Barbara Bolgiano

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.

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 degrees 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 degrees C, but one developed conformational changes in the protein carrier when incubated at 23 degrees 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 degrees 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.

Physico-chemical and immunological examination of the thermal stability of tetanus toxoid conjugate vaccines

The thermal stability of meningococcal C (MenC)- and Haemophilus influenzae b (Hib)-tetanus toxoid (TT) conjugate vaccines was investigated using spectroscopic and chromatographic techniques and immunogenicity assays in animal models. In this stability study, both the bulk concentrate and final fills were incubated at -20, 4, 23, 37 or 55 degrees C for 5 weeks or subjected to cycles of freeze-thawing. The structural stability, hydrodynamic size and molecular integrity of the treated vaccines were monitored by circular dichroism (CD), fluorescence and nuclear magnetic resonance (NMR) spectroscopic techniques, size exclusion chromatography (FPLC-SEC), and high performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD). Only storage at 55 degrees C for 5 weeks caused some slight unfolding and modification in the tertiary structure of the carrier protein in the MenC-TT conjugate. Substantial loss of saccharide content from the MenC conjugates was observed at 37 and 55 degrees C. Unexpectedly, the experimental immunogenicity of MenC-TT vaccine adsorbed to Alhydrogel was significantly reduced only by repeated freeze-thawing, but not significantly decreased by thermal denaturation. Neither the molecular integrity nor the immunogenicity of the lyophilised Hib-TT vaccines was significantly affected by freeze-thawing or by storage at high temperature. In conclusion, the MenC- and Hib-TT conjugate vaccines were relatively stable when stored at higher temperatures, though when MenC-TT vaccine was adsorbed to Alhydrogel, it was more vulnerable to repeated freeze-thawing. When compared with CRM(197) conjugate vaccines studied previously using similar techniques, the tetanus toxoid conjugates were found to have higher relative thermal stability in that they retained immunogenicity following storage at elevated temperatures.

Characterization of size, structure and purity of serogroup X Neisseria meningitidis polysaccharide, and development of an assay for quantification of human antibodies.

Serogroup X Neisseria meningitidis (MenX) has recently emerged as a cause of localized disease outbreaks in sub-Saharan Africa. In order to prepare for vaccine development, MenX polysaccharide (MenX PS) was purified by standard methods and analyzed for identity and structure by NMR spectroscopy. This study presents the first full assignment of the structure of the MenX PS using (13)C, (1)H and (31)P NMR spectroscopy and total correlation spectroscopy (TOCSY) and (1)H-(13)C heteronuclear single quantum coherence (HSQC). Molecular size distribution analysis using HPLC-SEC with multi-angle laser light scattering (MALLS) found the single peak of MenX PS to have a weight-average molar mass of 247,000g/mol, slightly higher than a reference preparation of purified serogroup C meningococcal polysaccharide. MenX PS tended to be more thermostable than serogroup A PS. A method for the quantification of MenX PS was developed by use of high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). A novel and specific ELISA assay for quantification of human anti-MenX PS IgG based on covalent linkage of the MenX PS to functionally modified microtitre plates was developed and found valid for the assessment of the specific antibody concentrations produced in response to MenX vaccination or natural infection. The current work thus provides the necessary background for the development of a MenX PS-based vaccine to prevent meningococcal infection caused by bacteria bearing this capsule.

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.

Evaluation of meningococcal C oligosaccharide conjugate vaccines by size-exclusion chromatography/multi-angle laser light scattering

The mean molecular masses of three different meningococcal C saccharide (MenC)–protein conjugate vaccines and their constituent proteins were estimated using HPLC size‐exclusion chromatography (SEC) with multi‐angle laser light scattering (MALLS) and refractive‐index (RI) detection (SEC/MALLS). Chromatography of two CRM197 conjugates (MenC–CRM197‐A and MenC–CRM197‐B) and one tetanus toxoid (TT) conjugate (MenC–TT) was performed in PBS, pH 7.4, on TSK‐Gel® (TosoHaas) analytical columns [CRM197 is a non‐catalytic cross‐reacting mutant (CRM) of diphtheria toxin]. Analysis of the light‐scattering signal measured at 18 angles simultaneously, using the RI signal as a measure of concentration, gave absolute weight‐average‐molecular‐mass (M̄w) values for the CRM197 conjugates as follows: MenC‐CRM197‐A, ≈75000 g·mol−1 and MenC–CRM197‐B, ≈350000 g·mol−1, suggesting that MenC–CRM197‐A is a monomer (one carrier protein per conjugate molecule), while MenC‐CRM197‐B is largely composed of conjugates containing three or four CRM197 molecules. The MenC–TT conjugate eluted as a two‐component system with M̄w of 1.63×106 and 395000 g· mol−1, suggesting that some cross‐linked complexes contain up to six TT molecules. Comparison of results from MALLS/RI with those obtained using UV detection highlights the differences in size and relative composition of the various subpopulations of the MenC conjugates that can be obtained using different detection systems.

Modifications of the catalytic and binding subunits of pertussis toxin by formaldehyde: effects on toxicity and immunogenicity.

A panel of pertussis toxin (PT) preparations with varying levels of residual toxicity was prepared by treatment of native PT with formaldehyde (0-1.00% (w/v)) with the purpose of investigating the effects of residual toxicity on immunogenicity. The catalytically inactive mutant PT (PT-9K/129G) was used for comparison. Results from in vitro ADP-ribosyl transferase and Chinese hamster ovary (CHO)-cell toxicity assays demonstrated a formaldehyde-dependent reduction in PT toxicity, and implied that both A and B domain functions of PT were modified. The in vivo histamine sensitisation and leukocyte proliferation tests suggested that the formaldehyde-treated native PT preparations were subject to reversion to toxicity. Reversion was confirmed by in vitro toxicity assays, which demonstrated recovery of A and B domain functions. The presence of high molecular weight aggregated and cross-linked species of PT in these preparations did not appear to be detrimental to the production of a neutralising antibody response. IgG responses to native and non-catalytic mutant PT suggested that low levels of residual activity in the native PT enhanced the antibody response, while higher levels of activity inhibited the response. Using the non-catalytic mutant PT showed that formaldehyde-induced changes were not detrimental to the magnitude of the PT-specific antibody response but did reduce the PT-specific neutralising activity. In conclusion, the residual toxicity of PT preparations following formaldehyde treatment may play an important role in the immune response to pertussis vaccine, potentially altering the quality, class and magnitude of the antibodies produced to PT.

Solution stability studies of the subunit components of meningococcal C oligosaccharide–CRM197 conjugate vaccines

Spectroscopic methods were used to detect modifications in the structures of CRM197, the mutant diphtheria toxin, and meningococcal C capsular oligosaccharide following their conjugation and incubation at various temperatures. Meningococcal C oligosaccharide–CRM197 conjugate vaccines obtained from two different manufacturers were incubated at −20, 4, 23, 37 or 55 °C for 5 weeks or subjected to ten cycles of freeze–thawing. The CRM197 carrier protein and the saccharide components of the treated vaccines were monitored by CD and NMR spectroscopic techniques. CD data indicated incubation temperature‐dependent conformational changes in the carrier protein from vaccine A. Modifications appeared in both secondary and tertiary structures of the conjugated CRM197 when incubated at 23 °C or above. This was characteristic of the ‘open’ conformation previously observed for this protein component. The NMR spectra also indicated modification of the structure of the conjugated CRM197 component of vaccine A when incubated at 23 °C or above, but failed to show any modification in the conjugated oligosaccharide. On the other hand, the structure of the oligosaccharide chains in vaccine B appeared to be degraded following incubation at 55 °C, even though the thermal effect on the conjugated CRM197 was less apparent. Repeated freeze–thawing did not affect the CD or NMR spectra. In conclusion, the two meningococcal C oligosaccharide–CRM197 conjugate vaccines were stable when stored at their recommended temperatures, but were differently affected by elevated temperatures. The conjugates differ in their conjugation chemistry, attachment positions, oligosaccharide chain length and loading, as well as recommended pH and storage buffer, and their different stability properties can probably be attributed to a combination of these factors.

Reduction of the Ganglioside Binding Activity of the Tetanus Toxin HC Fragment Destroys Immunogenicity: Implications for Development of Novel Tetanus Vaccines

ABSTRACT In this study, the immunogenicities of the nontoxic HC fragment of tetanus toxin and derivatives lacking ganglioside binding activity were compared with that of tetanus toxoid after subcutaneous immunization of mice. Wild-type HC (HCWT) protein and tetanus toxoid both elicited strong antibody responses against toxoid and HC antigens and provided complete protection against toxin challenge. Mutants of HC containing deletions essential for ganglioside binding elicited lower responses than HCWT. HCM115, containing two amino acid substitutions within the ganglioside binding site, provided reduced protection against tetanus toxin challenge compared with HCWT, consistent with lower anti-HC and anti-toxoid antibody titers. Circular-dichroism spectroscopy and intrinsic fluorescence spectroscopy showed minimal structural perturbation in HCM115. We conclude that the presence of the ganglioside binding site within HC may be essential for induction of a fully protective anti-tetanus response comparable to that induced by tetanus toxoid by subcutaneous injection.

Novel configurations of high molecular weight species of the pertussis toxin vaccine component.

Pertussis toxin (PT) is used in its formaldehyde-detoxified form in acellular pertussis vaccines for preventing whooping cough in children. The effects of formaldehyde treatment (up to 0.5% (w/v) formaldehyde) on the size, molecular association, folding and monoclonal antibody (mAb) binding of PT were studied to further define the structural nature of the high molecular weight species as related to their epitope integrity. Analytical ultracentrifugation (AUC) demonstrated that formaldehyde treatment of PT prevented the dissociation of the holotoxin. Together with results from size exclusion chromatography (SEC), SEC/multi-angle laser light scattering (MALLS) and immunoblotting it was demonstrated that PT increased in molecular weight and heterogeneity as a function of formaldehyde concentration, caused at least in part by covalent cross-linking. Five mAbs specific for PT subunits (S1-S5) bound to the cross-linked species, although there was some loss of epitopes in the larger aggregates. Intrinsic fluorescence spectroscopy gave evidence of progressive unfolding and re-association of PT. These findings demonstrate that a favourable balance between protein stabilisation and denaturation may be achieved by the treatment of pertussis toxin with formaldehyde, and provides a basis for determining the significance of high molecular weight cross-linked species of pertussis toxin in protection against whooping cough.