Comparative Molecular Biology Approaches for the Production of Poliovirus Virus-Like Particles Using Pichia pastoris

Abstract

For enteroviruses such as poliovirus (PV), empty capsids, which are antigenically indistinguishable from mature virions, are produced naturally during viral infection. The production of such capsids recombinantly, in heterologous systems such as yeast, have great potential as virus-like particle (VLP) vaccine candidates. Here, using PV as an exemplar, we show the production of VLPs in Pichia pastoris by coexpression of the structural precursor protein P1 and the viral protease 3CD. The level of expression of the potentially cytotoxic protease relative to that of the P1 precursor was modulated by three different approaches: expression of the P1 precursor and protease from different transcription units, separation of the P1 and protease proteins using the Thosea asigna virus (TaV) 2A translation interruption sequence, or separation of the P1 and protease-coding sequences by an internal ribosome entry site sequence from Rhopalosiphum padi virus (RhPV). We also investigate the antigenicity of VLPs containing previously characterized mutations when produced in Pichia Finally, using transmission electron microscopy and two-dimensional classification, we show that Pichia-derived VLPs exhibited the classical icosahedral capsid structure displayed by enteroviruses.IMPORTANCE Although the current poliovirus immunization program has been extremely successful in reducing the number of cases of paralytic polio worldwide, now more cases are caused by vaccine-derived polioviruses than by wild poliovirus. Switching to inactivated poliovirus vaccines will reduce this over time; however, their production requires the growth of large amounts of virus. This biosafety concern can be addressed by producing just the virus capsid. The capsid serves to protect the genetic material, which causes disease when introduced into a cell. Therefore, empty capsids (virus-like particles [VLPs]), which lack the viral RNA genome, are safe both to make and to use. We exploit yeast as a versatile model expression system to produce VLPs, and here we specifically highlight the potential of this system to supply next-generation poliovirus vaccines to secure a polio-free world for the future.

Keywords: Pichia pastoris; enterovirus; poliovirus; virus-like particle.

FIG 1

Schematic of the poliovirus genome, Pichia expression systems, and correct processing of PV P1. (A) Schematic of the poliovirus genome highlighting the P1 structural region and the nonstructural proteins, including the viral protease 3CD. UTR, untranslated region; ssRNA, single-stranded RNA. (B) Schematic of the pPink HC plasmid and the position in which the different expression constructs were cloned. (C) Immunoblot for PV VP1 and 3D. The virus control sample was prepared from HeLa cell lysates 8 h postinfection. Yeast samples were collected 48 h postinduction and lysed using 0.1 M NaOH. All samples were boiled in 2× Laemmli buffer and separated by SDS-PAGE prior to analysis by immunoblotting using mouse monoclonal α-VP1 and rabbit monoclonal α-3D antibody. Two exposures of the same α-3D immunoblot are shown to aid interpretation.

FIG 2

Purification of virions and VLPs. All samples were purified by ultracentrifugation and fractioned from top to bottom in 1-ml aliquots. A 12-μl sample from each fraction then was taken and boiled in 5× Laemmli buffer and separated by SDS-PAGE prior to analysis by immunoblotting using mouse monoclonal α-VP1. Shown is a representative example of three separate experiments for each construct.

FIG 3

Electron microscopy and 2D class averages of PV VLPs. (a) Representative micrographs from different preparations of PV VLPs and infectious PV (PV-1). White arrowheads indicate ∼33-nm-diameter particles. Red arrowheads indicate smaller particles (∼22-nm diameter). Scale bar shows 100 nm. (b) 2D class averages from the twenty most populated classes of ∼33-nm-diameter particles in each data set, following removal of the smaller particles. Class averages are shown with a sigma contrast of 3. Average particle size, n = 20 for each preparation.

FIG 4

Antigenicity of Pichia-derived VLPs. Shown are the antigenicity of Pichia-derived PV-1 wt and PV57δ VLPs and the reactivity of gradient fractions with MAb 1588 (C antigen) and MAb 234 (D antigen) in ELISA. The pink dashed line represents the positive control, BRP, for the D antigen ELISA. The optical density at 492 nm (OD 492 nm) is represented in arbitrary units (n = 2).