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Published March 18, 2023 | Supplemental Material + Submitted
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ESCRT recruitment to mRNA-encoded SARS-CoV-2 spike induces virus-like particles and enhanced antibody responses


Prime-boost regimens for COVID-19 vaccines elicit poor antibody responses against Omicron-based variants and employ frequent boosters to maintain antibody levels. We present a natural infection-mimicking technology that combines features of mRNA- and protein nanoparticle-based vaccines through encoding self-assembling enveloped virus-like particles (eVLPs). eVLP assembly is achieved by inserting an ESCRT- and ALIX-binding region (EABR) into the SARS-CoV-2 spike cytoplasmic tail, which recruits ESCRT proteins to induce eVLP budding from cells. Purified spike-EABR eVLPs presented densely-arrayed spikes and elicited potent antibody responses in mice. Two immunizations with mRNA-LNP encoding spike-EABR elicited potent CD8+ T-cell responses and superior neutralizing antibody responses against original and variant SARS-CoV-2 compared to conventional spike-encoding mRNA-LNP and purified spike-EABR eVLPs, improving neutralizing titers >10-fold against Omicron-based variants for three months post-boost. Thus, EABR technology enhances potency and breadth of vaccine-induced responses through antigen presentation on cell surfaces and eVLPs, enabling longer-lasting protection against SARS-CoV-2 and other viruses.

Additional Information

The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license. We thank J. Vielmetter and the Caltech Protein Expression Center for assistance with protein production, K. Dam for biotinylated proteins for ELISAs, M. Anaya for a BirA expression plasmid, and J. Bloom (Fred Hutchinson) and P. Bieniasz (Rockefeller University) for neutralization assay reagents. We thank J. Keeffe, A. West, Y. Tam (Acuitas Therapeutics), C. Barnes (Stanford), H. Kleanthous (Bill and Melinda Gates Foundation), B. Wold, G. Tolomiczenko, and the Caltech Merkin Institute for Translational Research for helpful discussions and A. West for careful reading of the manuscript. Electron tomography was performed in the Caltech Cryo-EM Center with assistance from S. Chen. We thank Labcorp Drug Development–Antibody Reagents and Vaccines (Denver, PA) (formerly Covance, Inc.) for mouse immunization studies, BIOQUAL, Inc. (Rockville, MD) for PRNT50 assays, and R. Sukhovershin and RNAcore (Houston Methodist Research Institute) for synthesis of mRNAs and helpful discussion. Figures 1A and 3A were created with Biorender.com. This work was supported by the Bill and Melinda Gates Foundation INV-034638 (P.J.B.), the Caltech Merkin Institute (P.J.B.), the George Mason University Fast Grants (P.J.B.), the Rothenberg Innovation Initiative (RI2 475 ) (P.J.B.), and Wellcome Leap (P.J.B.). M.A.G.H. was supported by an NIH Director's Early Independence Award (FAIN# DP5OD033362). Author contributions: M.A.G.H. and P.J.B. conceived the study, acquired funding, analyzed the data, and wrote the manuscript with contributions from other authors (Z.Y., P.J.C.L.). M.A.G.H. and K.E.H.T. generated, expressed, and evaluated EABR constructs by Western blot and flow cytometry analysis. M.A.G.H., K.E.H.T., P.N.P.G., L.M.K., and K.N.S. evaluated serum antibody responses from immunized mice by ELISA and neutralization assays. Z.Y. performed cryo-electron tomography and interpreted results. A.A.C. prepared S-mi3 immunogens for immunization studies in mice. W.J.M. and P.J.C.L. prepared mRNA-LNP for immunization studies in mice. Data availability. All data are available in the main text or the supplementary information. Materials are available upon request to the corresponding authors with a signed material transfer agreement. This work is licensed under a Creative Commons Attribution 4.0 International (CC BY 4.0) license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. To view a copy of this license, visit https://creativecommons.org/licenses/by/4.0/. This license does not apply to figures/photos/artwork or other included in the article that is credited to a third party; obtain authorization from the rights holder before using such material. Competing Interest Statement. M.A.G.H. and P.J.B. are inventors on a US patent application filed by the California Institute of Technology that covers the EABR technology described in this work. W.J.M. and P.J.C.L. are employees of Acuitas Therapeutics, a company developing lipid nanoparticle delivery technology; P.J.C.L. holds equity in Acuitas Therapeutics.

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October 9, 2023
January 18, 2024