Levamisole, as a viral vaccine adjuvant, induces robust host defense through the modulation of innate and adaptive immune responses

Abstract

Introduction: An effective vaccination policy must be implemented to prevent foot-and-mouth disease (FMD). However, the currently used vaccines for FMD have several limitations, including induction of humoral rather than cellular immune responses.

Methods: To overcome these shortcomings, we assessed the efficacy of levamisole, a small-molecule immunomodulator, as an adjuvant for the FMD vaccine. We conducted in vitro studies using murine peritoneal exudate cells (PECs) and porcine peripheral blood mononuclear cells (PBMCs) and in vivo studies using mice (experimental animals) and pigs (target animals). We evaluated levamisole-mediated modulation of the innate and adaptive immune responses; early, mid-term, and long-term immune-inducing effects; modes of action; and host defense against viral infection.

Results: Levamisole treatment promoted IFNγ secretion in murine PECs and porcine PBMCs. Additionally, it induced robust and long-lasting immune responses by eliciting high antibody titers and high virus-neutralizing antibody titers. By activating downstream signaling pathways of various pattern-recognition receptors, levamisole stimulated the expression of multiple cytokines and costimulatory molecules. Owing to these immunostimulatory effects, levamisole elicited host defense against viral infections in pigs. Our findings demonstrate the potential of levamisole as an immunostimulatory agent.

Discussion: The results also indicate that levamisole, as an adjuvant for animal vaccines, can elicit robust innate and adaptive immune responses, thereby enhancing host defense against viral infections. This study provides a promising approach for the development of improved FMD vaccine strategies in the future.

Keywords: host defense; immunomodulation; innate and adaptive immunity; levamisole; vaccine adjuvant.

Figure 1

Levamisole mediates innate immune response via IFNγ secretion in murine PECs and porcine PBMCs. To evaluate the innate immune response to levamisole, with or without inactivated foot-and-mouth disease virus (FMDV) type O (O PA) or A (A YC) antigen, IFNγ secretion was determined using the enzyme-linked immunosorbent spot (ELISpot) assay. (A) Number of IFNγ-secreting cell spots in murine peritoneal exudate cells (PECs). (B) Images of IFNγ-secreting murine PECs. (C) Number of IFNγ-secreting cell spots in porcine peripheral blood mononuclear cells (PBMCs). (D) Images of IFNγ-secreting porcine PBMCs. Data are presented as the mean ± SEM of the number of spot-forming cells (SFCs) obtained from triplicate measurements (n = 3/group). Statistical analyzes were conducted using a one-way ANOVA followed by Tukey’s post-hoc test. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, and ^****p < 0.001.

Figure 2

Levamisole enhances the efficacy of FMD vaccine and protects mice against FMDV infection. C57BL/6 mice (n = 5/group) were administered foot-and-mouth disease (FMD) vaccine containing inactivated FMD virus (FMDV) type O (O PA2) and A (A YC) antigens (0.375 + 0.375 μg/dose/100 μL; 1/40 of the dose for pigs), 100 μg levamisole/dose/mouse, ISA 206 (50%, w/w), 10% Al(OH)₃, and 15 μg Quil-A. The positive control (PC) group received the same volume and composition of vaccines as did the experimental (Exp) group but without the addition of levamisole as an adjuvant. The negative control (NC) group was injected with an equal volume of phosphate-buffered saline (PBS). The test vaccines were injected via the intramuscular route into mice that were later challenged with FMDV O (100 lethal dose 50%, LD₅₀ O/VET/2013) or FMDV A (100 LD₅₀ A/Malay/97), at 7 dpv via the intraperitoneal route. The survival rates and body weights were monitored for 7 days post-challenge (dpc). (A–E) Experimental strategy (A); survival rates post-challenge with O/VET/2013 (B) and A/Malay/97 (C); changes in body weight post-challenge with O/VET/2013 (D) and A/Malay/97 (E). Data are presented as the mean ± SEM of values from triplicate measurements (n = 5/group).

Figure 3

FMD vaccine containing levamisole elicits potent humoral immune response in mice. C57BL/6 mice (n = 5/group) were administered foot-and-mouth disease (FMD) vaccine containing inactivated FMD virus (FMDV) type O (O PA2) and A (A YC) antigens (0.375 + 0.375 μg/dose/100 μL; 1/40 of the dose for pigs), 100 μg levamisole/dose/mouse, 50% ISA 206 (w/w), 10% Al(OH)₃, and 15 μg Quil-A. The positive control (PC) group received the same volume and composition of vaccines as did the experimental (Exp) group but without the addition of levamisole as an adjuvant. The negative control (NC) group was injected with an equal volume of phosphate-buffered saline (PBS). Mice were vaccinated with the test vaccine via the intramuscular route, and blood was collected at 0, 7, 28, 56, and 84 days post-vaccination (dpv) for serological analysis using SP O and A ELISA and VN titers for O/PKA/44/2008 (O PA2) and A/SKR/YC/2017 (A YC). (A–E) Experimental strategy: (A) Ab titers, determined using SP O (B) and SP A (C) ELISA; and VN titers for O PA2 (D) or A YC (E) determined using the VN test. Data are presented as the mean ± SEM of values from triplicate measurements (n = 5/group). Statistical analyzes were performed using a two-way ANOVA followed by Tukey’s post-hoc test. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, and ^****p < 0.0001.

Figure 4

FMD vaccine containing levamisole elicits potent humoral immune response in pigs. Foot-and-mouth disease virus (FMDV) type O and A antibody seronegative pigs (8–9 weeks old) were used. The pigs were divided into three groups (n = 5–6/group) and administered inactivated bivalent FMD vaccine without (positive control (PC) group) or with 1 mg/dose/pig levamisole (experimental (Exp) group). The PC group received FMDV type O (O PA2) and type A (A YC) antigens (15 + 15 μg/dose/mL, one dose for cattle and pig use) with ISA 206 (50%, w/w), 10% Al(OH)₃, and 150 μg Quil-A. Vaccination was performed twice at 28-day intervals, with 1 mL vaccine (one dose) injected via the deep intramuscular route into the neck of the animals. The negative control (NC) group was injected with an equal volume of phosphate-buffered saline (PBS). Blood samples were collected from pigs at 0, 7, 14, 28, 42, 56, and 84 days post-vaccination (dpv) for serological assays. (A–E) Experimental strategy (A); Abs titers, determined using SP O (B) and SP A (C) ELISA; and VN titers for O PA2 (D) or A YC (E), determined using the VN test. Data are presented as means ± SEM of triplicate measurements (n = 5–6/group). Statistical analyzes were performed using two-way ANOVA followed by the Tukey’s post-hoc test. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, and ^****p < 0.0001.

Figure 5

FMD vaccine containing levamisole increases the levels of IgG, IgA, and IgM in pigs. The experimental strategy and method used were the same as those described in the Figure 4 legend. (A–C) IgG (A), IgA (B), and IgM (C) concentrations. Data are presented as the means ± SEM of triplicate measurements (n = 5–6/group). Statistical analyzes were performed using two-way ANOVA followed by the Tukey’s post-hoc test. ^***p < 0.001, and ^****p < 0.0001.

Figure 6

FMD vaccine containing levamisole mediates the expression of immunoregulatory genes in PBMCs from vaccinated pigs. Porcine peripheral blood mononuclear cells (PBMCs) isolated from the whole blood of vaccinated pigs (n = 5–6/group), as described in Figure 4A legend, were used for qRT-PCR assays. Gene expression levels were normalized against HPRT levels and are presented as a ratio compared to the control levels. (A–Z) Expression levels of RIG-I (A); TLR9 (B); dectin-1 (C); dectin-2 (D); SYK (E); CARD9 (F); CARD11 (G); NF-κB (H); BCL10 (I); MALT1 (J); STAT1 (K); IFNα (L); IFNβ (M); IFNγ (N); IL-1β (O); IL-6 (P); IL-12p40 (Q); IL-17A (R); IL-18 (S), IL-23p19 (T); CD80 (U); CD86 (V); CD28 (W); CD19 (X); CD21 (Y); and CD81 (Z). Data are presented as means ± SEM of triplicate measurements (n = 5–6/group). Statistical analyzes were conducted using one-way ANOVA followed by the Tukey’s test. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, and ^****p < 0.001.

Figure 7

FMD vaccine containing levamisole elicits potent host defense against FMDV infection in pigs. For the challenge experiments, foot-and-mouth disease virus (FMDV) type O and A antibody-seronegative pigs (8–9 weeks old, n = 3–4/group) were administered the FMD vaccine containing FMDV type O (O PA2) and type A (A YC) antigens (15 + 15 μg/dose/mL, one dose for cattle and pig use) with levamisole (1 mg/dose/pig), ISA 206 (50%, w/w), 10% Al(OH)3, and 150 μg Quil-A. One-milliliter vaccine was prepared as a single dose and administered to the animals, via an intramuscular injection. The positive (PC) and negative (NC) controls were treated with an equal volume of a commercial FMD vaccine (O Primorsky+A Zabaikalski, ARRIAH-VAC^®; FGBI “ARRIAH”) and phosphate-buffered saline (PBS), respectively, via the same route. Blood samples were collected at 0 and 28 days post-vaccination (dpv) for serological assays. Vaccinated pigs were challenged with FMDV type O (O/SKR/JC/2014) on the heel bulb, at a dose of 10⁵ TCID₅₀/100 μL, at 28 dpv. (A–J) Experimental workflow (A); antibody titers, determined using SP O (B) and SP A (C) ELISA; VN titers for O PA2 (D), A YC (E), O JC (F), A GP (G), determined using the VN test; and clinical score and viral load in serum samples and oral swabs from the NC (n = 4/group) (H), PC (commercial FMD vaccine, n = 3/group) (I), and experimental (Exp; O PA2 + A YC + levamisole, n = 4/group) groups (J) infected with the FMDV type O (O/SKR/JC/2014). The left Y-axis of the graph depicts the viral load in the serum and oral swab samples, represented as log₁₀ values, whereas the right Y-axis depicts the clinical index as the maximum value of 10 points. Data are presented as the means ± SEM of triplicate measurements (n = 3–4/group). Statistical analyzes were performed using a two-way ANOVA followed by the Tukey’s test. ^****p < 0.0001.