Neutralizing Antibody Screening Using NanoBiT-Based Virus-like Particles of Foot-and-Mouth Disease Type Asia1 Enhances Biosafety and Sensitivity

Figures

• 

  Figure 1. Schematic and validation of HiBiT-tagged Asia1 Shamir (AS-HiBiT) plasmids and luciferase activity for the nano-luciferase binary technology (NanoBiT) assay. (A) Schematic representation of the HiBiT-tagged AS virus-like particles (AS-HiBiT VLPs). The AS-HiBiT plasmid includes the HiBiT inserted into the VP1 protein (153S) of the pCDNA3.1-AS P1-2A/3C (AS) plasmid. (B) Western blot analysis confirming the expression of LgBiT, HiBiT, and AS proteins of HEK293T cells transfected with the respective plasmids; β-actin was used as an internal control. (C) Luminescence assay showing luciferase activity in cells transfected with plasmids. Cells co-transfected with LgBiT and AS-HiBiT plasmids exhibited a significant increase in luminescence compared with cells transfected with LgBiT or AS-HiBiT alone. The results are presented as means ± SEM of triplicate wells. Statistical significance was analyzed using one-way ANOVA followed by Tukey’s multiple comparisons test. ****, p < 0.0001; ns, no significant difference.

• 

  Figure 2. Layout of U-bottom 96-well plates for the NanoBit-based VNT using HiBiT-tagged AS VLPs. (A) Serial dilutions of different serum samples across rows A to G of a plate. Row H contains no serum as a control. (B) AS-HiBiT VLPs were added to columns 2, 3, 5, 6, 8, 9, 11, and 12. Serum controls were set in columns 1, 4, 7, and 10, and cell controls were set in row H. (C) The plate setup and results of the interaction between serum and AS-HiBiT VLPs. Red wells represent serum controls, blue wells indicate AS-HiBiT VLPs, and purple wells show the serum-AS-HiBiT VLP reactions.

• 

  Figure 3. Production of AS and AS-HiBiT VLPs. (A) Sucrose density gradient ultra-centrifugation of AS and AS-HiBiT VLPs. Absorbance was measured at 280 nm, and fractions 6-9 corresponding to the 75S peak were collected for further analysis. (B) SDS-PAGE and Western blot analysis of the collected fractions. The silver-stained SDS-PAGE gel confirms the presence of AS and AS-HiBiT VLP proteins. (C) Transmission electron microscopy images of purified AS (left) and AS-HiBiT VLPs (right) showing the VLPs. Scale bar = 100 nm.

• 

  Figure 4. AS-HiBiT VLP binding and entry into stable LgBiT-expressing cells. (A) The effect of AS-HiBiT VLP incubation time in stable cells seeded at 5000, 10,000, and 20,000 cells/well at 37 °C, with measurements taken at multiple time points up to 24 h. The dotted line represents the detection threshold of 104 RLU for luminescence activity. (B,C) The effect of AS-HiBiT VLP dilution and medium conditions on luciferase activity in stable LgBiT cells (20,000 cells/well) at 37 °C for 1h. All data represent the means ± SEM of triplicate wells. Statistical significance was analyzed using two-way ANOVA followed by Tukey’s multiple comparisons test. *, p < 0.05; ****, p < 0.0001; and ns, p > 0.05.

• 

  Figure 5. Application of the AS-HiBiT VLPs with guinea pig sera for NanoBiT assay. (A) Sera from guinea pigs (n = 4) vaccinated with a commercial FMD vaccine (including AS) were tested to measure the neutralizing antibody titers of FMD AS using the traditional VNT. The sera collected at 28 dpv showed high neutralizing antibody titers against AS and were classified into the positive group, whereas the sera collected at 0 dpv did not show neutralizing antibody titers and were classified into the negative group. (B) The negative and positive sera were applied to the NanoBiT assay. All data represent means ± SEM analyzed using two-way ANOVA following Sidak’s multiple comparisons test. ****, p < 0.0001; ns, no significant difference.

• 

  Figure 6. Time-dependent neutralizing antibody responses after vaccination and correlation between traditional VNT and NanoBiT-VNT in pig sera. (A) FMDV SP Asia1 ELISA results for five pigs collected at 0, 28, 42, and 56 dpv. The PI (%) gradually increased after vaccination, indicating the induction of antibody responses over time. (B) VN titers against live AS virus for five pigs measured at 0, 28, 42, and 56 dpv. A clear increase in VN titers was observed, especially after 28 dpv. (C) Inhibition (%) of AS-HiBiT VLPs using negative and positive sera in stable LgBiT cells analyzed as indicated in Section 2.8. The serum samples were diluted, starting from 1:16 to 1:1024. Positive sera showed higher inhibition (%) at various serum dilutions, indicating the presence of neutralizing antibodies. (D) Comparison of VN titers (log₁₀) between the traditional VNT and NanoBiT-VNT for sera collected at 28, 42, and 56 dpv. No significant differences were observed between the two methods, suggesting comparable accuracy. Statistical significance was analyzed using two-way ANOVA, followed by Sidak’s multiple comparisons test. N.s., no significant difference. (E) Correlation analysis between VN titers…

• 

  Figure 7. Comparison of VNTs between live FMD AS and AS-HiBiT VLPs in various anti-AS pig sera (n = 58). VN titers (log10) against FMDV AS were measured using both the traditional VNT and the AS-HiBiT VLP-based NanoBiT-VNT in pig sera. Correlation analysis between VN titers measured through the traditional VNT and the AS-HiBiT VLP-based NanoBiT-VNT. The regression line indicates a strong positive correlation (R2 = 0.8881), further supporting the consistency of the two methods.