Explore the vast range of titles available.

Chimeric fusion proteins comprising a single domain antibody (V H H) fused to a crystallizable fragment (Fc) of an immunoglobulin are modular glycoproteins that are becoming increasingly in demand because of their value as diagnostics, research reagents and passive immunization therapeutics. Because ER-associated degradation and misfolding may potentially be limiting factors in the oxidative folding of V H H-Fc fusion proteins in the ER, we sought to explore oxidative folding in an alternative sub-compartment, the chloroplast thylakoid lumen, and determine its viability in a molecular farming context. We developed a set of in-house expression vectors for transient transformation of Nicotiana benthamiana leaves that target a V H H-Fc to the thylakoid lumen via either secretory (Sec) or twin-arginine translocation (Tat) import pathways. Compared to stromal [6.63 ± 3.41 mg/kg fresh weight (FW)], cytoplasmic (undetectable) and Tat-import pathways (5.43 ± 2.41 mg/kg FW), the Sec-targeted V H H-Fc showed superior accumulation (30.56 ± 5.19 mg/kg FW), but was less than that of the ER (51.16 ± 9.11 mg/kg FW). Additionally, the introduction of a rationally designed de novo disulfide bond enhances in planta accumulation when introduced into the Sec-targeted Fc fusion protein from 50.24 ± 4.08 mg/kg FW to 110.90 ± 6.46 mg/kg FW. In vitro immunofluorescent labeling assays on V H H-Fc purified from Sec, Tat, and stromal pathways demonstrate that the antibody still retains V H H functionality in binding Escherichia coli O157:H7 and neutralizing its intimate adherence to human epithelial type 2 cells. These results overall provide a proof of concept that the oxidative folding environment of the thylakoid lumen may be a viable compartment for stably folding disulfide-containing recombinant V H H-Fc proteins.

We previously isolated a single domain antibody (V H H) that binds Enterohemorrhagic Escherichia coli (EHEC) with the end-goal being the enteromucosal passive immunization of cattle herds. To improve the yield of a chimeric fusion of the V H H with an IgA Fc, we employed two rational design strategies, supercharging and introducing de novo disulfide bonds, on the bovine IgA Fc component of the chimera. After mutagenizing the Fc, we screened for accumulation levels after transient transformation in Nicotiana benthamiana leaves. We identified and characterized five supercharging and one disulfide mutant, termed ‘(5 + 1)Fc’, that improve accumulation in comparison to the native Fc. Combining all these mutations is associated with a 32-fold increase of accumulation for the Fc alone, from 23.9 mg/kg fresh weight (FW) to 599.5 mg/kg FW, as well as a twenty-fold increase when fused to a V H H that binds EHEC, from 12.5 mg/kg FW tissue to 236.2 mg/kg FW. Co-expression of native or mutated V H H-Fc with bovine joining chain (JC) and bovine secretory component (SC) followed by co-immunoprecipitation suggests that the stabilizing mutations do not interfere with the capacity of V H H-Fc to assemble with JC and FC into a secretory IgA. Both the native and the mutated V H H-Fc similarly neutralized the ability of four of the seven most prevalent EHEC strains (O157:H7, O26:H11, O111:Hnm, O145:Hnm, O45:H2, O121:H19 and O103:H2), to adhere to HEp-2 cells as visualized by immunofluorescence microscopy and quantified by fluorometry. These results collectively suggest that supercharging and disulfide bond tethering on a Fc chain can effectively improve accumulation of a V H H-Fc fusion without impacting V H H functionality.

Enterohemorrhagic Escherichia coli (EHEC) has consistently been one of the foremost foodborne pathogen threats worldwide based on the past 30 years of surveillance. EHEC primarily colonizes the bovine gastrointestinal (GI) tract from which it can be transmitted to nearby farm environments and remain viable for months. There is an urgent need for effective and easily implemented pre-harvest interventions to curtail EHEC contamination of the food and water supply. In an effort to address this problem, we isolated single-domain antibodies (VHHs) specific for intimin, an EHEC adhesin required for colonization, and designed chimeric VHH fusions with secretory IgA functionality intended for passive immunotherapy at the mucosal GI surface. The antibodies were produced in leaves of Nicotiana benthamiana with production levels ranging between 1 and 3% of total soluble protein. in vivo assembly of all subunits into a hetero-multimeric complex was verified by co-immunoprecipitation. Analysis of multivalent protection across the most prevalent EHEC strains identified one candidate antibody, VHH10-IgA, that binds O145:Hnm, O111:Hnm, O26:H11, and O157:H7. Fluorometric and microscopic analysis also indicated that VHH10-IgA completely neutralizes the capacity of the latter three strains to adhere to epithelial cells in vitro. This study provides proof of concept that a plant-produced chimeric secretory IgA can confer cross-serotype inhibition of bacterial adhesion to epithelial cells.

Type 3 secretion systems (T3SSs) are utilized by pathogenic to infect their hosts and many proteins from these systems are affected by chaperones specific to T3SS-containing bacteria. Toward developing a recombinant vaccine against enterohaemorrhagic (EHEC), we expressed recombinant T3SS and related proteins from predominant EHEC serotypes in chloroplasts. were transiently transformed to express chloroplast-targeted Tir, NleA, and EspD from the EHEC serotype O157:H7; a fusion of EspA proteins from serotypes O157:H7 and O26:H11; and a fusion of epitopes of Tir (Tir-ep) from serotypes O157:H7, O26:H11, O45:H2, and O111:H8. C-terminal GFP reporter fusion constructs were also developed and transiently expressed to confirm subcellular localization and quantify relative expression levels . Recombinant proteins were co-expressed with chaperones specific to each T3SS protein with the goal of increasing their accumulation in the chloroplast. We found that co-expression with the chloroplast-targeted chaperone CesT significantly increases accumulation of recombinant Tir when the latter is either transiently expressed in the nucleus and targeted to the chloroplast of or stably expressed in transplastomic . CesT also helped maintain higher levels of Tir:GFP fusion protein over time both and , indicating that the favorable effect of CesT on accumulation of Tir is not specific to a single time point or to fresh material. By contrast, T3SS chaperones CesT, CesAB, CesD, and CesD2 did not increase accumulation of NleA:GFP, EspA:GFP, or EspD:GFP, which suggests dissimilar functioning of these chaperone-substrate combinations. CesT did not increase accumulation of Tir-ep:GFP, which may be due to the absence of the CesT binding domain from this fusion protein. The fusion to GFP improved accumulation of Tir-ep relative to the unfused protein, but not for the other recombinant proteins. These results emphasize the importance of native chaperones and stabilizing fusions as potential tools for the production of higher levels of recombinant proteins in plants; and may have implications for understanding interactions between T3SS chaperones and their substrates. In particular, our findings highlight the potential of T3SS chaperones to increase accumulation of recombinant T3SS proteins in heterologous systems.

[This corrects the article on p. 283 in vol. 8, PMID: 28321227.].

We previously isolated a single domain antibody (V H) that binds Enterohemorrhagic (EHEC) with the end-goal being the enteromucosal passive immunization of cattle herds. To improve the yield of a chimeric fusion of the V H with an IgA Fc, we employed two rational design strategies, supercharging and introducing disulfide bonds, on the bovine IgA Fc component of the chimera. After mutagenizing the Fc, we screened for accumulation levels after transient transformation in leaves. We identified and characterized five supercharging and one disulfide mutant, termed '(5 + 1)Fc', that improve accumulation in comparison to the native Fc. Combining all these mutations is associated with a 32-fold increase of accumulation for the Fc alone, from 23.9 mg/kg fresh weight (FW) to 599.5 mg/kg FW, as well as a twenty-fold increase when fused to a V H that binds EHEC, from 12.5 mg/kg FW tissue to 236.2 mg/kg FW. Co-expression of native or mutated V H-Fc with bovine joining chain (JC) and bovine secretory component (SC) followed by co-immunoprecipitation suggests that the stabilizing mutations do not interfere with the capacity of V H-Fc to assemble with JC and FC into a secretory IgA. Both the native and the mutated V H-Fc similarly neutralized the ability of four of the seven most prevalent EHEC strains (O157:H7, O26:H11, O111:Hnm, O145:Hnm, O45:H2, O121:H19 and O103:H2), to adhere to HEp-2 cells as visualized by immunofluorescence microscopy and quantified by fluorometry. These results collectively suggest that supercharging and disulfide bond tethering on a Fc chain can effectively improve accumulation of a V H-Fc fusion without impacting V H functionality.

Enterohemorrhagic (EHEC) has consistently been one of the foremost foodborne pathogen threats worldwide based on the past 30 years of surveillance. EHEC primarily colonizes the bovine gastrointestinal (GI) tract from which it can be transmitted to nearby farm environments and remain viable for months. There is an urgent need for effective and easily implemented pre-harvest interventions to curtail EHEC contamination of the food and water supply. In an effort to address this problem, we isolated single-domain antibodies (V Hs) specific for intimin, an EHEC adhesin required for colonization, and designed chimeric V H fusions with secretory IgA functionality intended for passive immunotherapy at the mucosal GI surface. The antibodies were produced in leaves of with production levels ranging between 1 and 3% of total soluble protein. assembly of all subunits into a hetero-multimeric complex was verified by co-immunoprecipitation. Analysis of multivalent protection across the most prevalent EHEC strains identified one candidate antibody, V H10-IgA, that binds O145:Hnm, O111:Hnm, O26:H11, and O157:H7. Fluorometric and microscopic analysis also indicated that V H10-IgA completely neutralizes the capacity of the latter three strains to adhere to epithelial cells . This study provides proof of concept that a plant-produced chimeric secretory IgA can confer cross-serotype inhibition of bacterial adhesion to epithelial cells.

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 twentyfold. 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 neutralizat efficacy in in vitro assays. Additionally, the introduction of a rationally designed de novodisulfide 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.