mRNA-based prime-and-pull vaccination combines benefits of subcutaneous and mucosal BCG vaccination

Problem Statement
The current licensed tuberculosis (TB) vaccine, Mycobacterium bovis Bacillus Calmette-Guérin (BCG), effectively protects children against severe forms of the disease but offers variable and often insufficient protection against pulmonary TB, the most transmissible form. While mucosal vaccination strategies have emerged as a promising approach to generate robust local lung immunity and tissue-resident memory (TRM) cells, they face significant safety hurdles. Specifically, mucosal delivery of live-attenuated vaccines (LAVs), such as aerosolized BCG, carries risks of uncontrolled replication and excessive inflammation, which can compromise tissue integrity and gas exchange in the lungs. There is an urgent need for a vaccination strategy that balances the high immunogenicity of mucosal delivery with the safety profile of non-replicating, parenteral vaccines.

Methodology
The authors developed and evaluated an mRNA-based "prime-and-pull" vaccination strategy. This approach involves two steps:

1. Prime: Systemic immunity is established via subcutaneous (SC) vaccination with BCG (either standard BCG or recombinant BCG expressing the ESX-1 locus, BCG::RD1).
2. Pull: Local lung immunity is induced via intratracheal (IT) administration of lipid nanoparticle (LNP)-formulated mRNA.

The study utilized "stealth" LNPs (sLNPs) composed of DOTAP, DOPE, cholesterol, and C16 PEG-ceramide to deliver mRNA to the lungs. Two distinct antigenic models were tested:

• Proof-of-Concept Model: A single antigen, ESAT-6 (a key M. tuberculosis antigen), was used to optimize dosing regimens (single high dose vs. multiple low doses) and compare outcomes against SC and IT BCG controls.
• Multivalent Model: Four mRNA constructs were generated, each encoding two antigens (totaling eight proteins: HBHA, HRP1, HspX, CFP10, Ag85B, VapB47, PPE18, and PE13). These were tested in hybrid F1 (C57BL/6 × BALB/c) mice to broaden MHC compatibility.

Vaccinated mice were challenged with aerosolized M. tuberculosis H37Rv 60 days post-vaccination. Outcomes were assessed via bacterial burden (CFU) in lungs and spleen, histological analysis of lung pathology, flow cytometry for T cell subsets (including TRM and stem cell-like memory T cells), and cytokine profiling.

Key Contributions and Results

• Delivery and Expression: The study demonstrated that sLNPs successfully deliver mRNA to lung cells via the intratracheal route, supporting protein expression in dendritic cells and neutrophils within 48 hours.
• Optimization of Dosing: In the ESAT-6 model, a single high-dose (10 μg) IT administration of mRNA following SC BCG priming induced significantly higher numbers of antigen-specific T cells in the lungs and broncho-alveolar lavage fluid (BALF) compared to multiple low-dose regimens. This single-dose prime-and-pull regimen achieved T cell frequencies comparable to those induced by IT BCG vaccination.
• Safety Profile: While IT BCG vaccination induced the highest levels of antigen-specific T cells, it also caused significant lung pathology and inflammation. In contrast, the prime-and-pull strategy induced robust local immunity with minimal inflammatory cell infiltration and significantly reduced lung tissue damage compared to IT BCG.
• Multivalent Efficacy: The multivalent mRNA approach (targeting eight antigens) enhanced humoral responses, significantly increasing IgG titers against CFP-10, Ag85b, and HspX compared to BCG-only groups. Crucially, this strategy induced higher frequencies of stem cell-like memory T (TSCM) cells in the lungs and mediastinal lymph nodes compared to IT BCG, suggesting potential for more durable immunity.
• Protective Immunity: Upon M. tuberculosis challenge, the prime-and-pull strategy conferred protection comparable to IT BCG in reducing lung bacterial burden and pathology, while maintaining a lower inflammatory profile than IT BCG and superior protection to SC BCG alone.

Significance and Claims
The paper presents the prime-and-pull strategy as a "modular" translational approach that bridges the gap between the improved immunogenicity of mucosal BCG vaccination and the safety of parenteral BCG administration. The authors claim that this strategy successfully "uncouples" immune recruitment from tissue-destructive inflammation. By using non-replicating mRNA to "pull" systemic immune cells into the lung, the platform achieves protective immunity against pulmonary TB without the risks of uncontrolled replication or excessive inflammation associated with live-attenuated mucosal vaccines.

The study concludes that this approach provides a proof-of-principle for a balanced vaccination regimen that limits excessive inflammation while maintaining immunogenicity and protection. The authors position this not as a final universal formulation, but as a flexible platform where antigen composition and delivery can be further optimized to address the specific challenges of respiratory TB vaccine development.