ABSTRACT
Background: Vaccine-induced immune thrombocytopenia (VITT) is a rare but serious complication of SARS-COV- 2 vaccination and its diagnosis is based on both clinical and biological criteria. Aim(s): To evaluate the performance of these criteria in a prospective cohort of patients with suspected VITT. Method(s): Among 105 consecutive patients (Table 1), 79 had received one (n = 71) or two (n = 8) injections of adenoviral vector vaccine, and 26 one (n = 18) or two (n = 8) doses of an mRNA vaccine. Most patients presented thrombosis either isolated (n = 40), or associated with thrombocytopenia (T+T = 45). Other patients had isolated thrombocytopenia (n = 15), or variable clinical manifestations. ELISA (PVS/PF4 HAT45G) detected anti-PF4 antibodies, and when present VITT diagnosis was confirmed using PF4 serotonin release assay. Result(s): VITT was confirmed in 26 patients (both PVS/PF4 ELISA and PF4/SRA were positive) and probable in one case (PVS/PF4 ELISA OD = 2.4 but PF4/SRA negative). VITT was excluded in all patients who had received an mRNA vaccine, or in those with isolated thrombocytopenia or thrombosis or with other symptoms. In contrast, the presence of both thrombocytopenia and thrombosis 5 to 30 days after injection was highly suggestive of VITT with a specificity and PPV of 85% and 69%, respectively, these values increasing to 93.6% and 85% if patients had received adenoviral vector vaccine. Importantly, the PPV reached in these cases 100% when OD > 1 in ELISA. (Table) Conclusion(s): The occurrence of thrombocytopenia and thromboses, 5 to 30 days after vaccination with an adenoviral vector is highly predictive of VITT. PF4/PVS ELISA allows to rule out VITT when OD is < 0.4 (NPV 100%) and to confirm the diagnosis when OD is > 1 without necessitating a platelet activation test. Moreover, the follow-up of patients with ELISA is mandatory since anti-PF4 antibodies may remain several weeks after VITT. (Table Presented).
ABSTRACT
[Formula presented] Introduction Vaccine-induced thrombotic thrombocytopenia (VITT) is a severe complication of recombinant adenoviral vector vaccines used to prevent COVID-19, likely due to anti-platelet factor 4 (PF4) IgG antibodies. The specificity and platelet-activating activity of VITT antibodies strikingly resemble that of antibodies detected in “autoimmune” heparin-induced thrombocytopenia (HIT), but their features remain poorly characterized. In particular, a better knowledge of these antibodies should help to understand the mechanisms leading to hypercoagulability and the particular thrombotic events observed in VITT, but rarely in typical HIT. We have recently developed a chimeric IgG1 anti-PF4 antibody, 1E12, which strongly mimics “autoimmune” HIT antibodies in terms of specificity and cellular effects. Therefore, we assessed whether 1E12 could mimic VITT antibodies. We then evaluated the capability of DG-1E12, a deglycosylated form of 1E12 unable to bind FcγR, to inhibit cellular activation induced by VITT antibodies. Methods and Results Using a PF4-sensitized serotonin release assay (PF4-SRA) (Vayne C, New Engl J Med, 2021), we demonstrated that 1E12 (5 and 10 μg/mL) strongly activated platelets, with a pattern similar to that obtained with human VITT samples (n=7), i.e. in a PF4-dependent manner and without heparin. This platelet activation was inhibited by low heparin concentration (0.5 IU/mL), an effect also observed with VITT samples. Serotonin release induced by 1E12 was also fully inhibited by IV-3, a monoclonal antibody blocking FcγRIIa, or by IdeS, a bacterial protease that cleaves IgG and strongly inhibits the binding of IgG antibodies to FcγRIIa. This inhibitory effect of IV-3 and IdeS strongly supports that interactions between pathogenic anti-PF4 IgG and FcγRIIa play a central role in VITT. Incubation of 1E12 or VITT samples with isolated neutrophils (PMN) and platelets with PF4 (10 µg/mL) strongly induced DNA release and NETosis, supporting that PMN are involved in the processes leading to thrombosis in VITT. Furthermore, when whole blood from healthy donors incubated with 1E12 or VITT plasma was perfused in capillaries coated with von Willebrand Factor, numerous large platelet/leukocyte aggregates containing fibrin(ogen) were formed. To investigate whether 1E12 and VITT antibodies recognize overlapping epitopes on PF4, we then performed competitive assays with a deglycosylated form of 1E12 (DG-1E12), still able to bind PF4 but not to interact with Fcγ receptors. In PF4-SRA, pre-incubation of DG-1E12 (50 µg/mL) dramatically reduced platelet activation induced by VITT antibodies, which was fully abrogated for 9 of the 14 VITT samples tested. Additional experiments using a whole blood PF4-enhanced flow cytometry assay recently designed for VITT diagnosis (Handtke et al, Blood 2021), confirmed that DG-1E12 fully prevented platelet activation induced by VITT antibodies. Moreover, when platelets and neutrophils were pre-incubated with DG-1E12 (100 µg/mL), NETosis and thus DNA release, nuclear rounding, and DNA decondensation induced by VITT antibodies were completely inhibited. Finally, DG-1E12 (100 µg/mL) also fully abolished VITT antibody-mediated thrombus formation in whole blood in vitro under vein flow conditions. Comparatively, DG-1E12 did not inhibit ALB6, a murine monoclonal anti-CD9 antibody, which also strongly activates platelets in a FcγRIIa-dependent manner. Conclusions Our results show that 1E12 exhibits features similar to those of human VITT antibodies in terms of specificity, affinity and cellular effects, and could therefore be used as a model antibody to study the pathophysiology of VITT. Our data also demonstrate that DG-1E12 prevents blood cell activation and thrombus formation induced by VITT antibodies, likely due to the competitive effect of its Fab fragment on antibody binding to PF4. DG-1E12 may allow the development of a new drug neutralizing the pathogenic effect of autoimmune anti-PF4 antibodies, such as those associated with VITT. Disclosures: T iele: Bristol Myers Squibb: Honoraria, Other;Pfizer: Honoraria, Other;Bayer: Honoraria;Chugai Pharma: Honoraria, Other;Novo Nordisk: Other;Novartis: Honoraria;Daichii Sankyo: Other. Pouplard: Stago: Research Funding. Greinacher: Macopharma: Honoraria;Biomarin/Prosensa: Other, Research Funding;Sagent: Other, Research Funding;Rovi: Other, Research Funding;Gore inc.: Other, Research Funding;Bayer Healthcare: Other, Research Funding;Paringenix: Other, Research Funding;BMS: Honoraria, Other, Research Funding;MSD: Honoraria, Other, Research Funding;Boehringer Ingelheim: Honoraria, Other, Research Funding;Aspen: Honoraria, Other, Research Funding;Portola: Other;Ergomed: Other;Instrument Laboratory: Honoraria;Chromatec: Honoraria. Gruel: Stago: Other: symposium fees, Research Funding. Rollin: Stago: Research Funding.