A Bivalent Inactivated Vaccine Prevents Enterovirus 71 and Coxsackievirus A16 Infections in the Mongolian Gerbil

Figures

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  Fig. 1. Immunization with a bivalent inactivated vaccine increases Ag-specific antibodies and helper T cell cytokines. (A) Mongolian gerbils were immunized twice with a bivalent EV71 C4a/CVA16 vaccine via the intramuscular route (n=6-8). (B) At 1 week following the final immunization, Ag-specific IgG levels in the sera were determined. A Student’s t-test was performed, comparing the vaccine group with the vehicle group. (C) The gene expression of T cell cytokines was analysed in the spleen. Graphs show the mean ± SEM. Analysis was based on a one-way ANOVA with multiple comparisons; ns, not significant; *p<0.05, ***p<0.001, compared with vehicle group.

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  Fig. 2. Immunization with the bivalent inactivated vaccine protects the host against a lethal EV71 C4a and CVA16 challenge. Mongolian gerbils were immunized twice with a bivalent EV71 C4a/CVA16 vaccine via the intramuscular route (n=8). (A, B) At 1 week following the final immunization, gerbils were challenged with a lethal dose of (A) EV71 C4a and (B) CVA16 via the intraperitoneal route. Body weight, paralytic signs, and survival were monitored daily for 20 days. Graphs show the mean ± SEM. One-way ANOVA was performed; ns, not significant; ***p<0.001, virus-infected group vs immunized group. ###p<0.001, healthy controls vs virus-infected group. A log-rank (Mantel-Cox) test was performed for the survival rate.

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  Fig. 3. Immunization with the bivalent vaccine effectively prevents viral amplification in infected tissues. Mongolian gerbils were immunized twice with the bivalent EV71 C4a/CVA16 vaccine via the intramuscular route (n=3). (A, B) At 1 week following the final immunization, (A) EV71 C4a and (B) CVA16 were used for infection via the intraperitoneal route. On day 5 after infection, viral RNA content was analysed in the brainstem, muscle, spleen, and heart. Graphs show the mean ± SEM. One-way ANOVA was performed; ###p<0.001, vehicle vs virus-infected group; ***p<0.001, virus-infected group vs immunized group.

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  Fig. 4. Immunization with bivalent inactivated vaccine prevents tissue damage caused by EV71 C4a infection. Mongolian gerbils were immunized twice with the bivalent inactivated EV71 C4a/CVA16 vaccine via the intramuscular route (n=4). At 1 week following the final immunization, 3-week-old gerbils were infected with EV71 C4a via the intraperitoneal route. (A) On day 5 after infection, the muscle and heart were stained with haematoxylin-eosin. (B, C) The severity of tissue damage was scored in the (B) muscle and (C) heart. Graphs show the mean ± SEM. Non-parametric test (Kruskal-Wallis followed by Dunn’s multiple comparison test) was performed; ns, not significant; *p<0.05, **p<0.01, ***p<0.001.

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  Fig. 5. Immunization with the bivalent inactivated vaccine prevents tissue damage caused by CVA16 infection. Mongolian gerbils were immunized twice with the bivalent inactivated EV71 C4a/CVA16 vaccine via the intramuscular route (n=4). At 1 week following the final immunization, 3-week-old gerbils were infected with CVA16 via the intraperitoneal route. (A) On day 5 after infection, the muscle and heart were stained with haematoxylin-eosin. (B, C) The severity of tissue damage was scored in the (B) muscle and (C) heart. Graphs show the mean ± SEM. Non-parametric test (Kruskal-Wallis followed by Dunn’s multiple comparison test) was performed; *p<0.05, **p<0.01, ***p<0.001.