Immunogenicity and protective efficacy of recombinant chimeric antigens based on surface proteins of Toxoplasma gondii

Abstract

Introduction: Toxoplasmosis is caused by the opportunistic, cosmopolitan protozoan Toxoplasma gondii is one of the most common parasitoses in the world. This parasite can pose a threat to people with immunodeficiency but also to the fetus, since the invasion can lead to miscarriages. Moreover, this parasite can contribute to economic losses in livestock farming. These problems lead to the implementation of new, safe solutions for the development of effective toxoplasmosis immunoprophylaxis.

Methods: In this work, newly produced recombinant trivalent chimeric proteins of T. gondii, based on SAG1-SAG2 recombinant chimeric antigen that differ in one terminal antigenic component, were tested in terms of their ability to induce an effective post-vaccination response. Antigens were tested in vitro to assess their ability to elicit APC cells response and further mice of the C3H/HeOuJ strain were immunized using those antigens, to evaluate their immunogenicity and immunoprotective effect in vivo. Two weeks after the last dose mice were either sacrificed to assess selected parameters of the immune response or infected with T. gondii DX strain to determine the degree of protection one month later.

Results: The results of serological tests revealed a high level of serum IgG antibodies specific for the native T. gondii TLA antigens. TLA-stimulated splenocytes produced cytokines that are important in inhibiting protozoal invasion. Additionally, CD3⁺ CD4⁺ and CD3⁺ CD8⁺ T cell subpopulations of splenocytes were analysed by flow cytometry. One month after experimental infection mice were sacrificed, and their brains were isolated to count T. gondii tissue cyst. Immunization of mice with recombinant trivalent chimeric proteins of T. gondii resulted in reduction of tissue cyst burden rates reaching even 74%.

Discussion: The obtained results demonstrate strong immunogenicity of the studied proteins and will allow to select candidates for further research aimed at increasing the immunoprotective properties of experimental vaccines against toxoplasmosis based on T. gondii chimeric antigens.

Keywords: T. gondii experimental vaccine; Toxoplasma gondii; immunoprotection; murine experimental model; recombinant chimeric antigen.

Figure 1

In vitro stimulation of APC cells. (A) Relative activation of the NF-κB pathway (OD 650 nm) after stimulation of THP1-Blue monocytes with recombinant chimeric antigens, TLA RH, TLA Me49, LPS or unstimulated cells (medium). Data are presented as mean and SD (n ≥ 3 independent experiments). (B) T. gondii clearance assay shows T. gondii RH GFP viability in recombinant chimeric antigen, TLA Me49, LPS (control) stimulated ANA1 macrophages or unstimulated cells (medium). Data are presented as mean and SD (n ≥ 5 independent experiments). (C) Selected cytokines production levels by ANA1 macrophages after stimulation with recombinant antigens, TLA Me49, LPS (control), or unstimulated cells (medium). Data are presented as mean and SD (n ≥ 3 independent experiments). The dotted line (A-C) represents the mean of the medium. Analysis for data (A-C) was performed using the one-way ANOVA followed by the FDR two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli to compare each mean with every other mean, excluding LPS. Q-value is shown above brackets.

Figure 2

Antibody response following vaccination. (A) TLA RH specific IgG and IgM relative antibody levels. Sera dilution 1:500 (IgG) and 1:100 (IgM). The dotted line represents the cut-off value of the PBS group (mean + 2*SD). Data are presented as mean and SD (n = 4 mice). (B) Antigen specific IgG1 and IgG2a titers. Antibody titers were logarithmically transformed. Data are presented as mean and SD (n = 4 mice). IgG1/IgG2a graph shows the ratio of logarithmically transformed IgG1 and IgG2a titers. Data are presented as mean and SD (n = 4 mice). (C) Antibody functional assay indicates T. gondii viability after incubation with vaccinated and control (PBS) mouse sera in the presence (Comp.+) or absence (Comp.-) of complement factors. Data are presented as mean and SD (n = 4 mice). (D) Antigen specific IgG titer. Terminal antigen represents the single antigen used in a particular recombinant chimeric construct, such as MAG1 in SS-MAG1. Full chimera refers to the antigen used for vaccination. Antibody titers were logarithmically transformed. Data are presented as mean and SD (n = 4 mice). Analysis was performed using Brown-Forsythe and Welch’s ANOVA followed by Dunnett’s T3 multiple comparison test to compare each mean with every other mean of TLA RH specific IgG and IgM antibody level (A) and to compare each mean to the SS group of IgG1/IgG2a ratio (B). Two-way ANOVA followed by Tukey`s multiple comparison test was used to compare IgG1 IgG2a titers within the antigen group and between the antigens within the antibody isotype (B); to compare the means between each antigen with or without addition of complement factors, and to compare the means between Comp- and Comp+ in each antigen (C); to compare the titers of individual chimeric component antigens within a group of test sera and between different groups of test sera within an individual chimeric component antigen (D). ns – p_adj > 0.05, (*) – p_adj ≤ 0.05, (**) – p_adj ≤ 0.01, (***) – p_adj ≤ 0.001, (****) – p_adj ≤ 0.0001.

Figure 3

Post-vaccination cellular response. (A) Selected cytokine production levels by mouse splenocytes after vaccination. Data are presented as mean and SD (n = 4 mice). (B) Lymphoproliferation was assessed using the MTT assay and cell viability was calculated relative to unstimulated cells for each mouse, and then stimulation index (SI) was calculated based on antigen/PBS groups values. Data are presented as mean and SD (n = 4 mice). (C) Immunophenotyping of splenocytes after vaccination. Data are presented as mean and SD (n = 4-7 mice). The dotted line represents the mean of the PBS group (A, B). Analysis was performed using one-way ANOVA followed by the FDR two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli to compare each mean with PBS as a control (A, C). Q-value is shown above brackets. One-way ANOVA test followed by the Dunnet test to compare each mean of stimulation index with PBS as a control (B). (*) – p_adj ≤ 0.05, (**) – p_adj ≤ 0.01.

Figure 4

T. gondii tissue cyst burden in mice infected after vaccination. Data were calculated as the percentage of burden relative to PBS value. Data are presented as mean (value above dots) and SD (n = 10-20 mice). Analysis was performed using Brown-Forsythe and Welch’s ANOVA followed by Dunnett’s T3 multiple comparison test to compare each mean with every other mean. (*) – p_adj ≤ 0.05, (**) – p_adj ≤ 0.01, (***) – p_adj ≤ 0.001, (****) – p_adj ≤ 0.0001.