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Cited by 37Toxoplasma gondii Research Studiesvaccines and immunoinformatics approachesCoccidia and coccidiosis research

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GRA24-Based DNA Vaccine Prolongs Survival in Mice Challenged With a Virulent Toxoplasma gondii Strain

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Zheng, Bin, Lou, Di, Ding, Jianzu, Zhuo, Xunhui, Ding, Haojie, Kong, Qingming, Lu, Shaohong·2019·Frontiers in Immunology
DOI 10.3389/fimmu.2019.00418PMC PMC6414464
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Abstract

Toxoplasma gondii causes zoonotic infections in a wide range of intermediate hosts and remains a threatening disease worldwide because of the lack of effective drugs and vaccines. Dense granule protein 24 (GRA24) is a novel essential virulence factor that is transferred into the nucleus of host cells from the parasitophorous vacuole to regulate gene expression. In the present study, bioinformatic analysis showed that GRA24 had a high score for B-cell and T-cell epitopes compared with surface antigen 1 (SAG1), which has been studied as a promising vaccine candidate. As a DNA vaccine, pVAX1-GRA24 was injected it intramuscularly into BALB/c mice and the induced immune response was evaluated. pVAX1-GRA24 induced high levels of a mixed Th1/ Th2 cytokines at 6 weeks after immunization. Antibody determinations, cytokines (interferon gamma (IFN-γ), interleukin (IL)-12, IL-4, IL-10), antigen-specific lymphocyte proliferation, CD4+ and CD8+ T lymphocytes, and cytotoxic T lymphocyte activity showed that mice immunized with pVAX1-GRA24 produced specific humoral and cellular immune responses. Activation of the interferon regulatory factor 8 (IRF8), nuclear factor kappa B (NF-κB), and T-Box 21 (T-bet) pathways was analyzed in the host immune response. Survival times were prolonged significantly (24.6 ± 5.5 days) in the mice immunized with pVAX1-GRA24 compared with the mice in the control groups, which died within 7 days of T. gondii challenge (p < 0.05). The results of the present study showed that pVAX1-GRA24 induced a T. gondii-specific immune response and thus represents a promising candidate vaccine to treat toxoplasmosis.

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Figure 1 Plot of the DNASTAR-predicted hydrophilicity, flexible regions, antigenic index, and surface probability plot of the linear-B cell epitopes of GRA24 compared with those of SAG1.
Figure 1 Plot of the DNASTAR-predicted hydrophilicity, flexible regions, antigenic index, and surface probability plot of the linear-B cell epitopes of GRA24 compared with those of SAG1.CC BY · PubMed Central
Figure 2 Expression of pET-28a- GRA24 in E. coli BL21(DE3) and pVAX1- GRA24 in HEK293 cell. (A) PCR amplified gra24 gene detected by 1.5% agarose gel. M: Markers; 1: Blank control; 2: coding region (CDS) of T. gondii. (B) Analysis of purified rGRA24 by SDS-PAGE. (C) The expression of pVAX1-GRA24 in HEK293 cells by an indirect immunofluorescence assay. (D) Western blotting detection of the expression of GRA24 in HEK293 cell lysates and STAg. Blank: non-transfected cells; pVAX1: empty pVAX1 transfected cells; pVAX1-GRA24 transfected cells; soluble antigen of T. gondii; rGRA24 acts as a positive control.
Figure 2 Expression of pET-28a- GRA24 in E. coli BL21(DE3) and pVAX1- GRA24 in HEK293 cell. (A) PCR amplified gra24 gene detected by 1.5% agarose gel. M: Markers; 1: Blank control; 2: coding region (CDS) of T. gondii. (B) Analysis of purified rGRA24 by SDS-PAGE. (C) The expression of pVAX1-GRA24 in HEK293 cells by an indirect immunofluorescence assay. (D) Western blotting detection of the expression of GRA24 in HEK293 cell lysates and STAg. Blank: non-transfected cells; pVAX1: empty pVAX1 transfected cells; pVAX1-GRA24 transfected cells; soluble antigen of T. gondii; rGRA24 acts as a positive control.CC BY · PubMed Central
Figure 3 DNA vaccination with pVAX1-GRA24 induced dynamic changes in humoral responses in BALB/c mice. (A) Total IgG; (B) IgG1, IgG2a. The OD450 value of total IgG were detected on 0, 2, 4, and 6 weeks post-vaccination. The OD450 value of IgG subclass (IgG1, IgG2a) were detected at 6 weeks post-vaccination. Data are expressed as mean ± SD (n = 20) from three independent experiments. Asterisks (*) indicate statistically significant differences (p < 0.05) compared with the control groups.
Figure 3 DNA vaccination with pVAX1-GRA24 induced dynamic changes in humoral responses in BALB/c mice. (A) Total IgG; (B) IgG1, IgG2a. The OD450 value of total IgG were detected on 0, 2, 4, and 6 weeks post-vaccination. The OD450 value of IgG subclass (IgG1, IgG2a) were detected at 6 weeks post-vaccination. Data are expressed as mean ± SD (n = 20) from three independent experiments. Asterisks (*) indicate statistically significant differences (p < 0.05) compared with the control groups.CC BY · PubMed Central
Figure 4 Percentages of CD4+ T cells and CD8+ T cells subsets in immunized BALB/c mice. (A) CD4+ T cells; (B) CD8+ T cells. Results are shown as the mean ± SD (n = 5 mice in each group) from three independent experiments. *p < 0.05 compared with the controls. PBS, phosphate-buffered saline.
Figure 4 Percentages of CD4+ T cells and CD8+ T cells subsets in immunized BALB/c mice. (A) CD4+ T cells; (B) CD8+ T cells. Results are shown as the mean ± SD (n = 5 mice in each group) from three independent experiments. *p < 0.05 compared with the controls. PBS, phosphate-buffered saline.CC BY · PubMed Central
Figure 5 The mRNA and protein expression levels of p65, IRF8, and T-bet. (A–C) The mRNA levels of p65, IRF8, and T-bet; (D) The protein levels of p65, IRF8 and T-bet. Data are expressed as the mean ± SD (n = 5) from three independent experiments. Asterisks (*) indicate statistically significant differences (p < 0.05) compared with the control groups. PBS, phosphate-buffered saline.
Figure 5 The mRNA and protein expression levels of p65, IRF8, and T-bet. (A–C) The mRNA levels of p65, IRF8, and T-bet; (D) The protein levels of p65, IRF8 and T-bet. Data are expressed as the mean ± SD (n = 5) from three independent experiments. Asterisks (*) indicate statistically significant differences (p < 0.05) compared with the control groups. PBS, phosphate-buffered saline.CC BY · PubMed Central
Figure 6 CTL activities of spleen lymphocytes in pVAX1-GRA24 immunized mice. The vertical axis shows T. gondii-specific lysis as a percentage of the total possible lysis (%). CTL, cytotoxic T lymphocyte; PBS, phosphate-buffered saline. Asterisks (*) indicate statistically significant differences (p < 0.05) compared with the control groups.
Figure 6 CTL activities of spleen lymphocytes in pVAX1-GRA24 immunized mice. The vertical axis shows T. gondii-specific lysis as a percentage of the total possible lysis (%). CTL, cytotoxic T lymphocyte; PBS, phosphate-buffered saline. Asterisks (*) indicate statistically significant differences (p < 0.05) compared with the control groups.CC BY · PubMed Central
Figure 7 Survival rate of mice immunized with pVAX1-GRA24, pVAX1, PBS, and untreated mice. Each group comprised 10 mice. PBS, phosphate-buffered saline.
Figure 7 Survival rate of mice immunized with pVAX1-GRA24, pVAX1, PBS, and untreated mice. Each group comprised 10 mice. PBS, phosphate-buffered saline.CC BY · PubMed Central

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Metadata

Authors
Zheng, B., Lou, D., Ding, J., Zhuo, X., Ding, H., Kong, Q., & Lu, S.
Affiliations
Zhejiang Academy of Medical Sciences (CN)
Corresponding author
Lu, Shaohong
ORCID
Venue
Frontiers in Immunology
Publisher
Frontiers Media SA
Published
2019-03-06
Volume
10
Pages
418-418
ISSN
1664-3224 (ISSN-L)
DOI
10.3389/fimmu.2019.00418
PMC
PMC6414464
OpenAlex
W2922290179
Cited by
37
References
2
Citations by year
2025: 4 · 2024: 5 · 2023: 8 · 2022: 5 · 2021: 8 · 2020: 5 · 2019: 1
FWCI
3.66
Type
journal article
Language
en
Open access
Licence
Locations
  • Frontiers in Immunology (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
National Natural Science Foundation of China
Topics
  • Life Sciences › Immunology and Microbiology › Parasitology › Toxoplasma gondii Research Studies
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › vaccines and immunoinformatics approaches
  • Life Sciences › Agricultural and Biological Sciences › Animal Science and Zoology › Coccidia and coccidiosis research
Keywords
Toxoplasma gondii, toxoplasmosis, DNA vaccine, pVAX1, vaccine candidate, Th1/Th2 cytokines, humoral and cellular immune response, cytotoxic T lymphocytes, IRF8, NF-κB, T-bet, survival, SAG1
MeSH terms
Animals, Female, Mice, Inbred BALB C, Toxoplasma, Toxoplasmosis, Animal, Protozoan Proteins, Protozoan Vaccines, Vaccines, DNA, Virulence Factors, Mice

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