Assessment of Pfs25 expressed from multiple soluble expression platforms for use as transmission-blocking vaccine candidates
Assessment of Pfs25 expressed from multiple soluble expression platforms for use as transmission-blocking vaccine candidates
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DOI：
10.1186/s12936-016-1464-6
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发表时间：
2016-08-11
期刊：
影响因子：
3
通讯作者：
King, C. Richter
中科院分区：
文献类型：
--
作者：
Lee, Shwu-Maan;Wu, Chia-Kuei;King, C. Richter
背景资料：传播阻断疫苗（TBV）已成为控制并最终消除疟疾的战略重点，因为它们针对斯氏按蚊进入性阶段，从而防止传播，这是寄生虫生命周期的重要组成部分。这些疫苗被设想为靶向人类感染的疫苗的补充，例如RTS、S以及药物治疗和载体控制策略。包括Pfs 25在内的许多保守蛋白已被鉴定为研究或早期开发中有希望的TBV靶标。Pfs 25是恶性疟原虫的一种25 kDa的蛋白质，表达于受精卵和动合子的表面。其复杂的三级结构（包括许多半胱氨酸）导致难以表达同质的、具有适当构象且不含糖基化的重组蛋白，这是在天然寄生虫机器中未发现的现象。虽然Pfs 25在各种系统中的表达和纯化先前已被独立报道，在此，提供了Pfs 25的平行分析，以告知Pfs 25的生化特征及其对功能的影响。使用三种可扩展的表达系统在体外和体内表达、纯化和评估Pfs 25，包括通过标准膜饲养试验每种蛋白产生功能抗体的能力。结果：经过多次尝试，未能获得来源于大肠杆菌的可溶性单体Pfs 25，而巴斯德毕赤酵母将Pfs 25呈现为具有糖基化的非均质产物。相比之下，杆状病毒产生了一个纯的，单体蛋白质的糖基化自由。存在于毕赤酵母产生的Pfs 25中的糖基化在功能评价中显示出引发传递减少抗体的能力没有显著降低，而还原和烷基化的Pfs 25（来源于植物并用作对照）发现具有显著降低的传递减少活性，强调了在疫苗设计和生产期间确保正确的二硫键稳定构象的重要性。在这项研究中，Pfs25，从不同的表达系统产生的生化特征，描述沿着与他们的影响的蛋白质的能力，引发功能性抗体。使用杆状病毒和毕赤酵母表达的Pfs25显示出作为疫苗开发候选物的希望。
Background: Transmission-blocking vaccines (TBVs) have become a focus of strategies to control and eventually eliminate malaria as they target the entry of sexual stage into the Anopheles stephensi mosquito thereby preventing transmission, an essential component of the parasite life cycle. Such vaccines are envisioned as complements to vaccines that target human infection, such as RTS, S as well as drug treatment, and vector control strategies. A number of conserved proteins, including Pfs25, have been identified as promising TBV targets in research or early stage development. Pfs25 is a 25 kDa protein of Plasmodium falciparum expressed on the surface of zygotes and ookinetes. Its complex tertiary structure, including numerous cysteines, has led to difficulties in the expression of a recombinant protein that is homogeneous, with proper conformation, and free of glycosylation, a phenomenon not found in native parasite machinery.Methods: While the expression and purification of Pfs25 in various systems, has been previously independently reported, here a parallel analysis of Pfs25 is presented to inform on the biochemical features of Pfs25 and their impact on functionality. Three scalable expression systems were used to express, purify, and evaluate Pfs25 both in vitro and in vivo, including the ability of each protein to produce functional antibodies through the standard membrane feeding assay.Results: Through numerous attempts, soluble, monomeric Pfs25 derived from Escherichia coli was not achieved, while Pichia pastoris presented Pfs25 as an inhomogeneous product with glycosylation. In comparison, baculovirus produced a pure, monomeric protein free of glycosylation. The glycosylation present for Pichia produced Pfs25, showed no notable decrease in the ability to elicit transmission reducing antibodies in functional evaluation, while a reduced and alkylated Pfs25 (derived from plant and used as a control) was found to have significantly decreased transmission reducing activity, emphasizing the importance of ensuring correct disulfide stabilized conformation during vaccine design and production.Conclusions: In this study, the biochemical features of Pfs25, produced from different expression systems, are described along with their impact on the ability of the protein to elicit functional antibodies. Pfs25 expressed using baculovirus and Pichia showed promise as candidates for vaccine development.