Doctoral Thesis
DOI
https://doi.org/10.11606/T.9.2012.tde-21052013-170007
Document
Author
Full name
Elaine Cristina Matos Vicentin
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2011
Supervisor
Committee
Soares, Irene da Silva (President)
Costa, Fábio Trindade Maranhão
Ho, Paulo Lee
Nascimento, João Roberto Oliveira do
Rosa, Daniela Santoro
Title in Portuguese
Imunizações pré-clínicas contra malária baseadas no Antígeno 1 de Membrana Apical (PvAMA-1): testes de protocolos homólogos e heterólogos de indução e reforço utilizando DNA plasmidial e/ou proteína recombinante
Keywords in Portuguese
Imunização
Malária humana
Plasmodium vivax
PvAMA-1
Abstract in Portuguese
O Antígeno 1 de Membrana Apical (AMA-1) é um dos mais promissores candidatos a vacina contra a fase sanguínea da malária. No presente estudo, geramos uma nova proteína recombinante baseada no ectodomínio de AMA-1 de P. vivax (PvAMA-1) a partir do gene sintético com códon otimizado para expressão secretada na levedura Pichia pastoris. Por ELISA, a proteína PvAMA-1 foi reconhecida por 62,5% dos soros de pacientes da Amazônia brasileira infectados por P. vivax. A imunogenicidade de PvAMA-1 foi avaliada em camundongos BALB/c, utilizando protocolos homólogos e heterólogos de indução e reforço com DNA plasmidial e/ou proteína recombinante, emulsificada em Adjuvante de Freund. Os resultados mostraram que a eficiência da imunização é dependente da presença da proteína emulsificada em adjuvante forte. As imunizações subseqüentes foram realizadas com a proteína recombinante emulsificada em diferentes adjuvantes: sais de alumínio (Alum), Monophosphoryl Lipid A (MPLA) de Bordetella pertussis, flagelina FliC de Salmonella Typhimurium, saponina Quil A e Adjuvante Incompleto de Freund (AIF). Os resultados demonstraram que as imunizações na presença de Quil A e AIF foram capazes de induzir altos títulos de anticorpos IgG e uma resposta de isotipos de IgG mais balanceada, caracterizando um padrão do tipo Th1/Th2. Títulos de anticorpos mais baixos, mas também expressivos, foram obtidos na presença de Alum, MPLA, Alum + MPLA e Alum + FliC. A análise da resposta proliferativa, por citometria de fluxo utilizando marcação com CFSE, mostrou que células esplênicas CD3+CD4+, assim como CD3+CD8+, de animais imunizados com PvAMA-1 na presença dos adjuvantes Alum, Alum + MPLA, Quil A e AIF foram capazes de proliferar especificamente in vitro. Por imunofluorescência, soros policlonais de camundongos imunizados com o antígeno na presença de MPLA e Quil A foram capazes de reconhecer a proteína nativa expressa em isolados de P. Vivax da Ásia. Visando futuros testes pré clínicos em primatas não humanos, a proteína PvAMA-1 foi produzida em boas práticas de laboratório (BPL) por uma companhia especializada e o potencial imunogênico da proteína foi confirmado em imunizações posteriores. Em conjunto, nossos resultados demonstram que PvAMA-1 é um antígeno promissor para ser explorado em protocolos de imunização contra malária vivax, individualmente, ou em combinação com outros antígenos.
Title in English
Pre-clinical immunizations against malaria based on the Apical Membrane Antigen 1 (PvAMA-1): testing prime-boost homologous and heterologous protocols using DNA and/or recombinant protein
Keywords in English
Human malaria
Immunization
Plasmodium vivax
PvAMA-1
Abstract in English
The Apical Membrane Antigen 1 (AMA-1) is one of the most promissing vaccine candidates against the erythrocytic stage of malaria. In the present study we generated a new recombinant protein based on AMA-1 ectodomain of P. vivax (PvAMA-1) using a synthetic codon optimized gene for yeast secreted expression. By ELISA, the protein PvAMA-1 was recognized by 62,5% of the sera from Brazilian Amazonia patients infected by P. vivax. The immunogenicity of PvAMA-1 was evaluated in BALB/c mice using homologous and heterologous protocols of prime and boost with plasmidial DNA and/or recombinant protein emulsified in Freund adjuvant. The results showed that the efficiency of immunization is dependent on the presence of a strong adjuvant. The following immunizations were conducted using the protein emulsified in different adjuvants: aluminium salts (Alum), Monophosphoryl Lipid A (MPLA) of Bordetella pertussis, flagelin FliC of Salmonella Typhimurium, saponin Quil A and Incomplete Freund's Adjuvant (IFA). The results showed that immunizations in the presence of Quil A e IFA were able to induce high IgG titers and a more balanced IgG isotypes response, featuring a standard type Th1/Th2. Lower antibody titers, but also significant, were obtained in the presence of Alum, MPLA, Alum + MPLA and Alum + FliC. The proliferative cellular analysis, by flow cytometry using CFSE staining, showed that splenic cells CD3+CD4+, as well as CD3+CD8+ from immunized mice that received PvAMA-1 with Alum, Alum + MPLA, Quil A and IFA were specifically able to proliferate in vitro. By immunofluorescence, the polyclonal sera from mice immunized with the antigen in the presence of MPLA and Quil A were able to recognize native protein expressed in Asian P. vivax isolates. Aiming future pre-clinical assays in non-human primates, the protein PvAMA-1 was produced in good laboratory practices (GLP) conditions by a specialized company and its immunogenic efficiency was confirmed in later immunizations. Altogether, our results showed that PvAMA-1 is a promissor antigen to be explored in immunization protocols against vivax malaria, individually, or combined with other antigens.
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Publishing Date
2013-07-03
WARNING: The material described below relates to works resulting from this thesis or dissertation. The contents of these works are the author's responsibility.
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- VICENTIN, E. C., et al. Immunogenicity of a malaria vaccine candidate expressed in Pichia pastoris and administered in mice using different adjuvant formulations. In XII Reunião Nacional de Pesquisa em Malária, Ouro Preto, MG, 2010. Livro de Resumos., 2010. Abstract.
- VICENTIN, E. C., et al. Immunogenicity of the Plasmodium vivax Apical Membrane Antigen 1 (PvAMA-1): comparison of homologous and heterologous prime-boost strategies using naked DNA and a recombinant protein produced in Pichia pastoris. In XIV Science and Pharmaceutical Technology Meetingof the Faculty of the Pharmaceutical Sciences of the University of Sao Paulo, São Paulo, 2009. Brazilian Journal of Pharmaceutical Sciences., 2009. Abstract.