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.

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 epitope of at least one antigen, determining the specificity of an immune response modulated by such iMAT molecule, wherein in the entire iMAT molecule all cysteine residues are substituted with a different amino acid residue,   wherein the at least one second module is selected from the group consisting of: the invariant chain selected from the canine and/or feline species' or a partial sequence thereof, and   wherein the at least one antigen module comprises at least one epitope derived from at least one allergen eliciting an immune response in animals is selected from members of the genus  Canis  or  Felis , in response to flea bites, allergens derived from fleas and/or mites food allergies, atopic dermatitis, atopic dermatitis caused by flea bites, allergic asthma, allergic airway inflammation and/or obstruction.   
     
     
         2 . (canceled) 
     
     
         3 . The iMAT molecule according to  claim 1 , wherein the modules are covalently linked to each other, and wherein no additional spacer module(s) between two or more adjacent modules of said first, second, and/or third modules are present. 
     
     
         4 . 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. 
     
     
         5 - 7 . (canceled) 
     
     
         8 . The iMAT molecule according to  claim 1 , wherein said at least one allergen comprises Der f 15 allergen according to SEQ ID NO: 11 and/or 18, and/or Cte f1 allergen according to SEQ ID NO: 13 and/or 20. 
     
     
         9 . (canceled) 
     
     
         10 . The iMAT molecule according to  claim 1 , further comprising at least one tag module, wherein said at least one tag module is present N-terminally and/or C-terminally. 
     
     
         11 . (canceled) 
     
     
         12 . The iMAT molecule according to  claim 10 , wherein said at least one tag module is a His-tag module which 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. 
     
     
         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 - 18 . (canceled) 
     
     
         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, and   (iii) at least one third module as antigen module being an amino acid sequence derived from at least one epitope of at least one antigen, determining the specificity of an immune response modulated by such iMAT molecule,   wherein said first module comprises SEQ ID NO. 1,   wherein the second module consists of SEQ ID NOs: 4 or 5, and   wherein the third module consists of any one of SEQ ID NOs: 14 to 23.   
     
     
         20 - 22 . (canceled) 
     
     
         23 . The amino acid sequence according to  claim 19 , wherein the third module comprises SEQ ID NO: 18. 
     
     
         24 . The amino acid sequence according to  claim 19 , wherein the 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, wherein said Hid-tag module is present N-terminally and/or C-terminally. 
     
     
         26 . (canceled) 
     
     
         27 . The amino acid sequence according to  claim 25 , 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 - 31 . (canceled) 
     
     
         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), 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, and   (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 all cysteine residue are substituted with a different amino acid residue.   
     
     
         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 - 42 . (canceled)