HLA-restricted peptides
The HLA-restricted peptides UC-100 (KLNSGDSKV) and UC-152 (KLNSGLSKV) were designed by Vacino Biotech Taiwan based on the S2 domain of the SARS-CoV-2 spike protein. The UC-100 and UC-152 peptides were synthesized by GenScript (NJ, USA) with 98% purity, and the pH value was adjusted to 7.2–7.4 for the in vitro and in vivo studies.
HLA-A201-peptide complex refolding assay
The easYmer kit (ImmunAware Aps, Copenhagen, Denmark) was used to determine the binding ability of the peptide to the HLA-A201 molecule. The detailed methods were performed according to the kit’s manual instructions. Briefly, the peptide was incubated with the β2m-microglobulin (β2m) light chain subunit and biotin-tagged recombinant HLA-A201 heavy chain at 18°C for 48 h to form a 500 nM folded HLA complex. The folded complexes were diluted with dilution phosphate-buffered saline (PBS) buffer containing 5% glycerol to serial concentrations of 8.9 nM, 3.0 nM and 1.0 nM. Then, 20 µL of 45-fold diluted streptavidin-coated beads (6–8 µm beads, Cat SVP-60-5, Spherotech, IL, USA) was added to each well. The beads were suspended in the solution with PE-labelled anti-human β2m monoclonal antibody and incubated for 30 min at 4°C. The fluorescence intensity was measured by flow cytometry (BD LSRFortessaTMX20, Franklin Lakes, NJ, USA). An HLA-A201 binding peptide, NLVPMVATV (CMV pp65495–503), which was reported to have an IC50 of 45 nM for HLA-A201, was used as the positive control44. Complex formation without peptide addition was used as the negative control.
In vivo study of HLA-restricted peptides
The mouse immunogenicity studies were performed by Super Laboratory Co., Ltd. (Taipei, Taiwan), and the animals were supplied by BioLASCO Taiwan Co., Ltd. (Taipei, Taiwan). For the immunogenicity study of the UC-100 peptide antigen, three female BALB/c mice (7–9 weeks old) were injected intramuscularly with 3 doses of 50 µg peptide 14 days apart. For the immunogenicity study of UC-152 peptide antigen, four female BALB/c mice (7–9 weeks old) were injected intramuscularly with 3 doses of 400 µg peptide 14 days apart. For the oral ability study of the UC-100 peptide antigen, five female BALB/c mice (7–9 weeks old) were orally administered 3 doses of 200 µg of peptide formulated with 1% PLCL-PEG-PLCL polymer (Sigma‒Aldrich, USA). The serum was collected before the first dose of immunization (predose) and at 14 days after the last dose immunization (postdose). The serum was stored at -80°C prior to antibody measurement and functional assay. The animal protocol was reviewed and approved by the Super Laboratory Company's Institutional Animal Care and Use Committee (approval IACUC number 111-14c). All animal experiments in this study were performed in accordance with the relevant guidelines and regulations. This study was reported in accordance with the ARRIVE guidelines.
Measurement of IgG and IgA levels
To determine the total IgG and IgA levels, the IgG (Total) Mouse Uncoated ELISA Kit (Cat., No. 88-50400, Thermo Fisher Scientific, MA, USA) and Mouse IgA ELISA Kit (Cat., E-90A, Immunology Consultants Laboratory, Inc., OR, USA) were used according to the manufacturers’ protocols. For total IgG measurement, a Nunc™ MaxiSorp™ 9018 ELISA plate was coated with 100 µL of the coating buffer containing capture antibody and incubated overnight at 4°C. The plate was then washed twice with 400 µL of wash buffer. After the wells were blocked with 250 µL of blocking buffer (PBS, 1% bovine serum albumin (BSA), 0.1% Tween 20) and incubated at RT for 2 h, the plate was washed twice. Then, 100 µL of the diluted samples and standards were added to the reaction wells. Then, 50 µL of diluted detection antibody was added to all wells. The plate was covered and incubated at RT for 2 h. The samples were washed four times, 100 µL of substrate solution provided in the kit was added, and the samples were incubated at RT for 15 min. Finally, 100 µL of stop solution provided in the kit was added, and the plate was read at an optical density (OD) of 450 nm by a microplate reader (SpectraMax iD5, Molecular Devices, CA, USA). For total IgA measurement, 4000-fold diluted samples (100 µL) were added to an IgA capture antibody-precoated plate. The procedures followed the kit’s instructions. The signal was read at OD 450 nm by a microplate reader (SpectraMax iD5, Molecular Devices, CA, USA).
To measure the levels of anti-UC peptide-specific IgG, a 96-well plate was coated with 100 µL of PBS buffer containing 5 µg of UC-100 and UC-152 peptide and incubated overnight at 4°C. The following steps for anti-peptide IgG measurement were performed as described for total IgG measurement. Briefly, after incubation with anti-mouse HRP antibody, the reaction microplate was washed four times, 100 µL of TMB Solution (ab171522, Abcam, Cambridge, UK) was added, and the plate was incubated at RT for 20 min. Finally, 100 µL of Stop Solution (Cat No. 5150-0020, SeraCare, MA, USA) was added, and the plate was read at OD 450 nm by a microplate reader (SpectraMax iD5, Molecular Devices, CA, USA). For specific IgA antibody measurement, UC-100 was coated onto a 96-well microplate. The experimental procedures were performed as previously described for IgG.
To measure the anti-spike trimer IgG antibody, a mouse SARS-CoV-2 spike trimer-coated 96-well plate kit (RAS-T023, Acro Biosystems, DE, USA) was used. The procedure was performed according to the kit’s instructions. 100 µL of diluted sample was added to an anti-spike trimer-coated plate, and 100 µL dilution buffer was used as a blank. The reaction was stopped by adding 50 µL of stop solution provided in the kit and read at OD 450 nm by a microplate reader (SpectraMax iD5, Molecular Devices, CA, USA).
Th1 and Th2 cytokine release assay
Briefly, 1 x 105 HLA-A201+ human PBMCs (STEMCELL Technologies Inc., Vancouver, British Columbia, Canada) were seeded in a 96-well microplate45. The PBMCs were treated with 5 different concentrations of antigen peptides at final concentrations of 0.4, 2, 10, 50 and 250 nM and cultured with X-VIVO 15 medium for 12 days. The culture medium was refreshed every 5 days. To analyse the surface marker and intracellular cytokine levels, the suspended cells were collected and stained with anti-CD4-PerCP-Cy5.5 conjugated (Cat. 566923, BD Pharmingen, USA), IFN-γ-FITC chrome conjugated (Cat. 554700, BD Pharmingen, USA), and IL4-PE chrome conjugated (Cat. 559333, BD Pharmingen, USA) antibodies. The cytokine levels in CD4+ T-helper cells was measured by flow cytometry (BD LSRFortessaTMX20, Franklin Lakes, NJ, U.S.)
HLA-DR-peptide engagement assay
A U-Load Dextramer kit MHC II HLA-DRB1*0101/APC (Immudex, Denmark) was used to determine the binding ability of a peptide to the HLA-DR molecule. The method was performed according to the kit’s protocol. The 10 µL MHC-II-peptide monomer was prepared by mixing the reagents of a 2 µL of 1 mM peptide (UC-100 or UC-152), a 3 µL of dissolved U-load MHC-II peptide loading component and a 5 µL of U-load MHC-II (1 mg/mL). PBMCs (2 x 105) suspended in 50 µL of wash buffer composed of 1% FCS in phosphate-buffered saline (PBS), pH 7.4. The 10 µL of prepared MHC dextramer as described in the protocol was added to the PBMCs and incubated in the dark for 30 min at RT, followed by staining with the anti-CD4-PerCP-Cy5.5 conjugated antibody and further incubation in the dark for 20 min at RT. The cells were washed twice with 2 mL of wash buffer, and the cell pellet was resuspended in wash buffer for analysis by flow cytometry (BD LSRFortessaTMX20, Franklin Lakes, NJ, U.S.).
Peptide-specific CTL engagement assay
The easYmer kit (ImmunAware Aps, Denmark) was used for a tetramer assay to determine the peptide-specific CTL binding level to the peptide-HLA-A201 complex46. The methods were performed according to the kit’s protocol. Briefly, the peptide was incubated with the β2-microglobulin (β2m) light chain subunit and biotin-tagged recombinant HLA-A201 heavy chain for 48 h at 18°C to form a 50 µL 500 nM folded HLA complex. A tetramer was produced by incubating the equivalent of 2.1 µL of 0.2 mg/mL streptavidin-APC (Cat., 554067, BD Pharmingen, USA) and 50 µL of 500 nM folded HLA complex in the dark at 4°C overnight. PBMCs (1 x 105, HLA-A201+) (STEMCELL Technologies Inc., Vancouver, British Columbia, Canada) were costained with the APC-labelled tetramer complex and FITC-conjugated anti-CD8 antibody (Cat., 555634, BD Pharmingen, USA) and analysed by flow cytometry (BD LSRFortessaTMX20, Franklin Lakes, NJ, U.S.) for the percentage of tetramer-stained CD8+ cells.
Cytotoxic activity assay of specific T-cells
HEK293 cells expressing the SARS-CoV-2 spike protein (D614) (Cat No. 293-cov2-s, InvivoGen, CA, USA) were used as target cells, which were seeded into a 96-well plate47. HLA-A201+ PBMCs (STEMCELL Technologies Inc., Vancouver, British Columbia, Canada) were cocultured with 3,000 SARS-CoV-2 spike-expressing cells at ratios of 27:1, 9:1, 3:1 and 1:1, and the culture was treated with UC-100 and UC-152 peptides at a final concentration of 9 nM or 27 nM for 5 days at 37°C and 5% CO2. Cytotoxicity was measured by using an LDH-Cytox™ assay kit (Cat No. 426401, BioLegend, CA, USA). SARS-CoV-2 spike-expressing cells without PBMCs and peptide treatment were used as the negative control, while lysis buffer added to the well containing only spike-expressing cells was used as the positive control. Fifty microlitres of LDH substrate was added to each well and incubated for 30 min at RT. Finally, the OD at 490 nm was read by a microplate reader (EnVision Xcite Multilabel Reader, PerkinElmer, MA, USA). The percentage of specific cell lysis was calculated according to the manufacturer’s instructions.
Complement-dependent cytotoxicity assay
To determine the functions of the antibodies induced by the UC peptide, complement cytotoxicity was measured in SARS-CoV-2 spike protein (D614)-expressing HEK293 cells (Cat No. 293-cov2-s, InvivoGen CA, USA)48. The cytotoxicity mediated by the antibodies was measured by using an LDH-Cytox™ assay kit (BioLegend, CA, USA). The 4 x 103 SARS-CoV-2 spike-expressing cells were suspended in 45 µL of serum-free DMEM in each well of a 96-well microplate. The serum samples were serially diluted twofold for 9 dilutions with serum-free DMEM starting at a ratio of 1:20 to 1:5120. Five microlitres of diluted serum was added to the wells containing SARS-CoV-2-expressing cells and incubated for 15 min at 37°C and 5% CO2. A final concentration of 10% young rabbit complement (CL3441-S50-R, Cedarlane, Ontario, Canada) was added to each well and incubated for an additional 30 min at 37°C and 5% CO2. The plate was then cooled at RT for 15 min for measurement of the cytotoxicity by LDH release. The sample wells containing serum were used as background controls, while lysis buffer was added to the sample wells containing SARS-CoV-2-expressing cells, and complement was used as the positive control. Then, 50 µL of LDH substrate was added to each well and incubated for 30 min at RT. Finally, the OD at 490 nm was read by a microplate reader (EnVision Xcite Multilabel Reader, PerkinElmer, MA, USA). The percentage of specific cell lysis was calculated according to the manufacturer’s instructions.
Statistical analysis
Groups were compared using an unpaired one-tailed Student’s t test embedded in Prism 5 (GraphPad Software Inc., San Diego, CA, USA) for the measurement of IgG antibody, IgA antibody, tetramer and dextramer. A p value < 0.05 was considered significant (*p < 0.05, **p < 0.01, and ***p < 0.001).