Burkholderia pseudomallei Capsular Polysaccharide Recognition by a Monoclonal Antibody Reveals Key Details toward a Biodefense Vaccine and Diagnostics against Melioidosis

Abstract

Burkholderia pseudomallei is the bacterium responsible for melioidosis, an infectious disease with high mortality rates. Since melioidosis is a significant public health concern in endemic regions and the organism is currently classified as a potential biothreat agent, the development of effective vaccines and rapid diagnostics is a priority. The capsular polysaccharide (CPS) expressed by B. pseudomallei is a highly conserved virulence factor and a protective antigen. Because of this, CPS is considered an attractive antigen for use in the development of both vaccines and diagnostics. In the present study, we describe the interactions of CPS with the murine monoclonal antibody (mAb) 4C4 using a multidisciplinary approach including organic synthesis, molecular biology techniques, surface plasmon resonance, and nuclear magnetic spectroscopy. Using these methods, we determined the mode of binding between mAb 4C4 and native CPS or ad hoc synthesized capsular polysaccharide fragments. Interestingly, we demonstrated that the O-acetyl moiety of CPS is essential for the interaction of the CPS epitope with mAb 4C4. Collectively, our results provide important insights into the structural features of B. pseudomallei CPS that enable antibody recognition that may help the rational design of CPS-based vaccine candidates. In addition, our findings confirm that the mAb 4C4 is suitable for use in an antibody-based detection assay for diagnosis of B. pseudomallei infections.

Genome-scale analysis of the genes that contribute to Burkholderia pseudomallei biofilm formation identifies a crucial exopolysaccharide biosynthesis gene cluster

“…Cross reactivity with purified CPSI suggested that the acidic exopolysaccharide-specific antibody reacts with a constituent of CPSI (Fig 7A). We also confirmed that the polysaccharide preparations produced by the becA-R biosynthetic cluster were not CPSI (Fig 7B), since the purified CPSI [14] and preparations from Δ becA-R are reactive to the CPSI-specific 4C4 IgG1 antibody [46, 60, 61], whereas the preparations from Δ wcbR-A mutant and the Δ wcbR-A Δ becA-R double mutants are not reactive to the CPSI-specific 4C4 IgG1 antibody.…”

“…Immunoblots were probed with either 1:2,000 primary B . pseudomallei -specific CPSl (4C4 IgG1) [46] or 1:2,000 acidic exopolysaccharide (mAb 3015) [15] and detected with goat anti-mouse poly HRP secondary (1:50,000) (Pierce). The immunoblots were visualized using a Clarity Western ECL Blotting Substrate.…”

Detailed Investigation of the Immunodominant Role of O‐Antigen Stoichiometric O‐Acetylation as Revealed by Chemical Synthesis, Immunochemistry, Solution Conformation and STD‐NMR Spectroscopy for Shigella flexneri 3a

“…Additional insights on the implication of bacterial polysaccharide O‐acetylation in mAb recognition emerged from a recent study on Burkholderia pseudomallei . Molecular investigation on a di‐ O ‐acetylated disaccharide/mAb 4C4 system by means of STD‐NMR and tr‐NOESY analysis confirmed that the O ‐acetyl moiety of the capsular polysaccharide was essential for its interaction with the mAb, as suggested from an investigation on the purified antigen . It is of note that the capsular polysaccharides of S. typhi and B. pseudomallei are O ‐acetylated homopolymers, possibly featuring less antigenic diversity than the more complex SF O‐Ags.…”

In vivo Distribution and Clearance of Purified Capsular Polysaccharide from Burkholderia pseudomallei in a Murine Model

“…Isolation of mAb 4C4 was described previously [17]. Microtiter plates were coated overnight with 100 μL of mAb 4C4 (2.5 μg/mL in PBS) at room temperature.…”