Mice immunized with a synthetic peptide construct corresponding to an epitope present on a Plasmodium falciparum antigen are protected against Plasmodium chabaudi challenge

Inhibitory monoclonal antibody (MoAb) 8E7/55 recognizes a parasitophorous vacuole membrane (PVM) antigen in Plasmodium falciparum. Previous studies have identified the epitope, DNNLVSGP, recognized by the MoAb. A synthetic peptide containing this sequence was synthesized and coupled to diphtheria toxoid (DT) and was found capable of generating antibodies when used as an immunogen in mice which recognize the native antigen exp-1. In this study we demonstrate the ability of the MoAb and antisera generated against the peptide construct to recognize a 54 kD PVM antigen in Plasmodium chabaudi. The P. chabaudi antigen is synthesized in trophozoites and released to the surrounding culture media outside the parasitized erythrocyte. Mice immunized with the peptide conjugate are protected when challenged with a lethal strain of P. chabaudi. Protection in the mice correlated with the antibody titre prior to challenge. If the PVM antigen from P. chabaudi is a homologue of exp-1 from P. falciparum, then these experiments may provide a guide to the antibody titres required in human trials before antibody mediated protection could be expected. The discovery that a PVM localized antigen is secreted into the surrounding in vitro culture media provides us with a valuable model system for further investigation of protein trafficking pathways in malaria-infected erythrocytes.     

Asparagine repeat function in a Plasmodium falciparum protein assessed via a regulatable fluorescent affinity tag

One in four proteins in Plasmodium falciparum contains asparagine repeats. We probed the function of one such 28-residue asparagine repeat present in the P. falciparum proteasome lid subunit 6, Rpn6. To aid our efforts, we developed a regulatable, fluorescent affinity (RFA) tag that allows cellular localization, manipulation of cellular levels, and affinity isolation of a chosen protein in P. falciparum. The tag comprises a degradation domain derived from Escherichia coli dihydrofolate reductase together with GFP. The expression of RFA-tagged proteins is regulated by the simple folate analog trimethoprim (TMP). Parasite lines were generated in which full-length Rpn6 and an asparagine repeat-deletion mutant of Rpn6 were fused to the RFA tag. The knockdown of Rpn6 upon removal of TMP revealed that this protein is essential for ubiquitinated protein degradation and for parasite survival, but the asparagine repeat is dispensable for protein expression, stability, and function. The data point to a genomic mechanism for repeat perpetuation rather than a positive cellular role. The RFA tag should facilitate study of the role of essential genes in parasite biology.     

An antigen specific to the liver stage of rodent malaria recognized by a monoclonal antibody

Summary Vaccines currently being evaluated against malaria are based on proteins derived from the blood, sporozoite and sexual stages. Antigens from the liver stage, which is now recognized as the major target of protective sporozoite induced immunity, have received comparatively little attention. This paper describes the generation of a monoclonal antibody (MoAb), which recognizes an antigen specific to the liver stage of the rodent malaria Plasmodium berghei. The antigen is expressed throughout liver stage development and appears to be localized to the parasitophorous vacuole membrane. The MoAb did not affect the growth of liver stages cultured in vitro nor could protection be demonstrated in vivo following passive transfer of the antibody.     

Protective immune response to Plasmodium chabaudi, developed by mice after drug controlled infection or vaccination with parasite extracts: analysis of stage specific antigens from the asexual blood cycle

Summary The protective immune response to asexual blood infection by Plasmodium chabaudi was studied in mice immunized either by drug controlled infection or by vaccination with preparations of merozoïtes or free parasites at different stages of development. Animals immunized by the first method developed a sterile immunity. The passive transfer of their serum protected naive recipients from the lethal development of the infection, but affected only moderately the initial course of the parastiaemia. Animals immunized with either ring, schizont or merozoi'te preparations exhibited a limited but significant resistance to infection: when challenged with 10⁶ parasites of the homologous strain they exhibited a reduced parasitaemia as compared to control mice, and in addition, 50% of them recovered from the infection. Immunochemical analysis of parasite antigens showed that a family of high molecular weight proteins synthesized essentially at the schizont stage and conserved in the merozoites are important immunogens. Quantitative rather than qualitative differences were observed in the pattern of parasite proteins immunoprecipitated by serum of animals exhibiting sterile immunity or moderate protective immunity. A schizont specific polypeptide of mol. wt 82 Kd which is found in the surface of the merozoite is preferentially immunoprecipited by serum from animals exhibiting sterile immunity.     

Torins are potent antimalarials that block replenishment of Plasmodium liver stage parasitophorous vacuole membrane proteins

Residence within a customized vacuole is a highly successful strategy used by diverse intracellular microorganisms. The parasitophorous vacuole membrane (PVM) is the critical interface between Plasmodium parasites and their possibly hostile, yet ultimately sustaining, host cell environment. We show that torins, developed as ATP-competitive mammalian target of rapamycin (mTOR) kinase inhibitors, are fast-acting antiplasmodial compounds that unexpectedly target the parasite directly, blocking the dynamic trafficking of the Plasmodium proteins exported protein 1 (EXP1) and upregulated in sporozoites 4 (UIS4) to the liver stage PVM and leading to efficient parasite elimination by the hepatocyte. Torin2 has single-digit, or lower, nanomolar potency in both liver and blood stages of infection in vitro and is likewise effective against both stages in vivo, with a single oral dose sufficient to clear liver stage infection. Parasite elimination and perturbed trafficking of liver stage PVM-resident proteins are both specific aspects of torin-mediated Plasmodium liver stage inhibition, indicating that torins have a distinct mode of action compared with currently used antimalarials.The population at risk for developing malaria is vast, comprising some 3.3 billion people particularly in sub-Saharan Africa and Southeast Asia, with mortality estimates ranging from 655,000 to 1,200,000 (1). Widespread resistance has limited the therapeutic utility of most existing antimalarial drugs, and artemisinin, the highly efficacious cornerstone of artemisinin combination therapies, appears to be at risk for the same fate (2). The need for new antimalarial chemotherapeutic strategies is thus acute.Plasmodium spp., the causative agents of malaria, have a complex life cycle with alternating motile-nonreplicative and sessile-replicative forms in both mammal and mosquito. In the mammalian host, Plasmodium invades and replicates inside two very distinct cell types: hepatocytes and red blood cells (RBCs). In mammals, the Plasmodium life cycle is initiated by a motile sporozoite that invades a hepatocyte, where it resides for 2–14 d, multiplying into >10,000 merozoites in a single cycle (3). Once released into the bloodstream, each of these motile merozoites will infect an RBC and, within 1–3 d, generate 10–30 new merozoites, which will contribute to the continuous cycle of blood stage infection that causes the symptoms, morbidity, and mortality of malaria.These two stages of mammalian infection, despite taking place in distinct cell types and having an orders-of-magnitude difference in parasite replication, do share common features. In both, the motile “zoite” invades the host cell through formation of a parasitophorous vacuole (PV). Both stages grow and replicate exclusively within the confines of the PV, and the parasitophorous vacuole membrane (PVM), which is populated with parasite proteins, constitutes the physical host–parasite interface throughout development. Unlike the vacuoles of many intracellular pathogens including Leishmania, Chlamydia, Mycobacteria, and Legionella (4, 5), the Plasmodium vacuole, like that of Toxoplasma gondii, does not fuse with host lysosomes and is not acidified (6). This is not unsurprising in the context of Plasmodium development in an RBC, which lacks endomembrane system trafficking and, indeed, lysosomes. The highly polarized hepatocyte, however, has extensive vesicular transport networks (7) and can target intracellular pathogens residing in a vacuole (8), suggesting that the exoerythrocytic form (EEF) may need to resist host cell attack.Although the PVM is thought to be critical for Plasmodium growth in both the hepatocyte and the RBC contexts, its cellular roles remain elusive. The importance of several Plasmodium PVM-resident proteins, however, has been conclusively demonstrated in both blood and liver stages. Attempts to generate exported (exp)1 and Plasmodium translocon of exported protein (ptex)150 knockout parasites in Plasmodium falciparum failed (9, 10), revealing that these are both essential proteins for the blood stage, whereas Plasmodium berghei and Plasmodium yoelii mutants lacking up-regulated in sporozoites (uis)3 or uis4 fail to complete liver stage development (11, 12). These PVM-resident proteins, and thus the PVM itself, are performing functions that are crucial for Plasmodium growth, but delineating the functions of individual PVM-resident proteins has proven as difficult as identifying the cellular processes mediated by the PVM.The one process in which both the centrality of the PVM is known and evidence for the participation of specific PVM proteins exists is the export of parasite proteins to the RBC. A cohort of parasite proteins that are involved in extensive physiological and structural modifications of the infected RBC (iRBC) is exported into the iRBC cytoplasm and beyond (13). Five proteins have been identified as components of PTEX, the proposed export machinery at the iRBC PVM (9). Although liver stage protein export has been shown for the Circumsporozite (CS) protein (14) and PTEX components are expressed in P. falciparum EEFs (15), a role for parasite protein export into the hepatocyte remains speculative; the host hepatocyte may not require the extensive structural remodeling that the iRBC does.Conversely, however, the hepatocyte, with its extensive vesicular transport network, intuitively constitutes a more hostile host environment than the RBC, and there is evidence that the liver stage PVM may play a crucial role in preventing host cell-mediated parasite killing, as it does in Toxoplasma gondii (16). Support for a protective role for the liver stage PVM comes from knockout parasites that fail in the earliest steps of PVM formation and remodeling. Sporozoites lacking the p52/p36 gene pair invade hepatocytes successfully, but fail in PVM formation (17, 18) and are severely reduced in abundance midway through liver stage development. Parasites lacking slarp/sap (19, 20), a regulator of early liver stage development, fail to express UIS4 and exported protein 1 (EXP-1), along with other parasite proteins, and are also eliminated at the beginning of infection.Acquisition of resources from the host-cell environment, an unambiguous requirement for an obligate intracellular parasite like Plasmodium, is a function ascribed to the PVM in both mammalian stages. The PVM allows the free passage of molecules (21, 22), presumably through proteinaceous pores, which may contribute to acquisition of host nutrients and disposal of parasite waste products. Members of the early transcribed membrane protein (ETRAMP) family, single-pass transmembrane proteins conserved among Plasmodium spp., which are highly expressed and developmentally regulated in both blood and liver stage parasites (23, 24), could be candidates for mediating uptake of host resources. Such a role in lipid uptake has indeed been proposed for the P. berghei ETRAMP UIS3 on the basis of its interaction with host-cell L-FABP (liver fatty acid binding protein) (25).Although Plasmodium parasites must use host resources to support their own growth in both mammalian stages, the single cycle replicative output of the liver stage parasite is vastly greater than that of the blood stage, which may reflect a similarly increased need for host resources. In this respect, the hepatocyte constitutes far superior “raw material” compared with the RBC; hepatocytes are not only metabolically active, but also highly versatile cells, which are capable of altering uptake, storage, production, and degradation of a wide array of macromolecules in response to cellular and organismal requirements. The presence of a growing Plasmodium parasite is sensed by the host hepatocyte, which responds with activation of cellular stress responses and altered metabolism (26, 27). The mammalian target of rapamycin (mTOR) kinase integrates signals from amino acids, stress, oxygen, energy, and growth factors and responds by altering cellular protein and lipid synthesis, as well as autophagy (28). As such, we sought to determine how inhibition of host mTOR signaling would affect Plasmodium liver stage development. Here we show that torins, a single structural class of mTOR inhibitors, are highly potent antiplasmodial compounds targeting both mammalian stages in vitro and in vivo. Independent of host-cell mTOR, torins impair trafficking of Plasmodium liver stage PVM-resident proteins, revealing the fast turnover of these proteins at the liver stage PVM, and provoke elimination of liver stage parasites.     

Molecular analysis of chloroquine resistance in Plasmodium falciparum in Yunnan Province, China

Resistance of Plasmodium falciparum to chloroquine (CQ) is determined by the mutation at K76T of the P. falciparum chloroquine resistance transporter (pfcrt) gene and modified by other mutations in this gene and in the P. falciparum multidrug resistance 1 (pfmdr1) gene. To determine the extent of polymorphisms in these genes in field P. falciparum isolates from Yunnan province of China, we genotyped the pfcrt codon 76, pfmdr1 codons 86 and 1246. Our results showed that although CQ has been withdrawn from treating falciparum malaria for over two decades, 90.3% of the parasites still carried the pfcrt K76T mutation. In contrast, mutations at pfmdr1 codons 86 and 1246 were rare. Sequencing analysis of the pfcrt gene in 34 parasite field isolates revealed CVIET at positions 72-76 as the major type, consistent with the theory of Southeast Asian origin of CQ resistance in the parasite. In addition, two novel pfcrt haplotypes (75D/144Y/220A and 75E/144Y/220A) were identified. Real-time polymerase chain reaction was used to determine pfmdr1 gene amplification, which is associated with mefloquine resistance. Our result indicated that in agreement with that mefloquine has not been used in this area, most (>90%) of the parasites had one pfmdr1 copy. Genotyping at two hypervariable loci showed relatively low levels of genetic diversity of the parasite population. Meanwhile, 28.4% of cases were found to contain mixed clones, which favour genetic recombination. Furthermore, despite a unique history of antimalarial drugs in Yunnan, its geographical connections with three malarious countries facilitate gene flow among parasite populations and evolution of novel drug-resistant genotypes. Therefore, continuous surveillance of drug resistance in this area is necessary for timely adjustment of local drug policies and more effective malaria control.     

Positive selection on the Plasmodium falciparum clag2 gene encoding a component of the erythrocyte-binding rhoptry protein complex

A protein complex of high-molecular-mass proteins (PfRhopH) of the human malaria parasite Plasmodium falciparum induces host protective immunity and therefore is a candidate for vaccine development. Clarification of the level of polymorphism and the evolutionary processes is important both for vaccine design and for a better understanding of the evolution of cell invasion in this parasite. In a previous study on 5 genes encoding RhopH1/Clag proteins, positive diversifying selection was detected in clag8 and clag9 but not in the paralogous clag2, clag3.1 and clag3.2. In this study, to extend the analysis of clag polymorphism, we obtained sequences surrounding the most polymorphic regions of clag2, clag8, and clag9 from parasites collected in Thailand. Using sequence data obtained newly in this study and reported previously, we classified clag2 sequences into 5 groups based on the similarity of the deduced amino acid sequences and number of insertions/deletions. By the sliding window method, an excess of nonsynonymous substitutions over synonymous substitutions was detected in the group 1 and group 2 clag2 and clag8 sequences. Population-based analyses also detected a significant departure from the neutral expectation for group 1 clag2 and clag8. Thus, two independent approaches suggest that clag2 is subject to a positive diversifying selection. The previously suggested positive selection on clag8 was also supported by population-based analyses. However, the positive selection on clag9, which was detected by comparing the 5 sequences, was not detected using the additional 34 sequences obtained in this study.     

The distinctive role of membrane fibrinogen-like protein 2 in the liver stage of rodent malaria infections

Viral infection often induce the expression of murine fibrinogen-like protein 2 (mFGL2) triggering immune coagulation, which causes severe liver pathogenesis via increased fibrin deposition and thrombosis in the microvasculature. We aimed to investigate the role of mFGL2 in the liver stage of malaria infections. We reveal that infection with malaria sporozoites also induces increased expression of mFGL2 and that this expression is primarily located within the liver Kupffer and endothelial cells. In addition, we report that inhibition of FGL2 has no significant effect on immune coagulation but increases the expression of inflammatory cytokines in the livers of infected mice. Interestingly, FGL2 deficiency had no significant impact on the development of liver stage malaria parasites or the pathogenesis of the infected liver. In contrast to viral infections, we conclude that mFGL2 does not contribute to either parasite development or liver pathology during these infections, revealing the unique features of this protein in liver-stage malaria infections.     

Coordinate estrogen induction of vitellogenin and a small serum protein mRNA in Xenopus laevis liver

We have used plus-minus hybridization to identify Xenopus liver cDNA clones of mRNAs whose levels are regulated by estrogen. One clone identified in this way was shown to be a nearly full-length cDNA clone of the mRNA coding for a small 22 000 dalton estrogen-inducible serum protein (EISP). Quantitation of EISP mRNA levels by in vitro translation and by hybridization to the cloned DNA demonstrated a 7-12-fold estrogen induction of EISP mRNA, both in vivo and in primary Xenopus liver cultures. The kinetics of induction of EISP mRNA closely parallel those of the mRNA coding for the abundant estrogen-inducible serum protein, vitellogenin. In contrast, the massive, and toxic, estrogen-mediated accumulation of vitellogenin in serum of male Xenopus laevis is accompanied by a sharp decline in the levels of albumin mRNA and in the levels of the mRNAs coding for several other serum proteins.     

Opsonization by antigen-specific antibodies as a mechanism of protective immunity induced by Plasmodium falciparum circumsporozoite protein-based vaccine

Recently conducted trials involving the Plasmodium falciparum circumsporozoite (CS) protein-based RTS,S malaria vaccine yielded unprecedented protection against a challenge with infectious sporozoites (spzs). The RTS,S vaccine induced high titres of CS protein-specific antibodies (Abs) in many of the protected volunteers, but the contribution of these Abs to protection remains unknown. Because opsonization by Ab promotes the uptake and destruction of spzs by monocytes and macrophages in both rodent and primate malaria, we asked if the RTS,S-induced Abs have antigen-specific opsonizing activity. Screening plasma from a large number of subjects using spzs was impractical, therefore we developed an alternative assay based on cytofluorometry that allowed the detection of fluoresceinated-Ag-Ab complexes endocytosed by the FcR+ THP-1 human monocyte line. The results showed that plasma samples from RTS,S-immunized subjects contained opsonizing CS protein-specific Abs and the endocytic activity of these Abs in protected subjects was significantly higher than in subjects who were susceptible to infection with spzs. We also demonstrated by electron microscopy that live spzs exposed to RTS,S-immune plasma could be internalized by the THP-1 cells. These results suggest that opsonization by CS protein-specific Abs might be one of the mechanisms that contributes to RTS,S-induced protective immunity.     

Expression of Plasmodium falciparum G6PD-6PGL in laboratory parasites and in patient isolates in G6PD-deficient and normal Nigerian children

As the production of NADPH in the pentose phosphate pathway is the main antioxidant defence mechanism available to the Plasmodium falciparum, we have studied the expression of P. falciparum glucose 6-phosphate dehydrogenase-6-phosphogluconolactonase (PfG6PD-6PGL) in G6PD-deficient and normal erythrocyte host cells. Both erythrocytes infected in vitro with a laboratory isolate and erythrocytes from natural human infections were used. Total RNA was prepared from parasites collected from five G6PD-deficient and nine G6PD-normal children in Ibadan, Nigeria, selected after screening 189 rural schoolchildren and 68 clinical malaria patients, and was subjected to Northern blot analysis. The probe was a cDNA fragment of the G6PD domain of the PfG6PD-6PGL gene, with an internal control probe of P. falciparum 18S ribosomal RNA. Quantification was performed using a phosphoimager. Relative to internal control, the abundance of PfG6PD-6PGL mRNA (mean +/- standard deviation) was lower in parasites from G6PD-deficient children (0.29 +/- 0.27) than in G6PD-normal control subjects (0.74 +/- 0.26) (P = 0.014, Mann-Whitney U-test). Although confirmation in a larger study is required, our results suggest a lower relative abundance of PfG6PD-6PGL, and presumably antioxidant activity, in malaria parasites from G6PD-deficient hosts, thus extending the current knowledge of the mechanism of G6PD-deficiency related host protection.     

用无细胞蛋白合成系统重组表达恶性疟原虫蛋白的研究

目的探索无细胞麦芽体外蛋白合成系统重组表达恶性疟原虫蛋白可行性,并检测用重组蛋白制备的免疫血清对原虫蛋白的特异性反应。方法将目的蛋白PfRON2的部分片断克隆连接到重组表达载体后,用无细胞麦芽体外蛋白合成系统进行重组表达并纯化,用纯化的重组蛋白免疫小鼠制备免疫血清,并用免疫斑点实验(Western blot)和免疫荧光抗体实验(IFA)检测该血清对恶性疟蛋白的特异性反应。结果成功克隆的重组质粒能在无细胞麦芽体外蛋白合成系统中生成大小相符的GST融合蛋白,并能较好地纯化。免疫斑点实验中,用该重组蛋白制备的免疫血清能检测到重组蛋白和恶性疟目标蛋白,免疫荧光抗体实验显示该免疫血清能成功地标记恶性疟目标蛋白所在部位。结论无细胞麦芽体外蛋白合成系统可应用于恶性疟原虫蛋白的重组表达,获得的免疫血清能特异性识别疟原虫蛋白。     

细粒棘球绦虫TSP11基因在不同发育期的差异表达及生物信息学分析

目的研究细粒棘球绦虫(Echinococcus granulosus,Eg)TSP基因家族TSP11基因的分子特性及其在虫体不同发育期的差异表达,为细粒棘球绦虫疫苗的研发奠定基础。方法从NCBI GenBank数据库中获得TSP11基因序列并设计特异性引物,以Eg原头节RNA为模板进行RT-PCR。将PCR产物克隆到pMD19-T载体后测序,通过生物信息学软件分析预测TSP11基因编码蛋白的结构与功能;通过SYBR GreenⅠqRT-PCR检测TSP11基因在虫体原头蚴以及成虫mRNA相对转录情况。结果生物信息学分析TSP11基因全长765个核苷酸,编码蛋白含254个氨基酸,理论等电点为8.91,为稳定蛋白分子。编码TSP11基因的氨基酸序列共含有3个跨膜区域,推测含有7个优势B抗原表位,与已登录的细粒棘球绦虫TSP11序列(XP024352489.1)同源性为99.61%。qRT-PCR显示TSP11基因在原头蚴及成虫阶段均有表达,且表达水平差异无统计学义(P>0.05)。结论成功克隆了细粒棘球绦虫TSP11基因,该基因在Eg原头蚴及成虫期均有表达,其编码蛋白为稳点蛋白,含有B细胞抗原表位,为进一步揭示其分子生物学功能奠定了基础。     

Structural basis for discriminatory recognition of Plasmodium lactate dehydrogenase by a DNA aptamer

DNA aptamers have significant potential as diagnostic and therapeutic agents, but the paucity of DNA aptamer-target structures limits understanding of their molecular binding mechanisms. Here, we report a distorted hairpin structure of a DNA aptamer in complex with an important diagnostic target for malaria: Plasmodium falciparum lactate dehydrogenase (PfLDH). Aptamers selected from a DNA library were highly specific and discriminatory for Plasmodium as opposed to human lactate dehydrogenase because of a counterselection strategy used during selection. Isothermal titration calorimetry revealed aptamer binding to PfLDH with a dissociation constant of 42 nM and 2:1 protein:aptamer molar stoichiometry. Dissociation constants derived from electrophoretic mobility shift assays and surface plasmon resonance experiments were consistent. The aptamer:protein complex crystal structure was solved at 2.1-Å resolution, revealing two aptamers bind per PfLDH tetramer. The aptamers showed a unique distorted hairpin structure in complex with PfLDH, displaying a Watson–Crick base-paired stem together with two distinct loops each with one base flipped out by specific interactions with PfLDH. Aptamer binding specificity is dictated by extensive interactions of one of the aptamer loops with a PfLDH loop that is absent in human lactate dehydrogenase. We conjugated the aptamer to gold nanoparticles and demonstrated specificity of colorimetric detection of PfLDH over human lactate dehydrogenase. This unique distorted hairpin aptamer complex provides a perspective on aptamer-mediated molecular recognition and may guide rational design of better aptamers for malaria diagnostics.Aptamers are artificially selected oligonucleotides that bind to molecular targets, typically proteins, with high specificity and avidity (1–3). DNA aptamers have been selected against dozens of targets for biomedical applications both as therapeutics (4, 5) and diagnostics (6, 7). Despite their widespread application, few DNA aptamer-target complex structures have been solved (8)–the best studied of which is the G-quadruplex aptamer that binds to thrombin (9–12). A DNA aptamer that binds to von Willebrand factor showed a three-stem structure of mainly B-form DNA with some noncanonical base pairing (13). Most recently, the structure of an innovative Slow Off-rate Modified Aptamer (SOMAmer) bound to platelet-derived growth factor B was solved, revealing binding via a hydrophobic surface that mimics how the factor binds to its receptor (14). Generally, the lack of DNA aptamer-target structures has limited our understanding of the mechanisms by which DNA aptamers attain their specificity (15), resulting in a bias in aptasensor development (16).Better point-of-care tests are critically needed for malaria, a disease which continues to claim more than 1 million lives globally every year (17). Antimalarial drugs have been administered presumptively to patients with fever for decades, leading to drug resistance and poor management of other febrile illness. The cost of newer, more effective treatments has led to a situation whereby improved diagnostics has become a major factor that could reduce the burden of malaria in the developing world (17). Antibody-based rapid diagnostic tests have greatly benefitted malaria management, but significant issues with cost (17) and stability in tropical climates (18) remain that are intrinsically associated with the use of protein antibodies. DNA aptamers compare favorably to antibodies for diagnostic applications (19) with particular advantages that could be critical for diagnostic tests of the developing world: thermal stability, convenient chemical synthesis, and potentially lower costs of production (16). Here, we report the crystal structure and application of a unique DNA aptamer against an established malaria pan-species diagnostic target, Plasmodium falciparum lactate dehydrogenase (PfLDH) (20), and a mechanism of molecular recognition by a distorted hairpin DNA aptamer.     

A prospective analysis of the Ab response to Plasmodium falciparum before and after a malaria season by protein microarray

Abs are central to malaria immunity, which is only acquired after years of exposure to Plasmodium falciparum (Pf). Despite the enormous worldwide burden of malaria, the targets of protective Abs and the basis of their inefficient acquisition are unknown. Addressing these knowledge gaps could accelerate malaria vaccine development. To this end, we developed a protein microarray containing ∼23% of the Pf 5,400-protein proteome and used this array to probe plasma from 220 individuals between the ages of 2–10 years and 18–25 years in Mali before and after the 6-month malaria season. Episodes of malaria were detected by passive surveillance over the 8-month study period. Ab reactivity to Pf proteins rose dramatically in children during the malaria season; however, most of this response appeared to be short-lived based on cross-sectional analysis before the malaria season, which revealed only modest incremental increases in Ab reactivity with age. Ab reactivities to 49 Pf proteins measured before the malaria season were significantly higher in 8–10-year-old children who were infected with Pf during the malaria season but did not experience malaria (n = 12) vs. those who experienced malaria (n = 29). This analysis also provided insight into patterns of Ab reactivity against Pf proteins based on the life cycle stage at which proteins are expressed, subcellular location, and other proteomic features. This approach, if validated in larger studies and in other epidemiological settings, could prove to be a useful strategy for better understanding fundamental properties of the human immune response to Pf and for identifying previously undescribed vaccine targets.     

Decline in the expression of C4 binding protein alpha-chain gene during ageing of the rat liver

It appears that consistent changes in the levels of activity of a small cohort of genes(probably less than 1% of all active genes)occur in all mammalian cells during ageing. We have studied this phenomenon in rat liver using an optimised form of differential display. During this investigation we observed one gene which exhibited a decline in expression in livers from young adult (6 months) to aged adult (24 months) animals. The differential expression of this gene was confirmed by single strand conformational polymorphism (SSCP) gel analysis and Northern blotting. Densitometry of the latter indicated that there was adecline of 35% in its expression with age. Characterisation of the isolated PCR fragment demonstrated it to code for the alpha subchain of the complement 4 binding protein (C4BP). The C4BP is a key regulatory protein of the complement system and this observation therefore indicates that a decline in the efficiency of the complement system may be an important factor in the overall decline in immune function that has been observed during ageing. This revised version was published online in July 2006 with corrections to the Cover Date.     

Domain III of Plasmodium falciparum apical membrane antigen 1 binds to the erythrocyte membrane protein Kx

Plasmodium falciparum apical membrane antigen 1 (AMA1) is located in the merozoite micronemes, an organelle that contains receptors for invasion, suggesting that AMA1 may play a role in this process. However, direct evidence that P. falciparum AMA1 binds to human erythrocytes is lacking. In this study, we determined that domain III of AMA1 binds to the erythrocyte membrane protein, Kx, and that the rate of invasion of Kx(null) erythrocytes is reduced, indicating a significant but not unique role of AMA1 and Kx in parasite invasion of erythrocytes. Domains I/II/III, domains I/II and domain III of AMA1 were expressed on the surface of CHO-K1 cells, and their ability to bind erythrocytes was determined. We observed that each of these domains failed to bind untreated human erythrocytes. In contrast, domain III, but not the other domains of AMA1, bound to trypsin-treated human erythrocytes. We tested the binding of AMA1 to trypsin-treated genetically mutant human erythrocytes, missing various erythrocyte membrane proteins. AMA1 failed to bind trypsin-treated Kx(null) (McLeod) erythrocytes, which lack the Kx protein. Furthermore, treatment of human erythrocytes with trypsin, followed by alpha-chymotrypsin, cleaved Kx and destroyed the binding of AMA1 to human erythrocytes. Lastly, the rate of invasion of Kx null erythrocytes by P. falciparum was significantly lower than Kx-expressing erythrocytes. Taken together, our data suggest that AMA1 plays an important, but not exclusive, role in invasion of human erythrocytes through a process that involves exposure or modification of the erythrocyte surface protein, Kx, by a trypsin-like enzyme.