Investigators: Antje Mueller, Ph.D., Elena Csernok, Ph.D. and Prof. Peter Lamprecht, MD

Location: Department of Rheumatology, University Hospital of Schleswig-Holstein, Campus Luebeck, and Rheumaklinik Bad Bramstedt, Germany

Timeline: January 1, 2006 – December 31, 2006

Abstract
Autoantibodies against human neutrophilic cytoplasmic antigens (ANCA) have been a major diagnostic hallmark for small vessel vasculitides such as Wegener`s granulomatosis (WG) since their discovery more than 20 years ago. Animal models demonstrated that ANCA targeting either myeloperoxidase or proteinase 3 (PR3 = “Wegener`s autoantigen”) induce vasculitis. Binding of ANCA to an exposed epitope of the autoantigen PR3 is one of the determining steps in their mechanism of action. Therefore, knowledge of binding epitopes on the autoantigen is necessary to understand subsequent pathologic events and develop inhibitory strategies. The aim of this study is to identify and characterize immunodominant, conformational epitopes binding to ANCA directed against proteinase 3 and/or related molecules such as other neutrophilic enzymes and complementary PR3 (cPR3). A random peptide (12-mers) library expressed in E. coli will be employed to screen for antigen-antibody interactions. We hypothesize that by screening immunoglobulin G (IgG) preparations of patients with PR3-ANCA+ WG we will find and characterize novel antigenic peptide molecules with homologies to PR3 and/or other antigens, e.g. cross-reactive antigens. This approach differs from previous experiments, because the ANCA antibodies can bind conformational, i.e. non-linear epitopes. We shall isolate peptide clones that bind to PR3-ANCA-IgG pooled from WG patients (n=25) and synthesize and further characterize such peptides. The analysis allows to detect immunodominant, conformationally expressed antigenic peptide epitopes of PR3. Blocking such epitopes and thereby subsequent events might provide a future therapeutic approach of selective intervention in WG.

Specific aims
The aim of the study is to identify and characterize immunodominant, conformational epitopes binding to anti-neutrophil cytoplasmic antibodies in WG. Such an analysis will also disclose cross-reactive antigens and related molecules (cPR3), respectively. The study might generate new tools for the detection of disease-inducing epitopes and therapeutic intervention. The study will also provide new clues for the analysis of the impact of PR3-specific T cells (reacting with linear peptides) in the pathogenesis of WG.

1. Rationale of proposal and relevance to Wegener’s Granulomatosis.
a) Background

Several linear antigenic epitopes/areas of PR3 and, recently, complementary PR3 (cPR3) binding to antibodies have been characterized, mainly by looking at proliferative responses.

b) Own studies

As inferred by renowned immunologist R.M. Zinkernagel, many autoimmune diseases display lymphoid tissue-like structures in their inflamed target organs, where autoantigen recognition and presentation is thought to be maintained. We have shown that PR3+ cells, B cells, plasma cells, T cells and potential autoantigen-presenting cells are seen in granulomatous lesions of WG patients (11,12,13), thus permitting and sustaining an ongoing (auto)immune response.

Further, we know that monoclonal antibodies directed against PR3, like WGM2, generated by our group (14), recognize conformational epitopes of PR3. In contrast, T cells recognize linear peptide sequences as shown by incubation with four linear HLA-restricted PR3 peptides resulting in the detection of TNFα-producing PR3-specific T cells in WG (15). To detect conformational epitopes binding to PR3-ANCA+ IgG we searched for an approach that allows for antibody binding to non-linear epitopes (16). By now, we have established a random peptide library screening, which can identify binding to linear and/or non-linear antigenic epitopes. Preliminary findings yielded a potentially PR3-ANCA-binding epitope that shows some consensus sequence to PR3 (depicted in bold), “SVSTVHNHETVR” (one-letter-code of amino acids). When compared to the crystal structure of PR3 (17), the contiguous area seems to be accessible for antibody binding (Fig. 2). These findings warrant further analyses of a larger patient group.
The necessity and the potential of finding relevant ANCA-binding epitopes is also supported by a study showing that the interaction of PR3 and PR3-ANCA can be targeted in an interventional approach by inhibition of PR3-ANCA binding and destruction of ANCA-producing B cells by a fusion molecule of PR3 with a toxin (18).

c) Rationale and relevance

The pathogenetic role and relevance of a PR3-specific immune response in WG is still not fully elucidated. One major open question relates to (an) antigenic motif(s) on PR3 or other potential antigens, which via binding to ANCA induce(s) the well-described chain of inflammatory events leading eventually to subsequent endothelial destruction and full-blown small vessel vasculitis, i.e. WG (19). Once this question is answered, a more precise examination of a corresponding PR3-specific T cell response should be possible, as well as a monitoring of a potential epitope change. For systemic sclerosis, the use of a random peptide library not only led to the detection of an immunodominant epitope with homologies to a viral protein as well as to an autoantigen, but this recognition also initiated a potential pathologic mechanism, namely apoptosis of endothelial cells (20). Therefore, we hypothesize that such an approach will allow us to confirm known and identify still unknown immunodominant antigenic epitopes binding to ANCA IgG. Thus, the rationale of this proposed study is to obtain information about WG-relevant and less-WG-relevant peptide epitopes binding to ANCA, which could then be used for elucidating (auto)antigen presentation and antigenic T cell responses in WG and even for PR3-PR3-ANCA binding inhibition experiments in search for specific therapeutic agents.

2. Research methods and procedures including, if applicable, proposed statistical analysis.

IgG from PR3-ANCA+ sera of WG patients (n=25) will be purified by protein G column liquid chromatography. IgG preparations will be examined for PR3-ANCA titer (U/ml) by ELISA. Screening of potentially ANCA binding epitopes will be done employing a commercially available random peptide library, based upon a fusion protein consisting of a flagellar protein and thioredoxin, which is expressed at the surface of E. coli. In brief, the pooled IgG preparations will be coated onto a tissue culture plate at 20 µg/ml and then an incubation with the peptide-expressing E. coli will follow. After washing the bound bacteria are eluted, multiplied and incubated again with the IgG preparation to select for frequent epitopes. Following each so-called panning step the bound bacteria are seeded on agar plates as well to identify single clones. These clones are then grown in liquid culture and the plasmid DNA is isolated, purified and subjected to sequencing. Follwowing sequencing, data will be analysed using appropriate alignment and structure tools from NCBI (Blast etc., 21) to identify potential peptides, i.e. if mimics can be found. Then, a number of peptides will be synthesized and tested for binding to the patient’s IgG in ELISA, in comparison to PR3. Statistics will be analyzed using the SPSS software (Munich, Germany).

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