Dissecting complement regulatory sites of the smallpox inhibitor of complement and abrogating function with a monoclonal antibody (44.37)

Abstract

Abstract Public health concerns have emerged regarding use of smallpox as a bioterrorist weapon and the possibility of natural emerging infection with monkeypox. Stockpiles of variola in rogue nations and the outbreak of monkeypox in the US in 2003 underpin these concerns. Poxviruses express virulence factors that down-modulate the host’s innate immune system. We previously constructed the smallpox inhibitor of complement enzymes, SPICE, from its homolog in vaccinia, VCP. Additionally, we cloned and functionally characterized MOPICE, the highly homologous monkeypox inhibitor. SPICE had enhanced activity over both poxviral inhibitors (PICES). The goals of our current study were to dissect the complement regulatory sites of SPICE andto characterize a monoclonal antibody that binds to and abrogates function of the PICES. We created SPICE-VCP chimeras and SPICE point mutations. Generally, function increased for human C3b and C4b as more SPICE amino acids were present. We also identified SPICE residues that were critical for ligand binding and cofactor activities. Additionally, we characterized a mAb that binds SPICE, MOPICE and VCP. This mAb served as a capture reagent for ELISA to quantify PICES and as a detection mAb in Western blots. Importantly, the mAb blocked cofactor and decay accelerating activities, suggesting its possible use as a therapeutic. Thus, our studies add novel data relative to the complement inhibitory profile of SPICE. Further, a cocktail of such mAbs that inhibit virulence factors could serve in both diagnostic and therapeutic applications for poxviruses.