N-Glycosylation of Cholera Toxin B Subunit: Serendipity for Novel Plant-Made Vaccines?

Abstract

The non-toxic B subunit of cholera toxin (CTB) has attracted considerable interests from vaccinologists due to strong mucosal immunomodulatory effects and potential utility as a vaccine scaffold for heterologous antigens. Along with other conventional protein expression systems, various plant species have been used as production hosts for CTB and its fusion proteins. However, it has recently become clear that the protein is N-glycosylated within the endoplasmic reticulum of plant cells—a eukaryotic post-translational modification that is not present in native CTB. While functionally active aglycosylated variants have been successfully engineered to circumvent potential safety and regulatory issues related to glycosylation, this modification may actually provide advantageous characteristics to the protein as a vaccine platform. Based on data from our recent studies, I discuss the unique features of N-glycosylated CTB produced in plants for the development of novel vaccines.

Hydroponic Treatment of Nicotiana benthamiana with Kifunensine Modifies the N-glycans of Recombinant Glycoprotein Antigens to Predominantly Man9 High-Mannose Type upon Transient Overexpression

“…Thus, it is evident that a minor fraction of gp120-HDEL escaped from the ER into the Golgi apparatus. This in turn highlights the limitation of the H/KDEL signal-based ER-retention strategy to restrict glycosylation heterogeneity, as we have previously shown with gCTB-KDEL (Matoba, 2015 ). A previous study by Rosenberg et al showed that the glycan composition of a plant-produced gp140-KDEL had a similar HMG-rich profile, but with no detectable plant-specific glycoforms (Rosenberg et al, 2013 ).…”

“…We have recently shown that CTB is N -glycosylated when expressed in N. benthamiana (Hamorsky et al, 2013b , 2015 ). The N -glycosylated CTB (gCTB) bound to cell-surface DC-SIGN in addition to GM1-ganglioside receptors, indicating that the glycosylated vaccine antigen may elicit additional immunomodulatory activity via several C-type lectin receptors (Matoba, 2015 ). Furthermore, preliminary results showed that gCTB's DC-SIGN-binding affinity could be significantly enhanced when the protein was produced under kifunensine treatment (Hamorsky et al, 2015 ), providing a basis for the present study and for the development of novel C-type lectin receptor-targeting vaccines.…”

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].…”

Therapeutic potential of a plant-made cholera toxin b subunit variant for the treatment of ulcerative colitis.

“…Studies using anti-inflammatory agents, such as anti-TNFα agents, have led to a general consensus that mucosal healing is the most important treatment goal in UC [80,81]. In two randomized, double-blind, placebo-controlled studies (ACT1 and 2), there was a ~5-fold higher rate of clinical remission at week 30 in patients treated with the anti-TNFα infliximab who exhibited mucosal healing at week 8 than those without mucosal healing [16]. Additionally, mucosal healing was significantly associated with a lower risk of future colectomy in a study of 513 Norwegian patients diagnosed with UC, [17].…”

“…Alternatively, in plant expression systems, rCTB 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 (CTBp; plant-made CTB; Table 1), with the aim to economically mass-produce the vaccine antigen to facilitate global cholera vaccination [14][15][16]. CTBp 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 [14,15].…”