RESUMO
The basis of Haemophilus influenzae type b (Hib) and Neisseria meningitidis serogroup C (MenC) glycoconjugates binding to aluminum-containing adjuvants was studied. By measuring the amount of polysaccharide and protein in the non-adsorbed supernatant, the adjuvant, aluminum phosphate, AlPO4, was found to be less efficient than aluminum hydroxide, Al(OH)3 at binding to the conjugates, at concentrations relevant to licensed vaccine formulations and when equimolar. At neutral pH, binding of TT conjugates to AlPO4 was facilitated through the carrier protein, with only weak binding of AlPO4 to CRM197 being observed. There was slightly higher binding of either adjuvant to tetanus toxoid conjugates, than to CRM197 conjugates. This was verified in AlPO4 formulations containing DTwP-Hib, where the adsorption of TT-conjugated Hib was higher than CRM197-conjugated Hib. At neutral pH, the anionic Hib and MenC polysaccharides did not appreciably bind to AlPO4, but did bind to Al(OH)3, due to electrostatic interactions. Phosphate ions reduced the binding of the conjugates to the adjuvants. These patterns of adjuvant adsorption can form the basis for future formulation studies with individual and combination vaccines containing saccharide-protein conjugates.
Assuntos
Adjuvantes Imunológicos/metabolismo , Alumínio/análise , Vacinas Bacterianas/imunologia , Haemophilus influenzae tipo b/imunologia , Vacinas Meningocócicas/imunologia , Adjuvantes Imunológicos/química , Adsorção , Proteínas de Transporte/imunologiaRESUMO
Protein oligomerisation is a prerequisite for the toxicity of a number of bacterial toxins. Examples include the pore-forming cytotoxin streptolysin O, which oligomerises to form large pores in the membrane and the protective antigen of anthrax toxin, where a heptameric complex is essential for the delivery of lethal factor and edema factor to the cell cytosol. Binding of the clostridial neurotoxins to receptors on neuronal cells is well characterised, but little is known regarding the quaternary structure of these toxins and the role of oligomerisation in the intoxication process. We have investigated the oligomerisation of the receptor binding domain (H(C)) of tetanus toxin, which retains the binding and trafficking properties of the full-length toxin. Electrophoresis, size exclusion chromatography and mass spectrometry were used to demonstrate that H(C) undergoes concentration-dependent oligomerisation in solution. Reducing agents were found to affect H(C) oligomerisation and, using mutagenesis, Cys869 was shown to be essential for this process. Furthermore, the oligomeric state and quaternary structure of H(C) in solution was assessed using synchrotron small-angle X-ray scattering. Ab initio shape analysis and rigid body modelling coupled with mutagenesis data allowed the construction of an unequivocal model of dimeric H(C) in solution. We propose a possible mechanism for H(C) oligomerisation and discuss how this may relate to toxicity.
Assuntos
Estrutura Quaternária de Proteína , Toxina Tetânica/química , Toxina Tetânica/metabolismo , Sítios de Ligação , Cromatografia em Gel , Cisteína/metabolismo , Dimerização , Eletroforese em Gel de Poliacrilamida , Gangliosídeos , Modelos Moleculares , Mutagênese , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Raios XRESUMO
UNLABELLED: ⦠BACKGROUND: Patients are satisfied with their kidney care but want more support in making dialysis choices. Predialysis leaflets vary across services, with few being sufficient to enable patients' informed decision making. We describe the acceptability of a patient decision aid and feasibility of evaluating its effectiveness within usual predialysis practice. ⦠METHODS: Prospective non-randomized comparison design, Usual Care or Usual Care Plus Yorkshire Dialysis Decision Aid Booklet (+YoDDA), in 6 referral centers (Yorkshire-Humber, UK) for patients with sustained deterioration of kidney function. Consenting (C) patients completed questionnaires after predialysis consultation (T1), and 6 weeks later (T2). Measures assessed YoDDA's utility to support patients' decisions and integration within usual care. ⦠RESULTS: Usual Care (n = 105) and +YoDDA (n = 84) participant characteristics were similar: male (62%), white (94%), age (mean = 62.6; standard deviation [SD] 14.4), kidney disease severity (glomerular filtration rate [eGFR] mean = 14.7; SD 3.7); decisional conflict was < 25; choice-preference for home versus hospital dialysis approximately 50:50. Patients valued receiving YoDDA, reading it on their own (96%), and sharing it with family (72%). The +YoDDA participants had higher scores for understanding kidney disease, reasoning about options, feeling in control, sharing their decision with family. Study engagement varied by center (estimated range 14 - 49%; mean 45%); participants varied in completion of decision quality measures. ⦠CONCLUSIONS: Receiving YoDDA as part of predialysis education was valued and useful to patients with worsening kidney disease. Integrating YoDDA actively within predialysis programs will meet clinical guidelines and patient need to support dialysis decision making in the context of patients' lifestyle.
Assuntos
Técnicas de Apoio para a Decisão , Falência Renal Crônica/terapia , Aceitação pelo Paciente de Cuidados de Saúde , Educação de Pacientes como Assunto , Diálise Renal , Idoso , Comportamento de Escolha , Tomada de Decisões , Feminino , Humanos , Falência Renal Crônica/psicologia , Masculino , Pessoa de Meia-Idade , Folhetos , Estudos Prospectivos , Encaminhamento e Consulta , Inquéritos e QuestionáriosRESUMO
Physico-chemical analysis of pneumococcal polysaccharide (PS)-protein conjugate vaccine components used for two commercially licensed vaccines was performed to compare the serotype- and carrier protein-specific stabilities of these vaccines. Nineteen different monovalent pneumococcal conjugates from commercial vaccines utilizing CRM197, diphtheria toxoid (DT), Protein D (PD) or tetanus toxoid (TT) as carrier proteins were incubated at temperatures up to 56°C for up to eight weeks or were subjected to freeze-thawing (F/T). Structural stability was evaluated by monitoring their size, integrity and carrier protein conformation. The molecular size of the vaccine components was well maintained for Protein D, TT and DT conjugates at -20°C, 4°C and F/T, and for CRM197 conjugates at 4°C and F/T. It was observed that four of the eight serotypes of Protein D conjugates tended to form high molecular weight complexes at 37°C or above. The other conjugated carrier proteins also appeared to form oligomers or 'aggregates' at elevated temperatures, but rarely when frozen and thawed. There was evidence of degradation in some of the conjugates as evidenced by the formation of lower molecular weight materials which correlated with measured free saccharide. In conclusion, pneumococcal-Protein D/TT/DT and most CRM197 bulk conjugate vaccines were stable when stored at 2-8°C, the recommended temperature. In common between the conjugates produced by the two manufacturers, serotypes 1, 5, and 19F were relatively less stable and 6B was the most stable, with types 7F and 23F also showing good stability.
Assuntos
Proteínas de Transporte/química , Fenômenos Químicos , Vacinas Pneumocócicas/química , Estabilidade de Medicamentos , Humanos , Estabilidade Proteica , Temperatura , Vacinas Conjugadas/químicaRESUMO
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 oligosaccharide-CRM(197) (Cross-Reacting Material 197) and MenC-CRM(197), 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-CRM(197) eluted significantly earlier than MenC-CRM(197) 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-CRM(197) and MenC-CRM(197) in saline-based buffers, pH 7.2.
Assuntos
Vacinas Anti-Haemophilus/química , Vacinas Meningocócicas/química , Polissacarídeos Bacterianos/química , Vacinas Conjugadas/química , Anticorpos Antibacterianos/imunologia , Físico-Química , Criança , Cromatografia Líquida de Alta Pressão , Interações Medicamentosas , Vacinas Anti-Haemophilus/administração & dosagem , Vacinas Anti-Haemophilus/imunologia , Haemophilus influenzae tipo b/imunologia , Humanos , Lactente , Substâncias Macromoleculares , Vacinas Meningocócicas/administração & dosagem , Vacinas Meningocócicas/imunologia , Neisseria meningitidis/imunologia , Oligossacarídeos/química , Oligossacarídeos/imunologia , Polissacarídeos Bacterianos/imunologia , Refratometria , Vacinas Conjugadas/administração & dosagem , Vacinas Conjugadas/imunologiaRESUMO
Following the reduction in efficacy of Hib-TT vaccines in the primary immunization schedule observed in the UK between 1999 and 2003, batches of vaccine manufactured by two different companies were retrospectively examined by the National Institute for Biological Standards and Control. The study evaluated 41 batches of the Hib-TT vaccines manufactured between 1994 and 2003, assaying potency (total PRP saccharide content), integrity (% free saccharide), consistency (molecular sizing), and immunogenicity, as well as reviewing data previously obtained at the time of release. The study indicated the stability of the lyophilized final fill vaccines to extend well past their assigned shelf-lives, and found no trends in the endotoxin content, total saccharide or % free saccharide content. A trend towards slightly larger conjugates was observed over time in Hib-TT A, evidenced in both the manufacturer's data obtained at the time that samples were submitted for testing and in data obtained from the retrospective analysis. The study confirmed that that there had been no significant change in the quality of the Hib vaccines that could possibly account for the change reported in their protective efficacy in the UK. The study also demonstrated the value of independent testing of vaccines from the time of licensure and in the ongoing monitoring and re-examination of selected batches, as necessary, to assure their continuing quality, safety and consistency.
Assuntos
Vacinas Anti-Haemophilus/imunologia , Vacinas Anti-Haemophilus/normas , Polissacarídeos Bacterianos/imunologia , Cápsulas Bacterianas , Cromatografia em Gel , Vacinas Anti-Haemophilus/efeitos adversos , Inquéritos Epidemiológicos , Polissacarídeos Bacterianos/efeitos adversos , Estudos Retrospectivos , Toxoide Tetânico/efeitos adversos , Toxoide Tetânico/imunologia , Resultado do Tratamento , Reino UnidoRESUMO
Haemophilus influenzae b conjugate vaccines (Hib) are almost entirely evaluated by physico-chemical methods to ensure the consistency of manufacture of batches. As different assays are employed for the quantification of Hib capsular polysaccharide PRP (polyribosyl ribitol phosphate; 5-D-ribitol-(1-->1)-beta-D-ribose-3-phosphate) in final formulations and bulk components, there was deemed a need for an International Standard of Hib PRP polysaccharide to be made available. Ten laboratories from 8 different countries participated in a collaborative study to determine the PRP content and assess the suitability of a candidate International Standard PRP preparation (02/208). The results illustrate that a reduction in between-laboratory variability could be achieved by use of a common reference preparation and data analysis showed no significant differences in the values obtained by the different assays: ribose, phosphorus, and high performance anion exchange chromatography-pulsed amperometric detection (HPAEC-PAD), suggesting the suitability of the proposed reference for use across these assays for quantification of PRP content in Hib vaccines. On the basis of the results of this study, the First International Standard for PRP, NIBSC Code 02/208, has been established by the Expert Committee of Biological Standards of the World Health Organisation, with a content of 4.933+/-0.267mg/ampoule, as determined by the ribose assays carried out by 7 of the participating laboratories.
Assuntos
Vacinas Anti-Haemophilus/química , Vacinas Anti-Haemophilus/normas , Haemophilus influenzae tipo b/química , Polissacarídeos Bacterianos/química , Cápsulas Bacterianas , Carboidratos/análise , Comportamento Cooperativo , Estabilidade de Medicamentos , Cooperação Internacional , Padrões de Referência , Organização Mundial da SaúdeRESUMO
In this study, the immunogenicities of the nontoxic H(C) fragment of tetanus toxin and derivatives lacking ganglioside binding activity were compared with that of tetanus toxoid after subcutaneous immunization of mice. Wild-type H(C) (H(C)WT) protein and tetanus toxoid both elicited strong antibody responses against toxoid and H(C) antigens and provided complete protection against toxin challenge. Mutants of H(C) containing deletions essential for ganglioside binding elicited lower responses than H(C)WT. H(C)M115, containing two amino acid substitutions within the ganglioside binding site, provided reduced protection against tetanus toxin challenge compared with H(C)WT, consistent with lower anti-H(C) and anti-toxoid antibody titers. Circular-dichroism spectroscopy and intrinsic fluorescence spectroscopy showed minimal structural perturbation in H(C)M115. We conclude that the presence of the ganglioside binding site within H(C) may be essential for induction of a fully protective anti-tetanus response comparable to that induced by tetanus toxoid by subcutaneous injection.
Assuntos
Anticorpos Antibacterianos/sangue , Gangliosídeos/metabolismo , Fragmentos de Peptídeos/imunologia , Toxina Tetânica/imunologia , Toxoide Tetânico/imunologia , Animais , Feminino , Deleção de Genes , Imunização , Camundongos , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Tétano/prevenção & controle , Toxina Tetânica/administração & dosagem , Toxina Tetânica/genética , Toxina Tetânica/metabolismo , Toxoide Tetânico/administração & dosagem , Toxoide Tetânico/genética , Toxoide Tetânico/metabolismo , Toxinas BiológicasRESUMO
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.