Project description:Though mRNA vaccines against COVID-19 have revolutionized vaccinology and have been administered in billions of doses, we know incredibly little about how mRNA vaccines are metabolized in vivo. Here we implemented enhanced nanopore Direct RNA sequencing (eDRS), to enable the analysis of single Moderna’s mRNA-1273 molecules, giving in vivo information about the sequence and poly(A) tails. We show that mRNA-1273, with all uridines replaced by N1-methylpseudouridine (mΨ), is terminated by a long poly(A) tail (~100 nucleotides) followed by an mΨCmΨAG sequence. In model cell lines, mRNA-1273 is swiftly degraded in a process initiated by the removal of mΨCmΨAG, followed by CCR4-NOT-mediated deadenylation. In contrast, intramuscularly inoculated mRNA-1273 undergoes more complex modifications. Notably, mRNA-1273 molecules are re-adenylated after mΨCmΨAG removal. Detailed analysis of immune cells involved in antigen production revealed that in macrophages, after mΨCmΨAG removal, vaccine mRNA is very efficiently re-adenylated, and poly(A) tails can reach up to 200A. In contrast, in dendritic cells, vaccine mRNA undergoes slow deadenylation-dependent decay. We further demonstrate that enhancement of mRNA stability in macrophages is mediated by TENT5 poly(A) polymerases, whose expression is induced by the vaccine itself. Lack of TENT5-mediated re-adenylation results in lower antigen production and severely compromises specific immunoglobulin production following vaccination. Together, our findings provide an unexpected principle for the high efficacy of mRNA vaccines and open new possibilities for their improvement. They also emphasize that, in addition to targeting a protein of interest, the design of mRNA therapeutics should be customized to its cellular destination.
Project description:Development of Raman spectroscopy-based microbial desorption technology for high-speed separation of functional single cells and genome analysis (KAP220340)
Project description:Messenger RNA vaccines based on lipid nanoparticles (mRNA-LNPs) are promising vaccine modalities. However, mRNA-LNP vaccines frequently cause adverse reactions such as swelling and fever in humans, partly due to the inflammatory nature of LNP. Modification of the ionizable lipids used in LNP is one approach to avoid these adverse reactions. Herein, we report the development of mRNA-LNP vaccines with better protective immunity and reduced adverse reactions using LNP, including SS-cleavable and pH-activated lipid-like materials with oleic acid (ssPalmO) as an ionizable lipid (LNPssPalmO). We used mRNA expressing H5N1 subtype high-pathogenicity avian influenza virus-derived hemagglutinin or neuraminidase to generate mRNA-LNP vaccines against H5N1 influenza. Compared with conventional LNP, mRNA-LNPssPalmO induced comparable antigen-specific antibodies and better IFN--producing Th1 responses in mice. Both mRNA-LNPssPalmO and conventional mRNA-LNP conferred strong protection against homologous H5N1 virus challenge. In addition, mRNA-LNPssPalmO showed better cross-protection against heterologous H5N1 virus challenge compared to conventional mRNA-LNPs. Furthermore, we observed that mRNA-LNPssPalmO induced less inflammatory responses (e.g., inflammatory cytokine production and vascular hyperpermeability) and fewer adverse reactions (e.g., weight loss and fever) compared with conventional mRNA-LNP. These results suggest that mRNA-LNPssPalmO would be a safe alternative to conventional vaccines to overcome mRNA-LNP vaccine hesitancy.
Project description:Messenger RNA vaccines based on lipid nanoparticles (mRNA-LNPs) are promising vaccine modalities. However, mRNA-LNP vaccines frequently cause adverse reactions such as swelling and fever in humans, partly due to the inflammatory nature of LNP. Modification of the ionizable lipids used in LNP is one approach to avoid these adverse reactions. Herein, we report the development of mRNA-LNP vaccines with better protective immunity and reduced adverse reactions using LNP, including SS-cleavable and pH-activated lipid-like materials with oleic acid (ssPalmO) as an ionizable lipid (LNPssPalmO). We used mRNA expressing H5N1 subtype high-pathogenicity avian influenza virus-derived hemagglutinin or neuraminidase to generate mRNA-LNP vaccines against H5N1 influenza. Compared with conventional LNP, mRNA-LNPssPalmO induced comparable antigen-specific antibodies and better IFN--producing Th1 responses in mice. Both mRNA-LNPssPalmO and conventional mRNA-LNP conferred strong protection against homologous H5N1 virus challenge. In addition, mRNA-LNPssPalmO showed better cross-protection against heterologous H5N1 virus challenge compared to conventional mRNA-LNPs. Furthermore, we observed that mRNA-LNPssPalmO induced less inflammatory responses (e.g., inflammatory cytokine production and vascular hyperpermeability) and fewer adverse reactions (e.g., weight loss and fever) compared with conventional mRNA-LNP. These results suggest that mRNA-LNPssPalmO would be a safe alternative to conventional vaccines to overcome mRNA-LNP vaccine hesitancy.