Glycan Array Evaluation of Synthetic Epitopes between the Capsular Polysaccharides from Streptococcus pneumoniae 19F and 19A

Abstract

Vaccination represents the most effective way to prevent invasive pneumococcal diseases. The glycoconjugate vaccines licensed so far are obtained from capsular polysaccharides (CPSs) of the most virulent serotypes. Protection is largely limited to the specific vaccine serotypes, and the continuous need for broader coverage to control the outbreak of emerging serotypes is pushing the development of new vaccine candidates. Indeed, the development of efficacious vaccine formulation is complicated by the high number of bacterial serotypes with different CPSs. In this context, to simplify vaccine composition, we propose the design of new saccharide fragments containing chemical structures shared by different serotypes as cross-reactive and potentially cross-protective common antigens. In particular, we focused on Streptococcus pneumoniae (Sp) 19A and 19F. The CPS repeating units of Sp 19F and 19A are very similar and share a common structure, the disaccharide ManNAc-β-(1→4)-Glc (A-B). Herein, we describe the synthesis of a small library of compounds containing different combinations of the common 19F/19A disaccharide. The six new compounds were tested with a glycan array to evaluate their recognition by antibodies in reference group 19 antisera and factor reference antisera (reacting against 19F or 19A). The disaccharide A-B, phosphorylated at the upstream end, emerged as a hit from the glycan array screening because it is strongly recognized by the group 19 antisera and by the 19F and 19A factor antisera, with similar intensity compared with the CPSs used as controls. Our data give a strong indication that the phosphorylated disaccharide A-B can be considered a common epitope among different Sp 19 serotypes.

Figure 1

(a) Natural repeating units of S. pneumoniae 19F and 19A CPSs highlighting their common structures, the disaccharide A-B. (b) Synthesized oligomers related to different combinations of the common disaccharide A-B [ManNAc-β-(1→4)-Glc].

Scheme 1. Synthetic Approach to Target Compounds 1–6

Reagents and conditions: (a) TESOTf, DCM, 4 Å molecular sieves (MS), −20 °C, 74%; (b) MeONa, DCM/MeOH, 93%; (c) SO₂Im₂, NaH_60%, DMF, −40 °C, 75%; (d) NaN₃, DMF dry, 80 °C, 90%; (e) Zn, CuSO₄·5H₂O, THF/Ac₂O/AcOH, 66%; (f) Pd(OH)₂/C, H₂, EtOAc/MeOH/HCl, 99%; (g) Et₃SiH, BF₃·Et₂O, DCM, 0 °C, 4 Å MS, 80%; (h) (BnO)₂PN(iPr)₂, tetrazole, DCM, then mCPBA, −20 to 0 °C, 80%; (i) Pd/C, H₂, MeOH/H₂O, 80%; (j) TMSOTf, DCM, 4 Å MS, 0 °C to room temperature (rt), 55%; (k) Pd(OH)₂/C, H₂, EtOAc/MeOH/HCl, 95%; (l) TESOTf, DCM, 4 Å MS, −20 °C, 85%; (m) Zn, CuSO₄·5H₂O, THF/Ac₂O/AcOH, 30–47%; (n) Pd(OH)₂/C, H₂, EtOAc/MeOH/HCl, quant; (o) Cl(iPr₂N)P(OCH₂CH₃CN), DIPEA, DCM, 90%; (p) DCI, DCM, 95%; (q) tBuOOH, ACN, 0 °C to rt, 73% (55% α); (r) TEA, DCM, 4 days, 80%; (s) Pd/C, H₂, MeOH/H₂O, 90%. DIPEA, N,N-diisopropylethylamine; DCI, 4,5-dicyanoimidazole; ACN, acetonitrile.

Figure 2

IgG binding of reference group 19 antisera recognizing the common epitope to all CPSs belonging to Sp group 19. The vertical axis represents the averaged serum IgG binding as relative fluorescence units (corrected over background). The horizontal axis shows the different synthetic structures printed on the glycan microarray. CPSs from Sp 19A and Sp 19F were used as positive controls as both contain the group 19 common epitope. Each bar corresponds to the median value from three replicates (represented as individual values with the circles) of the IgG binding to the printed Sp 19 common epitopes. Raw data of glycan array analyses are reported in Figure SI-3.

Figure 3

IgG in infected reference sera can recognize different synthetic Sp 19 common epitopes printed on the glycan microarray. The vertical axis represents the averaged serum IgG binding as relative fluorescence units (corrected over background). The horizontal axis shows the different synthetic structures printed on the glycan microarray. CPSs from Sp 19F and Sp 19A were used as positive or negative controls, depending on the tested sera. Each bar corresponds to the median value from three replicates (represented as individual values with the circles) of the IgG binding to the printed Sp 19 common epitopes. (a) IgG binding of reference sera from rabbits immunized with Sp 19F. (b) IgG binding of reference sera from rabbits immunized with Sp 19A. Raw data of glycan array analyses are reported in Figure SI-3.