RESUMO
ABSTRACT Objectives: The plague, which is an infectious disease caused by Yersinia pestis, still threatens many populations in several countries. The worldwide increase in human plague cases and the potential use of the bacteria as a biological weapon reinforce the need to study the immunity that is induced by potential vaccine candidates. To determine the immunogenicity of antigenic preparations based on the F1 protein and the total extract from Y. pestis, we assessed the role of these antigens in inducing an immune response. Methods: The immunogenicity of antigenic preparations based on the Y. pestis (YP) total extract and the Y. pestis fraction 1 capsular antigen protein (F1) was determined in Swiss-Webster mice immunized with 40 µg or 20 µg for each preparation. Immunophenotyping was performed by flow cytometry. Results: Animals immunized with the YP total extract did not elicit detectable anti-F1 antibodies (Ab) in the hemaglutination/inhibition (HA/HI) test. Animals immunized with 40 µg or 20 µg of the F1 protein produced anti-F1 Abs, with titres ranging from 1/16 to 1/8132. The average of CD3+-CD4+ and CD3+-CD8+ T cells did not differ significantly between the groups. Neither YP total extract nor F1 protein induced a significant expression of IFN-γ and IL-10 in CD4+ T lymphocytes. In addition, F1 failed to induce IFN-γ expression in CD8+ T cells, unlike the YP total extract. Conclusion: The results showed that F1 protein is not an immunogenic T cell antigen, although the YP total extract (40 µg dose) favoured CD8+ T cell-mediated cellular immunity.
Assuntos
Animais , Feminino , Ratos , Baço/imunologia , Yersinia pestis/imunologia , Vacina contra a Peste/imunologia , Imunogenicidade da Vacina , Antígenos de Bactérias/imunologia , Peste/prevenção & controle , Baço/citologia , Linfócitos T CD4-Positivos/imunologia , Imunofenotipagem , Interferon gama/imunologia , Interleucina-10/imunologia , Relação CD4-CD8 , Linfócitos T CD8-Positivos , Citometria de Fluxo , Imunidade CelularRESUMO
OBJECTIVES: The plague, which is an infectious disease caused by Yersinia pestis, still threatens many populations in several countries. The worldwide increase in human plague cases and the potential use of the bacteria as a biological weapon reinforce the need to study the immunity that is induced by potential vaccine candidates. To determine the immunogenicity of antigenic preparations based on the F1 protein and the total extract from Y. pestis, we assessed the role of these antigens in inducing an immune response. METHODS: The immunogenicity of antigenic preparations based on the Y. pestis (YP) total extract and the Y. pestis fraction 1 capsular antigen protein (F1) was determined in Swiss-Webster mice immunized with 40µg or 20µg for each preparation. Immunophenotyping was performed by flow cytometry. RESULTS: Animals immunized with the YP total extract did not elicit detectable anti-F1 antibodies (Ab) in the hemaglutination/inhibition (HA/HI) test. Animals immunized with 40µg or 20µg of the F1 protein produced anti-F1 Abs, with titres ranging from 1/16 to 1/8132. The average of CD3+-CD4+ and CD3+-CD8+ T cells did not differ significantly between the groups. Neither YP total extract nor F1 protein induced a significant expression of IFN-γ and IL-10 in CD4+ T lymphocytes. In addition, F1 failed to induce IFN-γ expression in CD8+ T cells, unlike the YP total extract. CONCLUSION: The results showed that F1 protein is not an immunogenic T cell antigen, although the YP total extract (40µg dose) favoured CD8+ T cell-mediated cellular immunity.
Assuntos
Antígenos de Bactérias/imunologia , Imunogenicidade da Vacina , Vacina contra a Peste/imunologia , Baço/imunologia , Yersinia pestis/imunologia , Animais , Relação CD4-CD8 , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos , Feminino , Citometria de Fluxo , Imunidade Celular , Imunofenotipagem , Interferon gama/imunologia , Interleucina-10/imunologia , Camundongos , Peste/prevenção & controle , Baço/citologiaRESUMO
Expression of the protective F1 and V antigens of Yersinia pestis, as a fusion protein, in carrot was pursued in an effort to develop an alternative vaccine production system against the serious plague disease. Transgenic carrot plants carrying the F1-V encoding gene were developed via Agrobacterium-mediated transformation. Presence, integration, and expression of the F1-V encoding gene were confirmed by polymerase chain reaction (PCR), DNA gel blot analysis, and reverse-transcriptase (RT)-PCR analyses, respectively. An ELISA assay confirmed the antigenicity of the plant-derived F1-V fusion protein. Immunogenicity was evaluated subcutaneously in mice using a soluble protein extract of freeze-dried transgenic carrot. Significant antibody levels were detected following immunization. These results demonstrated that the F1-V protein could be expressed in carrot tap roots, and that the carrot F1-V recombinant protein retained its antigenicity and immunogenicity.
Assuntos
Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Daucus carota/metabolismo , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Yersinia pestis/metabolismo , Animais , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Daucus carota/genética , Ensaio de Imunoadsorção Enzimática , Camundongos , Camundongos Endogâmicos BALB C , Peste/imunologia , Vacina contra a Peste/genética , Vacina contra a Peste/imunologia , Vacina contra a Peste/metabolismo , Raízes de Plantas/genética , Plantas Geneticamente Modificadas/genética , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Recombinantes de Fusão/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Yersinia pestis/genéticaRESUMO
Yersinia pestis is a pathogenic agent that causes the bubonic and pneumonic plague. The development of an efficient and low-cost oral vaccine against these diseases is highly desirable. In this study, the immunogenic fusion protein F1-V from Y. pestis was introduced into lettuce via Agrobacterium-mediated transformation, and putative transgenic lines were developed. The presence of the transgene in these putative transgenic lines was determined using polymerase chain reaction (PCR), and transgene integration and transgene copy number were confirmed following Southern blot analysis. The presence of specific F1-V transcripts was confirmed by reverse-transcriptase (RT)-PCR. Using monoclonal antibodies, ELISA and western blot analysis revealed that the expected antigenic F1-V protein was successfully expressed in transgenic lines. Mice immunized subcutaneously with lettuce expressing the F1-V antigen developed systemic humoral responses as 'proof of concept' of using lettuce as a production platform for the F1-V immunogen that could be used as a candidate plant-based vaccine against plague.