Research

Wider Research Context: Secretory IgA (SIgA) has emerged as a promising candidate for therapeutic interventions owing to its unique structural features including heightened pathogen neutralization efficacy, anti-inflammatory characteristics, and enhanced stability in mucosal secretions. Its interaction with commensal bacteria and mucosal components presents an innovative avenue for novel therapeutic applications. A pivotal determinant of SIgA's unique properties is its extensive glycosylation, yet the glycosylation profile across different tissues and its biological significance remains largely unexplored. Hypotheses: We hypothesize that specific glycan modifications on SIgA are functionally crucial for interactions with components of the mucosa, commensal bacteria and host cell receptors. We aim to fill the knowledge gap on human SIgA glycosylation through a comprehensive analysis of SIgA glycan profiles in different human tissues and subsequent glyco-engineering approaches on recombinant SIgA to elucidate structure-function relationships of distinct glycoforms. Approach: We will elucidate site-specific glycosylation profiles on human SIgA isolated from different tissues, based on which we will employ glyco-engineering tools in the plant-based transient production platform Nicotiana benthamiana a proven and highly suitable system for efficiently producing this intricate and multimeric protein with tailored N- and O-glycans. Generated recombinant SIgA glycoforms will be subjected to extensive biochemical and biophysical characterization as well as in vitro and cell-based binding assays, activation assays and proteolytic stability assessments in mucosal fluids to shed light on the implication of SIgA glycosylation for mucosal immunity and therapeutic development. Innovation: This project innovatively leverages the plant-based transient production platform in Nicotiana benthamiana for glycoengineering, enabling the production of fully assembled and functional SIgA with tailored N- and O-glycans. This approach distinguishes itself from previous studies that relied on heterogeneous glycosylation profiles. The capability to produce large amounts of recombinant human SIgA with homogenous glycoforms allows for unprecedented insights into the structure-function relationships of SIgA, advancing its potential for therapeutic applications. Primary Researchers Involved: Kathrin Göritzer, based at BOKU Vienna as a postdoctoral research fellow (FWF Schrödinger fellowship) has expertise in protein and glyco-engineering of therapeutic antibodies, and has undergone extensive training in immunology. Julian Ma, based at St. George’s University of London is an internationally renowned plant biotechnologist and immunologist.

We aim to generate a panel of recombinant neutralising IgA monoclonal antibodies against SARS-CoV-2 by transient expression in plants, a manufacturing platform that would facilitates rapid production and scale-up to levels sufficient to treat up several million people in a very short time-scale. We will evaluate the stability of our antibody candidates in aerosol formulation for simplified topical delivery to the lungs and determine their efficacy in cell-based neutralisation assays and animal models. This passive immunotherapy approach using a plant-based production systems could provide a massively scalable, affordable solution on a global scale, something that is hardly achievable would be impossible using traditional antibody manufacturing platforms.

In all eukaryotic cells, the endoplasmic reticulum (ER) has a central role in protein biosynthesis and maturation. Folding of newly synthesized secretory and membrane proteins takes place in the lumen of the ER. Properly folded and assembled proteins exit the ER and continue their journey in the secretory pathway. However, protein folding is error-prone and the accumulation of misfolded or surplus proteins endangers the cellular homeostasis and subsequently the survival of organisms especially under adverse environmental conditions. Consequently, cells have established sophisticated quality control processes that ensure the export of biologically active proteins and the elimination of non-native ones. These quality control processes involve proteins that sense folding-defective proteins and target them for destruction by a precisely controlled protein breakdown process known as ER-associated degradation (ERAD). The degradation of folding-defective glycoproteins is initiated in the lumen of the ER and the specific proteins that recognize the misfolded glycoproteins and determine their fate are unknown. We hypothesize that protein complexes consisting of alpha-mannosidases and thioredoxin-fold containing proteins are key factors in this process. The role of these complexes will be investigated using genetic, biochemical and cell biology approaches. The project will help to better understand protein quality control mechanisms in plants, which, in the long run, will lead to new strategies to improve plant fitness under constantly changing environmental conditions.