Efficient Removal of Non-Structural Protein Using Chloroform for Foot-and-Mouth Disease Vaccine Production

Abstract

To differentiate foot-and-mouth disease (FMD)-infected animals from vaccinated livestock, non-structural proteins (NSPs) must be removed during the FMD vaccine manufacturing process. Currently, NSPs cannot be selectively removed from FMD virus (FMDV) culture supernatant. Therefore, polyethylene glycol (PEG) is utilized to partially separate FMDV from NSPs. However, some NSPs remain in the FMD vaccine, which after repeated immunization, may elicit NSP antibodies in some livestock. To address this drawback, chloroform at a concentration of more than 2% (v/v) was found to remove NSP efficiently without damaging the FMDV particles. Contrary to the PEG-treated vaccine that showed positive NSP antibody responses after the third immunization in goats, the chloroform-treated vaccine did not induce NSP antibodies. In addition to this enhanced vaccine purity, this new method using chloroform could maximize antigen recovery and the vaccine production time could be shortened by two days due to omission of the PEG processing phase. To our knowledge, this is the first report to remove NSPs from FMDV culture supernatant by chemical addition. This novel method could revolutionize the conventional processes of FMD vaccine production.

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

Figure 1

Effect of chloroform treatment on viral proteins of FMD virus (FMDV). (a) The O/Boeun/SKR/2017 and O/Andong/SKR/2010 virus culture supernatants were treated with various concentrations of chloroform (upper panel) and trichloroethylene (lower panel). The structural (VP1) and non-structural (3AB) proteins were detected by Western blot analysis using anti-FMDV type O VP1 and 3B monoclonal antibodies. (b) The O/Boeun/SKR/2017 (left panel) and O/Andong/SKR/2010 (right panel) virus culture supernatants were treated with various concentrations of chloroform. Viral titers were determined on baby hamster kidney-21 (BHK-21) cells, and the titers were expressed as log10 TCID₅₀ per milliliter. All experiments were performed in duplicate.

Figure 1

Effect of chloroform treatment on viral proteins of FMD virus (FMDV). (a) The O/Boeun/SKR/2017 and O/Andong/SKR/2010 virus culture supernatants were treated with various concentrations of chloroform (upper panel) and trichloroethylene (lower panel). The structural (VP1) and non-structural (3AB) proteins were detected by Western blot analysis using anti-FMDV type O VP1 and 3B monoclonal antibodies. (b) The O/Boeun/SKR/2017 (left panel) and O/Andong/SKR/2010 (right panel) virus culture supernatants were treated with various concentrations of chloroform. Viral titers were determined on baby hamster kidney-21 (BHK-21) cells, and the titers were expressed as log10 TCID₅₀ per milliliter. All experiments were performed in duplicate.

Figure 2

Comparison of non-structural proteins (NSPs) in FMD vaccine antigens prepared by the PEG and chloroform treatment. The O/Boeun/SKR/2017 virus supernatant was treated with 7.5% (w/v) of PEG and 10% (v/v) of chloroform. The chloroform-treated virus supernatant was subsequently concentrated by ultrafiltration. After the preparation of vaccine antigen, NSP (3AB) was detected by Western blot analysis using anti-FMDV 3B monoclonal antibody. (a) FMD vaccine antigens prepared by PEG or chloroform plus ultrafiltration methods. (b) The retentate and permeate samples obtained by ultrafiltration of FMDV culture supernatant without chloroform treatment.

Figure 3

Antibody responses elicited by immunizations with PEG and chloroform treated FMD vaccines. (a) Scheme of animal experiment. Goats were divided into 2 groups and vaccinated three times at a 4-week interval with PEG (n = 10) and chloroform (n = 10) treated FMD vaccines. Blood samples were collected every 4 weeks after vaccination. (b) Antibody titers of structural protein were measured by ELISA. The antibody values were expressed as percentage inhibition (PI). PI value of ≥50% (dotted line) was considered positive. (c) Homologous virus-neutralizing (VN) antibody titers were measured by virus neutralization test. The VN titers were expressed as a log 10 value. A titer of ≥1.653 log 10 (dotted line) was considered positive. (d) Antibody titers of non-structural protein were measured by ELISA. The antibody values were expressed as PI. PI value of ≥50% (dotted line) was considered positive. The full blue and red lines represent the mean of the PI values in each group. A statistically significant difference of the mean values between the two groups in the third vaccination was indicated. *** p < 0.001.

Figure 3

Antibody responses elicited by immunizations with PEG and chloroform treated FMD vaccines. (a) Scheme of animal experiment. Goats were divided into 2 groups and vaccinated three times at a 4-week interval with PEG (n = 10) and chloroform (n = 10) treated FMD vaccines. Blood samples were collected every 4 weeks after vaccination. (b) Antibody titers of structural protein were measured by ELISA. The antibody values were expressed as percentage inhibition (PI). PI value of ≥50% (dotted line) was considered positive. (c) Homologous virus-neutralizing (VN) antibody titers were measured by virus neutralization test. The VN titers were expressed as a log 10 value. A titer of ≥1.653 log 10 (dotted line) was considered positive. (d) Antibody titers of non-structural protein were measured by ELISA. The antibody values were expressed as PI. PI value of ≥50% (dotted line) was considered positive. The full blue and red lines represent the mean of the PI values in each group. A statistically significant difference of the mean values between the two groups in the third vaccination was indicated. *** p < 0.001.

Figure 4

The diagram of FMD vaccine antigen manufacturing process utilizing conventional and new methods. The dotted arrows indicate the turnaround time for the production of FMD vaccine antigen concentrates.