Degradomics-Based Analysis of Tetanus Toxoids as a Quality Control Assay

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Abstract

Currently, batch release of toxoid vaccines, such as diphtheria and tetanus toxoid, requires animal tests to confirm safety and immunogenicity. Efforts are being made to replace these tests with in vitro assays in a consistency approach. Limitations of current in vitro assays include the need for reference antigens and most are only applicable to drug substance, not to the aluminum adjuvant-containing and often multivalent drug product. To overcome these issues, a new assay was developed based on mimicking the proteolytic degradation processes in antigen-presenting cells with recombinant cathepsin S, followed by absolute quantification of the formed peptides by liquid chromatography-mass spectrometry. Temperature-exposed tetanus toxoids from several manufacturers were used as aberrant samples and could easily be distinguished from the untreated controls by using the newly developed degradomics assay. Consistency of various batches of a single manufacturer could also be determined. Moreover, the assay was shown to be applicable to Al(OH) ₃ and AlPO ₄-adsorbed tetanus toxoids. Overall, the assay shows potential for use in both stability studies and as an alternative for in vivo potency studies by showing batch-to-batch consistency of bulk toxoids as well as for aluminum-containing vaccines.

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Enhancing immunogenicity by limiting susceptibility to lysosomal proteolysis

Lélia Delamarre, Rachael Couture, Ira Mellman … (2006)

T cells recognize protein antigens as short peptides processed and displayed by antigen-presenting cells. However, the mechanism of peptide selection is incompletely understood, and, consequently, the differences in the immunogenicity of protein antigens remain largely unpredictable and difficult to manipulate. In this paper we show that the susceptibility of protein antigens to lysosomal proteolysis plays an important role in determining immunogenicity in vivo. We compared the immunogenicity of proteins with the same sequence (same T cell epitopes) and structure (same B cell epitopes) but with different susceptibilities to lysosomal proteolysis. After immunizing mice with each of the proteins adsorbed onto aluminum hydroxide as adjuvant, we measured serum IgG responses as a physiological measure of the antigen's ability to be presented on major histocompatibility complex class II molecules and to prime CD4+ T cells in vivo. For two unrelated model antigens (RNase and horseradish peroxidase), we found that only the less digestible forms were immunogenic, inducing far more efficient T cell priming and antibody responses. These findings suggest that stability to lysosomal proteolysis may be an important factor in determining immunogenicity, with potential implications for vaccine design.

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Identification of formaldehyde-induced modifications in proteins: reactions with model peptides.

Hugo Meiring, Daan Crommelin, Hans A M Timmermans … (2004)

Formaldehyde is a well known cross-linking agent that can inactivate, stabilize, or immobilize proteins. The purpose of this study was to map the chemical modifications occurring on each natural amino acid residue caused by formaldehyde. Therefore, model peptides were treated with excess formaldehyde, and the reaction products were analyzed by liquid chromatography-mass spectrometry. Formaldehyde was shown to react with the amino group of the N-terminal amino acid residue and the side-chains of arginine, cysteine, histidine, and lysine residues. Depending on the peptide sequence, methylol groups, Schiff-bases, and methylene bridges were formed. To study intermolecular cross-linking in more detail, cyanoborohydride or glycine was added to the reaction solution. The use of cyanoborohydride could easily distinguish between peptides containing a Schiff-base or a methylene bridge. Formaldehyde and glycine formed a Schiff-base adduct, which was rapidly attached to primary N-terminal amino groups, arginine and tyrosine residues, and, to a lesser degree, asparagine, glutamine, histidine, and tryptophan residues. Unexpected modifications were found in peptides containing a free N-terminal amino group or an arginine residue. Formaldehyde-glycine adducts reacted with the N terminus by means of two steps: the N terminus formed an imidazolidinone, and then the glycine was attached via a methylene bridge. Two covalent modifications occurred on an arginine-containing peptide: (i) the attachment of one glycine molecule to the arginine residue via two methylene bridges, and (ii) the coupling of two glycine molecules via four methylene bridges. Remarkably, formaldehyde did not generate intermolecular cross-links between two primary amino groups. In conclusion, the use of model peptides enabled us to determine the reactivity of each particular cross-link reaction as a function of the reaction conditions and to identify new reaction products after incubation with formaldehyde.

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Fold stability during endolysosomal acidification is a key factor for allergenicity and immunogenicity of the major birch pollen allergen

Yoan Machado, Regina Freier, Sandra Scheiblhofer … (2016)

Background The search for intrinsic factors, which account for a protein's capability to act as an allergen, is ongoing. Fold stability has been identified as a molecular feature that affects processing and presentation, thereby influencing an antigen's immunologic properties. Objective We assessed how changes in fold stability modulate the immunogenicity and sensitization capacity of the major birch pollen allergen Bet v 1. Methods By exploiting an exhaustive virtual mutation screening, we generated mutants of the prototype allergen Bet v 1 with enhanced thermal and chemical stability and rigidity. Structural changes were analyzed by means of x-ray crystallography, nuclear magnetic resonance, and molecular dynamics simulations. Stability was monitored by using differential scanning calorimetry, circular dichroism, and Fourier transform infrared spectroscopy. Endolysosomal degradation was simulated in vitro by using the microsomal fraction of JAWS II cells, followed by liquid chromatography coupled to mass spectrometry. Immunologic properties were characterized in vitro by using a human T-cell line specific for the immunodominant epitope of Bet v 1 and in vivo in an adjuvant-free BALB/c mouse model. Results Fold stabilization of Bet v 1 was pH dependent and resulted in resistance to endosomal degradation at a pH of 5 or greater, affecting presentation of the immunodominant T-cell epitope in vitro. These properties translated in vivo into a strong allergy-promoting TH2-type immune response. Efficient TH2 cell activation required both an increased stability at the pH of the early endosome and efficient degradation at lower pH in the late endosomal/lysosomal compartment. Conclusions Our data indicate that differential pH-dependent fold stability along endosomal maturation is an essential protein-inherent determinant of allergenicity.

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Journal

Journal ID (nlm-ta): Vaccines (Basel)

Journal ID (iso-abbrev): Vaccines (Basel)

Journal ID (publisher-id): vaccines

Title: Vaccines

Publisher: MDPI

ISSN (Electronic): 2076-393X

Publication date (Electronic): 01 December 2020

Publication date Collection: December 2020

Volume: 8

Issue: 4

Electronic Location Identifier: 712

Affiliations

[1 ]Leiden Academic Centre for Drug Research (LACDR), Division of BioTherapeutics, Leiden University, 2333 CC Leiden, The Netherlands; w.jiskoot@ 123456lacdr.leidenuniv.nl

[2 ]Intravacc, Institute for Translational Vaccinology, 3721 MA Bilthoven, The Netherlands; wichard.tilstra@ 123456intravacc.nl (W.T.); martin.hamzink@ 123456intravacc.nl (M.R.J.H.); justin.de.ridder@ 123456intravacc.nl (J.W.d.R.); Maarten.Danial@ 123456intravacc.nl (M.D.); hugo.meiring@ 123456intravacc.nl (H.D.M.); bernard.metz@ 123456intravacc.nl (B.M.)

Author notes

[* ]Correspondence: Thomas.michiels@ 123456intravacc.nl (T.J.M.M.); g.f.a.kersten@ 123456lacdr.leidenuniv.nl (G.F.A.K.)

Author information

Thomas J. M. Michiels https://orcid.org/0000-0003-1517-0312

Hugo D. Meiring https://orcid.org/0000-0003-0455-8899

Gideon F. A. Kersten https://orcid.org/0000-0001-8884-0685

Wim Jiskoot https://orcid.org/0000-0002-1012-3456

Bernard Metz https://orcid.org/0000-0001-6814-7656

Article

Publisher ID: vaccines-08-00712

DOI: 10.3390/vaccines8040712

PMC ID: 7712181

PubMed ID: 33271767

SO-VID: a1b5b451-91e2-4718-b74f-9057c07c419e

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Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

Degradomics-Based Analysis of Tetanus Toxoids as a Quality Control Assay

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Abstract

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Malaria vaccine development

Most cited references 25

Enhancing immunogenicity by limiting susceptibility to lysosomal proteolysis

Identification of formaldehyde-induced modifications in proteins: reactions with model peptides.

Fold stability during endolysosomal acidification is a key factor for allergenicity and immunogenicity of the major birch pollen allergen

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