Application of Heparin Affinity Chromatography to Produce a Differential Vaccine without Eliciting Antibodies against the Nonstructural Proteins of the Serotype O Foot-and-Mouth Disease Viruses

Figures

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  Figure 1. Chromatogram of heparin affinity chromatography. The three strains of the foot-and-mouth disease virus culture supernatant were loaded onto a HiTrap HP heparin column and traced by UV monitoring at 280 nm. The chromatogram shows the fractions obtained during the purification process. The colored lines represent each as follows: blue, O/Boeun/SKR/2017 strain; green, O/Jincheon/SKR/2014 strain; red, Om-O-PanAsia2 recombinant strain; gray, conductivity.

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  Figure 2. Identification of the purified protein after heparin affinity chromatography. The protein samples of each fraction obtained in the purification process were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis. (a) The gel was revealed by Coomassie brilliant blue staining. Lane M, molecular weight marker; Lane 1, foot-and-mouth disease virus serotype O Boeun virus culture supernatant (before column loading); Lane 2, flow-through fraction from the heparin column; Lane 3, washed fraction with the heparin column; Lane 4, eluted fraction from the heparin column (10-fold concentrated); Lane 5, Lane P, positive control (purified FMDV O/Boeun/SKR/2017 by sucrose density gradient ultracentrifugation). Western blot analysis was performed with (b) anti-foot-and-mouth disease virus serotype O VP1. structural protein monoclonal antibodies and (c) anti-foot-and-mouth disease virus 3B nonstructural protein monoclonal antibodies. Lane C, foot-and-mouth disease virus serotype O/Boeun/SKR/2017 virus supernatant; Lane E, eluted fraction from the heparin column (10-fold concentrated).

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  Figure 3. Morphological observation on a transmission electron microscope. The heparin affinity column purified serotype O foot-and-mouth disease viruses were negatively stained and analyzed for morphology. The bars represent 50 nm.

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  Figure 4. Antibody responses elicited by immunizations with polyethylene glycol (PEG)-treated and heparin affinity column purified serotype O foot-and-mouth disease (FMD) Boeun/SKR/2017 vaccines. (a) Scheme of the animal experiment. Twenty goats were vaccinated five times at a four-week interval with PEG-treated (n = 10) and heparin affinity column purified (n = 10) FMD vaccines. Blood samples were collected every four weeks after vaccination. (b) The antibody titers of the structural proteins were measured via enzyme-linked immunosorbent assay (ELISA). The antibody values were expressed as percentage inhibition (PI) values. A PI value of ≥50% (dotted line) was considered to indicate positivity. (c) The homologous virus-neutralizing (VN) antibody titers were measured using the virus neutralization test. The VN titers were expressed as a log10 value. A titer of ≥1.65 log10 (dotted line) was considered to indicate positivity. (d) The antibody titers of the nonstructural proteins were measured via ELISA. The antibody values were expressed as PI values. A PI value of ≥50% (dotted line) was considered to indicate positivity. The full blue and red lines represent the mean PI values in each…