Chemical evidence for covalent linkages of a semi-synthetic glycoconjugate vaccine for Haemophilus influenzae type B disease.
We have defined the nature of the covalent linkages in a Haemophilus influenzae type b oligosaccharide-CRM197 conjugate vaccine, designated HbOC. The conjugate was acid hydrolyzed to release a novel amino-acid derivative, N epsilon-(2-hydroxyethyl)lysine (OHEt-Lys), identifiable with an amino-acid analyzer. This amino-acid derivative was formed by reduction of Schiff bases formed between H. influenzae type b oligosaccharides (HbO) and the lysyl epsilon-amino groups of CRM197 (a non-toxic, cross-reactive variant of diphtheria toxin), followed by acid hydrolysis of HbOC. Quantification of OHEt-Lys per CRM197 molecule allowed the determination of a covalency ratio, a useful parameter for evaluating the stoichiometry and consistency of HbOC preparations. Covalent association between HbO and CRM197 was also demonstrated by the coincidence of immunoreactivity of gel-electrophoresed HbOC on a Western blot probed with anti-CRM197 and anti-saccharide antisera.
Seid
R C
RC
Praxis Biologics, Inc., Rochester, NY 14623.
Boykins
R A
RA
Liu
D F
DF
Kimbrough
K W
KW
Hsieh
C L
CL
Eby
R
R
D000596:Amino Acids / Q000032:analysis; D001428:Bacterial Vaccines / Q000737:chemistry*; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D006001:Glycoconjugates / Q000737:chemistry*; D006192:Haemophilus Infections / Q000276:immunology*; D006193:Haemophilus influenzae / Q000276:immunology*; D006801:Humans; D009844:Oligosaccharides / Q000737:chemistry; D014614:Vaccines, Synthetic / Q000737:chemistry*
D000596:Amino Acids; D001428:Bacterial Vaccines; D006001:Glycoconjugates; D009844:Oligosaccharides; D014614:Vaccines, Synthetic