An Ultralong Bovine CDRH3 that Targets a Conserved, Cryptic Epitope on SARS-CoV and SARS-CoV-2

Abstract

The ability of broadly neutralising antibodies to target conserved epitopes gives them huge potential as antibody-based therapeutics, particularly in the face of constant viral antigen evolution. Certain bovine antibodies are highly adept at binding conserved, glycosylated epitopes, courtesy of their ultralong complementarity determining region (CDR)H3. Here, we used a SARS-naïve, bovine ultralong CDRH3 library and mammalian cell display, to isolate a bovine paratope that engages the SARS-CoV and SARS-CoV-2 receptor-binding domain (RBD). This neutralises viruses pseudo-typed with SARS-CoV Spike protein but not by competition with RBD binding to ACE2. Instead, using differential hydrogen-deuterium exchange mass spectrometry and site-directed mutagenesis, we demonstrate that this ultralong CDRH3 recognises a rarely identified, conserved, cryptic epitope that overlaps the target of pan-sarbecovirus antibodies (7D6/6D6). The epitope is glycan-shielded and becomes accessible only transiently via inter-domain movements. This represents the first bovine anti-sarbecovirus paratope and highlights the power of this approach in identifying novel tools to combat emerging pathogens.

SciScore for 10.1101/2022.04.06.487306: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Ethics	not detected.
Sex as a biological variable	not detected.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
After binding, samples were washed twice in sort buffer and incubated with a 1:100 dilution of α-Myc-FITC (Abcam, #Ab1263) and α-His-PE (Abcam, #Ab72467) antibody at room temperature for 10 minutes.	α-His-PE ( Abcam , #Ab72467 ) suggested: None
After 1 hour at 4 °C, cells were washed twice in sort buffer and stained with α-His-PE antibody (1:100).	α-His-PE suggested: None
Experimental Models: Cell Lines
Sentences	Resources
Briefly, 293T cells expressing scFv on the cell surface were detached …

SciScore for 10.1101/2022.04.06.487306: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Ethics	not detected.
Sex as a biological variable	not detected.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
After binding, samples were washed twice in sort buffer and incubated with a 1:100 dilution of α-Myc-FITC (Abcam, #Ab1263) and α-His-PE (Abcam, #Ab72467) antibody at room temperature for 10 minutes.	α-His-PE ( Abcam , #Ab72467 ) suggested: None
After 1 hour at 4 °C, cells were washed twice in sort buffer and stained with α-His-PE antibody (1:100).	α-His-PE suggested: None
Experimental Models: Cell Lines
Sentences	Resources
Briefly, 293T cells expressing scFv on the cell surface were detached with trypsin-EDTA (Thermo Fisher Scientific) and washed twice in prechilled sort buffer (1% FCS, 25 mM HEPES-KOH pH 7.9, 1 mM EDTA in PBS).	293T suggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)
Recombinant DNA
Sentences	Resources
An expression vector for the secretion and purification of His-tagged proteins from mammalian cells was generated by insertion of DNA encoding an IGK leader and 8xHis tag into pCS2-MT+.	pCS2-MT+ suggested: None
A DNA fragment encoding the SARS-CoV-2 RBD (aa 319-591) was amplified from pCAGGS-SARS-CoV-2-Spike vector (a kind gift from Keith Grehan) and cloned in frame with the N-terminal IGK leader sequence and C-terminal 8xHis tag using NheI and XhoI sites.	pCAGGS-SARS-CoV-2-Spike suggested: None
The round 3 enriched ultralong scFv library was transferred from pBovShow into this lentiviral vector and lentiviruses were generated by transient transfection of 293T cells.	pBovShow suggested: None
The next day, 4 μg Lenti-BovShow-IRES-PuroR, 4 μg of pCMVR8.74 packaging vector (Addgene plasmid #22036) and 2 μg of pMD2.	pCMVR8.74 suggested: RRID:Addgene_22036) pMD2 suggested: None
G coat protein vector (Addgene plasmid #12259; both gifts from Didier Trono) were mixed with PEI at a 1:3 molar ratio and added to the 10 cm2 dish.	#12259 suggested: None
Pseudotype Neutralisation assays: Pseudotyped lentiviral particles were generated by transfecting 3 × 106 293T cells in a 10 cm2 dish with a lentivirus backbone plasmid encoding a luciferase reporter gene (BEI: NR-52516 pHAGE-CMV-Luc2-IRES-ZsGreen-W), a plasmid encoding either the SARS-CoV Spike (pCAGGS-SARS-CoV-Spike_Urbani), SARS-CoV-2 Spike (BEI: NR-52514) or VSV glycoprotein (VSV-G; pMD2.G) and the packaging vectors HDM-Hgpm2 (BEI: NR-52517), HDM-tat1b (BEI: NR-52518) and pRC-CMV-Rev1b (BEI: NR-52519).	pHAGE-CMV-Luc2-IRES-ZsGreen-W suggested: RRID:Addgene_164432) pCAGGS-SARS-CoV-Spike_Urbani suggested: None pMD2 . G suggested: None pRC-CMV-Rev1b suggested: RRID:Addgene_164443)
293T cells were transfected with either B9-WT or B9-Mut plasmid DNA and cell-surface expressed scFvs were tested for binding to purified SARS-CoV RBD (200 nM) using the standard staining protocol.	B9-Mut suggested: None
Software and Algorithms
Sentences	Resources
The raw HDX-MS data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset: PXD032965.	PRIDE suggested: (Pride-asap, RRID:SCR_012052)
Disruption of the B9-scFv knob domain: The sequence encoding B9-scFv was mutated in the BovShow cell surface expression vector; residues 123YNCRPAVWY131 of the B9-scFv knob domain (B9-WT) were replaced with the irrelevant amino acid sequence 123ETCYYGSGL131 by site-directed mutagenesis (B9-Mut) with Q5 polymerase (New England Bioloabs)	New England Bioloabs suggested: None
The KD for interactions between cell surface scFvs and recombinant RBD proteins was estimated by non-linear analyses of the log(molarity)-response plots on GraphPad.	GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)

Results from OddPub: Thank you for sharing your data.

Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

Results from rtransparent:

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No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

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An Ultralong Bovine CDRH3 that Targets a Conserved, Cryptic Epitope on SARS-CoV and SARS-CoV-2

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