Site-Specific Glycosylation of Recombinant Viral Glycoproteins Produced in Nicotiana benthamiana.

Emmanuel Margolin,Michiel Van Diepen,Max Crispin,Anna-Lise Williamson,Matthew Verbeek,Edward Rybicki,Rosamund Chapman,Phindile Ximba,Ann Meyers,Joel D Allen

doi:10.3389/fpls.2021.709344

Abstract

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 Nicotiana benthamiana 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 N. benthamiana is an important step toward producing heterologous viral glycoproteins in plants with authentic human-like glycosylation.

Highlights

Plant-based expression systems are gaining increased attention for the production of recombinant proteins, including subunit vaccines and therapeutic monoclonal antibodies (Sack et al, 2015; Group et al, 2016)
Viral envelope glycoproteins from HIV-1, EpsteinBarr virus (EBV), and Marburg virus (MARV) were selected for this study as they are all extensively glycosylated and are ideal models to interrogate the impact of the plant cellular machinery on glycosylation
Both HIV gp140 and EBV gp350 TM were successfully expressed in plants as part of a previous study exploring the impact of coexpressing human chaperones on viral glycoprotein production in N. benthamiana (Margolin et al, 2020c)

Summary

Introduction

Plant-based expression systems are gaining increased attention for the production of recombinant proteins, including subunit vaccines and therapeutic monoclonal antibodies (Sack et al, 2015; Group et al, 2016) This is largely driven by the potential for more rapid implementation timelines and capacity for rapid production scale-up when compared to established expression systems. The upfront capital investment for a plantbased manufacturing facility is considerably lower than for mammalian cell production, and the production costs for the raw material are reduced (Nandi et al, 2016) These features are appealing to low-and middle-income countries where biomanufacturing capacity is urgently needed, but is mostly absent (Margolin et al, 2020a). Products from plantbased expression systems may not always display desired critical quality attributes: for example, when a biologic requires extensive post-translational modifications, that are not performed in plants as in other cell types, there is a need to further develop plant-based expression technologies to better support the production of complex pharmaceutical products such as glycosylated biologics (Margolin et al, 2020b)

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