We hypothesized that disparity in A33 and B5 protein density on the EV Chlorhexidine hydrochloride surface contributed to the difference in mechanism. Galmiche et al. showed that total EV lysate had A33 and B5 protein amounts of,5 mg/mg and 30 mg/mg, respectively. The reduced amount of A33 protein on the EV surface could decrease the amount of antibody bound to EV to the point where coating with C1q and C3b/C4b in the area around the bound antibody is still insufficient to completely opsonize the EV virion. Under this scenario, formation of even one or two membrane attack complexes on the EV virion could be enough to disrupt the outer membrane and allow access of neutralizing MV antibody. This model would predict that further limiting the amount of anti-B5 antibody bound to the EV surface would switch the 4-(Benzyloxy)phenol mechanism of C’mediated neutralization from opsonization to lysis. To test this hypothesis, we used a novel approach whereby EV was generated with the incorporation of different human regulators of C’. We found that when EV was generated in cells that would result in the inclusion of CD59 on EV, CD59 could not provide additional protection from C’-mediated neutralization at high concentrations of anti-B5 antibody as neutralization could occur through opsonization. However, at low concentrations of anti-B5 antibody, CD59 was protective against C’-mediated neutralization to the same degree as EV containing CD55, likely indicating the mechanistic switch from opsonization to lysis. Additionally, we found that under the right experimental conditions, human regulators of C’ on the VACV EV surface can block C’ activation by antibody, and not just activation by C’ alone. These findings provide new insight into interactions of antibody, C’, and viral protein and how those interactions impact neutralization of virus. The finding that A33 requires virolysis for C’-mediated neutralization while B5 does not may also explain differences in protection we observed after vaccinating with A33 or B5/CpG/ alum. At the challenge doses we used, the ability of B5 to provide at least partial protection could be explained by the ability to neutralize EV in the absence of an anti-MV antibody response, which A33 is incapable. A33 antibody and C’ would simply release MV particles which could propagate the infection, albeit that some anti-A33 effect could be gained by allowing C’ free access to the C’ sensitive MV particle or A33 antibody-dependent lysis of infected cells. This may also explain why a vaccine that adds L1 to A33 improves protection from disease 
compared to A33 or L1 alone. To examine more closely which effector functions of antibodies are important for protection in vivo, we studied the role of C’ and FcRs in the protection we observed with B5 antibody. The rabbit anti-B5 pAb used in neutralization experiments had been previously shown to be protective in vivo by passive immunization and the ability to neutralize EV in the presence of C’ potentially contributed to this observation. To confirm this, we examined the ability of anti-B5 antibody to protect mice in the absence of the central C’ component C3. We found that both passive immunization with rabbit anti-B5 antibody and active immunization with B5/CpG/alum partially relied on C’ for protection. Similar to previously reported studies that transiently depleted C’ in challenged animals, we found that antibody could still provide partial protection even in the genetic absence of C3, which abrogates the function of the C’ system. Somewhat unexpectedly, we found that vaccinated C3KO mice generated antibody responses similar to that of wild-type mice.