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[Studies on the antigens of invasive stages of Plasmodium yoelii and Plasmodium berghei].

OBJECTIVE: To detect the rhoptry and surface proteins of invasive stages of Plasmodium yoelii and P. berghei with monoclonal antibodies. METHODS: Subcellular localization of antigens was detected by IFA. The antigens of different stages of the two species malaria parasites were analyzed by Western blotting. RESULTS: The antigens of rhoptry are very complicated. There are similar epitopes of the rhoptry proteins detected between the two species of Plasmodium. The similar epitopes were also detected between ookinetes and merozoites of P. yoelii, and ookinete antigens between the two species. But there are different antigens detected between merozoites and ookinetes in P. yoelii. The sporozoite surface antigen of P. yoelii was not detected in the ookinetes and merozoites in the same species. CONCLUSION: There are similar epitopes in the rhoptry and surface antigens of different stages and different species of rodent malaria parasites. There are also distinct antigens among them.

Animals↗

A T cell clone directed at the circumsporozoite protein which protects mice against both Plasmodium yoelii and Plasmodium berghei.

Clone B is a cytotoxic T cell clone induced by immunization with Plasmodium yoelii sporozoites which recognizes an epitope on both the P. yoelii and Plasmodium berghei circumsporozoite proteins. It is CD8, uses the V beta 8.1 TCR, and is Kd restricted. When adoptively transferred, it protects mice against infection by both species of malaria sporozoites, and this protection is dependent on IFN-gamma. Clone B cells are more broadly reactive and protective than previously described murine T cell clones against malaria. Clone B may be an important model for immune protection against the spectrum of variant parasites in nature.

Amino Acid Sequence↗

Use of polymerase chain reaction technique to confirm VecTest screening results in Plasmodium falciparum and Plasmodium vivax VK 210 laboratory-infected Anopheles stephensi mosquitoes.

We evaluated polymerase chain reaction (PCR) to confirm immunoassays for malaria parasites in mosquito pools after a failure to detect malaria with PCR during an outbreak in which pools tested positive using VecTest and enzyme-linked immunosorbent assay (ELISA). We combined VecTest, ELISA, and PCR to detect Plasmodium falciparum and Plasmodium vivax VK 210. Each mosquito pool, prepared in triplicate, consisted of 1 exposed Anopheles stephensi and up to 9 unfed mosquitoes. The results of VecTest and ELISA were concordant. DNA from a subset of the pools, 1 representative of each ratio of infected to uninfected mosquitoes, was extracted and used as template in PCR. All P. vivax pools were PCR positive but some needed additional processing for removal of apparent inhibitors before positive results were obtained. One of the pools selected for P. falciparum was negative by PCR, probably because of losses or contamination during DNA extraction; 2 remaining pools at this ratio were PCR positive. Testing pools by VecTest, ELISA, and PCR is feasible, and PCR is useful for confirmation of immunoassays. An additional step might be needed to remove potential inhibitors from pools prior to PCR.

Animals↗

Dramatic difference in diversity between Plasmodium falciparum and Plasmodium vivax reticulocyte binding-like genes.

Malaria parasite proteins involved in erythrocyte invasion are considered important vaccine targets. Members of the reticulocyte binding-like (RBL) family of Plasmodium merozoite proteins are found in human, simian, and rodent malaria parasites and function in the initial steps of erythrocyte selection and invasion. The RBL genes are large, ranging in size from 7.7 to 10 kb, and the extent of any sequence diversity in parasite populations is unknown. We present the first assessment of sequence diversity within RBL genes from the two major human malaria parasites: Plasmodium falciparum and P. vivax. Polymorphism within the RBL genes is generally limited, except for P. vivax reticulocyte binding protein 2 (PvRBP2), which has nucleotide diversity levels 25-fold higher than the other RBL genes. The PvRBP2 haplotypes appear to fall into two distinct classes of alleles, suggesting large-scale dimorphism in this gene. Polymorphisms were frequently clustered, suggesting that different RBL domains may be evolving under different selection and functional pressures.

Amino Acid Sequence↗

Circumsporozoite protein gene from Plasmodium reichenowi, a chimpanzee malaria parasite evolutionarily related to the human malaria parasite Plasmodium falciparum.

We have cloned and sequenced the gene encoding the circumsporozoite (CS) protein of Plasmodium reichenowi a Plasmodium falciparum-like malaria parasite of chimpanzees. Comparison of the two CS proteins reveals both similarities and differences in these two evolutionarily related parasites that have adapted to different hosts. The P. reichenowi CS protein has a new repeat sequence, NVNP, in addition to the P. falciparum-like NANP and NVDP repeats. In the immunodominant TH2R and TH3R regions of the CS protein, the amino acid sequences are similar in both parasite proteins. The differences in the two proteins exist in domains around the conserved regions, Region I and Region II, which are otherwise conserved in the CS proteins of P. falciparum analyzed to date. Studies of parasite protein genes of evolutionarily related malaria parasites, together with other immunologic and biologic characteristics, will help better understand the evolution and host parasite relationship of malaria parasites and may provide a tool for identifying protein determinants for malaria vaccine development.

Amino Acid Sequence↗

Evaluation of the antimalarial activity of new compounds against Plasmodium falciparum in vitro, and Plasmodium berghei in vivo.

Various hydrazones of thiophene carboxaldehyde were tested in vitro on two Plasmodium falciparum strains and in vivo on mice experimentally infected with Plasmodium berghei. These hydrazones were obtained by condensation of appropriate hydrazines with thiophene-2-carboxaldehyde (series 1), thiophene-3- carboxaldehyde (series 2) and 5-Nitrothiophene-2-carboxaldehyde (series 3). Compounds of series 3, 5-Nitrothiophene-2-carboxaldehyde presented significant effects in vitro. In vivo tests confirmed the antimalarial activity observed in vitro with two compounds of this series.

Animals↗

A merozoite receptor protein from Plasmodium knowlesi is highly conserved and distributed throughout Plasmodium.

The 66-kDa merozoite surface antigen (PK66) of Plasmodium knowlesi, a simian malaria, possesses vaccine-related properties that are thought to originate from a receptor-like role in parasite invasion of erythrocytes. We report the complete sequence of PK66 which allowed the demonstration that highly conserved analogues exist throughout Plasmodium including a recently reported gene from P. falciparum (Peterson, M. G., Marshall, V. M., Smythe, J. A., Crewther, P. E., Lew, A., Silva, A., Anders, R. F., and Kemp, D. J. (1989) Mol. Cell. Biol. 9, 3151-3155). These analogues are highly promising vaccination candidates. The distribution of PK66 changes after schizont rupture in a coordinate manner associated with merozoite invasion. The protein is concentrated at the apical end prior to rupture, following which it can distribute itself entirely across the surface of the free merozoite. During invasion, immunofluorescence studies suggest that, PK66 is excluded from the erythrocyte at, and behind, the invasion interface.

Amino Acid Sequence↗

Immunological cross-reactivity of the histidine-rich protein of Plasmodium lophurae and the knob protein of Plasmodium falciparum.

Rabbits were immunized with the histidine-rich protein (HRP) of the avian malaria parasite, Plasmodium lophurae. The anti-HRP immunoprecipitated the knob protein (KP) from extracts of the human parasite, Plasmodium falciparum. The anti-HRP did not react with any antigens of a laboratory-derived strain of P. falciparum (K-) that does not form knobs. Antisera raised against a membrane-enriched fraction of P. falciparum-infected erythrocytes as well as sera from humans exposed to P. falciparum infections immunoprecipitated HRP from P. lophurae extracts.

Animals↗

Detection of CD4+CD45RO+ T lymphocytes producing IL-4 in response to antigens on Plasmodium falciparum erythrocytes: an in vitro correlate of protective immunity induced with attenuated Plasmodium falciparum sporozoites.

Malaria is caused by Plasmodium spp. and is one of the major infectious diseases leading to morbidity and mortality in tropical areas of the world. The model of protective immunity induced by immunization with radiation-attenuated Plasmodia sporozoites (SPZ) has become the framework for the elucidation of protective immune mechanisms and the prototype for a promising vaccine strategy. We have previously reported that although considered stage specific based on antibody and CD8+ cytolytic T lymphocyte responses directed against preerythrocytic stage antigens, in particular, the circumsporozoite protein and sporozoite surface protein 2, protective immunity induced in humans by attenuated Plasmodium falciparum SPZ may also involve CD4+ T cell responding to antigens present on parasitized red blood cells (pRBC). In this study we examined the functional role of pRBC responding CD4+ T cells by comparing in vitro pRBC-stimulated responses of CD4+ T cells from persons during preimmunity to irradiated SPZ, during induction of protection, and infection induced with SPZ. The results reported herein corroborate previously published observations that antigens associated with pRBC induce proliferative CD4+ lymphocytes responses in subjects exposed to malaria parasite-derived antigens and not malaria-naive persons; however, now we demonstrate that pRBC-proliferative CD4+ T cells did not coincide with protective immunity. Similarly, pRBC-induced IFN-gamma levels did not distinguish malaria protected from susceptible persons, although IFN-gamma was observed only in lymphocyte cultures from malaria parasite-exposed volunteers and not in lymphocyte cultures from malaria-naive persons. In contrast, we noted an increase in the IL-4-producing CD4+ T cells that also exhibited the memory phenotype, CD45RO, and an upregulated expression of CD25 in cultures from malaria protected persons as compared to malaria naive persons and subjects who became parasitemic. Hence, these observations suggest that the induction of memory CD4+ T cell subset distinguished by the expression of CD45RO and CD25 and production of IL-4 coincides with protective immune responses generated by immunization with attenuated SPZ.

Animals↗

Immunoglobulin isotype distribution of malaria-specific antibodies produced during infection with Plasmodium chabaudi adami and Plasmodium yoelii.

The antibody response of mice to Plasmodium chabaudi adami and Plasmodium yoelii has been compared using a solid phase isotype-specific radioimmunoassay and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Serological cross-reactivity between these parasites was substantial. Studies using a radioimmunoassay detecting all classes of malaria-specific antibody demonstrated that during the early part of infection it was not possible to distinguish between homologous and heterologous reactions. Immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that 50% or more of the protein antigens detected were apparently shared by both parasites although the intensity of bands was always greater with homologous reactions. However, the distribution of isotypes in the antibody (Ab) response differed in the two infections. P. chabaudi infections were characterized by a predominant and persistent IgM response, moderate IgG2 and IgG3 and little significant IgG1 response during a primary infection. By contrast, IgM antibodies were transient in P. yoelii infection, IgG2 was the predominant isotype, and both IgG1 and IgG3 antibodies were present during a primary infection. These differences in isotypes were also detected when sera were tested on the heterologous antigen extracts suggesting that antigens shared by P. chabaudi and P. yoelii do not necessarily induce similar antibody responses in the two infections.

Animals↗

Distribution of immunoglobulin isotypes in the nonspecific B-cell response induced by infection with Plasmodium chabaudi adami and Plasmodium yoelii.

The nonspecific B-cell response induced by infecting mice with two nonlethal malaria parasites, Plasmodium chabaudi adami and Plasmodium yoelii, was analyzed in an isotype-specific reverse plaque assay. Our results showed different isotypic patterns in the two infections, although cells secreting immunoglobulin of all isotypes were increased to some extent. P. yoelii induced large increases in secreting cells of all isotypes; IgG2a-secreting cells were increased out of proportion to those of the other IgG classes. P. chabaudi induced large increases in secreting cells of all isotypes except IgG1. In addition, there was not a disproportionate increase in cells secreting IgG2a. The data show that these "polyclonal" responses are different during each infection. There are marked similarities between the distribution of "nonspecific isotypes" and the specific antibodies formed in each infection.

Animals↗

Possible roles of Ca2+ and cGMP as mediators of the exflagellation of Plasmodium berghei and Plasmodium falciparum.

The roles of Ca2+ and cyclic nucleotides as secondary, intracellular messengers for exflagellation of Plasmodium berghei and Plasmodium falciparum were investigated. Treatment with Ca2+ antagonists such as TMB-8 (an inhibitor of intracellular Ca2+ release) or W-7 (a calmodulin inhibitor) strongly inhibited exflagellation induced by alkaline medium at pH 8.0 whereas EGTA (a Ca2+ chelator) or nicardipine and nifedipine (Ca2+ channel inhibitors) had no effect. These results may indicate that mobilization of parasites' internal resources of Ca2+ is a prerequisite for exflagellation. Agents which increase cAMP levels did not induce exflagellation at the non-permissive pH of 7.3, and had no significant inhibitory effect at the permissive pH of 8.0. IBMX (cAMP/cGMP-phosphodiesterase inhibitor), however, enhanced exflagellation at pH 7.3, indicating the possibility that cGMP, but not cAMP, may be involved in the induction of exflagellation. Furthermore, cGMP or agents which increase cGMP levels such as nitroprusside (a potent activator of guanylate cyclase), enhanced exflagellation at pH 7.3, whereas N-methyl-hydroxylamine (guanylate cyclase inhibitor) inhibited the exflagellation at pH 8.0. From these results, it may be concluded that the induction of exflagellation requires both Ca2+ mobilization and an increase in cGMP levels.

Animals↗

Conservation of the Plasmodium falciparum sporozoite surface protein gene, STARP, in field isolates and distinct species of Plasmodium.

The extent of structural conservation of the Plasmodium falciparum sporozoite surface protein gene, STARP, recently characterized in the T9/96 clone, has been analyzed using the polymerase chain reaction. Results from Ivory Coast and Thai clones, field isolates originating from Brazil and Kenya and laboratory-maintained strains strongly suggest that this gene has a highly conserved structure throughout this species. This structure includes a complex repetitive central domain consisting of a mosaic region followed by tandem 45-amino acid-encoding (Rp45) and 10-amino acid-encoding (Rp10) repeat regions. Limited size variation in this domain appeared to result from highly localized duplication events in the Rp45 and Rp10 regions. No size variation was observed in the 5' and 3' coding non-repetitive regions, but minor size polymorphism was found in the single intron at the 5' end of the gene. No evidence was found of distinct families of polymorphic types, as has been observed with the blood-stage MSA-1, MSA-2 and S-antigens. The sequence of the STARP homologue in the phylogenetically close chimpanzee parasite, Plasmodium reichenowi, has also been elucidated and reveals high sequence conservation, although interesting differences were detected in the composition of the Rp10 region, known in P. falciparum to contain B- and T-cell epitopes. Finally, DNA hybridization reveals the presence in rodent malaria species of sequences containing homology to the STARP non-repetitive (though not the repetitive) regions, which would suggest that a similar, conserved gene may exist in these species.

Amino Acid Sequence↗

The A/T-specific DNA alkylating agent adozelesin inhibits Plasmodium falciparum growth in vitro and protects mice against Plasmodium chabaudi adami infection.

There is an urgent need for new anti-malarial drugs to combat the resurgence of resistance to current therapies. To exploit the A/T richness of malaria DNA as a potential target for anti-malarial drugs we tested an A/T-specific DNA synthesis inhibitor, adozelesin, for activity against Plasmodium falciparum in vitro and Plasmodium chabaudi adami in mice. Adozelesin is a DNA alkylating agent that exhibits specificity for the motif A/T, A/T and A. In P. falciparum 3D7 cultures, adozelesin acts as a powerful inhibitor of parasite growth (IC(50) of 70 pM) and is equally potent at killing the drug-resistant strains FCR3 and 7G8. Using a real-time PCR assay, we show that treatment with adozelesin in vitro results in damage of P. falciparum genomic DNA. In synchronized cultures, adozelesin exhibits a concentration-dependent effect on parasitemia and on the development of parasites through the asexual cycle. In asynchronous cultures, parasites arrest at all stages of the asexual cycle suggesting that adozelesin exerts other anti-parasitic effects in addition to inhibiting DNA replication. These anti-parasite effects are irreversible since cultures exposed to adozelesin for more than 6h fail to recover upon removal of the drug. Furthermore, adozelesin is very effective at suppressing malaria infection in vivo; growth of P. c. adami DK in mice was highly impaired by a single injection of adozelesin (25 microg/kg) at 4 days post-infection. These results demonstrate that adozelesin irreversibly blocks parasite growth in vitro and suppresses parasite infection in vivo, suggesting that A/T-specific DNA damaging agents represent a new class of compounds with potential as anti-malarials.

Adenine↗

Simultaneous identification of the four human Plasmodium species and quantification of Plasmodium DNA load in human blood by real-time polymerase chain reaction.

The incidence of imported malaria cases in travellers returning from endemic areas has considerably increased over the last few years. The microscopical examination of stained blood films is the gold standard method to confirm clinical suspicion of malaria but diagnosis is difficult in the case of mixed infections, low-grade parasitaemia, or forms altered by uncompleted treatment. We have developed a real-time polymerase chain reaction (PCR) for the simultaneous identification of the 4 human Plasmodium spp. and quantification of Plasmodium DNA in human blood. The rapid turnaround and reduction in the risk of PCR product carryover are major advantages compared with conventional PCR. In combination with conventional tests, this method could be a powerful tool for the diagnosis of malaria infections among travellers from endemic areas and during the follow-up of patients in reference centres involved in travel and tropical medicine. Quantitative real-time PCR could also be used for the follow-up of patients during drug resistance studies managed by national malaria programmes, the testing of new drugs, and vaccine trials.

Animals↗

Identification and characterization of the protective hepatocyte erythrocyte protein 17 kDa gene of Plasmodium yoelii, homolog of Plasmodium falciparum exported protein 1.

We recently reported the discovery of a 17-kDa Plasmodium yoelii protein expressed in infected hepatocytes and erythrocytes, P. yoelii hepatocyte erythrocyte protein 17 (PyHEP17), and have demonstrated that this protein is a target of protective antibodies and T cells. Here, we report the identification and characterization of the gene encoding this protein and reveal that it is composed of two exons. Immunization of mice with PyHEP17 plasmid DNA induces antibodies, cytotoxic T lymphocytes, and protective immunity directed against the infected hepatocyte. Based on extensive sequence homology, expression pattern, and antigenic cross-reactivity, the Plasmodium falciparum homolog of PyHEP17 is identified as the protein known as exported protein-1 (PfExp-1), also called antigen 5.1, circumsporozoite related antigen, or QF116. Identity between PyHEP17 and PfExp-1 is 37% at the amino acid level (60/161 residues), mapping primarily to two regions within the second exon of 73% (16/22 residues) and 71% (25/35 residues) identity. On this basis, PfExp-1 is proposed as an important component of pre-erythrocytic human malaria vaccines.

Amino Acid Sequence↗

PlasmoDB: An integrative database of the Plasmodium falciparum genome. Tools for accessing and analyzing finished and unfinished sequence data. The Plasmodium Genome Database Collaborative.

The PLASMODIUM: falciparum Genome Database (http://PlasmoDB.org) integrates sequence information, automated analyses and annotation data emerging from the P.falciparum genome sequencing consortium. To date, raw sequence coverage is available for >90% of the genome, and two chromosomes have been finished and annotated. Data in PlasmoDB are organized by chromosome (1-14), and can be accessed using a variety of tools for graphical and text-based browsing or downloaded in various file formats. The GUS (Genomics Unified Schema) implementation of PlasmoDB provides a multi-species genomic relational database, incorporating data from human and mouse, as well as P.falciparum. The relational schema uses a highly structured format to accommodate diverse data sets related to genomic sequence and gene expression. Tools have been designed to facilitate complex biological queries, including many that are specific to PLASMODIUM: parasites and malaria as a disease. Additional projects seek to integrate genomic information with the rich data sets now becoming available for RNA transcription, protein expression, metabolic pathways, genetic and physical mapping, antigenic and population diversity, and phylogenetic relationships with other apicomplexan parasites. The overall goal of PlasmoDB is to facilitate Internet- and CD-ROM-based access to both finished and unfinished sequence information by the global malaria research community.

Animals↗

Plasmodium chabaudi antigen Pch105, Plasmodium falciparum antigen Pf155, and erythrocyte band 3 share cross-reactive epitopes.

By immunoblotting with a number of monoclonal antibodies raised in human and murine malaria systems, we have been able to establish the presence of cross-reactive epitopes on the Plasmodium falciparum vaccine candidate antigen Pf155/RESA and its proposed Plasmodium chabaudi analog Pch105. These findings were confirmed when the same antibodies were tested in an immunofluorescence assay. By using short synthetic peptides corresponding to repeated sequences in the C terminus of the Pf155 and enzyme-linked immunosorbent assays, the cross-reacting epitope was found to be localized to this repeat segment. Furthermore, a monoclonal antibody to murine erythrocyte band 3 which also cross-reacted with human band 3 bound to both Pch105 and Pf155 as well as to the synthetic peptides, suggesting that these proteins share a related epitope. The cross-reactions reflect the existence of sequence homologies of band 3 with these plasmodial proteins. This molecular similarity may be used by the parasite to disturb the rigidity of the erythrocyte membrane, thereby facilitating its entrance into the cell.

Animals↗