A fusion protein LT25 containing Rv2660c failed to provide protection against Mycobacterium bovis infection due to limited humoral immune responses.

Rv2660c is present in both Mycobacterium tuberculosis (M. tuberculosis) and Mycobacterium bovis (M. bovis) and has been reported as a protective antigen. In this study, Rv2660c was linked with another M. tuberculosis specific antigen Rv2645, which is present in M. tuberculosis but not in M. bovis, to construct a fusion protein Rv2645-Rv2660c (LT25 in short). LT25 failed to confer protection following M. bovis challenge. Structural predictions via AlphaFold 3 revealed that LT25 retained the native conformation of Rv2645, whereas Rv2660c underwent significant conformational alterations. LT25 elicited strong Rv2645 and Rv2660c-specific cellular immune responses and high levels of Rv2645-specific antibodies, but lower levels of Rv2660c-specific antibodies than Rv2660c did. Further investigation showed that the Rv2660c-induced polyclonal antibody possessed stronger phagocytosis-promoting activity than LT25-induced antibodies did. These findings suggest that the structural integrity of Rv2660c is essential for its protective function.