夏氏疟原虫裂殖子表面蛋白和表面抗原的鉴定

裂殖子提取液已成功地作为猴和鼠的免疫原,另外,已发现与裂殖子表面组分起反应的抗体能阻碍诺氏疟原虫裂殖子侵入红细胞或在体外抑制恶性疟原虫红内期发育。这表明在抵抗疟原虫的保护性免疫中有裂殖子表面抗原的参与。作者为了解夏氏疟原虫裂殖子膜的抗原成分,通过碘标记技术,用~(125)I标记夏氏疟原虫活裂殖子表面膜蛋白及总的裂殖子蛋白,然后与正常或免疫鼠血清反应,反应前后均通过SDS-聚丙烯酰胺凝胶电泳分析。结果:     

伯氏疟原虫两个主要抗原在斯氏按蚊内表达的动态

伯氏疟原虫动合子表面抗原(Pbs21)是在雌配子内合成。配子生殖开始后10h在未成熟的动合子表面达到高峰。该抗原能与单克隆抗体MAb13.1发生特异反应。环子孢子蛋白(CSP)则是子孢子的主要蛋白由成熟子孢子合成。存在于所有的疟原虫属原虫中。该蛋白能特异地与单克隆抗体MAb8.1反应。目前对疟原虫蚊内期抗原的生化特性、结     

抗约氏疟原虫血清控制鼠疟感染有效成分的分析

用约氏疟原虫(P.y.)裂殖体、裂殖子、全虫等抗原和淆虫免疫小鼠,制备多种免疫血清。小鼠体内被动转移上述血清发现只有活虫感染或经氯喹治疗恢复后的小鼠血清可以使受者获得保护,并可在体外抑制裂殖子侵入红细胞。实验表明氯喹治疗血清中起保护作用的成分是抗体。血清中的其它因子和可能残存的氯喹没有显示控制疟原虫感染的作用。用同位素标记、免疫沉淀、SDS-PAGE和自显影技术发现氯喹治疗血清和其它几种免疫血清沉淀的抗原有明显差异。氯喹治疗血清可以特异识别245、210、190、156和130KD抗原,因此这些特异抗体很可能是氯喹治疗血清中对约氏疟原虫感染有阻断作用的成分。     

伯氏疟原虫裂殖子入侵红细胞的电镜观察

用透射电镜研究了伯氏疟原虫入侵红细胞的过程,并与约氏疟原虫进行了比较。结果表明,入侵开始时,裂殖子多以顶端对着红细胞,并显示出调整方向的能力;红细胞也有部分突出等主动性反应。裂殖子接触后,红细胞即形成凹陷,逐渐将其包围。在此过程中,裂殖子通过顶端和表被两种不同的附着方式与凹陷壁保持联系。包围完成后,凹陷口融合封闭,变成一个包着入侵裂殖子的游离囊泡。与此同时,某些细胞成分也发生一定的变化。 约氏疟原虫多感染幼龄红细胞,且虫体被凹陷口勒出缢痕。伯氏疟原虫则多入侵成熟红细胞,无缢痕现象。     

大鼠感染约氏疟原虫后的保护性免疫

前已报道,约氏疟原虫子孢子入侵大鼠肝细胞与枯氏细胞的吞噬活性水平呈正相关,而与其异化代谢水平呈负相关。体外试验证明,伯氏疟原虫子孢子可主动侵入小鼠腹腔巨噬细胞 在正常情况下,巨噬细胞很快死亡而子孢子则完好无损,但在免疫血清条件下,巨噬细胞中子孢子迅即退化。大鼠感染伯氏疟原虫急性期,其血清在体外有抑制腹腔巨噬细胞的吞噬作用。以往研究提示红内期感染条件下有可能通过抑制或刺激单核巨噬细胞系统而影响子孢子的入侵。现用对约氏疟原虫子孢子敏感的Wistar大鼠,观察其在感染约氏疟原虫急     

用间接荧光抗体方法鉴别疟原虫子孢子

本文报道用血清学方法,鉴别获自现场蚊虫中的子孢子种类。抗原制备:按Pacheco等(1979)方法,将新杀死或冰冻保存的蚊子,经不连续的梯度高速离心,分离子孢子,并以子孢子50～150/μl的浓度悬于199培养液中,取上述悬液20μl置于玻片上,室温(24℃)干燥(1～5天内使用)或冰冻(-80℃)保存供以后使用。制备伯氏疟原虫、约氏疟原虫、间日疟原虫及鸡疟原虫子孢子等4种抗原。抗血清的制备:新分离的或钴~(60)照射的伯氏疟原虫、约氏疟原虫子孢子静脉接种小白鼠,或用感染蚊叮咬兔;用伯氏疟原虫的红内期,经静脉注射大白鼠,约氏疟原虫红内期免疫小白鼠,制备抗血清。接种子孢子的动     

恶性疟原虫FCC1／HN株185 kDa和8241kDa保护性抗原的超微结构定位

用LR White树脂低温包埋感染人恶性疟原虫FCC1／HN株的红细胞，用保护性单克隆抗体F6－D3和F6－C2并结合蛋白A－胶体金探针免疫标记恶性疟原虫红内期185kDa和82／41kDa蛋白。结果表明单克隆抗体F6－D3识别的185kDa蛋白定位于游离的和细胞内的裂殖子表面以及未成熟裂殖体的细胞质、质膜及带虫泡膜。而单克隆抗体F6－C2识别的81／41kDa蛋白则定位于未成熟裂殖体及成熟殖子的棒状体中。从超微结构上表明185kDa和82／41kDa保护性抗原分别为恶性疟原虫FCC1／HN株的裂殖子表面抗原和裂殖子棒状体抗原。     

用单克隆抗体分析约氏疟原虫抗原的研究 Ⅰ.约氏疟原虫红内期与人疟原虫的共同抗原

用粗提的约氏疟裂殖子免疫BALB/c小鼠,取其脾细胞与Sp 2/0瘤细胞融合,获得13株分泌抗约氏疟红内期单克隆抗体的杂交瘤。这些McAb分别属于小鼠:IgG_1,IgG_(2a),IgG_(2b)及IgG_3亚类。据免疫荧光观察,13株McAb可分为4类:1.与红内期各发育阶段的原虫能出现荧光反应;2.针对晚期滋养体及裂殖体;3.抗裂殖体及裂殖子;4.单纯抗裂殖子。有5株McAb与人疟原虫发生荧光反应,其中4株只与恶性疟原虫交叉,M_(26-32)则不仅与恶性疟原虫且与间日疟原虫均有强的交叉反应,,表明约氏疟与人的两种疟原虫间有共同抗原,并提示恶性疟原虫与间日疟原虫间也有共同抗原。共同抗原在不同种间的分布和含量不尽相同。用未经固定的感染红细胞加McAb作间接荧光试验,在感染红细胞表面未观察到荧光反应。     

红内期恶性疟原虫的一种主要表面抗原—P190的加工、多晶形及生物学意义

Hold和Freenen成功地用单克隆抗体方法在约氏疟原虫中分离出了分子量为23.5万和23万的两种裂殖子蛋白,并用这二种蛋白做成疫苗预防约氏疟原虫的大量感染。同时也鉴定了分子量为4.2万和4.4万的诺氏疟原虫和伯氏疟原虫子孢子蛋白,其单克隆抗体对它们具有防止感染作用,由此可见,在疟疾研究中,单克隆抗体已作为一种工具用来鉴定抗原。本文报道有两种单克隆抗体     

以间接免疫荧光试验或免疫过氧化物酶抗体试验检测体外培养的伯氏疟原虫红外期的血清反应性

以有人胎肺细胞的盖玻片置于含10%小牛血清的NCTC-135培养基培养至细胞生长接近汇合成片时,加入按蚊唾腺的伯氏疟原虫子孢子,放5%CO_2培养箱中37℃孵育,于不同时间,取培养物用Hanks缓冲盐水(HBSS)洗涤,以冷甲醇固定,再以HBSS洗后即成培养的红外期抗原片,存4℃备用。经84拉德~(60)Co照射的伯氏疟原虫子孢子,按每次30,000个子孢子的剂量静脉免疫小鼠3次,获得与红细胞期无交叉反应的抗子孢子血清。用氯喹控制小鼠的感染,取得抗红细胞期的血清。单克隆抗体有二:其一是能与伯氏疟原虫子孢子保护性表面抗原(Pb44)反应的,腹水液纯化的IgG_1,在体内可中和子孢子感染;另一种是能与子孢子表面抗原相反应并     

Ultrastructural localization of protective antigens of Plasmodium yoelii merozoites by the use of monoclonal antibodies and ultrathin cryomicrotomy

The production of two hybridoma cell lines secreting monoclonal antibodies (MAb), both of which react specifically with erythrocytic merozoites of Plasmodium yoelii in the indirect immunofluorescence assay, has been reported earlier. MAb 25.77 was reactive with a localized region within each merozoite, while MAb 25.1 appeared to be specific for the plasma membrane of schizonts and merozoites. The parasite antigens recognized by antibodies 25.77 and 25.1 are proteins of 235,000 and 230,000 molecular weight, respectively, both of which induce protective immunity against P. yoelii in mice. In order to establish the precise localization of these protective antigens within erythrocyte merozoites, ultrathin cryomicrotomy was used in conjunction with the MAb and protein A-gold. This technique showed that gold particles were exclusively concentrated over the rhoptries when erythrocytic merozoites were incubated with MAb 25.77. On the other hand, gold particles were distributed uniformly over the merozoite surface when parasites were incubated with MAb 25.1. These results demonstrate, for the first time, that a protective antigen of the erythrocytic stage of P. yoelii is localized within the rhoptries as well as on the merozoite surface.     

Merozoite surface coat precursor protein completely protects Aotus monkeys against Plasmodium falciparum malaria

Groups of Aotus (owl) monkeys were immunized with either the Plasmodium falciparum merozoite surface-coat precursor protein and its processing fragments or a complex of high molecular mass rhoptry proteins and challenged with a lethal infection of the homologous P. falciparum Uganda Palo Alto (FUP) strain. No patent parasitemia could be detected on thick blood films of monkeys immunized with the merozoite surface antigens; however, only one of three monkeys immunized with the rhoptry proteins was partially protected, while two required drug therapy. The experiment clearly demonstrates that the merozoite surface-coat precursor protein can completely protect Aotus monkeys against a lethal infection of the human malaria parasite.     

Two Plasmodium falciparum genes express merozoite proteins that are related to Plasmodium vivax and Plasmodium yoelii adhesive proteins involved in host cell selection and invasion

Two related Plasmodium falciparum genes and their encoded proteins have been identified by comparative analyses with Plasmodium vivax reticulocyte binding protein 2 (PvRBP-2). The P. falciparum genes have a structure which suggests that they may be the result of an evolutionary duplication event, as they share more than 8 kb of closely related nucleotide sequence but then have quite divergent unique 3' ends. Between these shared and unique regions is a complex set of repeats, the nature and number of which differs between the two genes, as well as between different P. falciparum strains. Both genes encode large hydrophilic proteins, which are concentrated at the invasive apical end of the merozoite and are predicted to be more than 350 kDa, with an N-terminal signal sequence and a single transmembrane domain near their C termini. Importantly, they also share gene structure and amino acid homology with the Plasmodium yoelii 235-kDa rhoptry protein family, which is also related to PvRBP-2. Together these Plasmodium proteins define an extended family of proteins that appear to function in erythrocyte selection and invasion. As such, they may prove to be essential components of malaria vaccine preparations.     

Anti-mosquito midgut antibodies block development of Plasmodium falciparum and Plasmodium vivax in multiple species of Anopheles mosquitoes and reduce vector fecundity and survivorship

The mosquito midgut plays a central role in the sporogonic development of malaria parasites. We have found that polyclonal sera, produced against mosquito midguts, blocked the passage of Plasmodium falciparum ookinetes across the midgut, leading to a significant reduction of infections in mosquitoes. Anti-midgut mAbs were produced that display broad-spectrum activity, blocking parasite development of both P. falciparum and Plasmodium vivax parasites in five different species of mosquitoes. In addition to their parasite transmission-blocking activity, these mAbs also reduced mosquito survivorship and fecundity. These results reveal that mosquito midgut-based antibodies have the potential to reduce malaria transmission in a synergistic manner by lowering both vector competence, through transmission-blocking effects on parasite development, and vector abundance, by decreasing mosquito survivorship and egg laying capacity. Because the intervention can block transmission of different malaria parasite species in various species of mosquitoes, vaccines against such midgut receptors may block malaria transmission worldwide.     

Antigenic diversity of Plasmodium vivax and their geographic distribution in Thailand.

Fifty-eight monoclonal antibodies (MAbs) raised against the erythrocytic stages of Plasmodium vivax were selected for typing of 501 P. vivax isolates from different geographic locations throughout Thailand. Based on their reactivities in the indirect fluorescent antibody test, these MAbs were classified into five groups: group I MAbs showing generalized staining of all blood stages; group II MAbs reacting with merozoites and their organelles; group III MAbs reacting with the surface membrane of merozoites; Group V MAbs reacting with the surface membrane of trophozoites and schizonts; and group VII MAbs reacting with internal components of the parasites. Sixteen MAbs reacted with more than 95% of the isolates; the epitopes recognized by these MAbs were considered as being invariant. The remaining MAbs reacted with 30-90% of the isolates, and the epitopes recognized by these MAbs were regarded as being variable. The variant epitopes were associated with > 200-, 135-, and 100-kilodalton (kDa) molecules of all blood stages, the 95-kDa molecule on merozoite organelles, the 200-kDa molecule on the surface of trophozoites and schizonts, and the 85-kDa molecule of the parasite internal components. Antigenic diversity occurred among the P. vivax population in the endemic areas of Thailand and was shown to vary from place to place and was highest in the area with the highest rate of transmission along the Myanmar border in western Thailand and along the Cambodian border in eastern Thailand, including Trat (48.4%), Tak (41.7%), Chantaburi (36.5%), and Mae Hong Son (36.4%). Demonstration of antigenic diversity of P. vivax parasites signals a note of caution in the development of vaccines for vivax malaria. The vaccines should be directed against protective, conserved and not against variant epitopes.     

Effect of dietary p-aminobenzoic acid on murine Plasmodium yoelii infection

Plasmodia species, unlike humans, can utilize p-aminobenzoic acid (PABA) for the de novo generation of folate. Plasmodial enzymes for the synthesis of PABA via the shikimate pathway are being investigated as novel targets for malaria chemotherapy. We show that, despite the presence of biosynthetic machinery to synthesize PABA, Plasmodium yoelii, a rodent malaria species, requires exogenous dietary PABA for survival. Mice fed low-PABA diets do not die from lethal doses of P. yoelii. The initiation of a PABA-deficient diet after P. yoelii infection is established leads to the clearance of parasites and subsequent resistance to infection by P. yoelii. An intact immune system is not necessary for protection, given that mice with severe combined immunodeficiency were also protected by PABA-deficient diet. Our studies suggest that the PABA content in the diet will affect the host clearance of malaria parasites and may affect the efficacy of treatments that target the shikimate pathway.     

Plasmodium liver stage developmental arrest by depletion of a protein at the parasite-host interface

Plasmodium parasites of mammals, including the species that cause malaria in humans, infect the liver first and develop there into clinically silent liver stages. Liver stages grow and ultimately produce thousands of first-generation merozoites, which initiate the erythrocytic cycles causing malaria pathology. Here, we present a Plasmodium protein with a critical function for complete liver stage development. UIS4 (up-regulated in infective sporozoites gene 4) is expressed exclusively in infective sporozoites and developing liver stages, where it localizes to the parasitophorous vacuole membrane. Targeted gene disruption of UIS4 in the rodent model malaria parasite Plasmodium berghei generated knockout parasites that progress through the malaria life cycle until after hepatocyte invasion but are severely impaired in further liver stage development. Immunization with UIS4 knockout sporozoites completely protects mice against subsequent infectious WT sporozoite challenge. Genetically attenuated liver stages may thus induce immune responses, which inhibit subsequent infection of the liver with WT parasites.     

Linkage maps from multiple genetic crosses and loci linked to growth-related virulent phenotype in Plasmodium yoelii

Plasmodium yoelii is an excellent model for studying malaria pathogenesis that is often intractable to investigate using human parasites; however, genetic studies of the parasite have been hindered by lack of genome-wide linkage resources. Here, we performed 14 genetic crosses between three pairs of P. yoelii clones/subspecies, isolated 75 independent recombinant progeny from the crosses, and constructed a high-resolution linkage map for this parasite. Microsatellite genotypes from the progeny formed 14 linkage groups belonging to the 14 parasite chromosomes, allowing assignment of sequence contigs to chromosomes. Growth-related virulent phenotypes from 25 progeny of one of the crosses were significantly associated with a major locus on chromosome 13 and with two secondary loci on chromosomes 7 and 10. The chromosome 10 and 13 loci are both linked to day 5 parasitemia, and their effects on parasite growth rate are independent but additive. The locus on chromosome 7 is associated with day 10 parasitemia. The chromosome 13 locus spans ~220 kb of DNA containing 51 predicted genes, including the P. yoelii erythrocyte binding ligand, in which a C741Y substitution in the R6 domain is implicated in the change of growth rate. Similarly, the chromosome 10 locus spans ~234 kb with 71 candidate genes, containing a member of the 235-kDa rhoptry proteins (Py235) that can bind to the erythrocyte surface membrane. Atypical virulent phenotypes among the progeny were also observed. This study provides critical tools and information for genetic investigations of virulence and biology of P. yoelii.     

Inhibition of Plasmodium yoelii blood-stage malaria by interferon alpha through the inhibition of the production of its target cell, the reticulocyte

The effect of a recombinant hybrid human interferon alpha (IFN-alpha) (which cross-reacts with murine cells) on C57BL/6 mice infected with Plasmodium yoelii sporozoites or parasitized erythrocytes was determined. IFN-alpha did not inhibit the development of the parasite in the liver, but it did reduce the blood parasite load and the hepatosplenomegaly induced by the infection in mice injected with blood-stage parasites. The extent of anemia in IFN-alpha-treated and control mice was similar, despite the lower parasite load in the IFN-alpha-treated mice. The reduced blood parasite load in IFN-alpha-treated mice was associated with reduced erythropoiesis and reticulocytosis. As reticulocytes are the preferred target cells for the strain of P yoelii used (P yoelii yoelii 265 BY), it was postulated that the inhibition of reticulocytosis in IFN-alpha-treated mice was causally related to the observed decreased blood parasite load. This was supported by the finding that IFN-alpha inhibited a different strain of P yoelii (17X clone A), which also displays a tropism for reticulocytes, but not a line of Plasmodium vinckei petteri, which infects only mature red blood cells. As human malaria species also display different tropism for reticulocytes, these findings could be relevant for people coinfected with multiple Plasmodium species or strains or coinfected with Plasmodium and virus. (Blood. 2001;97:3966-3971)