RESUMO
BACKGROUND: In this phase 1 clinical trial, healthy adult, malaria-naïve subjects were immunized with radiation-attenuated Plasmodium falciparum sporozoites (PfRAS) by mosquito bite and then underwent controlled human malaria infection (CHMI). The PfRAS model for immunization against malaria had previously induced >90 % sterile protection against homologous CHMI. This study was to further explore the safety, tolerability and protective efficacy of the PfRAS model and to provide biological specimens to characterize protective immune responses and identify protective antigens in support of malaria vaccine development. METHODS: Fifty-seven subjects were screened, 41 enrolled and 30 received at least one immunization. The true-immunized subjects received PfRAS via mosquito bite and the mock-immunized subjects received mosquito bites from irradiated uninfected mosquitoes. Sera and peripheral blood mononuclear cells (PBMCs) were collected before and after PfRAS immunizations. RESULTS: Immunization with PfRAS was generally safe and well tolerated, and repeated immunization via mosquito bite did not appear to increase the risk or severity of AEs. Local adverse events (AEs) of true-immunized and mock-immunized groups consisted of erythaema, papules, swelling, and induration and were consistent with reactions from mosquito bites seen in nature. Two subjects, one true- and one mock-immunized, developed large local reactions that completely resolved, were likely a result of mosquito salivary antigens, and were withdrawn from further participation as a safety precaution. Systemic AEs were generally rare and mild, consisting of headache, myalgia, nausea, and low-grade fevers. Two true-immunized subjects experienced fever, malaise, myalgia, nausea, and rigours approximately 16 h after immunization. These symptoms likely resulted from pre-formed antibodies interacting with mosquito salivary antigens. Ten subjects immunized with PfRAS underwent CHMI and five subjects (50 %) were sterilely protected and there was a significant delay to parasitaemia in the other five subjects. All ten subjects developed humoral immune responses to whole sporozoites and to the circumsporozoite protein prior to CHMI, although the differences between protected and non-protected subjects were not statistically significant for this small sample size. CONCLUSIONS: The protective efficacy of this clinical trial (50 %) was notably less than previously reported (>90 %). This may be related to differences in host genetics or the inherent variability in mosquito biting behavior and numbers of sporozoites injected. Differences in trial procedures, such as the use of leukapheresis prior to CHMI and of a longer interval between the final immunization and CHMI in these subjects compared to earlier trials, may also have reduced protective efficacy. This trial has been retrospectively registered at ISRCTN ID 17372582, May 31, 2016.
Assuntos
Anticorpos Antiprotozoários/sangue , Culicidae/fisiologia , Mordeduras e Picadas de Insetos , Vacinas Antimaláricas/efeitos adversos , Vacinas Antimaláricas/imunologia , Malária Falciparum/prevenção & controle , Plasmodium falciparum/imunologia , Adolescente , Adulto , Animais , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/epidemiologia , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/patologia , Humanos , Vacinas Antimaláricas/administração & dosagem , Masculino , Pessoa de Meia-Idade , Plasmodium falciparum/efeitos da radiação , Esporozoítos/imunologia , Esporozoítos/efeitos da radiação , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/efeitos adversos , Vacinas Atenuadas/imunologia , Adulto JovemRESUMO
We have evaluated a technology called transcriptionally active PCR (TAP) for high throughput identification and prioritization of novel target antigens from genomic sequence data using the Plasmodium parasite, the causative agent of malaria, as a model. First, we adapted the TAP technology for the highly AT-rich Plasmodium genome, using well-characterized P. falciparum and P. yoelii antigens and a small panel of uncharacterized open reading frames from the P. falciparum genome sequence database. We demonstrated that TAP fragments encoding six well-characterized P. falciparum antigens and five well-characterized P. yoelii antigens could be amplified in an equivalent manner from both plasmid DNA and genomic DNA templates, and that uncharacterized open reading frames could also be amplified from genomic DNA template. Second, we showed that the in vitro expression of the TAP fragments was equivalent or superior to that of supercoiled plasmid DNA encoding the same antigen. Third, we evaluated the in vivo immunogenicity of TAP fragments encoding a subset of the model P. falciparum and P. yoelii antigens. We found that antigen-specific antibody and cellular immune responses induced by the TAP fragments in mice were equivalent or superior to those induced by the corresponding plasmid DNA vaccines. Finally, we developed and demonstrated proof-of-principle for an in vitro humoral immunoscreening assay for down-selection of novel target antigens. These data support the potential of a TAP approach for rapid high throughput functional screening and identification of potential candidate vaccine antigens from genomic sequence data.
Assuntos
Antígenos de Protozoários/genética , Vacinas Antimaláricas/imunologia , Plasmodium falciparum/genética , Plasmodium yoelii/genética , Reação em Cadeia da Polimerase/métodos , Vacinas de DNA/imunologia , Animais , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/imunologia , Citocinas/biossíntese , Feminino , Vacinas Antimaláricas/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Plasmodium falciparum/imunologia , Plasmodium yoelii/imunologia , Baço/imunologia , Linfócitos T/imunologia , Vacinas de DNA/genéticaRESUMO
Effective vaccines against infectious diseases and biological warfare agents remain an urgent public health priority. Studies have characterized the differentiation of effector and memory T cells and identified a subset of T cells capable of conferring enhanced protective immunity against pathogen challenge. We hypothesized that the kinetics of T cell differentiation influences the immunogenicity and protective efficacy of plasmid DNA vaccines, and tested this hypothesis in the Plasmodium yoelii murine model of malaria. We found that increasing the interval between immunizations significantly enhanced the frequency and magnitude of CD8+ and CD4+ T cell responses as well as protective immunity against sporozoite challenge. Moreover, the interval between immunizations was more important than the total number of immunizations. Immunization interval had a significantly greater impact on T cell responses and protective immunity than on antibody responses. With prolonged immunization intervals, T cell responses induced by homologous DNA only regimens achieved levels similar to those induced by heterologous DNA prime/ virus boost immunization at standard intervals. Our studies establish that the dosing interval significantly impacts the immunogenicity and protective efficacy of plasmid DNA vaccines.
Assuntos
Esquemas de Imunização , Malária/imunologia , Malária/prevenção & controle , Plasmídeos/imunologia , Plasmodium yoelii/imunologia , Vacinas de DNA/administração & dosagem , Vacinas de DNA/imunologia , Animais , Anticorpos Antiprotozoários/sangue , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Feminino , Imunização , Imunização Secundária , Malária/parasitologia , Vacinas Antimaláricas/administração & dosagem , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Plasmodium yoelii/patogenicidadeRESUMO
The present study has evaluated the immunogenicity of single or multiple Plasmodium falciparum (Pf) antigens administered in a DNA prime/poxvirus boost regimen with or without the poloxamer CRL1005 in rhesus monkeys. Animals were primed with PfCSP plasmid DNA or a mixture of PfCSP, PfSSP2/TRAP, PfLSA1, PfAMA1 and PfMSP1-42 (CSLAM) DNA vaccines in PBS or formulated with CRL1005, and subsequently boosted with ALVAC-Pf7, a canarypox virus expressing the CSLAM antigens. Cell-mediated immune responses were evaluated by IFN-gamma ELIspot and intracellular cytokine staining, using recombinant proteins and overlapping synthetic peptides. Antigen-specific and parasite-specific antibody responses were evaluated by ELISA and IFAT, respectively. Immune responses to all components of the multi-antigen mixture were demonstrated following immunization with either DNA/PBS or DNA/CRL1005, and no antigen interference was observed in animals receiving CSLAM as compared to PfCSP alone. These data support the down-selection of the CSLAM antigen combination. CRL1005 formulation had no apparent effect on vaccine-induced T cell or antibody responses, either before or after viral boost. In high responder monkeys, CD4+IL-2+ responses were more predominant than CD8+ T cell responses. Furthermore, CD8+ IFN-gamma responses were detected only in the presence of detectable CD4+ T cell responses. Overall, this study demonstrates the potential for multivalent Pf vaccines based on rational antigen selection and combination, and suggests that further formulation development to increase the immunogenicity of DNA encoded antigens is warranted.
Assuntos
Antígenos de Protozoários/imunologia , Imunização Secundária/métodos , Vacinas Antimaláricas/administração & dosagem , Malária Falciparum/imunologia , Plasmodium falciparum/imunologia , Poxviridae/imunologia , Vacinas de DNA/imunologia , Animais , Anticorpos Antiprotozoários/biossíntese , Antígenos de Protozoários/administração & dosagem , Antígenos de Protozoários/genética , Imunização , Macaca mulatta , Vacinas Antimaláricas/imunologia , Plasmídeos , Vacinas de DNA/administração & dosagemRESUMO
Plasmodium falciparum intraerythrocytic development is a complex process. Development proceeds rapidly from the trophozoite phase of nutrient acquisition and growth through to the synthetic and reproductive schizont phase, which ends with production of new invasive merozoites. During this process, the malaria parasite must express a series of different gene products, depending on its metabolic and synthetic needs. We are particularly interested in the development of the merozoite's organelles in the apical complex, which form during the later schizont stages. We have used quantitative real-time RT-PCR fluorogenic 5' nuclease assays (TaqMan) for the first time on malaria parasites for analysis of erythrocytic stage-specific gene expression. We analyzed transcripts of the P.falciparum eba-175 and other erythrocyte binding-like (ebl) family genes in temperature-synchronized parasites and found ebl genes have tightly controlled, stage-specific transcription. As expected, eba-175 transcripts were abundant only at the end of schizont development in a pattern most common among ebl, including baebl, pebl and jesebl. The maebl transcript pattern was unique, peaking at mid-late trophozoite stage, but absent in late-stage schizonts. ebl-1 demonstrated another pattern of expression, which peaked during mid-schizont stage and then significantly diminished in late-stage schizonts. Our analysis demonstrates that using real-time RT-PCR fluorogenic 5' nuclease assays is a sensitive, quantitative method for analysis of Plasmodium transcripts.
Assuntos
Antígenos de Protozoários , Genes de Protozoários/genética , Plasmodium falciparum/genética , Proteínas de Protozoários , Animais , Southern Blotting , Proteínas de Transporte/genética , DNA de Protozoário/genética , Regulação da Expressão Gênica no Desenvolvimento , Plasmodium falciparum/crescimento & desenvolvimento , Reação em Cadeia da Polimerase/métodos , RNA de Protozoário/genética , RNA de Protozoário/metabolismo , Receptores de Superfície Celular/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Temperatura , Fatores de Tempo , Transcrição GênicaRESUMO
The sexual stages of malarial parasites are essential for the mosquito transmission of the disease and therefore are the focus of transmission-blocking drug and vaccine development. In order to better understand genes important to the sexual development process, the transcriptomes of high-purity stage I-V Plasmodium falciparum gametocytes were comprehensively profiled using a full-genome high-density oligonucleotide microarray. The interpretation of this transcriptional data was aided by applying a novel knowledge-based data-mining algorithm termed ontology-based pattern identification (OPI) using current information regarding known sexual stage genes as a guide. This analysis resulted in the identification of a sexual development cluster containing 246 genes, of which approximately 75% were hypothetical, exhibiting highly-correlated, gametocyte-specific expression patterns. Inspection of the upstream promoter regions of these 246 genes revealed putative cis-regulatory elements for sexual development transcriptional control mechanisms. Furthermore, OPI analysis was extended using current annotations provided by the Gene Ontology Consortium to identify 380 statistically significant clusters containing genes with expression patterns characteristic of various biological processes, cellular components, and molecular functions. Collectively, these results, available as part of a web-accessible OPI database (http://carrier.gnf.org/publications/Gametocyte), shed light on the components of molecular mechanisms underlying parasite sexual development and other areas of malarial parasite biology.
Assuntos
Perfilação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos , Plasmodium falciparum/genética , Transcrição Gênica , Animais , Sequência de Bases , DNA de Protozoário/genética , Feminino , Genoma de Protozoário , Masculino , Família Multigênica , Plasmodium falciparum/crescimento & desenvolvimento , Alinhamento de Sequência , Maturidade SexualRESUMO
The transcriptional repertoire of the in vivo liver stage of Plasmodium has remained largely unidentified and seemingly not amenable to traditional molecular analysis because of the small number of parasites and large number of uninfected hepatocytes. We have overcome this obstruction by utilizing laser capture microdissection to provide a high quality source of parasite mRNA for the construction of a liver stage cDNA library. Sequencing and annotation of this library demonstrated expression of 623 different Plasmodium yoelii genes during development in the hepatocyte. Of these genes, 25% appear to be unique to the liver stage. This is the first comprehensive analysis of in vivo gene expression undertaken for the liver stage of P. yoelii, and provides insights into the differential expression of P. yoelii genes during this critical stage of development.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Fígado/parasitologia , Malária/parasitologia , Plasmodium yoelii/crescimento & desenvolvimento , Proteínas de Protozoários/metabolismo , Animais , Etiquetas de Sequências Expressas , Biblioteca Gênica , Hepatócitos/parasitologia , Fígado/citologia , Camundongos , Camundongos Endogâmicos BALB C , Plasmodium yoelii/genética , Plasmodium yoelii/metabolismo , Proteínas de Protozoários/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição GênicaRESUMO
The promise of new interventions against malaria and other infectious diseases of global health importance derived from pathogen genomic sequence data may only be realized through the coordinated effort of genomic and post-genomics scientists, vaccine and drug developers along with lab- and field-based investigators. With the availability of the Plasmodium falciparum genome and the genomes of related species, post-genomics research can now be applied to the development of new interventions against malaria and may provide a more complete understanding of complex parasite biology. The vast amount of data that are generated through these new approaches must be organized, assembled and made accessible in a useful manner. By establishing a set of "credentials" for each gene in the genome, which captures information about gene expression, diversity, function and other information, the time and resources needed to test and evaluate candidate drugs and vaccines can be substantially reduced and a more complete picture of parasite biology can be created.
Assuntos
Genômica/tendências , Vacinas Antimaláricas , Malária/genética , Plasmodium falciparum/imunologia , Animais , Variação Genética , Humanos , Malária/prevenção & controle , Plasmodium falciparum/genéticaRESUMO
Efficient activation of specific immune responses requires a concerted interaction between T cells and antigen-presenting cells. A requirement for bystander participation of CD4+ T cells for expansion and maintenance of memory CD8+ T cells has been noted in several models, but a role with regard to effector CD8+ T responses has not been well-defined. In this report, the requirement of bystander participation for optimal induction of antigen-specific CD8+ T cell effector function was determined by directly quantitating antigen-specific interferon-gamma (IFN-gamma) CD8+ T cell responses by enzyme-linked immunospot assays, and by indirectly evaluating induction of the chemokine monokine induced by IFN-gamma as a marker for IFN-gamma-mediated effector function. Our results demonstrate that bystander cell participation, mediated by CD4+ T cell and natural killer (NK) cells, is required for optimal induction of antigen-specific CD8+ T cell effector responses. Our data further establish a novel role for NK cells in the activation of antigen-specific immune responses.
Assuntos
Antígenos Virais/imunologia , Efeito Espectador/imunologia , Linfócitos T CD8-Positivos/imunologia , Peptídeos e Proteínas de Sinalização Intercelular , Adulto , Linfócitos T CD4-Positivos/imunologia , Quimiocina CXCL9 , Quimiocinas CXC/biossíntese , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interferon gama/farmacologia , Células Matadoras Naturais/imunologia , Pessoa de Meia-IdadeRESUMO
The concept behind the first Molecular Approaches to Malaria meeting, held 1-5 February 2000 in Lorne, Australia, was ahead of its time; to convene a meeting of malaria researchers, database developers and genomics scientists, and to discuss how genomic sciences and their relevant disciplines could be applied to solve important problems in malaria research. The success of the second Molecular Approaches to Malaria meeting, held 1-5 February 2004 in the same place, together with the influence of genomics on malaria research, is testament to the vision that the organizers had at the first meeting. This review attempts to capture some of the current efforts in the post-genomics era of malaria research and highlights the approaches discussed at the Molecular Approaches to Malaria 2004 meeting.
Assuntos
Genoma de Protozoário , Malária/parasitologia , Plasmodium falciparum/genética , Plasmodium yoelii/genética , Proteômica/métodos , Animais , Bases de Dados Genéticas , Humanos , Plasmodium falciparum/metabolismo , Plasmodium yoelii/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Transcrição GênicaRESUMO
The Rodent Malaria Genomics Symposium: Current Status and Future Directions was held on 15-16 November 2001 in Atlanta, GA, USA.
Assuntos
Genoma de Protozoário , Malária/parasitologia , Plasmodium/genética , Roedores/parasitologia , Animais , Modelos Animais de DoençasRESUMO
Scientists from several organizations worldwide are working together to develop a multistage, multigene DNA-based vaccine against Plasmodium falciparum malaria. This collaborative vaccine development effort is named Multi-Stage DNA-based Malaria Vaccine Operation. An advisory board of international experts in vaccinology, malariology and field trials provides the scientific oversight to support the operation. This article discusses the rationale for the approach, underlying concepts and the pre-clinical development process, and provides a brief outline of the plans for the clinical testing of a multistage, multiantigen malaria vaccine based on DNA plasmid immunization technology.
Assuntos
Vacinas Antimaláricas/administração & dosagem , Malária Falciparum/prevenção & controle , Plasmodium falciparum/imunologia , Vacinas de DNA/administração & dosagem , Animais , Humanos , Cooperação Internacional , Vacinas Antimaláricas/imunologia , Vacinas de DNA/imunologiaRESUMO
Proteins on the surface of parasite-infected erythrocytes (PIESPs) have been one of the major focuses of malaria research due to their role in pathogenesis and their potential as targets for immunity and drug intervention. Despite intense scrutiny, only a few surface proteins have been identified and characterized. We report the identification of two novel surface proteins from Plasmodium falciparum-infected erythrocytes. Surface proteins were fractionated through biotin-streptavidin interaction and analyzed by shotgun proteomics. From a list of 36 candidates, two were selected for further characterization. The surface location of both proteins was confirmed by confocal microscopy using specific antibodies. PIESP1 and PIESP2 are unlikely to be associated with knobs, the protrusions on the parasite-infected erythrocyte (PIE) surface. In contrast to other known PIESPs, such as PfEMP1 and Rifin, these novel proteins are encoded by single copy genes, highly conserved across Plasmodium ssp., making them good targets for interventions with a broad specificity to various P. falciparum isolates.
Assuntos
Antígenos de Protozoários/isolamento & purificação , Eritrócitos/química , Eritrócitos/parasitologia , Proteínas de Membrana/isolamento & purificação , Plasmodium falciparum/química , Proteômica , Proteínas de Protozoários/isolamento & purificação , Sequência de Aminoácidos , Animais , Anticorpos Antiprotozoários , Antígenos de Protozoários/química , Antígenos de Protozoários/genética , Sequência Conservada , Membrana Eritrocítica/química , Genes de Protozoários , Proteínas de Membrana/química , Proteínas de Membrana/genética , Microscopia de Fluorescência , Dados de Sequência Molecular , Plasmodium falciparum/genética , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
With the entire genomic sequence of several species of Plasmodium soon to be available, researchers are now focusing on methods to study gene and protein expression at the whole organism level. Traditional methods of characterising and identifying large numbers of proteins from a complex protein mixture have relied predominantly on two-dimensional gel electrophoresis combined with N-terminal sequencing or mass spectrometry of individually prepared proteins. New proteomics methods are now available that are based on resolving small peptides derived from complex protein mixtures by high-resolution liquid chromatography and directly identifying them by tandem mass spectrometry (LC/LC/MS/MS) and sophisticated computer search algorithms against whole genome sequence databases. These newer proteomic methods have the potential to accelerate the reproducible identification of large numbers of proteins from various life cycle stages of Plasmodium and may help to better understand parasite biology and lead to the identification of new targets of vaccines and drugs.
Assuntos
Plasmodium/genética , Proteômica/métodos , Animais , Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas/métodos , ProteomaRESUMO
Using bioinformatic, proteomic, immunofluorescence, and genetic cross methods, we have functionally characterized a family of putative parasite ligands as potential mediators of cell-cell interactions. We name these proteins the Limulus clotting factor C, Coch-5b2, and Lgl1 (LCCL)-lectin adhesive-like protein (LAP) family. We demonstrate that this family is conserved amongst Plasmodium spp. It possesses a unique arrangement of adhesive protein domains normally associated with extracellular proteins. The proteins are expressed predominantly, though not exclusively, in the mosquito stages of the life cycle. We test the hypothesis that these proteins are surface proteins with 1 member of this gene family, lap1, and provide evidence that it is expressed on the surface of Plasmodium berghei sporozoites. Finally, through genetic crosses of wild-type Pblap1+ and transgenic Pblap1- parasites, we show that the null phenotype previously reported for sporozoite development in a Pblap1- mutant can be rescued within a heterokaryotic oocyst and that infectious Pblap1 sporozoites can be formed. The mutant is not rescued by coparasitization of mosquitoes with a mixture Pblap1+ and Pblap1- homokaryotic oocysts.
Assuntos
Lectinas/genética , Família Multigênica , Plasmodium berghei/genética , Proteínas de Protozoários/genética , Sequência de Aminoácidos , Animais , Anopheles , Western Blotting , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Expressão Gênica , Lectinas/química , Camundongos , Microscopia de Contraste de Fase , Dados de Sequência Molecular , Plasmodium berghei/química , Reação em Cadeia da Polimerase , Proteínas de Protozoários/químicaRESUMO
We evaluated the capacity of the cationic lipid based formulation, Vaxfectin, to enhance the immunogenicity and protective efficacy of DNA-based vaccine regimens in the Plasmodium yoelii murine malaria model. We immunized Balb/c mice with varying doses (0.4-50 microg) of plasmid DNA (pDNA) encoding the P. yoelii circumsporozoite protein (PyCSP), either in a homologous DNA/DNA regimen (D-D) or a heterologous prime-boost DNA-poxvirus regimen (D-V). At the lowest pDNA doses, Vaxfectin substantially enhanced IFA titers, ELISPOT frequencies, and protective efficacy. Clinical trials of pDNA vaccines have often used low pDNA doses based on a per kilogram weight basis. Formulation of pDNA vaccines in Vaxfectin may improve their potency in human clinical trials.
Assuntos
Vacinas Antimaláricas/imunologia , Malária/prevenção & controle , Fosfatidiletanolaminas/farmacologia , Plasmodium yoelii/imunologia , Proteínas de Protozoários/imunologia , Vacinas de DNA/imunologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Anticorpos Antiprotozoários/sangue , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Imunização Secundária , Linfócitos/imunologia , Vacinas Antimaláricas/administração & dosagem , Vacinas Antimaláricas/genética , Camundongos , Camundongos Endogâmicos BALB C , Fosfatidiletanolaminas/administração & dosagem , Proteínas de Protozoários/genética , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genética , Vaccinia virus/genéticaRESUMO
Glycosylphosphatidylinositol (GPI)-anchored proteins coat the surface of extracellular Plasmodium falciparum merozoites, of which several are highly validated candidates for inclusion in a blood-stage malaria vaccine. Here we determined the proteome of gradient-purified detergent-resistant membranes of mature blood-stage parasites and found that these membranes are greatly enriched in GPI-anchored proteins and their putative interacting partners. Also prominent in detergent-resistant membranes are apical organelle (rhoptry), multimembrane-spanning, and proteins destined for export into the host erythrocyte cytosol. Four new GPI-anchored proteins were identified, and a number of other novel proteins that are predicted to localize to the merozoite surface and/or apical organelles were detected. Three of the putative surface proteins possessed six-cysteine (Cys6) motifs, a distinct fold found in adhesive surface proteins expressed in other life stages. All three Cys6 proteins, termed Pf12, Pf38, and Pf41, were validated as merozoite surface antigens recognized strongly by antibodies present in naturally infected individuals. In addition to the merozoite surface, Pf38 was particularly prominent in the secretory apical organelles. A different cysteine-rich putative GPI-anchored protein, Pf92, was also localized to the merozoite surface. This insight into merozoite surfaces provides new opportunities for understanding both erythrocyte invasion and anti-parasite immunity.
Assuntos
Antígenos de Protozoários/química , Proteína 1 de Superfície de Merozoito/química , Plasmodium falciparum/metabolismo , Motivos de Aminoácidos , Animais , Antígenos de Protozoários/metabolismo , Antígenos de Superfície/química , Membrana Celular/metabolismo , Cisteína/química , Detergentes/farmacologia , Fator de Crescimento Epidérmico/química , Eritrócitos/metabolismo , Glicosilfosfatidilinositóis/química , Proteínas de Fluorescência Verde/química , Microdomínios da Membrana/química , Modelos Biológicos , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas , Proteômica , Proteínas de Protozoários/químicaRESUMO
Plasmodium berghei and Plasmodium chabaudi are widely used model malaria species. Comparison of their genomes, integrated with proteomic and microarray data, with the genomes of Plasmodium falciparum and Plasmodium yoelii revealed a conserved core of 4500 Plasmodium genes in the central regions of the 14 chromosomes and highlighted genes evolving rapidly because of stage-specific selective pressures. Four strategies for gene expression are apparent during the parasites' life cycle: (i) housekeeping; (ii) host-related; (iii) strategy-specific related to invasion, asexual replication, and sexual development; and (iv) stage-specific. We observed posttranscriptional gene silencing through translational repression of messenger RNA during sexual development, and a 47-base 3' untranslated region motif is implicated in this process.