Detailed functional characterization of glycosylated and nonglycosylated variants of malaria vaccine candidate PfAMA1 produced in Nicotiana benthamiana and analysis of growth inhibitory responses in rabbits
Detailed functional characterization of glycosylated and nonglycosylated variants of malaria vaccine candidate PfAMA1 produced in Nicotiana benthamiana and analysis of growth inhibitory responses in rabbits
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DOI：
10.1111/pbi.12255
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发表时间：
2015-02-01
影响因子：
13.8
通讯作者：
Fischer, Rainer
中科院分区：
文献类型：
--
作者：
Boes, Alexander;Spiegel, Holger;Fischer, Rainer
恶性疟原虫根尖膜抗原1 （PfAMA1）是最有希望的疟疾疫苗候选抗原之一。一些研究表明，这种血期抗原可以诱导强烈的寄生虫生长抑制抗体反应。PfAMA1包含多达6个n -链糖基化的识别位点，这是恶性疟原虫中不存在的翻译后修饰。为了防止n -糖基化的任何潜在负面影响，在真核宿主中表达的大多数PfAMA1变异中，识别位点被敲除。然而，n -链糖基化可以通过提高免疫原性和/或通过聚糖掩膜将反应集中到相关表位来提高疗效。我们描述了本烟中糖基化和非糖基化PfAMA1的生产，并在产量、完整性和保护功效方面对其进行了详细的表征。瞬时表达后，两种PfAMA1变异体均积累到高水平（50 - 510g/g鲜叶重），并且糖基化变异体被证实为高甘露糖型n -聚糖。在构象敏感的配体结合研究中，benthamiana和Pichia pastoris的PfAMA1变异没有发现显著差异。在家兔中诱导了bbb20 × 10(6)的特异性滴度，免疫荧光实验观察到bbb20对恶性疟原虫分裂体有很强的反应性，对两种变异的寄生虫生长抑制率高达100%，IC50值接近35g/mL。竞争分析表明，n -聚糖屏蔽了许多表位，使其无法被免疫识别，这就需要进一步的研究来确定糖基化如何用于免疫应答的定向靶向。这些结果突出了植物瞬时表达系统作为候选疫苗生产平台的潜力。
One of the most promising malaria vaccine candidate antigens is the Plasmodium falciparum apical membrane antigen 1 (PfAMA1). Several studies have shown that this blood-stage antigen can induce strong parasite growth inhibitory antibody responses. PfAMA1 contains up to six recognition sites for N-linked glycosylation, a post-translational modification that is absent in P.falciparum. To prevent any potential negative impact of N-glycosylation, the recognition sites have been knocked out in most PfAMA1 variants expressed in eukaryotic hosts. However, N-linked glycosylation may increase efficacy by improving immunogenicity and/or focusing the response towards relevant epitopes by glycan masking. We describe the production of glycosylated and nonglycosylated PfAMA1 in Nicotiana benthamiana and its detailed characterization in terms of yield, integrity and protective efficacy. Both PfAMA1 variants accumulated to high levels (>510g/g fresh leaf weight) after transient expression, and high-mannose-type N-glycans were confirmed for the glycosylated variant. No significant differences between the N.benthamiana and Pichia pastoris PfAMA1 variants were detected in conformation-sensitive ligand-binding studies. Specific titres of >2x10(6) were induced in rabbits, and strong reactivity with P.falciparum schizonts was observed in immunofluorescence assays, as well as up to 100% parasite growth inhibition for both variants, with IC50 values of similar to 35g/mL. Competition assays indicated that a number of epitopes were shielded from immune recognition by N-glycans, warranting further studies to determine how glycosylation can be used for the directed targeting of immune responses. These results highlight the potential of plant transient expression systems as a production platform for vaccine candidates.