Functional Evaluation of Neutralizing Antibodies Against Foot-and-Mouth Disease Virus Serotype O Using a Luciferase-Based Surrogate Neutralization Assay

Figures

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  Figure 1. Schematic representation of HiBiT-tagged FMDV serotype O VLP expression constructs: (A) Schematic representation of the control plasmid without a HiBiT tag (pcDNA3.1-O-P1-2A/3C and VLP). (B–D) Plasmids containing the HiBiT tag inserted at amino acid position 37 of VP4 ((B) VP4-HiBiT), position 51 within the VP1 BC loop ((C) VP1 BC-HiBiT), and position 153 within the VP1 GH loop ((D) VP1 GH-HiBiT). The HiBiT insertion sites are indicated by arrows.

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  Figure 2. Expression and luciferase activity of HiBiT-tagged FMDV serotype O VLPs: (A) Protein expression of HiBiT-tagged VLPs in cell lysates and culture supernatants of HEK293T cells co-transfected with HiBiT-tagged VLP and LgBiT expression plasmids. (B) NanoBiT reporter activity measured after incubation of culture supernatants containing HiBiT-tagged VLPs with stable LgBiT-expressing LF-BK cells. Data are presented as the mean ± SEM from three independent experiments (n = 3). Statistical significance was analyzed using one-way ANOVA followed by a multiple-comparison test (ns, not significant; ****, p < 0.0001).

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  Figure 3. Purification and characterization of HiBiT-tagged FMDV serotype O VLPs: (A,B) Distribution of capsid proteins and HiBiT-tagged proteins in sucrose density gradient fractions, detected using anti-VP2 and anti-HiBiT antibodies, respectively. (C) Detection of HiBiT-tagged proteins in pooled peak fractions using an anti-HiBiT antibody. (D) Representative transmission electron microscopy (TEM) images of purified VLPs, VP4-HiBiT VLPs, and VP1 GH-HiBiT VLPs. Scale bars = 100 nm.

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  Figure 4. Evaluation of the NanoBiT-VNT using porcine serum samples: (A) FMDV serotype O SP ELISA results for negative sera (Neg.) and two groups of positive sera (Pos. 1 and Pos. 2). The dashed line indicates the ELISA cutoff value. (B) Neutralization of VP4-HiBiT VLPs by negative and positive porcine sera. Serum samples were serially diluted from 1:16 to 1:512, and percent inhibition was determined using the NanoBiT assay. The dashed line indicates the 50% inhibition threshold. (C) Comparison of neutralizing antibody titers determined by the conventional VNT and the NanoBiT-VNT using porcine serum samples. Statistical significance was analyzed using two-way ANOVA followed by Sidak’s multiple-comparison test. ns, not significant.

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  Figure 5. Correlation between neutralizing antibody titers determined by the conventional virus neutralization test (VNT) and the VP4-HiBiT VLP-based NanoBiT-VNT. Neutralizing antibody titers against FMDV serotype O were measured using the conventional VNT and the VP4-HiBiT VLP-based NanoBiT-VNT in 66 porcine serum samples. The correlation between the titers quantified by the two assays was analyzed by linear regression.