Glycan Masking of Plasmodium vivax Duffy Binding Protein for Probing Protein Binding Function and Vaccine Development

Glycan masking is an emerging vaccine design strategy to focus antibody responses to specific epitopes, but it has mostly been evaluated on the already heavily glycosylated HIV gp120 envelope glycoprotein. Here this approach was used to investigate the binding interaction of Plasmodium vivax Duffy Binding Protein (PvDBP) and the Duffy Antigen Receptor for Chemokines (DARC) and to evaluate if glycan-masked PvDBPII immunogens would focus the antibody response on key interaction surfaces. Four variants of PVDBPII were generated and probed for function and immunogenicity. Whereas two PvDBPII glycosylation variants with increased glycan surface coverage distant from predicted interaction sites had equivalent binding activity to wild-type protein, one of them elicited slightly better DARC-binding-inhibitory activity than wild-type immunogen. Conversely, the addition of an N-glycosylation site adjacent to a predicted PvDBP interaction site both abolished its interaction with DARC and resulted in weaker inhibitory antibody responses. PvDBP is composed of three subdomains and is thought to function as a dimer; a meta-analysis of published PvDBP mutants and the new DBPII glycosylation variants indicates that critical DARC binding residues are concentrated at the dimer interface and along a relatively flat surface spanning portions of two subdomains. Our findings suggest that DARC-binding-inhibitory antibody epitope(s) lie close to the predicted DARC interaction site, and that addition of N-glycan sites distant from this site may augment inhibitory antibodies. Thus, glycan resurfacing is an attractive and feasible tool to investigate protein structure-function, and glycan-masked PvDBPII immunogens might contribute to P. vivax vaccine development.     

Whole Genome Sequencing of Field Isolates Reveals a Common Duplication of the Duffy Binding Protein Gene in Malagasy Plasmodium vivax Strains

Background

Plasmodium vivax is the most prevalent human malaria parasite, causing serious public health problems in malaria-endemic countries. Until recently the Duffy-negative blood group phenotype was considered to confer resistance to vivax malaria for most African ethnicities. We and others have reported that P. vivax strains in African countries from Madagascar to Mauritania display capacity to cause clinical vivax malaria in Duffy-negative people. New insights must now explain Duffy-independent P. vivax invasion of human erythrocytes.

Methods/Principal Findings

Through recent whole genome sequencing we obtained ≥70× coverage of the P. vivax genome from five field-isolates, resulting in ≥93% of the Sal I reference sequenced at coverage greater than 20×. Combined with sequences from one additional Malagasy field isolate and from five monkey-adapted strains, we describe here identification of DNA sequence rearrangements in the P. vivax genome, including discovery of a duplication of the P. vivax Duffy binding protein (PvDBP) gene. A survey of Malagasy patients infected with P. vivax showed that the PvDBP duplication was present in numerous locations in Madagascar and found in over 50% of infected patients evaluated. Extended geographic surveys showed that the PvDBP duplication was detected frequently in vivax patients living in East Africa and in some residents of non-African P. vivax-endemic countries. Additionally, the PvDBP duplication was observed in travelers seeking treatment of vivax malaria upon returning home. PvDBP duplication prevalence was highest in west-central Madagascar sites where the highest frequencies of P. vivax-infected, Duffy-negative people were reported.

Conclusions/Significance

The highly conserved nature of the sequence involved in the PvDBP duplication suggests that it has occurred in a recent evolutionary time frame. These data suggest that PvDBP, a merozoite surface protein involved in red cell adhesion is rapidly evolving, possibly in response to constraints imposed by erythrocyte Duffy negativity in some human populations.     

Structural Analysis of the Synthetic Duffy Binding Protein (DBP) Antigen DEKnull Relevant for Plasmodium vivax Malaria Vaccine Design

The Plasmodium vivax vaccine candidate Duffy Binding Protein (DBP) is a protein necessary for P. vivax invasion of reticulocytes. The polymorphic nature of DBP induces strain-specific immune responses that pose unique challenges for vaccine development. DEKnull is a synthetic DBP based antigen that has been engineered through mutation to enhance induction of blocking inhibitory antibodies. We determined the x-ray crystal structure of DEKnull to identify if any conformational changes had occurred upon mutation. Computational and experimental analyses assessed immunogenicity differences between DBP and DEKnull epitopes. Functional binding assays with monoclonal antibodies were used to interrogate the available epitopes in DEKnull. We demonstrate that DEKnull is structurally similar to the parental Sal1 DBP. The DEKnull mutations do not cause peptide backbone shifts within the polymorphic loop, or at either the DBP dimerization interface or DARC receptor binding pockets, two important structurally conserved protective epitope motifs. All B-cell epitopes, except for the mutated DEK motif, are conserved between DEKnull and DBP. The DEKnull protein retains binding to conformationally dependent inhibitory antibodies. DEKnull is an iterative improvement of DBP as a vaccine candidate. DEKnull has reduced immunogenicity to polymorphic regions responsible for strain-specific immunity while retaining conserved protein folds necessary for induction of strain-transcending blocking inhibitory antibodies.     

Improvement in Yield and Purity of a Recombinant Malaria Vaccine Candidate Based on the Receptor-Binding Domain of Plasmodium vivax Duffy Binding Protein by Codon Optimization

A recombinant blood-stage vaccine for Plasmodium vivax malaria based on the functional receptor-binding domain of PvDBP (PvRII) has been developed. A synthetic gene coding for PvRII was expressed in Escherichia coli using codon optimization. Expression level of recombinant PvRII was 10% of the total cellular proteins. Truncated PvRII products, seen when the native PvRII gene was expressed, were absent in case of synthetic gene.     

Plasmodium simium,a Plasmodium vivax-Related Malaria Parasite: Genetic Variability of Duffy Binding Protein II and the Duffy Antigen/Receptor for Chemokines

Plasmodium simium is a parasite from New World monkeys that is most closely related to the human malaria parasite Plasmodium vivax; it also naturally infects humans. The blood-stage infection of P. vivax depends on Duffy binding protein II (PvDBPII) and its cognate receptor on erythrocytes, the Duffy antigen receptor for chemokines (hDARC), but there is no information on the P. simium erythrocytic invasion pathway. The genes encoding P. simium DBP (PsDBP_II) and simian DARC (sDARC) were sequenced from Southern brown howler monkeys (Alouatta guariba clamitans) naturally infected with P. simium because P. simium may also depend on the DBPII/DARC interaction. The sequences of DBP binding domains from P. vivax and P. simium were highly similar. However, the genetic variability of PsDBPII was lower than that of PvDBPII. Phylogenetic analyses demonstrated that these genes were strictly related and clustered in the same clade of the evolutionary tree. DARC from A. clamitans was also sequenced and contained three new non-synonymous substitutions. None of these substitutions were located in the N-terminal domain of DARC, which interacts directly with DBPII. The interaction between sDARC and PvDBPII was evaluated using a cytoadherence assay of COS7 cells expressing PvDBPII on their surfaces. Inhibitory binding assays in vitro demonstrated that antibodies from monkey sera blocked the interaction between COS-7 cells expressing PvDBPII and hDARC-positive erythrocytes. Taken together, phylogenetic analyses reinforced the hypothesis that the host switch from humans to monkeys may have occurred very recently in evolution, which sheds light on the evolutionary history of new world plasmodia. Further invasion studies would confirm whether P. simium depends on DBP/DARC to trigger internalization into red blood cells.     

Natural Selection Mediated Association of the Duffy (FY) Gene Polymorphisms with Plasmodium vivax Malaria in India

The Duffy (Fy) antigens act as receptors for chemokines as well as for Plasmodium vivax to invade human RBCs. A recent study has correlated the occurrence of the FY*A allele of Duffy gene with decreased susceptibility to vivax malaria, but no epidemiological correlation between the distribution of FY*A allele and incidences of vivax malaria has been established so far. Furthermore, if such correlations exist, whether natural selection has mediated the association, is an important question. Since India is highly endemic to P. vivax malaria with variable eco-climatic and varying vivax malaria epidemiology across different regions, such a question could well be answered in Indians. For this, we have genotyped the FY gene at the −33^rd and the 125^th nucleotide positions in 250 Indians sampled from six different zonal plus one tribal population covering the whole of India and studied possible correlations with eco-climatic and vivax malaria incidences. No FY*O allele was found, however, both the FY*A and FY*B alleles forming FY*A/FY*A, FY*A/FY*B and FY*B/FY*B genotypes were widely distributed among Indians. Five out of seven population samples significantly deviated from the Hardy-Weinberg equilibrium expectation, and two alleles (FY*A and FY*B) and the homozygote genotype, FY*B/FY*B were clinally distributed over the population coordinates. Furthermore, vivax malaria incidences over the past five years were significantly negatively and positively associated with the frequencies of the FY*A and FY*B alleles, respectively. The Northern Indians were highly differentiated from the other zonal population samples at the FY gene, as evidenced from the reconstructed Neighbor-Joining phylogenetic tree. The results specify the role of natural selection in the distribution of FY gene polymorphism in India. Furthermore, the hypotheses on the part of the FY*A allele in conferring protection to vivax malaria could be validated following population genetic studies in a vivax malaria epidemiological setting, such as India.     

Analysis of polymorphic regions of Plasmodium vivax Duffy binding protein of Korean isolates.

The present study was designed to investigate polymorphism in Duffy binding protein (DBP) gene of Plasmodium vivax isolates of Korea. Thirty samples were obtained from P. vivax patients in Yonchon-gun, Kyonggi-do in 1998. The PCR products of the samples were subjected to sequencing and hybridization analyses of the regions II and IV of P. vivax DBP gene. Two genotypes, SK-1 and SK-2, were identified on the basis of amino acid substitution and deletion. The genotype of 10 isolates was SK-1 and that of 20 isolates was SK-2. Most of the predicted amino acids in the region II of DBP gene were conserved between the Korean isolates and Belem strain except for 4-5 amino acid substitutions. In the region IV of DBP, a 6-bp insert that was shown in the Sal-1 allele type was found in SK-1, and a 27-bp insert that was shown in the Papua New Guinea allele type was found in SK-2. In conclusion, the present findings suggest that two genotypes of P. vivax coexist in the endemic area of Korea.     

Worldwide genetic variability of the Duffy binding protein: insights into Plasmodium vivax vaccine development

The dependence of Plasmodium vivax on invasion mediated by Duffy binding protein (DBP) makes this protein a prime candidate for development of a vaccine. However, the development of a DBP-based vaccine might be hampered by the high variability of the protein ligand (DBP(II)), known to bias the immune response toward a specific DBP variant. Here, the hypothesis being investigated is that the analysis of the worldwide DBP(II) sequences will allow us to determine the minimum number of haplotypes (MNH) to be included in a DBP-based vaccine of broad coverage. For that, all DBP(II) sequences available were compiled and MNH was based on the most frequent nonsynonymous single nucleotide polymorphisms, the majority mapped on B and T cell epitopes. A preliminary analysis of DBP(II) genetic diversity from eight malaria-endemic countries estimated that a number between two to six DBP haplotypes (17 in total) would target at least 50% of parasite population circulating in each endemic region. Aiming to avoid region-specific haplotypes, we next analyzed the MNH that broadly cover worldwide parasite population. The results demonstrated that seven haplotypes would be required to cover around 60% of DBP(II) sequences available. Trying to validate these selected haplotypes per country, we found that five out of the eight countries will be covered by the MNH (67% of parasite populations, range 48-84%). In addition, to identify related subgroups of DBP(II) sequences we used a Bayesian clustering algorithm. The algorithm grouped all DBP(II) sequences in six populations that were independent of geographic origin, with ancestral populations present in different proportions in each country. In conclusion, in this first attempt to undertake a global analysis about DBP(II) variability, the results suggest that the development of DBP-based vaccine should consider multi-haplotype strategies; otherwise a putative P. vivax vaccine may not target some parasite populations.     

Characterization of Plasmodium vivax Early Transcribed Membrane Protein 11.2 and Exported Protein 1

In Plasmodium, the membrane of intracellular parasites is initially formed during invasion as an invagination of the red blood cell surface, which forms a barrier between the parasite and infected red blood cells in asexual blood stage parasites. The membrane proteins of intracellular parasites of Plasmodium species have been identified such as early-transcribed membrane proteins (ETRAMPs) and exported proteins (EXPs). However, there is little or no information regarding the intracellular parasite membrane in Plasmodium vivax. In the present study, recombinant PvETRAMP11.2 (PVX_003565) and PvEXP1 (PVX_091700) were expressed and evaluated antigenicity tests using sera from P. vivax-infected patients. A large proportion of infected individuals presented with IgG antibody responses against PvETRAMP11.2 (76.8%) and PvEXP1 (69.6%). Both of the recombinant proteins elicited high antibody titers capable of recognizing parasites of vivax malaria patients. PvETRAMP11.2 partially co-localized with PvEXP1 on the intracellular membranes of immature schizont. Moreover, they were also detected at the apical organelles of newly formed merozoites of mature schizont. We first proposed that these proteins might be synthesized in the preceding schizont stage, localized on the parasite membranes and apical organelles of infected erythrocytes, and induced high IgG antibody responses in patients.     

Plasmodium vivax invasion of human erythrocytes inhibited by antibodies directed against the Duffy binding protein

Background

Plasmodium vivax invasion requires interaction between the human Duffy antigen on the surface of erythrocytes and the P. vivax Duffy binding protein (PvDBP) expressed by the parasite. Given that Duffy-negative individuals are resistant and that Duffy-negative heterozygotes show reduced susceptibility to blood-stage infection, we hypothesized that antibodies directed against region two of P. vivax Duffy binding protein (PvDBPII) would inhibit P. vivax invasion of human erythrocytes.

Methods and Findings

Using a recombinant region two of the P. vivax Duffy binding protein (rPvDBPII), polyclonal antibodies were generated from immunized rabbits and affinity purified from the pooled sera of 14 P. vivax–exposed Papua New Guineans. It was determined by ELISA and by flow cytometry, respectively, that both rabbit and human antibodies inhibited binding of rPvDBPII to the Duffy antigen N-terminal region and to Duffy-positive human erythrocytes. Additionally, using immunofluorescent microscopy, the antibodies were shown to attach to native PvDBP on the apical end of the P. vivax merozoite. In vitro invasion assays, using blood isolates from individuals in the Mae Sot district of Thailand, showed that addition of rabbit anti-PvDBPII Ab or serum (antibodies against, or serum containing antibodies against, region two of the Plasmodium vivax Duffy binding protein) (1:100) reduced the number of parasite invasions by up to 64%, while pooled PvDBPII antisera from P. vivax–exposed people reduced P. vivax invasion by up to 54%.

Conclusions

These results show, for what we believe to be the first time, that both rabbit and human antibodies directed against PvDBPII reduce invasion efficiency of wild P. vivax isolated from infected patients, and suggest that a PvDBP-based vaccine may reduce human blood-stage P. vivax infection.     

Sulphated tyrosines mediate association of chemokines and Plasmodium vivax Duffy binding protein with the Duffy antigen/receptor for chemokines (DARC)

Plasmodium vivax is one of four Plasmodium species that cause human malaria. P. vivax and a related simian malaria parasite, Plasmodium knowlesi, invade erythrocytes by binding the Duffy antigen/receptor for chemokines (DARC) through their respective Duffy binding proteins. Here we show that tyrosines 30 and 41 of DARC are modified by addition of sulphate groups, and that the sulphated tyrosine 41 is essential for association of the Duffy binding proteins of P. vivax (PvDBP) and P. knowlesi (PkDaBP) with DARC-expressing cells. These sulphated tyrosines also participate in the association of DARC with each of its four known chemokine ligands. Alteration of tyrosine 41 to phenylalanine interferes with MCP-1, RANTES and MGSA association with DARC, but not with that of IL8. In contrast, alteration of tyrosine 30 to phenylalanine interferes with the association of IL8 with DARC. A soluble sulphated amino-terminal domain of DARC, but not one modified to phenylalanine at residue 41, can be used to block the association of PvDBP and PkDaBP with red blood cells, with an IC50 of approximately 5 nM. These data are consistent with a role for tyrosine sulphation in the association of many or most chemokines with their receptors, and identify a key molecular determinant of erythrocyte invasion by P. vivax.     

Identification of Immunodominant B-cell Epitope Regions of Reticulocyte Binding Proteins in Plasmodium vivax by Protein Microarray Based Immunoscreening

Plasmodium falciparum can invade all stages of red blood cells, while Plasmodium vivax can invade only reticulocytes. Although many P. vivax proteins have been discovered, their functions are largely unknown. Among them, P. vivax reticulocyte binding proteins (PvRBP1 and PvRBP2) recognize and bind to reticulocytes. Both proteins possess a C-terminal hydrophobic transmembrane domain, which drives adhesion to reticulocytes. PvRBP1 and PvRBP2 are large (> 326 kDa), which hinders identification of the functional domains. In this study, the complete genome information of the P. vivax RBP family was thoroughly analyzed using a prediction server with bioinformatics data to predict B-cell epitope domains. Eleven pvrbp family genes that included 2 pseudogenes and 9 full or partial length genes were selected and used to express recombinant proteins in a wheat germ cell-free system. The expressed proteins were used to evaluate the humoral immune response with vivax malaria patients and healthy individual serum samples by protein microarray. The recombinant fragments of 9 PvRBP proteins were successfully expressed; the soluble proteins ranged in molecular weight from 16 to 34 kDa. Evaluation of the humoral immune response to each recombinant PvRBP protein indicated a high antigenicity, with 38-88% sensitivity and 100% specificity. Of them, N-terminal parts of PvRBP2c (PVX_090325-1) and PvRBP2 like partial A (PVX_090330-1) elicited high antigenicity. In addition, the PvRBP2-like homologue B (PVX_116930) fragment was newly identified as high antigenicity and may be exploited as a potential antigenic candidate among the PvRBP family. The functional activity of the PvRBP family on merozoite invasion remains unknown.     

The Origin of Trypsin: Evidence for Multiple Gene Duplications in Trypsins

The trypsin family of serine proteases is one of the most studied protein families, with a wealth of amino acid sequence information available in public databases. Since trypsin-like enzymes are widely distributed in living organisms in nature, likely evolutionary scenarios have been proposed. A novel methodology for Fourier transformation of biological sequences (FOTOBIS) is presented. The methodology is well suited for the identification of the size and extent of short repeats in protein sequences. In the present paper the trypsin family of enzymes is analyzed with FOTOBIS and strong evidence for tandem gene duplication is found. A likely evolutionary path for the development of present-day trypsins involved an intrinsic extensive tandem gene duplication of a small DNA fragment of 15–18 nucleotides, corresponding to five or six amino acids. This ancestral trypsin gene was subsequently duplicated, leading to the earliest version of a full-sized trypsin, from which the contemporary trypsins have developed. Received: 22 November 1997 / Accepted: 26 January 1998     

Defining the Erythrocyte Binding Domains of Plasmodium vivax Tryptophan Rich Antigen 33.5

Tryptophan-rich antigens play important role in host-parasite interaction. One of the Plasmodium vivax tryptophan-rich antigens called PvTRAg33.5 had earlier been shown to be predominantly of alpha helical in nature with multidomain structure, induced immune responses in humans, binds to host erythrocytes, and its sequence is highly conserved in the parasite population. In the present study, we divided this protein into three different parts i.e. N-terminal (amino acid position 24–106), middle (amino acid position 107–192), and C-terminal region (amino acid position 185–275) and determined the erythrocyte binding activity of these fragments. This binding activity was retained by the middle and C-terminal fragments covering 107 to 275 amino acid region of the PvTRAg33.5 protein. Eight non-overlapping peptides covering this 107 to 275 amino acid region were then synthesized and tested for their erythrocyte binding activity to further define the binding domains. Only two peptides, peptide P4 (at 171–191 amino acid position) and peptide P8 (at 255–275 amino acid position), were found to contain the erythrocyte binding activity. Competition assay revealed that each peptide recognizes its own erythrocyte receptor. These two peptides were found to be located on two parallel helices at one end of the protein in the modelled structure and could be exposed on its surface to form a suitable site for protein-protein interaction. Natural antibodies present in the sera of the P. vivax exposed individuals or the polyclonal rabbit antibodies against this protein were able to inhibit the erythrocyte binding activity of PvTRAg33.5, its fragments, and these two synthetic peptides P4 and P8. Further studies on receptor-ligand interaction might lead to the development of the therapeutic reagent.     

Plasmodium vivax interaction with the human Duffy blood group glycoprotein: identification of a parasite receptor-like protein

The interaction between merozoites of the human pathogen, Plasmodium vivax, and the Duffy blood group glycoprotein on the surface of human erythrocytes is essential for the invasion of erythrocytes and the survival of the parasite. We have identified a P. vivax protein of 135 to 140 kDa which binds with receptor-like specificity to the human Duffy blood group glycoprotein. This interaction can be specifically inhibited by purified Duffy glycoprotein and by pretreating erythrocytes with a monoclonal antibody directed against a novel Duffy determinant. A protein with similar specificity for the Duffy glycoprotein from the phylogenetically related simian malaria, P. knowlesi, is shown to be immunologically related by the generation of cross-reactive antibodies. Despite their shared properties, these two Duffy associating proteins from P. vivax and P. knowlesi differ in some aspects of their interaction with the Duffy glycoprotein. The identification of these proteins will help elucidate the molecular mechanisms of erythrocyte invasion by Plasmodium.     

The association of Duffy binding protein region II polymorphisms and its antigenicity in Plasmodium vivax isolates from Thailand

Plasmodium vivax Duffy binding protein II (DBPII) plays an important role in reticulocyte invasion and is a potential vaccine candidate against vivax malaria. However, polymorphisms in DBPII are a challenge for the successful design of a broadly protective vaccine. In this study, the genetic diversity of DBPII among Thai isolates was analyzed from Plasmodium vivax-infected blood samples and polymorphism characters were defined with the MEGA4 program. Sequence analysis identified 12 variant residues that are common among Thai DBPII haplotypes with variant residues L333F, L424I, W437R and I503K having the highest frequency. Variant residue D384K occurs in combination with either E385K or K386N/Q. Additionally, variant residue L424I occurs in conjunction with W437R in most Thai DBPII alleles and these variants frequently occur in combination with the I503K variant. The polymorphic patterns of Thai isolates were defined into 9 haplotypes (Thai DBL-1, -2, -3, etc.…). Thai DBL-2, -5, -6 haplotypes are the most common DBPII variants in Thai residents. To study the association of these Thai DBPII polymorphisms with antigenic character, the functional inhibition of anti-DBPII monoclonal antibodies against a panel of Thai DBL variants was characterized by an in vitro erythrocyte binding inhibition assay. The functional inhibition of anti-DBPII monoclonal antibodies 3C9, 2D10 and 2C6 against Thai variants was significantly different, suggesting that polymorphisms of Thai DBPII variants alter the antigenic character of the target epitopes. In contrast, anti-DBPII monoclonal antibody 2H2 inhibited all Thai DBPII variants equally well. Our results suggest that the immune efficacy of a DBPII vaccine will depend on the specificity of the anti-DBPII antibodies induced and that it is preferable to optimize responses to conserved epitopes for broadly neutralizing protection against P. vivax.     

Purification and characterization of an erythrocyte membrane protein complex carrying Duffy blood group antigenicity. Possible receptor for Plasmodium vivax and Plasmodium knowlesi malaria parasite

A murine monoclonal antibody, named anti-Fy6, which agglutinates all human red cells except those of Fy(a-b) phenotype was used for purification and characterization of Duffy antigens. Duffy antigens are multimeric red cell membrane proteins composed of different subunits of which only one, designated pD protein, reacts in immunoblots with the murine monoclonal antibody anti-Fy6. Affinity-purified detergent-soluble antigen-antibody complex obtained from red cells, surface-labeled with 125I yielded a complex pattern of bands when separated by polyacrylamide gel electrophoresis. Proteins that react with anti-Fy6 in immunoblots are: pA and pB (greater than 100 kDa) and pD (36-46 kDa). Electroeluted pD protein aggregates and generates bands of similar molecular mass to pA and pB proteins. Electroeluted pA and pB proteins disaggregate yielding pD protein. Oligomers and monomers of pD protein are present in red cells carrying Duffy antigens and absent in Fy(a-b-) cells. Six other proteins of molecular weight ranging from 68 to 21 kDa either associate or co-purify with pD protein. These proteins are only present in Duffy antigen positive cells. The pD protein is different in Fy(a+b-) and Fy(a-b+) cells by fingerprint analysis. Human antisera identify the same proteins in red cell carrying Duffy antigens as the murine monoclonal antibody anti-Fy6.     

Relationship between Antibody-Positive Rate against Plasmodium vivax Circumsporozoite Protein and Incidence of Malaria

The relationship between anti-Plasmodium vivax circumsporozoite protein (CSP) antibody levels and the prevalence of malaria in epidemic areas of South Korea was evaluated. Blood samples were collected from inhabitants of Gimpo-si (city), Paju-si, and Yeoncheon-gun (county) in Gyeonggi-do (province), as well as Cheorwon-gun in Gangwon-do from November to December 2004. Microscopic examinations were used to identify malaria parasites. ELISA was used to quantitate anti-circumsporozoite protein (CSP) antibodies against P. vivax. A total of 1,774 blood samples were collected. The overall CSP-ELISA-positive rate was 7.7% (n=139). The annual parasite incidences (APIs) in these areas gradually decreased from 2004 to 2005 (1.09 and 0.80, respectively). The positive rate in Gimpo (10.4%, 44/425) was the highest identified by CSP-ELISA. The highest API was found in Yeoncheon, followed by Cheorwon, Paju, and Gimpo in both years. The positive rates of CSP-ELISA were closely related to the APIs in the study areas. These results suggest that seroepidemiological studies based on CSP may be helpful in estimating the malaria prevalence in certain areas. In addition, this assay can be used to establish and evaluate malaria control and eradication programs in affected areas.     

Distinct Binding and Immunogenic Properties of the Gonococcal Homologue of Meningococcal Factor H Binding Protein

Neisseria meningitidis is a leading cause of sepsis and meningitis. The bacterium recruits factor H (fH), a negative regulator of the complement system, to its surface via fH binding protein (fHbp), providing a mechanism to avoid complement-mediated killing. fHbp is an important antigen that elicits protective immunity against the meningococcus and has been divided into three different variant groups, V1, V2 and V3, or families A and B. However, immunisation with fHbp V1 does not result in cross-protection against V2 and V3 and vice versa. Furthermore, high affinity binding of fH could impair immune responses against fHbp. Here, we investigate a homologue of fHbp in Neisseria gonorrhoeae, designated as Gonococcal homologue of fHbp (Ghfp) which we show is a promising vaccine candidate for N. meningitidis. We demonstrate that Gfhp is not expressed on the surface of the gonococcus and, despite its high level of identity with fHbp, does not bind fH. Substitution of only two amino acids in Ghfp is sufficient to confer fH binding, while the corresponding residues in V3 fHbp are essential for high affinity fH binding. Furthermore, immune responses against Ghfp recognise V1, V2 and V3 fHbps expressed by a range of clinical isolates, and have serum bactericidal activity against N. meningitidis expressing fHbps from all variant groups.     

Prevalence of Drug Resistance-Associated Gene Mutations in Plasmodium vivax in Central China

Resistance of Plasmodium spp. to anti-malarial drugs is the primary obstacle in the fight against malaria, and molecular markers for the drug resistance have been applied as an adjunct in the surveillance of the resistance. In this study, we investigated the prevalence of mutations in pvmdr1, pvcrt-o, pvdhfr, and pvdhps genes in temperate-zone P. vivax parasites from central China. A total of 26 isolates were selected, including 8 which were previously shown to have a lower susceptibility to chloroquine in vitro. For pvmdr1, pvcrt-o, and pvdhps genes, no resistance-conferring mutations were discovered. However, a highly prevalent (69.2%), single-point mutation (S117N) was found in pvdhfr gene. In addition, tandem repeat polymorphisms existed in pvdhfr and pvdhps genes, which warranted further studies in relation to the parasite resistance to antifolate drugs. The study further suggests that P. vivax populations in central China may still be relatively susceptible to chloroquine and sulfadoxine-pyrimethamine.