Monoclonal and oligoclonal anti-platelet factor 4 antibodies mediate VITT.
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ABSTRACT:
SUBMITTER: Kanack AJ
PROVIDER: S-EPMC9262283 | biostudies-literature | 2022 Jul
REPOSITORIES: biostudies-literature
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Monoclonal and oligoclonal anti-platelet factor 4 antibodies mediate VITT.
Kanack Adam J AJ Bayas Antonios A George Gemlyn G Abou-Ismail Mouhamed Yazan MY Singh Bandana B Kohlhagen Mindy C MC Splinter Noah P NP Christ Monika M Naumann Markus M Moser Karen A KA Smock Kristi J KJ Grazioli Alison A Wen Renren R Wang Demin D Murray David L DL Padmanabhan Anand A
Blood 20220701 1
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Project description:BackgroundCOVID-19 vaccines have been associated with a rare thrombotic and thrombocytopenic reaction, Vaccine-induced immune thrombotic thrombocytopenia (VITT) characterized by platelet-activating anti-PF4 antibodies. This study sought to assess clonality of VITT antibodies and evaluate their characteristics in antigen-based and functional platelet studies.MethodsAnti-PF4 antibodies were isolated from five patients with VITT secondary to ChAdOx1 nCoV-19 (n=1) or Ad26.COV2.S (n=4) vaccination. For comparative studies with heparin-induced thrombocytopenia (HIT), anti-PF4 antibodies were isolated from one patient with spontaneous HIT, another with "classical" HIT, and two patients with non-pathogenic (non-platelet activating) anti-PF4 antibodies. Isolated antibodies were subject to ELISA and functional testing, and mass spectrometric evaluation for clonality determination.ResultsAll five VITT patients had oligoclonal anti-PF4 antibodies (3 monoclonal, one bi- and one tri-clonal antibodies), while HIT anti-PF4 antibodies were polyclonal. Notably, like VITT antibodies, anti-PF4 antibodies from a spontaneous HIT patient were monoclonal. The techniques employed did not detect non-pathogenic anti-PF4 antibodies. The ChAdOx1 nCoV-19-associated VITT patient made an excellent recovery with heparin treatment. In vitro studies demonstrated strong inhibition of VITT antibody-induced platelet activation with therapeutic concentrations of heparin in this and one Ad26.COV2.S-associated VITT patient. Oligoclonal VITT antibodies with persistent platelet-activating potential were detected at 6 and 10 weeks after acute presentation in two patients tested. Two of the 5 VITT patients had recurrence of thrombocytopenia and one patient had focal seizures several weeks after acute presentation.ConclusionOligoclonal anti-PF4 antibodies mediate VITT. Heparin use in VITT needs to be further studied.
| S-EPMC8491860 | biostudies-literature
Project description:Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a severe adverse effect of ChAdOx1 nCoV-19 COVID-19 vaccine (Vaxzevria) and Janssen Ad26.COV2.S COVID-19 vaccine, and it is associated with unusual thrombosis. VITT is caused by anti-platelet factor 4 (PF4) antibodies activating platelets through their FcγRIIa receptors. Antibodies that activate platelets through FcγRIIa receptors have also been identified in patients with COVID-19. These findings raise concern that vaccination-induced antibodies against anti-SARS-CoV-2 spike protein cause thrombosis by cross-reacting with PF4. Immunogenic epitopes of PF4 and SARS-CoV-2 spike protein were compared using in silico prediction tools and 3D modeling. The SARS-CoV-2 spike protein and PF4 share at least 1 similar epitope. Reactivity of purified anti-PF4 antibodies from patients with VITT was tested against recombinant SARS-CoV-2 spike protein. However, none of the affinity-purified anti-PF4 antibodies from 14 patients with VITT cross-reacted with SARS-CoV-2 spike protein. Sera from 222 polymerase chain reaction-confirmed patients with COVID-19 from 5 European centers were tested by PF4-heparin enzyme-linked immunosorbent assays and PF4-dependent platelet activation assays. We found anti-PF4 antibodies in sera from 19 (8.6%) of 222 patients with COVID-19. However, only 4 showed weak to moderate platelet activation in the presence of PF4, and none of those patients developed thrombotic complications. Among 10 (4.5%) of 222 patients who had COVID-19 with thrombosis, none showed PF4-dependent platelet-activating antibodies. In conclusion, antibodies against PF4 induced by vaccination do not cross-react with the SARS-CoV-2 spike protein, indicating that the intended vaccine-induced immune response against SARS-CoV-2 spike protein is not the trigger of VITT. PF4-reactive antibodies found in patients with COVID-19 in this study were not associated with thrombotic complications.
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Project description:Antibodies recognizing complexes of the chemokine platelet factor 4 (PF4/CXCL4) and polyanions (P) opsonize PF4-coated bacteria hereby mediating bacterial host defense. A subset of these antibodies may activate platelets after binding to PF4/heparin complexes, causing the prothrombotic adverse drug reaction heparin-induced thrombocytopenia (HIT). In autoimmune-HIT, anti-PF4/P-antibodies activate platelets in the absence of heparin. Here we show that antibodies with binding forces of approximately 60-100 pN activate platelets in the presence of polyanions, while a subset of antibodies from autoimmune-HIT patients with binding forces ≥100 pN binds to PF4 alone in the absence of polyanions. These antibodies with high binding forces cluster PF4-molecules forming antigenic complexes which allow binding of polyanion-dependent anti-PF4/P-antibodies. The resulting immunocomplexes induce massive platelet activation in the absence of heparin. Antibody-mediated changes in endogenous proteins that trigger binding of otherwise non-pathogenic (or cofactor-dependent) antibodies may also be relevant in other antibody-mediated autoimmune disorders.
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Project description:Staphylococcus aureus pneumonia is one of the most common invasive diseases caused by this human pathogen. S. aureus alpha-hemolysin, a pore-forming cytotoxin, is an essential virulence factor in the pathogenesis of pneumonia. Vaccine-based targeting of this toxin provides protection against lethal staphylococcal pneumonia in a murine model system, suggesting that a monoclonal antibody-based therapy may likewise prove to be efficacious for prevention and treatment of this disease. We report the generation of two distinct anti-alpha-hemolysin monoclonal antibodies that antagonize toxin activity, preventing human lung cell injury in vitro and protecting experimental animals against lethal S. aureus pneumonia. Each of these two monoclonal antibodies recognized an epitope within the first 50 amino acid residues of the mature toxin and blocked the formation of a stable alpha-hemolysin oligomer on the target cell surface. Active immunization with the first 50 amino acids of the toxin also conferred protection against S. aureus pneumonia. Together, these data reveal passive and active immunization strategies for prevention or therapy of staphylococcal pneumonia and highlight the potential role that a critical epitope may play in defining human susceptibility to this deadly disease.
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Project description:BackgroundVaccine-induced thrombotic thrombocytopenia (VITT) is a rare complication of adenovirus-based vaccines aimed to prevent and minimize COVID-19 and related pathophysiology.ObjectivesTo describe patterns of testing for anti-platelet factor 4 (PF4) antibodies using various ELISA assays in a large Australian cohort and comparative functional platelet activation assays in a subset.Patients/methodsAsserachrom HPIA IgG ELISA was performed in 1284 patients over a period of 12 months, supplemented in select cohorts by comparative ELISA using three other methods (n = 78-179), three different functional assays (flow cytometry, serotonin release assay, and/or Multiplate; n = 476), and rapid immunological chemiluminescence anti-PF4 assay (n = 460), in a multicenter study.ResultsFor first episode presentations, 190/1284 (14.8%) ELISA tests were positive. Conversely, most (445/460; 96.7%) chemiluminescence anti-PF4 test results were negative. All functional assays showed associations of higher median ELISA optical density with functional positivity and with high rates of ELISA positivity (64.0% to 85.2%). Data also identified functional positivity in 14.8%-36.0% of ELISA negative samples, suggesting false negative VITT by HPIA IgG ELISA in upward of one third of assessable cases.ConclusionTo our knowledge, this is the largest multicenter evaluation of anti-PF4 testing for investigation of VITT. Discrepancies in test results (ELISA vs. ELISA or ELISA vs. functional assay) in some patients highlighted limitations in relying on single methods (ELISA and functional) for PF4 antibody detection in VITT, and also highlights the variability in phenotypic test presentation and pathomechanism of VITT.
| S-EPMC9828670 | biostudies-literature
Project description:Vaccination is crucial in combatting the severe acute respiratory syndrome coronavirus 2 pandemic. The rare complication of thrombocytopenia and thrombotic complications at unusual sites after ChAdOx1 nCov-19 vaccination is caused by platelet-activating antibodies directed against platelet factor 4 (PF4). We present a widely applicable whole-blood standard flow cytometric assay to identify the pathogenic antibodies associated with vaccine-induced immune-mediated thrombotic thrombocytopenia (VITT) after ChAdOx1 nCov-19 vaccination. This assay will enable rapid diagnosis by many laboratories. This trial was registered at www.clinicaltrials.gov as #NCT04370119.
| S-EPMC8105122 | biostudies-literature
Project description:Background/objectivesAdenoviral vector-based vaccines against COVID-19 rarely cause vaccine-induced immune thrombocytopenia and thrombosis (VITT), a severe adverse reaction caused by IgG antibodies against platelet factor 4 (PF4). To study VITT, patient samples are crucial but have become a scarce resource. Recombinant antibodies (rAbs) derived from VITT patient characteristic amino acid sequences of anti-PF4 IgG are an alternative to study VITT pathophysiology.MethodsAmino acid sequences of the variable region of immunoglobulin light and heavy chain of anti-PF4 IgG derived from VITT patients were obtained by mass spectrometry sequencing and rAbs were synthetized by reverse-engineering. Six different rAbs were produced: CR23003, CR23004, and CR23005 (from a patient vaccinated with Jcovden, Johnson & Johnson-Janssen (Beerse, Belgium)), CR22046, and CR22050 and CR22066 (from two different patients vaccinated with Vaxzevria, AstraZeneca (Cambridge, UK)). These rAbs were further characterized using anti-PF4 and anti-PF4/heparin IgG ELISAs, rapid anti-PF4 and anti-PF4/polyanion chemiluminescence assays, and PF4-induced platelet activation assay (PIPA) and their capacity to induce procoagulant platelets.ResultsrAbs bound to PF4 alone, but not to PF4/polyanion complexes in rapid chemiluminescence assays. Chemiluminescence assays and both anti-PF4 IgG and anti-PF4 IgG/heparin ELISA showed concentration-dependent PF4 binding of all six rAbs, however, with different reactivities among them. PIPA showed a similar, concentration-dependent platelet activation pattern. rAbs varied in their reactivity and the majority of the tested rAbs were able to induce procoagulant platelets.ConclusionsThe six rAbs derived from VITT patients reflect VITT-typical binding capacities and the ability to activate platelets. Therefore, these rAbs offer an attractive new option to study VITT pathophysiology.
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