Dengue envelope-based \u2018four-in-one\u2019 virus-like particles produced using Pichia pastoris induce enhancement-lacking, domain III-directed tetravalent neutralising antibodies in mice

Ravi Kant Rajpoot,Upasana Arora,Navin Khanna,Rahul Shukla,Sathyamangalam Swaminathan

doi:10.1038/s41598-018-26904-5

Abstract

Dengue is a significant public health problem worldwide, caused by four antigenically distinct mosquito-borne dengue virus (DENV) serotypes. Antibodies to any given DENV serotype which can afford protection against that serotype tend to enhance infection by other DENV serotypes, by a phenomenon termed antibody-dependent enhancement (ADE). Antibodies to the viral pre-membrane (prM) protein have been implicated in ADE. We show that co-expression of the envelope protein of all four DENV serotypes, in the yeast Pichia pastoris, leads to their co-assembly, in the absence of prM, into tetravalent mosaic VLPs (T-mVLPs), which retain the serotype-specific antigenic integrity and immunogenicity of all four types of their monomeric precursors. Following a three-dose immunisation schedule, the T-mVLPs elicited EDIII-directed antibodies in mice which could neutralise all four DENV serotypes. Importantly, anti-T-mVLP antibodies did not augment sub-lethal DENV-2 infection of dengue-sensitive AG129 mice, based on multiple parameters. The ‘four-in-one’ tetravalent T-mVLPs possess multiple desirable features which may potentially contribute to safety (non-viral, prM-lacking and ADE potential-lacking), immunogenicity (induction of virus-neutralising antibodies), and low cost (single tetravalent immunogen produced using P. pastoris, an expression system known for its high productivity using simple inexpensive media). These results strongly warrant further exploration of this vaccine candidate.

Highlights

Dengue is a mosquito-borne viral disease which threatens nearly half the world population[1]
As we already had monovalent[16,17,18,19] and bivalent[20] P. pastoris clones at hand, we created a tetravalent P. pastoris clone, capable of co-expressing all four dengue virus (DENV) E proteins, as a prelude to making head-to-head comparison of the immunogenicity of the resultant tetravalent mosaic VLPs (mVLPs) (T-mVLPs) with that of M-virus-like particles (VLPs) mix and B-mVLP mix. The purpose of this comparative analysis was to ascertain if the T-mVLPs would retain the antigenic integrity and immunogenicity of their four monovalent precursors and serve as a single tetravalent dengue immunogen
We found that pre-incubating the anti-T-mVLP antisera with recombinant EDIII-maltose-binding protein (MBP) fusion proteins corresponding to the four DENV serotypes resulted in large losses in homotypic, but not heterotypic, neutralising antibodies (nAbs) titres (Fig. 6d)

Summary

Introduction

Dengue is a mosquito-borne viral disease which threatens nearly half the world population[1]. In the light of the role of anti-prM antibodies in ADE, prM is an undesirable component of these VLPs. A serendipitous discovery we made a few years ago showed that the DENV-2 E ectodomain (referred to hereafter as ‘E2’), expressed in the absence of prM, in P. pastoris, possessed the inherent ability to self-assemble into highly immunogenic VLPs16. The inherent safety (lack of prM), immunogenicity (the capacity to elicit virus-neutralising anti-EDIII antibodies), and the low cost of production (P. pastoris expression system) make the E-based VLPs ideally suited to development of tetravalent dengue vaccine candidates. We describe the co-expression and co-purification strategy adopted to obtain these tetravalent mVLPs (T-mVLPs) and present data on the comparison of their immunogenicity with that of two other tetravalent E-based VLP formulations: a physical mixture containing four monovalent VLPs (M-VLP mix) and a physical mixture of two bivalent mVLPs (B-mVLP mix)

Methods

Results

Conclusion