Enrichment of Antigen-Specific Class-Switched B Cells from Individuals Naturally Immunized by Infection with Group A Streptococcus

ABSTRACT The low frequency of circulating antigen-specific memory B cells is a considerable obstacle in the discovery and development of human monoclonal antibodies for therapeutic application. Here, we evaluate two solid-phase isolation methods to enrich the number of antigen-specific B cells from...

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Autores principales:	Cheri L. Lamb, Emily Price, Kevin P. Field, Christopher Dayton, Eric R. McIndoo, Eva J. Katahira, Dennis L. Stevens, Sarah E. Hobdey
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2019
Materias:	group A streptococcus antigen-specific B cells antimicrobial immune response solid-phase enrichment Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/eb054233c1604f348cbb5d94c5de138a
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spelling	oai:doaj.org-article:eb054233c1604f348cbb5d94c5de138a2021-11-15T15:22:24ZEnrichment of Antigen-Specific Class-Switched B Cells from Individuals Naturally Immunized by Infection with Group A Streptococcus10.1128/mSphere.00598-192379-5042https://doaj.org/article/eb054233c1604f348cbb5d94c5de138a2019-12-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00598-19https://doaj.org/toc/2379-5042ABSTRACT The low frequency of circulating antigen-specific memory B cells is a considerable obstacle in the discovery and development of human monoclonal antibodies for therapeutic application. Here, we evaluate two solid-phase isolation methods to enrich the number of antigen-specific B cells from individuals naturally immunized against streptolysin O (SLO), a key virulence factor and known immunogen of group A streptococcus (GAS). Class-switched B cells obtained from individuals with a history of GAS infection were separated from peripheral blood mononuclear cells (PBMCs) by immunomagnetic methods. SLO-specific B cells were further enriched directly by binding to SLO monomers and captured by streptavidin-coated magnetic microbeads or indirectly by binding a fluorescently labeled SLO-streptavidin tetramer and captured by anti-fluorophore immunomagnetic microbeads. SLO-bound B cells were quantitated by flow cytometry and/or expanded in batch culture to determine IgG specificity. From individuals who have suffered a GAS infection ≥2 years prior, only the direct method enriched SLO-specific B cells, as determined by flow cytometry. Likewise, in batch culture, B cells isolated by the direct method resulted in an average of 375-fold enrichment in anti-SLO IgG, while no enrichment was observed for B cells isolated by the indirect method. The direct method established here provides a simple approach to increase low-frequency antigen-specific B cell populations supporting many downstream applications, such as immortalization of B cells, cloning of immunoglobulin genes, or purification of antibodies from supernatant for future study. Overall, this process is efficient, is inexpensive, and can be applied to many naturally immunogenic antigens. IMPORTANCE Bacteria called group A streptococci can cause a variety of skin and soft tissue infections ranging from mild pharyngitis (“strep throat”) to deadly necrotizing fasciitis (sometimes called “flesh-eating” disease). In each case, the development of disease and the degree of tissue damage are mediated by toxins released from the bacteria during infection. Consequently, novel therapies aimed at clearing bacterial toxins are greatly needed. One promising new treatment is the utilization of monoclonal antibodies delivered as an immunotherapeutic for toxin neutralization. However, current methods of antibody development are laborious and costly. Here, we report a method to enrich and increase the detection of highly desirable antigen-specific memory B cells from individuals previously exposed to GAS using a cost-effective and less-time-intensive strategy. We envision that this method will be incorporated into many applications supporting the development of immunotherapeutics.Cheri L. LambEmily PriceKevin P. FieldChristopher DaytonEric R. McIndooEva J. KatahiraDennis L. StevensSarah E. HobdeyAmerican Society for Microbiologyarticlegroup A streptococcusantigen-specific B cellsantimicrobial immune responsesolid-phase enrichmentMicrobiologyQR1-502ENmSphere, Vol 4, Iss 6 (2019)
institution	DOAJ
collection	DOAJ
language	EN
topic	group A streptococcus antigen-specific B cells antimicrobial immune response solid-phase enrichment Microbiology QR1-502
spellingShingle	group A streptococcus antigen-specific B cells antimicrobial immune response solid-phase enrichment Microbiology QR1-502 Cheri L. Lamb Emily Price Kevin P. Field Christopher Dayton Eric R. McIndoo Eva J. Katahira Dennis L. Stevens Sarah E. Hobdey Enrichment of Antigen-Specific Class-Switched B Cells from Individuals Naturally Immunized by Infection with Group A Streptococcus
description	ABSTRACT The low frequency of circulating antigen-specific memory B cells is a considerable obstacle in the discovery and development of human monoclonal antibodies for therapeutic application. Here, we evaluate two solid-phase isolation methods to enrich the number of antigen-specific B cells from individuals naturally immunized against streptolysin O (SLO), a key virulence factor and known immunogen of group A streptococcus (GAS). Class-switched B cells obtained from individuals with a history of GAS infection were separated from peripheral blood mononuclear cells (PBMCs) by immunomagnetic methods. SLO-specific B cells were further enriched directly by binding to SLO monomers and captured by streptavidin-coated magnetic microbeads or indirectly by binding a fluorescently labeled SLO-streptavidin tetramer and captured by anti-fluorophore immunomagnetic microbeads. SLO-bound B cells were quantitated by flow cytometry and/or expanded in batch culture to determine IgG specificity. From individuals who have suffered a GAS infection ≥2 years prior, only the direct method enriched SLO-specific B cells, as determined by flow cytometry. Likewise, in batch culture, B cells isolated by the direct method resulted in an average of 375-fold enrichment in anti-SLO IgG, while no enrichment was observed for B cells isolated by the indirect method. The direct method established here provides a simple approach to increase low-frequency antigen-specific B cell populations supporting many downstream applications, such as immortalization of B cells, cloning of immunoglobulin genes, or purification of antibodies from supernatant for future study. Overall, this process is efficient, is inexpensive, and can be applied to many naturally immunogenic antigens. IMPORTANCE Bacteria called group A streptococci can cause a variety of skin and soft tissue infections ranging from mild pharyngitis (“strep throat”) to deadly necrotizing fasciitis (sometimes called “flesh-eating” disease). In each case, the development of disease and the degree of tissue damage are mediated by toxins released from the bacteria during infection. Consequently, novel therapies aimed at clearing bacterial toxins are greatly needed. One promising new treatment is the utilization of monoclonal antibodies delivered as an immunotherapeutic for toxin neutralization. However, current methods of antibody development are laborious and costly. Here, we report a method to enrich and increase the detection of highly desirable antigen-specific memory B cells from individuals previously exposed to GAS using a cost-effective and less-time-intensive strategy. We envision that this method will be incorporated into many applications supporting the development of immunotherapeutics.
format	article
author	Cheri L. Lamb Emily Price Kevin P. Field Christopher Dayton Eric R. McIndoo Eva J. Katahira Dennis L. Stevens Sarah E. Hobdey
author_facet	Cheri L. Lamb Emily Price Kevin P. Field Christopher Dayton Eric R. McIndoo Eva J. Katahira Dennis L. Stevens Sarah E. Hobdey
author_sort	Cheri L. Lamb
title	Enrichment of Antigen-Specific Class-Switched B Cells from Individuals Naturally Immunized by Infection with Group A Streptococcus
title_short	Enrichment of Antigen-Specific Class-Switched B Cells from Individuals Naturally Immunized by Infection with Group A Streptococcus
title_full	Enrichment of Antigen-Specific Class-Switched B Cells from Individuals Naturally Immunized by Infection with Group A Streptococcus
title_fullStr	Enrichment of Antigen-Specific Class-Switched B Cells from Individuals Naturally Immunized by Infection with Group A Streptococcus
title_full_unstemmed	Enrichment of Antigen-Specific Class-Switched B Cells from Individuals Naturally Immunized by Infection with Group A Streptococcus
title_sort	enrichment of antigen-specific class-switched b cells from individuals naturally immunized by infection with group a streptococcus
publisher	American Society for Microbiology
publishDate	2019
url	https://doaj.org/article/eb054233c1604f348cbb5d94c5de138a
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Enrichment of Antigen-Specific Class-Switched B Cells from Individuals Naturally Immunized by Infection with Group A Streptococcus

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