Advanced In vitro Immune Profiling of GMMA-based vaccines

Generalized Modules for Membrane Antigens (GMMA) are outer membrane vesicles derived from engineered Gram-negative bacteria. GMMA, resembling bacteria outer membrane surface, provide a flexible platform for vaccine development. Indeed, GMMA-based vaccines induced functional antibodies with a good safety profile. Previous findings demonstrated that Shigella GMMA activate Antigen Presenting Cells (APCs) through self- adjuvanticity potential. However, we still have a lack of information about how GMMA interact with the immune cells as well as additional cell subset at the injection site, influencing their function and development. Using multi-parametric approach (high-dimension flow cytometry combined with cytokine detection assay), we characterized the Shigella GMMA-based vaccine Mode of Action (MoA) on human peripheral blood mononuclear cells (hPBMCs), in vitro differentiated Monocyte-derived Dendritic Cell (MoDC) and human Skeletal Muscle Cell (hSkMC). Moreover, we developed an in vitro Naïve CD4+ T cell polarization assay to investigate the impact of GMMA-mediated APC activation on adaptive immune response. Data from hPBMCs showed that NK cells, monocytes, and dendritic cells (DCs) are activated upon stimulation with Shigella vaccine and produced pro-inflammatory cytokines and chemokines (e.g. IL-6, TNFα, IL-8 and MIP-1α). Moreover, MoDC in vitro model allowed us to demonstrate that GMMA-based vaccine leads activation and maturation of DC by inducing cytokine production and upregulation of activation markers (e.g. CD40, CD83). Furthermore, we showed that GMMA induced the production of myokines on hSkMC after in vitro stimulation suggesting that GMMA influence the activation of muscle cells at the injection site. Finally, supernatants from GMMA-stimulated MoDC lead naïve CD4 T cells toward Th1 phenotype as showed by the up-regulation of T-bet and secretion of IFNy in the T cell polarization system. In conclusion, our in vitro models provide a tool to define an immune profile giving a new insight into the human response to GMMA-based vaccines, allowing the development of more effective vaccines.