Distinguishing the antibodies induced by the live attenuated vaccine (LC16m8) and orthopoxvirus infections

[Introduction] The vaccinia virus (VACV) LC16m8 strain is a highly attenuated vaccine against smallpox and mpox, licensed in Japan. Unlike its parental strain (LC16mO) or ancestral vaccines, the B5R protein of the LC16m8 strain has a frameshift mutation, resulting in a truncated B5R protein.

[Objective] Our objective was to determine the B5R protein antigenic domains and develop an assay to distinguish LC16m8 vaccine-induced antibodies from orthopoxvirus infections.

[Methods] Four recombinant proteins, located at the N-terminal and C-terminal region of the B5R, were expressed in E.coli, and their antigenicity was examined by immunoblot analysis. They were then used to produce polyclonal antibodies in SPF rabbits. Next, the sensitivity of the anti-B5R antibodies was evaluated by immunofluorescence assay using orthopoxvirus-infected cells, or by expressing their B5R homologous regions. Furthermore, a Luminex bead-peptide assay was developed to target the B5R domains, and its specificity was evaluated using anti-orthopoxvirus antibodies. Finally, we screened the sera of 45 healthy human donors and 5 Mpox patients.

[Results] The expressed B5R N-terminal and C-terminal proteins were specifically recognized by anti-LC16mO B5R antibodies. The anti-B5R N-terminal antibodies cross-reacted with orthopoxvirus-infected cells or their expressed B5R homologous region. Nonetheless, the C-terminal antibody cross-reacted with infected cells of VACV, monkeypox and cowpox virus, but did not with the LC16m8 strain or the expressed LC16m8 B5R protein. Both B5R peptides showed cross-reaction with antibodies against VACV and monkeypox virus (Clade I, IIa & IIb). Furthermore, seven human serum samples showed neutralizing antibodies against VACV, from which two human donors and one Mpox patient showed affinity to the C-terminal peptide and recognized the full-length-B5R protein.

[Conclusion] The expressed B5R proteins, anti-B5R antibodies and Luminex bead-peptide assay might be useful for detecting orthopoxvirus infections among the LC16m8-vaccinated population.