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. 2021 Sep 29;13(10):1582.
doi: 10.3390/pharmaceutics13101582.

Nano DNA Vaccine Encoding Toxoplasma gondii Histone Deacetylase SIR2 Enhanced Protective Immunity in Mice

Zhengqing Yu  1 , Yujia Lu  1 , Wandi Cao  1 , Muhammad Tahir Aleem  1 , Junlong Liu  2 , Jianxun Luo  2 , Ruofeng Yan  1 , Lixin Xu  1 , Xiaokai Song  1 , Xiangrui Li  1
Affiliations

Nano DNA Vaccine Encoding Toxoplasma gondii Histone Deacetylase SIR2 Enhanced Protective Immunity in Mice

Zhengqing Yu et al. Pharmaceutics. .

Abstract

The pathogen of toxoplasmosis, Toxoplasma gondii (T. gondii), is a zoonotic protozoon that can affect the health of warm-blooded animals including humans. Up to now, an effective vaccine with completely protection is still inaccessible. In this study, the DNA vaccine encoding T. gondii histone deacetylase SIR2 (pVAX1-SIR2) was constructed. To enhance the efficacy, chitosan and poly (d, l-lactic-co-glycolic)-acid (PLGA) were employed to design nanospheres loaded with the DNA vaccine, denoted as pVAX1-SIR2/CS and pVAX1-SIR2/PLGA nanospheres. The pVAX1-SIR2 plasmids were transfected into HEK 293-T cells, and the expression was evaluated by a laser scanning confocal microscopy. Then, the immune protections of pVAX1-SIR2 plasmid, pVAX1-SIR2/CS nanospheres, and pVAX1-SIR2/PLGA nanospheres were evaluated in a laboratory animal model. The in vivo findings indicated that pVAX1-SIR2/CS and pVAX1-SIR2/PLGA nanospheres could generate a mixed Th1/Th2 immune response, as indicated by the regulated production of antibodies and cytokines, the enhanced maturation and major histocompatibility complex (MHC) expression of dendritic cells (DCs), the induced splenocyte proliferation, and the increased percentages of CD4+ and CD8+ T lymphocytes. Furthermore, this enhanced immunity could obviously reduce the parasite burden in immunized animals through a lethal dose of T. gondii RH strain challenge. All these results propose that pVAX1-SIR2 plasmids entrapped in chitosan or PLGA nanospheres could be the promising vaccines against acute T. gondii infections and deserve further investigations.

Keywords: PLGA; SIR2; Toxoplasma gondii; chitosan; histone deacetylase; immune protection.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
(a) Double digestion analysis of the recombinant plasmid pVAX1-SIR2. Line M: DL5000 marker; Line 1: Double digestion with EcoRI and XhoI. (b) Fluorescent microscopy imagines of HEK 293-T cells transfected with pVAX1-SIR2 and pVAX1 plasmids. Cells were detected by sera from rats against rTgSIR2 protein. Bar represents 25 μm.
Figure 2
Figure 2
SEM images of PLGA (a) and chitosan nanospheres (b) loaded with pVAX1-SIR2 plasmids. PLGA and chitosan nanospheres were synthesized by double emulsion evaporation technique (w/o/w) and ionic gelation technique. After being totally freeze-dried, nanospheres were imagined at magnification of ×30,000 (bar represented 500 nm).
Figure 3
Figure 3
The release characteristics of PLGA and chitosan nanosphere loaded with pVAX1-SIR2 plasmids in vitro. The amount of free pVAX1-SIR2 plasmids in the supernatant was measured by the nanodrop microvolume spectrophotometer. Each group had three replications and each replication was investigated once. Values were represented as mean ± SD (n = 3).
Figure 4
Figure 4
Toxicity analysis of PLGA and chitosan nanospheres loaded with pVAX1-SIR2 plasmids. Five serum samples from each group were separated and the levels of BUN were investigated by the spectrophotometry immediately after blood collection. Each replication was investigated once, and values were analyzed by one-way ANOVA analysis followed by Dunnett’s test. Values were shown as mean ± SD (n = 5).
Figure 5
Figure 5
Secretions of IgG (a), isotypes IgG1 (b), and IgG2a (c) in the sera separated from immunized animals at week zero, two, and four. The dilution ratio of tested sera was 1:100 in ELISA analysis, and each serum was investigated once. Values were analyzed by one-way ANOVA analysis followed by Dunnett’s test. Values between the pVAX1-SIR2/CS and pVAX1-SIR2/PLGA group were estimated by the independent t-test. Values were shown as mean of OD450 ± SD (n = 5). * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with blank or control group.
Figure 6
Figure 6
Cytokine secretions in animals. Using the commercial ELISA kits, the secretions of IFN-γ (a), IL-4 (b), IL-10 (c), and IL-17 (d) in animals’ sera were investigated at week zero, two, and four. Each serum was investigated once, and values were analyzed by one-way ANOVA analysis followed by Dunnett’s test. Values between the pVAX1-SIR2/CS and pVAX1-SIR2/PLGA group were estimated by the independent t-test. Values were shown as mean ± SD (n = 5). * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with blank or control group.
Figure 7
Figure 7
Analysis of splenic DC maturation at week zero, two, and four. The expression of CD83 and CD86 molecules on DCs was evaluated by flow cytometry. Bar graph showed the ratio of CD83 (a) and CD86 (b) molecules on the surface of splenic DCs, and the dot plots (c) showed the percentage of CD11c+ CD83+ and CD11c+ CD86+ cells. Each sample was investigated once, and values were analyzed by one-way ANOVA analysis followed by Dunnett’s test. Values between the pVAX1-SIR2/CS and pVAX1-SIR2/PLGA group were estimated by the independent t-test. Values were shown as mean ± SD (n = 5). * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with blank or control group.
Figure 8
Figure 8
Analysis of MHC molecules on the surface of DCs at week zero, two, and four. The expression of MHC-I and MHC-II molecules was evaluated by flow cytometry. Bar graph showed the ratio of MHC-I (a) and MHC-II (b) molecules on splenic DCs, and the dot plots (c) showed the percentage of CD11c+MHC-I+ and CD11c+MHC-II+ cells. Each sample was investigated once, and values were analyzed by one-way ANOVA analysis followed by Dunnett’s test. Values between the pVAX1-SIR2/CS and pVAX1-SIR2/PLGA group were estimated by the independent t-test. Values were shown as mean ± SD (n = 5). ** p < 0.01 and *** p < 0.001 compared with blank or control group.
Figure 9
Figure 9
Splenocyte proliferation of immunized animals. Three mice from each group were euthanized, the spleen lymphocytes were collected, and the lymphocytes from one mouse were randomized into four replications. Then, the lymphocytes were cultured with 20 μg/mL rTgSIR2 protein. Each replication was investigated once, and values were analyzed by one-way ANOVA analysis followed by Dunnett’s test. Values between the pVAX1-SIR2/CS and pVAX1-SIR2/PLGA group were estimated by the independent t-test. Values were shown as mean ± SD (n = 3). ** p < 0.01 and *** p < 0.001 compared with blank or control group.
Figure 10
Figure 10
Proportions of CD4+ and CD8+ T lymphocytes at week zero, two, and four. Stained with CD3e-PE, CD4-FITC (a) or CD3e-PE, CD8-FITC (b), the separated lymphocytes were evaluated by flow cytometry. Each sample was investigated once, and values were analyzed by one-way ANOVA analysis followed by Dunnett’s test. Values between the pVAX1-SIR2/CS and pVAX1-SIR2/PLGA group were estimated by the independent t-test. Values were shown as mean ± SD (n = 5). * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with blank or control group.
Figure 10
Figure 10
Proportions of CD4+ and CD8+ T lymphocytes at week zero, two, and four. Stained with CD3e-PE, CD4-FITC (a) or CD3e-PE, CD8-FITC (b), the separated lymphocytes were evaluated by flow cytometry. Each sample was investigated once, and values were analyzed by one-way ANOVA analysis followed by Dunnett’s test. Values between the pVAX1-SIR2/CS and pVAX1-SIR2/PLGA group were estimated by the independent t-test. Values were shown as mean ± SD (n = 5). * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with blank or control group.
Figure 11
Figure 11
Copy number of T. gondii 529 bp repeat element in cardiac muscles. Immunized animals were challenged with 103 tachyzoites two weeks after the second immunization (week four). Seven days after the challenge, cardiac muscles were harvested and investigated. Each sample was investigated three times and values were analyzed by one-way ANOVA analysis followed by Dunnett’s test. Values between the pVAX1-SIR2/CS and pVAX1-SIR2/PLGA group were estimated by the independent t-test. Values were shown as mean ± SD (n = 5). *** p < 0.001 compared with blank or control group.
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