# Improved immune responses and tuberculosis protection by aerosol vaccination with recombinant BCG expressing ESX-1 from Mycobacterium marinum

## TL;DR

A new BCG vaccine that boosts immune responses through aerosol delivery offers better protection against tuberculosis in mice.

## Contribution

A recombinant BCG vaccine expressing ESX-1 from Mycobacterium marinum improves immune responses and protection when delivered via aerosol.

## Key findings

- Aerosol vaccination with BCG::ESX-1Mmar increases CD4+ and CD8+ T cell activation in the lungs compared to subcutaneous vaccination.
- Aerosol delivery induces stronger humoral and mucosal immune responses and reduces lung pathology in TB-infected mice.
- BCG::ESX-1Mmar provides superior protection against M. tuberculosis compared to standard BCG Pasteur for both vaccination routes.

## Abstract

The currently licensed anti-tuberculosis (TB) vaccine, Mycobacterium bovis BCG, provides limited protection against pulmonary TB in adolescents and adults, the main cause of TB transmission and mortality. To obtain an improved BCG-based vaccine candidate with increased immune signalling but still low virulence, we have previously generated a recombinant BCG strain named BCG::ESX-1Mmar, which is heterologously expressing ESX-1 functions of Mycobacterium marinum and thereby modulates the host innate immune responses via phagosomal rupture-associated induction of type I interferon production and enhanced inflammasome activity, leading to superior protection against TB disease in murine infection models. As protection may also vary with the route of vaccination, here, we have explored aerosol vaccination relative to subcutaneous vaccination, using BCG Pasteur and BCG::ESX-1Mmar. We found that mice vaccinated via the aerosol route with BCG Pasteur or BCG::ESX-1Mmar both yielded higher frequencies of CD4+ and CD8+ Th1 activated effectors and T effector memory cells in the lungs compared to subcutaneously immunised mice, whereas comparable polyfunctional Th1 (IL-2, TNF-α and IFN-γ) cytokine-producing subsets were observed in the spleens of all vaccinated mice. Significantly higher IL-17A responses without severe lung pathology were seen in the lungs of aerosol-vaccinated mice associated to local and transient inflammatory cytokine responses and immune cell infiltrations. In contrast to the subcutaneous route, aerosol vaccination elicited high amounts of humoral IgG and IgA responses in the bronchoalveolar lavage fluid and induced a substantial number of lung CD4+ and CD8+ T cells expressing CD69+ CD103+ tissue-residency markers. These effects led to significant improved protection against M. tuberculosis and reduced lung pathology in aerosol-vaccinated mice compared to subcutaneously vaccinated mice. Moreover, BCG::ESX-1Mmar vaccine induced enhanced T-cell immunity and superior protection compared to parental BCG Pasteur for both vaccination routes and thereby represents an interesting candidate for developing improved vaccination strategies against TB.

Anti-tuberculosis vaccine efficacy is influenced by multiple parameters, including the immunogenicity of the vaccine strain, the type of preclinical host model used, and the route of vaccination. Given recent advances in the field of mucosal vaccination, in the current study we were particularly interested to explore and compare aerosol-based vaccination with standard subcutaneous vaccination in a C57BL/6J mouse model using our recently developed recombinant BCG::ESX-1Mmar vaccine candidate in comparison with parental BCG Pasteur. Our results show that in this setting the protective efficacy of mucosal vaccination was superior to subcutaneous vaccination for both vaccine strains, whereby the use of BCG::ESX-1Mmar induced additional benefits in terms of bacterial load reduction compared to standard BCG Pasteur. Taken together, we propose that aerosol vaccination using BCG::ESX-1Mmar as live-attenuated vaccine candidate is a promising and powerful combination for obtaining improved protection against an M. tuberculosis challenge, a concept that can now be tested in other animal models in a perspective of a putative clinical trial.