Rapid and Scalable Plant-based Production of a Cholera Toxin B Subunit Variant to Aid in Mass Vaccination against Cholera Outbreaks

Abstract

IntroductionCholera toxin B subunit (CTB) is a component of an internationally licensed oral cholera vaccine. The protein induces neutralizing antibodies against the holotoxin, the virulence factor responsible for severe diarrhea. A field clinical trial has suggested that the addition of CTB to killed whole-cell bacteria provides superior short-term protection to whole-cell-only vaccines; however, challenges in CTB biomanufacturing (i.e., cost and scale) hamper its implementation to mass vaccination in developing countries. To provide a potential solution to this issue, we developed a rapid, robust, and scalable CTB production system in plants.Methodology/Principal FindingsIn a preliminary study of expressing original CTB in transgenic Nicotiana benthamiana, the protein was N-glycosylated with plant-specific glycans. Thus, an aglycosylated CTB variant (pCTB) was created and overexpressed via a plant virus vector. Upon additional transgene engineering for retention in the endoplasmic reticulum and optimization of a secretory signal, the yield of pCTB was dramatically improved, reaching >1 g per kg of fresh leaf material. The protein was efficiently purified by simple two-step chromatography. The GM1-ganglioside binding capacity and conformational stability of pCTB were virtually identical to the bacteria-derived original B subunit, as demonstrated in competitive enzyme-linked immunosorbent assay, surface plasmon resonance, and fluorescence-based thermal shift assay. Mammalian cell surface-binding was corroborated by immunofluorescence and flow cytometry. pCTB exhibited strong oral immunogenicity in mice, inducing significant levels of CTB-specific intestinal antibodies that persisted over 6 months. Moreover, these antibodies effectively neutralized the cholera holotoxin in vitro.Conclusions/SignificanceTaken together, these results demonstrated that pCTB has robust producibility in Nicotiana plants and retains most, if not all, of major biological activities of the original protein. This rapid and easily scalable system may enable the implementation of pCTB to mass vaccination against outbreaks, thereby providing better protection of high-risk populations in developing countries.

A plant-made cholera toxin B subunit enhances mucosal wound healing and protects against ulcerative colitis and colon cancer.

“…Given that CTB appears to provide additional efficacy to killed bacteria-based cholera vaccines, development of alternative rCTB production and delivery methods may significantly contribute to cholera prevention and control. We have therefore developed a novel CTB variant that can be rapidly mass produced in Nicotiana benthamiana plants (CTBp) to aid in reactive vaccination in response to outbreaks (76). Also, because of the capacity to induce potent mucosal humoral immune responses, antigen-CTB fusion provides a promising strategy for vaccines against enteric pathogens and mucosally transmitted diseases.…”

“…Previously, our lab has attempted to produce recombinant CTB in plants (Nicotiana benthamiana) using a plant virus vector overexpression system, with the aim of economically manufacturing the protein at a large scale to facilitate global cholera vaccination (76). When the original CTB was expressed in N.…”

“…Allergic inflammation in mouse airways has also been shown to be reduced by CTB administration, not only in a preventative sense but also in mice that have already been sensitized to airway inflammation (60 A) Commercial non-recombinant CTB containing a trace amount of CT (CTB+CT) significantly reduces the production of TNF-α due to LPS stimulation. Raw 264.7 cells were pretreated with 10 μg/ml rCTB (produced in E. coli (76)), CTB+CT (Sigma-Aldrich, catalog no. C9903), or PBS, and a final concentration of 1 μg/ml LPS was added and incubated for 24 hours.…”

“…For the characterization of the global impacts of CTBp oral administration, animals were orally administered with PBS or 30 µg CTBp twice at a 2-week interval after neutralization of stomach acids with a sodium bicarbonate solution, as described previously (76). CTBp was produced in N. benthamiana and purified to >95% homogeneity as a pentamer molecule with an endotoxin level of <1 endotoxin units (EU)/mg, as described previously (76).…”

“…CTBp was produced in N. benthamiana and purified to >95% homogeneity as a pentamer molecule with an endotoxin level of <1 endotoxin units (EU)/mg, as described previously (76). One or two weeks after the second dose, mice were sacrificed using carbon dioxide inhalation followed by a thoracotomy.…”