Department of Pathogenic Biology, Second Military Medical University, 800 Xiang Yin Road, Shanghai 200433, China.
Malar J. 2010 Apr 12;9:94. doi: 10.1186/1475-2875-9-94.
Apical membrane antigen 1 (AMA-1) and merozoite surface protein 1 (MSP1) of Plasmodium falciparum are two leading blood-stage malaria vaccine candidates. A P. falciparum chimeric protein 2.9 (PfCP-2.9) has been constructed as a vaccine candidate, by fusing AMA-1 domain III (AMA-1 (III)) with a C-terminal 19 kDa fragment of MSP1 (MSP1-19) via a 28-mer peptide hinge. PfCP-2.9 was highly immunogenic in animal studies, and antibodies elicited by the PfCP-2.9 highly inhibited parasite growth in vitro. This study focused on locating the distribution of epitopes on PfCP-2.9.
A panel of anti-PfCP-2.9 monoclonal antibodies (mAbs) were produced and their properties were examined by Western blot as well as in vitro growth inhibition assay (GIA). In addition, a series of PfCP-2.9 mutants containing single amino acid substitution were produced in Pichia pastoris. Interaction of the mAbs with the PfCP-2.9 mutants was measured by both Western blot and enzyme-linked immunosorbent assay (ELISA).
Twelve mAbs recognizing PfCP-2.9 chimeric protein were produced. Of them, eight mAbs recognized conformational epitopes and six mAbs showed various levels of inhibitory activities on parasite growth in vitro. In addition, seventeen PfCP-2.9 mutants with single amino acid substitution were produced in Pichia pastoris for interaction with mAbs. Reduced binding of an inhibitory mAb (mAb7G), was observed in three mutants including M62 (Phe491-->Ala), M82 (Glu511-->Gln) and M84 (Arg513-->Lys), suggesting that these amino acid substitutions are critical to the epitope corresponding to mAb7G. The binding of two non-inhibitory mAbs (mAbG11.12 and mAbW9.10) was also reduced in the mutants of either M62 or M82. The substitution of Leu31 to Arg resulted in completely abolishing the binding of mAb1E1 (a blocking antibody) to M176 mutant, suggesting that the Leu residue at this position plays a crucial role in the formation of the epitope. In addition, the Asn15 residue may also play an important role in the global folding of PfCP-2.9, as its substitution by Arg lead to reduced binding of most mAbs and abolishing the binding of mAb6G and mAbP5-W12.
This study provided valuable information on epitopes of PfCP-2.9 vaccine candidate through generation of a panel of mAbs and a series of PfCP-2.9 mutants. The information may prove to be useful for designing more effective malaria vaccines against blood-stage parasites.
恶性疟原虫顶膜抗原 1（AMA-1）和裂殖子表面蛋白 1（MSP1）是两种主要的血阶段疟疾疫苗候选物。恶性疟原虫嵌合蛋白 2.9（PfCP-2.9）已被构建为一种疫苗候选物，通过将 AMA-1 结构域 III（AMA-1（III））与 MSP1 的 C 端 19 kDa 片段（MSP1-19）通过 28 个氨基酸肽铰链融合。PfCP-2.9 在动物研究中具有高度的免疫原性，由 PfCP-2.9 引起的抗体在体外高度抑制寄生虫的生长。本研究集中于定位 PfCP-2.9 上的表位分布。
产生了一组抗 PfCP-2.9 单克隆抗体（mAb），并通过 Western blot 以及体外生长抑制试验（GIA）检查其特性。此外，还在毕赤酵母中产生了一系列含有单个氨基酸取代的 PfCP-2.9 突变体。通过 Western blot 和酶联免疫吸附试验（ELISA）测量 mAb 与 PfCP-2.9 突变体的相互作用。
产生了 12 种识别 PfCP-2.9 嵌合蛋白的 mAb。其中，8 种 mAb 识别构象表位，6 种 mAb 表现出不同程度的体外抑制寄生虫生长的活性。此外，在毕赤酵母中产生了 17 个含有单个氨基酸取代的 PfCP-2.9 突变体，用于与 mAb 相互作用。在包括 M62（Phe491-->Ala）、M82（Glu511-->Gln）和 M84（Arg513-->Lys）在内的三个突变体中，观察到抑制性 mAb（mAb7G）的结合减少，表明这些氨基酸取代对于与 mAb7G 对应的表位至关重要。两个非抑制性 mAb（mAbG11.12 和 mAbW9.10）的结合也在 M62 或 M82 的突变体中减少。Leu31 取代为 Arg 导致 mAb1E1（阻断抗体）与 M176 突变体的结合完全被阻断，表明该位置的亮氨酸残基在表位形成中起着关键作用。此外，Asn15 残基可能在 PfCP-2.9 的整体折叠中也起着重要作用，因为其取代为 Arg 导致大多数 mAb 的结合减少，并阻断 mAb6G 和 mAbP5-W12 的结合。
本研究通过产生一组 mAb 和一系列 PfCP-2.9 突变体，为 PfCP-2.9 疫苗候选物的表位提供了有价值的信息。这些信息可能有助于设计针对血阶段寄生虫的更有效的疟疾疫苗。
