As expected, IgG2a antibody was only produced in BALB/c mice given the CpG/alum combination adjuvant (Fig. anti-A33 antibodies likely result in virolysis, while anti-B5 antibodies with complement can neutralize by opsonization (coating). In vivo studies found that mice lacking the C3 protein of complement were less protected than wild-type mice after passive transfer of anti-B5 pAb or vaccination with B5. Passive transfer of anti-B5 pAb or monoclonal antibody into mice lacking Fc receptors (FcRs) found that FcRs were also important in mediating protection. These results demonstrate that both complement and FcRs are important effector mechanisms for antibody-mediated protection from VACV challenge in mice. == Introduction == In the 1970s, the World Health Organization led a successful campaign to eradicate smallpox using live TAPI-2 TAPI-2 vaccinia virus (VACV) Rabbit Polyclonal to CHRM4 vaccines[1]. However, recent concern over the intentional or accidental release of variola virus has led some of the world’s nations to stockpile live VACV vaccines[2][4]. With the risk of variola virus release minimal, concerns regarding live VACV vaccine’s rare but serious side effects and many contraindications[5][7]have led to the pursuit of safer smallpox vaccine strategies[8][10]. Modified vaccinia virus Ankara (MVA), a highly attenuated VACV-derived vaccine, has been under development and will likely soon become a safer alternative[11],[12]. However, subunit vaccination is an approach that does not rely on production of a virus. We evaluated the efficacy and mechanism by which a protein-based subunit vaccine can protect against orthopoxvirus infection. After vaccination, protection from orthopoxvirus disease heavily depends on antibody responses in animal models[13][15]and humans[16],[17]. Many of the responses are directed against viral surface proteins on the two virion forms, mature virus (MV) and extracellular virus (EV). The MV form is the most abundant virion form in infected cells[18]and is believed to mediate spread between hosts. The EV form mediates dissemination within an infected host[19][22]. The MV form contains a large set of surface proteins, while the EV form contains an TAPI-2 extra membrane and an additional, unique subset of surface proteins. Antibody against certain proteins of either form can be partially protective, such as L1 on MV[23][27]and B5 or A33 on EV[15],[23],[26],[28][30], though optimal protection is seen when antibodies are directed against both forms[23][26],[31],[32]. Subunit protein vaccination including target antigens from both forms achieves protection from lethal orthopoxvirus challenge in mouse and non-human primate challenge models[23],[32][35]. 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 immune responses and provide protection from lethal disease. Serum from vaccinated animals or humans is capable of efficiently neutralizing the MV form of VACV[23],[32],[34],[36],[37]; however, direct antibody neutralization of the EV form has been suboptimal at even TAPI-2 high concentrations of anti-EV antibody[15],[29],[38][41]. 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 (mAb) against the B5 protein called B126 can neutralize EV in the presence of complement (C’) and utilizes C’ to partially mediate protection in vivo[42],[43]. This evidence suggests that antibody against EV would be more effective if it TAPI-2 was of an isotype that mediated effector functions such as activation of C’ and/or Fc receptor (FcR) dependent activity (e.g. antibody dependent cellular cytotoxicity (ADCC)). 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[33],[34]. This suggests that antibody with specific Fc activities might be beneficial for safety. By utilizing a high PFU luciferase reporter EV neutralization assay, we find that polyclonal antibody reactions against the EV proteins A33 and B5 use C’ to neutralize disease in vitro, though in mechanistically different ways. These findings shed light on how differing viral proteins dictate the requirements for the sponsor to neutralize incoming disease with C’. Additionally, we display that.