A Multidisciplinary Structural Approach to the Identification of the Haemophilus influenzae Type b Capsular Polysaccharide Protective Epitope

Glycoconjugate vaccines so far licensed are generally composed of a native or size-reduced capsular polysaccharide conjugated to carrier proteins. Detailed information on the structural requirements necessary for CPS recognition is becoming the key to accelerating the development of next-generation improved glycoconjugate vaccines. Structural glycobiology studies using oligosaccharides (OS) complexed with functional monoclonal antibodies represent a powerful tool for gaining information on CPS immunological determinants at the atomic level. Herein, the minimal structural epitope of Haemophilus influenzae type b (Hib) CPS recognized by a functional human monoclonal antibody (hmAb) is reported. Short and well-defined Hib oligosaccharides originating from the depolymerization of the native CPS have been used to elucidate saccharide–mAb interactions by using a multidisciplinary approach combining surface plasmon resonance (SPR), saturation transfer difference-nanomagnetic resonance (STD-NMR), and X-ray crystallography. Our study demonstrates that the minimal structural epitope of Hib is comprised within two repeating units (RUs) where ribose and ribitol are directly engaged in the hmAb interaction, and the binding pocket fully accommodates two RUs without any additional involvement of a third one. Understanding saccharide antigen structural characteristics can provide the basis for the design of innovative glycoconjugate vaccines based on alternative technologies, such as synthetic or enzymatic approaches.


B.
n.d.=not detected *obtained from the deconvoluted spectrum

Figure
Figure S1.A) 1 H-NMR spectra (left) and 31 P-NMR spectra (right) of the produced Hib oligosaccharide fragments: DP2 (blue), DP3 (red), DP4 (green), DP5 (purple).Signals of H3-ribose within the chain and at the reducing terminal units are indicated in light blue and yellow boxes, respectively (left panel).The relative intensity of the reducing terminal H3-ribose signal decreased with increasing chain length.Signals of phosphate groups of terminal RU and within the chain are circled yellow and light blue, respectively (right panel).B) DP2-DP5 mass detected by negative mode ESI-MS.

Figure
Figure S2.A) Binding kinetics of Fab CA4 to avDP80, DP4 and DP3 fragments by SPR.B) Sugar forms in water [25] (a) and biotinylation (b)

Figure S4 .
Figure S4.Zoom of the 1 H-13 C HSQC spectrum of Hib DP3 1 mM in D 2 O on the anomeric region.The peak assignment is reported, together with the calculated 3 J H1-H2 couplings, the integrals for each signal and the relative percentage for each anomer.The experiment was acquired with 500 μL of sample transferred into a 5 mm NMR tube.

Figure
Figure S5.A) STD NMR experiments performed for the complex Hib DP2-CA4 hmAb.Off-resonance spectrum (irradiation at δ 100 ppm, top panel) and STD spectra obtained from aliphatic irradiation (δ 0.6 ppm) and processed applying exponential line-broadening.The main 1 H NMR signals are annotated in the STD spectrum and the pictorial notation for each residue is reported.The antibody/ligand molar ratio was 1:50, with the CA4 hmAb being at a concentration of 10 μM.The STD NMR experiments were acquired with 2 s of saturation time, 3 s of relaxation delay, and 2880 scans at 298 K. B) Expansion of the STD in the anomeric region of protons H1 Ribfβ int and Ribpβ OH processed omitting the exponential multiplication for resolution enhancement.C) Representation of the epitope map disclosed by the analysis of the STD-NMR experiments of Hib DP2-CA4 hmAb and Hib DP3-CA4 hmAb complexes.The color legend of associated to STD% values is also reported.

Figure S6 .
Figure S6.STD NMR experiments performed for the complex Hib DP3-CA4 hmAb.Off-resonance spectrum (irradiation at δ 100 ppm, top panel) and STD spectra obtained from aliphatic irradiation (δ 0.6 ppm) and processed applying exponential line-broadening.The main 1 H NMR signals are annotated in the STD spectrum and the pictorial notation for each residue is reported.The antibody/ligand molar ratio was 1:50, with the CA4 hmAb being at a concentration of 10 μM.The STD NMR experiments were acquired with 2 s of saturation time, 3 s of relaxation delay, and 2880 scans at 298 K. B) Representation of the epitope map disclosed by the analysis of the STD-NMR experiments of Hib DP3-CA4 hmAb and Hib DP3-CA4 hmAb complexes.The color legend associated to the STD% values is also reported.

FigureFigure S8 .
Figure S7.A) Study of the interconversion of ring conformations performed through J coupling analysis.Representation of the possible 3 T 2 and 2 T 3 conformations for Ribf (on the left) and Ribfα (on the right) with the 3 J H1-H2 expected values reported.B) Table reporting the measured experimental 3 J H1-H2 and the relative population for each moiety.