Incellulosite-specific unnatural amino acid incorporation based on amber stop codon reassignment is a powerful tool to modify protein at defined positions. This technique is herein applied to the selective functionalization of the Pneumococcal surface adhesin A protein at three distinct positions. N-propargyloxycarbonyl-L-lysine residues were incorporated and their alkyne groups reacted using click-chemistry with a synthetic azido-functionalized tetrasaccharide representative of one repeat unit of the Streptococcus pneumoniaeserotype 14 capsular polysaccharide. Anti-PsaA antibody response induced in mice by the trivalent glycoconjugate was determined in comparison with corresponding monovalent and randomly functionalized conjugates. Our results suggest that controlled was superior to random conjugation for preserving antigenicity. In definitive, the reported strategy offers a unique opportunity to study the impact of carbohydrate antigen-carrier protein connectivity on immunogenicity.