Glycoconjugate vaccines are composed of a weak antigen, generally represented by a microbial poly- or oligosaccharide, covalently linked to a carrier protein to increase its immunogenicity1.The commercial glyconjugate vaccine against Haemophilus influenzae type b (Hib) has been the first example of the success of carbohydrate-based vaccination based on microbial polysaccharides2.The identification of minimal saccharide epitopes recognized by protective antibodies is crucial to instruct the rational design of next generation glycoconjugate vaccines3. H.influenzae is a Gram-negative bacterium which can be present in encapsulated and uncapsulated forms. The six serotypes of encapsulated strains are named a-f and among them, Hib represents a major cause of bacterial respiratory tract morbidity that can lead to severe diseases such as pneumonia, sepsis, and meningitis4.The surface of Hib is coated with a capsular polysaccharide composed of a linear copolymer of polyribosyl-ribitol-phosphate repeating units.To identify the minimal saccharidic length able to elicit a functional immune response, short Hib oligosaccharide fragments, composed from 2 to 5 repeating units (DP2 to DP5, degree of polymerization), obtained from acid hydrolysis of the native polysaccharide, have been used to map the area of interaction with a human protective anti-Hib monoclonal antibody (hmAb)5.This interaction has been investigated by using three principal techniques: SPR (Surface Plasmon Resonance) STD-NMR (Saturation Transfer Difference NMR) X-ray crystallography Competitive SPR showed that Hib DP2, DP3, DP4 and DP5 are well recognized by hmAb. STD-NMR experiments carried out on the DP2-hmAb complex highlighted the involvement of both ribose and ribitol in the binding with the hmAb. X-ray diffraction of a crystal of Hib DP2-Fab complex showed the interaction of the sugar exclusively with the Fab fragment Heavy chain, through a groove-type paratope. A crystal of Hib DP3-Fab has recently been obtained and X-ray diffraction studies are ongoing. These structural results will be correlated with in vivo studies in which the Hib oligosaccharide of different length conjugated to carrier proteins will be tested for their immunological properties.

Hib epitope mapping studies: towards modern glycoconjugate vaccine design

ACS Spring Meeting 2021

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