Advanced in vitro human immune profiling of GMMA-based vaccines

Generalized Modules for Membrane Antigens (GMMA) are outer membrane vesicles derived from engineered Gram-negative bacteria. GMMA, resembling bacterial surface, present antigens in their native conformation and provide self-adjuvanticity, establishing them as a flexible vaccine platform. Despite previous studies demonstrated that GMMA-based vaccines, administered intramuscularly in healthy subjects, elicited a strong specific antibody response, our understanding of how GMMA-based vaccines influence immune cell function and development, as well as activation of other cell subsets at the injection-site remains limited. Using multi-parametric flow cytometry combined with cytokine detection assays, we define the Mode of Action (MoA) of Shigella and N. gonorrhoeae (Ng) GMMA-based vaccines on immune cells. Specifically, we developed in vitro models which recapitulate the peripheral blood, hPBMCs and a specific subpopulation of innate immune cells, the dendritic cells (DCs) which are known to drive T cell differentiation and function. Moreover, to gain insight into the role of GMMA on the cellular crosstalk occurring at the site of injection, which is instrumental for the priming of adaptive immunity, a primary muscle cell model (PMCM), which combines immune cells with myotubes, was developed. Both GMMA-based vaccines activated immune cells, including hPBMCs and Mo-DCs, eliciting a strong innate pro-inflammatory response. Furthermore, Mo-DCs internalized GMMA and secreted factors that skewed naïve CD4+ T cells toward a Th1 phenotype. GMMA-based vaccines also activated non-immune muscle cells, revealing, in the PMCM cultures, a distinct cytokine milieu compared to hPBMCs in vitro model. In conclusion, our data demonstrated that GMMA-based vaccines are internalized by both professional antigen presenting cells and muscle cells and promoted the secretion of pro-inflammatory mediators, thereby shaping the development and function of T cells independently of the bacterial origin.