1. Plasmid construction

1. Insert DNA encoding SpyCatcher sequence (Figure 1) into an ampicillin-resistant pThioHisA-Cytolysin A (ClyA) plasmid (see Table of Materials) between the BamH I and Sal I sites to construct the plasmid pThioHisA ClyA-SC following a previously published report.
2. Ligate the synthesized SpyTag-SARS-CoV-2 receptor-binding domain (RBD)-polyhistidine tag (Histag) fusion gene (Figure 1) into a pcDNA3.1 plasmid (see Table of Materials) between the BamH I and EcoR I sites to construct the plasmid pcDNA3.1 RBD-ST following a previously published report.

2. OMV-SC preparation

1. Perform ClyA-SC transformation following the steps below.
   1. Add 5 µL of pThioHisA ClyA-SC plasmid solution (50 ng/µL) to 50 µL of BL21 competent strain, gently blow, and let cool on ice for 30 min.
   2. Place the solution for 90 s in the water bath at 42 °C, and then immediately put the mixed solution on ice for 3 min.
   3. Add 500 µL of Luria-Bertani (LB) medium to the bacterial suspension and, after mixing, culture at 220 rpm at 37 °C for 1 h.
   4. Plate all the transformation onto an LB agar plate containing ampicillin (100 µg/mL, see Table of Materials) and culture overnight at 37 °C.
      NOTE: In subsequent experiments, the ampicillin was kept at the same concentration in the medium. If not, this may cause loss of plasmids in the bacteria.
2. For OMV-SC production, perform the steps below.
   1. Isolate a single colony from the plate (step 2.1.4.) to 20 mL of LB (ampicillin-resistant) medium and culture overnight at 220 rpm at 37 °C.
   2. Inoculate the bacterial solution (from step 2.2.1.) into 2 L medium, culture at 220 rpm, 37 °C for 5 h until the logarithmic growth stage (the OD₆₀₀nm is between 0.6 to 0.8).
   3. When the OD at 600 nm of the bacterial solution reaches 0.6-0.8, add isopropyl beta-D-thiogalactopyranoside (IPTG, see Table of Materials) to make the final concentration of the bacterial solution to be 0.5 mM, and then culture overnight at 220 rpm at 25 °C.
3. Perform OMV-SC purification.
   1. Centrifuge the bacterial solution at 7,000 x g at 4 °C for 30 min.
   2. Filter the supernatant with a 0.22 µm membrane filter, then concentrate it using a 100 kD ultrafiltration membrane or hollow fiber column (see Table of Materials).
   3. Filter the concentrate through a 0.22 µm membrane filter, then centrifuge it at 150,000 x g at 4 °C for 2 h using an ultracentrifuge (see Table of Materials), and discard the supernatant with a pipette.
   4. Resuspend the precipitation with PBS and store it at −80 °C. The solution can maintain long-term stability at −80 °C.

3. RBD-ST preparation

1. Perform RBD-ST transfection following the steps below.
   1. Select an appropriate eukaryotic expression system (e.g., HEK293F) and culture the cells overnight at 130 rpm at 37 °C after recovery.
   2. Add 20 µL of HEK293F cell solution into the automated cell counter (see Table of Materials), record the number of cells, adjust the concentration to 1 x 106 cells/mL, and then culture the cells at 130 rpm at 37 °C for 4 h.
   3. Filter RBD-ST plasmid through a 0.22 µm membrane filter, and add 300 µg of plasmids into the cell culture medium (see Table of Materials) until the final volume is 10 mL; shake for 10 s.
   4. Heat polyethylenimine (PEI, 1 mg/mL, see Table of Materials) to 65 °C in the water bath, mix 0.7 mL of PEI with 9.3 mL of cell culture medium, and shake intermittently for 10 s.
      NOTE: Do not shake the solution vigorously. Otherwise, the resulting bubbles may affect the transfection efficiency.
   5. Add plasmid solution to the PEI solution, shake the mixture intermittently for 10 s, and incubate it at 37 °C for 15 min.
   6. Add the mixture to 280 mL of cell culture medium and culture at 130 rpm at 37 °C for 5 days.
2. Perform RBD-ST purification.
   1. Centrifuge the cells at 6,000 x g at 25 °C for 20 min and use a pipette to collect the supernatant.
   2. Fill the column with 2 mL of Ni-NTA agarose (see Table of Materials) and wash it 3x with 3x PBS.
   3. Add imidazole (see Table of Materials) into the cell supernatant to make the final concentration of 20 mM, and load the cell supernatant 2x.
   4. Add 3 column volumes (CV) of PBS containing 20 mM of imidazole for washing, and collect the washing fraction.
   5. Gradient elute with 3 CV of PBS containing low to high concentrations (e.g., 0.3 M, 0.4 M, 0.5 M) of imidazole; elute 2x for each concentration.
   6. Use SDS-PAGE to identify the RBD-ST in different concentration gradients.

4. OMV-RBD bioconjugation and purification

1. Determine the protein concentration by the bicinchoninic acid assay (BCA) method (see Table of Materials).
   1. Serially dilute the standard bovine serum albumin (BSA) protein solution from 2 mg/mL to 0.0625 mg/mL, dilute the purified OMV-SC and RBD-ST 10x, then mix BCA working solutions A and B (provided in the assay kit) at a ratio of 50:1 (v/v).
   2. Add diluted protein solution (25 µL/well) and mix with BCA working solution (200 µL/well); incubate at 37 °C for 30 min.
   3. Measure the absorbance (OD) at 562 nm of each well and calculate the protein concentration from the standard curve.
2. Perform bioconjugation of OMV-SC and RBD-ST following the steps below.
   1. Mix OMV-SC and RBD-ST in PBS at a 40:1 (w/w) ratio.
   2. Vertically rotate to blend the mixture overnight at 15 rpm at 4 °C.
      NOTE: Different antigens may react in different proportions. One could try different reaction ratios based on the characteristics of the antigen.