Immunotoxicity Assessment of a VLP-Based Vaccine for the Prevention of Rotavirus Infection

Objective: This study evaluated the potential immunotoxicity and safety of a novel hexavalent virus-like particle (VLP)-based rotavirus vaccine candidate by assessing the t-cell-dependent antibody response (TDAR) to keyhole limpet hemocyanin (KLH) in Sprague-Dawley (SD) rats. Methods: SD rats (24 males, 24 females) received three intramuscular vaccine injections at antigen doses of 30, 120, or 600 µg at 14 d intervals, followed by two KLH immunizations (on d 29 and d 35). Safety was monitored via clinical signs, body weight, food consumption, gross pathology, organ weight, and plasma KLH-specific immunoglobulin M (KLH-IgM) and immunoglobulin G (KLH-IgG) levels measured by enzyme-linked immunosorbent assay (ELISA) on d 35, d 42, and d 49. Results: Repeated-dose administration of the "Gam-VLP-rota" vaccine was tolerated, causing no mortality, adverse clinical signs, gross tissue pathology, or systemic organ toxicity. Absolute body weight and food intake remained within physiological norms. Comprehensive gravimetric analysis of internal organs revealed no adverse effects on the metabolic, reproductive, or central nervous systems in either sex. One-way ANOVA followed by Dunnett’s post-hoc test demonstrated that female organ weights remained stable, whereas male rats at the 600 μg dose exhibited a localized enlargement of the thymus (p<0.05). To systematically control type I error inflation across multi-dose longitudinal body weight data, post-hoc pairwise comparisons were subjected to Benjamini–Hochberg FDR adjustments, under which somatic variations did not exceed critical thresholds (adjusted q ≥ 0.3195). However, two-way ANOVA on log-transformed datasets revealed highly significant sex ⨯ treatment interactions for KLH-IgG on d 35 (p = 0.007) and KLH-IgM on d 49 (p = 0.028). Within-sex post-hoc testing with Bonferroni correction revealed a dose-dependent humoral response in females: on d 49, all vaccine groups (30, 120, and 600 µg) significantly exceeded the vehicle control group for KLH-IgM (padj ≤ 0.0125), validated by a monotonic Jonckheere–Terpstra trend test (p = 0.00097). Conclusion: The hexavalent VLP-based vaccine was well-tolerated with no overt immunosuppression or systemic toxicity observed. However, the toxicological significance of the observed thymus enlargement and antibody fluctuations requires further investigation.