engleski

PEPTIDOGLYCAN MONOMER DERIVATIVE COUPLED TO POLYSACCHARIDE ANTIGENS; PREPARATION AND CHARACTERIZATION OF CONJUGATES

Polysaccharides in currently used vaccines are mostly week immunogens. To improve their immunogenicity conjugates of covalently coupled polysaccharides to protein carriers are being developed. Our approach is different: we are trying to prepare a conjugate by coupling the small immunomodulatory molecule - N-tert-butyloxycarbonyl-L-tyrosyl-peptidoglycan monomer (BocTyrPGM) (1) to polysaccharides of interest, assuming that its immunoadjuvant properties could improve the response to a polysaccharide part of the molecule. The aim of present work was to prepare the conjugates of polysaccharides and BocTyrPGM which could be further tested in various experimental models and their immunogenic properties assesed. We have used as polyssacharide antigens the water-soluble dextran FP 70, (Mw 70 kDa) and the major capsular polysaccharide from the fungus Cryptococcus neoformans var. gatii, glucuronoxylomannan (GXM), serotype B (Mw 700 kDa) (2). The native form of GXM is insoluble in water, so we reduced its size by ultrasonic irradiation, and separated water-soluble fragments of approx. 80 kDa on the Sepharose 4FF column. Both sugars were derivatized by coupling the adipic acid dihydrazide (ADH) to respective polysaccharides activated with cyanogen bromide. The AH-sugar derivatives were separated from the reaction mixture on the Sephadex G25 column and their sugar and ADH contents were analysed by the phenol-sulfuric and picryl-sulfonic acid method, respectively. The peptide bonds were subsequently formed between -NH2 groups of the AH-sugar derivatives and -COOH group of the BocTyrPGM in the presence of N-ethyl-N’-(3-dimethylaminopropyl)-carbodiimid-hydrochloride. The conjugates formed were separated from the reaction mixture by chromatography on the Sephadex G25 column and characterized with respect to their sugar and peptide content. References: 1. Vranešić, B. et al. (1991) A competitive radioimmunoassay for peptidoglycan monomer, Clin. Chim. Acta 202, 23-38 2. Bhattacharjee, A.K. et al. (1984) Capsular polysaccharides of Cryptococcus neoformans, Rev. Infect. Dis. 6, 619-624

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