Microsatellite loci: determining the genetic variability of Plasmodium vivax.

OBJECTIVE: To describe the genetic diversity of Plasmodium vivax isolates from different areas in the Brazilian Amazon using 11 polymorphic microsatellites and to evaluate the correlation between microsatellite variation and repeat array length. METHODS: Microsatellites with variable repeat units and array lengths were selected using in silico search of the P. vivax genome. We designed primers and amplified the selected loci in DNA obtained from patients with P. vivax acute infections. RESULTS: Positive correlation between repeat array length and microsatellite variation was detected independently of the size of repeat unit (di, tri, or tetranucleotide). We used these markers to describe the genetic variability of P. vivax isolates from four geographic regions of the Brazilian Amazon. Substantial variability was observed among P. vivax isolates within populations, concurrent with high levels of multiple-clone infections and high linkage disequilibrium. Overall, structured populations were observed with moderate to high genetic differentiation. CONCLUSION: The markers studied are useful tools for assessing population structure of P. vivax, as demonstrated for Brazilian populations and for searching for evidence of recent selection events associated with different phenotypes, such as drug resistance.

Naturally acquired inhibitory antibodies to Plasmodium vivax Duffy binding protein are short-lived and allele-specific following a single malaria infection.

The Duffy binding protein of Plasmodium vivax (DBP) is a critical adhesion ligand that participates in merozoite invasion of human Duffy-positive erythrocytes. A small outbreak of P. vivax malaria, in a village located in a non-malarious area of Brazil, offered us an opportunity to investigate the DBP immune responses among individuals who had their first and brief exposure to malaria. Thirty-three individuals participated in the five cross-sectional surveys, 15 with confirmed P. vivax infection while residing in the outbreak area (cases) and 18 who had not experienced malaria (non-cases). In the present study, we found that only 20% (three of 15) of the individuals who experienced their first P. vivax infection developed an antibody response to DBP; a secondary boosting can be achieved with a recurrent P. vivax infection. DNA sequences from primary/recurrent P. vivax samples identified a single dbp allele among the samples from the outbreak area. To investigate inhibitory antibodies to the ligand domain of the DBP (cysteine-rich region II, DBP(II)), we performed in vitro assays with mammalian cells expressing DBP(II) sequences which were homologous or not to those from the outbreak isolate. In non-immune individuals, the results of a 12-month follow-up period provided evidence that naturally acquired inhibitory antibodies to DBP(II) are short-lived and biased towards a specific allele.

Inhibitory properties of the antibody response to Plasmodium vivax Duffy binding protein in an area with unstable malaria transmission.

The function of the Plasmodium vivax Duffy binding protein (DBP) during the erythrocyte invasion process is critical for successful parasite growth and pathogenesis in human infections. Although DBP is the subject of intensive malaria vaccine research, investigations on the functional proprieties of anti-DBP antibodies in the human population have been limited [Infect Immun68 (2000) 3164]. In the present study, we examined the ability of sera from different populations of the Brazilian Amazon--an area of markedly unstable malaria transmission--to inhibit the erythrocyte-binding function of the DBP ligand domain (region II, DBP(II)). We found that long-term exposure to malaria in the Amazon area elicits DBP-specific antibodies that inhibit the binding of different DBP(II) variants to erythrocytes. Despite the great variability of inhibitory antibody responses observed among study participants, we observed a positive correlation between erythrocyte binding-inhibitory activity and enzyme-linked immunosorbent assay anti-DBP antibodies. Of importance, there was a non-significant tendency towards increased levels of anti-DBP antibodies among individuals with asymptomatic P. vivax infections.

Real-time multiplex allele-specific polymerase chain reaction for genotyping of the Duffy antigen, the Plasmodium vivax invasion receptor.

BACKGROUND AND OBJECTIVES: Duffy blood group is of major interest in clinical medicine as it is not only involved in blood-transfusion risks and occasionally in neonatal haemolytic disease, but it is also the receptor for the human malaria parasite Plasmodium vivax in the erythrocyte invasion. The aim of this study was to develop a rapid and inexpensive approach for high-throughput Duffy genotyping. MATERIALS AND METHODS: This paper reported the development of a Duffy genotyping assay based on multiplex real-time polymerase chain reaction (PCR) using SYBR Green I fluorescent dye. RESULTS: By using this approach for Duffy genotyping we obtained the same results as that for the conventional allele-specific PCR, however, in a high-throughput assay. The Duffy genotyping of field samples demonstrated that P. vivax-infected individuals showed a significantly higher prevalence of two functional alleles than Plasmodium falciparum-infected and non-infected individuals. This finding corroborates the hypothesis that the presence of two functional alleles increases the risk of P. vivax infection. CONCLUSION: This methodology may be suitable for epidemiological studies, particularly for exploring the relationship between Duffy alleles and malaria susceptibility, and also for identification of transfusional incompatibility in blood banks.

Sm14 gene expression in different stages of the Schistosoma mansoni life cycle and immunolocalization of the Sm14 protein within the adult worm.

Sm14 is a 14-kDa vaccine candidate antigen from Schistosoma mansoni that seems to be involved in cytoplasmic trafficking of fatty acids. Although schistosomes have a high requirement for lipids, they are not able to synthesize fatty acids and sterols de novo. Thus, they must acquire host lipids. In order to determine whether Sm14 is present in different stages of the life cycle of the parasite, we performed RT-PCR. Sm14 mRNA was identified in all stages of the life cycle studied, mainly schistosomulum, adult worm and egg. Additionally, we used a rabbit anti-Sm14 polyclonal antibody in an indirect immunofluorescence assay to localize Sm14 in adult worm sections. The basal lamella of the tegument and the gut epithelium were strongly labeled. These tissues have a high flow of and demand for lipids, a finding that supports the putative role of Sm14 as an intracellular transporter of fatty acids from host cells.

Sm14 gene expression in different stages of the Schistosoma mansoni life cycle and immunolocalization of the Sm14 protein within the adult worm

Sm14 is a 14-kDa vaccine candidate antigen from Schistosoma mansoni that seems to be involved in cytoplasmic trafficking of fatty acids. Although schistosomes have a high requirement for lipids, they are not able to synthesize fatty acids and sterols de novo. Thus, they must acquire host lipids. In order to determine whether Sm14 is present in different stages of the life cycle of the parasite, we performed RT-PCR. Sm14 mRNA was identified in all stages of the life cycle studied, mainly schistosomulum, adult worm and egg. Additionally, we used a rabbit anti-Sm14 polyclonal antibody in an indirect immunofluorescence assay to localize Sm14 in adult worm sections. The basal lamella of the tegument and the gut epithelium were strongly labeled. These tissues have a high flow of and demand for lipids, a finding that supports the putative role of Sm14 as an intracellular transporter of fatty acids from host cells

Immunological properties of gene vaccines delivered by different routes

Gene vaccines represent a new and promising approach to control infectious diseases, inducing a protective immune response in the appropriate host. Several routes and methods of genetic immunization have been shown to induce antibody production as well as T helper (Th) cell and cytotoxic T lymphocyte activation. However, few studies have compared the nature of the immune responses generated by different gene vaccination delivery systems. In the present study we reviewed some aspects of immunity induced by gene immunization and compared the immune responses produced by intramuscular (im) DNA injection to gene gun-mediated DNA transfer into the skin of BALB/c mice. Using a reporter gene coding for ß-galactosidase, we have demonstrated that im injection raised a predominantly Th1 response with mostly IgG2a anti-ßgal produced, while gene gun immunization induced a mixed Th1/Th2 profile with a balanced production of IgG2a and IgG1 subclasses. Distinct types of immune responses were generated by different methods of gene delivery. These findings have important implications for genetic vaccine design. Firstly, a combination between these two systems may create optimal conditions for the induction of a broad-based immune response. Alternatively, a particular gene vaccine delivery method might be used according to the immune response required for host protection. Here, we describe the characteristics of the immune response induced by gene vaccination and the properties of DNA involved in this process