COMPARISON OF THE DIPHTHERIA MUTANT TOXIN, CRM197, WITH A HAEMOPHILUS-INFLUENZAE TYPE-B POLYSACCHARIDE CRM197 CONJUGATE BY OPTICAL SPECTROSCOPY

A Haemophilus influenzae type-b capsular polysaccharide-CRM197 protein conjugate vaccine was compared with unconjugated CRM197 and diphtheri a toxin, its parent molecule. Using CD and fluorescence spectroscopy, it has been possible to observe differences in structure and stability to pH and temperature due to the G52-->E mutation in CRM197 and the ' glycosylation' of CRM197 in the conjugate. CRM197 resembles the 'open' conformation of diphtheria toxin [Blewitt, M. G., Chung, L. A. & Lond on, E. (1985) Biochemistry 24, 5458-5464] and the attachment of poly(r ibosyl-ribitol phosphate) carbohydrate chains results in a still 'more open' state, although only a small decrease in the amount of ordered structure was observed. Fluorescence spectra of gel-filtration column fractions of the conjugate suggest that material of higher apparent mo lecular size is in the 'more open' conformation. Conjugated CRM197 beg ins unfolding at slightly lower temperatures (25-35 degrees C) than na tive material (>35 degrees C). In the conjugate, tryptophan residues a re more accessible to the non-ionic fluorescence quencher acrylamide a t 35 degrees C. The conformational change observed at pH4-6 for diphth eria toxin is also observed for CRM197, but in the conjugate begins at higher pH. This may result from the presence of charged oligosacchari de residues on the surface or the conjugation methods used. The conseq uences of these changes in conformation and solution behaviour of the carrier protein in terms of its ability to induce a protective, T-cell -dependent response to H. influenzae, polysaccharide remain to be dete rmined.