High Titer Persistent Neutralizing Antibodies Induced by TSST-1 Variant Vaccine Against Toxic Shock Cytokine Storm.

Staphylococcal superantigen toxins lead to a devastating cytokine storm resulting in shock and multi-organ failure. We have previously assessed the safety and immunogenicity of a recombinant toxic shock syndrome toxin 1 variant vaccine (rTSST-1v) in clinical trials (NCT02971670 and NCT02340338). The current study assessed neutralizing antibody titers after repeated vaccination with escalating doses of rTSST-1v. At study entry, 23 out of 34 subjects (67.6%) had neutralizing antibody titers inhibiting T cell activation as determined by 3H-thymidine incorporation at a serum dilution of ≤1:100 with similar figures for inhibition of IL-2 activation (19 of 34 subjects, 55.9%) as assessed by quantitative PCR. After the first vaccination, numbers of subjects with neutralization titers inhibiting T cell activation (61.7% ≥ 1:1000) and inhibiting IL-2 gene induction (88.2% ≥ 1:1000) increased. The immune response was augmented after the second vaccination (inhibiting T cell activation: 78.8% ≥ 1:1000; inhibiting IL-2 induction: 93.9% ≥ 1:1000) corroborated with a third immunization months later in a small subgroup of subjects. Assessment of IFNγ, TNFα and IL-6 inhibition revealed similar results, whereas neutralization titers did not change in placebo participants. Antibody titer studies show that vaccination with rTSST-1v in subjects with no/low neutralizing antibodies can rapidly induce high titer neutralizing antibodies persisting over months.

Safety, tolerability, and immunogenicity of a recombinant toxic shock syndrome toxin (rTSST)-1 variant vaccine: a randomised, double-blind, adjuvant-controlled, dose escalation first-in-man trial.

BACKGROUND: Staphylococcal toxic shock syndrome is a superantigen-driven potentially life-threatening disease affecting mainly young and otherwise healthy individuals. Currently, no specific treatment or preventive measure is available. We aimed to assess the safety, tolerability, and immunogenicity of a recombinant detoxified toxic shock syndrome toxin-1 variant (rTSST-1v) vaccine in adult volunteers. METHODS: In this randomised, double-blind, adjuvant-controlled, dose-escalation first-in-human trial, healthy adults aged 18-64 years were enrolled from the Medical University of Vienna, Austria. Participants were randomly assigned (2:1 and 3:1) by block randomisation (block sizes of three and 12) to receive increasing doses of rTSST-1v (100 ng to 30 µg) or the adjuvant comparator aluminium hydroxide (Al(OH)3) (200 µg, 600 µg, or 1 mg). Investigators and participants were masked to group allocation. The per-protocol population received a booster immunisation 42 days after the first vaccination. The primary endpoint was safety and tolerability of rTSST-1v. The per-protocol population included all participants who had adhered to the study protocol without any major protocol deviations. The per-protocol population was the primary analysis population for immunogenicity. The trial is registered with EudraCT, number 2013-003716-50, and ClinicalTrials.gov, number NCT02340338. FINDINGS: Between Aug 19, 2014, and April 14, 2015, 46 participants were enrolled (safety population), of whom three were assigned to cohort 1 (two to receive 100 ng rTSST-1v and one to receive 200 µg Al(OH)3), three to cohort 2 (two to receive 300 ng rTSST-1v and one to receive 600 µg Al(OH)3), four to cohort 3 (three to receive 1 µg rTSST-1v and one to receive 1 mg Al(OH)3), 12 to cohort 4 (nine to receive 3 µg rTSST-1v and three to receive 1 mg Al(OH)3), 12 to cohort 5 (nine to receive 10 µg rTSST-1v and three to receive 1 mg Al(OH)3), and 12 to cohort 6 (nine to receive 300 µg rTSST-1v and three to receive 1 mg Al(OH)3). 45 participants (98%) were included in the per-protocol population. rTSST-1v had a good safety profile, and no vaccination-related severe or serious adverse events occurred. Adverse event rates were similar between participants who received rTSST-1v and those who received placebo (26 [76%] vs 10 [83%]; p=0·62) independent of pre-existing TSST-1 immunity. INTERPRETATION: rTSST-1v was safe, well-tolerated, and immunogenic. This study represents an important step in vaccine development to prevent or treat a potentially lethal disease. FUNDING: Biomedizinische Forschungs GmbH.

Toxic shock syndrome toxin-1-mediated toxicity inhibited by neutralizing antibodies late in the course of continual in vivo and in vitro exposure.

Toxic shock syndrome (TSS) results from the host's overwhelming inflammatory response and cytokine storm mainly due to superantigens (SAgs). There is no effective specific therapy. Application of immunoglobulins has been shown to improve the outcome of the disease and to neutralize SAgs both in vivo and in vitro. However, in most experiments that have been performed, antiserum was either pre-incubated with SAg, or both were applied simultaneously. To mirror more closely the clinical situation, we applied a multiple dose (over five days) lethal challenge in a rabbit model. Treatment with toxic shock syndrome toxin 1 (TSST-1) neutralizing antibody was fully protective, even when administered late in the course of the challenge. Kinetic studies on the effect of superantigen toxins are scarce. We performed in vitro kinetic studies by neutralizing the toxin with antibodies at well-defined time points. T-cell activation was determined by assessing T-cell proliferation (3H-thymidine incorporation), determination of IL-2 release in the cell supernatant (ELISA), and IL-2 gene activation (real-time PCR (RT-PCR)). Here we show that T-cell activation occurs continuously. The application of TSST-1 neutralizing antiserum reduced IL-2 and TNFα release into the cell supernatant, even if added at later time points. Interference with the prolonged stimulation of proinflammatory cytokines is likely to be in vivo relevant, as postexposure treatment protected rabbits against the multiple dose lethal SAg challenge. Our results shed new light on the treatment of TSS by specific antibodies even at late stages of exposure.

Staphylococcal superantigen (TSST-1) mutant analysis reveals that t cell activation is required for biological effects in the rabbit including the cytokine storm.

Staphylococcal superantigens (sAgs), such as toxic shock syndrome toxin 1 (TSST-1), induce massive cytokine production, which may result in toxic shock syndrome (TSS) and sepsis. Recently, we reported that in vitro studies in human peripheral blood mononuclear cells (PBMC) do not reflect the immunological situation of the host, because after exposure to superantigens (sAgs) in vivo, mononuclear cells (MNC) leave the circulation and migrate to organs, e.g., the spleen, liver and lung. Our experimental model of choice is the rabbit because it is comparable to humans in its sensitivity to sAg. T cell activation has been assessed by lymphocyte proliferation and IL-2 gene expression after in vivo challenge with TSST-1 and the mutant antigens; expression of the genes of proinflammatory cytokines were taken as indicators for the inflammatory reaction after the combined treatment with TSST-1 and LPS. The question as to whether the biological activities of TSST-1, e.g., lymphocyte extravasation, toxicity and increased sensitivity to LPS, are mediated by T cell activation or activation by MHC II-only, are unresolved and results are contradictory. We have addressed this question by studying these reactions in vivo, with two TSST-1 mutants: one mutated at the MHC binding site (G31R) with reduced MHC binding with residual activity still present, and the other at the T cell binding site (H135A) with no residual function detectable. Here, we report that the mutant G31R induced all the biological effects of the wild type sAg, while the mutant with non-functional TCR binding did not retain any of the toxic effects, proving the pivotal role of T cells in this system.

Analysis of the early response to TSST-1 reveals Vbeta-unrestricted extravasation, compartmentalization of the response, and unresponsiveness but not anergy to TSST-1.

Staphylococcal toxic shock syndrome toxin 1 (TSST-1) is the major cause of toxic shock syndrome and is important in the pathophysiology of staphylococcal septic shock. Our study about the biological effects of TSST-1 in the rabbit 3 and 6 h and 7 days postinjection provides evidence that TSST-1 induces leukopenia, lymphopenia, and monocytopenia as a result of extravasation of cells in a Vss-unrestricted manner. Cells in the circulation, reduced significantly in numbers, show the same phenotypic distribution as before TSST-1 injection. Three hours post-in vivo TSST-1 injection, we demonstrated compartmentalization of the response. By quantitative RT-PCR, the induction of mRNA expression of TH1 and inflammatory cytokines in the spleen and lung and a complete lack of induction in PBMC could be shown. Proliferation assays revealed that 3 h after TSST-1, PBMC were neither activated nor responsive to in vitro restimulation, even when IL-2 was added. In contrast, 7 days later, PBMC and spleen cells were anergic: showing no response to TSST-1 but a vigorous response upon addition of IL-2. The results presented extend our understanding of the pathophysiology of toxic and septic shock as a result of superantigen toxin-producing Staphylococcus aureus. Demonstration of compartmentalization of the response proves that erroneous conclusions could be drawn by the exclusive analysis of PBMCs. The results reveal further that in nonresponsiveness to the antigen, different immunological mechanisms may be operational. Measurements of the induction of cytokine gene activation provide important complementary information to that of serum cytokine levels.

Tuning the setup of sputter-coated multilayers in nanocluster-based signal-enhancing biochips for optimal performance in protein and DNA assays.

In biochip development two issues are critical: stable and specific immobilization of the ligand and achievement of high signal-to-background ratio. In this work we have addressed these issues for the development of biochips, produced by sputtering multilayers of thin metal films, metal oxides, and metal nitrides (tens to hundreds of nanometers thick) onto glass wafers. Optimized surfaces have shown good results in genomic and proteomic experiments with biochips based on surface-enhanced fluorescence and absorption techniques.

Phage display antibody-based proteomic device using resonance-enhanced detection.

The combination of phage display antibody arrays with a novel nanotransducer technique based on resonant nanoparticles in a nanosandwiched film enables the sensitive parallel screening of proteins. Using the resonance of nanoparticles with their induced mirror dipoles in a thin-film structure, limitations of fluorophores, such as unspecific background and nonvisibility to the eye, can be overcome, thereby leading to an optical signal significantly more sensitive than that of standard colloid techniques. The signal can be both directly observed as a color change of a microdot at the sensor surface and tuned throughout the visible range of the spectrum. Here we report the application of an optical chip using scFv-antibody-antigen interactions. Artificial scFv-antibodies against a variety of proteins, including yeast enzymes and bovine serum albumin (as a standard), were constructed via Phage Display. These scFv-antibodies were then coated onto metal nanoclusters and bound to their antigens that were arrayed as nanodroplets at the resonance layer of the chip. ScFv-Antibody-antigen interaction resulted in a visible array of microdots. Using resonance-enhanced absorption, the absorption signal of the spots was amplified by one to two orders of magnitude (compared to colloid-based techniques). For quantitative analysis, either an 8-micron scanner or a CCD camera (resolution 4 microns) was employed to gain direct-reflection spectra rather than unspecific scatter data (prone to dust and unspecific interaction). Our results demonstrate that this device enables high-throughput proteomics to overcome some limitations of fluorescence, enzyme labels, and colloid techniques.