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Crosslinked flagella as a stabilized vaccine adjuvant scaffold

BACKGROUND: Engineered vaccine proteins incorporating both antigen and adjuvant components are constructed with the aim of combining functions to induce effective protective immunity. Bacterial flagellin is a strong candidate for an engineered vaccine scaffold as it is known to provide adjuvant acti...

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Autores principales: Gries, Casey M., Mohan, Rohith R., Morikis, Dimitrios, Lo, David D.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6637544/
https://www.ncbi.nlm.nih.gov/pubmed/31319823
http://dx.doi.org/10.1186/s12896-019-0545-3
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author Gries, Casey M.
Mohan, Rohith R.
Morikis, Dimitrios
Lo, David D.
author_facet Gries, Casey M.
Mohan, Rohith R.
Morikis, Dimitrios
Lo, David D.
author_sort Gries, Casey M.
collection PubMed
description BACKGROUND: Engineered vaccine proteins incorporating both antigen and adjuvant components are constructed with the aim of combining functions to induce effective protective immunity. Bacterial flagellin is a strong candidate for an engineered vaccine scaffold as it is known to provide adjuvant activity through its TLR5 and inflammasome activation. Moreover, polymerized flagellin filaments can elicit a more robust immunoglobulin response than monomeric flagellin, and the multimeric antigen form can also promote T cell-independent antibody responses. Here, we aim to produce and test a covalently stabilized polymerized flagellar filament, providing additional immune efficacy through stabilization of its polymeric filament structure, as well as stabilization for long-term storage. RESULTS: Computational modeling of monomer packing in flagellin filaments helped identify amino acids with proximity to neighboring flagella protofilaments. Paired cysteine substitutions were made at amino acids predicted to form inter-monomer disulfide cross-links, and these substitutions were capable of forming flagella when transfected into a flagellin-negative strain of Salmonella enterica subspecies Typhimurium. Interestingly, each paired substitution stabilized different helical conformational polymorphisms; the stabilized filaments lost the ability to transition between conformations, reducing bacterial motility. More importantly, the paired substitutions enabled extensive disulfide cross links and intra-filament multimer formation, and in one of the three variants, permitted filament stability in high acidic and temperature conditions where wild-type filaments would normally rapidly depolymerize. In addition, with regard to potential adjuvant activity, all crosslinked flagella filaments were able to induce wild-type levels of epithelial NF-κB in a cell reporter system. Finally, bacterial virulence was unimpaired in epithelial adherence and invasion, and the cysteine substitutions also appeared to increase bacterial resistance to oxidizing and reducing conditions. CONCLUSIONS: We identified amino acid pairs, with cysteine substitutions, were able to form intermolecular disulfide bonds that stabilized the resulting flagellar filaments in detergent, hydrochloric acid, and high temperatures while retaining its immunostimulatory function. Flagellar filaments with disulfide-stabilized protofilaments introduce new possibilities for the application of flagella as a vaccine adjuvant. Specifically, increased stability and heat tolerance permits long-term storage in a range of temperature environments, as well as delivery under a range of clinical conditions. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12896-019-0545-3) contains supplementary material, which is available to authorized users.
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spelling pubmed-66375442019-07-25 Crosslinked flagella as a stabilized vaccine adjuvant scaffold Gries, Casey M. Mohan, Rohith R. Morikis, Dimitrios Lo, David D. BMC Biotechnol Research Article BACKGROUND: Engineered vaccine proteins incorporating both antigen and adjuvant components are constructed with the aim of combining functions to induce effective protective immunity. Bacterial flagellin is a strong candidate for an engineered vaccine scaffold as it is known to provide adjuvant activity through its TLR5 and inflammasome activation. Moreover, polymerized flagellin filaments can elicit a more robust immunoglobulin response than monomeric flagellin, and the multimeric antigen form can also promote T cell-independent antibody responses. Here, we aim to produce and test a covalently stabilized polymerized flagellar filament, providing additional immune efficacy through stabilization of its polymeric filament structure, as well as stabilization for long-term storage. RESULTS: Computational modeling of monomer packing in flagellin filaments helped identify amino acids with proximity to neighboring flagella protofilaments. Paired cysteine substitutions were made at amino acids predicted to form inter-monomer disulfide cross-links, and these substitutions were capable of forming flagella when transfected into a flagellin-negative strain of Salmonella enterica subspecies Typhimurium. Interestingly, each paired substitution stabilized different helical conformational polymorphisms; the stabilized filaments lost the ability to transition between conformations, reducing bacterial motility. More importantly, the paired substitutions enabled extensive disulfide cross links and intra-filament multimer formation, and in one of the three variants, permitted filament stability in high acidic and temperature conditions where wild-type filaments would normally rapidly depolymerize. In addition, with regard to potential adjuvant activity, all crosslinked flagella filaments were able to induce wild-type levels of epithelial NF-κB in a cell reporter system. Finally, bacterial virulence was unimpaired in epithelial adherence and invasion, and the cysteine substitutions also appeared to increase bacterial resistance to oxidizing and reducing conditions. CONCLUSIONS: We identified amino acid pairs, with cysteine substitutions, were able to form intermolecular disulfide bonds that stabilized the resulting flagellar filaments in detergent, hydrochloric acid, and high temperatures while retaining its immunostimulatory function. Flagellar filaments with disulfide-stabilized protofilaments introduce new possibilities for the application of flagella as a vaccine adjuvant. Specifically, increased stability and heat tolerance permits long-term storage in a range of temperature environments, as well as delivery under a range of clinical conditions. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12896-019-0545-3) contains supplementary material, which is available to authorized users. BioMed Central 2019-07-18 /pmc/articles/PMC6637544/ /pubmed/31319823 http://dx.doi.org/10.1186/s12896-019-0545-3 Text en © The Author(s). 2019 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research Article
Gries, Casey M.
Mohan, Rohith R.
Morikis, Dimitrios
Lo, David D.
Crosslinked flagella as a stabilized vaccine adjuvant scaffold
title Crosslinked flagella as a stabilized vaccine adjuvant scaffold
title_full Crosslinked flagella as a stabilized vaccine adjuvant scaffold
title_fullStr Crosslinked flagella as a stabilized vaccine adjuvant scaffold
title_full_unstemmed Crosslinked flagella as a stabilized vaccine adjuvant scaffold
title_short Crosslinked flagella as a stabilized vaccine adjuvant scaffold
title_sort crosslinked flagella as a stabilized vaccine adjuvant scaffold
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6637544/
https://www.ncbi.nlm.nih.gov/pubmed/31319823
http://dx.doi.org/10.1186/s12896-019-0545-3
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