Inactivated mycobacterium paragordonae delivered via microneedle patches as a novel tuberculosis booster vaccine

<i>Tuberculosis</i> (TB) remains a significant global health challenge with approximately 8.2 million new cases reported in 2023, despite the century-old Bacillus Calmette-Guérin (BCG) vaccine. BCG's protective efficacy diminishes over time, especially against pulmonary TB in adults. This study evaluates ethanol-inactivated <i>Mycobacterium paragordonae</i> (<i>M.pg</i>) delivered via Microneedle Array Patches (MAPs) as a novel booster strategy to enhance BCG vaccination efficacy. Various inactivation methods including heat treatment, formalin, and ethanol were compared, with ethanol-inactivated <i>M.pg</i> selected for optimal preservation of morphology and immunologically significant proteins. MAPs were fabricated using the droplet extension technique (DEN). Immunological assessment was conducted in a mouse model receiving either BCG alone or BCG followed by one or two administrations of inactivated <i>M.pg</i> MAP. Protective efficacy was evaluated through <i>M. tuberculosis</i> H37Rv challenge. Ethanol inactivation uniquely preserved morphology and maintained protein integrity, particularly Ag85B. Two administrations of inactivated <i>M.pg</i> following BCG priming significantly enhanced protective immune responses compared to BCG alone, inducing strong Th1-polarized immunity characterized by elevated IFN-γ, TNF-α, and IL-2 production in both CD4+ and CD8+ T cells. This vaccination strategy effectively generated effector memory T cells in lung and spleen, contributing to significant reduction in bacterial burden following challenge, with the BCG+Inactivated M.pg^2nd group demonstrating the greatest reduction. Inactivated <i>M.pg</i> delivered via microneedle patches represents an effective booster strategy for enhancing BCG-induced protection against tuberculosis, with a two-dose schedule demonstrating optimal efficacy. This approach combines the safety advantages of an inactivated vaccine with the practical benefits of MAPs, addressing key limitations of tuberculosis vaccination strategies.