406. Cloning Antibodies Against Kawasaki Disease from Acute Plasmablast Responses

Abstract Background Kawasaki Disease (KD) is a childhood vasculitis, marked by prolonged fevers and coronary artery inflammation/aneurysms in near one-quarter of those untreated. The cause remains unknown; however, epidemiologic and demographic data support a single preceding infectious agent may lead to KD. Plasmablasts (PBs) are a stage of transitional B-cells that lead to plasma cells, the long-lived antibody-producing cells of the bone marrow. After initial infection, peripherally circulating PB populations are enriched for cells with antibodies against the preceding infection. We have recently published data showing children with KD have similar PB responses to children with infections. We sought to define the antibody characteristics, including clonality, of these PBs during KD. Methods We used antibody repertoire next-generation sequencing to characterize memory and PB populations. Additionally, pairing of heavy and light chains was performed with Chromium Single Cell Gene Expression (10x Genomics, Pleasanton, CA) using the Human B cell Single Cell V(D)J Enrichment Kit. Results From subject 24, antibody sequences using VH4-34 and a 19 amino acid length complementarity determining region 3 showed a massive expansion between day 4 and 6 of fever. Chromium single-cell sequencing produced over 946 heavy and light chain paired sequences. Sequence comparison showed 40% of sequences demonstrated markers of clonal expansion, which represented 100 clonal groups. Seven other KD subjects are being processed and comparative analysis will be presented. Conclusion This clonal expansion within plasmablast populations supports that Kawasaki disease is caused by an infection. Antigen targeting of these monoclonal antibodies is currently being explored. Disclosures All authors: No reported disclosures.

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Single-cell sequencing reveals clonally expanded plasma cells during chronic viral infection produce virus-specific and cross-reactive antibodies

10.1101/2021.01.29.428852 ◽

2021 ◽

Author(s):

Daniel Neumeier ◽

Alessandro Pedrioli ◽

Alessandro Genovese ◽

Ioana Sandu ◽

Roy Ehling ◽

...

Keyword(s):

Viral Infection ◽

Single Cell ◽

Plasma Cell ◽

Plasma Cells ◽

Clonal Expansion ◽

Chronic Viral Infection ◽

Antibody Repertoire ◽

Single Cell Sequencing ◽

Cell Transcriptome ◽

Single Cell Transcriptome

AbstractPlasma cells and their secreted antibodies play a central role in the long-term protection against chronic viral infection. However, due to experimental limitations, a comprehensive description of linked genotypic, phenotypic, and antibody repertoire features of plasma cells (gene expression, clonal frequency, virus specificity, and affinity) has been challenging to obtain. To address this, we performed single-cell transcriptome and antibody repertoire sequencing of the murine bone marrow plasma cell population following chronic lymphocytic choriomeningitis virus infection. Our single-cell sequencing approach recovered full-length and paired heavy and light chain sequence information for thousands of plasma cells and enabled us to perform recombinant antibody expression and specificity screening. Antibody repertoire analysis revealed that, relative to protein immunization, chronic infection led to increased levels of clonal expansion, class-switching, and somatic variants. Furthermore, antibodies from the highly expanded and class-switched (IgG) plasma cells were found to be specific for multiple viral antigens and a subset of clones exhibited cross-reactivity to non-viral- and auto-antigens. Integrating single-cell transcriptome data with antibody specificity suggested that plasma cell transcriptional phenotype was correlated to viral antigen specificity. Our findings demonstrate that chronic viral infection can induce and sustain plasma cell clonal expansion, combined with significant somatic hypermutation, and can generate cross-reactive antibodies. Graphical abstract.Single-cell sequencing reveals clonally expanded plasma cells during chronic viral infection produce virus-specific and cross-reactive antibodies.

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Abstract P472: Edil3 Autoantibodies Do Not Distinguish Kawasaki Disease (KD) From Febrile Controls, But Are Acutely Lower In Children With Cardiac Involvement.

Circulation Research ◽

10.1161/res.129.suppl_1.p472 ◽

2021 ◽

Vol 129 (Suppl_1) ◽

Author(s):

R. Ross Welliver ◽

Mark Hicar

Keyword(s):

Coronary Artery ◽

Kawasaki Disease ◽

Demographic Data ◽

Infectious Agent ◽

Autoimmune Response ◽

Attractive Potential ◽

Plasma Samples ◽

Tissue Samples ◽

Autoantibody Response ◽

Serial Samples

Kawasaki Disease (KD) is a childhood vasculitis, marked by prolonged fevers and coronary artery inflammation/aneurysms. Epidemiologic and demographic data support that a single preceding infectious agent may lead to KD, however, the cause remains unknown. The leading theory on pathogenesis is a post-infectious self-limiting autoimmune response, but this is still controversial. A recent study of individuals with KD demonstrated an association with autoantibody response to EDIL3 (also known as DEL-1). EDIL3 is a structural glycoprotein expressed by both macrophages and vascular endothelium. This presents an attractive potential target for investigation, as EDIL3 is known to modulate inflammation via leukocyte binding and infiltration through vessel walls. Additionally, EDIL3 is highly expressed in aortic and coronary artery tissue samples, showing some of the highest non-neural expression. Genetic variants of Del-1 have been associated with risk of intracranial aneurysms. Due to this burgeoning literature, we interrogated our cohort of plasma samples from febrile children, including Kawasaki disease, to confirm specificity of targeting to EDIL3. Plasma samples from febrile children and children with KD, including timecourse samples, were interrogated with ELISA assays using full-length recombinant EDIL3 (LSBio). In contrary to the published report, EDIL3 binding does not appear to be specific to KD when compared to febrile controls inclusive of a variety of disease etiologies (viral, bacterial, etc.). On serial samples (pre-IVIG, post-IVIG and convalescent) from the two different patient cohorts, antibodies binding EDIL3 are elevated in post-IVIG samples. This further supports general autoantibody reactivity against EDIL3 as IVIG is a pooled blood product from thousands of individuals typically. Intriguingly, in comparison of samples from children with or without any coronary artery Z scores above 2.5, EDIL3 autoantibody responses were actually lower in those with dilated coronary arteries. Current efforts are focused on identify specific human autoantibodies against EDIL3. Implications of these findings will be explored.

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ShinyCell: Simple and sharable visualisation of single-cell gene expression data

10.1101/2020.10.25.354100 ◽

2020 ◽

Author(s):

John F. Ouyang ◽

Uma S. Kamaraj ◽

Elaine Y. Cao ◽

Owen J. L. Rackham

Keyword(s):

Single Cell ◽

Rna Sequencing ◽

Bioinformatic Analysis ◽

R Package ◽

Published Data ◽

Expression Data ◽

Single Cell Rna Sequencing ◽

Cell Gene Expression ◽

Interactive Interfaces ◽

Cell Gene

AbstractMotivationAs the generation of complex single-cell RNA sequencing datasets becomes more commonplace it is the responsibility of researchers to provide access to these data in a way that can be easily explored and shared. Whilst it is often the case that data is deposited for future bioinformatic analysis many studies do not release their data in a way that is easy to explore by non-computational researchers.ResultsIn order to help address this we have developed ShinyCell, an R package that converts single-cell RNA sequencing datasets into explorable and shareable interactive interfaces. These interfaces can be easily customised in order to maximise their usability and can be easily uploaded to online platforms to facilitate wider access to published data.AvailabilityShinyCell is available at https://github.com/SGDDNB/[email protected]

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