Validation of Pretreatment Methods for the In-Process Quantification of Foot-and-Mouth Disease Vaccine Antigens.

Ah-Young Kim,Jae Young Kim,Sun Young Park,Sang Hyun Park,Jong-Hyeon Park,Young-Joon Ko,Jong Sook Jin,Eun-Sol Kim

doi:10.3390/vaccines9111361

Abstract

Foot-and-mouth disease (FMD), caused by the FMD virus (FMDV), is controlled by vaccine policy in many countries. For vaccine potency, the content of intact virus particles (146S antigens) is critical, and the sucrose density gradient (SDG) fractionation is the gold standard for the quantification of 146S antigens. However, this method has several drawbacks. Although size-exclusion high-performance liquid chromatography (SE-HPLC) was introduced to replace the classic method, its application is generally confined to purified samples owing to the interfering signals. Therefore, we aimed to develop optimal pretreatment methods for SE-HPLC quantification in less purified samples. Crude virus infection supernatant (CVIS) and semi-purified samples with PEG precipitation (PEG-P) were used. Chloroform pretreatment was essential to remove a high level of non-specific signals in CVIS, whereas it caused loss of 146S antigens without the distinctive removal of non-specific signals in PEG-P. Benzonase pretreatment was required to improve the resolution of the target peak in the chromatogram for both CVIS and PEG-P. Through spiking tests with pure 146S antigens, it was verified that the combined pretreatment with chloroform and benzonase was optimal for the CVIS, while the sole pretreatment of benzonase was beneficial for PEG-P.

Highlights

Foot-and-mouth disease (FMD), caused by the FMD virus (FMDV), is a highly contagious animal disease threatening the livestock industry [1]
With the addition of chloroform, both chloroform extraction (C+)B+ and C+ samples exhibited the clearance of non-target protein bands except for the FMDV structural proteins, regardless of the quantitation method, even though the structural protein bands did not show the strongest signal intensity in silver staining (Figures 1e and S1b)
The removal rate of host cell-derived dsDNA contamination in the target peak fraction was significantly lower in the non-pretreated control (NPC) and C+ samples of size-exclusion highperformance liquid chromatography (SE-HPLC) fractions, the B+ and C+B+ samples exhibited a dsDNA removal rate of more than 95%, regardless of the quantitation method (Figure 1g)

Summary

Introduction

Foot-and-mouth disease (FMD), caused by the FMD virus (FMDV), is a highly contagious animal disease threatening the livestock industry [1]. The virus affects cloven-hoofed animals, resulting in characteristic vesicle formation in the mucosa around the foot and mouth [2]. To control this disease, vaccination policies utilizing inactivated FMD vaccines have been implemented worldwide. The intact virion of FMDV, comprising 60 copies each of four structural proteins and a single-stranded RNA genome [3], is called a 146S particle based on its sedimentation coefficient [4]. As 146S particles are so unstable that they can be dissociated into less immunogenic 12S particles under mild changes in pH or temperature [5], it is important to know the amount of intact particles remaining in the vaccine to guarantee vaccine efficacy [6]. Identifying the 146S content is critical for in-process quality control during the FMD vaccine production process

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Methods

Results

Conclusion