Free Saccharide Research Articles

Assessment of the stability and immunogenicity of meningococcal oligosaccharide C-CRM197 conjugate vaccines

In this stability study, meningococcal C-CRM 197 conjugate vaccines from two different manufacturers that differ in oligosaccharide chain length, number of conjugation sites, conjugation chemistry, manufacturing process and formulation were used. Both the bulk concentrated and final fill preparations were incubated at −20, 4, 23, 37 or 55°C for 5 weeks or subjected to ten cycles of freeze-thawing. The structural stability, hydrodynamic size and integrity of the treated vaccines were monitored by size exclusion chromatography (FPLC-SEC), high performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) and fluorescence spectroscopy techniques. The data showed that the structural stability of the oligosaccharide chains and of the protein carrier varied between the two conjugates. The experimental immunogenicity was not severely affected by repeated freeze-thawing, incubation at −20 or 4°C, but one developed conformational changes in the protein carrier when incubated at 23°C or above, although the integrity of the oligosaccharide structure was maintained. This was not associated with any reduction in primary IgG or IgM antibody responses to meningococcal C polysaccharide. In the other conjugate vaccine, exposure to 55°C resulted in the release of a substantial proportion of free saccharide that was accompanied by significant reduction in both IgG and IgM antibody responses to immunisation in the model system. In conclusion, the two meningococcal C-CRM 197 conjugate vaccines were stable when stored at the recommended temperatures, although their structural stability and subsequent immunogenicity were influenced by their conjugation chemistry and formulation.

Effect of proteasome inhibitors on the release into the cytosol of free polymannose oligosaccharides from glycoproteins.

Prompted by previous observations which suggested that the release of polymannose oligosaccharides shortly after the cotranslational N-glycosylation of proteins is a function of the ER-associated quality control system (Moore and Spiro (1994) J. Biol. Chem., 269, 12715-12721), we evaluated the effect which proteasome inhibitors have on the appearance of these free saccharide components. Employing as a model system castanospermine-treated BW5147 mouse T-lymphoma cells in which accelerated degradation of the T-cell receptor (TCR) alpha subunit takes place (Kearse et al. (1994) EMBO J., 13, 3678-3686), we noted that both lactacystin and N-acetyl-L-leucyl-L-leucyl-L-norleucinal, but not leupeptin, brought about a rapid and substantial reduction in the release of free polymannose oligosaccharides into the cytosol during pulse-chase studies, while the oligosaccharides in the intravesicular compartment remained unchanged, as measured by streptolysin O permeabilization. This inhibition was furthermore selective in that it affected solely the components terminating in a single N-acetylglucosamine residue (OS-GlcNAc(1)) and not the oligosaccharides terminating in a di-N-acetylchitobiose sequence (OS-GlcNAc(2)), which reside primarily in the intravesicular compartment. Despite the quantitative effect of the proteasome inhibitors on the cytosolic oligosaccharides, the molar distribution of the triglucosyl OS-GlcNAc(1) species was unaffected. The decrease in cytosolic oligosaccharides brought about by proteasome inhibition was reflected in a pronounced increase in the stability of the TCRalpha subunit. Our findings suggest that the N-deglycosylation and proteasome mediated degradation are coupled events. On the basis of our data and those of others we propose that the quality control mechanism involves proteasomes associated with the cytosolic side of the endoplasmic reticulum acting in concert with a membrane situated N-glycanase. Such a complex by removing the carbohydrate units could facilitate the retrograde ER to cytosol translocation of glycoproteins.

Quantitative determination of saccharide in Haemophilus influenzae type b glycoconjugate vaccines, alone and in combination with DPT, by use of high-performance anion-exchange chromatography with pulsed amperometric detection

The stability and integrity of glycoconjugate vaccines requires determination of the total saccharide and quantification of the unbound or free saccharide present. The traditional assay for Hib conjugates, based on colorimetric determination of ribose, has been much improved by the use of base hydrolysis and analysis of the Hib subunit generated using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The production of this subunit was confirmed by NMR analysis. However, quantification of free Hib saccharide using this method was not possible in the combination vaccines evaluated due to interferences emanating from DPT. Thus a method based on TFA hydrolysis followed by the chromatographic separation and quantification of ribitol on a CarboPac MA1 column was developed. The method is selective, and with the use of ED40 electrode, requires only nanomole amounts for the chromatographic step, thereby ensuring that free saccharide can be monitored accurately in the formulated Hib-CRM vaccine alone and when in combination with other vaccines.

The lipopolysaccharide of Helicobacter mustelae type strain ATCC 43772 expresses the monofucosyl A type 1 histo-blood group epitope

The lipopolysaccharide of Helicobacter mustelae type strain ATCC 43772 was obtained by phenol-water extraction of bacterial cells. Structural investigations were made on the lipid A free saccharide moiety released from the lipopolysaccharide by mild acetic acid hydrolysis. Nuclear magnetic resonance, gas liquid chromatography-mass spectrometry and fast atom bombardment-mass spectrometry were employed in the characterization of products from chemical manipulations. A monoclonal antibody specific for blood group A reacted strongly with lipopolysaccharide of H. mustelae. Chemical and serological data showed that the outer core region of the lipopolysaccharide from H. mustelae ATCC 43772 expresses the monofucosyl A type 1, α- d-GalNAc-(1→3)-[α- l-Fuc-(1→2]-β- d-Gal-(1→3)-β- d-GlcNAc, blood group determinant, a mimic of animal cell surface glycolipids and glycoproteins.

Immunogenicity of a Streptococcus pneumoniae type 4 polysaccharide--protein conjugate vaccine is decreased by admixture of high doses of free saccharide.

In this study we report that the priming capacity of a Streptococcus pneumoniae type 4 polysaccharide-protein conjugate to booster immunizations with the native capsular polysaccharide is dose dependent. Furthermore, it is shown by admixture experiments that simultaneous administration of high doses of free saccharide (0.5-25 micrograms) of different chain lengths (varying from M(r) 1.6-120 kDa) decreases the anti-polysaccharide antibody response. Presence of low doses of saccharide (up to 10%), which are usually present in conjugates prepared by the carbodiimide coupling procedure, did not influence the anti-polysaccharide antibody response in adult and neonatal mice.

Binding of 4-methyl umbelliferyl-α-D-glucoPyranoside to Vicia faba lectin: Fluorescence-quenching studies

On binding toVicia faba lectin, the fluorescence of 4-methylumbelliferyl-α-D-glucoPyranoside was quantitatively quenched showing that the interaction of 4-methylumbelliferyl-α-D-glucoPyranoside took Place in a binding environment. The binding of the fluorescent sugar was saccharide sPecific as evidenced by the reversal of 4-methylumbelliferyl-α-D-glucoPyranoside fluorescence quenching by D-fructose. The association constant,K a, values for the 4-methylumbelliferyl-α-D-glucoPyranoside was determined by comPetition study emPloying reversal of fluorescence quenching of 4-methylumbelliferyl-α-D-glucoPyranoside by D-fructose. TheK a value obtained for D-fructose was 1.07 ±0.03 X 104 M-1 and for 4-methylumbelliferyl-α-D-glucoPyranoside was 1.60 ±0.05 X 104 M-1 at 15°C. TheK a values of 2.51 ±0.06 X 104M-1, l.26 ±0.02 X 104 M-1 and 0.56 ±0.01 X 104M-1, resPectively at 10°, 20° and 30°C were obtained from the ChiPman equation. The relative fluorescence quenching, ΔF a, at infinite concentration of the free saccharide sites ofVicia faba lectin [P′] was 93.5% at 30°C and the binding constant for 4-methylumbelliferyl-α-D-glucoPyranoside lectin interaction as derived by Yank and Hanaguchi equation was 0.63 ±0.01 X 104M-1.

Non-antibiotic prevention of urinary tract infection.

The identification of several glycoconjugates as receptors for attaching bacteria has resulted in optimism regarding the use of receptor analogues in preventing infection. The structure of receptor-active oligosaccharides and the patient groups which might benefit from such treatment are most thoroughly understood for urinary tract infections. Prevention of adhesion and delay or decrease of infection was achieved using oligosaccharides from the globoseries of glycolipids containing Gal alpha 1----4Gal. This minimal receptor structure as a free saccharide was, however, not sufficient. It was the intact P, P1 and pk determinants which showed optimal activity in vitro. In addition, protection against experimental UTI results from previous exposure to whole bacteria or isolated antigens. The mechanism(s) of protection and their relevance in clinical UTI remain to be defined.

Non-antibiotic prevention of urinary tract infection.

The identification of several glycoconjugates as receptors for attaching bacteria has resulted in optimism regarding the use of receptor analogues in preventing infection. The structure of receptor-active oligosaccharides and the patient groups which might benefit from such treatment are most thoroughly understood for urinary tract infections. Prevention of adhesion and delay or decrease of infection was achieved using oligosaccharides from the globoseries of glycolipids containing Gal alpha 1----4Gal. This minimal receptor structure as a free saccharide was, however, not sufficient. It was the intact P, P1 and pk determinants which showed optimal activity in vitro. In addition, protection against experimental UTI results from previous exposure to whole bacteria or isolated antigens. The mechanism(s) of protection and their relevance in clinical UTI remain to be defined.