Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, SE-17177, Sweden.
Malar J. 2013 Jan 24;12:32. doi: 10.1186/1475-2875-12-32.
Rosette-formation of Plasmodium falciparum parasitized erythrocytes is of importance in the development of severe malaria. The parasite-derived molecule PfEMP1 (Plasmodium falciparum erythrocyte membrane protein 1), central to rosetting, is suggested to be included in a multimeric vaccine targeting severe disease.
Three recombinant NTS-DBL1α-domains of PfEMP1 were generated in Escherichia coli, purified and used for immunization of rats and goats. Antibody titres were determined in ELISA assays and responses were compared in-between different individual animals and species. Reactivity with the parasites was tested in live pRBC using FACS. B-cell epitopes prediction was carried out in silico and compared to the results obtained by peptide microarray. Screening for serological cross-reactivity with heterologous NTS-DBL1α variants was carried out by ELISA, peptide array and FACS on pRBC of different laboratory strains and patient isolates.
All three NTS-DBL1α-domains induced high titres of antibodies that were biologically active with no apparent difference between constructs covering slightly different parts of the DBL1α-sequence. The different animal species showed comparable titres of antibodies, while variations within individuals of the species could be observed.Mapping of the recognized epitopes revealed that most parts of the molecule were able to induce an antibody response with a tendency for the N and C terminal parts of the molecule for slightly higher recognition. Important differences to the epitopes predicted were found as some of the most conserved parts of the DBL1α-domain contained the main epitopes for antibody reactivity. ELISA assays and peptide microarray demonstrated substantial cross-reactivity to heterologous variants, while binding to native PfEMP1 was observed only in few combinations on the pRBC surface, underlining that mainly internal, conserved and not surface exposed parts of the DBL1α-domain are responsible for this observation.
Biologically active antibodies can be induced consistently, with high titres, in different animal species and the antibodies elicited by different constructs react with similar epitopes. Induced antibodies recognize epitopes localized in all subdomains of the DBL1α-sequence. Cross-reactivity between NTS-DBL1α-variants is common in ELISA, but rare with live pRBC emphasizing that also internal, conserved areas of PfEMP1 carry important highly immunogenic epitopes of the molecule.
恶性疟原虫寄生红细胞的玫瑰花形成在严重疟疾的发展中很重要。寄生虫衍生的分子 PfEMP1（恶性疟原虫红细胞膜蛋白 1）是玫瑰花形成的核心，被认为包含在针对严重疾病的多价疫苗中。
在大肠杆菌中生成了三个重组 PfEMP1 的 NTS-DBL1α 结构域，进行了纯化并用于大鼠和山羊的免疫接种。通过 ELISA 测定抗体滴度，并在不同个体动物和物种之间进行比较。使用 FACS 在活 pRBC 上测试与寄生虫的反应。通过计算机进行 B 细胞表位预测，并与肽微阵列的结果进行比较。通过 ELISA、肽阵列和 FACS 在不同实验室株和患者分离株的 pRBC 上筛选与异源 NTS-DBL1α 变体的血清学交叉反应性。
所有三个 NTS-DBL1α 结构域都诱导了高滴度的抗体，这些抗体具有生物活性，构建物覆盖的 DBL1α 序列略有不同，但没有明显差异。不同的动物物种显示出可比的抗体滴度，而在物种内的个体中可以观察到变化。识别表位的映射表明，分子的大部分区域都能够诱导抗体反应，分子的 N 和 C 末端区域略有更高的识别。与预测的表位存在重要差异，因为 DBL1α 结构域的一些最保守部分包含抗体反应的主要表位。ELISA 测定和肽微阵列显示与异源变体具有实质性的交叉反应性，而在 pRBC 表面仅观察到少数组合与天然 PfEMP1 结合，这表明主要是内部、保守的而不是表面暴露的 DBL1α 结构域负责这种观察结果。
可以在不同的动物物种中一致地诱导具有高滴度的生物活性抗体，并且不同构建物诱导的抗体与类似的表位反应。诱导的抗体识别 DBL1α 序列的所有亚结构域中的表位。NTS-DBL1α 变体之间的交叉反应性在 ELISA 中很常见，但与活 pRBC 很少见，这强调 PfEMP1 的内部、保守区域也携带该分子的重要高度免疫原性表位。
