Advanced Foot-And-Mouth Disease Vaccine Platform for Stimulation of Simultaneous Cellular and Humoral Immune Responses

Figures

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  Figure 1. Construction of the immunopotent foot-and-mouth disease virus (FMDV) vaccine strains A-3A and A-HSP70. (A,B) represent: (A) A-3A; (B) A-HSP70. FMDV T cell epitope 3A (the 15 amino acid residues in VP1) (A) and enhanced long-term immunity protein HSP70 (the 20 amino acid residues of VP1) (B) were selected as target molecules for preparation of immunopotent virus. The A22 (A22-R) P1, where the P1 region of O1 Manisa is substituted with A22 P1, was used as the backbone for the preparation of immunopotent FMD vaccine strains. The detailed strategy is presented in the Materials and Methods.

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  Figure 2. A-3A- and A-HSP70-mediated vaccine efficacy and protective effects in mice. (A–F) represent: (A–C) A-3A; (D–F) A-HSP70. C57BL/6 mice were administered the test vaccine at 1/10, 1/40, 1/160, 1/640 doses of A-3A or A-HSP70 antigen for cattle or pig use, ISA 206 (oil-based emulsion, 50%, w/w), 10% Al(OH)3, and 15 µg Quil-A. A negative control (NC) group was injected with the same volume of PBS. The test vaccines were injected intramuscularly into mice that were later challenged with FMDV (100 LD50 A/Malay/97) at 7 dpv. The survival rates and body weights were monitored for 7 dpc. Experimental strategy (A,D); survival rates post-challenge with A/Malay/97 (B,E); and changes in body weight post-challenge with A/Malay/97 (C,F). The data represent the mean ± SEM of triplicate measurements (n = 5/group). Statistical analyses were performed using two-way ANOVAs with a Bonferroni correction and a one-way ANOVA followed by a Tukey’s post-hoc test. ns, not significant. Statistical analyses are summarized in Table S2.

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  Figure 3. A-3A and A-HSP70 mediated a long-lasting memory response in mice. C57BL/6 mice were administered with oil emulsion-free test vaccines, including A-3A antigen alone, A-HSP70 antigen alone, or combined A-3A and A-HSP70 antigens based on their positive control composition. The negative control group received PBS in the same volume as the vaccine. A positive control group received 1.5 μg (1/10 dose for cattle and pig use) of naïve A antigen, without (w/o) oil emulsion, 10% Al(OH)3, and 15 μg Quil-A. Briefly, vaccination was performed twice at a 35-day interval. The mice were vaccinated intramuscularly in the thigh muscle. Later, at 84 dpv or 168 dpv, the mice were challenged with FMDV (100 LD50 A/Malay-97, SEA topotype) by intraperitoneal injection. Survival rates and body weights were monitored for 7 dpc. In addition, serum sampled from mice at 0, 7, 14, 28, 56, 84, and 168 dpv was analyzed via SP A ELISA and VN assay. (A–G) represent: (A) the strategy for this study; (B) antibody titers by SP A ELISA; (C) VN titers; (D) survival rate at 84 dpv challenge; (E) changes of body weight at 84 dpv challenge; (F) survival rate at 168 dpv challenge; (G) changes of body weight at 168 dpv…

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  Figure 4. A-3A and A-HSP70 mediated-immune response in pigs. For the pig experiments, FMD antibody-seronegative animals were used (the pigs were 10–12 weeks old). Pigs were divided into 3 groups (n = 5/group). Pigs were administrated oil emulsion free-test vaccines, including A-HSP70 antigen alone, or combined A-3A and A-HSP70 antigens based on their positive control composition. A positive control group of pigs received 15 µg (1 dose for cattle and pig use) naïve A antigen, without (w/o) oil emulsion, 10% Al(OH)3, and 150 µg Quil-A. The vaccination was performed twice at a 28-day interval, and 1 ml vaccine (1 dose) was injected via a deep intramuscular route at the animals’ necks. Blood samples were collected at 0, 7, 14, 28, 42, 56, 70, and 84 dpv in pigs for the serological assays and at 0, 14, 28, and 56 dpv for PBMC isolation. (A–C) represent: (A) the strategy for this study; (B) antibody titers by SP A ELISA; (C) VN titers. The data represent the means ± SEM of triplicate measurements (n = 5/group). Statistical analyses were performed using two-way ANOVA with Bonferroni correction. * p < 0.05; ** p < 0.01; *** p < 0.001.

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  Figure 5. A-3A and A-HSP70 induced co-stimulatory molecule and cytokine gene expression in porcine PBMCs. Porcine peripheral blood mononuclear cells (PBMCs) isolated from the whole blood of vaccinated pigs (n = 5/group) individually, as described in Figure 4, were used for the qRT-PCR assay. Gene expression levels were normalized to hprt levels and are presented as a relative ratio compared to control levels. (A–K) represent: gene expression levels of (A) CD40; (B) CD80; (C) CD86; (D) CTLA-4; (E) MHC class I; (F) MHC class II; (G) IFNα; (H) IFNβ; (I) IFNγ; (J) IL-12p40; (K) IL-17A in porcine PBMCs were analyzed. The data represent the means ± SEM of triplicate measurements (n = 5/group). Statistical analyses were performed using one-way ANOVA with Tukey’s post-hoc test. *, # p < 0.05; **, ## p < 0.01; ***, ### p < 0.001.