Conformational flexibility in neutralization of SARS-CoV-2 by naturally elicited anti-SARS-CoV-2 antibodies

Abstract

As new variants of SARS-CoV-2 continue to emerge, it is important to assess the cross-neutralizing capabilities of antibodies naturally elicited during wild type SARS-CoV-2 infection. In the present study, we evaluate the activity of nine anti-SARS-CoV-2 monoclonal antibodies (mAbs), previously isolated from convalescent donors infected with the Wuhan-Hu-1 strain, against the SARS-CoV-2 variants of concern (VOC) Alpha, Beta, Gamma, Delta and Omicron. By testing an array of mutated spike receptor binding domain (RBD) proteins, cell-expressed spike proteins from VOCs, and neutralization of SARS-CoV-2 VOCs as pseudoviruses, or as the authentic viruses in culture, we show that mAbs directed against the ACE2 binding site (ACE2bs) are more sensitive to viral evolution compared to anti-RBD non-ACE2bs mAbs, two of which retain their potency against all VOCs tested. At the second part of our study, we reveal the neutralization mechanisms at high molecular resolution of two anti-SARS-CoV-2 neutralizing mAbs by structural characterization. We solve the structures of the Delta-neutralizing ACE2bs mAb TAU-2303 with the SARS-CoV-2 spike trimer and RBD at 4.5 Å and 2.42 Å resolutions, respectively, revealing a similar mode of binding to that between the RBD and ACE2. Furthermore, we provide five additional structures (at resolutions of 4.7 Å, 7.3 Å, 6.4 Å, 3.3 Å, and 6.1 Å) of a second antibody, TAU-2212, complexed with the SARS-CoV-2 spike trimer. TAU-2212 binds an exclusively quaternary epitope, and exhibits a unique, flexible mode of neutralization that involves transitioning between five different conformations, with both arms of the antibody recruited for cross linking intra- and inter-spike RBD subunits. Our study provides additional mechanistic understanding about how antibodies neutralize SARS-CoV-2 and its emerging variants and provides insights on the likelihood of reinfections.

SciScore for 10.1101/2022.04.03.486854: (What is this?)

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

Table 1: Rigor

Ethics	Field Sample Permit: Data collection, structure determination, and refinement: The X-ray diffraction data were collected at the beamlines BL18U (RBD-Fab2303) and BL02U (Fab2212) of the Shanghai Synchrotron Research Facility.
Sex as a biological variable	not detected.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Cell Line Authentication	Authentication: Immunofluorescence imaging and analysis: For viral nucleocapsid detection in Vero-TMPRSS2 cells by immunofluorescence, cells were washed twice with PBS x1 and fixed in 4% formaldehyde for 30 min at RT.

Table 2: Resources

Antibodies
Sentences	Resources
The plates were then washed 3 times with washing buffer before adding …

SciScore for 10.1101/2022.04.03.486854: (What is this?)

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

Table 1: Rigor

Ethics	Field Sample Permit: Data collection, structure determination, and refinement: The X-ray diffraction data were collected at the beamlines BL18U (RBD-Fab2303) and BL02U (Fab2212) of the Shanghai Synchrotron Research Facility.
Sex as a biological variable	not detected.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Cell Line Authentication	Authentication: Immunofluorescence imaging and analysis: For viral nucleocapsid detection in Vero-TMPRSS2 cells by immunofluorescence, cells were washed twice with PBS x1 and fixed in 4% formaldehyde for 30 min at RT.

Table 2: Resources

Antibodies
Sentences	Resources
The plates were then washed 3 times with washing buffer before adding secondary anti-IgG (Jackson ImmmunoResearch) antibody conjugated to horseradish peroxidase (HRP) diluted 1:5000 in blocking buffer, and incubation for 1 h at RT.	anti-IgG suggested: (Vector Laboratories Cat# FI-5000, RRID:AB_2336128)
Monolayers were observed for at least 4 days for appearance of CPE and then fixed as above and stained with antibody against SARS-CoV-2 Nucleocapsid.	antibody against SARS-CoV-2 Nucleocapsid. suggested: None
Fixed cells were washed with PBS x1 and permeabilized for immunofluorescence using BD Cytofix/Cytoperm according to the manufacturer’s protocol for fixed cells, and then stained for SARS-CoV-2 with a primary nucleocapsid antibody (GeneTex GTX135357) and a secondary anti-rabbit AF647 antibody (ThermoFisher A20185), The nuclei were counterstained with Sytox Green.	anti-rabbit AF647 suggested: None
Expression and purification of soluble RBD, spike and antibodies for crystallization: SARS-CoV-2 RBD (residues Arg319 to Lys529) was expressed by using the Bac-to-Bac Baculovirus System (Invitrogen)	Lys529 suggested: None
Supernatant containing S2P was collected four days after transfection, S2P was affinity purified by anti-Flag antibody beads and was eluted by 0.1 mg/mL 3×Flag peptide in 20 mM HEPES at pH 7.6 and 150 mM NaCl.	anti-Flag suggested: None
Experimental Models: Cell Lines
Sentences	Resources
HEK-293 cells stably expressing hACE2 were seeded into 0.1% gelatin-coated 96-well plates (Greiner) at an initial density of 0.75 × 105 cells per well.	HEK-293 suggested: None
Virus titers were validated using a combination of fluorescent focus assay and tissue culture infectious dose (TCID)50 assays on Vero-TMPRSS2 and Calu-3 (ATCC) monolayers.	Calu-3 suggested: KCLB Cat# 30055, RRID:CVCL_0609)
For TCID50 assays, Vero-TMPRSS2 or Calu3 cells were infected as above and 100µL DMEM or MEM with 2% FBS was subsequently added per well.	Calu3 suggested: RRID:CVCL_EQ19)
Immunofluorescence imaging and analysis: For viral nucleocapsid detection in Vero-TMPRSS2 cells by immunofluorescence, cells were washed twice with PBS x1 and fixed in 4% formaldehyde for 30 min at RT.	Vero-TMPRSS2 suggested: JCRB Cat# JCRB1818, RRID:CVCL_YQ48)
The S2P or S6P pCMV plasmids were used to transiently transfect HEK293F cells with polyethylenimine (PEI) (Polysciences, #24765).	HEK293F suggested: None
Experimental Models: Organisms/Strains
Sentences	Resources
Expression and purification of soluble RBD, spike and antibodies for crystallization: SARS-CoV-2 RBD (residues Arg319 to Lys529) was expressed by using the Bac-to-Bac Baculovirus System (Invitrogen)	SARS-CoV-2 RBD suggested: None
Data collection, structure determination, and refinement: The X-ray diffraction data were collected at the beamlines BL18U (RBD-Fab2303) and BL02U (Fab2212) of the Shanghai Synchrotron Research Facility.	RBD-Fab2303 suggested: None
Recombinant DNA
Sentences	Resources
Antibody inhibition of hACE2 binding to cell-expressed spike: Expi293F cells were transfected with pcDNA 3.1 containing SΔC19 of wild type, Alpha, Beta, or Delta variants, using the ExpiFectamine 293 Transfection Kit (Thermo Fisher).	pcDNA 3.1 suggested: RRID:Addgene_20407)
Pseudo-particle preparation and neutralization assays: SARS-CoV-2-spike pseudo-particles were obtained by co-transfecting Expi293F cells with pCMV delta R8.2, pLenti-GFP (Genecopoeia), and pcDNA 3.1 SΔC19 (Thermo Fisher) at a ratio of 1:2:1, respectively, according to the manufacturer’s instructions.	pCMV suggested: RRID:Addgene_16459) pLenti-GFP suggested: RRID:Addgene_172394)
RBD containing the gp67 signal peptide and a C-terminal 6×His tag was inserted into pFastBac1 to form the plasmid pFastBac1-RBD.	pFastBac1 suggested: RRID:Addgene_1956) pFastBac1-RBD suggested: None
Software and Algorithms
Sentences	Resources
Fixed cells were washed with PBS x1 and permeabilized for immunofluorescence using BD Cytofix/Cytoperm according to the manufacturer’s protocol for fixed cells, and then stained for SARS-CoV-2 with a primary nucleocapsid antibody (GeneTex GTX135357) and a secondary anti-rabbit AF647 antibody (ThermoFisher A20185), The nuclei were counterstained with Sytox Green.	BD Cytofix/Cytoperm suggested: None
Data were logged from the Incucyte analysis modules and graphed with GraphPad Prism 8.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)
SerialEM was used for the data collection.	SerialEM suggested: (SerialEM, RRID:SCR_017293)
The CTF parameters of the micrographs were determined by using the program Gctf with local defocus variations taken into consideration59.	Gctf suggested: (GCTF, RRID:SCR_016500)
90569 particles were picked by Gautomatch and then were subjected to 2D classifications with RELION 3.0.	RELION suggested: (RELION, RRID:SCR_016274)

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

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

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Conformational flexibility in neutralization of SARS-CoV-2 by naturally elicited anti-SARS-CoV-2 antibodies

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