Humanised monoclonal antibodies neutralise pertussis toxin by receptor blockade and reduced retrograde trafficking

Abstract

Pertussis toxin (PTx) is a major protective antigen produced by Bordetella pertussis that is included in all current acellular vaccines. Of several well-characterized monoclonal antibodies binding this toxin, the humanised hu1B7 and hu11E6 antibodies are highly protective in multiple in vitro and in vivo assays. In this study, we determine the molecular mechanisms of protection mediated by these antibodies. Neither antibody directly binds the B. pertussis bacterium nor supports antibody-dependent complement cytotoxicity. Both antibodies, either individually or as a cocktail, form multivalent complexes with soluble PTx that bind the FcγRIIb receptor more tightly than antibody alone, suggesting that the antibodies may accelerate PTx clearance via immune complex formation. However, a receptor binding assay and cellular imaging indicate that the main mechanism used by hu11E6 is competitive inhibition of PTx binding to its cellular receptor. In contrast, the main hu1B7 neutralising mechanism appears to be inhibition of PTx internalisation and retrograde trafficking. We assessed the effects of hu1B7 on PTx retrograde trafficking in CHO-K1 cells using quantitative immunofluorescence microscopy. In the absence of hu1B7 or after incubation with an isotype control antibody, PTx colocalizes to organelles in a manner consistent with retrograde transport. However, after preincubation with hu1B7, PTx appears restricted to the membrane surface with colocalization to organelles associated with retrograde transport significantly reduced. Together, these data support a model whereby hu11E6 and hu1B7 interfere with PTx receptor binding and PTx retrograde trafficking, respectively.

An intranasally administered monoclonal antibody cocktail abrogates ricin toxin-induced pulmonary tissue damage and inflammation

“…13,14 We reasoned that combining PB10 with a second MAb might improve overall in vivo toxin-neutralizing activity, especially in light of other reports in the literature demonstrating additive and often synergistic benefits associated with MAb cocktails in vitro and in vivo. 22,26,[30][31][32][33] PB10 is directed against an immunodominant epitope near RTA's active site 34 and evidence indicates that PB10 neutralizes ricin by interfering with trafficking of the toxin from the plasma membrane to the TGN. 35 Therefore, coupling PB10 with a second MAb that affects ricin attachment to target cells might afford the greatest combined benefit.…”

Neutralization of pertussis toxin by a single antibody prevents clinical pertussis in neonatal baboons

“…Our prior work suggested that hu1B7 does not rely heavily on Fc functions to neutralize PTx activities (18). To formally evaluate this hypothesis and understand the potential for Fc changes such as YTE to affect protection, we generated an aglycosylated hu1B7 variant and compared it to hu1B7 in a mouse B. pertussis challenge model.…”

“…We previously described the anti-PTx antibody hu1B7 and determined that it protects against toxin activities by altering intracellular transport steps required for PTx to access its target (18). When administered to mice, hu1B7 prevented leukocytosis in intoxication (19) and infection models (20) and reduced B. pertussis lung colonization levels ~30-fold (20).…”

Discovery of compounds inhibiting the ADP-ribosyltransferase activity of pertussis toxin

“…In this respect, several drug modes of action could be envisioned, including to inhibit – i) secretion from the bacterium, ii) host cell recognition, iii) endocytosis, iv) intracellular trafficking, and v) ADP-ribosylation activity inside the host cell. Humanized monoclonal antibodies have been developed that block pertussis toxin cell surface receptor interaction or the subsequent internalization and retrograde trafficking 32 . These humanized antibodies prevented the characteristic signs of whooping cough in mouse and baboon models 33 .…”