Structural characterization of a pathogenic antibody underlying vaccine-induced immune thrombotic thrombocytopenia (VITT)

Nguyen, S. N.; Le, S.-H.; Ivanov, D. G.; Ivetic, N.; Nazy, I.; Kaltashov, I. A.

doi:10.1101/2023.05.28.542636

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bioRxiv · 10.1101/2023.05.28.542636

Structural characterization of a pathogenic antibody underlying vaccine-induced immune thrombotic thrombocytopenia (VITT)

Nguyen, S. N.·Le, S.-H.·Ivanov, D. G.·Ivetic, N.·Nazy, I.·Kaltashov, I. A.

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Abstract

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare but extremely dangerous side effect that has been reported for several adenoviral (Ad)-vectored COVID-19 vaccines. VITT pathology had been linked to production of antibodies that recognize platelet factor 4 (PF4), an endogenous chemokine. In this work we characterize anti-PF4 antibodies obtained from a VITT patients blood. Intact-mass MS measurements indicate that a significant fraction of this ensemble is comprised of antibodies representing a limited number of clones. MS analysis of large antibody fragments (the light chain, as well as the Fc/2 and Fd fragments of the heavy chain) confirms the monoclonal nature of this component of the anti-PF4 antibodies repertoire, and reveals the presence of a fully mature complex biantennary N-glycan within its Fd segment. Peptide mapping using two complementary proteases and LC-MS/MS analysis were used to determine the amino acid sequence of the entire light chain and over 98% of the heavy chain (excluding a short N-terminal segment). The sequence analysis allows the monoclonal antibody to be assigned to IgG2 subclass and verify that the light chain belongs to the {lambda}-type. Incorporation of enzymatic de-N-glycosylation into the peptide mapping routine allows the N-glycan in the Fab region of the antibody to be localized to the framework 3 region of the VH domain. This novel N-glycosylation site (absent in the germline sequence) is a result of a single mutation giving rise to an NDT motif in the antibody sequence. Peptide mapping also provides a wealth of information on lower-abundance proteolytic fragments derived from the polyclonal component of the anti-PF4 antibody ensemble, revealing the presence of all four subclasses (IgG1 through IgG4) and both types of the light chain ({lambda} and {kappa}). The structural information reported in this work will be indispensable for understanding the molecular mechanism of VITT pathogenesis.

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Nguyen, S. N., Le, S.-H., Ivanov, D. G., Ivetic, N., Nazy, I., Kaltashov, I. A.. 2023-05-29. Structural characterization of a pathogenic antibody underlying vaccine-induced immune thrombotic thrombocytopenia (VITT). https://doi.org/10.1101/2023.05.28.542636

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