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ABSTRACTRespiratory syncytial virus (RSV) is a highly contagious virus that affects the lungs and respiratory passages of many vulnerable people. It is a leading cause of lower respiratory tract infections and clinical complications, particularly among infants and elderly. It can develop into serious complications such as pneumonia and bronchiolitis. The development of RSV vaccine or immunoprophylaxis remains highly active and a global health priority. Currently, GSK’s Arexvy™ vaccine is approved for the prevention of lower respiratory tract disease in older adults (>60 years). Palivizumab and currently nirsevimab are the approved monoclonal antibodies (mAbs) for RSV prevention in high-risk patients. Many studies are ongoing to develop additional therapeutic antibodies for preventing RSV infections among newborns and other susceptible groups. Recently, additional antibodies have been discovered and shown greater potential for development as therapeutic alternatives to palivizumab and nirsevimab. Plant expression platforms have proven successful in producing recombinant proteins, including antibodies, offering a potential cost-effective alternative to mammalian expression platforms. Hence in this study, an attempt was made to use a plant expression platform to produce two anti-RSV fusion (F) mAbs 5C4 and CR9501. The heavy-chain and light-chain sequences of both these antibodies were transiently expressed in Nicotiana benthamiana plants using a geminiviral vector and then purified using single-step protein A affinity column chromatography. Both these plant-produced mAbs showed specific binding to the RSV fusion protein and demonstrate effective viral neutralization activity in vitro. These preliminary findings suggest that plant-produced anti-RSV mAbs are able to neutralize RSV in vitro.

Abstract Coronavirus disease of 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is an ongoing outbreak, disrupting human life worldwide. Vaccine development was prioritized to obtain a biological substance for combating the viral pathogen and lessening disease severity. In vaccine production, biological origin and relevant materials must be carefully examined for potential contaminants in conformity with good manufacturing practice. Due to fast mutation, several SARS-CoV-2 variants and sublineages have been identified. Currently, most of COVID-19 vaccines are developed based on the protein sequence of the Wuhan wild type strain. New vaccines specific for emerging SARS-CoV-2 strains are continuously needed to tackle the incessant evolution of the virus. Therefore, in vaccine development and production, a reliable method to identify the nature of subunit vaccines is required to avoid cross-contamination. In this study, liquid chromatography-mass spectrometry using quadrupole-time of flight along with tryptic digestion was developed for distinguishing protein materials derived from different SARS-CoV-2 strains. After analyzing the recombinantly produced receptor-binding domain (RBD) of the SARS-CoV-2 spike protein, nine characteristic peptides were identified with acceptable limits of detection. They can be used together to distinguish 14 SARS-CoV-2 strains, except Kappa and Epsilon. Plant-produced RBD-Fc protein derived from Omicron strains can be easily distinguished from the others with 4–5 unique peptides. Eventually, a peptide key was developed based on the nine peptides, offering a prompt and precise flowchart to facilitate SARS-CoV-2 strain identification in COVID-19 vaccine manufacturing.

The production of cannabinoid compounds such as Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabichromene (CBC) with potential pharmaceutical applications is growing sharply. However, challenges such as the low yield of minor cannabinoids, legal restrictions on cultivation, and the complexity and cost of purification from the Cannabis sativa plant necessitate a biotechnological approach. Since the biosynthetic pathway is disclosed, cannabinoids have been produced in yeast, insect cells and plants mainly by the heterologous expression of tetrahydrocannabinol acid synthase (THCAS). THCAS and cannabidiolic acid synthase (CBDAS) use cannabigerolic acid (CBGA) as a substrate. In this study, we transiently expressed recombinant forms of THCAS and CBDAS in leaves of Nicotiana benthamiana. Our results demonstrate that efficient expression in the secretory pathway relies on replacing the endogenous signal peptide with a heterologous one. Both proteins were successfully secreted to the apoplast. MS-based analysis of the purified proteins revealed that they are heavily glycosylated with mainly Golgi-processed complex type N-glycans. In planta enzymatic removal of N-glycans indicated that glycosylation plays a role for CBDAS protein folding or stability. Finally, in vitro assays with CBGA showed that the plant-made recombinant CBDAS and THCAS are enzymatically active.

Plant-based manufacturing has the advantage of post-translational modifications. While plant-specific N-glycans have been associated with allergic reactions, their effect on the specific immune response upon vaccination is not yet understood. In this study, we produced an RBD-Fc subunit vaccine in both wildtype (WT) and glycoengineered (∆XF) Nicotiana benthamiana plants. The N-glycan analysis: RBD-Fc carrying the ER retention peptide mainly displayed high mannose. When produced in WT RBD-Fc displayed complex-type (GnGnXF) N-glycans. In contrast, ∆XF plants produced RBD-Fc with humanized complex N-glycans that lack potentially immunogenic xylose and core fucose residues (GnGn). The three recombinant RBD-Fc glycovariants were tested. Immunization with any of the RBD-Fc proteins resulted in a similar titer of anti-RBD antibodies in mice. Likewise, antisera from subunit RBD-Fc vaccines also demonstrated comparable neutralization against SARS-CoV-2. Thus, we conclude that N-glycan modifications of the RBD-Fc protein have no impact on their capacity to activate immune responses and induce neutralizing antibody production.

Dengue virus (DENV), transmitted by mosquitoes, is classified into four serotypes (DENV1-4) and typically causes mild, self-limiting symptoms upon initial infection. However, secondary infection can lead to severe symptoms due to antibody-dependent enhancement (ADE). To address this, anti-DENV antibodies are being developed with the goal of neutralizing infection without ADE activity. Previous attempts using a 54_hG1 antibody from CHO-K1 mammalian cells resulted in ADE induction, increasing viral infection. This study aimed to express the D54 monoclonal antibody in Nicotiana benthamiana. The plant-produced antibody had a similar neutralizing profile to the previous 54_hG1 antibody. Notably, the ADE activities of the plant-derived antibody were successfully eliminated, with no sign of viral induction. These findings suggest that N. benthamiana could be a source of therapeutic DENV antibodies. The method offers several advantages, including lower ADE, cost-effectiveness, simple facility requirements, scalability, and potential industrial-scale production in GMP facilities.

Cobra (Naja kaouthia) venom contains many toxins including α-neurotoxin (αNTX) and phospholipase A2 (PLA2), which can cause neurodegeneration, respiratory failure, and even death. The traditional antivenom derived from animal serum faces many challenges and limitations. Heavy-chain-only antibodies (HCAb), fusing VHH with human IgG Fc region, offer advantages in tissue penetration, antigen binding, and extended half-life. This research involved the construction and transient expression of two types of VHH-FC which are specific to α-Neurotoxin (VHH-αNTX-FC) and Phospholipase A2 (VHH-PLA2-FC) in Nicotiana benthamiana leaves. The recombinant HCAbs were incubated for up to six days to optimize expression levels followed by purification by affinity chromatography and characterization using LC/Q-TOF mass spectrometry (MS). Purified proteins demonstrated over 92 % sequence coverage and an average mass of around 82 kDa with a high-mannose N-glycan profile. An antigen binding assay showed that the VHH-αNTX-Fc has a greater ability to bind to crude venom than VHH-PLA2-Fc.

The transition of IgA antibodies into clinical development is crucial because they have the potential to create a new class of therapeutics with superior pathogen neutralization, cancer cell killing, and immunomodulation capacity compared to IgG. However, the biological role of IgA glycans in these processes needs to be better understood. This study provides a detailed biochemical, biophysical, and structural characterization of recombinant monomeric human IgA2, which varies in the amount/locations of attached glycans. Monomeric IgA2 antibodies were produced by removing the N-linked glycans in the CH1 and CH2 domains. The impact of glycans on oligomer formation, thermal stability, and receptor binding was evaluated. In addition, we performed a structural analysis of recombinant IgA2 in solution using Small Angle X-Ray Scattering (SAXS) to examine the effect of glycans on protein structure and flexibility. Our results indicate that the absence of glycans in the Fc tail region leads to higher-order aggregates. SAXS, combined with atomistic modeling, showed that the lack of glycans in the CH2 domain results in increased flexibility between the Fab and Fc domains and a different distribution of open and closed conformations in solution. When binding with the Fcα-receptor, the dissociation constant remains unaltered in the absence of glycans in the CH1 or CH2 domain, compared to the fully glycosylated protein. These results provide insights into N-glycans’ function on IgA2, which could have important implications for developing more effective IgA-based therapeutics in the future.

Abstract Immune checkpoint inhibitors are a well-known class of immunotherapeutic drugs that have been used for effective treatment of several cancers. Atezolizumab (Tecentriq) was the first antibody to target immune checkpoint PD-L1 and is now among the most commonly used anticancer therapies. However, this anti-PD-L1 antibody is produced in mammalian cells with high manufacturing costs, limiting cancer patients’ access to the antibody treatment. Plant expression system is another platform that can be utilized, as they can synthesize complex glycoproteins, are rapidly scalable, and relatively cost-efficient. Herein, Atezolizumab was transiently produced in Nicotiana benthamiana and demonstrated high expression level within 4–6 days post-infiltration. After purification by affinity chromatography, the purified plant-produced Atezolizumab was compared to Tecentriq and showed the absence of glycosylation. Furthermore, the plant-produced Atezolizumab could bind to PD-L1 with comparable affinity to Tecentriq in ELISA. The tumor growth inhibitory activity of plant-produced Atezolizumab in mice was also found to be similar to that of Tecentriq. These findings confirm the plant’s capability to serve as an efficient production platform for immunotherapeutic antibodies and suggest that it could be used to alleviate the cost of existing anticancer products.

Abstract As a response to invasion by pathogens, the secretion of interleukin 6 (IL-6) which is a cytokine, activates IL-6/JAKs/STAT3 intracellular signaling via., phosphorylation. Over expression of pSTAT3 induces IL-6 positive feedback loop causing cytokine release syndrome or cytokine storm. Plants have gained momentum as an alternative expression system. Hence, this study aims to produce mAb targeting human IL-6 receptor (hIL-6R) in Nicotiana benthamiana for down regulating its cellular signaling thus, decreasing the expression of pSTAT3. The variable regions of heavy and light chains of anti-hIL-6R mAb were constructed in pBYK2e geminiviral plant expression vector and transiently co-expressed in N. benthamiana. The results demonstrate the proper protein assembly of anti-hIL-6R mAb with highest expression level of 2.24 mg/g FW at 5 dpi, with a yield of 21.4 µg/g FW after purification. The purity and N-glycosylation of plant produced antibody was analyzed, including its specificity to human IL-6 receptor by ELISA. Additionally, we investigated the effect to pSTAT3 expression in human PBMC’s by flow cytometry wherein, the results confirmed lower expression of pSTAT3 with increasing concentrations of plant produced anti-hIL-6R mAb. Although, further in vivo studies are key to unveil the absolute functionality of anti-hIL-6R, we hereby show the potential of the plant platform and its suitability for the production of this therapeutic antibody.

Introduction: Prolyl-4-hydroxylases (P4H) catalyse the irreversible conversion of proline to hydroxyproline, constituting a common posttranslational modification of proteins found in humans, plants, and microbes. Hydroxyproline residues can be further modified in plants to yield glycoproteins containing characteristic O-glycans. It is currently unknown how these plant endogenous modifications impact protein functionality and they cause considerable concerns for the recombinant production of therapeutic proteins in plants. In this study, we carried out host engineering to generate a therapeutic glycoprotein largely devoid of plant-endogenous O-glycans for functional characterization.Methods: Genome editing was used to inactivate two genes coding for enzymes of the P4H10 subfamily in the widely used expression host Nicotiana benthamiana. Using glycoengineering in plants and expression in human HEK293 cells we generated four variants of a potent, SARS-CoV-2 neutralizing antibody, COVA2-15 IgA1. The variants that differed in the number of modified proline residues and O-glycan compositions of their hinge region were assessed regarding their physicochemical properties and functionality.Results: We found that plant endogenous O-glycan formation was strongly reduced on IgA1 when transiently expressed in the P4H10 double mutant N. benthamiana plant line. The IgA1 glycoforms displayed differences in proteolytic stability and minor differences in receptor binding thus highlighting the importance of O-glycosylation in the hinge region of human IgA1.Discussion: This work reports the successful protein O-glycan engineering of an important plant host for recombinant protein expression. While the complete removal of endogenous hydroxyproline residues from the hinge region of plant-produced IgA1 is yet to be achieved, our engineered line is suitable for structure-function studies of O-glycosylated recombinant glycoproteins produced in plants.

Plants are being increasingly recognized for the production of complex human proteins, including monoclonal antibodies (mAbs). Various methods have been applied to boost recombinant expression, with DNA codon usage being an important approach. Here, we transiently expressed three complex human mAbs in Nicotiana benthamiana, namely one IgG3 and two IgM directed against SARS-CoV-2 as codon optimized(CO) and non-codon optimized (NCO) variants. qRT-PCR exhibited significantly increased mRNA levels of all CO variants compared to the non-codon optimized orthologues, in line with increased protein expression. Purified CO and NCO mAbs did not exhibit obvious biochemical differences, as determined by SDS-PAGE and antigen binding activities. By contrast, enhanced production selectively impacts on glycosite occupancy and N-glycan processing, with increased mannosidic structures. The results point to a careful monitoring of recombinant proteins upon enhancing expression. Especially if it comes to therapeutic application even subtle modifications might alter product efficacy or increase immunogenicity.

IntroductionThe Golgi apparatus of plants is the central cellular organelle for glycan processing and polysaccharide biosynthesis. These essential processes are catalyzed by a large number of Golgi-resident glycosyltransferases and glycosidases whose organization within the Golgi is still poorly understood.MethodsHere, we examined the role of the stem region of the cis/medial Golgi enzyme N-acetylglucosaminyltransferase I (GNTI) in homomeric complex formation in the Golgi of Nicotiana benthamiana using biochemical approaches and confocal microscopy.ResultsTransient expression of the N-terminal cytoplasmic, transmembrane, and stem (CTS) regions of GNTI leads to a block in N-glycan processing on a co-expressed recombinant glycoprotein. Overexpression of the CTS region from Golgi α-mannosidase I, which can form in planta complexes with GNTI, results in a similar block in N-glycan processing, while GNTI with altered subcellular localization or N-glycan processing enzymes located further downstream in the Golgi did not affect complex N-glycan processing. The GNTI-CTS-dependent alteration in N-glycan processing is caused by a specific nine-amino acid sequence motif in the stem that is required for efficient GNTI-GNTI interaction.DiscussionTaken together, we have identified a conserved motif in the stem region of the key N-glycan processing enzyme GNTI. We propose that the identified sequence motif in the GNTI stem region acts as a dominant negative motif that can be used in transient glycoengineering approaches to produce recombinant glycoproteins with predominantly mannosidic N-glycans.

Subunit vaccines based on recombinant viral antigens are valuable interventions to fight existing and evolving viruses and can be produced at large-scale in plant-based expression systems. The recombinant viral antigens are often derived from glycosylated envelope proteins of the virus and glycosylation plays an important role for the immunogenicity by shielding protein epitopes. The receptor-binding domain (RBD) of the SARS-CoV-2 spike is a principal target for vaccine development and has been produced in plants, but the yields of recombinant RBD variants were low and the role of the N-glycosylation in RBD from different SARS-CoV-2 variants of concern is less studied. Here, we investigated the expression and glycosylation of six different RBD variants transiently expressed in leaves of Nicotiana benthamiana. All of the purified RBD variants were functional in terms of receptor binding and displayed almost full N-glycan occupancy at both glycosylation sites with predominately complex N-glycans. Despite the high structural sequence conservation of the RBD variants, we detected a variation in yield which can be attributed to lower expression and differences in unintentional proteolytic processing of the C-terminal polyhistidine tag used for purification. Glycoengineering towards a human-type complex N-glycan profile with core α1,6-fucose, showed that the reactivity of the neutralizing antibody S309 differs depending on the N-glycan profile and the RBD variant.

Cytotoxic T lymphocyte-associated protein 4 (CTLA-4) is an immune checkpoint regulator exclusively expressed on T cells that obstructs the cell’s effector functions. Ipilimumab (Yervoy®), a CTLA-4 blocking antibody, emerged as a notable breakthrough in modern cancer treatment, showing upfront clinical benefits in multiple carcinomas. However, the exhilarating cost of checkpoint blockade therapy is discouraging and even utmost prominent in developing countries. Thereby, affordability of cancer care has become a point of emphasis in drug development pipelines. Plant expression system blossomed as a cutting-edge platform for rapid, facile to scale-up, and economical production of recombinant therapeutics. Here, we describe the production of an anti-CTLA-4 2C8 antibody in Nicotiana benthamiana. ELISA and bio-layer interferometry were used to analyze antigen binding and binding kinetics. Anticancer responses in vivo were evaluated using knocked-in mice implanted with syngeneic colon tumor. At 4 days post-infiltration, the antibody was transiently expressed in plants with yields of up to 39.65 ± 8.42 μg/g fresh weight. Plant-produced 2C8 binds to both human and murine CTLA-4, and the plant-produced IgG1 also binds to human FcγRIIIa (V158). In addition, the plant-produced 2C8 monoclonal antibody is as effective as Yervoy® in inhibiting tumor growth in vivo. In conclusion, our study underlines the applicability of plant platform to produce functional therapeutic antibodies with promising potential in cancer immunotherapy.

N-Glycosylation of immunoglobulin G1 (IgG1) at the heavy chain Fc domain (Asn297) plays an important role for antibody structure and effector functions. While numerous recombinant IgG1 antibodies have been successfully expressed in plants, they frequently display a considerable amount (up to 50%) of unglycosylated Fc domain. To overcome this limitation, we tested a single-subunit oligosaccharyltransferase from the protozoan Leishmania donovani (LdOST) for its ability to improve IgG1 Fc glycosylation. LdOST fused to a fluorescent protein was transiently expressed in Nicotiana benthamiana and confocal microscopy confirmed the subcellular location at the endoplasmic reticulum. Transient co-expression of LdOST with two different IgG1 antibodies resulted in a significant increase (up to 97%) of Fc glycosylation while leaving the overall N-glycan composition unmodified, as determined by different mass spectrometry approaches. While biochemical and functional features of “glycosylation improved” antibodies remained unchanged, a slight increase in FcγRIIIa binding and thermal stability was observed. Collectively, our results reveal that LdOST expression is suitable to reduce the heterogeneity of plant-produced antibodies and can contribute to improving their stability and effector functions.

Immunoglobulin M (IgM) is the largest antibody isotype with unique features like extensive glycosylation and oligomerization. Major hurdles in characterizing its properties are difficulties in the production of well-defined multimers. Here we report the expression of two SARS-CoV-2 neutralizing monoclonal antibodies in glycoengineered plants. Isotype switch from IgG1 to IgM resulted in the production of IgMs, composed of 21 human protein subunits correctly assembled into pentamers. All four recombinant monoclonal antibodies carried a highly reproducible human-type N-glycosylation profile, with a single dominant N-glycan species at each glycosite. Both pentameric IgMs exhibited increased antigen binding and virus neutralization potency, up to 390-fold, compared to the parental IgG1. Collectively, the results may impact on the future design of vaccines, diagnostics and antibody-based therapies and emphasize the versatile use of plants for the expression of highly complex human proteins with targeted posttranslational modifications.

SARS-CoV-2 infects human cells via binding of the viral spike glycoprotein to its main cellular receptor, angiotensin-converting enzyme 2 (ACE2). The spike protein-ACE2 receptor interaction is therefore a major target for the development of therapeutic or prophylactic drugs to combat coronavirus infections. Various engineered soluble ACE2 variants (decoys) have been designed and shown to exhibit virus neutralization capacity in cell-based assays and in vivo models. Human ACE2 is heavily glycosylated and some of its glycans impair binding to the SARS-CoV-2 spike protein. Therefore, glycan-engineered recombinant soluble ACE2 variants might display enhanced virus-neutralization potencies. Here, we transiently co-expressed the extracellular domain of ACE2 fused to human Fc (ACE2-Fc) with a bacterial endoglycosidase in Nicotiana benthamiana to produce ACE2-Fc decorated with N-glycans consisting of single GlcNAc residues. The endoglycosidase was targeted to the Golgi apparatus with the intention to avoid any interference of glycan removal with concomitant ACE2-Fc protein folding and quality control in the endoplasmic reticulum. The in vivo deglycosylated ACE2-Fc carrying single GlcNAc residues displayed increased affinity to the receptor-binding domain (RBD) of SARS-CoV-2 as well as improved virus neutralization activity and thus is a promising drug candidate to block coronavirus infection.

Molecular farming of vaccines has been heralded as a cheap, safe and scalable production platform. In reality, however, differences in the plant biosynthetic machinery, compared to mammalian cells, can complicate the production of viral glycoproteins. Remodelling the secretory pathway presents an opportunity to support key post-translational modifications, and to tailor aspects of glycosylation and glycosylation-directed folding. In this study, we applied an integrated host and glyco-engineering approach, NXS/T Generation™, to produce a SARS-CoV-2 prefusion spike trimer in Nicotiana benthamiana as a model antigen from an emerging virus. The size exclusion-purified protein exhibited a characteristic prefusion structure when viewed by transmission electron microscopy, and this was indistinguishable from the equivalent mammalian cell-produced antigen. The plant-produced protein was decorated with under-processed oligomannose N-glycans and exhibited a site occupancy that was comparable to the equivalent protein produced in mammalian cell culture. Complex-type glycans were almost entirely absent from the plant-derived material, which contrasted against the predominantly mature, complex glycans that were observed on the mammalian cell culture-derived protein. The plant-derived antigen elicited neutralizing antibodies against both the matched Wuhan and heterologous Delta SARS-CoV-2 variants in immunized hamsters, although titres were lower than those induced by the comparator mammalian antigen. Animals vaccinated with the plant-derived antigen exhibited reduced viral loads following challenge, as well as significant protection from SARS-CoV-2 disease as evidenced by reduced lung pathology, lower viral loads and protection from weight loss. Nonetheless, animals immunized with the mammalian cell-culture-derived protein were better protected in this challenge model suggesting that more faithfully reproducing the native glycoprotein structure and associated glycosylation of the antigen may be desirable.

Human polioviruses are highly infectious viruses that are spread mainly through the fecal-oral route. Infection of the central nervous system frequently results in irreversible paralysis, a disease called poliomyelitis. Children under five years are mainly affected if they have not acquired immunity through natural infection or via vaccination. Current polio vaccines comprise the injectable inactivated polio vaccine (IPV, also called the Salk vaccine) and the live-attenuated oral polio vaccine (OPV, also called the Sabin vaccine). The main limitations of the IPV are the reduced protection at the intestinal mucosa, the site of virus replication, and the high costs for manufacturing due to use of live viruses. While the OPV is more effective and stimulates mucosal immunity, it is manufactured using live-attenuated strains that can revert into pathogenic viruses resulting in major safety concerns and vaccine-derived outbreaks. During the last fifteen years, plant-based poliovirus vaccines have been explored by several groups as a safe and low-cost alternative, and promising results in protection against challenges with viruses and induction of neutralizing antibodies have been obtained. However, low yields and a high frequency in dose administration highlight the need for improvements in polioviral antigen production. In this review, we provide insights into recent efforts to develop plant-made poliovirus candidates, with an emphasis on strategies to optimize the production of viral antigens.

Asparagine (N)-linked protein glycosylation is a ubiquitous co- and posttranslational modification which has a huge impact on the biogenesis and function of proteins and consequently on the development, growth, and physiology of organisms. In mammals, N-glycan processing carried out by Golgi-resident glycosidases and glycosyltransferases creates a number of structurally diverse N-glycans with specific roles in many different biological processes. In plants, complex N-glycan modifications like the attachment of β1,2-xylose, core α1,3-fucose, or the Lewis A-type structures are evolutionary highly conserved, but their biological function is poorly known. Here, I highlight recent developments that contribute to a better understanding of these conserved glycoprotein modifications and discuss future directions to move the field forward.

Glycosylation of viral envelope proteins is important for infectivity and immune evasion. The SARS-CoV-2 spike protein is heavily glycosylated and host-derived glycan modifications contribute to the formation of specific immunogenic epitopes, enhance the virus-cell interaction or affect virus transmission. On recombinant viral antigens used as subunit vaccines or for serological assays, distinct glycan structures may enhance the immunogenicity and are recognized by naturally occurring antibodies in human sera. Here, we performed an in vivo glycoengineering approach to produce recombinant variants of the SARS-CoV-2 receptor-binding domain (RBD) with blood group antigens in Nicotiana benthamiana plants. SARS-CoV-2 RBD and human glycosyltransferases for the blood group ABH antigen formation were transiently co-expressed in N. benthamiana leaves. Recombinant RBD was purified and the formation of complex N-glycans carrying blood group A antigens was shown by immunoblotting and MS analysis. Binding to the cellular ACE2 receptor and the conformation-dependent CR3022 antibody showed that the RBD glycosylation variants carrying blood group antigens were functional. Analysis of sera from RBD-positive and RBD-negative individuals revealed further that non-infected RBD-negative blood group O individuals have antibodies that strongly bind to RBD modified with blood group A antigen structures. The binding of IgGs derived from sera of non-infected RBD-negative blood group O individuals to blood group A antigens on SARS-CoV-2 RBD suggests that these antibodies could provide some degree of protection from virus infection.

The striking innovation and clinical success of immune checkpoint inhibitors (ICIs) have undoubtedly contributed to a breakthrough in cancer immunotherapy. Generally, ICIs produced in mammalian cells requires high investment, production costs, and involves time consuming procedures. Recently, the plants are considered as an emerging protein production platform due to its cost-effectiveness and rapidity for the production of recombinant biopharmaceuticals. This study explored the potential of plant-based system to produce an anti-human PD-1 monoclonal antibody (mAb), Pembrolizumab, in Nicotiana benthamiana. The transient expression of this mAb in wild-type N. benthamiana accumulated up to 344.12 ± 98.23 μg/g fresh leaf weight after 4 days of agroinfiltration. The physicochemical and functional characteristics of plant-produced Pembrolizumab were compared to mammalian cell-produced commercial Pembrolizumab (Keytruda®). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis results demonstrated that the plant-produced Pembrolizumab has the expected molecular weight and is comparable with the Keytruda®. Structural characterization also confirmed that both antibodies have no protein aggregation and similar secondary and tertiary structures. Furthermore, the plant-produced Pembrolizumab displayed no differences in its binding efficacy to PD-1 protein and inhibitory activity between programmed cell death 1 (PD-1) and programmed cell death ligand 1 (PD-L1) interaction with the Keytruda®. In vitro efficacy for T cell activation demonstrated that the plant-produced Pembrolizumab could induce IL-2 and IFN-γ production. Hence, this proof-of-concept study showed that the plant-production platform can be utilized for the rapid production of functional mAbs for immunotherapy.

The receptor binding domain (RBD) of the SARS-CoV-2 spike protein plays a key role in the virus-host cell interaction, and viral infection. The RBD is a major target for neutralizing antibodies, whilst recombinant RBD is commonly used as an antigen in serological assays. Such assays are essential tools to gain control over the pandemic and detect the extent and durability of an immune response in infected or vaccinated populations. Transient expression in plants can contribute to the fast production of viral antigens, which are required by industry in high amounts. Whilst plant-produced RBDs are glycosylated, N-glycan modifications in plants differ from humans. This can give rise to the formation of carbohydrate epitopes that can be recognized by anti-carbohydrate antibodies present in human sera. For the performance of serological tests using plant-produced recombinant viral antigens, such cross-reactive carbohydrate determinants (CCDs) could result in false positives. Here, we transiently expressed an RBD variant in wild-type and glycoengineered Nicotiana benthamiana leaves and characterized the impact of different plant-specific N-glycans on RBD reactivity in serological assays. While the overall performance of the different RBD glycoforms was comparable to each other and to a human cell line produced RBD, there was a higher tendency toward false positive results with sera containing allergy-related CCD-antibodies when an RBD carrying β1,2-xylose and core α1,3-fucose was used. These rare events could be further minimized by pre-incubating sera from allergic individuals with a CCD-inhibitor. Thereby, false positive signals obtained from anti-CCD antibodies, could be reduced by 90%, on average.

The Arabidopsis ER-α-mannosidase I MNS3 generates N-glycan structures typical of ER-resident glycoproteins. Here Schoberer et al. identify a novel motif that anchors MNS3 to the cis-Golgi, spatially separating MNS3 from ER-localized mannose trimming associated with the ER-associated degradation pathway.

Abstract Background The allotetraploid tobacco species Nicotiana benthamiana native to Australia has become a popular host for recombinant protein production. Although its usage grows every year, little is known on this plant’s genomic and transcriptomic features. Most N. benthamiana accessions currently used in research lack proper documentation of their breeding history and provenance. One of these, the glycoengineered N. benthamiana line ΔXT/FT is increasingly used for the production of biopharmaceutical proteins. Results Based on an existing draft assembly of the N. benthamiana genome we predict 50,516 protein –encoding genes (62,216 transcripts) supported by expression data derived from 2.35 billion mRNA-seq reads. Using single-copy core genes we show high completeness of the predicted gene set. We functionally annotate more than two thirds of the gene set through sequence homology to genes from other Nicotiana species. We demonstrate that the expression profiles from leaf tissue of ΔXT/FT and its wild type progenitor only show minimal differences. We identify the transgene insertion sites in ΔXT/FT and show that one of the transgenes was inserted inside another predicted gene that most likely lost its function upon insertion. Based on publicly available mRNA-seq data, we confirm that the N. benthamiana accessions used by different research institutions most likely derive from a single source. Conclusions This work provides gene annotation of the N. benthamiana genome, a genomic and transcriptomic characterization of a transgenic N. benthamiana line in comparison to its wild-type progenitor, and sheds light onto the relatedness of N. benthamiana accessions that are used in laboratories around the world.

Nicotiana benthamiana is used worldwide as production host for recombinant proteins. Many recombinant proteins such as monoclonal antibodies, growth factors or viral antigens require posttranslational modifications like glycosylation for their function. Here, we transiently expressed different variants of the glycosylated receptor binding domain (RBD) from the SARS-CoV-2 spike protein in N. benthamiana. We characterized the impact of variations in RBD-length and posttranslational modifications on protein expression, yield and functionality. We found that a truncated RBD variant (RBD-215) consisting of amino acids Arg319-Leu533 can be efficiently expressed as a secreted soluble protein. Purified RBD-215 was mainly present as a monomer and showed binding to the conformation-dependent antibody CR3022, the cellular receptor angiotensin converting enzyme 2 (ACE2) and to antibodies present in convalescent sera. Expression of RBD-215 in glycoengineered ΔXT/FT plants resulted in the generation of complex N-glycans on both N-glycosylation sites. While site-directed mutagenesis showed that the N-glycans are important for proper RBD folding, differences in N-glycan processing had no effect on protein expression and function.

Denosumab, an anti-receptor activator of nuclear factor-kappa B ligand antibody (anti-RANKL), is a fully human monoclonal antibody (mAb) available for the treatment of osteoporosis. In the present study, an anti-RANKL mAb was transiently expressed using the geminiviral expression system in Nicotiana benthamiana, and the functional activity of the plant-produced mAb was determined. The highest expression level of the plant-produced mAb was found at 8 days post-infiltration, and it was estimated to be 0.5 mg/g leaf fresh weight. The recombinant mAb from the plant crude extracts was purified by using Protein A affinity column chromatography. The plant-produced mAb demonstrated good in vitro affinity binding with human RANKL, as determined by RANKL-ELISA binding. The function of the plant-produced mAb was evaluated in vitro. CD14-positive cells isolated from human peripheral blood mononuclear cells (PBMCs) were cultured in vitro in the presence of human RANKL and macrophage-colony-stimulating factor (M-CSF) to stimulate osteoclastogenesis. The results demonstrated that plant-produced mAb could significantly decrease the number of osteoclasts compared to commercial denosumab. These results demonstrated that the plant-produced mAb has the potential to inhibit osteoclast differentiation and that it could be considered for osteoporosis treatment.

Background: Antibody tests are essential tools to investigate humoral immunity following SARS-CoV-2 infection or vaccination. While first-generation antibody tests have primarily provided qualitative results, accurate seroprevalence studies and tracking of antibody levels over time require highly specific, sensitive and quantitative test setups. Methods: We have developed two quantitative, easy-to-implement SARS-CoV-2 antibody tests, based on the spike receptor binding domain and the nucleocapsid protein. Comprehensive evaluation of antigens from several biotechnological platforms enabled the identification of superior antigen designs for reliable serodiagnostic. Cut-off modelling based on unprecedented large and heterogeneous multicentric validation cohorts allowed us to define optimal thresholds for the tests’ broad applications in different aspects of clinical use, such as seroprevalence studies and convalescent plasma donor qualification. Findings: Both developed serotests individually performed similarly-well as fully-automated CE-marked test systems. Our described sensitivity-improved orthogonal test approach assures highest specificity (99.8%); thereby enabling robust serodiagnosis in low-prevalence settings with simple test formats. The inclusion of a calibrator permits accurate quantitative monitoring of antibody concentrations in samples collected at different time points during the acute and convalescent phase of COVID-19 and disclosed antibody level thresholds that correlate well with robust neutralization of authentic SARS-CoV-2 virus. Interpretation: We demonstrate that antigen source and purity strongly impact serotest performance. Comprehensive biotechnology-assisted selection of antigens and in-depth characterisation of the assays allowed us to overcome limitations of simple ELISA-based antibody test formats based on chromometric reporters, to yield comparable assay performance as fully-automated platforms. Funding: WWTF, Project No. COV20–016; BOKU, LBI/LBG

Plant glycoproteins display a characteristic type of O-glycosylation where short arabinans or larger arabinogalactans are linked to hydroxyproline. The conversion of proline to 4-hydroxyproline is accomplished by prolyl-hydroxylases (P4Hs). Eleven putative Nicotiana benthamiana P4Hs, which fall in four homology groups, have been identified by homology searches using known Arabidopsis thaliana P4H sequences. One member of each of these groups has been expressed in insect cells using the baculovirus expression system and applied to synthetic peptides representing the O-glycosylated region of erythropoietin (EPO), IgA1, Art v 1 and the Arabidopsis thaliana glycoprotein STRUBBELIG. Unlike the situation in the moss Physcomitrella patens, where one particular P4H was mainly responsible for the oxidation of erythropoietin, the tobacco P4Hs exhibited rather similar activities, albeit with biased substrate preferences and preferred sites of oxidation. From a biotechnological viewpoint, this result means that silencing/knockout of a single P4H in N. benthamiana cannot be expected to result in the abolishment of the plant-specific oxidation of prolyl residues in a recombinant protein.

N-glycosylation is a highly abundant protein modification present in all domains of life. Terminal sugar residues on complex-type N-glycans mediate various crucial biological processes in mammals such as cell-cell recognition or protein-ligand interactions. In plants, the Lewis A trisaccharide constitutes the only known outer-chain elongation of complex N-glycans. Lewis A containing complex N-glycans appear evolutionary conserved, having been identified in all plant species analyzed so far. Despite their ubiquitous occurrence, the biological function of this complex N-glycan modification is currently unknown. Here, we report the identification of Lewis A bearing glycoproteins from three different plant species: Arabidopsis thaliana, Nicotiana benthamiana, and Oryza sativa. Affinity purification via the JIM84 antibody, directed against Lewis A structures on complex plant N-glycans, was used to enrich Lewis A bearing glycoproteins, which were subsequently identified via nano-LC-MS. Selected identified proteins were recombinantly expressed and the presence of Lewis A confirmed via immunoblotting and site-specific N-glycan analysis. While the proteins identified in O. sativa are associated with diverse functions, proteins from A. thaliana and N. benthamiana are mainly involved in cell wall biosynthesis. However, a Lewis A-deficient mutant line of A. thaliana showed no change in abundance of cell wall constituents such as cellulose or lignin. Furthermore, we investigated the presence of Lewis A structures in selected accessions from the 1001 genome database containing amino acid variations in the enzymes required for Lewis A biosynthesis. Besides one relict line showing no detectable levels of Lewis A, the modification was present in all other tested accessions. The data provided here comprises the so far first attempt at identifying Lewis A bearing glycoproteins across different species and will help to shed more light on the role of Lewis A structures in plants.

Secretions of parasitic worms (helminths) contain a wide collection of immunomodulatory glycoproteins with the potential to treat inflammatory disorders, like autoimmune diseases. Yet, the identification of single molecules that can be developed into novel biopharmaceuticals is hampered by the limited availability of native parasite-derived proteins. Recently, pioneering work has shown that helminth glycoproteins can be produced transiently in Nicotiana benthamiana plants while simultaneously mimicking their native helminth N-glycan composition by co-expression of desired glycosyltransferases. However, efficient “helminthization” of N-glycans in plants by glyco-engineering seems to be hampered by the undesired truncation of complex N-glycans by β-N-acetyl-hexosaminidases, in particular when aiming for the synthesis of N-glycans with antennary GalNAcβ1-4GlcNAc (LacdiNAc or LDN). In this study, we cloned novel β-hexosaminidase open reading frames from N. benthamiana and characterized the biochemical activity of these enzymes. We identified HEXO2 and HEXO3 as enzymes responsible for the cleavage of antennary GalNAc residues of N-glycans on the model helminth glycoprotein kappa-5. Furthermore, we reveal that each member of the HEXO family has a distinct specificity for N-glycan substrates, where HEXO2 has strict β-galactosaminidase activity, whereas HEXO3 cleaves both GlcNAc and GalNAc. The identification of HEXO2 and HEXO3 as major targets for LDN cleavage will enable a targeted genome editing approach to reduce undesired processing of these N-glycans. Effective knockout of these enzymes could allow the production of therapeutically relevant glycoproteins with tailor-made helminth N-glycans in plants.

Glycosylation is a fundamental co-translational and/or post-translational modification process where an attachment of sugars onto either proteins or lipids can alter their biological function, subcellular location and modulate the development and physiology of an organism. Glycosylation is not a template driven process and as such produces a vastly larger array of glycan structures through combinatorial use of enzymes and of repeated common scaffolds and as a consequence it provides a huge expansion of both the proteome and lipidome. While the essential role of N- and O-glycan modifications on mammalian glycoproteins is already well documented, we are just starting to decode their biological functions in plants. Although significant advances have been made in plant glycobiology in the last decades, there are still key challenges impeding progress in the field and, as such, holistic modern high throughput approaches may help to address these conceptual gaps. In this snapshot, we present an update of the most common O- and N-glycan structures present on plant glycoproteins as well as (1) the plant glycosyltransferases (GTs) and glycosyl hydrolases (GHs) responsible for their biosynthesis; (2) a summary of microorganism-derived GHs characterized to cleave specific glycosidic linkages; (3) a summary of the available tools ranging from monoclonal antibodies (mAbs), lectins to chemical probes for the detection of specific sugar moieties within these complex macromolecules; (4) selected examples of N- and O-glycoproteins as well as in their related GTs to illustrate the complexity on their mode of action in plant cell growth and stress responses processes, and finally (5) we present the carbohydrate microarray approach that could revolutionize the way in which unknown plant GTs and GHs are identified and their specificities characterized.

The current COVID-19 pandemic very dramatically shows that the world lacks preparedness for novel viral diseases. In addition to newly emerging viruses, many known pathogenic viruses such as influenza are constantly evolving, leading to frequent outbreaks with severe diseases and deaths. Hence, infectious viruses are a recurrent burden to our daily life, and powerful strategies to stop the spread of human pathogens and disease progression are of utmost importance. Transient plant-based protein expression is a technology that allows fast and highly flexible manufacturing of recombinant viral proteins and, thus, can contribute to infectious disease detection and prevention. This review highlights recent progress in the transient production of viral glycoproteins in N. benthamiana with a focus on SARS-CoV-2-derived viral antigens.

More than 200 diverse secretory proteins from Arabidopsis thaliana carry a glycosylphosphatidylinositol (GPI) lipid anchor covalently attached to their carboxyl-terminus. The GPI-anchor contains a lipid-linked glycan backbone that is preassembled in the endoplasmic reticulum (ER) of plants and subsequently transferred to distinct proteins, which provides them with specific features. The GPI-anchored proteins exit the ER and are transported through the Golgi apparatus to the plasma membrane. In the Golgi, the glycan moiety can be further modified by the specific attachment of sugar residues. While these biosynthetic steps are already quite well understood in mammals and yeast, comparatively little is known in plants. In this perspective, we discuss the current knowledge about the biosynthesis of the GPI-anchor glycan moiety in the light of recent findings for mammalian GPI-anchor glycan modifications.

Production of monomeric IgA in mammalian cells and plant expression systems such as Nicotiana benthamiana is well-established and can be achieved by co-expression of the corresponding light and heavy chains. In contrast, the assembly of dimeric IgA requires the additional expression of the joining chain and remains challenging especially in plant-based systems. Here, we examined factors affecting the assembly and expression of HER2 binding dimeric IgA1 and IgA2m(2) variants transiently produced in N. benthamiana. While co-expression of the joining chain resulted in efficient formation of dimeric IgAs in HEK293F cells, a mixture of monomeric, dimeric and tetrameric variants was detected in plants. Mass-spectrometric analysis of site-specific glycosylation revealed that the N-glycan profile differed between monomeric and dimeric IgAs in the plant expression system. Co-expression of a single-subunit oligosaccharyltransferase from the protozoan Leishmania major in N. benthamiana increased the N-glycosylation occupancy at the C-terminal heavy chain tailpiece and changed the ratio of monomeric to dimeric IgAs. Our data demonstrate that N-glycosylation engineering is a suitable strategy to promote the formation of dimeric IgA variants in plants.

N-glycosylation is an essential protein modification that plays roles in many diverse biological processes including protein folding, quality control and protein interactions. Despite recent advances in characterization of the N-glycosylation and N-glycan processing machinery our understanding of N-glycosylation related processes in plant development is limited. In Arabidopsis thaliana, failure of mannose trimming from oligomannosidic N-glycans in the endoplasmic reticulum (ER) and cis/medial-Golgi leads to a defect in root development in the mns123 triple mutant. Here, we show that the severe root phenotype of mns123 is restored in asparagine-linked glycosylation (ALG)-deficient plants with distinct defects in the biosynthesis of the lipid-linked oligosaccharide precursor. The root growth of these ALG-deficient plants is not affected by the α-mannosidase inhibitor kifunensine. Genetic evidence shows that the defect is uncoupled from the glycan-dependent ER-associated degradation (ERAD) pathway that removes misfolded glycoproteins with oligomannosidic N-glycans from the ER. Restoration of mannose trimming using a trans-Golgi targeted α-mannosidase suppresses the defect of mns123 roots. These data suggest that processing of terminal mannose residues from oligomannosidic N-glycans is important for an unknown late-Golgi or post-Golgi process that is implicated in proper root formation.