A Human Monoclonal Antibody Blocking Sars-cov-2 Infection

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Salient features :

This article focusses on:

  • Results :
    • Identification of SARS-CoV-2 reactive antibodies.
    • Antiviral and biochemical properties of the human mAb 47D11.
    • 47D11 targets a conserved epitope in the SARS2-S-S1B domain.
  • Methods :
    • Expression and purification of coronavirus spike proteins
    • Generation of H2L2 mAbs
    • Production of human monoclonal antibody 47D11.
    • Immunofluorescence microscopy
    • Flow cytometry-based receptor-binding inhibition assay.
    • Pseudotyped virus neutralization assay
    • Virus neutralization assay.
    • ELISA analysis of antibody binding to CoV spike antigens

Detailed summary:

  • Monoclonal antibodies target vulnerable sites on viral surface proteins and are recognized as a promising class of drugs against infectious diseases and have shown  therapeutic efficacy for a number of viruses.
  • Coronavirus-neutralizing antibodies target the trimeric spike (S) glycoproteins on the viral surface that mediate entry into host cells. 
  • S protein has two functional subunits that 
  • mediate cell attachment (the S1 subunit, existing of four core domains S1A through S1D)
  • fusion of the viral and cellular membrane (the S2 subunit). 
  • Potent neutralizing antibodies target the receptor interaction site in S1 and disable  receptor  interactions.
  • The spike proteins of SARS-CoV-2 and SARS-CoV are 77.5% identical by having same primary amino acid sequence and structure.
  • These proteins bind to the human angiotensin converting enzyme 2 (ACE2) protein as a host receptor through their S1B domain.
  • The receptor interaction triggers an irreversible conformational changes in coronavirus spike proteins which enables membrane fusion.

RESULTS:

  • Identification of SARS-CoV-2 reactive antibodies
    • 51 SARS-S hybridoma’s were derived from immunized transgenic H2L2 mice that encode chimeric immunoglobulins with human variable heavy and light chains and constant regions of rat origin and ELISA-(cross) reactivity was done.
    • Four out of 51 SARS-S hybridoma supernatants displayed ELISA-cross-reactivity with SARS2-S1.
    • One of them (47D11) exhibited cross-neutralizing activity of SARS-S and SARS2-S pseudotyped VSV infection. 
    • The chimeric 47D11 H2L2 antibody was reformatted to a fully human immunoglobulin, by cloning the human variable heavy and light chain regions into a human IgG1 isotype backbone. 
  • Antiviral and biochemical properties of the human mAb 47D11
    • Binds to cells and expresses the full-length spike proteins of SARS-CoV and SARS-CoV-2.
    • Inhibits infection of VeroE6 cells with SARS-S and SARS2-S pseudotyped VSV with IC50 values of 0.061 and 0.061 μg/ml.
    • Authentic infection of VeroE6 cells with SARS-CoV and SARS-CoV-2 was neutralized with IC50 values of 0.19 and 0.57 μg/ml.
    • Targets S1B receptor-binding domain (RBD) of SARS-S and SARS2-S.
    • Binds  the S1B of both viruses with similar affinities i.e (EC50) values (0.02 and 0.03 μg/ml, respectively). 
    • ELISA based binding affinity of 47D11 for the spike ectodomain (Secto) of SARS-CoV was higher relative to that of SARS-CoV-2 (EC50values: 0.018 and 0.15 μg/ml, respectively.
  • 47D11 targets a conserved epitope in the SARS2-S-S1B domain.
    • The SARS2-S1B RBD (residues 338–506) consists of a core domain and a receptor-binding subdomain (residues 438–498) which loops out from the antiparallel betasheet core domain structure and directly engages the receptor. 
    • Protein sequence identity of the S1B receptor interacting subdomain of SARS-S and SARS2-S is substantially lower when compared to SIB core domain.
    • Potent neutralizing antibodies target this receptor-binding subdomain.
    • These antibodies are virus-specific and bind and neutralize related viruses
    • Because of the cross-reactive nature of 47D11, the antibody targets the conserved core structure of the S1B RBD.
    • SARS-CoV- neutralizing antibody CR3022 targeting the S1B core domain was recently found to cross-bind SARS-CoV-2.
    • The inability to compromise spike–receptor interaction explains S1B binding by 47D11 further away from the receptor-binding interface.
    • Antibody combinations which target non-overlapping epitopes may act synergistically resulting in lower dosage and mitigate risk of immune escape.
    • 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 other diseases caused by viruses from the Sarbecovirus subgenus.

METHODS:

Expression and purification of coronavirus spike proteins: 

  • Coronavirus spike ectodomains (Secto) of SARS-CoV-2 and HCoV-OC43 were expressed transiently in HEK-293T cells with a C-terminal trimerization motif and Strep-tag using the pCAGGS expression plasmid.
  • Coronavirus spike ectodomain of MERS-CoV  and SARS-CoV fused with an C-terminal trimerization motif, a thrombin cleavage site and a strep-tag purification tag were cloned into pMT\Bip\V5\His expression vector. 
  • Furin cleavage site at the S1/S2 junction was mutated to prevent cleavage by furin at this position. 
  • Spike ectodomains were stably produced in Drosophila S2 cell line.
  • Recombinant proteins were affinity purified from the culture supernatant by protein-A sepharose beads or streptactin beads purification. 
  • Purity and integrity of all purified recombinant proteins was checked by coomassie stained SDS-PAGE.

Generation of H2L2 mAbs. 

  • H2L2 mice were sequentially immunized in 2 weeks intervals with purified Secto 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 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 are then screened in antigenspecific ELISA and those selected for further development, subcloned and produced on a small scale (100 ml of medium). 
  • 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 are purified from hybridoma culture supernatants using Protein-G affinity chromatography.
  •  Purified antibodies were stored at 4°C until use. 

Production of human monoclonal antibody 47D11

  • For recombinant human mAb production, the cDNA’s encoding the 47D11 H2L2 mAb variable regions of the heavy and light chains were cloned into expression plasmids containing the human IgG1 heavy chain and Ig kappa light chain constant regions respectively (InvivoGen). 
  • Both plasmids contain the interleukin-2 signal sequence which enables efficient secretion of recombinant antibodies. 
  • Recombinant human 47D11 mAb and previously described isotype control (anti-strep-tag mAb) or 7.7G6 mAb were produced in HEK-293T cells following transfection with pairs of the IgG1 heavy and light chain expression plasmids.
  • Human antibodies were purified from cell culture supernatants using Protein-A affinity chromatography. 
  • Purified antibodies were stored at 4°C until use.

Immunofluorescence microscopy:

  • Antibody binding to cell surface spike proteins of SARS-CoV, SARS-CoV-2, and MERS-CoV was measured by immunofluorescence microscopy. 
  • HEK-293T (ATCC#CRL-3216) cells seeded on glass slides were transfected with plasmids encoding SARS-S, SARS2-S, or MERS-S – Cterminally fused to the green fluorescence protein (GFP) using Lipofectamine 2000.
  • 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.
  • 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 for 45 min at room temperature. 
  • The fluorescence images were recorded using a Leica SpeII confocal microscope.

Flow cytometry-based receptor-binding inhibition assay:

  • Antibody interference of S1B 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 × 105 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. 
  • In all, 2.5 μg/ml of human Fc tagged SARS-S1B and SARS2-S1B was pre-incubated with mAb at the indicated mAb:S1B 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 S1B and mAb mixture for 1 hour on ice, followed by incubation with 1:200 diluted Alexa Fluor 594 conjugated goat anti-human IgG antibodies  for 45 min at room temperature. 
  • Cells were subjected to flow cytometric analysis with a CytoFLEX Flow Cytometer (Beckman Coulter). 
  • The results were analyzed by FlowJo (version 10). FSC/SSC gates were used to select mononuclear cells. 
  • Control antibody staining was used to define positive/negative cell populations.

Pseudotyped virus neutralization assay:

  • Production of VSV pseudotyped with SARS-S and SARS2-S was performed.
  • 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. 
  • 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-luciferin as a substrate (Promega). 
  • The percentage of infectivity was calculated as ratio of luciferase readout in the presence of mAbs normalized to luciferase readout in the absence of mAb. 
  • The half maximal inhibitory concentrations (IC50) were determined using 4-parameter logistic regression.

Virus neutralization assay:

  • Neutralization of authentic SARS-CoV and SARSCoV- 2 was performed using a plaque reduction neutralization test.
  • mAbs were twofold serially diluted in culture medium starting at 40 μg/ml and 50 μl was mixed with 50 μl (500 TCID50) SARS-CoV or SARS-CoV-2 for 1 hour. 
  • The mixture was then added to VeroE6 cells and incubated for 1 hour, after which the cells were washed and further incubated in medium for 8 hours. 
  • The cells were then fixed and stained using a rabbit anti-SARS-CoV serum (Sino Biological) and a secondary peroxidase-labeled goat anti-rabbit IgG (Dako). 
  • The signal was developed using a precipitate forming TMB substrate (True Blue, KPL) and the number of infected cells per well were counted using the ImmunoSpot Image analyzer (CTL Europe GmbH). 
  • The half maximal inhibitory concentrations (IC50) were determined using 4-parameter logistic regression.

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 then washed three times and incubated with horseradish peroxidase (HRP)-conjugated goat anti-human secondary antibody diluted 1:2000 in blocking buffer for 1 hour at room temperature. 
  • HRP-conjugated anti-StrepMAb 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 (EC50) binding values were calculated by non-linear regression analysis on the binding curves using GraphPad Prism.

Reference Link : https://www.biorxiv.org/content/10.1101/2020.03.11.987958v1.abstract

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