Cultures were incubated in 384-well culture plates for 10 days and then expanded using cell culture medium containing irradiated heterologous human PBMCs (Nashville Red Cross). MAbs to Poxviruses, Related to Figure 1 Cross-reactivity of individual mAbs to different poxviruses were assessed by ELISA using infected cell lysates or purified recombinant protein antigens.(A) Examples of mAbs within the panel that exhibited cross-reactivity to VACV, CPXV, MPXV, and VARV-infected cell lysates. Reactivity to VARV-infected cell lysate was measured at single mAb dilution as detailed in Table S3, ND indicates not determined. (B) Examples of four mAbs within the panel that exhibited cross-reactivity to VACV and VARV protein antigen orthologs. These four mAbs were included in mAb mixtures Mix4 and Mix6, which later were assessed for protective capacity genus, cowpox virus (CPXV), monkeypox virus (MPXV) and variola virus (VARV), cause severe infections in humans. VARV exclusively causes human infections, with an estimated 300C500 million deaths during the 20th century before the initiation of the global smallpox vaccination campaign (Smith and McFadden, 2002). MPXV and CPXV are emerging zoonotic infections with a sporadic occurrence worldwide (McCollum et al., 2015; Reed et al., 2004; Vorou et al., 2008). There is no licensed specific treatment for these infections, and the only method of prevention is vaccination using vaccinia virus (VACV). Vaccination against smallpox was discontinued in the late 1970s, leaving a large proportion of the current human population vulnerable to orthopoxviruses. The fear that smallpox could potentially re-emerge following a bioterror or biowarfare action (Smith Probucol and McFadden, 2002), the sporadic outbreaks of zoonotic MPXV and CPXV, and the increasing prevalence of immunocompromised individuals who cannot be vaccinated safely (Kemper et al., 2002), has stimulated renewed interest in research on orthopoxvirus protective immunity and treatment. Poxviruses have a large and complex proteome containing over 200 proteins. During infection, the virus exists in two antigenically distinct forms, designated mature virions (MV) or enveloped virions (EV), which contain ~25 or 6 surface proteins, respectively (Moss, 2011). MPXV and VARV are select agents and subject to the select agent regulations under (42 CFR Probucol 73). Various poxvirus species share many genetic and antigenic features (Hughes et al., 2010; Ichihashi and Oie, 1988; Stanford et al., 2007), and an infection with an orthopoxvirus of any one species may confer substantial protection against infection with the other orthopoxviruses (McConnell et al., 1964). Vaccination with VACV protects against disease caused by VARV, MPXV, or CPXV (Hammarlund et al., 2005). The immunologic mechanisms underlying cross-protection by immunization with VACV likely are diverse, but include neutralizing antibodies (Moss, 2011). A critical role for antibodies (Abs) in poxvirus immunity was suggested by historical cases in which passive transfer of serum from VARV- or VACV-immune subjects protected exposed individuals against smallpox (Kempe et al., 1961). Recent studies in non-human primate or murine models of experimental infection showed that polyclonal Abs are necessary and sufficient for protection against lethal challenge with Hdac8 MPXV or VACV (Belyakov et al., 2003; Edghill-Smith et al., 2005). The level of neutralizing activity in immune serum is thought to be the best laboratory predictor of protective immunity to orthopoxvirus infections in humans (Mack et al., 1972). Human vaccinia immune globulin (VIG) has been used for the prevention and treatment of some smallpox and vaccine-related complications with limited success Probucol (Wittek, 2006); FDA indications only address use for complications following vaccinia inoculation. The level of efficacy is uncertain due to lot-to-lot variation in potency and a lack of understanding of the molecular determinants of protection. Percutaneous inoculation with VACV elicits a.