Nunc cell factory system

P2 virus stock was propagated in Vero-E6 cells cultured in a Nunc cell factory system (Thermo Fisher Scientific, Waltham, MA, USA) at multiplicity of infection (MOI) 0.005, then cells were microscopically investigated daily. The virus-infected culture supernatant was clarified by centrifugation at 4000 rpm for 15 min at 4 °C twice. The harvested virus was titrated by plaque titration assay [5 (link)]. Harvested SARS-CoV-2 was inactivated with 0.1% β-Propiolactone (Invitrogen), then the treated virus was incubated at 4 °C for two days in a cooling shaking incubator. The β-Propiolactone treated virus was tested for loss of its infectivity by inoculating it in Vero-E6 cells for seven days. No cytopathic effect (CPE) was observed on cells infected with inactivated virus. Virus harvest (1000 mL) was concentrated by ultra-filtration system (Amersham Bioscience, Amersham, UK) hollow fiber cartridge of 50 KDa pore size and a circulation rate of 150 rpm. The concentrated virus was further concentrated by ultracentrifugation (80,000× g, 4 °C, 1 h with 20% sucrose as a cushion). Total protein content was measured by Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific).

Vero E-6 cells were grown in a Nunc cell factory system (Thermo Fisher Scientific, Waltham, MA, USA) using DMEM containing 10% FBS. P4 viral stock was used to infect Vero E6 cells at a multiplicity of infection (MOI) 0.01. The supernatant was harvested at 72–96 h post-infection. The virus-containing supernatant was clarified by centrifugation at 3500 rpm for 20 min at 4 °C. The virus was inactivated with ß-propiolactone 1:1500 (v/v) at 4 °C and was further incubated at 37 °C for 2 h. Inactivation was confirmed by the inoculation of ß-propiolactone-treated samples on Vero E-6 cells. The inactivated virus was filtered using a 0.45 um filter (Millipore-sigma) following polyethylene glycol (PEG-8000, Promega, Madison, WI, USA) precipitation. Precipitated viral supernatants were treated with 20 U/mL of benzonase (Millipore; Burlington, MA, USA) overnight at 2–8 °C to digest host cell DNA. Column chromatography (Acta avant 150) was used for the purification following a tangential flow filtration system (Millipore cogent). After sterile filtration, an inactivated whole-virion SARS CoV-2 (ERUCoV-VAC) vaccine was formulated with aluminium hydroxide adjuvant (Alhydrogel, 250 µg per dose) (InvivoGen, San Diego, CA, USA). Different antigen concentrations (2.5 μg, 3 μg, 5 μg and 6 μg) were prepared in phosphate-buffered saline (PBS) and aluminium hydroxide adjuvant.