FIG 4.
Antibodies targeting the epitope of S1399–410 effectively neutralized TiLV. (A) Schedule for immunization, sample collection, and challenge of tilapia. Tilapias (70 fish per group for nonmutated peptides, 40 fish per group for mutated peptides) were prime vaccinated at D0, with a 14-day interval before a booster vaccination at a dose of 20 μg per fish of synthetic peptide vaccine, which was mixed with Freund's adjuvant and administered by intraperitoneal injection. At D35, 33 fish were randomly selected for the TiLV challenge test (the mutant peptide vaccine did not perform), and survival rates were calculated at D51. (B) Specific antibody analysis against the S1399–410 epitope peptide in serum was collected at different time points in the KLH-S1399–410, S1, and PBS immunization groups. Serum samples (5 fish per group) were serially diluted 2-fold (initial as 1:100) and the peptide-specific antibody response of the serum was measured by the absorbance at 450 nm. Each well was coated with 2 μg of peptide. The values are presented as means absorbance ± SD for each dilution with triplicates. (C) Endpoint titers of antibodies targeting S1399–410 in serum samples at different time points, shown as the reciprocal of the highest dilution when the OD450 (samples)was ≥2× OD450 of preimmunization serum. (D) Specific antibody analysis against the S1 protein in serum collected at different time points in the KLH-S1399–410, S1, and PBS immunization groups. Serum samples (5 fish per group) were serially diluted 2-fold (initial as 1:100), and the S1-specific antibody response of the serum was measured by the absorbance at 450 nm. Each well was coated with 0.5 μg of S1 protein. The values are mean absorbance ± SD for each dilution with triplicates. (E) Endpoint titers of antibodies targeting S1 protein in serum samples at different time points, shown as the reciprocal of the highest dilution when the OD450 (samples) was ≥2× OD450 of preimmunization serum. (F) Dose-response neutralization of live TiLV titration curves and IC50 heat map of serum samples with different vaccination groups at D21, D28, D35, and D42. Serum was diluted from 1:20 to 1:1,280 at 2-fold ratios, and the test was repeated 3 times per serum sample (5 fish mix serum). V are expressed as means ± SD. (G and H) Depletion or nondepletion of antibodies to specific peptide or protein in the serum at D35 after primary immunization was validated by ELISA. Anti-S1399–410 IgM reactive after depletion of serum antibodies specific to S1399–410 (left) or S1 protein (right) (G). Anti-S1 IgM reactive after depletion of serum antibodies specific to S1399–410 (left) or S1 protein (right) (H). (I) Neutralization efficacy of pooled sera not depleted or depleted of specific peptide or protein antibodies against live TiLV. Data are expressed as percent neutralization relative to a blank group of unincubated serum and virus. Data are from an experiment performed in triplicate. Data are expressed as means. Statistical significance was tested by one-way ANOVA and Tukey’s multiple-comparison tests. ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05; ns, no significant difference.