Protective human anti-poxvirus monoclonal antibodies are generated from rare memory B cells isolated by multicolor antigen tetramers

Smallpox, an epidemic disease caused by Orthopoxvirus variola, was eradicated worldwide through immunization. The immunization against smallpox was discontinued in 1980. However, incidences of monkeypox virus infection in humans have occurred sporadically, and there is also great fear that engineered forms of poxvirus could be used as biological weapons. Therefore, monoclonal antibodies against poxvirus are in urgent need for the detection and treatment of poxvirus infection. The vaccinia virus extracellular envelope protein A33 is a potential candidate for subunit vaccine. We used multi-fluorescence-labeled tetrameric A33 antigen to identify rare poxvirus specific memory B cells from PBMC of volunteers with vaccinia virus immunization more than 40 years ago. Despite extremely low frequencies of the poxvirus specific memory B cells, we successfully sorted A33 tetramer-labeled single memory B cells and reconstructed antibodies with single-cell RT-PCR of the B-cell receptor. Among the monoclonal antibodies, one clone H2 exhibited high specificity and affinity with A33. H2 efficiently inhibited viral infection and spread in cells. Passive immunotherapy of H2 in mice protected mice from lethal infection when administered either prophylactically or therapeutically. These results suggest the potential of the anti-A33 human antibody-based detection and therapeutics for poxvirus infection.