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.

Preclinical evaluation of group B Neisseria meningitidis and Escherichia coli K92 capsular polysaccharide-protein conjugate vaccines in juvenile rhesus monkeys.

We reported the first use of group B meningococcal conjugate vaccines in a nonhuman primate model (S. J. N. Devi, C. E. Frasch, W. Zollinger, and P. J. Snoy, p. 427-429, in J. S. Evans, S. E. Yost, M. C. J. Maiden, and I. M. Feavers, ed., Proceedings of the Ninth International Pathogenic Neisseria Conference, 1994). Three different group B Neisseria meningitidis capsular polysaccharide (B PS)-protein conjugate vaccines and an Escherichia coli K92 capsular polysaccharide-tetanus toxoid (K92-TT) conjugate vaccine are here evaluated for safety and relative immunogenicities in juvenile rhesus monkeys with or without adjuvants. Monkeys were immunized intramuscularly with either B PS-cross-reactive material 197 conjugate, B PS-outer membrane vesicle (B-OMV) conjugate, or N-propionylated B PS-outer membrane protein 3 (N-pr. B-OMP3) conjugate vaccine with or without adjuvants at weeks 0, 6, and 14. A control group of monkeys received one injection of the purified B PS alone, and another group received three injections of B PS noncovalently complexed with OMV. Antibody responses as measured by enzyme-linked immunosorbent assay varied among individual monkeys. All vaccines except B PS and the K92-TT conjugate elicited a twofold or greater increase in total B PS antibodies after one immunization. All vaccines, including the K92-TT conjugate, elicited a rise in geometric mean B PS antibody levels of ninefold or more over the preimmune levels following the third immunization. Antibodies elicited by N-pr. B-OMP3 and B-OMV conjugates were directed to the N-propionylated or to the spacer-containing B PS antigens as well as to the native B PS complexed with methylated human serum albumin. None of the vaccines caused discernible safety-related symptoms.

Immunogenicity of meningococcal B polysaccharide conjugated to tetanus toxoid or CRM197 via adipic acid dihydrazide.

Vaccine development against Group B Neisseria meningitidis is complicated by the nature of the capsular polysaccharide, which is alpha 2-8-linked poly-sialic acid, identical in structure to the poly-sialic acid found in many mammalian tissues during development. To test the feasibility of a vaccine based on this polysaccharide, we synthesized several conjugates of meningococcal B polysaccharide linked to a carrier protein (tetanus toxoid or diphtheria CRM197), via an adipic acid dihydrazide (ADH) spacer. All conjugates induced a strong immune response. However, most of the antibodies were not directed against the Meningococcus B polysaccharide and could not be inhibited by the purified polysaccharide alone. Further investigations showed that the antibodies recognized an epitope composed by the junction between the spacer and the polysaccharide and protein, that is not present in the native polysaccharide and is generated during the coupling reaction. This epitope becomes immunodominant with respect to the poorly immunogenic polysaccharide. While the majority of the immune response is directed against the above epitope, the conjugates induced also an immune response against the Meningococcus B polysaccharide. The anti-Meningococcus B antibodies elicited are of the IgM and IgG class and are inhibitable by the polysaccharide. Moreover, they are bactericidal, thus suggesting that they would induce protection against disease.

Synthesis and applications of phosphonoacetic derivatives.

In this paper, the synthesis of new phosphonoacetic acid derivatives and their applications in fields of biotechnological interest are discussed. Phosphonoacetic acids are competitive inhibitors of alkaline phosphatase, an enzyme widely used in diagnostics, as colorimetric detection tool. The phosphonoacetic acid's inhibition activity has been exploited by us for the obtainment of an innovative technique for non-radioactive DNA probes detection, the last being based on DNA labeling with the enzyme inhibitor, followed by detection by means of the chromogenic enzyme and substrate. Moreover, we have found a further application of phosphonoacetic acids, by the preparation of an affinity chromatography support that has been revealed to be very effective in the purification of alkaline phosphatase. Finally, phosphonoacetic acid derivatives have been tested also for their antiviral activity. Some of them, examined in preliminary in vitro experiments, have been found very active against Herpes simplex virus.