A component of an antigenic rhoptry complex of Plasmodium falciparum is modified after merozoite invasion

Human antibodies affinity purified on an adsorbent prepared from a cDNA clone (Ag44) expressing a portion of a rhoptry antigen were used to characterize the synthesis and fate of the antigen in the asexual blood stages of Plasmodium falcipartum. The rhoptry antigen is synthesized in the mature trophozoite-stage parasites as a 103 kDa polypeptide, is present in the schizonts and merozoites as a 105 kDa polypeptide, is discharged from the rhoptries and found in the newly invaded red cells as a 110 kDa polypeptide. Anti-Ag44 antibodies immunoprecipitate the antigen and two additional polypeptides of 135 and 150 kDa from lysates of infected cells and from culture supernatants. The three polypeptides are associated in a non-covalent complex that persists in the newly invaded red cells. All the components of the high molecular weight rhoptry complex are antigenic and can be precipitated with immune human serum. The 135 kDa polypeptide is identical to a 140 kDa rhoptry antigen previously identified by a monoclonal antibody.     

Plasmodium falciparum sporozoite invasion is inhibited by naturally acquired or experimentally induced polyclonal antibodies to the STARP antigen

Antibody(Ab)-mediated inhibition of sporozoite invasion of hepatocytes is a mechanism that has been clearly demonstrated to act upon Plasmodium falciparum pre-erythrocytic stages in humans. Consequently we have analyzed the Ab response to a recently identified P. falciparum sporozoite surface protein, STARP, in malaria-exposed individuals and tested the inhibitory effect of these Ab upon hepatocyte invasion in vitro. STARP-specific IgG were detected in 90 and 61 % of sera from regions where individuals were exposed to 100 and 1–5 infectious bites per year, respectively. These IgG were predominantly of the cytophilic IgG1 or IgG3 type. STARP and the major sporozoite surface protein, CS, elicited equivalent IgG levels in adults. When affinity purified from either African immune sera or the serum of an individual experimentally protected by irradiated sporozoite immunization, STARP-specific Ab prevented up to 90% of sporozoites from invading human hepatocytes. The dose-dependent and reproducible inhibition was more pronounced than that observed with human CS-specific Ab affinity purified under identical conditions. Substantial reduction of sporozoite invasion was also observed with Ab induced by artificial immunization with recombinant STARP protein and reactive with the native protein. Taken together with recent findings of human cytotoxic T lymphocytes specific for this antigen, these results promote the interest of studying the efficacy of STARP as a target for immune effector mechanisms operating upon preerythrocytic stages.     

自制免疫胶体金层析条检测恶性疟原虫的初步研究

目的：建立一种简易快速，适合于基层使用的自测式免疫胶体金层析（GICA）法用于恶性疟原虫的检测。方法：采用柠檬酸三钠还原法制备胶体金颗粒，标记抗恶性疟原虫乳酸脱氢酶（LDHpf)单抗2A5，并以另外4株单抗作为包被抗体，制成诊断恶性疟原虫的免疫层析条，探索最佳配对模式及估计最低检测量，以镜检和PCR法为对照，用GICA条检测门诊“四热”病人血样，评价其敏感性和特异性，并对其稳定性进行了初步研究，结果：GICA检测重组LDHpf，最低检测量为1ng，以镜检法和PCR法为标准，GICA条检测恶性疟原虫的敏感性分别为88．37％和86．67％，GICA与镜检法的符合率均为91．55％，并且当虫体密度＞200个／μl时，GICA的敏感性为100％，GICA条在4℃下可保存3个月以上。结论：GICA检测恶性疟原虫简易快速，灵敏度高，无需特殊仪器设备，有望成为一种恶性疟原虫快速免疫诊断试剂盒。     

Isolation of a Plasmodium falciparum rhoptry protein

A monoclonal antibody raised against the malaria parasite Plasmodium falciparum recognised a protein of 140000 molecular weight which was synthesized during schizogony. The protein has been purified by monoclonal antibody affinity chromatography from extracts of parasitized red cells. Antibodies against the protein have been used to determine its subcellular location. The protein is not expressed on the merozoite surface and has been located in the rhoptries, the apical organelles of the merozoite.     

The role of calcium in the invasion of human erythrocytes by Plasmodium falciparum.

The role of calcium in the invasion of human erythrocytes by Plasmodium falciparum merozoites has been investigated using a variety of techniques. It has been demonstrated using calcium-depleted medium that invasion is dependent upon the presence of calcium and that neither magnesium, manganese or zinc may substitute for it, suggesting that the effect is calcium specific and not dependent upon a non-specific, charge-based mechanism. Using resealed erythrocyte ghosts and altering the internal and external concentrations of calcium and the chelator EGTA, it has been shown that the role of calcium in invasion, at least as far as the target cell is concerned, is in the extracellular environment. Similarly, loading either the schizontinfected, or target erythrocyte with the membrane permeant calcium chelator Indo-1, at concentrations sufficient to chelate approximately 100 times the concentration of resting cell calcium, produced no change in the parasite invasion rate. Consequently we conclude that calcium plays an extra-cellular role in merozoite invasion of the human erythrocyte.     

Classification of adhesive domains in the Plasmodium falciparum erythrocyte membrane protein 1 family

The Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) family of cytoadherent proteins has a central role in disease from malaria infection. This highly diverse gene family is involved in binding interactions between infected erythrocytes and host cells and is expressed in a clonally variant pattern at the erythrocyte surface. We describe by sequence analysis the structure and domain organization of 20 PfEMP1 from the GenBank database. Four domains comprise the majority of PfEMP1 extracellular sequence: the N-terminal segment (NTS) located at the amino terminus of all PfEMP1, the C2, the Cysteine-rich Interdomain Region (CIDR) and the Duffy Binding-like (DBL) domains. Previous work has shown that CIDR and DBL domains can possess adhesive properties. CIDR domains grouped as three distinct sequence classes (, β, and γ) and DBL domains as five sequence classes (, β, γ, δ, and ). Consensus motifs are described for the different DBL and CIDR types. Whereas the number of DBL and CIDR domains vary between PfEMP1, PfEMP1 domain architecture is not random in that certain tandem domain associations — such as DBLCIDR, DBLδCIDRβ, and DBLβC2 — are preferentially observed. This conservation may have functional significance for PfEMP1 folding, transport, or binding activity. Parasite binding phenotype appears to be a determinant of infected erythrocyte tissue tropism that contributes to parasite survival, transmission, and disease outcome. The sequence classification of DBL and CIDR types may have predictive value for identifying PfEMP1 domains with a particular binding property. This information might be used to develop interventions targeting parasite binding variants that cause disease.     

Cytokine-induced inhibition of Plasmodium falciparum erythrocytic growth in vitro.

The addition of recombinant cytokines to Plasmodium falciparum in vitro cultures retarded the growth of the parasite with the effect of recombinant IL-2 (rIL-2) > interferon-gamma (IFN-gamma) > tumour necrosis factor-beta (TNF-beta). The process was concentration dependent, being greatest at 30,000 U/ml and required a 72-h period of continuous exposure for maximum effect. Growth inhibition, as determined morphologically and radiometrically, was a consequence of defective schizont maturation rather than inhibition of merozoite invasion. It was cumulative and detectable within one erythrocytic (48 h) growth cycle.     

抗恶性疟原虫HRP-II单链抗体的筛选与特性分析

目的　筛选抗恶性疟原虫富含组氨酸蛋白 2 (HRP II)单链抗体 (ScFv)并对其进行鉴定。方法从抗恶性疟原虫HRP IIScFv库中 ,挑取单菌落鉴定重组噬粒 ,采用ELISA法筛选能与重组抗原HRP II结合的阳性克隆 ,并选其中 2株强阳性克隆进行序列分析。结果筛选到 8株具有HRP II结合活性的阳性克隆 ,所测 2株克隆的基因序列一致 ,其重链及轻链分别属于鼠抗体基因家族中第I及κVI亚群。结论从抗体库中成功地筛选出阳性克隆 ,为基因工程抗体在恶性疟诊断试剂中的应用奠定了基础     

Anti-malaria antibody-producing B cell frequencies in adults after a Plasmodium falciparum outbreak in Madagascar.

The central highlands of Madagascar offer a unique opportunity to explore the malaria immune memory, as the last murderous epidemic in the study area occurred 8 years ago. Quantification of the circulating memory B lymphocytes reacting to Plasmodium falciparum was assessed among 14 Madagascans by using a limiting dilution assay, applied to the EL4 culture system, which leads to activation, proliferation and differentiation into antibody-secreting cells (ASC) of most peripheral B cells. This system allowed us to observe, without any malaria-specific restimulation, a geometric mean frequency of one anti-P. falciparum ASC among 2992 circulating B cells, except for one Madagascan who did not have any detectable ASC. A geometric mean frequency of one anti-P. falciparum ASC among 1403 was obtained for six malaria hyperimmune Cameroonians, but conversely, no anti-malaria ASC was detected in the blood of six malaria non-immune French control subjects. Anti-P. falciparum ASC frequencies and serum specific antibodies were strongly related. Our results indicate that anti-malaria ASC are still present in peripheral blood of Madagascan subjects, who have not been exposed to P. falciparum for several years. These responder B cells reflect the malaria B cell memory acquired during the last epidemic.     

恶性疟原虫裂殖子表面蛋白1的17区片段基因复合核酸疫苗诱导小鼠抗体免疫性的研究

目的　检测以恶性疟原虫裂殖子表面蛋白 1的 17区片段基因为基础的复合核酸疫苗(分泌性的VR10 12 /TPA/HG MSP1 17和非分泌性的VR10 12 /HG MSP1 17)诱导小鼠的体液免疫反应和免疫血清对疟原虫生长的抑制能力。方法　以 2 0 0 μg/10 0 μl或 10 0 μg/10 0 μl每次每只VR10 12 /HG MSP1 17或VR10 12 /TPA/HG MSP1 17肌注免疫BALB/c或C5 7BL/6小鼠。用ELISA间接法测定小鼠血清的特异性抗体 ,用体外抑制试验检测免疫血清抑制疟原虫生长效果。结果　经 3次 10 0 μg/10 0 μl每次每只VR10 12 /HG MSP1 17免疫后 ,BALB/c小鼠和C5 7BL/6小鼠均产生了明显的HG和YMSP119抗体。但总体抗体水平不高。BALB/c小鼠经 3次 2 0 0 μg/10 0 μl每次每只的VR10 12 /HG MSP1 17免疫后 ,产生了较高的HG抗体 ,但MSP1 17的抗体无明显变化 ,经 3次 2 0 0 μg/10 0 μl每次每只VR10 12 /TPA/HG MSP1 17免疫后 ,仅产生较低的HG抗体 ,无MSP1 17抗体的产生。用 2 0 0 μg/10 0 μlVR10 12 /HG MSP1 17免疫的BALB/c小鼠血清做体外抑制试验 ,结果抑制效果明显。结论　VR10 12 /HG MSP1 17比VR10 12 /TPA/HG MSP 17具有更强的免疫原性 ,其免疫鼠血清能明显地抑制疟原虫生长     

Plasmodium falciparum ring-infected erythrocyte surface antigen is released from merozoite dense granules after erythrocyte invasion.

Electron microscopy was used to study the fate of Plasmodium falciparum ring-infected erythrocyte surface antigen after merozoite invasion by using postembedding immunolabeling. The antigen was localized to small dense granules located centrally or laterally in free merozoites. In newly invaded erythrocytes, labeling was found in pockets of the parasitophorous vacuole space or in aggregates closely associated with the parasitophorous vacuole. These patterns indicate that ring-infected erythrocyte surface antigen is contained in merozoite dense granules that are released after merozoite invasion and not via apical rhoptry ducts at the time of merozoite attachment.     

Allelic family-specific humoral responses to merozoite surface protein 2 (MSP2) in Gabonese residents with Plasmodium falciparum infections

Merozoite surface protein 2 (MSP2) expressed by Plasmodium falciparum asexual blood stages has been identified as a promising vaccine candidate. In order to explore allelic family-specific humoral responses which may be responsible for parasite neutralization during natural infections, isolates from individuals with either asymptomatic infections or uncomplicated malaria and residing in a Central African area where Plasmodium transmission is high and perennial, were analysed using MSP2 as polymorphic marker. The family-specific antibody responses were assessed by ELISA using MSP2 synthetic peptides. We observed an age-dependence of P. falciparum infection complexity. The decrease of infection complexity around 15 years of age was observed simultaneously with an increase in the mean number of MSP2 variants recognized. No significant difference in the P. falciparum genetic diversity and infection complexity was found in isolates from asymptomatic subjects and patients with uncomplicated malaria. The longitudinal follow-up showed a rapid development of immune responses to various regions of MSP2 variants within one week. Comparing humoral responses obtained with the other major antigen on the merozoite surface, MSP1, our findings suggest that different pathways of responsiveness are involved in antibody production to merozoite surface antigens.     

Stage-specific protein synthesis by asexual blood stage parasites of Plasmodium falciparum

Highly synchronised cultures of cloned Plasmodium falciparum (clone T9-94) were metabolically radiolabelled with [35S]methionine during eight consecutive non-overlapping intervals, while parasites developed from young rings to mature schizonts. Analysis of equal amounts of trichloroacetic acid precipitable radioactivity from each interval by sodium dodecyl sulphate-polyacrylamide gel electrophoresis fluorography allowed the stage specificity of protein synthesis to be investigated. More than forty polypeptides with molecular weights of 20 000 to 200 000 can be distinguished. While some proteins are synthesised throughout erythrocytic schizogony many are shown to be stage-specific. Among these are a range of high molecular weight proteins synthesised only during nuclear division. Detailed morphological information permits correlations to be made between synthesis of particular polypeptides and parasite structure.     

恶性疟复合多价抗原基因在小鼠中免疫应答的初步研究

目的探索恶性疟复合多价ＤＮＡ疫苗的可行性。方法把带有ＡＴＧ的接头与人工合成的恶性疟原虫复合多价抗原基因ＡＢ相连后，构建分别带有ＳＶ４０或ＲＳＶ启动子的真核表达载体ｐＳＶ２／ＡＢ及ｐＲＥＰ９／ＡＢ，重组表达质粒经肌肉注射免疫ＢＡＬＢ／ｃ小鼠后，检测其诱发特异性体液和细胞免疫应答水平及毒副作用。结果ｐＳＶ２／ＡＢ及ｐＲＥＰ９／ＡＢ免疫ＢＡＬＢ／ｃ小鼠后均诱发了一定水平的细胞及体液免疫应答，带ＲＳＶ启动子的ｐＲＥＰ９／ＡＢ免疫原性略强于带ＳＶ４０启动子的ｐＳＶ２／ＡＢ，ＤＮＡ免疫后未见明显的毒副作用。结论恶性疟复合多价ＤＮＡ疫苗可诱发特异的免疫应答，为疟疾ＤＮＡ疫苗的研究提供了一定的理论及实验依据     

TNFalpha-308A associated with shorter intervals of Plasmodium falciparum reinfections

A mutation at position -308 of the tumor necrosis factor alpha (TNF-308A) gene promoter has previously been associated with particular manifestations of infectious and non-infectious diseases. In a longitudinal study on malariological parameters in Gabon, TNF promoter variants of 98 children initially presenting with severe Plasmodium falciparum malaria, followed by a total of 504 reinfection events within 52 months, and 100 children initially presenting with mild malaria followed by a total of 342 reinfections were analyzed. Symptomatic P. falciparum reinfections occurred more quickly (median 11 weeks) in carriers of the TNF-308A allele, with severe malaria at enrollment, compared to carriers of other TNF promoter variants (median 16 weeks). The results show that this particular TNF promoter allele increases the risk of reinfection with the malaria parasite P. falciparum.     

Secondary processing of the Plasmodium falciparum merozoite surface protein-1 (MSP1) by a calcium-dependent membrane-bound serine protease: shedding of MSP133 as a noncovalently associated complex with other fragments of the MSP1.

Merozoites of the malaria parasite Plasmodium falciparum possess on their surface proteolytically processed fragments of the merozoite surface protein-1 (MSP1). Secondary processing of one of these fragments, MSP1₄₂, always occurs prior to, or at the point of successful erythrocyte reinvasion. It is shown that a product of this secondary processing, MSP1₃₃, is shed in the form of a noncovalently-associated complex with a number of other proteins, including the MSP1-derived species MSP1₃₈ and MSP1₈₃. Secondary processing of MSP1₄₂, is inhibited by the chelating agents ethylenediaminetetraacetic acid (EDTA) and ethyleneglycol-bis-(β-aminoethyl ether)-tetraacetic acid (EGTA), and this inhibition is reversible by addition of excess calcium. Secondary processing occurs in preparations of washed, disrupted merozoites, and is inhibited by the protease inhibitors phenylmethylsulphonyl fluoride (PMSF) and diisopropyl fluorophosphate (DFP), indicating that the protease responsible is a membrane-associated serine protease.     

含恶性疟原虫Pf70基因片段表达载体DNA的免疫学特性

目的 研究恶性疟原虫Pf70 DNA片段作为DNA疫苗候选抗原基因的可能性。方法 应用PCR方法从含有恶性疟原虫Pf70基因DNA片段的质粒pGEX-Pf70中扩增出包含Pf70 DNA片段的长度为957bP的PCR产物。将其插人带有乙肝表面抗原基因的真核表达载体pCMV-S中，构建出重组质粒pCMV-S-pf70。用提纯的重组质粒pCMV-S-Pf70作为DNA免疫制剂，以质粒pCMV-S DNA和经过谷胱甘肽亲和层析法纯化的融合蛋白GST-Pf70作为对照，免疫昆明种小白鼠。每隔14d加强免疫1次，加强免疫2次后第7天采小鼠全血，用流式细胞仪检测CD8 T淋巴细胞和CD4 T淋巴细胞的水平，以检测体液免疫和细胞免疫状况。结果 接种了重组质粒pCMV-S-Pf70小鼠CD8 T淋巴细胞的绝对量增加，其百分比含量增加了39％；CD4 T淋巴细胞的相对含量保持恒定，绝对量稍有增加。用ELISA法分别检测经重组质粒pCMV-S-Pf70免疫的小鼠和经融合蛋白GST-Pf70免疫的小鼠血清中抗Pt70蛋白质的抗体滴度，发现前者抗体滴度约为1：800，远远低于后者的滴度(1：5000)。研究结果证明，重组质粒pCMV-S-Pf70 DNA可以诱导小鼠产生很强的细胞免疫和相对于蛋白质免疫而言较弱的体液免疫；Pt70蛋白质可以诱导小鼠产生很强的体液免疫。结论 恶性疟原虫红内期Pf70 DNA片段是有前景的红内期DNA疫苗候选抗原基因片段。     

Uniparental inheritance of the mitochondrial gene cytochrome b in Plasmodium falciparum

The inheritance of an extrachromosomal 6-kb element has been examined in the human malaria parasite Plasmodium falciparum. A single base pair difference in the cytochrome b gene from the 6-kb element of two different cloned lines of the parasite was identified, and used as a marker in a cross in the mosquito stage of the life cycle. Analysis of 59 individual hybrid oocysts resulting from this cross clearly demonstrated that inheritance of the cytochrome b gene was uniparental. This observation makes it possible to investigate the inheritance and evolution of cytoplasmic traits, including certain forms of drug resistance, in natural populations of this parasite.