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GMMA has been proposed as a potent technology platform for the design of safe, effective and affordable vaccines. As GMMA are vesicles blebbing out of the outer membrane of Gram-negative bacteria, they contain lipopolysaccharides, lipoproteins and peptidoglycans that stimulate immune cells via Toll-like Receptors 4 (TLR4) or TLR2. Being basically nanoparticles, GMMA can be efficiently captured by Follicular Dendritic Cells (FDC) for antigen presentation to cognate B cells. GMMA have shown to be highly immunogenic in preclinical and clinical studies and the engagement of TLR4 and TLR2 or antigen presentation by FDC may have a prominent role in GMMA immunogenicity, which is well worth investigating. By using GMMA derived from Shigella sonnei and Salmonella Typhimurium, we show for the first time that the antigen presentation by FDC to cognate B cells plays a major role in the induction of an effective humoral immune response upon immunization with GMMA by using both models. The engagement of TLR4 is critical to elicit an optimal antibody production, but its effect on antibody functionality is dependent on GMMA type and is dispensable when immunizing with Alum adjuvant, whereas TLR2 does not have any role for GMMA immunogenicity. Our findings represent a substantial advancement of the knowledge on GMMA mode of action and shed a light on novel perspectives for the design of safer and more effective GMMA-based vaccines. ONE SENTENCE SUMMARY: The study demonstrated that the antigen presentation by FDC to cognate B cells plays a major role for GMMA immunogenicity.

Self-amplifying mRNAs (SAM)-based vaccines have been shown to induce a robust immune response in various animal species against both viral and bacterial pathogens. Due to their synthetic nature and to the versatility of the manufacturing process, SAM technology may represent an attractive solution for rapidly producing novel vaccines, which is particularly critical in case of pandemic infections or diseases mediated by newly emerging pathogens. Recent published data support the hypothesis that Antigen Presenting Cells (APCs) are responsible for CD8+ T-cell priming after SAM vaccination, suggesting cross-priming as the key mechanism for antigen presentation by SAM vaccines. In our study we investigated the possibility to enhance the immune response induced in mice by a single immunization with SAM by increasing the recruitment of APCs at the site of injection. To enhance SAM immunogenicity, we selected murine granulocyte-macrophage colony-stimulating factor (GM-CSF) as a model chemoattractant for APCs, and developed a SAM-GM-CSF vector. We evaluated whether the use of SAM-GM-CSF in combination with a SAM construct encoding the Influenza A virus nucleoprotein (NP) would lead to an increase of APC recruitment and NP-specific immune response. We indeed observed that the administration of SAM-GM-CSF enhances the recruitment of APCs at the injection site. Consistently with our hypothesis, co-administration of SAM-GM-CSF with SAM-NP significantly improved the magnitude of NP-specific CD8+ T-cell response both in terms of frequency of cytotoxic antigen-specific CD8+ T-cells and their functional activity in vivo. Furthermore, co-immunization with SAM-GM-CSF and SAM-NP provided an increase in protection against a lethal challenge with influenza virus. In conclusion, we demonstrated that increased recruitment of APCs at the site of injection is associated with an enhanced effectiveness of SAM vaccination and might be a powerful tool to potentiate the efficacy of RNA vaccination.