Bestatin, A Pluripotent Immunomodulatory Small Molecule, Drives Robust and Long-Lasting Immune Responses as an Adjuvant in Viral Vaccines

Abstract

An inactivated whole-virus vaccine is currently used to prevent foot-and-mouth disease (FMD). Although this vaccine is effective, it offers short-term immunity that requires regular booster immunizations and has several side effects, including local reactions at the vaccination site. To address these limitations, herein, we evaluated the efficacy of bestatin as a novel small molecule adjuvant for inactivated FMD vaccines. Our findings showed that the FMD vaccine formulated with bestatin enhanced early, intermediate-, and particularly long-term immunity in experimental animals (mice) and target animals (pigs). Furthermore, cytokines (interferon (IFN)α, IFNβ, IFNγ, and interleukin (IL)-29), retinoic acid-inducible gene (RIG)-I, and T-cell and B-cell core receptors (cluster of differentiation (CD)28, CD19, CD21, and CD81) markedly increased in the group that received the FMD vaccine adjuvanted with bestatin in pigs compared with the control. These results indicate the significant potential of bestatin to improve the efficacy of inactivated FMD vaccines in terms of immunomodulatory function for the simultaneous induction of potent cellular and humoral immune response and a long-lasting memory response.

Keywords: adjuvant; bestatin; cellular and humoral immunity; foot-and-mouth disease; inactivated vaccine.

Figure 1

Foot-and-mouth virus (FMDV) antigen (O PA2 or A YC) was treated with bestatin to induce interferon (IFN)γ secretion in murine peritoneal exudate cells (PECs) and porcine peripheral blood mononuclear cells (PBMCs). IFNγ secretion was assayed to evaluate the innate immune responses induced by bestatin with or without inactivated FMDV (iFMDV) antigens using the enzyme-linked immune absorbent spot (ELISpot) assay. (a–d) Mean number of IFNγ spot-forming cells (SFCs) stimulated under different conditions on PECs (a); mean number of IFNγ SFCs stimulated under different conditions on PBMCs (b); ELISpot representative images of IFNγ activated with negative control (NC), antigen (O PA2 or A YC) only, bestatin only, and antigen with bestatin for each concentration on PECs (c); ELISpot representative images of IFNγ activated with NC, antigen (O PA2 or A YC) only, bestatin only, and antigen with bestatin for each concentration on PBMCs (d). Data are presented as mean ± standard error of the mean (SEM) of SFCs from triplicate measurements (n = 3/group). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 (one-way analysis of variance (ANOVA) followed by Tukey’s test).

Figure 2

FMDV vaccine containing bestatin showed favorable vaccine efficacy and conferred protective effects on mice. C57BL/6 mice (6–7 weeks-old, n = 5/group) were observed for mortality for 7 days, and protection of each group was represented by Kaplan–Meier survival curves. (a–e) Experimental workflow (a); survival rates post-challenge with O/VET/2013 (b) and A/Malay/97 (d), respectively; changes in body weight post-challenge with O/VET/2013 (c) and A/Malay/97 (e), respectively. Data are presented as mean ± SEM of triplicate measurements (n  =  5/group). Abbreviations: dpv, days post-vaccination; I.M., intramuscular injection; LD₅₀, lethal dose, 50%.

Figure 3

FMDV vaccine containing bestatin upregulated early, intermediate-, and long-term immune responses in mice. C57BL/6 mice (6–7 weeks-old, n = 5/group) were vaccinated via I.M. injection. (a–e) Experimental workflow (a); antibody titers using SP ELISA type O (b) or type A (c); VN titers for O PA2 (d) or A YC (e) using the VN test. Data are presented as mean ± SEM of triplicate measurements (n = 5/group). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 (two-way ANOVA with Tukey’s test).

Figure 4

FMDV vaccine containing bestatin upregulated the early, intermediate-, and long-term immune response in pigs. For the pig experiments, FMDV (O and A) antibody-seronegative animals (8–9 weeks-old, n = 5–6/group) were used. (a–e) Experimental workflow (a); antibody titers using SP ELISA type O (b) or type A (c); VN titers for O PA2 (d) or A YC (e) using the VN test. Data are presented as mean ± SEM of triplicate measurements (n = 5–6/group). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 (two-way ANOVA with Tukey’s test).

Figure 5

Immune responses mediated by the FMDV vaccine with bestatin measured using the levels of immunoglobulin subtypes IgG, IgA, and IgM in pigs. For the pig experiments, FMDV type O and A antibody-seronegative animals (8–9 weeks-old, n = 5–6/group) were used. Single-dose vaccination was administered I.M. twice at 28-day intervals. (a–c) IgG (a); IgA (b); and IgM (c) concentrations. Data are presented as mean ± SEM of triplicate measurements (n = 5–6/group). ** p < 0.01, and **** p < 0.0001 (two-way ANOVA with Tukey’s test).

Figure 6

FMDV vaccine with bestatin induced the immunomodulatory gene expression in porcine PBMCs. In Figure 4a, PBMCs isolated from the whole blood of vaccinated pigs (n  =  5–6/group) were used for real-time quantitative PCR. Gene expression levels were normalized to HPRT levels and are presented as relative ratios compared with control levels. (a–j) Gene expression of IFNα (a); IFNβ (b); IFNγ (c); IL-29 (d); RIG-I (e); CD28 (f); CD19 (g); CD21 (h); CD81 (i); and C3d (j). Data are presented as mean ± SEM of triplicate measurements (n = 5–6/group). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 (one-way ANOVA followed by Tukey’s test).