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.

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.

Weight, temperature and humidity sensor data of honey bee colonies in Germany, 2019-2022.

Humans have kept honeybees as livestock to harvest honey, wax and other products for thousands of years and still continue doing so. Today however, beekeepers in many parts of the world report unprecedented high numbers of colony losses. Sensor data from honey bee colonies can contribute to new insights about development and health factors for honey bee colonies. The data can be incorporated in smart decision support systems and warning tools for beekeepers. In this paper, we present sensor data from 78 honey bee colonies in Germany collected as part of a citizen science project. Each honey bee hive was equipped with five temperature sensors within the hive, one temperature sensor for outside measurements, a combined sensor for temperature, ambient air pressure and humidity, and a scale to measure the weight. During the data acquisition period, beekeepers used a web app to report their observations and beekeeping activities. We provide the raw data with a measurement interval of up to 5 s as well as aggregated data, with per minute, hourly or daily average values. Furthermore, we performed several preprocessing steps, removing outliers with a threshold based approach, excluding changes in weight that were induced by beekeeping activities and combining the sensor data with the most important meta-data from the beekeepers' observations. The data is organised in directories based on the year of recording. Alternatively, we provide subsets of the data structured based on the occurrence or non-occurrence of a swarming event or the death of a colony. The data can be analysed using methods from time series analysis, time series classification or other data science approaches to form a better understanding of specifics in the development of honey bee colonies.

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.

Simple Routes to Stable Isotope-Coded Native Glycans.

Understanding the biological role of protein-linked glycans requires the reliable identification of glycans. Isomer separation and characterization often entail mass spectrometric detection preceded by high-performance chromatography on porous graphitic carbon. To this end, stable isotope-labeled glycans have emerged as powerful tools for retention time normalization. Hitherto, such standards were obtained by chemoenzymatic or purely enzymatic methods, which introduce, e.g., 13C-containing N-acetyl groups or galactose into native glycans. Glycan release with anhydrous hydrazine opens another route for heavy isotope introduction via concomitant de-N-acetylation. Here, we describe that de-N-acetylation can also be achieved with hydrazine hydrate, which is a more affordable and less hazardous reagent. Despite the slower reaction rate, complete conversion is achievable in 72 h at 100 °C for glycans with biantennary glycans with or without sialic acids. Shorter incubation times allow for the isolation of intermediate products with a defined degree of free amino groups, facilitating introduction of different numbers of heavy isotopes. Mass encoded glycans obtained by this versatile approach can serve a broad range of applications, e.g., as internal standards for isomer-specific studies of N-glycans, O-glycans, and human milk oligosaccharide by LC-MS on either porous graphitic carbon or─following permethylation─on reversed phase.

The tobacco GNTI stem region harbors a strong motif for homomeric protein complex formation.

Introduction: The 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. Methods: Here, 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. Results: Transient 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. Discussion: Taken 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.

Harnessing Attenuation-Related Mutations of Viral Genomes: Development of a Serological Assay to Differentiate between Capripoxvirus-Infected and -Vaccinated Animals.

Sheeppox, goatpox, and lumpy skin disease caused by the sheeppox virus (SPPV), goatpox virus (GTPV), and lumpy skin disease virus (LSDV), respectively, are diseases that affect millions of ruminants and many low-income households in endemic countries, leading to great economic losses for the ruminant industry. The three viruses are members of the Capripoxvirus genus of the Poxviridae family. Live attenuated vaccines remain the only efficient means for controlling capripox diseases. However, serological tools have not been available to differentiate infected from vaccinated animals (DIVA), though crucial for proper disease surveillance, control, and eradication efforts. We analysed the sequences of variola virus B22R homologue gene for SPPV, GTPV, and LSDV and observed significant differences between field and vaccine strains in all three capripoxvirus species, resulting in the truncation and absence of the B22R protein in major vaccines within each of the viral species. We selected and expressed a protein fragment present in wildtype viruses but absent in selected vaccine strains of all three species, taking advantage of these alterations in the B22R gene. An indirect ELISA (iELISA) developed using this protein fragment was evaluated on well-characterized sera from vaccinated, naturally and experimentally infected, and negative cattle and sheep. The developed wildtype-specific capripox DIVA iELISA showed >99% sensitivity and specificity for serum collected from animals infected with the wildtype virus. To the best of our knowledge, this is the first wildtype-specific, DIVA-capable iELISA for poxvirus diseases exploiting changes in nucleotide sequence alterations in vaccine strains.

Impact of mutations on the plant-based production of recombinant SARS-CoV-2 RBDs.

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.

An oligosaccharyltransferase from Leishmania donovani increases the N-glycan occupancy on plant-produced IgG1.

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.

Recombinant Human CD19 in CHO-K1 Cells: Glycosylation Patterns as a Quality Attribute of High Yield Processes.

CD19 is an essential protein in personalized CD19-targeting chimeric antigen receptor (CAR)-T cell-based cancer immunotherapies and CAR-T cell functionality evaluation. However, the recombinant expression of this "difficult to-express" (DTE) protein is challenging, and therefore, commercial access to the protein is limited. We have previously described the successful stable expression of our soluble CD19-AD2 fusion protein of the CD19 extracellular part fused with human serum albumin domain 2 (AD2) in CHO-K1 cells. The function, stability, and secretion rate of DTE proteins can be improved by culture conditions, such as reduced temperature and a shorter residence time. Moreover, glycosylation, as one of the most important post-translational modifications, represents a critical quality attribute potentially affecting CAR-T cell effector function and thus impacting therapy's success. In this study, we increased the production rate of CD19-AD2 by 3.5-fold through applying hypothermic culture conditions. We efficiently improved the purification of our his-tagged CD19-AD2 fusion protein via a Ni-NTA-based affinity column using a stepwise increase in the imidazole concentration. The binding affinity to commercially available anti-CD19 antibodies was evaluated via Bio-Layer Interferometry (BLI). Furthermore, we revealed glycosylation patterns via Electrospray Ionization Mass Spectrometry (ESI-MS), and five highly sialylated and multi-antennary N-glycosylation sites were identified. In summary, we optimized the CD19-AD2 production and purification process and were the first to characterize five highly complex N-glycosylation sites.

In planta deglycosylation improves the SARS-CoV-2 neutralization activity of recombinant ACE2-Fc.

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.

Improving the efficacy of plant-made anti-HIV monoclonal antibodies for clinical use.

Introduction: Broadly neutralising antibodies are promising candidates for preventing and treating Human Immunodeficiency Virus/Acquired Immunodeficiency Syndrome (HIV/AIDS), as an alternative to or in combination with antiretroviral therapy (ART). These mAbs bind to sites on the virus essential for virus attachment and entry, thereby inhibiting entry into the host cell. However, the cost and availability of monoclonal antibodies, especially combinations of antibodies, hampers implementation of anti-HIV bNAb therapies in low- to middle- income countries (LMICs) where HIV-1 prevalence is highest. Methods: We have produced three HIV broadly neutralizing antibodies (bNAbs), 10-1074, VRC01 and 3BNC117 in the Nicotiana benthamiana transient expression system. The impact of specific modifications to enhance potency and efficacy were assessed. To prolong half-life and increase bioavailability, a M252Y/S254T/T256E (YTE) or M428L/N434S (LS) mutation was introduced. To increase antibody dependent cellular cytotoxicity (ADCC), we expressed an afucosylated version of each antibody using a glycoengineered plant line. Results: The majority of bNAbs and their variants could be expressed at yields of up to 47 mg/kg. Neither the expression system nor the modifications impacted the neutralization potential of the bNAbs. Afucosylated bNAbs exhibit enhanced ability to bind to FcγRIIIa and trigger ADCC, regardless of the presence of Fc amino acid mutations. Lastly, we demonstrated that Fc-modified variants expressed in plants show enhanced binding to FcRn, which results in a favourable in vivo pharmacokinetic profile compared to their unmodified counterparts. Conclusion: Tobacco plants are suitable expression hosts for anti-HIV bNAbs with increased efficacy and an improved pharmacokinetic profile.

Quantitative proteomic analysis of extracellular vesicles in response to baculovirus infection of a Trichoplusia ni cell line.

Due to its outstanding suitability to produce complex biopharmaceutical products including virus-like particles and subunit vaccines, the baculovirus/insect cell expression system has developed into a highly popular production platform in the biotechnological industry. For high productivity, virus-cell communication and an efficient spreading of the viral infection are crucial, and, in this context, extracellular vesicles (EVs) might play a significant role. EVs are small particles, utilized by cells to transfer biologically active compounds such as proteins, lipids as well as nucleic acids to recipient cells for intracellular communication. Studies in mammalian cells showed that the release of EVs is altered in response to infection with many viruses, ultimately either limiting or fostering infection spreading. In this study we isolated and characterized EVs, from both uninfected and baculovirus infected Tnms42 insect cells. Via quantitative proteomic analysis we identified more than 3000 T. ni proteins in Tnms42 cell derived EVs, of which more than 400 were significantly differentially abundant upon baculovirus infection. Subsequent gene set enrichment analysis revealed a depletion of proteins related to the extracellular matrix in EVs from infected cultures. Our findings show a significant change of EV protein cargo upon baculovirus infection, suggesting a major role of EVs as stress markers. Our study might serve in designing new tools for process monitoring and control to further improve biopharmaceutical production within the baculovirus/insect cell expression system.

Pseudomonas syringae DC3000 infection increases glucosylated N-glycans in Arabidopsis thaliana.

Studying the interaction between the hemibiotrophic bacterium Pseudomonas syringae pv. tomato DC3000 and Arabidopsis thaliana has shed light onto the various forms of mechanisms plants use to defend themselves against pathogen attack. While a lot of emphasis has been put on investigating changes in protein expression in infected plants, only little information is available on the effect infection plays on the plants N-glycan composition. To close this gap in knowledge, total N-glycans were enriched from P. syringae DC3000-infected and mock treated Arabidopsis seedlings and analyzed via MALDI-TOF-MS. Additionally, fluorescently labelled N-glycans were quantified via HPLC-FLD. N-glycans from infected plants were overall less processed and displayed increased amounts of oligomannosidic N-glycans. As multiple peaks for certain oligomannosidic glycoforms were detected upon separation via liquid chromatography, a porous graphitic carbon (PGC)-analysis was conducted to separate individual N-glycan isomers. Indeed, multiple different N-glycan isomers with masses of two N-acetylhexosamine residues plus 8, 9 or 10 hexoses were detected in the infected plants which were absent in the mock controls. Treatment with jack bean α-mannosidase resulted in incomplete removal of hexoses from these N-glycans, indicating the presence of glucose residues. This hints at the accumulation of misfolded glycoproteins in the infected plants, likely because of endoplasmic reticulum (ER) stress. In addition, poly-hexose structures susceptible to α-amylase treatment were found in the DC3000-infected plants, indicating alterations in starch metabolism due to the infection process.

A fast and easy one-step purification strategy for plant-made antibodies using Protein A magnetic beads.

A major difficulty to reach commercial- scale production for plant-made antibodies is the complexity and cost of their purification from plant extracts. Here, using Protein A magnetic beads, two monoclonal antibodies are purified in a one-step procedure directly from non-clarified crude plant extracts. This technique provides significant savings in terms of resources, operation time, and equipment.

Engineering the N-glycosylation pathway of Nicotiana tabacum for molecular pharming using CRISPR/Cas9.

Molecular pharming in plants offers exciting possibilities to address global access to modern biologics. However, differences in the N-glycosylation pathway including the presence of ß(1,2)-xylose and core α(1,3)-fucose can affect activity, potency and immunogenicity of plant-derived proteins. Successful glycoengineering approaches toward human-like structures with no changes in plant phenotype, growth, or recombinant protein expression levels have been reported for Arabidopsis thaliana and Nicotiana benthamiana. Such engineering of N-glycosylation would also be desirable for Nicotiana tabacum, which remains the crop of choice for recombinant protein pharmaceuticals required at massive scale and for manufacturing technology transfer to less developed countries. Here, we generated N. tabacum cv. SR-1 ß(1,2)-xylosyltransferase (XylT) and α(1,3)-fucosyltransferase (FucT) knockout lines using CRISPR/Cas9 multiplex genome editing, targeting three conserved regions of the four FucT and two XylT genes. These two enzymes are responsible for generating non-human N-glycan structures. We confirmed full functional knockout of transformants by immunoblotting of total soluble protein by antibodies recognizing ß(1,2)-xylose and core α(1,3)-fucose, mass spectrometry analysis of recombinantly produced VRC01, a broadly neutralizing anti-HIV-1 hIgG1 antibody, and Sanger sequencing of targeted regions of the putative transformants. These data represent an important step toward establishing Nicotiana tabacum as a biologics platform for Global Health.

Absolute and relative quantitation of amylase/trypsin-inhibitors by LC-MS/MS from wheat lines obtained by CRISPR-Cas9 and RNAi.

Quantitation of wheat proteins is still a challenge, especially regarding amylase/trypsin-inhibitors (ATIs). A selection of ATIs was silenced in the common wheat cultivar Bobwhite and durum wheat cultivar Svevo by RNAi and gene editing, respectively, in order to reduce the amounts of ATIs. The controls and silenced lines were analyzed after digestion to peptides by LC-MS/MS with different approaches to evaluate changes in composition of ATIs. First, a targeted method with stable isotope dilution assay (SIDA) using labeled peptides as internal standards was applied. Additionally, four different approaches for relative quantitation were conducted, in detail, iTRAQ labeled and label free quantitation (LFQ) combined with data dependent acquisition (DDA) and data independent acquisition (DIA). Quantitation was performed manually (Skyline and MASCOT) and with different proteomics software tools (PLGS, MaxQuant, and PEAKS X Pro). To characterize the wheat proteins on protein level, complementary techniques as high-performance liquid chromatography (HPLC) and gel electrophoresis were performed. The targeted approach with SIDA was able to quantitate all ATIs, even at low levels, but an optimized extraction is necessary. The labeled iTRAQ approach revealed an indistinct performance. LFQ with low resolution equipment (IonTrap) showed similar results for major ATIs, but low abundance ATIs as CM1, were not detectable. DDA measurements with an Orbitrap system and evaluation using MaxQuant showed that the relative quantitation was dependent on the wheat species. The combination of manual curation of the MaxQuant search with Skyline revealed a very good performance. The DIA approach with analytical flow found similar results compared to absolute quantitation except for some minor ATIs, which were not detected. Comparison of applied methods revealed that peptide selection is a crucial step for protein quantitation. Wheat proteomics faces challenges due to the high genetic complexity, the close relationship to other cereals and the incomplete, redundant protein database requiring sensitive, precise and accurate LC-MS/MS methods.

From strain engineering to process development: monoclonal antibody production with an unnatural amino acid in Pichia pastoris.

BACKGROUND: Expansion of the genetic code is a frequently employed approach for the modification of recombinant protein properties. It involves reassignment of a codon to another, e.g., unnatural, amino acid and requires the action of a pair of orthogonal tRNA and aminoacyl tRNA synthetase modified to recognize only the desired amino acid. This approach was applied for the production of trastuzumab IgG carrying p-azido-L-phenylalanine (pAzF) in the industrial yeast Pichia pastoris. Combining the knowledge of protein folding and secretion with bioreactor cultivations, the aim of the work was to make the production of monoclonal antibodies with an expanded genetic code cost-effective on a laboratory scale. RESULTS: Co-translational transport of proteins into the endoplasmic reticulum through secretion signal prepeptide change and overexpression of lumenal chaperones Kar2p and Lhs1p improved the production of trastuzumab IgG and its Fab fragment with incorporated pAzF. In the case of Fab, a knockout of vacuolar targeting for protein degradation further increased protein yield. Fed-batch bioreactor cultivations of engineered P. pastoris strains increased IgG and IgGpAzF productivity by around 50- and 20-fold compared to screenings, yielding up to 238 mg L-1 and 15 mg L-1 of fully assembled tetrameric protein, respectively. Successful site-specific incorporation of pAzF was confirmed by mass spectrometry. CONCLUSIONS: Pichia pastoris was successfully employed for cost-effective laboratory-scale production of a monoclonal antibody with an unnatural amino acid. Applying the results of this work in glycoengineered strains, and taking further steps in process development opens great possibilities for utilizing P. pastoris in the development of antibodies for subsequent conjugations with, e.g., bioactive payloads.

CRISPR/Cas9-mediated knockout of a prolyl-4-hydroxylase subfamily in Nicotiana benthamiana using DsRed2 for plant selection.

The properties of host plants used for molecular farming can be modified by CRISPR/Cas9 genome editing to improve the quality and yield of recombinant proteins. However, it is often necessary to target multiple genes simultaneously, particularly when using host plants with large and complex genomes. This is the case for Nicotiana benthamiana, an allotetraploid relative of tobacco frequently used for transient protein expression. A multiplex genome editing system incorporating the DsRed2 fluorescent marker for the identification and selection of transgenic plants was established. As proof of principle, NbP4H4 was targeted encoding a prolyl-4-hydroxylase involved in protein O-linked glycosylation. Using preselected gRNAs with efficiencies confirmed by transient expression, transgenic plant lines with knockout mutations in all four NbP4H4 genes were obtained. Leaf fluorescence was then used to screen for the absence of the SpCas9 transgene in T1 plants, and transgene-free lines with homozygous or biallelic mutations were identified. The analysis of plant-produced recombinant IgA1 as a reporter protein revealed changes in the number of peptides containing hydroxyproline residues and pentoses in the knockout plants. The selection of efficient gRNAs combined with the DsRed2 marker reduces the effort needed to generate N. benthamiana mutants and simplifies the screening processes to obtain transgene-free progeny.

Shut-Down of Type IX Protein Secretion Alters the Host Immune Response to Tannerella forsythia and Porphyromonas gingivalis.

Tannerella forsythia and Porphyromonas gingivalis target distinct virulence factors bearing a structurally conserved C-terminal domain (CTD) to the type IX protein secretion system (T9SS). The T9SS comprises an outer membrane translocation complex which works in concert with a signal peptidase for CTD cleavage. Among prominent T9SS cargo linked to periodontal diseases are the TfsA and TfsB components of T. forsythia's cell surface (S-) layer, the bacterium's BspA surface antigen and a set of cysteine proteinases (gingipains) from P. gingivalis. To assess the overall role of the bacterial T9SS in the host response, human macrophages and human gingival fibroblasts were stimulated with T. forsythia and P. gingivalis wild-type bacteria and T9SS signal peptidase-deficient mutants defective in protein secretion, respectively. The immunostimulatory potential of these bacteria was compared by analyzing the mRNA expression levels of the pro-inflammatory mediators IL-6, IL-8, MCP-1 and TNF-α by qPCR and by measuring the production of the corresponding proteins by ELISA. Shot-gun proteomics analysis of T. forsythia and P. gingivalis outer membrane preparations confirmed that several CTD-bearing virulence factors which interact with the human immune system were depleted from the signal peptidase mutants, supportive of effective T9SS shut-down. Three and, more profoundly, 16 hours post stimulation, the T. forsythia T9SS mutant induced significantly less production of cytokines and the chemokine in human cells compared to the corresponding parent strain, while the opposite was observed for the P. gingivalis T9SS mutant. Our data indicate that T9SS shut-down translates into an altered inflammatory response in periodontal pathogens. Thus, the T9SS as a potential novel target for periodontal therapy needs further evaluation.