Structural characterisation, stability and antibody recognition of chimeric NHBA-GNA1030: an investigational vaccine component against Neisseria meningitidis.

A new generation multi-component vaccine, principally directed against serogroup B Neisseria meningitidis (4CMenB), has recently been developed. One of its components, identified through reverse vaccinology, is the neisserial heparin-binding antigen (NHBA) which is included in the formulation as a novel NHBA-GNA1030 fusion protein (NHBA-FP). We describe here the biophysical characteristics of this vaccine antigen to understand better its structural properties in solution and concurrent immunogenicity prior to formulation.

Structural organization of NadADelta(351-405), a recombinant MenB vaccine component, by its physico-chemical characterization at drug substance level.

The physico-chemical characterization of NadADelta(351-405), a recombinant protein discovered by reverse vaccinology, component of a candidate vaccine against Neisseria meningitidis serotype B is presented. Analytical methods like mass spectrometry, electrophoresis, optical spectroscopy and SEC-MALLS have been applied to unveil the structure of NadADelta(351-405), and to evaluate Product-Related Substances. Moreover, analysis of the protein after intentional denaturation has been applied in order to challenge the chosen methods and to determine their appropriateness and specificity.

Physicochemical characterisation of glycoconjugate vaccines for prevention of meningococcal diseases.

Bacterial capsular polysaccharides covalently linked to an appropriate carrier protein represent the best tool to induce a protective immune response against a wide range of bacterial diseases, such as meningococcal infections. We describe here the physico-chemical characterisation of glycoconjugate molecules designed to prepare a vaccine against Neisseria meningitidis serogroups A, C, W135 and Y. The use of a selective conjugation chemistry resulted in well characterised, reproducible and traceable glycoconjugate that can be consistently manufactured at large scale.

Isolation of oligosaccharides from a partial-acid hydrolysate of pneumococcal type 3 polysaccharide for use in conjugate vaccines.

A series of well-defined oligosaccharide fragments of the capsular polysaccharide of Streptococcus pneumoniae type 3 has been generated. Partial-acid hydrolysis of the capsular polysaccharide, followed by fractionation of the oligosaccharide mixture by Sepharose Q ion-exchange chromatography yielded fragments containing one to seven [-->3)-beta-D-GlcpA-(1-->4)-beta-D-Glcp-(1-->] repeating units. The isolated fragments were analysed for purity by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) using an IonPac AS11 column, and their structures were verified by 1H NMR spectroscopy and nano-electrospray mass spectrometry.

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.

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.

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.

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.

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.

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.

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).