Bi-paratopic and multivalent human VH domains neutralize SARS-CoV-2 by targeting distinct epitopes within the ACE2 binding interface of Spike
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (ScreenIT)
Abstract
Neutralizing agents against SARS-CoV-2 are urgently needed for treatment and prophylaxis of COVID-19. Here, we present a strategy to rapidly identify and assemble synthetic human variable heavy (VH) domain binders with high affinity toward neutralizing epitopes without the need for high-resolution structural information. We constructed a VH-phage library and targeted a known neutralizing site, the angiotensin-converting enzyme 2 (ACE2) binding interface of the trimeric SARS-CoV-2 Spike receptor-binding domain (Spike-RBD). Using a masked selection approach, we identified 85 unique VH binders to two non-overlapping epitopes within the ACE2 binding site on Spike-RBD. This enabled us to systematically link these VH domains into multivalent and bi-paratopic formats. These multivalent and bi-paratopic VH constructs showed a marked increase in affinity to Spike (up to 600-fold) and neutralization potency (up to 1400-fold) on pseudotyped SARS-CoV-2 virus when compared to the standalone VH domains. The most potent binder, a trivalent VH, neutralized authentic SARS-CoV-2 with half-minimal inhibitory concentration (IC 50 ) of 4.0 nM (180 ng/mL). A cryo-EM structure of the trivalent VH bound to Spike shows each VH domain bound an RBD at the ACE2 binding site, explaining its increased neutralization potency and confirming our original design strategy. Our results demonstrate that targeted selection and engineering campaigns using a VH-phage library can enable rapid assembly of highly avid and potent molecules towards therapeutically important protein interfaces.
Article activity feed
-
SciScore for 10.1101/2020.08.08.242511: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: 36 Samples were collected in accordance with the Declaration of Helsinki using protocols approved by the UCSF Institutional Review Board (Protocol 20-30338).
Consent: All patients provided written consent.Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Preparation of SARS-CoV-2 pseudotyped virus and HEK-ACE2 overexpression cell line: HEK293T-ACE2 cells were a gift from Arun Wiita’s laboratory at the University of California, San Francisco. HEK293T-ACE2suggested: None48 Briefly, plasmids at … SciScore for 10.1101/2020.08.08.242511: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: 36 Samples were collected in accordance with the Declaration of Helsinki using protocols approved by the UCSF Institutional Review Board (Protocol 20-30338).
Consent: All patients provided written consent.Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Preparation of SARS-CoV-2 pseudotyped virus and HEK-ACE2 overexpression cell line: HEK293T-ACE2 cells were a gift from Arun Wiita’s laboratory at the University of California, San Francisco. HEK293T-ACE2suggested: None48 Briefly, plasmids at the designated concentrations were added to OptiMEM media with FuGENE HD Transfection Reagent (Promega) at a 3:1 FuGENE:DNA ratio, incubated for 30 min, and subsequently transfected into HEK-293T cells. HEK-293Tsuggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)HEK-ACE2 were seeded at 10,000 cells/well on 96-well white plates (Corning, cat. 354620). HEK-ACE2suggested: NoneVirus was incubated in infection media (EMEM 0% FBS) containing different concentrations of binders for 1 hr at 37°C and subsequently added to VeroE6 cells for a low volume infection for 1 hr. VeroE6suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)Software and Algorithms Sentences Resources 54 The final figures were prepared using ChimeraX. ChimeraXsuggested: (UCSF ChimeraX, RRID:SCR_015872)Results from OddPub: Thank you for sharing your code.
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 found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
-
SciScore for 10.1101/2020.08.08.242511: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement 36 Samples were collected in accordance with the Declaration of Helsinki using protocols approved by the UCSF Institutional Review Board (Protocol 20-30338). Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Preparation of SARS-CoV-2 pseudotyped virus and HEK-ACE2 overexpression cell line HEK293T-ACE2 cells were a gift from Arun Wiita’s laboratory at the University of California, San Francisco. HEK293T-ACE2suggested: None…SciScore for 10.1101/2020.08.08.242511: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement 36 Samples were collected in accordance with the Declaration of Helsinki using protocols approved by the UCSF Institutional Review Board (Protocol 20-30338). Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Preparation of SARS-CoV-2 pseudotyped virus and HEK-ACE2 overexpression cell line HEK293T-ACE2 cells were a gift from Arun Wiita’s laboratory at the University of California, San Francisco. HEK293T-ACE2suggested: None48 Briefly, plasmids at the designated concentrations were added to OptiMEM media with FuGENE HD Transfection Reagent (Promega) at a 3:1 FuGENE:DNA ratio, incubated for 30 min, and subsequently transfected into HEK293T cells. HEK293Tsuggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)HEK-ACE2 were seeded at 10,000 cells/well on 96-well white plates (Corning, cat. 354620). HEK-ACE2suggested: NoneVirus was incubated in infection media (EMEM 0% FBS) containing different concentrations of binders for 1 hr at 37°C and subsequently added to VeroE6 cells for a low volume infection for 1 hr. VeroE6suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)Software and Algorithms Sentences Resources 54 The final figures were prepared using ChimeraX. ChimeraXsuggested: (UCSF ChimeraX, RRID:SCR_015872)Results from OddPub: Thank you for sharing your code.
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 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.
About SciScore
SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
-
