Versatile RHDV virus-like particles: Incorporation of antigens by genetic modification and chemical conjugation
Versatile RHDV virus-like particles: Incorporation of antigens by genetic modification and chemical conjugation
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DOI：
10.1002/bit.21518
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发表时间：
2007-12-01
影响因子：
3.8
通讯作者：
Ward, Vernon K.
中科院分区：
文献类型：
--
作者：
Peacey, Matthew;Wilson, Sarah;Ward, Vernon K.
病毒样颗粒已被证明是优异的分子支架，但针对每个 VLP 产生的个体特征和免疫反应需要开发各种衣壳以用作疫苗、分子递送容器和纳米级模板。在这里，我们描述了兔出血性疾病病毒 (RHDV) 样颗粒作为快速通用分子工作台的开发，克服了已建立的嵌合 VLP 遗传抗原掺入程序所带来的限制。在杆状病毒系统中生产 RHDV 衣壳蛋白导致 VLP 自组装，其纯度超过 99%，并可进行遗传和化学操作。小肽序列与 RHDV VLP 的融合具有良好的耐受性，形成嵌合衣壳，将外源肽向杂交瘤 T 辅助细胞的呈递增强了 700 倍。采用异双功能化学接头磺基-SMCC 的快速、简单的缀合技术使小肽和整个蛋白质能够缀合到 RHDV VLP 的表面，克服了嵌合 VLP 对 VLP 形成和产量的限制。施用VLP/卵清蛋白缀合物在小鼠中激发高滴度卵清蛋白特异性抗体，证明衣壳的免疫刺激特性被赋予缀合的外源抗原。 VLP 促进结合抗原向树突状细胞的递送，在初始 TCR 转基因 T 辅助细胞中引发增殖反应，其增殖反应比单独递送的卵清蛋白抗原至少高 10 倍。
Virus-like particles have proved to be excellent molecular scaffolds, yet the individual characteristics and immune responses generated against each VLP requires the development of a wide range of capsids for use as vaccines, molecular delivery vessels, and nanoscale templates. Here we describe the development of Rabbit haemorrhagic disease virus (RHDV)-like particles as a rapidly versatile molecular workbench, overcoming limitations imposed by established genetic antigen incorporation procedures with chimeric VLP. Production of the RHDV capsid protein in a baculovirus system led to the self-assembly of VLP which were recovered at over 99% purity and manipulated both genetically and chemically. Fusion of small peptide sequences to RHDV VLP was well tolerated, forming chimeric capsids that enhanced the presentation of foreign peptide to hybridoma T helper cells 700-fold. Rapid and simple conjugation techniques employing the hetero-bifunctional chemical linker sulfo-SMCC enabled both small peptides and whole proteins to be conjugated to the surface of RHDV VLP, overcoming limitations imposed on VLP formation and yield experienced with chimeric VLP. Administration of VLP/ovalbumin conjugate provoked high titre ovalbumin-specific antibody in mice, demonstrating the immune stimulatory properties of the capsid were conferred to conjugated foreign antigen. VLP facilitated delivery of conjugated antigen to dendritic cells, eliciting prolifera tive responses in naive TCR transgenic T helper cells that were at least 10-fold greater than ovalbumin antigen delivered alone.