A Rationally Designed Bovine IgA Fc Scaffold Enhances in planta Accumulation of a VHH-Fc Fusion Without Compromising Binding to Enterohemorrhagic E. coli

Abstract

We previously isolated a single domain antibody (VHH) 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 VHH 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 VHH that binds EHEC, from 12.5 mg/kg FW tissue to 236.2 mg/kg FW. Co-expression of native or mutated VHH-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 VHH-Fc to assemble with JC and FC into a secretory IgA. Both the native and the mutated VHH-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 VHH-Fc fusion without impacting VHH functionality.

A VHH-Fc Fusion Targeted to the Chloroplast Thylakoid Lumen Assembles and Neutralizes Enterohemorrhagic E. coli O157:H7

“…We also introduced a de novo disulfide pair G196C/R219C into the V H H-Fc targeted with the Sec signal and found a significant yield improvement. Compared to the ten-fold yield improvement observed previously when targeted to the ER (Chin-Fatt et al, 2021), the yield improvement is substantially less at only about a two-fold improvement. This may possibly be due to the availability of relevant chaperones across the two compartments.…”

“…We have previously identified by rational design a residue pair (G196C/R219C) on the Fc that if mutated to cysteines would form a de novo disulfide bond that enables a ten-fold improvement in yield of the V H H-Fc when targeted to the ER (Chin-Fatt et al, 2021). The pair forms an intra-chain disulfide between strand G and strand F on the CH3 domain in oxidative folding conditions (Figures 4A,B).…”

“…Given that intimin mediates the attachment of E. coli O157:H7 to intestinal epithelial cells, and that V H H-Fc targeted to the ER neutralizes E. coli's ability to adhere to those cells (Chin-Fatt et al, 2021), we tested if thylakoid targeting of the V H H-Fc impacted its ability to neutralize the bacterium from adhering to epithelial cells by blocking intimin. HEp-2 cells were incubated with E. coli O157:H7 in the presence or absence of purified V H H-Fc from each of the compartments.…”

“…Structurally, they differ from the native IgA monomer in that the antigen binding fragment (Fab) has been replaced by a camelid-derived V H H that is smaller (∼15 kDa), more stable and does not require multi-chain assembly as is the case for Fab heavy and light chains (De Meyer et al, 2014). We have previously produced a V H H-Fc in Nicotiana benthamiana leaf tissue by targeting it to the endoplasmic reticulum (ER) for folding and demonstrated its functionality in vitro in binding and neutralizing four serotypes of enterohemorrhagic Escherichia coli (EHEC), including O157:H7, the most prevalent serotype in North America (Saberianfar et al, 2019;Chin-Fatt et al, 2021). The national food surveillance programs for foodborne pathogens in the United States and Canada, FoodNet and FoodNet Canada, respectively, reported that the average incidence rate for O157:H7 was most recently estimated at 0.8 per 100,000 persons in the United States (CDC, 2020) and 1.52 per 100,000 persons in Canada (PHAC, 2019).…”

Development of a plant-based oral vaccine candidate against the bovine respiratory pathogen Mannheimia haemolytica

“…Depending on the protein’s stability, conformation, and post-translational modifications, some proteins will become more or less abundant over the first week or two before they disappear completely. Some proteins remain stable over 3-8 days ( Chin-Fatt et al., 2021 ; VanderBurgt et al., 2023 ), some increase their accumulation with time ( Saberianfar et al., 2019 ), and others reach their highest accumulation at 4-6 days and then go down with time ( Garabagi et al., 2012 ). Here, we found that accumulation levels are highest four days post-infiltration, and this is the time we subsequently harvested the infiltrated leaf material.…”