TREATMENT OF STAPHYLOCOCCUS RELATED DISEASES

Abstract

The present invention relates to antibodies for treating or preventing infections of Staphylococcus genus bacteria and/or Staphylococcus genus bacteria-related diseases. Especially, the invention relates to S. intermedius group bacteria infections and diseases. Furthermore, the invention relates to respective pharmaceutical compositions and methods of manufacturing a medicament comprising anti-S. aureus alpha-hemolysin protein antibodies.

Claims

1-17. (canceled)

18. A pharmaceutical composition comprising an antibody, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier selected from phosphate-buffered saline, emulsions, oil/water emulsions, creams, ointments, and gels, and wherein the antibody is a naturally occurring antibody effective in treating or preventing an infection or disease caused by Staphylococcus bacteria.

19. The pharmaceutical composition according to claim 18, wherein the pharmaceutical composition comprises a hydrogel.

20. The pharmaceutical composition of claim 18, wherein the antibody that binds at least one epitope selected from epitopes comprising amino acid sequence KIGGLIG (SEQ ID NO: 2), ATKQQSN (SEQ ID NO: 3), KKILVIRTK (SEQ ID NO: 4), IDVIYERV (SEQ ID NO: 5), KAADNFLDP (SEQ ID NO: 6), and/or DSDINIK (SEQ ID NO: 7).

21. The pharmaceutical composition of claim 18, wherein the antibody is obtained from a naturally occurring antibody source.

22. The pharmaceutical composition of claim 18, wherein the antibody is obtained by a process which does not involve the immunization of an animal with isolated Staphylococcus genus bacteria, isolated part thereof, an isolated Staphylococcus genus bacteria protein, and/or isolated parts or fractions thereof as described above.

23. The pharmaceutical composition of claim 18, wherein concentration of the antibody in the pharmaceutical composition is increased in comparison to a pharmaceutical composition from which the antibody is obtained.

24. The pharmaceutical composition of claim 18, wherein the antibody is obtained from an animal which was not immunized with isolated Staphylococcus genus bacteria, isolated part thereof, an isolated Staphylococcus genus bacteria protein, and/or isolated part or fraction thereof.

25. The pharmaceutical composition of claim 18, wherein the antibody is a polyclonal antibody.

26. The pharmaceutical composition of claim 18, wherein the pharmaceutical composition is formulated for topical administration.

27. The pharmaceutical composition of claim 18, wherein the Staphylococcus bacteria comprise S. aureus bacteria or S. intermedius bacteria.

28. The pharmaceutical composition of claim 18, wherein the antibody is immunoreactive with alpha-toxin, beta-toxin, LukD, LukE, LukF, and/or HlgB of S. aureus bacteria.

29. The pharmaceutical composition of claim 25, wherein the polyclonal antibody is immunoreactive with alpha-toxin, beta-toxin, LukD, LukE, LukF, and/or HlgB of S. aureus bacteria.

30. The pharmaceutical composition of claim 25, wherein the Staphylococcus bacteria comprise an antibiotic resistant Staphylococcus bacterial strain.

31. The pharmaceutical composition of claim 18, wherein the antibody is obtained from a sheep, goat, rabbit, equine or bovine; and/or wherein the antibody is obtained from bovine colostrum or milk.

32. The pharmaceutical composition of claim 18, wherein the disease caused by Staphylococcus bacteria is pyoderma.

33. The pharmaceutical composition of claim 18, wherein the antibody binds at least one epitope selected from epitopes comprising amino acid sequence ATKQQSN (SEQ ID NO: 3), KKILVIRTK (SEQ ID NO: 4), IDVIYERV (SEQ ID NO: 5), KAADNFLDP (SEQ ID NO: 6) and/or DSDINIK (SEQ ID NO: 7).

34. The pharmaceutical composition of claim 18, wherein the pharmaceutical composition is formulated for treatment of infections or diseases in a dog, cat, horse, or human.

35. A formulation for treatment of infections or diseases in a dog, cat, horse, or human, wherein the formulation comprises the pharmaceutical composition of claim 18.

36. The pharmaceutical composition of claim 20, wherein the epitope comprises amino acid sequence KIGGLIG (SEQ ID NO: 2).

37. A method of treating or preventing infections or diseases caused by Staphylococcus, wherein the method comprises treating a subject in need thereof with the pharmaceutical composition of claim 18.

Description

FIGURES

[0131] FIG. 1 shows a chromatogram of an affinity chromatography of a polyclonal anti S. aureus Hla antibody on a S. aureus Ha H35L column.

[0132] FIG. 2 shows the binding of polyclonal anti S. aureus Hla antibody samples to S. aureus Ha H35L in an ELISA assay.

[0133] FIG. 3A shows the inhibition of Hla induced lysis of rabbit red blood cells by polyclonal antibodies obtained from an immunized cow before and after formulation.

[0134] FIGS. 3B and 3C, show the lysis of rabbit red blood cells induced by supernatants of S. pseudintermedius strains cultures 69687 (FIG. 3B) and 4639949 (FIG. 3C) and the inhibition of lysis by different IgG preparations FIGS. 4 A) and B) show the FACS diagrams of the binding of polyclonal anti S. aureus Ha antibodies to the surface of different S. pseudintermedius strains.

[0135] FIG. 5 shows the penetration of a polyclonal anti S. aureus Ha antibody into piglet skin. A) Fluorescence stain of intact skin. b) HE stain of laserporated skin. c) Fluorescence stain of laserporated skin.

[0136] FIG. 6 shows a rheogramm of hydrogels with different CMC concentrations.

[0137] FIG. 7 shows the spray forces required to activate the spray process of different CMC concentration from an Ursatec 3K horizontal spray system.

[0138] FIG. 8 shows viscosities of placebo and antibody containing hydrogels at storage temperature (8 C.) and skin temperature (32 C.) directly after preparation and two or six months after storage.

[0139] FIG. 9 shows relative content of monomer, dimer and larger aggregates as well as recovery of pIgGs in CMCs hydrogels of two different batches directly after preparation and after storage.

[0140] FIG. 10 shows CD spectra of antibodies in PBS and hydrogel before and after storage for 7 weeks at 2 C. to 8 C.

[0141] FIG. 11 shows the effect of S. aureus and anti-AT antibody on morphology and cell viability of human skin.

[0142] FIG. 12 shows a microarray analysis for pre-staining and secondary antibodies of Example 10. A) Pre-staining, 1:2000; B) Adjusted scan; C) Anti-bovine IgG (H+L) DyLight680 (1:2000), anti-bovine IgG (Fc) DyLight800 (1:2000)

[0143] FIG. 13 shows a microarray of sample A of Example 10. A) Sample A, 10 g/ml (IgG); B) Sample A, 10 g/ml (Ig); C) Sample A (polyclonal antibody pool before immunization), 10 g/ml.

[0144] FIG. 14 shows a microarray of sample B of Example 10. A) Sample B, 10 g/ml (IgG); B) Sample B, 10 g/ml (Ig); C) Sample B (polyclonal antibody pool before immunization), 10 g/ml.

[0145] FIG. 15 shows a microarray of sample C of Example 10. A) Sample C, 10 g/ml (IgG); B) Sample C, 10 g/ml (Ig); C) Sample C (polyclonal antibody pool before immunization), 10 g/ml.

[0146] FIG. 16 shows a comparison of the microarray results of Example 10.

[0147] FIG. 17 shows the top 35 interactions of polyclonal bovine antibody Sample A intensities from Example 11.

[0148] FIG. 18 shows the top 35 interactions of polyclonal bovine antibody Sample B intensities from Example 11.

[0149] FIG. 19 shows a comparison of the microarray results of Example 11.

[0150] FIG. 20 shows the quantitative results of a the dog skin infection model from Example 12 for Sample A (A) and Sample B (B)

[0151] FIG. 21 shows the effect of bovine IgG on the growth of antibiotic resistant S. pseudintermedius in a solo and combinational approach.

[0152] FIG. 22 shows scanning electron microscopy images of skin biopsies cultivated with S. pseudintermedius (A) and treated with 10 mg/mL bovine IgG (B.) FIG. 23 shows Colony forming unit (CFU) of adherent bacteria on canine skin.

[0153] FIG. 24 shows Expression of Th2 cytokine IL-13 (A.) and Th1 cytokine TNF (B.) in dog skin with bacterial strain 69687. BovgG=bovine IgG, CM=clindamycin

EXAMPLES

Example 1: Isolation and Purification of IgG

[0154] 1.1 Collection of Milk

[0155] Milk was collected over 7 days after delivery. The collected milk was stored at about 20 C.

[0156] The milk was either obtained from cows that were not immunized with an isolated antigen preparation or from cows immunized with aureus Hla H35 protein.

[0157] For immunization, a pregnant cow was immunized with 300 g of recombinant mature S. aureus Hla H35L protein in PBS with Quil-A (Invitrogen, San Diego, USA) as adjuvant. S. aureus Hla comprising a H35L mutation was obtained by cloning, expressing in E coli and purification as disclosed in Menzies and Kernodle, 1994 and Wardenburg & Schneewind 2008. In total, a volume of 2 ml was injected subcutaneously. The priming immunization was administered on day 0 and two boost immunizations were administered on days 14 and 21. The cow delivered on day 42.

[0158] 1.2 Milk Processing

[0159] The frozen milk was thawn and defatted at 50 C. in a disk separator at 8,000 g to a fat content of less than 0.1% w/w according to commonly known methods.

[0160] The defatted milk was subjected to 7 cycles of diafiltration with a microfiltration membrane of 0.14 m pore size and a utrafiltration membrane with a cut-off size of 10 kD at a constant pressure difference (pTM) of 2 bar. During this step casein micelles, bacteria and small molecular compounds were separated.

[0161] The whey obtained from diafiltration was stored at about 20 C.

[0162] 1.3 Capture-Chromatography

[0163] To capture the polyclonal antibodies from the whey fraction the whey was thawn over 12 h at room temperature.

[0164] The whey (20 g/l Immunglobulin) was adjusted to 20 mM NatriumKaliumphosphatbuffer pH 7.5; 250 mM NaCl and loaded on a Capto-MMC column (GE Healthcare Bio-Sciences, Pittsburgh, USA) with a bed volume of 101 (40 cm8 cm). The flow through comprised the polyclonal antibodies whereas proteins like lactoperoxidase and lactoferrin were bound to the column. The collected flow through was subsequently loaded on a MEP HyperCel column (Pall Corporation, New York, USA) with a bed volume of 30 l (40 cm24 cm). The column was washed with 5 column volumes of Buffer A (20 mM NaKCO3 pH 7.5; 250 mM NaCl). Subsequently, pre-elution was performed with 2 column volumes of buffer B (50 mm MES/NaOH pH 6.0). Subsequently the bound Ig was eluted with 3 column volumes of buffer C (50 mM Na acetate pH 4). MEP HyperCel column chromatography was performed at a flow rate of 5 l/min. The IgG comprising elution fractions were pooled.

[0165] Alternatively, the whey was subjected to Sepharose Q chromatography to separate beta-Lactoglobulin from the whey. In brief, one volume of whey was diluted with three volumes of Na-PFA-Buffer (30 mM Na.sub.2HPO.sub.4, 30 mM Na-formiat, 60 mM Na-acetate, pH 5.5). The diluted whey was loaded on a HiTrap Q FF (GE Healthcare) column at a flowrate of 1 ml/min. The flow-through comprising the IgG was further processed, whereas beta-Lactoglobulin was bound to the column.

[0166] A buffer exchange was performed by dialyzing the pooled fractions against PBS (20 mM Na2HPO4 pH 7.5; 150 mM NaCl) over a PES Membrane (100 kD cutoff) at 7 C. Subsequently, the pooled polyclonal anti S. aureus Ha H35L IgG fractions were sterile filtered over a Millipore Express SHC 0.5/0.2 m double membrane and frozen at 20 C.

Example 2: Hydrogel Formulation

[0167] 2.1. Formulation

[0168] 2.5% w/w of Sodium Carboxymethylcellulose (CMC) Blanose 7H4F (Ashland, Ky., USA) was added to PBS pH 7.4 (137 mM NaCl, 2.7 mM KCl, 9.0 mM Na2HPO4.2 H2O, 3.5 mM KH2PO4) under vigorous stirring. The mixture was heated to 40 C. and stirred over 24 h. Afterwards, the hydrogel was steam sterilized (121 C., 2 bar, 15 min).

[0169] Thawed polyclonal anti S. aureus Ha H35L IgG as obtained in Example 1 with a protein concentration of 40-80 mg/ml was added to the CMC stock solution under stirring over 5 to 10 min at room temperature to obtain a hydrogel with a CMC concentration of 1.5% w/w and a protein concentration of 10 to 12 mg/g.

[0170] 2.2. Analysis of Hydrogel Formulation

[0171] 2.2.1. Rheological Behaviour

[0172] Gel characteristics of hydrogels comprising 0%, 0.5%, 1%, 1.5%, 2% and 2.5% w/w CMC were determined with a MCR 100 rheometer (Anton Paar, Graz, Austria) with a stainless steel plate/plate system (diameter 25 mm). Viscosity measurements were performed in oscillation mode with a constant deformation of 0.5% and a constant angular frequency of 10 s.sup.1. The samples were equilibrated to a temperature of 8 C. (storage temperature) and 32 C. (skin temperature), respectively. The rheological properties of hydrogels were measured in rotation mode with a logarithmic increase in shear rate from 1-900 s.sup.1 followed by a logarithmic decrease in shear rate from 900-10 s.sup.1. The rheological properties investigated at a room temperature of 20 C. as well as at storage temperature of 8 C. The respective rheogramms are shown in FIG. 6. By an increasing shear rate the shear stress increases, whereas with a decreasing shear rate the process is reversed. The hysteresis loop, the area enclosed by the up and down curve, is a typical characteristic for thixotropic behavior. This behavior of the hydrogel allows shear thinning during the spraying process and afterwards a rearrangement of the hydrogel structure and its viscous properties on top of the skin.

[0173] 2.2.2 Spraying Forces

[0174] Spraying forces were tested using a TA.XT Plus Texture Analyser (Stable Micro Systems). A stainless steel plate was attached to the machine and used to compress the Ursatec 3K spray pump and determine their force needed to release the gel. The results for hydrogels having CMC concentrations of 0%, 0.5%, 1%, 1.5%, 2% and 2.5% CMC are shown in FIG. 7. Data are presented as meanSD (n=3).

[0175] 2.2.3 Viscosity

[0176] The viscosity at 8 C. and 32 C. of a hydrogel formulation comprising 1.5% w/w CMC and 12.5 mg/ml polyclonal antibody obtained as described was determined after preparation and after storage for 2 and 6.5 months. For comparison, the viscosity of a placebo formulation without antibody was determined. The results are shown in FIG. 8. Data is presented as meanSD (n=3).

[0177] 2.2.4 SEC Analysis

[0178] The potential aggregation was evaluated by size exclusion chromatography (SEC). SEC analytics was conducted on a Waters 2695 system (Waters GmbH, Eschborn, Germany). The flow rate of the running buffer (50 mM phosphate, 300 mM NaCl, pH 7.0) was 0.5 ml/min and 25 l of each sample was injected onto an YMC Pack-diol 300 column and detected with UV-detection at 280 nm. Protein concentrations were determined by SEC using a standard curve. The results are shown in FIG. 9. Aggregation in a freshly prepared batch of hydrogel, as prepared according to 2.1, was determined at to and after storage for 3, 5 and 11 weeks at 2 C. to 8 C., and for a second hydrogel batch after storage for 6.5 months. The results are shown in FIG. 9.

[0179] The relative amount of dimers only slightly varies over time. Only a minor amount of aggregated was observed even after >6 months of storage.

[0180] From this data it can be concluded that the secondary structure of the pIgG is stable in the gel formulation at the concentration desired for in vivo studies (10 mg/g).

[0181] 2.2.4. Circular Dichroism (CD) Analysis

[0182] Secondary structure is an important hallmark for activity of an antibody. Prevention of secondary structure in the hydrogel compared to PBS after preparation and upon storage for 7 weeks at 2 C. to 8 C. was analysed. The far-UV CD region (180-240 nm) corresponds to the peptide bond absorption and gives information on the secondary structure of a protein. For far-UV CD, the antibody hydrogel formulations were diluted to concentrations between 0.159 mg/ml and 0.209 mg/ml with phosphate buffered saline pH 7.4 and measured with a Jasco J-715 spectropolarimeter (Jasco International, Tokyo, Japan) in a quartz cuvette with a path length of 0.1 cm at 20 C. Far-UV CD spectra were collected in a continuous scanning method from 190 to 250 nm at a scanning speed of 50 nm/min, a response time of 1 s, a bandwidth of 1 nm, steps of 0.1 nm and an accumulation of 4 scans. Using the Spectra Analysis Software (Jasco International, Tokyo, Japan), the spectra were background corrected for the spectrum of the respective buffer or placebo gel and curves were smoothed. Data is recorded in millidegrees of ellipticity as a function of wavelength. The resulting spectra are shown in FIG. 10. Data shows the far-UV spectra of the antibodies in PBS directly after preparation as described above and after seven weeks of storage at 2-8 C. and the respective samples formulated in 1.5% CMC gel.

[0183] The spectra show typical curves of immunoglobulins representing high beta-sheet content. This can be concluded from the negative maximum217 nm, a zero ellipticity at 210 nm and a positive maximum200 nm. Differences in the amplitudes are caused by variation in the concentrations and the heterogeneity of antibody batches.

[0184] It can be concluded that there are no changes in secondary structure of the pIgG upon storage in PBS pH 7.4 or 1.5% CMC gel.

[0185] From CD and the SEC data it can be concluded that the secondary structure of the antibodies according to the invention are stable in the gel formulation according to the invention.

Example 3. Affinity Chromatography

[0186] 3.1 Column Preparation

[0187] A 1 ml affinity column comprising S. aureus Hla H35L prepared as follows. HiTrap NHS-activated HP Sepharose (GE Healthcare), 1 ml; with 10 mol NHS/ml Sepharose was washed with 6 ml of cold 1 mM HCl. Subsequently, the column was equilibrated twice and 1 ml recombinant alpha-hemolysin H35L protein solution (1 mg/ml in PBS) was added and incubated for 15 min at RT. Subsequently, the column was subjected to sequential washing with, 1 ml PBS, 6 ml 0.5 M Ethanolamine (pH 8.1, 0.5 M NaCl), 6 ml 0.1 M Natriumacetatebuffer (pH 4.0, 0.5 M NaCl), 6 ml 0.5 M Ethanolamine (pH 8.1, 0.5 M NaCl) and incubated for 15 min at RT. The sequential washing was repeated with a final washing step with 6 ml PBS. All flow rates were about 1 ml/min. Before chromatography, the column was equilibrated with 20 ml of Buffer A (0.1 M Glycin/Tris pH 8.0). Sample preparation: 1.6 ml of polyclonal S. aureus Hla H35L antibody obtained in Example 1 (75 mg/ml protein in PBS) were diluted with 10.4 ml Buffer A.

[0188] 3.2 Chromatography

[0189] Chromatography was performed at a flowrate of 1 ml/min. 10 ml of the sample solution were loaded on the affinity column. The flow through during loading was collected in fraction 1. Subsequently, the column was washed with 10 ml of Buffer A. The flow through during washing was collected in fraction 2. Elution was subsequently performed with Buffer B (0.1 M Glycin/HCl pH 2.7, 0.5 M NaCl). The eluate was collected in samples 3 to 12 in 1 ml fractions. Protein concentration of the flow through was monitored as absorbance at 280 nm. The two peak fractions 5 and 6 were pooled. 15 l 1.5 M Tris/HCl pH 8.8 was added to adjust the pH 7. The samples were concentrated with an Amicon Ultra-2, 100K (Millipore) cell by centrifugation over 5 min at 4800 rpm, Rotor S4180 (Beckman).

[0190] 3.3 Result

[0191] The chromatogram is shown in FIG. 1. It can be concluded from the relation of the AUC at 280 nm of the flow through during loading and the AUC of the elution fraction of the affinity column that the polyclonal antibody sample comprised about 3% of polyclonal IgG which specifically binds to the S. aureus Ha H35L protein.

Example 4: Determination of Toxin Binding by ELISA

[0192] 96-wells plates were coated with 0.3 g/ml recombinant S. aureus Ha. After blocking with BSA and washing with PBS with 0.05% Tween-20 the purified polyclonal anti S. aureus Hla IgG pool obtained from an immunized cow according to Example 1 (see. FIG. 2 Antibody solution) was added to the plate S. aureus starting with 100 g/ml and diluted in 1:2 dilution steps. Detection was done with a peroxidase conjugate Goat-anti-bovine Ig antibody (Jackson Immunoresearch). TMB was used as substrate and the reaction was stopped with H2SO4 (1N). Absorbance was measured at 450 nm. The assay was repeated with antibodies from a hydrogel formulation as obtained in Example 2.1 (see FIG. 2 Antibody Gel).

[0193] As shown in FIG. 2, the polyclonal S. aureus Hla IgG exhibits a concentration dependent binding to the recombinant S. aureus Hla.

[0194] The ELISA assay was repeated with S. aureus. beta-hemolysin, leukotoxins LukA, LukB, LukC, LukD, LukE, LukS, gamma-hemolysin components A (HlgA) and B (HlgB). The results for the ID50 values obtained in theses assays is summarized in Table 3.

TABLE-US-00003 TABLE 3 Toxin binding of purified polyclonal antibody Toxin ID50 in g/ml alpha-toxin 3 beta-toxin 25 LukA 0 LukB 0 LukD 25 LukE 50 LukF 25 LukS 0 HlgA 0 HlgB 25

[0195] Surprisingly, the IgG pool obtained from a cow immunized with alpha-hemolysin did not only comprise significant amounts of anti-alpha-hemolysin antibodies, but also antibodies reactive to beta-hemolysin, LukD, LukE, LukF, and HlgB.

Example 6: Neutralization of Hla Dependent Lysis of Rabbit Red Blood Cells

[0196] 6.1 Inhibition of Recombinant Alpha-Hemolysin Induced Lysis

[0197] The purified IgG obtained from a cow immunized with S. aureus Hla H35L as obtained in Example 1 and 2 was analyzed in a red blood cell-based neutralization assay. The ability of polyclonal anti S. aureus Hla IgG (PIgGs) before (FIG. 3 A, Antibody Solution) and after formulation (FIG. 3 A, Antibody Gel) to lyse rabbit erythrocytes (RBC) was tested in a 96-well format. Specifically, 140 l from each antibody sample was loaded into the first well and then serially diluted 2-fold, up to 1: 2048. After the dilution of each sample, 140 l of Hla (10 ng/ml) was added to each well incubated at RT for 2 h. 510.sup.6 rabbit RBC in 1PBS was added to each well and following incubation at RT for 2 h, plates were centrifuged for 5 min, 100 l of the supernatant was removed gently to a new microtiter plate, and absorbance was read at 415 nm.

[0198] As shown in FIG. 3A, the polyclonal anti S. aureus Hla IgG (PIgGs) exhibit a concentration dependent inhibition of RBC. Based on the Hla concentration in the assay and the amount of added polyclonal IgG, it can be concluded 37.5% of the anti S. aureus Hla IgG (PIgGs) are neutralizing.

[0199] 6.2 Inhibition of S. pseudintermedius Supernatant Induced Lysis

[0200] The purified IgG from a cow which was not immunized with an isolated antigen (natural IgG pool) was compared with the purified IgG from a cow immunized with S. aureus Ha H35L (aAT pool) obtained in Example 1 and Ha H35L affinity purified IgG obtained in Example 3 (aAT spec AB) in a red blood cell-based neutralization assay as described above in 6.1. In contrast to the assay of 6.1, culture supernatants from cultures of S. pseudintermedius strains 69687 and 4639949 were used instead of recombinant Hla for induction of lysis.

[0201] As shown in FIGS. 3A and 3B, the IgG from a cow which was not immunized with an isolated antigen (natural IgG pool) and the purified IgG from a cow immunized with S. aureus Ha H35L (aAT pool) surprisingly show a similar inhibition of S. pseudintermedius induced cell lysis, whereas the inhibition by Hla H35L affinity purified IgG obtained in Example 3 (aAT spec AB) exhibited a lower inhibition of cell lysis. Thus, the inhibition of hemolysis is only partially dependent on the anti Hla activity of the polyclonal antibodies.

Example 7: Antibody Binding to Drug Resistant S. pseudintermedius Strains

[0202] For surface staining of S. pseudintermedius with polyclonal antibody derived in Example 1, the bacteria were diluted to 5107 c/ml, in 15 l per well (OD0.5=5108 bacteria/ml) and Ab dilutions were prepared in PBS (0.1 BSA) to a 2 final concentration.

[0203] 15 l antibody solution were added to 15 l bacteria solution and incubated for 30 min at 4 C. and shaking at 750 rpm 170 l. PBS buffer were added to wash. After centrifugation 7 min at 3500 rpm, supernatants were taken off. Subsequently, 25 l of staining solution comprising anti-bovine IgG Alexa Fluor 647 1/350 (Jackson) was added and samples were incubated for 30 min at 4 C. and shaking at 750 rpm. The samples were washed with 200 l buffer. After centrifugation 7 min at 3500 rpm, supernatants were taken off pellets were fixed with 150 l 1% PFA ( 1/10 from stock 10%). Subsequently, the FACS measurement was performed. Table 4 shows the S. pseudintermedius analyzed:

TABLE-US-00004 TABLE 4 Drug resistant S. pseudintermedius strains Strain Source Phenotye Resistance Profile 23939 Ireland/2008/skin DR-MRSP OXA-PEN-AMP-LEX- ST68 SCC KAN-ERY-CLI-TET- mecV(T) SXT-CIP 69687 UK/2012/skin MDR-MRSP OXA-PEN-AMP-AMC- ST71 LEX-GEN-KAN-ERY- SCCmec11-111 CLI-SXT-CI P MRSPHH15 Germany/2012/ski MDR-MRSP OXA-PEN-AMP-AMC- ST71 LEX-GEN-KAN-ERY- SCCmec11-111 CLI-TET-SXT-CIP GL151A Germany/2012/wound MDR-MRSP AMP-AMC-LEX-GEN- ST71 KAN-ERY-CLI-TET - SCCmec11-111 SXT-CIP BNG I UK/2011/skin MRSP ST260 OXA-PEN-AMP-LEX- SCCmecV TET GL118B Germany/2012/skin MDR-MSSP PEN-AMP-KAN-TET ST262 4639949 USA/2012/skin MSSP ST309 PEN-AMP-TET GL117B Germany/2012/ear MDR-MSSP PEN-AMP-KAN-ERY ST263

[0204] Result: The anti-Hla antibody binds on the surface of various s. pseudintermedius strains in a concentration dependent manner. The resulting FACS diagrams are depicted in FIGS. 4 A and B.

Example 8: In Vivo Administration on Piglet Skin and Human Skin

[0205] 8.1 Piglet Skin

[0206] 8.1.1. Material and Methods:

[0207] A polyclonal anti-S. aureus Hla antibody as obtained in Example 2 was biotinylated and formulated in a hydrogel in accordance with Example 3. The hydrogel was applied in explanted piglet skin. Prior to application of the hydrogel, a part of the skin sample was laser porated with a P.L.E.A.S.E device (Pantec Biosolution, Ruggell, Liechtenstein) in a depth of 107 m.

[0208] The skin was subsequently cultivated over 24 h in a Franz-Cell. After 24 h the skin was cryo-conserved and sliced in a cryotome. The biotinylated antibody was visualized with streptavidin-Alexa Fluor 488. Subsequently, skin slides were examined by confocal laser microscopy.

[0209] 8.1.2. Results:

[0210] As shown the antibody comprised in the administered hydrogel does not penetrate intact skin and is localized on the skin surface (FIG. 5A). In the laser-porated parts of the skin, the antibody showns penetration and distribution into the dermis (FIG. 5C). FIG. 3B shows the skin morphology by HE stain.

[0211] 8.2. Human Skin

[0212] The effect of the polyclonal antibody according to the invention on the structure of epidermis and epidermal barrier in human skin sections colonialized with the methicillin-resistant Staphylococcus aureus strain USA300 was investigated. After colonization of S. aureus USA300 on 8 mm biopsies of normal human skin and co-application of 10 mg/ml polyclonal IgG as described above in PBS, the skin was cultivated over 24 h in a transwel system in a CO2 incubator. Afterwards the biopsies were frozen at 140 and kryosections were stained to investigate the morphology. The results are shown in FIG. 10. FIG. 10 a) shows a H&E stain of skin sections to investigate the morphology of epidermis and dermis, Magnification: 20FIG. 10 b) shows an immunofluorescence staining of E-cadherin on skin sections to make damage on cell-cell junctions visible. In green: goat-anti-mouse E-Cadherin+donkey-anti goat Alexa Fluor 488. In red: TO-PRO-3 nucleic acid (nucleus) counterstain. Scale bar: 50 m Magnification

Example 9: Clinical Trial

[0213] A clinical study was performed to assess the effect of the polyclonal antibody formulation obtained according to Example 2 for the treatment of intertrigo and pyotraumatic dermatitis in dogs. The study was performed according to the following protocol:

[0214] 9.1 Study Protocol

[0215] Introduction

[0216] The aim of this study is to evaluate the use of anti-polyclonal S. aureus antibodies in the treatment of two disorders, namely intertrigo (part 1) and pyotraumatic dermatitis (part 2).

[0217] Study Protocol:

[0218] Study DesignPart 1:

[0219] This is a study using polyclonal anti-polyclonal S. aureus Hla antibodies for the treatment of intertrigo in the dog. Owners will sign an informed consent form (Appendix 1) prior to inclusion in the study.

[0220] Inclusion Criteria:

[0221] Twenty dogs with intertrigo will be included in this study. Intertrigo will be diagnosed by history, clinical examination and cytology of impression smears of the folds showing neutrophils and numerous coccal bacteria present.

[0222] Exclusion Criteria:

[0223] Dogs will be withdrawn from the study if there is no improvement within the first two weeks after inclusion. Furthermore, dogs will be excluded with any severe adverse effects associated with the treatment. Lastly, a lack of owner compliance will be considered a reason for exclusion.

[0224] Intervention:

[0225] All owners will be asked to fill out a questionnaire regarding the clinical history and development of Intertrigo of the dog. Before start of the treatment blood is taken to analyze pre-existing antibodies against Staphylococcus. The intertrigo lesions will be treated with a polyclonal anti-S. aureus Hla antibody formulation as obtained in Example 2 twice daily. A clinician not involved in the treatment will judge the improvement clinically and cytologically. Improvement will be judged for clinical signs of erythema and exudate between 0 and 3 (0normal; 1Mild; 2Moderate; 3Severe), cytology results for the treated and control area for cocci, rods and yeast between 0 and 4 (0=No bacteria/yeast/inflammatory cells; 1=Occasional bacteria/yeast/inflammatory cells present, but slide must be scanned carefully for detection; 2=Bacteria/yeast/inflammatory cells present in low numbers, but detectable rapidly without difficulties; 3=Bacteria/yeast/inflammatory cells present in larger numbers and detectable rapidly without any difficulties; 4=Massive amounts of bacteria/yeast/inflammatory cells present and detectable rapidly without difficulties).

[0226] Clinical Evaluation:

[0227] Photographs will be taken from each affected area and labeled with intertrigo_ownername_dogname_date.jpg. Cutaneous cytology will be obtained from each affected area at each visit. The cytology specimens will be air dried and stained with Diff Quick. They will be evaluated by a veterinarian not involved in the treatment in a blinded fashion using a previously reported semiquantitative scale (Budach et al 2012).

[0228] Study DesignPart 2:

[0229] This is a study using polyclonal anti-polyclonal S. aureus Hla antibodies for the treatment of pyotraumatic dermatitis in the dog. Owners will sign an informed consent form prior to inclusion in the study.

[0230] Inclusion Criteria:

[0231] Ten dogs with pyotraumatic dermatitis will be included in the study. The condition will be diagnosed by history, clinical examination and cytology of the lesion showing neutrophils and coccal bacteria.

[0232] Medications to treat other concurrent diseases such as flea allergy dermatitis can be continued during the study.

[0233] Exclusion Criteria:

[0234] Dogs will be withdrawn from the study if there is no improvement within the first two weeks after inclusion. Furthermore, dogs will be excluded with any severe adverse effects associated with the treatment. Lastly, a lack of owner compliance will be considered a reason for exclusion.

[0235] Intervention:

[0236] All owners will be asked to fill out a questionnaire regarding the clinical history and development of hot spot of the dog. The affected area will be clipped with a sterilized no #40 clipper blade and cleaned with an antiseptic. All dogs will be treated with prednisolon at 0.5-1 mg/kg daily for three days and all dogs will be treated twice daily with anti-polyclonal S. aureus Hla in a hydrogel spray comprising 10 mg/ml polyclonal anti S. aureus Ha as obtained in Example 3 for 14 days. After the treatment period is finished blood will be taken to analyze anti-Staphylococcus antibodies.

[0237] Clinical Evaluation:

[0238] Photographs will be taken from the lesion and labeled with podo_ownername_dogname_date.jpg. Cutaneous cytology will be obtained from the lesion prior to inclusion and after two weeks. The cytology specimen will be air dried and stained with Diff Quick. They will be evaluated in a blinded fashion by a veterinarian using a previously reported semiquantitative scale (Budach et al 2012)).

[0239] 9.2. Results

[0240] The first dog with hot spot was included in the study showed an improvement of symptoms and no side effects after treatment. The effect of the treatment was confirmed in 8 other canine patients.

Example 10: Conformational Epitope Mappings of Polyclonal Antibody

[0241] 10.1. Material and Methods

[0242] Microarray Content: The sequence of Staphylococcus aureus alpha toxin was elongated by neutral GSGSGSG (SEQ ID NO: 185) linkers at the C- and N-terminus to avoid truncated peptides. The elongated antigen sequence was translated into linear 7, 10 and 13 amino acid peptides with peptide-peptide overlaps of 6, 9 and 12 amino acids. After peptide synthesis, all peptides were cyclized via a thioether linkage between a C-terminal cysteine and an appropriately modified N-terminus. The resulting conformational alpha toxin peptide microarrays contained 963 different peptides printed in duplicate (1,926 peptide spots), and were framed by additional HA (YPYDVPDYAG (SEQ ID NO: 186), 98 spots) control peptides. [0243] Samples: Polyclonal antibody obtained from cows not immunized with isolated antigen according to Example 2 (Sample A); polyclonal antibody obtained from cows immunized with aureus Hla H35 protein; affinity purified polyclonal antibody obtained according to Example 3 (Sample C). [0244] Washing Buffer: PBS, pH 7.4 with 0.005% Tween 20 (210 sec after each assay) [0245] Blocking Buffer: Rockland blocking buffer MB-070 (30 min before the first assay) [0246] Incubation Buffer: Washing buffer with 10% blocking buffer [0247] Assay Conditions: Antibody concentration of 10 g/ml in incubation buffer; incubation for 16 h at 4 C. and shaking at 140 rpm [0248] Secondary Antibodies: Rabbit anti-bovine IgG (Fc) DyLight800 (1:2000) and anti-bovine IgG (H+L) DyLight680 (1:2000); 45 min staining in incubation buffer at RT [0249] Control Antibody: Mouse monoclonal anti-HA (12CA5) DyLight680 (1:2000) and mouse monoclonal anti-HA (12CA5) DyLight800 (1:2000); 45 min staining in incubation buffer at RT [0250] Scanner: LI-COR Odyssey Imaging System; scanning offset 0.65 mm, resolution 21 m, scanning intensities of 7/7 (red=680 nm/green=800 nm)

[0251] Pre-staining of a conformational alpha toxin peptide microarray was performed with secondary rabbit anti-bovine IgG (Fc) DyLight800 antibody (1:2000) for IgG analysis or with secondary anti-bovine IgG (H+L) DyLight680 antibody (1:2000) and with control mouse monoclonal anti-HA (12CA5) DyLight800 antibody (1:2000) for total Ig analysis to investigate background interactions with the cyclic constrained antigen-derived peptides that could interfere with the main assays. Subsequent incubation of other alpha toxin peptide microarrays with the polyclonal antibody samples A, B and C at a concentration of 10 g/ml in incubation buffer was followed by staining with the secondary and control antibodies as well as read-out at scanning intensities of 7/7 (red/green). The additional HA peptides framing the peptide microarrays were simultaneously (rabbit anti-bovine IgG (Fc) DyLight800 antibody) or subsequently (anti-bovine IgG (H+L) DyLight680 antibody) stained as internal quality control to confirm the assay quality and the peptide microarray integrity.

[0252] Quantification of spot intensities and peptide annotation were based on the 16-bit gray scale tiff files at scanning intensities of 7/7 that exhibit a higher dynamic range than the 24-bit colorized tiff files. Microarray image analysis was performed with PepSlide Analyzer. A software algorithm broke down fluorescence intensities of each spot into raw, foreground and background signal, and calculated averaged median foreground intensities and spot-to-spot deviations of spot duplicates. Based on averaged median foreground intensities, an intensity map was generated and interactions in the peptide map highlighted by an intensity color code with red for high and white for low spot intensities. A maximum spot-to-spot deviation of 40% was tolerated, otherwise the corresponding intensity value was zeroed.

[0253] Additionally, averaged spot intensities of the assays with the antibody samples were plotted against the antigen sequence from the N- to the C-terminus of Staphylococcus aureus alpha toxin was to visualize overall spot intensities and signal-to-noise ratios. The intensity plots were correlated with peptide and intensity maps as well as with visual inspection of the microarray scans to identify epitopes that were recognized by the antibody samples. In case it was not clear if a certain amino acid contributed to antibody binding, the corresponding letters were written in gray. For a better data overview, the baselines of the intensity plots were leveled.

[0254] 10.2 Anti-Bovine IgG (H+L) DyLight680 (1:2000), Anti-Bovine IgG (Fc) DyLight800 (1:2000)

[0255] The alpha toxin peptide microarray was incubated with (1) secondary rabbit anti-bovine IgG (Fc) DyLight800 antibody (1:2000) and (2) with secondary anti-bovine IgG (H+L) DyLight680 antibody (1:2000) and control mouse monoclonal anti-HA (12CA5) DyLight800 antibody (1:2000) was followed by read-out at a scanning intensities of 7/7 (red/green). As shown in FIG. 12, no background interaction of secondary anti-bovine IgG (H+L) DyLight680 antibody in the red channel at 700 nm; weak background interactions of secondary rabbit anti-bovine IgG (Fc) DyLight800 antibody with basic peptides with the consensus motifs KKILVIRTK (SEQ ID NO: 4) and KIDWEKKK (SEQ ID NO: 187) in the green channel at 800 nm was observed. A well-defined staining of HA control peptides was observed.

[0256] 10.3. Sample A (Polyclonal Antibody Pool Before Immunization), 10 g/ml

[0257] The alpha toxin peptide microarray was incubated with sample A at a concentration of 10 g/ml was followed by staining with the secondary and control antibodies as well as read-out at a scanning intensities of 7/7 (red/green). As shown in FIG. 13, a weak IgG response against a epitope-like spot patterns formed by adjacent peptides with the consensus motifs KIGGLIG (SEQ ID NO: 2) with all peptide lengths, weak background interaction of the secondary antibody with peptides with the basic KKILVIRTK (SEQ ID NO: 4); additional weak and presumably less specific non-IgG response against peptides with the C-terminal ATKQQSN (SEQ ID NO: 3) motif was observed. A moderate signal-to-noise ratios was observed. Also, a well-defined subsequent staining of HA control peptides was observed.

[0258] 10.4 Sample B (Polyclonal Antibody Pool after Alpha Toxin Immunization), 10 g/ml

[0259] The alpha toxin peptide microarray was incubated with sample B at a concentration of 10 g/ml was followed by staining with the secondary and control antibodies as well as read-out at a scanning intensities of 7/7 (red/green). As shown in FIG. 14, a very strong IgG response against a epitope-like spot patterns formed by adjacent peptides with the consensus motifs KIGGLI (SEQ ID NO: 198) with all peptide lengths, weak background interaction of the secondary antibody with peptides with the basic KKILVIRTK (SEQ ID NO: 4) motif; no additional non-IgG response was observed. Also, a high signal-to-noise ratios and a well-defined subsequent staining of HA control peptides was observed.

[0260] 10.5 Sample C (Polyclonal Antibody Pool after Alpha Toxin Immunization), 10 g/ml

[0261] The alpha toxin peptide microarray was incubated with sample C at a concentration of 10 g/ml was followed by staining with the secondary and control antibodies as well as read-out at a scanning intensities of 7/7 (red/green). As shown in FIG. 15, a very strong IgG response against peptides with the consensus motifs KIGGLI (SEQ ID NO: 198) with all peptide lengths, weak additional IgG response against peptides with the IDVIYERV (SEQ ID NO: 5) motif, weak background interaction of the secondary antibody with peptides with the basic KKILVIRTK (SEQ ID NO: 4); additional weak non-IgG responses against peptides with the consensus motifs KAADNFLDP (SEQ ID NO: 6) and DSDINIK (SEQ ID NO: 7) was observed. Also, a high signal-to-noise ratios and well-defined subsequent staining of HA control peptides was observed.

[0262] 10.6 Summary of Results

[0263] The PEPperMAP Conformational Epitope Mappings of polyclonal antibodies obtained from cows not immunized with isolated antigen according to Example 2 (Sample A), polyclonal antibodies obtained from cows immunized with S. aureus Ha H35L protein and affinity purified polyclonal antibodies obtained according to Example 3 (Sample C) were performed against alpha toxin of Staphylococcus aureus translated into cyclic constrained 7, 10 and 13 amino acid peptides with peptide-peptide overlaps of 6, 9 and 12 amino acids. The corresponding alpha toxin peptide microarrays were incubated with the antibody samples at a concentration of 10 g/ml in incubation buffer followed by staining with secondary and control antibodies as well as read-out with a LI-COR Odyssey Imaging System. Quantification of spot intensities and peptide annotation were done with PepSlide Analyzer.

[0264] Pre-staining of an alpha toxin peptide microarray with secondary rabbit anti-bovine IgG (Fc) DyLight800 antibody or with secondary anti-bovine IgG (H+L) DyLight680 antibody and control mouse monoclonal anti-HA (12CA5) DyLight800 antibody highlighted very weak background interaction of the secondary rabbit anti-bovine IgG (Fc) DyLight800 antibody with basic peptides with the consensus motifs KKILVIRTK (SEQ ID NO: 4) and KIDWEKKK (SEQ ID NO: 187). Incubation of the antibody samples resulted in the following observations:

[0265] Sample A (polyclonal antibodies obtained from cows not immunized with isolated antigen) showed a weak IgG response against peptides with the consensus motif KIGGLIG (SEQ ID NO: 2), albeit with 20 fold lower spot intensities and signal-to-noise ratios compared to samples B and C; moreover, we observed a weak and presumably less specific non-IgG response against peptides with the C-terminal ATKQQSN (SEQ ID NO: 3) motif

[0266] Sample B (polyclonal antibodies obtained from cows immunized with aureus Hla H35 protein) showed a very strong IgG response against peptides with the consensus motif KIGGLI (SEQ ID NO: 198); otherwise we only observed a weak background interaction of the secondary rabbit anti-bovine IgG (Fc) DyLight800 antibody with peptides with the basic consensus motif KKILVIRTK (SEQ ID NO: 4); additional IgA or IgM responses were not identified

[0267] Sample C (affinity purified polyclonal S. aureus Hla H35 antibodies) also showed a very strong IgG response against peptides with the consensus motif KIGGLI (SEQ ID NO: 198) as well as a weaker IgG response against peptides with the consensus motif IDVIYERV (SEQ ID NO: 5); moreover, we observed additional weak IgA or IgM responses against peptides with the consensus motifs KAADNFLDP (SEQ ID NO: 6) and DSDINIK (SEQ ID NO: 7).

Example 11: IgG Response Profiling of Bovine Polyclonal Antibody Pool B and a Placebo Control Against 122 Staphylococcus Aureus Antigens

[0268] 11.1: Materials and Methods

[0269] Microarray Content: The IgG response profiling was done against 122 Staphylococcus aureus antigens that were elongated with neutral GSGSGSG (SEQ ID NO: 185) linkers to avoid truncated peptides. The linked and elongated antigen sequences were translated into 13 amino acid peptides with a peptide-peptide overlap of 11 amino acids. After peptide synthesis, all peptides were cyclized by thioether formation between a C-terminal cysteine side chain and an appropriately modified N-terminus. The resulting conformational PEPperCHIP Staphylococcus aureus Discovery Microarrays contained 29,519 different peptides printed in duplicate (59,038 peptide spots), and were framed by additional HA control peptides (782 spots). Positive control antigen alpha-hemolysin (UniProt ID: P09616) was incorporated five times.

[0270] Samples: Bovine antibody Sample A (polyclonal antibody pool before immunization) and Sample B (polyclonal antibody pool after alpha toxin immunization).

[0271] Washing Buffer: PBS, pH 7.4 with 0.005% Tween 20 (230 sec or 210 sec after each assay)

[0272] Blocking Buffer: Rockland blocking buffer MB-070 (30 min before the first assay)

[0273] Incubation Buffer: Washing buffer with 10% blocking buffer

[0274] Assay Conditions: Antibody concentration of 10 g/ml in incubation buffer; incubation for 16 h at 4 C. and shaking at 140 rpm

[0275] Secondary Antibody: Rabbit anti-bovine IgG (Fc) DyLight800 (1:2000); 45 min staining in incubation buffer at RT

[0276] Control Antibody: Mouse monoclonal anti-HA (12CA5) DyLight680 (1:2000); 45 min staining in incubation buffer at RT

[0277] Scanner: LI-COR Odyssey Imaging System; scanning offset 0.65 mm, resolution 21 m,

[0278] scanning intensities of 7/7 (red=700 nm/green=800 nm)

[0279] Microarray ID: 001846_01V, 001846_02V

[0280] After 15 min pre-swelling in washing buffer and 30 min in blocking buffer, a PEPperCHIP Staphylococcus Aureus Discovery Microarray was initially incubated with secondary rabbit anti-bovine IgG (Fc) DyLight800 antibody (1:2000) to analyze background interactions with the cyclic constrained antigen-derived peptides that could interfere with the main assays. Subsequent incubation of the PEPperCHIP Staphylococcus Aureus Discovery Microarrays with polyclonal bovine antibodies Sample B and Placebo ColBiot at a concentration of 10 g/ml in incubation buffer was followed by staining with secondary and control antibodies as well as read-out with a LI-COR Odyssey Imaging System at scanning intensities of 7/7 (red/green). The additional HA peptides framing the peptide microarrays were subsequently stained as internal quality control to confirm the assay quality and the peptide microarray integrity.

[0281] Quantification of spot intensities and peptide annotation were based on the 16-bit gray scale tiff files at a scanning intensity of 7 (green) that exhibit a higher dynamic range than the 24-bit colorized tiff files; microarray image analysis was done with PepSlide. A software algorithm broke down fluorescence intensities of each spot into raw, foreground and background signal, and calculated averaged median foreground intensities and spot-to-spot deviations of spot duplicates. A maximum spot-to-spot deviation of 40% was tolerated, otherwise the corresponding intensity value was zeroed. Based on averaged median foreground intensities, intensity maps were generated and interactions in the peptide maps highlighted by an intensity color code with green for high and white for low spot intensities.

[0282] To identify the top IgG responses of polyclonal bovine antibodies of Sample A and Sample B, the averaged and corrected intensity values were sorted by decreasing spot intensities. The averaged spot intensities of the assays were spotted with the bovine samples against the microarray content from left on top to right on bottom of the chip to visualize overall spot intensities and signal-to-noise ratios. The intensity plots were correlated with the peptide and intensity maps as well as with visual inspection of the microarray scans to identify epitopes that were recognized by the polyclonal cow antibodies Sample A and Sample B.

[0283] 11.2. Sample a (Polyclonal Antibody Pool Before Immunization), 10 g/ml

[0284] Data quantification was followed by generation of an intensity plot that highlighted the IgG response profile of polyclonal bovine antibody Sample A (10 g/ml) with the 29,519 cyclic constrained peptides of the PEPperCHIP S. Aureus Discovery Microarray.

[0285] A number of moderate and few strong IgG responses against peptides with the annotated consensus motifs next to the corresponding signal in the intensity plot were observed.

[0286] The top 35 interactions of polyclonal bovine antibody Sample A were sorted by decreasing spot intensities and are shown in FIG. 17.

[0287] A very strong IgG responses with lysine-rich peptides with the consensus motif AAKKKKK (SEQ ID NO: 188) (extracellular matrix and plasma binding protein Ebh) as well as other moderate responses with predominantly basic epitopes of immunoglobulin-binding protein sbi, penicillin-binding protein 2 Meca, serine-aspartate repeat protein C Sdrc and hyarulonate lyase was observed.

[0288] 11.3 Sample B (Polyclonal Antibody Pool after Alpha Toxin Immunization), 10 g/ml

[0289] Data quantification was followed by generation of an intensity plot that highlighted the IgG response profile of polyclonal bovine antibody Sample B (10 g/ml) with the 29,519 cyclic constrained peptides of the PEPperCHIP S. Aureus Discovery Microarray.

[0290] A number of strong to very strong IgG responses against peptides with the annotated consensus motifs next to the corresponding signal in the intensity plot were observed.

[0291] The top 35 interactions of polyclonal bovine antibody Sample B were sorted by decreasing spot intensities and are shown in FIG. 18.

[0292] A complex response with strong to very strong IgG interactions with epitopes of positive control alpha-hemolysin as well as epitopes of clumping factor A, serine-aspartate repeat protein E, collagen adhesin, Staphylococcal enterotoxin K, iron-regulated surface determinant protein B Isdb or extracellular matrix and plasma binding protein Ebh was observed.

[0293] 11.4 Summary

[0294] Comparison of the IgG responses of Sample B (polyclonal antibody pool after alpha toxin immunization) and Sample A (polyclonal antibody pool before immunization) control assayed at a concentration of 10 g/ml in incubation buffer with the background interactions of the secondary antibody.

[0295] The background interactions of the secondary antibody turned out to be widely negligible; Sample B exhibited a stronger and more complex IgG response compared to Sample A, the main responses were annotated in the intensity plot. Comparison of the top 40 interactions of Sample B with Sample A and the secondary antibody.

[0296] Most of the top IgG responses of Sample B were exclusively found in, or exhibited significantly higher spot intensities compared to Sample A (epitopes of clumping factor A, serine-aspartate repeat protein E, collagen adhesin); background interactions of the secondary antibody did not interfere at all.

[0297] The IgG response profiling of polyclonal bovine antibodies Sample B and Sample B was done with a multiplexed conformational epitope mapping of 122 Staphylococcus aureus antigens translated into cyclic constrained 13 amino acid peptides with a peptide-peptide overlap of 11 amino acids. The resulting conformational PEPperCHIP Staphylococcus Aureus Discovery Microarrays contained 29,519 antigen-derived cyclic constrained peptides printed in duplicate as well as additional HA control peptides (782 spots). As positive control, the peptides of alpha-hemolysin were incorporated five times. The PEPperCHIP Staphylococcus Aureus Discovery Microarrays were incubated with the bovine antibody samples at a concentration of 10 g/ml in incubation buffer followed by staining with secondary and control antibodies as well as by read-out with a LI-COR Odyssey Imaging System. Quantification of spot intensities and peptide annotation were done with PepSlide Analyzer.

[0298] Pre-staining of a PEPperCHIP Staphylococcus Aureus Discovery Microarray with secondary rabbit anti-bovine IgG (Fc) DyLight800 antibody highlighted few weak background interactions of the secondary antibody with arginine-rich and lysine-rich basic peptides like KPIPVRLIKRKKY (SEQ ID NO: 189), RKIKKVSKNKKRV (SEQ ID NO: 190), KSTAPKRLNTRMR (SEQ ID NO: 191), YLTRHYLVKNKKL (SEQ ID NO: 192), ALGSLLLFGRRKK (SEQ ID NO: 193), KHLPLLFAKRRRK (SEQ ID NO: 194), KVKHTPFFLPKRR (SEQ ID NO: 195) and SSIVAFVLPRKRK (SEQ ID NO: 196) presumably due to non-specific ionic binding. Compared to the IgG responses of the bovine antibody samples, these background interactions turned out to be widely negligible.

[0299] Polyclonal bovine antibody Sample A exhibited a very strong IgG response with basic peptides with the consensus motif AAKKKKK (SEQ ID NO: 188) (extracellular matrix and plasma binding protein Ebh) as well as other moderate responses with predominantly basic epitopes of immunoglobulin-binding protein sbi (ASENTQQTSTK, SEQ ID NO: 14), penicillin-binding protein 2 Meca (RKIKKVSKNKK, SEQ ID NO: 15), serine-aspartate repeat protein C Sdrc (TANQSTTKT, SEQ ID NO: 16), hyarulonate lyase (LNTDENK, SEQ ID NO: 17) and superantigen-like protein set 1/superantigen-like protein set 3 (LQTNRMS, SEQ ID NO: 18).

[0300] Sample B exhibited a significantly more complex and stronger IgG response than Sample A. The strongest responses were attributed to peptides with the consensus motif TGKIGGLIG (SEQ ID NO: 197) of positive control alpha-hemolysin. Other strong IgG responses were assigned to presumed epitopes of clumping factor A (VPEQPDEPG, SEQ ID NO: 8), serine-aspartate repeat protein E (EKKAPNNTNND, SEQ ID NO: 9), collagen adhesin (YTTHVDNND, SEQ ID NO: 10), Staphylococcal enterotoxin K (TFHLNNNDT, SEQ ID NO: 11), iron-regulated surface determinant protein B Isdb (DSKPEIELG, SEQ ID NO: 12) or extracellular matrix and plasma binding protein Ebh (TAMPTNLAGGSTT, SEQ ID NO: 13). Most of the top IgG responses of Sample B were exclusively found in this sample, or exhibited significantly higher spot intensities compared to bovine polyclonal antibody from Sample B (epitopes of clumping factor A, serine-aspartate repeat protein E, collagen adhesin).

[0301] A comparison of the antibody responses is shown in FIG. 19.

Example 12: Dog Skin Infection Model: Epidermal Skin Damage on Histological Slides

[0302] Methodology:

[0303] Punching of biopsies with a perimeter of 8 mm from healthy ex vivo dog skin were obtained. The biopsies were transferred into a transwell system (Corning) with a diameter of 6.5 mm per well and a membrane pore size of 0.4 m. Subsequently 110.sup.8 CFU/ml Staphylococcus pseudintermedius (eithertoxigenic strain 69687 or non-toxigenic strain GL151A) and/or 10 mg/ml IgG from cows without or with immunization with H35L (Samples A and B respectively) were applied to the cultures. Cultivation with Williams E medium in the lower chamber over 48 in a CO.sub.2 incubator.

[0304] The skin biopsy were removed from the transwell system and fixing with formalin and embedding in paraffin.

[0305] Staining of skin sections was performed with H&E to stain nuclei of cells, extracellular material and cytoplasmic proteins. The H&E stained slides were scanned and at a 30 fold magnification 10 areas with a perimeter of 500 m and an area of 0.016 mm.sup.2 were randomly picked at the number of viable and dead cells and cells showing pyknotic nuclei were counted.

[0306] Result:

[0307] Quantitative results of cell counting is show in FIG. 20. In summary, the antibody from cows without H35L shows better protection of skin damage by non-toxigenic strain, whereas the anti-alpha toxin antibody from cows immunized with H35L shows better protection of skin damage by toxigenic strain.

Example 13: Effect of Bovine IgG on the Growth of Antibiotic Resistant S. pseudintermedius

[0308] 13.1 Method: Minimal Inhibitory Concentration Assay

[0309] In order to test the antibiotic resistance of Staphylococcus pseudintermedius against Clindamycin, which is the standard antibiotic used in clinics, the minimal inhibiting concentration was assessed. Bacteria from strains 69687, HH15, GL151A and 23929 were grown overnight in 12 mL tubes (Greiner Bio-One International GmbH, Germany) with sterile T-Hewitt medium at 37 C. (Elbanton LT650 incubator, Gembini BV, The Netherlands) and 600 rpm (VWR Mini Shaker, VWR International, United States of America). The OD600 was measured and the bacterial concentration adjusted to 10.sup.6 bacteria/mL. 20 g/mL clindamycin was weighed in and dissolved in T-Hewitt medium. The triple assay was performed in 96-well u-bottom microplates (Greiner Bio-One International GmbH, Germany). The clindamycin was serially diluted (1:2) with a Pipet-Lite XLS+ (Mettler-Toledo International Inc., United States of America) multichannel pipet and the bacteria were added. As alternatives bovine IgG or BSA as control were titrated with a starting concentration of 6.3 mg/ml as well as a constant concentration (6.3 mg/ml) of bovine IgG or BSA as control were co-incubated with the dilution series of clindamycin. The positive control contained no antibiotic and the negative control contained neither antibiotics nor bacteria. The plates were incubated for 24 h at 37 C. without shaking. After the incubation the microplates were scanned with the Epson Perfection V700 Photo scanner (Seiko Epson K.K, Japan) and the minimal inhibitory concentration was calculated according to the dilution step of clindamycin.

[0310] Result:

[0311] To assess the effect of bovine IgG against canine S. pseudintermedius a combination of MIC assay and pharmacological testing was developed. Within this assay the growth-inhibiting effect of clindamycin, the combinational therapy of antibiotics and bovine IgG and the solo effect of bovine IgG in a concentration dependent manner could be determined. As a control bovine serum albumin (BSA) was added to the assay.

[0312] The MDR MRSP minimal inhibiting concentrations against clindamycin could be determined. 69687, HH15, GL151A and 23929 show their breakthrough points at 0,625 mg/ml clindamycin (FIG. 21A, upper panel). When a constant concentration of 6.3 mg/mL bovine IgG was added, the colonies showed deformation and a different morphology (FIG. 21A, lower panel). This suggests, that the antibodies interfered with the bacterial growth. The effect of bovine IgG on the bacterial growth is concentration dependent. The growth of 69687 was changed throughout the entire dilution series of bovine IgG. HH15, GL151A and 23929 showed altered and reduced colony formation at certain antibody dilutions (FIG. 21B). The combination of clindamycin and BSA and the dilution of BSA showed no alteration and leads to the assumption, that the growth inhibiting effect of bovine IgG is specific (FIG. 21C).

Example 14: Scanning Electron Microscope Analysis of Skin

[0313] 14.1. Method: Scanning Electron Microscopy

[0314] Skin biopsies cultivated with S. pseudintermedius and alternatively treated with 10 mg/mL bovine IgG in accordance with Example 12. The skin tissue was fixed in 1% (v/v) glutaraldehyde (Sigma-Aldrich Corporation, United States of America) in PBS at 4 C. In the next step the samples were consecutively dehydrated in 30 minutes incubations with 10% (v/v), 25% (v/v) and 50% (v/v) ethanol (Merck KGaA, Germany) diluted in PBS. Subsequently incubation with 75% (v/v) and 90% (v/v) ethanol-water dilutions occurred, followed by two incubations with 96% ethanol. Lastly the tissue was treated with 50% (v/v) ethanol-hexamethyldisilazane (HMDS, Sigma-Aldrich Corporation, United States of America) and 100% HMDS. The samples were air-dried at room temperature overnight and mounted onto 12 mm specimen stubs (Agar Scientific Ltd., United Kingdom). The coating with 5 nm gold was performed using a Quorum Q150R sputter coater (Quorum Technologies Ltd., United Kingdom). The images were taken with the scanning electron microscope (SEM) Scios DualBeam (Thermo Fisher Scientific, United States of America) and colored using the Adobe Photoshop CS6 software.

[0315] 14.2. Result:

[0316] For understanding of the localization and colony formation of Staphylococci on canine skin scanning electron microscopy was utilized. After a 24 h colonization with bacteria with or without antibody treatment the skin samples were serially dehydrated and coated with gold. As shown in FIG. 22 A, the skin samples without treatment showed massive bacterial colonies. S. pseudintermedius was rarely found in form of individual cells but rather as comprehensive patches. Furthermore, they appeared close to hair follicles. As shown in FIG. 22 B, in presence of 10 mg/mL bovine IgG, the bacteria showed an altered behavior. Comprehensive bacterial colonization could not be identified. Contrariwise, small patches of bacteria very often hidden underneath dead cells of the stratum corneum.

Example 15: Inhibition of Bacterial Colony Formation on Skin

[0317] 15.1. Method: Bacterial Adhesion Assay with Canine Skin Tissue

[0318] The canine skin was obtained as waste material from terminal animal experiments from the Veterinary Clinic at the University of Utrecht and biopsies were cut out as previously described. The tissue was incubated with 110.sup.6 GL 151A S. pseudintermedius bacteria alone or in combination with 10 mg/ml bovine IgG or as a control with an antibody directed against the chemical 2,4-Dinitrophenol (DNP) for four hours at 37 C. and 5% CO.sub.2. After cultivation the biopsies were washed 3 with sterile PBS to remove non-adherent bacteria. Subsequently, the samples were placed in cryotubes containing twelve sterile bead beater beads and 1 mL of sterile physiological salt solution was added. The cells were lysed for 45 seconds at full speed with the help of the Bead Bug Microtube Homogenizer (Benchmark Scientific, United States of America). After the homogenization the lysed tissue was diluted 1:10 and 1:100 in sterile MQ-water. 100 L of these dilutions were plated on MRSA Colorex Chromogenic Media (bioTRADING Benelux B.V., The Netherlands). The plates were incubated overnight at 37 C. The colony forming units were counted and according to the dilutions the amount of bacteria adherent to the mammalian cells were calculated.

[0319] 15.2. Result:

[0320] Adhesion assays were performed to quantify the adherent bacteria on canine skin. The graph in FIG. 23 shows a reduction in colony forming units in presence of bovine IgG. The anti-DNP control antibody also showed an unspecific decrease in bacterial colonies.

Example 16: Expression of Th2 Cytokine IL-13 and Th1 Cytokine TNF

[0321] 16.1. Method:

[0322] Skin biopsies of dogs were co-cultured with 110.sup.6 69687 S. pseudintermedius bacteria alone or in combination with 10 mg/ml bovine IgG or Clindamycin. After 30 h the biopsies were frozen at 150 C. and cryosection of 20 m size were cut. RNA was isolated from 50-60 slices of 20 m canine skin sections using the RNeasy Micro Kit (QUAGEN, Germany). Before starting with the isolation, the working space and used non-sterile materials were cleaned with RNAseZAP to reduce the presence of RNases. Prior following the kit-protocol, the samples were thawed on ice, vortexed and syringed up and down six times through a 1 mL syringe with 0.6 mm needle to homogenize the sample. In the next step 580 L of RNAse free water were pipetted to the sample and 20 L of a 10 mg/mL proteinase K were added. The sections were incubated for ten minutes at 55 C. on a heating block and spinned down for eight minutes at 12000 rpm (Hettich MIKRO 120 centrifuge, Hettich Benelux B.V., United States of America). The supernatant was transferred into new 2 mL tubes and the standard protocol for the RNeasy Micro Kit was executed.

[0323] The isolated RNA, which originated from either canine skin tissue was transcribed into cDNA with the iScript cDNA Synthesis Kit (Bio-Rad Laboratories Inc., United States of America). The RNA of all samples was diluted to the concentration of the samples with the lowest RNA content.

[0324] Real-time quantitative PCR was performed with the Step One Plus Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific, Unites States of America), using iQ SYBR Green Supermix kit (Bio-Rad Laboratories Inc., United States of America). The genomic DNA was diluted to a concentration of 10 g/L.

[0325] For the analysis, the cycle threshold (Ct) values of the samples were referred to a housekeeping gene (RPS19). To understand the effect of the bovine IgG or clindamycin treatment, the calculated Ct values of treatment samples were compared to non-treatment values (Ct). The fold-expression compared to the untreated samples, could be determined by calculating 2Ct

C_t=C_(t Target)C_(t Housekeeping gene)

C_t=C_(t Treated)C_(t Untreated)

Fold increase=2{circumflex over ()}(C_t)

[0326] 16.1. Result:

[0327] With the help of qPCR the expression of cytokines, produced by canine explant skin during 30 h cultivation with S. pseudintermedius, were studied. Quantification of the fold expression compared to the reference sample was calculated. Dog ex vivo skin was infected with S. pseudintermedius strain 69687. As shown in FIG. 24 B, the expression of the pro-inflammatory cytokine TNF was elevated approximately 3 in comparison to the uninfected and untreated reference sample. Treatment with bovine IgG could downregulate the expression level, clindamycin lowered the expression to a higher extend. As shown in FIG. 24 A, the Th2 cytokine IL-13 showed a different expression pattern. Infection with 69687 upregulated its expression 4. Bovine IgG strongly downregulated the cytokine quantities, whereas clindamycin treatment upregulated the production. Thus, TNF was produced in a lower quantity than IL-13 during infection with 69687. Clindamycin had a downregulating effect for TNF, whereas IL-13 was upregulated during infection with 69687.

REFERENCES

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