A point-mutated Ag85B antigen improves recombinant bacterial expression and protects mice from aerosol M. tuberculosis challenge

Abstract

Tuberculosis (TB) remains a global health problem, providing motivation for improved vaccine approaches, such as subunit vaccines targeting specific Mycobacterium tuberculosis (M. tuberculosis) antigens. Ag85B is a protein involved in cell wall biosynthesis, is abundant in M. tuberculosis culture supernatants, and has been incorporated in several TB vaccines candidates. We observed low expression yields of Ag85B when expressed recombinantly in E. coli using a histidine-tag purification approach. To address this, we utilized the ThermoMPNN protein structure algorithm to predict several stabilizing mutations in Ag85B. Of these, a single mutation, D52W, significantly enhanced expression yield in E. coli with good storage stability. Ag85B-52W exhibited rapid binding to liposomes incorporating cobalt-porphyrin via his-tag interaction, resulting in suppression in reactivity with an anti-his-tag antibody (due to anchoring of the his-tag in the bilayer), while the surface-displayed protein remained reactive towards anti-Ag85B antibodies. Immunization with Ag85B-52W in a liposomal vaccine elicited antigen-specific antibody and T cell responses, resulting in reduced lung bacterial burden in mice following aerosol M. tuberculosis challenge.

Significance statement

A point mutation in the M. tuberculosis Ag85B protein, predicted by ThermoMPNN algorithm, enhanced its antigen expression yield in E. coli with good storage stability. Immunization with the Ag85B mutant with a liposome vaccine system in mice resulted in antigen-specific humoral and cellular response that protected mice against M. tuberculosis infection. This approach could facilitate the use of recombinant Ag85B in TB vaccine development.