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Interruption of gametocyte to ookinete development
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A malária é uma doença provocada por um parasita do género Plasmodium, que ceifa aproximadamente 900.000 vidas por ano. Principalmente mulheres grávidas e crianças menores de 5 anos da África Subsaariana são vítimas deste parasita. Uma questão subsequente relacionada ao controlo da disseminação da doença ou mesmo erradicação da mesma, tem sido a resistência do parasita aos antimaláricos e a resistência do mosquito Anopheles (vetor responsável pela transmissão da malária), aos inseticidas. Desenvolver uma vacina eficiente ou compostos capazes de bloquear o desenvolvimento do parasita no vetor são estratégias promissoras para reduzir a transmissão e infectividade do parasita. As vacinas de bloqueio de transmissão têm sido desenvolvidas como uma tática para bloquear a transmissão do parasita, do hospedeiro humano para o vetor, baseando-se nos estadios do ciclo sexuado e esporogónico do parasita, tais como, os gametócitos e os oocineto – o principal foco desde trabalho. Os oocinetos são formas móveis que penetram no epitélio do intestino médio do mosquito e se desenvolveram em oocistos, que contém milhares de esporozoítos que serão responsáveis por infetar o hospedeiro humano durante uma subsequente refeição de sangue do mosquito. A criação de vacinas com o objetivo de atingir o oocineto bloqueando o seu desenvolvimento, ou a sua invasão do epitélio do intestino do mosquito, dificultam a sua progressão para os estágios seguintes e posteriormente a sua transmissão. Cultivar oocinetos in vitro tem sido um desafio. Produzir oocinetos de P. falciparum e de P. berghei, in vitro, permite-nos obter o parasita fora do hospedeiro e facilita o estudo da sua fisiologia, metabolismo e mecanismos biológicos, para que possam ser desenvolvidos diversos compostos que combatam o parasita. Além disso, o conhecimento dos antigénios envolvidos é fundamental para o desenvolvimento das vacinas de bloqueio de transmissão. Neste trabalho, foi possível produzir oocinetos de P. falciparum e P. berghei in vitro submetendo os parasitas ao meio de oocineto e a temperaturas de 21ºC e 26ºC. Da cultura de P. falciparum foram obtidos 500-800 oocinetos/mL em ambas as temperaturas e da cultura de P. berghei foram obtidos 1×106 oocinetos/mL. O SMFA (o ensaio padrão de alimentação por membrana) usando a cultura de oocinetos de P. berghei resultou numa taxa de infeção de 11.1%, e de 72% usando todo o sangue. Comparando os resultados deste trabalho com os de outros autores, a concentração de oocinetos que obtiveram foi de 5-60×105 oocinetos/mL., e de 1.5×106. Em ambas as nossas experiências os números de oocinetos obtidos in vitro foi muito superior ao número obtido nesta experiência (500-800 oocinetos/mL). No entanto, quando observados ao microscópio os oocinetos apresentavam alguma deformidade morfológicas provavelmente causadas por condições inadequadas. Em relação á cultura de P. berghei feito em estudos prévios, o número de oocinetos obtido foi de 3.75±0.48×106/mL, comparativamente maior do que o número de oocinetos presentes neste trabalho, ainda assim, foi possível obter oocinetos de P. berghei in vitro. A tentativa de produzir oocinetos de P. falciparum e de P. berghei por cultura in vitro, bem-sucedida, mas ainda assim, os protocolos precisam ser melhorados.
Malaria is one of the most life-threatening and deadliest diseases caused by a parasite from genus Plasmodium, reaping approximately 900,000 lives per year. Mostly pregnant woman and children under 5 years from sub-Saharan Africa are victims of this parasite. A subsequent issue related to control disease dissemination or its eradication, has been the parasite resistance to anti-malarial drugs and the resistance of, Anopheles mosquito´s (the vector responsible for malaria transmission) to the insecticides. The development of an efficient vaccine or the design of compounds capable of blocking the parasite development in the vector are promising strategies to reduce parasite transmission and infectivity. Transmission-blocking vaccines (TBV) have been developed as a tactic to block the development of sexual/sporogonic stages from the human host to the vector basing it on the stages of the sexual and sporogonic cycle of the parasite, such as, gametocytes and ookinete forms – the main subject of this work. Ookinetes are motile forms that penetrate the mosquito midgut epithelium and develop into oocysts carrying thousands of sporozoites which are responsible for infecting the human host during a subsequent mosquito blood meal. Creating vaccines aiming to target ookinetes and, by doing so, blocking its development and/or the egress in the mosquito midgut, is critical to hamper their progression to the next stages and their transmission. The cultivation of ookinete in vitro has been a great challenge. Generating P. falciparum and P. berghei ookinetes in vitro, allows us to obtain the parasite from the host and facilitates the study of their physiology, metabolism, and biological mechanisms to design several compounds against the parasite and fight the infection. Also, the knowledge of antigens involved is fundamental to develop transmission-blocking vaccines. In this work, we were able to produce P. falciparum and P. berghei ookinetes in vitro, by submitting the parasites to ookinete medium, at 21ºC and 26ºC. From the P. falciparum culture were obtained 500-800 ookinetes/mL at both temperatures and from P. berghei culture were obtained 1×106 ookinetes/mL. The SMFA (Standard Membrane Feeding Assay) using the P. berghei ookinete culture resulted in an infection rate of 11.1% and, for whole blood tested, 72%. Comparing the results from this work with other studies, the ookinete concentration obtained was the equivalent to 5-60×105 ookinetes/mL and to 1.5×106 ookinetes/mL. In both our experiments, the number of ookinetes obtained is much higher than in our experiment (500-800 ookinetes/mL). However, the ookinetes found were morphologically different from the expected, with some of them showing deformities probably due to inadequate conditions. Regarding to P. berghei in vitro culture, in previous studies, the ookinete concentration obtained was 3.75±0.48×106/mL, comparatively higher to the ookinete concentration present in this work, still we were able to obtain P. berghei ookinetes by in vitro culture. The attempt to induce P. falciparum and P. berghei ookinetes development, in vitro, was well accomplish, however the protocols used need to be improved.
Malaria is one of the most life-threatening and deadliest diseases caused by a parasite from genus Plasmodium, reaping approximately 900,000 lives per year. Mostly pregnant woman and children under 5 years from sub-Saharan Africa are victims of this parasite. A subsequent issue related to control disease dissemination or its eradication, has been the parasite resistance to anti-malarial drugs and the resistance of, Anopheles mosquito´s (the vector responsible for malaria transmission) to the insecticides. The development of an efficient vaccine or the design of compounds capable of blocking the parasite development in the vector are promising strategies to reduce parasite transmission and infectivity. Transmission-blocking vaccines (TBV) have been developed as a tactic to block the development of sexual/sporogonic stages from the human host to the vector basing it on the stages of the sexual and sporogonic cycle of the parasite, such as, gametocytes and ookinete forms – the main subject of this work. Ookinetes are motile forms that penetrate the mosquito midgut epithelium and develop into oocysts carrying thousands of sporozoites which are responsible for infecting the human host during a subsequent mosquito blood meal. Creating vaccines aiming to target ookinetes and, by doing so, blocking its development and/or the egress in the mosquito midgut, is critical to hamper their progression to the next stages and their transmission. The cultivation of ookinete in vitro has been a great challenge. Generating P. falciparum and P. berghei ookinetes in vitro, allows us to obtain the parasite from the host and facilitates the study of their physiology, metabolism, and biological mechanisms to design several compounds against the parasite and fight the infection. Also, the knowledge of antigens involved is fundamental to develop transmission-blocking vaccines. In this work, we were able to produce P. falciparum and P. berghei ookinetes in vitro, by submitting the parasites to ookinete medium, at 21ºC and 26ºC. From the P. falciparum culture were obtained 500-800 ookinetes/mL at both temperatures and from P. berghei culture were obtained 1×106 ookinetes/mL. The SMFA (Standard Membrane Feeding Assay) using the P. berghei ookinete culture resulted in an infection rate of 11.1% and, for whole blood tested, 72%. Comparing the results from this work with other studies, the ookinete concentration obtained was the equivalent to 5-60×105 ookinetes/mL and to 1.5×106 ookinetes/mL. In both our experiments, the number of ookinetes obtained is much higher than in our experiment (500-800 ookinetes/mL). However, the ookinetes found were morphologically different from the expected, with some of them showing deformities probably due to inadequate conditions. Regarding to P. berghei in vitro culture, in previous studies, the ookinete concentration obtained was 3.75±0.48×106/mL, comparatively higher to the ookinete concentration present in this work, still we were able to obtain P. berghei ookinetes by in vitro culture. The attempt to induce P. falciparum and P. berghei ookinetes development, in vitro, was well accomplish, however the protocols used need to be improved.
Descrição
Palavras-chave
Hibridomas malaria Gametócito Oocineto Cultura in vitro Atividade de bloqueio de transmissão