Ookinete Surface Protein Research Articles

P25 and P28 proteins of the malaria ookinete surface have multiple and partially redundant functions.

The ookinete surface proteins (P25 and P28) are proven antimalarial transmission-blocking vaccine targets, yet their biological functions are unknown. By using single (Sko) and double gene knock-out (Dko) Plasmodium berghei parasites, we show that P25 and P28 share multiple functions during ookinete/oocyst development. In the midgut of mosquitoes, the formation of ookinetes lacking both proteins (Dko parasites) is significantly inhibited due to decreased protection against lethal factors, including protease attack. In addition, Dko ookinetes have a much reduced capacity to traverse the midgut epithelium and to transform into the oocyst stage. P25 and P28 are partially redundant in these functions, since the efficiency of ookinete/oocyst development is only mildly compromised in parasites lacking either P25 or P28 (Sko parasites) compared with that of Dko parasites. The fact that Sko parasites are efficiently transmitted by the mosquito is a compelling reason for including both target antigens in transmission-blocking vaccines.

Presence of three distinct ookinete surface protein genes, Pos25, Pos28- 1, and Pos28-2, in Plasmodium ovale

Presence of three distinct ookinete surface protein genes, Pos25, Pos28- 1, and Pos28-2, in Plasmodium ovale

Interaction of Plasmodium gallinaceum ookinetes and oocysts with extracellular matrix proteins.

Plasmodium ookinetes are elongate, motile and invasive while inside the mosquito gut but promptly metamorphose into spherical immobile oocysts upon coming in contact with the basement membrane surrounding the midgut. There they begin a prolonged growth period characterized by massive DNA synthesis for the production of sporozoites. Living Plasmodium gallinaceum ookinetes attached avidly to the murine extracellular matrix proteins, laminin and collagen type IV. In ELISA-type assays, the main ookinete surface protein, Pgs28 was implicated as a mediator of parasite attachment to these basement membrane constituents. Laminin and collagen IV adhered to ookinete and oocyst lysates spotted onto nitrocellulose membranes. Receptor-ligand blot assays demonstrated that Pgs28 and an oocyst-specific antigen recognized by the mAb 10D6 interact with murine collagen IV and laminin. 10D6 antigen was also recognized by monospecific antiserum against the human epidermal growth factor receptor. Mosquito-derived laminin was incorporated into oocyst capsules of P. gallinaceum growing in Aedes aegypti. We hypothesize that contact with the mosquito basement membrane triggers the transformation of ookinetes into oocysts. Coalescence of basement membrane proteins onto the capsules masks developing oocysts from the mosquito's immune system and facilitates their prolonged extracellular development in the mosquito body cavity.

Detection of low level Plasmodium falciparum gametocytes using reverse transcriptase polymerase chain reaction

The polymerase chain reaction (PCR) is widely used to detect and identify low numbers of blood forms of different species of human malaria parasites [1]. The method has demonstrated a high prevalence of sub-microscopic asymptomatic Plasmodium falciparum infections among inhabitants of malaria-endemic countries [2–5]. Absence of patent parasites from the peripheral blood is often associated with sub-patent parasitaemias that can last for several months, even when the possibility of reinfection can be ruled out [5,6]. However, it is not known whether such sub-patent long lasting infections can produce sexual forms and thus be capable of infecting mosquitoes. Some studies have indicated that the minimum density of gametocytes capable of infecting mosquitoes can be remarkably low (reviewed by Carter and Gwadz [7]), and it has been demonstrated that sexual forms are infectious to mosquitoes even when they occur at levels not detectable by microscopy [8]. Here we describe a reverse transcriptase polymerase chain reaction (RT-PCR) for detection of gametocytes of P. falciparum. Unlike asexual parasites, the mature gametocytes of P. falciparum are metabolically relatively inactive [9]. However, almost immediately mature gametocytes are activated to undergo gametogenesis, either upon ingestion by a mosquito or upon stimulation in vitro, they enter a period of intense protein synthesis [10]. Among the proteins which are produced rapidly and in large amounts following such activation, is the zygote and ookinete surface protein Pfs25 [10]. This protein and, as we show here, its mRNA molecules are specific to the activated gametocyte and to the zygote and ookinete stages into which it transforms. Both Pfs25 and its mRNA are totally absent from the asexual stage parasites of P. falciparum. We have exploited this property of the mRNA of Pfs25 in the activated gametocytes of P. falciparum in or* Corresponding author. Tel.: +44-131-6508658; fax: +44131-6673210; e-mail: H.Babiker@ed.ac.uk.

Sequence Polymorphism in Two Novel Plasmodium vivax Ookinete Surface Proteins, Pvs25 and Pvs28, That Are Malaria Transmission-blocking Vaccine Candidates

For many malarious regions outside of Africa, development of effective transmission-blocking vaccines will require coverage against both Plasmodium falciparum and P. vivax. Work on P. vivax transmission-blocking vaccines has been hampered by the inability to clone the vaccine candidate genes from this parasite. To search for genes encoding the ookinete surface proteins from P. vivax, the DNA sequences of the eight known proteins in the P25 subfamily (Pfs25, Pgs25, Pys25, Pbs25) and in the P21/28 subfamily (Pfs28, Pgs28, Pys21, Pbs21) of zygote/ookinete surface proteins were aligned. Regions of highest identity were used to design degenerate PCR oligonucleotides. Genomic DNA from the Sal I strain of P. vivax and genomic and splinkerette DNA libraries were used as PCR templates. To characterize the polymorphisms of Pvs25 and Pvs28, these two genes were PCR amplified and the DNA sequences were determined from genomic DNA extracted from patients infected with P. vivax. Analysis of the deduced amino acid sequence of Pvs28 revealed a secretory signal sequence, four epidermal growth factor (EGF)-like domains, six copies of the heptad amino acid repeat (GSGGE/D), and a short hydrophobic region. Because the fourth EGF-like domain has four rather than six cysteines, the gene designated Pvs28 is the presumed homologue of P21/28 subfamily members. Analysis of the deduced amino acid sequence of Pvs25 revealed a similar structure to that of Pvs28. The presence of six rather than four cysteines in the fourth EGF-like domain suggested that Pvs25 is the homologue of P25 subfamily members. Several regions of genetic polymorphisms in Pvs25 and Pvs28 were identified in field isolates of P. vivax. The genes encoding two ookinete surface proteins, Pvs28 and Pvs25, from P. vivax have been isolated and sequenced. Comparison of the primary structures of Pvs25, Pvs28, Pfs25, and Pfs28 suggest that there are regions of genetic polymorphism in the P25 and P21/28 subfamilies.

Antigenic polymorphism of novel ookinete surface proteins, PVS25 and PVS28 from Plasmodium vivax that are candidates of transmission-blocking vaccine

Antigenic polymorphism of novel ookinete surface proteins, PVS25 and PVS28 from Plasmodium vivax that are candidates of transmission-blocking vaccine

Two antigens on zygotes and ookinetes of Plasmodium yoelii and Plasmodium berghei that are distinct targets of transmission-blocking immunity.

We have developed transmission-blocking monoclonal antibodies (MAbs) against Plasmodium yoelii 21-kDa (Pys21) and 28-kDa (Pys25) ookinete surface proteins. These MAbs block infectivity of P. yoelii to Anopheles stephensi. One MAb, 14, cross-reacted by Western blotting with a 28-kDa surface protein (Pbs25) of P. berghei ookinetes and blocked oocyst development, as assayed by direct mosquito feeds on passively immunized P. berghei-infected mice. In total, we have identified two ookinete surface proteins in P. yoelii, one of which is also present in P. berghei. The transmission-blocking activity of the anti-Pys25 MAb 4 was complete and more potent than that of the anti-Pys21 MAb 2. Moreover, Fab fragments of MAb 4 had transmission-blocking activity in mice. In comparison, Fab fragments of MAb 2 did not have detectable transmission-blocking effect, although F(ab')2 did. Furthermore, MAb 2 and MAb 4 appeared to block the in vitro formation and development of zygotes as well.

Primary structure of a novel ookinete surface protein from Plasmodium berghei

Primary structure of a novel ookinete surface protein from Plasmodium berghei

Induction of anti-malarial transmission blocking immunity with a recombinant ookinete surface antigen of Plasmodium berghei produced in silkworm larvae using the baculovirus expression vector system

We have studied Pbs21, a major ookinete surface protein of Plasmodium berghei, for the development of a model transmission blocking immunogen. In the mouse, recombinant Pbs21 expressed in the Escherichia coli expression system (EcrPbs21) is not as effective in inducing transmission blocking antibodies as native Pbs21 (nPbs21), possibly because of differences in post-translational processing between EcrPbs21 and nPbs21. In an attempt to improve the efficacy of the recombinant molecule, we describe here the use of a baculovirus expression vector system in the silkworm Bombyx mori. Following an injection of recombinant baculovirus containing Pbs21 cDNA, B. mori larvae produced recombinant Pbs21 (BmrPbs21) with a molecular weight indistinguishable from nPbs21. Fifty micrograms of BmrPbs21 could be purified from the hemolymph of each infected larva using affinity chromatography. Immunization of Balb c mice with BmrPbs21 induced high anti-BmrPbs21 and anti-ookinete antibodies but low anti-EcrPbs21 antibody. In contrast, EcrPbs21 induced high anti-EcrPbs21 antibody but low anti-BmrPbs21 and anti-ookinete antibodies. This suggests that most B-cell epitopes on nPbs21 are conformational and that many of the linear epitopes in EcrPbs21 are not normally exposed in nPbs21. Oocyst formation in Anopheles stephensi mosquitoes, which fed on mice immunized with purified BmrPbs21 and infected with P. berghei, was blocked by 85.5–97.1%. These results suggest that the baculovirus-silkworm system produces useful quantities of recombinant Pbs21 which in limited studies is structurally and immunogenically indistinguishable from the native molecule.

Differential expression in blood stages of the gene coding for the 21-kilodalton surface protein of ookinetes of Plasmodium berghei as detected by RNA in situ hybridisation

The developmentally regulated transcription of the gene encoding the ookinete surface protein, Pbs21, has been investigated in the rodent malaria parasite, Plasmodium berghei, by RNA in siu hybridisation using fluorescently labelled DNA probes. We used a procedure that will allow the visualisation of cytoplasmic mRNA in the parasite and of high copy DNA repeats in the nucleus. Specific hybridisation to Pbs21 mRNA occurred in the cyptoplas of female gametocytes, zygotes and ookinetes, while asexual blood stages, male gemetocytes and gametes showed no fluorescence. Analysis of the transcription of the Pbs21 gene during blood stage development in two tightkt synchronised parasite clones using the same methodology revealed that transcription is restricted to sexual stages and is initiated in immature gametocytes at 19 h post invasion (hpi). At this point in development it is not yet possible to discriminate between the morphology of asexual trophozoites and immature gametocytes. At 24 hpi approximate 50% of the gametocytes transcribed the Pbs21 gene and the morphology of these gametocytes was identical and female. The distribution of hte mRNA encoding Pbs21 confirmed that post-transcriptional control of expression occurred in the cytoplasm by repression of translation and not through delayed transport of the message to the cytoplasm., The transcription of the Pbs21 gene is the earliest demonstrated event in gametocytogenesis in rodent malaria species to date.

Sequential expression of antigens on sexual stages of Plasmodium falciparum accessible to transmission-blocking antibodies in the mosquito.

Plasmodium falciparum gametocytes contain specific antigens, some of which (Mr 230,000, 48,000, 45,000) are expressed on the surface of the newly emerged macrogamete. A different antigen (Mr 25,000) surrounds the surface of the ookinete and, although present to some extent in the developing gametocyte, is synthesized in high quantities by the macrogamete/zygote and expressed progressively on the transforming zygote surface. These antigens are targets of transmission blocking antibodies that are effective at two distinct points after gametogenesis: fertilization of the macrogamete and ookinete to oocyst development. The antigens involved in the fertilization blockade are the Mr 48 and 45 proteins, which are expressed on the macrogamete surface. The Mr 230 K coprecipitating protein probably plays no part in transmission block. mAb directed against the Mr 25 K ookinete surface protein blocked transmission without inhibiting ookinete formation, indicating that this protein has an important role in the transformation of ookinete into oocyst. A combination of mAb recognizing different epitopes on the same protein molecule acted synergistically in inhibiting oocyst formation. Using a mixture of two blocking mAb reacting against the Mr 48/45 and 25 K proteins, respectively, an additive blocking effect could be demonstrated.