β propiolactone

Vero cells were obtained from the American Type Culture Collection (ATCC) and cultured in Dulbecco Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific) and 1% penicillin and streptomycin at 5% CO₂, 37 ℃. Chikungunya/human/China/GD134/2010 (GenBank Accession: HQ846359) was isolated from the serum of an infected female patient at Guangdong Provincial Center for Disease Control and Prevention. Zika/human/China/GD01/2016 (GenBank Accession: KU740184) was isolated from Guangdong inbound passengers. All experiments involving the CHIKV and ZIKA authentic virus were conducted in biosafety level 2 or higher laboratories. Virus virions were inactivated using β-propiolactone (Sigma-Aldrich) with a concentration of 1:2000 at 4 ℃ for 24 h. Complete inactivation of the virus was confirmed by the lack of replication in a Vero infection experiment. Supernatants containing virus particles were concentrated using PEG-it (SBI Biosciences) overnight at 4 °C and resuspended in phosphate-buffered solution (PBS, pH7.4) for further use.

Swine influenza vaccine strains (CA09, MN21, and GA19 viruses) grown in MDCK cells were inactivated by β-propiolactone (Sigma-Aldrich, St. Luis, MO) treatment as previously described (27 (link)). Sufficient virus inactivation was demonstrated by failure of virus replication in two-serial passages in MDCK cells (28 (link)). Following inactivation, vaccine preparations were formulated with a commercial mineral oil-in-water (O/W) emulsion (Montanide™ ISA 15 VG), which served as the adjuvant solution. WIV vaccine formulations were mixed at a volume-to-volume ratio of 85:15 virus suspension to adjuvant, prepared under a low shear rate, and stored at 4°C according to the manufacturer’s instructions, 1 day prior to each vaccination. Monovalent vaccine formulations (CA09, MN21, and GA19) contained prior to adjuvant addition approximately 128 hemagglutination units (HAU)/mL of WIV while bivalent vaccine formulations (CA09-MN21) contained approximately 256 HAU/mL (128 HAU of each WIV). In assessing the HAU of the vaccine virus stocks, we conducted a hemagglutination assay for each virus, as previously described (29 (link)). After calculating the HA units within the stock, a subsequent dilution was performed, to achieve a final HA concentration of either 128 or 256 HA units/mL.

Benzonase^® (Sigma, St. Louis, MO, USA) was added to the bioreactor at 10 units/mL and incubated for 1 h before the clarification step. Depth filtration was evaluated as an alternative to the commonly used centrifugation as a cell removing step. From each harvest, 1 L of the harvested material after Benzonase^® treatment was centrifuged at 4000× g for 10 min and another 1 L was filtered through MilliStack D0HC^® (Millipore, Rockville, MD, USA) depth filter at 7.66 mL/min in sterile conditions as previously described [28 (link),29 (link)]. Before filtration, the depth filter was equilibrated with the filter buffer (50 mM HEPES, 300 mM NaCl at pH 7.2). The clarified material was supplemented with HEPES (Sigma, St. Louis, MO, USA) to a final concentration of 70 mM to maintain the pH at 7.2 during the inactivation. The material was inactivated by adding 0.1% (v/v) β-propiolactone (Sigma, St. Louis, MO, USA) and then stirred for 24 h at 4 °C. The inactivated supernatant was frozen at −80 °C and was thawed upon loading to the AKTA^TM Avant 25 system (Cytiva, Marlborough, MA, USA) for evaluation of the ion exchange chromatography membranes.