Enhanced Efficacy of Immunization with a Foot-and-Mouth Disease Multi-Epitope Subunit Vaccine Using Mannan-Decorated Inulin Microparticles

Abstract

Background: Despite the many advantages of recombinant subunit vaccines, they have critical weaknesses that include a low efficacy for promoting cellular and humoral immune responses against antigens because of their poor immunogenicity, and a rapidly cleared properties as a result of proteolytic enzymes in the body. To circumvent these problems, we developed mannan-decorated inulin acetate microparticles (M-IA MPs) that functioned as carriers and adjuvants for immunization with the recombinant foot-and-mouth disease multi-epitope subunit vaccine (M5BT).

Methods: The M5BT-loaded M-IA MPs were obtained by a double-emulsion solvent-evaporation method. Their properties including morphology, size and release ability were determined by field emission scanning electron microscope, dynamic light-scattering spectrophotometer and spectrophotometer. To assess the immunization efficacy of the MPs, mice were immunized with MPs and their sera were analyzed by ELISA.

Results: The M-IA MPs obtained by a double-emulsion solvent-evaporation method were spherical and approximately 2-3 µm, and M5BT was encapsulated in the M-IA MPs. The M5BT-loaded M-IA MPs showed higher antigen-specific IgG, IgG1, IgG2a and anti-FMDV antibodies than the M5BT-loaded IA MPs and the Freund's adjuvant as a control.

Conclusion: The M-IA MPs showed a powerful and multifunctional polymeric system that combined two toll-like receptor agonists compared to the conventional adjuvant.

Keywords: Food-and-mouth disease; Inulin acetate; Mannan; Microparticle; Subunit vaccine.

Fig. 1

Chemical reaction scheme for synthesis of inulin acetate

Fig. 2

¹H 600 MHz NMR spectra of A inulin and B inulin acetate. The asterisk indicates three hydrogens in an acetyl group with 2 ppm

Fig. 3

Analysis of the morphology of MPs by FE-SEM. A M5BT-loaded IA MPs and B M5BT-loaded M-IA MPs (Magnification 2000 X and scale bar (white bar): 10 µm)

Fig. 4

Analysis of dynamic light scattering for determining the diameter of the MPs. A IA MPs, B M-IA MPs, C M5BT-loaded IA MPs and D M5BT-loaded M-IA MPs. (d) means average diameter of MPs and (P.I.) is polydispersity Index

Fig. 5

In vitro release profile of the M5BT protein from M5BT loaded IA and M-IA MPs at serum pH. MPs (10 mg/ml) were suspended in PBS (pH 7.4). The protein concentration was measured by micro BCA assay. All values represent the mean ± SD (n = 3)

Fig. 6

Confirmation of mannan-decoration of INAC MPs by CLSM. FITC-labeled mannan (FITC-mannan) was used to decorate the IA MPs. A M-IA MPs and B FITC-M-IA MPs

Fig. 7

FMDV serotype O-specific antibody assay using SP competition ELISA. Test sera were collected at week 4 postimmunization. PI (%) means the percent inhibition. Each symbol represents the value for an individual animal. The dotted line describes 50 PI (%). Horizontal lines describe the mean value for each group of animals

Fig. 8

M5BT-specific IgG and IgG subtype immune responses at 4 weeks postimmunization. The anti-M5BT serum immunoglobulin titers were measured using blocking ELISA. Note that the endpoint titers of the result are represented on a log scale on the y-axis. A Anti-M5BT total IgG titers, B IgG1 titers and C IgG2a titers. All values represent the mean ± SD (n = 5). Significant differences were compared to the untreated group. *: p < 0.05; **: p < 0.01; ***: p < 0.001, one-way ANOVA

Fig. 9

In vitro released cytokines in the splenocyte culture supernatant of vaccinated animals when restimulated with antigen. The white bar is IL-4, and the gray bar is IFNγ. Relative A IFNγ and B IL-4 levels. All values represent the mean ± SD (n = 5). Significant differences were compared to the untreated group. **: p < 0.01; ***: p < 0.001, one-way ANOVA