Broadly neutralizing and protective nanobodies against diverse sarbecoviruses
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Abstract
As SARS-CoV-2 Omicron and other variants of concern continue spreading around the world, development of antibodies and vaccines to confer broad and protective activity is a global priority. Here, we report on the identification of a special group of nanobodies from immunized alpaca with exceptional breadth and potency against diverse sarbecoviruses including SARS-CoV-1, Omicron BA.1, and BA.2. Crystal structure analysis of one representative nanobody, 3-2A2-4, revealed a highly conserved epitope between the cryptic and the outer face of the receptor binding domain (RBD). The epitope is readily accessible regardless of RBD in “up” or “down” conformation and distinctive from the receptor ACE2 binding site. Passive delivery of 3-2A2-4 protected K18-hACE2 mice from infection of authentic SARS-CoV-2 Delta and Omicron. This group of nanobodies and the epitope identified should provide invaluable reference for the development of next generation antibody therapies and vaccines against wide varieties of SARS-CoV-2 infection and beyond.
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SciScore for 10.1101/2022.04.12.488087: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics Field Sample Permit: Immunization of alpaca, construction of yeast display VHH library, and isolation of VHH yeasts specific for SARS-CoV-2 and SARS-CoV-1 spikes: The animal experiment protocol involving immunization, collection of blood samples, and construction of VHH library was approved by IACUC at NBbiolab, Inc. in Chengdu, China.
IACUC: Immunization of alpaca, construction of yeast display VHH library, and isolation of VHH yeasts specific for SARS-CoV-2 and SARS-CoV-1 spikes: The animal experiment protocol involving immunization, collection of blood samples, and construction of VHH library was approved by IACUC at NBbiolab, Inc. in Chengdu, China.Sex as a biological variable Eight-week-ol… SciScore for 10.1101/2022.04.12.488087: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics Field Sample Permit: Immunization of alpaca, construction of yeast display VHH library, and isolation of VHH yeasts specific for SARS-CoV-2 and SARS-CoV-1 spikes: The animal experiment protocol involving immunization, collection of blood samples, and construction of VHH library was approved by IACUC at NBbiolab, Inc. in Chengdu, China.
IACUC: Immunization of alpaca, construction of yeast display VHH library, and isolation of VHH yeasts specific for SARS-CoV-2 and SARS-CoV-1 spikes: The animal experiment protocol involving immunization, collection of blood samples, and construction of VHH library was approved by IACUC at NBbiolab, Inc. in Chengdu, China.Sex as a biological variable Eight-week-old female K18-hACE2 transgenic mice (InVivos Ptd Ltd, Lim Chu Kang, Singapore) were used for this study. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources After extensive wash with cold PBS+1%FBS, the yeast clones were incubated with HA-Tag (6E2) mouse monoclonal antibody conjugated with Alexa Fluor® 488 (1:100 dilution) and eBioscience™ streptavidin conjugated with PE Conjugate (1:200 dilution) on ice for 30 min. HA-Tagsuggested: (Cell Signaling Technology Cat# 2350, RRID:AB_491023)The cells were then fixed, permeabilized, and incubated with cross-reactive rabbit anti-SARS-CoV-N IgG (Sino Biological, Inc., China) for 1 h at room temperature before adding an HRP-conjugated goat anti-rabbit IgG antibody (Jackson ImmunoResearch, USA). anti-SARS-CoV-N IgGsuggested: Noneanti-rabbit IgGsuggested: NoneSections were then covered with rabbit anti-SARS-CoV-2 N protein monoclonal antibody (Abcam; 1:1000) for 1 h at room temperature. anti-SARS-CoV-2 N proteinsuggested: NoneExperimental Models: Cell Lines Sentences Resources Cell lines: HEK293T cells (ATCC, CRL-3216) and HeLa cells expressing hACE2 were kindly provided by Dr. Qiang Ding at Tsinghua University. HEK293Tsuggested: NoneHeLasuggested: CLS Cat# 300194/p772_HeLa, RRID:CVCL_0030)Sf9 cells (ATCC) were maintained at 27°C in Sf-900 II SFM medium. Sf9suggested: RRID:CVCL_4U10)Expression and production of nanobodies were conducted by transfecting the expression vectors into the HEK293F cells using polyethyleneimine (PEI) (Polysciences). HEK293Fsuggested: RRID:CVCL_6642)Specifically, human immunodeficiency virus backbones expressing firefly luciferase (pNL4-3-R-E-luciferase) and pcDNA3.1 vector encoding either SARS-CoV-2 or sarbecovirus spike proteins were co-transfected into the HEK-293T cells (ATCC). HEK-293Tsuggested: NoneHeLa-ACE2 cells were then added to the mixture of nanobody-pseudovirus, incubated at 37°C for additional 48 h, and lysed for measuring luciferase-activity. HeLa-ACE2suggested: JCRB Cat# JCRB1845, RRID:CVCL_B3LW)Tissues were homogenized with 0.5 mL DMEM supplemented with antibiotic and antimycotic (Gibco, Waltham, MA, USA) and titrated in Vero E6 cells using plaque assays. Vero E6suggested: RRID:CVCL_XD71)Experimental Models: Organisms/Strains Sentences Resources Eight-week-old female K18-hACE2 transgenic mice (InVivos Ptd Ltd, Lim Chu Kang, Singapore) were used for this study. K18-hACE2suggested: RRID:IMSR_GPT:T037657)Recombinant DNA Sentences Resources Cell lines: HEK293T cells (ATCC, CRL-3216) and HeLa cells expressing hACE2 were kindly provided by Dr. Qiang Ding at Tsinghua University. hACE2suggested: RRID:Addgene_1786)VHH sequences were amplified by PCR, cloned into a yeast surface display vector pYD1, and introduced into the electrocompetent EBY100 cells. pYD1suggested: RRID:Addgene_73447)For the former, VHH genes were cloned into the multiple cloning sites of pMD18T containing the upstream CMV promoter, the secretory signal sequence from the mouse Ig heavy chain, and the downstream human IgG1 Fc gene fragment and SV40 poly (A) signal sequence. pMD18Tsuggested: NoneFor the latter, selected VHH genes were cloned into pVRC8400 vector with a 6xHis tag. pVRC8400suggested: RRID:Addgene_63163)Specifically, human immunodeficiency virus backbones expressing firefly luciferase (pNL4-3-R-E-luciferase) and pcDNA3.1 vector encoding either SARS-CoV-2 or sarbecovirus spike proteins were co-transfected into the HEK-293T cells (ATCC). pNL4-3-R-E-luciferasesuggested: NonepcDNA3.1suggested: RRID:Addgene_79663)Software and Algorithms Sentences Resources The IC50 values were calculated based on the reduction of 50% relative light units (Bright-Glo Luciferase Assay Vector System, Promega, USA) compared to the virus-only control, using Prism 8.0 (GraphPad Software Inc., USA). Prismsuggested: (PRISM, RRID:SCR_005375)GraphPadsuggested: (GraphPad Prism, RRID:SCR_002798)Phylogenetic tree and genetic analysis of nanobodies: Neighbor-joining phylogenetic trees were generated using MEGA version 10.1.8 with 1000 bootstrap replicates 68. MEGAsuggested: (Mega BLAST, RRID:SCR_011920)Chord diagrams showing the germline gene usages and V/J gene pairing were analyzed and presented by the R package circlize version 0.4.13 69. circlizesuggested: (circlize, RRID:SCR_002141)Sequence logo were plotted using Python package Logomaker 70 Pythonsuggested: (IPython, RRID:SCR_001658)Subsequent model building and refinement were performed using COOT (PMID: 15572765) and PHENIX (PMID: 12393927), respectively. COOTsuggested: (Coot, RRID:SCR_014222)PHENIXsuggested: (Phenix, RRID:SCR_014224)All structure figures were generated with ChimeraX and Pymol (PMID: 28158668) ChimeraXsuggested: (UCSF ChimeraX, RRID:SCR_015872)Pymolsuggested: (PyMOL, RRID:SCR_000305)Half-maximal inhibitory concentration (IC50) of nanobodies was calculated by the equation of four-parameter dose inhibition response using Graphpad Prism 8.0. Graphpad Prismsuggested: (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:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
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- No protocol registration statement was detected.
Results from scite Reference Check: We found no unreliable references.
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