Gene targeting in the rodent malaria parasite Plasmodium yoelii

It is anticipated that the sequencing of Plasmodium falciparum genome will soon be completed. Rodent models of malaria infection and stable transformation systems provide powerful means of using this information to study gene function in vivo. To date, gene targeting has only been developed for one rodent malaria species, Plasmodium berghei. Another rodent species, Plasmodium yoelii, however, is favored to study the mechanisms of protective immunity to the pre-erythrocytic stages of infection and vaccine development. In addition, it offers the opportunity to investigate unique aspects of pathogenesis of blood stage infection. Here, we report on the stable transfection and gene targeting of P. yoelii. Purified late blood stage schizonts were used as targets for electroporation with a plasmid that contains a pyrimethamine-resistant form of the P. berghei dihydrofolate reductase-thymidylate synthase (Pbdhfr-ts) fused to green fluorescent protein (gfp) gene. After drug selection, fluorescent parasites contained intact, non-rearranged plasmids that remain stable under drug-pressure. In addition, we used another dhfr-ts/gfp based plasmid to disrupt the P. yoelii trap (thrombospondin-related anonymous protein) locus by site-specific integration. The phenotype of P. yoelii TRAP knockout was identical to that previously reported for the P. berghei TRAP knockout. In the absence of TRAP, the erythrocytic cycle, gametocyte and oocyst development of the mutant parasites were indistinguishable from wild type (WT). Although the sporozoites appeared morphologically normal, they failed to glide and to invade the salivary glands of mosquitoes.     

Improved transfection and new selectable markers for the rodent malaria parasite Plasmodium yoelii

The rodent malaria Plasmodium yoelii is a useful model to study protective immunity to pre-erythrocytic stages of infection, pathogenesis of erythrocytic stages, and vaccine development. However, the utility of the P. yoelii model system has not been fully realized because transfection and genetic manipulation methodologies for this rodent species are less developed than that of another rodent species Plasmodium berghei. Here we report improved transfection efficiency using the AMAXA nucleofector system compared to conventional transfection methodologies. We also show that heterologous promoters from P. berghei can be used to drive expression of a green fluorescent protein (GFP) reporter protein in P. yoelii. In an effort to develop additional selectable markers for this parasite, we also tested positive selectable markers that have been used successfully in P. falciparum and P. berghei. Human dihydrofolate reductase (hdhfr) and Toxoplasma gondii dihydrofolate reductase-thymidylate synthase (Tgdhfr-ts) conferred drug resistance to WR99210 and pyrimethamine, respectively, when introduced as episomes. These improvements should make genetic manipulation of P. yoelii more amenable and facilitate further studies of host-parasite interactions using this attractive rodent model.     

Human antibodies against Plasmodium falciparum liver-stage antigen 3 cross-react with Plasmodium yoelii preerythrocytic-stage epitopes and inhibit sporozoite invasion in vitro and in vivo

The Plasmodium falciparum liver-stage antigen 3 (LSA3), a recently identified preerythrocytic antigen, induces protection against malaria in chimpanzees. Using antibodies from individuals with hyperimmunity to malaria affinity purified on recombinant or synthetic polypeptides of LSA3, we identified four non-cross-reactive B-cell epitopes in Plasmodium yoelii preerythrocytic stages. On sporozoites the P. yoelii protein detected has a molecular mass similar to that of LSA3. T-cell epitopes cross-reacting with P. yoelii were also demonstrated using peripheral blood lymphocytes from LSA3-immunized chimpanzees. In contrast, no cross-reactive epitopes were found in Plasmodium berghei. LSA3-specific human antibodies exerted up to 100% inhibition of in vitro invasion of P. yoelii sporozoites into mouse hepatocytes. This strong in vitro activity was reproduced in vivo by passive transfer of LSA3 antibodies. These results indicate that the homologous epitopes may be biologically functional and suggest that P. yoelii could be used as a model to assess the antisporozoite activity of anti-LSA3 antibodies.     

Plasmodium yoelii sporozoites with simultaneous deletion of P52 and P36 are completely attenuated and confer sterile immunity against infection

Malaria infection starts when sporozoites are transmitted to the mammalian host during a mosquito bite. Sporozoites enter the blood circulation, reach the liver, and infect hepatocytes. The formation of a parasitophorous vacuole (PV) establishes their intracellular niche. Recently, two members of the 6-Cys domain protein family, P52 and P36, were each shown to play an important albeit nonessential role in Plasmodium berghei sporozoite infectivity for the rodent host. Here, we generated p52/p36-deficient Plasmodium yoelii parasites by the simultaneous deletion of both genes using a single genetic manipulation. p52/p36-deficient parasites exhibited normal progression through the life cycle during blood-stage infection, transmission to mosquitoes, mosquito-stage development, and sporozoite infection of the salivary glands. p52/p36-deficient sporozoites also showed normal motility and cell traversal activity. However, immunofluorescence analysis and electron microscopic observations revealed that p52/p36-deficient parasites did not form a PV within hepatocytes in vitro and in vivo. The p52/p36-deficient parasites localized as free entities in the host cell cytoplasm or the host cell nucleoplasm and did not develop as liver stages. Consequently, they did not cause blood-stage infections even at high sporozoite inoculation doses. Mice immunized with p52/p36-deficient sporozoites were completely protected against infectious sporozoite challenge. Our results demonstrate for the first time the generation of two-locus gene deletion-attenuated parasites that infect the liver but do not progress to blood-stage infection. The study will critically guide the design of Plasmodium falciparum live attenuated malaria vaccines.     

Species-specific inhibition of cerebral malaria in mice coinfected with Plasmodium spp

Recent epidemiological observations suggest that clinical evolution of Plasmodium falciparum infections might be influenced by the concurrent presence of another Plasmodium species, and such mixed-species infections are now known to occur frequently in residents of most areas of endemicity. We used mice infected with P. berghei ANKA (PbA), a model for cerebral malaria (CM), to investigate the influence of experimental mixed-species infections on the expression of this pathology. Remarkably, the development of CM was completely inhibited by the simultaneous presence of P. yoelii yoelii but not that of P. vinckei or another line of P. berghei. In the protected coinfected mice, the accumulation of CD8(+) T cells in the brain vasculature, a pivotal step in CM pathogenesis, was found to be abolished. Protection from CM was further found to be associated with species-specific suppression of PbA multiplication. These observations establish the concept of mixed Plasmodium species infections as potential modulators of pathology and open novel avenues to investigate mechanisms implicated in the pathogenesis of malaria.     

Role of host cellular response in differential susceptibility of nonimmunized BALB/c mice to Plasmodium berghei and Plasmodium yoelii sporozoites.

We found BALB/c mice to be on the order of 2,000 times more susceptible to Plasmodium yoelii than Plasmodium berghei sporozoites, as measured by the ability of these sporozoites to differentiate into microscopically detectable hepatic schizonts in the livers of immunologically naive mice. One of the factors that determine the relative insusceptibility of mice to P. berghei sporozoites is the innate cellular inflammatory response that the mice mount after injection with sporozoites. The cellular inflammatory response against P. berghei is initiated soon after sporozoite injection; by 24 h, substantial histopathological changes have developed within the liver. There is considerably less of a cellular inflammatory response against P. yoelii; significant histopathological changes within the liver are not observed until well after hepatic schizonts have begun to rupture at around 44 h postinjection of sporozoites. These differences in the cellular inflammatory response against two different, closely related species of sporozoites are of considerable interest. The data strongly suggest that the BALB/c-P. berghei sporozoite system is a relatively poor biological model for sporozoite immunization studies.     

A conserved peptide sequence of the Plasmodium falciparum circumsporozoite protein and antipeptide antibodies inhibit Plasmodium berghei sporozoite invasion of Hep-G2 cells and protect immunized mice against P. berghei sporozoite challenge.

Minutes after injection into the circulation, malaria sporozoites enter hepatocytes. The speed and specificity of the invasion process suggest that it is receptor mediated. The region II sequence of Plasmodium falciparum circumsporozoite (CS) protein includes a nonapeptide (WSPCSVTCG) which is highly conserved in all of the CS proteins sequenced to data, including the one from Plasmodium berghei. We have found that two peptides based on the P. falciparum region II sequence, P18 (EWSPCSVTCGNGIQVRIK) and P32 (IEQYLKKIKNS ISTEWSPCSVTCGNGIQVRIK), significantly inhibited P. berghei sporozoite invasion into Hep-G2 cells in vitro. This inhibition was enhanced if either peptide was preincubated with Hep-G2 cells prior to sporozoite invasion. We confirm that region II is a sporozoite ligand for the hepatocyte receptor; moreover, despite the few differences between P. falciparum and P. berghei region II sequences around the nonapeptide sequence (66% homology), the functional characteristics of the motif sequences are not affected. Since the conserved motifs represent a crucial sequence involved in Plasmodium sporozoite invasion of hepatocytes, antibodies to region II should inhibit sporozite invasion into hepatocytes. Indeed, we found that polyclonal antibodies generated to the P. falciparum-based peptide P32 inhibited P. berghei sporozoite invasion of Hep-G2 cells. Furthermore, inbred mice (C57BL/6) immunized with P32 were protected against a lethal challenge of P. berghei sporozoites. Our results suggest that the conserved region II of the CS protein contains crucial B- and T-cell epitopes, that such peptide sequences from the human malaria parasite P. falciparum can be screened in the P. berghei rodent model, and, finally, that region II can be considered useful as one of the components of a malaria vaccine.     

Class II-restricted protective immunity induced by malaria sporozoites

The irradiated-sporozoite vaccine elicits sterile immunity against Plasmodium parasites in experimental rodent hosts and human volunteers. Based on rodent malaria models, it has been proposed that CD8+ T cells are the key protective effector mechanism required in sporozoite-induced immunity. To investigate the role of class II-restricted immunity in protective immunity, we immunized beta2-microglobulin knockout (beta2M-/-) mice with irradiated Plasmodium yoelii or P. berghei sporozoites. Sterile immunity was obtained in the CD8+-T-cell-deficient mice immunized with either P. berghei or P. yoelii sporozoites. beta2M-/- mice with the BALB/c (H-2d) genetic background as well as those with the C57BL (H-2b) genetic background were protected. Effector mechanisms included CD4+ T cells, mediated in part through the production of gamma interferon, and neutralizing antibodies that targeted the extracellular sporozoites. We conclude that in the absence of class I-restricted CD8+ T cells, sporozoite-induced protective immunity can be effectively mediated by class II-restricted immune effector mechanisms. These results support efforts to develop subunit vaccines that effectively elicit high levels of antibody and CD4+ T cells to target Plasmodium pre-erythrocytic stages.     

Stage-specific expression of aldolase isoenzymes in the rodent malaria parasite Plasmodium berghei.

We have cloned two gene (aldo-1 and aldo-2) encoding the glycolytic enzyme aldolase of the rodent malaria parasite Plasmodium berghei. The amino acid sequence of one gene product, ALDO-1, is virtually identical to P. falciparum aldolase whereas ALDO-2, the second gene product, is different and has 13% sequence diversity to ALDO-1. We expressed ALDO-2 as an active enzyme in Escherichia coli and compared the biochemical and kinetic properties to that of P. falciparum recombinant aldolase (ALDO-1 type). Based on the Km and Vmax constants for FMP and FBP, neither ALDO-1 nor ALDO-2 can be clearly assigned to any of the known mammalian isoenzyme classes. We demonstrate that expression of the two isoenzymes is developmentally regulated: specific antibody probes detect ALDO-1 in sporozoite stages of P. berghei and ALDO-2 is found in blood stage parasites.     

Merozoite surface protein 4/5 provides protection against lethal challenge with a heterologous malaria parasite strain

Immunization with merozoite surface protein 4/5 (MSP4/5), the murine malaria homologue of Plasmodium falciparum MSP4 and MSP5, has been shown to protect mice against challenge by parasites expressing the homologous form of the protein. The gene encoding MSP4/5 was sequenced from a number of Plasmodium yoelii isolates in order to assess the level of polymorphism in the protein. The gene was found to be highly conserved among the 13 P. yoelii isolates sequenced, even though many of the same isolates showed pronounced variability in their MSP1(19) sequences. Nonsynonymous mutations were detected only for the isolates Plasmodium yoelii nigeriensis N67 and Plasmodium yoelii killicki 193L and 194ZZ. Immunization and challenge of BALB/c mice showed that the heterologous MSP4/5 proteins were able to confer a level of protection against lethal Plasmodium yoelii yoelii YM challenge infection similar to that induced by immunization with the homologous MSP4/5 protein. To explore the limits of heterologous protection, mice were immunized with recombinant MSP4/5 protein from Plasmodium berghei ANKA and Plasmodium chabaudi adami DS and challenged with P. y. yoelii YM. Interestingly, significant protection was afforded by P. berghei ANKA MSP4/5, which shows 81% sequence identity with P. y. yoelii YM MSP4/5, but it was abolished upon reduction and alkylation. Significant protection was not observed for mice immunized with recombinant P. c. adami DS MSP4/5, which shows 55.7% sequence identity with P. y. yoelii YM MSP4/5. This study demonstrates the robustness of MSP4/5 in conferring protection against variant forms of the protein in a murine challenge system, in contrast to the situation found for other asexual-stage proteins, such as MSP1(19) and AMA1.     

Characterization of the merozoite surface protein 4/5 gene of Plasmodium berghei and Plasmodium yoelii

The genes encoding merozoite surface protein 4/5 (MSP4/5) from Plasmodium berghei and Plasmodium yoelii have been cloned and completely sequenced. Comparisons of the predicted protein sequences with those of Plasmodium chabaudi MSP4/5 and Plasmodium falciparum MSP4 and MSP5 show general structural similarities. All predicted proteins contain hydrophobic signal sequences, potential GPI attachment sequences and a single epidermal growth factor (EGF)-like domain at the C-terminus. The amino acid sequence of the EGF-like motif is highly conserved in rodent malaria species and also shows a considerable degree of similarity with the EGF-like domains found in the P. falciparum proteins. Both the P. yoelii and P. berghei genes show evidence of both spliced and unspliced mRNA at steady state. This phenomenon is similar to that seen for the P. chabaudi MSP4/5 gene, and is believed to be involved in regulation of protein expression. We describe here the construction of clones expressing full length recombinant protein. Antibodies directed against recombinant MSP4/5 proteins recognize a single polypeptide on parasite material and show crossreactivity between MSP4/5 from different murine malaria species, but do not crossreact with either MSP4 or MSP5 from P. falciparum. The various antisera show reactivity against reduction sensitive epitopes as well as reduction insensitive epitopes.     

Use of a two-sited monoclonal antibody assay to detect a heat-stable malarial antigen in the sera of mice infected with Plasmodium yoelii.

Antigens, circulating in the blood during malarial infections, have been implicated in immune protection, immunosuppression, and immune-complex formation. We used a monoclonal antibody (MAb 7H8) to identify an antigen (Ag-7H8) in the sera of mice infected with Plasmodium yoelii. The major form of the antigen has a molecular weight of approximately 120,000 in P. yoelii, with minor components of 220,000; 65,000 to 75,000; and 45,000. Ag-7H8 remains antigenic after boiling for 5 min. A two-sited assay was developed with MAb 7H8 that demonstrated that the Ag-7H8 has at least two similar epitopes per molecule. The two-sited assay was used to follow Ag-7H8 in the blood of mice during lethal (strain 17XL) and nonlethal (strain 17XNL) P. yoelii infections. Ag-7H8 appeared on days 6 and 7 after infection with 10(6) and 10(4) 17XL P. yoelii parasites, respectively, and remained until the animals died. It was in plasma samples between days 6 and 14 after 17XNL P. yoelii injections in several inbred strains of mice, regardless of the course of parasitemia. Thus, the kinetics of antigenemia correspond with early stages of infection and not with the number of circulating parasites. Indirect immunofluorescence assays demonstrated that MAb 7H8 detects a cross-reactive antigen in other malarial parasites, including Plasmodium berghei and Plasmodium falciparum. Thus, this two-sited assay may have general application for the serodiagnosis of malaria and may be beneficial in determining the relationship of circulating antigens to malarial immunity.     

Tandemly arranged gene clusters of malarial parasites that are highly conserved and transcribed

A molecular clone containing a 5.8 kb Eco RI fragment was isolated from a genomic library of the rodent malarial parasite Plasmodium yoelii. The P. yoelii genome contains about 150 copies of this sequence, making up almost 3% of the DNA. These sequences are tandemly arrayed in head-to-tail configurations with the unit length of the repeat being 5.8 kb. Several poly(A+) RNAs of P. yoelii ranging from 1.6 to 0.3 kb are recognized by the 5.8 kb clone. Five additional species of malarial parasites (P. chabaudi, P. berghei, P. falciparum, P. knowlesi, and P. cynomolgi) contain tandemly repeated arrays of sequences having the same unit length of 5.8 kb, which readily hybridize to the sequence cloned from P. yoelii.     

Truncation of Plasmodium berghei merozoite surface protein 8 does not affect in vivo blood-stage development

Merozoite surface protein 8 (MSP8) has shown promise as a vaccine candidate in the Plasmodium yoelii rodent malaria model and has a proposed role in merozoite invasion of erythrocytes. However, the temporal expression and localisation of MSP8 are unusual for a merozoite antigen. Moreover, in Plasmodium falciparum the MSP8 gene could be disrupted with no apparent effect on invitro growth. To address the invivo function of full-length MSP8, we truncated MSP8 in the rodent parasite Plasmodium berghei. PbDeltaMSP8 disruptant parasites displayed a normal blood-stage growth rate but no increase in reticulocyte preference, a phenomenon observed in P. yoelii MSP8 vaccinated mice. Expression levels of erythrocyte surface antigens were similar in P. berghei wild-type and PbDeltaMSP8-infected erythrocytes, suggesting that a parasitophorous vacuole function for MSP8 does not involve global trafficking of such antigens. These data demonstrate that a full-length membrane-associated form of PbMSP8 is not essential for blood-stage growth.     

鼠疟模型作为恶性疟原虫多表位疫苗保护性的探索试验

目的为寻找恶性疟原虫基因工程多表位疫苗保护性评价的模型动物，利用小鼠约氏疟原虫(Plasmodiumyoelii)及小鼠伯氏疟原虫(Plasmodiumberghei)模型对本实验室构建的恶性疟原虫(Plasmodiumfalciparum)多阶段、多表位基因工程候选疫苗进行了体内保护性及免疫模式探索试验。结果在约氏疟原虫模型，先用该疫苗基因的大肠杆菌表达蛋白免疫，后用含同一基因的重组痘苗病毒加强免疫组，与野生型痘苗病毒免疫组及空白对照组相比，小鼠的死亡时间延长(P<0.01)。结果初步显示，约氏疟原虫鼠疟模型可用作该基因工程多表位恶性疟原虫候选疫苗的保护性评价。     

Identification, cloning, expression, and characterization of the gene for Plasmodium knowlesi surface protein containing an altered thrombospondin repeat domain

Proteins present on the surface of malaria parasites that participate in the process of invasion and adhesion to host cells are considered attractive vaccine targets. Aided by the availability of the partially completed genome sequence of the simian malaria parasite Plasmodium knowlesi, we have identified a 786-bp DNA sequence that encodes a 262-amino-acid-long protein, containing an altered version of the thrombospondin type I repeat domain (SPATR). Thrombospondin type 1 repeat domains participate in biologically diverse functions, such as cell attachment, mobility, proliferation, and extracellular protease activities. The SPATR from P. knowlesi (PkSPATR) shares 61% and 58% sequence identity with its Plasmodium falciparum and Plasmodium yoelii orthologs, respectively. By immunofluorescence analysis, we determined that PkSPATR is a multistage antigen that is expressed on the surface of P. knowlesi sporozoite and erythrocytic stage parasites. Recombinant PkSPATR produced in Escherichia coli binds to a human hepatoma cell line, HepG2, suggesting that PkSPATR is a parasite ligand that could be involved in sporozoite invasion of liver cells. Furthermore, recombinant PkSPATR reacted with pooled sera from P. knowlesi-infected rhesus monkeys, indicating that native PkSPATR is immunogenic during infection. Further efficacy evaluation studies in the P. knowlesi-rhesus monkey sporozoite challenge model will help to decide whether the SPATR molecule should be developed as a vaccine against human malarias.     

Cloning of Plasmodium yoelii genes expressing three different sporozoite-specific antigens

Genomic DNA isolated from Plasmodium yoelii (strain #17XNL) was prepared by partial digestion and cloned in Escherichia coli TB-1 with pUC18 plasmid. Antigen-producing recombinants were detected by a battery of monoclonal antibodies against antigens of the sporozoite stages. Four clones producing stage-specific sporozoite antigens were identified. One produced P. yoelii circumsporozoite protein, and three produced other P. yoelii sporozoite antigens.     

Immune responses to Plasmodium vivax pre-erythrocytic stage antigens in naturally exposed Duffy-negative humans: a potential model for identification of liver-stage antigens

Duffy antigen is the receptor used by Plasmodium vivax to invade erythrocytes. Consequently, individuals lacking Duffy antigen [Fy(-)] do not develop blood-stage infections. We hypothesized that naturally exposed Fy(-) humans may develop immune responses mainly to pre-erythrocytic stages and could be used to study acquired immunity to P. vivax and to identify liver-stage antigens. We report here that antibody and IFN-gamma responses to known sporozoite antigens were significantly induced by natural exposure in Fy(-) humans, whereas responses to blood-stage antigens were significantly induced in Fy(+) humans. IFN-gamma responses to sporozoite antigens were lower in Fy(+) than in Fy(-) humans, indicating that in Fy(+) humans blood-stage infections may have suppressed T cell responses to pre-erythrocytic stages. We evaluated the immune responses to 18 novel P. vivax homologs of P. falciparum sporozoite proteins identified from the P. vivax genome sequence. Eight proteins recalled IFN-gamma responses in P. vivax-exposed but not in unexposed individuals. Of these, 3 antigens elicited IFN-gamma responses in Fy(-) but not in Fy(+) individuals. These results suggest that differential immune responses observed in naturally exposed Fy(-) and Fy(+) individuals can be exploited to identify P. vivax stage-specific antigens.