Article Figures & Data
Figures
- Fig 1
Purification, quantification, and stability of soy-mSEB. Soybean-derived mSEB (soy-mSEB) was expressed in transgenic soybean seeds and purified by affinity chromatography as outlined in Materials and Methods. (A) Representative Coomassie blue-stained SDS-PAGE gel. (B) Representative Western blot of crude soybean extract (10 μg) next to purified extract (1 μg), each demonstrating a single band of soy-mSEB at approximately 28 kDa. (C) Representative Western blot used for quantification of soy-mSEB purified from seed extracts relative to recombinant SEB standards. (D) (Left) Western blot analysis of soymilk protein (10 μg) extracted from T1 seeds expressing mSEB. Seeds were harvested in 2009 and stored for 3 years at ambient temperature. (Right) Digestion of recombinant mSEB (2 μg) with increasing amounts of protease. The migration of molecular size standard proteins is shown in kilodaltons in all panels.
- Fig 2
Soy-mSEB lacks superantigen activity in pig lymphocytes. Pig splenic leukocytes were isolated and then cultured in the presence of the indicated concentrations of native SEB (nSEB) or soy-mSEB. After 48 h of culture, culture supernates were taken and porcine IFN-γ secretion determined using an ELISA. Results are presented as mean values (plus SEM) for triplicate determinations. Levels of gamma interferon that were below the 50-pg/ml detection limit for this ELISA were designated nondetectable (ND).
- Fig 3
Immunization of pigs with soy-mSEB and E. coli-mSEB results in high antitoxin serum titers. Groups of 25-day-old pigs (n = 5) were immunized intramuscularly (day 0) and then boosted on days 14 and 28 with E. coli-mSEB or soy-mSEB in alum. Control pigs were immunized with an equal amount of buffer containing alum. (A) Representative Coomassie blue-stained SDS-PAGE gel and accompanying Western blot, demonstrating the purity of each immunogen. Each animal was bled just prior to immunization on days 0, 14, 28, and 38. (B) ELISAs were performed to determine serum IgG anti-native SEB reactivity. These data are presented as mean anti-native SEB titers (plus SEM).
- Fig 4
Antibodies from pigs immunized with soy-mSEB and E. coli-mSEB effectively neutralized native SEB activity in vitro. Sera collected at day 38 postimmunization were used in an in vitro neutralization assay to demonstrate the ability to limit native SEB-induced cytokine responses. Dilutions (1:33 or 1:100) of sera from soy-mSEB-immunized, E. coli-mSEB-immunized, or control animals were mixed with 0.5 μg/ml of native SEB for 30 min prior to the addition of the mixtures to cultures of pig splenic leukocytes. Forty-eight hours after coincubation, culture supernates were taken and porcine IFN-γ secretion determined using an ELISA. Results are presented as mean values (plus SEM) for triplicate determinations for each immunogen group (n = 3 or 4). Asterisks indicate statistically significant differences (P < 0.01) compared to control animals.
- Fig 5
Reduced cytokine responses in pigs immunized with soy-mSEB and E. coli-mSEB following challenge with a sublethal dose of native SEB. At 36 to 38 days postimmunization, pigs had intravenous catheters surgically implanted to facilitate toxin administration and to allow for rapid and frequent blood collection. At days 38 to 40 postimmunization, pigs (n = 3 to 5) were challenged intravenously with a sublethal dose of native SEB (25 μg/kg). Blood was taken at the indicated hours postchallenge for quantification of IL-1β (A), IL-6 (B), IL-8 (C), IL-12p40 (D), IFN-γ (E), and TNF-α (F). For each time postchallenge (0, 1, 2, 3, 4, 8, 12, and 24 h), cytokines in sera (ng/ml) are presented as mean levels (plus SEM) for each group (n = 3 to 5) of immunized pigs. Asterisks indicate P values of <0.05 for comparing control values to values obtained for soy-mSEB- or E. coli-mSEB-immunized animals.
Tables
- Table 1
Comparison of E. coli- and soybean-derived mSEB starting materials and yields
Expression systema Vol of starting material (liters)b Starting biomassc Total soluble protein from biomassd Recoverable yield of vaccine (g)e E. coli cell culture 56.5 (from fermentor) 2,543 g of bacterial cell pellet 28.127 g 8.297 Transgenic soybean seeds 1.0 (soy powder) 800 g of soy powder per liter 320.000 g of protein per liter (theoretical) 12.800 (theoretical) ↵a E. coli-derived mutant SEB (L45R/Y89A/Y94A) was produced as described previously (6). Soy-mSEB was produced as described in Materials and Methods.
↵b The volume of E. coli cell culture was taken from a previous report (41). One liter of soy powder made by grinding transgenic seeds expressing soy-mSEB is used as a theoretical comparison.
↵c The starting biomass of E. coli-mSEB required medium filtration and concentration of the cell pellet. The size of the pellet mass was taken from a previous report (41). One liter of soy powder equals approximately 800 g and required no additional concentration or manipulation.
↵d The total soluble protein extracted from the E. coli cell pellet was taken from a previous report (41). Theoretically, 320 g of protein can be extracted from 800 g of soy powder, since approximately 40% of the total mass is protein (42). This number assumes minimal loss of protein during the extraction procedure.
↵e The total recoverable yield of E. coli-mSEB was taken from a previous report (41). Theoretically, for transgenic seeds expressing soy-mSEB at 4%, 320 g of total protein contains approximately 12.8 g of soy-mSEB (42). This number does not account for any losses during the purification procedures.
