Plasmodium falciparum: distribution of msp2 genotypes among symptomatic and asymptomatic individuals from the Wosera region of Papua New Guinea

The merozoite surface protein 2 (MSP2) is a leading asexual-stage malaria vaccine candidate that has already proven to have an effect in phase I/IIb vaccine trials, where it was tested in combination with other antigens. Alleles of msp2 fall within two major allelic families, 3D7 and FC27. We analyzed the msp2 genotype in 306 asymptomatic and 63 symptomatic infections from the Wosera region of Papua New Guinea. The multiplicity of infection and the distribution of msp2 alleles was similar to that found in previous studies in the region, but there was no association found between FC27-type or 3D7-type forms of MSP2 and clinical malaria.     

Solution conformation, backbone dynamics and lipid interactions of the intrinsically unstructured malaria surface protein MSP2

Merozoite surface protein 2 (MSP2), one of the most abundant proteins on the surface of the merozoite stage of Plasmodium falciparum, is a potential component of a malaria vaccine, having shown some efficacy in a clinical trial in Papua New Guinea. MSP2 is a GPI-anchored protein consisting of conserved N- and C-terminal domains and a variable central region. Previous studies have shown that it is an intrinsically unstructured protein with a high propensity for fibril formation, in which the conserved N-terminal domain has a key role. Secondary structure predictions suggest that MSP2 contains long stretches of random coil with very little α-helix or β-strand. Circular dichroism spectroscopy confirms this prediction under physiological conditions (pH 7.4) and in more acidic solutions (pH 6.2 and 3.4). Pulsed field gradient NMR diffusion measurements showed that MSP2 under physiological conditions has a large effective hydrodynamic radius consistent with an intrinsic pre-molten globule state, as defined by Uversky. This was supported by sedimentation velocity studies in the analytical ultracentrifuge. NMR resonance assignments have been obtained for FC27 MSP2, allowing the residual secondary structure and backbone dynamics to be defined. There is some motional restriction in the conserved C-terminal region in the vicinity of an intramolecular disulfide bond. Two other regions show motional restrictions, both of which display helical structure propensities. One of these helical regions is within the conserved N-terminal domain, which adopts essentially the same conformation in full-length MSP2 as in corresponding peptide fragments. We see no evidence of long-range interactions in the full-length protein. MSP2 associates with lipid micelles, but predominantly through the N-terminal region rather than the C terminus, which is GPI-anchored to the membrane in the parasite.     

Multilocus population genetic analysis of the Southwest Pacific malaria vector Anopheles punctulatus

The population structure and history of the cryptic malaria vector species, Anopheles punctulatus (Doenitz), was investigated throughout Papua New Guinea and the Solomon Islands with the aim of detailing genetic subdivisions and the potential for movement through this biogeographically complex region. We obtained larval collections from over 80 sites and utilised a diverse array of molecular markers that evolve through different processes. Individuals were initially identified to species and genotyped using the ribosomal DNA second internal transcribed spacer. DNA sequencing of a single copy nuclear ribosomal protein S9 and the mitochondrial cytochrome oxidase I loci were then investigated and 12 nuclear microsatellite markers were developed and analysed. Our data revealed three genetically distinct populations – one in Papua New Guinea, the second on Buka Island (Bougainville Province, Papua New Guinea), and the third on Guadalcanal Island (Solomon Islands). Genetic differentiation within Papua New Guinea was much lower than that found in studies of other closely related species in the region. The data does suggest that A. punctulatus has undergone a population bottleneck followed by a recent population and range expansion in Papua New Guinea. Humans and regional economic growth may be facilitating this population expansion, as A. punctulatus is able to rapidly occupy human modified landscapes and traverse unsealed roads. We therefore anticipate extensive movement of this species through New Guinea – particularly into the highlands, with a potential increase in malaria frequency in a warming climate – as well as relatively unrestricted gene flow of advantageous alleles that may confound vector control efforts.     

Quantifying the importance of MSP1-19 as a target of growth-inhibitory and protective antibodies against Plasmodium falciparum in humans

Background

Antibodies targeting blood stage antigens are important in protection against malaria, but the key targets and mechanisms of immunity are not well understood. Merozoite surface protein 1 (MSP1) is an abundant and essential protein. The C-terminal 19 kDa region (MSP1-19) is regarded as a promising vaccine candidate and may also be an important target of immunity.

Methodology/Findings

Growth inhibitory antibodies against asexual-stage parasites and IgG to recombinant MSP1-19 were measured in plasma samples from a longitudinal cohort of 206 children in Papua New Guinea. Differential inhibition by samples of mutant P. falciparum lines that expressed either the P. falciparum or P. chabaudi form of MSP1-19 were used to quantify MSP1-19 specific growth-inhibitory antibodies. The great majority of children had detectable IgG to MSP1-19, and high levels of IgG were significantly associated with a reduced risk of symptomatic P. falciparum malaria during the 6-month follow-up period. However, there was little evidence of PfMSP1-19 specific growth inhibition by plasma samples from children. Similar results were found when testing non-dialysed or dialysed plasma, or purified antibodies, or when measuring growth inhibition in flow cytometry or microscopy-based assays. Rabbit antisera generated by immunization with recombinant MSP1-19 demonstrated strong MSP1-19 specific growth-inhibitory activity, which appeared to be due to much higher antibody levels than human samples; antibody avidity was similar between rabbit antisera and human plasma.

Conclusions/Significance

These data suggest that MSP1-19 is not a major target of growth inhibitory antibodies and that the protective effects of antibodies to MSP1-19 are not due to growth inhibitory activity, but may instead be mediated by other mechanisms. Alternatively, antibodies to MSP1-19 may act as a marker of protective immunity.     

New approaches to the serotypic analysis of the epidemiology of Plasmodium falciparum

Considerable antigenic heterogeneity of Plasmodium falciparum has been demonstrated in natural parasite populations. However, very little is known about the relative virulence, transmission efficiency and prevalence over space and time of parasites expressing different serotypes of variant antigens. The recent application of recombinant DNA techniques to express a wide range of P. falciparum antigens in Escherichia coli has led to a better understanding of the molecular basis of antigenic diversity of a number of parasite proteins including the precursor to the major merozoite surface antigen (PMMSA) and the heat-stable S-antigens. Highly specific reagents such as DNA probes, monoclonal antibodies and polyclonal antisera to either cloned antigens or synthetic peptides have become available for serotypic analysis of natural parasite populations. With these reagents important epidemiological questions can now be asked concerning the population biology of different serotypes of P. falciparum. The use of the polymorphic S-antigen system as a serotypic marker to analyse the transmission dynamics of P. falciparum in Madang, Papua New Guinea (PNG) is discussed. Results of serotyping studies with the S-antigen system highlight the complexities of malaria transmission, which require consideration in the design of malaria vaccine trials.     

Health research in Papua New Guinea

The Papua New Guinea Institute of Medical Research (PNGIMR) is one of the most respected health research institutions in the developing world, and its studies of the local health problems of PNG have consistently had international relevance. This article examines the structural and philosophical factors that have enabled the success of the PNGIMR, and presents the PNGIMR approach to research as a potential model for other disease-endemic countries. An overview of PNGIMR research into malaria and filariasis is given with selected examples as an introduction to a Trends in Parasitology series on health research in Papua New Guinea.     

Allelic dimorphism in a surface antigen gene of the malaria parasite Plasmodium falciparum

Merozoites of the malaria parasite Plasmodium falciparum carry surface proteins processed from a precursor termed p190 or p195. Polymorphism has been reported in this protein. Since the protein is a candidate for a malaria vaccine, it is important to understand the nature of this polymorphism. We have determined the complete nucleotide sequence of the p190 gene from the MAD20 strain (a Papua New Guinea isolate). Comparisons of the gene with that from other strains of P. falciparum allowed us to study the genetic basis of the antigen's polymorphism. The gene consists of sequences distributed in variable blocks, which are separated by conserved or semi-conserved sequences. Variable sequences occur both in regions that code for tripeptide repeats and in regions with no apparent repeats. Interestingly, according to the present data, variable sequences are not widely polymorphic but fall into two distinct types. We argue that the p190 protein is encoded by dimorphic alleles capable of limited genetic exchange and present evidence at the nucleotide level documenting intragenic recombination in Plasmodium.     

Engaging the community in research: lessons learned from the malaria vaccine trial

When staff of the Papua New Guinea Institute of Medical Research first went into Wosera in 1979, to try and interest the community into participating in malaria research, the traditional warning sign of crossed bamboo sticks constantly blocked their path as they tried to enter the villages. In 2003, the site is a thriving research centre, with many projects under its belt, a successfully executed malaria vaccine trial to its credit, and approximately 13000 people from 29 villages currently participating in demographic surveillance and various research projects. It has been a long road from community suspicion to sustainable community participation, and the field workers have learned many important lessons along the way that can be applicable to research programs in other disease-endemic areas.     

Molecular parasitology of malaria in Papua New Guinea

Research into the molecular biology of infectious diseases is mostly associated with well-developed countries. But in the midst of tropical Papua New Guinea, highly sophisticated molecular research has being conducted over years to understand and fight malaria and other tropical diseases. Here, we review such research carried out at the Papua New Guinea Institute of Medical Research. This Institute has considerably shaped research on molecular epidemiology through its analysis of the diversity and structure of the Plasmodium falciparum population. In addition, research has been conducted on human host factors and, more recently, the molecular analysis of drug resistance and the underlying molecular mechanisms of host-parasite interactions have been investigated.     

Musaespora,a genus of pyrenocarpous lichens with campylidia,and other additions to the foliicolous lichen flora of New Guinea

Abstract: The new lichen genus Musaespora is described from Java and Papua New Guinea. It is the first known pyrenocarpous lichen genus with campylidia, and appears to belong to the Aspidotheliaceae. Three species are described, two foliicolous species from Papua New Guinea and one corticolous species from Java. In addition, a list is given of the foliicolous lichen flora of the site in Papua New Guinea, where the foliicolous Musaespora species have been found, two more new foliicolous species are described, Echinoplaca hispida and Sporopodium lucidum, and 19 species are recorded for the first time form Papua New Guinea.     

Antipyretic effect of ibuprofen in Gabonese children with uncomplicated falciparum malaria: a randomized, double-blind, placebo-controlled trial

Background

The protection afforded by human erythrocyte polymorphisms against the malaria parasite, Plasmodium falciparum, has been proposed to be due to reduced ability of the parasite to invade or develop in erythrocytes. If this were the case, variable levels of parasitaemia and rates of seroconversion to infected-erythrocyte variant surface antigens (VSA) should be seen in different host genotypes.

Methods

To test this hypothesis, P. falciparum parasitaemia and anti-VSA antibody levels were measured in a cohort of 555 asymptomatic children from an area of intense malaria transmission in Papua New Guinea. Linear mixed models were used to investigate the effect of α⁺-thalassaemia, complement receptor-1 and south-east Asian ovalocytosis, as well as glucose-6-phosphate dehydrogenase deficiency and ABO blood group on parasitaemia and age-specific seroconversion to VSA.

Results

No host polymorphism showed a significant association with both parasite prevalence/density and age-specific seroconversion to VSA.

Conclusion

Host erythrocyte polymorphisms commonly found in Papua New Guinea do not effect exposure to blood stage P. falciparum infection. This contrasts with data for sickle cell trait and highlights that the above-mentioned polymorphisms may confer protection against malaria via distinct mechanisms.     

Plasmodium falciparum erythrocyte invasion through glycophorin C and selection for Gerbich negativity in human populations

Geographic overlap between malaria and the occurrence of mutant hemoglobin and erythrocyte surface proteins has indicated that polymorphisms in human genes have been selected by severe malaria. Deletion of exon 3 in the glycophorin C gene (called GYPCDeltaex3 here) has been found in Melanesians; this alteration changes the serologic phenotype of the Gerbich (Ge) blood group system, resulting in Ge negativity. The GYPCDeltaex3 allele reaches a high frequency (46.5%) in coastal areas of Papua New Guinea where malaria is hyperendemic. The Plasmodium falciparum erythrocyte-binding antigen 140 (EBA140, also known as BAEBL) binds with high affinity to the surface of human erythrocytes. Here we show that the receptor for EBA140 is glycophorin C (GYPC) and that this interaction mediates a principal P. falciparum invasion pathway into human erythrocytes. EBA140 does not bind to GYPC in Ge-negative erythrocytes, nor can P. falciparum invade such cells using this invasion pathway. This provides compelling evidence that Ge negativity has arisen in Melanesian populations through natural selection by severe malaria.     

Population genetic analyses of Plasmodium falciparum chloroquine receptor transporter gene haplotypes reveal the evolutionary history of chloroquine-resistant malaria in India

Inferring the origin and dispersal of the chloroquine-resistant (CQR) malaria parasite, Plasmodium falciparum, is of academic and public health importance. The Pfcrt gene of P. falciparum is widely known as the CQR gene and two major haplotypes of this gene (CVIET and SVMNT) occur widely across CQR-endemic regions of the globe. In India, studies to date of the Pfcrt gene have indicated the widespread prevalence of the SVMNT haplotype (prevalent in the South America and Papua New Guinea), whereas the CVIET haplotype, primarily found in southeast Asia, was not detected at a high frequency in India. This distribution pattern of the two most common CQR-Pfcrt haplotypes in India is quite surprising. Thus, in order to understand probable evolutionary and migration patterns of the CQR-Pfcrt haplotypes into India, we generated new sequence data of exon 2 of the Pfcrt gene and collected published information on the CQR-Pfcrt haplotype data from India, Papua New Guinea, southeast Asia and South America, and performed several population and evolutionary genetic analyses. Among several interesting findings, statistically significant longitudinal clines for the CVIET and SVMNT haplotypes (in opposite directions) in India, and the clustering of India and Papua New Guinea under the SVMNT-specific clade in the phylogenetic tree, are the two most remarkable aspects of the data. It also appears that both the SVMNT and CVIET haplotypes in India have migrated from southeast Asia. In particular, whereas the Indian CVIET haplotype has a southeast Asian origin, the SVMNT haplotype, prevalent in India, seems to have originated in Papua New Guinea and entered India through southeast Asia.     

Contributions to the Cladocera fauna from Papua New Guinea

Twenty-eight taxa of the Cladocera are identified in collections from Papua New Guinea, 17 being new records for New Guinea, bringing the total number of Cladocera taxa reported for this region to 39. Most of the taxa are circumtropical. One species (Sarsilatona papuana) is endemic to Papua New Guinea and northern Australia. The species list includes two species that are normally listed as Holarctic:Alonella nana andAlona rustica. Widespread genera such asDaphnia, Pleuroxus, Disparalona, Acroperus were strikingly absent from the Papua New Guinean material.     

Conformational Dynamics and Antigenicity in the Disordered Malaria Antigen Merozoite Surface Protein 2

Merozoite surface protein 2 (MSP2) of Plasmodium falciparum is an abundant, intrinsically disordered protein that is GPI-anchored to the surface of the invasive blood stage of the malaria parasite. Recombinant MSP2 has been trialled as a component of a malaria vaccine, and is one of several disordered proteins that are candidates for inclusion in vaccines for malaria and other diseases. Nonetheless, little is known about the implications of protein disorder for the development of an effective antibody response. We have therefore undertaken a detailed analysis of the conformational dynamics of the two allelic forms of MSP2 (3D7 and FC27) using NMR spectroscopy. Chemical shifts and NMR relaxation data indicate that conformational and dynamic properties of the N- and C-terminal conserved regions in the two forms of MSP2 are essentially identical, but significant variation exists between and within the central variable regions. We observe a strong relationship between the conformational dynamics and the antigenicity of MSP2, as assessed with antisera to recombinant MSP2. Regions of increased conformational order in MSP2, including those in the conserved regions, are more strongly antigenic, while the most flexible regions are minimally antigenic. This suggests that modifications that increase conformational order may offer a means to tune the antigenicity of MSP2 and other disordered antigens, with implications for vaccine design.     

Characterization of population structure from the mitochondrial DNA vis‐à‐vis language and geography in Papua New Guinea

Situated along a corridor linking the Asian continent with the outer islands of the Pacific, Papua New Guinea has long played a key role in understanding the initial peopling of Oceania. The vast diversity in languages and unique geographical environments in the region have been central to the debates on human migration and the degree of interaction between the Pleistocene settlers and newer migrants. To better understand the role of Papua New Guinea in shaping the region's prehistory, we sequenced the mitochondrial DNA (mtDNA) control region of three populations, a total of 94 individuals, located in the East Sepik Province of Papua New Guinea. We analyzed these samples with a large data set of Oceania populations to examine the role of geography and language in shaping population structure within New Guinea and between the region and Island Melanesia. Our results from median‐joining networks, star‐cluster age estimates, and population genetic analyses show that while highland New Guinea populations seem to be the oldest settlers, there has been significant gene flow within New Guinea with little influence from geography or language. The highest genetic division is between Papuan speakers of New Guinea versus East Papuan speakers located outside of mainland New Guinea. Our study supports the weak language barriers to genetic structuring among populations in close contact and highlights the complexity of understanding the genetic histories of Papua New Guinea in association with language and geography. Am J Phys Anthropol 142:613–624, 2010. © 2010 Wiley‐Liss, Inc.     

Intersterility between populations of <Emphasis Type="Italic">Lentinula edodes</Emphasis> from Papua New Guinea

Mating tests among strains of Lentinula edodes distributed in Asia-Australasia were conducted. As a result, 26 strains were classified into three groups: 2 strains from Mt. Wilhelm in Papua New Guinea (PN1 group) showed intersterility with 7 strains from Mt. Albert Edward and Mt. Kaisenik in Papua New Guinea (PN2 group) and semicompatibility (clamp formation restricted to contact zone between paired monokaryons) with 17 strains from Asia-Australasia (AA group), whereas the strains of the PN2 group showed compatibility with the AA group. These results suggest that the shiitake populations distributed in Asia-Australasia including Papua New Guinea are in the process of speciation. Contribution no. 391 from the Tottori Mycological Institute     

Lipid interactions of the malaria antigen merozoite surface protein 2

With more than half the world's population living at risk of malaria infection, there is a strong demand for the development of an effective malaria vaccine. One promising vaccine candidate is merozoite surface protein 2 (MSP2), which is among the most abundant antigens of the blood stage of the Plasmodium falciparum parasite. In solution, MSP2 is intrinsically unstructured, but little is known about the conformation of native MSP2, which is GPI-anchored to the merozoite surface, or of the implications of that conformation for the immune response induced by MSP2. Initial NMR studies have shown that MSP2 interacts with lipid micelles through a highly conserved N-terminal domain. We have further developed these findings by investigating how different lipid environments affect the protein structure. All of the tested lipid preparations perturbed only the N-terminal part of MSP2. In DPC micelles this region adopts an α-helical structure which we have characterized in detail. Our findings suggest a possible mechanism by which lipid interactions might modulate immune recognition of the conserved N-terminus of MSP2, potentially explaining the apparent immunodominance of the central variable region of this important malaria antigen.     

The Strongylophthalmyiidae (Diptera) of Papua New Guinea,with descriptions of five new species and a world checklist

Eight species of the Strongylophthalmyiidae are reported from Papua New Guinea. Of these, five species (Strongylophthalmyia gigantica sp. nov., S. papuana sp. nov., S. rubella sp. nov., S. sedlaceki sp. nov. and S. shatalkini sp. nov.) are described as new to science. Strongylophthalmyia puncticollis Frey is recorded for the first time from Papua New Guinea. Faunistic remarks, a key to the species of the Strongylophthalmyia in Papua New Guinea and a world checklist are provided.     

Population genetics of Anopheles koliensis through Papua New Guinea: New cryptic species and landscape topography effects on genetic connectivity

New Guinea is a topographically and biogeographically complex region that supports unique endemic fauna. Studies describing the population connectivity of species through this region are scarce. We present a population and landscape genetic study on the endemic malaria‐transmitting mosquito, Anopheles koliensis (Owen). Using mitochondrial and nuclear sequence data, as well as microsatellites, we show the evidence of geographically discrete population structure within Papua New Guinea (PNG). We also confirm the existence of three rDNA ITS2 genotypes within this mosquito and assess reproductive isolation between individuals carrying different genotypes. Microsatellites reveal the clearest population structure and show four clear population units. Microsatellite markers also reveal probable reproductive isolation between sympatric populations in northern PNG with different ITS2 genotypes, suggesting that these populations may represent distinct cryptic species. Excluding individuals belonging to the newly identified putative cryptic species (ITS2 genotype 3), we modeled the genetic differences between A. koliensis populations through PNG as a function of terrain and find that dispersal is most likely along routes with low topographic relief. Overall, these results show that A. koliensis is made up of geographically and genetically discrete populations in Papua New Guinea with landscape topography being important in restricting dispersal.