Nucleic acid-based vaccines such as viral vectors, plasmid DNA and mRNA have been developed as a means to address limitations of both live attenuated and subunit vaccines. Among them, Self Amplifying mRNA vaccine (SAM) has been widely evaluated in different animal models and has been confirmed to be well tolerated and able to drive in vitro antigen expression. However, the molecular mechanism of action of SAM approach has not been fully elucidated. To address this gap, we employed a quantitative mass spectrometry (MS) approach to investigate the molecular fate of vaccine antigens encoded by SAM, from RNA delivery, until MHC-peptide presentation. In this work, we investigated the quantitative correlation between the antigen expression and epitope presentation on MHC class I molecules in a dose-range and time-lapse assay using myoblast cell cultures. Two delivery systems were compared, viral replicon particles (VRPs) and lipid nanoparticle, both already successfully tested in vivo with many different vaccine candidates. The data obtained show that the rate of intracellular antigen expression driven by VRPs is faster compared to the expression driven by SAM encapsulated in LNPs. Moreover we observed a tight correlation between the onset of protein expression and MHC class I epitope presentation for both delivery systems, providing strong evidence that epitope presentation is temporally linked to antigen translation. Furthermore, after detection, no evident differences in the intracellular amount of protein antigen and in the level of epitope peptide were observed, assuming that the main difference between VRPs and LNPs is only related to the mechanism of cellular uptake. Then we applied this technology to quantify the SAM encoded antigen in the muscle and lymph nodes of vaccinated mice at the site of injection, where this new type of vaccine are able to generate amount of antigen lower compare to the standard dose given by classical vaccines. Moreover, we move to an in vitro model of co-culture dendritic cells through, we were able to definitively confirm that this cells are not directly transfected by SAM but are able to up take antigen encoded by transfected myoblast cells. In this study we demonstrated the powerful use of Mass Spectrometry to better understand the mechanism of action of new kind of vaccines during the immune response.
|Titolo:||Fate of antigens encoded by Self-amplifying mRNA vaccines|
|Data di discussione:||17-feb-2017|
|Appartiene alla tipologia:||07a Tesi di Dottorato|