Multiple mRNA vaccine, first report of protein expression inhibition phenomenon
Multiple mRNA vaccines, the hidden pitfall of translation inhibition
mRNA vaccines have achieved great success in preventing infectious diseases, but as viral variants become more severe, vaccines containing multiple antigens are needed. However, we hypothesized that simultaneous administration of several mRNA species could interfere with the translation of the target proteins.
Inhibitory phenomenon revealed by LC‑MS/MS and immunological validation
After introducing the transcriptome into human cells, we measured protein amounts by liquid chromatography‑tandem mass spectrometry (LC‑MS/MS) and found that multiformulations exhibited markedly lower antigen expression compared with dose‑matched monovalent formulations. The same result was confirmed by Western blot and intracellular immunostaining using type‑specific antibodies, and the untargeted proteome data obtained without additional preprocessing showed signals that high‑dose mRNA reduces the overall translational capacity of cells.
A novel potency assessment method feasible without antibodies
This study demonstrates that LC‑MS/MS can serve as a practical alternative to quantify vaccine potency without reliance on antibodies. In other words, it provides a basis for generating reference materials required for quality control of complex multivalent vaccines and for precise dose optimization.
Implications and future outlook
We learned that interactions among individual mRNAs must be rigorously evaluated during multivalent mRNA vaccine development. Minimizing translation inhibition in future vaccine design and manufacturing processes will enable safer and more effective multivalent vaccines.
Messenger RNA (mRNA) vaccines are highly effective against infectious diseases, but viral diversity has underscored the necessity for multivalent and combination vaccines. We hypothesize there could be interference of target protein translation in multivalent mRNA vaccines in cell-based assays used for potency testing. Following transfection into human cells, we observed that antigen protein translation is inhibited in multivalent formulations compared to their dose-matched monovalent counterparts using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Inhibited expression was further validated using Western blot and intra-cellular immunostaining with type-specific antibodies. Beyond direct antigen-specific analysis, LC-MS/MS proteomics data from the same assay without additional sample preparation provided a broad view of host cell responses, revealing proteome-level changes indicative of impaired global translation capacity in cells transfected with high dose mRNA(s). To the best of our knowledge, we report for the first time in literature that antagonistic interactions among mRNA constructs can reduce protein expression in multivalent drug products. Our study also demonstrates that LC-MS/MS is a viable alternative for potency testing without dependence on antibodies. These findings highlight the need for careful cell-based assay design, multivalent drug product reference standards for potency assessment, and deliberate mRNA dose optimization in multivalent formulations.
It identified that multiple mRNA vaccines interfere with each other, preventing sufficient protein production. This will help accurately assess vaccine efficacy and enable everyone to receive safer immunizations.