Oral vaccine formulations stimulate mucosal and systemic antibody responses against staphylococcal enterotoxin B in a piglet model

Abstract

Despite the potential for its use as an agent of biowarfare or bioterrorism, no approved vaccine against staphylococcal enterotoxin B (SEB) exists. Nontoxic, mutant forms of SEB have been developed; however, it has been difficult to determine the efficacy of such subunit vaccine candidates due to the lack of superantigen activity of native SEB in rodents and due to the limitations of primate models. Since pigs respond to SEB in a manner similar to that of human subjects, we utilized this relevant animal model to investigate the safety and immunogenicity of a triple mutant of SEB carrying the amino acid changes L45R, Y89A, and Y94A. This recombinant mutant SEB (rmSEB) did not possess superantigen activity in pig lymphocyte cultures. Furthermore, rmSEB was unable to compete with native SEB for binding to pig leukocytes. These in vitro studies suggested that rmSEB could be a safe subunit vaccine. To test this possibility, piglets immunized orally with rmSEB formulations experienced no significant decrease in food consumption and no weight loss during the vaccination regimen. Oral vaccination with 1-mg doses of rmSEB on days 0, 7, 14, and 24 resulted in serum IgG and fecal IgA levels by day 36 that cross-reacted with native SEB. Surprisingly, the inclusion of cholera toxin adjuvant in vaccine formulations containing rmSEB did not result in increased antibody responses compared to formulations using the immunogen alone. Taken together, these studies provide additional evidence for the potential use of nontoxic forms of SEB as vaccines.

FIG. 1.

Characterization and quantification of rmSEB. Recombinant mutant SEB (rmSEB) was expressed in E. coli and purified as a C-terminal six-histidine-tagged fusion protein. (A) A Coomassie blue-stained SDS-PAGE gel of 1, 2, and 5 μg of purified rmSEB showed a single band migrating at approximately 28 kDa compared to the total E. coli protein lysate (lane L). Migration of protein standards are shown to the right of the gel. (B) Western blot analyses demonstrated that rmSEB (rm) could be recognized by the same antibodies used to detect native SEB (n). Note that rmSEB migrates at a slightly higher molecular weight due to the amino acid additions made to this recombinant protein. (C) Known quantities of standard proteins (bovine albumin, egg albumin, and lysozyme) were coelectrophoresed on a Coomassie blue-stained SDS-PAGE gel to quantify the amounts of rmSEB used in these studies.

FIG. 2.

rmSEB lacks superantigen activity for pig leukocytes. Pig splenic leukocytes were isolated and cultured in the presence of the indicated concentrations of native SEB (nSEB) or rmSEB. After 40 h of culture, supernatants were taken, and porcine gamma interferon (IFN gamma) secretion was determined using an ELISA. It should be noted that no results are shown for 100 μg/ml of native SEB, as such a high concentration of toxin was nearly 100% toxic to pig leukocytes under these culture conditions. Results are presented as mean values (±standard errors of the mean [SEM]) for triplicate determinations. Levels of gamma interferon that were below the 50-ng/ml detection limit for this ELISA were designated nondetectable (ND). This study was performed twice, with similar results.

FIG. 3.

rmSEB is not a competitive inhibitor of native SEB for pig leukocytes. Pig splenic leukocytes were isolated and cocultured in the presence of 1.0 μg/ml of native SEB (nSEB) with the indicated concentrations of rmSEB. After 40 h of culture, supernatants were taken, and porcine gamma interferon (IFN gamma) secretion was determined using an ELISA. Results are presented as mean values (±SEM) for triplicate determinations.

FIG. 4.

Oral administration of rmSEB results in significant antibody responses. Groups of 7-day-old piglets (n = 8) were orally immunized (day 0) and then boosted 7, 14, and 24 days later with formulations of 1 mg of rmSEB in soy milk (rmSEB) or with soy milk alone (vehicle). Each animal was bled at days 0, 7, 14, 24, and 36 postimmunization to obtain sera. Fecal samples were also collected at the time of euthanasia (day 36) for ELISA to determine serum IgG (A) or fecal IgA (B) anti-SEB reactivity. Results are presented as mean absorbance values (±SEM) using a serum dilution of 1:200 or a total fecal IgA level of 1 μg/ml. Asterisks indicate a statistically significant difference (P < 0.05) compared to all other determinations.

FIG. 5.

Oral administration of rmSEB plus cholera toxin adjuvant does not augment the antibody response. Groups of 7-day-old piglets (n = 8) were orally immunized (day 0) and then boosted 7, 14, and 24 days later with formulations of 1 mg of rmSEB in soy milk (rmSEB) with (+) or without (−) 100 μg of cholera toxin (CT). On day 36 postimmunization, piglets were euthanized and sera collected for ELISA to determine serum IgG anti-CT (A) or serum IgG anti-SEB (B) titers. Endpoint titers were defined as the last serum dilution with an absorbance double that of sera from animals which received the vehicle only. Results are presented as mean titers (±SEM). Asterisks indicate a statistically significant difference (P < 0.05) compared to the result for animals receiving the vehicle only.