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Identification and characterization of Toxoplasma gondii aspartic protease 1 as a novel vaccine candidate against toxoplasmosis.

ABSTRACT: BACKGROUND: Toxoplasma gondii is an obligate intracellular parasite that can pose a serious threat to human health by causing toxoplasmosis. There are no drugs that target the chronic cyst stage of this infection; therefore, development of an effective vaccine would be an important advance. Aspartic proteases play essential roles in the T. gondii lifecycle. The parasite has four aspartic protease encoding genes, which are called toxomepsin 1, 2, 3 and 5 (TgASP1, 2, 3 and 5, respectively). METHODS: Bioinformatics approaches have enabled us to identify several promising linear-B cell epitopes and potential Th-cell epitopes on TgASP1, thus supporting its potential as a DNA vaccine against toxoplasmosis. We expressed TgASP1 in Escherichia coli and used the purified protein to immunize BALB/c mice. The antibodies obtained were used to determine where TgASP1 was localized in the parasite. We also made a TgASP1 DNA vaccine construct and evaluated it for the level of protection conferred to mice against infection with the virulent RH strain of T. gondii. RESULTS: TgASP1 appears to be a membrane protein located primarily at the tip of the T. gondii tachyzoite. Investigation of its potential as a DNA vaccine showed that it elicited strong humoral and cellular immune responses in mice, and that these responses were mediated by Th-1 cells. Mice immunized with the vaccine had greater levels of protection against mortality following challenge with T. gondii RH tachyzoites than did those immunized with PBS or the empty vector control. CONCLUSIONS: TgASP1 is a novel candidate DNA vaccine that merits further investigation.

SUBMITTER: Zhao G

PROVIDER: S-EPMC3691725 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Identification and characterization of Toxoplasma gondii aspartic protease 1 as a novel vaccine candidate against toxoplasmosis.

Zhao Guanghui G Zhou Aihua A Lu Gang G Meng Min M Sun Min M Bai Yang Y Han Yali Y Wang Lin L Zhou Huaiyu H Cong Hua H Zhao Qunli Q Zhu Xing-Quan XQ He Shenyi S

Parasites & vectors 20130614

<h4>Background</h4>Toxoplasma gondii is an obligate intracellular parasite that can pose a serious threat to human health by causing toxoplasmosis. There are no drugs that target the chronic cyst stage of this infection; therefore, development of an effective vaccine would be an important advance. Aspartic proteases play essential roles in the T. gondii lifecycle. The parasite has four aspartic protease encoding genes, which are called toxomepsin 1, 2, 3 and 5 (TgASP1, 2, 3 and 5, respectively). ...[more]

PMID: 23768047

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Project description:The neurotropic parasite Toxoplasma gondii (T. gondii) infection can change the behavior of rodents and cause neuropsychological symptoms in humans, which may be related to the change in neurotransmitter dopamine in the host brain caused by T. gondii infection. T. gondii tyrosine hydroxylase (TgTH) is an important factor in increasing the neurotransmitter dopamine in the host brain. In this study, the enzyme activity of TgTH catalytic substrate for dopamine production and the molecular characteristics of TgTH were identified. In order to amplify the open reading frame (ORF), the designing of the specific primers for polymerase chain reaction (PCR) was on the basis of the TgTH sequence (GenBank Accession No. EU481510.1), which was inserted into pET-32a (+) for the expression of recombined TgTH (rTgTH). The sequence analysis indicated that the gene of TgTH directed the encoding of a 62.4-kDa protein consisting of 565 amino acid residues, which was predicted to have a high antigen index. The enzyme activity test showed that rTgTH and the soluble proteins extracted separately from T. gondii RH strain and PRU strain could catalyze the substrate to produce dopamine in a dose-dependent manner, and the optimum catalytic temperature was 37 °C. The result of the Western Blotting assay revealed that the rTgTH and the native TgTH extracted from somatic of T. gondii RH tachyzoite were successfully detected by the sera of mice infected with T. gondii and the rat serum after rTgTH immune, respectively. Immunofluorescence analysis using antibody against rTgTH demonstrated that the protein was expressed and located on the surface of T. gondii RH tachyzoite. Freund's adjuvant was used to emulsify the rTgTH, which was subsequently applied to BALB/c mouse immune thrice on week 0, week 2, and week 4, respectively. The result of the animal challenge experiments showed an integral increase in IgG, IgG2a, IgG1, and IFN-γ, IL-4, and IL17 were as well significantly increased, and that the rTgTH vaccinated animals apparently had a prolonged survival time (14.30 ± 2.41) after infection with the RH strain of T. gondii compared with that of the non-vaccinated control animals, which died within 11 days. Additionally, in the rTgTH vaccination group, the number of brain cysts (1275 ± 224) significantly decreased (p < 0.05) compared to the blank control group (2375 ± 883), and the size of the brain cysts in the animals immunized with rTgTH vaccination was remarkably smaller than that of the control mice. All the findings prove that TgTH played an important role in increasing the neurotransmitter dopamine in the host brain and could be used as a vaccine candidate antigen to mediate cell-mediated and humoral immunity.

Project description:Toxoplasma gondii is a ubiquitous protozoan intracellular parasite, the causative agent of toxoplasmosis, and a worldwide zoonosis for which an effective vaccine is needed. Actin is a highly conserved microfilament protein that plays an important role in the invasion of host cells by T. gondii. This study investigated the immune responses elicited by BALB/c mice after nasal immunisation with a recombinant T. gondii actin (rTgACT) and the subsequent protection against chronic and lethal T. gondii infections. We evaluated the systemic response by proliferation, cytokine and antibody measurements, and we assessed the mucosal response by examining the levels of TgACT-specific secretory IgA (SIgA) in nasal, vaginal and intestinal washes. Parasite load was assessed in the liver and brain, and the survival of mice challenged with a virulent strain was determined. The results showed that the mice immunised with rTgACT developed high levels of specific anti-rTgACT IgG titres and a mixed IgG1/IgG2a response with a predominance of IgG2a. The systemic immune response was associated with increased production of Th1 (IFN-γ and IL-2), Th2 (IL-4) and Treg (IL-10) cytokines, indicating that not only Th1-type response was induced, but also Th2- and Treg-types responses were induced, and the splenocyte stimulation index (SI) was increased in the mice immunised with rTgACT. Nasal immunisation with rTgACT led to strong mucosal immune responses, as seen by the increased secretion of SIgA in nasal, vaginal and intestinal washes. The vaccinated mice displayed significant protection against lethal infection with the virulent RH strain (survival increased by 50%), while the mice chronically infected with RH exhibited lower liver and brain parasite loads (60.05% and 49.75%, respectively) than the controls. Our data demonstrate, for the first time, that actin triggers a strong systemic and mucosal response against T. gondii. Therefore, actin may be a promising vaccine candidate against toxoplasmosis.

Dataset Information

Identification and characterization of Toxoplasma gondii aspartic protease 1 as a novel vaccine candidate against toxoplasmosis.

Publications

Identification and characterization of Toxoplasma gondii aspartic protease 1 as a novel vaccine candidate against toxoplasmosis.

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