A classical swine fever virus E2 fusion protein produced in plants elicits a neutralizing humoral immune response in mice and pigs

Abstract

Classical swine fever (CSF) is one of the most important viral diseases of swine worldwide. Although live or attenuated virus vaccines have been used to control CSFV, it is difficult to distinguish vaccinated pigs from infected pigs; this leads to restrictions on import and export. Subunit vaccines based on the CSFV E2 glycoprotein have been developed using baculovirus or insect cell systems, but some weaknesses remain. Here, we describe production of an E2 recombinant protein using a Nicotiana benthamiana plant expression system. To do this, we took advantage of the ability of the swine Fc domain to increase solubility and stability of the fusion protein and to strengthen immune responses in target animals. N. benthamiana expressed high amounts of pFc2-fused E2 proteins, which were isolated and purified by affinity chromatography to yield a high pure recombinant protein in a cost-effective manner. Native-polyacrylamide gel electrophoresis and size exclusion chromatography confirmed that the pmE2:pFc2 fusion exists as a multimer rather than as a dimer. Injection of recombinant pmE2 protein into mice or piglets generated anti-pmE2 antibodies with efficient neutralizing activity against CSFV. These results suggest that a purified recombinant E2 protein produced in N. benthamiana generates high titers of neutralizing antibodies in vivo; as such, the protein could be developed as a subunit vaccine against CSFV. Keywords Nicotiana benthamiana Á Classical swine fever virus Á DIVA concept Á Subunit vaccine Á Molecular farming Á Fc-fusion protein Abbreviations CSFV Classical swine fever virus DIVA Differentiating infected from vaccinated animals ER Endoplasmic reticulum CBD Cellulose-binding domain BSA Bovine serum albumin MLV Modified live vaccines PCR Polymerase chain reaction Electronic supplementary material The online version of this article (

A Novel E2 Glycoprotein Subunit Marker Vaccine Produced in Plant Is Able to Prevent Classical Swine Fever Virus Vertical Transmission after Double Vaccination

“…The glycosylation of E2 protein produced in plant tissue was confirmed elsewhere [16] and correct glycosylation of antigenic proteins has been known to affect antigen efficacy [37]. However, the effect of xylose and fucose epitopes on N-glycans produced in plants is controversial and how plant-specific glycans on E2 protein affect antigenicity of the subunit vaccine in pigs remains elusive.…”

Future perspectives on swine viral vaccines: where are we headed?

“…Examples of plant-expressed vaccines include CSFV, for which the structural glycoprotein E2 is commonly used [ 59 , 60 ]. For example, when using the Nicotiana benthamiana plant expression system, purified recombinant E2 protein generated high titers of neutralizing antibodies in pigs and mice when injected intramuscularly [ 60 ]. Furthermore, transplastomic tobacco was used to produce a subunit vaccine candidate against PEDV [ 61 ].…”

Research Progress and Challenges in Vaccine Development against Classical Swine Fever Virus

“…A single-dose vaccination (200 μg of E2 protein) induced a strong neutralizing antibody response, providing complete protection in pigs against CSFV challenge. Park et al further investigated the production of a fusion of the CSFV E2 protein and the pFc2 domain of porcine IgG, which was designated as pmE2:pFc2, in transgenic N. benthamiana [ 144 ]. Fusing proteins to the Fc domain could enhance the solubility and half-life of an antigenic protein [ 145 , 146 ].…”