mRNA-based SARS-CoV-2 vaccines: intracellular processing and aggregation of the encoded spike protein as a mechanistic contributor to cardiac cellular stress.

Introduction: The trimeric spike (S) protein on the envelope of the SARS-CoV-2 virus is the primary target structure for currently approved corona vaccines. For this reason, the two mRNA-based corona vaccines Comirnaty (BNT162b2, Pfizer/BioNTech) and Spikevax (mRNA-1273, Moderna) first induce the production of a spike monomer in body cells. After enzymatic cleavage by the endoprotease furin, two S subunits are formed, which are supposed to trigger the desired immune response following secretion. Based on this concept, a preventive measure against symptomatic SARS-CoV-2 infections became available within one year of the pandemic's onset. mRNA-based vaccines have proven highly effective in reducing severe disease and mortality. However, both the virus itself and mRNA vaccines have been associated with cardiac symptoms, which are commonly classified as myocarditis, pericarditis, or a combination thereof based on clinical presentation. Although vaccine-induced myocarditis remains a rare adverse event, recent longitudinal studies have raised questions regarding its long-term impact. Objective: To better understand the molecular mechanisms potentially involved in vaccine-associated cardiac side effects, we investigated the translation and proteolytic processing of the encoded spike monomers in human AC16 cardiomyocytes, as well as (for comparative purposes) in HEK-293 and HeLa cells. Results: In all three cell types, both BNT162b2 and mRNA-1273 produced two divergently sized monomer translation products from which one S1 subunit was formed after enzymatic cleavage. However, the number of identified S2 subunits varied between two and four depending on the cell line and mRNA used. Within a few hours, covalently bonded high-molecular complexes formed from both the spike monomers and their subunits. The arrangement of these complexes always adhered to a consistent pattern in each cell type. Particularly in AC16 cardiomyocytes, the various spike protein derivatives impaired not only cell proliferation, but also induced a pro-inflammatory response and oxidative stress. Only the secreted S1 subunit was detected as an immunogen in the supernatant of all three cell lines. Conclusion: Our findings may help to improve the safety and specificity of future mRNA platform technologies by emphasizing the importance of evaluating intracellular protein processing and the potential cellular effects of translated immunogens already during preclinical development.