Oral MucoRice expressing double-mutant cholera toxin A and B subunits induces toxin-specific neutralising immunity
Introduction
To prepare for the successful execution of future global vaccination programs, it is essential that we consider creating a new generation of vaccines that do not require refrigeration storage and traditional syringes and needles for vaccination. The use of transgenic plant-derived recombinant protein is a promising strategy that combines innovation and knowledge of mucosal immunology and plant biotechnology to produce such suitable plant-based vaccines for global immunisation [1], [2]. The potential benefits include cost-effective and rapid up-scaling of production, expression of multiple genes at one time, and lower risk of contamination with human pathogens in the preparation of vaccine antigens. Furthermore, plants are suitable for foreign protein production and storage and as oral delivery options for subunit-type vaccines to induce protective immunity against infectious diseases via the mucosal immune system [1], [2]. Among several plant-based vaccines developed, grains such as corn, wheat and rice have recently attracted interest for vaccine production, storage and delivery systems for oral immunisation. As a vaccine antigen production system, rice seed has advantages over other grains, including easier storage and processing and greater yield; moreover, the rice plant has self-crossing ability [3]. In addition, a rice transformation system has been established and the full genome sequence elucidated, enabling rice genetic information to be easily applied to the creation of a gene-manipulated product [3], [4].
We recently developed a rice-based oral cholera toxin (CT) B (CTB)-subunit vaccine (MucoRice-CTB) that has many practical advantages over most traditional injection-type vaccines and other plant-based oral vaccines [3]. The rice-based oral vaccine is stable at room temperature for several years and is protected from digestive enzymes in the harsh conditions of the gastrointestinal tract. When MucoRice-CTB was given orally, the vaccine induced antigen-specific antibodies with toxin-neutralising activity [3]. Here, to demonstrate the development of a multicomponent vaccine as part of a rice-based vaccine antigen expression system, we produced transgenic rice seed expressing the A and B subunits of a nontoxic double-mutant cholera toxin (dmCT), which contained two amino acid substitutions, of the ADP-ribosyltransferase active centre (E112K) and carboxyl-terminal KDEL (E112K/KDGL) in the A subunit (dmCTA) [5], [6]. We then examined whether oral vaccination with this seed would effectively induce enterotoxin-neutralising immunity. Although dmCT is considered safe and nontoxic, exhibiting no ADP-ribosyltransferase activity and participating in normal intracellular trafficking [6], it retains the biological capacity to enhance antibody immune responses against co-administered antigens [6]. Our strategy was aimed at utilising these unique characteristics of dmCT by inserting a dmCTA-specific gene into the rice genome to develop rice expressing dmCTA in addition to the original CTB, thus yielding a multicomponent vaccine, MucoRice-dmCT.
Section snippets
DNA construction and transformation of rice plants
A double mutant of the CT gene (dmCT E112K/KDGL) was modified to a suitable codon optimisation form for rice seed by introducing two potent mutations into the ADP-ribosylation activity centre and C-terminal KDEL [6], [7]. The modified dmCTA subunit and B-subunit genes for the dmCT gene were cloned as individual ORFs flanked with plant elements to facilitate the transcription of each subunit. The dmCTA subunit and dmCT cassettes were assembled. The dmCTA subunit cassette consisted of a GluB-4
Development of rice-expressed nontoxic double-mutant cholera toxin (dmCT)
The two genes encoding dmCTA and CTB were generated as shown in Fig. 1. We chose to introduce the dmCTA and CTB genes separately into the same rice as rice-expressed dmCT in order to prove the antigenicity, and lack of adjuvanticity, of dmCT. Using codons preferentially used for translation of several rice-seed-protein genes, both genes were optimised for expression in the transgenic rice seed. Rice seed storage protein glutelin 1.4-kb GluB-4 promoter/signal peptides and 2.3-kb GluB-1
Discussion
It is well known that oral administration of CT to mice induces strong protective immunity against CT-induced diarrhoea [16]. Although appropriate oral administration of CT itself can protect mice from toxin-induced diarrhoea, it is unsuitable for use in humans because of its toxicity, causing severe diarrhoea if given orally. To overcome this obstacle, a killed Vibrio cholerae vaccine combined with recombinant CTB was successfully developed and approved by the European Union in 2004 and in
Acknowledgments
We thank Nippon Paper Group Inc. and Rohto Pharmaceutical Co. Ltd. for their contribution to the MucoRice development project. This work was supported by the Research and Development Program for New Bio-industry Initiatives of the Bio-oriented Technology Research Advancement Institution (Y.Y.); a Research Fellowship from the Japan Society for the Promotion of Science (T.N.); a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) and the Ministry and Health
References (21)
- et al.
Plant-based vaccines: unique advantages
Vaccine
(2001) - et al.
Plant-based vaccines
Int J Parasitol
(2003) - et al.
Oral cholera vaccines: use in clinical practice
Lancet Infect Dis
(2006) - et al.
Needle-free vaccine delivery
Adv Drug Deliv Rev
(2006) - et al.
Rice-based mucosal vaccine as a global strategy for cold-chain- and needle-free vaccination
Proc Natl Acad Sci USA
(2007) - et al.
A rice-based edible vaccine expressing multiple T cell epitopes induces oral tolerance for inhibition of Th2-mediated IgE responses
Proc Natl Acad Sci USA
(2005) - et al.
Mutants in the ADP-ribosyltransferase cleft of cholera toxin lack diarrheagenicity but retain adjuvanticity
J Exp Med
(1997) - et al.
A second generation of double mutant cholera toxin adjuvants: enhanced immunity without intracellular trafficking
J Immunol
(2006) - et al.
Cholera toxin genes: nucleotide sequence, deletion analysis and vaccine development
Nature
(1983) - et al.
Evaluation of tissue specificity and expression strength of rice seed component gene promoters in transgenic rice
Plant Biotechnol J
(2004)
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These authors contributed equally to this work and share first authorship.