• Vaccine Name: soybean-expressed E. coli LTB vaccine
• Target Pathogen: Escherichia coli
• Target Disease: Hemorrhagic colitis
• Vaccine Ontology ID: VO_0000466
• Type: Subunit vaccine
• Antigen: Subunti B of E. coli heat labile enterotoxin LTB (Moravec et al., 2007).
• eltB gene engineering:
  • Type: Expression of protein subunit
  • Description: The B subunit of the heat labile toxin of enterotoxigenic Escherichia coli (LTB) was used as a model immunogen for production in soybean seed. LTB expression was directed to the endoplasmic reticulum (ER) of seed storage parenchyma cells for sequestration in de novo synthesized inert protein accretions derived from the ER. Pentameric LTB accumulated to 2.4% of the total seed protein at maturity and was stable in desiccated seed(Moravec et al., 2007) .
  • Detailed Gene Information: Click Here.
• FaeG gene engineering:
  • Type: Seed-specific protein expression
  • Description: A synthetic plant codon-optimized LTB gene and AAC60441, generously provided by A. Walmsley (Arizona Biodesign Institute) was modified by substitutions of the bacterial signal peptide with a 20 aa signal peptide from A. thaliana basic chitinase. A 14 aa extension comprising the FLAG epitope and KDEL ER retention signal, and flanking Bsp120 restriction sites were introduced by PCR. The final sequence encoded a 137 aa protein of 15.5 kDa that yielded a 13.3 kDa LTB-FLAG protein after signal peptide cleavage. Following subcloning into pGEM T/A (Promega) for sequence verification, the Bsp120 LTB gene fragment was subcloned into the pGly vector, placing it under the control of soybean seed-specific glycinin promoter and terminator [35]. The final soybean transformation vector pGly::ER-LTB contained a hygromycin selection marker (kindly provided by N. Murai, Lousiana State University) under the control of potato ubiquitin 3 promoter and terminator.
    LT is a hetero-oligomeric AB5 type enterotoxin composed of a 27 kDa A subunit with toxic ADP ribosyl transferase activity and a stable noncovalent-linked pentamer of 11.6 kDa B subunits. ETEC infection and colonization of the small intestine, and the production of LT, causes acute diarrhea that can be fatal without intervention. The ADP-ribosylation of Gsα, catalyzed by the A subunit, triggers increased intracellular cAMP levels that induce chloride efflux and fluid loss from intoxicated cells lining the small intestine. The B subunit pentamer mediates holotoxin binding to ganglioside GM1 on intestinal epithelial cells, with lower affinity for GD1B, asialoGM1 and lactosylceramide gangliosides (Moravec et al., 2007).
  • Detailed Gene Information: Click Here.
• Adjuvant:
  • Adjuvant name:
  • VO adjuvant ID: VO_0000139
  • Description: In the event of s.c. immunization, LTB was administered in complete Freund's adjuvant (Moravec et al., 2007).
• Preparation: For immunization, transgenic LTB-laden soybean seeds were ground in 5 vol. of PBS at 4 °C, the extracts were clarified by microcentrifugation at 20,000 × g for 5 min, and the total protein concentration was measured using the Bradford method (Moravec et al., 2007).
• Virulence: Soy LTB was biochemically stable, functionally active and highly immunogenic (Moravec et al., 2007).
• Description: Effective needle-free immunization strategies are needed to accommodate large-scale vaccination programs and avoid injection-related risks. To improve the efficacy of oral vaccination, antigens can be co-administered, or fused with a strong mucosal adjuvant. LT is a potent immunogen whose adjuvant active dose is well below its immunogenic dose. LT and detoxified mutants of LT trigger a stronger antibody response than LTB to co-administered antigens on a dose-for-dose basis. However, recombinant LTB is safely and commonly used as an adjuvant to stimulate antibody responses to co-administered protein antigens. LTB has also been used experimentally for the prevention and treatment of autoimmune diseases. Importantly, LTB has been shown to protect against the development of oral tolerance to co-fed soluble vaccine proteins, a serious consideration in the food-based delivery of vaccines. Transgenic plants offer the possibility to both produce and deliver an oral immunogen on a large-scale with low production costs and minimal purification or enrichment, and the potential exists for direct formulation of vaccines into animal feed and human consumables. Soybean has great potential as a vaccine delivery platform because of its naturally high protein content, nutritional value and multiple product streams (Moravec et al., 2007).

Mouse Response

• Host Strain: Inbred female C57BL/6J mice (Jackson Laboratory).
• Vaccination Protocol: Mice were immunized with soluble protein extracts from LTB transgenic soybean seed or nontransgenic cv. Jack seed. Mice were fasted for 12 h, but allowed water ad libitum prior to oral immunization by gavage using a ball-tip feeding needle. Five mice were used per group. Group 1 was immunized s.c. with soybean extract, followed by secondary s.c. immunization after 14 days. Group 2 was primed with soybean LTB by s.c. immunization, then followed by immunization at weekly intervals by oral gavage. Group 3 was immunized by oral gavage at weekly intervals. Control mice were vaccinated by mock s.c. primary immunization followed by oral gavage or by oral gavage alone with a soluble protein extract made from nontransgenic soybean seed (Moravec et al., 2007).
• Persistence: Not noted.
• Immune Response: Immunization of mice with LTB transgenic soybean extracts elicited robust systemic anti-LTB IgG and IgA antibody responses, as well as significant levels of intestinal anti-LTB IgA. The serum anti-LTB IgG titer from mice immunized by parenteral primary immunization followed by a series of oral gavage boosts was approximately four-fold higher than in mice immunized by oral gavage only. Likewise, serum anti-LTB IgA titers rose more rapidly over the 35-day experimental period in mice undergoing prime-boost immunization than oral gavage. Following a final oral boost at day 48, serum IgA titers in both cases rose almost equivalently when measured at day 60, and significantly exceeded IgA levels elicited by parenteral immunization alone. These results demonstrate that systemic IgA responses were enhanced by oral mucosal immunization. Importantly, the fecal anti-LTB IgA titer in mice immunized by prime-boost was twice as high as that in mice immunized solely by gavage following the final boost at day 48. A comparison of the antibody responses in parenterally-immunized mice, mice immunized using a prime-boost regime, and mice immunized solely by oral gavage indicated that a more optimal balance of systemic IgG/IgA immunity, and mucosal sIgA immunity was achieved using a parenteral prime-oral gavage boost strategy.
• Side Effects: Not noted.
• Challenge Protocol: Following oral LTB immunization, protection against toxin challenge was determined using the patent mouse assay. Challenge of immunized mice was performed on day 64. Briefly, mice were fasted for 12 h and challenged by oral gavage with 200 μl of 0.9% saline containing 25 μg purified LT, or saline alone, using five mice per group. Intragastric delivery was performed using a ball-tip feeding needle. Water was available ad libitum. Three hours after toxin administration, mice were euthanized by CO2 inhalation (Moravec et al., 2007).
• Efficacy: Partial protection against fluid accumulation in the gut was achieved following LT challenge of mice orally-immunized with soy LTB.