title: Control of antigen-binding to aluminum adjuvants and the immune response with a novel phosphonate linker.
abstract: The strongest mechanism for adsorption of antigens to aluminum adjuvants is ligand exchange, which involves the replacement of a surface hydroxyl on the adjuvant by a terminal phosphate group of the antigen. A novel phosphonate linker was developed that allows the addition of phosphonate (C-PO3) groups to proteins under controlled and chemically mild conditions. Increasing the number of linkers per protein molecule progressively increased the adsorption strength to aluminum hydroxide adjuvant (AH) as measured by elution in serum. The effect of phosphonate conjugation on the antibody response was determined with hen egg lysozyme (HEL), a protein that has the same charge as AH at neutral pH and does not adsorb to AH. The phosphonylated form of HEL (HEL-P) adsorbed to AH, indicating that the ligand exchange interaction could overcome the electrostatic repulsion. Mice injected with HEL-P/AH had a higher antibody titer to HEL than mice injected with HEL/AH, especially at lower antigen doses, suggesting that adsorption of antigen has a dose-sparing effect. Conjugation of CRM197, an antigen that adsorbs electrostatically to AH, with phosphonate linkers did not enhance the antibody response, indicating that adsorption by either electrostatic or ligand exchange to AH is sufficient to enhance the antibody response.
authors.affiliation: Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, IN 47907, USA.
authors.forename: Fangjia
authors.initials: F
authors.lastname: Lu
authors.forename: Irene
authors.initials: I
authors.lastname: Boutselis
authors.forename: Richard F
authors.initials: RF
authors.lastname: Borch
authors.forename: Harm
authors.initials: H
authors.lastname: Hogenesch
mesh_terms: D000276:Adjuvants, Immunologic / Q000138:chemical synthesis / Q000737:chemistry*; D000327:Adsorption; D000536:Aluminum Hydroxide / Q000737:chemistry* / Q000276:immunology; D000818:Animals; D000906:Antibodies / Q000097:blood; D000917:Antibody Formation / Q000276:immunology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000276:immunology; D005260:Female; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D009113:Muramidase / Q000737:chemistry / Q000276:immunology*; D063065:Organophosphonates / Q000737:chemistry*; D011485:Protein Binding / Q000276:immunology; D051381:Rats
chemical_list: D000276:Adjuvants, Immunologic; D000906:Antibodies; D001426:Bacterial Proteins; D063065:Organophosphonates; C028581:CRM197 (non-toxic variant of diphtheria toxin); D000536:Aluminum Hydroxide; C421864:hen egg lysozyme; D009113:Muramidase
keywords: 3-(N-morpholino)propane-sulfonic acid; AH; AP; Adsorption; Aluminum adjuvant; DT; HEL; Ligand exchange; MOPS; N-hydroxysuccinimide; NHS; PBS-T; Phosphate; TT; aluminum hydroxide adjuvant; aluminum phosphate adjuvant; diphtheria toxoid; hen egg lysozyme; isoelectric point; pI; phosphate buffered saline with 0.05% Tween 20; rPA; recombinant protective antigen; tetanus toxoid