Overexpression and oral immunogenicity of a dengue antigen transiently expressed in Nicotiana benthamiana

The feasibility of a safe and cost-effective oral dengue vaccine approach was investigated. Consensus domain III of the dengue virus (DENV) serotypes 1–4 envelope glycoprotein was fused with cholera toxin B subunit (CTB–cEDIII) and cloned into TMV-based 3′ pro-module for high level expression. The construct was co-infiltrated with TMV-based 5′ pro-module and Integrase module into Nicotiana benthamiana leaves using vacuum agroinfiltration. CTB–cEDIII fusion protein directed to endoplasmic reticulum by C-terminal SEKEDL sequence was expressed at up to 8.4% of total soluble protein at 5 days-post infiltration (dpi). After oral immunization of mice with the transiently expressed plant-derived CTB–cEDIII, high titers of serum IgG and sIgA against both cEDIII and CTB were observed, peaking at week four. Sera from immunized mice reacted with each of the four DENV serotype-specific envelope proteins domain III. These results show that (i) the TMV-based pro-vector system is suitable for high-level expression of antigen in plant cells and (ii) the CTB fusion with cEDIII could be exploited as an oral vaccine candidate for induction of both systemic and mucosal immune responses to DENV.