Repeated-dose toxicity and immunogenicity evaluation of norovirus P particle-based rotavirus vaccine in juvenile cynomolgus monkeys

Figures

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  Figure 1. Characterization of particle size, purity, and homogeneity of PP-P[6]-VP8 and PP-P[8]-VP8 monovalent virus-like particles (VLPs). (A) Transmission electron microscopy images demonstrate that both PP-P[6]-VP8 and PP-P[8]-VP8 rotavirus multimeric particles are well-assembled, structurally intact, and highly uniform. Scale bar=100 nm. (B) Particle size distribution analysis indicates that the diameters of both PP-P[6]-VP8 and PP-P[8]-VP8 particles are approximately 20 nm, with high uniformity. (C) Reducing SDS-PAGE analysis shows a single band, corresponding to a molecular mass of 45–55 kDa, indicating high purity of the assembled particles.

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  Figure 2. Overview of the repeat-dose toxicity study design for the bivalent subunit rotavirus vaccine. Cynomolgus monkeys received four intramuscular injections of either sodium chloride (blank control), aluminum hydroxide adjuvant (adjuvant control), or the bivalent subunit vaccine (60 μg or 120 μg protein per dose).

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  Figure 3. Body weight changes and weight gain (A, B) in cynomolgus monkeys following four administrations of the bivalent rotavirus vaccine. n = 10 per group from day −14 to day 84; n = 4 per group from Dr7 to Dr42. Comparisons were made with the control group (P > 0.05).

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  Figure 4. Humoral immune response to the bivalent rotavirus vaccine in cynomolgus monkeys. (A, B) Serum IgG and IgA binding antibodies specific to P[6]-VP8 and P[8]-VP8 on day 57 were quantified using indirect ELISA. (C, D) Serum IgG and IgA binding antibodies specific to P[6]-VP8 and P[8]-VP8 on day 57 were quantified using indirect ELISA. (E, F) Neutralizing antibody titers against the Wa (G1P[8]), DS-1(G2P[4]) strains were measured in serum samples collected on days 56 and 84 using a virus neutralization assay. n = 10 animals/group. Comparisons were made using one-way analysis of variance (ANOVA). **P < 0.01 compared to the adjuvant control group.

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  Figure 5. Kinetics of serum-specific IgG antibodies. Sample size: n = 10 animals/group on days 28, 56, and 84; n = 4 animals/group on Dr14 and Dr42. Results are expressed as mean ± SD. (A) Serum IgG levels against P[6]-VP8. (B) Serum IgG levels against P[8]-VP8.

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  Figure 6. Cellular immune response to the bivalent rotavirus vaccine in cynomolgus monkeys on Dr2. (A) Splenocytes were plated in ELISpot wells, and IFN-γ, IL-2 and IL-4 cytokine-producing cells were detected. (B) IFN-γ-producing cells, (C) IL-2-producing cells, and (D) IL-4-producing cells were detected by ELISpot after stimulation with either P[6]-VP8 or P[8]-VP8. Splenocytes were collected on Dr2 following the fourth immunization. n = 6 animals/group. Comparisons were performed using one-way ANOVA. **P < 0.01 compared to the adjuvant control group.

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  Figure 7. Histopathological analysis of major organs in cynomolgus monkeys following repeated bivalent vaccine administration. No significant histopathological abnormalities were observed in the heart (A, a), brain (B, b), kidney (C, c), liver (D, d), lung (E, e), intestine (F, f), or spleen (G, g) between the control group (A–G) and the high-dose vaccine group (a–g). Representative gross and histological images of the injection site muscle and inguinal lymph nodes are shown for the control group (H, I, J) and the high-dose group (h, i, j). The arrow indicates yellowish patchy deposits at the injection site (h). Hematoxylin and eosin staining: (A–i), ×200; (J–j), ×20.