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and many other members of the CCP group. Culture supernatant con-
taining VCP and partially purified VCP has previously been shown to
inhibit C'-mediated lysis of sheep erythrocytes, bind to C4b and C3b,
and decay the classical and alternative pathway C3 convertases [63,64].
Further studies have indicated that VCP acts as a cofactor in the prote-
olytic inactivation of C4b and C3b by factor I [64].
It has been suggested that all four SCRs are required for VCP's
complement-neutralizing activity. To elucidate which SCR domains are
involved in abolishing complement-enhanced neutralization of vac-
cinia virus virions and further define the mechanisms of complement
inactivation by VCP, we have developed monoclonal antibodies that
react with VCP [65]. We used the recombinant VCP expressed in the
yeast
Pichia pastoris
to vaccinate mice for the development of VCP-
specific hybridomas. Ten Mabs were isolated and all recognized VCP
on Western blots under reducing conditions as well as native-bound
VCP in a sandwich enzyme-linked immunosorbent assay. Three of the
10 MAbs (2E5, 3D1, and 3F11) inhibited VCP's abolition of complement-
enhanced neutralization of vaccinia virus virions. These MAbs blocked
the interaction of VCP with C3b/C4b. The seven remaining MAbs did
not alter VCP function in the complement neutralization assay and
recognized VCP bound to C3b/C4b.
To understand MAb specificity and mode of interaction with VCP,
we mapped the MAb binding regions on VCP. The seven nonblocking
MAbs all bound to the first SCR of VCP. One of the blocking MAbs
recognized SCR 2 while the other two recognized either SCR 4 or the
junction between SCRs 3 and 4, indicating that structural elements
involved in the interaction of VCP with C3b/C4b are located within
SCR domains 2 and 3 and 4. These anti-VCP MAbs may have impor-
tant clinical applications serving as potential therapeutic inhibitors of
VCP's complement control activity, and may also offer a novel thera-
peutic platform for managing vaccinia virus vaccine complications
that occur from smallpox vaccination. We also hope to use these
monoclonals to further characterize the contribution of VCP to viral
pathogenesis.
STUDIES ON THE VCP
/
SPICE INTERACTION WITH COMPLEMENT
COMPONENT C
3b
The smallpox inhibitor of complement enzymes (SPICE) is encoded by
variola virus, the causative agent of smallpox, and was found to have
complement-modulating activity that is similar to that of VCP [17].
Strikingly, despite the fact that it is 100-fold more potent than VCP in
inactivating human C3b, SPICE differs from VCP in only 11 amino acid
residues. In order to identify the amino acid residues that account for
the significant difference in function between the two molecules,
chimeric proteins consisting of VCP and SPICEs CCP modules were
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