Capto core 700

Recombinant baculovirus encoding untagged full length BKV VP1 or JCV VP1 were used to infect Sf9 insect cells in suspension at 1.5 × 10⁶ cells/mL, the cells were incubated at 27°C with shaking at 120 RPM for 72 hr then harvested by centrifugation and stored at −80°C. Cells were re-suspended in lysis buffer (20 mM Tris-HCl pH 7.5, 1 M NaCl, 1X Roche EDTA free protease inhibitor cocktail) at a ratio of 10 mL lysis buffer per gram of cell pellet and lysed by sonication on ice then the lysate was centrifuged at 16,000 x g for 20 min at 4°C. The supernatant was layered onto 3 mL of 40% glucose made up in 1X PBS and centrifuged at 116,000 x g for 2.5 hr at 4°C. Dissolved pellet in IEX buffer A (25 mM Tris-HCl pH 8.0, 25 mM NaCl) and loaded onto a 10 mL Sepharose Q-HP (GE) column equilibrated in IEX buffer A. Column was washed with 3 CV IEX buffer A and eluted with a linear NaCl gradient from 25 mM to 700 mM NaCl across 25 CV. Pooled peak fractions and loaded onto 10 mL Capto Core 700 (GE) resin equilibrated in SEC buffer (25 mM Tris-HCl pH 8.0, 100 mM NaCl) collecting the flow-through fraction. Loaded onto a Sephacryl S500 26/60 column (GE) and collected peak fractions, concentrated with a 100,000 MWCO Amicon concentrator.

HPV 6, −11, −16, −18, −26, −31, −33, −35, −39, −45, −51, −52, −53, −56, −58, −59, −66, −68, −69, and −70 pseudoviruses (PsVs) were produced in 293FT cells^{65 (link)–67 (link)}. The HPV16 L1 and L2 expression vector, and the pN31-EGFP used in the experiment were kindly provided by Dr. J. T. Schiller^{34 (link)}. 293FT cells were harvested at 72 h after transfection, lysed in a Dulbecco’s PBS-Mg solution cell lysis buffer comprising 0.5% Brij58 (Sigma-Aldrich), 0.2% Benzonase (Merck), 0.2% PlasmidSafe ATP-Dependent DNase (EPICENTRE Biotechnologies), and incubated at 37 °C for 24 h. Afterwards, 5 M NaCl solution was added to the samples to extract the cell lysate. The tissue culture infective dose (TCID₅₀) of the supernatant was then measured to determine the titers of the PsVs, calculated according to the classical Reed-Muench method^{68 (link)}. For chPsVs, the mutant pAAV-HPV16L1-C175A and pAAV-HPV52L1-C428A plasmids were mixed in equal proportions for transfection into 293FT cells. These PsVs were purified using a Capto Core 700 (GE Healthcare, America). After purification, the PsVs were analyzed by TEM.

Purified inactivated ZIKV vaccine was produced at the virus vaccine pilot facility at Bharat Biotech, Hyderabad. Virus inoculation in Vero cells was carried out at a standardized MOI of 0.01 PFU/cell and the virus was harvested at 5–6 days post-inoculation. The virus harvest was clarified with 0.45 micron filter using a tangential flow filtration (TFF) system and purified on Capto^TM Core 700 (GE Healthcare Life Sciences, Pittsburg, USA) column, concentrated by TFF and inactivated with 0.04% of formalin for 7 days at 22 °C in the presence of added stabilizers. Formalin was removed by diafiltration. Residual formalin content in the inactivated vaccine was estimated with acetylacetone reagent as described in the monograph 2.3.20, IP, 7^th edition, p99, 2014 and was below the specified limit of 0.01% w/v (WHO, TRS 963, Annexe 1, 2011). Inactivation was considered as complete when no infectious particle could be detected by plaque assay after three serial amplifications of the sample (1:3 diluted with DMEM) in vitro in Vero cells for 7 days. The purified inactivated ZIKV antigen was formulated with 0.25 mg aluminum per dose using aluminum hydroxide (2% Alhydrogel, Brenntag Biosector, Denmark).

sMDCK cells were cultured in a 40 L bioreactor (Bio-510, NBS, USA) for 72 h and inoculated with virus prepared in SPF eggs at 33 ± 1°C. To remove host cell protein and DNA, the harvest was clarified by depth filtration (PALL, USA), treated with benzonase, and purified by CaptoTM Core 700 (GE, USA) and CaptoTM Q (GE, USA). β-Propiolactone (Serva, Germany) was added to inactivate the virus. The solution (containing 128 μg/mL hemagglutinin [HA]) was stored at 4°C in phosphate-buffered saline (PBS). The placebo in this study was the PBS that was used to dilute the solution.