Pichia pastoris-expressed dengue virus type 2 envelope domain III elicits virus-neutralizing antibodies

A tetravalent dengue vaccine that can protect against all four serotypes of dengue viruses is a global priority. The host-receptor binding, multiple neutralizing epitope-containing carboxy-terminal region of the dengue envelope protein, known as domain III (EDIII), has emerged as a promising subunit vaccine antigen. One strategy to develop a tetravalent dengue subunit vaccine envisages mixing recombinant EDIIIs, corresponding to the four dengue virus serotypes. Towards this objective, a recombinant clone of the methylotrophic yeast Pichia pastoris, harboring the EDIII gene of dengue virus type 2 (EDIII-2) for its intracellular expression, was created. Recombinant EDIII-2 protein, expressed by this clone was purified to near homogeneity by affinity chromatography, with final yields of >50mg/l culture. Groups of Balb/c mice were immunized with this protein, separately formulated in two adjuvants, alum and montanide ISA 720. The EDIII-2 antigen, formulated in either adjuvant, elicited high levels of neutralizing antibodies to dengue virus type 2 in mice as analyzed by Plaque Reduction Neutralization Test (PRNT). This study demonstrates the feasibility of using P. pastoris to produce EDIII antigens capable of eliciting potent virus-neutralizing antibodies.