IL-24 Is a Promising Molecular Adjuvant for Enhancing Protective Immunity Induced by DNA Vaccination Against Toxoplasma gondii

Abstract

Toxoplasma gondii, a parasitic protozoan, causes zoonotic infections with severe health impacts in humans and warm-blooded animals, underscoring the urgent need for effective vaccines to control these infections. In this study, a DNA vaccine encoding TgROP5, TgROP18, TgGRA7, TgGRA15, and TgMIC6 was formulated using the eukaryotic expression vector pVAX I. IL-24 was delivered as a molecular adjuvant using plasmid pVAX-IL-24. BALB/c, C57BL/6, and Kunming mouse strains received the DNA immunization, after which antibody levels, cytokine production, and lymphocyte surface markers were analyzed to assess immune responses. Additionally, survival rates and brain cyst counts were measured 1 to 2 months post-vaccination in experimental models of toxoplasmosis. As a result, compared to controls, the DNA vaccine cocktail significantly increased serum IgG levels, Th1 cytokine production, and proportions of CD4+/CD8+ T cells, leading to extended survival and reduced brain cyst counts post-challenge with T. gondii ME49. Furthermore, the five-gene DNA vaccine cocktail conferred greater protection compared to single-gene immunizations. Co-administration of IL-24 significantly enhanced the immune efficacy of the multi-gene DNA vaccination. Our findings suggest that IL-24 is an effective molecular adjuvant, enhancing the protective immunity of DNA vaccines against T. gondii, supporting its potential role in vaccine strategies targeting other apicomplexan parasites.

Keywords: T-cell activation; Th1 immunity; protective efficacy; protozoan vaccine; serological markers.

Figure 1

Determination of the expression of pVAX-IL-24 in vitro in 293-T cells by ELISA and immunization. (A) Flow chart of mice immunization and immunological analyses. (B) 293-T cells were transfected with empty pVAX I or pVAX-IL-24. Statistical significance is indicated as **** p < 0.0001.

Figure 2

Detection of specific anti-T. gondii humoral immune responses induced by DNA immunization with single or multiple genes in different mouse strains. (A) Measurement of IgG antibodies in the sera of Kunming mice at 0, 2, 4, and 6 weeks post-immunization. (B) Quantification of IgG1 and IgG2a antibodies in immunized mice two weeks after the final vaccination. Statistical significance is indicated as *** p < 0.001, ** p < 0.01, * p < 0.05. Data are presented as means ± SD.

Figure 3

Splenocyte proliferative response in immunized and control mice across different mouse strains. (A) Stimulation index (SI) for lymphocyte proliferation in immunized and control BALB/c mice, n = 3/group. (B) Stimulation index (SI) for lymphocyte proliferation in immunized and control Kunming mice, n = 3/group. (C) Stimulation index (SI) for lymphocyte proliferation in immunized and control C57BL/6 mice, n = 3/group. Statistical significance is indicated as *** p < 0.001, ** p < 0.01, * p < 0.05. Data are presented as means ± SD.

Figure 4

Percentages of CD4+ and CD8+ T cells in immunized and control mice across different mouse strains. (A) The proportion of CD4+ T cells is shown for both immunized and control groups in BALB/c, C57BL/6, and Kunming mice, n = 3/group in each mouse strain. (B) The proportion of CD8+ T cells is shown for both immunized and control groups in BALB/c, C57BL/6, and Kunming mice, n = 3/group in each mouse strain. Statistical significance is indicated as *** p < 0.001. Data are presented as means ± SD.

Figure 5

Cytokine production by splenocytes from mice immunized with single or multiple genes across different mouse strains. Statistical significance is indicated as *** p < 0.001, ** p < 0.01, * p < 0.05, and ns: no significance. Data are presented as means ± SD.

Figure 6

Survival curves of immunized BALB/c, C57BL/6, and Kunming mice two weeks after the final immunization. (A) Survival rates of immunized mice (BALB/c, C57BL/6, and Kunming mice) challenged with 1 × 10³ tachyzoites of the RH strain, n = 8/group in each mouse strain. (B) Survival rates of immunized mice (BALB/c, C57BL/6, and Kunming mice) challenged with 100 cysts of the ME49 strain, n = 8/group in each mouse strain 100 cysts of the ME49 strain.

Figure 7

Protection against chronic toxoplasmosis in immunized mice two weeks after the final booster immunization. (A) Cyst reduction in immunized BALB/c mice, n = 6/group. (B) Cyst reduction in immunized C57BL/6 mice, n = 6/group. (C) Cyst reduction in immunized Kunming mice, n = 6/group. Bars represent the mean cyst burden per mouse brain following an oral challenge with 10 cysts of the ME49 strain. Cyst load was determined from whol-brain homogenates collected four weeks post-challenge. Data are presented as means ± SD (representative of three experiments). Statistical significance is indicated as *** p < 0.001, and ** p < 0.01 compared to control groups.