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Generation of protective immunity by inactivated recombinant staphylococcal enterotoxin B vaccine in nonhuman primates and identification of correlates of immunity

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Abstract

At this time there are no vaccines or therapeutics to protect against staphylococcal enterotoxin B (SEB) exposure. Here, we report vaccine efficacy of an attenuated SEB in a nonhuman primate model following lethal aerosol challenge and identify several biomarkers of protective immunity. Initial in vitro results indicated that the mutation of key amino acid residues in the major histocompatibility complex (MHC) class II binding sites of SEB produced a nontoxic form of SEB, which had little to no detectable binding to MHC class II molecules, and lacked T-cell stimulatory activities. When examined in a mouse model, we found that the attenuated SEB retained antigenic structures and elicited protective immune responses against wild-type SEB challenge. Subsequently, a vaccine regimen against SEB in a nonhuman primate model was partially optimized, and investigations of immune biomarkers as indicators of protection were performed. SEB-naïve rhesus monkeys were vaccinated two or three times with 5 or 20 μg of the attenuated SEB and challenged by aerosol with wild-type SEB toxin. Unlike exposure to the native toxin, the vaccine did not trigger the release of inflammatory cytokines (TNFα, IL6, or IFNγ). All rhesus monkeys that developed anti-SEB serum titers ≥104 and elicited high levels of neutralizing antibody survived the aerosol challenge. These findings suggest that the attenuated SEB is fully protective against aerosolized toxin when administered to unprimed subjects. Moreover, experiments presented in this study identified various biomarkers that showed substantial promise as correlates of immunity and surrogate endpoints for assessing in vivo biological responses in primates, and possibly in humans, to vaccines against SEs.

Introduction

Staphylococcus aureus-produced bacterial superantigens (BSAgs)—staphylococcal enterotoxins (SEs), and toxic shock syndrome toxin 1—belong to a growing family of pyrogenic exotoxins [1], [2]. The unprocessed toxins act as ligands for major histocompatibility complex (MHC) class II molecules, and form a ternary complex with the variable β chain of the T-cell receptor (TCR) when presented by antigen-presenting cells (APC). This unusually promiscuous cross-ligation of T cells and APC may allow these virulence determinants to help the bacterium escape cell surface proteins that serve as restricting elements for the cell-mediated immune recognition and trigger the overproduction of pyrogenic and inflammatory cytokines [1], [2], [3].

Following BSAg encounter, T cells may become unresponsive to subsequent challenge by the same BSAg, but not by other members of the BSAg family. Furthermore, triggered T cells down-regulate their TCR and may exit the peripheral circulation [4]. As a result these cells would not be readily available to contest viral or other infections. As an opposing hazard, a pool of silent autoreactive T cells could be overstimulated by SAgs and may find inappropriate targets to attack, causing autoimmune disorders [2], [5]. More importantly staphylococcal enterotoxin A and B (SEA and SEB, respectively) release during sepsis or intentional delivery by aerosol causes increased lung endothelial cell injury and enhanced development of pathologies associated with acute respiratory distress syndrome, leading to death [6], [7], [8], [9], [10], [11]. Because inhaled SEs cause remarkable toxicity when delivered via aerosol, SEB is considered a significant threat as a biological weapon and a “dual-threat” agent [12], [13], [14]. Consequently, there is a great need to develop protective vaccines and/or therapeutic regimens for use to protect both military and civilian populations.

It is now clear that BSAgs bind as unprocessed proteins outside of the peptide-binding groove of MHC class II molecules and in the case of SEB, little to no contact with the bound peptide has been observed [15], [16], [17], [18]. Furthermore, the integral binding sites of SEB to MHC class II consist of two structurally conserved surfaces [18]. Based on these observations, and our previous experience, we hypothesized that silenced BSAg proteins, i.e., BSAgs that contain mutations of critical amino acids that participate in BSAg molecular interactions with class II molecules, should provide an excellent avenue to generate efficacious vaccines against BSAg-associated pathology [19].

Previously we showed that SEB vaccine containing three site mutations in hydrophobic binding loop, polar binding pocket, and disulfide loop (L45R, Y89A, and Y94A, respectively) retained its antigenic characteristics [20]. This attenuated SAg-produced anti-SEB antibodies in mice and rhesus monkeys [20]. Here, we further expanded our previous work and showed efficacy of various vaccination regimens of an attenuated SEB mutant protein containing SEB L45R Y89A Y94A in a primate model against aerosolized wild-type (wt) SEB toxin. Additionally, in this study, we show a clear correlation between antibody titers and neutralizing antibody and survival in rhesus monkeys. These studies have identified immune-correlate biomarkers in primates that could be measured in the human population, thereby predicting not only the vaccine efficacy, but also pointing to the human dose schedules that may be most effective.

Section snippets

Bacterial superantigens and lipopolysaccharides (LPS)

SEA, SEB, SEC1, and TSST1 were purchased from Toxin Technology (Sarasota, FL). Each toxin was judged to be greater than 95% pure by electrophoresis on 5–20% gradient sodium dodecyl sulfate (SDS)–polyacrylamide gels. The toxins were prepared in phosphate-buffered saline (PBS; 140 mM NaCl, 50 mM Na2H2PO3, pH 7.4). Escherichia coli 055:B5-derived LPS was obtained from Difco Laboratories (Detroit, MI) and reconstituted with PBS. Aliquots were stored at −70°C for future use.

Animals

Pathogen-free BALB/c

Evaluation of rSEB binding to MHC class II, T-cell stimulation, and cytokine induction

We examined cell surface binding of rSEB to MHC class II of human lymphoblastoid B cell line LG2 by using flow cytometery, and compared to wt SEB. As seen in Fig. 1A, substantial SEB binding to LG2 cells was detected at 10 μg/ml after a short incubation with the wt toxin. Higher binding of SEB was observed at 250 μg/ml. By comparison, there was no detectable binding of rSEB to LG2 cells greater than the control values.

As a second measure of rSEB attenuation, the effect of different

Conclusions

SEs are believed to be part of the biological weapons arsenals of certain countries, and to pose a real bioterrorism threat. At present there are no licensed vaccines available to protect humans against the toxic effects of BSAgs, such as SEA and SEB. We showed previously that inactivated BSAgs may be used as vaccine candidates (19). In those studies, the approach to producing an effective and safe SAg-vaccine candidate centered on designing attenuated nonsuperantigenic proteins with the

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