5 and 67.9 showed inhibition; neither 67.11 nor 67.13 could inhibit this activity (Fig. 3A). Essentially similar results were obtained for inhibition of C4b cofactor activity by the monoclonal antibodies. Only 67.5 and 67.9 showed inhibition, Selleck LGK974 while 67.11 and 67.13 failed to inhibit the C4b cofactor activity (Fig. 3B). These data therefore revealed that CCP domain 3 and/or linker between CCPs 3 and 4 of VCP play an essential role in imparting the cofactor activities. Besides acting as a cofactor for C3b and C4b inactivation, VCP is also an efficient

decay accelerator of the classical/lectin pathway C3-convertase C4b,2a. Thus, to examine the effect of mAbs on VCP-mediated decay of the convertase, we utilized a hemolytic assay. In this assay, C4b,2a was formed on antibody sensitized sheep erythrocytes using purified complement components and then the enzyme was allowed to decay in the presence of rVCP or rVCP pre-incubated with each of

the mAbs. The activity of the remaining enzyme was assayed by adding EDTA-sera (a source of C3-C9) and measuring hemolysis. Interestingly, the antibodies that inhibited the C3b and C4b cofactor activities (67.5 and 67.9) also inhibited the decay-accelerating activity of VCP, albeit with 67.5 having much less effect compared to 67.9. Among the remaining two antibodies 67.11 and 67.13, which bound to CCP 4 domain, only the former had moderate inhibitory activity while the latter did not Selleckchem Metabolism inhibitor inhibit the decay activity. Terminal deoxynucleotidyl transferase The C3-convertase decay inhibition efficiency of the monoclonals followed the order 67.9 ≈ 67.11 > 67.5 with 67.13 having negligible inhibitory potential (Fig. 4). Since mAbs differentially inhibited the VCP functions it was intriguing to know if blocking VCP function in vivo with these mAbs would translate into differences in viral pathogenesis. For in vivo disabling of VCP using mAbs, a prerequisite is that they should be retained at the site of injection until VCP is secreted by the infected cells. To verify this, we determined their half-life. The mAbs (67.5 and 67.9) were labeled with 131I, injected intradermally on either

flanks of New Zealand White rabbits and imaging was carried out with a γ-ray camera. The results showed that the labeled antibodies were retained at the site of injection even after 72 h. The half-life was found to be 8 h for both the antibodies (Fig. 5; data not shown for 67.9). Next, in order to determine whether disabling of VCP using neutralizing mAb affects VACV pathogenicity, we used a rabbit skin lesion model. In these experiments, VACV-WR was injected intradermally (104 pfu) either alone or in combination with mAbs and the lesion size was measured over a period of time. Initially, the two blocking antibodies (67.5 and 67.9) were titrated with VACV-WR to identify the optimal concentration required for reduction in lesion response. When varying concentrations of 67.5 (Fig. 6A) or 67.