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Characterisation of Plasmodium falciparum antigens for the development of serological assay or vaccine candidates for malaria
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A malária causada por Plasmodium falciparum continua a ser um grande problema de saúde nas regiões tropicais e subtropicais em todo o mundo. Várias proteínas do parasita expressas durante o ciclo replicativo no hospedeiro vertebrado, foram descritas nos últimos anos e estão sendo consideradas como potenciais candidatos à vacina anti-malária; no entanto ainda existem algumas proteínas que não foram identificadas como potenciais alvos de diagnostico, terapêuticos e/ou candidatos a vacina. Este estudo retrospetivo descreve a identificação e caracterização de antigénios imunogénicos de P. falciparum como possíveis candidatos para o desenvolvimento de novos testes serológicos ou candidatos a vacina para malaria. Os métodos: Um extrato total de P. falciparum (PfET) do clone 3D7, foi selecionado como o nosso alvo de estudo. A antigenicidade do PfET foi avaliada usando soro humano de indivíduos anteriormente expostos à malária. Foram analisadas 436 amostras de soro, das quais 419 soros de indivíduos, com história clínica de malária, oriundos de alguns países de Africa, América e Asia, previamente diagnosticados na Unidade de Clínica Tropical do IHMT no 2012; e 17 soros de indivíduos sem história clínica de malária foram usados como controle negativo. Foram determinados anticorpos Totais, IgM, IgG, IgG1, IgG2, IgG3, IgG4 anti-P. falciparum por ELISA. Foi feita a caracterização imunoquímica de antigénios de P. falciparum responsáveis por tais reatividades serológicas por immunoblotting. Foi efetuada uma análise in silico de frações proteicas de P. falciparum previamente identificadas por espectrometria de massa. Resultados: Anticorpos anti-P. falciparum reconheceram os antigénios do PfET, com maior contribuição de anticorpos tipo IgG (72%), IgG1 (43%) e IgG3 (64%). O immunoblotting revelou frações proteicas de P. falciparum com massa molecular entre 20 -190 kDa, das quais bandas com massa molecular de 25, 35, 40, 45, 75, 80, 100 e 150 kDa apresentaram maior reatividade antigénica. A análise in silico identificou 29 epitopes correspondentes a seis proteínas de P. falciparum previamente identificadas: Elongation factor 1 alpha; Protein disulphide isomerase (PDI), Phosphoglycerate kinase; Glucose regulated protein homologue; Rhoptry associated protein 2 (RAP-2); Rhoptry-associated protein 3 (RAP-3); a análise in silico sugere que RAP-2, RAP-3, PDI, apresentam características imunogénicas essenciais como epitopes consensos, signal peptidica, domínio transmembrana. Conclusão: Este estudo mostra a caracterização de antigénios do PfET, tendo em conta que algumas das suas proteínas apresentaram várias características imunogénicas importantes, como aqui se mostra, estas proteínas podem ser consideradas como um bom candidato à vacina. São assim recomendados estudos complementares destinados a avaliar a sua capacidade de imunogenicidade e indução de proteção contra a malaria. Futuramente espera-se potencializar a ação destas proteínas e contribuir na caracterização de potenciais novos alvos antigénicos de P. falciparum importantes para o diagnóstico sorológico ou candidatos a vacina para malaria.
Plasmodium falciparum malaria remains a major health problem in tropical and subtropical regions worldwide. Several parasite proteins expressed during the replicative cycle in the vertebrate host have been described in recent years, are being considered as potential candidates for anti-malaria vaccine; there are still some proteins that have not been identified as potential diagnostic, therapeutic and/or vaccine candidates. This retrospective study describes the identification and characterization of immunogenic P. falciparum antigens as possible candidates for the development of new serological tests or candidates for the malaria vaccine. Methods: A of P. falciparum total extract 3D7 (PfTE) was selected as our study target. The antigenicity of PfTE was evaluated using human sera from individuals previously exposed to malaria. We analyzed 436 serum samples, of which 419 sera from individuals with a clinical history of malaria, from some countries of Africa, America and Asia, previously diagnosed at the Tropical Clinic Unit of the IHMT in 2012; and 17 sera from healthy Portuguese individuals, who had never been in malaria-endemic area, were used as negative control. Total antibodies, IgM, IgG, IgG1, IgG2, IgG3, IgG4 anti-P. falciparum were determined by ELISA. Immunochemical characterization of P. falciparum antigens responsible for such serological reactivities was performed by immunoblotting. An in silico analysis of protein fractions of P. falciparum previously identified by mass spectrometry was performed. Results: Anti-P. falciparum antibodies recognised antigens of PfET, with a higher contribution of IgG (72%), IgG1 (43%) and IgG3 (64%) antibodies. Immunoblotting revealed protein fractions of P. falciparum with molecular mass between 20 -190 kDa, of which bands with molecular mass of 25, 35, 40, 45, 75, 80, 100 and 150 kDa showed higher antigenic reactivity. In silico analysis identified 29 epitopes corresponding to six P. falciparum proteins previously identified: Elongation factor 1 alpha; Protein disulphide isomerase (PDI), Phosphoglycerate kinase; Glucose regulated protein homologue; Rhoptry associated protein 2 (RAP-2); Rhoptry-associated protein 3 (RAP-3); in silico analysis suggests that RAP-2, RAP-3, PDI, present essential immunogenic characteristics as consensus epitopes, signal peptide, transmembrane domain. Conclusion: This study shows the characterization of PfTE antigens, taking into account that some of its proteins presented several important immunogenic characteristics, as shown here, it could be considered as a good vaccine candidate. Further studies aimed at assessing its immunogenicity and protection-inducing ability against malaria are thus recommended. It is expected to potentiate the action of these proteins and contribute to the characterization of potential new antigenic targets of P. falciparum important for the serological diagnosis or vaccine candidates for malaria
Plasmodium falciparum malaria remains a major health problem in tropical and subtropical regions worldwide. Several parasite proteins expressed during the replicative cycle in the vertebrate host have been described in recent years, are being considered as potential candidates for anti-malaria vaccine; there are still some proteins that have not been identified as potential diagnostic, therapeutic and/or vaccine candidates. This retrospective study describes the identification and characterization of immunogenic P. falciparum antigens as possible candidates for the development of new serological tests or candidates for the malaria vaccine. Methods: A of P. falciparum total extract 3D7 (PfTE) was selected as our study target. The antigenicity of PfTE was evaluated using human sera from individuals previously exposed to malaria. We analyzed 436 serum samples, of which 419 sera from individuals with a clinical history of malaria, from some countries of Africa, America and Asia, previously diagnosed at the Tropical Clinic Unit of the IHMT in 2012; and 17 sera from healthy Portuguese individuals, who had never been in malaria-endemic area, were used as negative control. Total antibodies, IgM, IgG, IgG1, IgG2, IgG3, IgG4 anti-P. falciparum were determined by ELISA. Immunochemical characterization of P. falciparum antigens responsible for such serological reactivities was performed by immunoblotting. An in silico analysis of protein fractions of P. falciparum previously identified by mass spectrometry was performed. Results: Anti-P. falciparum antibodies recognised antigens of PfET, with a higher contribution of IgG (72%), IgG1 (43%) and IgG3 (64%) antibodies. Immunoblotting revealed protein fractions of P. falciparum with molecular mass between 20 -190 kDa, of which bands with molecular mass of 25, 35, 40, 45, 75, 80, 100 and 150 kDa showed higher antigenic reactivity. In silico analysis identified 29 epitopes corresponding to six P. falciparum proteins previously identified: Elongation factor 1 alpha; Protein disulphide isomerase (PDI), Phosphoglycerate kinase; Glucose regulated protein homologue; Rhoptry associated protein 2 (RAP-2); Rhoptry-associated protein 3 (RAP-3); in silico analysis suggests that RAP-2, RAP-3, PDI, present essential immunogenic characteristics as consensus epitopes, signal peptide, transmembrane domain. Conclusion: This study shows the characterization of PfTE antigens, taking into account that some of its proteins presented several important immunogenic characteristics, as shown here, it could be considered as a good vaccine candidate. Further studies aimed at assessing its immunogenicity and protection-inducing ability against malaria are thus recommended. It is expected to potentiate the action of these proteins and contribute to the characterization of potential new antigenic targets of P. falciparum important for the serological diagnosis or vaccine candidates for malaria
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Ciências biomédicas Biologia molecular Doenças tropicais Malária Plasmodium falciparum Antigénio Vacina