Malaria vaccine research has been battling with persistent challenges, including polymorphisms of vaccine antigens, difficulties with production processes, and limited immune protection against the disease. Intrinsically unstructured proteins (IUPs) are a fairly newly classified group of proteins that have no stable 3D structure and are generally heat-resistant. They usually contain low complexity regions and repetitive sequences, both of which are distinct characteristics of the malaria proteome. Surprisingly, some of the vaccine candidates that have been extensively studied were later reported to have unstructured regions, some of which serve as targets of protective immunity. In keeping with their interesting immunological profiles and their unique properties, which are exceptionally beneficial for vaccine production, malarial IUP antigens may be good vaccine candidates. This PhD project has the following aims:- 1) to develop a synthetic unstructured protein antigen based on the Block 2 region of MSP-1, named the MSP-1 hybrid 2) to characterize a novel vaccine antigen derived from the MSP-3.3 protein, namely an IUP region of PF10_0347 gene product, for its potential as a vaccine candidate 3) to develop a second-generation vaccine by combining the MSP-1 hybrid, with two allelic variants of MSP-2, to overcome antigenic polymorphism and strain-specific immune responses 4) to validate protocols for IUP identification from proteins extracted from the malaria parasite. This study showed that 1) MSP-1 hybrid production was scalable, yielding high protein yields with comparable immunological properties to small-scale production. MSP-1 hybrid was shown to be compatible with different adjuvants, and elicited specific antibodies covering the whole range of Block 2 allelic diversities. 2) A novel antigen, MSP-3.3C, an IUP based on the 3’ region of the PF10_0347 gene, was cloned, expressed and purified. Anti-MSP3.3C antibodies showed very strong parasite growth inhibitory effects in vitro. 3) The MSP-multihybrid antigen was expressed using simple techniques, but only at low levels. It contains epitopes from all three parasite antigen components, and is recognized by specific naturally acquired antibodies. 4) an unconventional 2D gel technique was tested as a method of malaria parasite IUP identification. Plans for further validation of this technique were discussed.

Apresenta-se os resultados de estudo de coorte em um assentamento rural no Acre, Brasil, onde 509 indivíduos contribuíram com 489,7 pessoas-ano de seguimento. A incidência de malária por Plasmodium vivax e Plasmodium falciparum foi de 30,0/100 e 16,3/100 pessoas-ano, respectivamente. A morbidade por malária se associou fortemente ao desmatamento e agropecuária e diminuiu após 5 anos de residência no local; além disso ocorreram conglomerados espaciais significantes de casos de malária vivax e falciparum em áreas de ocupação recente. Não houve associação entre alótipos do receptor FcgRIIa, promotor Duffy ou anticorpos contra a PvMSP-1, e malária no seguimento. As respostas humorais e celulares foram mais freqüentes contra as porções C- e N-terminal da PvMSP-1 respectivamente, porém sem relação com as variantes de PvMSP-1 infectantes. O espectro clínico dos episódios de malária foi variado; enquanto cefaléia, febre e mialgia foram sintomas freqüentes, 29,4% dos episódios eram assintomáticos. Os resultados mostram que a aquisição de imunidade clínica é adquirida em áreas de baixa transmissão e que mudanças ambientais causadas nos assentamentos rurais perpetuam a transmissão de malária. Estes achados são de extrema importância para o controle da malária na Amazônia. / Here a cohort study in a frontier settlement in Acre, Brazil, where 509 subjects contributed 489.7 person-years of follow-up, is described. Incidence rates for Plasmodium vivax and Plasmodium falciparum malaria were 30.0/100 and 16.3/100 person-years at risk, respectively. Malaria morbidity was strongly associated with land clearing and farming, and decreased after 5 years of local residence. Besides, there was significant spatial clustering of vivax and falciparum malaria in areas of recent occupation. No significant association was found between FcgRIIa allotype, Duffy promoter type or presence of antibodies against PvMSP-1 and malaria in the follow-up. Humoral and cellular responses were more common against the C- and N-terminal portions of PvMSP-1 respectively, but did not match the PvMSP-1 variants found in infecting parasites. The clinical spectrum of malaria episodes varied widely; while headache, fever and myalgia were the most frequent symptoms, 29.4% of the episodes were asymptomatic. The results show that clinical immunity is acquired under low malaria transmission and environmental changes occurring in the settlements perpetuate malaria transmission. These findings are of utmost importance for malaria control in the Amazon.

Apresenta-se os resultados de estudo de coorte em um assentamento rural no Acre, Brasil, onde 509 indivíduos contribuíram com 489,7 pessoas-ano de seguimento. A incidência de malária por Plasmodium vivax e Plasmodium falciparum foi de 30,0/100 e 16,3/100 pessoas-ano, respectivamente. A morbidade por malária se associou fortemente ao desmatamento e agropecuária e diminuiu após 5 anos de residência no local; além disso ocorreram conglomerados espaciais significantes de casos de malária vivax e falciparum em áreas de ocupação recente. Não houve associação entre alótipos do receptor FcgRIIa, promotor Duffy ou anticorpos contra a PvMSP-1, e malária no seguimento. As respostas humorais e celulares foram mais freqüentes contra as porções C- e N-terminal da PvMSP-1 respectivamente, porém sem relação com as variantes de PvMSP-1 infectantes. O espectro clínico dos episódios de malária foi variado; enquanto cefaléia, febre e mialgia foram sintomas freqüentes, 29,4% dos episódios eram assintomáticos. Os resultados mostram que a aquisição de imunidade clínica é adquirida em áreas de baixa transmissão e que mudanças ambientais causadas nos assentamentos rurais perpetuam a transmissão de malária. Estes achados são de extrema importância para o controle da malária na Amazônia. / Here a cohort study in a frontier settlement in Acre, Brazil, where 509 subjects contributed 489.7 person-years of follow-up, is described. Incidence rates for Plasmodium vivax and Plasmodium falciparum malaria were 30.0/100 and 16.3/100 person-years at risk, respectively. Malaria morbidity was strongly associated with land clearing and farming, and decreased after 5 years of local residence. Besides, there was significant spatial clustering of vivax and falciparum malaria in areas of recent occupation. No significant association was found between FcgRIIa allotype, Duffy promoter type or presence of antibodies against PvMSP-1 and malaria in the follow-up. Humoral and cellular responses were more common against the C- and N-terminal portions of PvMSP-1 respectively, but did not match the PvMSP-1 variants found in infecting parasites. The clinical spectrum of malaria episodes varied widely; while headache, fever and myalgia were the most frequent symptoms, 29.4% of the episodes were asymptomatic. The results show that clinical immunity is acquired under low malaria transmission and environmental changes occurring in the settlements perpetuate malaria transmission. These findings are of utmost importance for malaria control in the Amazon.

Die Malaria ist heute noch immer die bedeutendste parasitäre Infektionskrankheit des Menschen. Hiervon sind in Endemiegebieten neben Kleinkindern insbesondere schwangere Frauen betroffen. P. falciparum weist eine hohe genetische Diversität auf. So sind in Endemiegebieten Infektionen mit P. falciparum in der Regel polyklonal. Man spricht in diesen Fällen von der Multiplizität der Infektion. Bei Schwangeren sequestrieren mit P. falciparum infizierte Erythrozyten, die spezifische Oberflächenproteine exprimieren, in der Plazenta. Hierdurch bedingt können pathologische Schwangerschafts-verläufe klinische Manifestationsformen der Malaria darstellen. Um die Diversität von P. falciparum und die Multiplizität der Infektion bei schwangeren Frauen zu erforschen, wurden in einer Querschnittsstudie im holoendemischen Malariagebiet von Agogo in Ghana über den Zeitraum von einem Jahr 474 Gebärende mit einer nachgewiesenen plazentaren Infektion von P. falciparum untersucht. Hierzu wurden die Gene, die für das "Merozoiten-Oberflächen-Protein-1" (msp-1) und "Merozoiten-Oberflächen-Protein-2" (msp-2) kodieren, aus peripher und plazentar gewonnen Isolaten typisiert. Plazentar gewonnene Isolate waren im Vergleich zu peripher gewonnenen mit einer signifikant höheren Prävalenz an polyklonalen Infektionen und einer höheren Multiplizität der Infektion assoziiert. Die höchste Multiplizität der Infektion wurde bei Erstgebärenden und jüngeren Patientinnen beobachtet. Mit zunehmendem Alter und einer höheren Anzahl an vorangegangenen Schwangerschaften fielen signifikant sowohl die Multiplizität der Infektion als auch die Parasitendichte. Zudem wurde eine hohe Korrelation zwischen der Multiplizität der Infektion und der Parasitendichte nachgewiesen. Weder das Alter noch die Parität beeinflussten diese Korrelation. Der Einfluss von Alter und Parität auf die Multiplizität der Infektion konnte somit nicht unabhängig von der Parasitendichte nachgewiesen werden. Multivariate Analysen zeigten aber, dass es unabhängig von der Parasitendichte bei plazentaren Infektionen mit zwei und mehr als zwei Klonen im Vergleich zu monoklonalen plazentaren Infektionen mit einer höheren Wahrscheinlichkeit zu einer Frühgeburt kam. Dies betraf insbesondere Erstgebärende und Frauen mit submikroskopischen plazentaren Infektionen. Ob bei polyklonalen Infektionen eine Sequestration von P. falciparum in der Plazenta durch alle oder nur durch einen Teil der zahlreichen Genotypen geschieht, die an einer Infektion bei Schwangeren beteiligt sind, ist nicht entgültig geklärt. Es wurden aus zusammengehörenden plazentar und peripher gewonnenen P.-falciparum-Isolaten die Verteilungsmuster der Genotypen verglichen. Zwar korrelierte die Multiplizität der Infektion plazentarer und peripherer Isolate, die Genotypenmuster der Plazenta und der Peripherie waren jedoch deutlich unterschiedlich. Nur in 12% der Fälle konnte eine Genotypisierung eines peripher gewonnenen Isolates das klonale Gesamtbild der Infektion nachweisen. In 67% der Fälle waren neben identischen Genotypen wenigstens in einem der beiden Isolate unterschiedliche Genotypen nachweisbar. Einzelne spezifische Genotypen traten in der gesamten Untersuchungsgruppe öfter in der Plazenta als in der peripheren Blutprobe auf. Bei Frauen, die mit den Genotypen der Allelfamilie FC27 infiziert waren, lagen signifikant häufiger klinischen Manifestationen der Malaria vor. So konnte in multivariaten Analysen eine Assoziation zwischen FC27 und einer Frühgeburtlichkeit nachgewiesen werden. Darüber hinaus war FC27 zumindest in univariater Analyse mit einer Anämie und einem verminderten Geburtsgewicht assoziiert. Dies konnte insbesondere für Primiparae und für Gebärende mit submikroskopischen plazentaren Infektionen beobachtet werden. / Malaria is still one of the most considerable parasite infections of the human being. Pregnant women are at an increased risk in endemic areas. P. falciparum shows a high genetic diversity. In endemic areas infections with P. falciparum are very often polyclonal. They are described as multiple Infections or as the multiplicity of infection. In pregnant women P.-falciparum-infected-erythrocytes which exprimate specific surface proteins sequester in the placental tissue. Often this is the course of preterm delivery, low birth weight and anaemia. To investigate the diversity of P. falciparum and the multiplicity of infection in pregnant women a cross-sectional study was conducted in the holoendemic area of Agogo in Ghana. In this study over a period of one year 474 labouring women infected with placental P.-falciparum where investigated. To examine the diversity and the multiplicity of infection merozoite surface protein-1 (msp1) block 2 and merozoite surface protein-2 (msp2) genotypes were determined in Isolates from peripheral and placental blood samples. The study showed that in comparision to isolates of peripheral blood samples isolates of placental blood samples where associated with a significant higher prevalence of polyclonal infections and a higher multiplicity of infection. The highest multiplicity of infection was found among primiparae and young women. With age and parity multiplicity of infection as well as parasite density decreased. In addition a high correlation between the multiplicity of infection and parasite density could be demonstrated. Age and parity did not influence this correlation. Thus the influence of age independent from parity on the multiplicity of infection could not be proved. However, multivariate analyses showed, that independently from parasite density placental Infections with two or more clones were in comparison to monoclonal Infections associated with a higher probability of preterm delivery. This was the case especially in primiparae and in women with submicroscopical placental Infection. Presently it is not clear, whether all or only a subset of co-infecting genotypes sequester in the placental tissue. To address this issue the genotype distribution of matched placental and peripheral P. falciparum isolates where investigated. While the multiplicity of infection of placental and peripheral isolates correlated the genotype pattern of the placenta and the periphery differed extensively. Only 12% genotyping of a peripheral Isolate showed the entire picture of the infection. In 67% of the cases despite finding identical genotypes differing genotypes in at least one of the two Isolates were detectable. Specific genotypes appeared more often in the placental than in the peripheral Isolate. In women, who were infected with genotypes of the allelic family FC27 clinical manifestation of malaria were observed more often. In multivariate analysis an association between FC27 and a preterm delivery was established. Beside this at least in univariate analyses FC27 was associated with low birth weight and anaemia. This was the case especially for primiparae and labouring women with submicroscopic placental infections.