Malaria merozoite invasion of human erythrocytes depends on recognition of specific erythrocyte surface receptors by parasite ligands. Plasmodium vivax merozoite invasion is totally dependent on the recognition of the Duffy blood group antigen by the parasite ligand Duffy-binding protein (DBP). Receptor recognition by P. vivax relies on a cysteine-rich domain, the DBL domain or region II, at the N terminus of the extracellular portion of DBP. The minimal region of the DBP implicated for receptor recognition lies between cysteines 4 and 8 of the DBL domain, which is a region that also has the highest rate of allelic polymorphisms among parasite isolates. We previously found that allelic polymorphisms in this region altered the P. vivax DBL domain antigenic character, which contrasts with changes in receptor specificity attributed to polymorphisms in some homologous ligands of Plasmodium falciparum. To further investigate the relative importance of conserved and polymorphic residues within this DBL central region, we identified residues critical for receptor recognition by site-directed mutagenesis. Seventy-seven surface-predicted residues of the Sal-1 DBL domain were substituted with alanine and assayed for erythrocyte binding activity by expression of the mutant proteins on the surface of transiently transfected COS cells. The functional effect of alanine substitution varied from nil to complete loss of DBL erythrocyte-binding activity. Mutations that caused loss of ligand function mostly occurred in discontinuous clusters of conserved residues, whereas nearly all mutations in polymorphic residues did not affect erythrocyte binding. These data delineate DBL domain residues essential for receptor recognition.malaria ͉ erythrocyte-binding protein M alaria caused by Plasmodium vivax is responsible for substantial morbidity in developing countries, accounting for 70-90% of infections acquired in Asia and the Americas as well as 12.4% of infections acquired in Africa (1). Malaria merozoites must attach to and invade new blood cells to begin each cycle of parasite development, making this brief event a critical phase of the parasite's life cycle. Invasion must occur quickly through a complex, multistep process with a distinct progression of events, involving numerous molecules expressed on the surface of the merozoite and in the apical organelles (2-7). Merozoites of P. vivax and Plasmodium knowlesi are not able to invade human erythrocytes lacking the Duffy blood group antigen because they are unable to proceed through the critical step of junction formation between the merozoite and erythrocyte. The ligands that mediate this are the Duffy-bindinglike (DBL) family of erythrocyte-binding proteins (DBL-EBP), expressed from the erythrocyte-binding-like (ebl) genes (8). The DBL-EBP are type I membrane proteins sequestered in the micronemes of invasive merozoites during late schizont development and then presumably released during the invasion process (9-12). Total reliance of P. vivax merozoite recognition on a single er...

Plasmodium vivax Duffy binding protein (DBP) is a merozoite microneme ligand vital for blood-stage infection, which makes it an important candidate vaccine for antibody-mediated immunity against vivax malaria. A differential screen with a linear peptide array compared the reactivities of noninhibitory and inhibitory high-titer human immune sera to identify target epitopes associated with protective immunity. Naturally acquired anti-DBP-specific serologic responses observed in the residents of a region of Papua New Guinea where P. vivax is highly endemic exhibited significant changes in DBP-specific titers over time. The anti-DBP functional inhibition for each serum ranged from complete inhibition to no inhibition even for high-titer responders to the DBP, indicating that epitope specificity is important. Inhibitory immune human antibodies identified specific B-cell linear epitopes on the DBP (SalI) ligand domain that showed significant correlations with inhibitory responses. Affinity-purified naturally acquired antibodies on these epitopes inhibited the DBP erythrocyte binding function greatly, confirming the protective value of specific epitopes. These results represent an important advance in our understanding of part of blood-stage immunity to P. vivax and some of the specific targets for vaccine-elicited antibody protection.

Childhood gut dysfunction (enteropathy) is common in resource-poor environments. Stunting is its presumed major consequence. Identification of biomarkers of gut dysfunction could identify the presence of, and, ideally, assess interventions for, enteropathy. Classically, enteropathy has been identified histopathologically. However, less invasive assays may be more sensitive for detecting earlier perturbations reflecting specific functional derangements. The most commonly used test has been the urinary lactulose to mannitol ratio (L:M), which primarily assesses gut leakiness, and which also measures absorption. We systematically reviewed the L:M literature published from 2000 to 2010 pertinent to children in developing country settings, and identified 25 relevant publications representing heterogeneous studies. We conclude that the L:M test has many attributes, including reflecting 2 physiologic processes (absorption and permeability) and likely correlation with growth failure consequent to child gut dysfunction. However, improved test technical performance, data reporting, and correlation with host phenotypes are needed to maximize the utility of this test.

Falciparum malaria is initiated when Anopheles mosquitoes transmit the Plasmodium sporozoite stage during a blood meal. Irradiated sporozoites confer sterile protection against subsequent malaria infection in animal models and humans. This level of protection is unmatched by current recombinant malaria vaccines. However, the live-attenuated vaccine approach faces formidable obstacles, including development of accurate, reproducible attenuation techniques. We tested whether Plasmodium falciparum could be attenuated at the early liver stage by genetic engineering. The P. falciparum genetically attenuated parasites (GAPs) harbor individual deletions or simultaneous deletions of the sporozoiteexpressed genes P52 and P36. Gene deletions were done by double-cross-over recombination to avoid genetic reversion of the knockout parasites. The gene deletions did not affect parasite replication throughout the erythrocytic cycle, gametocyte production, mosquito infections, and sporozoite production rates. However, the deletions caused parasite developmental arrest during hepatocyte infection. The double-gene deletion line exhibited a more severe intrahepatocytic growth defect compared with the single-gene deletion lines, and it did not persist. This defect was assessed in an in vitro liver-stage growth assay and in a chimeric mouse model harboring human hepatocytes. The strong phenotype of the double knockout GAP justifies its human testing as a whole-organism vaccine candidate using the established sporozoite challenge model. GAPs might provide a safe and reproducible platform to develop an efficacious whole-cell malaria vaccine that prevents infection at the preerythrocytic stage.genetically attenuated parasites ͉ malaria vaccine ͉ P36 ͉ P52 ͉ sporozoite M alaria is a formidable global health problem, affecting 300 million to 500 million people worldwide annually (1). The resulting Ϸ1 million deaths per year are mainly caused by Plasmodium falciparum infections. Eradication of malaria will in large part depend on an effective vaccine that prevents infection by Plasmodium, but such a vaccine has remained elusive. The parasites' preerythrocytic stages, encompassing the mosquito-inoculated sporozoites and liver stages that develop from sporozoites after their invasion of hepatocytes, are attractive targets for antiinfection vaccines, because at this stage the number of infected host cells is low, and further transmission of the parasite is not yet possible. Occurrence of blood-stage infection after sporozoite challenge is completely preventable by immunization with radiation-attenuated sporozoites in mouse models of malaria (2). This was a landmark finding that set the standards for malaria preerythrocytic vaccine development. Radiation-attenuated sporozoites arrest in development during hepatocyte infection, but their safety and efficacy are dependent on a precise irradiation dose. Humans immunized with P. falciparum radiation-attenuated sporozoites have been effectively protected from subsequent challenge with homologous an...

Interaction of the Duffy binding protein (DBP) with its erythrocyte receptor is critical for maintaining Plasmodium vivax blood-stage infections, making DBP an appealing vaccine candidate. The cysteine-rich region II is the ligand domain of DBP and a target of vaccine development. Interestingly, most of the allelic diversity observed in DBP is due to the high rate of nonsynonymous polymorphisms in this critical domain for receptor recognition. Similar to the hypervariability in influenza hemagglutinin, this pattern of polymorphisms in the DBP ligand domain suggests that this variation is a mechanism to evade antibody neutralization. To evaluate the role that dbp allelic diversity plays in strain-specific immunity, we examined the ability of an anti-Sal1 DBP serum to inhibit the erythrocyte-binding function of variant dbp alleles expressed on COS cells. We observed that the PNG-7.18 allele was significantly less sensitive to immune inhibition of its erythrocyte-binding activity than were the Sal1 and PNG-27.16 alleles. This result suggested that the unique polymorphisms of resistant PNG-7.18 were part of a protective epitope on the DBP ligand. To confirm this, Sal1 was converted to the refractory phenotype by introduction of 3 polymorphisms unique to PNG-7.18, via site-directed mutagenesis. The results of the present study indicate that linked polymorphisms have an additive, synergistic effect on DBP antigenic character.

BackgroundA major limitation to understanding the etiopathogenesis of environmental enteric dysfunction (EED) is the lack of a comprehensive, reproducible histologic framework for characterizing the small bowel lesions. We hypothesized that the development of such a system will identify unique histology features for EED, and that some features might correlate with clinical severity. Data Availability Statement: All relevant analyses are within the manuscript and its Supporting was assessed. The histology index was further used to identify within-and between-child variations as well as features common across and unique to each cohort, and those that correlated with host phenotype. ResultsEight of the 11 histologic scoring parameters showed useful degrees of variation. The overall concordance across all parameters was 96% weighted agreement, kappa 0.70, and Gwet's AC 0.93. Zambian and Pakistani tissues shared some histologic features with GSE, but most features were distinct, particularly abundance of intraepithelial lymphocytes in the Pakistani cohort, and marked villous destruction and loss of secretory cell lineages in the Zambian cohort. ConclusionsWe propose the first EED histology index for interpreting duodenal biopsies. This index should be useful in future clinical and translational studies of this widespread, poorly understood, and highly consequential disorder, which might be caused by multiple contributing processes, in different regions of the world. Author summaryThe study of EED has been limited by the lack of a rigorously tested, reproducible histology index that can provide insight to the pathogenesis of this entity. In this study we report the first duodenal histology index that was developed using an unbiased approach, with excellent inter-observer reproducibility, for the study of EED. The EED histology index readily identified histologic features that are common or unique to cohorts of distinct geographic locations. Incorporating the histology index into future clinical studies will provide useful insight into the pathogenesis and for intervention strategy development.

Environmental enteropathy is a major contributor to growth faltering in millions of children in Africa and South Asia. We carried out a longitudinal, observational and interventional study in Lusaka, Zambia, of 297 children with stunting (aged 2–17 months at recruitment) and 46 control children who had good growth (aged 1–5 months at recruitment). Control children contributed data only at baseline. Children were provided with nutritional supplementation of daily cornmeal-soy blend, an egg and a micronutrient sprinkle, and were followed up to 24 months of age. Children whose growth did not improve over 4–6 months of nutritional supplementation were classified as having non-responsive stunting. We monitored microbial translocation from the gut lumen to the bloodstream in the cohort with non-responsive stunting (n = 108) by measuring circulating lipopolysaccharide (LPS), LPS-binding protein and soluble CD14 at baseline and when non-response was declared. We found that microbial translocation decreased with increasing age, such that LPS declined in 81 (75%) of 108 children with non-responsive stunting, despite sustained pathogen pressure and ongoing intestinal epithelial damage. We used confocal laser endomicroscopy and found that mucosal leakiness also declined with age. However, expression of brush border enzyme, nutrient transporter and mucosal barrier genes in intestinal biopsies did not change with age or correlate with biomarkers of microbial translocation. We propose that environmental enteropathy arises through adaptation to pathogen-mediated epithelial damage. Although environmental enteropathy reduces microbial translocation, it does so at the cost of impaired growth. The reduced epithelial surface area imposed by villus blunting may explain these findings.

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