Synthetic active peptide fragments

Abstract

The present invention relates to peptide fragments which have one or more shared and/or similar amino acid sequences to amino acid sequences of specific portions of the 14 kDa protein of S. mansoni (Sm14) or related FABPs (Fatty Acid Binding Proteins), the said peptide fragments functioning as continuous or discontinuous epitopic regions of the molecule or mimicking its biological activity. More particularly, the present invention relates to a method for constructing active peptide fragments, peptide fragments, immunogenic composition and diagnostic kit using said peptide fragments.

1 ) Method for constructing peptides on the basis of the three-dimensional structures of homologous molecules wherein said peptides are able to mimic or prevent the interaction between helminth pathogens and receptors, said method consisting of: 
 (i) selecting regions of the parent protein which contain a high spatial density of residues which form continuous or discontinuous epitopes;    (ii) give priority to maintaining the previously predicted epitopic residues, responsible for stimulating the desired immuno/antigenic response, within the selected peptide sequence;    (iii) elaborate sequences which, whilst maintaining a high percentage of previously identified epitopic residues, are of limited size varying from 8 to 28 residues;    (iv) two peptides may be chosen, which may correspond to regions which are distant in the primary structure (amino acid sequence) but spatially close in the tertiary (three-dimensional) structure, the said regions are chosen on the basis that the three-dimensional structure indicates that they can be readily united assuming that they retain their original structures.    (v) modifications may be made to the peptide, be it derived from a single continuous stretch of amino acids or be it derived in the manner described in (iv), so as to favour the three-dimensional conformation as seen in the original protein, such modifications may include substitutions of amino acids as well as insertions or deletions of amino acids;    (vi) residues may be used in order to restrict the conformational freedom of the final peptide.    
     
     
         2 ) Method according to  claim 1  wherein in step (vi) 01/2-cystines residues are used to restrict the conformational freedom of the final peptide.  
     
     
         3 ) Method according to  claim 1  wherein the three-dimensional structure selected is that corresponding to a molecule of a Fatty Acid Binding Protein—FABP.  
     
     
         4 ) Method according to  claim 1  wherein the three-dimensional structure selected is that of a molecule/protein from helminths, more preferentially, Sm14 and homologous molecules.  
     
     
         5 ) Peptide fragments comprising a sequence of amino acids derived from a 14 kDa protein of  Schistosoma mansoni  (Sm14) or its recombinant (rSm14) or related FABPs wherein the said peptide fragments are preferentially but not exclusively selected from the cross-reactive epitopic regions in the C-terminal third of the Sm14 or rSm14 molecule.  
     
     
         6 ) Peptide fragments according to  claim 5  wherein said fragments are composed of parts of the β-strands 9 and 10 of the Sm14 molecule and the connection between these β-strands.  
     
     
         7 ) Peptide fragments according to  claim 6  wherein the peptide fragment is contained within the amino acid sequence VTVGDVTA and its functional equivalents.  
     
     
         8 ) Peptide fragments according to  claim 5  wherein said fragments are composed of an α-helix located in the large connection between β-strands 1 and 2 of the Sm14 molecule.  
     
     
         9 ) Peptide fragments according to  claim 8  wherein the peptide fragment is contained within the amino acid sequence NFDAVMSKLG and its functional equivalents.  
     
     
         10 ) Peptide fragments according to  claim 5  wherein the fragments are a direct fusion of the β-hairpin between and including strands 9 and 10 together with the first α-helix located in the large connection between β-strands 1 and 2 of the Sm14 molecule.  
     
     
         11 ) Peptide fragments according to  claim 10  wherein the peptide fragment is contained within the amino acid sequence VTVGDVTANFDAVMSKLG and its functional equivalents.  
     
     
         12 ) Peptide fragments according to  claim 5  wherein the fragments are a direct fusion of the β-hairpin between and including strands 9 and 10 together with the first α-helix located in the large connection between β-strands 1 and 2 of the Sm14 molecule, said fragments derived by the substitution of selected amino acid residues.  
     
     
         13 ) Peptide fragments according to  claim 12  wherein the peptide fragment is contained within the amino acid sequence VTVGDVTGGSDAVMSKLG and its functional equivalents.  
     
     
         14 ) Peptide fragments according to  claim 12  wherein the fragment is modified to introduce glycine residues at its centre.  
     
     
         15 ) Peptide fragments according to  claim 5  wherein said fragments are derived from two β-hairpins wherein the first β-hairpin is composed of β-strands 6 and 7 and the second β-hairpin is composed of β-strands 8 and 9, in both cases together with their connecting loops.  
     
     
         16 ) Peptide fragments according to  claim 15  wherein said fragments are derived from the first β-hairpin, between the strands 6 and 7.  
     
     
         17 ) Peptide fragments according to  claim 16  wherein the peptide fragment is contained within the amino acid sequence EKNSESKLTQ and its functional equivalents.  
     
     
         18 ) Peptide fragments according to  claim 15  wherein the said fragments are derived from the second β-hairpin, between the strands 8 and 9.  
     
     
         19 ) Peptide fragments according to  claim 18  wherein the peptide fragment is contained within the amino acid sequence IVREVDGDTMKTT and its functional equivalents.  
     
     
         20 ) Peptide fragments according to  claim 15  wherein the said fragments are a direct fusion of the first β-hairpin between strands 6 and 7 and the second β-hairpin between strands 8 and 9.  
     
     
         21 ) Peptide fragments according to  claim 20  wherein the peptide fragment is contained within the amino acid sequence EKNSESKLTQIVREVDGDTMKTT and its functional equivalents.  
     
     
         22 ) Peptide fragments according to  claim 15  wherein the fragments are a direct fusion of the first β-hairpin between and including strands 6 and 7 and the second β-hairpin between and including strands 8 and 9, wherein said fragments undergo a substitution of at least one amino acid at a position previously chosen and/or an insertion of at least one residue in its main chain.  
     
     
         23 ) Peptide fragments according to  claim 22  wherein the peptide fragment is contained within the amino acid sequence EKFSESKLTSDPTGIVREVDGATMKTT and its functional equivalents.  
     
     
         24 ) Peptide fragments according to  claim 22  wherein the peptide fragment is contained within the amino acid sequence EKFSESKLTFDGIVREVDGATMKTT and its functional equivalents.  
     
     
         25 ) Immunogenic compositions consisting of the peptide fragments obtained according to the method described in  claim 1  and a pharmaceutical carrier, said immunogenic compositions being able to confer at least partial protection against infection with pathogenic helminths, and thus serve as vaccines against same.  
     
     
         26 ) Immunogenic composition according to  claim 25  where the pathogenic helminths are  Schiostosoma mansoni  and/or  Fasciola hepatica .  
     
     
         27 ) Immunogenic composition consisting of peptide fragments selected from the cross-reactive epitopic regions in the C-terminal third of the Sm14 or rSm14 molecule, said immunogenic compositions being able to confer at least partial protection against infection with pathogenic helminths, and thus serve as vaccines against same.  
     
     
         28 ) Immunogenic composition according to  claim 27  where the pathogenic helminths are  Schiostosoma mansoni  and/or  Fasciola hepatica.    
     
     
         29 ) Direct or indirect diagnostic test for helminth infection diagnoses using peptides fragments obtained as described in  claim 1  as the active ingredient wherein the said peptide fragments have similar or conservatively substituted amino acid sequences to those found in epitopic portions of the Sm14 antigen or related FABPs.  
     
     
         30 ) Direct or indirect diagnostic test according to  claim 29  where the helminth is  Schiostosoma mansoni  and/or  Fasciola hepatica.    
     
     
         31 ) Direct or indirect diagnostic test for helminth infection diagnoses using mono- or policlonal antibodies of any origin raised against fragments obtained as described in  claim 1  wherein the said peptide fragments have similar or conservatively substituted amino acid sequences to those found in epitopic portions of the Sm14 antigen or related FABPs.  
     
     
         32 ) Direct or indirect diagnostic test according to  claim 31 , where the helminth is  Schiostosoma mansoni  and/or  Fasciola hepatica.    
     
     
         33 ) Direct or indirect diagnostic test for helminth infection diagnostics according to one of claims  29  through  32  wherein the said diagnostic test can be a parasitic or immunological test using any kind of substrate including excrement, urine, serum, saliva.  
     
     
         34 ) Direct or indirect diagnostic test according to  claim 33 , where the helminth is  Schiostosoma mansoni  and/or  Fasciola hepatica.    
     
     
         35 ) Diagnostic kit comprising at least a peptide fragments obtained as described in precedent claims.  
     
     
         36 ) Diagnostic kit according to  claim 35  wherein it is further used medical and veterinary vehicles.