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.

Exposure of iron nanoparticles to Arabidopsis thaliana enhances root elongation by triggering cell wall loosening.

In this study, we investigated the effect of nZVI on plant root elongation in Arabidopsis thaliana and showed, for the first time, that nZVI enhanced root elongation by inducing OH radical-induced cell wall loosening. Exposure of plants to 0.5 g/L nZVI enhanced root elongation by 150-200% over that in the control, and further mechanistic studies showed that this occurred via nZVI-mediated OH radical-induced cell wall loosening. The oxidation capacity of nZVI, leading to release of H2O2, allowed it to cause OH radical-induced cell wall loosening in roots. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometers (MALDI-TOFMS)-based analysis clearly revealed that pectin-polysaccharides in roots were degraded; they are one of the main matrix-polysaccharide-connecting and load-bearing polymers in cell walls. Rapid root elongation led to structural changes in root cell walls: reduction of cell wall thickness and a bias on the orientation of cellulose microfibrils. Additionally, the asymmetrical distribution of tensional strength resulted from the OH radical-induced cell wall loosening enhanced endocytosis. These findings emphasize that OH radical-induced cell wall loosening is important for mechanical regulation of the cell wall and provide new insights into the cellular responses of plants exposed to reactive metal nanoparticles.

Field Application of a New CSF Vaccine Based on Plant-Produced Recombinant E2 Marker Proteins on Pigs in Areas with Two Different Control Strategies.

A classical swine fever virus (CSFV)-modified live LOM (low-virulence strain of Miyagi) vaccine (MLV-LOM) to combat CSF has been used in places where the disease is prevalent around the world, including in Korea, except in Jeju Island. In general, modified live virus-based vaccines (MLV) are known to be highly effective in inducing immune responses. At the same time, MLVs also have potential dangers such as a circulation in the field. There is still a need for safer and more effective vaccines to control CSF in the field. In this study, we applied a new CSF vaccine based on plant-produced recombinant E2 marker proteins at two different locations, Jeju Island and a suburb of Pohang, using different CSF control strategies. The result suggested that vaccinated sows in Jeju Island highly developed immunogenicity and maintained stably until 102 days post-vaccination (dpv). Its piglets that received maternal antibodies were shown to carry high serological values and maintained them until 40 days of age, which was the end of the follow-up. Naïve piglets vaccinated at 40 days of age showed high serological values and these were maintained until 100 days of age (60 dpv), which was the end of the follow-up. The vaccine was also effective in inducing immune responses in newborn piglets that carried maternal antibodies received from MLV-LOM vaccine-immunized mother sows.

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

The efficacy of a novel subunit vaccine candidate, based in the CSFV E2 glycoprotein produced in plants to prevent classical swine fever virus (CSFV) vertical transmission, was evaluated. A Nicotiana benthamiana tissue culture system was used to obtain a stable production of the E2-glycoprotein fused to the porcine Fc region of IgG. Ten pregnant sows were divided into three groups: Groups 1 and 2 (four sows each) were vaccinated with either 100 µg/dose or 300 µg/dose of the subunit vaccine at 64 days of pregnancy. Group 3 (two sows) was injected with PBS. Groups 1 and 2 were boosted with the same vaccine dose. At 10 days post second vaccination, the sows in Groups 2 and 3 were challenged with a highly virulent CSFV strain. The vaccinated sows remained clinically healthy and seroconverted rapidly, showing efficient neutralizing antibodies. The fetuses from vaccinated sows did not show gross lesions, and all analyzed tissue samples tested negative for CSFV replication. However, fetuses of non-vaccinated sows had high CSFV replication in tested tissue samples. The results suggested that in vaccinated sows, the plant produced E2 marker vaccine induced the protective immunogenicity at challenge, leading to protection from vertical transmission to fetuses.

Development of Recombinant Protein-Based Vaccine Against Classical Swine Fever Virus in Pigs Using Transgenic Nicotiana benthamiana.

Classical swine fever virus (CSFV) is highly contagious, and fatal to infected pigs. Vaccines against CSFV have been developed from attenuated or modified live viruses. These vaccines are effective for immunization of animals, but they are associated with problems such as the accidental spreading of viruses to animals in the field, and with barriers to trade following vaccination. Here, we report the generation of transgenic Nicotiana benthamiana plants for large-scale, cost-effective production of E2 fusion protein for use as a recombinant vaccine against CSFV in pigs. Transgenic N. benthamiana plants harboring an intergenic, single-copy insertion of a chimeric gene encoding E2 fusion protein had high levels of transgene expression. For large-scale production of E2 fusion protein from leaf tissues, we developed a protein-purification protocol consisting of cellulose-binding domain (CBD)-cellulose-based affinity purification and size-exclusion gel-filtration chromatography. E2 fusion proteins showed high immunogenicity in piglets and provided protection against CSFV challenge. The CBD in the E2 fusion protein was also highly immunogenic. These results suggest that plant-produced recombinant E2 fusion proteins can be developed into cost-effective vaccines against CSFV, with the CBD as a marker antigen to differentiate between vaccination and natural infection.