Determinants of mRNA-LNP vaccine efficacy

PROJECT SUMMARY We aim to better understand how mRNA lipid nanoparticle (mRNA-LNP) vaccines elicit immune responses. Broadly, our proposal seeks to address one of the apparent paradoxes of the mRNA-LNP vaccines, namely, how does a membrane-anchored immunogen, delivered to skeletal muscle, elicit germinal center responses in distant lymphoid organs? Our proposal builds upon two recent findings from other groups: (1) Hassett et al. showed that skeletal myocytes are not efficiently transduced by intramuscularly administered mRNA-LNPs. Instead, resident and infiltrating immune cells at the injection site as well as macrophages, dendritic cells, and lymphocytes, albeit fewer, in the draining lymph nodes appear to be the primary targets. (2) Hoffmann et al. showed that cells transduced with mRNA-LNP shed extracellular vesicles (EVs) containing the immunogen, and that modifying the immunogen to enhance EV release improves vaccine efficacy. We hypothesize that transduced cells in the draining lymph node, most likely professional antigen presenting cells (APCs), drives the immune response, and that EVs contribute to immunogenicity by delivering intact immunogen from the injection site to APCs in the draining lymph nodes. To investigate this hypothesis in its entirety is beyond the scope of this application. Instead, we will address two specific aspects. In Aim 1, we will determine the importance of transducing specific cell type(s) on mRNA-LNP vaccine efficacy, using a reporter and then the SARS-CoV-2 S protein as an immunogen. Although Hassett et al. already investigated biodistribution, their report is the only one to date. We will therefore verify their finding that macrophages are the major target cells and that muscle cells do not express the immunogen. To do so, we will carry out a more detailed characterization of leukocyte subsets transduced during vaccination, both at the injection site and in the draining lymph nodes. Using a reporter in conjunction with flow cytometry and immunohistochemistry, we will immunophenotype transduced cells to complement the approaches used in the previous study. We will also determine the functional importance of key cell types employing miRNA-mediated de-targeting in APCs, skeletal muscle cells, and liver cells. This will involve inserting cell type-specific miRNA target sequences into the 3' UTR of reporter and SARS-CoV-2 S protein mRNA vaccine constructs. Aim 2 builds on the study by Hoffmann et al., which showed modifying the carboxy terminus of the SARS-CoV- 2 S protein to promote EV shedding improves mRNA vaccine efficacy. EVs bearing S protein resemble enveloped virions released during infection. Unlike virions, however, these EVs lack "danger signals" such as viral RNA, which acts as a TLR7 agonist. As a result, once EVs have migrated from their cell of origin, they are unlikely to be recognized in a pro-inflammatory context. To address this limitation, we will introduce a danger signal or co-stimulatory molecules to the vaccinal EVs to enhance their immunogenicity and that of the overall vaccine. We will evaluate three such strategies in vitro and in mice.