Validation of an mRNA vaccine platform expressing eGFP and development of a recombinant Streptococcus gordonii as live vaccine vector

Thesis project involved the development of an mRNA and a recombinant Streptococcus gordonii vaccine model. The mRNA vaccine was produced starting from the design of a DNA plasmid template encoding for the enhanced Green Fluorescent Protein (eGFP) (Chapter 2). The mRNA was transcribed, modified and complexed with a delivery system. The vaccine complex obtained was evaluated for the transfection efficiency and the protein expression in two mammalian cell lines: HeLa cells and C2C12 cells. The expression of the protein was already detectable 1 hour after transfection in both cell lines, with an increase after 6h and 12h respectively. Observation at inverted microscope showed a progressive detachment of the transfected cells from the surface of the wells, indicating a possible cell death after transfection. In addition, it was observed that cell death occurs massively only when the mRNA-lipofectamine complex is transfected, but not when mRNA or lipofectamine alone is transfected, suggesting a direct correlation with the expression of the heterologous protein. These results led us to investigate cell death phenomenon by analyzing with annexin V and propidium iodide. Obtained results confirmed the cell death, whereas phagocytosis assays revealed that transfected cells were more efficiently engulfed by macrophage like THP-1 cell respect the untreated ones, suggesting a possible immunogenic action of the cells expressing the heterologous protein. The second part of this thesis (Chapter 3) describes the development of a Streptococus gordonii strain expressing the hemagglutinin subunit HA1 of influenza A/Puerto Rico 8/1934 for vaccination purposes. For the construction of the vaccine vector, the previously established host-vector system for heterologous gene expression in S. gordonii was used. A synthetic DNA encoding for the HA1 subunit (PR8) was custom synthesized and cloned into pSMB55 shuttle vector for the construction of emm6::ha1(PR8) gene fusion, that was subsequently PCR amplified and transformed into S. gordonii GP1295 recipient strain. The obtain recombinant clone, TP484C2, was first sequenced to confirm chromosomal integration of the genetic construct and then used for M6-HA1(PR8) protein expression analysis performed by western blot and flow cytometry assay, that confirmed the heterologous protein expression at bacterial surface. Preliminary data obtained through these studies provide basis for future in vivo studies on heterologous immunization strategy using both, mRNA vaccine and recombinant Streptococcus gordonii. Lastly, Chapter 4 aimed to briefly describe the methodology learnt in my in-company period in VisMederi Group in Siena.