Preventative Vaccines for Zika Virus Outbreak: Preliminary Evaluation

Kim, Eun; Erdos, Geza; Huang, Shaohua; Kenniston, Thomas; Falo, Louis D.; Gambotto, Andrea

For construction of pAd/ZIKV-Efl, the gene encoding human secretory signal peptide hidden Markov model (SP-HMM, MWWRLWW LLLLLLLLWPMVWA), the extracellular portion of the ZIKV strain BeH815744 envelope gene (GenBank KU365780, defined as amino acids 216-794 of the polyprotein), BamH I-linked T4 fibritin foldon trimerization domain (GSGYIPEAPRDGQAYVRKDGEWVLLSTFL), Tobacco Etch Virus Protease (Tp) (ENLYFEG), and six histidine tag were codon-optimized for optimal expression in mammalian cells using the UpGene codon optimization algorithm (Gao et al., 2004) . pAd/ZIKV-Efl was generated by subcloning the codon-optimized ZIKV-Efl gene into the shuttle vector, pAd (GenBank U62024) at SalI/NotI sites. Subsequently, replication-defective adenovirus 5, designated as Ad5.ZIKV-Efl, was generated by loxP homologous recombination. Moreover, we also purified recombinant proteins named ZIKV-rEfl from the supernatant using His60 Ni Superflow Resin (Clontech) under native conditions to be used as a subunit vaccine. Briefly, the supernatant of Human Embryonic Kidney (HEK) 293 cells infected with Ad5.ZIKV-Efl was heatinactivated at 65°C for 30 min and mixed with the same volume of binding buffer (40 mM imidazole, 900 mM NaCl, 100 mM sodium phosphate, pH 7.4). His60 Ni Superflow Resin (Clontech) previously equilibrated with equilibration buffer (20 mM imidazole, 500 mM NaCl, 50 mM sodium phosphate, pH 7.4) was added and the mixture was incubated overnight at 4°C with rotation. The next day, the settled resin mix was packed into an empty column. The column was washed with 10 ml of equilibration buffer three times followed by 10 ml of wash buffer (40 mM imidazole, 500 mM NaCl, 50 mM sodium phosphate, pH 7.4) three times and eluted in 10 ml of elution buffer (500 mM imidazole, 500 mM NaCl, 50 mM sodium phosphate, pH 7.4). The eluate was concentrated and desalted with phosphate buffered saline (PBS) in an Amicon Ultra-15 filter (Millipore). This desalting step was repeated three times. The concentrations of the purified recombinant ZIKV-Efl were determined by the Bradford assay using bovine serum albumin (BSA) as a protein standard.