MODULAR ANTIGEN TRANSPORTATION MOLECULES AND USES THEREOF IN ANIMALS

Abstract

The present invention relates to (isolated) recombinant proteins, also referred to as improved MAT (iMAT) molecules, comprising at least one translocation module, at least one targeting module and at least one antigen module, wherein at least one cysteine residue is substituted with a different amino acid residue. Such iMAT molecules are useful specifically as vaccines, e.g. for therapy and/or prevention of allergies and/or infectious diseases and/or prevention of transmission of infectious diseases in animals, more preferably ruminants, pigs, dogs and/or cats, but excluding equines. The present invention further relates to nucleic acids encoding such iMAT molecules, corresponding vectors and primary cells or cell lines.

Claims

1. An improved MAT (iMAT) molecule, comprising: (i) at least one first module being an amino acid sequence allowing the translocation of the iMAT molecule from the extracellular space into the interior of cells, (ii) at least one second module being an amino acid sequence allowing species-specific intracellular targeting of the iMAT molecule to the cell organelles which are involved in the processing of antigens and/or the loading of MHC molecules with antigens, and (iii) at least one third module as antigen module being an amino acid sequence derived from at least one full or partial epitope of at least one antigen, determining the specificity of an immune response modulated by such iMAT molecule, wherein in the antigen modules at least one cysteine residue is substituted with a different amino acid residue.

2. The iMAT molecule according to claim 1, wherein in the at least one antigen module all cysteine residues are substituted with a different amino acid residue selected from serine, leucine, isoleucine, arginine, methionine, aspartic acid and combinations thereof.

3. The iMAT molecule according to claim 1, wherein all of such modules are covalently linked to each other, and wherein no additional spacer module(s) between two or more adjacent modules of such first, second and/or third modules are present.

4. The iMAT molecule according to claim 1, wherein the at least one second module comprises the invariant chain selected from the canine, feline, bovine, ovine, caprine and/or porcine species' or a partial sequence thereof, provided that such at least one second module is functional as a module allowing species-specific intracellular targeting of the iMAT molecule to the cell organelles which are involved in the processing of antigens and/or loading of MHC molecules with antigens, preferably processed antigens.

5. The iMAT molecule according to claim 1, wherein the at least one second module comprises, one or more of the amino acid sequences of SEQ ID NO: 4 (canine) or SEQ ID NO: 5 (feline) or fragments thereof, provided such fragments maintain their intracellular transport function.

6. The iMAT molecule according to claim 1, wherein the at least one antigen module comprises at least one full or partial epitope derived from at least one allergen eliciting an allergy in animals excluding equines.

7. The iMAT molecule according to claim 6, wherein at least one full or partial epitope of at least one allergen is derived from allergies to flea bites, allergies to certain food components, atopic dermatitis, allergic airway inflammation and/or obstruction.

8. The iMAT molecule according to claim 6, wherein such at least one antigen, preferably at least one allergen, is Der f 15 allergen (SEQ ID NO: 11 and/or 18).

9. The iMAT molecule according to claim 1, wherein the at least one antigen module comprises at least one full or partial epitope derived from at least one antigen of a pathogen eliciting one or more infectious diseases in animals excluding equines, selected from the genera Campylobacter, Dirofilaria, Ehrlichia, Leishmania, Trypanosoma, Borrelia, Orthobunyavirus, Orbivirus, Flavivirus, Rotavirus, Coronavirus, Trichophyton, Microsporum; Cooperia, Haemonchus, Ostertagia, Trichostrongylus, Dictyocaulus, Metastrongylus; Eimeria, Isospora, Cryptosporidium, Giardia, wherein preferably the at least one antigen module may also be an antigen of a vector involved in the transmission of one or more infectious diseases in animals excluding equines, preferably vectors selected from blood feeding bugs, flies, midges, ticks and/or mosquitos.

10. The iMAT molecule according to claim 1, further comprising at least one tag module, wherein such at least one tag module is present N-terminally and/or C-terminally.

11. The iMAT molecule according to claim 10, wherein said at least one tag molecule is at least one His-tag.

12. The iMAT molecule according to claim 11, is present N-terminally after one methionine residue.

13. The iMAT molecule according to claim 1, wherein the at least one first module comprises, the amino acid sequence of HIV-tat, VP22 and/or Antennapedia or a partial sequence thereof, provided that such at least one first module is functional as a module for translocation of the iMAT molecule from the extracellular space into the interior of cells.

14. The iMAT molecule according to claim 13, wherein, such at least one first module comprises SEQ ID NO: 1.

15. The iMAT molecule according to claim 1, wherein the third module comprises any one of SEQ ID NOs: 14 to 23.

16. The iMAT molecule according to claim 1, comprising any one of SEQ ID NOS: 24 to 83.

17. The iMAT molecule according to claim 1, wherein the animal is selected from ruminants, members of the genus Canis, members of the genus Felis, or members of the genus Sus.

18. The iMAT molecule according to claim 17, wherein the animal is selected from members of the genus Canis or Felis.

19. An amino acid sequence of an improved MAT (iMAT) molecule, comprising: (i) at least one first module being an amino acid sequence allowing the translocation of the iMAT molecule from the extracellular space into the interior of cells, (ii) at least one second module being an amino acid sequence allowing species-specific intracellular targeting of the iMAT molecule to the cell organelles which are involved in the processing of antigens and/or the loading of MHC molecules with antigens, (iii) at least one third module as antigen module being an amino acid sequence derived from at least one full or partial epitope of at least one antigen, determining the specificity of an immune response modulated by such iMAT molecule, wherein in the antigen modules at least one cysteine residue is substituted with a different amino acid residue, preferably serine, leucine, isoleucine, arginine, methionine, and/or aspartic acid.

20. The amino acid sequence according to claim 19, wherein said first module comprises SEQ ID NO: 1.

21. The amino acid sequence according to claim 19, wherein the second module consists of SEQ ID NOs: 4 or 5.

22. The amino acid sequence according to claim 19, wherein the third module consists of any one of SEQ ID NOs: 14 to 23.

23. The amino acid sequence according to claim 22, wherein the third module comprises SEQ ID NO: 18.

24. The amino acid sequence according to claim 19, wherein all of said modules are covalently linked to each other, and wherein no additional spacer module(s) between two or more adjacent modules of such first, second and/or third modules are present at all.

25. The amino acid sequence according to claim 19, further comprising at least one His-tag module.

26. The amino acid sequence according to claim 25, wherein said His-tag module is present N-terminally and/or C-terminally.

27. The amino acid sequence according to claim 26, wherein said at least one His-tag module is present N-terminally after one methionine residue.

28. The amino acid sequence according to claim 19, wherein said amino acid sequence comprises any one of SEQ ID NOs: 24-83.

29. The amino acid sequence according to claim 28, wherein said amino acid sequence any one of SEQ ID NOs: 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, or 81.

30. The amino acid sequence according to claim 28, wherein said amino acid sequence comprises any one of SEQ ID NOs: 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, or 82.

31. The amino acid sequence according to claim 28, wherein said amino acid sequence comprises any one of SEQ ID NOs: 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, or 83.

32. The amino acid sequence according to claim 28, wherein said amino acid sequence comprises SEQ ID NO: 36 (Der f15 iMAT molecule cat), SEQ ID NO: 37 (Der f15 iMAT molecule cat), SEQ ID NO: 38 (Der f15 iMAT molecule cat), or SEQ ID NO: 57 (Der f15 iMAT molecule dog), SEQ ID NO: 58 (Der f15 iMAT molecule dog), or SEQ ID NO: 59 (Der f15 iMAT molecule dog).

33. The amino acid sequence according to claim 28, wherein said amino acid sequence/iMAT molecule comprises SEQ ID NO: 68 or SEQ ID NO: 77.

34. An amino acid sequence, comprising: (i) at least one first module being an amino acid sequence allowing the translocation of the iMAT molecule from the extracellular space into the interior of cells, comprising SEQ ID NO: 1, (ii) at least one second module being an amino acid sequence allowing species-specific intracellular targeting of the iMAT molecule to the cell organelles which are involved in the processing of antigens and/or the loading of MHC molecules with antigens, comprising SEQ ID NO: 4 or 5, (iii) at least one third module as antigen module being an amino acid sequence derived from at least one full or partial amino acid sequence, of any combination of two or more antigens selected from the group consisting of: SEQ ID NO: 7, 8, 9, 10, 11, 12, 84, 85, 86, 87, and 88, determining the specificity of an immune response modulated by such iMAT molecule, wherein at least in the antigen modules at least one cysteine residue is substituted with a different amino acid residue, preferably serine, leucine, isoleucine, arginine, methionine, and/or aspartic acid.

35. The amino acid sequence/improved MAT (iMAT) molecule according to claim 34, wherein the antigen module is an amino acid sequence derived from at least one full or partial amino acid sequence, of any combination of two or more antigens selected from the group consisting of: SEQ ID NO: 10, 11, 84, 85, 86, 87, and 88 (Hybrid 1), or wherein the antigen module is an amino acid sequence derived from at least one full or partial amino acid sequence, of any combination of two or more antigens selected from the group consisting of: SEQ ID NO: 7, 8, 9, 10, 11, and 12 (Hybrid 2) or wherein the antigen module is an amino acid sequence derived from at least one full or partial amino acid sequence, of any combination of two or more antigens selected from the group consisting of: SEQ ID NO: 7, 8, 10, and 11 (Hybrid 3).

36. The amino acid sequence according to claim 35, wherein the antigen module is an amino acid sequence based on a backbone derived from SEQ ID NO: 84 comprising any combination of one or more of the peptides according to SEQ ID NOs: 91-96 embedded into said backbone sequence, or wherein the antigen module is an amino acid sequence derived from any combination of two or more of the peptides according to SEQ ID NOs: 97-102, or wherein the antigen module is an amino acid sequence derived from any combination of two or more of the peptides according to SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 101, and SEQ ID NO: 102.

37. An amino acid sequence comprising, any of SEQ ID NO: 36 (Der f15 iMAT molecule cat), SEQ ID NO: 37 (Der f15 iMAT molecule cat), SEQ ID NO: 38 (Der f15 iMAT molecule cat), SEQ ID NO: 57 (Der f15 iMAT molecule dog), SEQ ID NO: 58 (Der f15 iMAT molecule dog), SEQ ID NO: 59 (Der f15 iMAT molecule dog), SEQ ID NO: 66 (hybrid 1 iMAT cat), SEQ ID NO: 67 (hybrid 1 iMAT cat), SEQ ID NO: 68 (hybrid 1 iMAT cat), SEQ ID NO: 75 (hybrid 1 iMAT dog), SEQ ID NO: 76 (hybrid 1 iMAT dog) and/or SEQ ID NO: 77 (hybrid 1 iMAT dog).

38. A vaccine or immunogenic composition or a pharmaceutical composition comprising the amino acid sequence according to claim 19.

39. A nucleic acid encoding the amino acid sequence according to claim 19.

40. A vector comprising at least one nucleic acid according to claim 39.

41. A method of prevention and/or therapy of one or more allergies in animals excluding equines comprising administering an effective amount of the iMAT molecule according to claim 1.

42. A method of prevention and/or therapy of one or more infectious diseases in animals excluding equines comprising administering an effective amount of the iMAT molecule according to claim 1.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0297] FIG. 1A: shows an SDS-PAGE of MAT-Fel d1 (IVN201) under reducing conditions; FIG. 1A shows the Original SDS-PAGE (10 g protein per lane, Coomassie staining).

[0298] FIG. 1B: shows an SDS-PAGE of MAT-Fel d1 (IVN201) under reducing conditions with bands that have been cut out of the SDS-PAGE gel shown in FIG. 1A and reloaded on SDS-PAGE (silver staining).

[0299] FIG. 2: shows an SDS-PAGE of Fel d1 under reducing conditions; NUPAGE 4-12% Bis-Tris Gel. Lanes: 1) Marker: SeeBlue Plus2 Pre-Stained Standard 2) 5 g Fel d1; 3) 5 g Fel d1.

[0300] FIG. 3A: shows an RP-HPLC chromatogram 0.1% TFA/Acetonitrile gradient of native protein (MAT-Fel d1) without additives.

[0301] FIG. 3B: shows an RP-HPLC chromatogram 0.1% TFA/Acetonitrile gradient of MAT-Fel d1 denaturated by addition of guanidinium chloride plus DTT.

[0302] FIG. 4: depicts MAT-Fel d1 molecule and its Kyte Doolittle hydrophobicity plot.

[0303] FIG. 5: shows a NUPAGE SDS-PAGE-System (4-12% Bis-Tris Gels, 1MES-Running buffer, 35 min, 200 V). Lanes: 1) Lysozyme, 1 g Protein ox. 2) PageRuler Prestained Protein Ladder; 3) MAT-Fel d1 (5 g) oxidized with Iodacetamide (ox.); 4) iMAT-Cul o4 (ox.); 5) PageRuler Prestained Protein Ladder; 6) MAT-Fel d1 (5 g) reduced; 7) iMAT-Cul o4 reduced; 8) Lysozyme reduced.

[0304] FIG. 6: shows an RP-HPLC chromatogram 0.1% TFA/Acetonitrile gradient. The peak reflects native (oxidized) protein (iMAT-Cul o4) without additives.

[0305] FIG. 7: shows the results of the following experiment: Proteins and ADJU-PHOS that were incubated at RT for 30 min while mixing gently. After incubation, the samples were centrifuged for 3 min. and subsequently analyzed by SDS-Page Lane 1) pageRuler Prestained Protein Ladder; 2) iMAT-Cul o3 Supernatant; 3) iMAT-Cul o3 Pellet in Urea 4) iMAT-Cul o3 Supernatant; 5) iMAT-Cul o3 Pellet in Urea; 6) Empty; 7) iMAT-Cul o2 Supernatant 8) iMAT-Cul o2 Pellet in Urea 9) iMAT-Cul o2 Supernatant 10) iMAT-Cul o2 Pellet in Urea; 11) Empty; 12) iMAT-Cul o4 Supernatant; 13) iMAT-Cul o4 Pellet in Urea 14) iMAT-Cul o4 Supernatant; 15) iMAT-Cul o4 Pellet in Urea; (2, 3, 7, 8, 12, 13 w/o freeze thaw); (4, 5, 9, 10, 14, 15 after two times freeze thaw process).

[0306] FIG. 8: shows the Kyte-Doolittle hydrophobicity plot of MAT molecules versus iMAT molecules in the generic part of the respective fusion proteins [i.e. without the respective antigen module(s)]. The hydrophobicity index is shown on the Y-axis versus the amino acid position on the X-axis. Positive numbers in the index indicate hydrophobicity. The shift of the graph of the iMAT molecule at the beginning of the hydrophobicity plot as compared to the MAT molecule is due to the additional N-terminal presence of a His-tag and one methionine residue in the iMAT molecule as well as the absence of any spacer module(s).

[0307] FIG. 9A: shows examples of D. pteronyssinus allergens of the genus dermatophagoides identifying the species, the allergen and the uniprot accession number.

[0308] FIG. 9B: shows examples of D. farinae allergens of the genus dermatophagoides identifying the species, the allergen and the uniprot accession number.

[0309] FIG. 10: shows the N-terminus sequence alignment of invariant chains from dog and cat. The CLIP sequence is shaded in grey.

[0310] FIG. 11A: shows Histamine Release Test (Details of assay in Example 2) of 5 concentrations of iMAT-Cul o2 and the respective allergens in a polysensitized horse (Horse 1).

[0311] FIG. 11B: shows Histamine Release Test (Details of assay in Example 2) of 5 concentrations of iMAT-Cul o3 and the respective allergens in a polysensitized horse (Horse 1).

EXAMPLES

[0312] The following examples serve to further illustrate the present invention; but the same should not be construed as a limitation of the scope of the invention disclosed herein.

Example 1Surrogate Marker for Immunity/Duration of Immunity

[0313] Administration of an (isolated) recombinant protein, as disclosed and claimed herein, to an animal, e.g. ruminants, pigs, more preferably a dog and/or a cat, but excluding an equine, produces an immunological response to the allergen and/or epitope present in the antigen module. Additionally, the C- or N-terminal tag, e.g. a HIS-tag, the TAT module together with the adjacent amino acid residues from the adjacent module is used in order to detect a unique product-specific immunological signal (e.g. an antibodyor a T-cell response) in a target subject that is used as a surrogate marker for immunity or duration of immunity. This surrogate marker, as a treatment-specific immunological parameter, enables the assessment of immunity or immune modulation or the duration of immunity or the duration of immune modulation after the administration. Thus, a specific indicator for an immune response triggered by the (isolated) recombinant protein according to the invention is the induction of terminal-tag (optionally with the adjacent amino acid residues) specific antibodies, such as IgG antibodies. Alternatively or additionally, the indicator is the induction of antibodies specific to the junction of a spacer and a module as described and claimed herein or the junction between two modules.

[0314] A single iMAT molecule such as SEQ ID NO: 57 (Der f 15 iMAT molecule (dog) with N-terminal Hexa-Histidine tag) or a combination of one or several iMAT molecules selected from SEQ ID NOs: 45, 51, 54, 57, 60, 63, 66, 69, 72 containing different antigen modules according to the present invention are employed for treating prophylactically or therapeutically a dog suffering from or being at risk of allergic diseases, especially atopic dermatitis. In such dogs the above iMAT molecules are administered as described in Example 3. In serum samples derived from a blood draw from these iMAT treated dogs, the immunological reaction upon iMAT treatment against the N-terminus of the iMAT proteins with respect to antibody production more specifically the specific IgG can be measured. The measurement employs standard ELISA (Enzyme Linked Immunosorbent Assay) techniques whereby sufficient amounts of a synthetized peptide comprising SEQ ID NO: 6 is coated on the surface of ELISA plates. Serum samples of treated animals are then incubated on such plates and the specific binding of IgG to the said peptide is detected by a secondary biotinylated antibody, specific for IgG in cats or dogs respectively, followed by application of the corresponding detection system e.g. streptavidin-peroxidase and 3,3,5,5-tetramethylbenzidine (TMB) as substrate.

[0315] With such ELISA test the onset of immunity elicited by iMAT immunotherapy as well as the duration of immunity is determined and observed over time. The therapeutic vaccination regime, i.e. number and schedule of booster injections, is determined by this surrogate parameter during clinical development.

Example 2Hypoallergenicity

[0316] The allergenicity of a therapeutic allergen is of utmost importance, it is a measure of the potential to induce adverse events, e.g. provoke anaphylaxis. Exemplarily for an allergy in mammals, allergen specific IgE mediated hypersensitivity is studied in procedures as the allergen provocation tests, in particular such tests targeting the skin [Griffin C E. Diagnosis of canine atopic dermatitis DOI: 10.1002/9781118738818.ch10].

[0317] Intradermal skin tests have been used for the biological evaluation of recombinant allergens and for validation of genetically engineered hypoallergenic derivatives.

[0318] Intradermal testing in a dog is performed by administering injections of small amounts of allergen solutions directly into the dog's dermis. This is usually done with small-gauge (27 gauge) needles and injections of 0.05 to 0.1 mL at each site. The positive reactions are arbitrarily interpreted by the presence of erythema, turgidity, height, and size of the wheal.

[0319] The advantages of the intradermal tests are high sensitivities. This is of particular importance if the test shall deliver a quantitative measure for hypoallergenicity. In said tests the MAT molecules show a 10-, 100- to 1000-times or even higher molar concentration of the allergenic component as compared to the natural, native allergen applied in the same test to reach a positive reaction in sensitized individuals, as cats and dogs.

[0320] With regard to MAT molecules conflicting results about their allergenicity in comparison to the corresponding native allergens have been reported in the prior art. Senti G et al. (J Allergy Clin Immunol. 2012, 129(5): 1290-1296) demonstrated hypoallergenicity of a MAT-Fel d1 in the Cellular Antigen Stimulation Test (CAST) assay as well as in the intradermal and in the intracutaneous test. The quantitative difference in sensitivity between the allergen and the MAT molecule comprising the Fel d1 was 100-, 23- and 16-fold, respectively. Though MAT-Fel d1 was clearly hypoallergenic, some allergenicity remained. In contrast Zhao et al. Int J Clin Exp Med 2015; 8(4):6436-6443 describe their MAT-Der p1 construct to exhibit an even stronger allergenicity (hyperallergenicity) as compared to the native Der p1 protein.

[0321] Surprisingly the improved MAT molecules, as disclosed and claimed herein show clear superiority in this respect. The safety of 2 iMAT molecules manufactured according to the present invention comprising Cul o2 and Cul o3 in the antigen module, respectively, is tested. Freshly withdrawn blood of a horse sensitized to these allergens and suffering from insect bite hypersensitivity (IBH) is employed in the histamine release test (HRT) as described below.

[0322] As shown in FIG. 11B and FIG. 11A, respectively, the native allergens elicit a strong histamine release whereas surprisingly the two different iMAT molecules (iMAT-Cul o3 and iMAT-Cul o2) show virtually no response at all.

[0323] Thus, iMAT molecules show clear superiority in respect to safety as compared with the MAT molecule as described in the prior art (see above).

[0324] Histamine release test (HRT): Freshly withdrawn blood of subjects is prepared to test the basophil reactivity to (isolated) recombinant proteins/iMAT molecules, as disclosed and claimed herein. Briefly, a 10-fold dilution series (e.g. ranging from 10 nM to 0.001 nM final allergen concentration) of iMAT molecules and/or recombinant allergens is prepared in PIPES buffer (AppliChem, Darmstadt, Germany), pH 7.4. Washed red and white blood cells obtained from Na-EDTA coagulation-inhibited blood are incubated with individual dilutions for 1 h at 37 C. The reaction is stopped by incubation on ice for 20 min and the supernatant containing the released histamine is collected from each sample after centrifugation. Maximal histamine content is obtained by boiling the blood cells for 10 min in a water bath (maximal release). The incubation of releasing buffer with washed blood cells serves as a negative control (spontaneous release). Histamine concentrations are determined using a competitive RIA (LDN Nordhorn, Germany) as per the manufacturer's instructions.

[0325] Alternatively, another basophil activation test is the Cellular Antigen Stimulation Test CAST ELISA which can also be considered as an in vitro allergy provocation test. This assay is done according to the manufacturer's instructions (Biihlmann Laboratories AG, Allschwil, Switzerland). In the CAST, sedimented leukocytes from allergic subjects' blood are simultaneously primed with the cytokine IL-3 and stimulated with iMAT molecules and/or recombinant allergens. Basophilic cells among others generate the allergic mediator, sulfidoleukotriene LTC4, and its metabolites LTD4 and LTE4. These freshly synthesized sulfidoleukotrienes (sLT) are subsequently measured in an ELISA test (Enzyme Linked Immunosorbent Assay).

[0326] The potential of iMAT molecules to induce adverse events, e.g. provoke anaphylaxis as a side effect of administration can be evaluated in vitro with these assays by comparing the effects of the iMAT molecules (containing an allergen) to the respective recombinant allergen alone.

[0327] A reduced basophil degranulation, e.g. histamine and/or sulfidoleukotriene release by iMAT molecules as compared to the recombinant allergen indicates a lower potential for adverse effects, i.e. a better safety profile of the iMAT molecules.

[0328] Said HRT (or CAST) can be used as an in-vitro provocation test for type 1 allergic reactions in a subject. Allergen specific histamine release indicates the relevance of the respective allergen for the basophilic cell activation and thus can be used as a quantitative parameter for the allergen specific sensitization of a subject.

[0329] It can be expected that no allergen related adverse reactions occur if subjects suffering from allergen specific IgE mediated hypersensitivity are treated with the corresponding iMAT molecules comprising relevant allergens in the iMAT-antigen module. This makes a desensitization therapy applying iMAT proteins specifically appropriate for treatment of life threatening diseases.

[0330] The consequence of this surprising safety property of iMAT molecules in contrast to MAT molecules is, that iMAT molecules used as desensitizing proteins can be used similar to vaccines against pathogens. No up-dosing as with classical therapeutic allergens is needed, since vaccines comprising iMAT molecules do not show allergen properties with respect to allergic adverse events. Already the dose of the first injection of the iMAT molecule in a treatment course is selected based on efficacy considerations only and one does not have to consider potential allergic adverse reactions. This could not be performed using MAT molecules described in the prior art since the allergenicity of MAT, compared to the native allergen, was only reduced to a certain level. However, MAT molecules still are allergens; iMAT molecules in contrast are not. The advantage of this improved property renders a more efficacious treatment regime possible with e.g. three subcutaneous or intralymphatic injections with a high biopharmaceutical content (e.g. 3 times 1 g to 100 g, preferably 3 times 10 g to 50 g iMAT protein).

[0331] The lack of allergenicity of the iMAT molecules can be explained by the fact that in contrast to the MAT molecules described in the prior art no linker amino acid residues [i.e. spacer module(s) between the first, second and/or third module(s)] are used to separate the different modules in such iMAT molecules. It is known in the prior art that engineered fusion proteins containing two or more functional polypeptides joined by a peptide or protein linker are important for the function (e.g. epitope recognition by the immune system) of the proteins [Klein J S et al., Protein Eng Des Sel. 2014, 27(10): 325-330]. The separation distance between functional units can impact epitope access and the ability to bind with avidity. If the missing amino acid residue linkers between the modules, in particular between the targeting domain and the antigen module, lead to a more rigid structure, conformational epitopes of the allergen module might not be formed due to incorrect folding. A cross linking of antibodies bound on the surface of basophils (e.g. IgE) by its high affinity receptors is necessary to induce activation and histamine release. However, misfolded allergens might not be able to induce such cross linking. Thus, an iMAT molecule without linker may not form conformational IgE epitopes which renders the iMAT molecules non-allergenic.

Example 3Therapeutic Vaccine/Prophylaxis of Atopic Dermatitis in Dogs and/or Cats

[0332] A single iMAT molecule or a combination of iMAT molecules containing different antigen modules according to the present invention is employed for treating prophylactically or therapeutically a dog and/or a cat suffering from or to be at risk of atopic dermatitis (AD).

[0333] In first example the iMAT molecule according to SEQ ID NO: 36 (Der f 15) is administered into the popliteal lymph node of cats suffering from atopic dermatitis.

[0334] In a second example the iMAT molecule according to SEQ ID NO: 66 (Hybrid 1) is administered into the popliteal lymph node of cats suffering from atopic dermatitis.

[0335] In a third example the iMAT molecule according to SEQ ID NO: 57 (Der f 15) is administered into the popliteal lymph node of dogs suffering from atopic dermatitis.

[0336] In a fourth example the iMAT molecule according to SEQ ID NO: 81 (Hybrid 3) is administered into the popliteal lymph node of dogs suffering from atopic dermatitis.

[0337] In each case, the hair over the lymph node of the affected animal is clipped and surgically prepared. Using palpation and/or ultrasound for guidance a 25 G needle is inserted into the lymph node. The injected iMAT molecule is adsorbed to an adjuvant. The adjuvant consists e.g. of aluminum phosphate (ADJU-PHOS, Brenntag Biosector, Denmark). The iMAT molecule stock is a frozen solution of e.g. 375 g/mL protein concentration in vials, each containing 500 L to be thawed before use.

[0338] After thawing the iMAT molecule solution, 400 L of the solution are mixed with e.g. 200 L of the adjuvant. This final formulation is left at room temperature e.g. for 60 minutes prior to the intralymphatic injection to allow for absorption of the iMAT molecule to e.g. ADJU-PHOS, e.g. 50 L of the mixture containing 12.5 g iMAT molecule is removed into a 500 L syringe for lymph node injection. This preparation is first administered typically on day 0, day 28 and day 56 in a dose between 10 g and 50 g (referring to the weight of solely the one or more antigen modules) per injection and iMAT molecule.

[0339] Throughout the treatment period and/or thereafter the efficacy of a therapy or the prevention of AD is investigated clinically by quantitative, semi-quantitative or qualitative assessment of pruritus, skin lesions and a medication score (Hobi S, Mueller R S; Tierarztliche Praxis. Ausgabe K, Kleintiere/Heimtiere 2014, 42(3):167-173).

[0340] These clinical parameters are compared to clinical signs of the individual dog and/or cat prior to the start of a therapeutic intervention. Alternatively, a comparison to AD affected dogs and/or cats that are not treated or treated with placebo can demonstrate the efficacy of the iMAT molecule-mediated treatment and/or prevention of clinical signs of AD.

[0341] Alternatively or in addition an intradermal provocation test with certain dermatophagoides allergens can be employed in said dogs and/or cats. A reduced response (immediate and/or late phase reactivity) indicates a therapy and/or prevention effect of the iMAT molecule administration.

[0342] Furthermore, the modulation of the different components of the immune system are monitored, e.g. changes in allergen specific IgE and IgG antibody titers indicate therapy and/or prevention effects.

[0343] Apart from changes in IgE levels, an increase in allergen-specific IgG is surprisingly found when treating a dog and/or cat for AD with such iMAT molecules. These antibodies block IgE-mediated anaphylaxis in vivo and seem to inhibit not only the allergen-induced release of inflammatory mediators from basophils and mast cells, but also IgE-mediated allergen presentation to T cells. Among the iMAT-induced IgG antibodies specifically binding to the allergen, some allergen-specific subtypes have been suggested to play an important protective role, as they compete with allergen-specific IgE antibodies and can prevent the activation of CD4+ T cells, by inhibiting the IgE-mediated antigen presentation. Furthermore, the IgG subset which is secreted promotes a significant reduction in mast cells and eosinophils, accompanied by a diminished release of inflammatory mediators.

[0344] Allergen-specific immunotherapy can modulate different components of the immune system. Cellular modifications consist of a reduction in allergen-induced T-cell proliferation, indicating the induction of peripheral tolerance in allergen-specific T cells and a decrease in antigen-specific Th2-dominated immune response in favor of a Th1 reaction with increased IFN- production. The key cell type responsible for coordinating this immunological switch is a heterogeneous T-cell population, called regulatory T cells (T,). At the cellular level, the crucial factor for successful allergen immunotherapy is the peripheral induction of type 1 T, cells. Functional studies on type 1 T, cells, specific in recognizing antigens, revealed that the modulation of Th1 and Th2 responses by type 1 T, cells mostly depends on the secretion of the cytokine IL-10, which has immunosuppressive properties. In fact, IL-10 inhibits the proliferative response of peripheral T cells against specific allergens and plays a central role in the induction of T-cell anergy. In vitro, IL-10 enhances the expression of the regulatory factor FoxP3, modulates eosinophilic function and reduces pro-inflammatory mediators released by mast cells.

[0345] Another possible marker of the outstanding clinical efficacy of said iMAT molecules-mediated immunotherapy is the detection of changes in the number or the nature of allergen-specific T cells. On the basis of, for example, Bet v tetramer staining studies, the levels and characteristics of circulating birch pollen-specific CD4.sup.+ T cells can potentially be compared before and after SIT. Recently, transforming growth factor (TGF)- has also been identified as a key cytokine in successful SIT. Many actions may account for its relevance, such as the suppression of specific Th1, Th2 and Th17 cells, the induction of FoxP3 and the suppressive function of Tregs. In addition, TGF-3 downregulates FceRI expression on Langerhans cells and suppresses IgE synthesis. These immunological relevant T cell mediated immunological responses can be measured in ex vivo experiments applying peripheral blood mononuclear cell (PBMC) cultures and cytokine detection thereof as described by Nuttall et al. (T. J. Nuttall et al., Veterinary immunology and Immunopathology 84 (2002) 143-150).

Example 4Comparison of iMAT Molecules According to the Present Invention with Prior Art MAT Molecules According to WO 2004/035793 (US Equivalent US 2005/0281816)

[0346] For assessment of purity of a MAT protein, a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) test procedure has been established (Thompson J et al., J Biol Chem 2002, 277: 34310-34331). The method, including sample preparation with a reducing agent, Lithium Dodecylsulphate (LDS) and heating at 75 C., resulted in reproducible multiple sharp bands after electrophoretic separation. Staining with Coomassie blue gives linear quantitative (densitometry) features in gels loaded with 200 to 1000 ng protein. Using a monoclonal antibody, detecting the allergen module in a MAT molecule with Fel d 1 as allergen module (MAT-Fel d1) it has been shown, that the main band and 13 minor bands all contain the MAT-Fel d1 protein. The small bands migrate at the same position as on the original gel also after re-loading on the gel (FIG. 1A and FIG. 1B). Several different methods, such as different temperature and buffer composition protocols, for sample preparation prior to PAGE generated the same band pattern.

[0347] In all of these bands the presence of the full length (complete) MAT-Fel d1 protein and only traces of host cell proteins could be demonstrated after each band was cut out of the gel, digested by trypsin and subsequently analyzed by mass spectrometry (nanoLC/ESI-MS). From these experiments an anomalous feature (gel shifting), e.g. of different folding variants, of MAT-Fel d1 in the SDS-PAGE can be concluded. This means that MAT-Fel d1 in the analyzed preparation is not suitable as biopharmaceutical molecule, in particular for clinical and/or commercial biopharmaceutical manufacturing, since its purity could not be determined with standard methods (e.g. SDS-PAGE), but only with the modified procedure explained above.

[0348] In contrast to this gel shifting phenomenon of the MAT-Fel d1 molecule, the Fel d1 as such does not show such anomalous feature in SDS PAGE (FIG. 2, lanes 2 and 3). The Fel d1 displays a single sharp band at the expected molecular weight (19.6 kD).

[0349] A further anomalous feature could be observed in RP-HPLC analysis. No single peak of the MAT-Fel d1 was seen in this analytical method (FIG. 3A and FIG. 3B), neither in the native conformation of the protein (FIG. 3A) nor in the denatured form (FIG. 3B) induced by chaotropic and reducing conditions. However, for GMP certified biopharmaceutical manufacturing a single isoform of the biomolecule in marketed pharmaceutical preparations is mandatory.

[0350] These observations in SDS-PAGE and RP-HPLC analysis may be explained by the physicochemical properties based on the amino acid sequence. Analysis in the Kyte & Doolittle hydrophobicity plot [Kyte J, Doolittle R F, Journal of Molecular Biology 1982, 157(1), 105-132] revealed adjacent extreme hydrophobic and hydrophilic domains (FIG. 4) which may be responsible for this anomalous behavior.

[0351] In particular, the hydrophobic region of the targeting domain of the fusion protein is similar to the transmembrane segments of membrane proteins which are known in the art to cause such anomalous feature [Rath A et al., Proc Natl Acad Sci USA. 2009, 106(6): 1760-1765].

[0352] Migration on SDS-PAGE, which does not correlate with formula's molecular weights, termed gel shifting appears to be common for membrane proteins. This means, that the prerequisite of the SDS-PAGE method, which is a separation of molecules solely according to their molecular weight, independent on their native 2D- or 3D-structure does not apply in these cases. In the above cited work (PNAS article), the authors investigate the anomalous gel mobility of helical membrane proteins using a library of wild-type and mutant helix-loop-helix (hairpin) sequences derived from transmembrane segments 3 and 4 of the human cystic fibrosis transmembrane conductance regulator (CFTR), including disease-phenotypic residue substitutions. They found that these hairpins migrate at rates of minus 10% to plus 30% vs. their actual formula's molecular weights on SDS-PAGE and load detergent at ratios ranging from 3.4-10 g SDS/g protein. They additionally demonstrated that mutant gel shifts strongly correlate with changes in hairpin SDS loading capacity, and with hairpin helicity, indicating that gel shift behavior originates in altered detergent binding. In some cases, this differential solvation by SDS may result from replacing protein-detergent contacts with protein-protein contacts, implying that detergent binding and folding are intimately linked.

[0353] The SDS PAGE (FIG. 5) as well as the RP-HPLC analysis (FIGS. 3A, 3B, and 6) of MAT and iMAT proteins reveal substantial differences in the migration pattern or elution, respectively. The oxidized form of MAT-Fel d1 does not show a single sharp band on the SDS-PAGE gel (Lane 3) but several diffuse bands with larger and smaller apparent molecular weights than the actual 32.2 kD of MAT-Fel d1. In contrast, as an example an iMAT molecule with an antigen module of a culicoides obsoletus allergen (iMAT-Cul o4) exhibits a single sharp band (M=41.6 kD) under oxidized conditions. Also the RP-HPLC chromatogram shows a single peak (FIG. 6).

[0354] Under reducing conditions, the MAT-Fel d1 in the SDS-PAGE reveals a main band migrating approximately at the known molecular weight but in addition some of the minor bands described in FIG. 1A and FIG. 1B known to contain the complete sequence of MAT-Fel d1 emerge again (FIG. 5, lane 6). This is an attribute, which is characteristic for the anomalous feature of MAT-Fel d1. Additionally, the RP-HPLC chromatogram of MAT-Fel d1 under reducing conditions (FIG. 3B) exhibits at least 3 different isoforms of the MAT-Fel d1. In contrast the iMAT molecules reveal characteristics evidently indicative for a single isoform in the SDS-PAGE (FIG. 5, lane 7) as well as in the RP-HPLC (FIG. 6).

[0355] The reducing conditions lead to a cleavage of the disulfide bridges in the MAT molecule, thus the MAT and the iMAT molecules should behave alike under reducing conditions if the disulphide bridges are solely responsible for the anomalous feature of MAT. However, this is not the case, since the anomalous gel shifting and the occurrence of isoforms in RP-HPLC of MAT molecules is still present under reducing conditions.

[0356] However, the iMAT molecule does not show such gel shifting and exhibits a peak in RP-HPLC chromatogram in the native (oxidized) form of the protein. Furthermore, the Kyte-Doolittle plots of MAT and iMAT molecules are nearly identical at the N-terminus covering the sequence of His-tag, TAT and targeting domain (FIG. 8). Consequently, a person skilled in the art would not be motivated to construct a MAT molecule according to the prior art with cysteine residues substituted with other amino acid residues in order to overcome the disadvantages of the prior art.

[0357] iMAT molecules can be constructed by the bioinformatical engineering procedures according to Example 5 below and produced by recombinant expression technology in E. coli. As an example three iMAT molecules, as shown in FIG. 7 are stable in buffer (20 mM citrate, 1 M arginine, pH 6.0) after freezing and thawing twice and could be adsorbed to ADJU-PHOS (Brenntag, Denmark) as adjuvant, so that the iMAT molecules can be used as a vaccine. The proteins are desorbed from ADJU-PHOS without degradation in the same buffer system (FIG. 7).

Example 5Bioinformatical Engineering of iMAT Molecules: Selection of Proteins for the Antigen Module and Optimization of the Full iMAT Molecule

[0358] In order to, for example, treat dogs and/or cats with allergic disorders using the iMAT technology effectively, it is further advisable:

a) to select a protein as allergen module in iMAT molecules that is an allergen and thus has a high potential to cause hypersensitivity in affected subjects and thus can also be the target for tolerance induction, and
b) to construct an iMAT molecule with said allergens that is thermodynamically stable and can be produced efficiently by protein engineering and can additionally be analyzed with standard methods to ensure sufficient enough quality (i.e. identity, purity and potency).

[0359] In order to fulfil these requirements a bioinformatics approach is chosen for the selection of the allergen to be included into the iMAT molecules according to the invention. The objective of the selection is (i) to choose one or more allergens to be expected to be of relevance in a given allergic disorder, i.e. that the majority of individuals suffering from allergic disorder are sensitized to the respective allergen, and (ii) to choose the allergen with the highest probability of comprising linear epitopes of allergen characteristics, i.e. comprising high numbers of short peptide sequences (7 to 13 amino acid residues) homologue to those in published allergens.

[0360] To select appropriate antigens for pharmaceutical preparations, a homology comparison based on local sequence alignments to known allergens is chosen. Often epitope detection for antibody recognition (mostly conformational epitopes) is achieved by functional analysis (e.g. peptide microarrays) or for T-cells epitopes (linear epitopes) by calculation of peptide binding probabilities to MHC molecules. The therapeutic principle of the iMAT technology inter alia is based on endocytosis and degradation by acid-dependent proteases in endosomes followed by MHC Class II binding and antigen presentation.

[0361] Thus a differentnon experimental but bioinformaticsapproach for allergen selection is chosen that is based on local homology searches of peptides derived from given proteins to known allergenic proteins, most of which are known to raise allergies in humans. Amino acid sequences of proteins suspected to have allergenic properties are exported from publicly available databases (e.g. UNIPROT) and redundancies are determined by analysis of sequences homologies within the exported dataset. Highly homologue sequence counterparts are eliminated and the resulting remainder of sequences served as the canonical sequence database of probable valid antigens for subsequent analyses. To determine proteins with putative high allergenic potential, proteins are in silico cleaved into peptides with lengths of 6 to 15 amino acids with a one amino acid shifting. Next, local-pairwise alignments of e.g. dermatophagoides proteins and the corresponding peptides to the canonical sequence database are performed. Following this, a scaling of obtained alignment hits is conducted by setting the self-alignment score of a given protein to one and alignment hits of the corresponding peptides accordingly. Thereafter the number of alignment hits exceeding a given threshold are counted for each peptide and compared by local-pairwise alignment to a randomly generated database of protein sequences with no known allergenic properties, and subsequently scaled and counted. The resulting non allergic protein counts are subtracted from those of the allergen results and cumulative hit scores for each protein based on the number of hits for all corresponding peptides are calculated. Proteins with highest counts are selected as iMAT antigen module candidates.

[0362] Each of the selected allergens are integrated into separate iMAT molecules as the antigen module and subsequently the full iMAT molecule is optimized for thermodynamic stability by iterative modeling of three-dimensional protein structures and calculation of changes of free energies after substitution of single amino acids. Physicochemical properties and stability is influenced by substituting different amino acid residue(s) within the primary amino acid sequence.

[0363] The results of the herein described analyses (antigen search and modeling) are transformed into an iMAT amino acid sequence suitable for pharmacological production and application.

[0364] In a specific example, this bioinformatics engineering approach identifies Zen 1 and Der f 15 to be of relevance in the allergic disorder atopic dermatitis elicited by proteins derived from the mite species dermatophagoides farinae. Furthermore, the bioinformatics analysis reveals stable iMAT molecules with cysteines substituted by other amino acid residues. Examples of such stable iMAT molecules are SEQ ID NO: 39 (Dog Zenl) and SEQ ID NO: 57 (Dog Der f 15).

Example 6Construction of Mosaic-Like iMAT Molecules According to the Invention

[0365] It is expected that iMAT molecules according to the invention is further improved if components of more than one allergen are included into the antigen module. For this purpose, it is possible to apply the basic principle of the above described bioinformatics selection approach (Example 5) in a different way. Instead of selecting complete allergens based on the hit count of allergen peptides found in the allergen data base, only the most abundant peptides of several of such allergens are used to engineer an iMAT antigen module. Thus, such an iMAT molecule consists of an antigen module of peptides that stem from several allergens. This allows broadening of the spectrum of a single iMAT molecule with respect to its targeted allergic profile and is thus beneficial for pharmacological drug development.

[0366] In order to find short peptide sequences that qualify for such mosaic-like iMAT molecule proteins from e.g. dermatophagoides are analyzed by homology comparison as described above. Briefly in silico cleaved proteins with peptide lengths of 6-15 amino acid residues are locally aligned to a canonical sequence database of allergen-related proteins and a random database of non-allergy-related proteins. The number of differences of significant homologies for each peptide found within the canonical database is determined. Subsequently, each peptide is locally aligned to a random database triple the size of the canonical database to reduce false positive hits. The top (e.g. tenth percentile) of remaining homologies for each peptide length is especially suitable to serve as a base for construction of a mosaic-like or hybrid allergen carrying iMAT molecule. To construct a mosaic-like iMAT molecule a protein precursor is chosen (for example from the list of precursor proteins corresponding to top ranking peptides) as a scaffold protein for embedding top ranking peptides. The signal peptide sequence is removed from the scaffold protein and top ranking peptides, optionally with additional adjacent N- or C-terminal amino acids, can be inserted within the original sequence of the scaffold protein or can replace parts of the original sequence of the scaffold protein. The position for insertion or replacement is determined using similarity alignments or the reference position of the peptide in the corresponding precursor protein. As a next step, His-Tag, the TAT and targeting domain are added. Finally, cysteine residues are replaced by most stabilizing residues as described above.

[0367] In a specific example (hybrid 1), this bioinformatics engineering approach identifies a combination of SEQ ID NO: 84 (A1KXC1_DERFA DFP1 OS=Dermatophagoides farina), SEQ ID NO: 10 (Q86R84_DERFA 60 kDa allergen Der f 18p OS=Dermatophagoides farinae GN=Der f 18 PE=2 SV=1), SEQ ID NO: 85 (A0A088SAS1_DERFA Der f 28 allergen OS=Dermatophagoides farinae PE=2 SV=1), SEQ ID NO: 88: (A7XXV2_DERFA Der f 2 allergen OS=Dermatophagoides farinae PE=4 SV=1), SEQ ID NO: 86 (B7U5T1_DERFA Der f 6 allergen OS=Dermatophagoides farinae PE=2 SV=1), SEQ ID NO: 87 (T2B4F3_DERPT LytFM OS=Dermatophagoides pteronyssinus GN=lytFM PE=4 SV=1), SEQ ID NO: 11 (Q9U6R7_DERFA 98 kDa HDM allergen OS=Dermatophagoides farinae PE=2 SV=1)

[0368] to be of relevance in the allergic disorder atopic dermatitis elicited by proteins derived from the mite species dermatophagoides farinae. Furthermore, the bioinformatics analysis reveals stable iMAT molecules with cysteines substituted by other amino acid residues. Examples of such stable mosaic-like iMAT molecules are

[0369] SEQ ID NOS: 75 (Dog Hybrid 1) and SEQ ID NO: 66 (Cat Hybrid 1).

Example 7Therapeutic Vaccine/Prophylaxis of Allergic Asthma in Cats

[0370] A single iMAT molecule or a combination of iMAT molecules containing different antigen modules according to the present invention can be employed for treating prophylactically or therapeutically a cat suffering from or being at risk of allergic asthma. In cats iMAT molecules according to the present invention are administered as described in Example 3.

[0371] Adult cats with a known history of allergic asthma will be included in the study, i.e. cats reported to exhibit clinical signs as spastic coughing episodes, wheezing and expiratory dyspnea.

[0372] Bronchoalveolar lavage fluids (BALF) are collected prior to treatment start and e.g. 2, 3, and 6 months during/after treatment. BALF is used for cytologic examination and nucleated cell counts.

[0373] Cats are sedated with e.g. Ketamine HCl intravenously. Bronchoalveolar lavage fluid is collected by gently inserting e.g. a 7 Fr polypropylene catheter through the endotracheal tube. When resistance is felt, an up to 20 ml aliquot of warmed sterile saline is lavaged through the catheter and retrieved by manual suction. After centrifugation and resuspension, a smear cytology of the collected BALF cells is prepared, the presence of significant numbers of eosinophils support a diagnosis of feline asthma. Differential cell counts can quantitatively evaluate the ratio (%) of eosinophils in BAL fluids.

[0374] Alternatively or in addition, employing certain recombinant allergens an intradermal provocation test, skin prick test or also allergen specific IgE and/or IgG determination in BAL fluid or serum can be monitored in said cats (Norris et al., Vet Immunol Immunopathol. 2003, 96(3-4): 119-127). A reduced response (immediate and/or late phase reactivity) and/or changes of the antibody titers indicate a therapy and/or prevention effects of the iMAT molecule treatment.

[0375] Clinical signs as the respiratory rate and scores to account for respiratory effort/difficulty are employed. Said respiratory scoring system can be employed also e.g. in response to an aerosol challenge. Briefly, awake, spontaneously breathing cats in a sealed chamber are exposed for different time length and/or different concentrations of aerosolized recombinant allergens. Alternatively, quantitative measures of the airway hyper-responsiveness can be performed in anesthetized cats. Pneumotachograph measurements can be done baseline and in a broncho-provocation protocol e.g. a dose response of the pulmonary resistance to methacholine and/or selected recombinant allergens.

[0376] Thus, throughout the treatment period and/or thereafter the efficacy of a therapy or the prevention of allergic asthma is investigated clinically by quantitative, semi-quantitative or qualitative assessments.

[0377] The parameters can be compared in the individual cat to the severity prior to the start of a therapeutic intervention. Alternatively, a comparison to cats with allergic asthma that are not treated or treated with placebo demonstrates the efficacy of the iMAT molecule-mediated treatment and/or prevention of clinical signs of feline allergic asthma.

Example 8Therapeutic Vaccine/Prophylaxis of Flea Allergy in Cats and/or Dogs

[0378] A single iMAT molecule or a combination of iMAT molecules containing different antigen modules according to the present invention can be employed for treating prophylactically or therapeutically a cat and/or a dog suffering from or being at risk of flea atopic dermatitis.

[0379] In a first example the iMAT molecule according to SEQ ID NO: 42 (Cat Cte f 1) is administered into the popliteal lymph node of cats suffering from or being at risk of flea atopic dermatitis.

[0380] In a second example the iMAT molecule according to SEQ ID NO: 63 (Dog Cte f 1) is administered into the popliteal lymph node of dogs suffering from or being at risk of flea atopic dermatitis.

[0381] The further treatment details are as described in Example 3 above.

[0382] The efficacy of said iMAT treatment is evaluated by an intradermal test (IDT), T-cell analyses and measurement of flea allergen specific IgE and IgG (Gerber, J. D. Vaccine 1990-12-8(6):536-542) in treated cats and/or dogs before and after iMAT treatment as described by Jin (Jin J et al., Vaccine 28 (2010) 1997-2004). Intradermal tests (IDTs) are done following the protocol from Hillier and DeBoer, (DeBoer, D. J., Hillier, A. Veterinary Immunology and Immunopathology 2001, 81 (3-4), 271-276). 4 weeks after the last immunization, the cats and/or dogs are injected with 100 l PBS containing 100 g of flea extract on the lateral thorax skin of the cats and/or dogs intradermally; histamine is used as positive control, BSA used as an irrelevant stimulator, and saline used as the negative control. The size of reactive bleb on the skin is marked with a marker pen and measured perpendicularly and horizontally within 20 min after the challenge by a micrometer. The results are calculated as an average of the three measurements. A reduced response (immediate and/or late phase reactivity) and/or changes of the antibody titers or Th1 or Treg skewed T-cell responses indicate a therapy and/or prevention effects of the iMAT molecule treatment.

REFERENCES

[0383] (1) 3.sup.rd Havemeyer workshop, Hlar, Iceland, June 2007, Veterinary Immunology and Immunotherapy 2008, 126: 351-361 [0384] (2) Allergome (www.allergome.org) [0385] (3) Crameri R. et al., Allergy 2007, 62: 197-206 [0386] (4) DeBoer, D. J., Hillier, A. Veterinary Immunology and Immunopathology 2001, 81(3-4): 271-276 [0387] (5) Gadermaier G et al., Molecular Immunology 2010, 47: 1292-1298 [0388] (6) Gerber J. D. Vaccine 1990-12-8(6):536-542 [0389] (7) Griffin C E. Diagnosis of canine atopic dermatitis in Veterinary Allergy DOI: 10.1002/9781118738818.ch10 [0390] (8) Guaguere E et al. EJCAP, 2009, 19 (3), 234-241 [0391] (9) Hill et al. Vet Immunol Immunopathol, 2001; 81(3-4): 169-186 [0392] (10) Hobi S, Mueller R S; Tierarztliche Praxis. Ausgabe K, Kleintiere/Heimtiere 2014, 42(3):167-173 [0393] (11) Jackson H A, EJCAP, 2009, 19 (3), 230-233 [0394] (12) Jin J. et al. Vaccine 2010, 28: 1997-2004 [0395] (13) Klein J S et al., Protein Eng Des Sel 2014, 27(10): 325-330 [0396] (14) Kyte J, Doolittle R F, Journal of Molecular Biology 1982, 157(1): 105-132 [0397] (15) Martnez-Gmez J M et al., Allergy 2009, 64(1): 172-178 [0398] (16) McCall C et al., Vet Immunol Immunopath 2001; 78:231-247 [0399] (17) McDermott M J et al. Molecular Immunology 2000, 37: 361-375 [0400] (18) Norris et al., Vet Immunol Immunopathol. 2003, 96(3-4): 119-127 [0401] (19) Nuttall T. J. et al., Veterinary immunology and Immunopathology 2002; 84: 143-150 [0402] (20) Pires D E et al. Bioinformatics 2014, 30(3): 335-342 [0403] (21) Prost C, Rev Fr Allergol Immunol Clin, 2008, 48(5), 409-413 [0404] (22) Rath A et al., PNAS 2009, 106(6): 1760-1765 [0405] (23) Rose H, Arb Paul Ehrlich Inst Bundesinstitut Impfstoffe Biomed Arzneim Langen Hess 2009, 96: 319-327 [0406] (24) Senna G et al., Curr Opin Allergy Clin Immunol. 2011, 11(4): 375-380 [0407] (25) Senti G et al., J Allergy Clin Immunol. 2012, 129(5): 1290-1296 [0408] (26) SIAF Annual Report 2010 [0409] (27) SIAF Annual Report 2011 [0410] (28) Thomas W R et al., Chang Gung Med J 2004; 27: 563-569 [0411] (29) Thompson J et al., J Biol Chem 2002, 277: 34310-34331 [0412] (30) US 2005/0281816 [0413] (31) U.S. Pat. No. 7,629,446 [0414] (32) U.S. Pat. No. 7,653,866 [0415] (33) WHO PD-VAC 2014Status of Vaccine Research and Development of Vaccines for Leishmaniasis [0416] (34) WO 2004/035793 [0417] (35) Zhao et al. Int J Clin Exp Med 2015; 8(4):6436-6443