Chimeric Virus-Like Particles of Prawn Nodavirus Displaying Hepatitis B Virus Immunodominant Region: Biophysical Properties and Cytokine Response

Abstract

Hepatitis B is a major global health challenge. In the absence of an effective treatment for the disease, hepatitis B vaccines provide protection against the viral infection. However, some individuals do not have positive immune responses after being vaccinated with the hepatitis B vaccines available in the market. Thus, it is important to develop a more protective vaccine. Previously, we showed that hepatitis B virus (HBV) 'a' determinant (aD) displayed on the prawn nodavirus capsid (Nc) and expressed in Spodoptera frugiperda (Sf9) cells (namely, Nc-aD-Sf9) self-assembled into virus-like particles (VLPs). Immunisation of BALB/c mice with the Nc-aD-Sf9 VLPs showed significant induction of humoral, cellular and memory B-cell immunity. In the present study, the biophysical properties of the Nc-aD-Sf9 VLPs were studied using dynamic light scattering (DLS) and circular dichroism (CD) spectroscopy. Enzyme-linked immunosorbent assay (ELISA) was used to determine the antigenicity of the Nc-aD-Sf9 VLPs, and multiplex ELISA was employed to quantify the cytokine response induced by the VLPs administered intramuscularly into BALB/c mice (n = 8). CD spectroscopy of Nc-aD-Sf9 VLPs showed that the secondary structure of the VLPs predominantly consisted of beta (β)-sheets (44.8%), and they were thermally stable up to ~52 °C. ELISA revealed that the aD epitope of the VLPs was significantly antigenic to anti-HBV surface antigen (HBsAg) antibodies. In addition, multiplex ELISA of serum samples from the vaccinated mice showed a significant induction (p < 0.001) of IFN-γ, IL-4, IL-5, IL-6, IL-10, and IL-12p70. This cytokine profile is indicative of natural killer cell, macrophage, dendritic cell and cytotoxic T-lymphocyte activities, which suggests a prophylactic innate and adaptive cellular immune response mediated by Nc-aD-Sf9 VLPs. Interestingly, Nc-aD-Sf9 induced a more robust release of the aforementioned cytokines than that of Nc-aD VLPs produced in Escherichia coli and a commercially used hepatitis B vaccine. Overall, Nc-aD-Sf9 VLPs are thermally stable and significantly antigenic, demonstrating their potential as an HBV vaccine candidate.

Keywords: ELISA; Sf9 cells; capsid protein; circular dichroism; cytokine; hepatitis B virus; prawn nodavirus; virus-like particles; ‘a’ determinant.

Figure 1

Construction of recombinant bacmid DNA harbouring the coding regions of nodavirus capsid (Nc) protein and HBV ‘a’ determinant (aD). (A) Restriction enzyme digestion of recombinant pFastBac HT C plasmid harbouring the Nc-aD gene insert. Lane 1: the undigested recombinant plasmid; lane 2: recombinant plasmid linearised with XhoI yielded an ~6 kb product; lane 3: the recombinant plasmid digested with BglI and HindIII yielded fragments with sizes 2508 bp, 1600 bp, 1258 bp and 626 bp. (B) A schematic representation of the recombinant bacmid DNA harbouring the Nc-aD gene insert. (C) Western blot of Sf9 cells transfected with the recombinant bacmid bearing the Nc-aD gene shows a protein band of ~52 kDa, corresponding to the expected size of the Nc-aD protein. (D) Primary structure of the chimeric protein consisting of MrNV capsid protein (Nc) fused to HBV ‘a’ determinant (aD). The protein consists of the HBV aD fused to the C-terminal end of the Nc. The chimeric protein is flanked on both sides by 6x histidine (His) tags.

Figure 2

SDS-PAGE and western blot analysis of Nc-aD-Sf9 purified using sucrose density gradient ultracentrifugation. (A) SDS-PAGE and (B) Western blotting of cell lysate separated on 10–40% (w/v) sucrose density gradient ultracentrifugation with anti-His monoclonal antibody. This antibody was used because the chimeric protein contains His-tags at its N- and C-terminal ends. Arrows indicate the ~52 kDa Nc-aD-Sf9 protein band. Protein fractions in lanes 15 to 18 were pooled, dialysed and concentrated because they contained less impurities than samples in fractions 1 to 14. (C) SDS-PAGE of the concentrated Nc-aD-Sf9 protein (D) Western blotting of the concentrated Nc-aD protein with anti-His monoclonal antibody. Lane M: Molecular mass markers in kDa.

Figure 3

Dynamic light scattering (DLS) analysis of the Nc-aD-Sf9 VLPs. The vertical dotted line intersecting the red peak represents the mean diameter of the Nc-aD-Sf9 VLPs, 56.4 nm. DLS analysis showed that the largest population of the VLPs (13.1%) had a diameter of ~43.8 nm.

Figure 4

Circular dichroism (CD) spectra of the chimeric Nc-aD VLPs from wavelengths 240 nm to 190 nm. (A) The CD spectra showed the α-helix structure for Nc-aD-Sf9 as indicated by the negative band at wavelength 208 nm (arrow head) and a positive band at wavelength 192 nm (arrow head). The CD spectra showed a β-sheet structure for Nc-aD-E. coli as indicated by the negative band at wavelength 216 nm (arrow head) and a positive band at wavelength 195 nm (arrow head). (B) Thermal denaturation spectra for Nc-aD VLPs. The Nc-aD-Sf9 VLPs were stable from 20 to 51.9 °C (absorbance 0.844-0.849) with a Tm of 56.2 °C. Nc-aD-E. coli VLPs were stable from 20 to 45 °C (absorbance 0.859-0.870) with a Tm of 68.6 °C.

Figure 5

Antigenicity of the chimeric Nc-aD VLPs. The 96 well plates were coated with 0.5, 1, 2, 5, 10, 15 and 20 µg/mL of Nc-aD-Sf9, the negative controls (BSA, Nc-Sf9 and Nc-E. coli), and the positive control (Nc-aD-E. coli). The symbol * indicates p < 0.001 when compared to the BSA negative control. The error bars represent the mean (±) standard deviation. BSA: bovine serum albumin, Nc-Sf9: Nc produced in Sf9 cells, Nc-E. coli: Nc produced in E. coli, Nc-aD-Sf9: Nc-aD produced in Sf9 cells, and Nc-aD-E. coli: Nc-aD produced in E. coli.

Figure 6

Cytokine quantification in sera of immunised mice. For each cytokine quantified, multiplex ELISA was performed using sera from mice in the test group (Nc-aD-Sf9), the positive control groups (Nc-aD-E. coli and Engerix B) and the negative control groups (Buffer only, Buffer and alum, Nc-Sf9 and Nc-E. coli). The symbols * and ** indicate p < 0.001 and p < 0.0001, respectively, when cytokine concentrations from the test group (Nc-aD-Sf9) and positive control groups (Nc-aD-E. coli and Engerix B) are compared to that of the ‘Buffer only’ negative control group. The error bars represent the mean (±) standard deviation. Nc-aD-Sf9: Nc-aD produced in Sf9 cells, Nc-aD-E. coli: Nc-aD produced in E. coli, Nc-Sf9: Nc produced in Sf9 cells, and Nc-E. coli: Nc produced in E. coli.