Plant-based vaccine development: tobacco chloroplast engineering for Canine distemper virus antigen expression

Abstract

Canine distemper virus (CDV) causes a highly contagious and potentially lethal disease in domestic dogs and other carnivores. Currently, a safer and more effective CDV vaccine is required to control the increasing incidence of the disease. In this study, we produced the nucleocapsid protein (NP) from an Argentinian wild-type strain (Arg26) of CDV in transplastomic tobacco plants. The NP expression cassette was introduced into the plastid genome by biolistic transformation. Stable integration of NP transgene and homoplasmy were confirmed by Southern blot analysis. No phenotypic differences were observed between NP and WT plants. NP was mainly expressed in its soluble form and was detected in leaves of different ages. Although NP was partially degraded, full-length recombinant protein accumulated at high levels with a concentration of 0.64 mg/g fresh weight in mature leaves, equivalent to 4.82% of total proteins. NP was purified using nickel affinity chromatography and the protein yield was 0.09 mg/g fresh weight. Finally, the immunogenicity of NP was assessed in a heterologous subcutaneous prime-oral boost immunization. Mice primed with chloroplast-purified NP and boosted with NP tissue suspensions without enrichment, purification or addition of mucosal adjuvants elicited a strong systemic humoral immune response, characterized by a Th2 IgG isotypes profile. Our findings demonstrate that transplastomic plants expressing high levels of the NP antigen are a promising approach for future development of an affordable subunit vaccine against CDV.