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We are grateful for the participation of Courtney Yoshiyama, @youngjunpark.bsky.social @dabiophysicist.bsky.social Jack Brown & our amazing collaborators in the @tylernstarr.bsky.social lab & @virbiotechnology.bsky.social 12/12

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Using #cryoEM and functional assays, we showed that MB11 binds to the PDCoV RBD as designed and inhibits viral entry by preventing engagement of the host receptor through steric hindrance, explaining its potent neutralizing activity 11/12

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MB11 is endowed with exceptionally favorable biophysical properties (resistance to proteases and pH changes) which are ideally suited for cost-effective manufacturing and distribution for use as a countermeasure for outbreak response! 10/12

Since PDCoV is an enteropathogenic virus, counter-measures would likely need to be delivered to the intestinal mucosa to prevent viral entry at the initial site of infection. We therefore subjected MB11 to a bunch of biochemical stresses to assess if it would be a viable option 9/12

We systematically evaluated if viral evolution may affect MB11 and only a few S mutations dampen recognition, most of which reduce receptor binding (viral fitness) and/or require several nucleotide changes (rare). Accordingly, only one of these mutations are found in known PDCoV isolates 8/12

Accordingly, MB11 neutralized a panel of deltacoronaviruses, outperforming antibodies we previously described against PDCoV both in terms of neutralization potency and breadth 7/12 www.sciencedirect.com/science/arti...

The diversity of deltacoronavirus RBD amino acid sequences is a grand challenge for the design of broadly neutralizing countermeasures. MB11 (and the related MB10) bound to a wide range of RBDs in 3 clades, including an RBD with only 59% sequence identity with the PDCoV strain used for design 6/12

One minibinder, designated MB11, stoud out from the 17 we experimentally evaluated by binding to the PDCoV RBD with picomolar affinity straight out of the computer! 5/12

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To prepare for possible future PDCoV outbreaks, we de novo designed miniproteins targeting the PDCoV receptor-binding domain (RBD) in the spike (S) glycoprotein which promotes viral entry into cells and is the main target of coronavirus neutralizing antibodies 4/12

No vaccines or specific therapeutics are approved for use in humans against PDCoV 3/12

Independent infections of porcine deltacoronavirus among Haitian children - Nature

Porcine deltacoronavirus (PDCoV) was shown to be able to use human APN for cell entry and was recently associated with multiple zoonotic transmission events in children with acute undifferentiated febrile illness 2/12 www.nature.com/articles/s41... www.pnas.org/doi/abs/10.1...

nature.com

The presence of porcine deltacoronavirus has been detected in three children from Haiti that could have originated from zoonotic spillover.

Proceedings of the National Academy of Sciences (PNAS), a peer reviewed journal of the National Academy of Sciences (NAS) - an authoritative source of high-impact, original research that broadly spans...

Can we use #proteindesign to develop countermeasures before viruses cause pandemics? We describe the computational design of an ultrapotent and broadly neutralizing deltacoronavirus miniprotein in our latest work led by Nathan Avery @hhmi-science.bsky.social 1/12 www.pnas.org/doi/10.1073/...

pnas.org

PNAS

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We are incredibly grateful to everyone involved including Jimin Lee, Cecily Gibson, Cameron Stewart, Ale Tortorici @dabiophysicist.bsky.social & our wonderful collaborators Lisa Purcell, @turville.bsky.social Nick Riley Federica Sallusto Jun Siong Low ... 19/19

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Prophylactic administration of H1H7 to human TMPRSS2 transgenic mice protected them against challenge with SARS-CoV-2 by reducing lung viral titers to level comparable to genetic ablation of TMPRSS2 18/19

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We show that H1H7 recognizes an epitope distant from the catalytic site and does not inhibit cleavage of a peptide substrate, suggesting that inhibition occurs through stabilization of a TMPRSS2 conformation incompatible with E-FIC cleavage 17/19

We describe a human monoclonal antibody (H1H7) targeting TMPRSS2 and inhibiting all SARS-CoV-2 variants we evaluated, and even SARS-CoV-1 and MERS-CoV! 16/19

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Based on these results, we wondered if one could target the host instead of the virus to block viral infections. Specifically, we aimed to prevent TMPRSS2-mediated activation of membrane fusion & viral entry 15/19

We summarize our findings in an animation made in partnership with @jiwasa.bsky.social detailing the process of coronavirus entry into cells 14/19

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Binding of E-FIC to HR2 was markedly enhanced by TMPRSS2, due to proteolytic processing, relative to inactive TMPRSS2 or untreated. Therefore, cleavage of E-FIC at the S2′ site promotes efficient access of HR2 to its binding site on HR1 and refolding to postfusion S leading to viral entry 13/19

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As the S2′ site is the target of the most broadly neutralizing CoV antibodies, we used #proteindesign to yield a stable E-FIC that would overcome its transient nature. Our stabilized E-FIC design recapitulates native E-FIC antibody recognition and paves the way for future studies 12/19

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These findings show how VN01H1, and similar S2′ site-directed antibodies, would sterically hinder TMPRSS2 binding to E-FIC (through recognition of an overlapping site on S) and in turn proteolytic activation of membrane fusion and viral entry! 11/19

Given that E-FIC is the target of TMPRSS2, we hypothesized that S2′ site-directed antibodies also recognize this state. We thus characterized the complex between SARS-CoV-2 E-FIC and the VN01H1 monoclonal antibody which neutralizes several human alpha- and beta-coronaviruses using #cryoEM 10/19

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To visualize TMPRSS2-mediated recognition of a coronavirus S2′ site and overcome transient binding, we determined #cryoEM structures of TMPRSS2 bound to the HCoV-NL63 S2′ peptide, providing a blueprint of the interactions that lead to viral entry 9/19

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Our results show that TMPRSS2 recognizes and cleaves E-FIC at position R815 , promoting efficient fusion of the host membrane (in which the fusion peptide is embedded) and the viral membrane (tethered to the S transmembrane domain) leading to viral entry 8/19

We set out to test this hypothesis by reconstituting the entry process using purified recombinant glycoproteins and using molecular imaging with electron microscopy to obtain snapshots at various points of the entry process 7/19

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Based on the exposure of the S2' site and its proximity to the host membrane, we hypothesized that E-FIC is the conformational state recognized by host proteases, including the canonical TMPRSS2 which participate in promoting entry of many coronaviruses 6/19

We previously described antibodies with near pan-coronavirus reactivity targeting the S2' site. We found that binding of these antibodies to S is markedly enhanced by preincubation with the receptor, pointing to the existence of a conformational intermediate 4/19 www.science.org/doi/10.1126/...

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It has been known for ~25 years that receptor engagement induces allosteric conformational changes exposing the S2' site but a molecular understanding of this step has remained elusive. 3/19

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