Viruses and cells
Vero E6 cells and HEK293 cells (American Type Culture Collection, ATCC) were cultured at 37 °C in Dulbecco's Modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES pH 7.4, 1 mM sodium pyruvate, 1 × non-essential amino acids, and 100 U/mL of penicillin-streptomycin as our previously studies [18, 19]. SARS-CoV-2 strain 2019n-CoV/USA_WA1/2020 and SARS-CoV-2 variants, including Alpha, Beta, Gamma and Delta were obtained from the Guangdong Provincial Center for Disease Control and Prevention and Institute of Medical Laboratory Animals of Chinese Academy of Medical Sciences. All experiments with infectious SARS-CoV-2 were performed in BSL3 facilities approved by Institutional Biosafety Committee.
Animals in the experiments
Six weeks-old, 36 weeks-old female BALB/c mice and 6–8 weeks female Wistar rats were purchased from the Laboratory Animal Center of Southern Medical University (Guangdong, China). The Animal Care and Use Committee at Guangzhou University of Chinese Medicine approved the research. All animals were given a commercial mouse food and water ad libitum and housed in a temperature-controlled environment with a 12-h light-dark cycle.
All animals were immunized with rAAV5-based vaccines or rAAV5-GFP (control group) via intramuscular (IM) injection in the hind leg or intranasal inoculation at a single dose (five to eight animals for each group). Sera were collected for cytokines analyses performed by the Macau University of Science and Technology using a Non-Human Primate Cytokine Panel kit (Merck Millipore, Billerica, USA, Cat# PCYTMG-40K-PX23) on a Bio-Plex 200 instrument (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s protocol. For the virus attacking test, mice were challenged on day 40 after immunization with 3.6Log PFU of SARS-CoV-2 (HRB26M strain) via intranasal route. Animals were euthanized, and tissues were harvested for further analysis.
Construction and titration of rAAV5-based vaccines
The recombinant type 5 adenoviral-vectored SARS-CoV-2 (Accession Number: MN985325.1) vaccines encoding RBD domain (residues 319-541), RBD-plus domain (residues 319-583), S1 protein (residues 14-685), full-length of S protein (residues 14-1213) and NTD domain (residues 14-305 of the S protein N-terminal domain) of SARS-CoV-2 were produced by PackGene Biotech (Guangzhou, China). Briefly, rAAV5 packaging plasmids were transfected into HEK293T cells using PEI transfection reagent, according to the manufacturer’s protocol. The transfected cells and supernatants were harvested 72 h post transfection. rAAV5-vaccine was purified and titrated by real-time quantitative PCR. rAAV5-vaccine was adjusted to 1012 GCs/mL in PBS and used for the following vaccinations.
ELISA
Specific IgG and IgM against COVID-19 in mouse sera were tested by ELISA. Briefly, serially diluted mouse sera were added to 96-well microtiter plates pre-coated with RBD-His or S1-His protein. The plates were incubated at 37 °C for 30 min, followed by four washes with PBS containing 0.1% Tween 20 (PBST). Bound Abs were then reacted with HRP-conjugated goat anti-mouse IgG or IgM (Southern Biotech, Birmingham, USA, Cat.#1030-05) at 37 °C for 20 min. After four washes, the substrate 3,3,5,5-tetramethylbenzidine (Southern Biotech, Cat.#1030-05) was added to the plates, and the reaction was stopped by adding 1 N H2SO4. The absorbance at 450 nm was measured by an ELISA plate reader (Bio-Rad, Hercules, CA, USA). The endpoint serum dilution was calculated with a curve to fit the analysis of optical density (OD) values for serially diluted sera with a cut-off value of negative control.
Neutralization assay
Titers of NA in sera of mice immunized with rAAV5-GFP or rAAV5-vaccines were detected in Vero E6 cells as in our previous study [18]. Vero E6 cells were seeded at 1×104/well in 96-well culture plates and cultured at 37 °C to form a monolayer. Serial 4-fold dilutions of serum samples were mixed separately with 100 TCID50 (50% tissue-culture infectious dose) of SARS-CoV-2 strain (Guangdong Provincial Center for Disease Control and Prevention and institute of medical laboratory animals of Chinese Academy of Medical Sciences), incubated at 37 °C for 1 h, and added to the monolayer of Vero E6 cells in tetrad. Cells infected with or without 100 TCID50 SARS-CoV-2 were applied as positive and negative controls. Each well's cytopathic effect (CPE) was observed daily and recorded on day 3 post infection. The neutralizing titers of mouse antisera that completely prevented CPE in 50% of the wells were calculated by the Reed-Muench method.
Quantitative RT-PCR
The viral RNA copies in nasal or lung tissues of challenged mice were determined by Quantitative RT-PCR according to the protocol. Total RNA was extracted from 20 mg of lung tissues using a RNeasy Mini kit (Qiagen, Hilden, Germany, Cat.#74104). Then cDNA was synthesized using random primers and the SuperScript II RT kit (Invitrogen, Waltham, MA, USA, Cat.#18064014). Extracted RNA (10 µL) was reverse transcribed in a 20-µL reaction mixture containing 1 × first strand buffer, 100 mM DTT, 10 mM each dNTP, 50 ng of random primers, 40 U of RNaseOUT, and 200 U of SuperScript II RT at 42 °C for 50 min, followed by 15 min at 70 °C. The solution was incubated with RNase H (Invitrogen, Waltham, MA, USA, Cat.# 18021071) at 37 °C for 20 min. Synthesized cDNA was quantified using Power SYBR Green PCR Master Mix (Life Technologies, Carlsbad, California, United States, Cat.#4309155) in a 20-µL mixture containing 5 µL of cDNA (1/10), 10 µL of 2 × Power SYBR Green PCR Master Mix, 3 µL of RNase-free H2O, 10 µM forward primer and reverse primer in a Mx3000 QPCR System (Agilent, Santa Clara, California, USA).
Cell surface markers/intracellular cytokines staining
Single-cell suspensions (3 × 106) from spleens of the vaccinated mice were harvested and stimulated with or without SARS-CoV-2 S-specific peptide (S full-length peptide, residues 1-1213, 1 µg/mL) plus anti-mouse IL-2 (20 U/mL). Cells with stimulatory agents were incubated for 72 h at 37 °C with 5% CO2. The cells were harvested and stained directly with conjugated mAbs specific for cell surface markers including CD45, CD3, CD4, CD8, CD44, CD62L (BioLegend, San Diego, USA) for 30 min at 4 °C. Intracellular antigens including IFN-γ, IL-4 and TNF-α were stained for 20 min in the dark at room temperature. The stained cells were analyzed using a flow cytometer (BD Biosciences, San Jose, CA, USA). Data were analyzed by Cell Quest software (BD Biosciences, San Jose, CA, USA).
Statistical analysis
Statistical significance among different vaccination groups was calculated by the student t test using statistical software (GraphPad Prism 8). Values were presented as mean with SEM. Values of P < 0.05 were considered significant.