Exploring the Effect of Conjugation Site and Chemistry on the Immunogenicity of an anti-Group B Streptococcus Glycoconjugate Vaccine Based on GBS67 Pilus Protein and Type V Polysaccharide

• Published in: Bioconjugate chemistry Vol. 26; no. 8; pp. 1839 - 1849
• Main Authors: Nilo, Alberto, Passalacqua, Irene, Fabbrini, Monica, Allan, Martin, Usera, Aimee, Carboni, Filippo, Brogioni, Barbara, Pezzicoli, Alfredo, Cobb, Jennifer, Romano, Maria Rosaria, Margarit, Immaculada, Hu, Qi-Ying, Berti, Francesco, Adamo, Roberto
• Format: Journal Article
• Language: English
• Published: WASHINGTON American Chemical Society 19.08.2015; Amer Chemical Soc
• Subjects: Amino Acid Sequence; Animals; Bacterial Proteins - chemistry; Bacterial Proteins - immunology; Bacterial Vaccines - immunology; Bacterial Vaccines - pharmacology; Binding sites; Biochemical Research Methods; Biochemistry; Biochemistry & Molecular Biology; Carbohydrate Sequence; Carbohydrates; Chemistry; Chemistry, Multidisciplinary; Chemistry, Organic; Enzyme-Linked Immunosorbent Assay; Female; Glycoconjugates - immunology; Glycoconjugates - pharmacology; Gram-positive bacteria; Immunization; Immunoglobulins; Life Sciences & Biomedicine; Mice; Molecular Sequence Data; Physical Sciences; Polysaccharides - chemistry; Polysaccharides - immunology; Proteins; Science & Technology; Streptococcal Infections - immunology; Streptococcal Infections - prevention & control; Streptococcus; Streptococcus - immunology; Tyrosine - chemistry; Tyrosine - immunology; Vaccines, Conjugate - immunology; Vaccines, Conjugate - pharmacology; ANTIBODIES; IMMUNIZATION; PREGNANT-WOMEN; IDENTIFICATION; CARRIER
• ISSN: 1043-1802; 1520-4812
• DOI: 10.1021/acs.bioconjchem.5b00365

We have recently described a method for tyrosine-ligation of complex glycans that was proven efficient for the site selective coupling of GBS capsular polysaccharides (PSs). Herein, we explored the effect of conjugation of type V polysaccharide onto predetermined lysine or tyrosine residues of the GBS67 pilus protein with the dual role of T-cell carrier for the PS and antigen. For the preparation of a conjugate at predetermined lysine residues of the protein, we investigated a two-step procedure based on microbial Transglutaminase (mTGase) catalyzed insertion of a tag bearing an azide for following copper-free strain-promoted azide–alkyne [3 + 2] cycloaddition (SPAAC) with the polysaccharide. Two glycoconjugates were obtained by tyrosine-ligation through the known SPAAC and a novel thiol-maleimide addition based approach. Controls were prepared by random conjugation of PSV to GBS67 and CRM197, a carrier protein present in many commercial vaccines. Immunological evaluation in mice showed that all the site-directed constructs were able to induce good levels of anti-polysaccharide and anti-protein antibodies inducing osponophagocytic killing of strains expressing individually PSV or GBS67. GBS67 randomly conjugated to PSV showed carrier properties similar to CRM197. Among the tested site-directed conjugates, tyrosine-directed ligation and thiol-malemide addition was elected as the best combination to ensure production of anti-polysaccharide and anti-protein functional antibodies (in vitro opsonophagocytic killing titers) comparable to the controls made by random conjugation, while avoiding anti-linker antibodies. Our findings demonstrate that (i) mTGase based conjugation at lysine residues is an alternative approach for the synthesis of large capsular polysaccharide–protein conjugates; (ii) GBS67 can be used with the dual role of antigen and carrier for PSV; and (iii) thiol-maleimide addition in combination with tyrosine-ligation ensures the production of anti-polysaccharide and anti-protein functional antibodies while maintaining low levels of anti-linker antibodies. Site-specific conjugation methods aid in defining conjugation site and chemistry in carbohydrate–protein conjugates.