Margolin Emmanuel, Allen Joel D, Verbeek Matthew, van Diepen Michiel, Ximba Phindile, Chapman Rosamund, Meyers Ann, Williamson Anna-Lise, Crispin Max, Rybicki Edward
Division of Medical Virology, Department of Pathology, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa.
Wellcome Trust Centre for Infectious Disease Research in Africa, University of Cape Town, Cape Town, South Africa.
Front Plant Sci. 2021 Jul 22;12:709344. doi: 10.3389/fpls.2021.709344. eCollection 2021.
There is an urgent need to establish large scale biopharmaceutical manufacturing capacity in Africa where the infrastructure for biologics production is severely limited. Molecular farming, whereby pharmaceuticals are produced in plants, offers a cheaper alternative to mainstream expression platforms, and is amenable to rapid large-scale production. However, there are several differences along the plant protein secretory pathway compared to mammalian systems, which constrain the production of complex pharmaceuticals. Viral envelope glycoproteins are important targets for immunization, yet in some cases they accumulate poorly in plants and may not be properly processed. Whilst the co-expression of human chaperones and furin proteases has shown promise, it is presently unclear how plant-specific differences in glycosylation impact the production of these proteins. In many cases it may be necessary to reproduce features of their native glycosylation to produce immunologically relevant vaccines, given that glycosylation is central to the folding and immunogenicity of these antigens. Building on previous work, we transiently expressed model glycoproteins from HIV and Marburg virus in and mammalian cells. The proteins were purified and their site-specific glycosylation was determined by mass-spectrometry. Both glycoproteins yielded increased amounts of protein aggregates when produced in plants compared to the equivalent mammalian cell-derived proteins. The glycosylation profiles of the plant-produced glycoproteins were distinct from the mammalian cell produced proteins: they displayed lower levels of glycan occupancy, reduced complex glycans and large amounts of paucimannosidic structures. The elucidation of the site-specific glycosylation of viral glycoproteins produced in is an important step toward producing heterologous viral glycoproteins in plants with authentic human-like glycosylation.
非洲迫切需要建立大规模生物制药生产能力，因为该地区生物制品生产基础设施严重受限。分子农业是指在植物中生产药物，它为主流表达平台提供了一种更廉价的替代方案，并且适合快速大规模生产。然而，与哺乳动物系统相比，植物蛋白分泌途径存在一些差异，这限制了复杂药物的生产。病毒包膜糖蛋白是免疫接种的重要靶点，但在某些情况下，它们在植物中的积累不佳，可能无法得到正确加工。虽然人类伴侣蛋白和弗林蛋白酶的共表达已显示出前景，但目前尚不清楚植物特异性糖基化差异如何影响这些蛋白质的生产。在许多情况下，鉴于糖基化对于这些抗原的折叠和免疫原性至关重要，可能有必要重现其天然糖基化特征以生产具有免疫相关性的疫苗。基于先前的工作，我们在植物和哺乳动物细胞中瞬时表达了来自HIV和马尔堡病毒的模型糖蛋白。对这些蛋白质进行了纯化，并通过质谱法确定了它们的位点特异性糖基化。与同等的哺乳动物细胞衍生蛋白相比，这两种糖蛋白在植物中产生时产生的蛋白质聚集体数量增加。植物产生的糖蛋白的糖基化谱与哺乳动物细胞产生的蛋白质不同：它们显示出较低的聚糖占有率、减少的复杂聚糖和大量的寡甘露糖结构。阐明植物中产生的病毒糖蛋白的位点特异性糖基化是在植物中生产具有真实人源化糖基化的异源病毒糖蛋白的重要一步。
