Adenoviral Inciting Antigen and Somatic Hypermutation in VITT

BACKGROUND: Vaccine-induced immune thrombocytopenia and thrombosis (VITT) is a rare prothrombotic complication that occurs after adenoviral vector-based vaccination against coronavirus disease 2019; in rare cases, it can also occur after natural adenovirus infection. VITT is mediated by platelet-activating antibodies against the highly cationic protein platelet factor 4 (PF4). The underlying inciting antigen trigger and immunopathogenesis remain unknown. METHODS: We used antibody proteomics to determine the amino acid sequences of anti-PF4 antibodies from 21 patients with VITT and sequenced the genes encoding the immunoglobulin light-chain hypervariable region from 100 patients with VITT. To identify an adenoviral trigger, we used the antigen-binding fingerprints of anti-PF4 and anti-adenovirus protein antibodies to identify a shared serum clonotype and subsequently used adenovirus protein peptides and recombinant anti-PF4 VITT antibodies to map the mimicking linear epitope. RESULTS: Genomic and proteomic profiling of VITT antibodies revealed a shared immunoglobulin light-chain allele, IGLV3-21*02 or *03, harboring a critical somatic hypermutation, K31E. Only antibodies purified against adenoviral core protein VII (pVII) contained anti-PF4 species matching the VITT fingerprint; antibodies against intact virions or other adenoviral proteins did not. Cross-reactive IgGs were mapped to a basic linear epitope on pVII. A pathogenic anti-PF4 VITT antibody, back-mutated to germline (K31), lost its prothrombotic activity in vitro and in vivo and preferentially bound pVII, a finding that directly supported the role of the hypermutation in the antigenic shift from adenovirus pVII to PF4. CONCLUSIONS: The results of our study indicate that VITT occurs when, in persons with immunoglobulin light-chain allele IGLV3-21*02 or *03, a specific somatic hypermutation develops that affects antibodies that recognize a specific epitope on the adenoviral core protein pVII, which results in misdirection of antibody targeting toward PF4. (Funded by Deutsche Forschungsgemeinschaft and others; German Clinical Trials Register number, DRKS00025738; EU Post-Authorization Study Register number, EUPAS45098.).