A Roadmap for the Molecular Farming of Viral Glycoprotein Vaccines: Engineering Glycosylation and Glycosylation-Directed Folding

Abstract

Immunization with recombinant glycoprotein-based vaccines is a promising approach to induce protective immunity against viruses. However, the complex biosynthetic maturation requirements of these glycoproteins typically necessitate their production in mammalian cells to support their folding and post-translational modification. Despite these clear advantages, the incumbent costs and infrastructure requirements with this approach can be prohibitive in developing countries, and the production scales and timelines may prove limiting when applying these production systems to the control of pandemic viral outbreaks. Plant molecular farming of viral glycoproteins has been suggested as a cheap and rapidly scalable alternative production system, with the potential to perform post-translational modifications that are comparable to mammalian cells. Consequently, plant-produced glycoprotein vaccines for seasonal and pandemic influenza have shown promise in clinical trials, and vaccine candidates against the newly emergent severe acute respiratory syndrome coronavirus-2 have entered into late stage preclinical and clinical testing. However, many other viral glycoproteins accumulate poorly in plants, and are not appropriately processed along the secretory pathway due to differences in the host cellular machinery. Furthermore, plant-derived glycoproteins often contain glycoforms that are antigenically distinct from those present on the native virus, and may also be under-glycosylated in some instances. Recent advances in the field have increased the complexity and yields of biologics that can be produced in plants, and have now enabled the expression of many viral glycoproteins which could not previously be produced in plant systems. In contrast to the empirical optimization that predominated during the early years of molecular farming, the next generation of plant-made products are being produced by developing rational, tailor-made approaches to support their production. This has involved the elimination of plant-specific glycoforms and the introduction into plants of elements of the biosynthetic machinery from different expression hosts. These approaches have resulted in the production of mammalian N-linked glycans and the formation of O-glycan moieties in planta. More recently, plant molecular engineering approaches have also been applied to improve the glycan occupancy of proteins which are not appropriately glycosylated, and to support the folding and processing of viral glycoproteins where the cellular machinery differs from the usual expression host of the protein. Here we highlight recent achievements and remaining challenges in glycoengineering and the engineering of glycosylation-directed folding pathways in plants, and discuss how these can be applied to produce recombinant viral glycoproteins vaccines.

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

“…The inherent ease of biomass production also offers the prospect of rapid vaccine production in response to pandemic viral outbreaks (Margolin et al, 2018), as has recently been demonstrated by Medicago Inc., in Canada for a SARS-CoV-2 virus-like particle (VLP) vaccine candidate (Ward et al, 2020a). However, 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 consequently 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).…”

“…In contrast, native-like glycosylation is widely regarded as an essential requirement for an HIV envelope-based vaccine, and subtle differences in glycosylation can dictate the quality of the immune response (Behrens and Crispin, 2017). Additionally, glycosylation is intrinsically linked to protein folding, and aberrant glycosylation and particularly under-glycosylation could compromise the efficient production of viral glycoproteins in a heterologous system (Margolin et al, 2020b).…”

“…Although these extensions do not occur in mammalian cells and might therefore be expected to appear foreign to mammalian immune systems, they do not seem to impair the immunogenicity of plant-produced influenza vaccines in humans, nor is there any evidence that they pose safety concerns in individuals with pre-existing plant allergies (Ward et al, 2014). However, the observation that these vaccines induced transient IgG and IgE responses to glyco-epitopes cannot be discounted (Ward et al, 2014) and some concerns remain, especially in the context of more heavily glycosylated proteins or where repeated administration may be necessary (Margolin et al, 2020b). Paucimannosidic glycans, comprising truncated structures where the terminal N-acetylglucosamines have been removed from the glycan core, also occur in plants (Liebminger et al, 2011).…”

“…Encouragingly, a recent study reported similar glycan occupancy for recombinant human cytomegalovirus glycoprotein B when produced in Nicotiana tabacum BY-2 and CHO cells. However, under-glycosylation could account for the poor yields of other glycoproteins in the system, given that inadequate glycan occupancy could impair protein folding and result in the degradation of aberrantly folded protein (Wormald and Dwek, 1999;Margolin et al, 2020b). This could also result in suboptimal immunogenicity, or the induction of antibodies against epitopes that are not accessible on the wild type virus if glycosylation occludes these regions (Zhou et al, 2017;Margolin et al, 2020b).…”

Production of Gloeophyllum trabeum Endoglucanase Cel12A in Nicotiana benthamiana for Cellulose Degradation

“…To avoid the proteolytic degradation of target proteins in the cytosol, expressed proteins can be sequestered to subcellular organelles such as endoplasmic reticulum (ER) or chloroplasts by incorporating organellar targeting signals that are proteolytically removed by organellar peptidase after organellar targeting of proteins (Lee and Hwang, 2018;Islam et al, 2019;Muthamilselvan et al, 2019;Margolin et al, 2020b). ER is an organelle in which several posttranslational modifications such as glycosylation or disulfide bond formation occur, thereby contributing to the functionality of produced proteins (Margolin et al, 2018(Margolin et al, , 2020a. Recently, Oh et al (2019) isolated and characterized the acidic endoglucanase GtCel12A (Gloeophyllum trabeum Cel12A).…”