Poxviral infection elicits human neutralizing antibodies recognizing diverse epitopes of the major vaccinia virus surface protein D8 (VIR4P.1016)

Immunization with vaccinia virus (VACV) confers cross-protective immunity to variola, the causative agent of smallpox, and other clinically important poxviral species, such as cowpox and monkeypox viruses. Vaccine-mediated protection has been correlated with elicitation of broad neutralizing antibody (Ab) responses, however the specificity of human neutralizing antibodies to poxviruses is poorly understood. We used a highly-optimized human hybridoma technology to generate large panels of anti-VACV monoclonal Abs (mAbs) from vaccinia-immunized subjects or from subjects who had naturally acquired monkeypox infection. Immunologic characterization and DNA sequencing of the panel revealed 16 individual mAbs that recognized the immunodominant surface protein antigen D8. Several of anti-D8 mAbs possessed neutralizing activity against VACV, cowpox and monkeypox viruses. These Abs exhibited mainly a complement-dependent pattern of neutralization and they formed at least four cross-blocking groups, suggesting the existence of several non-overlapping neutralizing epitopes for D8. The results suggest that D8 is an important target for human neutralizing Abs generated following poxviral infection or vaccination, and suggest the existence of novel epitopes targeted by anti-D8 Abs that contribute to cross-protective immunity following smallpox vaccination.