PLoS One
[Accessed 9 March 2013]
http://www.plosone.org/

Rapid and Scalable Plant-based Production of a Cholera Toxin B Subunit Variant to Aid in Mass Vaccination against Cholera Outbreaks
Krystal Teasley Hamorsky, J. Calvin Kouokam, Lauren J. Bennett, Keegan J. Baldauf, Hiroyuki Kajiura, Kazuhito Fujiyama, Nobuyuki Matoba
Research Article | published 07 Mar 2013 | PLOS Neglected Tropical Diseases 10.1371/journal.pntd.0002046

Abstract
Introduction
Cholera 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 Findings
In 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/Significance
Taken 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.

Author Summary
Cholera sporadically causes outbreaks in regions where safe water supply and sanitation systems are not sufficient. As currently available vaccines are only effective for 2 to 3 years, reactive mass vaccination has been proposed to reduce mortality during outbreaks. Cholera toxin B subunit (CTB), when combined with killed whole-cell bacteria, has been shown to provide superior short-term protection, but manufacturing challenges of the protein limit its availability for mass vaccination programs in developing countries. Our work presented herein developed a rapid, robust, and scalable bioproduction system in plants for a CTB variant, pCTB. The system allowed for the accumulation of pCTB at >1 g per kg of fresh leaf of tobacco-related plants within 5 days, which accounts for over 1000 doses of original CTB included in the World Health Organization-prequalified vaccine Dukoral. We further analyzed in depth the integrity of pCTB using a series of biochemical, biophysical, and immunological experiments, demonstrating that the plant-made protein is feasible as a cholera vaccine antigen. Thus, pCTB plus killed bacteria may be ideal for reactive vaccination against cholera outbreaks.