Development, Interlaboratory Evaluations, and Application of a Simple, High-Throughput <italic toggle="yes">Shigella</italic> Serum Bactericidal Assay

ABSTRACT Shigella is an important cause of diarrhea worldwide, with serotypes Shigella flexneri 2a, S. flexneri 3a, and Shigella sonnei demonstrating epidemiological prevalence. Many development efforts are focused on Shigella lipopolysaccharide (LPS)-based vaccines, as O antigen-specific conjugate...

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Autores principales:	Moon H. Nahm, Jigui Yu, Hailey P. Weerts, Heather Wenzel, Chitradevi S. Tamilselvi, Lakshmi Chandrasekaran, Marcela F. Pasetti, Sachin Mani, Robert W. Kaminski
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2018
Materias:	Shigella antibody functional assay immunoassay Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/d8d798810ea7405b957210f4d056ca42
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spelling	oai:doaj.org-article:d8d798810ea7405b957210f4d056ca422021-11-15T15:24:22ZDevelopment, Interlaboratory Evaluations, and Application of a Simple, High-Throughput <italic toggle="yes">Shigella</italic> Serum Bactericidal Assay10.1128/mSphere.00146-182379-5042https://doaj.org/article/d8d798810ea7405b957210f4d056ca422018-06-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00146-18https://doaj.org/toc/2379-5042ABSTRACT Shigella is an important cause of diarrhea worldwide, with serotypes Shigella flexneri 2a, S. flexneri 3a, and Shigella sonnei demonstrating epidemiological prevalence. Many development efforts are focused on Shigella lipopolysaccharide (LPS)-based vaccines, as O antigen-specific conjugate vaccines are immunogenic and efficacious. Immunization with Shigella vaccines containing LPS can elicit antibodies capable of killing Shigella in a serotype-specific manner. Thus, to facilitate Shigella vaccine development, we have developed a serum bactericidal assay (SBA) specific for three Shigella serotypes that measures killing of target bacteria at multiple serum dilutions and in the presence of exogenous complement. The SBA has a high analytical throughput and uses simple technologies and readily available reagents. The SBA was characterized with human sera with bactericidal antibodies against S. flexneri 2a, S. flexneri 3a, and S. sonnei. Purified LPS of a homologous serotype, but not a heterologous serotype, inhibited bacterial killing. Assessment of precision found median intra-assay precision to be 13.3% and median interassay precision to be 19 to 30% for the three serotypes. The SBA is linear, with slight deviations for samples with low (~40) killing indices. The SBA was sensitive enough to allow about 100-fold predilution of serum samples. Repeat assays yielded results with less than 2-fold deviations, indicating the robustness of the assay. Assay results from four different laboratories were highly comparable when normalized with a reference serum. The Shigella SBA, combined with a reference serum, should facilitate the development of Shigella vaccines across the field. IMPORTANCE Shigella is an important cause of diarrhea worldwide, and efforts are ongoing to produce a safe and effective Shigella vaccine. Although a clear immune correlate of protection has not been established, antibodies with bactericidal capacity may provide one means of protecting against shigellosis. Thus, it is important to measure the functional capacity of antibodies, as opposed to only binding activity. This article describes a simple, robust, and high-throughput serum bactericidal assay capable of measuring Shigella-specific functional antibodies in vitro. We show for the first time that this assay was successfully performed by multiple laboratories and generated highly comparable results, particularly when SBA titers were normalized using a reference standard. The serum bactericidal assay, along with a reference serum, should greatly facilitate Shigella vaccine development.Moon H. NahmJigui YuHailey P. WeertsHeather WenzelChitradevi S. TamilselviLakshmi ChandrasekaranMarcela F. PasettiSachin ManiRobert W. KaminskiAmerican Society for MicrobiologyarticleShigellaantibodyfunctional assayimmunoassayMicrobiologyQR1-502ENmSphere, Vol 3, Iss 3 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	Shigella antibody functional assay immunoassay Microbiology QR1-502
spellingShingle	Shigella antibody functional assay immunoassay Microbiology QR1-502 Moon H. Nahm Jigui Yu Hailey P. Weerts Heather Wenzel Chitradevi S. Tamilselvi Lakshmi Chandrasekaran Marcela F. Pasetti Sachin Mani Robert W. Kaminski Development, Interlaboratory Evaluations, and Application of a Simple, High-Throughput <italic toggle="yes">Shigella</italic> Serum Bactericidal Assay
description	ABSTRACT Shigella is an important cause of diarrhea worldwide, with serotypes Shigella flexneri 2a, S. flexneri 3a, and Shigella sonnei demonstrating epidemiological prevalence. Many development efforts are focused on Shigella lipopolysaccharide (LPS)-based vaccines, as O antigen-specific conjugate vaccines are immunogenic and efficacious. Immunization with Shigella vaccines containing LPS can elicit antibodies capable of killing Shigella in a serotype-specific manner. Thus, to facilitate Shigella vaccine development, we have developed a serum bactericidal assay (SBA) specific for three Shigella serotypes that measures killing of target bacteria at multiple serum dilutions and in the presence of exogenous complement. The SBA has a high analytical throughput and uses simple technologies and readily available reagents. The SBA was characterized with human sera with bactericidal antibodies against S. flexneri 2a, S. flexneri 3a, and S. sonnei. Purified LPS of a homologous serotype, but not a heterologous serotype, inhibited bacterial killing. Assessment of precision found median intra-assay precision to be 13.3% and median interassay precision to be 19 to 30% for the three serotypes. The SBA is linear, with slight deviations for samples with low (~40) killing indices. The SBA was sensitive enough to allow about 100-fold predilution of serum samples. Repeat assays yielded results with less than 2-fold deviations, indicating the robustness of the assay. Assay results from four different laboratories were highly comparable when normalized with a reference serum. The Shigella SBA, combined with a reference serum, should facilitate the development of Shigella vaccines across the field. IMPORTANCE Shigella is an important cause of diarrhea worldwide, and efforts are ongoing to produce a safe and effective Shigella vaccine. Although a clear immune correlate of protection has not been established, antibodies with bactericidal capacity may provide one means of protecting against shigellosis. Thus, it is important to measure the functional capacity of antibodies, as opposed to only binding activity. This article describes a simple, robust, and high-throughput serum bactericidal assay capable of measuring Shigella-specific functional antibodies in vitro. We show for the first time that this assay was successfully performed by multiple laboratories and generated highly comparable results, particularly when SBA titers were normalized using a reference standard. The serum bactericidal assay, along with a reference serum, should greatly facilitate Shigella vaccine development.
format	article
author	Moon H. Nahm Jigui Yu Hailey P. Weerts Heather Wenzel Chitradevi S. Tamilselvi Lakshmi Chandrasekaran Marcela F. Pasetti Sachin Mani Robert W. Kaminski
author_facet	Moon H. Nahm Jigui Yu Hailey P. Weerts Heather Wenzel Chitradevi S. Tamilselvi Lakshmi Chandrasekaran Marcela F. Pasetti Sachin Mani Robert W. Kaminski
author_sort	Moon H. Nahm
title	Development, Interlaboratory Evaluations, and Application of a Simple, High-Throughput <italic toggle="yes">Shigella</italic> Serum Bactericidal Assay
title_short	Development, Interlaboratory Evaluations, and Application of a Simple, High-Throughput <italic toggle="yes">Shigella</italic> Serum Bactericidal Assay
title_full	Development, Interlaboratory Evaluations, and Application of a Simple, High-Throughput <italic toggle="yes">Shigella</italic> Serum Bactericidal Assay
title_fullStr	Development, Interlaboratory Evaluations, and Application of a Simple, High-Throughput <italic toggle="yes">Shigella</italic> Serum Bactericidal Assay
title_full_unstemmed	Development, Interlaboratory Evaluations, and Application of a Simple, High-Throughput <italic toggle="yes">Shigella</italic> Serum Bactericidal Assay
title_sort	development, interlaboratory evaluations, and application of a simple, high-throughput <italic toggle="yes">shigella</italic> serum bactericidal assay
publisher	American Society for Microbiology
publishDate	2018
url	https://doaj.org/article/d8d798810ea7405b957210f4d056ca42
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Development, Interlaboratory Evaluations, and Application of a Simple, High-Throughput <italic toggle="yes">Shigella</italic> Serum Bactericidal Assay

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