Identification and characterization of the Plasmodium vivax thrombospondin-related apical merozoite protein.

BACKGROUND: Malaria caused by Plasmodium vivax is a major public health problem worldwide that affects 70-80 million people in the Middle East, Asia, Western Pacific, South America and the Caribbean. Despite its epidemiological importance, few antigens from this parasite species have been characterized to date compared to Plasmodium falciparum, due in part to the difficulties of maintaining an in vitro culture of P. vivax. This study describes the identification of the P. falciparum thrombospondin-related apical merozoite protein homologue in P. vivax (PvTRAMP) and examines its potential to be further evaluated as vaccine candidate. METHODS: The gene encoding PvTRAMP was identified through an extensive search of the databases hosting the genome sequence of P. vivax. Genes adjacent to pvtramp were identified in silico to determine the degree of similarity between the protein sequences encoded by equivalent chromosomic fragments in P. falciparum and Plasmodium knowlesi. The pvtramp gene was amplified from cDNA of P. vivax schizont stages, cloned and expressed in Escherichia coli. Anti-PvTRAMP antisera was obtained by inoculating rabbits with PvTRAMP B cell epitopes produced as synthetic peptides in order to assess its recognition in parasite lysates by Western blot and in intact parasites by indirect immunofluorescence. The recognition of recombinant PvTRAMP by sera from P. vivax-infected individuals living in endemic areas was also assessed by ELISA. RESULTS: The PfTRAMP homologue in P. vivax, here denoted as PvTRAMP, is a 340-amino-acid long antigen encoded by a single exon that could have a potential role in cytoadherence, as indicated by the presence of a thrombospondin structural homology repeat (TSR) domain. According to its transcription and expression profile, PvTRAMP is initially located at the parasite's apical end and later on the parasite surface. Recombinant PvTRAMP is recognized by sera from infected patients, therefore, indicating that it is targeted by the immune system during a natural infection with P. vivax. CONCLUSIONS: The results of this work support conducting further studies with PvTRAMP to evaluate its immunogenicity and protection-inducing ability in the Aotus animal model.

Characterization and antigenicity of the promising vaccine candidate Plasmodium vivax 34kDa rhoptry antigen (Pv34).

This study describes the identification of the Plasmodium vivax rhoptry antigen Pv34 whose sequence was obtained based on homology comparison with the Plasmodium falciparum Pf34. The pv34 gene product was characterized by molecular biology and immunological techniques. Additionally, association of Pv34 to detergent-resistant microdomains (DRMs), expression in late blood-stage parasites and recognition of recombinant Pv34 (rPv34) by sera from P. vivax-infected Aotus monkeys and patients was assessed. Lymphoproliferation and cytokine secretion was also evaluated in individuals living in malaria endemic areas. Altogether, the data support carrying out further studies to assess the immunogenicity and protection-inducing ability of rPv34 as component of a multi-antigenic, multi-stage vaccine against vivax malaria.

The Plasmodium vivax Pv41 surface protein: identification and characterization.

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.

Characterisation of the Plasmodium vivax Pv38 antigen.

This study describes the identification and characterisation of Pv38, based on the available genomic sequence of Plasmodium vivax and previous studies done with its Plasmodium falciparum homologue: Pf38. Pv38 is a 355 amino acid long peptide encoded by a single exon gene, for which orthologous genes have been identified in other Plasmodium species by bioinformatic approaches. As for Pf38, Pv38 was found to contain a s48/45 domain which is usually found in proteins displayed on gametocytes surface. The association of Pv38 with detergent-resistant membranes (DRMs), its expression in mature blood stages of the parasite (mainly schizonts) and the detection of its recombinant protein by sera from Aotus monkeys previously exposed to the parasite, were here assessed to further characterise this new antigen.

Identifying and characterising the Plasmodium falciparum RhopH3 Plasmodium vivax homologue.

Four Plasmodium species cause malaria in humans, Plasmodium falciparum being the most widely studied to date. All Plasmodium species have paired club-shaped organelles towards their apical extreme named rhoptries that contain many lipids and proteins which are released during target cell invasion. P. falciparum RhopH3 is a rhoptry protein triggering important immune responses in patients from endemic regions. It has also been shown that anti-RhopH3 antibodies inhibit in vitro invasion of erythrocytes. Recent immunisation studies in mice with the Plasmodium yoelii and Plasmodium berghei RhopH3 P. falciparum homologue proteins found that they are able to induce protection in murine models. This study described identifying and characterising RhopH3 protein in Plasmodium vivax; it is encoded by a seven exon gene and expressed during the parasite's asexual stage. PvRhopH3 has similar processing to its homologue in P. falciparum and presents a cellular immunolocalisation pattern characteristic of rhoptry proteins.

Cutaneous tuberculosis diagnosis in an inhospitable Amazonian region by means of telemedicine and molecular biology.

We report on a 13-year-old boy who displayed a chronic granulomatous inflammatory reaction of 5 years duration. The lesion was resistant to different antibiotic schemes; his routine laboratory tests and chest radiographs were normal. Teledermatologic consultation and histopathologic study of skin biopsy suggested scrofulodermal tuberculosis. Polymerase chain reaction amplification of DNA extracted from lymph node biopsy was taken as starting material for dot-blot hybridization using Mtp-40 and IS 6110 as probes for detecting either Mycobacterium tuberculosis or any mycobacteria belonging to the M tuberculosis complex, respectively. Positive results in both hybridizations were further confirmed by culturing in BACTEC MGIT 960 system. The lesion greatly diminished following isoniazid, rifampin, and ethambutol treatment. Telemedicine allowed a cutaneous tuberculosis diagnosis to be made of a patient living in a remote town located in the Amazon jungle by using molecular biology techniques.