Molecular signature of highly stereotyped clonotypic anti-PF4

VITT is a rare thromboembolic event facilitated by Abs targeting platelet factor 4 (PF4) in adenoviral-vectored severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccinations such as Ad26.COV2.S (Johnson & Johnson/Janssen) and ChAdOx1 nCoV-19 (AstraZeneca). Ongoing studies are investigating the pathways by which adenoviral deoxyribonucleic acid (DNA) vectors damage PF4 immunity and induce B cell clonal expansion and anti-PF4 immunoglobulin (Ig) secretion. Available data suggest that this event likely encompasses the emergence of immunogenic PF4 complexes comprising vaccine elements in a pro-inflammatory environment.

Understanding the antigenic targets and molecular structure of anti-PF4 Abs is essential for establishing improved treatments and diagnostics due to their causal involvement in VITT. In addition, it is also important for the accurate monitoring of secreted clonotypes and PF4-specific B cell clones.

In the present study, researchers used a novel proteomics discovery workflow to assess the composition of the Ig variable part (IgV) of anti-PF4 antibodies at the secreted proteome level. Serum anti-PF4 IgG Abs were isolated by affinity using PF4-linked magnetic beads and sequenced by mass spectrometry in five individuals with VITT caused by COVID-19 ChAdOx1 nCoV-19 immunization. All study volunteers submitted written informed consent before enrolling in the survey.

Prior to sequencing, the monospecificity of anti-PF4 Abs was assessed using in-house enzyme immunoassays (ELISA). Anti-PF4 antibodies were detected using Lifecodes PF4 IgG Immucor and Asserachrom HP image assistant (HPIA) IgG Stago.

According to the United Kingdom (UK) Hematology Expert Panel (EHP) level 1 certainty determination for thrombosis with thrombocytopenia syndrome (TTS), all five patients with VITT met the endpoint of case definition for "Definite VITT". In four out of five patients with VITT, thrombosis was present at unusual sites, particularly splanchnic vein thrombosis (SVT) and cerebral venous sinus thrombosis (CVST).

Study results described the absence of cross-reactivity between eluted/purified anti-PF4 Ig and SARS-CoV-2 spike 1 (S1)/S2 proteins in VITT patient serum samples similar to a publication recent.

A single IgG heavy (H) chain species coupled to a single lambda light (L) chain species in each assessed VITT patient was revealed by anti-PF4 Ig mass spectrometry sequencing. A similar Ig L-chain variable 3-21*02 (IGLV3-21*02) gene subfamily with equivalent lengths of third L-chain complementarity determining region (LCDR3) peptides encoded all L chains, indicating a high degree of L chain stereotypy.