Anti-Platelet Factor 4 Antibodies Causing VITT do not Cross-React with SARS-CoV-2 Spike Protein

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a severe adverse effect of ChAdOx1 nCoV-19 COVID-19 vaccine (Vaxzevria) and COVID-19 vaccine Janssen (Ad26.COV2.S), and associated with unusual thrombosis. VITT is caused by anti-platelet factor 4 (PF4) antibodies activating platelets through their FcgRIIa receptors. Antibodies activating platelets through FcgRIIa receptors have also been identified in COVID-19 patients. 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-modelling. The SARS-CoV-2 spike protein and PF4 share at least one 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 VITT patients cross-reacted with SARS-CoV-2 spike protein. Sera from 222 PCR-confirmed COVID-19 patients from five European centers were tested by PF4/heparin ELISA and PF4-dependent platelet activation assays. We found anti-PF4 antibodies in 19 of 222 (8.6%) COVID-19 patient sera. However, only four showed weak to moderate platelet activation in the presence of PF4, and none of these patients developed thrombotic complications. Among 10 of 222 (4.5%) COVID-19 patients 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 COVID-19 patients of the present study were not associated with thrombotic complications.