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

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.