This study followed the principles of the Declaration of Helsinki, and was approved by the institutional review board of Osaka University Hospital (No. 885). Informed consent for the collection of blood samples was obtained from the patients or their relatives. The use of left-over specimen of daily test after anonymization was approved by the institutional review board of Habikino Medical Center (150-7), Louis Pasteur Center for Medical Research (LPC29), and Tokushukai Hospital (TGE01547). A brief summary of the protocol was disclosed. The use of human blood from healthy volunteers in this study was approved by the Research Ethics Committee of the Research Institute for Microbial Diseases, Osaka University, Japan (No. 2021-3).
The animal study was carried out in accordance with the recommendations in the guide for the care and use of laboratory animals of Osaka University, Japan. Our protocol was approved by the Committee on the Ethics of Animal Experiments of the Research Institute for Microbial Diseases, Osaka University, Japan (No. R01-17-0). All procedures in this animal experiment were conducted in a manner to avoid or minimize discomfort, distress or pain to the animals according to ARRIVE guidelines (https://arriveguidelines.org/), and the approved operating procedures and guidelines at the Research Institute for Microbial Diseases of Osaka University.
Viruses and cells
SARS-CoV-2 (KNG19-020) was kindly supplied by Dr. Tomohiko Takasaki of the Kanagawa Prefectural Institute of Public Health. SARS-CoV-2 (JPN-TY-WK-521) was obtained from the National Institute for Infectious Diseases, Japan. Clinical isolates hCoV-19/Japan/OIPH14/2020 and hCoV-19/Japan/OIPH21/2020 57 were propagated in TMPRSS2/VeroE6 cells 58 that were obtained from the National Institutes of Biomedical Innovation, Health and Nutrition, Japanese Collection of Research Bioresources Cell Bank, Japan. K-ML2 cells were established as described previously 59 and maintained in minimum essential medium supplemented with 10% fetal calf serum. Peripheral blood mononuclear cells were obtained from the blood buffy coats of healthy donors by Ficoll-Paque density gradient centrifugation, then plated in 24-well MULTIWELL™ PRIMARIA™ plates (Becton Dickinson, Franklin Lakes, NJ) containing RPMI 1640 supplemented with 10% fetal calf serum. Monocytes were differentiated into macrophages for 8 days in the presence of 100 ng/mL of GM-CSF (PeproTECH, Rocky Hill, NJ) or 50 ng/mL of M-CSF (PeproTECH). To prepare SARS-CoV-2 infected cell lysates, TMPRSS2/VeroE6 cells were infected with SARS-CoV-2 at a multiplicity of infection of 0.01, and the cells were harvested 16 h after infection. Subsequently, 1.6 × 105 infected cells were suspended in 200 µL of phosphate-buffered saline (PBS) and frozen at -20°C. After thawing, 10 µL of suspended cell lysate was added to 100 µL of culture medium in each well containing macrophage or K-ML2 cells with or without SARS-CoV-2 and diluted serum. After 4 h of incubation, macrophages were washed once with culture medium, then 500 µL of fresh medium was added, and the cells were cultivated for 2 days at 37°C under an atmosphere of 5% CO2.
Viral RNA was extracted from 140 µL of culture supernatant using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. Real-time quantitative reverse transcription PCR (RT-qPCR) assays were performed using the One Step TB Green® PrimeScript™ RT-PCR kit II (Takara, Shiga, Japan) and the SYBR Green assay 2 primer sets designed by MilliporeSigma (https://www.sigmaaldrich.com/US/en/technical-documents/protocol/research-and-disease-areas/immunology-research/ncov-coronavirus). The components of the RT-qPCR reaction mixture were as follows: 6.25 µL of 2× One Step SYBR® RT-PCR Buffer 4, 0.25 µL of forward primer (10 µM), 0.25 µL of reverse primer (10 µM), 0.25 µL of ROX reference Dye II (50×), 2.5 µL of deionized water, 0.5 µL of PrimeScript 1 step Enzyme Mix 2, and 2.5 µL of template RNA. Reverse transcription was performed at 42°C for 5 min, followed by denaturation at 95°C for 10 min, and 40 amplification cycles at 95°C for 5 s and 60°C for 34 s. A Quant Studio 3 Real-Time PCR System (Life Technologies, Carlsbad, CA) was used for the analysis.
Serially diluted S protein (Spike S1+S2 ECD-His Recombinant Protein, 40589-V08B1, Sino Biological, Beijing, China), N protein (40588-V08B or 40588-V07E, Sino Biological), or 10 µL of cell lysate suspended in 100 µL of culture medium was added to 100 µL of the K-ML2 cell suspension or monocyte-derived macrophages with 100 µL of culture medium and incubated at 37°C for 4 h. Then, 500 µL of fresh medium was added to the wells. Two days later, the culture supernatants were harvested, and the levels of IL-6 were measured by an enzyme-linked immunoassay (ELISA MAX Deluxe Set Human IL-6, BioLegend, San Diego, CA).
Plasmid construction and transfection
We used standard molecular biology techniques for cloning and plasmid construction. Most of the plasmid constructs were generated in the pLVX-EF1alpha-SARS-CoV-2-N-2xStrep-IRES-Puro vector backbone (for more details, see the Key Resources Table). A standard transfection reagent, TransIT-293 transfection reagent (V2704, Takara), was used.
Plasmid-transfected 293T cells (6 × 105 cells) were lysed in 100 µL of lysis buffer (50 mM Tris–HCl at pH 7.5, 150 mM NaCl, 1% Nonidet P-40, and 0.5% sodium deoxycholate). Proteins in the lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins in the gel were then electrically transferred to a membrane (Immobilion; Millipore, Billerica, MA). Blots were blocked and probed with N2 mAb (HM1057, EastCoast Bio, Maryland Hights, MO) or C2 mAb (CV15, CerTest Biotec, Zaragoza, Spain) overnight at 4°C. The blots were then incubated with peroxidase-linked anti-mouse IgG (H+L), and the bound antibodies were visualized with a Chemi-Lumi One chemiluminescent kit (Nacalai Tesque, Kyoto, Japan).
Immunization, fusion, and selection of mAb
SARS-CoV-2 (KNG19-020) was propagated in TMPRSS2/VeroE6 cells (JCBR1819) and purified by sucrose gradient centrifugation 60. Concentrated virus was then exposed to ultraviolet light (0.6 J/cm2) to inactivate the virus. We confirmed that the virus had completely lost its infectivity by this method. BALB/c mice (4 weeks old, female) were intraperitoneally immunized three times by the inactivated virion (corresponding to 3.8 × 107 TCID50/mouse) with adjuvant (1st immunization: Freund’s complete adjuvant (WAKO, Tokyo, Japan); 2nd and 3rd immunization: Freund’s incomplete adjuvant (WAKO)). Three days after the last immunization, splenic cells from the mice were used to prepare hybridomas. The hybridoma-producing mAbs were generated as described previously 61 using mouse myeloma PAI cells. The antibodies secreted by the hybridomas were screened by an indirect immunofluorescence assay using SARS-CoV-2-infected cells. SARS-CoV-2-infected and mock-infected TMPRSS2/VeroE6 cells were cultured for 18 h, fixed with 7% formaldehyde-PBS for 30 min, and then permeabilized with 1% Triton X-100 PBS for 5 min. The cells were incubated with hybridoma culture supernatant at 37°C for 1 h, followed by incubation with goat anti-mouse IgG conjugated to Alexa Flour 488 (1:1000; Invitrogen, Carlsbad, CA) for 30 min at 37°C. The cells were observed under a fluorescence microscope (ECLIPSE Ti2, Nikon, Tokyo, Japan). The isotype of the antibodies was determined by an IsoStripTM mouse Monoclonal Antibody Isotyping kit (Roche, Mannheim, Germany). The target of the antibodies was determined by the staining patterns using an indirect immunofluorescence assay and the reactivity to N protein-expressing cells.
ELISA for S protein
The amount of S protein in the SARS-CoV-2-infected cell lysates was measured by a SARS-CoV-2 Spike Protein ELISA kit (E-EL-E605, Elabscience, Houston TX) according to the manufacturer’s instructions.
In-house ELISA of anti-N antibodies
Ninety-six-well flat-bottom microplates were coated with 100 ng/well of N protein (40588-V08B, Sino Biological), NTD (40588-V07E10, Sino Biological), or CTD (40588-V07E5, Sino Biological) in 50 µL of carbonate-bicarbonate buffer (C-3041, Sigma, St. Louis, MZ), and incubated at 4°C overnight. After washing with 0.05% Tween 20 in PBS (PBS-T), wells were blocked with 200 µL of a 25% solution of BlockAce for 1 h at room temperature. After washing with PBS-T, 100-times diluted patient serum with PBS-T or mouse mAb (1 µg/mL), as listed in the Key Resources Table, was added and incubated for 1 h at room temperature. After washing with PBS-T, 50 µL of the secondary antibody solution of peroxidase-conjugated AffiniPure alpaca anti-Human IgG (H+L) (609-035-213, Jackson ImmunoResearch, Pennsylvania, PA) or peroxidase-labeled goat anti-mouse IgG (H+L) (5220-0341, CeraCare, Milford, MA) was added and incubated for 1 h at room temperature. A TMB substrate kit (34021, Thermo Fisher Scientific, Waltham, MA) was used for colorimetric detection, and the optical density at 450 nm was measured by a Multigrading Microplate Reader (SH-9500Lab, Corona, Hitachinaka, Ibaraki, Japan).
Multiplex cytokine measurement
Cytokine and chemokine biomarkers were quantified using the Bio-Plex 200 multiplex cytokine array system (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer’s instructions. Serum samples were collected from patients and centrifuged at 1600 g for 10 min. The serum samples were frozen at −80°C until they were analyzed.
We simultaneously quantified cytokines, chemokines, and soluble receptors using the Bio-Plex Human Cytokine 27-plex Panel (IL-1β, IL-1Rα, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 (p70), IL-13, IL-15, IL-17A, eotaxin, basic fibroblast growth factor, G-CSF, GM-CSF, IFN-γ, IP-10, MCP-1, MIP-1α, platelet-derived growth factor-BB, MIP-1β, RANTES, TNF-α, and vascular endothelial growth factor; (Bio-Rad Laboratories, Hercules, CA), the Inflammation Panel (a proliferation-inducing ligand (APRIL), B-cell activation factor, soluble CD30, soluble CD163, chitinase, soluble glycoprotein 130, IFN-α2, IFN-β, IFN-γ, IL-2, IL-8, soluble IL-6 receptor α, IL-10, IL-11, IL-12 (p40), IL-12 (p70), IL-19, IL-20, IL-22, IL-26, IL-27, IL-28A, IL-29, IL-32, IL-34, IL-35, lymphotoxin-like inducible protein that competes with glycoprotein D for herpesvirus entry on T cells (LIGHT), matrix metalloproteinase (MMP)-1, MMP-2, MMP-3, osteocalcin, osteopontin, pentraxin-3, soluble TNF receptor 1, soluble TNF receptor 2, thymic stromal lymphopoietin, and TNF-like weak inducer of apoptosis; Bio-Rad Laboratories), and the Bio-Plex pro (hepatocyte growth factor, IL-18, TNF-related apoptosis-inducing ligand, IL-2 receptor α, M-CSF, growth-related oncogene α, MCP-3, and monokine induced by IFN-γ; (Bio-Rad Laboratories, Hercules, CA). Data acquisition and analysis were performed using Bio-Plex Manager software version 5.0. (Bio-Rad Laboratories, Hercules, CA).
The disease severity of patients was determined at hospital admission according to The Guideline for Medical Treatment of COVID-19 in Japan (https://www.mhlw.go.jp/content/000785119.pdf)
Briefly, patients with “mild” illness showed one or some of the signs and symptoms of COVID-19 (e.g., fever, cough, sore throat, malaise, headache, muscle pain, nausea, vomiting, diarrhea, and loss of taste and smell), but lacked shortness of breath, dyspnea on exertion, and abnormal imaging findings. “Moderate I” cases showed evidence of lower respiratory disease with a percutaneous oxygen saturation (SpO2) of >93% on room air, and were compatible with “moderate illness” described in the Coronavirus Disease 2019 (COVID-19) Treatment Guidelines of the National Institutes of Health (NIH; https://www.covid19treatmentguidelines.nih.gov). “Moderate II” cases were supported with non-invasive mechanical ventilation or supplemental oxygen (including high-flow oxygen devices), and were compatible with “severe illness” described in the NIH guidelines. “Severe” cases were admitted into the intensive care unit or supported with invasive mechanical ventilation or extracorporeal membrane oxygenation, and were compatible with “critical illness” described in the NIH guidelines. In Japan, invasive ventilation was not applicable for several terminal cases. Aliquots of patient sera were collected from the leftover specimens of daily tests, and kept at -80°C until use. The median age among the 63 mild, 43 moderate I, 40 moderate II, and 55 severe cases was 57 (interquartile range, 37 ‒ 72.5), 70 (56.5 ‒ 80.75), 75.5 (58.25 ‒ 85), and 69 (58.25 ‒ 79) years, respectively. The number of males was 33 (52.4%), 22 (51.2%), 23 (57.5%), and 41 (74.5%), respectively.
Quantification and statistical analysis
The numbers of repetitions of specific experiments are shown in the figure legends. For multiple comparisons, statistical analysis was performed by the Kruskal-Wallis and Mann Whitney U tests (GraphPad Prism version 9.0.2, GraphPad Software, San Diego, CA) where applicable (Figure 4F).