Exaggerated Lung Inflammation Induced by Lung-Targeted mRNA-LNP Dampens Vaccines against Tuberculosis

The challenges in developing a tuberculosis (TB) vaccine stem from the complex life cycle of <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) and various bacterial proteins encoded by approximately 4000 genes. mRNA is easy to design and can accommodate multiple antigens, suggesting that it may be an effective TB vaccine technology. Here, we designed an mRNA encoding Ag85B and ESAT6 that was delivered by lung targeted lipid nanoparticles (LNP_lung-mRNA_A-E), intending to stimulate lung immunity to combat TB. To enhance the vaccine efficacy, we further cofabricated monophosphoryl lipid A (MPLA) with mRNA to evaluate the adjuvanted mRNA vaccine (LNP_lung-mRNA_A-E-MPLA). Both vaccines elicited robust CD4⁺ T cell response, resulting in markedly locally higher production of IFN-γ, TNF-α, and IL-2. As anticipated, the addition of MPLA further enhanced the immunogenicity of LNP_lung-mRNA_A-E. However, the <i>Mtb</i> challenge experiment showed that LNP_lung-mRNA_A-E-MPLA neither provided effective protection nor enhanced the immune protection primed by BCG (Bacillus Calmette-Guérin). The subsequent HE staining of the lung revealed that the LNP_lung-mRNA_A-E-MPLA induced pulmonary inflammation, leading to tissue damage. Moreover, the inflammatory cytokines including IL-6, IL-1β, and MCP-1 were significantly increased and the MPLA additive exacerbated the inflammatory process. Therefore, the lung targeted mRNA vaccine and MPLA adjuvant synergistically induced lung inflammation and weakened protection from <i>Mtb</i> infection. Thus, this work provides valuable implications for developing targeted lung vaccines: Addressing chronic lung inflammation induced by vaccine systems is critical for lung-targeted mRNA vaccines.