title: 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.
abstract: 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.
authors.forename: Alberto
authors.initials: A
authors.lastname: Nilo
authors.forename: Irene
authors.initials: I
authors.lastname: Passalacqua
authors.forename: Monica
authors.initials: M
authors.lastname: Fabbrini
authors.affiliation: ‡Novartis Institutes for BioMedical Research, 100 Technology Square, Cambridge, Massachusetts 02139, United States.
authors.forename: Martin
authors.initials: M
authors.lastname: Allan
authors.affiliation: ‡Novartis Institutes for BioMedical Research, 100 Technology Square, Cambridge, Massachusetts 02139, United States.
authors.forename: Aimee
authors.initials: A
authors.lastname: Usera
authors.forename: Filippo
authors.initials: F
authors.lastname: Carboni
authors.forename: Barbara
authors.initials: B
authors.lastname: Brogioni
authors.forename: Alfredo
authors.initials: A
authors.lastname: Pezzicoli
authors.affiliation: ‡Novartis Institutes for BioMedical Research, 100 Technology Square, Cambridge, Massachusetts 02139, United States.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Cobb
authors.forename: Maria Rosaria
authors.initials: MR
authors.lastname: Romano
authors.forename: Immaculada
authors.initials: I
authors.lastname: Margarit
authors.affiliation: ‡Novartis Institutes for BioMedical Research, 100 Technology Square, Cambridge, Massachusetts 02139, United States.
authors.forename: Qi-Ying
authors.initials: QY
authors.lastname: Hu
authors.forename: Francesco
authors.initials: F
authors.lastname: Berti
authors.forename: Roberto
authors.initials: R
authors.lastname: Adamo
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000276:immunology*; D001428:Bacterial Vaccines / Q000276:immunology / Q000494:pharmacology*; D002240:Carbohydrate Sequence; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006001:Glycoconjugates / Q000276:immunology / Q000494:pharmacology*; D007114:Immunization; D051379:Mice; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000737:chemistry / Q000276:immunology*; D013290:Streptococcal Infections / Q000276:immunology / Q000517:prevention & control*; D013291:Streptococcus / Q000276:immunology*; D014443:Tyrosine / Q000737:chemistry / Q000276:immunology; D018074:Vaccines, Conjugate / Q000276:immunology / Q000494:pharmacology*
chemical_list: D001426:Bacterial Proteins; D001428:Bacterial Vaccines; D006001:Glycoconjugates; D011134:Polysaccharides; D018074:Vaccines, Conjugate; D014443:Tyrosine