Molecular signature of highly stereotyped clonotypic anti-PF4 antibodies indicates shared pathways of antibody production in VITT patients

By Shanet Susan AlexApr 7 2022Reviewed by Danielle Ellis, B.Sc.

A recent article posted to the medRxiv* preprint server analyzed the antibody (Ab) that mediated the coronavirus disease 2019 (COVID-19) vaccine-induced thrombotic thrombocytopenia (VITT).

This news article was a review of a preliminary scientific report that had not undergone peer-review at the time of publication. Since its initial publication, the scientific report has now been peer reviewed and accepted for publication in a Scientific Journal. Links to the preliminary and peer-reviewed reports are available in the Sources section at the bottom of this article. View Sources

Background

VITT is an uncommon 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). The ongoing studies investigate the pathways through which the adenoviral deoxyribonucleic acid (DNA) vectors damage immunity to PF4 and induce B-cell clonal expansion and anti-PF4 immunoglobulin (Ig) secretion. The available data suggests that this event probably 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 vital for establishing improved treatments and diagnostics because of their causative involvement in VITT. In addition, it is also significant for the accurate surveillance of secreted clonotypes and B-cell clones specific for PF4.  

About the study

In the current study, the investigators utilized a new proteomic discovery workflow to assess the Ig variable (IgV) portion makeup of anti-PF4 Abs at the secreted proteome level. Serum anti-PF4 IgG Abs were affinity isolated using PF4-linked magnetic beads and were sequenced using 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 investigation.

Before sequencing, the monospecificity of anti-PF4 Abs was assessed using in-house enzyme-linked immunosorbent assays (ELISAs). Anti-PF4 Abs were detected using the Lifecodes PF4 IgG Immucor and the Asserachrom HP image assistant (HPIA) IgG Stago.

According to Level 1 Certainty Determination and the United Kingdom (UK) Expert Hematology Panel (EHP) for Thrombosis with Thrombocytopenia Syndrome (TTS), all five VITT patients met the case definition parameter for "Definite VITT". In four out of five VITT patients, thrombosis was present at unusual sites, especially splanchnic vein thrombosis (SVT) and cerebral venous sinus thrombosis (CVST). 

Results

The study results depicted the absence of cross-reactivity between eluted/purified anti-PF4 Igs and SARS-CoV-2 spike 1 (S1)/S2 proteins in the serum samples of VITT patients similar to a recent publication.

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

The authors pointed out that the common IGLV3-21*02 allele has an acidic DDxD motif in the CDR2 domain, which they believed might be significant for Ab adherence with the positively charged PF4 epitope. They mentioned that the LCDR3 area also had another common aa motif of relevance, named QxWD. A stereotypic IGLV3-21 B-cell receptor characterized a subset of chronic lymphocytic leukemia exhibiting poor prognosis. However, formal structural investigations are needed to verify the roles of the potential binding motifs.

The researchers discovered stereotypic traits of H-chain CDR3 (HCDR3) clonal barcode peptides, such as homologous sequences and similar HCDR3 lengths, along with common binding motif G/NLED in Ig heavy constant delta (IGHD) areas known to impart antigen-binding specificity.

Despite the convergent HCDR3 domains, anti-PF4 Ig proteomes of each patient were encoded by different Ig H-chain variable (IGHV) subfamilies, including 3-53, 3-48, 2-5, 3-7, and 7-4. This highlights the critical function of HCDR3s in PF4 epitope binding versus divergent IGHV region.

Individual patient LCDR3 and HCDR3 areas also had amino acid substitution mutations and aligned with a hypothesis of PF4 antigen-stimulated intraclonal diversification, which was observed in Sjögren's syndrome and Lupus. In the IgV regions of the L- and H-chains, additional aspartic acid (D) and glutamic acid (E) replacement mutations with possible binding relevance were discovered, implying recall immunogenicity of memory B cells specific to PF4.

Conclusions 

The authors of the present study established a novel proteomic methodology centered on de novo mass spectrometric sequencing of immunopurified serum anti-PF4 Abs collected from SARS-CoV-2 VITT cases. The researchers used this method to identify the anti-PF4 Abs IgV subfamily expression characteristics, clonotypic HCDR3 and LCDR3 aa segments as clonal monitoring barcodes, and V-region aa replacement mutational fingerprints as molecular indicators of antigen-mediated intraclonal diversification.

The study findings demonstrated that a novel single IgG H-chain species linked with a single lambda L-chain species were present in all five unrelated VITT patients. Surprisingly, the same IGLV3-21*02 gene subfamily comprising identical LCDR3 lengths encoded all L-chains. Furthermore, H-chain anti-PF4 Abs with similar homologous sequences and HCDR3 length demonstrated remarkable stereotypic characteristics.

To conclude, the present study deciphered the molecular fingerprint of highly stereotyped clonotypic anti-PF4 Abs, pointing to substantially convergent mechanisms of anti-PF4 Ab production in COVID-19 VITT patients. The study also indicated that the common CDR3 peptide barcodes might be translated into new molecular biomarkers for the anti-PF4 Abs highly pathogenic clonotypes. These findings were crucial for understanding the molecular underpinnings of the fatal condition, VITT, and devising new therapeutics to eliminate pathogenic clones.

This news article was a review of a preliminary scientific report that had not undergone peer-review at the time of publication. Since its initial publication, the scientific report has now been peer reviewed and accepted for publication in a Scientific Journal. Links to the preliminary and peer-reviewed reports are available in the Sources section at the bottom of this article. View Sources