Specific lysis of Plasmodium yoelii infected mouse erythrocytes with antibody and complement.

An assay for complement-dependent antibody-mediated cytolysis of Plasmodium yoelii infected mouse erythrocytes was designed. By means of a rat hyperimmune serum to P. yoelii the presence of parasite associated antigens was demonstrated on erythrocytes from P. yoelii infected mice. Tests with such erythrocytes fractionated on Percoll gradients showed that the target antigens for the cytotoxic antibodies were confined to the surface of only infected erythrocytes containing late stages of the parasite. The cytotoxic antibodies were of IgG class.     

CD8+ cytolytic T cell clones derived against the Plasmodium yoelii circumsporozoite protein protect against malaria.

Immunization of BALB/c mice with radiation-attenuated Plasmodium yoelii sporozoites induces cytotoxic T lymphocytes (CTL) specific for an epitope located within the amino acid sequence 277-288 of the P. yoelii circumsporozoite (CS) protein. Several CD8+ CTL clones were derived from the spleen cells of sporozoite-immunized mice, all displaying an apparently identical epitope specificity. All the clones induced high levels of cytolysis in vitro upon exposure to peptide-incubated MHC-compatible target cells. The adoptive transfer of two of these clones conferred complete protection against sporozoite challenge to naive mice. This protection is species and stage specific. Using P. yoelii specific ribosomal RNA probes to monitor the in vivo effects of the CTL clones, we found that their target was the intrahepatocytic stage of the parasite. The protective clones completely inhibited the development of the liver stages of P. yoelii. Some CTL clones were only partially inhibitory in vivo, while others failed completely to alter liver stage development and to confer any detectable degree of protection. The elucidation of the effector mechanism of this CTL mediated protection against rodent malaria should facilitate the design of an effective malaria vaccine. From a broader perspective this model may provide further insight into the mechanism(s) of CTL mediated killing of intracellular non-viral pathogens in general.     

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.     

Molecular clones of alpha-tubulin genes of Plasmodium yoelii reveal an unusual feature of the carboxy terminus

By using a Trypanosoma brucei alpha-tubulin cDNA probe under reduced stringency hybridization conditions, we have isolated two genomic clones that constitute portions of alpha-tubulin genes of the rodent malarial parasite Plasmodium yoelii. P. yoelii has two alpha-tubulin genes, the 3' portions of which were present in the two clones, Py alpha T1 and Py alpha T2, containing 1.3 kb and 6.6 kb EcoRI fragments respectively. The 1358 bp Py alpha T1 clone was completely sequenced and found to contain 591 nucleotides of uninterrupted coding sequence with a strong bias for AT-rich codons, starting with codon 254 of a consensus alpha-tubulin sequence. Numerous attempts to clone 5' portions of these genes were unsuccessful. A single mRNA of 2.3 kb was recognized by both the clones in the erythrocytic stages of P. yoelii. A probe constituting the untranslated sequences of Py alpha T1 also recognized this RNA but failed to hybridize with Py alpha T2 sequences, indicating that the gene represented by the Py alpha T1 clone was expressed during the erythrocytic stages. The deduced amino acid sequence of the Py alpha T1 gene terminates in Tyr-Glu instead of Glu-Tyr observed in alpha-tubulins of almost all other organisms. The difference observed may have implications for alpha-tubulin metabolism in malarial parasites.     

The Plasmodium falciparum knob-associated PfEMP3 antigen is also expressed at pre-erythrocytic stages and induces antibodies which inhibit sporozoite invasion

The expression of the pfemp3 gene and the corresponding PfEMP3 knob-associated protein in the pre-erythrocytic stages of Plasmodium falciparum was demonstrated by RT-PCR, Western blots, IFAT and IEM. The antigen was found on the surface of the sporozoite and in the cytoplasm of mature hepatic stage parasites. Immunological cross-reactivity was observed with sporozoites from the rodent malaria parasites Plasmodium yoelii yoelii and Plasmodium berghei and was exploited to assess a potential role of this protein at the pre-erythrocytic stages. Specific antibodies from immune individuals were found to inhibit P. yoelii yoelii and P. berghei sporozoite invasion of primary hepatocyte cultures. PfEMP3 should now be added to the small list of proteins expressed at the pre-erythrocytic stages of P. falciparum, and its vaccine potential now deserves to be investigated.     

C-reactive protein protects against preerythrocytic stages of malaria.

We previously reported that low doses of interleukin-1 strongly inhibited in vitro development of the hepatic stages of Plasmodium falciparum and P. yoelii. Among several hypotheses, we considered the role of C-reactive protein (CRP), a major acute-phase reactant whose concentration increases markedly in infectious disorders. We demonstrated that human hepatocytes cultured in the presence of interleukin-1 released, as early as 30 min after stimulation, an increased amount of CRP. We then established that CRP bound to the P. falciparum and P. yoelii sporozoite surface membranes, probably via a phosphorylcholine binding site. Experiments in which CRP was added to rat hepatocyte monolayers during or after inoculation confirmed that the target of the CRP-mediated inhibition was at the very early phase of infection. These in vitro functional activities were confirmed in an in vivo model; rats with increased levels of CRP in serum following an injection of turpentine oil were found to be largely protected against an inoculation of P. yoelii sporozoites. The same results were observed in animals inoculated with sporozoites previously incubated in purified CRP or in sera of rats pretreated with turpentine oil. The latter effect was inhibited after incubation of serum from turpentine-injected rats with anti-CRP serum.     

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.     

Serum antibody immunoglobulin G of mice convalescent from Plasmodium yoelii infection inhibits growth of Plasmodium falciparum in vitro: blood stage antigens of P. falciparum involved in interspecies cross-reactive inhibition of parasite growth.

We demonstrated that antibodies in the serum of BALB/c mice convalescent from Plasmodium yoelii infection inhibit the in vitro growth of Plasmodium falciparum. Blood stage P. falciparum antigens that cross-react with the convalescent-phase mouse serum antibodies were identified and partially characterized. Convalescent-phase mouse serum immunoglobulin G (IgG) reacted with P. falciparum lysates at up to a 1:15,000 dilution of the immune sera and bound to P. falciparum-parasitized erythrocytes at up to a 1:5,000 dilution of the sera. The cross-reactive moieties of antigens in parasite lysates were resistant to oxidation by periodate but sensitive to trypsinization. About 15 polypeptides (M(r)s of 15,000 to 110,000) of P. falciparum blood stages were recognized by the convalescent-phase mouse anti-P. yoelii sera; many of these antigens were metabolically 35S labeled and specifically immunoprecipitated. Also, virtually all of the cross-reactive antigens were recognized by human malaria-immune sera. The anti-P. yoelii serum antibodies bound, with high affinity, to at least five of the cross-reactive antigens (M(r)s of 107,000, 84,000, 53,000, 36,000, and 30,000). By phase separation in Triton X-114, eight interspecies cross-reactive antigens (M(r)s of 84,000, 76,000, 51,000, 31,000, 29,000, 28,000, 23,000, and 22,000) were found to be integral membrane proteins. Convalescent-phase mouse serum IgG strongly inhibited the differentiation of P. falciparum from schizonts to rings; 75 micrograms of IgG per ml caused 80% inhibition of release of merozoites and their invasion into erythrocytes. On the other hand, the anti-P. yoelii serum antibodies also inhibited intracellular development of P. falciparum from rings to schizonts; 25 micrograms of IgG per ml caused 50% inhibition. Inhibition of P. falciparum growth by anti-P. yoelii serum IgG suggests that some of the interspecies cross-reactive antigens contain important conserved epitopes and induce protective antibodies against P. falciparum.     

Inhibition by cyclosporin A of rodent malaria in vivo and human malaria in vitro.

The development and course of normally lethal parasitemias in mice inoculated intraperitoneally with erythrocytic stages of Plasmodium yoelii or Plasmodium berghei were markedly affected by treatment with the antilymphoid drug cyclosporin A (CS-A). When the first of four daily subcutaneous 25-mg/kg doses of CS-A was given at the time of parasite inoculation, patent infections failed to develop. If begun up to 5 days earlier, this same treatment regimen prolonged the prepatent period, attenuated parasitemia, and reduced mortality. In mice with patient infections, two consecutive daily 25-mg/kg doses of CS-A were sufficient to terminate parasitemias which, after several days, reappeared but were self-limiting. This pattern of apparent cure followed by transient recrudescence remained unaltered even when daily treatment with the same drug dose was continued for 3 weeks. Recrudescence was associated with the emergence of parasite populations that were relatively resistant to CS-A and, in the case of P. yoelii, of reduced virulence. In more limited experiments, CS-A was found to be active in vitro against erythrocytic stages of the human malarial parasite palsmodium falciparum. Depending on the concentration of drug in the culture medium, parasite growth was either prevented or inhibited.     

Host defenses in murine malaria: evaluation of the mechanisms of immunity to Plasmodium yoelii infection.

The immune response of random-bred mice to infection with a relatively avirulent strain of Plasmodium yoelii was measured in terms of parasitemia, splenomegaly, immediate and delayed hypersensitivity to a P. yoelii antigen preparation, resistance to challenge with a virulent variant of P. yoelii, and nonspecific resistance to L. monocytogenes. Avirulent P. yoelii produced a self limiting infection which resolved in 21 days. Peak parasitemia and splenomegaly were observed at 14 days, and infected mice were resistant to challenge with virulent P. yoelii from 7 days through at least 126 days. Mice infected with avirulent P. yoelii developed humoral immunity as judged by immediate hypersensitivity reactions and the capacity of their serum to passively protect normal mice against virulent P. yoelii. At no time did mice infected with the avirulent P. yoelii display evidence of cell-mediated immunity, as expressed by delayed-type hypersensitivity and increased resistance to L. monocytogenes. In fact, at the height of avirulent P. yoelii infection there was decreased resistance to L. monocytogenes in both liver and spleen, and the macrophages of the undisturbed peritoneal cavity were similarly defective. It was concluded that the defense mechanism of mice against P. yoelii is mediated by humoral factors in the absence of demonstrable cell-mediated immunity.     

Antibody recognition and isoelectrofocusing of antigens of the malaria parasite Plasmodium yoelii.

Inbred BALB/c mice were either immunized with Triton X-100-extracted antigens of blood-stage Plasmodium yoelii or infected with P. yoelii and cured in three successive schedules. Whereas the immunized BALB/c became only partially protected from subsequent challenge infection with blood-stage P. yoelii, the convalescent mice acquired total immunity. When total P. yoelii antigen extract was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and immunoblotted with anti-P. yoelii serum, five major protein bands of 150, 84, 40, 19, and 16 kDa were recognized by the sera of fully protected convalescent mice but not by the sera of partially protected mice. The utility of comparing reactivities of sera from fully protected and partially protected malaria hosts and the possibility that antigens uniquely recognized by the convalescent mouse sera may contribute to immunity against P. yoelii infection are discussed. Although previously reported to be an effective adjuvant for immunization against P. yoelii infection in (BALB/c x C57BL)F1 hybrid mice, saponin did not promote protection any better than did Freund adjuvant in BALB/c mice immunized with detergent-extracted P. yoelii antigen. Most of the P. yoelii proteins (14 to 250 kDa) found in Triton X-100 extracts of P. yoelii-parasitized erythrocytes isoelectrofocused as a single peak in the pH region 4.4 to 4.6, suggesting a rationale for previous findings that the most anti-P. yoelii protective and T-helper activities are induced by antigens isoelectrically focused in a fraction of similar pH.     

Immunization with a recombinant C-terminal fragment of Plasmodium yoelii merozoite surface protein 1 protects mice against homologous but not heterologous P. yoelii sporozoite challenge.

It has been reported previously that immunization with recombinant protein containing the two epidermal growth factor (EGF)-like modules from merozoite surface protein 1 (MSP-1) of Plasmodium yoelii (strain YM) protects mice against a lethal blood-stage challenge with the same parasite strain. Since MSP-1 is expressed in both liver- and blood-stage schizonts and on the surface of merozoites, we evaluated the effectiveness of immunization with recombinant proteins containing either the individual or the two combined EGF-like modules in producing a protective response against a sporozoite challenge. The recombinant protein expressing the combined EGF-like modules of the YM strain protected mice against a homologous sporozoite challenge, and sterile protection, as defined by the absence of detectable blood-stage parasites, was observed in the majority of the mice. In contrast, mice immunized with recombinant P. yoelii YM MSP-1 were not protected against a heterologous challenge with sporozoites from strain 265 BY of P. yoelii. The lack of protection may be explained by differences identified in the amino acid sequences of MSP-1 for the two strains. A recombinant protein containing the two EGF-like modules of MSP-1 from P. yoelii 265 BY was produced and used to immunize mice. These mice were protected against a homologous challenge with sporozoites of P. yoelii 265 BY. The results suggest that a recombinant MSP-1 has potential as a vaccine against malaria, but its efficacy may be limited by sequence polymorphism and selection of variants.     

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.     

Molecular genetic characterization and subcellular localization of a putative<Emphasis Type="Italic"> Theileria annulata</Emphasis> membrane protein

A Theileria annulata protein (TaD) exhibiting an N-terminal signal sequence for endoplasmic reticulum membrane translocation and a conserved cysteine-rich region was isolated by screening the mRNA of a T. annulata-infected bovine lymphoblastoid cell line with degenerated primers directed against T. annulata-targeting sequences. The TaD-coding sequence was found to be most closely related to the genomic DNA sequence of T. parva (TIGR database, 72%) and the amino acid sequence of Plasmodium falciparum (41%), P. yoelii yoelii (38%) and Cryptosporidium parvum (36%). The TaD mRNA is expressed within the sporozoite, schizont and merozoite stages of the parasite, implying that it is constitutively transcribed throughout the parasites life cycle. Allelic variants were found between isolates originating from different geographical regions, however not affecting conserved cysteines. The open reading frame encoded a protein of 19.5 kDa and non-reducing SDS-PAGE analysis demonstrated a homodimeric protein. Using confocal microscopy, the protein was found to be both located in the parasite cytoplasm and to colocalize with a transmembrane protein of the schizonts within infected cells.     

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.     

Ultrastructure and cytochemistry study of Eimeria sparis (Protozoa: Apicomplexa) stages from the intestine of gilthead sea bream Sparus aurata L. (Pisces: Teleostei)

The ultrastructure and cytochemistry of merogonial, gamogonial, and early sporogonial stages of Eimeria sparis in the intestine of Sparus aurata were studied. Mature stages showed the typical pellicle, which was lost in some transitional stages. An apparent unit membrane was seen in some immature stages. A parasitophorous vacuole, sometimes with membrane vesicular invaginations, was usually observed. We propose that the stages located over the epithelium and the so-called epicellular stages be termed supraepithelial stages. Endomerogony was observed in intraepithelial and supraepithelial positions. Intraepithelial stages apparently starting ectomerogony were also detected. Electron-dense granules similar to wall-forming-like bodies of types 1 and 2 were observed. Microgametes exhibited two flagella. Cytochemistry study revealed scarce polysaccharides, if any, in merogonial stages and in microgamonts. The occurrence of polysaccharides and amylopectin granules increased progressively in macrogamonts, macrogametes, zygotes, and early oocysts. Lipidic droplets were scarce or absent in merogonial stages and abundant in maturing macrogamonts. Some glycoproteins were demonstrated in certain merogonial stages. Received: 30 January 1996 / Accepted: 12 August 1996     

Characteristics of the protective response of BALB/c mice immunized with a purified Plasmodium yoelii schizont antigen

The response of BALB/c mice to immunization with a 230,000 mol. wt protective antigen purified from the blood stages of Plasmodium yoelii was analysed. The protective response to immunization was adjuvant-dependent, and saponin was found to be the most effective adjuvant tested. The intraperitoneal route was found to be superior to the subcutaneous route for protective immunization. Maximal protection was achieved using two immunizations with greater than 12.5 micrograms of antigen plus saponin, but even under these conditions challenge infection developed normally for 5 days, followed by the appearance of crisis forms and rapid clearance of parasites. Effective immunization induced high antibody titres, but serum from immunized mice was not protective on passive transfer. On the other hand, hyperimmune serum from mice recovered and rechallenged with live P. yoelii was protective on passive transfer, even though the titre of antibody specific for the 230,000 mol. wt antigen in this serum was the same as that in the serum from mice immunized with the purified antigen. It was concluded that the 230,000 mol. wt antigen may provide protection against P. yoelii via the cell-mediated immune effector pathway described by Playfair et al. (1979), and that hyperimmune serum contains protective antibodies specific for P. yoelii antigens other than the 230,000 mol. wt antigen.     

Ultrastructure and cytochemistry study of Goussia sparis (Protozoa: Apicomplexa) stages from the intestine of the gilthead sea bream Sparus aurata L. (Pisces: Teleostei)

 The ultrastructure and cytochemistry of merogonial and gamogonial stages and early unsporulated oocysts of Goussia sparis in the intestine of Sparus aurata were studied. The typical pellicle was observed in some stages. The different stages might appear in intraepithelial or supraepithelial positions, but they were always intracellular. First steps of two apparently different endomerogonies were observed in intra- and supraepithelial positions, respectively. An apparent ectomerogony also occurred in supraepithelial stages. Developing macrogamonts showed surface invaginations and were densely packed with ribosomes, well-developed rough endoplasmic reticulum, mitochondria, amylopectin granules, lipidic droplets, and wall-forming-like bodies. The latter could participate in the formation of the oocyst wall. Abundant and large mitochondria, together with residual nuclei, appeared in advanced microgamonts. Microgametes showed two flagella with microtubul arranged according to the typical pattern. An increase in polysaccharide content was observed with coccidian development, reaching a maximum in zygotes and unsporulated oocysts. Received: 30 January 1996 / Accepted: 8 July 1996