UCSF

The Neisseria meningitidis serogroup B (MenB) polysaccharide, α(2-8) polysialic acid (polySia), is essential for resistance to complement-mediated bacteriolysis and inhibition of opsonophagocytosis. The monoclonal antibody SEAM 3 binds de-N-acetyl polySia (dPSA) but not polySia, yet still recognizes encapsulated MenB, indicating the capsule contains dPSA derivatives. Since SEAM 3 also recognizes other pathogens as well as human cancers, we have explored its biological role and mapped the SEAM 3 epitope. We compared binding of N-acetylated and de-N-acetylated polySia and ganglioside GD3 derivatives to sialic-acid-binding immunoglobulin-like lectins (Siglecs) on leukocytes. Siglec-9 showed higher affinity for dPSA and de-N-acetyl GD3 (K_D ∼2.6-3.1 nM) than for their acetylated counterparts, while Siglec-5 bound polySia and GD3 derivatives with similar and moderate (K_D∼40-60 nM and 7.9 nM, respectively) affinity. Siglecs-2, -3, -7, -10, and -11 showed negligible binding. T cells, NK cells, and monocytes incubated with live dPSA-expressing MenB acquired dPSA and bacterial protein. A lipophilic dPSA derivative also suppressed endotoxin-induced secretion of IL-1β, IL-6, and TNFα from human peripheral blood mononuclear cells. To map the SEAM 3 epitope, we co-crystallized a humanized SEAM 3 Fab with a dPSA derivative, resolving the structure at 1.83 Å (Rfree = 0.230) with good geometry. The epitope comprises four residues, with a de-N-acetylated residue at the non-reducing end. These findings suggest that dPSA may help mediate MenB immune evasion by engaging the inhibitory Siglec receptors 5 and 9 and confirm the utility of SEAM 3 as a structural probe of dPSA biology and the role of Siglecs in MenB pathogenesis.