Biological Activity of Masked Endotoxin

Schwarz, Harald; Gornicec, Jan; Neuper, Theresa; Parigiani, Maria Alejandra; Wallner, Michael; Duschl, Albert; Horejs‐Hoeck, Jutta

doi:10.1038/srep44750

Cited by 70 publications

(58 citation statements)

References 19 publications

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“…7). Our finding is consistent with previous reports demonstrating that LPS, similar to HVEM-(Fc*), enhanced the expression of activation markers CD40, CD80, and CD83 but not the expression of CD86 (58,61). Recent human and mouse studies have shown that CD80 and CD86 have differential roles in different disease states (62)(63)(64)(65).…”

Section: Discussionsupporting

confidence: 93%

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Soluble Fc-Disabled Herpes Virus Entry Mediator Augments Activation and Cytotoxicity of NK Cells by Promoting Cross-Talk between NK Cells and Monocytes

Meng

Zaidi

Šedý

et al. 2019

The Journal of Immunology

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CD160 is highly expressed by NK cells and is associated with cytolytic effector activity. Herpes virus entry mediator (HVEM) activates NK cells for cytokine production and cytolytic function via CD160. Fc-fusions are a well-established class of therapeutics, where the Fc domain provides additional biological and pharmacological properties to the fusion protein including enhanced serum t1/2 and interaction with Fc receptor–expressing immune cells. We evaluated the specific function of HVEM in regulating CD160-mediated NK cell effector function by generating a fusion of the HVEM extracellular domain with human IgG1 Fc bearing CD16-binding mutations (Fc*) resulting in HVEM-(Fc*). HVEM-(Fc*) displayed reduced binding to the Fc receptor CD16 (i.e., Fc-disabled HVEM), which limited Fc receptor–induced responses. HVEM-(Fc*) functional activity was compared with HVEM-Fc containing the wild type human IgG1 Fc. HVEM-(Fc*) treatment of NK cells and PBMCs caused greater IFN-γ production, enhanced cytotoxicity, reduced NK fratricide, and no change in CD16 expression on human NK cells compared with HVEM-Fc. HVEM-(Fc*) treatment of monocytes or PBMCs enhanced the expression level of CD80, CD83, and CD40 expression on monocytes. HVEM-(Fc*)–enhanced NK cell activation and cytotoxicity were promoted via cross-talk between NK cells and monocytes that was driven by cell–cell contact. In this study, we have shown that soluble Fc-disabled HVEM-(Fc*) augments NK cell activation, IFN-γ production, and cytotoxicity of NK cells without inducing NK cell fratricide by promoting cross-talk between NK cells and monocytes without Fc receptor–induced effects. Soluble Fc-disabled HVEM-(Fc*) may be considered as a research and potentially therapeutic reagent for modulating immune responses via sole activation of HVEM receptors.

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Section: Discussionsupporting

confidence: 93%

“…7A) and flow-gated monocytes in PBMC cultures ( Fig. 7B), consistent with Schwarz et al (58) who also found upregulation of CD80, CD83, and CD40 but downregulation of CD86 in response to LPS. Similar to HVEM-(Fc*), HVEM-His also activated both monocytes and NK cells in PBMC cultures (Supplemental Fig.…”

Section: Hvem-(fc*) Promotes Activation Of Monocytessupporting

confidence: 89%

Soluble Fc-Disabled Herpes Virus Entry Mediator Augments Activation and Cytotoxicity of NK Cells by Promoting Cross-Talk between NK Cells and Monocytes

Meng

Zaidi

Šedý

et al. 2019

The Journal of Immunology

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“…[214] It has been shown that specific components, including citrate, Triton X-100, and bovine serum albumin hinder reliable LPS recognition by commercially available LPS detection assays. [215] From these data, we can conclude that effects of NM on immune cells must be analyzed very carefully to distinguish potential contaminating LPS effects from real NM-mediated effects. However, due to their ability to interact with endotoxin and due to their optical and physical properties, novel LPS detection platforms involving NM may be developed in the future.…”

Section: Interaction Of Bacterial Molecules With Nanomaterialsmentioning

confidence: 92%

Addressing Nanomaterial Immunosafety by Evaluating Innate Immunity across Living Species

Boraschi

Alijagić

Auguste

et al. 2020

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The interaction of a living organism with external foreign agents is a central issue for its survival and adaptation to the environment. Nanosafety should be considered within this perspective, and it should be examined that how different organisms interact with engineered nanomaterials (NM) by either mounting a defensive response or by physiologically adapting to them. Herein, the interaction of NM with one of the major biological systems deputed to recognition of and response to foreign challenges, i.e., the immune system, is specifically addressed. The main focus is innate immunity, the only type of immunity in plants, invertebrates, and lower vertebrates, and that coexists with adaptive immunity in higher vertebrates. Because of their presence in the majority of eukaryotic living organisms, innate immune responses can be viewed in a comparative context. In the majority of cases, the interaction of NM with living organisms results in innate immune reactions that eliminate the possible danger with mechanisms that do not lead to damage. While in some cases such interaction may lead to pathological consequences, in some other cases beneficial effects can be identified.

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“…Endotoxin content was determined by Limulus amebocyte assay (PYROTELL®-T, Associates of Cape Cod, MA, USA) according to the manufacturer’s instructions. Selected proteins were also tested for masked endotoxin using a NFκB reporter assay based on HEK293 cells overexpressing TLR4, MD-2, and CD14, as previously described 27, 28 .…”

Section: Methodsmentioning

confidence: 99%

In silico design of Phl p 6 variants with altered folding stability significantly impacts antigen processing, immunogenicity and immune polarization

Winter

Stubenvoll

Scheiblhofer

et al. 2020

Preprint

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AbstactIntroductionProtein fold stability has been proposed to represent an intrinsic feature contributing to immunogenicity and immune polarization by influencing the amount of peptide-MHC II complexes (pMHCII). Using in silico prediction, we introduced point mutations in proteins that either increase or decrease their fold-stability without altering immunodominant epitopes or changing the overall structure of the protein. Here, we investigated how modulation of the fold-stability of the grass pollen allergen Phl p 6 affects its ability to stimulate immune responses and T cell polarization.MethodsUsing the MAESTRO software tool, stabilizing or destabilizing mutations were selected and verified by molecular dynamics simulations. The mutants were expressed in E. coli, purified tag-free, and analyzed for thermal stability and resistance to endolysosomal proteases. The resulting peptides were analysed by degradome assay and mass spectrometry. The structure of the most stable mutant protein was obtained by X-ray crystallography. We evaluated the capacity of the mutants to stimulate T cell proliferation in vitro, as well as antibody responses and T cell polarization in vivo in an adjuvant-free BALB/c mouse model.ResultsFour stabilizing and two destabilizing mutations were identified by MAESTRO. Experimentally determined changes in thermal stability compared to the wild type protein ranged from -5 to +14 °C. Destabilization led to faster proteolytic processing in vitro, whereas highly stabilized mutants were degraded very slowly. However, the overall pattern of identified peptides remained very similar. This was confirmed in bone marrow derived dendritic cells that processed and presented the immune dominant epitope from a destabilized mutant more efficiently. In vivo, stabilization resulted in a shift in immune polarization as indicated by higher levels of IgG2a and increased secretion of TH1/TH17 cytokines.ConclusionMAESTRO was very efficient in detecting single point mutations that increase or reduce fold-stability. Thermal stability correlated well with susceptibility to protease resistance and presentation of pMHCII on the surface of dendritic cells in vitro. This change in processing kinetics significantly influenced the polarization of T cell responses in vivo. Modulating the fold-stability of proteins thus has the potential to optimize and polarize immune responses, which opens the door to more efficient design of molecular vaccines.

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Biological Activity of Masked Endotoxin

Cited by 70 publications

References 19 publications

Soluble Fc-Disabled Herpes Virus Entry Mediator Augments Activation and Cytotoxicity of NK Cells by Promoting Cross-Talk between NK Cells and Monocytes

Soluble Fc-Disabled Herpes Virus Entry Mediator Augments Activation and Cytotoxicity of NK Cells by Promoting Cross-Talk between NK Cells and Monocytes

Addressing Nanomaterial Immunosafety by Evaluating Innate Immunity across Living Species

In silico design of Phl p 6 variants with altered folding stability significantly impacts antigen processing, immunogenicity and immune polarization

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