Factors Involved in Removing the Non-Structural Protein of Foot-and-Mouth Disease Virus by Chloroform and Scale-Up Production of High-Purity Vaccine Antigens

Abstract

Foot-and-mouth disease (FMD) is an economically important and highly infectious viral disease, predominantly controlled by vaccination. The removal of non-structural proteins (NSPs) is very important in the process of FMD vaccine production, because vaccinated and naturally infected animals can be distinguished by the presence of NSP antibodies in the FMD serological surveillance. A previous study reported that 3AB protein, a representative of NSPs, was removed by chloroform treatment. Therefore, in this study, the causes of 3AB removal and factors affecting the effect of chloroform were investigated. As a result, the effectiveness of chloroform differed depending on the virus production medium and was eliminated by detergents. In addition, it was found that 3AB protein removal by chloroform is due to the transmembrane domain of the N-terminal region (59-76 amino acid domain). Further, industrial applicability was verified by applying the chloroform treatment process to scale-up FMD vaccine antigen production. A novel downstream process using ultrafiltration instead of polyethylene glycol precipitation for high-purity FMD vaccine antigen production was established. This result will contribute toward simplifying the conventional process of manufacturing FMD vaccine antigens and ultimately reducing the time and cost of vaccine production.

Keywords: chloroform; foot-and-mouth disease virus; non-structural protein; scale-up; vaccine purity.

Figure 1

Effect of chloroform treatment according to the type of medium or buffer. (a) The O/Boeun/SKR/2017 virus produced in different types of cell culture medium were treated with various concentrations of chloroform (upper panel). The O/Jincheon/SKR/2014, A₂₂ Iraq/24/64, and Asia1 Shamir/ISR/1989 virus were produced in Dulbecco’s modified Eagle’s medium (DMEM) and then exchanged with Tris-NaCl buffer. DMEM and Tris-NaCl buffer were each treated with 10% (v/v) chloroform (lower panel). (b) The O/Jincheon/SKR/2014 and O/Andong/SKR/2010 were produced in ProVero medium and then exchanged with radioimmunoprecipitation assay (RIPA) buffer. ProVero and RIPA buffer were each treated with 10% (v/v) of chloroform. The 3AB protein was detected by Western blot using anti-FMDV 3B monoclonal antibody.

Figure 2

Identification of 3AB protein regions affected by chloroform. (a) The topology of 3AB was estimated by the TMHMM program. The x-axis indicates the amino acid sequence position, and the y-axis shows the possibility of regions located in intracellular (blue line), extracellular (pink line), and transmembrane (red line and stripe) spaces. (b) The full-length and truncated 3AB amino acid regions for cloning into the pET28a vector were indicated. The yellow box represents a transmembrane domain. (c) Purified recombinant full-length and truncated 3AB proteins were treated with various concentrations of chloroform, and then, 3AB protein was detected by Western blot using anti-FMDV 3B monoclonal antibody.

Figure 2

Identification of 3AB protein regions affected by chloroform. (a) The topology of 3AB was estimated by the TMHMM program. The x-axis indicates the amino acid sequence position, and the y-axis shows the possibility of regions located in intracellular (blue line), extracellular (pink line), and transmembrane (red line and stripe) spaces. (b) The full-length and truncated 3AB amino acid regions for cloning into the pET28a vector were indicated. The yellow box represents a transmembrane domain. (c) Purified recombinant full-length and truncated 3AB proteins were treated with various concentrations of chloroform, and then, 3AB protein was detected by Western blot using anti-FMDV 3B monoclonal antibody.

Figure 3

Production of large-scale FMD vaccine antigen using chloroform treatment process. (a) The detailed procedure for manufacturing FMD vaccine antigen is shown in images. Description of images from left to right: (i) The O/Boeun/SKR/2017 virus was produced using a wave-type bioreactor. (ii) The 20 L of virus culture supernatant was treated with 2% (v/v) of chloroform and mixed with an electronic overhead stirrer. (iii) The supernatant was filtrated in the order of depth and sterile filters. (iv) The filtrate was concentrated 10-fold by ultrafiltration. (b) FMDV SP (VP1) and NSP (3AB) were detected by Western blot analysis using anti-FMDV VP1 and 3B monoclonal antibodies. (c) The 10-fold concentrated vaccine antigens were negatively stained and analyzed for morphology by a transmission electron microscope.

Figure 3

Production of large-scale FMD vaccine antigen using chloroform treatment process. (a) The detailed procedure for manufacturing FMD vaccine antigen is shown in images. Description of images from left to right: (i) The O/Boeun/SKR/2017 virus was produced using a wave-type bioreactor. (ii) The 20 L of virus culture supernatant was treated with 2% (v/v) of chloroform and mixed with an electronic overhead stirrer. (iii) The supernatant was filtrated in the order of depth and sterile filters. (iv) The filtrate was concentrated 10-fold by ultrafiltration. (b) FMDV SP (VP1) and NSP (3AB) were detected by Western blot analysis using anti-FMDV VP1 and 3B monoclonal antibodies. (c) The 10-fold concentrated vaccine antigens were negatively stained and analyzed for morphology by a transmission electron microscope.