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Thesis Format

Integrated Article


Doctor of Philosophy




Menassa, Rima


Agriculture and Agri-Food Canada

2nd Supervisor

Smith, David



Enterohemorrhagic Escherichia coli (EHEC) is a foodborne pathogen that harbors asymptomatically in a bovine intestinal reservoir and has been a consistent global health risk for the last thirty years. In this thesis, I have functionally characterized an anti-EHEC antibody and shown it to bind and neutralize four of the seven most prevalent EHEC strains. However, yield is arguably the greatest technical hurdle in advancing such an antibody from proof of principle to market. Although there have been many advances in the last thirty years in improving plant-based recombinant yields, little is still known about both the mutational potential in engineering the structure for better yield as well as the limitations of the antibody’s oxidative folding environment. This thesis explores two main strategies toward yield improvement: first, rational design of the antibody and second, targeting it to the thylakoid lumen. I have identified and characterised six rationally designed mutations that can be pyramided to improve the yield of the antibody by twenty-fold. I show that despite engineering its structure, the antibody retains its ability to assemble into a polymeric complex as well as its binding and neutralization efficacy against EHEC in in vitro assays. Because endoplasmic reticulum (ER)-associated degradation and misfolding may potentially be limiting factors in the oxidative folding of antibodies in the ER, I sought to explore oxidative folding in an alternative subcompartment, the chloroplast thylakoid lumen, and determine its viability in a molecular farming context. I developed a set of in-house expression vectors targeting the antibody to the thylakoid lumen via either Sec or Tat import pathways. Compared to stromal, cytoplasm and Tat-imported pathways, the Sec-targeted antibody showed superior accumulation, but about one third less than its ER-targeted counterpart. Sec-targeted antibodies also retain binding and neutralization efficacy in in vitro assays. Additionally, the introduction of a rationally designed de novo disulfide enhances in vivo accumulation when introduced into the Sec-targeted antibody. These results collectively provide a proof of concept on the viability of rational design and thylakoid targeting as novel, broadly applicable strategies for yield improvement and potentially advancing an anti-EHEC antibody closer toward market adoption.

Summary for Lay Audience

Enterohemorrhagic Escherichia coli (EHEC) is a pathogen that harbors in the intestines of cattle and can be transmitted through the food supply chain to cause foodborne illness in a population. An appealing intervention strategy is the use of antibodies that can be fed to the animal to clear EHEC from its intestines prior to harvest. In this thesis, I describe the design of such an antibody and demonstrate it to bind and neutralize four of the seven most prevalent EHEC strains that collectively account for 72% of related foodborne illnesses. However, because yield is a major hurdle preventing transitioning of this antibody from proof of principle to market, there is a need to explore yield optimization strategies. Therefore, this thesis explores two novel strategies for improving yield: engineering of the structure of the antibody and targeting it to a cellular compartment known as the thylakoid lumen.

I designed a screen of mutational candidates and assessed their yield when expressed in tobacco. Out of 24 candidates, I identified six mutations that individually improve the yield of the antibody. Furthermore, these mutations could be pooled to incrementally increase yield to give a twenty-fold improvement of the antibody. To ensure that the engineered antibody was still functional, I tested its binding and neutralization across the same seven strains and found that it retained efficacy.

The thylakoid lumen is a cellular subcompartment inside the chloroplast of plants that may potentially allow for higher antibody yields because of a different intracellular environment. However, when targeted to the thylakoid lumen, the antibody reached a yield about 60% of the conventional endoplasmic reticulum compartment. Still, functional assays indicated that the thylakoid-targeted antibody retained efficacy.

Overall, this thesis is notable because it demonstrates the viability of rational design and thylakoid targeting as strategic research directions for yield improvement of an anti-EHEC antibody.

Creative Commons License

Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.

Available for download on Monday, May 29, 2023

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