Plant-based production of recombinant antiviral antibodies in N. benthamiana

Background.

Treatment options for many zoonotic viral diseases are limited, highlighting the need for rapidly producible antivirals such as recombinant antibodies, which can be produced rapidly and flexibly in transient platforms based on Nicotiana benthamiana. Such high-level recombinant protein expression has been shown to induce stress in the plant endoplasmic reticulum (ER), triggering the unfolded protein response (UPR), which may affect protein folding, stability, and accumulation. It is also known that environmental conditions such as extreme temperatures modulate ER stress and protein production.

Objective.

We hypothesize that different environmental conditions may exacerbate or alleviate ER stress caused by recombinant protein production. The PhD will investigate how different antibody constructs and environmental conditions, particularly temperature, influence recombinant antibody production, ER stress, and UPR activation. The project aims to identify the most promising antibody formats and optimal conditions for production, providing both fundamental understanding of plant ER stress responses and practical guidance for developing plant-based antiviral biologics.

Approach.

Transient expression of antibody constructs in N. benthamiana leaves will allow the characterization of antibody integrity, stability, and functionality, while also generating detailed insights into the molecular mechanisms of plant ER stress under recombinant protein production. Defined environmental conditions, combined with ER stress readouts, will provide insights into the relationship between recombinant protein production, environmental stress, and ER homeostasis, supporting future development of optimized protocols.

Methodology.

A set of newly designed antibody fusion proteins will be produced by agroinfiltration of N. benthamiana leaves. Environmental and growth conditions, including temperature, will be optimized to maximize protein yield and minimize ER stress. Reporter constructs sensitive to unfolded protein response (UPR) activation will be co-expressed to monitor ER stress in combination with standard molecular analysis. Recombinant antibodies will be extracted from plant tissue and purified using affinity chromatography; Structural integrity, folding, aggregation, and stability will be analyzed using SDS-PAGE and size-exclusion chromatography. Post-translational modifications, such as glycosylation, will be characterized as needed. Antibody functionality will be evaluated through antigen-binding assays, including ELISA or surface plasmon resonance.

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