US2007141076A1PendingUtilityA1
Antigen fragments for the diagnosis of Toxoplasma gondii
Est. expiryMar 21, 2022(expired)· nominal 20-yr term from priority
A61K 39/00C07K 2319/00C07K 14/45
51
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Abstract
The invention described herein relates to a method for identifying antigen fragments of Toxoplasma gondii proteins, and their use as diagnostic and immunogenic agents. Said method is implemented by means of selection of DNA fragments libraries of the parasite with sera of subjects who have been infected, using the phage display technique, and is characterised in that it uses the expression/exposure vector λKM4. The method allows also to identify antigen fragments related to the time of the infection.
Claims
exact text as granted — not AI-modified1 . Method for the identification of antigen fragments and/or fragments containing epitopes of Toxoplasma gondii proteins, particularly phase-specific antigen fragments, by means of selection of libraries of cDNA or DNA fragments of specific genes of the parasite with sera of subjects who have been infected, using the phage display technique, characterised in that it uses the vector λKM4.
2 . Antigen fragments and/or fragments containing epitopes of Toxoplasma gondii proteins obtainable with the method according to claim 1 .
3 . Antigenic portions of the fragments according to claim 2 .
4 . Antigen fragment and/or fragment containing an epitope according to claim 3 with the following amino acid sequence:
RRTGCHAFRENCSPGRCIDDASHENGYTCECPTG
(SEQ ID NO: 31)
YSREVTSKAEESCVEGVEVTLAEKCEKEFGISAS
SCKCD
LNPIDDMLFETALTANEMMEDITWRPRVDVEFDS
(SEQ ID NO: 58)
KKKEMIILADLPGLQKDDVTIEVDNGAIVIKGEK
TSKEAEKVDDGKTKNILTERVSGYFARRFQLPSN
YKPDGISAAMDNGVLRVTIKVEDSGGAKQQISV
SGGTGQGLGIGESVDLEMMGNTYRVERPTGNPDL
(SEQ ID NO: 26)
LKIAIKASDGSYSEVGNVNVEEVIDTMKSMQRDE
DIFLRALNKGETVEEAIEDVAQAEGLNSEQTLQL
EDAVSAVASVVQDE
AALGGLAADQPENHQALAEPVTGVGEAGVSPVNE
(SEQ ID NO: 28)
AGESYSSATSGVQEATAPGAVLLDAIDAESDKVD
NQAEGGERMKKVEEELSLLRRELYDRTDRPG
FATAATASDDELMSRIRNSDFFDGQAPVDSLRPT
(SEQ ID NO: 29)
NAGVDSKGTDDHLTTSMDKASVESQLPRREPLET
EPDEQEEVHF
PQDAICSDWSAWSPCSVSCGDGSQIRTRTEVSAP
(SEQ ID NO: 64)
QPGTPTCPDCPAPMGRTCVEQGGLEEIRECSAGV
CAVDAGCGVWV
PCPINATCGQFEEWSTCSVSCGGGLKTRSRNPWN
(SEQ ID NO: 65)
EDQQHGGLSCEQQHPGGRTETVTCNPQACPVDER
PGEWAEWGECSVTCGDGVRERRRGKSLVEAKFGG
RTIDQQNEALPEDLKIKNVEYEPCSYPACGASCT
YVWSDWNK
LRGYRFGVWKKGRCLDYTELTDTVIERVESKAQC
(SEQ ID NO: 67)
WVKTFENDGVASDQPHTYPLTSQASWNDWWPLHQ
SDQPHSGGVGRNYGFYYVDTTGEGKCALSDQVPD
CLVSDSAAVSYTAAGSLSEETPNFIIPSNPSVTP
PTPETALQCTADKFPDSFGACDVQACKRQKTSCV
GGQIQSTSVDCTADEQNECGSNTA
NEQVALAQLSTFLELVEVPCNSVHVQGVMTPNQM
(SEQ ID NO: 66)
VKVTGAGWDNGVLEFYVTRPTKTGGDTSRSHLAS
IMCYSKDIDGVPSDKAGKCFLKNFSGEDSSEIDE
KEVSLPIKSHNDAFMFVCSSNDGSALQCDVFALD
NTNSSDGWKVNTVDLGVSVSPDLAFGLTADGVKV
KKLYASSGLTAINDDPSLGCKAPPHSPPAGEEPS
LPSPENSGSATPAEESPSESES
GLSQRVPELPEVEPFDEVGTGARRSGSIATLLPQ
(SEQ ID NO: 57)
DAVLYENSEDVAVPSDSASTPSYFHVESPSASVE
ATTGAVGEVVPDCEEQQEQGDTTLSDHDFH
PSVVNNVARCSYGADSTLGPVKLSAEGPTTMTLV
(SEQ ID NO: 34)
CGKDGVKVPQDNNQYCSGTTLTGCNEKSFKDILP
KLTENPWQGNASSDKGATLTIKKEAFPAESKSVI
IGCTGGSPEKHHCTVKLEFAGAAGSADSA
VMASDPPLVANQVVTCPDKKSTAAVILTPTENHF
(SEQ ID NO: 33)
TLKCPKTALTEPPTLAYSPNRQICPAGTTSSCTS
KAVTLSSLIPEAEDSWWTGDSASLDTAGIKLTVP
IEKFPVTTQTFVVGCIKGDDAQSCMVTVTVQARA
SSVVNNVARCSYGADS
APTQSEMKEFQEEIKEGVEETKHEDDPEMTRLMV
(SEQ ID NO: 32)
TEKQESKNFSKMAKSQSFSTRIEELGGSISFLTE
TGVTMIELPKTVSEHDMDQLLH
SANVTSSEPAKLDLSCAHSDNKGSRAPTIGEPVP
(SEQ ID NO: 68)
DVSLEQCAAQCKAVDGCTHFTYNDDSKMCHVKEG
KPDLYDLTGGKTAPRSCDRSCFEQHVSYEGAPDV
MTAMVTSQSADCQAACAADPSCEIFTYNEHDQKC
TFKGRGFSAFKERGVLGVTSGPKQFCDEGGKLT
ENPVRPPPPGFHPSVIPNPPYPLGTPAGMPQPEV
(SEQ ID NO: 30)
P
YSSPRIVVLIRYCFFSTYRLTMFAVKHCLLVVAV
(SEQ ID NO: 27)
GALVNVSVRAAEFSGVVNQGP.
5 . Collection of antigen fragments obtainable with the method according to claim 1 .
6 . Nucleotide sequence coding for an antigen fragment according to claim 2 .
7 . Nucleotide sequence coding for an antigen fragment according to claim 4 selected from the group consisting of:
AGGAGGACTGGATGTCATGCCTTCAGGGAGAACT
(SEQ ID NO: 22)
GCAGCCCTGGTAGATGTATTGATGACGCCTCGCA
TGAGAATGGCTACACCTGCGAGTGCCCCACAGGG
TACTCACGTGAGGTGACTTCCAAGGCGGAGGAGT
CGTGTGTGGAAGGAGTCGAAGTCACGCTGGCTGA
GAAATGCGAGAAGGAATTCGGCATCAGCGCGTCA
TCCTGCAAATGCGAT
TCTTCAGAAAGATGACGTAACCATAGAAGTCGAC
(SEQ ID NO: 56)
AACGGAGCCATCGTTATCAAAGGAGAGAAGACCT
CGAAAGAAGCGGAGAAAGTGGACGATGGCAAAAC
AAAGAACATTTTGACTGAGCGAGTGTCCGGTTAT
TTTGCGCGCCGGTTCCAGCTCCCGAGTAATTACA
AGCCCGACGGAATCAGTGCGGCAATGGACAACGG
CGTTCTACGTGTCACGATCAAGGTCGAGGATTCA
GGGGGCGCAAAGCAACAAATCAGCGTG
AGTGGAGGGACAGGGCAGGGATTAGGAATCGGAG
(SEQ ID NO: 17)
AATCTGTAGATTTGGAGATGATGGGGAACACGTA
TCGTGTGGAGAGACCCACAGGCAACCCGGACTTG
CTCAAGATCGCCATTAAAGCTTCAGATGGATCGT
ACAGCGAAGTCGGCAATGTTAACGTGGAGGAGGT
GATTGATACTATGAAAAGCATGCAGAGGGACGAG
GACATTTTCCTTCGTGCGTTGAACAAAGGCGAAA
CAGTAGAGGAAGCGATCGAAGACGTGGCTCAAGC
AGAAGGGCTTAATTCGGAGCAAACCCTGCAACTG
GAAGATGCAGTGAGCGCGGTGGCGTCTGTTGTTC
AAGACGAG
GCTGCCTTGGGAGGCCTTGCGGCGGATCAGCCTG
(SEQ ID NO: 19)
AAAATCATCAGGCTCTTGCAGAACCAGTTACGGG
TGTGGGGGAAGCAGGAGTGTCCCCCGTCAACGAA
GCTGGTGAGTCATACAGTTCTGCAACTTCGGGTG
TCCAAGAAGCTACCGCCCCAGGTGCAGTGCTCCT
GGACGCAATCGATGCCGAGTCGGATAAGGTGGAC
AATCAGGCGGAGGGAGGTGAGCGTATGAAGAAGG
TCGAAGAGGAGTTGTCGTTATTGAGGCGGGAATT
ATATGATCGCACAGATCGCCCTGGT
CAGTTCGCTACCGCGGCCACCGCGTCAGATGACG
(SEQ ID NO: 20)
AACTGATGAGTCGAATCCGAAATTCTGACTTTTT
CGATGGTCAAGCACCCGTTGACAGTCTCAGACCG
ACGAACGCCGGTGTCGACTCGAAAGGGACCGACG
ATCACCTCACCACCAGCATGGATAAGGCATCTGT
AGAGAGTCAGCTTCCGAGAAGAGAGCCATTGGAG
ACGGAGCCAGATGAACAAGAAGAAGTTCAT
CCCCAGGATGCCATTTGCTCGGATTGGTCCGCAT
(SEQ ID No: 59)
GGAGCCCCTGCAGTGTATCCTGCGGTGACGGAAG
CCAAATCAGGACGCGAACTGAGGTTTCTGCTCCG
CAACCTGGAACACCAACATGTCCGGACTGCCCTG
CGCCCATGGGAAGGACTTGCGTGGAACAAGGCGG
ACTTGAAGAAATCCGTGAATGCAGTGCGGGGGTA
TGTGCTGTTGACGCTGGATGTGGCGTCTGGGTT
CCGTGTCCAATTAATGCAACTTGCGGTCAGTTTG
(SEQ ID No: 60)
AAGAATGGAGTACATGCTCGGTCTCATGTGGTGG
TGGACTGAAAACGAGGTCGAGGAACCCTTGGAAT
GAAGACCAACAACATGGAGGACTATCCTGCGAGC
AGCAGCATCCTGGTGGGCGGACGGAAACGGTAAC
TTGCAATCCTCAAGCGTGTCCTGTGGATGAACGA
CCGGGGGAGTGGGCAGAGTGGGGGGAATGTAGTG
TCACGTGCGGCGACGGAGTGCGAGAGCGCAGGCG
CGGGAAAAGTCTAGTTGAGGCTAAATTCGGCGGA
CGCACCATTGATCAGCAGAATGAGGCTCTTCCGG
AAGACTTAAAAATCAAAAACGTCGAGTATGAGCC
ATGTTCGTATCCTGCTTGTGGAGCTTCCTGCACG
TACGTCTGGAGTGACTGGAACAAG
CTTCGCGGGTACAGGTTCGGTGTTTGGAAGAAAG
(SEQ ID No: 62)
GCCGTTGCCTCGACTACACTGAATTGACCGACAC
TGTGATAGAACGTGTTGAGTCAAAGGCACAGTGC
TGGGTGAAAACCTTTGAAAACGACGGGGTCGCGA
GTGACCAACCCCATACGTATCCACTGACGTCGCA
AGCATCATGGAACGATTGGTGGCCTCTCCACCAG
AGTGACCAACCTCACTCAGGTGGCGTTGGGCGTA
ATTACGGTTTCTACTACGTGGACACGACTGGAGA
GGGCAAGTGTGCACTCTCTGACCAGGTACCCGAC
TGCCTGGTGTCGGATTCTGCCGCCGTGTCGTATA
CAGCAGCGGGGAGTTTGTCTGAAGAGACGCCGAA
TTTCATAATTCCGTCAAATCCCTCTGTTACTCCG
CCAACGCCCGAGACGGCACTTCAGTGCACGGCCG
ACAAGTTCCCCGACTCTTTCGGTGCCTGCGACGT
TCAAGCCTGTAAAAGACAGAAGACGTCCTGCGTT
GGCGGACAGATTCAAAGTACTAGCGTCGACTGCA
CCGCGGACGAACAAAATGAATGTGGCTCTAACAC
TGCG
AACGAACCGGTGGCCCTAGCTCAGCTCAGCACAT
(SEQ ID NO: 61)
TCCTCGAGCTCGTCGAGGTGCCATGTAACTCTGT
TCATGTTCAGGGGGTGATGACCCCGAATCAAATG
GTCAAAGTGACTGGTGCAGGATGGGATAATGGCG
TTCTCGAGTTCTATGTCACGAGGCCAACGAAGAC
AGGCGGGGACACAAGCCGAAGCCATCTTGCGTCG
ATCATGTGTTATTCCAAGGACATTGACGGCGTGC
CGTCAGACAAAGCGGGAAAGTGCTTTCTGAAGAA
CTTTTCTGGTGAAGACTCGTCGGAAATAGACGAA
AAAGAAGTATCTCTACCCATCAAGAGCCACAACG
ATGCGTTCATGTTCGTTTGTTCTTCAAATGATGG
ATCCGCACTCCAGTGTGATGTTTTCGCCCTTGAT
AACACCAACTCTAGCGACGGGTGGAAAGTGAATA
CCGTGGATCTTGGCGTCAGCGTTAGTCCGGATTT
GGCATTCGGACTCACTGCAGATGGGGTCAAGGTG
AAGAAGTTGTACGCAAGCAGCGGCCTGACAGCGA
TCAACGACGACCCTTCCTTGGGGTGCAAGGCTCC
TCCCCATTCTCCGCCGGCCGGAGAGGAACCGAGT
TTGCCGTCGCCTGAAAACAGCGGGTCTGCAACAC
CAGCGGAAGAAAGTCCGTCTGAGTCTGAATCT
GGATTGAGCCAAAGGGTGCCAGAGCTACCAGAAG
(SEQ ID NO: 55)
TGGAGCCCTTTGATGAAGTAGGCACGGGAGCTCG
ACGGTCCGGGTCCATTGCGACCCTTCTTCCACAA
GACGCTGTTTTATATGAGAACTCAGAGGACGTTG
CCGTTCCGAGTGATTCAGCATCGACCCCGTCATA
CTTTCATGTGGAATCTCCAAGTGCTAGTGTGGAA
GCCGCGACTGGCGCTGTGGGAGAGGTGGTGCCGG
ACTGTGAAGAACAACAGGAACAGGGTGACACGA
CGTTATCCGATCACGATTTCCATTCA
CCATCGGTCGTCAATAATGTCGCAAGGTGCTCCT
(SEQ ID NO: 25)
ACGGTGCAGACAGCACTCTTGGTCCTGTCAAGTT
GTCTGCGGAAGGACCCACTACAATGACCCTCGTG
TGCGGGAAAGATGGAGTCAAAGTTCCTCAAGACA
ACAATCAGTACTGTTCCGGGACGACGCTGACTGG
TTGCAACGAGAAATCGTTCAAAGATATTTTGCCA
AAATTAACTGAGAACCCGTGGCAGGGTAACGCTT
CGAGTGATAAGGGTGCCACGCTAACGATCAAGAA
GGAAGCATTTCCAGCCGAGTCAAAAAGCGTCATT
ATTGGATGCACAGGGGGATCGCCTGAGAAGCATC
ACTGTACCGTGAAACTGGAGTTTGCCGGGGCTGC
AGGGTCAGCAAAATCGGCT
GTTATGGCATCGGATCCCCCTCTTGTTGCCAATC
(SEQ ID NO: 24)
AAGTTGTCACCTGCCCAGATAAAAAATCGACAGC
CGCGGTCATTCTCACACCGACGGAGAACCACTTC
ACTCTCAAGTGCCCTAAAACAGCGCTCACAGAGC
CTCCCACTCTTGCGTACTCACCCAACAGGCAAAT
CTGCCCAGCGGGTACTACAAGTAGCTGTACATCA
AAGGCTGTAACATTGAGCTCCTTGATTCCTGAAG
CAGAAGATAGCTGGTGGACGGGGGATTCTGCTAG
TCTCGACACGGCAGGCATCAAACTCACAGTTCCA
ATCGAGAAGTTCCCCGTGACAACGCAGACGTTTG
TGGTCGGTTGCATCAAGGGAGACGACGCACAGAG
TTGTATGGTCACGGTGACAGTACAAGCCAGAGCC
TCATCGGTCGTCAATAATGTCGCAAGGTGCTCCT
ATGGTGCGGACAGC
GCACCCACTCAATCTGAAATGAAAGAATTCCAAG
(SEQ ID NO: 23)
AGGAAATCAAAGAAGGGGTGGAGGAAACAAAGCA
TGAAGACGATCCTGAGATGACGCGGCTCATGGTG
ACCGAGAAGCAGGAGAGCAAAAATTTCAGCAAGA
TGGCGAAATCCCAGAGTTTTAGCACGCGAATCGA
AGAGCTCGGGGGATCCATTTCGTTTCTAACTGAA
ACGGGGGTCACAATGATCGAGTTGCCCAAAACTG
TCAGTGAACATGACATGGACCAACTACTCCAC
AGTGCCAACGTAACAAGTTCGGAGCCTGCAAAAC
(SEQ ID NO: 63)
TTGATCTCTCTTGTGCGCACTCTGACAATAAGGG
ATCAAGGGCTCCCACAATAGGCGAGCCAGTGCCA
GATGTGTCCCTGGAACAATGTGCTGCGCAATGCA
AGGCTGTTGATGGCTGCACACATTTCACTTATAA
TGACGATTCGAAGATGTGCCATGTGAAGGAGGGA
AAACCCGATTTATACGATCTCACAGGAGGCAAAA
CAGCACCGCGCAGTTGCGATAGATCATGCTTCGA
ACAACACGTATCGTATGAGGGAGCTCCTGACGTG
ATGACAGCGATGGTCACGAGCCAGTCAGCGGACT
GTCAGGCTGCGTGTGCGGCTGACCCGAGCTGCGA
GATCTTCACTTATAACGAACACGACCAGAAATGT
ACTTTCAAAGGAAGGGGGTTTTCTGCGTTTAAGG
AACGAGGGGTGTTGGGTGTGACTTCCGGGCCGAA
ACAGTTCTGCGATGAAGGCGGTAAATTAACT
GAGAACCCGGTGAGACCGCCTCCTCCCGGTTTCC
(SEQ ID NO: 21)
ATCCAAGCGTTATTCCCAATCCCCCGTACCCGCT
GGGCACTCCAGCGGGCATGCCACAGCCAGAGGTT
CC
TACTCTTCACCACGAATAGTTGTTTTGATTAGAT
(SEQ ID NO: 18)
ATTGCTTCTTCTCCACATATCGCCTCACAATGTT
CGCCGTAAAACATTGTTTGCTGGTTGTTGCCGTT
GGCGCCCTGGTCAACGTCTCGGTGAGGGCTGCCG
AGTTTTCCGGAGTTGTTAACCAGGGACCT
8 . Nucleotide sequences that hybridise with the sequences coding for the sequences according to claim 4 , under stringent hybridisation conditions.
9 . Nucleotide sequences that hybridise with the sequences coding for the antigen fragments according to claim 2 under stringent hybridisation conditions.
10 . Use of an antigen fragment according to claim 4 as active agents for the diagnosis of Toxoplasma gondii infections.
11 . Use according to claim 10 for the diagnosis of the time of infection.
12 . Use according to claim 11 , where said time of infection is determined by the IgG avidity assay.
13 . Specific ligand for an epitope according to claim 2 .
14 . Anti-epitope antibody raised against an epitope according to claim 2 .
15 . Ligands for the collection according to claim 5 .
16 . Anti-collection antibody raised against a collection according to claim 5 .
17 . Use of at least one ligand according to claim 13 for the preparation of means for the diagnosis of Toxoplasma gondii infection.
18 . Use of ligands according to claim 15 for the preparation of means for the diagnosis of Toxoplasma gondii infection.
19 . Method for the diagnosis of Toxoplasma gondii infection, comprising the selection of sera of subjects affected by or suspected of being affected by said infection with the antigen fragments of claim 1 and/or with at least one ligand of said antigen and/or at least one antibody to the same.
20 . Use of antigen fragments according to claim 2 as medicaments.
21 . Use of antigen fragments of claim 2 as active agents for the preparation of medicaments for the prevention or treatment of Toxoplasma gondii infections.
22 . Use of the sequences according to claim 6 as medicaments.
23 . Use of the sequences according to claim 6 for the preparation of medicaments useful for the treatment and prevention of Toxoplasma gondii infections.
24 . Diagnostic kit for the diagnosis of Toxoplasma gondii infection, containing at least one antigen fragment according to claim 2 .
25 . Kit for the diagnosis of an acute and/or chronic Toxoplasma gondii infection, containing at least one antigen fragment according to claim 4 .
26 . Pharmaceutical composition, particularly in the form of a vaccine, containing at least one antigen fragment according to claim 2 .
27 . Pharmaceutical composition, particularly in the form of a vaccine, containing at least one sequence according to claim 6 .
28 . Composition according to claim 26 suitable for human and/or veterinary use.Cited by (0)
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