RESUMEN
The secreted proteins intestinal trefoil factor (ITF, 59 residues), pS2 (60 residues), and spasmolytic polypeptide (SP, 106 residues) form a small family of trefoil domain-containing mammalian cell motility factors, which are essential for the maintenance of all mucous-coated epithelial surfaces. We have used 1H NMR spectroscopy to determine the high-resolution structure of human ITF, which has allowed detailed structural comparisons with the other trefoil cell motility factors. The conformation of residues 10-53 of hITF is determined to high precision, but the structure of the N- and C-terrminal residues is poorly defined by the NMR data, which is probably indicative of significant mobility. The core of the trefoil domain in hITF consists of a two-stranded antiparallel beta-sheet (Cys 36 to Asp 39 and Trp 47 to Lys 50), which is capped by an irregular loop and forms a central hairpin (loop 3). The beta-sheet is preceded by a short alpha-helix (Lys 29 to Arg 34), with the majority of the remainder of the domain contained in two loops formed from His 25 to Pro 28 (loop 2) and Ala 12 to Arg 18 (loop 1), which lie on either side of the central hairpin. The region formed by the surface of loop 2, the cleft between loop 2 and loop 3, and the adjacent face of loop 3 has previously been proposed to form the functional site of trefoil domains. Detailed comparisons of the backbone conformations and surface features of the family of trefoil cell motility factors (porcine SP, pS2, and hITF) have identified significant structural and electrostatic differences in the loop 2/loop 3 regions, which suggest that each trefoil protein has a specific target or group of target molecules.
Asunto(s)
Sustancias de Crecimiento/química , Mucinas , Proteínas Musculares , Neuropéptidos , Péptidos/química , Secuencia de Aminoácidos , Humanos , Intestinos/química , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Estructura Terciaria de Proteína , Alineación de Secuencia , Factor Trefoil-2 , Factor Trefoil-3RESUMEN
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.
Asunto(s)
Vacunas contra Haemophilus/química , Vacunas contra Haemophilus/inmunología , Polisacáridos Bacterianos/química , Polisacáridos Bacterianos/inmunología , Animales , Anticuerpos Antibacterianos/biosíntesis , Cápsulas Bacterianas , Proteínas Bacterianas/química , Proteínas Bacterianas/inmunología , Actividad Bactericida de la Sangre/inmunología , Fenómenos Químicos , Química Física , Dicroismo Circular , Almacenaje de Medicamentos , Femenino , Inmunoglobulina G/biosíntesis , Ratones , Ratones Endogámicos CBA , Peso Molecular , Oligosacáridos/química , Oligosacáridos/inmunología , Polisacáridos/química , Polisacáridos/inmunología , Conformación Proteica , Conejos , Espectrometría de Fluorescencia , Temperatura , Vacunas Conjugadas/química , Vacunas Conjugadas/inmunologíaRESUMEN
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 degrees 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 CRM(197) when incubated at 23 degrees 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 degrees 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 degrees 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.
Asunto(s)
Vacuna contra Difteria, Tétanos y Tos Ferina/química , Vacunas contra Haemophilus/química , Vacunas Meningococicas/química , Oligosacáridos/química , Vacunas Conjugadas/química , Dicroismo Circular , Estabilidad de Medicamentos , Neisseria meningitidis/inmunología , Resonancia Magnética Nuclear Biomolecular , Polisacáridos Bacterianos/química , Estructura Secundaria de Proteína , SolucionesRESUMEN
We describe the use of Nuclear Magnetic Resonance (NMR) spectroscopy to control the identity of purified bulk capsular polysaccharide [called poly(ribosylribitolphosphate) or PRP] from Haemophilus influenzae type b (Hib), and derivatised forms, used in the production of Hib polysaccharide-protein conjugate vaccines. We describe the approaches we have developed to validate this test.
Asunto(s)
Vacunas contra Haemophilus/normas , Haemophilus influenzae/inmunología , Polisacáridos Bacterianos/inmunología , Polisacáridos Bacterianos/aislamiento & purificación , Vacunas Conjugadas/normas , Vacunas contra Haemophilus/química , Haemophilus influenzae/clasificación , Resonancia Magnética Nuclear Biomolecular/métodos , Control de Calidad , Sensibilidad y Especificidad , Vacunas Conjugadas/químicaRESUMEN
Full proton, 13C and 31P NMR assignments for the capsular polysaccharide from Streptococcus pneumoniae Type 17F are reported, and a revised structure differing in the anomeric configuration of the sidechain beta-Galp residue proposed. This polysaccharide is a component of the current 23-valent polysaccharide vaccine. The implications of this revised structure for published work are discussed.
Asunto(s)
Polisacáridos/química , Streptococcus pneumoniae/química , Secuencia de Carbohidratos , Ensayo de Inmunoadsorción Enzimática , Espectroscopía de Resonancia Magnética , Metilación , Datos de Secuencia Molecular , Fosfatos/químicaAsunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/inmunología , Proteínas Portadoras/química , Proteínas Portadoras/inmunología , Glicoconjugados/química , Glicoconjugados/inmunología , Polisacáridos Bacterianos/química , Polisacáridos Bacterianos/inmunología , Vacunas Conjugadas/química , Vacunas Conjugadas/inmunología , Animales , Fenómenos Químicos , Química Física , Humanos , Hidrólisis , Espectroscopía de Resonancia MagnéticaRESUMEN
Two meningococcal C-CRM197 conjugates differing in oligosaccharide chain length, number of conjugation sites, conjugation chemistry and process were monitored for stability at various temperatures or after repeated freeze-thawing by physico-chemical assays. The results were compared with assessment of immunogenicity in mice, previously shown to correlate with performance of the vaccine in clinical trials. The structural stability of the oligosaccharide chains and the protein carrier varied between the two types of conjugates. Neither was adversely affected by repeated freeze-thawing but one developed conformational changes in the protein carrier, detected by optical (CD, fluorescence) and NMR spectroscopy, when incubated at 23 degrees C or above, although integrity of the oligosaccharide structure was maintained. This was not associated with any reduction in primary IgM or IgG antibody responses to meningococcal C polysaccharide. Exposure to more extreme conditions resulting in release of a substantial proportion of free saccharide from the other conjugate sample was accompanied by significant reduction in both IgG and IgM antibody responses. In conclusion, FPLC-SEC, HPAEC-PAD and NMR spectroscopy were found useful for monitoring the stability of meningococcal C-CRM197 conjugates. Although optical spectroscopy was a sensitive method for detecting modification of the protein carrier, the results did not correlate with reduced immunogenicity.
Asunto(s)
Vacunas Meningococicas/química , Vacunas Meningococicas/inmunología , Animales , Fenómenos Químicos , Química Física , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Estabilidad de Medicamentos , Almacenaje de Medicamentos , Ensayo de Inmunoadsorción Enzimática , Femenino , Espectroscopía de Resonancia Magnética , Ratones , Ratones Endogámicos BALB C/inmunología , Espectrometría de Fluorescencia , Espectrofotometría UltravioletaAsunto(s)
Antígenos Bacterianos/química , Polisacáridos Bacterianos/química , Salmonella typhi/química , Vacunas Tifoides-Paratifoides/química , Fenómenos Químicos , Química Física , Cromatografía en Gel , Dicroismo Circular , Electroforesis en Gel de Poliacrilamida , Espectroscopía de Resonancia Magnética , Rotación Óptica , Termogravimetría , ViscosidadRESUMEN
Modern physicochemical methods allow biological pharmaceuticals, particularly those arising from recombinant DNA technology, to be characterised with a degree of precision not previously possible. These techniques, which tell us what a material is (rather than what it does) provide an approach complementary to traditional bioassays for the control of biological pharmaceuticals. As we come to understand the mechanisms by which structural variation modulates the various biological activities of a product, structure-based assays will be able to replace biological identity and potency assays, although replacement of safety tests to find trace impurities (such as endotoxin) may be more difficult.
Asunto(s)
Vacunas contra Haemophilus/química , Vacunas contra Haemophilus/normas , Vacunas Conjugadas/química , Vacunas Conjugadas/normas , Alternativas a las Pruebas en Animales/métodos , Bioensayo/métodos , Proteínas Portadoras/química , Proteínas Portadoras/inmunología , Dicroismo Circular , Seguridad de Productos para el Consumidor , Vacunas contra Haemophilus/farmacología , Polisacáridos Bacterianos/química , Polisacáridos Bacterianos/inmunología , Vacunas Conjugadas/farmacologíaRESUMEN
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.
Asunto(s)
Indicadores y Reactivos/normas , Albúmina Sérica , Endotoxinas , Ácidos Grasos/metabolismo , Factor Estimulante de Colonias de Granulocitos/normas , Humanos , Interleucina-15/normas , Proteínas Recombinantes de Fusión/química , Estándares de Referencia , Albúmina Sérica/química , Temperatura , Tirotropina/normasRESUMEN
Aqueous solutions of cyclodextrins and inorganic metaphosphate at pH 4, upon drying and subsequent warming, produce mixtures of isomeric monophosphate esters which are amenable to separation by anion-exchange chromatography. The products are characterised by enzymatic, mass, and NMR spectroscopic analysis. The methodology provides a route to these derivatives by a single reaction.
Asunto(s)
Ciclodextrinas/síntesis química , Fosfatos/síntesis química , alfa-Ciclodextrinas , beta-Ciclodextrinas , Secuencia de Carbohidratos , Cromatografía por Intercambio Iónico , Ciclodextrinas/química , Ciclodextrinas/aislamiento & purificación , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Fosfatos/química , Ácidos Fosforosos/químicaRESUMEN
We report essentially complete 1H NMR assignments for the capsular polysaccharides from Neisseria meningitidis serotypes A, C, W-135, and Y. These polysaccharides are components of current polysaccharide vaccines against meningococcal infection and of the polysaccharide-protein conjugate vaccines under development. From these NMR data the pattern of O-acetylation was determined. O-Acetylation of the W-135 polysaccharide is reported for the first time. We also show that, for the Types C and W-135 polysaccharides a migration of O-acetyl groups occurs during storage in solution, and demonstrate that high field 1H NMR represents a simple and sensitive method to define the O-acetylation pattern of individual batches of these polysaccharides.
Asunto(s)
Neisseria meningitidis/química , Polisacáridos Bacterianos/química , Acetilación , Cápsulas Bacterianas/química , Espectroscopía de Resonancia Magnética , Polisacáridos Bacterianos/clasificación , Polisacáridos Bacterianos/inmunología , Secuencias Repetitivas de Ácidos Nucleicos , Serotipificación , Vacunas/inmunologíaRESUMEN
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.