Development of a new method for the quantitative analysis of the extracellular polysaccharide of Neisseria meningitidis serogroup A by use of high-performance anion-exchange chromatography with pulsed-amperometric detection.

A new method for the quantitative determination of Neisseria meningitidis group A (MenA) capsular polysaccharide (CPS) has been developed. The method is based on trifluoracetic acid (TFA) hydrolysis of the CPS (2 M at 80 degrees C for 3 h), followed by chromatographic separation and quantification of the liberated mannosamine-6-phosphate from the area of the peak obtained using an IonPac AS11 column coupled to the sensitive pulsed amperometric detector ED40. The highly selective nature of this method circumvents the interference problems associated with the classical method based on a colorimetric assay for phosphorus. Provided that suitable hydrolysis conditions can be found, this chromatographic approach might be applicable to the quantification of other bacterial antigens containing phosphorylated sugars such as meningococcal groups H, L, X and Z, and pneumococcal serotypes 6, 10A and 19.

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.

Physicochemical characterisation of the pertussis vaccine.

The characterisation of an acellular pertussis vaccine composed of a genetically modified pertussis toxin, filamentous haemagglutinin and pertactin is described. The three antigens are submitted to a mild treatment with formaldehyde in the presence of lysine before their use in vaccine formulation. Characterisation is performed by amino acid analysis, SDS-PAGE, analytical size exclusion chromatography and, in the case of pertactin, isoelectrofocusing. The effect of some variables on pertactin formaldehyde treatment has been studied by means of isoelectrofocusing and mouse immunogenicity.

Physicochemical characterisation of the oligosaccharide component of vaccines.

Glycoconjugate vaccines are being developed against Haemophilus influenzae (Hib) and meningococcal Type A and C micro-organisms; they consist of oligosaccharides of intermediate chain length conjugated to the carrier protein CRM (a non-toxic diphtheria toxin mutant). The oligosaccharides can be quantified using specific composition analyses and their structure and identity (and pattern of acetylation) evaluated by use of NMR spectroscopy. The average molecular-size (degree of polymerisation) can be determined using colorimetric assays, qualified by analysis of authentic standards. The molecular-size distribution of these anionic oligosaccharides can be achieved using ion exchange chromatography or application of the rapid and sensitive analytical HPAEC-PAD system (high performance anion-exchange chromatography with pulsed amperometric detection). Preparative ion exchange chromatography permits the isolation of purified oligomers, which can be well-characterised using the methods described above. Molecular size can be confirmed by use of mass spectrometry. These vaccines are semi-synthetic products and therefore their preparation involves several steps of chemical reaction, the detailed physicochemical characterisation of the oligosaccharide-components permits the consistent production of these well-defined glycoconjugate vaccines.

Size determination of bacterial capsular oligosaccharides used to prepare conjugate vaccines.

We recently described the use of ion exchange chromatography for analysis and the industrial scale preparation of pools of oligosaccharides of intermediate chain length from polysaccharides of Haemophilus influenzae type b (Hib) and Neisseria meningitidis groups A and C. These negatively charged "sized" oligosaccharides are activated and conjugated to the carrier protein (CRM197) to prepare the corresponding glycoconjugate vaccines. Characterization and accurate determination of the degree of polymerization (DP) of the pool of oligosaccharides is essential for the consistent production of these conjugate vaccines. This paper describes the colorimetric assays used for determination of the average DP of the Hib and meningococcal oligosaccharides, and the qualification of these assays achieved by size characterization of the respective oligosaccharides by use of physicochemical methods, including liquid chromatography, mass spectrometry (ionspray) and NMR spectroscopy.

Size fractionation of bacterial capsular polysaccharides for their use in conjugate vaccines.

We have developed a chromatographic method suitable for the fractionation of polysaccharides having a negatively charged group. The method permits the removal of all those polysaccharide fragments having a short sequence and which are likely unsuitable for conjugate vaccine construction. The selected polysaccharide fragments can be used to produce glycoconjugate vaccines containing a restricted saccharide polydispersion. We have applied this chromatographic method to three different antigens, Haemophilus influenzae type b and Neisseria meningitidis group A and group C polysaccharides. The method is easily adapted for manufacturing purposes.

Immunochemistry of capsular type polysaccharide and virulence properties of type VI Streptococcus agalactiae (group B streptococci).

The immunochemistry of capsular type polysaccharide and virulence characteristics of group B streptococci (GBS), type VI, were studied. By high-pressure anion-exchange chromatography and pulsed amperometric detection, as well as by 13C nuclear magnetic resonance analysis, both extracellular and cell-bound polysaccharides were found to contain glucose, galactose, and N-acetylneuraminic acid in the molar ratio of 2:2:1, respectively. At variance with all other GBS serotypes described to date (Ia, Ib, II, III, IV, and V), no N-acetylglucosamine was present, whatever the source of the material (secreted or cell bound; reference or clinical isolate). Sialic acid was probably involved in the immunodeterminant structure of this new serotype since cleavage of this sugar from the polysaccharide gave rise to an antigen which reacted very weakly with type VI antiserum and to a precipitation line in immunodiffusion with no identity with the native type VI polysaccharide. By using type VI antiserum and the protein A-gold technique, a large capsule was observed in the type VI GBS reference strain by electron microscopy. All type VI strains examined were lethal for CD-1 mice, the 50% lethal dose after intraperitoneal challenge ranging from 1.0 (+/- 0.9, standard deviation) x 10(5) to 2.5 (+/- 1.5, standard deviation) x 10(5) CFU per mouse. A rabbit antiserum against capsular type polysaccharide exhibited both protective activity for mice injected intraperitoneally with type VI reference strain or with clinical isolates and opsonic activity in a phagocytosis assay.