Mixture of Peptides Derived from E6 and/or E7 Papillomavirus Proteins and Uses Thereof

Abstract

The invention concerns a mixture of peptides derived from the E6 and/or E7 proteins of a papillomavirus involved in cervix of uterus cancer, such as HPV16, HPV18, HPV30, HPV31, HPV32, HPV33, HPV34, HPV35, HPV39, HPV40, HPV42, HPV43, HPV44, HPV45, HPV51, HPV52, HPV56, HPV57, and HPV58, for example, as well as its uses as medicine (in immunogenic compositions, capable of stimulating the production of anti-HPV T CD4+ lymphocytes in vivo and hence useful for vaccination against uterine of uterus cancer and in other cancers) or as diagnostic reagent of HPV-specific T lymphocytes, in particular for assessing the immune condition of patients. The invention also concerns a mixture of peptides derived from E6 and/or E7 proteins of a papillomavirus involved in benign skin lesions (for example warts), such as HPV10, HPV3 or HPV4 and its uses as medicine.

1 . A mixture of peptides derived from an E6 protein and/or from an E7 protein of an HPV involved in cervical cancer or benign lesions of the skin, characterized in that each of the peptides included in said mixture binds to at least one HLA-DRB1 (1st gene) molecule the frequency of which is greater than 5% in the Caucasian population and, optionally, to at least one HLA-DRB3, HLA-DRB4 or HLA-DRB5 (2nd gene) molecule, with a binding activity<1 000 nM, preferably <800 nM, said mixture of peptides binding to at least eight HLA class II molecules, the frequency of which is greater than 5% in the Caucasian population, encoded by the alleles selected from the group consisting of the alleles HLA DRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701, DRB1*1101, DRB1*1301 and DRB1*1501 (DR1, DR3, DR4, DR7, DR11, DR13 and DR15 molecules) (1st gene) and the alleles DRB3*0101, DRB4*0101 and DRB5*0101 (B3, B4 and B5) (2nd gene). 
     
     
         2 . The mixture of peptides according to  claim 1 , characterized in that the peptides derived from an E6 protein of HPV are derived from HPV16 and are selected from the group consisting of:
 (a) a peptide included between positions 14 and 16, selected from the group consisting of the peptide corresponding to positions 14-34 and the peptide corresponding to positions 14-46 (SEQ ID Nos. 8, 19),   (b) the peptide corresponding to positions 30-50 (SEQ ID No. 10),   (c) a peptide included between positions 44 and 67, selected from the group consisting of the peptide corresponding to positions 45-67 and the peptide corresponding to positions 44-67 (SEQ ID Nos. 26, 27),   (d) the peptide corresponding to positions 61-80 (SEQ ID No. 11),   (e) a peptide included between positions 76 and 119, selected from the group consisting of the peptide corresponding to positions 76-95, the peptide corresponding to positions 91-110 and the peptide corresponding to positions 91-119 (SEQ ID Nos. 12, 35, 13),   (f) a peptide included between positions 118-140, selected from the group consisting of the peptide corresponding to positions 118-140 and the peptide corresponding to positions 121-140 (SEQ ID Nos. 40, 41),   (g) the peptide corresponding to positions 135-158 (SEQ ID No. 44), of the E6 protein of HPV16, and   (h) the peptides, preferably of 15 to 20 amino acids, exhibiting an amino acid sequence having at least 60% identity or at least 80% similarity, and preferably at least 70% identity or at least 99% similarity, with the peptides defined in (a)-(g), said peptides being identical in size, included in the peptides defined in (a)-(g) or else completely or partially overlapping these peptides, with the exclusion of the peptides corresponding to positions 15-44, 46-67, 80-108 and 118-139 of the E6 protein of HPV16 (SEQ ID Nos. 53-56).   
     
     
         3 . The mixture of peptides according to  claim 1  or  claim 2 , characterized in that the peptides derived from an E7 protein are derived from HPV16 and are selected from the group consisting of the peptide corresponding to positions 1-20, the peptide corresponding to positions 7-27, the peptide corresponding to positions 65-87 and the peptide corresponding to positions 78-98 of said E7 protein of HPV16 and the peptides, preferably of 15 to 20 amino acids, exhibiting an amino acid sequence having at least 60% identity or at least 80% similarity, and preferably at least 70% identity or at least 99% similarity, with the above peptides, said peptides being identical in size, or included in the above peptides or else overlapping these peptides, with the exclusion of the peptides corresponding to positions 3-25 and 79-97 of the E7 protein of HPV16 (SEQ ID Nos. 57, 58). 
     
     
         4 . The mixture of peptides according to any one of  claims 1  to  3 , characterized in that it is selected from the group consisting of the following mixtures:
 a mixture of peptides derived from the E6 protein of HPV16, comprising: the peptide corresponding to positions 14-34 or to positions 14-46, the peptide corresponding to positions 30-50 and the peptide corresponding to positions 44-67 or to positions 45-67;   a mixture of peptides derived from the E6 protein of HPV16, comprising: the peptide corresponding to positions 61-80, the peptide corresponding to positions 76-95 and the peptide corresponding to positions 91-119;   a mixture of peptides derived from the E7 protein of HPV16, comprising: the peptide corresponding to positions 1-20 and the peptide corresponding to positions 7-27;   a mixture of peptides derived from the E7 protein of HPV16, comprising: the peptide corresponding to positions 65-87 and the peptide corresponding to positions 78-98;   and the mixtures comprising one of the mixtures of peptides derived from the E6 protein HPV16 and one of the mixtures derived from the E7 protein of HPV16.   
     
     
         5 . An immunogenic anti-HPV16 composition, characterized in that it comprises a mixture of peptides derived from an E6 protein of HPV and/or a mixture of peptides derived from an E7 protein of HPV, according to any one of  claims 1  to  4 , combined with at least one pharmaceutically acceptable vehicle and, optionally, with at least one adjuvant. 
     
     
         6 . The composition according to  claim 5 , characterized in that said peptides are either in the form of lipopeptides, or incorporated into a recombinant virus or a viral vector for gene therapy, or included in a protein, or chemically modified. 
     
     
         7 . The composition according to  claim 5  or  claim 6 , characterized in that said mixture of peptides is combined:
 with one or more peptides or lipopeptides containing one or more CD8+ epitopes, and more particularly the CD8+ epitopes derived from an HPV protein, in particular from an HPV16 protein, and/or,   with other peptides comprising multiple CD4+ epitopes, such as the tetanus toxin TT peptide (positions 830-846), the Influenza hemagglutinin HA peptide (positions 307-319), PADRE and the  Plasmodium falciparum  LSA3 peptide, and/or   with one or more peptides or lipopeptides containing one or more B epitopes, more particularly B epitopes derived from an HPV16 protein.   
     
     
         8 . An immunogenic composition, characterized in that it advantageously comprises the sequences encoding the peptides as defined in  claims 1  to  4 . 
     
     
         9 . A vaccine, characterized in that it includes an immunogenic composition according to any one of  claims 5  to  8 . 
     
     
         10 . A peptide derived from an E6 protein of HPV, in particular of HPV16, and/or from an E7 protein of HPV, in particular of HPV16, characterized:
 in that it contains a CD4+ epitope capable of having a binding activity<1 000 nM, preferably <800 nM, with respect to at least one HLA II (HLA-DR) molecule predominant in Caucasian populations (1st gene and/or 2nd gene), as defined in  claims 1  to  6 , of being recognized by CD4+ T lymphocytes specific for said peptides, and of stimulating CD4+ T lymphocytes specific for said peptides, and   in that it is selected from the group consisting of the fragments of sequence SEQ ID Nos. 8 to 18, 21-25, 28-34, 36-39, 42-43 and 45-52, corresponding respectively to the following peptides of the E6 protein of HPV16: the peptide E6 (14-34 or 14-45), the peptide E6 (30-50), the peptide E6 (61-80), the peptide E6 (76-95), the peptide E6 (91-119) and the peptides E6 (20-34, 24-38, 28-42, 31-45, 36-50, 42-56, 50-64, 55-69, 76-90, 78-92, 81-95, 84-98, 89-103, 93-107, 97-111, 101-115, 124-138, 130-144), or to the following peptides of the E7 protein of HPV16: the peptide E7 (1-20), the peptide E7 (7-27), the peptide E7 (60-74), the peptide E7 (65-87), the peptide E7 (78-98) and the peptides E7 (6-20, 9-23, 13-27, 65-79, 67-81, 72-86, 77-91, 84-98).   
     
     
         11 . A diagnostic reagent, characterized in that it is selected from the group consisting of the peptides as defined in  claims 1  to  4  or the peptides as claimed in  claim 10 , said peptides optionally being labeled or complexed, in the form of multimeric complexes. 
     
     
         12 . A method for evaluating the immune state of an individual, characterized in that it comprises a step of detecting the presence of CD4+ T cells specific for the E6 and/or E7 peptides as defined in  claims 1  to  4 , using an ELISPOT assay, a T cell proliferation assay or an assay using multimeric complexes. 
     
     
         13 . A method for sorting HPV16-specific T lymphocytes, characterized in that it comprises at least the following steps:
 incubating a suspension of cells to be sorted, or bringing it into contact, for 1 to 3 hours, with one or more tetramers formed from multimeric complexes made up of the E6 and/or E7 peptides as defined in  claims 1  to  4  and  10 /soluble and biotinylated HLA II molecule, and conjugated to streptavidin labeled with a fluorochrome,   analyzing by flow cytometry, and   sorting the tetramer-labeled cells.   
     
     
         14 . The mixture of peptides according to  claim 2 , characterized in that said peptides as defined in h) are selected from the group consisting of the peptides corresponding respectively to positions 20-34, 24-38, 28-42, 31-45, 36-50, 42-56, 50-64, 55-69, 76-90, 78-92, 81-95, 84-98, 89-103, 93-107, 97-111, 101-115, 124-138 and 130-144 of the E6 protein of HPV16 (SEQ ID Nos. 21-25, 28-34, 36-39, 42-43). 
     
     
         15 . The mixture of peptides according to  claim 3 , characterized in that said peptides are selected from the group consisting of the peptides corresponding respectively to positions 6-20, 9-23, 13-27, 65-79, 67-81, 72-86, 77-91 and 84-98 of the E7 protein of HPV16 (SEQ ID Nos. 45-52).