The COVID-19 pandemic has led to accelerated efforts to develop therapeutics and vaccines. A key target of these efforts is the spike (S) protein, which is metastable and difficult to produce recombinantly. Here, we characterized 100 structure-guided spike designs and identified 26 individual substitutions that increased protein yields and stability. Testing combinations of beneficial substitutions resulted in the identification of HexaPro, a variant with six beneficial proline substitutions exhibiting ~10-fold higher expression than its parental construct and the ability to withstand heat stress, storage at room temperature, and three freeze-thaw cycles. A 3.2 Å-resolution cryo-EM structure of HexaPro confirmed that it retains the prefusion spike conformation. High-yield production of a stabilized prefusion spike protein will accelerate the development of vaccines and serological diagnostics for SARS-CoV-2.

1The COVID-19 pandemic caused by the novel coronavirus SARS-CoV-2 has led to accelerated 2 efforts to develop therapeutics, diagnostics, and vaccines to mitigate this public health 3 emergency. A key target of these efforts is the spike (S) protein, a large trimeric class I fusion 4 protein that is metastable and difficult to produce recombinantly in large quantities. Here, we 5 designed and expressed over 100 structure-guided spike variants based upon a previously 6 determined cryo-EM structure of the prefusion SARS-CoV-2 spike. Biochemical, biophysical 7 and structural characterization of these variants identified numerous individual substitutions that 8 increased protein yields and stability. The best variant, HexaPro, has six beneficial proline 9 substitutions leading to ~10-fold higher expression than its parental construct and is able to 10 withstand heat stress, storage at room temperature, and multiple freeze-thaws. A 3.2 Å-resolution 11 cryo-EM structure of HexaPro confirmed that it retains the prefusion spike conformation. High-12 yield production of a stabilized prefusion spike protein will accelerate the development of 13 vaccines and serological diagnostics for SARS-CoV-2. 14 3 INTRODUCTION 15 Coronaviruses are enveloped viruses containing positive-sense RNA genomes. Four human 16 coronaviruses generally cause mild respiratory illness and circulate annually. However, SARS-17 CoV and MERS-CoV were acquired by humans via zoonotic transmission and caused outbreaks 18 of severe respiratory infections with high case-fatality rates in 2002 and 2012, respectively 1,2 . 19 SARS-CoV-2 is a novel betacoronavirus that emerged in Wuhan, China in December 2019 and 20 is the causative agent of the ongoing COVID-19 pandemic 3,4 . As of May 26, 2020, the WHO has 21 reported over 5 million cases and 350,000 deaths worldwide. Effective vaccines, therapeutic 22 antibodies and small-molecule inhibitors are urgently needed, and the development of these 23 interventions is proceeding rapidly. 24 Coronavirus virions are decorated with a spike (S) glycoprotein that binds to host-cell 25 receptors and mediates cell entry via fusion of the host and viral membranes 5 . S proteins are 26 trimeric class I fusion proteins that are expressed as a single polypeptide that is subsequently 27cleaved into S1 and S2 subunits by cellular proteases 6,7 . The S1 subunit contains the receptor-28 binding domain (RBD), which, in the case of SARS-CoV-2, recognizes the angiotensin-29 converting enzyme 2 (ACE2) receptor on the host-cell surface [8][9][10] . The S2 subunit mediates 30 membrane fusion and contains an additional protease cleavage site, referred to as S2′, that is 31 adjacent to a hydrophobic fusion peptide. Binding of the RBD to ACE2 triggers S1 dissociation, 32 allowing for a large rearrangement of S2 as it transitions from a metastable prefusion 33 conformation to a highly stable postfusion conformation 6,11 . During this rearrangement, the 34 fusion peptide is inserted into the host-cell membrane after cleavage at S2′, and two h...

Three pathogenic human coronaviruses have emerged, with SARS-CoV-2 causing a global pandemic. While therapeutic antibodies targeting the SARS-2 spike currently focus on the poorly conserved receptor-binding domain, targeting essential neutralizing epitopes on the more conserved S2 domain may provide broader protection. We report three antibodies, binding epitopes conserved on the pre-fusion MERS, SARS-1 and SARS-2 spike S2 domains. Antibody 3A3 binds a conformational epitope with ~2.5 nM affinity and neutralizes in in vitro SARS-2 cell fusion and pseudovirus assays. Hydrogen-deuterium exchange mass spectrometry identified residues 980-1006 in the flexible hinge region at the S2 apex as the 3A3 epitope, consistent with binding to natural and engineered spike variants. This location at the spike trimer interface suggests 3A3 prevents the S2 conformational rearrangements required for virus-host cell fusion. This work defines a highly conserved vulnerable site on the SARS-2 S2 domain and may help guide the design of pan-protective spike immunogens.TEASERA conserved, neutralizing epitope in the S2 domain of coronavirus spike was identified as a target for pan-coronavirus therapy and vaccination.

To address the ongoing SARS-CoV-2 pandemic and prepare for future coronavirus outbreaks, understanding the protective potential of epitopes conserved across SARS-CoV-2 variants and coronavirus lineages is essential. We describe a highly conserved, conformational S2 domain epitope present only in the prefusion core of β-coronaviruses: SARS-CoV-2 S2 apex residues 980–1006 in the flexible hinge. Antibody RAY53 binds the native hinge in MERS-CoV and SARS-CoV-2 spikes on the surface of mammalian cells and mediates antibody-dependent cellular phagocytosis and cytotoxicity against SARS-CoV-2 spike in vitro. Hinge epitope mutations that ablate antibody binding compromise pseudovirus infectivity, but changes elsewhere that affect spike opening dynamics, including those found in Omicron BA.1, occlude the epitope and may evade pre-existing serum antibodies targeting the S2 core. This work defines a third class of S2 antibody while providing insights into the potency and limitations of S2 core epitope targeting.

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