N-Glycosylation of cholera toxin B subunit in Nicotiana benthamiana: impacts on host stress response, production yield and vaccine potential

Abstract

Plant-based transient overexpression systems enable rapid and scalable production of subunit vaccines. Previously, we have shown that cholera toxin B subunit (CTB), an oral cholera vaccine antigen, is N-glycosylated upon expression in transgenic Nicotiana benthamiana. Here, we found that overexpression of aglycosylated CTB by agroinfiltration of a tobamoviral vector causes massive tissue necrosis and poor accumulation unless retained in the endoplasmic reticulum (ER). However, the re-introduction of N-glycosylation to its original or an alternative site significantly relieved the necrosis and provided a high CTB yield without ER retention. Quantitative gene expression analysis of PDI, BiP, bZIP60, SKP1, 26Sα proteasome and PR1a, and the detection of ubiquitinated CTB polypeptides revealed that N-glycosylation significantly relieved ER stress and hypersensitive response, and facilitated the folding/assembly of CTB. The glycosylated CTB (gCTB) was characterized for potential vaccine use. Glycan profiling revealed that gCTB contained approximately 38% plant-specific glycans. gCTB retained nanomolar affinity to GM1-ganglioside with only marginal reduction of physicochemical stability and induced an anti-cholera holotoxin antibody response comparable to native CTB in a mouse oral immunization study. These findings demonstrated gCTB's potential as an oral immunogen and point to a potential role of N-glycosylation in increasing recombinant protein yields in plants.

A recombinant cholera toxin b subunit variant (CTB-KDEL) exhibits unique colon mucosal healing effects that have therapeutic implications for inflammatory bowel disease.

“…Alternatively, in plant expression systems, CTB is expressed in whole plants grown in controlled growth rooms or greenhouses. Previously, our lab has produced recombinant CTB in tobacco plants (Nicotiana benthamiana) using a plant virus vector overexpression system (CTB-KDEL; plant-made CTB; Table 1), with the aim to economically mass-produce the vaccine antigen to facilitate global cholera vaccination [41][42][43]. CTB-KDEL was generated as a result of this endeavor; we genetically modified CTB to add an ER retention signal to the C-terminus, which was critical to mitigate ER stress/tissue necrosis upon overexpression and achieve high-level accumulation in leaf tissue [41,42].…”

“…Previously, our lab has produced recombinant CTB in tobacco plants (Nicotiana benthamiana) using a plant virus vector overexpression system (CTB-KDEL; plant-made CTB; Table 1), with the aim to economically mass-produce the vaccine antigen to facilitate global cholera vaccination [41][42][43]. CTB-KDEL was generated as a result of this endeavor; we genetically modified CTB to add an ER retention signal to the C-terminus, which was critical to mitigate ER stress/tissue necrosis upon overexpression and achieve high-level accumulation in leaf tissue [41,42]. Our analyses demonstrated that CTB-KDEL is virtually identical to original CTB in regards to GM1-ganglioside binding affinity, molecular stability and vaccine efficacy to induce anti-toxin IgG and IgA antibodies upon oral immunization in mice [41].…”

“…Using CTB-KDEL [42,89], we have shown that oral administration of the CTB variant significantly altered several immune cell populations in the colon lamina propria [50].…”

“…It is of importance to point out that many of the effects observed in the aforementioned studies using CTB-KDEL may be unique to the plant-made variant, as it has a mutation at amino acid position 4 and an ER retention signal sequence at the C-terminus (N4S-CTB-SEKDEL; [89]). The ER-retention sequence was added to CTB-KDEL to improve production in planta, while Asn4→Ser mutation was introduced to avoid N-glycosylation [42,89]. The addition of the KDEL sequence to N4S-CTB significantly reduced ER stress that otherwise caused poor production yield.…”

“…While investigating the mechanistic principle, we recently discovered that the epithelial wound repair effects observed in the aforementioned studies are unique to CTB-KDEL and not attained by CTB. The ER-retention motif was initially added to CTB to improve recombinant production in tobacco plants (Nicotiana benthamiana), which aided in reducing ER stress that otherwise caused poor production yield [42,89]. It was thought that the KDEL sequence helped prolong CTB-KDEL's residence time in the ER within plant cells to allow for proper folding and assembly.…”