The Modified Surface Killing Assay Distinguishes between Protective and Nonprotective Antibodies to PspA

ABSTRACT Pneumococcal surface protein A (PspA) elicits antibody protective against lethal challenge by Streptococcus pneumoniae and is a candidate noncapsular antigen for inclusion in vaccines. Evaluation of immunity to PspA in human trials would be greatly facilitated by an in vitro functional assa...

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Autores principales:	Kristopher R. Genschmer, Cintia F. M. Vadesilho, Larry S. McDaniel, Sang-Sang Park, Yvette Hale, Eliane N. Miyaji, David E. Briles
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2019
Materias:	PspA modified surface killing assay pneumococcal surface protein A Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/effb57108d0349dd800cafa469a29160
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spelling	oai:doaj.org-article:effb57108d0349dd800cafa469a291602021-11-15T15:22:24ZThe Modified Surface Killing Assay Distinguishes between Protective and Nonprotective Antibodies to PspA10.1128/mSphere.00589-192379-5042https://doaj.org/article/effb57108d0349dd800cafa469a291602019-12-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00589-19https://doaj.org/toc/2379-5042ABSTRACT Pneumococcal surface protein A (PspA) elicits antibody protective against lethal challenge by Streptococcus pneumoniae and is a candidate noncapsular antigen for inclusion in vaccines. Evaluation of immunity to PspA in human trials would be greatly facilitated by an in vitro functional assay able to distinguish protective from nonprotective antibodies to PspA. Mouse monoclonal antibodies (MAbs) to PspA can mediate killing by human granulocytes in the modified surface killing assay (MSKA). To determine if the MSKA can distinguish between protective and nonprotective MAbs, we examined seven MAbs to PspA. All bound recombinant PspA, as detected by enzyme-linked immunosorbent assay and Western blotting; four gave strong passive protection against fatal challenge, two were nonprotective, and the seventh one only delayed death. The four that were able to provide strong passive protection were also most able to enhance killing in the MSKA, the two that were not protective in mice were not effective in the MSKA, and the MAb that was only weakly protective in mice was weakly effective in the MSKA (P < 0.001). One of the four most protective MAbs tested reacted to the proline-rich domain of PspA. Two of the other most protective MAbs and the weakly protective MAb reacted with a fragment from PspA’s α-helical domain (αHD), containing amino acids (aa) 148 to 247 from the N terminus of PspA. The fourth highly protective MAb recognized none of the overlapping 81- or 100-aa fragments of PspA. The two nonprotective MAbs recognized a more N-terminal αHD fragment (aa 48 to 147). IMPORTANCE The most important finding of this study is that the MSKA can be used as an in vitro functional assay. Such an assay will be critical for the development of PspA-containing vaccines. The other important findings relate to the locations and nature of the protection-eliciting epitopes of PspA. There are limited prior data on the locations of protection-eliciting PspA epitopes, but those data along with the data presented here make it clear that there is not a single epitope or domain of PspA that can elicit protective antibody and there exists at least one region of the αHD which seldom elicits protective antibody. Moreover, these data, in concert with prior data, strongly make the case that protective epitopes in the αHD are highly conformational (≥100-amino-acid fragments of the αHD are required), whereas at least some protection-eliciting epitopes in the proline-rich domain are encoded by ≤15-amino-acid sequences.Kristopher R. GenschmerCintia F. M. VadesilhoLarry S. McDanielSang-Sang ParkYvette HaleEliane N. MiyajiDavid E. BrilesAmerican Society for MicrobiologyarticlePspAmodified surface killing assaypneumococcal surface protein AMicrobiologyQR1-502ENmSphere, Vol 4, Iss 6 (2019)
institution	DOAJ
collection	DOAJ
language	EN
topic	PspA modified surface killing assay pneumococcal surface protein A Microbiology QR1-502
spellingShingle	PspA modified surface killing assay pneumococcal surface protein A Microbiology QR1-502 Kristopher R. Genschmer Cintia F. M. Vadesilho Larry S. McDaniel Sang-Sang Park Yvette Hale Eliane N. Miyaji David E. Briles The Modified Surface Killing Assay Distinguishes between Protective and Nonprotective Antibodies to PspA
description	ABSTRACT Pneumococcal surface protein A (PspA) elicits antibody protective against lethal challenge by Streptococcus pneumoniae and is a candidate noncapsular antigen for inclusion in vaccines. Evaluation of immunity to PspA in human trials would be greatly facilitated by an in vitro functional assay able to distinguish protective from nonprotective antibodies to PspA. Mouse monoclonal antibodies (MAbs) to PspA can mediate killing by human granulocytes in the modified surface killing assay (MSKA). To determine if the MSKA can distinguish between protective and nonprotective MAbs, we examined seven MAbs to PspA. All bound recombinant PspA, as detected by enzyme-linked immunosorbent assay and Western blotting; four gave strong passive protection against fatal challenge, two were nonprotective, and the seventh one only delayed death. The four that were able to provide strong passive protection were also most able to enhance killing in the MSKA, the two that were not protective in mice were not effective in the MSKA, and the MAb that was only weakly protective in mice was weakly effective in the MSKA (P < 0.001). One of the four most protective MAbs tested reacted to the proline-rich domain of PspA. Two of the other most protective MAbs and the weakly protective MAb reacted with a fragment from PspA’s α-helical domain (αHD), containing amino acids (aa) 148 to 247 from the N terminus of PspA. The fourth highly protective MAb recognized none of the overlapping 81- or 100-aa fragments of PspA. The two nonprotective MAbs recognized a more N-terminal αHD fragment (aa 48 to 147). IMPORTANCE The most important finding of this study is that the MSKA can be used as an in vitro functional assay. Such an assay will be critical for the development of PspA-containing vaccines. The other important findings relate to the locations and nature of the protection-eliciting epitopes of PspA. There are limited prior data on the locations of protection-eliciting PspA epitopes, but those data along with the data presented here make it clear that there is not a single epitope or domain of PspA that can elicit protective antibody and there exists at least one region of the αHD which seldom elicits protective antibody. Moreover, these data, in concert with prior data, strongly make the case that protective epitopes in the αHD are highly conformational (≥100-amino-acid fragments of the αHD are required), whereas at least some protection-eliciting epitopes in the proline-rich domain are encoded by ≤15-amino-acid sequences.
format	article
author	Kristopher R. Genschmer Cintia F. M. Vadesilho Larry S. McDaniel Sang-Sang Park Yvette Hale Eliane N. Miyaji David E. Briles
author_facet	Kristopher R. Genschmer Cintia F. M. Vadesilho Larry S. McDaniel Sang-Sang Park Yvette Hale Eliane N. Miyaji David E. Briles
author_sort	Kristopher R. Genschmer
title	The Modified Surface Killing Assay Distinguishes between Protective and Nonprotective Antibodies to PspA
title_short	The Modified Surface Killing Assay Distinguishes between Protective and Nonprotective Antibodies to PspA
title_full	The Modified Surface Killing Assay Distinguishes between Protective and Nonprotective Antibodies to PspA
title_fullStr	The Modified Surface Killing Assay Distinguishes between Protective and Nonprotective Antibodies to PspA
title_full_unstemmed	The Modified Surface Killing Assay Distinguishes between Protective and Nonprotective Antibodies to PspA
title_sort	modified surface killing assay distinguishes between protective and nonprotective antibodies to pspa
publisher	American Society for Microbiology
publishDate	2019
url	https://doaj.org/article/effb57108d0349dd800cafa469a29160
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The Modified Surface Killing Assay Distinguishes between Protective and Nonprotective Antibodies to PspA

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