LAUSR

Acquired hemophilia A (AHA) is a rare bleeding disorder caused by functional insufficiency of coagulation factor VIII (FVIII). Autoantibodies targeting FVIII may neutralize its procoagulant effect, thereby causing severe bleeding. Such inhibitory autoantibodies have been detected in autoimmune diseases, pregnancy, infections, or malignant diseases. Older age and certain drugs are known corisk factors.1 To our knowledge, only two reported cases document AHA diagnosed 8 and 20 days after influenza vaccination.2,3 Vaccines have been rarely associated with autoimmune disease occurrence or disease flares. Recently, vaccineinduced immune thrombocytopenia and thrombosis (VITT) has been characterized as a new entity.4 Immunological studies established a pathogenetic role of plateletactivating autoantibodies targeting platelet factor 4 (PF4) in VITT. VITTassociated antiPF4IgG were not crossreactive with the SARSCoV2 spike antigen, suggesting that the vaccinespecific antibody response is not directly causing VITT.5 A recent study linked the occurrence of VITT to the interaction of the adenoviral vector with the coxsackie and adenovirus receptor and PF4, thus instigating memory B cell differentiation and the release of antiPF4 autoantibodies.6 Our group recently reported three cases of AHA occurring in temporal association with mRNA COVID19 vaccine immunizations.7 Statistically, we found no strong evidence that the AHA incidence during the COVID vaccination campaign in Switzerland was substantially higher than the background AHA incidence. In our previous report, we did not address the possibility of FVIII crossreactivity of the vaccineinduced antispike IgG (antiSIgG). Excluding crossreactivity of antiforeign IgG with a selfantigen is critical to refute ‘molecular mimicry’ in the immunopathogenesis of an autoimmune disease. Here, we studied the binding, function, and crossreactivity of the vaccineinduced antiSIgG in our previously reported three cases of AHA diagnosed in temporal association with COVID vaccination.7 The main goal was to address whether the vaccineinduced antibody response against the SARSCoV2 spike protein may exhibit FVIII inhibitory functions. The sequence alignment of the FVIII (UniProtKB accession number P00451) and the SARSCoV2 spike protein (UniProtKB accession number P0DTC2) revealed minimal sequence similarity. We identified one region (amino acid position 540– 570 within the A2 domain of FVIII) with 13/35(37%) amino acid sequence similarity using the NCBI blast sequence alignment tool. In silico antigenic peptide prediction (http://imed.med.ucm.es/Tools/ antig enic.pl) revealed 95 and 63 antigenic determinants in the FVIII and spike protein, respectively. Of those, a single overlapping potential epitope was present in both proteins, locating to the region with the sequence similarity (Figure 1A; SDPRCLTRYYSS in the FVIII sequence [FVIII 543– 554]; underlined amino acids indicate homology to the SARSCoV2 spike protein). Since only a few amino acids are shared between the FVIII and spike protein in this region, the likelihood of a crossreactive B cell epitope is, however, low. Next, we addressed this experimentally. The presence of vaccinespecific antibodies is a prerequisite for a potential crossreactivity to FVIII. Serological analyses proved considerable antiSpike IgG (antiSIgG) levels in the serum of all three vaccinated patients (Figure 1B). AntiSIgG is the only antigenspecificity induced by the mRNA COVID vaccines. To explore the FVIII inhibitory potential of the antiSIgG fraction, we performed a beadbased antibody pulldown to deplete and enrich for antiSIgG (Supplementary Data). The antiSIgG enrichment and depletion was confirmed in a Luminex assay using spike proteincoated beads (Figure 1C) and in western blot loaded with recombinant spike protein (Figure 1D). Despite efficient depletion and enrichment, the ‘antiSIgG enriched’ fraction contained residual nonantiSIgG based on total IgG measurements (mean totalIgG in the antiSenriched fraction 0.49g/l. Moreover, we found traces of other serum proteins as assessed by gel electrophoresis (Figure S1). To determine which of the serum fractions contained the FVIII inhibitory factor, we first performed a mixing FVIII assay (Supplementary Methods). The nonmanipulated serum and antiSIgGdepleted fractions showed similar FVIII inhibition of 75%. In