Broad ultra-potent neutralization of SARS-CoV-2 variants by monoclonal antibodies specific to the tip of RBD
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Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) continue to wreak havoc across the globe. Higher transmissibility and immunologic resistance of VOCs bring unprecedented challenges to epidemic extinguishment. Here we describe a monoclonal antibody, 2G1, that neutralizes all current VOCs and has surprising tolerance to mutations adjacent to or within its interaction epitope. Cryo-electron microscopy structure showed that 2G1 bound to the tip of receptor binding domain (RBD) of spike protein with small contact interface but strong hydrophobic effect, which resulted in nanomolar to sub-nanomolar affinities to spike proteins. The epitope of 2G1 on RBD partially overlaps with angiotensin converting enzyme 2 (ACE2) interface, which enables 2G1 to block interaction between RBD and ACE2. The narrow binding epitope but high affinity bestow outstanding therapeutic efficacy upon 2G1 that neutralized VOCs with sub-nanomolar half maximal inhibitory concentration in vitro. In SARS-CoV-2, Beta or Delta variant-challenged transgenic mice and rhesus macaque models, 2G1 protected animals from clinical illness and eliminated viral burden, without serious impact to animal safety. Mutagenesis experiments suggest that 2G1 is potentially capable of dealing with emerging SARS-CoV-2 variants in the future. This report characterized the therapeutic antibodies specific to the tip of spike against SARS-CoV-2 variants and highlights the potential clinical applications as well as for developing vaccine and cocktail therapy.
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SciScore for 10.1101/2021.09.24.461616: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics Field Sample Permit: Animal studies were carried out at Galveston National Laboratory at University of Texas Medical Branch at Galveston, Texas, an AAALAC accredited (November 24, 2020) and PHS OLAW approved (February 26, 2021) high-containment National Laboratory, based on a protocol approved by the Institutional Animal Care and Use Committee at UTMB at Galveston.
IACUC: Animal studies were carried out at Galveston National Laboratory at University of Texas Medical Branch at Galveston, Texas, an AAALAC accredited (November 24, 2020) and PHS OLAW approved (February 26, 2021) high-containment National Laboratory, based on a protocol approved by the Institutional Animal Care and Use Committee …SciScore for 10.1101/2021.09.24.461616: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics Field Sample Permit: Animal studies were carried out at Galveston National Laboratory at University of Texas Medical Branch at Galveston, Texas, an AAALAC accredited (November 24, 2020) and PHS OLAW approved (February 26, 2021) high-containment National Laboratory, based on a protocol approved by the Institutional Animal Care and Use Committee at UTMB at Galveston.
IACUC: Animal studies were carried out at Galveston National Laboratory at University of Texas Medical Branch at Galveston, Texas, an AAALAC accredited (November 24, 2020) and PHS OLAW approved (February 26, 2021) high-containment National Laboratory, based on a protocol approved by the Institutional Animal Care and Use Committee at UTMB at Galveston.
IRB: All animal procedures and operations were approved by the ethical committee of Wuhan Institute of Virology, Chinese Academy of Sciences.Sex as a biological variable Rhesus macaques were randomly divided into control group, low-dose (10 mg/kg of 2G1) and high-dose (50 mg/kg of 2G1) groups with one male and one female in each. Randomization Rhesus macaques were randomly divided into control group, low-dose (10 mg/kg of 2G1) and high-dose (50 mg/kg of 2G1) groups with one male and one female in each. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Biotin labeled RBD (Sino Biological), PE labeled streptavidin (ThermoFisher) and 7AAD (BD) Single memory B cells with potential SARS-CoV-2 antibody secretion were sorted out by gating 7AAD-, CD19+, CD27+, IgG+, and RBD+ using a BD Aria III cell sorter with fluorescence-activated cell sorting modules. CD27+ , IgG+suggested: NoneThe transfection efficiency was examined by flow cytometry using S1-mFc recombinant protein (Sino Biological) as primary antibody and FITC-AffiniPure Goat Anti-Mouse IgG (Jackson) as secondary antibody. Anti-Mouse IgGsuggested: NoneAntigen-binding ELISA: Enzyme-linked immunosorbent assays (ELISA) were applied to study the binding ability of antibodies with SARS-CoV-2 RBDs (Sino Biological) and S trimers (AcroBiosystems). Antigen-binding ELISAsuggested: (Ling-Chu Hung / Animal Health Research Institute, Council of Agriculture, Executive Yuan, Taiwan Cat# 12-Hung-03 m& 20 ORF3 7D3, RRID:AB_2827541)After pipetting off the unbound antibodies, plates were washed 4 times with PBST and further incubated with 100 μL per well of goat anti-human IgG (Fc specific)-Peroxidase antibody (1: 5000 dilution, Sigma) for 1 h at 37°C. anti-human IgG ( Fc specific)-Peroxidasesuggested: NoneACE2 competition ELISA: For experiments involving the competitive binding of antibodies to SARS-CoV-2 RBD or S trimer, recombinant hACE2-Fc protein was first biotinylated using EZ-Link Sulfo-NHS-Biotin (ThermoFisher) as the instruction described. Sulfo-NHS-Biotinsuggested: NoneAntibody-Dependent Cellular Phagocytosis (ADCP): In ADCP experiment, CD14+ monocytes (Allcells) were cultured and differentiated for 7 days to obtain macrophage cells. Antibody-Dependent Cellular Phagocytosis ( ADCP)suggested: NoneAfter washing, a Goat Anti-human Fc HRP (Sigma) was used as secondary antibody with 1:6000 dilutions. Anti-human Fc HRPsuggested: NoneExperimental Models: Cell Lines Sentences Resources In brief, HEK-293T cells (ATCC) with 70% - 80% confluence in a 10 cm dish were co-transfected with 12 μg of plasmid pHIV-puro encoding RRE and ACE2 genes, 8 μg of plasmid psPAX2 encoding gag and pol, and 4 μg of plasmid VSV-G encoding G glycoprotein of vesicular stomatitis virus(VSVG) using Lipofectamine 3000 Reagent (Invitrogen). HEK-293Tsuggested: None500 μL of filtered lentivirus supernatant was added in a 24-well plate with Jurkat cells (ATCC). Jurkatsuggested: TKG Cat# TKG 0209, RRID:CVCL_0065)The culture medium of ACE2-293T cells was removed and then replaced by the antibody-pseudovirus mixture. ACE2-293Tsuggested: NoneThe mixture was then added into a 96-well plate covered with Vero cells. Verosuggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)This recombinant S protein was overexpressed using the HEK 293F mammalian cells (Invitrogen) at 37°C under 5% CO2 in a Multitron-Pro shaker (Infors, 130 HEK 293Fsuggested: RRID:CVCL_6642)Experimental Models: Organisms/Strains Sentences Resources Pharmacokinetic study and toxicity test: For the pharmacokinetic study, BALB/c mice were tail intravenously injected with 2G1 (15, 30, or 60 mg/kg), or equivalent volume of PBS. BALB/csuggested: NoneRecombinant DNA Sentences Resources mAb preparation: Heavy chains and light chain genes were inserted separately into pcDNA3.4 and amplified in E. coli DH5α. pcDNA3.4suggested: RRID:Addgene_131198)In brief, HEK-293T cells (ATCC) with 70% - 80% confluence in a 10 cm dish were co-transfected with 12 μg of plasmid pHIV-puro encoding RRE and ACE2 genes, 8 μg of plasmid psPAX2 encoding gag and pol, and 4 μg of plasmid VSV-G encoding G glycoprotein of vesicular stomatitis virus(VSVG) using Lipofectamine 3000 Reagent (Invitrogen). pHIV-purosuggested: NonepsPAX2suggested: RRID:Addgene_12260)VSV-Gsuggested: RRID:Addgene_138479)S protein over-expression cells: The coding sequence for full-length wild-type S protein (GenBank: QHD43416.1) from Met1 to Thr1273 was inserted into plasmid pHIV-puro1.0, followed by an internal ribosome entry site (IRES) and puromycin resistance gene. pHIV-puro1.0suggested: NoneExpression and purification of S protein: The prefusion S extracellular domain (1-1208 a.a) (Genbank ID: QHD43416.1) was cloned into the pCAG vector (Invitrogen) with six proline substitutions at residues 817, 892, 899, 942, 986 and 98739, a “GSAS” substitution (instead of “RRAR”) at residues 682 to 685 and a C-terminal T4 fibritin trimerization motif followed by one Flag tag. pCAGsuggested: RRID:Addgene_74288)Software and Algorithms Sentences Resources The resulting bulk transfected population was sorted on a BD FACSJazz Cell Sorter (BD) with the BD FACS™ Sortware. BD FACS™suggested: (BD FACS Aria II, RRID:SCR_018091)Data were analyzed with GraphPad Prism Version 9.0.0 and EC50 values were determined using a four-parameter nonlinear regression. GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)SARS-CoV-2 RBD (Sino Biological), S trimer (AcroBiosystems), mutated RBDs (Sino Biological), and mutated S trimers (AcroBiosystems) were coated onto High Binding ELISA 96-Well Plate (BEAVER). AcroBiosystemssuggested: (ACRObiosystems, RRID:SCR_012550)The defocus values were estimated with Gctf43. Particles for S in complex with 2G1 were automatically picked using Relion 3.0.644–47 from manually selected micrographs. Relionsuggested: (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: Please consider improving the rainbow (“jet”) colormap(s) used on page 17. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.
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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