Synthetic human monoclonal antibodies toward staphylococcal enterotoxin B (SEB) protective against toxic shock syndrome
ABSTRACT Staphylococcal enterotoxin B (SEB) is a potent toxin that can cause toxic shock syndrome and act as a lethal and incapacitating agent when used as a bioweapon. There are currently no vaccines or immunotherapeutics available against this toxin. Using phage display technology, human antigen-binding fragments (Fabs) were selected against SEB, and proteins were produced in Escherichia coli cells and characterized for their binding affinity and their toxin neutralizing activity in vitro and in vivo. Highly protective Fabs were converted into full-length IgGs and produced in mammalian cells. Additionally, the production of anti-SEB antibodies was explored in the Nicotiana benthamiana plant expression system. Affinity maturation was performed to produce optimized lead anti-SEB antibody candidates with subnanomolar affinities. IgGs produced in N. benthamiana showed characteristics comparable with those of counterparts produced in mammalian cells. IgGs were tested for their therapeutic efficacy in the mouse toxic shock model using different challenge doses of SEB and a treatment with 200 μg of IgGs 1 h after SEB challenge. The lead candidates displayed full protection from lethal challenge over a wide range of SEB challenge doses. Furthermore, mice that were treated with anti-SEB IgG had significantly lower IFNγ and IL-2 levels in serum compared with mock-treated mice. In summary, these anti-SEB monoclonal antibodies represent excellent therapeutic candidates for further preclinical and clinical development.
- SourceAvailable from: Chao Wu
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- "Unfortunately, prophylactic or therapeutic treatments for this potential threat have yet to be developed. Many researchers have generated monoclonal antibodies against SEB that could prevent toxic shock syndrome that is induced by S. aureus infections –. Although SEB has a conserved sequence and stable structure, SEB is a potent toxin that acts as a lethal and incapacitating agent –, which has limited native SEB as a vaccine candidate. "
ABSTRACT: Staphylococcal enterotoxin B (SEB) is one of the most potent Staphylococcus aureus exotoxins (SEs). Due to its conserved sequence and stable structure, SEB might be a good candidate antigen for MRSA vaccines. Although cellular immune responses to SEB are well-characterized, much less is known regarding SEB-specific humoral immune responses, particularly regarding detailed epitope mapping. In this study, we utilized a recombinant nontoxic mutant of SEB (rSEB) and an AlPO4 adjuvant to immunize BALB/c mice and confirmed that rSEB can induce a high antibody level and effective immune protection against MRSA infection. Next, the antisera of immunized mice were collected, and linear B cell epitopes within SEB were finely mapped using a series of overlapping synthetic peptides. Three immunodominant B cell epitopes of SEB were screened by ELISA, including a novel epitope, SEB205-222, and two known epitopes, SEB97-114 and SEB247-261. Using truncated peptides, an ELISA was performed with peptide-KLH antisera, and the core sequence of the three immunodominant B cell epitopes were verified as SEB97-112, SEB207-222, and SEB247-257. In vitro, all of the immunodominant epitope-specific antisera (anti-SEB97-112, anti-SEB207-222 and anti-SEB247-257) were observed to inhibit SEB-induced T cell mitogenesis and cytokine production from splenic lymphocytes of BALB/c mice. The homology analysis indicated that SEB97-112 and SEB207-222 were well-conserved among different Staphylococcus aureus strains. The 3D crystal structure of SEB indicated that SEB97-112 was in the loop region inside SEB, whereas SEB207-222 and SEB247-257 were in the β-slice region outside SEB. In summary, the fine-mapping of linear B-cell epitopes of the SEB antigen in this study will be useful to understand anti-SEB immunity against MRSA infection further and will be helpful to optimize MRSA vaccine designs that are based on the SEB antigen.PLoS ONE 03/2014; 9(3):e90445. DOI:10.1371/journal.pone.0090445 · 3.23 Impact Factor
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ABSTRACT: In an effort to develop a sustainable platform for manufacturing protein-based vaccine candidates, we expressed a triple mutant of staphylococcal enterotoxin B carrying the L45R, Y89A, and Y94A modifications in transgenic soybean seeds (soy-mSEB). Soy-mSEB possessed no detectable superantigen activity when tested in vitro. We found that this soybean-derived, non-toxic mutant of SEB could be stably expressed, stored in seeds for extended periods of time at room temperature without degradation, and easily purified from contaminating soy proteins. Vaccination of pigs with purified soy-mSEB, or the identical triple mutant expressed in E. coli (E. coli-mSEB), resulted in high antibody titers against the native toxin in immunized animals. In fact, titers were indistinguishable regardless of the immunogen used, demonstrating the equivalence of soy-mSEB and E. coli-mSEB vaccinations. Antisera from either immunized group were able to block native SEB superantigen activity using an in vitro neutralization assay. Similar results were obtained when immunized animals were challenged with a sub-lethal dose of native toxin. Significant reductions in toxin-induced serum cytokine levels were observed in soy-mSEB and E. coli-mSEB immunized pigs when compared to control animals. The reduction in SEB-induced cytokine responses was similar regardless of the immunogen used for vaccination. Surprisingly, however, some clinical symptoms such as prostration, lethargy, emesis, and/or diarrhea were still observed in all immunized animals. These studies demonstrate the potential for soybean-derived proteins as a platform technology for sustainable vaccine manufacturing, and the usefulness of a sub-lethal challenge model in pigs for evaluating the efficacy of potential SEB vaccine candidates.Clinical and vaccine Immunology: CVI 10/2012; 20(1). DOI:10.1128/CVI.00526-12 · 2.37 Impact Factor
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ABSTRACT: Ebola virus (EBOV) is a highly pathogenic member of the Filoviridae family of viruses that causes severe hemorrhagic fever. Infection proceeds through fusion of the host cell and viral membranes, a process that is mediated by the viral envelope glycoprotein (GP). Following endosomal uptake, a key step in viral entry is the proteolytic cleavage of GP by host endosomal cysteine proteases. Cleavage exposes a binding site for the host cell receptor Niemann-Pick C1 (NPC1) and may induce conformational changes in GP leading to membrane fusion. However, the precise details of the structural changes in GP associated with proteolysis and the role of these changes in viral entry have not been established. Here, we have employed synthetic antibody technology to identify antibodies targeting EBOV GP prior to and following proteolysis (i.e. in the "uncleaved" [GP(UNCL) ] and "cleaved" [GP(CL) ] forms). We identified antibodies with distinct recognition profiles: Fab(CL) bound preferentially to GP(CL) (EC(50) =1.7 nM), whereas Fab(UNCL) bound specifically to GP(UNCL) (EC(50) =75 nM). Neutralization assays with GP-containing pseudotyped viruses indicated that these antibodies inhibited GP(CL) - or GP(UNCL) -mediated viral entry with specificity matching their recognition profiles (IC(50) : 87 nM for IgG(CL) ; 1 μM for Fab(UNCL) ). Competition ELISAs indicate that Fab(CL) binds an epitope distinct from that of KZ52, a well-characterized EBOV GP antibody, and from that of the luminal domain of NPC1. The binding epitope of Fab(UNCL) was also distinct from that of KZ52, suggesting that Fab(UNCL) binds a novel neutralization epitope on GP(UNCL) . Furthermore, the neutralizing ability of Fab(CL) suggests that there are targets on GP(CL) available for neutralization. This work showcases the applicability of synthetic antibody technology to the study of viral membrane fusion, and provides new tools for dissecting intermediates of EBOV entry.ChemBioChem 11/2012; 13(17). DOI:10.1002/cbic.201200493 · 3.06 Impact Factor