Stability Engineering of Recombinant Secretory IgA.

Secretory IgA (SIgA) presents a promising avenue for mucosal immunotherapy yet faces challenges in expression, purification, and stability. IgA exists in two primary isotypes, IgA1 and IgA2, with IgA2 further subdivided into two common allotypes: IgA2m(1) and IgA2m(2). The major differences between IgA1 and IgA2 are located in the hinge region, with IgA1 featuring a 13-amino acid elongation that includes up to six O-glycosylation sites. Furthermore, the IgA2m(1) allotype lacks a covalent disulfide bond between heavy and light chains, which is present in IgA1 and IgA2m(2). While IgA1 demonstrates superior epitope binding and pathogen neutralization, IgA2 exhibits enhanced effector functions and stability against mucosal bacterial degradation. However, the noncovalent linkage in the IgA2m(1) allotype raises production and stability challenges. The introduction of distinct single mutations aims to facilitate an alternate disulfide bond formation to mitigate these challenges. We compare four different IgA2 versions with IgA1 to further develop secretory IgA antibodies against SARS-CoV-2 for topical delivery to mucosal surfaces. Our results indicate significantly improved expression levels and assembly efficacy of SIgA2 (P221R) in Nicotiana benthamiana. Moreover, engineered SIgA2 displays heightened thermal stability under physiological as well as acidic conditions and can be aerosolized using a mesh nebulizer. In summary, our study elucidates the benefits of stability-enhancing mutations in overcoming hurdles associated with SIgA expression and stability.

The impact of N-glycans on the immune response of plant-produced SARS-CoV-2 RBD-Fc proteins.

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.

Nicotiana benthamiana as a potential source for producing anti-dengue virus D54 neutralizing therapeutic antibody.

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.

Characterization of plant produced VHH antibodies against cobra venom toxins for antivenom therapy.

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.

What comes next in glycobiology.

Glycans, with their variable compositions and highly dynamic conformations, vastly expand the heterogeneity of whatever factor or cell they are attached to. These properties make them crucial contributors to biological function and organismal health and also very difficult to study. That may be changing as we look to the future of glycobiology.

Effects of N-glycans on the structure of human IgA2.

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.

Suborganellar resolution imaging for the localisation of human glycosylation enzymes in tobacco Golgi bodies.

Plant cells are a capable system for producing economically and therapeutically important proteins for a variety of applications, and are considered a safer production system than some existing hosts such as bacteria or yeasts. However, plants do not perform protein modifications in the same manner as mammalian cells do. This can impact on protein functionality for plant-produced human therapeutics. This obstacle can be overcome by creating a plant-based system capable of 'humanising' proteins of interest resulting in a glycosylation profile of synthetic plant-produced proteins as it would occur in mammalian systems. For this, the human glycosylation enzymes (HuGEs) involved in N-linked glycosylation N-acetylglucosaminyltransferase IV and V (GNTIV and GNTV), ß-1,4-galactosyltransferase (B4GALT1), and α-2,6-sialyltransferase (ST6GAL) were expressed in plant cells. For these enzymes to carry out the stepwise glycosylation functions, they need to localise to late Golgi body cisternae. This was achieved by a protein targeting strategy of replacing the mammalian Golgi targeting domains (Cytoplasmic-Transmembrane-Stem (CTS) regions) with plant-specific ones. Using high-resolution and dynamic confocal microscopy, we show that GNTIV and GNTV were successfully targeted to the medial-Golgi cisternae while ST6GAL and B4GALT1 were targeted to trans-Golgi cisternae. Plant cells are a promising system to produce human therapeutics for example proteins used in enzyme replacement therapies. Plants can provide safer and cheaper alternatives to existing expression systems such as mammalian cell culture, bacteria or yeast. An important factor for the functionality of therapeutic proteins though are protein modifications specific to human cells. However, plants do not perform protein modifications in the same manner as human cells do. Therefore, plant cells need to be genetically modified to mimic human protein modifications patterns. The modification of importance here, is called N-linked glycosylation and adds specific sugar molecules onto the proteins. Here we show the expression of four human glycosylation enzymes, which are required for N-linked glycosylation, in plant cells. In addition, as these protein modifications are carried out in cells resembling a factory production line, it is important that the human glycosylation enzymes be placed in the correct cellular compartments and in the correct order. This is carried out in Golgi bodies. Golgi bodies are composed of several defined stacks termed cis-, medial and trans-Golgi body stacks. For correct protein function, two of these human glycosylation enzymes need to be placed in the medial-Golgi attacks and the other two in the trans-Golgi stacks. Using high-resolution laser microscopy in live plant cells, we show here that the human glycosylation enzymes are sent within the cells to the correct Golgi body stacks. These are first steps to modify plant cells in order to produce human therapeutics.

Quality control in SARS-CoV-2 RBD-Fc vaccine production using LC-MS to confirm strain selection and detect contaminations from other strains.

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.

Neutralizing activity of anti-respiratory syncytial virus monoclonal antibody produced in Nicotiana benthamiana.

Respiratory 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.

Implications of O-glycan modifications in the hinge region of a plant-produced SARS-CoV-2-IgA antibody on functionality.

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.

Analysis of Protein Glycosylation in the ER.

Protein N-glycosylation is an essential posttranslational modification which is initiated in the endoplasmic reticulum (ER). In plants, the N-glycans play a pivotal role in protein folding and quality control. Through the interaction of glycan processing and binding reactions mediated by ER-resident glycosidases and specific carbohydrate-binding proteins, the N-glycans contribute to the adoption of a native protein conformation. Properly folded glycoproteins are released from these processes and allowed to continue their transit to the Golgi where further processing and maturation of N-glycans leads to the formation of more complex structures with different functions. Incompletely folded glycoproteins are removed from the ER by a highly conserved degradation process to prevent the accumulation or secretion of misfolded proteins and maintain ER homeostasis. Here, we describe methods to analyze the N-glycosylation status and the glycan-dependent ER-associated degradation process in plants.

Recombinant neutralizing secretory IgA antibodies for preventing mucosal acquisition and transmission of SARS-CoV-2.

Passive delivery of antibodies to mucosal sites may be a valuable adjunct to COVID-19 vaccination to prevent infection, treat viral carriage, or block transmission. Neutralizing monoclonal IgG antibodies are already approved for systemic delivery, and several clinical trials have been reported for delivery to mucosal sites where SARS-CoV-2 resides and replicates in early infection. However, secretory IgA may be preferred because the polymeric complex is adapted for the harsh, unstable external mucosal environment. Here, we investigated the feasibility of producing neutralizing monoclonal IgA antibodies against SARS-CoV-2. We engineered two class-switched mAbs that express well as monomeric and secretory IgA (SIgA) variants with high antigen-binding affinities and increased stability in mucosal secretions compared to their IgG counterparts. SIgAs had stronger virus neutralization activities than IgG mAbs and were protective against SARS-CoV-2 infection in an in vivo murine model. Furthermore, SIgA1 can be aerosolized for topical delivery using a mesh nebulizer. Our findings provide a persuasive case for developing recombinant SIgAs for mucosal application as a new tool in the fight against COVID-19.

Codon optimization regulates IgG3 and IgM expression and glycosylation in N. benthamiana.

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.