Humoral immune response induced with dengue virus-like particles serotypes 1 and 4 produced in silkworm

Abstract

Dengue is an arboviral disease, which threatens almost half the global population, and has emerged as the most significant of current global public health challenges. In this study, we prepared dengue virus-like particles (DENV-LPs) consisting of Capsid-premembrane-envelope (CprME) and premembrane-envelope (prME) polypeptides from serotype 1 and 4, which were expressed in the silkworms using Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid. 1CprME, 1prME, 4CprME, and 4prME expressed proteins in hemolymph, and the molecular weight of the purified proteins was 55 kDa, respectively. The purified polypeptides formed spherical Dengue virus-like particles (DENV-LPs) with ~ 30-55 nm in diameter. The immunoelectron microscopy (IEM) images revealed antigens to the surface of a lipid bilayer of DENV-LPs. The heparin-binding assay shows a positive relationship between absorbance and E protein domain III (EDIII) quantity, which is supported by the isothermal titration calorimetry assay. This indicates a moderate binding affinity between heparin and DENV-LP. The high correlation between patient sera and DENV-LP reactivities revealed that these DENV-LPs shared similar epitopes with the natural dengue virus. IgG elicitation studies in mice have demonstrated that DENV-LPs/CPrMEs elicit a stronger immune response than DENV-LP/prMEs, which lends credence to this claim.

Keywords: Capsid; Dengue virus; Dengue virus-like particle; Envelope; Premembrane; Silkworm.

Fig. 1

Construction of recombinant dengue virus structural proteins expressed in this study. a 1CprME, b 1prME, c 4CprME, and d 4prME polypeptides of DENV-1 and 4 were expressed in silkworms as a fusion protein with HA + FLAG tags for DENV-1 and Strep-tag II + FLAG tags for DENV-4

Fig. 2

Western blot of purified (a) 1CprME, (b) 1prME, (c) 4CprME and (d) 4prME protein fractions (Fr). Each protein was purified from silkworm hemolymph using anti-FLAG tag protein purification gel column chromatography. E protein was verified using specific serotype monoclonal antibodies using (e) an anti-E E29 clone for DENV-1 constructs and (f) an anti-E E42 clone for DENV-4 antibodies. The C protein also confirmed using anti-capsid pAb (g)

Fig. 3

Western blot of purified (a) 1CprME, (b) 1prME, (c) 4CprME, and (d) 4prME polypeptides for the cross-reaction test was performed on the DENV-1 and DENV-4 constructs with specific serotype monoclonal antibodies for each serotype

Fig. 4

The purified (a) 1CprME, (b) 1prME, (c) 4CprME, and (d) 4prME polypeptides were immunogold labeled using an anti-E polyclonal antibody and analyzed by IEM. Black spots in A, B, C, and D indicate the immunogold label. Dynamic light scattering (DLS) was used to analyze the size distributions of (e) 1CprME, (f) 1prME, (g) 4CprME and (h) 4prME polypeptides

Fig. 5

a Binding assay of DENV-LPs/1CprME, /1prME, /4CprME, and /4prME to heparin using ELISA. Heparin (1.8 ng) was coated onto each well of an ELISA plate, and each amount of purified protein was used for this ELISA, which was carried out according to the protocol described in the Materials and methods (Welch t-test, *p < 0.05, **p < 0.01, ***p < 0.001). b–e The binding activities between DENV-LPs/1CprME, /1prME, /4CprME, and /4prME toward heparin were examined by ITC

Fig. 6

a The interactions between DENV-LPs/1CprME, /1prME, /4CprME and /4prME with mixed sera from dengue patients [rapid diagnostic test NS1(+)] were investigated (Welch t-test, *p < 0.05, **p < 0.01, ***p < 0.001). b Binding reactions were analyzed using direct ELISA as described. Specific IgG generation by DENV-LPs/1CprME, /1prME, /4CprME and /4prME. BALB/c mice were intraperitoneally immunized with 50 μg of monovalent DENV-LPs/1CprME, /1prME, /4CprME and /4prME. At 0, 2 and 4 weeks, sera were collected and used to test for binding of the specific IgG (Welch t-test, *p < 0.05, **p < 0.01, ***p < 0.001)