Recombinant rbd protein

Evaluation of IgG expression was performed by ELISA. The 96-well ELISA plates (Corning, New York, NJ, USA) were coated with 2 µg/mL purified recombinant RBD protein (Sino Biological, Beijing, China) at 4 °C overnight and were blocked in 5% BSA in PBST at 37 °C for 1 h. After plates were washed twice with PBST, mouse sera were diluted and incubated on the plate for 2 h at room temperature, which was followed by three washes. The plates were incubated with secondary antibody HRP-conjugated anti-mouse IgG antibody 1:10,000 (Abcam, Cambridge, UK), which was followed by incubation with TMB substrate (Beyotime). The absorbance was measured at 450 nm by Synergy HTX (BioTeK, Winooski, VT, USA) microplate reader.

Human antibody transgenic mice were generated by Boan Biotechnology. Mice were bred and kept under specific-pathogen-free conditions.
Three mice (named Q1–Q3) were immunized in 10-day intervals with the mixture of recombinant RBD protein (Cat 40592-V05H, Sino Biological), Spike S1 protein (Cat 40591-V02H, Sino Biological) and Spike S2 protein (Cat 40590-V08B, Sino Biological) and boosted by 105 μg mixtures (35 μg for each protein) after three rounds immunization. Two mice (named Q6 and Q7) were immunized in 10-day intervals with recombinant Spike S1 + S2 (Cat 40589-V08B1, Sino Biological) and boosted by 40 μg recombinant Spike S1 + S2 after 3 rounds of immunization. Another two mice (named Q14, Q15) were immunized in 10-day intervals with mixtures of Spike S1 protein (Cat 40591-V02H, Sino Biological) and RBD protein (Cat 40592-V05H, Sino Biological) and boosted by 70 μg mixtures (35 μg for each protein) after three rounds immunization. Genes of these proteins were derived from the SARS-CoV-2 strain (Wuhan-1 strain, GenBank: MN_908947). The antibody titers were tested by Elisa. After three rounds of immunization and one boost, spleen cells were harvested after 3 days of the last boost for phage libraries construction. Supplementary Table 1 showed details for the immunization process and Supplementary Fig. 2 showed serum antibody titers after three rounds of immunization.

Single-cell suspensions from bone marrow, BAL, and medLN cells were prepared from vaccinated mice. Cells were serially diluted in duplicate in complete media and incubated for 5 h at 37 °C on multiscreen cellulose filter ELISpot plates (Millipore-Sigma, Burlington, MA, USA) that were previously coated with purified recombinant RBD protein (Sino Biological, Wayne, PA, USA). RBD-specific antibodies secreted by plasma cells present in these tissues were detected using AP-conjugated goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA, USA) or AP-conjugated goat anti-mouse IgA (Jackson ImmunoReserch). ELISpots were imaged and counted using S6 Ultra-V Analyzer (Cellular Technology Limited, Shaker Heights, OH, USA).

SARS-CoV-2 spike protein and spike receptor-binding domain (RBD)-specific IgG and IgA titres were determined using ELISA. Briefly, Costar ELISA plates (Corning, Inc., Corning, NY, USA) were coated overnight with 0.2 μg SARS-CoV-2 spike protein or recombinant RBD protein (Sino Biological, Beijing, China). The plate was blocked with phosphate-buffered saline (PBS) containing 1% bovine serum albumin and 0.05% Tween 20 for 1 h at 37 °C. After washing the plates six times with PBS containing 0.05% Tween 20, sera were added to the wells at 4-fold serial dilutions. Plates were washed six times with PBS containing 0.05% Tween 20 and then incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (ZSGB-BIO, Beijing, China, 1:10,000) or horseradish peroxidase-conjugated goat anti-mouse IgA (Abcam, Cambridge, UK, 1:10,000) for 1 h at 37 °C. After washing, 3,3’,5,5'-tetramethylbenzidine (TMB) (Beyotime, Shanghai, China) was used as the substrate to detect the antibody responses at 450 and 630 nm. The endpoint of the serum antibody titre was calculated as the reciprocal of the highest dilution, which was 2.1-fold higher than the optical absorbance value of the negative control.

Splenocytes were isolated from the vaccinated mice (n = 5 per group) on days 12 and 25 after the initial vaccination (2 × 10⁵ cells/well) and cultured in RPMI 1640 medium supplied with 10% (v/v) FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, and 1 mM pyruvate. Recombinant RBD protein (2 µg/mL, Sino Biological, China) was used as a stimulus. After 36 h of incubation, 10 µL of the cell counting kit-8 (CCK-8) solution (Dojindo Laboratories, Kumamoto, Japan) was added to each well. The subsequent procedures were performed according to the manufacturer’s instructions. The plates were measured at 450 nm using a microplate reader (Bio-Tek, USA).

To investigate cell-mediated immune responses, the expression levels of interferon gamma (IFN-γ) and interleukin-4 (IL-4) were analyzed using ELISA kits (R&D Systems, USA) according to the manufacturer’s instructions. Briefly, splenocytes (1.0 × 10⁶ cells/well) described above were cultured in medium containing 10% (v/v) FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, 1 mM pyruvate, 50 µM β-mercaptoethanol and 20 U/mL IL-2 and stimulated with 2 µg/mL recombinant RBD protein (Sino Biological, China) for 72 h in a humidified incubator at 37 °C with 5% CO₂. The supernatants were collected for ELISA experiments to analyze the expression levels of cytokines.
The phenotypes of these cultured lymphocytes were determined by flow cytometry. In brief, these cultured lymphocytes were washed with sterile PBS and then stained for 30 min at 4 °C with monoclonal anti-CD8 and anti-CD4 antibodies. Afterwards, cells were fixed and permeabilized to facilitate intracellular staining with anti-IFN-γ and anti-IL-4 antibodies.