Cells lines and viruses
HEK293 cells (Human embryonic kidney, ATCC), Vero E6 cells (African green monkey kidney, ATCC) and ACE2-293T cells (ACE2-expressing cell line, constructed by hygromycin B screening) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Scientific, USA) supplemented with 10% foetal bovine serum (FBS) (Thermo Scientific, USA), penicillin (100 units/mL) and streptomycin (100 μg/mL) (complete medium) at 37 °C in 5% CO2. SARS-CoV-2/human/CHN/Beijing_IME-BJ01/2020 (Genbank No. MT291831) and SARS-CoV-2/HRB25/human/2020/CHN (HRB25, GISAID access no. EPI_ISL_467430) were respectively isolated from patients and propagated in Vero E6 cells. Mouse-adapted SARS-CoV-2/HRB26/human/2020/CHN (HRB26M, GISAID access no. EPI_ISL_459910) was generated by passaging the human patient isolate SARS-CoV-2/HRB26/human/2020/CHN (HRB26, GISAID access no. EPI_ISL_459909) in 4-6-week-old (young) female mice for 14 passages and propagated in Vero E6 cells. Virus titers were determined by standard plaque assay on Vero E6 cells, and virus stocks were stored in aliquots at -80 °C until use.
Construction of Ad5-nCoV expressing SARS-CoV-2 S
The full spike protein gene of SARS-CoV-2 based on the Wuhan-Hu-1 strain (NC_045512.2) was codon optimized by UpGene software, and the signal peptide was substituted with tPA for increased expression in mammalian cells. The gene was synthesized with EcoRI and HindIII upstream and downstream of the open reading frame, respectively, and cloned into the shuttle plasmid of the AdMax adenovirus system (Microbix Biosystem, Canada) by enzyme digestion and ligation. After sequencing identification, the shuttle plasmid with the target gene was cotransfected into HEK293 cells with the backbone plasmid (pBHGloxΔE1, 3Cre) by TurboFect transfection reagent (Thermo Scientific, USA) according to the manufacturer’s instructions. The transfected cells were passaged when they were overgrown and collected until Ad-related cytopathic effects were observed. The cells were lysed by three freeze-thaw cycles to release the recombinant viruses. The recombinant adenoviruses were confirmed by target gene sequencing, monocloned by agarose plaque selection, amplified by serial passage on HEK293 cells, and purified by ion exchange chromatography and size exclusion. The number of total VP was measured by ultraviolet spectrophotometer analysis, with one OD260 equal to approximately 1.1×1012 VP, and the infectious units (ifu) were titrated on HEK293 cells using an AdenoX™ Rapid Titer Kit (Clontech, USA) following the manufacturer’s instructions.
HEK293 cells in 6-well cell culture clusters were transiently transfected with 2 µg of plasmids expressing different S protein constructs by TurboFect transfection reagent (Thermo Scientific, USA). The culture supernatant was discarded, and cells were rinsed with phosphate-buffered saline pH 7.4 (PBS) and lysed by 150 µL of RIPA Lysis and Extraction Buffer (Thermo Scientific, USA) for each well at 48 h post transfection. The cell lysate was centrifuged, and the supernatant was collected, mixed with NuPAGE LDS sample buffer (Thermo Scientific, USA) and run on a SurePAGE 4-20% Bis-Tris protein gel (GenScript, China) with 20 μL per lane. Protein was transferred to a nitrocellulose membrane by an eBlot L1 transfer system (GenScript, China). Membranes were blocked for 1 h at room temperature (RT) in Tris-HCl-buffered saline pH 7.6 with 0.1% Tween 20 (TBST) containing 5% skim milk and then incubated overnight at 4 °C with a 1:2000 dilution of a polyclonal rabbit anti-SARS-CoV spike antibody (Sino Biological, China). After four washes with TBST, the membranes were incubated for 1 h at RT with a horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody (Cell Signaling Technology, USA). The membranes were developed with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, USA), and images were acquired with a Clinx ChemiScope imaging system (Clinx Science, China). As an internal parameter, β-actin was detected on the same membrane by an HRP-conjugated anti-β-actin antibody (Abcam, UK).
The experiments involving animals were approved by and carried out in accordance with the guidelines of the Institutional Experimental Animal Welfare and Ethics Committee. Specific pathogen-free (SPF) female BALB/c mice aged 6-8 weeks were obtained from Beijing Vital River Laboratory Animal Technologies Co., Ltd. (Beijing, China) and were housed and bred in the animal facility of the Animal Center, Academy of Military Medical Sciences, Beijing. Three- to four-month-old female ferrets purchased from Wuxi Cay Ferret Farm (Wuxi, China) were housed in the animal facility of Harbin Veterinary Research Institute (HVRI) of the Chinese Academy of Agricultural Sciences (CAAS). Mouse and ferret experiments with infectious SARS-CoV-2 were performed at the biosafety level 4 and animal biosafety level 4 facilities in HVRI, which is approved for such use by the Ministry of Agriculture and Rural Affairs of China.
Vaccination and challenge of mice
BALB/c mice (n = 10 per group) were immunized intramuscularly or intranasally with 5×109 VP (high dose), 5×108 VP (middle dose) or 5×107 VP (low dose) of Ad5-nCoV at day 0 or 5×109 VP of Ad5 vector as a control. Sera were collected for S-specific ELISA, SARS-CoV-2 NAb titration (MN50) and SARS-CoV-2 PNAb titration at different time points. Another three groups of mice immunized with a middle dose of Ad5-nCoV by the IM or IN route or with the control vaccine were euthanized at day 14 post immunization for splenic cellular immune response detection and trachea-lung wash antibody detection. At 10 weeks after vaccination, three of ten vaccinated mice in every group were euthanized for splenic T cell response detection and trachea-lung wash antibody and NAb detection. The remaining mice (seven per group) were challenged intranasally with SARS-CoV-2 HRB26M strain at a dosage of 103.6 PFU in a volume of 50 μL. Four and three out of seven mice in every dose group were sacrificed for viral load quantification in the lungs and turbinates at 3 and 5 dpi, respectively.
Vaccination and challenge of ferrets
Ferrets (6/group) were randomized by body weight, sex, and age and grouped into the IM vaccination group (5×1010 VP), the mucosal vaccination group (simultaneous oral delivery with 5×1010 VP and IN delivery with 5×1010 VP for one ferret) and the control group. Blood was collected at week 4 for analysis of antibody responses by ELISA, plaque reduction neutralization assays, and IFNγ ELISpot. The immunized animals were challenged intranasally with a dose of 105 PFU of SARS-CoV-2 HRB25 strain at day 28, and the viral load of the nasal wash was detected by qPCR and PFU assay every two days post infection.
Viral loads were determined by quantitative real-time PCR. Viral RNA was extracted by using a QIAamp vRNA Minikit (Qiagen, Germany). Reverse transcription was performed by using HiScript II Q RT SuperMix for qPCR (Vazyme, China). qPCR was conducted by using an Applied Biosystems QuantStudio 5 Real-Time PCR System (Thermo Scientific, USA) with Premix Ex Taq for probe qPCR (TaRaKa, China). N gene-specific primers (forward, 5’- GGG GAA CTT CTC CTG CTA GAA T-3’; reverse, 5'-CAG ACA TTT TGC TCT CAA GCT G-3') and probe (5'-FAM-TTG CTG CTG CTT GAC AGA TT-TAMRA-3') were utilized according to the information provided by the National Institute for Viral Disease Control and Prevention, China (http://nmdc.cn/nCoV). The amount of vRNA for the target SARS-CoV-2 N gene was normalized to the standard curve from a plasmid (pBluescript II SK-N, 4,221 bp) containing the full-length cDNA of the SARS-CoV-2 N gene. The assay sensitivity was 1000 copies/mL.
For SARS-CoV-2 S-specific IgG assays in mice, 96-well polystyrene high-binding microplates (Corning, USA) were coated with 2 μg/mL recombinant SARS-CoV-2 S protein purified from insect cells (Sino Biological, China) in carbonate-bicarbonate buffer pH 9.6, and the plates were incubated at 4 °C overnight. The plates were then blocked at 37 °C for 1 h with PBS pH 7.4 in 5% skim milk (blocking buffer) and washed with PBST. Serial dilutions of sera or trachea-lung washes of mice in dilution buffer were added to the plates and incubated at RT for 1 h. HRP-conjugated goat anti-mouse IgG (Abcam, UK) or HRP-conjugated goat anti-mouse IgA (Abcam, UK) was added to the plates, and the plates were incubated at RT for 1 h and washed with PBST. The assay was developed for 10 min at RT with 100 μL of TMB substrate solution (Solarbio, China), stopped by the addition of 50 μL of stop solution (Solarbio, China) and then measured at 450 nm/630 nm (SPECTRA MAX 190, Molecular Device, USA). The endpoint titre was defined as the highest reciprocal serum dilution that yielded an absorbance ≥2.1-fold over negative control serum values.
A double antigen sandwich ELISA kit (ProtTech, China) was used for SARS-CoV-2 S-specific IgG assays for the ferrets. Briefly, 100 μL of serum was added to an antigen-coated microtitre plate, and the plate was incubated at RT for 30 min and washed with PBST. Then, the plate was incubated with HRP-conjugated antigen at 37 °C for 30 min and washed with PBST. The optical density (OD) was measured at 450 nm after the addition of the substrate solution and the subsequent stop solution. Seropositivity was defined as an OD value ≥0.2.
SARS-CoV-2 neutralization assay
The neutralizing activity of sera from the mice was assessed using a microneutralization (MN) assay. Serial dilutions of heat-inactivated sera were incubated with 100 TCID50 of SARS-CoV-2 IME-BJ01 strain at 37 °C for 2 h. Antibody-virus complexes were added to pre-plated Vero E6 cell monolayers in 96-well plates and incubated for 48~72 h. The cells were stained with 0.05% crystal violet for 30 min. The OD was measured at 570 nm/630 nm after the addition of the decolorization solution. Neutralization results were analysed by Reed-Muench method to estimate the dilution of sera required for half-maximal neutralization of infection (EC50 titre). The initial dilution of sera (1:16) was set as the limit of confidence of the assay. Seropositivity was defined as a titre ≥16.
The neutralizing activity of heat-inactivated sera from the ferrets was assessed using a plaque reduction neutralization test (PRNT) assay. Serial dilutions of sera were incubated with 50 PFU of the SARS-CoV-2 HRB25 strain at 37 °C for 21 h. Antibody-virus complexes were added to pre-plated Vero E6 cell monolayers in 24-well plates and incubated for 48 h with agarose overlay. Neutralizing antibody titres were calculated as the maximum serum dilution yielding a 50% reduction in the number of plaques relative to that for control serum prepared from uninfected animals. Seropositivity was defined as a titre ≥ 8.
SARS-CoV-2 pseudovirus neutralization assay
SARS-CoV-2 pseudovirus bearing the full-length spike protein of SARS-CoV-2 was produced in an Env-defective, luciferase-expressing HIV-1 backbone. 7×106 293T cells were aliquoted into a 10-cm plate and co-transfected with 23 μg of pNL4-3.Luc-R-E- and 1 μg of CMV/SARS-CoV-2-S by TurboFect transfection reagent (Thermo Scientific, USA). At 48 h later, the supernatants containing pseudovirus were collected, filtered, aliquoted and frozen at -80 °C. Serial dilutions of heat-inactivated sera were mixed with the titrated pseudovirus, incubated for 60 min at 37 °C and added to ACE2-293T cells in duplicate in 96-well microplate. Cells were lysed 48 h later and luciferase activity was measured. EC50 neutralization titres were calculated for each individual mouse serum sample using Reed-Muench method.
SARS-CoV-2-specific cellular immune responses in ferrets were assessed by a Mabtech Ferret IFNγ ELISpot Kit (Mabtech, Sweden) following the manufacturer’s instructions. In brief, 1x105 of peripheral blood mononuclear cells (PBMCs) of vaccinated ferrets were produced by density gradient sedimentation and stimulated with inactivated SARS-CoV-2 in a pre-coated ELISpot plate for 16 h in a 37 °C humidified incubator with 5% CO2. The next day, the plate was washed 5 times with PBST, incubated for 1 h at RT with the biotin conjugated detection antibody, washed, incubated for 1 h at RT with streptavidin-HRP, washed, and developed with AEC substrate (BD Pharmingen, San Diego, CA). The plate was washed extensively in deionized water to stop color development and dried in the dark, and the spots were counted in an AID ELISPOT reader (AID GmbH, Strassberg, Germany). The result was expressed as the number of SARS-CoV-2-specific spots per 1 million PBMCs.
Intracellular cytokine staining
Splenocytes of BALB/c mice were prepared by pushing the spleen through a 70-μm cell strainer, followed by red blood cell lysis and several washes. The cells were stimulated for 6 h at 37 °C with or without 1 μg/mL of overlapping 15-amino-acid peptides covering the S protein and with BD GolgiStopTM and BD GolgiPlugTM to block cytokine secretion. Following peptide pool stimulation, the splenocytes were washed and stained with a mixture of antibodies against lineage markers, including anti-CD3 PerCP-Cy5.5 (clone 17A2), anti-CD4 Alexa Fluor 700 (clone RM4-5), and anti-CD8 FITC (clone 5H10-1), and the viability dye Near-IR to exclude dead cells from data analysis. After one wash with PBS, the cells were fixed and permeabilized with Cytofix/Cytoperm (BD Biosciences, USA), washed with Perm/Wash buffer (BD Biosciences, USA), and stained with anti-IFNγ PE (clone XMG1.2), anti-TNF PE-Cy7 (clone MP6-XT22) and anti-IL-2 Brilliant Violet 421 (clone JES6-5H4). The cells were washed successively with Perm/Wash buffer and PBS and resuspended in PBS, and data were acquired on a FACS CantoTM (BD Biosciences, USA). At least 200,000 events were collected for each sample, and the data were analysed by FACS Diva software. CD8+ and CD4+ T cells were gated from single cells (FSC-A vs FSC-H), lymphocytes (FSC-A vs SSC-A) and live CD3+ T cells (CD3+ vs Near-IR-), successively, and the detection results were defined as the percentage of cytokine-positive cells among CD8+ or CD4+ T cells.
The analyses were performed with GraphPad Prism v.8.0.2. Two-tailed nonparametric Mann-Whitney’s rank tests were conducted to compare differences between groups. Correlations were assessed by Spearman rank-correlation tests.