title: Identification of conformational epitopes for human IgG on Chemotaxis inhibitory protein of Staphylococcus aureus.
abstract: BACKGROUND
  The Chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS) blocks the Complement fragment C5a receptor (C5aR) and formylated peptide receptor (FPR) and is thereby a potent inhibitor of neutrophil chemotaxis and activation of inflammatory responses. The majority of the healthy human population has antibodies against CHIPS that have been shown to interfere with its function in vitro. The aim of this study was to define potential epitopes for human antibodies on the CHIPS surface. We also initiate the process to identify a mutated CHIPS molecule that is not efficiently recognized by preformed anti-CHIPS antibodies and retains anti-inflammatory activity.
  
  
  RESULTS
  In this paper, we panned peptide displaying phage libraries against a pool of CHIPS specific affinity-purified polyclonal human IgG. The selected peptides could be divided into two groups of sequences. The first group was the most dominant with 36 of the 48 sequenced clones represented. Binding to human affinity-purified IgG was verified by ELISA for a selection of peptide sequences in phage format. For further analysis, one peptide was chemically synthesized and antibodies affinity-purified on this peptide were found to bind the CHIPS molecule as studied by ELISA and Surface Plasmon Resonance. Furthermore, seven potential conformational epitopes responsible for antibody recognition were identified by mapping phage selected peptide sequences on the CHIPS surface as defined in the NMR structure of the recombinant CHIPS31-121 protein. Mapped epitopes were verified by in vitro mutational analysis of the CHIPS molecule. Single mutations introduced in the proposed antibody epitopes were shown to decrease antibody binding to CHIPS. The biological function in terms of C5aR signaling was studied by flow cytometry. A few mutations were shown to affect this biological function as well as the antibody binding.
  
  
  CONCLUSIONS
  Conformational epitopes recognized by human antibodies have been mapped on the CHIPS surface and amino acid residues involved in both antibody and C5aR interaction could be defined. This information has implications for the development of an effective anti-inflammatory agent based on a functional CHIPS molecule with low interaction with human IgG.
authors.affiliation: Alligator Bioscience AB, Scheelev. 19A, S-223 70 Lund, Sweden. erg@alligatorbioscience.com
authors.forename: Erika
authors.initials: E
authors.lastname: Gustafsson
authors.forename: Pieter-Jan
authors.initials: PJ
authors.lastname: Haas
authors.forename: Björn
authors.initials: B
authors.lastname: Walse
authors.forename: Marcel
authors.initials: M
authors.lastname: Hijnen
authors.forename: Christina
authors.initials: C
authors.lastname: Furebring
authors.forename: Mats
authors.initials: M
authors.lastname: Ohlin
authors.forename: Jos A G
authors.initials: JA
authors.lastname: van Strijp
authors.forename: Kok P M
authors.initials: KP
authors.lastname: van Kessel
mesh_terms: D000942:Antigens, Bacterial / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D001666:Binding Sites, Antibody / Q000235:genetics / Q000276:immunology; D002633:Chemotaxis; D018604:Epitope Mapping; D000939:Epitopes; D007074:Immunoglobulin G / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D008968:Molecular Conformation; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009504:Neutrophils / Q000378:metabolism / Q000473:pathology; D019151:Peptide Library; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology / Q000378:metabolism; D011485:Protein Binding; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement / Q000276:immunology / Q000378:metabolism; D044042:Receptors, Formyl Peptide / Q000276:immunology / Q000378:metabolism; D015398:Signal Transduction / Q000276:immunology; D013211:Staphylococcus aureus; D020349:Surface Plasmon Resonance
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C488033:C5AR1 protein, human; D000939:Epitopes; D007074:Immunoglobulin G; D019151:Peptide Library; D010455:Peptides; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement; D044042:Receptors, Formyl Peptide; C493134:chemotaxis inhibitory protein, Staphylococcus aureus