Engineering internally his-tagged ESX secretion system fusion antigens for tuberculosis nanoparticle vaccines

Improved tuberculosis (TB) vaccines are needed to combat the disease, a leading cause of infectious disease death worldwide. The culture filtrate TB antigens ESAT6 and CFP10 form dimeric complexes, serve as immune markers for TB exposure and are vaccine antigen candidates. Here, we report that expressing an ESAT6-CFP10 fusion protein with a conventional glycine-serine (GS) linker sequence significantly enhanced expression yield in E. coli compared to the single antigens. Substituting the GS linker with an internal His-tag generated the ESAT(Int7)CFP fusion, retained elevated expression levels while simultaneously enabled purification with immobilized metal chromatography. The internal His-tag also permitted rapid protein coupling with cobalt-porphyrin (CoPoP) liposomes. Immunization with these constructs elicited antigen-specific antibodies and T cell responses and reduced lung bacterial burden in mice following aerosol TB challenge. Another pair of TB culture filtrate antigens, EsxH and EsxG yielded no soluble protein as individual antigens. However, when linked with an internal His-tag, high soluble expression was observed for EsxH(Int7)EsxG, and this fusion antigen also conferred partial protective immunity in TB challenged mice after vaccination with CoPoP. These findings support the design and manufacturability of multivalent TB vaccine candidates through engineered fusion antigens and nanoparticle delivery platforms.