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© 2007 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved., Immunisation with live, radiation-attenuated sporozoites (RAS) or genetically attenuated sporozoites (GAS) of rodent plasmodial parasites protects against subsequent challenge infections. We recently showed that immunisation with Plasmodium berghei GAS that lack the microneme protein P36p protects mice for a period of up to 4 months. Here, we show that the period of full protection induced by p36p(-)-sporozoites lasts 12 and 18 months in C57Bl6 and BALB/c mice, respectively. Full protection is also achieved with three doses of only 1000 p36p(-) (but not RAS) sporozoites. Subcutaneous, intradermal or intramuscular routes of administration also lead to partial protection. In addition, immunisation with either P. berghei RAS- or, to a lesser extent, p36p(-)-sporozoites inhibits parasite intrahepatic development in mice challenged with Plasmodium yoelii sporozoites. Since naturally acquired malaria infections or subunit-based vaccines only induce short-term immune responses, the protection conferred by immunisation with p36p(-)-sporozoites described here further emphasises the potential of GAS as a vaccination strategy for malaria., This work has been funded by Fundação para a Ciência e Tecnologia (Portugal), European Science Foundation, The Netherlands Organisation for Health Research and Development (ZonMW) and The Wellcome Trust Functional Genomics Initiative. B.D. is recipient of a fellowship from Fundação para a Ciência e Tecnologia (SFRH/16813/BD/2004), and received support from European Science Foundation (COST STSM 857 00743) and from European Molecular Biology Organization (EMBO Short Term Fellowship ASTF 242-2005) for this work. M.M.M. is a EMBO YIP, is a recipient of a EURYI Award from European Science Foundation and is a Howard Hughes Medical Institute International Scholar.

Malaria parasites undergo a sporogonic cycle in the mosquito vector. Sporozoites, the form of the parasite injected into the host during a bloodmeal, develop inside oocysts in the insect midgut, then migrate to and eventually invade the salivary glands. The circumsporozoite protein (CS), one of the major proteins synthesized by salivary gland sporozoites, is a surface-associated molecule which is important in sporozoite infectivity to the host. Here, by gene targeting, we created Plasmodium berghei lines in which the single-copy CS gene was disrupted. The CS(-) and wild-type parasites produced similar numbers of oocysts of comparable size in the mosquito midgut. In the CS(-) oocysts, however, sporozoite formation was profoundly inhibited. CS therefore appears to have a pleiotropic role and to be vital for malaria parasites in both the vector and the host: in mosquitoes, CS is essential for sporozoite development within oocysts, and in the vertebrate host it promotes sporozoite attachment to hepatocytes.

Difficulties with inducing sterile and long lasting protective immunity against malaria with subunit vaccines has renewed interest in vaccinations with attenuated Plasmodium parasites. Immunizations with sporozoites that are attenuated by radiation (RAS) can induce strong protective immunity both in humans and rodent models of malaria. Recently, in rodent parasites it has been shown that through the deletion of a single gene, sporozoites can also become attenuated in liver stage development and, importantly, immunization with these sporozoites results in immune responses identical to RAS. The promise of vaccination using these genetically attenuated sporozoites (GAS) depends on translating the results in rodent malaria models to human malaria. In this study, we perform the first essential step in this transition by disrupting, p52, in P. falciparum an ortholog of the rodent parasite gene, p36p, which we had previously shown can confer long lasting protective immunity in mice. These P. falciparum P52 deficient sporozoites demonstrate gliding motility, cell traversal and an invasion rate into primary human hepatocytes in vitro that is comparable to wild type sporozoites. However, inside the host hepatocyte development is arrested very soon after invasion. This study reveals, for the first time, that disrupting the equivalent gene in both P. falciparum and rodent malaria Plasmodium species generates parasites that become similarly arrested during liver stage development and these results pave the way for further development of GAS for human use.

Contains fulltext : 185544.pdf (Publisher’s version ) (Closed access) Fertilization and zygote development are obligate features of the malaria parasite life cycle and occur during parasite transmission to mosquitoes. The surface protein PFS48/45 is expressed by male and female gametes of Plasmodium falciparum and PFS48/45 antibodies prevent zygote development and transmission. Here, gene disruption was used to show that Pfs48/45 and the ortholog Pbs48/45 from a rodent malaria parasite P. berghei play a conserved and important role in fertilization. p48/45- parasites had a reduced capacity to produce oocysts in mosquitoes due to greatly reduced zygote formation. Unexpectedly, only male gamete fertility of p48/45- parasites was affected, failing to penetrate otherwise fertile female gametes. P48/45 is shown to be a surface protein of malaria parasites with a demonstrable role in fertilization.

Pbs21 is a surface protein of the ookinete of Plasmodium berghei , which can induce a potent transmission-blocking immune response. Pbs21 is normally expressed only by parasite stages in the mosquito, i.e., female gametes/zygotes, ookinetes, and oocysts. However, the Pbs21 gene is transcribed in female gametocytes which circulate in the bloodstream of the host, where translation of the resulting mRNA is totally repressed. Episomal transfection has been used to investigate whether expression of Pbs21 protein could be achieved in blood stages of the parasite. By using plasmid pMD221, the complete mRNA-encoding region of Pbs21, flanked only by 218 nucleotides (nt) of its promoter region and 438 nt of its 3′ region downstream from the polyadenylation site, was introduced into the blood stages of gametocyte-producing and non-gametocyte-producing clones of P. berghei . In both of these transformed parasite lines, Pbs21 protein was expressed in asexual trophozoites, schizonts, and, when present, in both male and female gametocytes. Hence, the flanking regions present are sufficient to allow transcription but lack the elements that exert natural control of sex- and stage-specific transcription. The mRNA and the protein expressed by transformed blood stages were indistinguishable from the wild-type forms by the criteria tested, and the protein was recognized by both conformation-dependent and conformation-independent monoclonal antibodies raised against native Pbs21. In mice infected with transformed non-gametocyte-producing parasites, a Pbs21-specific immune response was induced and characterized with respect to isotype (IgG2a/IgG2b) and quantity (11.5 ± 10 μg/ml) of antibody produced. However, as found in previous studies, these antibody levels were insufficient to inhibit development of the parasites in the mosquito. The ability to express mosquito midgut-stage antigens in blood-stage parasites will facilitate further investigations of molecular and immunological properties of these proteins.

To fully exploit the transfection technology developed for Plasmodium we investigated the features of replication, expression and segregation of an episomally maintained DNA construct during a sexual blood stage development in genetically transformed parasites of P. berghei. Using DNA in situ hybridisation techniques we were able to show that the introduced DNA construct is located in the nucleus of the parasite and is not segregating uniformly during schizogony. Replication of the construct mainly takes place between 16 and 24 h after invasion of the merozoites, coinciding with chromosomal replication. Furthermore the plasmid-borne DHFR/TS gene is constitutively transcribed throughout the asexual blood stage development. Hence the DHFR/TS promoter would appear to be a useful tool in the study of (over)expression of introduced genes and performing complementation studies in transfected parasites during the complete a sexual blood stage development of P. berghei.

Dihydrofolate reductase thymidylate synthase (DHFR-TS) is a vital, bifunctional enzyme involved in the DNA synthesis of Plasmodium. The antifolate drugs pyrimethamine and cycloguanil competitively inhibit the function of the DHFR activity [1-3]. The molecular basis of resistance of malaria parasites against these drugs has been well studied and it appears that in Plasmodium falciparum and Plasmod ium chabaudi high level resistance to pyrimethamine is caused by a single point mutation in the DHFR-region of the enzyme [4-8]. We studied the molecular basis of pyrimethamine resistance in the rodent parasite Plasmodium berghei for several reasons. An understanding of mechanisms underlying resistance might help to develop strategies