Development of a subunit vaccine for prevention of Clostridium difficile associated diseases: Biophysical characterization of toxoids A and B.
Biochem Biophys Rep
; 9: 193-202, 2017 Mar.
Article
en En
| MEDLINE
| ID: mdl-28956005
ABSTRACT
Inactivation of bacterial toxins for use in human vaccines traditionally is achieved by treatment with formaldehyde. In contrast, the bivalent experimental vaccine for the prevention of C. difficile infections (CDI) that is currently being evaluated in clinical trials was produced using a different strategy. C. difficile toxins A and B were inactivated using site-directed mutagenesis and treatment with 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride/N-hydroxysulfosuccinimide (EDC/NHS). In the present work we investigate the effect of genetic and chemical modifications on the structure of inactivated toxins (toxoids) A and B. The far-UV circular dichroism (CD) spectra of wild type toxins, mutated toxins, and EDC/NHS-inactivated toxoids reveal that the secondary structure of all proteins is very similar. The near-UV CD spectra show that aromatic residues of all proteins are in a unique asymmetric environment, indicative of well-defined tertiary structure. These results along with the fluorescence emission maxima of 335 nm observed for all proteins suggest that the tertiary structure of toxoids A and B is preserved as well. Analytical ultracentrifugation data demonstrate that all proteins are predominantly monomeric with small fractions of higher molecular weight oligomeric species present in toxoids A and B. Differential scanning calorimetry data reveal that genetic mutations induce thermal destabilization of protein structures. Subsequent treatment with EDC/NHS results either in a minimal (1 °C) increase of apparent thermostability (toxoid B) or no change at all (toxoid A). Therefore, our two-step inactivation strategy is an effective approach for the preparation of non-toxic proteins maintaining native-like structure and conformation.
ANS, 1-anilinonaphtalene-8-sulfonic acid; Biophysical characterization; C. difficile; CD, circular dichroism spectroscopy; DSC, differential scanning calorimetry; EDC, 1-ethyl-3-[3dimethylaminopropyl]carbodiimide hydrochloride; NHS, N-hydroxysulfosuccinimide; PBS, Phosphate buffered saline; PM TcdB, D286A/D288A/C698A/E970K/E976K toxin B penta mutant; Subunit vaccine; TM TcdA, D285A/D287A/C700A toxin A triple mutant; TM TcdB, D286A/D288A/C698A toxin B triple mutant; TcdA, wild type toxin A; TcdB, wild type toxin B; Toxins/toxoids A and B; TxdA, EDC/NHS-treated D285A/D287A/C700A toxoid A triple mutant; TxdB, EDC/NHS-treated D286A/D288A/C698A toxoid B triple mutant
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Base de datos:
MEDLINE
Tipo de estudio:
Risk_factors_studies
Idioma:
En
Revista:
Biochem Biophys Rep
Año:
2017
Tipo del documento:
Article
País de afiliación:
Estados Unidos