Development and validation of an mRNA vaccine platform expressing the receptor binding domain of the SARS-CoV-2 spike protein or the enhanced green fluorescent protein.

In this thesis the development of an mRNA vaccine platform and its validation are described. The possibilities offered by mRNA vaccines platform are promising, due to their versatility, efficacy and rapid production. In Chapter 3 the production of an mRNA vaccine encoding as an antigen the SARS-CoV-2 Receptor Binding Domain (RBD) of the spike protein is described. mRNA was in vitro transcribed from a DNA template containing all the essential elements for the production of synthetic mRNA, and then post-transcriptionally modified with a 5’ cap and a 3’ poly-A tail. Purified mRNA was then transfected in human cell lines (HeLa and HEK 293) with two commercial cationic lipids (Lipofectamine™ 3000 or MessengerMAX™) as delivery systems. Then, heterologous protein expression was detected by Western-Blot and flow cytometric assays. In Chapter 4 the production of an mRNA encoding for the enhanced Aequorea victoria Green Fluorescent Protein (eGFP) is described, using the same platform and in vitro processing developed in Chapter 3. In this case, transfection was performed in HeLa cells and in a murine myoblast cell line (C2C12). Time course analyses of eGFP expression in both transfected cell lines showed the expression of the protein already at 1 hour, with an increase in time, following different kinetics. Expression of heterologous protein was detected by fluorescence microscopy and flow cytometry analysis. Transfection of both constructs complexed with lipofectamine induced detachment of the cells, which was investigated by annexin V and propidium iodide assays, suggesting that cells underwent apoptosis. Then, to get closer to in vivo conditions, phagocytosis assays were performed, showing that only transfected cells were efficiently engulfed by macrophage-like THP-1 cells, compared to the untreated ones. Therefore, cell death might enhance the response to mRNA vaccines. Further investigations through transcriptomic analyses may help elucidate this phenomenon.