Chemical evidence for covalent linkages of a semisynthetic glycoconjugate vaccine forHaemophilus influenzae type b disease

Abstract

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.

Carbohydrate-Based Vaccines

“…Some conjugation chemistries, such as reductive amination of lysine residues, result in markers which are stable to protein hydrolysis and can be quantifi ed chromatographically. For example after hydrolysis of a CRM197-Hib conjugate, where the Hib has been activated and depolymerized by periodate oxidation, N ε -(2-hydroxyethyl) lysine is produced and can be quantifi ed by amino acid analysis [ 56 ]. This data confi rms that the carrier protein has a consistent number of polysaccharide attachments sites, batch-to-batch.…”

“…For instance, the HIV-1 envelope glycoprotein gp120 is involved in binding to CD4 + T cells and subsequent virus entry [ 56 ]. The diffi culty in the development of antiviral carbohydrate-based vaccines is that viral glycans are produced by the glycosylation machinery of the host cell, and thus are usually tolerated by the host immune system.…”

“…As a consequence, novel adjuvants can be designed and selected on the basis of their molecular interactions. A new class of effective and safe vaccine adjuvants is based on PRR ligands that specifi cally activate Toll-like receptor (TLR) signaling [ 55 ] or C-type lectin receptor (CLR) signaling in APCs [ 56 ]. Owing to their high expression by pathogens, numerous glycan structures have been identifi ed to be recognized by PRRs.…”

Recent Developments in Synthetic Oligosaccharide-Based Bacterial Vaccines

“…The overwhelming success [34] of native [16] and degraded PS-based [35] conjugate vaccines against Hib raised the possibility that conjugates of proteins with relatively small OS's may also produce PS-specific antibodies. * An early support for this comes from the observations of Goebel who found that a protein conjugate of the disaccharide cellobiuronic acid, the repeating unit of the CPS of Streptococcus pneumonia type 3, elicited CPS-specific antisera in rabbits that developed resistance against the homologous organism [36].…”

Direct and Indirect Methods for Molar-Mass Analysis of Fragments of the Capsular Polysaccharide ofHaemophilus influenzaeType b

“…. and 3b) forms, which result from oxidative cleavage of ribitol residues (10). In this case, inaccuracy may result Isolation and Purification of PRP from the dissimilar reactivities in the reduction-oxidation chemistry of the reducing-end-group assay arising H. influenzae strain Eagan was obtained from Rockefeller University (New York, NY).…”