Publicação
Study of the immune responses induced by Toxoplasma gondii membrane extract using the mice model
| Resumo: | Toxoplasma gondii (T. gondii) is an obligate intracellular parasite with a complex life cycle, capable of infecting warm-blooded animals. About one third of the world's population is infected, with immunocompetent individuals usually asymptomatic, while immunocompromised individuals may develop cerebral or ocular toxoplasmosis. T. gondii infection poses a great risk during pregnancy, leading to miscarriage or congenital toxoplasmosis, which can cause malformations in the foetus, mental retardation, or even premature death. At the present there is no vaccine for humans. Therefore, the development of a vaccine capable of preventing toxoplasmosis in humans is necessary. This work aims to study humoral and cellular immune responses induced by immunization with a membrane extract from tachyzoite forms of T. gondii Me49 strain able to induce protection against infection. BALB/c mice were immunised by intranasal route, using T. gondii membrane proteins (TGMP) and Class B oligonucleotides that contain unmethylated CpG dinucleotides motifs adjuvant (CpG ODN or only CpG ODN (control). To assess the ability to induce protection, blood and intestinal lavage fluids were collected from animals after immunisation, and the levels of TGMP-specific IgG1 and IgG2a, and IgA, respectively, were measured by ELISA. Parasite load was quantified in tissues of immunized animals by detection of Surface Antigen-1 (SAG-1) by qPCR. In order to characterise the cellular response, spleen and lung cells were analysed by flow cytometry to quantify the CD4⁺ and CD8⁺ T-lymphocytes, as well as the cytokines produced by them after specific and non-specific stimuli, with and without infection. The results obtained showed a significant increase in the levels of TGMP-specific IgG1 and IgG2a, as well as TGMP-specific IgA in the intestinal mucosa of animals immunised with TGMP plus ODN, compared with control animals. Parasite load was significantly decreased in the spleen and peritoneal exudate (PE) of animals immunised with TGMP plus ODN when compared with the control group. In the analyses of cellular responses, no significant differences could be observed between the groups of animals under the defined experimental conditions. In the cellular response analysis in response to TGMP plus ODN immunisation and after T. gondii infection, it is possible to observe an increase of non-specific TCD8⁺IFNγ⁺ spleen cells at short term (3 weeks after the 2nd immunisation with TGMP plus ODN) and 5 days after infection, and an increase of TGMP-specific TCD4⁺IFNγ⁺ in the lung at long term (9 weeks after the 2nd immunisation with TGMP and ODN) and 5 days after infection with T. gondii. We conclude that the immunisation protocol used induces a significant protection against infection at short and long term, associated with an increase of a specific Th1/Th2 humoral responses and a significant increase in the TCD4⁺IFNγ⁺ or TCD8⁺IFNγ⁺ cell populations, determinant in the protective response against T. gondii infection. |
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| Autores principais: | Teixeira, Daniela do Carmo |
| Assunto: | Toxoplasmosis Vaccination Immune response Humoral immunity Cellular immunity |
| Ano: | 2022 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Aveiro |
| Idioma: | inglês |
| Origem: | RIA - Repositório Institucional da Universidade de Aveiro |
| Resumo: | Toxoplasma gondii (T. gondii) is an obligate intracellular parasite with a complex life cycle, capable of infecting warm-blooded animals. About one third of the world's population is infected, with immunocompetent individuals usually asymptomatic, while immunocompromised individuals may develop cerebral or ocular toxoplasmosis. T. gondii infection poses a great risk during pregnancy, leading to miscarriage or congenital toxoplasmosis, which can cause malformations in the foetus, mental retardation, or even premature death. At the present there is no vaccine for humans. Therefore, the development of a vaccine capable of preventing toxoplasmosis in humans is necessary. This work aims to study humoral and cellular immune responses induced by immunization with a membrane extract from tachyzoite forms of T. gondii Me49 strain able to induce protection against infection. BALB/c mice were immunised by intranasal route, using T. gondii membrane proteins (TGMP) and Class B oligonucleotides that contain unmethylated CpG dinucleotides motifs adjuvant (CpG ODN or only CpG ODN (control). To assess the ability to induce protection, blood and intestinal lavage fluids were collected from animals after immunisation, and the levels of TGMP-specific IgG1 and IgG2a, and IgA, respectively, were measured by ELISA. Parasite load was quantified in tissues of immunized animals by detection of Surface Antigen-1 (SAG-1) by qPCR. In order to characterise the cellular response, spleen and lung cells were analysed by flow cytometry to quantify the CD4⁺ and CD8⁺ T-lymphocytes, as well as the cytokines produced by them after specific and non-specific stimuli, with and without infection. The results obtained showed a significant increase in the levels of TGMP-specific IgG1 and IgG2a, as well as TGMP-specific IgA in the intestinal mucosa of animals immunised with TGMP plus ODN, compared with control animals. Parasite load was significantly decreased in the spleen and peritoneal exudate (PE) of animals immunised with TGMP plus ODN when compared with the control group. In the analyses of cellular responses, no significant differences could be observed between the groups of animals under the defined experimental conditions. In the cellular response analysis in response to TGMP plus ODN immunisation and after T. gondii infection, it is possible to observe an increase of non-specific TCD8⁺IFNγ⁺ spleen cells at short term (3 weeks after the 2nd immunisation with TGMP plus ODN) and 5 days after infection, and an increase of TGMP-specific TCD4⁺IFNγ⁺ in the lung at long term (9 weeks after the 2nd immunisation with TGMP and ODN) and 5 days after infection with T. gondii. We conclude that the immunisation protocol used induces a significant protection against infection at short and long term, associated with an increase of a specific Th1/Th2 humoral responses and a significant increase in the TCD4⁺IFNγ⁺ or TCD8⁺IFNγ⁺ cell populations, determinant in the protective response against T. gondii infection. |
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