Generation of Multiple Arbovirus-like Particles Using a Rapid Recombinant Vaccinia Virus Expression Platform

Abstract

As demonstrated by the 2015 Zika virus outbreak in the Americas, emerging and re-emerging arboviruses are public health threats that warrant research investment for the development of effective prophylactics and therapeutics. Many arboviral diseases are underreported, neglected, or of low prevalence, yet they all have the potential to cause outbreaks of local and international concern. Here, we show the production of virus-like particles (VLPs) using a rapid and efficient recombinant vaccinia virus (VACV) expression system for five tick- and mosquito-borne arboviruses: Powassan virus (POWV), Heartland virus (HRTV), severe fever with thrombocytopenia syndrome virus (SFTSV), Bourbon virus (BRBV) and Mayaro virus (MAYV). We detected the expression of arbovirus genes of interest by Western blot and observed the expression of VLPs that resemble native virions under transmission electron microscopy. We were also able to improve the secretion of POWV VLPs by modifying the signal sequence within the capsid gene. This study describes the use of a rapid VACV platform for the production and purification of arbovirus VLPs that can be used as subunit or vectored vaccines, and provides insights into the selection of arbovirus genes for VLP formation and genetic modifications to improve VLP secretion and yield.

Keywords: Bourbon virus; Heartland virus; Mayaro virus; Powassan virus; arboviruses; recombinant vaccinia virus vectors; severe fever with thrombocytopenia syndrome virus; virus-like particles.

Figure 1

Recombinant VACVs were generated by homologous recombination using the EPPIC VACV plaftorm. A lac-inducible parental VACV (top panel) contains the D6R promoter (P_D6R) followed by the lacO operator sequence (O) and the lacI and EGFP genes under back-to-back synthetic P_E/L promoters. The DNA shuttle vector (middle panel) contains the D6R gene under a tet operator (O₂)-controlled P_D6R promoter, tetR and an arbovirus gene of interest (GOI) under back-to-back P_E/L promoters, and the DsRed gene under a synthetic early/late promoter (P_sel) flanked by regions homologous to the D5R and D6R genes. Homologous recombination (dashed lines) between the shuttle vector (middle panel) and the lac-inducible parental VACV (top panel) generates tet-inducible rVACVs expressing DsRed (bottom panel), which is purified away from the parental virus within a week by switching inducers (IPTG to DOX). Schematics not drawn to scale.

Figure 2

vIND constructs express and secrete heterologous arboviral proteins of interest into cell culture supernatant. (A) Schematic representation of vIND constructs. All vIND constructs contained genes of interest placed in the D5R-D6R locus of VACV. P_E/L: synthetic early/late VACV promoter. P_sel: synthetic early/late VACV promoter. P_D6R: natural promoter of VACV D6R gene. tetR: tet repressor gene. O₂: tet operator sequence. IRES: internal ribosomal entry site sequence from encephalomyocarditis virus. DsRed: red fluorescent protein gene. SS-prM-E: POWV signal sequence, prM, and E gene within a single open reading frame. 3′-ORF: the 3′-end open reading frame of MAYV. GnGc: glycoprotein gene Gn and Gc of HRTV or SFTSV. NP: nucleoprotein gene of HRTV or SFTSV. E: BRBV envelope gene. M: BRBV matrix gene. (B) Detection of protein expression in Western blots. Protein samples (VLPs) were PEG precipitated from culture supernatant of Vero cells and detected after SDS-PAGE with specific secondary antibodies. A negative control (C) for each sample is also shown immediately to the right. A dashed line indicates that lanes were not continuous, and full-length blots with negative controls can be found in Figures S1 and S2.

Figure 3

Representative TEM images of VLPs produced in Vero cells infected by vINDs in the absence of tetracyclines. (A,B) POWV (prototype lineage) VLPs. (C,D) HRTV VLPs. (E,F) MAYV VLPs. (G,H) SFTSV VLPs. (I–L) BRBV VLPs.

Figure 4

Signal sequence (SS) modifications influenced POWV VLP secretion. (A) Depiction of modifications in the signal sequences. Amino acid sequences are shown. A methionine (underlined M) was added to the N-terminus of each signal peptide and each mutation is bolded accordingly. (B) Western blots of PEG-precipitated POWV VLPs produced in Vero cells in the absence of tetracyclines. A major band of 54 kDa and a minor band (not always detectable) of 60 kDa were observed. Full-length blots with negative controls are provided in Figure S2.