key: cord-0978044-pdjdsr5d
authors: Greinacher, Andreas; Selleng, Kathleen; Palankar, Raghavendra; Wesche, Jan; Handtke, Stefan; Wolff, Martina; Aurich, Konstanze; Lalk, Michael; Methling, Karen; Völker, Uwe; Hentschker, Christian; Michalik, Stephan; Steil, Leif; Reder, Alexander; Schönborn, Linda; Beer, Martin; Franzke, Kati; Büttner, Andreas; Fehse, Boris; Stavrou, Evi X.; Rangaswamy, Chandini; Mailer, Reiner K.; Englert, Hanna; Frye, Maike; Thiele, Thomas; Kochanek, Stefan; Krutzke, Lea; Siegerist, Florian; Endlich, Nicole; Warkentin, Theodore E.; Renné, Thomas
title: Insights in ChAdOx1 nCov-19 Vaccine-induced Immune Thrombotic Thrombocytopenia (VITT)
date: 2021-10-01
journal: Blood
DOI: 10.1182/blood.2021013231
sha: 6c0db9eee8c709d9e6cdafcf33ea3eed60cbde1b
doc_id: 978044
cord_uid: pdjdsr5d

SARS-CoV-2 vaccine ChAdOx1 nCov-19 (AstraZeneca) causes a thromboembolic complication termed vaccine-induced immune thrombotic thrombocytopenia (VITT). Using biophysical techniques, mouse models and analysis of VITT patient samples we identified determinants of this vaccine-induced adverse reaction. Super-resolution microscopy visualized vaccine components forming antigenic complexes with platelet factor 4 (PF4) on platelet surfaces to which anti-PF4 antibodies obtained from VITT patients bound. PF4/vaccine complex formation was charge-driven and increased by addition of DNA. Proteomics identified substantial amounts of virus production-derived T-REx HEK293 proteins in the EDTA-containing vaccine. Injected vaccine increased vascular leakage in mice leading to systemic dissemination of vaccine components known to stimulate immune responses. Together, PF4/vaccine complex formation and the vaccine-stimulated proinflammatory milieu trigger a pronounced B cell response that results in the formation of high-avidity anti-PF4 antibodies in VITT patients. The resulting high-titer anti-PF4 antibodies potently activated platelets in the presence of PF4 or DNA and polyphosphate polyanions. Anti-PF4 VITT patient antibodies also stimulated neutrophils to release NETs in a platelet PF4-dependent manner. Biomarkers of procoagulant NETs were elevated in VITT patient serum, and NETs were visualized in abundance by immunohistochemistry in cerebral vein thrombi obtained from VITT patients. Together, vaccine-induced PF4/adenovirus aggregates and proinflammatory reactions stimulate pathologic anti-PF4 antibody production that drive thrombosis in VITT. The data support a two-step mechanism underlying VITT that resembles the pathogenesis of (autoimmune) heparin-induced thrombocytopenia.

Anti-PF4 VITT patient antibodies also stimulated neutrophils to release NETs in a platelet PF4-dependent manner. Biomarkers of procoagulant NETs were elevated in VITT patient serum, and NETs were visualized in abundance by immunohistochemistry in cerebral vein thrombi obtained from VITT patients. Together, vaccine-induced PF4/adenovirus aggregates and proinflammatory reactions stimulate pathologic anti-PF4 antibody production that drive thrombosis in VITT. The data support a two-step mechanism underlying VITT that resembles the pathogenesis of (autoimmune) heparin-induced thrombocytopenia. Pathologic anti-PF4 antibodies were infrequently found in CVST patients prior to VITT, suggesting that the vaccine-induced antibodies drive these thrombotic complications. 4 PF4 opsonizes negatively charged surfaces of microbial pathogens, facilitating binding of anti-PF4 antibodies. 5 This is likely an evolutionary ancient immune defense mechanism. [6] [7] [8] However, a misdirected strong anti-PF4 antibody response underlies the thromboembolic disorder immune heparin-induced thrombocytopenia (HIT; caused by anti-PF4/heparin antibodies) and its most severe presentation, autoimmune HIT. [9] [10] [11] [12] This latter subtype of HIT is characterized by the formation of high-avidity platelet-activating anti-PF4 antibodies that are reactive even in the absence of heparin. 13 HIT proceeds by a two-step mechanism: initially, PF4/heparin complexes expose a neoantigen on PF4 that stimulate B cells to produce high-affinity anti-PF4/heparin antibodies in the presence of proinflammatory co-stimuli. Five to 10 days following heparin exposure, VITT closely mimics autoimmune HIT both clinically and serologically; 20 however the nature of neoantigens that trigger pathologic anti-PF4 antibodies, the "danger signal(s)" that prime for adverse immune reactions and prothrombotic mechanisms remain to be established in VITT. Here, we identify key components of VITT immunopathogenesis. The data suggest that VITT proceeds via a two-step mechanism: (i) vaccine components including the adenovirus hexon protein form complexes with PF4 leading to neoantigen exposure on PF4.

Vaccine components also have the capability to trigger proinflammatory responses that are "danger signals" known to amplify anti-PF4 antibody production in autoimmune HIT; (ii) between days 5-20 post vaccination, anti-PF4 antibodies from VITT patients activate platelets in a PF4-and polyanion-dependent manner. Additionally, anti-PF4 antibodies activate granulocytes in the presence of platelets to release procoagulant neutrophil extracellular traps (NETs) that are found in abundance in CVSTs of VITT patients. Together, the data highlight similarities in VITT and HIT pathogenesis and identify strategies to interfere with VITT-driven thrombotic events.

6

Detailed description of antibodies, reagents, and additional methods can be found in the Supplemental Methods section (available on the Blood website).

For Plano GmbH) and processed as above.

ChAdOx1 CoV-19 vaccine was analyzed by mass spectrometry, 1 H-nuclear magnetic resonance (NMR) spectroscopy, and 1D-SDS PAGE (detailed methods are given in the supplemental methods).

Platelet activation and aggregation by VITT patient serum were tested as previously described. 2 In brief, washed platelets of at least three healthy donors were incubated in the absence or presence of either PF4 (10 mg/mL), DNA (ds 25-mer, 1 µg/mL) or EDTA (0.2 µM) and serum from VITT patients under stirring conditions. Therefore, 75 µL platelet suspension was mixed with 20 µL serum prior to stirring. The time to aggregation was measured up to 45 min. The test was determined to be positive if platelets aggregated within 30 min.

For visualization of NETs within cerebral sinus vein thrombi, 5 µm paraffin sections were deparaffinized and rehydrated prior to antigen retrieval using sodium citrate. Slides were blocked in 2% BSA, 0.1% Triton X-100 in PBS for 45 min at RT, followed by incubation with 9

ChAdOx1 nCoV-19 vaccination is associated with delayed local and systemic reactions after the first administration indicating immunogenic reactions triggered by vaccine components. 23 We analyzed interactions of vaccine constituents with blood by biophysical techniques. 3Dsuper-resolution immunofluorescence microscopy visualized complexes formed between adenovirus-derived hexon protein and platelets ( Figure Figure S1 ). Both patient-purified and hybridoma-cell derived anti-PF4 antibodies, specifically detected PF4 alone or PF4 in a mixture with the vaccine;

however, there was no detectable signal with ChAdOx1 nCov-19 vaccine alone. Similarly, anti-AV hexon protein antibodies stained the ChAdOx1 nCov-19 vaccine alone or in mixture with PF4, however they did not bind to PF4 alone (Supplemental Figure S2) . The data reveal charge-driven complex formation of PF4 and adenovirus hexon proteins, to which VITT patient anti-PF4 IgG bound.

10

We hypothesized that ChAdOx1 nCoV-19 vaccine induces a proinflammatory "danger signal"

that promotes pathologic anti-PF4 antibody production in autoimmune HIT and possibly in VITT. 25, 26 Accordingly, we sought to characterize the vaccine composition. Unexpectedly, we found 70-80 µg protein/mL in four independent ChAdOx1 nCoV-19 vaccine lots analyzed.

Silver staining of ChAdOx1 nCoV-19 vaccine separated on SDS-PAGE showed numerous protein bands (Figure 2A) . Table S1 ).

We further analyzed the ChAdOx1 nCov-19 vaccine for small molecules by 1 H-NMR spectrometry and identified EDTA (~100 µM; Figure 3A) . Leaky vessels are a hallmark of inflammation and the Ca 2+ chelator EDTA increases vascular leakage by VE-cadherin endothelial junctional disassembly. 27 We therefore analyzed ChAdOx1 nCoV-19 vaccine for inducing proinflammatory reactions in mice using the Miles edema model ( Figure 3B ).

Intradermally injected vaccine triggered leakage in dermal vessels that was quantified by shown in Figure 3D . Taken together, the data are consistent with a model of neoantigen formation induced by vaccine constituents and PF4, that jointly stimulate pathologic anti-PF4 antibody formation facilitated by a vaccine-triggered inflammatory co-stimulus.

We next analyzed for thrombotic reactions triggered by VITT patient anti-PF4 antibodies.

Consistent with our previously published data, 2 all (14/14) sera of VITT patients analyzed, as well as their respective affinity-purified antibody fractions showed strong reactivity towards immobilized PF4/heparin in an ELISA assay. In a washed platelet aggregation-based assay, addition of PF4 amplified platelet activation triggered by VITT patient sera (up to a serum titer of 1:1000, Figure 4A) . Similarly, addition of short-chain polyphosphate (a platelet-derived inorganic polymer 30 , 0.2 µg/mL), or synthetic DNA (1 µg/ml) increased VITT patient seruminitiated platelet activation, albeit to a lower extent compared to PF4. VITT patient serum/PF4-stimulated platelet aggregation was completely inhibited by FcγRIIA receptor blockade using IV.3 antibody (Figure 4B) . Taken together, the data indicate a function of anti-PF4 IgG/PF4 complexes visualized in Figure 1 in mediating platelet activation.

Anti-PF4 antibodies of HIT patients activate neutrophils and are a major driver of thrombosis by the formation of procoagulant NETs (NETosis). 31 We tested pathologic anti-PF4

antibodies of VITT patients for their potency to trigger neutrophil activation and procoagulant NET formation in the presence of PF4 or platelets. Incubation of isolated human neutrophils and platelets with VITT patient serum-induced NET formation ( Figure 5A ) that was significantly increased by the addition of PF4 (Figures 5B, 5K) . In contrast, healthy control serum did not trigger NET formation (Figures 5C, 5D, 5K ). Similar to VITT patient serum, affinity-purified anti-PF4 antibodies from these respective samples triggered NETosis (Figures 5E, 5K ). PF4 addition strongly enhanced VITT patient serum-stimulated NET formation, confirming that PF4 in anti-PF4 IgG VITT antibodies initiates procoagulant NET formation (Figures 5F, 5K) . NETosis was virtually absent when neutrophils and platelets were co-incubated in buffer, in the absence or presence of added PF4 (Figures 5G, 5H) . In the absence of platelets, VITT patient serum failed to induce NETosis even in the presence of added PF4, suggesting that in VITT platelets play a key role in triggering NET formation (Figures 5I, 5J) .

Supporting a role of NETs in VITT, three NET biomarkers cell-free DNA, citrullinated histones and myeloperoxidase (MPO, marker for neutrophil activation), were elevated in VITT patient sera compared to healthy controls (Figures 6A-C) . CVST is a hallmark complication reported in many VITT patients. We analyzed the composition of cerebral sinus vein thrombi obtained by thrombectomy from one VITT patient or by autopsy from a second VITT patient, 12 respectively. Histology of the thrombectomy-derived tissue showed regions with amorphous material, likely representing fibrin deposition surrounded by nucleated cell-rich areas throughout the thrombi (Figure 6D ). Immunohistochemistry revealed that these cell-rich areas contained abundant activated neutrophils and NETs. Antibodies against the NET biomarker neutrophil elastase (NE, Figures 6E and 6F) and MPO (Figures 6G and 6H) visualized degranulated neutrophils releasing elongated DAPI-and chromatin-positive NETs into the platelet-rich (Figures 6I and J) 13

Based on a combination of biophysical imaging, mouse models and analysis of VITT patient material, our study suggests a two-step mechanism underlying VITT-driven thrombosis that is schematically shown in Figure 7 . VITT pathogenesis is reminiscent of autoimmune HIT pathology. Initially, neoantigens are generated by interaction of PF4 with vaccine components. As visualized by TEM, 3D super-resolution microscopy and DLS, in reconstituted systems PF4 has the capacity to bind to vaccine constituents leading to formation of complexes that contain adenovirus proteins (Figure 1, Supplemental Figures   S1, S2) . While PF4/vaccine aggregates are exposed on platelet surfaces and recognized ex vivo by VITT patient anti-PF4 antibodies, the precise sequence of events occurring in vivo at injection sites or in blood, requires additional studies. In humans, PF4 is enriched at the vessel wall and is locally released in high concentrations following platelet activation. 32, 33 Consistent with our imaging data, previous studies have shown that coronaviruses have the capacity to activate platelets 34, 35 and that adenoviruses binding to platelets can lead to platelet-activation and release of PF4. 36

ChAdOx1 nCov-19 vaccine-derived adenovirus aggregates bind to platelet surfaces and are transported via the bloodstream to the spleen where they are phagocytosed by macrophages, ultimately inducing a pronounced B-cell activation in the marginal zone and follicles in mice. 29 In line with the animal model findings, we visualized an interaction of adenovirus-derived hexon proteins, PF4, and VITT patient-derived anti-PF4 antibodies on platelet surfaces by three independent techniques (Figure 1 ). Our observations suggest that adenovirus binding to PF4 likely induces conformational changes in PF4 and creates potential neoantigen(s). Consistent with the hypothesis that VITT antibodies target neoantigen(s) in PF4, VITT patient anti-PF4 antibodies bind to PF4 following immobilization on plastic surfaces. PF4 binding to surfaces is known to induce conformational changes. 37 Additionally, mutagenesis studies have shown that VITT anti-PF4 antibodies, similarly to polyanions (Figure 4A) , induce PF4 clustering leading to platelet activation. 38 Anti-PF4 antibody stimulated platelet aggregation is not a new concept as high-affinity anti-PF4 antibodies also cluster PF4, even in the absence of polyanions and induce platelet activation in atypical HIT. 13 In addition to adenovirus proteins, we found substantial amounts (~43-60% contaminants have the potential to induce an acute inflammatory co-signal that enhances B cell responses (immunologic "'danger signal"). 15, 41, 42 Synergistically, disseminated viral proteins potently activate innate immune reactions supporting early inflammatory reactions following vaccination. 15, 41, 42 The inflammatory response provides a co-stimulus for anti-PF4 antibody production by preformed B-cells in HIT. 8, 14 Consistently, western blotting revealed that vaccination increased titers of preexisting antibodies that bind to an array of vaccine components separated by SDS-PAGE. Additionally, the ChAdOx1 nCov-19 vaccine contains EDTA, with the capacity for increasing capillary leakage at the inoculation site by a VEcadherin-dependent pathway. 27 Disruption of the endothelial barrier facilitates dissemination of vaccine constituents into the circulation (Figure 3) . Alternatively, accidental intravenous injection may contribute to vaccine dissemination. 39 (Figure 4B) . Anti-PF4 mediated platelet activation likely involves extracellular polyanions such as DNA and polyphosphate ( Figures   1C and 4A) . Clustering of PF4 by pathologic anti-PF4 auto-antibodies is also a central mechanism for platelet activation in autoimmune HIT. 13 Cross-talk of PF4, activated platelets, and VITT anti-PF4 antibodies activates neutrophils leading to NETs formation in VITT patient serum ( Figure 5 ). NETs are degraded by DNases 45 and extracellular circulating DNA was increased in VITT patients (Figure 6A) , which amplifies platelet activation in VITT (Figure 4) .

Furthermore, DNA within NETs binds PF4. 46 The resulting PF4/DNA complexes create an additional target for anti-PF4 antibodies and increase the resistance of NETs to DNasemediated degradation, further amplifying their procoagulant activity. 47 This sequence of events 31,47 culminates in massive Fcγ receptor-dependent activation of neutrophils, platelets 15 and-by analogy with autoimmune HIT-likely also monocytes and endothelial cells. 48 Consistent with our data in VITT patients, activated neutrophils and NETs contribute to venous thrombosis in HIT mouse models, and HIT antibodies selectively bind PF4-NET complexes. 47 The bimodal distribution of the neutrophil activation marker serum MPO observed in healthy controls ( Figure 6C ) may reflect the impact of smoking and hormonal contraceptive use, both of which increase MPO enzyme levels. 49 causing feto-maternal incompatibility with severe thrombocytopenia in calves. 55 In humans, narcolepsy has been associated with Pandemrix influenza vaccine, however, the underlying immune mechanisms remain to be completely understood. 56, 57 Our study has limitations; detailed specifications of the ChAdOx1 nCov-19 vaccine are not publicly available and we focused on identification of protein and some small molecule content without claiming completeness. Additionally, we have not investigated the specific roles of B-cells or T-cells in the VITT immune response, nor a potential contribution of the complement system that is known to contribute to immunogenicity and downstream thrombosis in HIT. [58] [59] [60] VITT is a rare adverse event with thrombosis occurring in 1 out of 30,000 -50,000 vaccinated people. We currently can only speculate on the low incidence of VITT by drawing parallels to HIT, an adverse reaction that occurs only in a small subset of heparin-exposed individuals (around 1 to 100 to 1 in 1,000 patients receiving UFH). VITT appears to share similarities with atypical HIT, as the latter is even more rare (<1 in 100 HIT cases) with similar approximate incidence (in the range of 1:100,000 cases) as VITT Dataset were compared using Wilcoxon matched-pairs signed rank test. 

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