Broadly neutralizing and protective nanobodies against diverse sarbecoviruses

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.

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-Tag suggested: (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 IgG suggested: None anti-rabbit IgG suggested: None
Sections 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 protein suggested: None
Experimental 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.	HEK293T suggested: None HeLa suggested: CLS Cat# 300194/p772_HeLa, RRID:CVCL_0030)
Sf9 cells (ATCC) were maintained at 27°C in Sf-900 II SFM medium.	Sf9 suggested: RRID:CVCL_4U10)
Expression and production of nanobodies were conducted by transfecting the expression vectors into the HEK293F cells using polyethyleneimine (PEI) (Polysciences).	HEK293F suggested: 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-293T suggested: None
HeLa-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-ACE2 suggested: 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 E6 suggested: 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-hACE2 suggested: 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.	hACE2 suggested: RRID:Addgene_1786)
VHH sequences were amplified by PCR, cloned into a yeast surface display vector pYD1, and introduced into the electrocompetent EBY100 cells.	pYD1 suggested: 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.	pMD18T suggested: None
For the latter, selected VHH genes were cloned into pVRC8400 vector with a 6xHis tag.	pVRC8400 suggested: 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-luciferase suggested: None pcDNA3.1 suggested: 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).	Prism suggested: (PRISM, RRID:SCR_005375) GraphPad suggested: (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.	MEGA suggested: (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.	circlize suggested: (circlize, RRID:SCR_002141)
Sequence logo were plotted using Python package Logomaker 70	Python suggested: (IPython, RRID:SCR_001658)
Subsequent model building and refinement were performed using COOT (PMID: 15572765) and PHENIX (PMID: 12393927), respectively.	COOT suggested: (Coot, RRID:SCR_014222) PHENIX suggested: (Phenix, RRID:SCR_014224)
All structure figures were generated with ChimeraX and Pymol (PMID: 28158668)	ChimeraX suggested: (UCSF ChimeraX, RRID:SCR_015872) Pymol suggested: (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 Prism 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:

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.

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

Read the original source

Broadly neutralizing and protective nanobodies against diverse sarbecoviruses

This article has been Reviewed by the following groups

Listed in

Abstract

Article activity feed