Long, Synthetic Staphylococcus aureus Type 8 Capsular Oligosaccharides Reveal Structural Epitopes for Effective Immune Recognition

Abstract

Staphylococcus aureus is a Gram-positive bacterium that is responsible for severe nosocomial infections. The rise of multidrug-resistant strains, which can pose significant health threats, prompts the development of new treatment interventions, and much attention has been directed at the development of prophylactic and therapeutic vaccination strategies. Capsular polysaccharides (CPs) are key protective elements of the S. aureus cell wall and have been proposed as promising candidate antigens. Thirteen different CP serotypes have been identified to date, of which types 5 and 8 are the most prominent. CP8 is composed of trisaccharide repeating units that are built up from an N-acetyl-4-O-acetyl-β-d-mannosaminuronic acid, that carries a C-4-O-acetyl, an N-acetyl-α-d-fucosamine, and an N-acetyl-α-l-fucosamine. Synthetic oligosaccharides are valuable tools to unravel the immunogenicity of bacterial oligosaccharides at the molecular level. However, the rare monosaccharides, cis-glycosidic linkages, and O-acetylation represent significant challenges for the synthesis of CP8 fragments. Here the stereoselective assembly of well-defined CP8 fragments, comprising a trimer, hexamer, nonamer, and dodecamer, is presented. This is the first time that fragments larger than a single repeating trisaccharide, which has been proven to be insufficient for antigenic activity, have been assembled. Structural studies have revealed a linear conformation for the oligosaccharides, with each trisaccharide repeat tilted ∼90° with respect to the flanking repeats, which is stabilized by the acetyl groups that prevent rotation around the glycosidic linkages. The N-acetyl groups in each repeating unit point in the same direction, generating a hydrophobic flank in the trisaccharide repeats. We applied the oligomers to generate model glycoconjugate vaccine modalities, which we then used to raise anti-CP8 antibodies. The antibody interaction and immunization studies have revealed a clear length dependent structure-activity relationship for the oligosaccharides, with an oligosaccharide of at least three repeating units required for an adequate immune response.

Figure 1

Representation of the common RUs of CP5 and CP8.

Figure 2

Previously synthesized trisaccharides and a dodecamer were synthesized in this work.

Scheme 1. Retrosynthetic Analysis of the Set of Target CP8 Oligosaccharides

Scheme 2. Synthesis of Target Oligosaccharides

Reaction conditions: a) DDQ, DCM/H₂O, 86%, b) 12a, NIS, TfOH, DCM, −78 °C → −10 °C, 44%, α:β = 25:75 or 12b, TfOH, DCM, −78 °C → −10 °C, 66%, α:β = 14:86, c) TBAF, AcOH, THF, 0 °C → rt °C, 84%, d) ClC(=NPh)CF₃, K₂CO₃, acetone, 93%, e) TMSI, Ph₃P=O, DCM/Et₂O, 83%, α:β = 75:25, f) DDQ, DCM/H₂O, 18 = 80%, 19 = 54%, 20 = 57%, g) 16, TBSOTf, DCM, 6 = 87%, 7 = 77%, 8 = 68%, h) Zn, AcOH, Ac₂O, THF, 50 °C, (i) Pd(OH)₂/C, AcOH, H₂, t-BuOH/H₂O, yield over two steps 1 = 45%, 2 = 37%, 3 = 57%, 4 = 33%, j) 1 M NaOH in H₂O, 2-deAc = 41%, 3-deAc = 46%.

Figure 3

A) Conjugation of the synthetic fragments. 1) Suberic acid bis(N-hydroxysuccinimide ester) 30 equiv for 1 and 15 equiv for 2–4 in DMSO/H₂O 9:1, 2) CRM₁₉₇ in PBS or HEPES 25 nM. B) SDS-page with conjugates CRM1–4. C) Western Blot performed with an anti-CP8 mAb showed recognition of CRM2–4. BSA-Pel-CRM was included as a negative control. D) Competitive ELISA with anti mAb-CP8. E) Competitive ELISA with polyclonal anti-CP8 serum.

Figure 4

Conformational analysis of trisaccharide 1 and of nonasaccharide 3 as established by NMR and MM calculations. A) Zoom area of the 2D NOESY spectrum of trisaccharide 1 (top) and its main conformation as defined by NOE analysis and MM calculations (bottom). B) Zoom area of the 2D NOESY spectrum of the nonasaccharide 3 (top) and its main conformation as defined by NOE analysis and MM calculations (bottom). Monosaccharidic residues are labeled with a letter code. The main conformation at each glycosidic linkage, the spatial orientation of the acetyl groups, and the average length are reported.

Figure 5

¹H STD-NMR spectra performed for the complexes of mAb-CP8 and the trisaccharide 1 (A), the deacetylated hexasaccharide 2-deAc (B), the hexasaccharide 2 (C), and the nonasaccharide 3 (D). Off-resonance spectra (in red) and the corresponding STD-NMR spectra at 310 K (in black) and at 288 K (in gray). The representation of the epitope map disclosed by the analysis of the relative STD-NMR signal intensities for each oligosaccharide is reported as a color legend associated with the STD% values.

Figure 6

A) Schematic illustration of the in vivo study. Injections were performed at day 1, day 22, and day 36, and a bleed was performed at day 0, day 35 (post 2), and day 50 (post 3). B) ELISA post 2 (P2) and post 3 (P3) IgG titers.