Synthesis And Immunogenicity Of PGL-tb1 Monovalent Glycoconjugate

TB is caused by the infection of mycobacterium tuberculosis (MTB),in which Beijing genotype strains have a strong ability to spread and drug resistance, including HN878, W4 and W451 strains. Phenolic glycolipid (PGL) is usually present in the outer layer of its cell wall, which plays a very important role in the process of Mycobacterium tuberculosis infection in the West of Beijing. In this paper, PGL-tbl oligosaccharide was synthesized in pefect yield, and then conjugated with CRM 197 to obtain PGL-tbl oligosaccharide-protein conjugate as candidate vaccine. Finally, PGL-tbl oligosaccharide-protein conjugate was taken preliminary testing of immunogenicity.Firstly, with L-rhamnose as the starting material, the block of p-iodophenyl 2-O-methyl-4-O-benzyl-a-L-rhamnopyranoside was obtained in yield of 35% by the 9-step reaction of protecting and deprotection, second, with L-rhamnose as the starting material,preparation of p-tolyl 2-O-benzoyl-3-O-allyl-4-O-benzyl-1-thio-a-L-rhamnopyranoside block with the yield of 38% needed the 8-step reaction. With L-fucose as the starting material, 7-step reaction in yield of 57% to give p-tolyl 2-O-methyl-3,4-di-O-acetyl-l-thio-L- fucopyranoside block. Then, the [1+1+1] glycosylation strategy with selecting the suitable protecting group and reaction condition was used to synthesize the PGL-tbl trisaccharide with 36% yield and the glycosidic linkage was a (1?3) which was designed for stereoselective glycosylation. We intrudced the linker to the reducing end of the PGI.-tbl trisaccharide by Sonogashira coupling reaction, then, with the reaction ofhydrogenation reduction, It will be noted that the alkyne function could be fully reduced to the corresponding aliphatic fragment together with the removal of the benzyl protecting groups and azido group reduction by only one step. Application of squaric acid chemistry for conjugation, we got PG-CRM197 conjugate effectively.The result of SDS-PAGE shows that the conjugation was succsessful. Carbohydrate loadings of the conjugates were determined by MALDI-TOF mass spectrometry which the binding ratio of the PGL-tbl trisaccharide to CRM197 was 3.0: 1 and the binding efficiency was 18%, similarly, PG-BSA was 13.2: 1 and 73%. Then the immune evaluation of PG-CRM197 candidate vaccine, after administered systemically in mice with different doses of PG-CRM197 candidate vaccine and without any adjuvant, the conjugate induced high antigen-specific IgG levels in serum. Mice given the PG-CRM197 vaccine with medium dose without adjuvant had high IgG titers against the synthetic PG hapten after 2 injections(median ?197,000),while the third injection resulted in a further increase of titer by more than a factor of 2 (median-430,000). In addition,following the third immunization,significant antibody titers frequently exceeding 0.8 million were observed in the sera of mice vaccinated with PG—CRM197 conjugate which showed the potential for preparation of TB vaccine.