Targeted Delivery of Toxoplasma gondii Antigens to Dendritic Cells Promote Immunogenicity and Protective Efficiency against Toxoplasmosis

Abstract

Toxoplasmosis is a major public health problem and the development of a human vaccine is of high priority. Efficient vaccination against Toxoplasma gondii requires both a mucosal and systemic Th1 immune response. Moreover, dendritic cells play a critical role in orchestrating the innate immune functions and driving specific adaptive immunity to T. gondii. In this study, we explore an original vaccination strategy that combines administration via mucosal and systemic routes of fusion proteins able to target the major T. gondii surface antigen SAG1 to DCs using an antibody fragment single-chain fragment variable (scFv) directed against DEC205 endocytic receptor. Our results show that SAG1 targeting to DCs by scFv via intranasal and subcutaneous administration improved protection against chronic T. gondii infection. A marked reduction in brain parasite burden is observed when compared with the intranasal or the subcutaneous route alone. DC targeting improved both local and systemic humoral and cellular immune responses and potentiated more specifically the Th1 response profile by more efficient production of IFN-γ, interleukin-2, IgG2a, and nasal IgA. This study provides evidence of the potential of DC targeting for the development of new vaccines against a range of Apicomplexa parasites.

Keywords: DEC-205; SAG1; single-chain fragment variable fragment antibody; toxoplasmosis; vaccination.

Figure 1

Design of vaccine proteins. (A) Structural 1YNT pdb model of SAG1 protein with two domains (D1 in N-terminal and D2 in C-terminal). (B) Schematic representation of the untargeted (SAG1t) and the targeted antigens (SA1 and SA2).

Figure 2

Structural characterization of the produced proteins. (A) Analysis of purified proteins from insect cells supernatant by Coomassie Blue staining and Western blot using rabbit polyclonal antibody anti-His or serum from an infected mouse under reducing and non-reducing conditions, respectively. (B) Representative elution profile of SAG1t, SA1, and SA2 size exclusion chromatography. 0.5 nmol of each purified protein was injected onto a Superdex 75 HR 10/30 column.

Figure 3

In vitro recognition and binding of the targeted protein to the cell surface. (A) Analysis by flow cytometry of the protein binding on bone marrow dendritic cells (BMDCs), CD11c+ splenic cells, and human foreskin fibroblast (HFF) cells. Cells were incubated overnight at 4°C with targeted or untargeted proteins, and the protein binding was assessed with anti-His FITC by flow cytometry. As a negative control, the cells were stained only with anti-His FITC. Binding was detected on CD11c+ gated cells (10,000 events). The percentages of cells labeled with anti-His FITC were calculated as change in fluorescence intensity compared to isotype control. (B) Immunofluorescence analysis of protein binding on BMDC cells. Coverslips were incubated overnight at 4°C with targeted or untargeted proteins, then fixed and blocked. Protein binding was revealed by immunofluorescence microscopy after incubation with anti-His FITC antibody.

Figure 4

Maturation and activation of bone marrow dendritic cells (BMDCs) following incubation with targeted or untargeted SAG1 protein. (A) Analysis of surface expression of maturation markers. BMDCs at day 10 of culture were stimulated 24 h with SA2 or SAG1t or left unstimulated (medium). Surface maturation markers (CD40, CD80, CD86, and MHCII) were assessed by flow cytometry. (B) Cytokines and chemokines secretion. Cytokines and chemokines were assayed by ELISA in the supernatants of stimulated cells. Results are expressed as median ± interquartile and represent one of two independent experiments. *P < 0.05; **P < 0.01.

Figure 5

Evaluation of the protection against chronic toxoplasmosis and humoral response induced following immunization. CBA/J mice (8/group) were primed and boosted twice with SA1, SA2, or SAG1t formulated with polyinosinique-polycytidylique acid (Poly I:C) by different administration routes. Control mice received Poly I:C by the combined routes. (A,B) Mice immunization with SA1 by intranasal, subcutaneous, and combined routes. (C,D) Mice immunization with SA1 and SA2 by combined routes. (D) Mice immunization with SAG1t and SA2 by combined routes. (A,C,G) Protection after vaccination. Protection was evaluated 1 month after challenge by analyzing the cyst load in brain tissue. Results are expressed as the mean ± SEM (n = 8) and represent one of two independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001. Detection of specific anti-SAG1 IgG antibodies and IgG subclasses in sera of immunized mice. Serum samples were tested by ELISA using SAG1t (B,D), P30 protein including the D1 domain and 17 additional amino acids (E) and SA1 (F) as the coating antigen. Results are expressed as the mean ± SEM (n = 8) and represent one of two independent experiments. *P < 0.05.

Figure 6

Specific antibody response in mice immunized with untargeted (SAG1t) or targeted (SA2) proteins. Detection of specific anti-SAG1 IgG antibodies (A) and IgG subclasses (B) in sera of mice (12/group) primed and boosted twice with SAG1t, SA2, or phosphate-buffered saline formulated with Poly I:C by combined routes. Serum samples collected after the last boost were tested by ELISA using SAG1t protein as the coating antigen. Results are expressed as the mean ± SEM (n = 12) of log2 titers and represent one of two independent experiments. ***P < 0.001. (C) Immunofluorescence assay of Toxoplasma gondii tachyzoites with sera of mice immunized with SAG1t or SA2 proteins. Tachyzoites were labeled with anti-Mouse IgG-TRITC antibody (1:200) after incubation with sera. Serum from non-infected and infected mice were used for negative and positive controls, respectively. (D) Western blot analysis of the IgA antibody response in nasal washes. IgA specific for SAG1 protein were analyzed 1 week after the last boost, in nasal washes from four mice of each group (control, SAG1t, and SA2).

Figure 7

Cellular response after vaccination with SAG1t or SA2 by the combined routes. CBA/J mice were primed and boosted twice by combined intranasal and subcutaneous routes with SAG1t, SA2, or phosphate-buffered saline formulated with polyinosinique-polycytidylique acid adjuvant. 7 days after the last immunization, mesenteric lymph node cells (A) and splenocytes (B) were collected from four mice in each group and restimulated with TE. Supernatants were collected after 24 (IL-2) or 72 h (IFN-γ, IL-10, and IL-13) for cytokines assay. Results are expressed as the mean ± SEM and represent one of two independent experiments. **P < 0.01.

Figure 8

Cytokine secretion by CD4+ and CD8+ T cells from mice immunized with untargeted (SAG1t) or targeted (SA2) proteins. CD4+ and CD8+ T cells were purified from control and immunized mice and incubated with SAG1t and SA2 prestimulated bone marrow dendritic cells (BMDCs). Supernatants were collected after 72 h for cytokine assay. (A) IFN-γ production by CD4+ T cells, (B) IL-2 production by CD4+ T cells, and (C) IFN-γ production by CD8+ T cells. Results are expressed as the mean ± SEM. *P < 0.05, ****P < 0.0001. (D) Seven days after the third immunization, spleen cell suspension from SA2 immunized mice was stimulated with TE, SAG1t, or SA2 in the absence or the presence of anti-CD4 or anti-CD8 mAb. IFN-γ was measured in supernatant after 72 h. Results are expressed as the mean ± SEM.