Ponente
Descripción
Finlay Vaccines Institute has a project for developing pneumococcal conjugates vaccines. In these, serotype-specific capsular polysaccharides are covalently linked to an immunogenic protein. So far, conjugation procedures for eleven serotypes have been developed. The 22F is epidemiologically relevant, been associated in some studies with serotype replacement. Now, a procedure for its capsular polysaccharide is under investigation. This is composed of repetitive units of a pentasaccharide decorated with a lateral glucose and an O-acetyl group potentially relevant for antigenicity. In this work we study reaction conditions of each step paying special attention to antigen conservation to obtain immunogenic conjugates. For this, a general methodology based in the fragmentation of the polysaccharide, periodate oxidation and conjugation to diphtheria toxoid via reductive-amination is used. Fragments evaluation by SEC-HPLC showed that KD work rank of 0.45-0.60 was reached 2-3 h of hydrolysis with TFA. For activation, it was determined that a periodate concentration of 2 mM best fit oxidation level work rank. Unspecific fragmentation occurred but this was minimized working with the tuning of temperature and buffer. Hetrin assay and 1H-NMR corroborated O-acetyl conservation after both reactions. Thus antigenicity was conserved all along the work ranks of KD and oxidation levels. It was determined that increasing carbohydrate concentrations and polysaccharide-protein ratio favored conjugation. Fragments with KD of 0.47 and 0.61 were effectively conjugated to diphtheria toxoid. These resulted immunogenic in BALB/c mice model. In conclusion, it was reached reaction conditions to establish a conjugation procedure that assure obtaining immunogenic conjugates.
