TY - GEN AU - Bakos, Tamás AU - Mészáros, Tamás AU - Kozma, Gergely Tibor AU - Petra, Berényi AU - Facskó, Réka AU - Henriette, Farkas AU - Dézsi, László AU - Carlo, Heirman AU - Stefaan, de Koker AU - Raymond, Schiffelers AU - Kathryn, Anne Glatter AU - Radovits, Tamás AU - Szénási, Gábor AU - Szebeni, János TI - mRNA-LNP COVID-19 vaccine lipids induce low level complement activation and production of proinflammatory cytokines: Mechanisms, effects of complement inhibitors, and relevance to adverse reactions PY - 2024 SP - & UR - https://m2.mtmt.hu/api/publication/34521339 ID - 34521339 AB - Messenger RNA-containing lipid nanoparticles (mRNA-LNPs) enabled widespread COVID-19 vaccination with a small fraction of vaccine recipients displaying acute or sub-acute inflammatory symptoms. The molecular mechanism of these adverse events (AEs) remains undetermined. Here we report that the mRNA-LNP vaccine, Comirnaty, triggers low-level complement (C) activation and production of inflammatory cytokines, which may be key underlying processes of inflammatory AEs. In serum, Comirnaty and the control PEGylated liposome (Doxebo) caused different rises of C split products, C5a, sC5b-9, Bb and C4d, indicating stimulation of the classical pathway of C activation mainly by the liposomes, while a stronger stimulation of the alternative pathway was equal with the vaccine and the liposomes. Spikevax had similar C activation as Comirnaty, but viral or synthetic mRNAs had no such effect. In autologous serum-supplemented peripheral blood mononuclear cell (PBMC) cultures, Comirnaty caused increases in the levels of sC5b-9 and proinflammatory cytokines in the following order: IL-1α < IFN-γ < IL-1β < TNF-α < IL-6 < IL-8, whereas heatinactivation of serum prevented the rises of IL-1α, IL-1β, and TNF-α. Clinical C inhibitors, Soliris and Berinert, suppressed vaccine-induced C activation in serum but did not affect cytokine production when applied individually. These findings suggest that the PEGylated lipid coating of mRNA-LNP nanoparticles can trigger C activation mainly via the alternative pathway, which may be causally related to the induction of some, but not all inflammatory cytokines. While innate immune stimulation is essential for the vaccine’s efficacy, concurrent production of C- and PBMC-derived inflammatory mediators may contribute to some of the AEs. Pharmacological attenuation of harmful cytokine production using C inhibitors likely requires blocking the C cascade at multiple points. LA - English DB - MTMT ER - TY - JOUR AU - Bakos, Tamás AU - Mészáros, Tamás AU - Kozma, Gergely Tibor AU - Berényi, Petra AU - Facskó, Réka AU - Farkas, Henriette AU - Dézsi, László AU - Heirman, Carlo AU - de Koker, Stefaan AU - Schiffelers, Raymond AU - Glatter, Kathryn Anne AU - Radovits, Tamás AU - Szénási, Gábor AU - Szebeni, János TI - mRNA-LNP COVID-19 Vaccine Lipids Induce Complement Activation and Production of Proinflammatory Cytokines: Mechanisms, Effects of Complement Inhibitors, and Relevance to Adverse Reactions JF - INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES J2 - INT J MOL SCI VL - 25 PY - 2024 IS - 7 PG - 17 SN - 1661-6596 DO - 10.3390/ijms25073595 UR - https://m2.mtmt.hu/api/publication/34754128 ID - 34754128 N1 - Nanomedicine Research and Education Center, Department of Translational Medicine, Semmelweis University, Budapest, 1085, Hungary SeroScience LCC., Budapest, 1089, Hungary Department of Cardiology, Heart and Vascular Center, Semmelweis University, Budapest, 1122, Hungary Department of Surgical Research and Techniques, Heart and Vascular Center, Semmelweis University, Budapest, 1089, Hungary Hungarian Center of Reference and Excellence, Department of Internal Medicine and Hematology, Semmelweis University, Budapest, 1088, Hungary Etherna Biopharmaceuticals, Niel, 2845, Belgium Division of Laboratories and Pharmacy, University Medical Center, Utrecht, 3584 CX, Netherlands Department of Education, Gratz College, Philadelphia, PA 19027, United States Department of Nanobiotechnology and Regenerative Medicine, Faculty of Health Sciences, Miskolc University, Miskolc, 3530, Hungary Translational Nanobioscience Research Center, Sungkyunkwan University, Suwon, 06351, South Korea Export Date: 16 May 2024 Correspondence Address: Szebeni, J.; Nanomedicine Research and Education Center, Hungary; email: szebeni.janos@med.semmelweis-univ.hu AB - A small fraction of people vaccinated with mRNA–lipid nanoparticle (mRNA-LNP)-based COVID-19 vaccines display acute or subacute inflammatory symptoms whose mechanism has not been clarified to date. To better understand the molecular mechanism of these adverse events (AEs), here, we analyzed in vitro the vaccine-induced induction and interrelations of the following two major inflammatory processes: complement (C) activation and release of proinflammatory cytokines. Incubation of Pfizer-BioNTech’s Comirnaty and Moderna’s Spikevax with 75% human serum led to significant increases in C5a, sC5b-9, and Bb but not C4d, indicating C activation mainly via the alternative pathway. Control PEGylated liposomes (Doxebo) also induced C activation, but, on a weight basis, it was ~5 times less effective than that of Comirnaty. Viral or synthetic naked mRNAs had no C-activating effects. In peripheral blood mononuclear cell (PBMC) cultures supplemented with 20% autologous serum, besides C activation, Comirnaty induced the secretion of proinflammatory cytokines in the following order: IL-1α < IFN-γ < IL-1β < TNF-α < IL-6 < IL-8. Heat-inactivation of C in serum prevented a rise in IL-1α, IL-1β, and TNF-α, suggesting C-dependence of these cytokines’ induction, although the C5 blocker Soliris and C1 inhibitor Berinert, which effectively inhibited C activation in both systems, did not suppress the release of any cytokines. These findings suggest that the inflammatory AEs of mRNA-LNP vaccines are due, at least in part, to stimulation of both arms of the innate immune system, whereupon C activation may be causally involved in the induction of some, but not all, inflammatory cytokines. Thus, the pharmacological attenuation of inflammatory AEs may not be achieved via monotherapy with the tested C inhibitors; efficacy may require combination therapy with different C inhibitors and/or other anti-inflammatory agents. LA - English DB - MTMT ER - TY - JOUR AU - Fekete, Nóra AU - Li, Luca Kamilla AU - Kozma, Gergely Tibor AU - Fekete, György AU - Pállinger, Éva AU - Kovács, Árpád Ferenc TI - Flow Cytometry-Based Assay to Detect Alpha Galactosidase Enzymatic Activity at the Cellular Level JF - CELLS J2 - CELLS-BASEL VL - 13 PY - 2024 IS - 8 PG - 10 SN - 2073-4409 DO - 10.3390/cells13080706 UR - https://m2.mtmt.hu/api/publication/34801936 ID - 34801936 N1 - Department of Genetics, Cell and Immunobiology, Semmelweis University, Budapest, 1085, Hungary For Human Genome Foundation, Budapest, 1094, Hungary Pediatrics Centre, Tűzoltó Street Department, Semmelweis University, Budapest, 1085, Hungary Nanomedicine Research and Education Center, Department of Translational Medicine, Semmelweis University, Budapest, 1085, Hungary SeroScience LCC, Budapest, 1089, Hungary Cited By :1 Export Date: 7 February 2025 Correspondence Address: Kovács, Á.F.; For Human Genome FoundationHungary; email: kovacs.arpad@semmelweis.hu AB - Background: Fabry disease is a progressive, X chromosome-linked lysosomal storage disorder with multiple organ dysfunction. Due to the absence or reduced activity of alpha-galactosidase A (AGAL), glycosphingolipids, primarily globotriaosyl-ceramide (Gb3), concentrate in cells. In heterozygous women, symptomatology is heterogenous and currently routinely used fluorometry-based assays measuring mean activity mostly fail to uncover AGAL dysfunction. The aim was the development of a flow cytometry assay to measure AGAL activity in individual cells. Methods: Conventional and multispectral imaging flow cytometry was used to detect AGAL activity. Specificity was validated using the GLA knockout (KO) Jurkat cell line and AGAL inhibitor 1-deoxygalactonojirimycin. The GLA KO cell line was generated via CRISPR-Cas9-based transfection, validated with exome sequencing, gene expression and substrate accumulation. Results: Flow cytometric detection of specific AGAL activity is feasible with fluorescently labelled Gb3. In the case of Jurkat cells, a substrate concentration of 2.83 nmol/mL and 6 h of incubation are required. Quenching of the aspecific exofacial binding of Gb3 with 20% trypan blue solution is necessary for the specific detection of lysosomal substrate accumulation. Conclusion: A flow cytometry-based assay was developed for the quantitative detection of AGAL activity at the single-cell level, which may contribute to the diagnosis of Fabry patients. LA - English DB - MTMT ER - TY - JOUR AU - Barta, Bálint András AU - Radovits, Tamás AU - Dobos, Attila Balázs AU - Kozma, Gergely Tibor AU - Mészáros, Tamás AU - Berényi, Petra AU - Facskó, Réka AU - Fulop, Tamas AU - Merkely, Béla Péter AU - Szebeni, János TI - Comirnaty-induced cardiopulmonary distress and other symptoms of complement-mediated pseudo-anaphylaxis in a hyperimmune pig model: Causal role of anti-PEG antibodies JF - VACCINE: X J2 - VACCINE X VL - 19 PY - 2024 PG - 11 SN - 2590-1362 DO - 10.1016/j.jvacx.2024.100497 UR - https://m2.mtmt.hu/api/publication/34877474 ID - 34877474 N1 - Heart and Vascular Center, Semmelweis University, Budapest, Hungary Nanomedicine Research and Education Center, Department of Translational Medicine, Semmelweis University, Budapest, Hungary SeroScience LCC, Budapest, Hungary TECOdevelopment GmbH, Rheinbach, Germany Department of Nanobiotechnology and Regenerative Medicine, Faculty of Health Sciences, Miskolc University, Miskolc, 2880, Hungary School of Chemical Engineering and Translational Nanobioscience Research Center, Sungkyunkwan University, Suwon, 16419, South Korea Export Date: 23 July 2024 Correspondence Address: Szebeni, J.; Heart and Vascular Center, Hungary; email: Szebeni.Janos@med.semmelweis-univ.hu Chemicals/CAS: complement, 9007-36-7; immunoglobulin M, 9007-85-6; thromboxane A2, 57576-52-0; thromboxane B2, 54397-85-2; tozinameran, 2417899-77-3 Tradenames: comirnaty, Pfizer; comirnaty, BioNTech Manufacturers: BioNTech; Pfizer Funding details: National Research, Development and Innovation Office Funding details: Horizon 2020, 825828, 952520 Funding details: Horizon 2020 Funding details: Nemzeti Kutatási Fejlesztési és Innovációs Hivatal, NKFIH, 2020-1.1.6-JÖVŐ-2021-00013, TKP2021-EGA-23 Funding details: Nemzeti Kutatási Fejlesztési és Innovációs Hivatal, NKFIH Funding details: Nemzeti Kutatási, Fejlesztési és Innovaciós Alap, NKFIA, ÚNKP-22-3-II-SE-30 Funding details: Nemzeti Kutatási, Fejlesztési és Innovaciós Alap, NKFIA Funding text 1: The financial support by the European Union Horizon 2020 projects 825828 (Expert) and 952520 (Biosafety) are acknowledged. This project was supported by a grant from the National Research, Development, and Innovation Office (NKFIH) of Hungary (2020-1.1.6-J\\u00D6V\\u0150-2021-00013). TKP2021-EGA-23 has been implemented with the support provided by the Ministry of Innovation and Technology of Hungary from the National Research, Development and Innovation Fund, financed under the TKP2021-EGA funding scheme. JS thanks the logistic support by the Applied Materials and Nanotechnology, Center of Excellence, Miskolc University, Miskolc, Hungary. The project was supported by the \\u00DANKP-22-3-II-SE-30 New National Excellence Program of the Ministry for Culture and Innovation from the source of the National Research, Development and Innovation Fund to BAB. LA - English DB - MTMT ER - TY - GEN AU - Dézsi, László AU - Kökény, Gábor AU - Révész, Csaba AU - Mészáros, Tamás AU - Bakos, Tamás AU - Kozma, Gergely Tibor AU - Dobos, Attila Balázs AU - Merkely, Béla Péter AU - Radovits, Tamás AU - Szebeni, János TI - LNP-mRNS vakcinák pszeudoallergiás és proinflammatorikus hatásainak vizsgálata sertés hiperszenzitivitási modellben PY - 2024 SP - 4 UR - https://m2.mtmt.hu/api/publication/35067737 ID - 35067737 AB - Bevezetés Korábban kimutattuk, hogy COVID-19 védőoltásként alkalmazott LNP-mRNS vakcinák sertés komplement-aktivációval kapcsolatos pszeudoallergiás (CARPA) modellben - humán adáshoz hasonló - korai hiperszenzitivitási reakcióhoz vezetnek. A jelenlegi vizsgálatban a fentiek mellett az LNP-mRNS vakcinák korai és késői proinflammatorikus mellékhatásait teszteltük. Anyag és módszer Kísérleteinkben a Pfizer SARS-CoV-2 elleni monovalens Comirnaty (CMT) és a bivalens Comirnaty/Omikron vakcinát altatott sertéseknek (n=10) 5x humán dózisban, 3-szori ismétléssel i.v. adtuk. Ezen kívül pegilált üres liposzóma (doxebo) előkezelés immunogén hatását, valamint az ismételt vakcinaadás általi tachyphylaxis (saját-tolerancia) kialakulását is vizsgáltuk. A vakcinabeadások előtt és után vérmintákat vettünk, melyekből perifériás mononukleáris sejteket (PBMC) izoláltunk. A kísérletek végén több szervből (pl. szív, vese, máj, lép, agy) szövetmintákat vettünk. A PBMC-ben, ill. a szövetekben a vakcinabeadást követő gyulladásos citokinek és a tüskefehérje (SP) expresszióját az őket kódoló mRNS szekvenciák qPCR módszerrel történő kimutatása alapján elemeztük. A paraméterek változását akut és krónikus (6-8 hét) kísérletekben (n=6) is követtük. Eredmények Az akut pszeudoallergiás reakció létrejöttét mindkét vakcina adásakor a sertés CARPA modell több (pl. hemodinamikai, hematológiai) markere jelezte. Doxebo előkezelés az anaphylaxiás sokk gyakoriságát növelte. Ismételt CMT adáskor esetenként tachypylaxist láttunk, a többi állatban mindhárom vakcinabeadás CARPA reakciót váltott ki. CMT adása PBMC-ben már a 15. percben gyulladásos citokinek mRNS expresszióját indukálta. Az mRNS szintek időbeli változása követte a 3-szori vakcinabeadást, és a kísérlet végén (6 óránál) volt a legmagasabb. CMT adása emellett a PBMC-ben tüskefehérje (SP1) mRNS expresszióhoz vezetett, ami 6 óra után a szívből, veséből, májból, lépből és az agyból vett szövetminták egy részében is kimutatható volt. Krónikus kísérleteinkben CMT adása után akár 2 hónappal az állatok szívében és veséjében kismértékű, de egyértelmű SP1 mRNS expressziót találtunk. Egyes állatokban immunfestéssel SP1-pozitív vesetubulusokat is kimutattunk. Következtetés Eredményeink szerint monovalens és bivalens CMT vakcina is pszeudolallergiás reakciót váltott ki. Emellett PBMC-ben a vakcinabeadás után pár perccel, a szövetmintákban 6 óra múlva citokin, ill. SP mRNS expressziót láttunk, ami akár 2 hónap után is fennállt. Ezáltal sertéseken a CMT adást kísérő gyulladásos mellékhatásokat sikeresen tudtuk modellezni. LA - Hungarian DB - MTMT ER - TY - GEN AU - Facskó, Réka AU - Mészáros, Tamás AU - Szebeni, János AU - Kozma, Gergely Tibor TI - A polietilén glikol (PEG) elleni antitestek aviditásának vizsgálata hagyományos ELISA módszerrel PY - 2024 UR - https://m2.mtmt.hu/api/publication/35740999 ID - 35740999 LA - Hungarian DB - MTMT ER - TY - GEN AU - Facskó, Réka AU - Mészáros, Tamás AU - Szebeni, János AU - Kozma, Gergely Tibor TI - Characterizing Antibody Avidity Against Polyethylene Glycol Using the Traditional ELISA Method PY - 2024 UR - https://m2.mtmt.hu/api/publication/35741006 ID - 35741006 LA - English DB - MTMT ER - TY - GEN AU - Facskó, Réka AU - Mészáros, Tamás AU - Szebeni, János AU - Kozma, Gergely Tibor TI - Characterizing Anti-PEG IgG Antibody Avidity Using the ELISA Equilibrium Titration Method PY - 2024 UR - https://m2.mtmt.hu/api/publication/35741008 ID - 35741008 LA - English DB - MTMT ER - TY - JOUR AU - Kökény, Gábor AU - Bakos, Tamás AU - Barta, Bálint András AU - Nagy, Georgina Viktória AU - Mészáros, Tamás AU - Kozma, Gergely Tibor AU - Szabó, András AU - Szebeni, János AU - Merkely, Béla Péter AU - Radovits, Tamás TI - Zymosan Particle-Induced Hemodynamic, Cytokine and Blood Cell Changes in Pigs: An Innate Immune Stimulation Model with Relevance to Cytokine Storm Syndrome and Severe COVID-19 JF - INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES J2 - INT J MOL SCI VL - 24 PY - 2023 IS - 2 PG - 18 SN - 1661-6596 DO - 10.3390/ijms24021138 UR - https://m2.mtmt.hu/api/publication/33548672 ID - 33548672 N1 - MTA Bolyai János Kutatási Ösztöndíj AB - Hemodynamic disturbance, a rise in neutrophil-to-lymphocyte ratio (NLR) and release of inflammatory cytokines into blood, is a bad prognostic indicator in severe COVID-19 and other diseases involving cytokine storm syndrome (CSS). The purpose of this study was to explore if zymosan, a known stimulator of the innate immune system, could reproduce these changes in pigs. Pigs were instrumented for hemodynamic analysis and, after i.v. administration of zymosan, serial blood samples were taken to measure blood cell changes, cytokine gene transcription in PBMC and blood levels of inflammatory cytokines, using qPCR and ELISA. Zymosan bolus (0.1 mg/kg) elicited transient hemodynamic disturbance within minutes without detectable cytokine or blood cell changes. In contrast, infusion of 1 mg/kg zymosan triggered maximal pulmonary hypertension with tachycardia, lasting for 30 min. This was followed by a transient granulopenia and then, up to 6 h, major granulocytosis, resulting in a 3–4-fold increase in NLR. These changes were paralleled by massive transcription and/or rise in IL-6, TNF-alpha, CCL-2, CXCL-10, and IL-1RA in blood. There was significant correlation between lymphopenia and IL-6 gene expression. We conclude that the presented model may enable mechanistic studies on late-stage COVID-19 and CSS, as well as streamlined drug testing against these conditions. LA - English DB - MTMT ER - TY - JOUR AU - Kozma, Gergely Tibor AU - Mészáros, Tamás AU - Berényi, Petra AU - Facskó, Réka AU - Patkó, Zsófia Panna AU - Oláh, Csaba Zsolt AU - Nagy, Adrienne AU - Fülöp, Tamás Gyula AU - Glatter, Kathryn Anne AU - Radovits, Tamás AU - Merkely, Béla Péter AU - Szebeni, János TI - Role of anti-polyethylene glycol (PEG) antibodies in the allergic reactions to PEG-containing Covid-19 vaccines: Evidence for immunogenicity of PEG JF - VACCINE J2 - VACCINE VL - 41 PY - 2023 IS - 31 SP - 4561 EP - 4570 PG - 10 SN - 0264-410X DO - 10.1016/j.vaccine.2023.06.009 UR - https://m2.mtmt.hu/api/publication/34039197 ID - 34039197 N1 - Funding Agency and Grant Number: Materials and Nanotechnology, Center of Excellence, Miskolc University, Miskolc, Hungary Funding text: The financial support by the European Union Horizon 2020 projects 825828 (Expert) and 952520 (Biosafety) are acknowledged. This project was supported by a grant from the National Research, Development, and Innovation Office (NKFIH) of Hungary (2020-1.1.6-JOEV}O-2021-00013). JS thanks the logistic support by the Applied & nbsp;Materials and Nanotechnology, Center of Excellence, Miskolc University, Miskolc, Hungary.r Materials and Nanotechnology, Center of Excellence, Miskolc University, Miskolc, Hungary. LA - English DB - MTMT ER -