Comparison of the Diphtheria Mutant Toxin, Crm197, with a <i>Haemophilus Influenzae</i> Type‐<i>b</i> Polysaccharide‐Crm197 Conjugate by Optical Spectroscopy

Crane, Dennis; Bolgiano, Barbara; Jones, Christopher

doi:10.1111/j.1432-1033.1997.00320.x

Cited by 32 publications

(19 citation statements)

References 39 publications

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“…These results are in agreement with previous studies performed on Hib and meningococcal group C CRM197 conjugates (Crane et al 1997;Ho et al 2001). Optical spectroscopy using circular dichroism (CD) and fluorescence spectroscopy were applied to CRM197 before and after conjugation.…”

Section: Conjugate Structuresupporting

confidence: 90%

Vaccine Analysis: Strategies, Principles, and Control

Nunnally¹,

Turula²,

Sitrin³

2015

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Section: Conjugate Structuresupporting

confidence: 90%

Vaccine Analysis: Strategies, Principles, and Control

Nunnally¹,

Turula²,

Sitrin³

2015

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“…polyribosyl ribitol phosphate). Previous observations with CD and fluorescence spectroscopic methods showed an increase in the mobility of the aromatic side chains in CRM 197 conjugated to Hib oligosaccharide when compared with that of the native CRM 197 [12]. These observations indicated that after conjugation, the CRM 197 was partially unfolded and adopted a slightly modified secondary structure and a more ‘open’ structure [17] in the conjugate molecule.…”

Section: Discussionmentioning

confidence: 91%

Solution stability studies of the subunit components of meningococcal C oligosaccharide–CRM₁₉₇ conjugate vaccines

Lemercinier

Bolgiano

et al. 2001

Biotech and App Biochem

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Spectroscopic methods were used to detect modifications in the structures of CRM197, the mutant diphtheria toxin, and meningococcal C capsular oligosaccharide following their conjugation and incubation at various temperatures. Meningococcal C oligosaccharide–CRM197 conjugate vaccines obtained from two different manufacturers were incubated at −20, 4, 23, 37 or 55 °C for 5 weeks or subjected to ten cycles of freeze–thawing. The CRM197 carrier protein and the saccharide components of the treated vaccines were monitored by CD and NMR spectroscopic techniques. CD data indicated incubation temperature‐dependent conformational changes in the carrier protein from vaccine A. Modifications appeared in both secondary and tertiary structures of the conjugated CRM197 when incubated at 23 °C or above. This was characteristic of the ‘open’ conformation previously observed for this protein component. The NMR spectra also indicated modification of the structure of the conjugated CRM197 component of vaccine A when incubated at 23 °C or above, but failed to show any modification in the conjugated oligosaccharide. On the other hand, the structure of the oligosaccharide chains in vaccine B appeared to be degraded following incubation at 55 °C, even though the thermal effect on the conjugated CRM197 was less apparent. Repeated freeze–thawing did not affect the CD or NMR spectra. In conclusion, the two meningococcal C oligosaccharide–CRM197 conjugate vaccines were stable when stored at their recommended temperatures, but were differently affected by elevated temperatures. The conjugates differ in their conjugation chemistry, attachment positions, oligosaccharide chain length and loading, as well as recommended pH and storage buffer, and their different stability properties can probably be attributed to a combination of these factors.

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“…Inhibition studies were performed both with synthetic OS structures and fragments from PS degradation to determine optimal OSs for conjugation. The protein carrier used in the OS conjugates was CRM197, a nontoxic, immunologically cross-reacting mutant protein of diphtheria toxin (9,23) containing 39 lysine residues with a free ␣ amino terminus for attachment of multiple OS chains. For the development of effective synthetic glycoconjugate vaccines, the effects of different OSs and different conjugation chemistries, as well as the effect of the ratio of protein to carbohydrate (OS loading), on the immunogenicity of the glycoconjugates were studied.…”

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confidence: 99%

Immunogenicity in a Mouse Model of a Conjugate Vaccine Made with a Synthetic Single Repeating Unit of Type 14 Pneumococcal Polysaccharide Coupled to CRM197

Mawas¹,

Niggemann

Jones

et al. 2002

Infect Immun

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Oligosaccharides (OSs) related to the pneumococcal type 14 capsular polysaccharide (Pn14PS) were studied for their ability to inhibit the binding between anti-PS14 antisera and native PS14. A synthetic tetrasaccharide corresponding to the repeating unit of the Pn14PS, a hexasaccharide mimic, and an octasaccharide fragment obtained by Pn14PS depolymerization were good inhibitors. CRM197 conjugates of the tetrasaccharide and an octasaccharide mimic were prepared by using either adipic acid diester or diethyl squarate linkers. The conjugate with the tetrasaccharide chains induced anti-Pn14PS antibodies when injected subcutaneously into mice, as determined by an enzyme-linked immunosorbent assay, and antibody titers increased with oligosaccharide loading. The adipic acid-linked tetrasaccharide conjugates elicited higher antibody titers than those prepared with a squarate spacer. The lower anti-Pn14PS antibody response of the octasaccharide mimic conjugate indicates the importance of the backbone galactose residue for an appropriate antibody response. The OS-CRM197 conjugate prepared from a single repeat unit of the Pn14PS is a potential vaccine candidate.Streptococcus pneumoniae is a major cause of morbidity and mortality in children and adults in both industrialized and developing countries (10). Although the licensed 14-and 23-valent pneumococcal capsular polysaccharide (PnPS) vaccines are safe and effective in reducing the incidence of invasive disease in healthy adults (5, 24), they are weakly immunogenic in children less than 2 years old and in the elderly, the two groups at highest risk (8,22,25,31). Whereas PSs are T-cellindependent immunogens, conjugates in which the PS is covalently attached to a protein carrier elicit a T-cell-dependent antisaccharide response even in infants, as evidenced by a booster effect upon subsequent immunizations. A number of PnPS serotypes have been conjugated to various carrier proteins and have been shown to be immunogenic and protective in various animal models (13,20,30) and in humans (1, 32). Intact PS, small oligosaccharides (OSs), or OSs of undefined length have been used to prepare those conjugates (3, 13, 36). The capsular polysaccharide of S. pneumoniae type 14 (Fig. 1) consists of a branched tetrasaccharide repeating unit (21) which is identical to the asialo core antigen of the type III group B Streptococcus PS (38). The poor immunogenicity of the Pn14PS compared to other PnPSs (18) may be due to structural similarities between antigenic determinants of the Pn14PS and human OS structures (e.g., human milk OSs and blood group carbohydrate structures). To avoid cross-reactivity with human tissue and the induction of autoreactive antibodies (6), we have been investigating the synthesis of well-defined OS fragments (26,27) corresponding to the Pn14PS to enable specific protective epitopes to be defined. Inhibition studies were performed both with synthetic OS structures and fragments from PS degradation to determine optimal OSs for conjugation. The protein carrier used in...

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Comparison of the Diphtheria Mutant Toxin, Crm197, with a Haemophilus Influenzae Type‐b Polysaccharide‐Crm197 Conjugate by Optical Spectroscopy

Cited by 32 publications

References 39 publications

Vaccine Analysis: Strategies, Principles, and Control

Vaccine Analysis: Strategies, Principles, and Control

Solution stability studies of the subunit components of meningococcal C oligosaccharide–CRM₁₉₇ conjugate vaccines

Immunogenicity in a Mouse Model of a Conjugate Vaccine Made with a Synthetic Single Repeating Unit of Type 14 Pneumococcal Polysaccharide Coupled to CRM197

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Comparison of the Diphtheria Mutant Toxin, Crm197, with a Haemophilus Influenzae Type‐b Polysaccharide‐Crm197 Conjugate by Optical Spectroscopy

Cited by 32 publications

References 39 publications

Vaccine Analysis: Strategies, Principles, and Control

Vaccine Analysis: Strategies, Principles, and Control

Solution stability studies of the subunit components of meningococcal C oligosaccharide–CRM197 conjugate vaccines

Immunogenicity in a Mouse Model of a Conjugate Vaccine Made with a Synthetic Single Repeating Unit of Type 14 Pneumococcal Polysaccharide Coupled to CRM197

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Product

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Solution stability studies of the subunit components of meningococcal C oligosaccharide–CRM₁₉₇ conjugate vaccines