Hrp conjugation kit

Purified VP2 protein (600 µL), derived from the induction of 3 T75 flasks infected with recombinant MVA, was placed on the membrane of a centrifugal filter device (Amicon^® Ultra 30K (Millipore, Burlington, MA, USA)) to remove the 3X FLAG peptide. Then, 100 µL of 100 mM NaCl was added to the purified VP2, before being centrifuged at 7000× g until the volume was less than 100 µL. One milliliter of anti-protease PBS with 10 mM NaCl was added to the membrane, and the column was centrifuged at 7000× g until a volume of 100 µL was obtained, which was then used to label the VP2 with an HRP Conjugation kit (Abcam, Cambridge, UK), following the manufacturer’s instructions.

Surrogate virus neutralization assay, which allows assessment of virus inhibition without the BSL-3 facility requirement, was performed as described previously [25 (link)]. HRP-conjugated RBD was provided by Optipharm (Cheongju, Republic of Korea), which was performed using the HRP conjugation kit (Abcam, Cambridge, UK). SARS-CoV-2 human angiotensin-converting enzyme 2 (hACE2) receptor protein was purchased from GenScript (Piscataway, NJ, USA). After coating the plates with 100 ng of hACE2 using carbonate coating buffer in 96-well plates overnight at 4 °C, plates were washed with PBST and subsequently blocked with BD OptEIA assay diluent (BD Bioscience, San Diego, CA, USA) for 1 h at RT. Sera collected from mice 1 week after the final immunization were serially diluted in PBS and incubated at 56 °C for 30 min for complement inactivation. After heat inactivation, equal volumes of inactivated sera and HRP-conjugated RBD were mixed and inoculated into each well to be incubated for 1 h at 37 °C. Wells were washed with PBST and chromogenic reactions were developed using TMB substrate (BD Bioscience, San Diego, CA, USA). Reactions were stopped with 2N H₂SO₄ and absorbance values at 450 nm were measured. Percentage of inhibition was calculated as follows: Inhibition (%) = (1 − sample OD₄₅₀/control OD₄₅₀) × 100.