The cDNAs of the SARS-CoV-2 spike protein were obtained by chemical synthesis with optimization for the humanized codon (Integrated DNA Technologies, Inc., Coralville, IA). The S cDNA of SARS-CoV-2 was cloned into the pCAGGS expression vector . The resulting plasmid was designated as pCAG-SARS-CoV-2. The plasmid, which contains the S protein gene with a 19 aa truncation at the C-terminus, was constructed using the cDNA of pCAG-SARS-CoV-2. The S proteins with the 19 aa deletion of coronaviruses were previously reported to show increased efficiency regarding incorporation into virions of VSV [9, 10].
Human (Huh7 and 293T), monkey (Vero), hamster (BHK and CHO), and mouse (NIH3T3) cell lines were obtained from the American Type Culture Collection (Summit Pharmaceuticals International, Tokyo, Japan). All cell lines were grown in Dulbecco’s modified Eagle’s medium (DMEM; Nacalai Tesque, Inc., Kyoto, Japan) containing 10% heat inactivated fetal bovine serum (FBS).
Generation of pseudotyped VSVs
Pseudotyped VSVs bearing the S protein, the 19 aa-truncated S protein of SARS-CoV-2, or VSV-G were generated as described below. Briefly, 293T cells were grown to 80% confluence on collagen-coated tissue culture plates and then transfected with each expression vector: pCAG-SARS-CoV-2 S-full, pCAG-SARS-CoV-2 S-t19, and pCAG-VSV-G. After 24 h of incubation, the cells transfected with each plasmid were infected with G-complemented (*G) VSV∆G/Luc (*G-VSV∆G/Luc) at a multiplicity of infection (MOI) of 0.5 per cell. Then, the virus was adsorbed and extensively washed four times with 10% FBS DMEM. After 24 h of incubation, to remove cell debris, the culture supernatants containing pseudotyped VSVs were centrifuged, and then, they stored at −80°C until ready for use. The pseudotyped VSV bearing SARS-CoV-2 S protein or SARS-CoV-2 truncated S protein are referred to as Sfullpv or St19pv, respectively. The infectivity of Sfullpv, St19pv, or VSVpv to 293T cells was assessed by measuring the luciferase activity. The value of the relative light unit (RLU) of luciferase was determined using a PicaGene Luminescence Kit (TOYO B-Net Co., LTD, Tokyo, Japan) and GloMax Navigator System G2000 (Promega Corporation, Madison, WI), according to the manufacturer’s protocol.
Coomassie Brilliant Blue (CBB) staining and Immunoblotting
Transfection of 293T cells occurred with pCAG-SARS-CoV-2 Sfull, pCAG-SARS-CoV-2 St19, or VSV-G. At 24 h post-transfection, the cells were collected and lysed in phosphate-buffered saline (PBS) containing 1% NP40. Then, the lysates were centrifuged to separate insoluble pellets from supernatants. The supernatants were used as samples. The Sfullpv or St19pv, which were generated as described above, were pelleted through a 20% (wt/vol) sucrose cushion at 25,000 rpm for 2 h in an SW41 rotor (Beckman Coulter, Tokyo, Japan). Then, the pellets were resuspended in PBS. Each sample that was boiled in loading buffer was subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). According to the manufacturer’s protocol, the proteins in the gel were stained with CBB Stain One (Nacalai Tesque, Inc.). Next, the proteins in another gel were electrophoretically transferred to a methanol-activated polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA) and reacted with COVID-19 hospitalized patient sera (#12). Then, immune complexes were visualized with SuperSignal West Dura Extended Duration Substrate (Pierce, Rockford, IL) and detected by an LAS3000 analyzer (Fuji Film, Tokyo, Japan).
Twenty-three serum samples were collected from hospitalized patients with COVID-19 who were admitted to the University of Toyama Hospital, Toyama, Japan. In addition, nineteen serum samples were collected from COVID-19 PCR-negative donors at the University of Toyama Hospital. The diagnosis of COVID-19 in all patients or donors was assessed using the real-time PCR method with specific primers, which were developed at the National Institute of Infectious Diseases, Japan .
By using a blood collection tube containing EDTA, whole blood samples were obtained from 5 hospitalized patients (the University of Toyama Hospital) with COVID-19.
Neutralization assays with patient blood samples
The patient sera used in this study were collected from participants after obtaining informed consent. To examine neutralization of the human serum or whole blood samples against pseudotyped viruses, Vero cells were treated with serially diluted sera or whole blood of convalescent patients with COVID-19 or PCR-negative donors and then inoculated with Sfullpv, St19pv, or VSVpv. To remove hematopoietic cells from whole blood samples, centrifugation was performed at 2,000 × g for 5 min. Infectivity of the pseudotyped viruses were determined by measuring luciferase activities after 24 h of incubation at 37 °C.
Immunofluorescence assay (IFA)
For the IFA, BHK cells transfected with pCAG-SARS-CoV-2 S-full were fixed with acetone-methanol (1:1) at 4 °C for 20 min. Fixed cells were reacted with the test serum samples, which were diluted at 1:100 with PBS. After an incubation for 1 h, the cells were rinsed with PBS and incubated with goat anti-human Alexa Fluor 488 (Invitrogen). After washing with PBS, staining was observed under a fluorescence microscope.
All of the samples, protocols, and procedures were approved by the Research Ethics Committee at the University of Toyama for the use of human subjects (approval number: R2019167).