Dengue virus-like particles mimic the antigenic properties of the infectious dengue virus envelope

Abstract

Background: The 4 dengue serotypes (DENV) are mosquito-borne pathogens that are associated with severe hemorrhagic disease. DENV particles have a lipid bilayer envelope that anchors two membrane glycoproteins prM and E. Two E-protein monomers form head-to-tail homodimers and three E-dimers align to form "rafts" that cover the viral surface. Some human antibodies that strongly neutralize DENV bind to quaternary structure epitopes displayed on E protein dimers or higher order structures forming the infectious virus. Expression of prM and E in cell culture leads to the formation of DENV virus-like particles (VLPs) which are smaller than wildtype virus particles and replication defective due to the absence of a viral genome. There is no data available that describes the antigenic landscape on the surface of flavivirus VLPs in comparison to the better studied infectious virion.

Methods: A large panel of well characterized antibodies that recognize epitope of ranging complexity were used in biochemical analytics to obtain a comparative antigenic surface view of VLPs in respect to virus particles. DENV patient serum depletions were performed the show the potential of VLPs in serological diagnostics.

Results: VLPs were confirmed to be heterogeneous in size morphology and maturation state. Yet, we show that many highly conformational and quaternary structure-dependent antibody epitopes found on virus particles are efficiently displayed on DENV1-4 VLP surfaces as well. Additionally, DENV VLPs can efficiently be used as antigens to deplete DENV patient sera from serotype specific antibody populations.

Conclusions: This study aids in further understanding epitopic landscape of DENV VLPs and presents a comparative antigenic surface view of VLPs in respect to virus particles. We propose the use VLPs as a safe and practical alternative to infectious virus as a vaccine and diagnostic antigen.

Keywords: Dengue virus; Epitopes; Serum depletions; VLP.

Fig. 1

Expression and characterization of DENV1–4 VLPs. Purified virus and VLPs were subjected to SDS-PAGE and analyzed with (a) CBB and (b) WB using anti-E 1 M7 and anti prM 2G3 mAbs. E-dimers (E^D), E-monomers (E^M), prM and capsid (c) proteins are indicated. The prM/E ratio is indicated below panel B and is determined using ImageJ software

Fig. 2

TEM analysis of DENV1–4 VLPs. a Particle shape and distribution was analyzed by TEM using negative staining. b VLP size distribution was determined by measuring the diameter of ~ 250 particles of each serotype

Fig. 3

Epitope analysis of DENV1–4 VLPs. a-d DENV VLPs and virus particles were subjected to a large panel of human and mouse derived, serotype specific (grey bars) or cross-reactive mAbs (black bars) that recognize epitopes of varying complexity (Table 1) by antigen capture ELISA. Quaternary epitope recognizing (Q) mAbs and binding regions are indicated

Fig. 4

Relative epitope binding on DENV virus vs VLPs. To demonstrate that certain epitopes are better represented on virus particles or VLPs, binding of each mAb within every DENV serotype was normalized to 1F4 (DENV1), 2D22 (DENV2), 5 J7 (DENV3) and 5H2 (DENV4). The normalized binding of each mAb to VLPs was subtracted from the normalized binding to purified virus, resulting in relative epitope binding (REB). Quaternary epitope recognizing (Q) mAbs and binding regions are indicated

Fig. 5

DENV infected patient sera is efficiently depleted by DENV VLPs. Purified DENV particles or DENV VLPs were coated onto magnetic beads and incubated with convalescent patient sera. BSA was used for the depletion handling control. a DENV specific IgG levels for all depletion samples were determined by ELISA. b DENV specific neutralizing antibody titers were analyzed by a Vero-cell flow cytometry based neutralization assay. Neutralizing titers are expressed as Neut₅₀ values indicating the serum dilution where 50% of the virus is neutralized