Immunogenicity and Protective Efficacy of A Novel DNA Vaccine Expressing Toxoplasma gondii SRS13 Protein Against Chronic Toxoplasmosis
Bildiriler > Özet Bildiri · 2023
Yazarlar: Ceren Gül, Aytül Gül, Tuğba Karakavuk, Sedef Erkunt Alak, Muhammet KARAKAVUK, Hüseyin CAN, Aysu DEĞİRMENCİ DÖŞKAYA, Adnan Yüksel GÜRÜZ, Mert DÖŞKAYA
23. Ulusal Parazitoloji Kongresi, Türkiye, Antalya, Ulusal, Poster, Özet Bildiri, 2023-2023, ss 228-229
Özet
<p>Toxoplasma gondii (T. gondii) is a parasite that can infect all warm-blooded animals, including humans. It causes toxoplasmosis, which has different forms, including chronic and acute toxoplasmosis. This parasite can lead to severe consequences, such as toxoplasmic encephalitis and abortions in pregnant individuals. While a vaccine exists for livestock animals but it's unsafe for humans. Herein, we describe to develop a DNA vaccine encoding SRS13 against all stages of T. gondii for the first time and determine the immunogenicity and protective efficacy of the DNA vaccine in BALB/c mice. In silico analysis revealed that the wild-type SRS13 protein, known to be immunogenic in all three forms of T. gondii, was found to be unstable. To address this issue, the protein was minimized containing on the T and B cell epitope regions, to design a soluble, stable, and highly antigenic protein. Subsequently, the designed SRS13 gene was cloned into the pVAX1 vector to generate the pVAX1-SRS13 DNA vaccine. The protein expression profile of the DNA vaccine was determined in human embryonic kidney (HEK293T) cells by Western blot and immunofluorescent antibody staining assays. To evaluate the vaccine's immunogenicity, BALB/c mice were immunized three times with naked pVAX1-SRS13, either through intramuscular (IM) injection or intradermally via electroporation (ID+EP). Humoral immune response was determined by Western blot and ELISA assays, while the cell-mediated immune response was determined by flow cytometry, ELISA, and MTT assays. Furthermore, to determine the protective efficacy of the DNA vaccine, BALB/c mice were challenged with T. gondii PRU strain tissue cysts orally and monitored for 42 days. At the end of 42 days mice were euthanized and the number of tissue cysts were investigated by microscopy and the T. gondii DNA concentration were analyzed by real-time polymerase chain reaction (RT-PCR). The pVAX1-SRS13 DNA vaccine expressed the SRS13 protein in HEK293T cells and induced antigenspecific IgG antibodies in BALB/c mice. In the ELISA test performed to determine the humoral immune response, it was observed that tachyzoites lysate antigen (TLA) and recombinant SRS13 (rSRS13) proteins formed complexes with immunoglobulin (IgG) antibodies in the serum of mice in the pVAX1SRS13 (IM) group, stimulating approximately 6-fold and 2-fold higher IgG responses, respectively (P<0.05). The percentage of IFN-γ-secreting CD8+ T cells in mice immunized with pVAX1-SRS13 (IM) and pVAX1-SRS13 (ID+EP) was approximately 2-fold and 4-fold higher compared to the control groups, respectively (P<0.05). Furthermore, the percentage of IFN-γ-secreting CD8+ T cells in mice immunized with pVAX1-SRS13 (ID+EP) was approximately 2-fold higher compared to mice immunized with pVAX1-SRS13 (IM) (P<0.05). The percentage of CD4+ T cells secreting IFN-γ was approximately 2-fold higher in the pVAX1-SRS13 (ID+EP) group compared to the control group (P<0.05). The IFN-γ levels in the spleen cell culture supernatants from both vaccine groups were significantly higher compared to the control groups (P<0.05; P<0.0001). Additionally, the IFN-γ levels in the pVAX1-SRS13 (ID+EP) group were found to be 8-fold higher than in the pVAX1-SRS13 (IM) group (P<0.05). It was observed that the extracellular IL-4 cytokine secretion levels were too low to be detected in all groups. In the pVAX1SRS13 (IM) vaccine group, a 1.3-fold higher cell viability compared to the control group was observed, while in the pVAX1-SRS13 (ID+EP) vaccine group, an approximately 1.5-fold higher cell viability compared to the control group was observed (P<0.0001). The cell viability in the pVAX1-SRS13 (ID+EP) vaccine group was found to be statistically higher than in the pVAX1-SRS13 (IM) vaccine group (P<0.05). According to the data obtained from microscopy, it was determined that the number of tissue cysts decreased by approximately 2-fold in both vaccine groups compared to the control groups (P<0.05). Consistent with the concentration values obtained from RT-PCR, a statistically lower T. gondii DNA concentration was found in the vaccine groups compared to the controls (P<0.05).</p><p>Key words: T. gondii, SRS13, vaccine </p><p>References </p><p>1) Can, H., Erkunt Alak, S., Köseoğlu, AE, Döşkaya, M., Ün, C., 2020a. Do Toxoplasma gondii apicoplast proteins have antigenic potential? 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