Rabbit anti-SARS-CoV-2 spike ECD polyclonal IgG, Rabbit anti-SARS-CoV-2 spike S1 polyclonal IgG, Rabbit anti-SARS-CoV-2 spike RBD polyclonal IgG was purchased from Sino Biological (Beijing, CN). HRP-conjugated goat anti-rabbit IgG was purchased from ZSGB-BIO (Beijing, CN). CF568 labeled Goat anti-rabbit IgG (H+L) was purchased from Biotium Inc. (Hayward, CA, USA). The luciferase assay system was obtained from Promega (Madison, WI, USA). Goat Anti-Rabbit IgG (whole molecule) Gold antibody was obtained from Sigma Aldrich (St. Louis, MO, USA).
Plasmids, bacterial strains and cell lines
The defective-rescue BmNPV BES reBmBac vector was constructed and maintained in our laboratory15. The pET-28a (+) vector was saved in our laboratory. The recombinant BmNPV transfer vector pBmPH (GenBank submission ID: 2426173) was constructed and maintained in our laboratory. BmN cells was preserved in our laboratory and cultured in TC100 insect cell culture medium (Applichem, Darmstadt, Germany) with 10% fetal bovine serum (FBS, Gibco, USA) at 27°C. HEK-293T cells was obtained from ATCC and cultured in DMEM (Thermo Fisher, Waltham, MA, USA) with 10% FBS at 37°C. The Escherichia coli strains Top10 and BL21 (DE3) were cultured in Luria-Bertani (LB) medium.
Recombinant BmNPV construction
Three peptide sequences of nanoparticle vaccines were designed according to the full-length sequence of S protein (RefSeq: YP_009724390.1). The sequences of ECD (14-1213 aa, R685N), S1 region (14-685 aa, R685N) and RBD (319-541 aa) were linked with Helicobacter pylori ferritin (RefSeq: WP_000949190.1, 5-167 aa, N19Q) via SGG linker. The human T-cell surface glycoprotein CD5 signal peptide (RefSeq: NP_055022.2, 1-24 aa) was used as signal peptide of these three sequences. After codon preference optimization, the three designed sequences were synthesized by GenScript (Nanjing, CN) with BamHI and EcoRI restriction sites at the upstream and downstream, respectively. The ECD-Ferritin sequence was inserted into pBmPH vector through BamHI/EcoRI digestion to construct the transfer vector pBmPH-ECD-Fe. The pBmPH-S1-Fe and pBmPH-RBD-Fe vectors were constructed the same way. The transfer vectors were then cotransfected in BmN cells with reBmBac respectively. The recombinant BmNPV baculovirus, reBm-ECD-Fe, reBm-S1-Fe and reBm-RBD-Fe, were obtained in the cell supernatant of cotransfection.
Nanoparticles expression and purification
Fifth instar silkworm larvae or pupae were injected with recombinant viruses (5 μL of the supernate, approximately 0.5×105 pfu) between the abdominal knobs on the backside and then reared or incubated at 25-27°C and 65% humidity. Larval haemolymph or pupae body expressing nanoparticles were harvested after 108-120 h. The crude samples were purified by gentle ultrasonic crushing and ultracentrifugation by 30% sucrose cushion at 120,000×g for 3 h. The white transparent nanoparticle pellet was resuspended in PBS overnight. The concentrations of the nanoparticles were detected by ELISA using standard samples of ECD, S1 and RBD (Sino Biological, Beijing, CN). Whether the nanoparticles were assembled successfully or not was observed by transmission electron microscope (TEM). Immunoelectron microscopy (IEM) was used to identify whether there was S protein on the surface of nanoparticles.
Animal ethics statement
The mouse immunogenicity studies were performed in Biotechnology Research Institute and Institute of Animal Sciences of Chinese Academy of Agricultural Science. The Ethics Review Board of Biotechnology Research Institute and Institute of Animal Sciences approved this study. All animal procedures were in strict accordance with the guidelines of the Animal Care and Use Committee of Biotechnology Research Institute and Institute of Animal Sciences.
Mouse study designs
Six BALB/c mice (6-8 weeks of age) were assigned in every group. Mice in Subcutaneous Administration (SA) group were subcutaneously immunized with 10μg dose of crude purified nanoparticle vaccines ECD-Nano, or equal moles dose of S1-Nano or RBD-Nano, formulated with aluminium hydroxide adjuvant. Mice in Oral Administration (OA) group were fed immunized with 50 μg dose of crude expression products ECD-Nano, or equal moles dose of S1-Nano or RBD-Nano, without adjuvant. All the mice were vaccinated with the above vaccines in a prime/boost manner which was vaccinating mice on days 0 and day 21. Sera were collected twice on days 14 and 35. Mice were euthanized at week 6. PBS administrator Mice were the control group.
In safety test groups, mice were fed with 50 μg dose of nanoparticles per day for five days continuously. Sera were collected on days 0 and 21. Mice were euthanized and dissected on day 21.
Identification of serum antibodies against the Spike in mice using an ELISA assay
Blood samples were collected from the retro-orbital plexus of mice on days 14 and 35. After coagulation at 37oC for 2 h, blood samples were centrifuged at 2,000 rpm for 10 min. The serum was collected and stored at -20oC. Recombinant ECD-His, S1-His, and RBD-His expressed and purified from E.coli BL21 (DE3), were used to coat flat-bottom 96-well plates at a final concentration of 1 μg/mL. After overnight incubation at 4oC, plates were washed three times with PBST and added with blocking buffer containing 1% BSA in PBST, followed by 1 h incubation at room temperature. Mouse sera were serially diluted and added to the plates. After 1 h incubation at 37oC, the plates were washed three times and added the HRP-conjugated Goat anti-mouse IgG antibody with a dilution of 1:5000. After incubation for 1 h at 37oC, the plates were washed three times with PBST and developed with TMB (Solarbio, Beijing, CN) for 10 min. The reactions were stopped with a stop solution (1M H2SO4). The absorbance at 450 nm was measured on a microplate reader.
Measurement of the inhibition of RBD binding to cell-surface ACE2
The hACE2 was co-expressed with EGFP using pEGFP-N3 vector in HEK-293T cells. The purified Spike nanoparticles were incubated with PBS or antisera for 1 h respectively. The mixtures were then added into the 293T-hACE2 cells, followed by incubating for 12 h. The cells were fixed with paraformaldehyde buffer, followed by incubating with Rabbit anti-SARS-CoV-2 spike RBD polyclonal IgG for 2 h at 37oC. After washing, the cells were incubated with CF568 labeled Goat anti-rabbit IgG (H+L) with a dilution of 1:5000 for 2 h at 37oC. Images in fluorescence were acquired by confocal scanning microscopy.
SARS-CoV-2 authentic virus neutralization assay
To assess the neutralization of the SARS-CoV-2 infection in vitro, Vero cells (5×104) were seeded in 96-well plates and grown overnight. SARS-CoV-2 origin stain and delta variants (approximately 100TCID50) was preincubated with an equal volume of sera (serially diluted from 1:2) from immunized mice. After incubation at 37oC for 1h, the mixture was added to Vero cells. After 3 days, cytopathogenic effects of the cells were recorded under the microscope. The neutralization assay was carried out in BSL-3.
All statistical analyses were carried out with GraphPad Prism software. P < 0.05 was considered significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.