Elucidating the Structural and Minimal Protective Epitope of the Serogroup X Meningococcal Capsular Polysaccharide

Abstract

Despite the considerable progress toward the eradication of meningococcal disease with the introduction of glycoconjugate vaccines, previously unremarkable serogroup X has emerged in recent years, recording several outbreaks throughout the African continent. Different serogroup X polysaccharide-based vaccines have been tested in preclinical trials, establishing the principles for further improvement. To elucidate the antigenic determinants of the MenX capsular polysaccharide, we generated a monoclonal antibody, and its bactericidal nature was confirmed using the rabbit serum bactericidal assay. The antibody was tested by the inhibition enzyme-linked immunosorbent assay and surface plasmon resonance against a set of oligosaccharide fragments of different lengths. The epitope was shown to be contained within five to six α-(1–4) phosphodiester mannosamine repeating units. The molecular interactions between the protective monoclonal antibody and the MenX capsular polysaccharide fragment were further detailed at the atomic level by saturation transfer difference nuclear magnetic resonance (NMR) spectroscopy. The NMR results were used for validation of the in silico docking analysis between the X-ray crystal structure of the antibody (Fab fragment) and the modeled hexamer oligosaccharide. The antibody recognizes the MenX fragment by binding all six repeating units of the oligosaccharide via hydrogen bonding, salt bridges, and hydrophobic interactions. In vivo studies demonstrated that conjugates containing five to six repeating units can produce high functional antibody levels. These results provide an insight into the molecular basis of MenX vaccine-induced protection and highlight the requirements for the epitope-based vaccine design.

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

“…Recently, it has been demonstrated that five to six RUs contain the minimal structural and immunogenic epitope of N. meningitidis serogroup X (MenX) capsular polysaccharide. 35 The study at the atomic level of the minimal epitope of group B Streptococcus type III revealed a sialylated epitope spread over two adjacent repeating units. 36 The same methodology applied to N. meningitidis serogroup A identified the O-acetylated trisaccharide as the minimal antigenic epitope.…”

Impact and Control of Sugar Size in Glycoconjugate Vaccines

“…Different conditions (temperature and reaction times) were used depending on the OS MW target. For example, to produce MenX OS with an average of 5 RU, the hydrolysis was performed in 50 mM NaOAc, with a saccharide concentration of 2.5 mg/mL at pH 4.0, 80 °C for ∼18 h, and two times overnight at RT [ 37 ].…”

“…When MenC OS of 2–4, 4–10, and 10–50 kDa, conjugated to P64k by reductive amination following saccharide oxidation, were tested in mice, the IgG antibody levels and bactericidal activity were not statistically different [ 36 ]. With a set of MenX OS of different length (average number of 5, 10 and 20 RU) conjugated to CRM197, the 20 RU OS conjugate induced in mice the optimal response in terms of both anti-MenX antibodies and SBA titers [ 37 ] ( Figure 1 C).…”

Synthetic Glycans to Improve Current Glycoconjugate Vaccines and Fight Antimicrobial Resistance

“…Recently, the structural epitope of the MenX CPS was mapped using a Fab derived from a murine bactericidal monoclonal antibody (mAb) obtained against the conjugated MenX PS. 98 Surface Plasmon Resonance (SPR), isothermal titration calorimetry (ITC), and STD-NMR showed binding for a fragment longer than 5 RUs. In silico docking with a hexamer and the structure of the Fab resolved by X-ray crystallography confirmed binding through a network of salt bridges and hydrophobic interactions.…”

“…Subsequently a tetramer fragment was generated, and a competitive ELISA showed that the corresponding TT conjugate was a much better competitor than the unconjugated form, indicating the need for longer length CPS fragments to sufficiently emulate the PS epitope. Recently, the structural epitope of the MenX CPS was mapped using a Fab derived from a murine bactericidal monoclonal antibody (mAb) obtained against the conjugated MenX PS . Surface Plasmon Resonance (SPR), isothermal titration calorimetry (ITC), and STD-NMR showed binding for a fragment longer than 5 RUs.…”