 (76) complexed with an anti PSIII rabbit Fab. (B) Combining data from STD-NMR and X-ray crystallography led to the identification of GBSIII structural minimal epitope highlighted in blue corresponding to hexasaccharide (76), which was synthesized accordingly. (C) The hexasaccharide (76) was conjugated to CRM₁₉₇ carrier protein through the SIDEA activation chemistry and tested in mice. (D) Hexa-CRM₁₉₇ conjugate elicited functional antibodies comparable to the positive control PSIII-CRM₁₉₇.

Figure 13

Synthetic routes for K. pneumoniae glycans for immunological testing: (A) Design of conjugates from synthetic fragments of the K2 capsule used for immunization of rabbits. (B) Conjugates from Kp ST238 CPS, that have been tested in rabbits, providing antibodies with opsonophagocytic killing (OPK) activity in vitro. (C) Structures related to O1 and O2 O-antigens used in the glycan array.

Figure 14

Chemical structure of N. gonorroheae LOS, composed of three characteristic α, β and γ chains. Highlighted is the epitope identified through the C27 mAb.

Figure 15

Synthetic strategy for P. aeruginosa Psl oligosaccharides and immune evaluation with class I, II, and II mAbs. Of these three mAbs, while class II and III allowed epitope mapping, class I did not map any epitope, suggesting that the structure is lacking motifs relevant for antibody recognition.

Figure 16

Synthetic approaches for conjugates from Salmonella oligosaccharides: (A) Fragments of S. Typhi capsular polysaccharide used to identify the minimal epitope length. (B) Design of conjugates against S. Enteritidis and Paratyphi and immunological evaluation in animal models.

Figure 17

Assembly and immunological evaluation of staphylococcal surface carbohydrates, including capsular polysaccharides and teichoic acids. (A) Repeating unit structure of capsular polysaccharides CP8 and CP5. (B) Synthesis of CP5 trisaccharide, which was recognized by murine anti-CP5 serum. (C) Synthesis of the CP8 trisaccharide repeating unit, which, conjugated to CRM₁₉₇, elicited anti-glycan antibodies in mice. (D) Assembly of a 10-mer glycerol phosphate (GroP) polymer and conjugation to TT for immunological evaluation. (E) Synthesis of ribitol-phosphate (RboP) oligomers. (F) Solution phase synthesis of 8-mer and 12-mer RboP oligomers and conjugation to BSA. (G) Example of WTA RboP substituted tetramer tested in mice. (H) Wall teichoic acid (WTA) fragments conjugated to recombinant P. aeruginosa exotoxin A (rEPA) induced murine antibodies providing in vitro opsonophagocytic killing (OPK).

Figure 18

(A) PNAG structure and design of a universal PNAG-based universal vaccine and therapeutic mAb. (B) Synthetic route to the enterobacterial common antigen (ECA) conjugate used to generate specific sera and mAbs.

Figure 19

Vaccine candidates against Candida infections: (A) Different types of synthesized β-(1–2)-mannan conjugates used tested in animal models. (B) β-(1–3)-Glucan conjugates of different lengths enabling identification of a minimal hexamer epitope.