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The elucidation of the molecular details underlying the immune properties of glycoconjugate vaccines has largely focused on the carbohydrate moiety, while very little is known on the effect of the corresponding conjugation sites. Herein we constructed a set of β-(1 → 3) glucan oligosaccharide conjugates with a well-defined glycan structure, connected to patterns of predetermined tyrosine or lysine residues onto the CRM197 carrier protein. To evaluate the effect of multivalent architecture in the glycan presentation, a novel linker enabling tyrosine-directed ligation of couples of oligosaccharides was prepared. The potential of these constructs as anti-Candida vaccines was evaluated in vivo, using as controls glycoconjugates prepared by a conventional random coupling strategy, and the structure–immune properties relationship was established. We found that: (i) the tyrosine-directed ligation resulted in higher anti-glycan IgG levels in comparison to the conjugation at predetermined lysine residues; (ii) the presentation of the carbohydrate antigen with a biantennary cluster of glycans onto specific tyrosine residues did not further increase the anti-glycan antibody level; (iii) the sera deriving from immunization with defined conjugates at tyrosine and, particularly at lysine residues, were proven stronger inhibitors of fungal adhesion to human epithelial cells in comparison to those from conjugates prepared by classic random chemistry; (iv) the presence of antibodies directed to the linkers did not affect the anti-glycan immune response. These findings suggest that a careful choice of the defined sites of conjugation and the loading density of antigens are important factors to raise high-quality anti-carbohydrate antibodies.