RESUMO
Apical membrane antigen 1 (AMA-1) is considered to be a major candidate antigen for a malaria vaccine. Previous immunoepidemiological studies of naturally acquired immunity to Plasmodium vivax AMA-1 (PvAMA-1) have shown a higher prevalence of specific antibodies to domain II (DII) of AMA-1. In the present study, we confirmed that specific antibody responses from naturally infected individuals were highly reactive to both full-length AMA-1 and DII. Also, we demonstrated a strong association between AMA-1 and DII IgG and IgG subclass responses. We analyzed the primary sequence of PvAMA-1 for B cell linear epitopes co-occurring with intrinsically unstructured/disordered regions (IURs). The B cell epitope comprising the amino acid sequence 290-307 of PvAMA-1 (SASDQPTQYEEEMTDYQK), with the highest prediction scores, was identified in domain II and further selected for chemical synthesis and immunological testing. The antigenicity of the synthetic peptide was identified by serological analysis using sera from P. vivax-infected individuals who were knowingly reactive to the PvAMA-1 ectodomain only, domain II only, or reactive to both antigens. Although the synthetic peptide was recognized by all serum samples specific to domain II, serum with reactivity only to the full-length protein presented 58.3% positivity. Moreover, IgG reactivity against PvAMA-1 and domain II after depletion of specific synthetic peptide antibodies was reduced by 18% and 33% (Pâ=â0.0001 for both), respectively. These results suggest that the linear epitope SASDQPTQYEEEMTDYQK is highly antigenic during natural human infections and is an important antigenic region of the domain II of PvAMA-1, suggesting its possible future use in pre-clinical studies.
Assuntos
Antígenos de Protozoários/imunologia , Epitopos de Linfócito B/imunologia , Proteínas de Membrana/imunologia , Plasmodium vivax/imunologia , Proteínas de Protozoários/imunologia , Adolescente , Adulto , Idoso , Sequência de Aminoácidos , Animais , Anticorpos Antiprotozoários/sangue , Anticorpos Antiprotozoários/genética , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/genética , Humanos , Malária Vivax/sangue , Malária Vivax/imunologia , Malária Vivax/microbiologia , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Dados de Sequência Molecular , Peptídeos/genética , Peptídeos/imunologia , Plasmodium vivax/citologia , Proteínas de Protozoários/genética , Adulto JovemRESUMO
Recently, Plasmodium vivax has been related to nearly 81% of malaria cases reported in Central America and the Mediterranean. Due to the difficulty of culturing this parasite species in vitro, most studies on P. vivax have focused on the identification of new antigens by homology comparison with P. falciparum vaccine candidate proteins. In this study, we have identified and characterized a Pf41 homologue in P. vivax, hence named Pv41, by following such approach and using web-available bioinformatics databases, molecular techniques and immunochemistry assays. Pv41 protein is a 384-amino-acid-long antigen encoded by a single exon that exhibits two s48/45 domains characteristic of gametocyte surface proteins. We have also demonstrated Pv41 transcription and expression during late intra-erythrocytic parasite stages and defined its subcellular localization on the parasite surface.
Assuntos
Antígenos de Protozoários/metabolismo , Eritrócitos/parasitologia , Proteínas de Membrana/metabolismo , Plasmodium vivax/crescimento & desenvolvimento , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Animais , Antígenos de Protozoários/genética , Membrana Celular/metabolismo , Clonagem Molecular , Éxons , Proteínas de Membrana/genética , Plasmodium vivax/citologia , Plasmodium vivax/genética , Estrutura Terciária de Proteína/genética , Proteínas de Protozoários/genética , Coelhos , Transcrição GênicaRESUMO
The diagnostic capacity of three malaria rapid diagnostic tests (RDTs), NOW-Malaria-ICT, OptiMAL-IT, and Paracheck-Pf, was evaluated against expert microscopy in Colombia. We tested 896 patients, of whom microscopy confirmed 139 P. falciparum, 279 P. vivax, and 13 mixed P.f/P.v infections and 465 negatives. Paracheck-Pf and NOW-malaria-ICT were more accurate in detecting P. falciparum (sensitivities 90.8% and 90.1%, respectively) in comparison with Optimal-IT (83.6%). NOW showed an acceptable Pf detection rate at low densities (< 500/microL), but resulted in a higher proportion of false positives. For P. vivax diagnosis, Optimal-IT had a higher sensitivity than NOW (91.0% and 81.4%, respectively). The choice between the two Pf/Pv detecting RDTs balances P. falciparum and P. vivax detection rates. Considering some degree of P. falciparum overtreatment and failure to detect all P. vivax cases as more acceptable than missing some cases of P. falciparum, we recommend careful implementation of NOW-malaria-ICT in areas where microscopy is lacking. The price is however still a constraint.
Assuntos
Malária Falciparum/diagnóstico , Malária Vivax/diagnóstico , Adolescente , Adulto , Idoso , Animais , Criança , Pré-Escolar , Colômbia , Feminino , Humanos , Lactente , Masculino , Microscopia/métodos , Pessoa de Meia-Idade , Plasmodium falciparum/citologia , Plasmodium falciparum/isolamento & purificação , Plasmodium vivax/citologia , Plasmodium vivax/isolamento & purificação , Controle de QualidadeRESUMO
The cdc2 gene product, a 34-kDa protein kinase, plays a universal role in the M phase of the eukaryotic cell cycle. To study the cell cycle regulation in malarial parasites, we have characterized a cdc2-related gene from the most widely distributed human malaria, Plasmodium vivax (Pvcrk2). The full-length Pvcrk2 revealed 90--99% homology with Crk2 proteins from other Plasmodium species and approximately 60% homology with p34(cdc2) proteins from higher eukaryotes. We used the temperature-sensitive Schizosaccharomyces pombe cdc2 mutant (cdc2-33(ts)) for gene complementation studies. Expression of the full-length 33-kDa PvCrk2 protein, a truncated 27-kDa version, and two chimeric proteins in which we exchanged the N- and C-terminal regions of PvCrk2 with their S. pombe counterparts at the restrictive temperature in the mutant cdc2-33(ts) did not complement the cell cycle defect. However, conditional expression of the Pvcrk2 genes or the chimera containing the C terminus from Spcdc2 in mutant cdc2-33(ts) cells produced cell-cycle-arrested phenotypes only in the induced state and at the permissive temperature. Our results thus provide the first compelling genetic evidence that the plasmodial Crk2 gene product(s) is capable of interfering with the well-conserved eukaryotic cell cycle machinery.