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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. CONCLUSION: 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.

SUBMITTER: Gustafsson E

PROVIDER: S-EPMC2662796 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

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Identification of conformational epitopes for human IgG on Chemotaxis inhibitory protein of Staphylococcus aureus.

Gustafsson Erika E Haas Pieter-Jan PJ Walse Björn B Hijnen Marcel M Furebring Christina C Ohlin Mats M van Strijp Jos A G JA van Kessel Kok P M KP

BMC immunology 20090311

<h4>Background</h4>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 ...[more]

PMID: 19284584

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Project description:BackgroundThe nature of epitopes on Bet v 1 recognized by natural IgG antibodies of birch pollen allergic patients and birch pollen-exposed but non-sensitized subjects has not been studied in detail.ObjectiveTo investigate IgE and IgG recognition of Bet v 1 and to study the effects of natural Bet v 1-specific IgG antibodies on IgE recognition of Bet v 1 and Bet v 1-induced basophil activation.MethodsSera from birch pollen allergic patients (BPA, n = 76), allergic patients without birch pollen allergy (NBPA, n = 40) and non-allergic individuals (NA, n = 48) were tested for IgE, IgG as well as IgG1 and IgG4 reactivity to folded recombinant Bet v 1, two unfolded recombinant Bet v 1 fragments comprising the N-terminal (F1) and C-terminal half of Bet v 1 (F2) and unfolded peptides spanning the corresponding sequences of Bet v 1 and the apple allergen Mal d 1 by ELISA or micro-array analysis. The ability of Bet v 1-specific serum antibodies from non-allergic subjects to inhibit allergic patients IgE or IgG binding to rBet v 1 or to unfolded Bet v 1-derivatives was assessed by competition ELISAs. Furthermore, the ability of serum antibodies from allergic and non-allergic subjects to modulate Bet v 1-induced basophil activation was investigated using rat basophilic leukaemia cells expressing the human FcεRI which had been loaded with IgE from BPA patients.ResultsIgE antibodies from BPA patients react almost exclusively with conformational epitopes whereas IgG, IgG1 and IgG4 antibodies from BPA, NBPA and NA subjects recognize mainly unfolded and sequential epitopes. IgG competition studies show that IgG specific for unfolded/sequential Bet v 1 epitopes is not inhibited by folded Bet v 1 and hence the latter seem to represent cryptic epitopes. IgG reactivity to Bet v 1 peptides did not correlate with IgG reactivity to the corresponding Mal d 1 peptides and therefore does not seem to be a result of primary sensitization to PR10 allergen-containing food. Natural Bet v 1-specific IgG antibodies inhibited IgE binding to Bet v 1 only poorly and could even enhance Bet v 1-specific basophil activation.ConclusionIgE and IgG antibodies from BPA patients and birch pollen-exposed non-sensitized subjects recognize different epitopes. These findings explain why natural allergen-specific IgG do not protect against allergic symptoms and suggest that allergen-specific IgE and IgG have different clonal origin.

Project description:Staphylococcus aureus is a well-recognized, clinically important cause of nosocomial infections, and as such, a vaccine to prevent S. aureus infections would be an important achievement. A Phase IIB/III study of V710, a vaccine containing iron-regulated surface determinant B (IsdB), demonstrated significant sero-conversion rates in cardiovascular surgery patients following a single pre-surgery immunization. However, the vaccine was not efficacious in preventing bacteremia or deep sternal wound infection post-surgery, thus raising the possibility that IsdB might not be available for immune recognition during infection. The purpose of the work described herein was to evaluate and quantify the naturally occurring anti-IsdB levels at baseline and over time during infection, to understand whether IsdB is expressed during a S. aureus infection in hospitalized non-vaccinated patients. We evaluated baseline and follow-up titers in 3 populations: (1) healthy subjects, (2) hospitalized patients with non-S. aureus infections, and (3) hospitalized patients with S. aureus infections. Baseline anti-IsdB levels generally overlapped between the 3 groups, but were highly variable within each group. In healthy subjects, baseline and follow-up levels were highly correlated (Spearman's rho = 0.93), and the geometric mean fold-rise (GMFR) in anti-IsdB levels between study entry and last value was 0.9-fold (95% confidence interval (CI): 0.8 to 1.0 ; p = 0.09), showing no trend over time. The convalescent GMFR in anti-IsdB levels from baseline was 1.7-fold (95% CI: 1.3 to 2.2, p = 0.0008) during S. aureus infection, significantly different from the 1.0-fold GMFR (95% CI: 0.9-1.2, p = 0.60) in non-S. aureus infection, p = 0.005. Additionally, S. aureus isolates (51) obtained from the hospitalized patient group expressed the IsdB protein in vitro. Collectively, these data suggest that IsdB expression levels rise substantially following infection with S. aureus, but not with other pathogens, and IsdB is likely well-conserved across S. aureus strains.

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Identification of conformational epitopes for human IgG on Chemotaxis inhibitory protein of Staphylococcus aureus.

Publications

Identification of conformational epitopes for human IgG on Chemotaxis inhibitory protein of Staphylococcus aureus.

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