Plasmids
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 [13]. 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 [14, 15].
Cells
Human (Huh7 and 293 T), 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, 293 T 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) [16] 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 293 T 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 293 T 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).
Blood samples
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. All of the sera were heat-inactivated at 56 °C for 30 min. 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 [17].
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 COVID-19 PCR-negative donors and then inoculated with Sfullpv, St19pv, or VSVpv. To remove hematopoietic cells from whole blood samples, centrifugation was performed at 2000×g for 5 min. Infectivity of the pseudotyped viruses were determined by measuring luciferase activities after 24 h of incubation at 37 °C.
Expression and purification of recombinant SARS-CoV-2 trimeric spike protein
SARS-CoV-2 monomeric spike (S) proteins were produced using a mammalian cell protein expression system. The S gene sequence (GenBank: MN908947) was commercially synthesized (Genewiz, Japan). The extracellular region of the S sequence (amino acids 1–1213; MFVF…IKWP) was codon optimized for mammalian cell expression and the polybasic cleavage site was removed (RRAR to A) with stabilizing mutations (K986P and V987P; wild-type numbering) added as described by Amanat et al. [18]. Expression plasmid of monomeric S comprise the extracellular domain of S that is C-terminally fused to the thrombin site, fibritin trimerization sequence, and a Strep-tag II plus a His tag cloned into a PCXSN vector. S proteins were expressed using the Expi293 Expression System (Thermo Fisher Scientific Inc., Waltham, MA, USA), according to the manufacturer’s instructions. Seven days post-transfection, the medium was clarified by centrifugation at 1200 × g, filtered, and purified with Ni–NTA Agarose (QIAGEN, Germantown, MD, USA). The purified S proteins were concentrated using Amicon Ultracell (Merck) centrifugation units with a cut-off of 50 kDa, and the buffer was changed to PBS (pH 7.4). The proteins were filtered through Cosmospin filter G with a pore size of 0.2 micron (Nacalai Tesque, Inc.) and stored at − 80 °C until use.
IgG-ELISA
The IgG-ELISA was performed as described below. Briefly, 96-well ELISA plates were coated with the predetermined optimal quantity of purified SARS-CoV-2 S protein (approximately 100 ng/well) at 4 °C overnight. Each well of the plates was then covered with 200 μL of PBS containing 3% BSA and 0.1% Tween 20 (PBST-B), followed by incubation for 1 h at 37 °C for blocking. The plates were washed three times with PBS containing 0.1% Tween 20 (PBST) and then incubated with test sera (50 μL/well), which were diluted 1:1000 with PBST-B. After a 1 h incubation period, the plates were washed three times with PBST and then were incubated with goat anti-human IgG antibody labeled with HRP (1:5000 dilution; Sigma-Aldrich, St. Louis, MO, USA). After a further 1 h incubation period, the plates were washed and 50 μL of TMB solution (SeraCare life Sciences Inc., Milford, MA, USA) was added to each well. The plates were incubated for 30 min at room temperature, 50 μl of TMB stop soln. (SeraCare life Sciences) was added to each well, and the optical density at 450 nm (OD450) was measured. The adjusted OD450 value was calculated by subtracting the OD450 value of the negative Ag-coated wells from that of the corresponding wells. The mean plus three standard deviations (mean ± 3SD) of the ELISA indices for the IgG-ELISAs was calculated using γ-globulin and was used as the cut-off value for the IgG-ELISAs.
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.
Statistical analysis
Unpaired t-test with Welch's correction or two-way ANOVA was used to determine significant differences in the data using the GraphPad Prism 7 software program (GraphPad software, La Jolla, CA).
Ethics statement
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).