Production and Purification of a Recombinant Staphylococcal Enterotoxin B Vaccine Candidate Expressed in Escherichia coli

The nasopharynx-associated lymphoid tissue (NALT) of humans and other mammals is associated with immunity against airborne infections, though it is generally considered to be a secondary component of the mucosa-associated lymphoid system. We found that protective immunity to a virulence factor of nasal mucosacolonizing Staphylococcus aureus, staphylococcal enterotoxin B (SEB), requires a functional NALT. We examined the role of NALT using intranasal (

Section: Methodsmentioning

confidence: 99%

Nasal Immunity to Staphylococcal Toxic Shock Is Controlled by the Nasopharynx-Associated Lymphoid Tissue

Fernandez

Cisney

Hall

et al. 2011

“…These mutant forms of SEB have been designed and tested based on the considerable data defining the structure-function relationships for this toxin (13,22,23). Specifically, several studies have focused on the role of particular amino acid residues in SEB that are important in toxinogenic activity (1,3,5,15,24,32,34). Most staphylococcal superantigens have common structures for binding to a subunit of the human MHC class II molecule (34).…”

mentioning

confidence: 99%

Oral Vaccine Formulations Stimulate Mucosal and Systemic Antibody Responses against Staphylococcal Enterotoxin B in a Piglet Model

Inskeep

Stahl

Odle

et al. 2010

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.Staphylococcus aureus produces several exotoxins that are important determinants of pathogenicity (7). The staphylococcal enterotoxins are among these exotoxins and are produced by S. aureus strains growing in contaminated food, with staphylococcal enterotoxin B (SEB) being the most potent of the exotoxins. SEB mediates its toxicity by linking major histocompatibility complex (MHC) class II molecules with T-cell receptors outside the antigen binding site (24). Several families of T lymphocytes expressing certain V beta T-cell receptors can be stimulated by this toxin, which can include up to 20% of the total T-cell population. The term "superantigen" has been given to SEB and similar toxins which have this ability to bridge MHC class II molecules and T-cell receptors, stimulating a large percentage of T lymphocytes in this unconventional manner (12). One result of this toxin-induced T-lymphocyte activation is the overproduction of certain cytokines which contribute to the clinical symptoms of SEB-induced toxicity and shock (8). S. aureus can produce SEB within the environment, but its production is most problematic following infection or when present in contaminated foodstuffs. Ingestion of the toxin results in symptoms which include anorexia, nausea, vomiting, and diarrhea, which may present with hypotension, tachycardia, and hyperperistalsis (18).Unfortunately, SEB has several characteristics which make it a candidate for possible use as an agent of biowarfare or bioterrorism. SEB has...

“…The recombinant staphylococcal enterotoxin B (rSEB) recombinant purified protein mutant derivative was manufactured under current good manufacturing practices (cGMP) at the Pilot Bioproduction Facility of the Walter Reed Army Institute of Research (Silver Spring, MD), using the same procedures previously reported (8,15). The study vaccine vials (lot no.…”

Section: Methodsmentioning

confidence: 99%

Safety and Immunogenicity of a Parenterally Administered, Structure-Based Rationally Modified Recombinant Staphylococcal Enterotoxin B Protein Vaccine, STEBVax

Chen

Pasetti

Adhikari

et al. 2016

S everal staphylococcal enterotoxins (SEs) are bacterial superantigens (SAgs) produced by Staphylococcus aureus. The serologically and genetically distinct SEs (A, A1, A2, B, C, C1, C2, C3, D, E, G, H, and I) bind to major histocompatibility complex class II (MHC-II) proteins and to T-cell antigen receptors (TCRs), bypassing processing by antigen-presenting cells (1). Since T-cell engagement with SAgs is independent of clonal specificity, picomolar concentrations of a SAg can result in the polyclonal activation of large subsets of peripheral T cells (up to 20%) (2-4). The nonspecific activation of lymphocytes and the subsequent overwhelming production of cytokines make the clinical effects of SAgs potentially lethal. During the 1960s, staphylococcal enterotoxin B (SEB) became a focus of interest because aerosolization of minute amounts could be used to produce an incapacitating biological weapon (5).Strategies to counteract the effects of SAgs are currently limited to treatment of the infection, which thereby limits toxin production. There are currently no approved vaccines for Staphylococcus aureus or SEB. There is evidence that anti-SEB antibodies capable of neutralizing the effect of the SAg can abrogate the superantigenic activation of the immune system and be protective for the host (6, 7). Nonetheless, efforts to develop SEB vaccines require caution because biological inactivation must be complete and the critical antigenic structural epitopes must be preserved.An Escherichia coli-expressed recombinant SEB (rSEB) mutant containing mutations in the hydrophobic binding loop (L45R), the polar binding pocket (Y89A), and the disulfide loop (Y94A) was developed using a structure-based rational approach (8) wherein site-specific mutagenesis was used to direct mutations at the MHC-II binding site (9-11). In a limited number of animal challenge studies, parenteral immunization with a nonadjuvanted rSEB mutant elicited anti-SEB IgG antibodies in mice and rhesus monkeys that could be measured by enzyme-linked immunosorbent assay (ELISA) and that correlated with protection (9). Nasal and oral immunization with the rSEB mutant, given with cholera toxin (CT) as an adjuvant, also elicited anti-SEB antibodies and protected against lethal challenge (10). In expanded studies, mice and rhesus monkeys parenterally immunized with the rSEB mutant plus an alum adjuvant demonstrated anti-SEB antibodies that correlated with protection; animals that achieved anti-SEB titers of Ն10 4 were 100% protected, those with titers of ϳ10 3 had partial