Epitope Presentation of Dengue Viral Envelope Glycoprotein Domain III on Hepatitis B Core Protein Virus-Like Particles Produced in Nicotiana benthamiana.

Ee Leen Pang,William M Rosenberg,Alex Ramirez,Kok-Song Lai,Hadrien Peyret,Hwei-San Loh,Chee-Mun Fang,George P Lomonossoff

doi:10.3389/fpls.2019.00455

Abstract

Dengue fever is currently ranked as the top emerging tropical disease, driven by increased global travel, urbanization, and poor hygiene conditions as well as global warming effects which facilitate the spread of Aedes mosquitoes beyond their current distribution. Today, more than 100 countries are affected most of which are tropical Asian and Latin American nations with limited access to medical care. Hence, the development of a dengue vaccine that is dually cost-effective and able to confer a comprehensive protection is ultimately needed. In this study, a consensus sequence of the antigenic dengue viral glycoprotein domain III (cEDIII) was used aiming to provide comprehensive coverage against all four circulating dengue viral serotypes and potential clade replacement event. Utilizing hepatitis B tandem core technology, the cEDIII sequence was inserted into the immunodominant c/e1 loop region so that it could be displayed on the spike structures of assembled particles. The tandem core particles displaying cEDIII epitopes (tHBcAg-cEDIII) were successfully produced in Nicotiana benthamiana via Agrobacterium-mediated transient expression strategy to give a protein of ∼54 kDa, detected in both soluble and insoluble fractions of plant extracts. The assembled tHBcAg-cEDIII virus-like particles (VLPs) were also visualized from transmission electron microscopy. These VLPs had diameters that range from 32 to 35 nm, presenting an apparent size increment as compared to tHBcAg control particles without cEDIII display (namely tEL). Mice immunized with tHBcAg-cEDIII VLPs showed a positive seroconversion to cEDIII antigen, thereby signifying that the assembled tHBcAg-cEDIII VLPs have successfully displayed cEDIII antigen to the immune system. If it is proven to be successful, tHBcAg-cEDIII has the potential to be developed as a cost-effective vaccine candidate that confers a simultaneous protection against all four infecting dengue viral serotypes.

Highlights

The alarming rise of dengue epidemics has been highlighted to affect over 40% of the world population (Brady et al, 2012)
N. benthamiana leaves were collected on 6 dpi and analyzed by Western blotting technique to check for expression
Western profiles indicated that the tHBcAgcEDIII proteins had been expressed well in N. benthamiana infiltrated with pEAQ-HT::tHBcAg-cEDIII (Figure 2), based on the band at around 54 kDa which was not present in the pEAQ-HT-infiltrated plant leaf sample

Summary

Introduction

The alarming rise of dengue epidemics has been highlighted to affect over 40% of the world population (Brady et al, 2012). Classified under the Flaviviridae virus family, dengue virus (DENV) is a single-stranded, positive-sense nonsegmented RNA virus with 40–50 nm enveloped particles (Guzman et al, 2010). The C protein encapsidates the viral RNA to form nucleocapsid particles whereas the prM assists the folding of surface-exposed E glycoprotein (Whitehead et al, 2017). The incidence rate has expanded 500-fold, spreading from Southeast Asia to the Americas and Western Pacific within just a-half century (Pang and Loh, 2016). It was reported that 500,000 people were hospitalized with severe dengue and approximately 2.5% of them would succumb to the disease (World Health Organization [WHO], 2017). The reported figure may be under-estimated due to the passive surveillance system adopted by many countries (Runge-Ranzinger et al, 2014)

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