Mincle and STING-Stimulating Adjuvants Elicit Robust Cellular Immunity and Drive Long-Lasting Memory Responses in a Foot-and-Mouth Disease Vaccine

Abstract

Conventional foot-and-mouth disease (FMD) vaccines exhibit several limitations, such as the slow induction of antibodies, short-term persistence of antibody titers, as well as low vaccine efficacy and safety, in pigs. Despite the importance of cellular immune response in host defense at the early stages of foot-and-mouth disease virus (FMDV) infection, most FMD vaccines focus on humoral immune response. Antibody response alone is insufficient to provide full protection against FMDV infection; cellular immunity is also required. Therefore, it is necessary to design a strategy for developing a novel FMD vaccine that induces a more potent, cellular immune response and a long-lasting humoral immune response that is also safe. Previously, we demonstrated the potential of various pattern recognition receptor (PRR) ligands and cytokines as adjuvants for the FMD vaccine. Based on these results, we investigated PRR ligands and cytokines adjuvant-mediated memory response in mice. Additionally, we also investigated cellular immune response in peripheral blood mononuclear cells (PBMCs) isolated from cattle and pigs. We further evaluated target-specific adjuvants, including Mincle, STING, TLR-7/8, and Dectin-1/2 ligand, for their role in generating ligand-mediated and long-lasting memory responses in cattle and pigs. The combination of Mincle and STING-stimulating ligands, such as trehalose-6, 6'dibehenate (TDB), and bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP), induced high levels of antigen-specific and virus-neutralizing antibody titers at the early stages of vaccination and maintained a long-lasting immune memory response in pigs. These findings are expected to provide important clues for the development of a robust FMD vaccine that stimulates both cellular and humoral immune responses, which would elicit a long-lasting, effective immune response, and address the limitations seen in the current FMD vaccine.

Keywords: PRR ligands; adjuvants; cytokines; foot-and-mouth disease; vaccine.

Figure 1

Adjuvanticity of PRR ligands and pro-inflammatory cytokines; significant enhancement of the memory response in mice against FMDV infection C57BL/6 mice were administered either a combination of PRR ligands or cytokines with the vaccine based on the vaccine composition of the positive control group. The PRR ligands and cytokines used in the experiment were as follows: TDB (Mincle agonist), c-di-GMP (STING agonist), Furfurman (Dectin-2 agonist), R848 (TLR-7/8 agonist), mIFNα, mIL-23, mIFNγ, mIL-2, mTNFα, mIL-15, and mIL-18. A negative control group of mice was injected with the same volume of PBS as the vaccine, and a positive control group received 11.7 ng (1/160 of the dose for cattle or pig use) of O/TWN/97-R Ag, ISA 206 (50%, w/w), 10% Al(OH)₃, and 15 μg Quil-A without PRR ligands and cytokines. The test vaccines were injected intramuscularly into mice that were later challenged with FMDV (100 LD₅₀ O VET 2013) at 56 dpv. Blood sampling was performed on 28 dpv and 56 dpv for the serological assays. The survival rates and body weights were monitored for 7 dpc. (A–E) represent (A) the strategy for this study; (B) antibody titer by SP O ELISA; (C) changes in body weight post vaccination; (D) survival rate against FMDV (O VET 2013); (E) changes in body weight post challenge with O VET 2013. The data are the mean ± SEM of triplicate measurements; statistical analyses were performed using two-way ANOVA with Bonferroni correction; ^#, *p < 0.05, ^##, **p < 0.01, and ***p < 0.001.

Figure 2

PRR ligands and cytokines promote the expansion of memory immune cells C57BL/6 mice were administered either a combination of PRR ligands or cytokines with the vaccine based on the vaccine composition of the positive control group. The PRR ligands and cytokines used in the experiment and the vaccination method are summarized in Figure 1. Peritoneal exudate cells (PEC) sampling was performed at 28 and 56 dpv for the flow cytometric assay. PEC was immunostained with fluorochrome-conjugated Abs to CD3, CD4, CD8a, CD44, CD62L, CD27, γδ TCR, CD335 (NKp46), CD11c, Anti-MHC Class II, CD11b, and anti-F4/80. Data were acquired by flow cytometry and analyzed by FlowJo software vX 0.7. (A–E) represent the expansion of immune cells; (A) CD4⁺ T cells; (B) CD8⁺ T cells; (C) CD44^high CD62^low T cells; (D) CD44^high CD27^low γδ T cells; (E) CD44⁺CD27⁺ B cells; (F) CD335 (NKp46)⁺CD27⁺ cells. The data are the mean ± SEM of triplicate measurements; statistical analyses were performed using two-way ANOVA with Bonferroni correction; *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3

PRR ligand-induced bovine and porcine PBMC proliferation, as assessed by a BrdU cell proliferation kit. Bovine and porcine PBMCs were co-incubated with either PRR ligands alone or a combination of PRR ligands or a mixture of oil, gel, and saponin. The PRR ligands used in the experiment were as follows: R848 (TLR-7/8 agonist), Curdlan (Dectin-1 agonist), Zymosan (Dectin-2/TLR-2 agonist), Furfurman (Dectin-2 agonist), TDB (Mincle agonist), c-di-GMP (STING agonist), MDP (NOD-2 agonist), MPLA-SM (TLR-4 agonist), chitosan (NLRP3 inflammasome inducer and MR agonist), poly(I:C) (TLR-3/MDA-5 agonist), poly(dA:dT), RIG-1/CDS agonist, and AIM2 inflammasome inducer. Gel alone, saponin alone, and a mixture of oil, gel, and saponin were also tested for comparison. At specific time points (96 h) after coincubation, cell proliferation was tested using a BrdU ELISA kit. (A–D) represent in vitro cell proliferation induced by the PRRs in bovine PBMCs; (A) PRR ligands alone; (B) PRR ligands with a mixture of oil, gel, and saponin; (C) combination of PRR ligands; (D) combination of PRR ligands with a mixture of oil, gel, and saponin. (E–H) represent in vitro cell proliferation induced by the PRRs in porcine PBMCs; (E) PRR ligands alone; (F) PRR ligands with a mixture of oil, gel, and saponin; (G) combination of PRR ligands; (H) combination of PRR ligands with a mixture of oil, gel, and saponin. The data are the mean ± SEM of triplicate measurements (n = 6); statistical analyses were performed using one-way ANOVA with Tukey's post-test; *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 4

PRR ligand-mediated long-lasting memory response in cattle. Cattle were administered either a combination of R848 (TLR-7/8 agonist) and TDB (Mincle agonist) or Curdlan (Dectin-1 agonist) and c-di-GMP (STING agonist) with the vaccine, based on the vaccine composition of the positive control group. A positive control group of cattle received 15 μg (1 dose for cattle use) of O/TWN/97-R Ag, ISA 206 (50%, w/w), 10% Al(OH)₃, and 150 μg Quil-A without PRR ligands. The vaccination was performed twice at a 28 days interval, and 1 ml vaccine (1 dose) was injected via the deep intramuscular route on the necks of the animals. Blood samples were collected at 0, 14, 28, 56, 84, 112, 140, and 168 dpv from the cattle for the serological assays. (A–C) represent (A) the strategy for this study; (B) antibody titers by SP O ELISA; (C) virus-neutralizing antibody titers. The data are the mean ± SEM of triplicate measurements; statistical analyses were performed using two-way ANOVA with Bonferroni correction; *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5

PRR ligand-mediated long-lasting memory response in pigs. Pigs were administered a combination of R848 (TLR-7/8 agonist) and TDB (Mincle agonist) or Furfurman (Dectin-2 agonist) and TDB (Mincle agonist) or TDB (Mincle agonist) and c-di-GMP (STING agonist) with the vaccine based on the vaccine composition of the positive control group. The positive control group of pigs received 15 μg (1 dose for pig use) of O/TWN/97-R Ag, ISA 206 (50%, w/w), 10% Al(OH)₃, and 150 μg Quil-A without PRR ligands. The vaccination was performed twice at a 28 days interval, and 1 ml vaccine (1 dose) was injected via the deep intramuscular route on the necks of the animals. Blood samples were collected at 0, 14, 28, 42, 56, 70, and 84 dpv from the pigs for the serological assays. (A–C) represent (A) the strategy for this study; (B) antibody titers by SP O ELISA; (C) virus-neutralizing antibody titers. The data are the mean ± SEM of triplicate measurements; statistical analyses were performed using two-way ANOVA with Bonferroni correction; *p < 0.05, **p < 0.01, ***p < 0.001.