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

Youngmin Park,Yongjik Lee,Soohong Park,Eun-Ju Sohn,Inhwan Hwang,Sungmin Gu,Seeun Choe,Jong Kook Kim,Sangmin Lee,Kyungmin Min,Minhee Park,Nam Hyung Kim,Hye-Yeon Kim,Dong-Jun An

doi:10.3389/fpls.2019.00624

Abstract

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.

Highlights

Classical swine fever (CSF) is one of the most important viral diseases of domestic and wild pigs (Edwards et al, 2000)
We investigated whether recombinant E2 fusion protein could be expressed at high levels in transgenic N. benthamiana plants by intergenic insertion of a single-copy transgene, and whether expressed fusion protein could be purified in a cost-effective manner for commercialization
N. benthamiana was assessed for protein production, because transgenic A. thaliana plants had previously been found to have a limitation for production of biomass (Sohn et al, 2018)

Summary

Introduction

Classical swine fever (CSF) is one of the most important viral diseases of domestic and wild pigs (Edwards et al, 2000). CSF is caused ppE2 Protects Against CSFV Virus by classical swine fever virus (CSFV), which belongs to the genus Pestivirus within the family Flaviviridae (Moennig, 2000). CSFV is an RNA virus with a single-stranded, positive RNA genome of approximately 12.3 kb that encodes a polypeptide of 3,898 amino acid residues (Rümenapf et al, 1993). This polypeptide is processed to produce 12 proteins, comprising four structural proteins (C, Erns, E1, and E2) and eight non-structural proteins (Npro, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) (Meyers et al, 1996). The genome contains a 5 untranslated region (UTR) and a 3 UTR

Results

Discussion

Conclusion