Chitosan Microsphere Used as an Effective System to Deliver a Linked Antigenic Peptides Vaccine Protect Mice Against Acute and Chronic Toxoplasmosis

Abstract

Multiple antigenic peptide (MAP) vaccines have advantages over traditional Toxoplasma gondii vaccines, but are more susceptible to enzymatic degradation. As an effective delivery system, chitosan microspheres (CS) can overcome this obstacle and act as a natural adjuvant to promote T helper 1 (Th1) cellular immune responses. In this study, we use chitosan microparticles to deliver multiple antigenic epitopes from GRA10 (G10E), containing three dominant epitopes. When G10E was entrapped within chitosan microparticles (G10E-CS), adequate peptides for eliciting immune response were loaded in the microsphere core and this complex released G10E peptides stably. The efficiency of G10E-CS was detected both in vitro, via cell culture, and through in vivo mouse immunization. In vitro, G10E-CS activated Dendritic Cells (DC) and T lymphocytes by upregulating the secretion of costimulatory molecules (CD40 and CD86). In vivo, Th1 biased cellular and humoral immune responses were activated in mice vaccinated with G10E-CS, accompanied by significantly increased production of IFN-γ, IL-2, and IgG, and decreases in IL-4, IL-10, and IgG1. Immunization with G10E-CS conferred significant protection with prolonged survival in mice model of acute toxoplasmosis and statistically significant decreases in cyst burden in murine chronic toxoplasmosis. The results from this study indicate that chitosan microspheres used as an effective system to deliver a linked antigenic peptides is a promising strategy for the development of efficient vaccine against T. gondii.

Keywords: Toxoplasma gondii; chitosan microspheres; epitopes; peptides; vaccine.

Figure 1

Construction of T. gondii G10E-CS vaccine and potential immune mechanism involved in vaccination with G10E-CS in vivo. In this study, dominant B cell, CD8⁺ and CD4⁺ T cell epitopes from GRA10 protein of T. gondii were selected by bioinformatics and immunological analysis. Then the G10E-CS microsphere vaccine formed by three dominant epitopes and linked by sequence GS was loaded onto chitosan microspheres by emulsion cross-linking method. When BALB/c mice were immunized with G10E-CS, the G10E peptides released from the microspheres induced specific humoral and cellular response. At the same time, chitosan as an attractive adjuvant could induce activation of dendritic cells (DCs) via cGAS-STING signaling pathway and then promote T helper 1 (Th1) cellular immune responses.

Figure 2

Screening of dominant epitopes from GRA10 and characteristic of G10E-CS. (A) Screening of the dominant B cell epitopes (a) and T cell epitopes (b) from TgGRA10. Positive serum samples from T. gondii infected mice were used to screen dominant B cell epitopes by ELISA analysis. Lymphocyte proliferation test was used to identify dominant CD8⁺ T and CD4⁺ T cell epitopes from spleen cells of infected mice by CCK-8 assay. P1(TQSPPESRKKRRRSGKKKRGKRSV)(B cell epitope), P5(LGYCALLPL) (CD8⁺ T cell epitope) and P7(SGFSLSSGSGVSVVE) (CD4⁺ T cell epitope) were selected to construct the multiple antigenic peptide (MAP) of T. gondii GRA10 (G10E). Values represent mean ±SD (n = 5). *p < 0.05. (B) Immunogenicity of P1, P5, P,7 and G10E peptides. Mice immunized with P1(B cell epitope), P5(CD4+ T cell epitope), P7(CD8+ T cell epitope) or G10E (P5-P7-P1) were tested for B cell antibody (a) and T cell proliferation ability (b).**p < 0.01. (C) The purity of G10E peptides detected by high performance liquid chromatography (HPLC) (a) and the molecular weight of G10E peptides analyzed by electrospray ionization mass spectrometry (ESI-MS) (b). (D) Scanning electron microscopy (SEM) images of G10E-CS microspheres prepared by emulsion crosslinking method. G10E-CS microspheres were then imaged at magnification of ×15,000 (bar represents 1 μm). (E) The diameter (a) and zeta of G10E-CS (b) microparticles was detected by Malvern particle size potentiometer. (F) Loading capacity (%LC) and encapsulation efficiency (%EE) of G10E-CS microspheres amongst 1–5 mg/ml concentration of total G10E peptides (a). Release profile of G10E peptides from free G10E peptides and crosslinked chitosan microparticles (G10E-CS) in vitro over a 15-day period (b). The amount of G10E in the supernatant was measured using BCA assay. Values represent mean ± SD (n = 3).

Figure 3

G10E-CS induce the activation of dendritic cells and lymphocyte in vitro. (A) The expression of the co-stimulatory molecules CD40 and CD86 on the surface of stimulated dendritic cells (DCs). DCs of C57BL/6 mice were incubated withG10E-CS, G10E, CS, CpG, or PBS for 48 h and the expression of CD40 and CD86 were detected by flow cytometry. Values represent mean ±SD (n = 5). *p < 0.05, **p < 0.01. (B) The analysis of T lymphocyte proliferation induced by stimulated DC cells. DCs of C57BL/6 mice were incubated with CpG, G10E-CS, CS, G10E, or PBS for 48 h and then they were cocultured with lymphocyte from C57BL/6 mice for 72 h. The lymphocyte proliferation was measured by CCK-8 assay. (C) The expression of cytokines (IFN-γ, IL-2) in the supernatant of lymphocytes induced by the stimulated DC cells. The supernatants of mixed cells were assayed for IL-2 and IFN-γ using ELISA assay. Data represent mean ±SD representative of three independent experiments. *p < 0.05, **p < 0.01. (D) Cytotoxicity of G10E-CS microparticles on dendritic cells (DCs) (a) and T cells (b). DCs and T cells were incubated with different concentrations (125, 250, and 500 μg/ml) of G10E-CS microsphere for different incubation times (48 and 72 h) and cytotoxicity of G10E-CS was assessed by CCK-8 assay (mean ± SD, n = 5).

Figure 4

Humoral immune response induced in BALB/C mice after vaccination with G10E-CS. (A) Detection of total anti-GRA10 IgG antibody in the sera of immunized BALB/C mice. The mice were immunized intramuscular injection three times at 2 weeks interval with G10E, CS, G10E-CS, or PBS. Sera were collected from mice by retro-orbital bleeding at 0, 2, 4, and 6 weeks after immunization. The levels of T. gondii-specific IgG antibody in the sera of samples was used to determine by ELISA assay. (B) Detection of IgG subclass IgG1 and IgG2a antibodies in the sera of the immunized mice. Sera were collected at 2 weeks after the last vaccination. The level of IgG1 and IgG2a antibodies were measured by ELISA. Results are expressed as the mean of OD450 ± SD (n = 15) and are representative of at least three independent experiments. *p < 0.05, **p < 0.01.

Figure 5

Cellular immune response induced in BALB/C mice after vaccination with G10E-CS. BALB/c mice were immunized with G10E-CS, CS, G10E, or PBS three times at 2-week intervals. Then splenocytes were harvested from three mice per group 2 weeks after the final immunization. Cellular immune responses were analyzed after stimulation with G10E peptides (10 μg/ml) for 72 h. (A) The lymphocyte proliferative response in immunized mice was measured by CCK-8 assay. The absorbance was detected at 450 nm and the stimulation index (SI) was calculated. (B) The percentages of T lymphocyte subsets (a) and ratio of CD8⁺/CD4⁺ (b) in immunized mice. The G10E stimulated splenocytes were stained with anti-mouse CD3-APC, anti- mouse CD4-FITC and anti-mouse CD8-PE for 30 min and cell population was analyzed by flow cytometry. (C,D) The production of cytokines (IFN-γ, IL-2) (C) and cytokines (IL-4, IL-10) (D) in the immunized mice. The splenocytes were stimulated with G10E peptides for 72 h in the presence of Cell Stimulation Cocktail to inhibit the secretion of cytokine into the extracellular and were fixed using an Intracellular Fixation & Permeabilization Buffer Set Kit. After stained with anti-mouse CD4-FITC, anti-mouse CD8-PE, anti-mouse IL-2 (APC), anti-mouse IFN-γ (PerCP-Cyanine 5.5), anti-mouse IL-4 (APC) and anti-mouse IL-10 (PerCP-Cyanine5.5) for 30 min, the cells population was analyzed by flow cytometry. Data represent the mean ± SD and splenocytes from three mice in each group were tested individually. *p < 0.05, **p < 0.01.

Figure 6

Protection of G10E-CS immunized mice against acute and chronic Toxoplasma gondii infection. (A) Survival rate of vaccinated mice after lethal RH strain tachyzoites challenge. Two weeks after the final immunization, 10 mice per group were intraperitoneally infected with 1 × 10³ tachyzoites of RH strain and observed daily for mortality. (B) The cyst number in the brain of mice after sublethal PRU strain cyst challenge. 10 mice per group were challenged orally with a sublethal dose of cysts (30 cysts) of the PRU strain (type II). Cyst load was counted from whole brain homogenates of mice 45 days after challenge. Data are representative results of three independent experiments and are represented as the means ± SD. *p < 0.05, **p < 0.01.