A study of Physico-Chemical Interactions between Haemophilus Influenzae Type b and Meningococcus group C Conjugate Vaccines

Background: Haemophilus influenzae type b (Hib) and Meningococcal group C (MenC) conjugate vaccines; which protect against otitis media; bacteremia and invasive diseases; including pneumonia and meningitis; are attractive candidates for combination; since they are both administered to infants and children.A Hib-MenC combination booster at 12 mo has recently been introduced in the U.K. Objectives: To rule out the possibility for the individual vaccine components in a Hib-MenC combination to interact; rendering one or both of them less effective; this work assessed whether these two saccharide-protein conjugates; namely; Hib oligosaccharideCRM197 (Cross-Reacting Material 197) and MenC-CRM197; interact in solution. Furthermore an evaluation of the size and integrity of the vaccines was also performed. Methods: HPLC Size-exclusion chromatography (SEC) with UV-adsorption and refractive index detection was performed with a phosphate and non-phosphate saline buffer to characterize the size of Hib and MenC conjugates as individual components or when combined. Results: Hib-CRM197 eluted significantly earlier than MenC-CRM197 in both phosphate-saline and MOPS-saline buffers on a TSK5000 PWXL column.When combined; there was no significant change in their elution. Refractive index monitoring showed no evidence of significant free saccharide or free protein. Conclusions: By size-exclusion chromatography and refractive index detection methods; there was no indication of degradation; and no evidence of significant associative interactions between Hib-CRM197 and MenC-CRM197 in saline-based buffers; pH 7.2

Effect of physico-chemical modification on the immunogenicity of Haemophilus influenzae type b oligosaccharide-CRM(197) conjugate vaccines.

Haemophilus influenzae type b (Hib) poly-ribosyl-ribityl phosphate (PRP) oligosaccharide-CRM(197) conjugate vaccines from two different manufacturers (Hib A and Hib B) were subjected to adverse storage conditions and used to establish correlates between physico-chemical characteristics and immunogenicity. There were manufacturer-specific differences in the effect of freezing or freeze-thawing on the carrier protein conformation and the anti-CRM(197) or anti-PRP IgG response in rabbits whereas both conjugates showed similar stability when stored at elevated temperatures. Both oligosaccharide-CRM(197) conjugate vaccines formed apparent 'aggregates' of non-specifically associated higher molecular weight material when subjected to elevated temperatures or repeated freeze-thawing. Following subcutaneous injection of samples into CBA mice and New Zealand White rabbits, the amount of IgG raised against CRM(197) was significantly lower for samples incubated at 37 or 55 degrees C compared with those kept at 4 degrees C, consistent with the less well-folded conformation of the carrier protein observed at elevated temperatures. Moreover, there was a parallel reduction in the amount of IgG raised against PRP and the level of bactericidal antibodies induced by vaccines A and B stored at 55 degrees C consistent with the observed depolymerisation of the oligosaccharide chains. Carrier protein conformational changes resulting from storage under adverse conditions did not affect the immunogenicity to Hib PRP in laboratory animals unless associated with loss of bound saccharide presumably because the carrier protein retains continuous T(H) cell epitopes which are unaffected by conformational changes.

Comparison of in vitro and in vivo methods to study stability of PLGA microencapsulated tetanus toxoid vaccines.

The purpose of this study was to investigate the utility of various in vitro and in vivo methods to assess the stability of experimental vaccines containing tetanus toxoid (TT) within PLGA microspheres. In vitro, the breakdown of the encapsulating polymers into their acid components led to changes in the structure of TT, as determined by the physico-chemical methods, rendering it undetectable by capture ELISA and altering its structural integrity. The changes in TT were directly related to increasing acidity of the vaccine supernate. Purified toxoid (not encapsulated) exposed to low pH (2.5) underwent similar changes but re-neutralisation of buffer containing free toxoid, even after one week at pH 2.5 led to some re-folding of protein as determined by fluorescence spectroscopy and gel filtration chromatography. The microencapsulated vaccines were still able to generate an antibody response in mice even after prolonged pre-incubation at 37 degrees C and the apparent absence of detectable toxoid in the vaccine supernate. Electron microscopy demonstrated differences in the amount of degradation between different formulations of microspheres. Vaccines that had retained their spherical morphology after incubation in vitro for up to 28 days were able to induce protective antibodies response equal to that of freshly prepared vaccines, which indicates that the toxoid within intact microspheres remained immunogenic. Immunochemical and physico-chemical detection methods, performed on antigen released from PLGA vaccines in vitro, are valuable in providing information on product characteristics but may not be able to predict effectiveness and should be used with in vivo methods to evaluate the stability of such formulations.

Monitoring of diphtheria, pertussis and tetanus toxoids by circular dichroism, fluorescence spectroscopy and size-exclusion chromatography.

A combination of spectroscopic and chromatographic methods has been used to monitor the quality and integrity of diphtheria, pertussis and tetanus toxoids (DTxd, PTxd and TTxd) which have been prepared from the toxins by formaldehyde treatment. Different processes for detoxifying all three toxins have yielded toxoids varying in their molecular size, including oligomers (associated monomers) and aggregates (high molecular weight complexes of non-specifically associated monomers). Changes in the intrinsic fluorescence spectra of the polypeptides have been observed in some sized fractions of DTxd and PTxd. Some physico-chemical changes have been observed to correlate with a loss of antigenicity. Spectroscopic and chromatographic methods are useful not only in monitoring the stability and consistency of vaccine starting materials, but can also be used to dissect heterogeneous toxoid preparations.

Characterisation of the calcium-binding C-terminal domain of Clostridium perfringens alpha-toxin.

Alpha-toxin is the key determinant in gas-gangrene. The toxin, a phospholipase C, cleaves phosphatidylcholine in eukaryotic cell membranes. Calcium ions have been shown to be required for the specific binding of toxin to membranes prior to phospholipid cleavage. Reported X-ray crystallographic structures of the toxin show that the C-terminal domain has a fold that is analogous to the eukaryotic calcium and membrane-binding C2 domains. We report the binding sites for three calcium ions that have been identified, by crystallographic methods, in the C-terminal domain of the protein close to the postulated membrane-binding surface. The position of these ions at the tip of the domain, and their function (to facilitate membrane binding) is similar to that of calcium ions observed bound to C2 domains. Using the optical spectroscopic techniques of circular dichroism (CD) and fluorescence spectroscopy, pronounced changes to both near and far-UV CD and tryptophan emission fluorescence upon addition of calcium to the C-terminal domain of alpha-toxin have been observed. The changes in near-UV CD, fluorescence enhancement and a 2 nm blue-shift in the fluorescence emission spectrum are consistent with tryptophan residue(s) becoming more immobilised in a hydrophobic environment. Calcium binding appears to be low-affinity: Kd approximately 175-250 microM at pH 8 assuming a 1:1 stoichiometry. as measured by spectroscopic methods.

Physico-chemical analysis of Bordetella pertussis antigens.

Physico-chemical methods are being developed for use in the control and standardization of acellular pertussis vaccines and their individual components. We have compared native and detoxified preparations of the B. pertussis antigens, pertussis toxin (PT), filamentous haemagglutinin (FHA), and the 69-kDa outer membrane protein (P69) using circular dichroism (CD), fluorescence spectroscopy, SDS-PAGE and FPLC gel filtration chromatography. Upon aldehyde detoxification, PT underwent a large change in its intrinsic fluorescence maximum (8-10 nm red-shift) and a large increase in its apparent size, detected by chromatography. Polyacrylamide gels showed individual subunits of the same apparent molecular weight (M(r)) as well as some polypeptides of higher M(r). FHA also changed conformation (5-nm red-shift in intrinsic fluorescence) upon aldehyde detoxification, with a resultant increase in the M(r)of its major constituent. The P69 protein appeared quite robust to formaldehyde treatment as measured by the same methods. Its near-UV CD spectrum contains a prominent tryptophan band; so this method may be more suitable for observing differences in conformation. We also examined an aluminium-desorbed DTaP preparation by these methods. When used in conjunction with immunochemical and toxicological assays, these methods are informative and useful in the characterization of candidate standards and should be valuable methods for ensuring the consistency of manufactured vaccines.

Recombinant human albumin as a stabilizer for biological materials and for the preparation of international reference reagents.

Recombinant human albumin expressed in Saccharomyces cerevisiae was compared with native human serum albumin in its physicochemical properties and in its use as a stabilizer in lyophilized preparations of thyroid-stimulating hormone (TSH), interleukin 15 (IL-15) and granulocyte colony-stimulating factor (G-CSF). Advantages of recombinant albumin include its lack of potential human contaminants and infectious agents. When used at concentrations of 0.1-0.2% (w/v), recombinant albumin was equivalent to native serum albumin in its capacity to protect immunological, biological and biochemical properties of TSH, IL-15 and G-CSF. Physicochemical characteristics of the two forms of albumin including their binding to fatty acids were also similar. The recombinant form of albumin used in this study should be considered as a suitable stabilizer in the preparation of lyophilized products and reference reagents.

Comparison of the diphtheria mutant toxin, CRM197, with a Haemophilus influenzae type-b polysaccharide-CRM197 conjugate by optical spectroscopy.

A Haemophilus influenzae type-b capsular polysaccharide-CRM197 protein conjugate vaccine was compared with unconjugated CRM197 and diphtheria toxin, its parent molecule. Using CD and fluorescence spectroscopy, it has been possible to observe differences in structure and stability to pH and temperature due to the G52-->E mutation in CRM197 and the 'glycosylation' of CRM197 in the conjugate. CRM197 resembles the 'open' conformation of diphtheria toxin [Blewitt, M. G., Chung, L. A. & London, E. (1985) Biochemistry 24, 5458-5464] and the attachment of poly(ribosyl-ribitol phosphate) carbohydrate chains results in a still 'more open' state, although only a small decrease in the amount of ordered structure was observed. Fluorescence spectra of gel-filtration column fractions of the conjugate suggest that material of higher apparent molecular size is in the 'more open' conformation. Conjugated CRM197 begins unfolding at slightly lower temperatures (25-35 degrees C) than native material (> 35 degrees C). In the conjugate, tryptophan residues are more accessible to the non-ionic fluorescence quencher acrylamide at 35 degrees C. The conformational change observed at pH4-6 for diphtheria toxin is also observed for CRM197, but in the conjugate begins at higher pH. This may result from the presence of charged oligosaccharide residues on the surface or the conjugation methods used. The consequences of these changes in conformation and solution behaviour of the carrier protein in terms of its ability to induce a protective, T-cell-dependent response to H. influenzae polysaccharide remain to be determined.

Physicochemical and immunological studies on the stability of free and microsphere-encapsulated tetanus toxoid in vitro.

The stability of tetanus toxoid released from experimental, slow-release microsphere vaccines was compared with that of free toxoid under different conditions over a 3 month period. The amount of antigenicity remaining, as measured by ELISA, correlated well with loss of toxoid structure, as determined by circular dichroism and fluorescence spectroscopy. At 37 degrees C and low pH, pH 2.5 for free toxoid, or under the acidic conditions generated by the hydrolysis of fast-release microspheres, a gradual unfolding of the polypeptide chain was observed within the first few weeks with more rapid denaturation beyond 30 days.

The interaction between heparin and polylysine: a circular dichroism and molecular modelling study.

The sulfated polysaccharide heparin interacts with both poly-L- and poly-D-lysine, promoting the alpha-helix conformation in the polypeptide. Chemically modified heparins in which one of the three sulfate groups per disaccharide has been removed form the same type of complex with poly-L-lysine; when two of the three sulfate groups are removed this property is lost. Heparin oligosaccharides as small as the octasaccharide can still promote alpha-helix in poly-L-lysine; the hexa- and tetrasaccharides do not, but they do disturb to a lesser extent the dynamic conformation equilibrium associated with poly-L-lysine at pH7 (22 degrees C). A three-dimensional structure for the heparin/polylysine complex is proposed in which the helical heparin molecule and the alpha-helical polypeptide are side-by-side, not intertwining. The regular periodicity of sulfate group clusters along one side of the polysaccharide chain matches the periodicity of the polypeptide alpha-helix, allowing electrostatic interactions every three peptide turns between a heparin sulfate cluster and a zeta-amino group of the polylysine. The heparin octasaccharide is the smallest even-numbered oligosaccharide long enough for two such interactions to take place.

Physicochemical studies of the structure and stability of polysaccharide-protein conjugate vaccines.

This short review highlights some areas in which spectroscopic methods have been applied to monitor the structure and stability of both the carbohydrate and protein moieties of polysaccharide-protein conjugate vaccines, such as those now in widespread clinical use for protection against Haemophilus influenzae infection.