U.S. patent application number 11/586694 was filed with the patent office on 2007-06-21 for antigen fragments for the diagnosis of toxoplasma gondii.
This patent application is currently assigned to Kenton S.r.I.. Invention is credited to Elisa Beghetto, Manlio Di Cristina, Franco Felici, Nicola Gargano.
Application Number | 20070141076 11/586694 |
Document ID | / |
Family ID | 28456094 |
Filed Date | 2007-06-21 |
United States Patent
Application |
20070141076 |
Kind Code |
A1 |
Gargano; Nicola ; et
al. |
June 21, 2007 |
Antigen fragments for the diagnosis of Toxoplasma gondii
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 .lamda.KM4. The method allows also to
identify antigen fragments related to the time of the
infection.
Inventors: |
Gargano; Nicola; (Pomezia
(RM), IT) ; Beghetto; Elisa; (Pomezia (RM), IT)
; Di Cristina; Manlio; (Pomezia (RM), IT) ;
Felici; Franco; (Pomezia (RM), IT) |
Correspondence
Address: |
NIXON & VANDERHYE, PC
901 NORTH GLEBE ROAD, 11TH FLOOR
ARLINGTON
VA
22203
US
|
Assignee: |
Kenton S.r.I.
Pomezia(Rome)
IT
|
Family ID: |
28456094 |
Appl. No.: |
11/586694 |
Filed: |
October 26, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10508622 |
Dec 10, 2004 |
7176286 |
|
|
PCT/IT03/00162 |
Mar 18, 2003 |
|
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11586694 |
Oct 26, 2006 |
|
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Current U.S.
Class: |
424/191.1 ;
435/258.1; 435/471; 435/69.3; 435/7.22; 530/350; 536/23.7 |
Current CPC
Class: |
A61K 39/00 20130101;
C07K 2319/00 20130101; C07K 14/45 20130101 |
Class at
Publication: |
424/191.1 ;
435/007.22; 435/069.3; 435/258.1; 435/471; 530/350; 536/023.7 |
International
Class: |
A61K 39/00 20060101
A61K039/00; G01N 33/569 20060101 G01N033/569; C07H 21/04 20060101
C07H021/04; C12N 15/74 20060101 C12N015/74; G01N 33/53 20060101
G01N033/53; A61K 39/002 20060101 A61K039/002; C07K 14/45 20060101
C07K014/45; C12N 1/10 20060101 C12N001/10 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 21, 2002 |
IT |
RM2002A000159 |
Nov 13, 2002 |
IT |
RM2002A000568 |
Claims
1. 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 .lamda.KM4.
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: TABLE-US-00011
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: TABLE-US-00012
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.
Description
[0001] This application is a divisional of Ser. No. 10/508,622,
filed Dec. 10, 2004, which is a U.S. national phase of
international application PCT/IT03/00162, filed Mar. 18, 2003,
which designated the U.S. and claims benefit of IT Application No.
RM02A000159, filed Mar. 21, 2002 and IT Application No.
RM02A000568, filed Nov. 13, 2002, the entire contents of each of
which is incorporated herein by reference.
[0002] 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 cDNA libraries of the parasite
or of DNA fragments of specific genes of the parasite with sera of
subjects who have been infected by the parasite, using the phage
display technique, and is characterised in that it uses the vector
.lamda.KM4.
[0003] The invention described herein also relates to the technical
field of the preparation of diagnostic means not applied directly
to the animal or human body and furnishes compounds, methods for
their preparation, methods for their use and compositions
containing them which are suitable for industrial application in
the pharmaceutical and diagnostic field, particularly for the
detection and diagnosis of Toxoplasma gondii infections, as well as
for the treatment and prevention of said infections.
BACKGROUND TO THE INVENTION
[0004] Early diagnosis is a priority and highly desirable objective
in all fields of medicament, particularly because it allows an
appreciable improvement in the patient's life and a concomitant
saving on the part of health care systems or on the part of the
actual patients. In the particular case of the invention described
herein, early diagnosis is that of potential or existing Toxoplasma
gondii infection in pregnant women, with particular concern for the
health of the foetus, and in infected subjects, particularly those
with impaired immunity.
[0005] Toxoplasma gondii is an obligate intracellular parasite that
infects all mammalian cells, including those of human subjects
(McCabe and Remington, N. Engl. J. Med. 1988, 318-313-5), and other
animal genera, e.g. birds. The life cycle of the parasite is
complex and one may distinguish between three stages of infection:
tachyzoite (asexual), bradyzoite (in tissue cysts, asexual) and
sporozoite (in oocysts, sexual reproduction). Transmission
typically occurs through ingestion of undercooked meat harbouring
tissue cysts or vegetables contaminated with oocysts shed by cats.
Human infection is generally asymptomatic and self-limiting in
immunocompetent hosts. In contrast, in subjects with impaired
immunity (particularly those affected by AIDS), toxoplasmosis is a
severe opportunist infection, which may give rise to encephalitis
with very serious outcomes (Luft, B. J., Remington J. S., 1992,
Clin. Infect. Dis. 15, 211-22). Moreover, contracting primary
infection during pregnancy may lead to miscarriages or to severe
foetal disease in mammals.
[0006] For an extensive overview of the problem of toxoplasmosis
the reader is referred to the specialistic medical literature.
[0007] Diagnosis of T. gondii infection is established by isolating
the micro-organism in the blood or body fluids, identifying the
parasite in tissues, detecting specific nucleotide sequences with
PCR, or by detecting specific anti-T. gondii immunoglobulins
produced by the host in response to the infection (Beaman et al.,
1995 Principles and Practice of Infectious Diseases 4th Ed.,
Churchill Livingstone Inc., New York, 2455-75; Remington J S et al.
1995, Infectious Diseases of the Fetus and Newborn Infant, W.B.
Saunders, Philadelphia, Pa., 140-267).
[0008] One of the main problems in diagnosing T. gondii infections
has to do with pregnant women. To implement suitable therapies in
good time and avoid possible damage to the foetus it is very
important to establish if parasitic infection occurred before or
after conception. This is generally done by attempting to detect
the presence of the various classes of anti-Toxoplasma
immunoglobulins (IgG, IgM, IgA, avidity of IgG). For this reason,
the availability of specific, sensitive diagnostic agents is
desirable.
[0009] T. gondii antigens have long been known and available, first
of all as antigen mixtures obtained in various ways (FR 2,226,468,
Merieux; SU 533376, Veterinary Research Institute; JP 54044016,
Nihon Toketsu Kanso), then as subsequent isolations of pure
antigens (EP 0 082 745, Merieux; EP 0 301 961, INSERM, Pasteur; WO
89/5658, Transgene) and their characterisation both as proteins,
and of their respective genes (WO 89/08700, U. Leland, Dartmouth
Coll.; U.S. Pat. No. 4,877,726, Res. Inst. Palo Alto; WO 89/12683,
INSERM, Pasteur; EP 0 391 319, Mochida Pharm.; IT 1,196,817, CNR;
EP 0 431 541, Behringwerke; WO 92/01067, CNRS; WO 92/02624, U.
Flinders; WO 92/11366, Innogenetics, Smithkline Beecham; U.S. Pat.
No. 5,215,917, Res. Inst. Palo Alto; WO 92/25689, FR 2702491,
INSERM, Pasteur; WO 96/02654, bioMerieux, Transgene; EP 0 710 724
Akzo; EP 0 724 016, bioMerieux; EP 0 751 147, Behringwerke; U.S.
Pat. No. 5,633,139, Res. Inst. Palo Alto; WO 97/27300,
Innogenetics; U.S. Pat. No. 5,665,542, U.S. Pat. No. 5,686,575,
Res. Inst. Palo Alto; WO 99/32633, Heska; JP 11225783, Yano; WO
99/61906, Abbott; WO 99/66043, Smithkline Beecham; JP 2000300278,
Yano; WO 00/164243, Virsol).
[0010] Numerous studies have found various different antigenic
proteins of T. gondii and the gene sequences of these have also
been determined.
[0011] Among the most interesting proteins both for diagnostic and
therapeutic purposes, in the form of vaccines, we should mention:
the surface antigens SAG1 (or P30) (WO 89/08700, Stanford
University; WO 89/12683 Pasteur, INSERM; WO 94/17813, WO 96/02654,
Transgene, bioMerieux; EP 0 724 016, WO 99/61906, U.S. Pat. No.
5,962,654, Harning et al., Clinical and Diagnostic Laboratory
Immunology, May 1996, 355-357); SAG2 (or P22) (Parmley et al.,
1992, J. Clin. Microbiol. 30, 1127-33); the dense granule proteins
GRA1 (or P24) (EP 0 301 961, Pasteur, INSERM; WO 89/05658,
Transgene, Cesbron-Delauw, et al., 1989 P.N.A.S. USA 86, 7537-41);
GRA2 (or P28) (WO 93/25689, INSERM, Pasteur; U.S. Pat. No.
5,633,139, U.S. Pat. No. 5,665,542, U.S. Pat. No. 5,686,575, Res.
Inst. Palo Alto; Prince et al., Mol. Biochem. Parasitol., 34 3-14);
GRA4 (Mevelec et al., Mol. Biochem. Parasitol. 56, 227-38); GRA6
(or P32) (FR 2,702,491, INSERM, Pasteur; Lecordier al., Mol.
Biochem. Parasitol. 70, 85-94); GRA7 (WO 99/61906, Abbott; Jacobs
et al., Mol. Biochem. Parasitol. 91, 237-49); GRA3 (Robben et al.
2002, J. Biol. Chem. 277, 17544-47): the rhoptry antigens ROP1 (or
P66) (U.S. Pat. No. 5,976,553, U. Leland; EP 0 431 541,
Innogenetics); ROP2 (or P54) (Sharma et al., J. Immunol., 131,
377-83).
[0012] As described in the above-mentioned references, the antigens
were obtained with well-known recombinant cDNA techniques in
expression vectors. For example, for the antigen SAG1, WO 98/08700
uses known expression vectors in phage .lamda.gt11. WO 98/12683
uses the same phage and transfects E. coli with a proprietary
plasmid, or by preparing a special expression cassette, as in WO
96/02654. EP 0 724 016 obtains mimotypes, using combinatorial
expression libraries of peptides. EP 0 301 961 describes how to
obtain excretion-secretion antigens with molecular weights ranging
from 20 kDa to 185 kDa. WO 89/05658 describes a protein containing
the epitopes of the 24 kDa protein recognised by the antibodies
produced against Toxoplasma excretion-secretion antigens; this
protein is obtained by transfection of cells by means of expression
vectors. The antigen P28 (GRA2) is described in U.S. Pat. No.
5,633,139 and the method of obtaining it is again implemented
through expression in phage .lamda.gt11. The antigen P32 (GRA6) is
described in patent FR 2,702,491, the antigen ROP1 (P66) in U.S.
Pat. No. 5,976,553, P35 (or GRA8) in EP 0 431 541, WO 99/57295 and
WO 99/61906, and lastly P68 in EP 0 431 541.
[0013] It should be stressed that all these antigens are obtained
by means of molecular biology techniques that use the expression of
proteins in bacterial cells. None of the documents cited describe
the technique of expression/exposure of libraries of cDNA deriving
from Toxoplasma gondii in the lambda phage (phage display) to
obtain fragments of antigens of the pathogen.
[0014] The invention described herein uses a new vector of DNA
expression and protein exposure as molecular fusion with the
amino-terminal part of protein D of the lambda bacteriophage capsid
(pD) (.lamda.KM4).
[0015] The expression/exposure vector was described for the first
time in patent application PCT/IT01/00405, filed on Jul. 26, 2001,
the most important part of which is incorporated herein. This
vector, called .lamda.KM4, differs from that used in expression
only experiments (.lamda.gt11) in that the recombinant protein
coded for by the DNA fragment is expressed as fusion with a protein
of the bacteriophage itself and then exposed on the capsid.
According to the vector project, the phage exposes the protein
fragment on the surface only if its open reading frame (ORF)
coincides with pD. The size of the fragments of DNA cloned in our
libraries was selected in order to represent a population of medium
size ranging from 300 to 1000 nucleotide base pairs (bp), and, for
statistical reasons, most of the out-of-frame sequences contain
stop codons which do not permit their translation and consequently
exposure on the surface of the phage.
SUMMARY OF THE INVENTION
[0016] It has now been found that the combination of the affinity
selection and phage display techniques, together with the use of
the vector .lamda.KM4, provides a method for the identification of
specific antigen fragments of Toxoplasma gondii by means of the
selection of display libraries of DNA fragments with sera of
infected individuals. DNA fragments are obtained either from cDNA
of whole parasite or from DNA encoding for known specific gene
products. With this method it proves possible to identify antigen
fragments from very large libraries (i.e. expressing a large number
of different sequences). The antigen fragments thus identified
enable specific ligands to be obtained, which in turn can be used
as diagnostic and therapeutic means.
[0017] Therefore, one object of the invention described herein is a
method for the identification of antigen fragments of Toxoplasma
gondii proteins, by means of the selection of libraries of DNA
fragments with sera of subjects who have been infected by the
parasite, using the phage display technique, characterised in that
it uses the vector .lamda.KM4.
[0018] Another object of the present invention are antigen
fragments obtainable with the above-mentioned method, both isolated
and characterised, and as sets of antigen fragments called
"collections". The invention described herein also extends to the
antigen portions of said fragments (epitopes).
[0019] The use of said antigen fragments as diagnostic agents and
the related diagnostic aids containing them, for example in the
form of kits or other supports, constitute a further object of the
present invention.
[0020] The use of said antigen fragments as active agents,
particularly with an immunogenic action, for the preparation of
medicaments for the prevention and therapy of Toxoplasma gondii
infection, constitute a further object of the present
invention.
[0021] Another object of the present invention are the gene
sequences coding for the above-mentioned antigen fragments, their
use as medicaments, particularly for the prevention and therapy of
Toxoplasma gondii infection, e.g. as gene therapy. The present
invention also extends to the gene sequences that hybridise with
the sequences of the above-mentioned fragments in stringent
hybridisation conditions.
[0022] Another object of the present invention are anti-epitope
antibodies and their use in the preparation of diagnostic,
preventive and therapeutic means, e.g. as conjugates with active
ingredients such as chemo-therapy agents. Antibodies can be
generated also against collections of said epitopes.
[0023] The method provided by the present invention makes it
possible to confirm the use of the Toxoplasma gondii antigens
described above as such as diagnostic agents and also to identify
in known antigens the epitopes that trigger an immune response in
human subjects; this portion is a further object of the present
invention; but it also makes it possible to identify the antigenic
function of proteins of Toxoplasma gondii, or of portions thereof,
which, though their structure and possibly their physiological
function may be known, are unknown as regards their antigenic
function, and such function comes within the framework of the
present invention; lastly, the method according to the present
invention also provides new antigen fragments of Toxoplasma gondii
proteins, that constitute yet another object of the present
invention.
[0024] Another object of the present invention is the use of the
antigen fragments thus identified for the preparation of means of
diagnosing the infection, as well as the actual diagnostic means
containing them. The use realtes also to the diagnosis of the time
of the infection, in particular by the IgG avidity assay.
[0025] Another object of the present invention is the use of the
antigen fragments thus identified as medicaments, particularly for
the preparation of formulations, and particularly in the form of
vaccines, which are useful for the prevention and cure of the
infection. The vaccines according to the present invention are
suitable for use in humans and other animals (particularly pigs,
cats, sheeps).
[0026] Another object of the present invention are ligands
generated from the above-mentioned antigen fragments and the
related collections and the use of such ligands for the preparation
of diagnostic means for the detection of the infection, with
particular reference to the time of infection, as well as
therapeutic means for the prevention and treatment of the infection
itself.
[0027] Another object of the present invention is a method for the
diagnosis of Toxoplasma gondii infection, comprising the selection
of sera of subjects affected or suspected of being affected by said
infection with the above mentioned antigen fragments and/or their
collection and/or at least one ligand and/or antibody.
[0028] These and other objects will be illustrated here below in
detail, also by means of examples and figures, where FIG. 1
represents the map of the vector .lamda.KM4.
DETAILED DESCRIPTION OF THE INVENTION
[0029] The present invention comprises the construction of
expression/exposure libraries of DNA fragments prepared from
Toxoplasma gondii cells, the selection of such libraries with the
sera of patients who have been infected by Toxoplasma gondii, the
characterisation of the antigen fragments, and the use of said
fragments for developing selective diagnostic means.
[0030] Optionally, the present invention may entail the generation
of specific ligands for said antigen fragments (e.g. human
recombinant antibodies or humanised murine recombinant antibodies)
and the construction of selective diagnostic means that incorporate
the ligands generated.
[0031] Antibodies and ligands of the present invention can be
obtained according according to the general common knowledge and
conventional methods.
[0032] The method according to the present invention advantageously
combines affinity selection and the power of phage display.
[0033] What is meant by phage display, as understood by the person
of ordinary skill in the art, is a strategy based on the selection
of expression/exposure libraries in which small protein domains are
exposed on the surface of bacteriophages containing the
corresponding genetic information.
[0034] The method implemented according to the present invention
for the first time provides new and advantageous analysis
possibilities: [0035] the use of small amounts of serum to identify
antigen fragments of the infectious agent, [0036] the possibility
of selecting only the domains responsible for the interaction with
the antibodies, without having to express the entire gene, the
product of which may be insoluble or toxic; lastly, the possibility
of effecting successive cycles of selection using sera from
different patients or mixtures of sera facilitates the
identification of cross-reactive antigens which represent one of
the main objectives of the present invention.
[0037] For the above-mentioned reasons, for each library, messenger
RNA was purified from an adequate number of cells (e.g. 10.sup.6
cells), using common commercially available means, from which the
corresponding cDNA was generated. The latter was fragmented (by
means of the bacterial enzyme DNaseI) and then cloned in the
expression/exposure vector .lamda.KM4 (see example).
[0038] In the other embodiment, relating to specific T. gondii
gene, each specific T. gondii gene, was amplified from the DNA of
the parasite (either cDNA or genomic DNA, both prepared by using
common commercially available kits) by means of PCR with specific
synthetic oligonucleotides. DNA of single genes was then fragmented
randomly by means of the bacterial enzyme DNaseI and then cloned as
a pool in the expression/exposure vector .lamda.KM4.
[0039] The amplification of the libraries was done by means of
normal techniques with which the expert in the field is familiar,
e.g. by plating, growth, elution, purification and concentration
(Sambrook et al., 1989, Molecular Cloning: a laboratory manual,
Cold Spring Harbor Laboratory Press, NY). The libraries were then
used to develop the selection conditions, screening and
characterisation of the sequences identified. Lastly, the phage
clones identified were characterised by immunoenzymatic assays.
[0040] A library of the phage display type, constructed using cDNA
deriving from cells of pathogenic organisms, makes it possible to
exploit affinity selection, which is based on incubation of
specific sera (reactive with the pathogen) with collections of
bacteriophages that express portions of proteins of the pathogen on
their capside and that contain the corresponding genetic
information. The bacteriophages that specifically bind the
antibodies present in the serum are easily recovered, remaining
bound (by the antibodies themselves) to a solid support (e.g.
magnetic beads); the non-specific ones, by contrast, are washed
away. Direct screening, i.e. the analysis of the ability of single
phage clones to bind the antibodies of a given serum, is done only
at a later stage, when the complexity of the library (i.e. the
different number of sequences) is substantially reduced, precisely
as a result of the selection.
[0041] The use of selection strategies allows faster analysis of a
large number of different protein sequences for the purposes of
identifying those that respond to a particular characteristic, e.g.
interacting specifically with antibodies present in the serum of
patients who have been infected by the pathogen. What is more, the
combination of affinity selection and phage display makes it
possible to use a smaller amount of serum for each analysis. The
direct screening of a classic cDNA library, in fact, entails the
use of large amounts of serum, which are not always easy to obtain.
For example, to analyse a library of approximately 10.sup.6
independent clones it would be necessary to incubate along with the
preselected serum the numerous filters containing a total of
approximately 10.sup.7 phage plaques transferred from the different
culture plates with the infected bacteria (e.g. a serum volume of
1-10 ml). The use of a display-type library, on the other hand,
permits affinity selection in small volumes (0.1-1 ml) prior to
direct screening, and from limited amounts of serum, such as, for
example, 10 .mu.l.
[0042] Lastly, given that a very large number of bacteriophages can
be contained in small volumes (e.g. 10.sup.11 phage particles are
normally contained in a volume of 0.1 ml), and affinity selection
is done in small volumes (0.1-1 ml), a further advantage of the use
of display-type libraries consists in analysing a number of
independent clones (particles of recombinant phages exposing
different cDNA sequences on their surfaces) 10-100 times greater
(e.g. 10.sup.8 different bacteriohages) than expression-alone
libraries where, as a result of technical problems, not more than
10.sup.6 independent clones are normally analysed.
[0043] As regards industrial applicability, one possible
realisation of the present invention is in the form of diagnostic
kits containing the antigen fragments and/or ligands and/or
antibodies described above.
[0044] The diagnostic kits which are the object of the present
invention are known to the expert in the field and do not require
any particular description. By way of an example, the reader is
referred to the patent literature cited above, to which may be
added U.S. Pat. No. 6,265,176 and WO 01/63283 as further
references.
[0045] Similar considerations hold good for the therapeutic
application, where the preparation of medicaments or vaccines comes
within the framework of general knowledge; for further reference
purposes the reader is again referred to the patent literature
cited in the present description.
[0046] The invention will now be illustrated in greater detail by
means of examples and figures, where FIG. 1 presents the map of the
vector .lamda.KM4.
EXAMPLE 1
Construction of the Vector .lamda.KM4
[0047] This technique is described in international patent
application N.sup.o PCT/IT01/00405, filed on Jul. 26, 2001 and
incorporated herein for reference purposes, explicitly mentioning
the references cited therein. FIG. 1 represents the map of the
vector .lamda.KM4. The plasmid pNS3785 (Sternberg and Hoess, 1995,
Proc. Natl. Acad. Sci. USA., 92:1609-1613) was amplified by inverse
PCR using the synthetic oligonucleotides
5'-TTTATCTAGACCCAGCCCTAGGAAGCTTCTCCTGAGTAGGACAAATCC-3' (SEQ ID No
1) bearing the sites XbaI and AvrII (underlined) for the subsequent
cloning of the lambda phage, and
5'-GGGTCTAGATAAAACGAAAGGCCCAGTCTTTC-3' (SEQ ID No 2) bearing the
site XbaI. In inverse PCR a mixture of Taq DNA polymerase and Pfu
DNA polymerase was used to increase the fidelity of the DNA
synthesis. Twenty-five amplification cycles were performed
(95.degree. C.--30 sec, 55.degree. C.--30 sec, 72.degree. C.--20
min). The autoligation of the PCR product, previously digested with
XbaI endonuclease gave rise to the plasmid pKM3. The lambda gene pD
was amplified with PCR from the plasmid pNS3785 using the primers
5'-CCGCCTTCCATGGGTACTAGTTTTAAATGCGGCCGCACGAGCAAAGAAACCTTTAC-3' (SEQ
ID No 3) e 5'-AGCTTCCTAGGGCTGGGTCTAG-3' (SEQ ID No 4) containing
the restriction sites NcoI, SpeI, NotI and EcoRI, respectively,
(underlined). The PCR product was then purified, digested with NcoI
and EcoRI endonuclease and recloned in sites NcoI and EcoRI of
pKM3, resulting in the plasmid pKM4 bearing only the restriction
sites SpeI and NotI at the 5' end of the protein gpD. The plasmid
was then digested with XbaI endonuclease and cloned in the XbaI
site of the lambda phage Dam15imm21nin5 (Sternberg and Hoess, 1995,
Proc. Natl. Acad. Sci. USA., 92:1609-1613).
Construction of cDNA Library from Tachyzoites of Toxoplasma
gondii
[0048] Tachyzoites of the protozoon Toxoplasma gondii (RH strain)
were grown in vitro in monkey kidney cells ("VERO" African green
monkey cells) using DMEM culture medium containing 10% foetal
bovine serum, 2 mM glutamine and 0.05 mg/ml gentamicin (Gibco BRL,
Canada). The parasites were collected after complete lysis of the
host cells and purified by filtration (filter porosity 3 .mu.m)
followed by centrifuging. 4 .mu.g of mRNA were isolated from
10.sup.7 tachyzoites using the "QuickPrep Micro mRNA Purification
Kit" (Amersham Pharmacia Biotech, Sweden) and following the
manufacturer's instructions. The double-helix cDNA was synthesised
from 200 ng of poly(A).sup.+ RNA using the "SMART cDNA Library
Construction Kit" (Clontech, CA, USA) and following the
manufacturer's instructions. 10 .mu.g of total cDNA were then
fragmented randomly using 0.5 ng of the endonuclease DNaseI
(Sigma-Aldrich, USA). The mixture of cDNA and DNaseI was incubated
for 20 minutes at 15.degree. C. and the cDNA fragments were
purified with extraction in phenol/chloroform and subsequent
purification by means of the "QIAquick PCR Purification Kit"
(Qiagen, CA, USA), following the manufacturer's instructions. The 3
.mu.g ends of the cDNA fragments were "flattened" by incubating the
DNA with 9 units of the enzyme T4 DNA polymerase (New England
Biolabs, MA, USA) for 60 minutes at 15.degree. C. The fragments
were then purified by means of extraction in phenol/chloroform and
subsequent precipitation in ethanol. 500 ng of the resulting DNA
were bound with a 20-fold molar excess of "synthetic adaptors" for
the purposes of adding the restriction sites SpeI and NotI to the
ends of the fragments. Six adaptors were used, obtained by
hybridisation of the following pairs of oligonucleotides: K185
5'-CTAGTCGTGCTGGCCAGC-3' (SEQ ID No 5) and K186
5'-GCTGGCCAGCACGA-3' (SEQ ID No 6); K187 5'-CTAGTCGTGCTGGCCAGCT-3'
(SEQ ID No 7) and K188 5'-AGCTGGCCAGCACGA-3' (SEQ ID No 8); K189
5'-CTAGTCGTGCTGGCCAGCTG-3' (SEQ ID No 9) and K190
5'-CAGCTGGCCAGCACGA-3' (SEQ ID No 10); K191 5'-TCTGGTGGCGGTAGC-3'
(SEQ ID No 11) and K192 5'-GGCCGCTACCGCCACCAGA-3' (SEQ ID No 12);
K193 5'-TTCTGGTGGCGGTAGC-3' (SEQ ID No 13) and K194
5'-GGCCGCTACCGCCACCAGAA-3' (SEQ ID No 14); K195
5'-TTTCTGGTGGCGGTAGC-3' (SEQ ID No 15) and K196
5'-GGCCGCTACCGCCACCAGAAA-3' (SEQ ID No 16). The excess of unligated
adaptors was removed from the ligation mixture by electrophoresis
on 2% agarose gel and the cDNA fragments with molecular weights
ranging from 300 bp to 1000 bp were excised from the gel and
purified by means of the "Qiaquick gel extraction kit" (Qiagen, CA,
USA) following the manufacturer's instructions. The vector
.lamda.KM4 was digested with SpeI/NotI and for the construction of
the library 6 ligation mixtures were performed, each containing 0.4
.mu.g of vector and approximately 7 ng of insert. After overnight
incubation at 4.degree. C. the ligation mixtures were packaged in
vitro with the "Ready-To-Go lambda packaging kit" (Amersham
Pharmacia Biotech, Sweden) and plated for infection of BB4 cells
(bacterial cells of E. coli strain BB4; Sambrook et al., 1989,
Molecular Cloning: a laboratory manual, Cold Spring Harbor
Laboratory Press, NY). After overnight incubation at 37.degree. C.
the phage was eluted from the plates with SM buffer (Sambrook et
al., 1989, Molecular Cloning: a laboratory manual, Cold Spring
Harbor Laboratory Press, NY), purified, concentrated and stored at
-80.degree. C. in SM buffer containing 7% dimethylsulphoxide. The
complexity of the library calculated as the number of total
independent clones with inserts was 10.sup.7 clones.
Affinity Selection
[0049] Two distinct methods were used for selecting the phage
library with human sera. In the first method 100 .mu.l of magnetic
beads coated with Protein G (Dynabeads Protein-G, Dynal, Norway)
were incubated with 10 .mu.l of human serum for 30 minutes at room
temperature. The beads were then incubated for 1 hour at 37.degree.
C. with blocking solution consisting of: 5% skimmed milk powder in
PBS (Sambrook et al., 1989, Molecular Cloning: a laboratory manual,
Cold Spring Harbor Laboratory Press, NY), 0.05% Tween 20, and
MgSO.sub.4 10 mM. Approximately 10.sup.10 phage particles of the
library were added to the beads and diluted in 1 ml of blocking
solution for a further 4-hour incubation at room temperature with
weak stirring. In the second method 40 .mu.l of "M280-Tosyl
activated" magnetic beads (Dynal, Norway) were coated with human
anti-IgM antibodies (Sigma-Aldrich, USA) following the
manufacturer's instructions. The beads were then washed with
PBS/TritonX-100 1% and incubated with 10 .mu.l of human serum in
300 .mu.l of blocking solution for 2 hours at room temperature.
After washing the beads three times with washing solution (PBS, 1%
TritonX100, 10 mM MgSO.sub.4), 10.sup.10 phage particles of the
library were added to the beads and diluted in 200 .mu.l of
blocking solution for a further 3-hour incubation at room
temperature with weak stirring.
[0050] With both selection methods, the beads were washed 10 times
with 1 ml of washing solution (PBS, 1% TritonX100, 10 mM
MgSO.sub.4). The bound bacteriophages were amplified for infection
of BB4 cells added directly to the beads (1.2 ml per selection) and
subsequent 30-minute incubation at room temperature. 12 ml of
NZY-Top Agar (Sambrook et al., 1989, Molecular Cloning: a
laboratory manual, Cold Spring Harbor Laboratory Press, NY) were
added to the mixture of beads and cells and immediately poured onto
NZY plates (2 15-cm Petri capsules for selection). The plates were
incubated for 12-16 hours at 37.degree. C. The next day the phages
were collected from the plates by means of the addition of 15 ml of
SM buffer per plate and stirring for 4 hours at room temperature.
The phages were purified by precipitation in PEG/NaCl (20%
polyethylene glycol, NaCl 1M) and finally resuspended in 5 ml of SM
and stored at +4.degree. C.
Selection of the Library with Human Sera
[0051] To identify the specific antigens of T. gondii an affinity
selection procedure was used consisting of two "panning" cycles
with one or more positive sera (that is to say sera deriving from a
patient who tested positive for the presence of antibodies directed
against the parasite), followed by an immunological screening
procedure carried out with the same sera or, alternatively, by
analysis of single clones taken at random from the mixture of
selected phages. Preferably, the library was selected with 10
positive sera (T1, T2, T3, T4, T5, T6, T7, T8, T9 and T10),
generating, after a single selection cycle, the corresponding
mixtures p1.sup.I, p2.sup.I, p3.sup.I, p4.sup.I, p5.sup.I,
p6.sup.I, p7.sup.I, p8.sup.I, p9.sup.I and p10.sup.I. Each mixture
was then subjected to a second affinity selection cycle with the
same serum, according to the first strategy mentioned above, giving
rise to a second series of mixtures (called p1.sup.II, p2.sup.II,
p3.sup.II, p4.sup.II, p5.sup.II, p6.sup.II, p7.sup.II, p8.sup.II,
p9.sup.II and p10.sup.II). The initial characterisation by means of
an enzyme-linked immunosorbent assay (Phage-ELISA) showed that some
of the mixtures were more reactive with the corresponding serum
used for the selection, thus confirming the efficacy of the library
and the affinity selection procedure. Various positive clones were
identified by means of immunoplate screening per plaque of reactive
mixtures.
Phage-ELISA
[0052] Multi-well plates (Immunoplate Maxisorb, Nunc, Denmark) were
coated, incubating 100 .mu.l/well of anti-lambda polyclonal
antibodies overnight at 4.degree. C. with a concentration of 0.7
.mu.g/ml in NaHCO.sub.3 50 mM, pH 9.6. After eliminating the
coating solution, the plates were incubated with 250 .mu.l of
blocking solution (5% skimmed milk powder in PBS, 0.05% Tween-20).
The plates were then washed twice with washing buffer (PBS, 0.05%
Tween-20). A mixture of 100 .mu.l of blocking solution containing
phage lysate (diluted 1:1) was added to each well and incubated for
60 minutes at 37.degree. C. 1 .mu.l of human serum was preincubated
for 30 minutes at room temperature with 10.sup.9 wild-type phage
particles, 1 .mu.l of rabbit serum, 1 .mu.l of bacterial extract of
BB4 cells, 1 .mu.l of foetal bovine serum in 100 .mu.l of blocking
solution. The plates were washed 5 times after incubation with the
phage lysate and then incubated with the serum solution for 60
minutes at 37.degree. C. The plates were then washed 5 times and
incubated in blocking solution containing human anti-immunoglobulin
antibodies conjugated with the enzyme peroxidase (Sigma-Aldrich,
USA) diluted 1:10000 and rabbit serum diluted 1:40. After 30
minutes' incubation the plates were washed 5 times and the
peroxidase activity was measured with 100 .mu.l of TMB liquid
substrate (Sigma-Aldrich, USA). After 15 minutes' development, the
reaction was stopped by adding 25 .mu.l of H.sub.2SO.sub.4 2M.
Lastly, the plates were analysed using an automatic ELISA reader
(Multiskan, Labsystem, Finland) and the results were expressed as
OD=OD.sub.450nm-OD.sub.620nm. The ELISA data were assessed as mean
values of two independent assays.
Immunoscreening
[0053] Phage plaques were transferred from the bacterial medium to
nitrocellulose filters (Schleicher & Schuell, Germany) by means
of incubation at room temperature for 60 minutes. The filters were
blocked for 60 minutes at room temperature in blocking solution (5%
skimmed milk powder in PBS, 0.05% Tween-20). 40 .mu.l of human
serum were preincubated with 40 .mu.l of bacterial extract of BB4
cells, 10.sup.9 wild-type lambda phage particles in 4 ml of
blocking solution. After eliminating the blocking solution, the
filters were incubated with the serum for 3 hours at room
temperature under stirring. The filters were then washed 5 times
with washing buffer (PBS, 0.05% Tween-20) and then incubated for 60
minutes at room temperature, alternatively with human anti-IgG
antibodies conjugated with alkaline phosphatase (Sigma-Aldrich,
USA), or with human anti-IgM antibodies conjugated with alkaline
phosphatase (Sigma-Aldrich, USA), both diluted 1:7500 in blocking
solution. After washing the filters 5 times, 5 ml of development
solution (substrates BCIP and NBT, Sigma-Aldrich, USA) were added
and the development was interrupted by washing the filters in
water.
Preparation of the Lambda Phase from Lysogenic Cells
[0054] Phage clones that proved positive at immunoscreening (direct
screening) were isolated from the respective phage plaques and then
amplified for subsequent characterisation. The bacterial BB4 cells
were grown under stirring at 37.degree. C. up to an optical density
OD.sub.600=1.0 in LB culture medium (Sambrook et al., 1989,
Molecular Cloning: a laboratory manual, Cold Spring Harbor
Laboratory Press, NY) containing 0.2% maltose and 10 mM MgSO.sub.4,
recuperated by centrifuging and resuspended in SM buffer at optical
density OD.sub.600=0.2. 100 .mu.l of cells were infected with
recombinant bacteriophages recovered from single plaques, incubated
for 20 minutes at room temperature, plated on LB medium with
ampicillin (100 .mu.g/ml) and then incubated for 18-20 hours at
32.degree. C. A single bacterial colony was then grown in 10 ml of
LB/ampicillin overnight at 32.degree. C. under stirring. 500 ml of
LB/ampicillin and MgSO.sub.4 10 mM were added to 5 ml of the
overnight culture and incubated at 32.degree. C. up to an optical
density OD.sub.600=0.6 under vigorous stirring. The culture was
then incubated for 15 minutes in a water bath at 45.degree. C. and
then at 37.degree. C. for a further 3 hours. After this, the
bacteriophages were purified of the bacterial culture according to
standard procedures (Sambrook et al., 1989, Molecular Cloning: a
laboratory manual, Cold Spring Harbor Laboratory Press, NY) and
stored at +4.degree. C.
[0055] Lastly, the phage clones were analysed by means of
phage-ELISA with a substantial panel of positive and negative sera.
Clones whose ELISA value exceeded the background value, as obtained
from the sum of the mean of the measurements of the negative sera
and three times the standard deviation, were judged to be
positive.
[0056] The following table 1 gives, by way of examples, the
reactivity of a number of the recombinant bacteriophages selected.
TABLE-US-00001 TABLE 1 Reactivity of phage Reactivity of phage
clone clone with positive with negative sera Name of clone sera
(positive/total pos.) (negative/total neg.) Tx-4.11 13/21 0/10
TxM-17.2 4/8 1/8 Tx-15.11 11/21 0/10 Tx-1.11 19/21 0/10 Tx-8.0 6/21
0/10 Tx-1.16 20/21 0/10 Tx-9.18 9/21 0/8 Tx-7.11 21/21 0/10
Characterisation of Positive Clones
[0057] The clones which showed multiple reactivity with the
Toxoplasma gondii positive sera and which presented no reactivity
to the negative sera were subsequently sequenced and compared with
various databases of sequences currently available (Non-Redundant
Genbank CDS, Non-Redundant Database of Genbank Est Division,
Non-Redundant Genbank+EMBL+DDBJ+PDB Sequences).
[0058] The sequences obtained can be classified in four groups:
[0059] sequences that code for known T. gondii antigen fragments;
[0060] sequences that code for known proteins which, however, are
not known to be involved in the human antibody response; [0061]
sequences that code for unknown proteins (e.g. EST); [0062] new
sequences, not yet figuring in the databases.
[0063] The following table 2 gives, by way of examples, the
sequences of some of the clones selected: TABLE-US-00002 TABLE 2
Name of clone Sequence Identification Classification Tx-4.11
AGTGGAGGGACAGGGC GRA 1 Dense granul (SEQ ID No 17) AGGGATTAGGAATCGG
known protein AGAATCTGTAGATTTG T. gondii GAGATGATGGGGAACA antigen
CGTATCGTGTGGAGAG ACCCACAGGCAACCCG GACTTGCTCAAGATCG CCATTAAAGCTTCAGA
TGGATCGTACAGCGAA GTCGGCAATGTTAACG TGGAGGAGGTGATTGA TACTATGAAAAGCATG
CAGAGGGACGAGGACA TTTCCTTCGTGCGTTG AACAAAGGCGAAACAG TAGAGGAAGCGATCGA
AGACGTGGCTCAAGCA GAAGGGCTTAATTCGG AGCAAACCCTGCAACT GGAAGATGCAGTGAGC
GCGGTGGCGTCTGTTG TTCAAGACGAG TxM-17.2 TACTCTTCACCACGAA GRA2 Dense
granul (SEQ ID No 18) TAGTTGTTTTGATTAG known protein
ATATTGCTTCTTCTCC T. gondii ACATATCGCCTCACAA antigen
TGTTCGCCGTAAAACA TTGTTTGCTGGTTGTT GCCGTTGGCGCCCTGG TCAACGTCTCGGTGAG
GGCTGCCGAGTTTTCC GGAGTTGATTAACCAG GGACCT Tx.15.11 GCTGCCTTGGGAGGCC
GRA 3 Dense granul (SEQ ID No 19) TTGCGGCGGATCAGCC protein-
TGAAAATCATCAGGCT unknown CTTGCAGAACCAGTTA as antigen i
CGGGTGTGGGGGAAGC human AGGAGTGTCCCCCGTC response AACGAAGCTGGTGAGT
CATACAGTTCTGCAAC TTCGGGTGTCCAAGAA GCTACCGCCCCAGGTG CAGTGCTCCTGGACGC
AATCGATGCCGAGTCG GATAAGGTGGACAATC AGGCGGAGGGAGGTGA GCGTATGAAGAAGGTC
GAAGAGGAGTTGTCGT TATTGAGGCGGGAATT ATATGATCGCACAGAT CGCCCTGGT
Tx-1.11 CAGTTCGCTACCGCGG GRA 7 Dense granul (SEQ ID No 20)
CCACCGCGTCAGATGA known protein CGAACTGATGAGTCGA T. gondii
ATCCGAAATTCTGACT antigen TTTTCGATGGTCAAGC ACCCGTTGACAGTCTC
AGACCGACGAACGCCG GTGTCGACTCGAAAGG GACCGACGATCACCTC ACCACCAGCATGGATA
AGGCATCTGTAGAGAG TCAGCTTCCGAGAAGA GAGCCATTGGAGACGG AGCCAGATGAACAAGA
AGAAGTTCAT Tx-8.0 GAGAACCCGGTGAGAC GRA8 Dense granul (SEQ ID No 21)
CGCCTCCTCCCGGTTT known protein CCATCCAAGCGTTATT T. gondii
CCCAATCCCCCGTACC antigen CGCTGGGCACTCCAGC GGGCATGCCACAGCCA GAGGTTCC
Tx-1.16 AGGAGGACTGGATGTC MIC 3 Microneme (SEQ ID No 22)
ATGCCTTCAGGGAGAA protein- CTGCAGCCCTGGTAGA unknown a
TGTATTGATGACGCCT antigen i CGCATGAGAATGGCTA human CACCTGCGAGTGCCCC
response ACAGGGTACTCACGTG AGGTGACTTCCAAGGC GGAGGAGTCGTGTGTG
GAAGGAGTCGAAGTCA CGCTGGCTGAGAAATG CGAGAAGGAATTCGGC ATCAGCGCGTCATCCT
GCAAATGCGAT Tx-9.18 GCACCCACTCAATCTG MIC 5 Microneme (SEQ ID No 23)
AAATGAAAGAATTCCA known protein AGAGGAAATCAAAGAA T. gondii
GGGGTGGAGGAAACAA antigen AGCATGAAGACGATCC TGAGATGACGCGGCTC
ATGGTGACCGAGAAGC AGGAGAGCAAAAATTT CAGCAAGATGGCGAAA TCCCAGAGTTTAGCAC
GCGAATCGAAGAGCTC GGGGGATCCATTTCGT TTCTAACTGAAACGGG GGTCACAATGATCGAG
TTGCCCAAAACTGTCA GTGAACATGACATGGA CCAACTACTCCAC Tx-7.11
GTTATGGCATCGGATC SAG 1 Surface (SEQ ID No 24) CCCCTCTTGTTGCCAA
known protein TCAAGTTGTCACCTGC T. gondii CCAGATAAAAAATCGA antigen
CAGCCGCGGTCATTCT CACACCGACGGAGAAC CACTTCACTCTCAAGT GCCCTAAAACAGCGCT
CACAGAGCCTCCCACT CTTGCGTACTCACCCA ACAGGCAAATCTGCCC AGCGGGTACTACAAGT
AGCTGTACATCAAAGG CTGTAACATTGAGCTC CTTGATTCCTGAAGCA GAAGATAGCTGGTGGA
CGGGGGATTCTGCTAG TCTCGACACGGCAGGC ATCAAACTCACAGTTC CAATCGAGAAGTTCCC
CGTGACAACGCAGACG TTTGTGGTCGGTTGCA TCAAGGGAGACGACGC ACAGAGTTGTATGGTC
ACGGTGACAGTACAAG CCAGAGCCTCATCGGT CGTCAATAATGTCGCA AGGTGCTCCTATGGTG
CGGACAGC Tx-4.18 CCATCGGTCGTCAATA SAG 1 Surface (SEQ ID No 25)
ATGTCGCAAGGTGCTC known protein CTACGGTGCAGACAGC T. gondii
ACTCTTGGTCCTGTCA antigen AGTTGTCTGCGGAAGG ACCCACTACAATGACC
CTCGTGTGCGGGAAAG ATGGAGTCAAAGTTCC TCAAGACAACAATCAG TACTGTTCCGGGACGA
CGCTGACTGGTTGCAA CGAGAAATCGTTCAAA GATATTTTGCCAAAAT TAACTGAGAACCCGTG
GCAGGGTAACGCTTCG AGTGATAAGGGTGCCA CGCTAACGATCAAGAA GGAAGCATTTCCAGCC
GAGTCAAAAAGCGTCA TTATTGGATGCACAGG GGGATCGCCTGAGAAG CATCACTGTACCGTGA
AACTGGAGTTTGCCGG GGCTGCAGGGTCAGCA AAATCGGCT
[0064] The clone Tx-4.11 constitutes a fragment of the antigen GRA1
(Cesbron-Delauw et al., 1989, Proc. Natl. Acad. Sci. USA
86:7537-7541) but has never been identified as an "antigen
fragment" of the protein in the human humoral response. Said clone
has the amino acid sequence (SEQ ID No 26)
SGGTGQGLGIGESVDLEMMGNTYRVERPTGNPDLLKIAIKASDGSYSEVGNVNVEEVIDTMKS-
MQRDEDIFLR ALNKGETVEEAIEDVAQAEGLNSEQTLQLEDAVSAVASVVQDE and its use
as a fragment containing an epitope is covered by the present
invention.
[0065] The clone TxM-17.2 constitutes a fragment of the antigen
GRA2 (Prince et al., 1989, Mol. Biochem. Parasitol., 34: 3-14) but
has never been identified as an "antigen fragment" of the protein
in the human humoral response. Said clone has the amino acid
sequence (SEQ ID No 27)
YSSPRIVVLIRYCFFSTYRLTMFAVKHCLLVVAVGALVNVSVRAAEFSGVVNQGP and its use
a fragment containing an epitope is covered by the present
invention.
[0066] The clone Tx-15.11 constitutes a fragment of the gene GRA3
(Bermudes et al., 1994, Mol. Biochem. Parasitol., 68: 247-257) and
has never been identified as an antigen in the human antibody
response. Said clone has the amino acid sequence (SEQ ID No 28)
AALGGLAADQPENHQALAEPVTGVGEAGVSPVNEAGESYSSATSG
VQEATAPGAVLLDAIDAESDKVDNQAEGGERMKKVEEELSLLRRE LYDRTDRPG and its use
as a fragment containing an epitope is covered by the present
invention.
[0067] The clone Tx-1.11 constitutes a fragment of the antigen GRA7
(Bonhomme et al., 1998, J. Histochem. Cytochem. 46, 1411-1421) and
has never been identified as an "antigen fragment" of the protein
in the human humoral response. Said clone has the amino acid
sequence (SEQ ID No 29)
FATAATASDDELMSRIRNSDFFDGQAPVDSLRPTNAGVDSKGTDDHLTTSMDKASVESQLPRREPLETE-
PDEQEEVHF and its use as a fragment containing an epitope is
covered by the present invention.
[0068] The clone Tx-8.0 constitutes a fragment of the antigen GRA8
(Kimberly et al., 2000, Mol. Biochem. Parasitol., 105: 25-37) and
has never been identified as an "antigen fragment" of the protein
in the human humoral response. Said clone has the amino acid
sequence (SEQ ID No 30) ENPVRPPPPGFHPSVIPNPPYPLGTPAGMPQPEVP and its
use as a fragment containing an epitope is covered by the present
invention.
[0069] The clone Tx-1.16 constitutes a fragment of the gene MIC3
(Garcia-Reguet et al., 2000, Cellular Microbiol., 2: 353-364) and
has never been identified as an antigen in the human antibody
response. Said clone has the amino acid sequence (SEQ ID No 31)
RRTGCHAFRENCSPGRCIDDASHENGYTCECPTGYSREVTSKAEESCVEGVEVTLAEKCEKE
FGISASSCKCD and its use as a fragment containing an epitope is
covered by the present invention.
[0070] The clone Tx-9.18 constitutes a fragment of the antigen MIC5
(Brydges et al., 2000, Mol. Biochem. Parasitol., 111: 51-66) but
has never been identified as an "antigen fragment" of the protein
in the human humoral response. Said clone has the amino acid
sequence (SEQ ID No 32)
APTQSEMKEFQEEIKEGVEETKHEDDPEMTRLMVTEKQESKNFSKMAKSQSFSTRIEELGGSISFLTET-
GVTMIELPKTVSEHDMDQLL H and its use as a fragment containing an
epitope is covered by the present invention.
[0071] The clone Tx-7.11 constitutes a fragment of the antigen SAG1
(Burg et al., 1988, J. Immunol., 141:3584-3591) but has never been
identified as an "antigen fragment" of the protein in the human
humoral response. Said clone has the amino acid sequence (SEQ ID No
33) VMASDPPLVANQVVTCPDKKSTAAVILTPTENHFTLKCPKTALTEPPTLAYSPNR
QICPAGTTSSCTSKAVTLSSLIPEAEDSWWTGDSASLDTAGIKLTVPI
EKFPVTTQTFVVGCIKGDDAQSCMVTVTVQARASSVVNNVARCSYG ADS and its use a
fragment containing an epitope is covered by the present
invention.
[0072] The clone Tx-4.18 constitutes a fragment of the antigen SAG1
(Burg et al., 1988, J. Immunol., 141:3584-3591) but has never been
identified as an "antigen fragment" of the protein in the human
humoral response. Said clone has the amino acid sequence (SEQ ID No
34) PSVVNNVARCSYGADSTLGPVKLSAEGPTTMTLVCGKDGVKVPQDNNQYCSGTT
LTGCNEKSFKDILPKLTENPWQGNASSDKGATLTIKEAFPAESKS
VIIGCTGGSPEKHHCTVLLEFAGAAGSAKSA and its use as a fragment
containing an epitope is covered by the present invention.
Expression of cDNA Fragments Selected from the Library as Fusion
Products with GST
[0073] The plasmid pGEX-SN was constructed by cloning the DNA
fragment deriving from the hybridisation of the synthetic
oligo-nucleotides K108 5'-GATCCTTACTAGTTTTAGTAGCGGCCGCGGG-3' (SEQ
ID No 35) and K109 5'-AATTCCCGCGGCCGCTACTAAAACTAGTAAG-3' (SEQ ID No
36) in the BamHI and EcoRI sites of plasmid pGEX-3X (Smith and
Johnson, 1988, Gene, 67, 31-40).
[0074] The phage clones for which specific reactivity with sera of
patients testing positive for Toxoplasma gondii was demonstrated,
were amplified and then analysed with a substantial panel of
positive and negative sera. After this ELISA study, DNA inserts of
clones that showed multiple reactivity with Toxoplasma
gondii-positive sera and presented no reactivity with the negative
sera were cloned as fusion products with the protein Glutathione
Sulphur Transferase (GST) and expressed in the cytoplasma of
bacterial cells, for the purposes of determining their specificity
and selectivity. To produce the fusion proteins each clone was
amplified from a single phage plaque by PCR, using the following
oligonucleotides: K47 5'-GGGCACTCGACCGGAATTATCG-3' (SEQ ID No 37)
and K85 5'-GGGTAAAGGTTTCTTTGCTCG-3' (SEQ ID No 38). The resulting
fragment was then purified by means of the "Qiagen Purification
Kit" (Qiagen, CA, USA), digested with the restriction enzymes SpeI
and NotI and cloned in the vector pGEX-SN to generate the fusion
with GST. The corresponding recombinant proteins were then
expressed in E. coli and purified by affinity using
Glutathione-Sepharose resin (Amersham Pharmacia Biotech, Sweden)
and following standard protocols (Sambrook et al., 1989, Molecular
Cloning, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor).
[0075] The following table 3, by way of examples, presents the
reactivity with negative and positive sera of a number of the
clones selected, assayed in the form of fusion proteins:
TABLE-US-00003 TABLE 3 Reactivity of GST fusion Reactivity of GST
fusion protein Name of protein with positive sera with negative
sera clone (pos./total neg.) (neg./total neg.) Tx-4.11 32/40 1/28
Tx-15.11 22/40 0/28 Tx-1.11 27/40 0/28 Tx-8.0 13/40 0/28 Tx-1.16
31/40 0/28 Tx-9.18 3/40 1/28 Tx-7.11 39/40 3/28 Tx-4.18 21/40
1/28
IgG Avidity for Determining the Time of Infection
[0076] Measurement of the binding force between immunoglobulin G
(IgG) and the T. gondii-specific antigens (IgG avidity) is a
diagnostic method used to establish the time of infection: IgG
avidity is lower during the acute phase of the infection and then
tends to increase in the course of time (Hedman et al. 1989 J.
Infect. Dis. 159, 736-740). Evaluation of IgG avidity is based on
an enzyme-linked immunosorbent assay (ELISA) in which the
immunoglobulins are "detached" from the antigen by washing with a
urea denaturing solution. A mathematical calculation based on the
reactivity before and after the denaturing washing makes it
possible to estimate serum IgG avidity (Jenum et al., 1997. J.
Clin. Microbiol. 35, 1972-1977). To evaluate the antigenic
properties of the protein fragments described in the present
invention, an IgG avidity test based on recombinant antigen
fragments was developed and the results obtained were compared with
the assay performed with a commercial kit (Toxo-IgG avidity kit,
bioMerieux, France) that employs the whole parasite extract as
antigen.
[0077] For the avidity analysis 27 sera coming from women who
contracted primary Toxoplasmosis during pregnancy and collected at
different times after infection were used. The infection was
diagnosed by seroconversion during gestation, taking into
consideration last negative and first positive samples. For each
sample the specific IgG and IgM levels for T. gondii and the IgG
avidity were determined by means of the use of commercial kits
(LDBIO Diagnostic, France; bioMerieux, France). Multi-well plates
(Nunc, Denmark) were incubated overnight at 4.degree. C. with a
solution of NaHCO.sub.3 50 mM, pH 9.6 containing the antigen
fragments expressed as GST fusion proteins at a final concentration
of 5 .mu.g/ml. The plates were blocked with 200 .mu.l of blocking
solution (5% skimmed milk powder in PBS, 0.05% Tween-20) and then
washed 5 times with washing buffer (PBS, 0.05% tween-20). Serial
dilutions of serum (1:50, 1:200, 1:800, 1:3200) in 100 .mu.l of
blocking solution were incubated on plates for 60 minutes at
37.degree. C. The plates were then incubated with a denaturing
solution of urea 6M in PBS/0.02% Tween-20 for 30 minutes at
37.degree. C. In parallel, for every sample, the same dilutions of
serum were effected and the wells concerned were incubated with
normal washing buffer. 100 .mu.l of blocking solution containing
human anti-IgG antibodies conjugated with the enzyme alkaline
phosphatase (diluted 1:10000) were added to the plates. After 30
minutes' incubation at 37.degree. C. the plates were washed and the
enzyme activity was determined with 100 .mu.l of development
solution (10% diethanolamine pH 9.8, 0.5 mM MgCl.sub.2, 0.05%
NaN.sub.3) containing the reaction substrate p-nitrophenylphosphate
(Sigma-Aldrich, USA). The enzyme activity was measured at optical
densities of 405 nm and 620 nm by means of an automatic ELISA OD
reader (Multiskan Labsystem, Finland) and the avidity calculation
was done according to the mathematical analysis described in the
literature (Jenum et al., 1997. J. Clin. Microbiol. 35,
1972-1977).
[0078] The following table 4 gives, by way of examples, the avidity
of the human sera for a number of the antigen fragments
selected.
[0079] The values should be interpreted as follows (commercial kit
criterion): TABLE-US-00004 TABLE 4 Time from Infection Commercial
kit Clones-GST Serum (months) bioMerleux tx-15.11 tx-1.11 tx-8.0
tx-4.18 tx-7.11 tx-1.16 T1 1 7.8% 20.0% 6.9% 12.0% -- -- 24.0% T2 1
5.7% 7.9% 2.3% 10.0% 9.8% 7.9% 2.7% T3 1-2 2.5% 5.4% 6.3% 9.5% 8.7%
11.5% 1.0% T4 1-2 8.1% 4.5% 9.0% 4.0% 10.3% 38.4% 9.5% T5 1-2 4.5%
2.4% 4.0% 9.3% 7.7% 6.7% 11.0% T6 1-2 2.6% 10.0% 4.7% 2.0% 10.0%
7.1% 12.4% T7 1-2 11.6% 9.1% 13.0% 8.0% 15.6% 4.2% 26.0% T8 1-2 6%
4.2% 8.7% 1.7% 7.5% 19.3% 26.2% T9 2-3 4.9% 17.0% 5.5% 3.0% 9.8%
4.8% 21.0% T10 2-3 13.7% 27.0% 30.8% 14.5% 28.1% 4.8% 52.0% T11 2-3
66.1% 41.0% 37.6% 15.7% 42.8% 59.0% 72.0% T12 3-4 16.2% 29.0% 25.1%
13.9% 28.7% 8.6% 56.0% T13 4-5 42.7% 20.1% 59.0% -- -- 28.6% 89.0%
T14 4-5 59.5% 22.0% 32.8% 67.5% 50.9% 75.0% 79.5% T15 5-6 14.7%
25.0% 9.5% 14.6% 19.2% 8.3% 59.0% T16 6 55.1% 35.0% 75.8% 70.0% --
63.5% 79.2% T17 6 15.6% -- -- -- 26.6% 10.2% 45.5% T18 6-7 45.8%
64.0% 59.1% -- 46.1% 50.7% 69.0% T19 6-7 25.7% 29.4% 23.0% 30.0%
24.6% 55.6% 62.3% T20 7-8 43.6% 44.0% 48.0% -- 63.9% 47.9% 90.0%
T21 7-8 52.2% 22.0% 31.0% 16.4% 49.7% 48.2% 69.0% T22 7-8 26.2%
2.5% -- 9.0% -- -- 36.0% T23 8 26.2% 47.3% 47.2% -- -- 21.0% 42.6%
T24 8 22.8% 26.5% 22.4% 82.8% -- 16.7% 40.5% T25 10 28.6% 46.0%
52.4% -- -- 23.2% 37.5% T26 24 25.6% 62.0% 35.3% -- -- -- 73.8% T27
38 41.8% 25.0% 34.1% 55.0% 35.2% 43.8% 58.0%
EXAMPLE 2
[0080] Using the vector .lamda.KM4 of Example 1, a library of DNA
fragments of known Toxoplasma gondii genes was constructed.
[0081] Cells of Toxoplasma gondii (10.sup.6 parasites, strain ME49)
were grown in vitro in monkey kidney cells ("VERO" African green
monkey cells) using DMEM culture medium containing 10% foetal
bovine serum, 2 mM glutamine and 0.05 mg/ml gentamicin (Gibco BRL,
Canada). To have both forms of the parasite (tachyzoites and
bradyzoites) present in the cell cultures, an experimental protocol
was used based on the change in pH of the culture medium (Soete et
al., 1994, Experimental Parasitology, 78, 361-370). The parasites
were collected after complete lysis of the host cells and purified
by filtration (filter porosity 3 .mu.m) followed by centrifuging. 2
.mu.g of mRNA were isolated from 5.times.10.sup.6 parasites using
the "QuickPrep Micro mRNA Purification Kit" (Amersham Pharmacia
Biotech, Sweden) and following the manufacturer's instructions.
cDNA was synthesised from 200 ng of poly(A)+ RNA using the "SMART
cDNA Library Construction Kit" (Clontech, CA, USA) and following
the manufacturer's instructions. Genomic DNA was purified from the
remaining 5.times.10.sup.6 cells using standard procedures
(Sambrook et al., 1989, Molecular Cloning: a laboratory manual,
Cold Spring Harbor Laboratory Press, NY) and stored at -20.degree.
C.
[0082] For the construction of the expression/exposure library the
following genes, expressed only in the bradyzoite stage, were
amplified by means of PCR with specific oligonucleotides: [0083]
1--SAG2D (Lekutis et al., 2000, Experimental Parasitology, 96,
89-96) was obtained from genomic DNA using the oligonucleotides
5'-ATGGCGGCTGCACACTCG-3' (SEQ ID No 39) and
5'-GAACATATTCCCTGTCACCAATG-3' (SEQ ID No 40); [0084] 2--SAG4
(Odberg-Ferragut et al., 1996, Molecular and Biochemical
Parasitology, 82, 237-244) was obtained from genomic DNA using the
oligonucleotides 5'-ATGACGAAAAATAAAATTCTTCTC-3' (SEQ ID No 41) and
5'-CATTGATATCAACACAAAGGCC-3' (SEQ ID No 42) [0085] 3--BSR4 (Manger
et al., 1998, Infection and Immunity, 66, 2237-2244) was obtained
from genomic DNA using the oligonucleotides
5'-ATGGTGATGATGGGCAGCATG-3' (SEQ ID No 43) and
5'-CGGCGGCCGCGCTAGAGG-3' (SEQ ID No 44); [0086] 4--MAG1 (Parmley et
al., 1994, Molecular and Biochemical Parasitology, 66, 283-296) was
obtained from genomic DNA using the oligonucleotides
5'-CGTTGGATCCTTGGATTGAGCCAAAGGGTGCCAG-3' (SEQ ID No 45) and
5'-CCCAGAATTCTCAAGCTGCCTGTTCCGCTAAGATCTG-3' (SEQ ID No 46); [0087]
5--LDH2 (Yang and Parmley, 1997, Gene, 184, 1-12) was obtained from
cDNA using the oligonucleotides 5'-ATGACGGGTACCGTTAGCAG-3' (SEQ ID
No 47) and 5'-ACCCAGCGCCGCTAAACTC-3' (SEQ ID No 48); [0088] 6--ENO1
(Dzierszinski et al., 2001, Journal of Molecular Biology, 309,
1017-1027) was obtained from genomic DNA using the oligonucleotides
5'-ATGGTGGTTATCAAGGACATCG-3' (SEQ ID No 49) and
5'-TTTTGGGTGTCGAAAGCTCTC-3' (SEQ ID No 50); [0089] 7--BAG1 (Bohne
et al., 1995, Molecular Microbiology, 16, 1221-1230) was obtained
from cDNA using the oligonucleotides 5'-ATGGCGCCGTCAGCATCG-3' (SEQ
ID No 51) and 5'-CTTCACGCTGATTTGTTGCTTTG-3' (SEQ ID No 52); [0090]
8--p-ATPase (Holpert et al., 2001, Molecular and Biochemical
Parasitology, 112, 293-296) was obtained from genomic DNA using the
oligonucleotides 5'-ATGGACGAAGCGAGCAGAAGG-3' (SEQ ID No 53) and
5'-ACGCGTGATCGAAGGAACCG-3' (SEQ ID No 54).
[0091] 10 .mu.g of DNA deriving from a mixture of the amplification
products of the above-mentioned genes were fragmented randomly
using 0.5 ng of the endonuclease DNaseI (Sigma-Aldrich, USA). The
mixture of DNA and DNaseI was incubated for 20 minutes at
15.degree. C. and the DNA fragments were purified by means of the
"QIAquick PCR Purification Kit" (Qiagen, CA, USA), following the
manufacturer's instructions. The 3 .mu.g ends of the cDNA fragments
were "flattened" by incubating the DNA with 9 units of the enzyme
T4 DNA polymerase (New England Biolabs, MA, USA) for 60 minutes at
15.degree. C. The fragments were then purified by means of
extraction in phenol/chloroform and subsequent precipitation in
ethanol. 500 ng of the resulting DNA were bound with a 20-fold
molar excess of "synthetic adaptors" for the purposes of adding the
restriction sites SpeI and NotI to the ends of the fragments. Six
adaptors were used, obtained by hybridisation of the following
pairs of oligonucleotides: K185 5'-CTAGTCGTGCTGGCCAGC-3' (SEQ ID No
5) and K186 5'-GCTGGCCAGCACGA-3' (SEQ ID No 6); K187
5'-CTAGTCGTGCTGGCCA GCT-3' (SEQ ID No 7) and K188
5'-AGCTGGCCAGCACGA-3' (SEQ ID No 8); K189 5'-CTAGTCGT
GCTGGCCAGCTG-3' (SEQ ID No 9) and K190 5'-CAGCTGGCCAGCACGA-3' (SEQ
ID No 10); K191 5'-TCTGGTGGCGGTAGC-3' (SEQ ID No 11) and K192
5'-GGCCGCTACCGCCACCAGA-3' (SEQ ID No 12); K193
5'-TTCTGGTGGCGGTAGC-3' (SEQ ID No 13) and K194
5'-GGCCGCTACCGCCACCAGAA-3' (SEQ ID No 14); K195
5'-TTTCTGGTGGCGGTAGC-3' SEQ ID No 15) and K196
5'-GGCCGCTACCGCCACCAGAAA-3' (SEQ ID No 16). The excess of unligated
adaptors was removed from the ligation mixture by electropheresis
on 2% agarose gel and the cDNA fragments with molecular weights
ranging from 250 bp to 1000 bp were excised from the gel and
purified by means of the "Qiaquick gel extraction kit" (Qiagen, CA,
USA) following the manufacturer's instructions. The vector
.lamda.KM4 was digested with SpeI/NotI and for the construction of
the library 6 ligation mixtures were performed, each containing 0.4
.mu.g of vector and approximately 7 ng of insert. After overnight
incubation at 4.degree. C. the ligation mixtures were packaged in
vitro with the "Ready-To-Go lambda packaging kit" (Amersham
Pharmacia Biotech, Sweden) and plated for infection of BB4 cells
(bacterial cells of E. coli strain BB4; Sambrook et al., 1989,
Molecular Cloning: a laboratory manual, Cold Spring Harbor
Laboratory Press, NY). After overnight incubation at 37.degree. C.
the phage was eluted from the plates with SM buffer (Sambrook et
al., 1989, Molecular Cloning: a laboratory manual, Cold Spring
Harbor Laboratory Press, NY), purified, concentrated and stored at
-80.degree. C. in SM buffer containing 7% dimethylsulphoxide. The
complexity of the library calculated as the number of total
independent clones with inserts was 10.sup.6 clones.
[0092] Affinity selection, phage-ELISA, immunoscreening and phage
clones preparation were performed exactly as described in Example
1
[0093] The following table 5 gives, by way of examples, the
reactivity of a number of the recombinant bacteriophages selected.
TABLE-US-00005 TABLE 5 Reactivity of phage clone with Reactivity of
phage positive sera (positive/total clone with negative sera Name
of clone positive) (negative/total negative) TxB-c121.2 10/20 1/10
TxB-c126.3 12/20 0/10 TxB-44.3 10/20 0/10 TxB-7.1 8/20 0/10 TxB-9.1
1/20 1/10 TxB-12.1 5/20 0/10
Characterisation of Positive Clones
[0094] The clones which showed multiple reactivity with the
Toxoplasma gondii positive sera and which presented no reactivity
with the negative sera were subsequently sequenced and compared
with the sequences of the genes used to construct the library
[0095] The following table 6 gives, by way of examples, the
sequences of some of the clones selected: TABLE-US-00006 TABLE 6
Name of clone Sequence TxB.26.3 GGATTGAGCCAAAGGGTGCCAGAGCTACCAGAAGT
(SEQ ID No 55) GGAGCCCTTTGATGAAGTAGGCACGGGAGCTCGAC
GGTCCGGGTCCATTGCGACCCTTCTTCCACAAGAC
GCTGTTTTATATGAGAACTCAGAGGACGTTGCCGT
TCCGAGTGATTCAGCATCGACCCCGTCATACTTTC
ATGTGGAATCTCCAAGTGCTAGTGTGGAAGCCGCG
ACTGGCGCTGTGGGAGAGGTGGTGCCGGACTGTGA
AGAACAACAGGAACAGGGTGACACGACGTTATCCG ATCACGATTTCCATTCA TxB-c17.1
TCTTCAGAAAGATGACGTAACCATAGAAGTCGACAA (SEQ ID No 56)
CGGAGCCATCGTTATCAAAGGAGAGAAGACCTCGAA
AGAAGCGGAGAAAGTGGACGATGGCAAAACAAAGAA
CATTTTGACTGAGCGAGTGTCCGGTTATTTTGCGCG
CCGGTTCCAGCTCCCGAGTAATTACAAGCCCGACGG
AATCAGTGCGGCAATGGACAACGGCGTTCTACGTGT
CACGATCAAGGTCGAGGATTCAGGGGGCGCAAAGCA ACAAATCAGCGTG
[0096] The clone TxB-cl26.3 constitutes a fragment of the gene
MAG1, a 65 kDa protein of the matrix and wall of T. gondii cysts
(Parmley et al., 1994, Molecular and Biochemical Parasitology, 66,
283-296), the protein product of which has never been identified as
an "antigen fragment" in the human humoral response. Said clone has
the amino acid sequence
GLSQRVPELPEVEPFDEVGTGARRSGSIATLLPQDAVLYENSEDVAVPSDSASTPSYFHVESPSASVEAATGA-
VGEVVPDCEE QQEQGDTTLSDHDFH (SEQ ID No 57) and its use as a fragment
containing an epitope is covered by the present invention.
[0097] The clone TxB-cl7.1 constitutes a fragment of the gene BAG1,
a 30 kDa protein of the heat shock protein family of T. gondii
(Bohne et al., 1995, Molecular Microbiology, 16, 1221-1230), the
protein product of which has never been identified as an "antigen
fragment" in the human humoral response. Said clone has the amino
acid sequence LNPIDDMLFETALTANEMMEDITWRPRVDVEFDSKKKEMIILADLP
GLQKDDVTIEVDNGAIVIKGEKTSKEAEKVDDGKTKNILTERVSGY
FARRFQLPSNYIDGISAAMDNGVLRVTIKVEDSGGAKQQISV (SEQ ID No 58) and its
use as a fragment containing an epitope is covered by the present
invention.
Expression of DNA Fragments Selected from the Library as fusion
Products with GST
[0098] The phage clones for which specific reactivity with sera of
patients testing positive for Toxoplasma gondii was demonstrated,
were amplified and then analysed with a substantial panel of
positive and negative sera. After this ELISA study, the clones that
showed multiple reactivity with Toxoplasma gondii-positive sera and
presented no reactivity with the negative sera were cloned as
fusion products with the protein Glutathione Sulphur Transferase
(GST) and expressed in bacterial cells, for the purposes of
determining their specificity and selectivity. To produce the
fusion proteins each clone was amplified from a single phage plaque
by PCR, using the following oligonucleotides: K47
5'-GGGCACTCGACCGGAATTATCG-3' (SEQ ID No 37) and K85
5'-GGGTAAAGGTTTCTTTGCTCG-3' (SEQ ID No 38). The resulting fragment
was then purified by means of the "Qiagen Purification Kit"
(Qiagen, CA, USA), digested with the restriction enzymes SpeI and
NotI and cloned in the vector pGEX-SN to generate the fusion with
GST. The corresponding recombinant proteins were then expressed in
E. coli and purified by affinity using Glutathione-Sepharose resin
(Amersham Pharmacia Biotech, Sweden) and following standard
protocols (Sambrook et al., 1989, Molecular Cloning, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor).
[0099] The following table 7, by way of examples, presents the
reactivity with negative and positive sera of a number of the
clones selected, assayed in the form of fusion proteins:
TABLE-US-00007 TABLE 7 Reactivity of GST fusion Reactivity of GST
fusion protein with positive portein with negative Name of clone
sera (pos./total neg.) sera (neg./totale neg.) TxB-c126.3 30/34
0/32 TxB-c17.1 17/34 0/32
EXAMPLE 3
[0100] By using the same strategy described in Example 2, a gene
collection of DNA encoding for protein products of the Toxoplasma
gondii microneme family was used to construct a "microneme-display
library".
[0101] For the construction of the microneme-library the following
genes were amplified by means of PCR with specific
oligonucleotides: [0102] 1--MIC2 (Wan et al, 1997, Mol. Biochem.
Parasitol. 84: 203-214) was obtained from single strand cDNA using
the oligonucleotides 5'-ATGAGACTCCAACCGAGGCC-3' (SEQ ID No 69) and
5'-CTGCCTGACTCTTTCTTGGACTG-3' (SEQ ID No 70); [0103] 2--M2AP
(Rabenau et al., 2001, Mol. Microbiol. 41: 537-547) was obtained
from single strand cDNA using the oligonucleotides
5'-GGAAAGTTGGAAATCCGGCGGC-3' (SEQ ID No 71) and
5'-CGCCTCATCGTCACTCGGC-3' (SEQ ID No 72) [0104] 3--MIC4 (Brecht et
al., 2001, J. Biol. Chem. 276:4119-412) was obtained from single
strand cDNA using the oligonucleotides 5'-ATGAGAGCGTCGCTCCCGG-3'
(SEQ ID No 73) and 5'-GTGTCTTTCGCTTCAAGCACCTG-3' (SEQ ID No 74);
[0105] 4--AMA1 (Hehl et al., 2000, Infect. Immun. 68:7078-7086) was
obtained from single strand cDNA using the oligonucleotides
5'-ATGGGGCTCGTGGGCGTAC-3' (SEQ ID No 75) and
5'-GATCAACGCAGTGTTAGAGCCAC-3' (SEQ ID No 76);
[0106] 10 .mu.g of DNA deriving from a mixture of the amplification
products of the above-mentioned genes were fragmented randomly
using 0.5 ng of the endonuclease DNaseI (Sigma-Aldrich, USA). The
mixture of DNA and DNaseI was incubated for 20 minutes at
15.degree. C. and the DNA fragments were purified by means of the
"QIAquick PCR Purification Kit" (Qiagen, CA, USA), following the
manufacturer's instructions. Consequent steps for the construction
of the microneme-library, and for the affinity selection were
performed by following the procedure described in Example 2.
Selection of the Microneme-Library with Sera of Infants Who Were
Infected by T. gondii During Pregnancy
[0107] To identify the antigenic domains of the T. gondii microneme
proteins an affinity selection procedure was used consisting of two
"panning" cycles with four sera collected from infants who were
congenitally infected by the parasite, followed by an immunological
screening procedure carried out with the same sera. The library was
selected with sera T1, T2, T3, T4, generating, after a single
selection cycle, the corresponding mixtures p1.sup.I, p2.sup.I,
p3.sup.I and p4.sup.I. Each mixture was then subjected to a second
affinity selection cycle with the same serum, giving rise to a
second series of mixtures (called p1.sup.IIp2.sup.II, p3.sup.II and
p4.sup.II). Various positive clones were identified by means of
immunoplate screening per plaque of reactive mixtures.
[0108] Phage-Elisa, immunoscreening, and the preparation of phage
clones were subsequently performed exactly as described in Examples
1 and 2.
[0109] The following table 8 gives, by way of examples, the
reactivity of a number of the recombinant bacteriophages selected.
TABLE-US-00008 TABLE 8 Reactivity of phage Reactivity of phage
clone with clone with positive sera negative sera (negative/total
Name of clone (positive/total positive) negative) Tx-2.a 13/16 0/10
Tx-1.b 11/16 0/10 Tx-11.b 12/16 0/10 Tx-13.b 9/16 0/10 Tx-15.b 9/16
0/10
Characterisation of Positive Clones
[0110] The following table 9 gives the sequences of the clones
selected: TABLE-US-00009 TABLE 9 Name of the clone Sequence
Identification Classification Tx-2.a CCCCAGGATGCCATTT MIC2
Microneme (SEQ ID No 59) GCTCGGATTGGTCCGC protein ATGGAGCCCCTGCAGT
unknown as GTATCCTGCGGTGACG antigen in GAAGCCAAATCAGGAC human
response GCGAACTGAGGTTTCT GCTCCGCAACCTGGAA CACCAACATGTCCGGA
CTGCCCTGCGCCCATG GGAAGGACTTGCGTGG AACAAGGCGGACTTGA AGAAATCCGTGAATGC
AGTGCGGGGGTATGTG CTGTTGACGCTGGATG TGGCGTCTGGGTT Tx-1.b
CCGTGTCCAATTAATG MIC2 Microneme (SEQ ID No 60) CAACTTGCGGTCAGTT
protein TGAAGAATGGAGTACA unknown as TGCTCGGTCTCATGTG antigen in
GTGGTGGACTGAAAAC human response GAGGTCGAGGAACCCT TGGAATGAAGACCAAC
AACATGGAGGACTATC CTGCGAGCAGCAGCAT CCTGGTGGGCGGACGG AAACGGTAACTTGCAA
TCCTCAAGCGTGTCCT GTGGATGAACGACCGG GGGAGTGGGCAGAGTG GGGGGAATGTAGTGTC
ACGTGCGGCGACGGAG TGCGAGAGCGCAGGCG CGGGAAAAGTCTAGTT GAGGCTAAATTCGGCG
GACGCACCATTGATCA GCAGAATGAGGCTCTT CCGGAAGACTTAAAAA TCAAAAACGTCGAGTA
TGAGCCATGTTCGTAT CCTGCTTGTGGAGCTT CCTGCACGTACGTCTG GAGTGACTGGAACAAG
Tx-11.b AACGAACCGGTGGCCC M2AP Microneme (SEQ ID No 61)
TAGCTCAGCTCAGCAC protein unknown ATTCCTCGAGCTCGTC as antigen in
GAGGTGCCATGTAACT human response CTGTTCATGTTCAGGG GGTGATGACCCCGAAT
CAAATGGTCAAAGTGA CTGGTGCAGGATGGGA TAATGGCGTTCTCGAG TTCTATGTCACGAGGC
CAACGAAGACAGGCGG GGACACAAGCCGAAGC CATCTTGCGTCGATCA TGTGTTATTCCAAGGA
CATTGACGGCGTGCCG TCAGACAAAGCGGGAA AGTGCTTTCTGAAGAA CTTTTCTGGTGAAGAC
TCGTCGGAAATAGACG AAAAAGAAGTATCTCT ACCCATCAAGAGCCAC AACGATGCGTTCATGT
TCGTTTGTTCTTCAAA TGATGGATCCGCACTC CAGTGTGATGTTTTCG CCCTTGATAACACCAA
CTCTAGCGACGGGTGG AAAGTGAATACCGTGG ATCTTGGCGTCAGCGT TAGTCCGGATTTGGCA
TTCGGACTCACTGCAG ATGGGGTCAAGGTGAA GAAGTTGTACGCAAGC AGCGGCCTGACAGCGA
TCAACGACGACCCTTC CTTGGGGTGCAAGGCT CCTCCCCATTCTCCGC CGGCCGGAGAGGAACC
GAGTTTGGCGTCGCCT GAAAACAGCGGGTCTG CAACACCAGGGGAAGA AAGTCCGTCTGAGTCT
GAATCT Tx-13.b CTTCGCGGGTACAGGT AMA1 Microneme (SEQ ID No 62)
TCGGTGTTTGGAAGAA protein AGGCCGTTGCCTCGAC unknown as
TACACTGAATTGACCG antigen in ACACTGTGATAGAACG human response
TGTTGAGTCAAAGGCA CAGTGCTGGGTGAAAA CCTTTGAAAACGACGG GGTCGCGAGTGACCAA
CCCCATACGTATCCAC TGACGTCGCAAGCATC ATGGAACGATTGGTGG CCTCTCCACCAGAGTG
ACCAACCTCACTCAGG TGGCGTTGGGCGTAAT TACGGTTTCTACTACG TGGACACGACTGGAGA
GGGCAAGTGTGCACTC TCTGACCAGGTACCCG ACTGCCTGGTGTCGGA TTCTGCCGCCGTGTCG
TATACAGCAGCGGGGA GTTTGTCTGAAGAGAC GCCGAATTTCATAATT CCGTCAAATCCCTCTG
TTACTCCGCCAACGCG CGAGACGGCACTTCAG TGCACGGCCGACAAGT TCCCCGACTCTTTCGG
TGCCTGCGACGTTCAA GCCTGTAAAAGACAGA AGACGTCCTGCGTTGG CGGACAGATTCAAAGT
ACTAGCGTCGACTGCA CCGCGGACGAACAAAA TGAATGTGGCTCTAAC ACTGCG Tx15.b
AGTGCCAACGTAACAA MIC4 Microneme (SEQ ID No 63) GTTCGGAGCCTGCAAA
protein- ACTTGATCTCTCTTGT unknown as GCGCACTCTGACAATA antigen in
AGGGATCAAGGGCTCC human response CACAATAGGCGAGCCA GTGCCAGATGTGTCCC
TGGAACAATGTGCTGC GCAATGCAAGGCTGTT GATGGCTGCACACATT TCACTTATAATGACGA
TTCGAAGATGTGCCAT GTGAAGGAGGGAAAAC CCGATTTATACGATCT CACAGGAGGCAAAACA
GCACCGCGCAGTTGCG ATAGATCATGCTTCGA ACAACACGTATCGTAT GAGGGAGCTCCTGACG
TGATGACAGCGATGGT CACGAGCCAGTCAGCG GACTGTCAGGCTGCGT GTGCGGCTGACCCGAG
CTGCGAGATCTTCACT TATAACGAACACGACC AGAAATGTACTTTCAA AGGAAGGGGGTTTTCT
GCGTTTAAGGAACGAG GGGTGTTGGGTGTGAC TTCCGGGCCGAAACAG TTCTGCGATGAAGGCG
GTAAATTAACT
[0111] The clones Tx-2.a e Tx-1.b represent two distinct fragments
of the MIC2 gene (Wan et al, 1997, Mol. Biochem. Parasitol. 84:
203-214) and have never been identified as antigens of the human
antibody response. Said clones have respectively the amino acid
sequences PQDAICSDWSAWSPCSVSCGDGSQIRTRTEVSAPQPGTPTCPDCPA
PMGRTCVEQGGLEEIRECSAGVCAVDAGCGVWV (SEQ ID No 64) and
PCPINATCGQFEEWSTCSVSCGGGLKTRSRNPWNEDQQHGGLSCE
QQHPGGRTETVTCNPQACPVDERPGEWAEWGECSVTCGDGVRER
RRGKSLVEAIFGGRTIDQQNEALPEDLKIKNVEYEPCSYPACGASC TYVWSDWNK (SEQ ID No
65) and their use as fragments containing an epitope is covered by
the present invention.
[0112] The clone Tx-11.b represents a distinct fragment of the M2AP
gene (Rabenau et al., 2001, Mol. Microbiol. 41: 537-547) and has
never been identified as antigen of the human antibody response.
Said clone has the amino acid sequence
NEPVALAQLSTFLELVEVPCNSVHVQGVMTPNQMVKVTGAGWDNGVLEFYVTRPTKTGGDTSRSHIASIMCYS-
K DIDGVPSDKAGKCFLKNFSGEDSSEIDEKEVSLPIKSHNDAFMFVC
SSNDGSALQCDVFALDNTNSSDGWKVNTVDLGVSVSPDLAFGLTA
DGVKVLYASSGLTAINDDPSLGCKAPPHSPPAGEEPSLPSPENS GSATPAEESPSESES (SEQ
ID No 66) and its use as fragment containing an epitope is covered
by the present invention.
[0113] The clone Tx-13.b represents a fragment of the AMA1 gene
(Hehl et al., 2000, Infect. Immun. 68:7078-7086). Said clone has
the amino acid sequence
LRGYRFGVWKKGRCLDYTELTDTVIERVESKAQCWVKTFENDGVASDQPHTYPLTSQASWNDWW-
PLHQSDQPHSGGVGRNYG FYYVDTTGEGKCALSDQVPDCLVSDSAAVSYTAAGSLSEETPNFIIP
SNPSVTPPTPETALQCTADKFPDSFGACDVQACKRQKTSCVGGQIQ STSVDCTADEQNECGSNTA
(SEQ ID No 67) and its use as fragment containing an epitope is
covered by the present invention.
[0114] The clone Tx-15.b represents a fragment of the MIC4 gene
(Brecht et al., 2001, J. Biol. Chem. 276:4119-4127). Said clone has
the amino acid sequence
SANVTSSEPAKLDLSCAHSDNKGSRAPTIGEPVPDVSLEQCAAQCKAVDGCTHFTYNDDSKMCHVKEGKPDLY-
DLTGGKTAPRS CDRSCFEQHVSYEGAPDVMTAIVTSQSADCQAACAADPSCEIFTY
NEHDQKCTFKGRGFSAFKERGVLGVTSGPKQFCDEGGKLT (SEQ ID No 68) and its use
as fragment containing an epitope is covered by the present
invention.
Expression of DNA Fragments Selected from the Microneme-Library as
Fusion Products with GST
[0115] Phage clones isolated from the microneme-library were cloned
as fusion products with GST protein and expressed in bacterial
cells, for the purposes of determining their specificity and
selectivity. The procedure described in Examples 1 and 2 was used
to produce the fusion proteins.
[0116] The following table 10, by way of example, presents the
reactivity with negative and positive sera of a number of the
clones selected, assayed in the form of fusion proteins:
TABLE-US-00010 TABLE 10 Reactivity of GST fusion Reactivity of GST
fusion protein with positive sera portein with negative sera Name
of clone (pos./total neg.) (neg./totale neg.) Tx-2.a 29/30 0/15
Tx-1.b 15/30 0/15 Tx-11.b 23/30 0/15 Tx-13.b 24/30 0/15 Tx-15.b
12/30 0/15
[0117]
Sequence CWU 1
1
76 1 48 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 1 tttatctaga cccagcccta ggaagcttct
cctgagtagg acaaatcc 48 2 32 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 2 gggtctagat
aaaacgaaag gcccagtctt tc 32 3 56 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 3
ccgccttcca tgggtactag ttttaaatgc ggccgcacga gcaaagaaac ctttac 56 4
22 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 4 agcttcctag ggctgggtct ag 22 5 18 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 5 ctagtcgtgc tggccagc 18 6 14 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 6 gctggccagc acga 14 7 19 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 7
ctagtcgtgc tggccagct 19 8 15 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 8 agctggccag cacga 15
9 20 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 9 ctagtcgtgc tggccagctg 20 10 16 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 10 cagctggcca gcacga 16 11 15 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 11 tctggtggcg gtagc 15 12 19 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 12 ggccgctacc gccaccaga 19 13 16 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 13 ttctggtggc ggtagc 16 14 20 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 14 ggccgctacc gccaccagaa 20 15 17 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 15 tttctggtgg cggtagc 17 16 21 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 16 ggccgctacc gccaccagaa a 21 17 348 DNA Toxoplasma
gondii 17 agtggaggga cagggcaggg attaggaatc ggagaatctg tagatttgga
gatgatgggg 60 aacacgtatc gtgtggagag acccacaggc aacccggact
tgctcaagat cgccattaaa 120 gcttcagatg gatcgtacag cgaagtcggc
aatgttaacg tggaggaggt gattgatact 180 atgaaaagca tgcagaggga
cgaggacatt ttccttcgtg cgttgaacaa aggcgaaaca 240 gtagaggaag
cgatcgaaga cgtggctcaa gcagaagggc ttaattcgga gcaaaccctg 300
caactggaag atgcagtgag cgcggtggcg tctgttgttc aagacgag 348 18 165 DNA
Toxoplasma gondii 18 tactcttcac cacgaatagt tgttttgatt agatattgct
tcttctccac atatcgcctc 60 acaatgttcg ccgtaaaaca ttgtttgctg
gttgttgccg ttggcgccct ggtcaacgtc 120 tcggtgaggg ctgccgagtt
ttccggagtt gttaaccagg gacct 165 19 297 DNA Toxoplasma gondii 19
gctgccttgg gaggccttgc ggcggatcag cctgaaaatc atcaggctct tgcagaacca
60 gttacgggtg tgggggaagc aggagtgtcc cccgtcaacg aagctggtga
gtcatacagt 120 tctgcaactt cgggtgtcca agaagctacc gccccaggtg
cagtgctcct ggacgcaatc 180 gatgccgagt cggataaggt ggacaatcag
gcggagggag gtgagcgtat gaagaaggtc 240 gaagaggagt tgtcgttatt
gaggcgggaa ttatatgatc gcacagatcg ccctggt 297 20 234 DNA Toxoplasma
gondii 20 cagttcgcta ccgcggccac cgcgtcagat gacgaactga tgagtcgaat
ccgaaattct 60 gactttttcg atggtcaagc acccgttgac agtctcagac
cgacgaacgc cggtgtcgac 120 tcgaaaggga ccgacgatca cctcaccacc
agcatggata aggcatctgt agagagtcag 180 cttccgagaa gagagccatt
ggagacggag ccagatgaac aagaagaagt tcat 234 21 104 DNA Toxoplasma
gondii 21 gagaacccgg tgagaccgcc tcctcccggt ttccatccaa gcgttattcc
caatcccccg 60 tacccgctgg gcactccagc gggcatgcca cagccagagg ttcc 104
22 219 DNA Toxoplasma gondii 22 aggaggactg gatgtcatgc cttcagggag
aactgcagcc ctggtagatg tattgatgac 60 gcctcgcatg agaatggcta
cacctgcgag tgccccacag ggtactcacg tgaggtgact 120 tccaaggcgg
aggagtcgtg tgtggaagga gtcgaagtca cgctggctga gaaatgcgag 180
aaggaattcg gcatcagcgc gtcatcctgc aaatgcgat 219 23 270 DNA
Toxoplasma gondii 23 gcacccactc aatctgaaat gaaagaattc caagaggaaa
tcaaagaagg ggtggaggaa 60 acaaagcatg aagacgatcc tgagatgacg
cggctcatgg tgaccgagaa gcaggagagc 120 aaaaatttca gcaagatggc
gaaatcccag agttttagca cgcgaatcga agagctcggg 180 ggatccattt
cgtttctaac tgaaacgggg gtcacaatga tcgagttgcc caaaactgtc 240
agtgaacatg acatggacca actactccac 270 24 456 DNA Toxoplasma gondii
24 gttatggcat cggatccccc tcttgttgcc aatcaagttg tcacctgccc
agataaaaaa 60 tcgacagccg cggtcattct cacaccgacg gagaaccact
tcactctcaa gtgccctaaa 120 acagcgctca cagagcctcc cactcttgcg
tactcaccca acaggcaaat ctgcccagcg 180 ggtactacaa gtagctgtac
atcaaaggct gtaacattga gctccttgat tcctgaagca 240 gaagatagct
ggtggacggg ggattctgct agtctcgaca cggcaggcat caaactcaca 300
gttccaatcg agaagttccc cgtgacaacg cagacgtttg tggtcggttg catcaaggga
360 gacgacgcac agagttgtat ggtcacggtg acagtacaag ccagagcctc
atcggtcgtc 420 aataatgtcg caaggtgctc ctatggtgcg gacagc 456 25 393
DNA Toxoplasma gondii 25 ccatcggtcg tcaataatgt cgcaaggtgc
tcctacggtg cagacagcac tcttggtcct 60 gtcaagttgt ctgcggaagg
acccactaca atgaccctcg tgtgcgggaa agatggagtc 120 aaagttcctc
aagacaacaa tcagtactgt tccgggacga cgctgactgg ttgcaacgag 180
aaatcgttca aagatatttt gccaaaatta actgagaacc cgtggcaggg taacgcttcg
240 agtgataagg gtgccacgct aacgatcaag aaggaagcat ttccagccga
gtcaaaaagc 300 gtcattattg gatgcacagg gggatcgcct gagaagcatc
actgtaccgt gaaactggag 360 tttgccgggg ctgcagggtc agcaaaatcg gct 393
26 116 PRT Toxoplasma gondii 26 Ser Gly Gly Thr Gly Gln Gly Leu Gly
Ile Gly Glu Ser Val Asp Leu 1 5 10 15 Glu Met Met Gly Asn Thr Tyr
Arg Val Glu Arg Pro Thr Gly Asn Pro 20 25 30 Asp Leu Leu Lys Ile
Ala Ile Lys Ala Ser Asp Gly Ser Tyr Ser Glu 35 40 45 Val Gly Asn
Val Asn Val Glu Glu Val Ile Asp Thr Met Lys Ser Met 50 55 60 Gln
Arg Asp Glu Asp Ile Phe Leu Arg Ala Leu Asn Lys Gly Glu Thr 65 70
75 80 Val Glu Glu Ala Ile Glu Asp Val Ala Gln Ala Glu Gly Leu Asn
Ser 85 90 95 Glu Gln Thr Leu Gln Leu Glu Asp Ala Val Ser Ala Val
Ala Ser Val 100 105 110 Val Gln Asp Glu 115 27 55 PRT Toxoplasma
gondii 27 Tyr Ser Ser Pro Arg Ile Val Val Leu Ile Arg Tyr Cys Phe
Phe Ser 1 5 10 15 Thr Tyr Arg Leu Thr Met Phe Ala Val Lys His Cys
Leu Leu Val Val 20 25 30 Ala Val Gly Ala Leu Val Asn Val Ser Val
Arg Ala Ala Glu Phe Ser 35 40 45 Gly Val Val Asn Gln Gly Pro 50 55
28 99 PRT Toxoplasma gondii 28 Ala Ala Leu Gly Gly Leu Ala Ala Asp
Gln Pro Glu Asn His Gln Ala 1 5 10 15 Leu Ala Glu Pro Val Thr Gly
Val Gly Glu Ala Gly Val Ser Pro Val 20 25 30 Asn Glu Ala Gly Glu
Ser Tyr Ser Ser Ala Thr Ser Gly Val Gln Glu 35 40 45 Ala Thr Ala
Pro Gly Ala Val Leu Leu Asp Ala Ile Asp Ala Glu Ser 50 55 60 Asp
Lys Val Asp Asn Gln Ala Glu Gly Gly Glu Arg Met Lys Lys Val 65 70
75 80 Glu Glu Glu Leu Ser Leu Leu Arg Arg Glu Leu Tyr Asp Arg Thr
Asp 85 90 95 Arg Pro Gly 29 78 PRT Toxoplasma gondii 29 Phe Ala Thr
Ala Ala Thr Ala Ser Asp Asp Glu Leu Met Ser Arg Ile 1 5 10 15 Arg
Asn Ser Asp Phe Phe Asp Gly Gln Ala Pro Val Asp Ser Leu Arg 20 25
30 Pro Thr Asn Ala Gly Val Asp Ser Lys Gly Thr Asp Asp His Leu Thr
35 40 45 Thr Ser Met Asp Lys Ala Ser Val Glu Ser Gln Leu Pro Arg
Arg Glu 50 55 60 Pro Leu Glu Thr Glu Pro Asp Glu Gln Glu Glu Val
His Phe 65 70 75 30 35 PRT Toxoplasma gondii 30 Glu Asn Pro Val Arg
Pro Pro Pro Pro Gly Phe His Pro Ser Val Ile 1 5 10 15 Pro Asn Pro
Pro Tyr Pro Leu Gly Thr Pro Ala Gly Met Pro Gln Pro 20 25 30 Glu
Val Pro 35 31 73 PRT Toxoplasma gondii 31 Arg Arg Thr Gly Cys His
Ala Phe Arg Glu Asn Cys Ser Pro Gly Arg 1 5 10 15 Cys Ile Asp Asp
Ala Ser His Glu Asn Gly Tyr Thr Cys Glu Cys Pro 20 25 30 Thr Gly
Tyr Ser Arg Glu Val Thr Ser Lys Ala Glu Glu Ser Cys Val 35 40 45
Glu Gly Val Glu Val Thr Leu Ala Glu Lys Cys Glu Lys Glu Phe Gly 50
55 60 Ile Ser Ala Ser Ser Cys Lys Cys Asp 65 70 32 90 PRT
Toxoplasma gondii 32 Ala Pro Thr Gln Ser Glu Met Lys Glu Phe Gln
Glu Glu Ile Lys Glu 1 5 10 15 Gly Val Glu Glu Thr Lys His Glu Asp
Asp Pro Glu Met Thr Arg Leu 20 25 30 Met Val Thr Glu Lys Gln Glu
Ser Lys Asn Phe Ser Lys Met Ala Lys 35 40 45 Ser Gln Ser Phe Ser
Thr Arg Ile Glu Glu Leu Gly Gly Ser Ile Ser 50 55 60 Phe Leu Thr
Glu Thr Gly Val Thr Met Ile Glu Leu Pro Lys Thr Val 65 70 75 80 Ser
Glu His Asp Met Asp Gln Leu Leu His 85 90 33 152 PRT Toxoplasma
gondii 33 Val Met Ala Ser Asp Pro Pro Leu Val Ala Asn Gln Val Val
Thr Cys 1 5 10 15 Pro Asp Lys Lys Ser Thr Ala Ala Val Ile Leu Thr
Pro Thr Glu Asn 20 25 30 His Phe Thr Leu Lys Cys Pro Lys Thr Ala
Leu Thr Glu Pro Pro Thr 35 40 45 Leu Ala Tyr Ser Pro Asn Arg Gln
Ile Cys Pro Ala Gly Thr Thr Ser 50 55 60 Ser Cys Thr Ser Lys Ala
Val Thr Leu Ser Ser Leu Ile Pro Glu Ala 65 70 75 80 Glu Asp Ser Trp
Trp Thr Gly Asp Ser Ala Ser Leu Asp Thr Ala Gly 85 90 95 Ile Lys
Leu Thr Val Pro Ile Glu Lys Phe Pro Val Thr Thr Gln Thr 100 105 110
Phe Val Val Gly Cys Ile Lys Gly Asp Asp Ala Gln Ser Cys Met Val 115
120 125 Thr Val Thr Val Gln Ala Arg Ala Ser Ser Val Val Asn Asn Val
Ala 130 135 140 Arg Cys Ser Tyr Gly Ala Asp Ser 145 150 34 131 PRT
Toxoplasma gondii 34 Pro Ser Val Val Asn Asn Val Ala Arg Cys Ser
Tyr Gly Ala Asp Ser 1 5 10 15 Thr Leu Gly Pro Val Lys Leu Ser Ala
Glu Gly Pro Thr Thr Met Thr 20 25 30 Leu Val Cys Gly Lys Asp Gly
Val Lys Val Pro Gln Asp Asn Asn Gln 35 40 45 Tyr Cys Ser Gly Thr
Thr Leu Thr Gly Cys Asn Glu Lys Ser Phe Lys 50 55 60 Asp Ile Leu
Pro Lys Leu Thr Glu Asn Pro Trp Gln Gly Asn Ala Ser 65 70 75 80 Ser
Asp Lys Gly Ala Thr Leu Thr Ile Lys Lys Glu Ala Phe Pro Ala 85 90
95 Glu Ser Lys Ser Val Ile Ile Gly Cys Thr Gly Gly Ser Pro Glu Lys
100 105 110 His His Cys Thr Val Lys Leu Glu Phe Ala Gly Ala Ala Gly
Ser Ala 115 120 125 Lys Ser Ala 130 35 31 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 35
gatccttact agttttagta gcggccgcgg g 31 36 31 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 36
aattcccgcg gccgctacta aaactagtaa g 31 37 22 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 37
gggcactcga ccggaattat cg 22 38 21 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 38
gggtaaaggt ttctttgctc g 21 39 18 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 39
atggcggctg cacactcg 18 40 23 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 40 gaacatattc
cctgtcacca atg 23 41 24 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 41 atgacgaaaa
ataaaattct tctc 24 42 22 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 42 cattgatatc
aacacaaagg cc 22 43 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 43 atggtgatga
tgggcagcat g 21 44 18 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 44 cggcggccgc
gctagagg 18 45 34 DNA Artificial Sequence Description of Artificial
Sequence Synthetic oligonucleotide 45 cgttggatcc ttggattgag
ccaaagggtg ccag 34 46 37 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 46 cccagaattc
tcaagctgcc tgttccgcta agatctg 37 47 20 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 47
atgacgggta ccgttagcag 20 48 19 DNA Artificial Sequence Description
of Artificial Sequence Synthetic oligonucleotide 48 acccagcgcc
gctaaactc 19 49 22 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 49 atggtggtta
tcaaggacat cg 22 50 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 50 ttttgggtgt
cgaaagctct c 21 51 18 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 51 atggcgccgt
cagcatcg 18 52 23 DNA Artificial Sequence Description of Artificial
Sequence Synthetic oligonucleotide 52 cttcacgctg atttgttgct ttg 23
53 21 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 53 atggacgaag cgagcagaag g 21 54 20 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 54 acgcgtgatc gaaggaaccg 20 55 297 DNA Toxoplasma
gondii 55 ggattgagcc aaagggtgcc agagctacca gaagtggagc cctttgatga
agtaggcacg 60 ggagctcgac ggtccgggtc cattgcgacc cttcttccac
aagacgctgt tttatatgag 120 aactcagagg acgttgccgt tccgagtgat
tcagcatcga ccccgtcata ctttcatgtg 180 gaatctccaa gtgctagtgt
ggaagccgcg actggcgctg tgggagaggt ggtgccggac 240 tgtgaagaac
aacaggaaca gggtgacacg acgttatccg atcacgattt ccattca 297 56 265 DNA
Toxoplasma gondii 56 tcttcagaaa gatgacgtaa ccatagaagt cgacaacgga
gccatcgtta tcaaaggaga 60 gaagacctcg aaagaagcgg agaaagtgga
cgatggcaaa acaaagaaca ttttgactga 120 gcgagtgtcc ggttattttg
cgcgccggtt ccagctcccg agtaattaca agcccgacgg 180 aatcagtgcg
gcaatggaca acggcgttct acgtgtcacg atcaaggtcg aggattcagg 240
gggcgcaaag caacaaatca gcgtg 265 57 98 PRT Toxoplasma gondii 57 Gly
Leu Ser Gln Arg Val Pro Glu Leu Pro Glu Val Glu Pro Phe Asp 1 5 10
15 Glu Val Gly Thr Gly Ala Arg Arg Ser Gly Ser Ile Ala Thr Leu Leu
20 25 30 Pro Gln Asp Ala Val Leu Tyr Glu Asn Ser Glu Asp Val Ala
Val Pro 35 40 45 Ser Asp Ser Ala Ser Thr Pro Ser Tyr Phe His Val
Glu Ser Pro Ser 50 55 60 Ala Ser Val Glu Ala Ala Thr Gly Ala Val
Gly Glu Val Val Pro Asp 65 70 75 80 Cys Glu Glu Gln Gln Glu Gln Gly
Asp Thr Thr Leu Ser Asp His Asp 85 90 95 Phe His 58 135 PRT
Toxoplasma gondii 58 Leu Asn Pro Ile Asp Asp Met Leu Phe Glu Thr
Ala Leu Thr Ala Asn 1 5 10 15 Glu Met Met Glu Asp Ile Thr Trp Arg
Pro Arg Val Asp Val Glu Phe 20
25 30 Asp Ser Lys Lys Lys Glu Met Ile Ile Leu Ala Asp Leu Pro Gly
Leu 35 40 45 Gln Lys Asp Asp Val Thr Ile Glu Val Asp Asn Gly Ala
Ile Val Ile 50 55 60 Lys Gly Glu Lys Thr Ser Lys Glu Ala Glu Lys
Val Asp Asp Gly Lys 65 70 75 80 Thr Lys Asn Ile Leu Thr Glu Arg Val
Ser Gly Tyr Phe Ala Arg Arg 85 90 95 Phe Gln Leu Pro Ser Asn Tyr
Lys Pro Asp Gly Ile Ser Ala Ala Met 100 105 110 Asp Asn Gly Val Leu
Arg Val Thr Ile Lys Val Glu Asp Ser Gly Gly 115 120 125 Ala Lys Gln
Gln Ile Ser Val 130 135 59 237 DNA Toxoplasma gondii 59 ccccaggatg
ccatttgctc ggattggtcc gcatggagcc cctgcagtgt atcctgcggt 60
gacggaagcc aaatcaggac gcgaactgag gtttctgctc cgcaacctgg aacaccaaca
120 tgtccggact gccctgcgcc catgggaagg acttgcgtgg aacaaggcgg
acttgaagaa 180 atccgtgaat gcagtgcggg ggtatgtgct gttgacgctg
gatgtggcgt ctgggtt 237 60 432 DNA Toxoplasma gondii 60 ccgtgtccaa
ttaatgcaac ttgcggtcag tttgaagaat ggagtacatg ctcggtctca 60
tgtggtggtg gactgaaaac gaggtcgagg aacccttgga atgaagacca acaacatgga
120 ggactatcct gcgagcagca gcatcctggt gggcggacgg aaacggtaac
ttgcaatcct 180 caagcgtgtc ctgtggatga acgaccgggg gagtgggcag
agtgggggga atgtagtgtc 240 acgtgcggcg acggagtgcg agagcgcagg
cgcgggaaaa gtctagttga ggctaaattc 300 ggcggacgca ccattgatca
gcagaatgag gctcttccgg aagacttaaa aatcaaaaac 360 gtcgagtatg
agccatgttc gtatcctgct tgtggagctt cctgcacgta cgtctggagt 420
gactggaaca ag 432 61 678 DNA Toxoplasma gondii 61 aacgaaccgg
tggccctagc tcagctcagc acattcctcg agctcgtcga ggtgccatgt 60
aactctgttc atgttcaggg ggtgatgacc ccgaatcaaa tggtcaaagt gactggtgca
120 ggatgggata atggcgttct cgagttctat gtcacgaggc caacgaagac
aggcggggac 180 acaagccgaa gccatcttgc gtcgatcatg tgttattcca
aggacattga cggcgtgccg 240 tcagacaaag cgggaaagtg ctttctgaag
aacttttctg gtgaagactc gtcggaaata 300 gacgaaaaag aagtatctct
acccatcaag agccacaacg atgcgttcat gttcgtttgt 360 tcttcaaatg
atggatccgc actccagtgt gatgttttcg cccttgataa caccaactct 420
agcgacgggt ggaaagtgaa taccgtggat cttggcgtca gcgttagtcc ggatttggca
480 ttcggactca ctgcagatgg ggtcaaggtg aagaagttgt acgcaagcag
cggcctgaca 540 gcgatcaacg acgacccttc cttggggtgc aaggctcctc
cccattctcc gccggccgga 600 gaggaaccga gtttgccgtc gcctgaaaac
agcgggtctg caacaccagc ggaagaaagt 660 ccgtctgagt ctgaatct 678 62 582
DNA Toxoplasma gondii 62 cttcgcgggt acaggttcgg tgtttggaag
aaaggccgtt gcctcgacta cactgaattg 60 accgacactg tgatagaacg
tgttgagtca aaggcacagt gctgggtgaa aacctttgaa 120 aacgacgggg
tcgcgagtga ccaaccccat acgtatccac tgacgtcgca agcatcatgg 180
aacgattggt ggcctctcca ccagagtgac caacctcact caggtggcgt tgggcgtaat
240 tacggtttct actacgtgga cacgactgga gagggcaagt gtgcactctc
tgaccaggta 300 cccgactgcc tggtgtcgga ttctgccgcc gtgtcgtata
cagcagcggg gagtttgtct 360 gaagagacgc cgaatttcat aattccgtca
aatccctctg ttactccgcc aacgcccgag 420 acggcacttc agtgcacggc
cgacaagttc cccgactctt tcggtgcctg cgacgttcaa 480 gcctgtaaaa
gacagaagac gtcctgcgtt ggcggacaga ttcaaagtac tagcgtcgac 540
tgcaccgcgg acgaacaaaa tgaatgtggc tctaacactg cg 582 63 507 DNA
Toxoplasma gondii 63 agtgccaacg taacaagttc ggagcctgca aaacttgatc
tctcttgtgc gcactctgac 60 aataagggat caagggctcc cacaataggc
gagccagtgc cagatgtgtc cctggaacaa 120 tgtgctgcgc aatgcaaggc
tgttgatggc tgcacacatt tcacttataa tgacgattcg 180 aagatgtgcc
atgtgaagga gggaaaaccc gatttatacg atctcacagg aggcaaaaca 240
gcaccgcgca gttgcgatag atcatgcttc gaacaacacg tatcgtatga gggagctcct
300 gacgtgatga cagcgatggt cacgagccag tcagcggact gtcaggctgc
gtgtgcggct 360 gacccgagct gcgagatctt cacttataac gaacacgacc
agaaatgtac tttcaaagga 420 agggggtttt ctgcgtttaa ggaacgaggg
gtgttgggtg tgacttccgg gccgaaacag 480 ttctgcgatg aaggcggtaa attaact
507 64 79 PRT Toxoplasma gondii 64 Pro Gln Asp Ala Ile Cys Ser Asp
Trp Ser Ala Trp Ser Pro Cys Ser 1 5 10 15 Val Ser Cys Gly Asp Gly
Ser Gln Ile Arg Thr Arg Thr Glu Val Ser 20 25 30 Ala Pro Gln Pro
Gly Thr Pro Thr Cys Pro Asp Cys Pro Ala Pro Met 35 40 45 Gly Arg
Thr Cys Val Glu Gln Gly Gly Leu Glu Glu Ile Arg Glu Cys 50 55 60
Ser Ala Gly Val Cys Ala Val Asp Ala Gly Cys Gly Val Trp Val 65 70
75 65 144 PRT Toxoplasma gondii 65 Pro Cys Pro Ile Asn Ala Thr Cys
Gly Gln Phe Glu Glu Trp Ser Thr 1 5 10 15 Cys Ser Val Ser Cys Gly
Gly Gly Leu Lys Thr Arg Ser Arg Asn Pro 20 25 30 Trp Asn Glu Asp
Gln Gln His Gly Gly Leu Ser Cys Glu Gln Gln His 35 40 45 Pro Gly
Gly Arg Thr Glu Thr Val Thr Cys Asn Pro Gln Ala Cys Pro 50 55 60
Val Asp Glu Arg Pro Gly Glu Trp Ala Glu Trp Gly Glu Cys Ser Val 65
70 75 80 Thr Cys Gly Asp Gly Val Arg Glu Arg Arg Arg Gly Lys Ser
Leu Val 85 90 95 Glu Ala Lys Phe Gly Gly Arg Thr Ile Asp Gln Gln
Asn Glu Ala Leu 100 105 110 Pro Glu Asp Leu Lys Ile Lys Asn Val Glu
Tyr Glu Pro Cys Ser Tyr 115 120 125 Pro Ala Cys Gly Ala Ser Cys Thr
Tyr Val Trp Ser Asp Trp Asn Lys 130 135 140 66 226 PRT Toxoplasma
gondii 66 Asn Glu Pro Val Ala Leu Ala Gln Leu Ser Thr Phe Leu Glu
Leu Val 1 5 10 15 Glu Val Pro Cys Asn Ser Val His Val Gln Gly Val
Met Thr Pro Asn 20 25 30 Gln Met Val Lys Val Thr Gly Ala Gly Trp
Asp Asn Gly Val Leu Glu 35 40 45 Phe Tyr Val Thr Arg Pro Thr Lys
Thr Gly Gly Asp Thr Ser Arg Ser 50 55 60 His Leu Ala Ser Ile Met
Cys Tyr Ser Lys Asp Ile Asp Gly Val Pro 65 70 75 80 Ser Asp Lys Ala
Gly Lys Cys Phe Leu Lys Asn Phe Ser Gly Glu Asp 85 90 95 Ser Ser
Glu Ile Asp Glu Lys Glu Val Ser Leu Pro Ile Lys Ser His 100 105 110
Asn Asp Ala Phe Met Phe Val Cys Ser Ser Asn Asp Gly Ser Ala Leu 115
120 125 Gln Cys Asp Val Phe Ala Leu Asp Asn Thr Asn Ser Ser Asp Gly
Trp 130 135 140 Lys Val Asn Thr Val Asp Leu Gly Val Ser Val Ser Pro
Asp Leu Ala 145 150 155 160 Phe Gly Leu Thr Ala Asp Gly Val Lys Val
Lys Lys Leu Tyr Ala Ser 165 170 175 Ser Gly Leu Thr Ala Ile Asn Asp
Asp Pro Ser Leu Gly Cys Lys Ala 180 185 190 Pro Pro His Ser Pro Pro
Ala Gly Glu Glu Pro Ser Leu Pro Ser Pro 195 200 205 Glu Asn Ser Gly
Ser Ala Thr Pro Ala Glu Glu Ser Pro Ser Glu Ser 210 215 220 Glu Ser
225 67 194 PRT Toxoplasma gondii 67 Leu Arg Gly Tyr Arg Phe Gly Val
Trp Lys Lys Gly Arg Cys Leu Asp 1 5 10 15 Tyr Thr Glu Leu Thr Asp
Thr Val Ile Glu Arg Val Glu Ser Lys Ala 20 25 30 Gln Cys Trp Val
Lys Thr Phe Glu Asn Asp Gly Val Ala Ser Asp Gln 35 40 45 Pro His
Thr Tyr Pro Leu Thr Ser Gln Ala Ser Trp Asn Asp Trp Trp 50 55 60
Pro Leu His Gln Ser Asp Gln Pro His Ser Gly Gly Val Gly Arg Asn 65
70 75 80 Tyr Gly Phe Tyr Tyr Val Asp Thr Thr Gly Glu Gly Lys Cys
Ala Leu 85 90 95 Ser Asp Gln Val Pro Asp Cys Leu Val Ser Asp Ser
Ala Ala Val Ser 100 105 110 Tyr Thr Ala Ala Gly Ser Leu Ser Glu Glu
Thr Pro Asn Phe Ile Ile 115 120 125 Pro Ser Asn Pro Ser Val Thr Pro
Pro Thr Pro Glu Thr Ala Leu Gln 130 135 140 Cys Thr Ala Asp Lys Phe
Pro Asp Ser Phe Gly Ala Cys Asp Val Gln 145 150 155 160 Ala Cys Lys
Arg Gln Lys Thr Ser Cys Val Gly Gly Gln Ile Gln Ser 165 170 175 Thr
Ser Val Asp Cys Thr Ala Asp Glu Gln Asn Glu Cys Gly Ser Asn 180 185
190 Thr Ala 68 169 PRT Toxoplasma gondii 68 Ser Ala Asn Val Thr Ser
Ser Glu Pro Ala Lys Leu Asp Leu Ser Cys 1 5 10 15 Ala His Ser Asp
Asn Lys Gly Ser Arg Ala Pro Thr Ile Gly Glu Pro 20 25 30 Val Pro
Asp Val Ser Leu Glu Gln Cys Ala Ala Gln Cys Lys Ala Val 35 40 45
Asp Gly Cys Thr His Phe Thr Tyr Asn Asp Asp Ser Lys Met Cys His 50
55 60 Val Lys Glu Gly Lys Pro Asp Leu Tyr Asp Leu Thr Gly Gly Lys
Thr 65 70 75 80 Ala Pro Arg Ser Cys Asp Arg Ser Cys Phe Glu Gln His
Val Ser Tyr 85 90 95 Glu Gly Ala Pro Asp Val Met Thr Ala Met Val
Thr Ser Gln Ser Ala 100 105 110 Asp Cys Gln Ala Ala Cys Ala Ala Asp
Pro Ser Cys Glu Ile Phe Thr 115 120 125 Tyr Asn Glu His Asp Gln Lys
Cys Thr Phe Lys Gly Arg Gly Phe Ser 130 135 140 Ala Phe Lys Glu Arg
Gly Val Leu Gly Val Thr Ser Gly Pro Lys Gln 145 150 155 160 Phe Cys
Asp Glu Gly Gly Lys Leu Thr 165 69 20 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 69
atgagactcc aaccgaggcc 20 70 23 DNA Artificial Sequence Description
of Artificial Sequence Synthetic oligonucleotide 70 ctgcctgact
ctttcttgga ctg 23 71 22 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 71 ggaaagttgg
aaatccggcg gc 22 72 19 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 72 cgcctcatcg
tcactcggc 19 73 19 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 73 atgagagcgt
cgctcccgg 19 74 23 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 74 gtgtctttcg
cttcaagcac ctg 23 75 19 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 75 atggggctcg
tgggcgtac 19 76 23 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 76 gatcaacgca
gtgttagagc cac 23
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