Staphylococcus aureus

Description

The present invention relates to a method for diagnosis of diseases, which are related to allergic reactions to Staphylococcus aureus, as well as diagnostic preparations and therapeutic applications.

Asthma is one of the most common chronic diseases throughout the world. About 300 million people currently suffer from asthma. Two forms of asthma can be distinguished: The allergic (exogenous) asthma and the so-called non-allergic (endogenous) asthma, which is also known as idiopathic or intrinsic asthma. Both forms are diseases of the respiratory system. Allergic asthma is mediated by IgE. It leads to the release of mast cell products which results in bronchial constriction and impaired respiration. Allergic asthma is triggered and maintained by typical inhalation allergies such as pollens, animal and herbal dusts, animal proteins and chemicals. On the contrary, non-allergic asthma is not caused by known allergens. Infections of the respiratory system, intolerance to drugs, and exposure to poisons are discussed as causes for this asthma form. Nevertheless, in at least 10 % of the endogenous, non-allergic asthma cases the causative agent is still unknown. These are referred to as cases of idiopathic or intrinsic asthma. However, an immune reaction to Staphylococcus aureus (S. aureus) could possibly explain a subgroup of intrinsic asthma, as IgE antibodies against S.aureus enterotoxins have recently been described in a group of severe asthmatics (Bachert, C, et al., Specific IgE against Staphylococcus aureus enterotoxins: an independent risk factor for asthma. J Allergy Clin Immunol, 2012. 130(2): p. 376 - 81 e. 8).

Chronic rhinosinusitis (CRS) is a heterogeneous group of inflammatory diseases of the nasal cavities. There are two forms of CRS: First CRS with nasal polyposis (CRSwNP) and second CRS without polyps (CRSsNP). A clinical study conducted in 23 centers throughout Europe showed that about 11 % of the European population suffers from CRS. This study also revealed that CRS was strongly associated with asthma, especially in patients with CRSwNP (Bachert, C. and N. Zhang, Chronic rhinosinusitis and asthma: novel understanding of the role of !gE 'above atopy'. J Intern Med, 2012. 272(2): p. 133 - 43). Factors within CRSwNP tissue which predict asthma comorbidity have been identified as I L5, SE-lgE, total IgE and ECP (Bachert, C, et al., see above).

S. aureus is a micro-organism colonizing about 20 % of mankind persistently as a commensal. The preferred niches are the interior nares and the pharynx. Besides this, S. aureus causes a broad range of infections like pneumonia, osteomyelitis, skin and soft tissue infections and sepsis.

There is a set of 21 identified superantigens (SAgs) in S. aureus. Genetic analysis of S. aureus clinical isolates, including whole genome sequencing, has shown that around 80 % of all S. aureus clinical isolates harbor SAg genes, on average five to six (Holtfreter, S., et al., egc- Encoded superantigens from Staphylococcus aureus are neutralized by human sera much less efficiently than are classical staphylococcal enterotoxins or toxic shock syndrome toxin. Infect Immun, 2004. 72(7): p. 4061 - 71; Jarraud, S., et. al., egc, a highly prevalent operon of enterotoxin gene, forms a putative nursery of superantigens in Staphylococcus aureus. J Immunol, 2001. 166(1): p. 669 - 77). Now SAgs are being discussed to modulate the immune response in chronic rhinosinusitis and thus cause this disease as well as asthma (Bachert, C. and N. Zhang, see above).

Clinical evidence points to an important role of allergic reactions to S. aureus (IgE-mediated hypersensitivities of type 1) in chronic severe airway inflammation. Especially patients with chronic rhinosinusitis accompanied by nasal polyposis are more frequently colonized by S. aureus than subjects without polyps (Gevaert, P., et al., Organization of secondary lymphoid tissue and local IgE formation to Staphylococcus aureus enterotoxins in nasal polyp tissue. Allergy, 2005. 60(1): p. 71 - 9). Bachert et al. (see above) have shown that nasal polyposis together with colonization of the polyps by 5. aureus is strongly associated with endogenous idiopathic asthma. IgE-antibodies with specificity for S. aureus enterotoxins (SAEs), namely the staphylococcal enterotoxin A (SEA), the staphylococcal enterotoxin B (SEB), the staphylococcal enterotoxin C (SEC), and the toxic shock syndrome toxin 1 (TSST1) indicate an allergic reaction to 5. aureus in some patient groups. Patients with IgE-antibodies specific for certain superantigens, namely SAEs, had a higher risk to develop idiopathic asthma than patients without such antibodies (see for example Gevaert, P., et al., see above). Besides the endogenous asthma, there is also an allergic tendency in other diseases associated with S. aureus colonization and/or infection, for example, in atopic dermatitis (AD) and allergic rhinitis. 90 % of AD patients are colonized by 5. aureus (Reginald, K., et al., Immunoglobulin E antibody reactivity to bacterial antigens in atopic dermatitis patients. Clin Exp Allergy, 2011. 41(3): p. 357 - 69).

Another group of S. aureus proteins is the so-called serine protease-like proteins (Spl). This is a group of six proteins (SplA-F) encoded on the spl operon (Reed, S. B., et. al., Molecular characterization of a novel Staphylococcus aureus serine protease operon. Infect Immun, 2001. 69(3): p. 1521 - 7). The Spls are not well characterized and their substrates are not known. For SplB and SpIC casein has been suggested as substrate (Dubin, G., et al., Enzymatic activity of the Staphylococcus aureus SplB serine protease is induced by substrates containing the sequence Trp-Glu-Leu-Gln. J Mol Biol, 2008. 379(2): p. 343 - 56).

Non-allergic or idiopathic asthma, chronic rhinosinusitis and nasal polyps as well as aspirin hypersensitivity are common chronic inflammatory conditions of the respiratory system. They tend to take a severe disease course. The situation is similar in the case of AD, an allergic disease affecting the skin. Nevertheless, the causes of these conditions are not known. The diseases are notoriously difficult to treat because they do not respond well to standard allergy treatments. Moreover, cases that are treatment refractory are common. There is no causative treatment of these diseases. Thus, the object of the present invention is to provide an effective approach to treat diseases, which are related to allergic reactions to S. aureus, especially for a treatment of non-allergic or idiopathic asthma, aspirin

hypersensitivity, chronic rhinosinusitis and nasal polyps as well as AD.

Additionally, there is no sensitive and specific tool to diagnose allergic reactions to S. aureus. Currently allergic reactions to S. aureus are diagnosed by measuring IgE binding to merely four selected S. aureus SAEs: SEA, SEB, SEC und TSST1. However, only one out of three clinical S. aureus strains are endowed with these SAEs, and 20 % of 5. aureus clinical isolates from the nose of patients do not own any SEA gene at all, whereby not all cases of endogenous idiopathic asthma, nasal polyposis or AD can be explained and whereby it is unlikely that the above-mentioned SEAs are the triggers of hypersensitivity type I reactions, i.e. allergies to S. aureus (Holtfreter, S., et al ., Clonal distribution of superantigen genes in clinical Staphylococcus aureus isolates. J Clin Microbiol, 2007. 45(8): p. 2669 - 80). Thus a further object of the present invention is to provide a sensitive and specific approach to diagnose allergic reactions to S. aureus.

These objects are solved by methods for diagnosis and treatment of diseases, which are related to allergic reactions to S. aureus, diagnostic and pharmaceutical preparations and a method to prepare a medicament for treatment of such diseases as it is set forth in the independent claims. Preferred embodiments of the methods and preparations are described in the dependent claims.

The claimed subject matters are based on the findings of the inventors that S. aureus serine proteases, especially SplA (SEQ ID NO. 1), SplB (SEQ ID NO. 2), SpIC (SEQ ID NO. 3), SpID (SEQ ID NO. 4), SplE (SEQ ID NO. 5), and SplF (SEQ ID NO. 6) act as allergens in humans resulting in diverse allergic diseases, especially non-allergic or idiopathic asthma, chronic rhinosinusitis, nasal polyps, aspirin hypersensitivity and AD. There is strong evidence that these serine proteases act as triggers and pace-makers of such allergic diseases in susceptible individuals. The claimed diagnosis and treatment is based on the well-supported assumption that the serine proteases SplA-F are major allergens of S. aureus. In susceptible individuals exposure to these proteases triggers allergic reactions and maintains them. These allergic

inflammatory reactions are the cause of frequent chronic diseases, which often take a severe course and are often refractory to symptomatic treatment strategies: Non-allergic or idiopathic asthma, aspirin hypersensitivity, rhinosinusitis with or without nasal polyps, and AD. Up until now, the pathogenesis of these diseases is largely unknown. The present invention enables for the first time a sensitive and specific diagnosis and a causal therapy.

As a first aspect the present invention claims a method for diagnosis of diseases, which are related to allergic reactions to S. aureus. The inventive method is characterized by an analysis or monitoring of an interaction of at least one serine protease like protein of S. aureus or a derived protein or peptide with the patient's body, especially with the immune system of the patient, by analyzing a blood sample or a serum sample or a plasma sample of the patient.

Preferably serum antibody binding to the serine protease like proteins or derived proteins or peptides is measured in the course of the diagnostic method. It is especially preferred to analyze IgE- and/or lgG4-antibodies of the patient. Advantageously, the inventive method is based on an assay for the determination of IgE- and/or lgG4-antibodies with specificity for one or several 5. aureus serine proteases or serine protease like proteins. IgE molecules are causative agents of allergies of the type I, also known as atopic allergies. Generally, they occur when the organism has developed an allergy to a certain antigen, called allergen, and trigger characteristic allergic immune reactions, e.g. mast cell degranulation (this type of allergy is TH2 driven in human response). The more allergen-specific IgE is present, the stronger the allergic reactions are. Consequently, the measurement of IgE-antibodies with specificity for S. aureus serine protease like proteins is particularly suitable for diagnosis of allergic diseases related to S. aureus according to the inventive method. lgG4-antibodies are so-called neutralizing antibodies. They are expressed after long-term exposure to allergens and counteract the IgE-driven inflammation. Their neutralizing capacity is due to binding to the specific allergen and to their inability to activate pro-inflammatory effector functions in the immune system. IgE- and lgG4-production both depend on IL4, a cytokine elaborated by TH2 cells. lgG4-producing B cells may switch to IgE production in response to repeated allergen contact (Aalberse, R. C. and T.A. Platts-Mills, How do we avoid developing allergy: modifications of the TH2 response from a B-cell perspective. J Allergy Clin Immunol, 2004. 113(5): p. 983 - 6). Therefore, also the analysis of lgG4-antibodies with specificity for one or several S. aureus serine protease like proteins is particularly suitable for diagnosis of allergic diseases according to the inventive method.

The serine protease like protein which is the basis of the inventive approach, is at least one protein selected from the group consisting of SplA (SEQ ID NO. 1), SplB (SEQ ID NO. 2), SpIC (SEQ ID NO. 3), SpID (SEQ ID NO. 4), SplE (SEQ ID NO. 5), and SplF (SEQ ID NO. 6). By the term serine protease like protein these serine proteases are comprised. Moreover, derived proteins or peptides, which are essentially based on the amino acid sequences of these proteases are also comprised. For example, the derived proteins or peptides may comprise only a part of the amino acid sequence of the original protein and/or some of the amino acids may be exchanged by similar amino acids. The resulting amino acid sequences of the derived proteins or peptides lacking enzymatic activity may preserve at least 90 % of the original protein sequences. With respect to the derived proteins or peptides it may be especially advantageous if the derived protein or peptide preserves some or most or all of the allergenic epitopes of the original protein, especially in view of diagnostic applications, and/or if the derived protein or peptide lacks enzymatic activity, especially in view of therapeutic applications as described below.

In a preferred embodiment of the claimed diagnostic and therapeutic methods the serine protease like proteins or the derived proteins or peptides are recombinant proteins or peptides. Advantageously the proteins or peptides may be produced by known genetic engineering methods. By genetic engineering the proteins or peptides may be produced in large scale if necessary. Moreover, the proteins or peptides may be adapted and engineered according to the requirements of the inventive method or application. For example, it may be advantageous to alter or to delete the enzymatic activity of the recombinant protein or peptide and/or to alter or to weaken or to strengthen the allergenic properties of the recombinant protein or peptide.

Before use of the proteins or peptides for the inventive diagnostic or therapeutic applications it may be advantageous to purify the proteins or peptides in order to enhance the specificity of the method.

The inventive diagnostic method may be performed as ELISA-procedure (enzyme-linked immune-sorbent assay). Therein at least one serine protease like protein of S. aureus or the derived protein or peptide may be adsorbed to a solid phase and exposed to the patient's serum. Then antibody binding can be measured with standard methods. Alternatively, the allergens, namely the serine protease like proteins or derived proteins or peptides, may be coupled to fluorescent particles, which enable multiple measurements in a single serum sample. This approach may be performed in a multiplex assay, using, for example, the known Luminex* system. Moreover, the inventive diagnostic test could also be developed in the form of a protein array. Numerous different S. aureus serine protease like proteins or derived proteins or peptides may be coupled to a solid phase in an array format. Upon incu bation with the patient's serum, the antibody binding to all these proteins can be determined simultaneously in a multiplex test. Another possibility is to first immobilize the antibodies, especially IgE-antibodies, from the patient's serum on a solid phase using, for example, affinity material for IgE, for example the known material IgE-AviQuant ^® of the company PLS- Design. Then the S. aureus serine protease like proteins or derived proteins or peptide, respectively allergens, which are labeled by genetic engineering e.g. with a streptavidin tag, are added. The binding of the labeled allergens may be visualized by using streptavidin for detection. Further assays for the measurement of serum antibody binding to serine protease like proteins of S. aureus or derived proteins or peptides are possible.

Further the invention comprises the use of at least one serine protease like protein of S. aureus or a derived protein or peptide for the manufacture of a diagnostic preparation for diagnosis of diseases, which are related to allergic reactions to S. aureus. The diagnostic preparation may be designed for ELISA-like procedures or other assays for the measurement of serum antibody binding as mentioned above, for example including labeling of the serine protease like proteins or derived proteins or peptides. With respect to further features of the claimed use it is referred to the description above.

Moreover, the diagnostic preparation may be designed to be intra-dermally introduced by pricking the skin. Minute amounts of the proteins or peptides are introduced intra-dermally in the course of the inventive diagnostic method. Then the local skin reaction is monitored. Especially a swelling at the prick site appearing after 30 to 90 minutes will be diagnostic for a positive reaction, especially a S. aureus allergy. Especially in view of a skin prick application it may be advantageous to apply genetically modified proteins without enzymatic activity. There may be concerns that the enzymatic activity of the serine proteases might have adverse effects upon intradermal application. By the use of derived proteins or peptides without enzymatic activity or with altered enzymatic activity, which can easily be derived by recombinant gene technology, this problem may be solved.

For the skin prick approach it is especially preferred to use recombinant proteins or peptides derived from S. aureus serine proteins SplA-F, because by using genetic engineering there are no problems in producing large scale amounts of the proteins or peptides. Moreover, there are no problems with inhomogeneity of the proteins or peptides. Preferably, the

recombinant s, aureus serine proteases or derived proteins or peptides are generated and purified using GMP protocols (good manufacturing practice).

The inventive diagnostic method is much more sensitive and more specific in the diagnosis of diseases which are related to allergic reactions to S. aureus than already known methods. Especially the diagnosis of asthma, especially non-allergic or idiopathic asthma, and/or chronic rhinosinusitis, and/or nasal polyps, and/or aspirin hypersensitivity, and/or AD is strongly improved by the inventive diagnostic method.

Moreover, the invention comprises a diagnostic preparation for diagnosis of diseases, which are related to allergic reactions to S. aureus, wherein the diagnostic preparation comprises at least one serine protease like protein of 5. aureus or a derived protein or peptide, as described above. Preferably, the diagnostic preparation also comprises at least one pharmaceutically acceptable vehicle.

The further main aspect of the present invention is a therapeutic approach on the basis of the findings of the inventors. Thus, the invention comprises the use of at least one serine protease like protein of S. aureus or a derived protein or peptide for the manufacture of a medicament for treatment of diseases, which are related to allergic reactions to 5. aureus, by a systemic immune therapeutic approach. It is known in the medical field that the systemic immune therapy is merely the only causal intervention strategy to terminate allergic reactions. In this therapeutic approach the allergens, which trigger the disease symptoms, are regularly applied to the patient. In the beginning minute amounts of the allergens are thus applied. Gradually the applied amounts are increased. Frequently this is a long-term therapy lasting several years before resolution or significant amelioration of a symptom. Generally there are two methods for systemic immune therapy. Firstly, the substances are applied intra-dermally and/or subcutaneously and/or intra-muscularly. Secondly, the substances are administered sublingually. Both methods have been shown to be highly effective in different forms of allergy. Up to now there was no systemic immune therapy approach for S. aureus allergies. As long as superantigens are considered to be the main molecular triggers of S. aureus allergies, a systemic immune therapy was not possible because of the known toxic effects of the superantigens. In contrast the inventive approach uses at least one serine protease like protein of 5. aureus or derived protein or peptide as allergen to be applied in the course of the systemic immune therapy. There are no known specific toxic effects of these proteins or peptides. Moreover, the enzymatic activity of the serine protease like protein of S. aureus or derived protein or peptide to be used for therapy can be eliminated by genetic engineering. Consequently, a systemic immune therapy can be done by use of these proteins or peptides based on the findings of the inventors, that these proteins or peptides are major allergens of S. aureus. With respect to further details of this aspect of the invention it is also referred to the above description.

The medicament for the systemic immune therapy comprises besides the serine protease like protein or a derived protein or peptide preferably at least one pharmaceutically acceptable vehicle. Preferably, the medicament is prepared to be applied intra-dermally and/or sublingually to the patient. The systemic immune therapy may be designed to apply the serine protease like protein or derived proteins or peptides a number of times beginning with a minute amount gradually increasing. Generally the therapy is designed to be applied for a longer term, preferably at least several months up to years.

It is especially preferred to employ the S. aureus allergens, namely the serine protease like proteins or derived proteins or peptides as recombinant proteins. The recombinant proteins may be generated by known genetic engineering methods and expressed in harmless bacteria or by other known methods and purified for example from bacterial culture supernatants by known biochemical methods. It is preferred to adhere to GMP standards for the production of the recombinant serine protease like protein or derived protein or peptide. The recombinant production of the protein or peptide is especially appropriate, because S. aureus releases a number of potent toxins that could otherwise contaminate the protein preparations and have adverse effects. In addition, such contaminating S. aureus toxins could induce an inflammatory reaction, which would counteract the therapeutic effects of the systemic immune therapy.

It may be preferred to genetically modify the serine protease like proteins or derived proteins or peptides, for example to eliminate their protease activity while preserving most allergenic epitopes. This can be achieved by known recombinant gene technology.

Generally, the causal therapy of allergy by systemic immune therapy encompasses two components: Firstly, the allergens, which are the triggers and pace-makers of the allergic symptoms, and secondly the particular preparation and/or application method which minimize inflammation and thereby facilitate the development of antigen-specific immune tolerance, which extinguishes the allergic inflammation in response to the bacteria.

Therefore, it is preferred to avoid pro-inflammatory adjuvants to the therapeutic preparation in order to minimize inflammation during the course of the inventive systemic immune therapy. This is a fundamental difference to classical immunization schemes aiming a protection against infection wherein pro-inflammatory adjuvants are frequently added to the immunizing antigen.

Finally, the invention comprises a pharmaceutical preparation for treatment of diseases, which are related to allergic reactions to S. aureus, by a systemic immune therapy, wherein the preparation comprises at least one serine protease like protein of 5. aureus or a derived protein or peptide as described above. By the pharmaceutical preparation respectively medicament it is possible for the first time to treat the causes of non-allergic or idiopathic asthma, aspirin hypersensitivity, chronic rhinosinusitis, nasal polyps, and AD especially in those cases that are caused or exacerbated by an allergic reaction against S. aureus.

Further features and advantages of the invention read from the following description of the experimental part in conjunction with the figures, wherein each of the features may be realized individually or in combination with each other. In the figures it is shown:

Fig. 1 graphical analysis of S. aureus specific lgG4 in sera of healthy carriers and non-carriers of

5. aureus;

Fig. 2 graphical analysis of enzyme-linked immuno-sorbent assays (ELISA) for IgE and lgG4 binding to SpIC, SpID and TSST-1 in sera from carriers and non-carriers of S. aureus;

Fig. 3 graphical analysis of IgE binding to 5. aureus SpIC and HIa in healthy individuals, patients with nasal polyps and asthma patients;

Fig. 4 graphical analysis of PBMC (peripheral blood mononuclear cells) stimulated with heat- inactivated recombinant antigens SpIC, SpID and HIa (a-Toxin);

Fig. 5 graphical analysis of frequencies of SpIC- and SplD-specific T cells in carriers and non- carriers of 5. aureus;

Fig. 6 graphical analysis of release of cytokines of PBMC stimulated with heat-inactivated HIa or SpID; and

Fig. 7 graphical analysis of cytokines released by SplD-specific T cell clones, splitted into Fig. 7-

1 to 7-4:

Fig.7-1: IL-4 and IL-9, Fig. 7-2: IL-12p70 and IL-lp, Fig. 7-3: IL-17A and TF-ct and Fig. 7-4: IFN-y and IL-10.

SUBSTITUTE SHEET (RULE 26) Experimental part

a. Most adults have lgG4 in their serum that binds to proteins released by

S. aureus.

The humoral immune response of five healthy S. aureus carriers to extracellular S. aureus proteins was examined by two-dimensional (2D) immune proteomics.

For antigen preparation, bacteria were inoculated in tryptic soy broth (TSB) to an optical density at 540 nm of 0.05 and cultivated in 100 ml cultures of TSB at 37 °C and 180 rpm. 3.5 h after the bacterial culture had entered the stationary phase, cultures were harvested, the extra-cellular proteins were extracted and protein concentration was determined as previously described (Holtfreter, S., et al., Human immune proteome in experimental colonization with Staphylococcus aureus. Clin Vaccine Immunol, 2009. 16(11): p. 1607-14). Extra-cellular proteins were stored in aliquots at -80 °C. Serum and plasma samples were obtained from five S. aureus carriers; they were stored in aliquots at -80 °C.

Two-dimensional polyacrylamide gel electrophoresis (2-DE) with mini 2-DE gels (2D gels) and 2-DE immunoblots (2D-IBs) were performed as described (Holtfreter, S., et al., see above). In short, isoelectric focusing was performed with 7 cm Immobiline Dry Strips (GE Healthcare, Munich, Germany). The pH range of 6-11 was chosen for analysis because most extra-cellular proteins resolved in this range, while protein A was excluded so that unspecific antibody binding, which obscured a large part of the blot at a pH range of 4-7, was avoided. The separated staphylococcal proteins were blotted onto a PVDF membrane (Immobilon-P, Millipore, Billerica, USA) with 1.33 mA/cm ² for 2 h (graphite blotter MilliBlot; Millipore, Billerica, USA) and incubated with the corresponding human sera at 1:10,000 dilution.

Binding of lgG4 was detected by horseradish peroxidase (H P)-conjugated goat anti-human lgG4 (Invitrogen, Darmstadt, Germany) and visualized with an ECL substrate (SuperSignal West Femto Maximum Sensitivity Substrate, Pierce, Rockford, USA). Three independent experiments were performed for each carrier.

Whereas all five S. aureus carriers showed strong binding of total serum IgG to the bacterial antigens, lgG4 binding to the same bacterial antigens was highly variable. These findings were corroborated by examining the binding of serum antibodies (total IgG and lgG4) in a larger cohort of healthy adults, 16 S. aureus carriers and 16 non-carriers. For this a partially automated one-dimensional (ID) immunoblot-based assay system was employed (Simon™, ProteinSimple, Santa Clara, CA, USA). Briefly, acrylamide gel matrix, dilutions of bacterial antigen (1 μg/μl), plasma (1:50), secondary goat-anti-human lgG4-H P antibody (1:100; Invitrogen) and luminol substrate (ProteinSimple, Santa Clara, CA, USA) were prepared and loaded into SIMON. According to the predefined experimental conditions, SIMON performed lD-immunoblots in capillaries. Raw data were subsequently analyzed with Compass software (ProteinSimple, Santa Clara, CA, USA) and GraphPad Prism 5 (GraphPad, La Jolla, CA, USA).

Approximately 80% of healthy adults exhibit measurable serum antibodies of the lgG4 subclass binding to S. aureus antigens, 20% did not. The binding intensity varied strongly between the individuals as shown in Fig. 1. This figure illustrates the results of an automated 1-dimensional immunoblots (SIMON) with extracellular S. aureus proteins. The immunoblots were used to determine S. aureus-specific lgG4 in sera of healthy adult S. aureus carriers and non-carriers. The proteins were separated by their molecular weight, incubated with the corresponding serum. lgG4 binding was detected via an anti-human-lgG4-HRP antibody preparation and a luminol substrate. Besides a great variance in binding intensity, nearly 80 % of all adults showed S. aureus-specific serum lgG4.

b. The major lgG4-binding proteins were identified as the S. aureus serine

protease-like proteins SpIC and SplD.

Bacterial proteins bound by serum antibodies were identified by overlaying the 2D immunoblot pictures with the corresponding 2D-PAGE. For protein identification 100 μg of extra-cellular proteins were loaded onto 11 cm Immobiline Dry Strips (GE Healthcare, Munich, Germany) with the pH range 6-11. After second dimension protein separation gels were stained with Flamingo ^® Fluorescent Gel Stain (BioRad, Munich, Germany) according to the manufacturer's instructions, except for fixation, which was performed twice for 1 h. Gels were scanned using a Typhoon 9400 scanner (GE Healthcare, Munich, Germany) in the fluorescence acquisition mode (532 nm) at a resolution of 100 μιτι. Subsequently, peptides were prepared for mass spectrometry by MALDI-MS by trypsin digestion as previously described (Holtfreter, S., et al., see above). The MALDI-TOF measurement of spotted peptide solutions was carried out on a Proteome-Analyzer 4700/4800 (Applied Biosystems, Foster City, CA, USA) as reported. Database searches were performed using the GPS explorer software version 3.6 (build 3329) with an organism-specific database. The combined MS and MS/MS peak lists were searched against a S. aureus 8325 protein database obtained from the ENTREZ genome database site (ftp://ftp.ncbi.nih.gov/genomes/Bacteria/) and a database containing protein sequences derived from the genome sequences of all completely sequenced S. aureus strains and, moreover, all additional protein sequences of S. aureus (continuously updated from www.uniprot.org) using the Mascot search engine version 2.104 (Matrix Science, London, UK). The following search criteria were applied:

Carbamidomethylation (C) and oxidation (M) were set as variable modifications; a peptide mass tolerance of ± 50 ppm and a fragment mass tolerance of ± 0.55 Da were used; the peptide charge state of +1 was accepted for the precursor peptides; the maximum number of missed cleavages was set to 1. The Mowse score for a significant identification of a protein spot had to exceed a value of 50, which corresponds to a P-value of 0.05 (Kolata, J., et al., Distinctive patterns in the human antibody response to Staphylococcus aureus bacteremia in carriers and non-carriers. Proteomics, 2011. 11(19): p. 3914-27).

Whereas total serum IgG binding on 2-dimensional immunoblots was observed to a variety of bacterial proteins, S. aureus specific lgG4 binding was strongest at a molecular mass of 32 kDa and 34 kDa. In some sera lgG4 binding was exclusively observed in this region. The corresponding S. aureus proteins were identified by mass spectrometry to be SpIC and SplD. Hence SpIC and SplD were identified to be the dominant lgG4-binding proteins of S. aureus.

This was corroborated by the 1-dimensional immunoblot analysis of serum lgG4 binding to S. aureus proteins in 16 S. aureus carriers and 16 non-carriers. If lgG4 binding was taking place at all, it was always highly abundant in a region corresponding to a molecular mass of 32 kDa and 34 kDa, which is the respective size of the Spls. lgG4 binding to bacterial proteins was monitored in all sera and binding to bacterial proteins with a molecular mass of 32 to 34 kDa was observed in 13 of 16 carriers and 12 of 16 non-carriers. This supports the results of the analysis of the 2-dimensional immunoblots, namely that Spl proteins are the dominant lgG4- binding proteins of S. aureus. c. The species S. aureus harbors six serine protease like genes, splA-F, and around 80% of clinical bacterial isolates have at least one of them in their genome

Up to six S. aureus serine protease genes are encoded on the sp/-operon (Reed, S.B., et al., Molecular characterization of a novel Staphylococcus aureus serine protease operon. Infect Immun, 2001. 69(3): p. 1521-7; Dubin, G., et al., Enzymatic activity of the Staphylococcus aureus SplB serine protease is induced by substrates containing the sequence Trp-Glu-Leu- Gln. J Mol Biol, 2008. 379(2): p. 343-56). The gene loci encoding each of the S. aureus serine protease like proteins, namely SplA (SEQ ID NO. 1), SplB (SEQ ID NO. 2), SpIC (SEQ ID NO. 3), SpID (SEQ ID NO. 4), SplE (SEQ ID NO. 5), and SplF (SEQ ID NO. 6), are highly conserved between the sequenced S. aureus strains (at least 95 % homology) (Zdzalik, M., et al., Prevalence of genes encoding extracellular proteases in Staphylococcus aureus - important targets triggering immune response in vivo. FEMS Immunol Med Microbiol, 2012. 66(2): p. 220-9).

However, the sequences of Spls A-F, which are encoded by different gene loci, are rather variable (Table 1).

Tab. 1: Comparison of the serine protease amino acid sequences (Reed, S.B., et al., Molecular characterization of a novel Staphylococcus aureus serine protease operon. Infect Immun, 2001. 69(3): p. 1521-7).

Clinical S. aureus strains (n = 135) isolated from healthy individuals and asthma patients were then tested for the prevalence of the genes encoding Spls A-F using a multiplex-PCR system. The primers used are shown in (Table 2). Spl gene forward primer reverse primer

spla taa tga tat ttt taa aaa tag agt caa acc ttc tgt act tgt t

splb tga aga gcg tgc aat aga a cag tat gcg ctg aat ata ca

splc atg aaa atg tec aag cat t ggc acc att ata ttc aga acc

spld gaa att aat tac caa cac ga tgc cag gtt gga caa cc

sple cgt tgc agg tat gga aat t gcg gtt tec acc aaa gtg

splf taa aca aat tac aaa tac aaa gat tac acc aat age ttc gtg t

Tab. 2: Primers for multiplex PC for spl a-f genes in S. aureus

The composition of the spl gene cluster was heterogenous, but 81% of the S. aureus isolates harboured at least one spl gene (Table 3). Table 3 also shows that the composition of the spl gene cluster was found to be tightly associated with the bacterial genetic background. S. aureus strains with similar genetic background are grouped within the same clonal lineage. This implies that Spl proteins can be generated and released by the vast majority of clinical S. aureus strains.

a) Lindsay, J. A., et al., Microarrays reveal that each of the ten dominant lineages of

Staphylococcus aureus has a unique combination of surface-associated and regulatory genes. J. Bacteriol. 188: 669-76 (2006)

Tab. 3: Prevalence of spl genes in S. aureus clinical isolates d. The S. aureus proteins SplA-F were produced as recombinant proteins and purified.

For the investigation of the interplay between S. aureus serine protease like proteins (Spls) and the immune system, two expression systems for the production of Spls were used. On the one hand, the SpIC and SpID were produced in recombinant form in Escherichia (E.) coli without signal peptide. They were expressed with a C-terminal Strep-tag, added by genetic engineering methods, for purification by affinity chromatography on columns coated with streptactin. On the other hand, SplA, SplB, SpIC, SpID, SplE and SplF were produced tag free in Bacillus (B.) subtilis. For this expression system the genetic information of the signal peptides were not removed and the recombinant, tag free proteins were released by the secretion system of B. subtilis in the supernatant. The recombinant proteins were purified by cation exchange chromatography.

To avoid non-specific immune cell activation by E. coli-LPS, the resulting protein preparations were subsequently depleted of lipopolysaccharide (LPS) using EndoTrap ^® red columns (Hyglos, Heidelberg, Germany) according to the manufacturer's instructions. The column material binds with high affinity to LPS and the protein of interest can be eluted while LPS remains bound in the column. Two rounds of purification yielded in final LPS concentrations below 10 ng LPS per mg protein.

The amino acid sequences of recombinant SpIC and SpID produced in E. coli without signal peptide are as follows, wherein a methionine was added to the N-terminus (bold) and the Strep-tag sequence (SAWSHPQFEK) was attached to the C-terminus (bold):

SpIC:

MVVEETQQIANAEKNVTQVKDTNIFPYNGVVSFKDATGFVIGKNTIITNKHVSKDYK VGD I TAHPNGDKGNGGIYKIKSISDYPGDEDISVMNIEEQAVERGPKGFNFNENVQAFNFAKDA K VDDKIKVIGYPLPAQNSFKQFESTGTIKRIKDNILNFDAYIEPGNSGSPVLNSNNEVIGV VYGG IGKIGSEYNGAVYFTPQIKDFIQKHIEQSAWSHPQFEK

SpID:

MENSVKLITNTNVAPYSGVTWMGAGTGFVVGNHTIITNKHVTYHMKVGDEIKAHPNG FY NNGGGLYKVTKIVDYPGKEDIAVVQVEEKSTQPKGRKFKDFTSKFNIASEAKENEPISVI GYP NPNGNKLQMYESTGKVLSVNGNIVTSDAVVQPGSSGSPILNSKREAIGVMYASDKPTGES T RSFAVYFSPEIKKFIADNLDKSAWSHPQFEK Spl gene forward primer reverse primer

splc atg gta ggt etc aaa tgg teg ttg aag aga cac atg gta ggt etc age get ttg ttc aat gtg ctt ttg aac aaa tag aat aaa ate

spld atg gta ggt etc aaa tgg aaa ata gtg tga aat atg gta ggt etc age get ttt ate taa att ate tgc taa tta cca ac aat aaa ttt ct

Tab.4: Primers used for spl gene expression cloning in E. coli

The amino acid sequences of recombinant SplA-F produced in B. subtilis are as follows:

SplA:

EKNVKEITDATKEPYNSVVAFVGGTGVVVGKNTIVTNKHIAKSNDIFKN VSAHHSSKGK GGGNYDVKDIVEYPGKEDLAIVHVHETSTEGLNFNKNVSYTKFADGAKVKDRISVIGYPK GAQTKYKMFESTGTINHISGTFMEFDAYAQPGNSGSPVLNSKHELIGILYAGSGKDESEK NFGVYFTPQLKEFIQNNIEK

SplB:

ENNVTKVKDTNIFPYTGVVAFKSATGFVVGKNTILTNKHVSKNYKVGDRITAHPNSD KGN GGIYSIKKIINYPGKEDVSVIQVEERAIERGPKGFNFNDNVTPFKYAAGAKAGERIKVIG YPHPYKNKYVLYESTGPVMSVEGSSIVYSAHTESGNSGSPVLNSNNELVGIHFASDVKND DNRNAYGVYFTPEIKKFIAENIDK

SpIC:

EKNVTQVKDTNIFPYNGVVSFKDATGFVIGKNTIITNKHVSKDYKVGDRITAHPNGD KGN GGIYKIKSISDYPGDEDISVMNIEEQAVERGPKGFNFNENVQAFNFAKDAKVDDKIKVIG YPLPAQNSFKQFESTGTIKRIKDNILNFDAYIEPGNSGSPVLNSNNEVIGVVYGGIGKIG SEYNGAVYFTPQIKDFIQKHIEQ

SpID:

ENSVKLITNTNVAPYSGVTWMGAGTGFVVGNHTIITNKHVTYHMKVGDEI KAHPNGFYNN GGGLYKVTKIVDYPGKEDIAVVQVEEKSTQPKGRKFKDFTSKFNIASEAKENEPISVIGY PNPNGNKLQMYESTGKVLSVNGNIVTSDAVVQPGSSGSPILNSKREAIGVMYASDKPTGE STRSFAVYFSPEIKKFIADNL.DK

SplE:

EHNVKLIKNTNVAPYNGVVSIGSGTGFIVGKNTIVTNKHVVAGMEIGAHIIAHPNGEYNN GGFYKVKKIVRYSGQEDIAILHVEDKAVHPKNRNFKDYTGILKIASEAKENERISIVGYP EPYINKFQMYESTGKVLSVKGNMIITDAFVEPGNSGSAVFNSKYEVVGVHFGGNGPGNKS TKGYGVYFSPEIKKFIADNTDK

SplF:

ENTVKQITNTNVAPYSGVTWMGAGTGFVVGNHTIITNKHVTYHMKVGDEI KAHPNGFYNN GGGLYKVTKIVDYPGKEDIAVVQVEEKSTQPKGRKFKDFTSKFNIASEAKENEPISVIGY PNPNGNKLQMYESTGKVLSVNGNIVSSDAIIQPGSSGSPILNSKHEAIGVIYAGNKPSGE STRGFAVYFSPEIKKFIADNLDK Spl gene forward primer reverse primer

spla gtg aac cct aaa tag aag gag gtt tag agg ccc caa ggg gtta gaa aca cat atg aat aaa aat tget aac tag ttt attt ttc aat att gta atg gtt aaa g att ttg

splb gtg aac ccta aat aga agg agg gtt tag agg ccc caa ggg gtt aaa cac ata tga aca aaa acg atg eta act agt tta ttt ate tat tag tea tea aga gtt ttc tg

splc gtg aac cct aaa tag aag gag gtt tag agg ccc caa ggg gtt gaa aca cat atg aat aaa aat atg eta act agt tta ttg ttc aat ata gtc att aaa age gtg ctt ttg

spld gtg aac cct aaa tag aag gag gtt tag agg ccc caa ggg gtt gaa aca cat atg aat aaa aat atg eta act agt tta ttt ate taa ata ate ate aaa agt att gcg att ate tgc aat a

sple gtg aac cct aaa tag aag gag gtt tag agg ccc caa ggg gtt gaa aca cat atg aat aaa aat atg eta act agt tta ttt ate tgt ata ate ate aaa agt att gca gtt ate tg

splf gtg aac cct aaa tag aag gag gtt tag agg ccc caa ggg gtt gaa aca cat atg aat aaa aat atg eta act agt tta ttt ate taa ata ate ate aaa agt att gga att ate tgc aat g

Tab. 5: Primers used for expression cloning of spl genes in B. subtilis

The sequences were taken from the genome of the fully sequenced S. aureus strain USA300. For recombinant production in E. coli the signal peptides - as predicted by SignalP 3.0 Server (http://www.cbs.dtu.dk/services/SignalP-3.07)- were removed.

e. The subclass composition of serum antibodies that bind to recombinant

S. aureus SpIC and SpID is shifted towards lgG4 in around 50% of healthy adults.

Serum antibody binding to recombinant SpIC and SpID was investigated by an enzyme linked immuno-sorbent assay (ELISA). Recombinant TSST-1 served as control. Sera of 32 healthy blood donors, 16 S. aureus carriers and 16 non-carriers, were analyzed for total serum IgG binding to SpIC, SpID, and TSST-1. Moreover, the binding of serum antibodies of the subclass lgG4 was also measured by ELISA. The lgG4/lgG ratio for binding was shifted towards lgG4 in half of the tested sera for SpIC (Fig. 2). On the other hand, there were also individuals who did not elaborate serum lgG4 with specificity for the tested bacterial antigens (Fig. 2). Fig. 2 shows the results of an enzyme-linked immune sorbent assays (ELISA) for IgG and lgG4 binding to SpIC, SpID and TSST-1. The assays were performed with sera from 16 S. aureus carriers and 16 non-carriers. Serum was diluted in blocking buffer (PBS/Tween, 2% milk powder) (serial dilutions 1:5; 1:50 to 1:31250) and tested in duplicate. Total IgG binding was detected with goat anti-human IgG antibodies (Jackson Immuno esearc) conjugated with HRP, diluted in blocking buffer to a concentration of 10 ng/ml. Binding of lgG4 was detected using goat anti-human lgG4 antibodies conjugated with HRP (Invitrogen) diluted in blocking buffer to a concentration of 1 ng/ml. ELISA plates (Maxisorp, Thermo Scientific) were coated with 2 μg/ml μΙ antigen over night at 4 °C. After washing three times with PBS/Tween20 (0,05%) each well was blocked with 100 μΙ blocking buffer for one hour. 50 μΙ/well of serum dilutions were incubated for 1 hour at 100 rpm and the ELISA plates were washed three times as previously described. 50 μΙ of the diluted secondary antibody were added to each well and incubated for 1 hour. After three rounds of washing, 50 μΙ/well substrate solution was added for 10 min and the chromogenic reaction was stopped with 20 μΙ/well 2 N H2SO4. The signal was measured at 450 nm.

Experiments were performed in duplicates; mean values are depicted. Area under the curve was calculated for each serum and individual ratios of lgG4/lgG-binding are depicted. Carrier sera are represented by open circles; non-carrier sera by filled circles.

f. Serum IgE with specificity for S. aureus SpIC and SpID is found in patients with cystic fibrosis, atopic dermatitis, asthma, and chronic rhinosinusitis with nasal polyps.

IgE binding to the staphylococcal proteins SpIC, SpID and a-toxin (Hla, used as control) was studied in sera of patients with cystic fibrosis (CF), atopic dermatitis (AD) and asthma using an ELISA assay as described above with the following modifications: Sera were diluted 1:100 in blocking buffer (PBS/10% fetal calf serum). IgE binding was measured by incubation with mouse anti-human IgE antibodies at a concentration of 10 μg/ml followed by incubation with rabbit anti-mouse IgG antibodies coupled with HRP at 3 μg/ml (secondary and tertiary antibody preparations were from Invitrogen). All five patients mounted an IgE response to SpID and four out of five mounted an IgE response to SpIC (Table 6). Patient Anti-SplC-lgE Anti-SplD-lgE

1 (CF) yes yes

2 (CF) yes yes

3 (AD) yes yes

4 (Asthma) yes yes

5 (Asthma) no yes

Tab. 6: Serine protease specific IgE in patient sera.

Following this pilot experiment, sera from 39 healthy individuals, 14 patients with nasal polyps and 48 asthma patients were tested for IgE binding to S. aureus SpIC and HIa using the modified ELISA-system described above.

The majority of healthy individuals did not exhibit IgE binding to any of the bacterial proteins. However, in most patients with nasal polyposis as well as in asthma IgE binding to SpIC was measurable. The differences between patients and control individuals were significant (Kruskal-Wallis ANOVA with Dunn's posttest; levels of significance: *, P < 0,05; **, P < 0.01; ***, P < 0,001) (Fig. 3). Only asthmatics but not patients with nasal polyposis had increased IgE binding to HIa in addition.

The results support a scenario with SpIC as a driver of allergic reactions to S. aureus as follows: Tolerance to this bacterial protein is broken early, as some adults, known as atopic individuals, exhibit SplC-specific IgE even before developing symptoms of allergy. Similar observations have been made with typical aeroallergens, which are known to trigger allergies. As the process continues, some individuals develop disease symptoms, e.g., nasal polyps or asthma. The symptomatic individuals have regularly lost their tolerance as reflected by IgE binding to SpIC. The disease typically progresses from nasal polyposis to asthma. At this late stage, allergic reactions are additionally directed at other s, aureus proteins, such as HIa, a phenomenon known in the field as epitope spreading. g. Human adults harbor S. aureus SpIC- and SplD-responsive human T cells in the blood.

Peripheral blood mononuclear cells (PBMC) from ten adult human volunteers were incubated with recombinant SpIC and SpID at increasing concentrations. The cells from most volunteers responded with a robust, concentration-dependent proliferation as measured by the incorporation of ³ H-methyl-thymidine using known methods. This indicates that SplC- and SplD-reactive T cells are present in the blood of most human adults as shown in Fig. 4. SpIC and SpID induced a robust proliferative response in PBMC. For the assay PBMC from ten healthy blood donors were isolated and stimulated with heat inactivated (65 °C, 45 min) recombinant antigens SpIC, SpID and a-toxin (Hla), which served as control. Cells were incubated with serially diluted antigens (10 - 0,08 μg/ml) for 7 d. Proliferation was determined by ³ H-methyl-thymidin incorporation. Area under the curve (AUC) was calculated for each donor.

The frequency of SpIC- and SplD-specific T cells in the peripheral blood was assessed in nine healthy adult volunteers by limiting dilution assays. Irradiated autologous antigen-presenting cells were pulsed with 10 μg/ml antigen (SpIC or SpID) and incubated (37 °C, 5 % CO2) with serially diluted T cell suspensions resulting in an input of between 10,000 to 78 T cells per culture. Supplementation with low doses of IL-2 (10 lU/ml) enhanced the proliferation of antigen-specific T cell clones. Proliferation was quantified by ³ H-methyl-thymidine incorporation after 10 days. The fraction of non-proliferating cultures was determined for each T cell number of input, and subsequently, the frequency of antigen-specific T cells was calculated from a regression curve. Frequencies of SpIC- and SplD-specific T cells ranged from 1 out of 3.000 to 1 out of 170.000 T cells as shown in Fig. 5. SpIC- and SplD-specific T cells are present at frequencies typical for memory T cells. This can be inferred from the observation that T cells responsive to the typical recall antigens tetanus toxoid (TT) and antigens from cytomegaly virus (CMV) are of similar frequency. In contrast, the superantigen TSST-1 activated a much larger fraction of T cells such as it is expected of a superantigen (Fig. 5). For the limiting dilution assays (LDA) autologous feeder cells were stimulated with 10 μg/ml of heat-inactivated antigen and serially diluted T cells (10 ⁴ -78 T cells per culture) were added. Cells were incubated for 10 d in total, and supplemented with IL-2 at d 5 and d 10 (10 lU/ml and 50 Ill/ml). Proliferation was determined by ³ H-methyl-thymidine incorporation. For calculation of T cell frequencies, the number of non-proliferative cultures per T cell concentration was determined. According to the Poisson distribution, a fraction of 0,368 of non-proliferative cultures is equal to the limiting dilution. Thus, regression equations were solved with f(x)=0,368. Frequencies of antigen-specific T cells from S. aureus carriers are depicted with open circles; cells from non-carriers with filled circles.

h. A subset of SpIC- and SplD-specific human T cells generates a cytokine profile typical of an allergic reaction.

Peripheral blood mononuclear cells (PBMC) from five to ten adult human volunteers were incubated with recombinant SpIC, SpID and a-toxin at 5 μg/ml. After 7 d the cytokines released by the activated cells were analysed by flow cytometry. The investigated cytokine panel covered the main cytokines which characterize TH1, TH2, TH9, TH17, TH22 and Tregs. Analysis of the cytokine response of these SplC-and SpID- stimulated T cells revealed secretion of the TH2 cytokine IL-5 (shown for SpID in Fig. 6). There was almost no IL-10, TNF- α nor IFN-y in these supernatants. The blood cell reaction to a-toxin was very different in that IL-10, TNF-a and IFN-γ were secreted at high concentrations (Fig. 6). This shows that SpIC- and SplD-trigger a specific response in T cells. Such responses are typical of allergies of type 1. For the assay PBMC from five to ten healthy blood donors were stimulated with heat- inactivated (65 °C, 45 min) a-toxin (right panel) or SpID (left panel) at concentrations of 5 μg/ml for 7 d. Cell culture supernatants were taken and analysed by flow cytometry for release of cytokines with flexible cytokine bead array (Becton Dickinson) according to the instruction manual. In contrast to the pro-inflammatory cytokines induced by a-toxin (e.g. IFN-γ), SplD-stimulated PBMC responded selectively with TH2 cytokines like IL-5. Every circle represents the concentration of a cytokine secreted in cell culture by the peripheral blood cells from one individual. The left Y-axis relates to the measured concentrations of IL5 (full circles), the right Y-axis to the measured concentrations of IL-10, TNF-a and IFN-γ (open circles).

SpIC- and SplD-specific T cell clones were activated with their respective antigens for 72 h. T cell clones responsive to the staphylococcal a-toxin (Hla) served as controls. The released cytokines in the supernatants were analyzed by a flow cytometric bead assay. The investigated cytokine panel covered the main cytokines which characterize TH1, TH2, TH9, TH17, TH22 and Tregs. It was observed that half of the SplD-specific T cell clones produced the cytokines IL-4, IL-9 and IL-12p70. These are characteristic of TH2 and TH9 cells. Small amounts of IL-Ιβ and IL-17 were noticed as well, but TNF-a and IFN-γ concentrations were below the detection limits. This is remarkable, because other well-known S. aureus virulence factors, like a-toxin (Hla), induced the specific T cell clones to release the pro-inflammatory cytokines TNF-a and IFN-γ in very large amounts (Fig. 7). Secretion of cytokines typical of regulatory T cells (Tregs), e.g. IL-10, was not observed in the Spl-specific T cell clones. Hence, a survey of S. aureus-reactive T cell clones revealed that a subset of T cell clones with specificity for SpIC and SpID is unique in that these T cells selectively secrete cytokines corresponding to a Th2/Th9 profile, namely IL-4, IL-5 and IL-9 (shown in black circles in Fig. 7). This supports the assumption that SpIC and SpID stimulation favors an allergic immune response profile even in healthy adults.

To obtain antigen-specific T cell clones, limiting dilution assays (LDA) were performed.

Therefore, autologous feeder cells were stimulated with 10 μg/ml of heat-inactivated antigen, SpIC, SpID, or Hla, and co-incubated with serially diluted T cells (10 ⁴ -78 T cells per culture). Cells were incubated for 10 d in total, and supplemented with IL-2 at d 5 and d 10 (10 lU/ml and 50 Ill/ml). Growing T cell clones were stimulated with their respective antigen again and 72 h later, supernatants were obtained for analysis of cytokines. Cytokines were determined by flow cytometry with the flexible cytokine bead array (Becton Dickinson) according to the instruction manual. Every circle represents the cytokine concentration elaborated by an individual T cell clone specific for the respective antigen as shown. Filled circles represent four independent T cell clones, specific for SpID, which showed the most pronounced pro-allergic cytokine profiles.

i. Conclusions

The results make a strong case for the concept that S. aureus serine protease like proteins act as allergens in humans. The concept is based on the following findings:

Many known allergens are proteases.

Some individuals develop a strong lgG4-antibody response against S. aureus proteins. This is not directed against all S. aureus products with the same intensity, but lgG4 binding is strongest to SpIC and SpID. It is assumed that adults with a strong lgG4 antibody binding to Spl proteins have an atopic predisposition. Healthy adults harbor T lymphocytes in their blood that specifically respond to SpIC or SplD. A significant subset of these T cells produces cytokines upon stimulation which are typical of allergic reactions of type 1: IL-4, IL-5 und IL-9. On the other hand it is remarkable that these Spl-specific T cells - in salient contrast to T cells with specificity for other S. aureus proteins - do not release cytokines that would counteract an allergic reaction: TNF-a, IFN-γ, IL-10. This is very different, for example, in a-toxin-reactive T cells.

From the previous points it follows that the serine protease like proteins of S. aureus favor the development of an immune response with a Th2 profile. A Th2-dominated immune response carries a high risk of development of allergic symptoms. Hence it is expected that the serine protease like proteins SplA-F act as triggers and pace-makers of allergies in susceptible individuals.

Some non-symptomatic individuals, presumably with atopic pre-disposition, show IgE binding to SpIC indicating that the tolerance to this bacterial protein can be broken early.

The majority of patients with chronic rhinosinusitis with nasal polyps as well as asthmatics have SplC-specific IgE.

Some asthmatics additionally show IgE binding to a-toxin, a phenomenon termed epitope spreading, which is typical of allergic disease progression.

From the above findings it is concluded that the serine protease like proteins SplA-F are major allergens of 5. aureus. Exposure to these proteases triggers allergic reactions and maintains them in susceptible individuals and these allergic inflammatory reactions are the cause of frequent chronic diseases like non-allergic or idiopathic asthma, aspirin

hypersensitivity, rhinosinusitis with or without nasal polyps, and AD. The findings lead to the concept of the invention to take serine protease like proteins of 5. aureus or derived proteins or peptides as targets for diagnosis and treatment of diseases, which are related to allergic reactions to S. aureus. The first main aspect of the invention is the diagnosis of such diseases by analyzing an interaction of S. aureus serine protease like proteins or derived proteins or peptides with the patient's body, especially with serum antibodies of the patient. The second main aspect of the invention is a therapeutic application based on systemic immune therapy, wherein small amounts of 5. aureus serine protease like proteins or derived proteins or peptides are administered to the patient, especially by intra-dermal, subcutaneous, intramuscular and/or by sublingual administration. Protein sequence information

The protein sequences are one allelic form each of the serine protease like proteins A-F as they are present in the genome of S. aureus strain USA300 according to the database UniProt (http://www.uniprot.org/).

NO 1: Staphylococcus aureus serine protease like protein A (SplA); Database accession no: Q2FFS9

MNKNVMVKGLTALTILTSLGFAENISNQPHSIAKAEKNVKEITDATKEPYNSVVAFV GGT GVVVGKNTIVTNKHIAKSNDIFKNRVSAHHSSKGKGGGNYDVKDIVEYPGKEDLAIVHVH ETSTEGLNFNKNVSYTKFADGAKVKDRISVIGYPKGAQTKYKMFESTGTINHISGTFMEF DAYAQPGNSGS PVLNSKHELIGILYAGSGKDESEKNFGVYFTPQLKEFIQNNIEK

NO 2: Staphylococcus aureus serine protease like protein B (SplB); Database accession no: Q2FFT0

MNKNVVIKSLAALTILTSVTGIGTTLVEEVQQTAKAENNVTKVKDTNIFPYTGVVAF KSA TGFVVGKNTILTNKHVSKNYKVGDRITAHPNSDKGNGGIYSIKKIINYPGKEDVSVIQVE ERAIERGPKGFNFNDNVTPFKYAAGAKAGERIKVIGYPHPYKNKYVLYESTGPVMSVEGS SIVYSAHTESGNSGSPVLNSNNELVGIHFASDVKNDDNRNAYGVYFTPEIKKFIAENIDK

NO 3: Staphylococcus aureus serine protease like protein C (SpIC); Database accession no: Q2FFT1

MNKNIVIKSMAALAILTSVTGINAAVVEETQQIANAEKNVTQVKDTNIFPYNGVVSF KDATGFVIGKNTIIT NKHVSKDYKVGDRITAHPNGDKGNGGIYKIKSISDYPGDEDISVMNIEEQAVERGPKGFN FNENVQAFNF AKDAKVDDKIKVIGYPLPAQNSFKQFESTGTIKRIKDNILNFDAYIEPGNSGSPVLNSNN EVIGVVYGGIGKI GSEYNGAVYFTPQIKDFIQKHIEQ

NO 4: Staphylococcus aureus serine protease like protein D (SpID); Database accession no: Q2FFT2

MNKNIIIKSIAALTILTSITGVGTTVVDGIQQTAKAENSVKLITNTNVAPYSGVTWM GAGTGFVVGNHTIIT NKHVTYHMKVGDEIKAHPNGFYNNGGGLYKVTKIVDYPGKEDIAVVQVEEKSTQPKGRKF KDFTSKFNIA SEAKENEPISVIGYPNPNGNKLQMYESTGKVLSVNGNIVTSDAVVQPGSSGSPILNSKRE AIGVMYASDKP TGESTRSFAVYFSPEIKKFIADNL.DK

NO 5: Staphylococcus aureus serine protease like protein E (SplE); Database accession no: Q2FFT3

MNKNIIIKSIAALTILTSVTGVGTTVVEGIQQTAKAEHNVKLIKNTNVAPYNGVVSI GSGTGFIVGKNTIVTN KHVVAGMEIGAHIIAHPNGEYNNGGFYKVKKIVRYSGQEDIAILHVEDKAVHPKNRNFKD YTGILKIASEA KENERISIVGYPEPYINKFQMYESTGKVLSVKGNMIITDAFVEPGNSGSAVFNSKYEVVG VHFGGNGPGN KSTKGYGVYFSPEIKKFIADNTDK NO 6: Staphylococcus aureus serine protease like protein F (SplF); Database accession no: Q2FFT4

MNKNIIIKSIGALTILTSITGVGTTMVEGIQQTAKAENTVKQITNTNVAPYSGVTWM GAGTGFVVGNHTII TNKHVTYHMKVGDEIKAHPNGFYNNGGGLYKVTKIVDYPGKEDIAVVQVEEKSTQPKGRK FKDFTSKFNI ASEAKENEPISVIGYPNPNGNKLQMYESTGKVLSVNGNIVSSDAIIQPGSSGSPILNSKH EAIGVIYAGNKPS GESTRGFAVYFSPEIKKFIADNL.DK