Humanized Recombinant Vaccinia Virus Complement Control Protein (hrVCP) with Three Amino Acid Changes, H98Y, E102K, and E120K Creating an Additional Putative Heparin Binding Site, Is 100‐fold More Active Than rVCP in Blocking Both Classical and Alternative Complement Pathways

Abstract

Vaccinia virus complement control protein (VCP) is able to modulate the host complement system by regulating both pathways of complement activation. Efficient downregulation of complement activation depends on the ability of the regulatory protein to effectively bind the activated third (C3b) and fourth (C4b) complement components. Based on native crystallographic structure, molecular modeling, and sequence alignment with other Orthopoxviral complement control proteins (CCPs) and their host homologs, putative sites have been found on VCP as contact points for C3b/C4b. Here, we report that using site-directed mutagenesis, modified proteins have been generated. In addition, we report that the generated modified proteins with postulated contact point substitutions have shown greater ability to regulate both the classical and the alternative pathways of complement activation than the recombinant Western Reserve VCP, with one modified protein showing nearly 100-fold more potency in regulating both complement activation pathways independently. The augmented in vitro inhibitory activity of the modified protein together with the newly created putative heparin binding site suggests its promising potential as a competent therapeutic agent in modulating various complement-mediated ailments, for example, traumatic brain injury, Alzheimer's disease, rheumatoid arthritis, multiple organ dysfunction syndrome, reperfusion injury, and xenorejection.

Inhibition of Complement and CD14 Attenuates theEscherichia coli-Induced Inflammatory Response in Porcine Whole Blood

“…Recombinant complement regulatory proteins from Orthopoxvirus. Vaccinia complement control protein (VCP), which has been described previously (18,19), was produced using the Pichia pastoris yeast expression system as described previously (10,28). Smallpox inhibitor of complement enzymes (SPICE), which also has been described previously (30), was produced by cloning and sitedirected mutagenesis of VCP (33).…”

Influence of glycosylation and oligomerization of vaccinia virus complement control protein on level and pattern of functional activity and immunogenicity

“…The lack of two amino acids in this region of the protein may not have effects on its interaction with either C3b or C4b components of the complement since generally SCR 1 has little or no influence on the complement inhibitory activity of VCP (Isaacs et al, 2003). Apart from lacking two residues, there are six differences in the primary sequence compared to WR VCP and 17 compared to SPICE; some of these substitutions could have functional significance (Ganesh et al, 2004;Ghebremariam et al, 2005;Sfyroera et al, 2005). When compared to WR VCP and SPICE only, most of these amino acid differences are found within SCR 2 and SCR 4 of the protein and particularly in the C4b binding region.…”

“…2). Even at low amounts, the native proteins were more potent by a factor of at least 20 in inhibiting lysis of ssRBCs than their recombinant yeast-expressed versions (not shown) (Ghebremariam, 2006). Lis-VCP is at least 4-fold less active than WR VCP in this assay (Fig.…”

“…A previous report suggested that the Lister strain of vaccinia virus encodes a functional VCP (Lis VCP) with an inferred amino acid sequence that is smaller by two residues compared to the WR VCP and SPICE, has two putative N-glycosylation sites and several other differences . The natural VCP or Lis VCP expressed by infection of African green monkey kidney (BSC-1) cell lines with vaccinia virus WR strain or Lister strain was found to inhibit complementmediated lysis of IgG-sensitized sheep erythrocytes several fold more efficiently than its respective recombinant version expressed in yeast cells (Ghebremariam, 2006). Taken together with earlier studies suggesting that in a natural infection the predominant active VCP that is secreted and shows a higher level of activity than the momoner is a dimer (Kotwal et al, 1990) while the recombinant yeast expressed protein is a monomer (Smith et al, 2002), we now suggest that the dimerization influences the higher level of observed activity of naturally expressed VCP.…”

Ectromelia Virus Inhibitor of Complement Enzymes Protects Intracellular Mature Virus and Infected Cells from Mouse Complement

“…The majority (five of nine) of unique residues in SPICE are located in CCP-2, and some of them mediate SPICE's enhanced activity against human complement compared to VCP's activity (25,42,64). IMP has a similar number of unique residues compared to EMICE, but they are concentrated in CCP-3 and CCP-4 (10 of 14).…”