Structural correlates of carrier protein recognition in tetanus toxoid-conjugated bacterial polysaccharide vaccines

Lockyer, Kay; Gao, Fang; Derrick, Jeremy P.; Bolgiano, Barbara

doi:10.1016/j.vaccine.2015.01.046

Cited by 28 publications

(18 citation statements)

References 37 publications

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“…The ability to do so for detergentsolubilized membrane proteins that cannot be characterized by traditional means is especially prized, and detailed protocols for this have been published 31,39,40,41,42,43 . Other common applications include establishing the degree of post-translational modification and polydispersity of glycoprotein, lipoproteins and similar conjugates 4,31,44,45,46,47 ; the formation (or lack thereof) and absolute stoichiometry (as opposed to stoichiometric ratio) of heterocomplexes including protein-protein, protein-nucleic acid and protein-polysaccharide complexes 24,46,48,49,50,51,52 ; determining the monomer-dimer equilibrium dissociation constant 49,53,54 ; and evaluating protein conformation 55,56 . Beyond proteins, SEC-MALS is invaluable for characterization of peptides 57,58 , broadly heterogeneous natural polymers such as heparins 59 and chitosans 60,61 , small viruses 62 and most types of synthetic or processed polymers 63,64,65,66 .…”

Section: Introductionmentioning

confidence: 99%

Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering (SEC-MALS)

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Amartely

Tsadok³

et al. 2019

JoVE

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Analytical size-exclusion chromatography (SEC), commonly used for the determination of the molecular weight of proteins and protein-protein complexes in solution, is a relative technique that relies on the elution volume of the analyte to estimate molecular weight. When the protein is not globular or undergoes non-ideal column interactions, the calibration curve based on protein standards is invalid, and the molecular weight determined from elution volume is incorrect. Multi-angle light scattering (MALS) is an absolute technique that determines the molecular weight of an analyte in solution from basic physical equations. The combination of SEC for separation with MALS for analysis constitutes a versatile, reliable means for characterizing solutions of one or more protein species including monomers, native oligomers or aggregates, and heterocomplexes. Since the measurement is performed at each elution volume, SEC-MALS can determine if an eluting peak is homogeneous or heterogeneous and distinguish between a fixed molecular weight distribution versus dynamic equilibrium. Analysis of modified proteins such as glycoproteins or lipoproteins, or conjugates such as detergent-solubilized membrane proteins, is also possible. Hence, SEC-MALS is a critical tool for the protein chemist who must confirm the biophysical properties and solution behavior of molecules produced for biological or biotechnological research. This protocol for SEC-MALS analyzes the molecular weight and size of pure protein monomers and aggregates. The data acquired serve as a foundation for further SEC-MALS analyses including those of complexes, glycoproteins and surfactant-bound membrane proteins.

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Section: Introductionmentioning

confidence: 99%

Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering (SEC-MALS)

Some

Amartely

Tsadok³

et al. 2019

JoVE

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“…13 In addition to multiple factors that can affect the immunogenicity of a vaccine during conjugation (e.g., polysaccharide composition, linker length, conjugation chemistry), the accessibility of specific side chains in the carrier protein can affect polysaccharide conjugation. 14 Therefore, ensuring the carrier protein is consistently manufactured and well characterized before conjugation is critical.…”

Section: Introductionmentioning

confidence: 99%

Analytical Comparability Assessments of 5 Recombinant CRM 197 Proteins From Different Manufacturers and Expression Systems

Hickey

Toprani

Kaur

et al. 2018

Journal of Pharmaceutical Sciences

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Cross-reacting material 197 (CRM), a single amino acid mutant of diphtheria toxoid, is a commonly used carrier protein in commercial polysaccharide protein conjugate vaccines. In this study, CRM proteins from 3 different expression systems and 5 different manufacturers were obtained for an analytical comparability assessment using a wide variety of physicochemical and in vitro antigenic binding assays. A comprehensive analysis of the 5 CRM molecules demonstrate that recombinant CRM's expressed in heterologous systems (Escherichia coli and Pseudomonas fluorescens) are overall highly similar (if not better in some cases) to those expressed in the traditional system (Corynebacterium diphtheriae) in terms of primary sequence/post-translational modifications, higher order structural integrity, apparent solubility, physical stability profile (vs. pH and temperature), and in vitro antigenicity. These results are an encouraging step to demonstrate that recombinant CRM expressed in alternative sources have the potential to replace CRM expressed in C diphtheriae as a source of immunogenic carrier protein for lower cost polysaccharide conjugate vaccines. The physicochemical assays established in this work to monitor the key structural attributes of CRM should also prove useful as complementary characterization methods (to routine quality control assays) to support future process and formulation development of lower cost CRM carrier proteins for use in various conjugate vaccines.

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“…Lockyer at al. [7] have analyzed structure-antibody recognition relationship in 9 licensed polysaccharide-tetanus toxoid conjugate vaccines. They found that high ratios of negatively charged conjugated polysaccharides did not interfere with tetanus toxoid tryptophan amino acid side-chain interaction and the recognition of epitopes on the H-chain domain or the holotoxoid was not necessarily hampered by the size of the molecule or extent of polysaccharide loading.…”

Section: Resultsmentioning

confidence: 99%

Potential protective immunogenicity of tetanus toxoid, diphtheria toxoid and Cross Reacting Material 197 (CRM197) when used as carrier proteins in glycoconjugates

Bröker¹

2015

Human Vaccines & Immunotherapeutics

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When tetanus toxoid (TT), diphtheria toxoid (DT) or Cross Reacting Material 197 (CRM197), a non-toxic diphtheria toxin mutant protein, are used as carrier proteins in glycoconjugate vaccines, these carriers induce a protein specific antibody response as measured by in vitro assays. Here, it was evaluated whether or not glycoconjugates based on TT, DT or CRM197 can induce a protective immune response as measured by potency tests according to the European Pharmacopoeia. It could be shown, that the conjugate carriers TT and DT can induce a protective immune response against a lethal challenge by toxins in animals, while glycoconjugates based on CRM197 failed to induce a protective immune response. Opportunities for new applications of glycoconjugates are discussed.

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Structural correlates of carrier protein recognition in tetanus toxoid-conjugated bacterial polysaccharide vaccines

Abstract: HighlightsA panel of tetanus toxoid conjugates had structure-mAb binding relationships in common.A large conjugate size or high saccharide-to-protein ratio does not hinder epitope accessibility.Saccharide-specific patterns of Trp and epitope accessibility can be localised to the HC domain.

Cited by 28 publications

References 37 publications

Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering (SEC-MALS)

Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering (SEC-MALS)

Analytical Comparability Assessments of 5 Recombinant CRM 197 Proteins From Different Manufacturers and Expression Systems

Potential protective immunogenicity of tetanus toxoid, diphtheria toxoid and Cross Reacting Material 197 (CRM197) when used as carrier proteins in glycoconjugates

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