Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Aug 27:12:726615.
doi: 10.3389/fimmu.2021.726615. eCollection 2021.

With Chitosan and PLGA as the Delivery Vehicle, Toxoplasma gondii Oxidoreductase-Based DNA Vaccines Decrease Parasite Burdens in Mice

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

With Chitosan and PLGA as the Delivery Vehicle, Toxoplasma gondii Oxidoreductase-Based DNA Vaccines Decrease Parasite Burdens in Mice

Zhengqing Yu et al. Front Immunol. .

Abstract

Toxoplasma gondii (T. gondii) is an intracellular parasitic protozoan that can cause serious public health problems. However, there is no effectively preventive or therapeutic strategy available for human and animals. In the present study, we developed a DNA vaccine encoding T. gondii oxidoreductase from short-chain dehydrogenase/reductase family (TgSDRO-pVAX1) and then entrapped in chitosan and poly lactic-co-glycolic acid (PLGA) to improve the efficacy. When encapsulated in chitosan (TgSDRO-pVAX1/CS nanospheres) and PLGA (TgSDRO-pVAX1/PLGA nanospheres), adequate plasmids were loaded and released stably. Before animal immunizations, the DNA vaccine was transfected into HEK 293-T cells and examined by western blotting and laser confocal microscopy. Th1/Th2 cellular and humoral immunity was induced in immunized mice, accompanied by modulated secretion of antibodies and cytokines, promoted the maturation and MHC expression of dendritic cells, and enhanced the percentages of CD4+ and CD8+ T lymphocytes. Immunization with TgSDRO-pVAX1/CS and TgSDRO-pVAX1/PLGA nanospheres conferred significant immunity with lower parasite burden in the mice model of acute toxoplasmosis. Furthermore, our results also lent credit to the idea that TgSDRO-pVAX1/CS and TgSDRO-pVAX1/PLGA nanospheres are substitutes for each other. In general, the current study proposed that TgSDRO-pVAX1 with chitosan or PLGA as the delivery vehicle is a promising vaccine candidate against acute toxoplasmosis.

Keywords: PLGA; Toxoplasma gondii; chitosan; immune protection; mouse; oxidoreductase.

PubMed Disclaimer

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
(A) Double digestion analysis of the recombinant plasmid TgSDRO-pVAX1. Line M: DL5000 marker; Line 1: Double digestion with EcoRI and XhoI. (B) Immunofluorescence analysis of the expression of TgSDRO-pVAX1 in HEK 293-T cells. Bar represented 25 μm. (C) Western blot was conducted to detect the expression of TgSDRO in HEK 293-T cells. Cell lysates of HEK 293-T cells transfected with TgSDRO-pVAX1 (Line 1) and pVAX1 (Line 2) detected by sera from T. gondii-infected rats. Line M: molecular weight (MW) marker proteins.
Figure 2
Figure 2
The morphology of TgSDRO-pVAX1/PLGA and TgSDRO-pVAX1/CS nanospheres. Freshly prepared TgSDRO-pVAX1/PLGA nanospheres (A) and those preserved over 3 months (C) were synthetized by a double emulsion solvent evaporation technique, while newly prepared TgSDRO-pVAX1/CS nanospheres (B) and those preserved over 3 months (D) were prepared by an ionic gelation technique (bar represented 500 nm).
Figure 3
Figure 3
The release profile of TgSDRO-pVAX1/PLGA and TgSDRO-pVAX1/CS nanospheres in vitro over a 14-day period. Each sample was conducted once. Bar represents mean ± SD (n = 3).
Figure 4
Figure 4
T. gondii detection in mouse tissues 7 days after infection. (A) T. gondii was detected by PCR based on ITS-1 sequence. Line M: DL5000 marker; Line P: positive control; Line N: negative control; Line 1-10: intestine, heart, leg muscle, brain, liver, spleen, lung, and kidney tissue. (B) T. gondii was detected by absolute qPCR based on a 529 bp repeat element. Results were showed as mean ± SD (n = 3).
Figure 5
Figure 5
Immunoglobulin determination of total IgG (A), isotypes IgG1 (B), and IgG2 (C) in the sera from immunized mice at weeks 0, 2, and 4. Each sample was conducted once, and values were estimated using one-way ANOVA followed by Dunnett’s test and shown as the mean of the OD450 ± SD (n = 5). Values between the TgSDRO-pVAX1/CS and TgSDRO-pVAX1/PLGA groups were compared with the independent t-test. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the blank or control group.
Figure 6
Figure 6
The dynamics of cytokine production in immunized mice. The mice sera were collected at weeks 0, 2, and 4, and the commercial ELISA kits were used to investigate the levels of IFN-γ (A), IL-4 (B), IL-10 (C), and IL-17 (D) in mice sera. Each serum was investigated once. Results were estimated using one-way ANOVA tracked by Dunnett’s test, and values were shown as mean ± SD (n = 5). Based on the independent t-test, values between the TgSDRO-pVAX1/CS and TgSDRO-pVAX1/PLGA groups were compared. **p < 0.01 and ***p < 0.001 compared with the blank or control group.
Figure 7
Figure 7
Analysis of DC maturation at weeks 0, 2, and 4 by flow cytometry. Five mice in each group were sacrificed; the bar graph showed the ratio of CD83 (A) and CD86 (B) molecules on splenic DCs, and the dot plots (C) showed the percentage of CD11c+ CD83+ and CD11c+ CD86+ cells. Each sample was conducted once. Results were estimated by one-way ANOVA followed by Dunnett’s test, and values were showed as mean ± SD (n = 5). Values between the TgSDRO-pVAX1/CS and TgSDRO-pVAX1/PLGA groups were compared with the independent t-test. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the blank group or the control group.
Figure 8
Figure 8
Analysis of MHC molecules at weeks 0, 2, and 4 by flow cytometry. Five mice in each group were sacrificed; the 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 conducted once. Results were estimated by one-way ANOVA followed by Dunnett’s test, and values were showed as mean ± SD (n = 5). Values between the TgSDRO-pVAX1/CS and TgSDRO-pVAX1/PLGA groups were compared with the independent t-test. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the blank group or the control group.
Figure 9
Figure 9
Splenocyte proliferation of different immunized mice. Three mice in each group were sacrificed, and spleen lymphocytes of each mouse were divided into four samples; the spleen lymphocytes were cultured using the medium from HEK 293-T lysate transfected with PBS (blank group), pVAX1 blank plasmid (control group), TgSDRO-pVAX1 recombinant plasmid (TgSDRO-pVAX1, TgSDRO-pVAX1/CS, and TgSDRO-pVAX1/PLGA groups). Each sample was measured once, and the bar graph was evaluated by one-way ANOVA followed by Dunnett’s test; values were showed as mean ± SD (n = 3). Values between the TgSDRO-pVAX1/CS and TgSDRO-pVAX1/PLGA groups were compared with the independent t-test. *p < 0.05 and ***p < 0.001 compared with the blank group or the control group.
Figure 10
Figure 10
The proportions of CD4+ and CD8+ T lymphocytes in all groups. Five mice in each group were sacrificed, and spleen lymphocytes from each animal were stained with CD3e-PE, CD4-FITC (A) or CD3e-PE, CD8-FITC (B). Determined by flow cytometry, each sample was conducted once. Results were estimated by one-way ANOVA followed by Dunnett’s test, and values were showed as mean ± SD (n = 5). Values between the TgSDRO-pVAX1/CS and TgSDRO-pVAX1/PLGA groups were compared with the independent t-test. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the blank group or the control group.
Figure 11
Figure 11
Copy number of a 529 bp repeat element in cardiac muscles from mice. Mice were intraperitoneally injected with 103 tachyzoites of the highly virulent T. gondii RH strain at week 4 (2 weeks after the last immunization). Seven days after the challenge, five mice in each group were sacrificed, and cardiac muscles from the tip of the heart were collected. Each sample was performed three times. Results were evaluated using one-way ANOVA followed by Dunnett’s test, and the values were shown as mean ± SD (n = 5). Values between the TgSDRO-pVAX1/CS and TgSDRO-pVAX1/PLGA groups were compared with the independent t-test. *p < 0.05 and ***p < 0.001 compared with the blank group or the control group.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Dubey JP, Hotea I, Olariu TR, Jones JL, Darabus G. Epidemiological Review of Toxoplasmosis in Humans and Animals in Romania. Parasitology (2014) 141(3):311–25. 10.1017/S0031182013001509 - DOI - PubMed
    1. Montoya JG, Liesenfeld O. Toxoplasmosis. Lancet (2004) 363(9425):1965–76. 10.1016/s0140-6736(04)16412-x - DOI - PubMed
    1. Weiss LM, Dubey JP. Toxoplasmosis: A History of Clinical Observations. Int J Parasitol (2009) 39(8):895–901. 10.1016/j.ijpara.2009.02.004 - DOI - PMC - PubMed
    1. Sroka J, Bilska-Zajac E, Wojcik-Fatla A, Zajac V, Dutkiewicz J, Karamon J, et al. . Detection and Molecular Characteristics of Toxoplasma Gondii DNA in Retail Raw Meat Products in Poland. Foodborne Pathog Dis (2019) 16(3):195–204. 10.1089/fpd.2018.2537 - DOI - PMC - PubMed
    1. Marques CS, Sousa S, Castro A, da Costa JMC. Detection of Toxoplasma Gondii Oocysts in Fresh Vegetables and Berry Fruits. Parasit Vectors (2020) 13(1):180. 10.1186/s13071-020-04040-2 - DOI - PMC - PubMed

Publication types

MeSH terms