Elsevier

Vaccine

Volume 27, Issue 43, 9 October 2009, Pages 5982-5988
Vaccine

Oral MucoRice expressing double-mutant cholera toxin A and B subunits induces toxin-specific neutralising immunity

https://doi.org/10.1016/j.vaccine.2009.07.071Get rights and content

Abstract

Rice-expressed cholera toxin B (CTB) subunit is a cold-chain-free oral vaccine that effectively induces enterotoxin-neutralising immunity. We created another rice-based vaccine, MucoRice, expressing nontoxic double-mutant cholera toxin (dmCT) with CTA and CTB subunits. Western-blot analysis suggested that MucoRice-dmCT had the shape of a multicomponent vaccine. Oral administration of MucoRice-dmCT induced CTB- but not CTA-specific serum IgG and mucosal IgA antibodies, generating protective immunity against cholera toxin without inducing rice-protein-specific antibody responses. The potency of MucoRice-dmCT was equal to that of MucoRice-CTB vaccine. MucoRice has the potential to be used as a safe multicomponent vaccine expression system.

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

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