Marked enhancement of the immunogenicity of plant-expressed IgG-Fc fusion proteins by inclusion of cholera toxin non-toxic B subunit within the single polypeptide.

Immunoglobulin G (IgG)-based fusion proteins have been widely exploited as a potential vaccine delivery platform but in the absence of exogenous adjuvants, the lack of robust immunity remains an obstacle. Here, we report on a key modification that overcomes that obstacle. Thus, we constructed an IgG-Fc vaccine platform for dengue, termed D-PCF, which in addition to a dengue antigen incorporates the cholera toxin non-toxic B subunit (CTB) as a molecular adjuvant, with all three proteins expressed as a single polypeptide. Following expression in Nicotiana benthamiana plants, the D-PCF assembled as polymeric structures of similar size to human IgM, a process driven by the pentamerization of CTB. A marked improvement of functional properties in vitro and immunogenicity in vivo over a previous iteration of the Fc-fusion protein without CTB [1] was demonstrated. These include enhanced antigen presenting cell binding, internalization and activation, complement activation, epithelial cell interactions and ganglioside binding, as well as more efficient polymerization within the expression host. Following immunization of mice with D-PCF by a combination of systemic and mucosal (intranasal) routes, we observed robust systemic and mucosal immune responses, as well as systemic T cell responses, significantly higher than those induced by a related Fc-fusion protein but without CTB. The induced antibodies could bind to the domain III of the dengue virus envelope protein from all four dengue serotypes. Finally, we also demonstrated feasibility of aerosolization of D-PCF as a prerequisite for vaccine delivery by the respiratory route.