Transcriptionally active PCR expression of neutralizing antibodies
The transcriptionally active PCR (TAP) expression of neutralizing antibodies (nAbs) was performed as previously described1,20. Antibodies heavy and light chain vectors were initially digested using restriction enzymes AgeI, SalI and Xho. PCR II products were ligated using the Gibson Assembly NEB into 25 ng of respective human Igγ1, Igκ and Igλ expression vectors30,31. TAP reaction was performed using 5 μl of Q5 polymerase (NEB), 5 μl of GC Enhancer (NEB), 5 μl of 5X buffer, 10 mM of dNTPs, 0.125 μl of forward/reverse primers and 3 μl of ligation product, using the following cycles: 98 °C for 2 min, 35 cycles 98 °C for 10 s, 61 °C for 20 s, 72 °C for 1 min and 72 °C for 5 min. TAP products were purified, quantified using the Qubit Fluorometric Quantitation assay (Invitrogen), and used for transient transfection in Expi293F cell line following manufacturer’s instructions.
SARS-CoV-2 authentic viruses neutralization assay
All SARS-CoV-2 authentic virus neutralization assays were performed in the biosafety level 3 (BSL3) laboratories at Toscana Life Sciences in Siena (Italy) and Vismederi Srl, Siena (Italy). BSL3 laboratories are approved by a Certified Biosafety Professional and are inspected every year by local authorities. To evaluate the neutralization activity of identified nAbs against SARS-CoV-2 and B.1.1.529 (Omciron) VoC a cytopathic effect-based microneutralization assay (CPE-MN) was performed1,20. Briefly, nAbs were co-incubated with a SARS-CoV-2 viral solution containing 100 median Tissue Culture Infectious Dose (100 TCID50) of virus for 1 hour at 37°C, 5% CO2. The mixture was then added to the wells of a 96-well plate containing a sub-confluent Vero E6 cell monolayer. Plates were incubated for 3-4 days at 37°C in a humidified environment with 5% CO2, then examined for CPE by means of an inverted optical microscope by two independent operators. All nAbs were tested at a starting dilution of 1:8, diluted step 1:2, and the IC100 evaluated based on their initial concentration. Technical duplicates for each experiment were performed. In each plate positive and negative control were used as previously described1,20.
SARS-CoV-2 virus variants CPE-MN neutralization assay
The SARS-CoV-2 Omicron (B.1.1.529) virus used to perform the CPE-MN neutralization assay was supplied and sequenced by the NRC UZ/KU Leuven (Leuven, Belgium). Sequence was deposited on GISAID with the following ID: EPI_ISL_6794907.
SARS-CoV-2 S protein competition assay
Competitive Flow cytometry-based assay was performed to characterize nAbs binding profiles to SARS-CoV-2 S-protein as previously described1. Briefly, magnetic beads (Dynabeads His-Tag, Invitrogen) were covered with His-tagged S-proteins, following manufacturers’ instructions. Then, 40 mg/mL of beads-bound-S-protein were incubated with unlabeled nAbs for 40 minutes at RT. Following incubation, samples were washed with PBS and incubated with fluorescently labeled Class 1/2 (J08-A647), Class 3 (S309-A488) or Class 4 (CR3022-A647) S-protein nAbs binders. Antibodies labelling was performed using Alexa Fluor NHS Ester kit (Thermo Scientific). Following 40 minutes of incubation at RT, beads-antibodies mix was washed with PBS, resuspended in 150 μL of PBS-BSA 1% and acquired using BD LSR II flow cytometer (Becton Dickinson). Results were analyzed using FlowJo™ Software (version 10). Beads with or without S-protein incubated with labeled antibodies were used as positive and negative controls respectively.
SARS-CoV-1 S protein binding assay
Expi293F cells (Thermo Fisher) were transiently transfected with SARS-CoV-1 S-protein expression vectors (pcDNA3.3_CoV1_D28) using Expifectamine Enhancer according to the manufacturer’s protocol (Thermo Fisher). Two days later, to exclude dead cells from analysis, Expi293F were harvested, dispensed into a 96-well plate (3x105 cell/well), and stained for 30 minutes at room temperature (RT) with Live/Dead Fixable Aqua reagent (Invitrogen; Thermo Scientific) diluted 1:500. Following Live/Dead staining, cells were washed with PBS and incubated with nAbs candidates for 40 minutes at RT. Next, to identify the SARS-CoV-1 S protein mAbs binders, cells were washed and stained with the Alexa Fluor 488-labelled secondary antibody Goat anti-Human IgG (H+L) secondary antibody (Invitrogen) diluted 1:500. After 40 minutes of incubation, labeled cells were washed, resuspended in 150 μL of PBS and analyzed using the BD LSR II flow cytometer (Becton Dickinson). Cells incubated with the SARS-CoV-1 nAb binder (S309) or incubated only with the secondary antibody were used as positive and negative controls respectively. Data were analyzed with FlowJo™ Software (version 10).
HEK293TN- hACE2 cell line generation
HEK293TN- hACE2 cell line was generated by lentiviral transduction of HEK293TN (System Bioscience) cells as described in Notarbartolo S. et al.32. Lentiviral vectors were produced following a standard procedure based on calcium phosphate co-transfection with 3rd generation helper and transfer plasmids. The transfer vector pLENTI_hACE2_HygR was obtained by cloning of hACE2 from pcDNA3.1-hACE2 (a gift from Fang Li, Addgene #145033) into pLenti-CMV-GFP-Hygro (a gift from Eric Campeau & Paul Kaufman, Addgene #17446). pLENTI_hACE2_HygR is now made available through Addgene (Addgene #155296). HEK293TN-hACE2 cells were maintained in DMEM, supplemented with 10% FBS, 1% glutamine, 1% penicillin/streptomycin and 250 μg/ml Hygromicin (GIBCO).
Production of SARS-CoV-1 pseudoparticles
SARS-CoV1 lentiviral pseudotype particles were generated as described in Conforti et al. for SARS-CoV-233. SARS-CoV1 SPIKE plasmid pcDNA3.3_CoV1_D28 is a gift from a gift from David Nemazee (Addgene plasmid # 170447).
SARS-CoV-1 neutralization assay
For neutralization assay, HEK293TN-hACE2 cells were plated in white 96-well plates in complete DMEM medium. 24h later, cells were infected with 0.1 MOI of SARS-CoV-1 pseudoparticles that were previously incubated with serial dilution of purified or not purified (cell supernatant) mAb . In particular, a 7-point dose-response curve (plus PBS as untreated control), was obtained by diluting mAb or supernatant respectively five-fold and three-fold. Thereafter, nAbs of each dose-response curve point was added to the medium containing SARS-CoV-1 pseudoparticles adjusted to contain 0.1 MOI. After incubation for 1h at 37°C, 50 µl of mAb/SARS-CoV-1 pseudoparticles mixture was added to each well and plates were incubated for 24h at 37°C. Each point was assayed in technical triplicates. After 24h of incubation cell infection was measured by luciferase assay using Bright-Glo™ Luciferase System (Promega) and Infinite F200 plate reader (Tecan) was used to read luminescence. Obtained relative light units (RLUs) were normalized to controls and dose response curve were generated by nonlinear regression curve fitting with GraphPad Prism to calculate Neutralization Dose 50 (ND50).
Functional repertoire analyses
nAbs VH and VL sequence reads were manually curated and retrieved using CLC sequence viewer (Qiagen). Aberrant sequences were removed from the data set. Analyzed reads were saved in FASTA format and the repertoire analyses was performed using Cloanalyst (http://www.bu.edu/computationalimmunology/research/software/)34,35.
Statistical analysis was assessed with GraphPad Prism Version 8.0.2 (GraphPad Software, Inc., San Diego, CA). Nonparametric Mann-Whitney t test was used to evaluate statistical significance between the two groups analyzed in this study. Statistical significance was shown as * for values ≤ 0.05, ** for values ≤ 0.01, *** for values ≤ 0.001, and **** for values ≤ 0.0001.
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31. Wardemann, H. & Busse, C.E. Expression Cloning of Antibodies from Single Human B Cells. Methods in molecular biology (Clifton, N.J.) 1956, 105-125 (2019).
32. Notarbartolo, S., et al. Integrated longitudinal immunophenotypic, transcriptional, and repertoire analyses delineate immune responses in patients with COVID-19. Science Immunology 6, eabg5021 (2021).
33. Conforti, A., et al. COVID-eVax, an electroporated DNA vaccine candidate encoding the SARS-CoV-2 RBD, elicits protective responses in animal models. Molecular therapy : the journal of the American Society of Gene Therapy 30, 311-326 (2022).
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