Highly synergistic combinations of nanobodies that target SARS-CoV-2 reduce viral load in hACE2 transgenic mice

ABSTRACT

The ongoing emergence of SARS-CoV-2 variants threatens current vaccines and renders current therapeutic antibodies obsolete, demanding powerful new treatments that can resist viral escape. We therefore generated a large nanobody repertoire to saturate the distinct and highly conserved available epitope space of SARS-CoV-2 spike, including the S1 receptor binding domain, N-terminal domain, and the S2 subunit, to identify new nanobody binding sites that may reflect novel mechanisms of viral neutralization. Structural mapping and functional assays show that these highly stable monovalent nanobodies potently inhibit SARS-CoV-2 infection, display numerous neutralization mechanisms, are effective against past and present emerging variants of concern, and are resistant to mutational escape. Rational combinations of these nanobodies that bind dissimilar sites within and between spike subunits exhibit extraordinary synergy and suggest multiple tailored therapeutic and prophylactic strategies. All mouse involved experiments were performed in compliance with the Institutional Animal Care and Use Committee and mice were housed and maintained in a specific pathogen-free conditions at Seattle Children's Research Institute. Infected mice with SARSCoV- 2 were housed in a Biosafety Level 3 facility in an Animal Biohazard Containment Suite. Prophylactic intranasal application of a synergistic pair of unmodified nanobodies in 10-12 week-old female K18-hACE2 transgenic mice, a mouse model of SARS-CoV-2 infection, showed significant reduction in viral load after 3 days post-challenge with SARS-CoV-2, the first demonstration of synergy in vivo. In summary, our results show that our diverse repertoire of nanobodies can neutralize current variants of live SARS-CoV-2, pairs of nanobodies that bind distinct sites on spike show tremendous synergy in neutralizing efficacy in vitro, and the application of synergizing pair of nanobodies translates to an in vivo mouse model of SARSCoV- 2. Research funded by the Mathers Foundation, Robertson Foundation, NIH P41GM109824.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.