RESUMEN
In this report we present the results of a collaborative study for the preparation and calibration of a replacement International Standard (IS) for Haemophilus influenzae type b polysaccharide (polyribosyl ribitol phosphate; 5-d-ribitol-(1 â 1)-ß-d-ribose-3-phosphate; PRP). Two candidate preparations were evaluated. Thirteen laboratories from 9 different countries participated in the collaborative study to assess the suitability and determine the PRP content of two candidate standards. On the basis of the results from this study, Candidate 2 (NIBSC code 12/306) has been established as the 2nd WHO IS for PRP by the Expert Committee of Biological Standards of the World Health Organisation with a content of 4.904 ± 0.185mg/ampoule, as determined by the ribose assays carried out by 11 of the participating laboratories.
Asunto(s)
Haemophilus influenzae tipo b/química , Polisacáridos Bacterianos/normas , Polisacáridos/normas , Organización Mundial de la Salud , Cápsulas Bacterianas/química , Bioensayo/normas , Calibración , Cromatografía Líquida de Alta Presión , Estabilidad de Medicamentos , Vacunas contra Haemophilus/química , Vacunas contra Haemophilus/normas , Concentración de Iones de Hidrógeno , Cooperación Internacional , Laboratorios/normas , Fósforo/análisis , Polisacáridos/análisis , Polisacáridos Bacterianos/análisis , Estándares de Referencia , Reproducibilidad de los Resultados , Ribosa/análisisRESUMEN
The physico-chemical characteristics and immunogenicity of a candidate vaccine against otitis media, prepared from recombinant lipidated outer membrane proteins (rLP4 and rLP6) from non-typeable Haemophilus influenzae (NTHi) and of the ubiquitous cell surface protein UspA2 from Moraxella catarrhalis, were evaluated. Optical spectroscopy, size exclusion chromatography and gel electrophoresis were used to characterise the purified protein components and assess their purity and molecular sizes. The results showed that the three proteins were highly purified. Possible dimers in rLP4, dimers and multimers in rLP6 and UspA2 were detected. Small amounts of rLP4 and rLP6 dimers and most of UspA2 complexes remained tightly bound even after SDS treatment under reducing conditions. Immunogenicity studies showed that all proteins induced substantial antibody responses in mice immunised with AlPO4-adsorbed rLP4, rLP6 or UspA2 or a combination of these proteins. However, combination of these proteins resulted in a reduced response to rLP4 and rLP6, but not to UspA2, suggesting interference between these proteins which should be taken into consideration during the development and evaluation of this vaccine.