Exploring the Potentiality of a Plant Platform for Monoclonal Antibody Production in Veterinary Medicine.

Canine atopic dermatitis (CAD) is an allergic, inflammatory, and pruritic skin disease associated with the production of IgE antibodies against environmental allergens and mainly house dust mite allergens. This complex dermatological pathology involves Interleukin 31 (IL-31) as a central itch mediator. One of the most effective CAD treatments is a caninized monoclonal antibody (mAb) called Lokivetmab. It is produced in CHO cells and targets specifically canine IL-31 (cIL-31) and blocks its cellular messaging. This treatment has undoubtedly contributed to a breakthrough in dermatitis-related pruritus. However, its production in mammalian cells requires time-consuming procedures, high production costs, and investment. Plants are considered an emerging protein production platform for recombinant biopharmaceuticals due to their cost-effectiveness and rapidity for production. Here, we use transient expression in Nicotiana benthamiana plants to produce recombinant canine Interleukin 31 (cIL-31) and an anti-IL-31 monoclonal antibody (M1). First, we describe the production and characterization of M1 and then its activity on an IL-31-induced pruritic model in dogs compared to its commercial homolog. Dogs treated with the plant-made M1 mAb have shown similar improvements to Lokivetmab-treated ones after different challenges using canine IL-31. Furthermore, M1 injections were not associated with any side effects. These results demonstrate the safety and efficacy of this plant-made Lokivetmab biosimilar to control dogs' pruritus in a well-established model. Finally, this study shows that the plant-production platform can be utilized to produce rapidly functional mAbs and bring hope to the immunotherapy field of veterinary medicine.

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.

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.

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.

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.

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.

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.