Analysis of Plasmablasts From Children With Kawasaki Disease Reveals Evidence of a Convergent Antibody Response to a Specific Protein Epitope

Abstract

Background: Kawasaki disease (KD) is a febrile illness of young childhood that can result in coronary artery aneurysms and death. Coronavirus disease 2019 (COVID-19) mitigation strategies resulted in a marked decrease in KD cases worldwide, supporting a transmissible respiratory agent as the cause. We previously reported a peptide epitope recognized by monoclonal antibodies (MAbs) derived from clonally expanded peripheral blood plasmablasts from 3 of 11 KD children, suggesting a common disease trigger in a subset of patients with KD.

Methods: We performed amino acid substitution scans to develop modified peptides with improved recognition by KD MAbs. We prepared additional MAbs from KD peripheral blood plasmablasts and assessed MAb characteristics that were associated with binding to the modified peptides.

Results: We report a modified peptide epitope that is recognized by 20 MAbs from 11 of 12 KD patients. These MAbs predominantly use heavy chain VH3-74; two-thirds of VH3-74 plasmablasts from these patients recognize the epitope. The MAbs were nonidentical between patients but share a common complementarity-determining region 3 (CDR3) motif.

Conclusions: These results demonstrate a convergent VH3-74 plasmablast response to a specific protein antigen in children with KD, supporting one predominant causative agent in the etiopathogenesis of the illness.

Keywords: Kawasaki disease; antigen; epitope; immune response; plasmablast.

Figure 1.

Approach to identify antigen-antibody response in KD patients. A, Scheme for analysis of plasmablast response in patients with KD. B, Diagram of the 2 approaches used to identify a consensus KD epitope and the convergent antibody response in plasmablasts from patients with KD. Abbreviations: CDR3, complementarity-determining region 3; Ig, immunoglobulin; KD, Kawasaki disease; MAb, monoclonal antibody; PB, plasmablast; RT-PCR, reverse transcription polymerase chain reaction; VH, variable heavy; VL, variable light.

Figure 2.

Amino acid substitution scans using monoclonal antibodies (MAbs) from multiple Kawasaki disease (KD) patients support a consensus PMLFQSIV epitope. The base peptide used is shown on the far left of each scan. Each amino acid of the base peptide was replaced by each of 20 amino acids and the relative binding of the peptide with each amino acid replacement is shown. Red indicates strong binding while blue indicates inhibition of binding relative to the base peptide. The base peptide differs slightly in individual scans. Scans using MAbs from KD patients 1, 3, 5, 6, 7, and 10 are shown; additional scans performed using MAbs from KD patients 2, 4, and 8 gave similar results.

Figure 3.

KD MAbs show preferential binding to KD peptide 3 over KD peptide 1. A, ELISA of a subset MAbs to the 3 peptides. For all antibodies recognizing KD peptide 3, binding to KD peptide 3 was significantly greater than KD peptide 1 in triplicate assays, P < .01. B, Western blots of KD peptide 3 (FP3) and KD peptide 1 (FP1) mouse Fc fusion proteins probed with KD MAbs KD4-2H4 and KD6-2H3. Human IgG (huIgG) was used as a positive control for the secondary anti-human IgG antibody, and stripped blots were reprobed with anti-mouse IgG (msIgG) to assess protein loading. Abbreviations: ELISA, enzyme-linked immunosorbent assay; Ig, immunoglobulin; KD, Kawasaki disease; MAb, monoclonal antibody; OD, optical density.

Figure 4.

Analysis of CDR3 sequences of KD MAbs supports a convergent antibody response in KD patients. A, Alignment of CDR3 amino acid sequences from KD MAbs that recognize KD peptide 3. B, Motif-based sequence analysis of CDR3 amino acid sequences of KD MAbs binding to KD peptide 3. Analysis was performed using the MEME suite (https://meme-suite.org/meme/), revealing a statistically significant consensus motif at 2.3e-6. Abbreviations: KD, Kawasaki disease; MAb, monoclonal antibody.

Figure 5.

A proposed model of the pathogenesis of Kawasaki disease (KD). In this model, the KD agent is acquired via the respiratory route and infects ciliated bronchial epithelial cells, where inclusion bodies can form. It can be picked up by monocyte/macrophages and presented to the immune system, resulting in innate and acquired immune responses. Monocyte/macrophages travel through the circulation and enter coronary arteries and other tissues, with the most significant damage occurring in the coronary arteries. The immune system ultimately clears the infection, although inclusion bodies can persist.