Expression of an Escherichia coli antigenic fusion protein comprising the heat labile toxin B subunit and the heat stable toxin, and its assembly as a functional oligomer in transplastomic tobacco plants

Abstract

). † These authors contributed equally to this work. SummaryEnterotoxigenic Escherichia coli (ETEC) strains are important pathogens in developing countries. Some vaccine formulations containing the heat labile toxin B subunit (LTB) have been used in clinical trials; however, the induction of neutralizing antibodies against the heat-stable toxin (ST), a poor immunogenic peptide, is necessary, as most ETEC strains can produce both toxins. In this study, a plant optimized synthetic gene encoding for the LTB-ST fusion protein has been introduced into plastids of tobacco leaf tissues, using biolistic microprojectile bombardment, in an effort to develop a single plant-based candidate vaccine against both toxins. Transplastomic tobacco plants carrying the LTB-ST transgene have been recovered. Transgene insertion into the plastid was confirmed by both PCR and Southern blot analysis. GM1-ELISA revealed that the LTB-ST fusion protein retained its oligomeric structure, and displayed antigenic determinants for both LTB and ST. Western blot analysis, using LTB antisera, confirmed the presence of a 17-KDa protein in transplastomic lines, with the correct antigenicity of the fusion protein. Expression levels of this fusion protein in different lines reached up to 2.3% total soluble protein. Oral immunization of mice with freeze-dried transplastomic tobacco leaves led to the induction of both serum and mucosal LTB-ST specific antibodies. Following cholera toxin challenge, a decrease of intestinal fluid accumulation was observed in mice immunized with LTB-ST-containing tobacco. These findings suggest that tobacco plants expressing LTB-ST could serve as a plant-based candidate vaccine model providing broad-spectrum protection against ETEC-induced diarrhoeal disease.

Heat-Stable Enterotoxin of EnterotoxigenicEscherichia colias a Vaccine Target

“…However, recombinant constructs of ST-CT-B and ST-LT-B have been reported to pentamerize, thereby creating a 5:1 haptento-carrier ratio (37,40).…”

Transgenic apple expressing an antigenic protein of the human respiratory syncytial virus

“…Higher transgene expression has been reported with chloroplast transformation (Koya et al, 2005;Ferná ndez-San Millá n et al, 2008;Rosales-Mendoza et al, 2009). Expression of the anthrax protective antigen (PA) in mature tobacco leaves driven by the constitutively expressed 16S rRNA promoter in the T0 transgenic line reached a maximum of 1.86 mg/g fresh tissue (Koya et al, 2005).…”

Is An Oral Plant-based Vaccine against Hepatitis B Virus Possible?

“…It was shown that not only monomeric or oligomeric antigens, as LT-B [104], but also HBs antigens assembled into VLPs preserved the native structure and immunogenicity in lyophilised tissue [67,70], similarly as purified HBsAg [103]. Furthermore, freeze-dried materials are characterised by durability and stability regarding their antigen content, especially SHBsAg, and make it possible to concentrate and standardize antigen content throughout vaccine doses, administered under a controlled regime [67,70].…”