Producing Vaccines against Enveloped Viruses in Plants: Making the Impossible, Difficult

Abstract

The past 30 years have seen the growth of plant molecular farming as an approach to the production of recombinant proteins for pharmaceutical and biotechnological uses. Much of this effort has focused on producing vaccine candidates against viral diseases, including those caused by enveloped viruses. These represent a particular challenge given the difficulties associated with expressing and purifying membrane-bound proteins and achieving correct assembly. Despite this, there have been notable successes both from a biochemical and a clinical perspective, with a number of clinical trials showing great promise. This review will explore the history and current status of plant-produced vaccine candidates against enveloped viruses to date, with a particular focus on virus-like particles (VLPs), which mimic authentic virus structures but do not contain infectious genetic material.

Keywords: Bunyavirales; Flaviviridae; Influenza virus; alphavirus; coronavirus; hepatitis B virus; human immunodeficiency virus; newcastle disease virus; plant molecular farming; plant-produced vaccines; rhabdovirus; virus-like particles.

Figure 1

Diagrams depicting the major structural elements of the virus clades described in this review. Background dark green band represents the lipid envelope, and other elements represent viral structural proteins. Blue represents capsid proteins while yellow/orange represents membrane-bound surface glycoproteins.

Figure 2

Transient co-expression of E, M and S coronavirus proteins leads to the formation of possible VLPs. Top: porcine epidemic diarrhoea virus, bottom: SARS-CoV-2. The E, M and S proteins are co-expressed in N. benthamiana and purified by sucrose cushion followed by a sucrose gradient then desalting column before concentration on a spin column concentrator. Left: anti-S Western blots on samples from co-expression of coronavirus proteins (E + M + S) or empty vector negative control (EV). Before: sample recovered from desalting column, FT: flow-through from spin-column concentrator, Conc: retentate from spin column concentrator, M: marker, COE: positive control. Right: transmission electron micrographs of Conc samples shown in Western blots. Samples stained with 2% (w/v) uranyl acetate, scale bars are 100 nm.

Figure 3

VHSV G protein produced in plants is glycosylated. His-tagged N- and C-terminally truncated G protein (VHSV-G^ΔNΔC) and C-terminally truncated G protein (VHSV-G^ΔC) were transiently expressed in N. benthamiana. The N-terminal truncation removes the native secretion signal and hydrophobic region, while the C-terminal truncation removes the transmembrane domain. (A) Anti-G Western blot of homogenate (H), flow-through (FT), wash (W) and elution (E) fractions of nickel column affinity purification for both constructs. (B) anti-G Western blot of affinity-purified VHSV-G^ΔC after mock deglycosylation treatment or treatment with PNGase A and F. The band shift is indicative of glycosylation.

Figure 4

ZIKV prM-E expression in plants yields possible VLPs. The prM-E region of ZIKV was transiently expressed in N. benthamiana and purified on a sucrose cushion followed by desalting and concentration on a spin filter concentrator. The concentrate was then further purified over a 30–60% (w/v) sucrose gradient and the fractions were assayed by anti-E Western blot (left). The positive fractions (indicated) were pooled, buffer-exchanged and concentrated on a spin filter concentrator and imaged by transmission electron microscopy (right), revealing possible VLPs 35–55 nm in size. Samples stained with 2% (w/v) uranyl acetate, scale bar is 100 nm.

Figure 5

Alphavirus structural proteins are expressed in plants and self-assemble into capsid-like structures. Proteins of CHIKV (A–D) and SAV (E,F) were transiently expressed in N. benthamiana from pEAQ-HT constructs for capsid protein, the entire open reading frame 2 (ORF2), or portions of ORF2 with C-terminal histidine tag. (A) Schematic representation of CHIKV-ORF2. (B) InstantBlue-stained SDS-PAGE gels of gradient fractions from CHIKV-C and CHIKV-ORF2-expressing tissue. CHIKV capsid protein band indicated. (C) Transmission electron micrograph of CHIKV-C sucrose gradient fraction. (D) Western blot of crude leaf extract probed with anti-his antibody, showing production of his-tagged CHIKV-E2. (E) InstantBlue-stained SDS-PAGE gel of Nycodenz gradient fractions for purification of SAV capsid protein (N-terminally his-tagged) or empty vector control. SAV capsid protein band indicated. (F) Transmission electron micrograph of N-terminally His-tagged SAV-C Nycodenz gradient fraction. Samples stained with 2% (w/v) uranyl acetate before TEM imaging, scale bars 100 nm.