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

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.

Keywords: dengue envelope glycoprotein; dengue vaccine; envelope glycoprotein domain III; epitope display; hepatitis B core antigen; tandem core technology; virus-like particles.

FIGURE 1

A schematic illustration of the recombinant vector, pEAQ-HT::tHBcAg-cEDIII. The cEDIII gene sequence was specifically inserted into the Core II c/el loop. The pEAQ-HT vector used in this study was based on Sainsbury et al. (2009), and the amino acid sequence of tHBcAg-cEDIII is shown in the Supplementary Material.

FIGURE 2

Expression profiles of tHBcAg-cEDIII protein in N. benthamiana analyzed at 6 dpi, which was predominantly obtained in insoluble form. Immunoblot detection of tHBcAg-cEDIII proteins at ∼54 kDa in size (black arrow) was performed using anti-HBcAg monoclonal antibody. Lane M: SeeBlue^® Plus2 Pre-Stained Standard; Lane 1: SP extracted from pEAQ-HT::tHBcAg-cEDIII-infiltrated leaf disks; Lane 2: IP extracted from pEAQ-HT::tHBcAg-cEDIII-infiltrated leaf disks; Lane 3: SP extracted from pEAQ-HT-infiltrated leaf disks; Lane 4: IP extracted from pEAQ-HT-infiltrated leaf disks.

FIGURE 3

Kinetic study of pEAQ-HT::tHBcAg-cEDIII-infiltrated N. benthamiana. (A) Physical appearances of the representative infiltrated leaf from 1 to 9 dpi. (B) Expression profiles of the corresponding tHBcAg-cEDIII proteins (∼54 kDa; black arrow) which were immunoblotted with anti-HBcAg monoclonal antibody. Lane M: SeeBlue^® Plus2 Pre-Stained Standard; Lanes 1–9: SP extracted from infiltrated N. benthamiana from 1 to 9 dpi, correspondingly. It was decided that 8 dpi to be the suitable harvest time for soluble tHBcAg-cEDIII protein.

FIGURE 4

Sucrose cushion and Nycodenz gradient fractionation of proteins extracted from pEAQ-HT::tHBcAg-cEDIII-infiltrated leaves. (A) Western blot profiles showing the desired tHBcAg-cEDIII VLPs (black arrow) were successfully detected using anti-HBcAg monoclonal antibody. Lane M: SeeBlue^® Plus2 Pre-Stained Standard; Lane 1: Clarified plant lysate; Lane 2: 70% sucrose fraction; Lane 3: Interface fraction. (B) A single grayish band along the Nycodenz gradient that can be visualized. (C) Western blot profiles of Nycodenz gradient fractions detected using anti-DENV 1–4 monoclonal antibody. Lane 1: 60% Nycodenz fraction; Lane 2: 50% Nycodenz fraction; Lane 3: 40% Nycodenz fraction; Lane 4: 30% Nycodenz fraction; Lane 5: 20% Nycodenz fraction. As indicated by the black arrow, desired tHBcAg-cEDIII VLPs were successfully recovered from the infiltrated N. benthamiana.

FIGURE 5

Electron micrographs showing the purified tandem core tHBcAg-cEDIII and tEL VLPs that were negatively stained with 2% (v/v) uranyl acetate. (A) tHBcAg-cEDIII VLPs visualized at 50,000× magnification. (B) Negative control tEL VLPs visualized at 50,000× magnification. Scale bar represents 100 nm. The chimeric tHBcAg VLPs with cEDIII epitopes had successfully assembled into viral particles.

FIGURE 6

Evaluation of cEDIII-specific IgG antibody responses in BALB/c mice by using ELISA. (A) cEDIII-specific IgG antibody levels of mouse sera at the dilutions from 1:100 to 1:3,200 following 4- and 9-week post-immunizations. (B) Comparison of mouse antibody responses following 4- and 9-week post-immunizations as tested by ELISA at the dilution of 1:100. Bars represent the mean ± standard error of mean and the graphs are representative of two independent animal immunization experiments (n = 2). Student’s t-test was used to determine the significance levels (p ≤ 0.001 denotes as ^∗∗ and p ≤ 0.0001 denotes as ^∗∗∗). Note that a indicates the comparison between the immunization groups of mice receiving cEDIII (positive control) against empty VLPs (tEL; negative control); b indicates the comparison between tHBcAg-cEDIII and tEL VLPs and c indicates the comparison between cEDIII and tHBcAg-cEDIII groups.