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Published March 22, 2023 | Supplemental Material + Submitted
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Multiviral Quartet Nanocages Elicit Broad Anti-Coronavirus Responses for Proactive Vaccinology


Defending against future pandemics may require vaccine platforms that protect across a range of related pathogens. The presentation of multiple receptor-binding domains (RBDs) from evolutionarily-related viruses on a nanoparticle scaffold elicits a strong antibody response to conserved regions. Here we produce quartets of tandemly-linked RBDs from SARS-like betacoronaviruses coupled to the mi3 nanocage through a SpyTag/SpyCatcher spontaneous reaction. These Quartet Nanocages induce a high level of neutralizing antibodies against several different coronaviruses, including against viruses not represented on the vaccine. In animals primed with SARS-CoV-2 Spike, boost immunizations with Quartet Nanocages increased the strength and breadth of an otherwise narrow immune response. Quartet Nanocages are a strategy with potential to confer heterotypic protection against emergent zoonotic coronavirus pathogens and facilitate proactive pandemic protection.

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 4.0 International license. We thank Dr. David Staunton from the University of Oxford Department of Biochemistry Biophysical Suite for help with biophysical analysis. We thank the Centre for the AIDS Programme of Research in South Africa (CAPRISA) and Gavin Screaton (University of Oxford) for supplying SARS-CoV-2 variant isolates. The BtKY72 K493Y/T498W Spike plasmid for generating pseudovirus was a kind gift to the Bjorkman lab from David Veesler (University of Washington). Funding for this study was provided by: Biotechnology and Biological Sciences Research Council (BBSRC BB/S007369/1) (A.H.K. and M.H.); Rhodes Trust (R.H.); EPA Cephalosporin Early Career Teaching and Research Fellowship (T.K.T.); Townsend-Jeantet Prize Charitable Trust (Charity Number 1011770) (T.K.T.); Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Science (CIFMS), China (grant no. 2018-I2M-2-002) (A.R.T.); University of Oxford COVID-19 Research Response Fund and its donors (reference 0009517) (M.H.) National Institutes of Health (NIH) NIH AI165075 (P.J.B.); Caltech Merkin Institute (P.J.B.); George Mason University Fast Grant (P.J.B.). Author Contributions: R.A.H. performed all experiments except T.K.T. performed mouse immunizations, J.R.K., P.N.P.G. and K.N.S. tested pseudovirus neutralization, and W.S.J., M.L.H., S.L., J.G-J., M.A. and A.N. tested virus neutralization. A.H.K. designed and purified initial Quartet constructs. R.A.H., T.T., A.A.C., W.S.J., P.J.B., A.R.T. and M.H. designed the project. R.A.H. and M.H. wrote the manuscript. All authors read and approved the manuscript. Data availability: Sequences of constructs are available in GenBank, as described in the section "Plasmids and Cloning". Plasmids encoding pDEST14-SpySwitch, pET28aSpyCatcher003-mi3, pET28a-SpyTag-MBP, pcDNA3.1-SpyTag-Quartet, pcDNA3.1-Alternate Quartet and pcDNA3.1-Quartet [SARS1] will be deposited before publication in the Addgene repository (https://www.addgene.org/Mark_Howarth/). Further information and request for resources and reagents should be directed to and will be fulfilled by the lead contact, M.H. (mh2186@cam.ac.uk). Competing Interest Statement. M.H. is an inventor on a patent on spontaneous amide bond formation (EP2534484) and a SpyBiotech co-founder and shareholder. M.H. and A.H.K. are inventors on a patent on SpyTag003:SpyCatcher003 (UK Intellectual Property Office 1706430.4). P.J.B. and A.A.C. are inventors on a US patent application filed by the California Institute of Technology that covers the methodology to generate cross-reactive antibodies using mosaic nanoparticles. P.J.B., and A.A.C. are inventors on a US patent application filed by the California Institute of Technology that covers the monoclonal antibodies elicited by vaccination with Mosaic nanoparticles described in this work. P.J.B., A.A.C. and J.R.K. are inventors on a US patent application filed by the California Institute of Technology that covers the methods of isolating cross-reactive antibodies by vaccination with mosaic nanoparticles. All other authors have no competing interests to declare.

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October 9, 2023
December 22, 2023