Double mutant and formaldehyde inactivated TSST-1 as vaccine candidates for TSST-1-induced toxic shock syndrome (original) (raw)
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Pasteur Institute of Iran, 2020
The development of a vaccine against Staphylococcus aureus has proven to be much more difficult than expected. In this study, we considered and analyzed a mutant Toxic Shock Syndrome Toxin-1 (TSST-1) as a potential vaccine candidate. Methods: An NCBI sequence of TSST-1 was analyzed bioinformatically by online tools such as Ensemble and Pubmlst. The protein sequence of TSST-1 was similarly analysed by Expasy ProtParam, Phyre2 and Vaxign databases. The protein functional class was predicted by VICMpred database while the Band T-cell epitopes were predicted by IEDB and BepiPred tools. The 3D structure was predicted by LOMETS, QMEAN, ProSA-web and ElliPro. The conservation of the epitopes was evaluated by ConSurf tool. Results: In silico analyses showed that this protein is present in high-prevalence sequence types of circulating clinical strains. It appears that TSST-1 has conserved liner and conformational B-cell epitopes. In addition, there are four potent of T-cell epitopes in this protein. Conclusion: This in silico data indicated that TSST-1 (and especially amino acid residues 81-221) is a promising vaccine target against S. aureus.
BMC Biotechnology, 2022
Background Staphylococcal superantigens are virulence factors that help the pathogen escape the immune system and develop an infection. Toxic shock syndrome toxin (TSST)-1 is one of the most studied superantigens whose role in toxic shock syndrome and some particular disorders have been demonstrated. Inhibiting TSST-1 production with antibiotics and targeting TSST-1 with monoclonal antibodies might be one of the best strategies to prevent TSST-1induced cytokines storm followed by lethality. Results A novel single-chain variable fragment (scFv), MS473, against TSST-1 was identified by selecting an scFv phage library on the TSST-1 protein. The MS473 scFv showed high affinity and specificity for TSST-1. Moreover, MS473 could significantly prevent TSST-1-induced mitogenicity (the IC 50 value: 1.5 µM) and cytokine production. Conclusion Using traditional antibiotics with an anti-TSST-1 scFv as a safe and effective agent leads to deleting the infection source and preventing the detrimental effects of the toxin disseminated into the whole body.
Improved purification and biologic activities of staphylococcal toxic shock syndrome toxin 1
Journal of Clinical Microbiology, 1993
An improved method for producing highly purified toxic shock syndrome toxin 1 (TSST-1) by preparative isoelectric focusing in a Bio-Rad Rotofor cell and then chromatofocusing is described. Purification to homogeneity was confirmed by silver staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 50 micrograms of protein was loaded), by immunoblotting with polyclonal rabbit antiserum raised against the crude culture supernatant used for purification, and by autoradiography after iodination and SDS-PAGE. Biologic activity was demonstrated by mitogenicity and cytokine induction (tumor necrosis factor alpha [TNF-alpha], interleukin 1-beta [IL-1 beta], and IL-6) of human peripheral blood mononuclear cells (PBMCs) and by lethality in New Zealand White rabbits following subcutaneous infusion. In contrast to commercial TSST-1 preparations, our TSST-1 preparation required the presence of both monocytes and T cells for the induction of TNF-alpha and IL-1 beta from ...
Infection and Immunity
Staphylococcus aureus toxic shock syndrome toxin 1 (TSST-1) is involved in the pathogenesis of toxic shock syndrome and perhaps other staphylococcal diseases. Recently, the C-terminal part of the TSST-1 toxin has been shown to be responsible for mitogenic activity in animal models. We studied the role of the C-terminal structural unit of TSST-1 with regard to proliferation, cytokine release (tumor necrosis factor alpha [TNF-␣], interleukin-6 [IL-6], and IL-8), mRNA expression for IL-6, IL-8, IL-10, TNF-␣, and CD40 ligand (CD40L), synthesis of immunoglobulin E (IgE), IgA, IgG, and IgM, CD23 expression, and soluble CD23 (sCD23) release from human peripheral blood mononuclear cells (PBMC). For this purpose, we used the recombinant wild-type TSST-1 (p17) mutant toxin Y115A (tyrosine residue modified to alanine) and toxin H135A (histidine residue modified to alanine). Unmodified toxin p17 and mutant toxin Y115A, at a concentration below 5 ng, to a lesser degree, induced a strong proliferation. Toxin p17 followed by toxin Y115A was the most pronounced inducer for mRNA expression for IL-10 and CD40L and cytokine generation (mRNA and protein) for TNF-␣, IL-6, and IL-8. Mutant protein H135A failed to activate human PBMC. Both toxins p17 and, to a lesser degree, Y115A significantly suppressed IL-4-and anti-CD40-induced synthesis of all four Igs as well as IL-4-induced CD23 expression and sCD23 release. Mutant toxin H135A failed to do so. Thus, our data show that a region in the C terminus of TSST-1 is responsible not only for mitogenic activity but also for additional immunomodulating biological activities of TSST-1. More specifically, histidine residue H135A of the 194-amino-acid toxin appears to be critical for the expression of biological activities in a human in vitro model.
Scientific Reports, 2019
superantigens (sAgs) play a major role in the pathogenesis of Staphylococcus aureus and are associated with several diseases, including food poisoning, bacterial arthritis, and toxic shock syndrome. Monoclonal antibodies to these SAgs, primarily TSST-1, SEB and SEA have been shown to provide protection in animal studies and to reduce clinical severity in bacteremic patients. Here we quantify the pre-existing antibodies against sAgs in many human plasma and IVIG samples and demonstrate that in a major portion of the population these antibody titers are suboptimal and IVIG therapy only incrementally elevates the anti-sAg titers. our in vitro neutralization studies show that a combination of antibodies against SEA, SEB,and TSST-1 can provide broad neutralization of staphylococcal SAgs. We report a single fusion protein (tBA 225) consisting of the toxoid versions of TSST-1, SEB and SEA and demonstrate its immunogenicity and protective efficacy in a mouse model of toxic shock. Antibodies raised against this fusion vaccine provide broad neutralization of purified SAgs and culture supernatants of multiple clinically relevant S. aureus strains. our data strongly supports the use of this fusion protein as a component of an anti-virulence based multivalent toxoid vaccine against S. aureus disease. Staphylococcus aureus (SA) is a leading cause of hospital and community-associated infections worldwide with no effective vaccines available 1. The remarkable ability of SA to cause a wide range of diseases from skin and soft tissue infections (SSTI) to life threatening sepsis and pneumonia is in part due to its ability to escape the immune response using a plethora of virulence factors: the superantigenic and pore-forming toxins, coagulase, capsular polysaccharide, adhesins, proteases, and complement inactivating exoproteins 2. Since its first emergence in the 1960s methicillin-resistant SA (MRSA) has become endemic in healthcare settings, and more recently also within the community, posing a major global challenge 3,4. There have hence been increasing efforts directed towards the development of vaccines and therapeutics for SA infections. However, to date, no successful vaccine or antibody against SA infections has been developed, and there has been a spate of clinical trial failures on this front 1,5-8. Targeting SA toxins represent an alternative approach as "anti-virulence" vaccine for prevention of severe SA disease. Staphylococcal pore forming toxins alpha and gamma hemolysins and leukotoxins play critical roles in immune evasion, by killing cells of the first line of defense such as neutrophils, monocytes, and macrophages, providing iron for bacterial growth by lysing red blood cells, or enabling dissemination of bacteria through killing of cells with critical barrier function such as epithelial cells 2. Pyrogenic superantigens (SAgs) represent a major family of SA toxins composed of staphylococcal enterotoxins (SEs) and toxic shock syndrome toxin 1 (TSST-1). In contrast to conventional antigens that undergo proteolytic processing by antigen presenting cells to be presented as MHC/peptide complex to T cells, SAgs cross-link T cell receptor (TCR) with MHC Class II and activate up to 30% of T cells 9 leading to a massive release of cytokines and chemokines, enhanced expression and activation of cell-adhesion molecules, increased T-cell proliferation, and eventually T cell apoptosis or anergy. This sequence of events can culminate in toxic shock syndrome (TSS), a life-threatening condition characterized by rash, hypotension, fever, and multisystem dysfunction 10. Antibodies play an important role in protection against TSS, thus individuals that do not seroconvert towards the offending toxin due to hypo responsive T-cells 11 and/ or T-cell dependent B-cell apoptosis 12 are more likely to experience recurring bouts. Furthermore, SAgs impact the virulence of SA strains through induction of a local excessive inflammatory response, immune subversion by
Journal of Clinical Microbiology, 1987
Toxic shock syndrome toxin 1 (TSST-1), an exotoxin produced by many Staphylococcus aureus strains, is implicated as the prime causal agent of toxic shock syndrome (TSS). A sensitive and specific noncompetitive enzyme-linked immunosorbent assay (ELISA) capable of detecting TSST-1 at concentrations from 0.5 to 16 ng/ml was developed. This assay did not detect other staphylococcal enterotoxins including A, B, C1, C2, C3, D, and E. Possible interactions with protein A were readily eliminated by pretreatment of test samples with 10% normal rabbit serum. The assay was adapted for rapid screening of TSST-1 production by S. aureus isolates in culture supernatants in vitro and for detection of TSST-1 in vaginal washings of TSS patients and healthy controls in vivo. All 35 S. aureus isolates confirmed to be TSST positive by Ouchterlony immunodiffusion and 59 of 60 isolates confirmed to be TSST-1 negative gave concordant results by ELISA. Interestingly, toxigenic S. aureus strains isolated fro...
Indian Journal of Microbiology, 2012
Staphylococcal enterotoxin B (SEB) and toxic shock syndrome toxin-1 are the super antigens responsible for diseases such as staphylococcal food poisoning and toxic shock syndrome. At low serum concentrations, SEB can trigger toxic shock, profound hypotension and multi organ failure and hence is recognized as biowarfare molecule. In this study, a multidomain fusion protein (r-TE) was generated with specificity for SEB and toxic shock syndrome toxin (Tsst-1). The fusion gene comprising the conserved regions of seb and the tsst genes was codonoptimized for expression in Escherichia coli and encoded a 26 kDa recombinant multidomain chimeric protein (r-TE). Hyperimmune antiserum raised against r-TE specifically reacted with SEB (*28 kDa) and Tsst-1 (*22 kDa) components during Western blot analysis and by plate ELISA in confirmed toxin producing strains of S. aureus. The antigenicity of the SEB component of the r-TE protein was also confirmed using TECRA kit. The described procedure of creating a single protein molecule carrying components of two different toxins whilst still retaining the original antigenic determinants of individual toxins proved highly advantageous in the development of rapid, reliable and cost effective immunoassays and may also have the potential to serve as candidate molecule for vaccine studies.
Infection and Immunity, 1984
Staphylococcal toxic shock syndrome toxin (TST) was labeled with 1251 under mild conditions without apparent destruction of the molecule. [125I]TST bound specifically to human epithelial (Chang) cells in culture; the binding was inhibited by a 100-fold excess of unlabeled toxin. Scatchard anaylsis of the binding data indicated about 104 receptor sites per cell and a dissociation constant (Kd) of 4 x 10-9 M. When cells pretreated with TST at 4°C were swiftly transferred to 37°C, the amount of surface-bound toxin rapidly declined, as determined by release of noninternalized label from the cell surface. Half-time (01,2) of internalization was about 1.5 min. Ultrastructural studies showed that toxin labeled with ferritin-conjugated antibodies entered the cytoplasm via coated pits forming coated vesicles in the first 2 min of incubation at 37°C. The coated vesicles coalesced with transport vesicles that are ultrastructurally unlike receptosomes. Thus, the unusual ultrastructural pattern of this internalization suggests that TST is initially internalized by receptor-mediated endocytosis and then enters an alternate pathway involving translocation in special transport vesicles, perhaps to other cells.