A human monoclonal antibody blocking SARS-CoV-2 infection methods
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47D11 targets a conserved epitope in the SARS2-S-S1 B domain
In conclusion, this is the first report of a (human) monoclonal antibody that neutralizes SARS-CoV-2. 47D11 binds a conserved epitope on the spike RBD explaining its ability to cross-neutralize SARS-CoV and SARS-CoV-2, using a mechanism that is independent of receptor-binding inhibition. This antibody will be useful for development of antigen detection tests and serological assays targeting SARS-CoV-2. Neutralizing antibodies can alter the course of infection in the infected host supporting virus clearance or protect an uninfected host that is exposed to the virus. Hence, this antibody-either alone or in combination-offers the potential to prevent and/or treat COVID-19, and possibly also other future emerging diseases in humans caused by viruses from the Sarbecovirus subgenus.
Generation of H2L2 mAbs
H2L2 mice were sequentially immunized in 2 weeks intervals with purified S ecto of different CoVs in the following order: HCoV-OC43, SARS-CoV, MERS-CoV, HCoV-OC43, SARS-CoV, and MERS-CoV. Antigens were injected at 20-25 µg/mouse using Stimune Adjuvant (Prionics) freshly prepared according to the manufacturer's instruction for first injection, whereas boosting was done using Ribi (Sigma) adjuvant. Injections were done subcutaneously into the left and right groin each (50 µl) and 100 µl intraperitoneally. Four days after the last injection, spleen and lymph nodes are harvested, and hybridomas made by standard method using SP 2/0 myeloma cell line (ATCC#CRL-1581) as a fusion partner. Hybridomas were screened in antigen-specific ELISA and those selected for further development, subcloned and produced on a small scale (100 ml of medium). For thi...
Immunofluorescence microscopy
Antibody binding to cell surface spike proteins of SARS-CoV, SARS-CoV-2, and MERS-CoV was measured by immunofluoresence microscopy. HEK-293T (ATCC#CRL-3216) cells seeded on glass slides were transfected with plasmids encoding SARS-S, SARS2-S, or MERS-S - C-terminally fused to the green fluorescence protein (GFP) using Lipofectamine 2000 (Invitrogen, Catalog# 11668019). Two days post transfection, cells were fixed by incubation with 2% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min at room temperature and stained for nuclei with 4,6-diamidino-2-phenylindole (Sigma, Catalog# D9542). Cells were subsequently incubated with mAbs at a concentration of 10 µg/ml for 1 hour at room temperature, followed by incubation with 1:200 diluted Alexa Fluor 594 conjugated goat anti-human IgG antibodies (Invitrogen, Thermo Fisher Scientific, Catalog# A-11014) for 45...
Flow cytometry-based receptor-binding inhibition assay
Antibody interference of S1 B binding to human ACE2 receptor on the cell surface was measured by flow cytometry. HEK-293T cells were seeded at a density of 2.5 × 10 5 cells per ml in a T75 flask. After reaching 70~80% confluency, cells were transfected with an expression plasmid encoding human ACE2 - C-terminally fused to the GFP using Lipofectamine 2000 (Invitrogen). Two days post transfection, cells were dissociated by cell dissociation solution (Sigma-aldrich, Merck KGaA; Catalog# C5914). In all, 2.5 µg/ml of human Fc tagged SARS-S1 B and SARS2-S1 B was pre-incubated with mAb at the indicated mAb:S1 B molar ratios for 1 hour on ice and subjected to flow cytometry. Single-cell suspensions in FACS buffer were centrifuged at 400 × g for 10 min. Cells were subsequently incubated with S1 B and mAb mixture for 1 hour on ice,...
Pseudotyped virus neutralization assay
Production of VSV pseudotyped with SARS-S and SARS2-S was performed as described previously with some adaptations. Briefly, HEK-293T cells were transfected with pCAGGS expression vectors encoding SARS-S or SARS2-S carrying a 28- or 18-a.a. cytoplasmic tail truncation, respectively. One day post transfection, cells were infected with the VSV-G pseudotyped VSVΔG bearing the firefly ( Photinus pyralis ) luciferase reporter gene. Twenty-four hours later, supernatants containing SARS-S/SARS2-S pseudotyped VSV particles were harvested and titrated on African green monkey kidney VeroE6 (ATCC#CRL-1586) cells. In the virus neutralization assay, mAbs were fourfold serially diluted at two times the desired final concentration in DMEM supplemented with 1% fetal calf serum (Bodinco), 100 U/ml Penicillin and 100 µg/ml Streptomycin (Lonza, Catalog# 17-602E). Diluted mAbs were...
ELISA analysis of antibody binding to CoV spike antigens
NUNC Maxisorp plates (Thermo Scientific) were coated with equimolar antigen amounts at 4 °C overnight. Plates were washed three times with PBS containing 0.05% Tween-20 and blocked with 3% bovine serum albumin (Bio-Connect) in PBS containing 0.1% Tween-20 at room temperature for 2 hours. Fourfolds serial dilutions of mAbs starting at 10 µg/ml (diluted in blocking buffer) were added and plates were incubated for 1 hour at room temperature. Plates were washed three times and incubated with horseradish peroxidase (HRP)-conjugated goat anti-human secondary antibody (ITK Southern Biotech) diluted 1:2000 in blocking buffer for 1 hour at room temperature. An HRP-conjugated anti-StrepMAb (IBA, Catalog# 2-1509-001) antibody was used to corroborate equimolar coating of the strep-tagged spike antigens. HRP activity was measured at 450 nanometer using tetramethyl...
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In conclusion, this is the first report of a (human) monoclonal antibody that neutralizes SARS-CoV-2. 47D11 binds a conserved epitope on the spike RBD explaining its ability to cross-neutralize SARS-CoV and SARS-CoV-2, using a mechanism that is independent of receptor-binding inhibition. This antibody will be useful for development of antigen detection tests and serological assays targeting SARS-CoV-2. Neutralizing antibodies can alter the course of infection in the infected host supporting virus clearance or protect an uninfected host that is exposed to the virus. Hence, this antibody-either alone or in combination-offers the potential to prevent and/or treat COVID-19, and possibly also other future emerging diseases in humans caused by viruses from the Sarbecovirus subgenus.
H2L2 mice were sequentially immunized in 2 weeks intervals with purified S ecto of different CoVs in the following order: HCoV-OC43, SARS-CoV, MERS-CoV, HCoV-OC43, SARS-CoV, and MERS-CoV. Antigens were injected at 20-25 µg/mouse using Stimune Adjuvant (Prionics) freshly prepared according to the manufacturer's instruction for first injection, whereas boosting was done using Ribi (Sigma) adjuvant. Injections were done subcutaneously into the left and right groin each (50 µl) and 100 µl intraperitoneally. Four days after the last injection, spleen and lymph nodes are harvested, and hybridomas made by standard method using SP 2/0 myeloma cell line (ATCC#CRL-1581) as a fusion partner. Hybridomas were screened in antigen-specific ELISA and those selected for further development, subcloned and produced on a small scale (100 ml of medium). For this purpose, hybridomas are cultured in serum- and protein-free medium for hybridoma culturing (PFHM-II (1 × ), Gibco) with addition of non-essential amino acids 100 × NEAA, Biowhittaker Lonza, Catalog# BE13-114E). H2L2 antibodies were purified from hybridoma culture supernatants using Protein-...
Antibody binding to cell surface spike proteins of SARS-CoV, SARS-CoV-2, and MERS-CoV was measured by immunofluoresence microscopy. HEK-293T (ATCC#CRL-3216) cells seeded on glass slides were transfected with plasmids encoding SARS-S, SARS2-S, or MERS-S - C-terminally fused to the green fluorescence protein (GFP) using Lipofectamine 2000 (Invitrogen, Catalog# 11668019). Two days post transfection, cells were fixed by incubation with 2% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min at room temperature and stained for nuclei with 4,6-diamidino-2-phenylindole (Sigma, Catalog# D9542). Cells were subsequently incubated with mAbs at a concentration of 10 µg/ml for 1 hour at room temperature, followed by incubation with 1:200 diluted Alexa Fluor 594 conjugated goat anti-human IgG antibodies (Invitrogen, Thermo Fisher Scientific, Catalog# A-11014) for 45 min at room temperature. The fluorescence images were recorded using a Leica SpeII confocal microscope.
Antibody interference of S1 B binding to human ACE2 receptor on the cell surface was measured by flow cytometry. HEK-293T cells were seeded at a density of 2.5 × 10 5 cells per ml in a T75 flask. After reaching 70~80% confluency, cells were transfected with an expression plasmid encoding human ACE2 - C-terminally fused to the GFP using Lipofectamine 2000 (Invitrogen). Two days post transfection, cells were dissociated by cell dissociation solution (Sigma-aldrich, Merck KGaA; Catalog# C5914). In all, 2.5 µg/ml of human Fc tagged SARS-S1 B and SARS2-S1 B was pre-incubated with mAb at the indicated mAb:S1 B molar ratios for 1 hour on ice and subjected to flow cytometry. Single-cell suspensions in FACS buffer were centrifuged at 400 × g for 10 min. Cells were subsequently incubated with S1 B and mAb mixture for 1 hour on ice, followed by incubation with 1:200 diluted Alexa Fluor 594 conjugated goat anti-human IgG antibodies (Invitrogen, Thermo Fisher Scientific, Catalog# A-11014) for 45 min at room temperature. Cells were subjected to flow cytometric analysis with a CytoFLEX Flow Cytometer (Beckman Coulter). The r...
Production of VSV pseudotyped with SARS-S and SARS2-S was performed as described previously with some adaptations. Briefly, HEK-293T cells were transfected with pCAGGS expression vectors encoding SARS-S or SARS2-S carrying a 28- or 18-a.a. cytoplasmic tail truncation, respectively. One day post transfection, cells were infected with the VSV-G pseudotyped VSVΔG bearing the firefly ( Photinus pyralis ) luciferase reporter gene. Twenty-four hours later, supernatants containing SARS-S/SARS2-S pseudotyped VSV particles were harvested and titrated on African green monkey kidney VeroE6 (ATCC#CRL-1586) cells. In the virus neutralization assay, mAbs were fourfold serially diluted at two times the desired final concentration in DMEM supplemented with 1% fetal calf serum (Bodinco), 100 U/ml Penicillin and 100 µg/ml Streptomycin (Lonza, Catalog# 17-602E). Diluted mAbs were incubated with an equal volume of pseudotyped VSV particles for 1 hour at room temperature, inoculated on confluent VeroE6 monolayers in 96-well plate, and further incubated at 37 °C for 24 hours. Luciferase activity was measured on a Berthold Centro LB 960 plate luminometer using D...
NUNC Maxisorp plates (Thermo Scientific) were coated with equimolar antigen amounts at 4 °C overnight. Plates were washed three times with PBS containing 0.05% Tween-20 and blocked with 3% bovine serum albumin (Bio-Connect) in PBS containing 0.1% Tween-20 at room temperature for 2 hours. Fourfolds serial dilutions of mAbs starting at 10 µg/ml (diluted in blocking buffer) were added and plates were incubated for 1 hour at room temperature. Plates were washed three times and incubated with horseradish peroxidase (HRP)-conjugated goat anti-human secondary antibody (ITK Southern Biotech) diluted 1:2000 in blocking buffer for 1 hour at room temperature. An HRP-conjugated anti-StrepMAb (IBA, Catalog# 2-1509-001) antibody was used to corroborate equimolar coating of the strep-tagged spike antigens. HRP activity was measured at 450 nanometer using tetramethylbenzidine substrate (BioFX) and an ELISA plate reader (EL-808, Biotek). Half-maximum effective concentration (EC 50 ) binding values were calculated by non-linear regression analysis on the binding curves using GraphPad Prism (version 8).
Machine-readable layer
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