The MV form contains a large set of surface proteins, while the EV form contains an extra membrane and an additional, unique subset of surface proteins. Tulathromycin B antibody against certain proteins of either form can be partially protective, such as L1 on MV and B5 or A33 on EV, though optimal protection is seen when antibodies are directed against both forms. Subunit protein vaccination including target antigens from both forms achieves protection from lethal orthopoxvirus challenge in mouse and non-human primate challenge models. In theory, antibody generated against the MV form would act to neutralize a portion of the initial infectious dose and antibody against the EV form could then prevent some spread of progeny virus within a host. Having these antibody responses present at the time of challenge could then allow the host time to generate additional 4-(Benzyloxy)phenol immune responses and provide protection from lethal disease. Serum from vaccinated animals or humans is capable of efficiently neutralizing the MV form of VACV; however, direct antibody neutralization of the EV form has been suboptimal at even high concentrations of anti-EV antibody. Therefore, understanding the mechanism by which anti-EV antibodies provide protection has been of interest. Recent mouse studies have elucidated that an IgG2a isotype monoclonal antibody against the B5 protein called B126 can neutralize EV in the presence of complement and utilizes C’ to partially mediate protection in vivo. This evidence suggests that antibody against EV would be more effective if it was of an isotype that mediated effector functions such as activation of C’ and/or Fc receptor dependent activity. Previous studies of antibody responses to protein vaccination found that formulations that included adjuvants that produced higher titers of IgG2a antibody in mice and IgG1 antibody in non-human primates were more effective at mediating protection than vaccines formulated without these adjuvants. This suggests that antibody with specific Fc activities might be beneficial for protection. By utilizing a high PFU luciferase reporter EV neutralization assay, we find that polyclonal antibody responses against the EV proteins A33 and B5 utilize C’ to neutralize virus in vitro, though in mechanistically different ways. These findings shed light on how differing viral proteins dictate the requirements for the host to neutralize incoming virus with C’. Additionally, we show that antibody against B5 utilizes C’ and FcRs to protect mice from lethal VACV challenge. These findings add to our understanding of how antibody can protect against orthopoxvirus disease and highlights the importance of understanding antibody effector functions necessary for protection to aid in the rational design of anti-viral vaccines and therapeutic antibodies. Proteins used in the vaccine formulations were purified recombinant baculovirus-expressed proteins that were previously described. Protein vaccines were prepared and used as described previously. Similar results were obtained when rabbit pAb against A33 and B5 was used. These results are consistent with the previously described findings that the mechanism of C’-mediated neutralization for A33 and B5 differ, with anti-A33 antibody relying on virolysis and anti-B5 antibody able to neutralize by opsonization. Benhnia hypothesized that antibody alone was unable to fulfill the basic occupancy model for EV neutralization because of the amount of B5 protein on the EV surface and that antibodyinduced C’ coating of the EV membrane allowed for the occupancy model to succeed.