Site-specific integration of canine distemper virus hemagglutinin protein in Expi293F cell lines contributes to protective neutralizing antibody responses in mice and dogs.

Despite worldwide applications of the modified live vaccines (MLV), canine distemper virus (CDV) remains a significant threat to highly susceptible wildlife animals and domestic dogs. Thus, to develop a novel completely safe vaccine is necessary. In this study, CDV hemagglutinin (H) protein as antigen target was expressed in a transient transfected Expi293F cells sandwiched with signal peptides and Twin strep II or Fc tag. The signal peptides HSA and mouse Ig kappa significantly produced the most secretory expressions of CDV H protein compared to the signal peptides TRY, AP, IL-2 and INS. Fc tag impaired CDV H protein secretion by dimer formation. The Expi293F cell pools harboring site-specific integration of the CDV H gene and GFP-T2A-BleoR gene in AAVS1 safe-harbor locus were generated by a dual- or single- sgRNA guided dCas9-piggyBac system with Zeocin screening. The dual-sgRNA provided higher transposition efficiency with 10.59 % or 10.32 % GFP+ cells than the single-sgRNA. Two monoclonal Expi293F cell lines E6 and G10 sorted by flow cytometry could stably produce 2.8 g per liter of CDV H protein in supernatant with a purity over 95 %. The secreted CDV H protein induced robust, dose-dependent neutralizing antibody responses in both mice and dogs. A 2 mg/kg dose of CDV H protein protected dogs 100 % survival against the virulent CDV HBF-1 challenge. This establishment of a transposon-mediated platform for stable expression of CDV H antigen protein in Expi293F cells demonstrates the incredible plasticity and provides a versatile framework for future subunit vaccine development.