GB2232675A - Stable forms of antigenic taenia ovis polypeptides - Google Patents
Stable forms of antigenic taenia ovis polypeptides Download PDFInfo
- Publication number
- GB2232675A GB2232675A GB9010357A GB9010357A GB2232675A GB 2232675 A GB2232675 A GB 2232675A GB 9010357 A GB9010357 A GB 9010357A GB 9010357 A GB9010357 A GB 9010357A GB 2232675 A GB2232675 A GB 2232675A
- Authority
- GB
- United Kingdom
- Prior art keywords
- thr
- peptide
- ovis
- val
- fragment
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 67
- 241000244154 Taenia ovis Species 0.000 title claims abstract description 48
- 230000000890 antigenic effect Effects 0.000 title claims description 26
- 229920001184 polypeptide Polymers 0.000 title claims description 23
- 102000004196 processed proteins & peptides Human genes 0.000 title claims description 23
- 108020004414 DNA Proteins 0.000 claims abstract description 33
- 241000282849 Ruminantia Species 0.000 claims abstract description 25
- 208000015181 infectious disease Diseases 0.000 claims abstract description 19
- 229960005486 vaccine Drugs 0.000 claims abstract description 18
- 239000012634 fragment Substances 0.000 claims description 39
- 108020001507 fusion proteins Proteins 0.000 claims description 39
- 102000037865 fusion proteins Human genes 0.000 claims description 38
- 238000000034 method Methods 0.000 claims description 28
- 150000001413 amino acids Chemical group 0.000 claims description 18
- 241000588724 Escherichia coli Species 0.000 claims description 17
- 102000053602 DNA Human genes 0.000 claims description 12
- 230000001681 protective effect Effects 0.000 claims description 12
- 239000013598 vector Substances 0.000 claims description 12
- 239000002773 nucleotide Substances 0.000 claims description 11
- 125000003729 nucleotide group Chemical group 0.000 claims description 11
- 230000001900 immune effect Effects 0.000 claims description 10
- 230000028993 immune response Effects 0.000 claims description 10
- 241000894006 Bacteria Species 0.000 claims description 9
- 239000002671 adjuvant Substances 0.000 claims description 8
- 239000013604 expression vector Substances 0.000 claims description 7
- 238000012217 deletion Methods 0.000 claims description 6
- 230000037430 deletion Effects 0.000 claims description 6
- 238000003780 insertion Methods 0.000 claims description 6
- 230000037431 insertion Effects 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 6
- 238000012258 culturing Methods 0.000 claims description 5
- 230000002163 immunogen Effects 0.000 claims description 5
- 238000006467 substitution reaction Methods 0.000 claims description 5
- 239000003085 diluting agent Substances 0.000 claims description 2
- STACJSVFHSEZJV-GHCJXIJMSA-N Ala-Asn-Ile Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O STACJSVFHSEZJV-GHCJXIJMSA-N 0.000 claims 1
- UHMQKOBNPRAZGB-CIUDSAMLSA-N Ala-Glu-Met Chemical compound C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCSC)C(=O)O)N UHMQKOBNPRAZGB-CIUDSAMLSA-N 0.000 claims 1
- OSRZOHXQCUFIQG-FPMFFAJLSA-N Ala-Phe-Pro Chemical compound C([C@H](NC(=O)[C@@H]([NH3+])C)C(=O)N1[C@H](CCC1)C([O-])=O)C1=CC=CC=C1 OSRZOHXQCUFIQG-FPMFFAJLSA-N 0.000 claims 1
- RMAWDDRDTRSZIR-ZLUOBGJFSA-N Ala-Ser-Asp Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(O)=O RMAWDDRDTRSZIR-ZLUOBGJFSA-N 0.000 claims 1
- ZUVDFJXRAICIAJ-BPUTZDHNSA-N Arg-Trp-Asp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](CCCN=C(N)N)N)C(=O)N[C@@H](CC(O)=O)C(O)=O)=CNC2=C1 ZUVDFJXRAICIAJ-BPUTZDHNSA-N 0.000 claims 1
- VKCOHFFSTKCXEQ-OLHMAJIHSA-N Asn-Asn-Thr Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O VKCOHFFSTKCXEQ-OLHMAJIHSA-N 0.000 claims 1
- KUYKVGODHGHFDI-ACZMJKKPSA-N Asn-Gln-Ser Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(O)=O KUYKVGODHGHFDI-ACZMJKKPSA-N 0.000 claims 1
- XLZCLJRGGMBKLR-PCBIJLKTSA-N Asn-Ile-Phe Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 XLZCLJRGGMBKLR-PCBIJLKTSA-N 0.000 claims 1
- QCVXMEHGFUMKCO-YUMQZZPRSA-N Asp-Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CC(O)=O QCVXMEHGFUMKCO-YUMQZZPRSA-N 0.000 claims 1
- GBSUGIXJAAKZOW-GMOBBJLQSA-N Asp-Ile-Arg Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O GBSUGIXJAAKZOW-GMOBBJLQSA-N 0.000 claims 1
- KNDCWFXCFKSEBM-AVGNSLFASA-N Asp-Tyr-Glu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCC(O)=O)C(O)=O KNDCWFXCFKSEBM-AVGNSLFASA-N 0.000 claims 1
- 241000206602 Eukaryota Species 0.000 claims 1
- RGRMOYQUIJVQQD-SRVKXCTJSA-N Gln-Arg-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCC(=O)N)N RGRMOYQUIJVQQD-SRVKXCTJSA-N 0.000 claims 1
- MTCXQQINVAFZKW-MNXVOIDGSA-N Gln-Ile-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CCC(=O)N)N MTCXQQINVAFZKW-MNXVOIDGSA-N 0.000 claims 1
- OREPWMPAUWIIAM-ZPFDUUQYSA-N Gln-Pro-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@@H]1CCCN1C(=O)[C@H](CCC(=O)N)N OREPWMPAUWIIAM-ZPFDUUQYSA-N 0.000 claims 1
- CGYDXNKRIMJMLV-GUBZILKMSA-N Glu-Arg-Glu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(O)=O CGYDXNKRIMJMLV-GUBZILKMSA-N 0.000 claims 1
- WOSRKEJQESVHGA-CIUDSAMLSA-N Glu-Arg-Ser Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(O)=O WOSRKEJQESVHGA-CIUDSAMLSA-N 0.000 claims 1
- SRZLHYPAOXBBSB-HJGDQZAQSA-N Glu-Arg-Thr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(O)=O SRZLHYPAOXBBSB-HJGDQZAQSA-N 0.000 claims 1
- BPLNJYHNAJVLRT-ACZMJKKPSA-N Glu-Ser-Ala Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O BPLNJYHNAJVLRT-ACZMJKKPSA-N 0.000 claims 1
- MRWYPDWDZSLWJM-ACZMJKKPSA-N Glu-Ser-Asp Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(O)=O MRWYPDWDZSLWJM-ACZMJKKPSA-N 0.000 claims 1
- 102000005720 Glutathione transferase Human genes 0.000 claims 1
- 108010070675 Glutathione transferase Proteins 0.000 claims 1
- RQZGFWKQLPJOEQ-YUMQZZPRSA-N Gly-Arg-Gln Chemical compound C(C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)CN)CN=C(N)N RQZGFWKQLPJOEQ-YUMQZZPRSA-N 0.000 claims 1
- LLZXNUUIBOALNY-QWRGUYRKSA-N Gly-Leu-Lys Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CCCCN LLZXNUUIBOALNY-QWRGUYRKSA-N 0.000 claims 1
- OQQKUTVULYLCDG-ONGXEEELSA-N Gly-Lys-Val Chemical compound CC(C)[C@H](NC(=O)[C@H](CCCCN)NC(=O)CN)C(O)=O OQQKUTVULYLCDG-ONGXEEELSA-N 0.000 claims 1
- FKESCSGWBPUTPN-FOHZUACHSA-N Gly-Thr-Asn Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(O)=O FKESCSGWBPUTPN-FOHZUACHSA-N 0.000 claims 1
- JVEKQAYXFGIISZ-HOCLYGCPSA-N His-Trp-Gly Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)NCC(O)=O)C1=CN=CN1 JVEKQAYXFGIISZ-HOCLYGCPSA-N 0.000 claims 1
- CYHYBSGMHMHKOA-CIQUZCHMSA-N Ile-Ala-Thr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(=O)O)N CYHYBSGMHMHKOA-CIQUZCHMSA-N 0.000 claims 1
- DVRDRICMWUSCBN-UKJIMTQDSA-N Ile-Gln-Val Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](C(C)C)C(=O)O)N DVRDRICMWUSCBN-UKJIMTQDSA-N 0.000 claims 1
- SVBAHOMTJRFSIC-SXTJYALSSA-N Ile-Ile-Asn Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(=O)N)C(=O)O)N SVBAHOMTJRFSIC-SXTJYALSSA-N 0.000 claims 1
- GLYJPWIRLBAIJH-UHFFFAOYSA-N Ile-Lys-Pro Natural products CCC(C)C(N)C(=O)NC(CCCCN)C(=O)N1CCCC1C(O)=O GLYJPWIRLBAIJH-UHFFFAOYSA-N 0.000 claims 1
- HQEPKOFULQTSFV-JURCDPSOSA-N Ile-Phe-Ala Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C)C(=O)O)N HQEPKOFULQTSFV-JURCDPSOSA-N 0.000 claims 1
- CZCSUZMIRKFFFA-CIUDSAMLSA-N Leu-Ala-Asn Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(O)=O CZCSUZMIRKFFFA-CIUDSAMLSA-N 0.000 claims 1
- REPPKAMYTOJTFC-DCAQKATOSA-N Leu-Arg-Asp Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(O)=O REPPKAMYTOJTFC-DCAQKATOSA-N 0.000 claims 1
- VGPCJSXPPOQPBK-YUMQZZPRSA-N Leu-Gly-Ser Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O VGPCJSXPPOQPBK-YUMQZZPRSA-N 0.000 claims 1
- JKSIBWITFMQTOA-XUXIUFHCSA-N Leu-Ile-Val Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C(C)C)C(O)=O JKSIBWITFMQTOA-XUXIUFHCSA-N 0.000 claims 1
- INCJJHQRZGQLFC-KBPBESRZSA-N Leu-Phe-Gly Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)NCC(O)=O INCJJHQRZGQLFC-KBPBESRZSA-N 0.000 claims 1
- RNYLNYTYMXACRI-VFAJRCTISA-N Leu-Thr-Trp Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O RNYLNYTYMXACRI-VFAJRCTISA-N 0.000 claims 1
- ULUQBUKAPDUKOC-GVXVVHGQSA-N Lys-Glu-Val Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O ULUQBUKAPDUKOC-GVXVVHGQSA-N 0.000 claims 1
- NKKFVJRLCCUJNA-QWRGUYRKSA-N Lys-Gly-Lys Chemical compound NCCCC[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCCN NKKFVJRLCCUJNA-QWRGUYRKSA-N 0.000 claims 1
- PLOUVAYOMTYJRG-JXUBOQSCSA-N Lys-Thr-Ala Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O PLOUVAYOMTYJRG-JXUBOQSCSA-N 0.000 claims 1
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- KZNQNBZMBZJQJO-UHFFFAOYSA-N N-glycyl-L-proline Natural products NCC(=O)N1CCCC1C(O)=O KZNQNBZMBZJQJO-UHFFFAOYSA-N 0.000 claims 1
- 108010047562 NGR peptide Proteins 0.000 claims 1
- 241000283898 Ovis Species 0.000 claims 1
- AYPMIIKUMNADSU-IHRRRGAJSA-N Phe-Arg-Asn Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(O)=O AYPMIIKUMNADSU-IHRRRGAJSA-N 0.000 claims 1
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- FRKBNXCFJBPJOL-GUBZILKMSA-N Pro-Glu-Glu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O FRKBNXCFJBPJOL-GUBZILKMSA-N 0.000 claims 1
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- YDTUEBLEAVANFH-RCWTZXSCSA-N Pro-Val-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H]1CCCN1 YDTUEBLEAVANFH-RCWTZXSCSA-N 0.000 claims 1
- UFKPDBLKLOBMRH-XHNCKOQMSA-N Ser-Glu-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CO)N)C(=O)O UFKPDBLKLOBMRH-XHNCKOQMSA-N 0.000 claims 1
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- IFPBAGJBHSNYPR-ZKWXMUAHSA-N Ser-Ile-Gly Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(O)=O IFPBAGJBHSNYPR-ZKWXMUAHSA-N 0.000 claims 1
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- ADPHPKGWVDHWML-PPCPHDFISA-N Thr-Ile-Leu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)O)NC(=O)[C@H]([C@@H](C)O)N ADPHPKGWVDHWML-PPCPHDFISA-N 0.000 claims 1
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Abstract
Stable forms of peptide antigens of molecular weight 23kD to 24kD of T.ovis suitable for use in vaccines to protect ruminants against infection by cestode parasites are described. The antigens are preferably obtained by expression of DNA coding therefor in a recombinant host cell.
Description
STABLE FORMS OF ANTIGENIC TAENIA OVIS POLYPEPTIDES
The Taenia ovis tapeworm exists in adult form in the small
intestine of its primary host, the dog. The cystic stage is carried in
the musculature of its secondary or intermediate hosts, notably sheep
and goats. Current control measures include prevention of feeding of
infected carcassesto dogs and treatment of dogs with cestocidal drugs,
notably praziquantel (Droncit, Bayer) to prevent transmission of the
parasite to ruminants. These controlmeasures are costly to implement
and are not effective in eradicating T.ovis.
Accordingly, as an adjunct to current control measures and to
effect eradication of the disease, it would be preferable to immunise
the secondary hosts to protect them from infection and also to preserve
carcass quality for the meat industry.
Previous investigations conducted into vaccination against T.ovis
infection with oncosphere antigens are reviewed by Rickard, M.D and
Williams, J.F, Hydatidosis/Cystercercosis: Immune mechanisms and
Immunisation against infection, Adv Parasitology 21, 230-296 (1982).
However, in the work reviewed no attempt was made to identify which
antigenic component of the oncospheres was responsible for the immune
response. As will be appreciated, T.ovis contains a large number of
antigenic components, most of which are not immunologically effective
against infection.
Earlier attempts have been made to identify a host protective
antigen for T.ovis (Howell, M. J & Hargreaves, J J Mol Biochem
Parasitol 28, 21-30 (1988)). A cDNA library was prepared using mRNA
extracted from adult T.ovis tape worms. Recombinants expressing
antigenic determinants as -B-galactosidase fusion proteins were
selected using antibodies in serum from sheep infected with T.ovis.
Some fusion proteins were shown to correspond with native antigens
(92.5 to 180kD) present in adult and oncosphere stages of T.ovis, but
trials of the host-protective nature of purified fusion proteins were
not reported.
In Johnson, K.S. et al, Nature 338, 585-587 (1989), the present
inventors have described the identification and cloning of a native
polypeptide of T.ovis capable of generating a protective immunological
response in ruminants against T.ovis infection. However, the
recombinant polypeptide described in this paper has been found to be
less stable than is optimal for the production of a commercial vaccine.
It is accordingly an object of the present invention to provide a stable form of protective antigen for use in vaccines for the protection of ruminants against T.ovis infection or at least to provide the public with a useful choice.
Accordingly, in one aspect the present invention may broadly be said to consist in a purified stable antigenic peptide comprising a fragment of a T.ovis polypeptide, which fragment
(a) has a molecular weight of from about 23 kD to about 24 kD; and
(b) is capable of generating a protective immunological response to T.ovis in a ruminant
or a subfragment or variant thereof having substantially equivalent stability and immunological activity thereto.
Preferably, the antigenic polypeptide fragment has the amino acid sequence set out in Figure 1.
Conveniently, the protective antigenic peptide of the invention is obtained by expression of the DNA sequence coding therefor in a host cell or organism.
In a further aspect, the invention consists in a stable antigenic peptide comprising a fragment of a T.ovis polypeptide which is capable of generating a protective immunological response to T.ovis in a ruminant, which fragment has the amino acid sequence encoded by the DNA sequence of Figure 1 herein, or a derivative or variant thereof having substantially equivalent stability and immunological activity thereto.
In still a further aspect, the invention consists in a composition of matter capable of generating a protective immunological response to
T.ovis infection in a ruminant which essentially consists of:
(a) a stable peptide comprising a polypeptide fragment havingthe amino acid sequence of Figure 1;
(b) a stable immunologically active subfragment of (a); or
(c) a variant of (a) or (b) which has been modified by the insertion, substitution or deletion of one or more amino acids and which has equivalent stability and immunological activity thereto.
In still a further aspect, the invention consists in a vaccine which includes a stable immunogenic peptide thereof as defined above in combination with a pharmaceutically acceptable carrier andlor adjuvant therefor.
In still a further aspect, the invention may be said to consist in a method of protecting a ruminant against infection by a cestode parasite comprising administering to said ruminant an immunologically effective amount of:
(a) a stable peptide as defined above;
(b) a composition as defined above; or
(c) a vaccine as defined above.
In additional aspects, the invention relates to a DNA isolate which comprises a DNA sequence encoding the immunologically effective antigenic peptide of the invention; to transformed hosts capable of expressing the antigenic-peptide encoded; and to methods of producing the antigenic peptide comprising culturing the said transformed hosts.
Although the invention is broadly as described above, it will be appreciated by those persons skilled in the art that the invention is not limited to the foregoing but also includes embodiments of which the following gives examples. In particular, certain aspects of the invention will be more clearly understood by having reference to the accompanying drawings wherein,
Figure 1 represents both the nucleotide sequence and the predicted amino acid sequence of the T.ovis polypeptide fragment.
Figure 2 is an auto radiograph showing that a monoclonal antibody generated against GST-45W fusion protein recognises the cloned B-X peptide of the invention.
Figure 3 is an immunoblot analysis showing that sheep vaccinated with the cloned GST 45W(B/X) peptide generate antibodies which recognise the native T.ovis antigen(s) having a molecular weight of 47-52 kD.
Figure 4 shows sheep antibody response following immunisation with
GST-45W (BIX) fusion protein extracts.
Previous investigations by the inventors have identified a 47-52 kD molecular weight fraction of the antigenic complement of T.ovis as a major antigen to which antibodies were present in immune sheep. The cloning and recombinant production of this native antigen in general and of a major fragment of the antigen in particular (termed the '"45W" antigen) has been reported (Johnson, K.S. et al, Nature (1989), supra), and forms the subject of USSN 07/349,723 (UK Application Nb. 8910822.9), the disclosure of which is specifically incorporated herein by reference.
However, during the course of subsequent investigations into the production of a commercial vaccine based upon this major fragment it was found that although it was perfectly possible to produce a vaccine composition protective against T.ovis, the stability of the fragment itself was less than ideal.
In further investigations, the inventors have now located a subfragment of the 45W antigen which exhibits enhanced stability while also having equivalent if not superior immunological activity as compared to the 45W antigen. It is to this more stable subfragment that the present invention is primarily directed.
The present invention therefore has as its first aspect an antigenic peptide comprising a fragment of a T.ovis polypeptide which is capable of generating a protective immunological response to
T.ovis. The molecular weight of this fragment has been calculated as about 20 kD from the DNA sequence determined for the molecule.
In a preferred form of this aspect of the invention, the immunologically active peptide includes a polypeptide fragment having the amino acid sequence of Figure 1. It will however be appreciated that modifications can be made to the native sequence of the polypeptide fragment whilst at least substantially retaining both its stability and biological activity. Such modifications to the native amino acid sequence to result in the insertion, substitution or deletion of one or more amino acids are specifically within the scope of this invention.
The antigenic polypeptide fragments of the invention can be prepared in a variety of ways. For example. they can be obtained by isolation from a natural source, by synthesis using any suitable known technique (such as by the stepwise solid phase approach described by
Merrifield (1963) J. Amer Chem. Soc 85 2149-2156) or, as is preferred, through employing recombinant DNA techniques.
The variants of the polypeptide fragments can similarly be made by any of those techniques known in the art. For example, variants can be prepared by site-specific mutagenesis of the DNA encoding the native amino acid sequence.
Site-specific mutagenesis allows the production of variants through the use of specific oligonucleotide sequences which encode the
DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence-complexity to form a stable duplex on both sides of the deletion junction being traversed. Typically, a primer of about 20 to 25 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction of the sequence being altered. In general, the technique of site-specific mutagenesis is well known in the art as exemplified by publications such as Adelman et al., DNA 2, 183 (1983).
In a further aspect, the invention accordingly relates to the recombinant production of the stable antigenic peptide defined above.
Stated generally, the production of the protective antigen of the invention by recombinant DNA techniques involves the transformation of a suitable host organism or cell with an expression vector including a
DNA sequence coding for the antigen, followed by the culturing of the transformed host and subsequent recovery of the antigen expressed.
An initial step in the method of recombinant production of the antigen involves the ligation of a DNA sequence encoding the antigen into a suitable expression vector containing a promoter and ribosome binding site operable in the host cell in which the coding sequence will be transformed. The most common examples of such expression vectors are plasmids which are double stranded DNA loops that replicate autonomously in the host cell. However, it will be understood that suitable vectors other than plasmids can be used in performing the invention.
Preferably, the host cell in which the DNA sequence encoding the peptide is cloned and expressed is a prokaryote such as E. coli. For example, E. coli DH5 (Raleigh E.A. et al Nucleic Acid Research 16 No 4 p 1563-1575 (1988)), E. coli K12 strain 294 (ATCC 31446), E. coli B, E.
coli X1776 (ATCC 31537) E. coli strain ST9 or E. coli JM 101 can be employed. Other prokaryotes can also be used, for example bacilli such as Bacillus subtilis and enterobacteriaceae such as Salmonella tynhimurium, Serratia marcesans or the attenuated strain Bacille
Calmette-Guerin (BCG) of Mycobacterium bovis.
In general, where the host cell is a prokaryote, expression or cloning vectors containing replication and control sequences which are derived from species compatible with the host cell are used. The vector may also carry marking sequences which are capable of providing phenotypic selection in transformed cells. For example, E. coli has commonly been transformed using pBR322, a plasmid derived from an E.
coli species (Bolivar, et al., Gene 2: 95 (1977)). pBR322 contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells.
For use in expression, the plasmid including the DNA to be expressed contains a promoter. Those promoters most commonly used in recombinant DNA construction for use with prokaryotic hosts include the 8-lactamase (penicillinase) and lactose promoter systems (Chang et al, Nature, 275: 615 (1978); Itakura, et al, Science, 198: 1056 (1977);
Goeddel, et al Nature 281: 544 (1979)) and a tryptophan (trp) promoter system (Goeddel, et al, Nucleic Acids Res., 8: 4057 (1980); EPO Publ
No. 0036776). While these are the most commonly used, other microbial promoters such as the tac promoter (Amann et al., Gene 25, 167-178 (1983)) have been constructed and utilised, and details concerning their nucleotide sequences have been published, enabling a skilled worker to ligate them functionally in operable relationship to genes in vectors (Siebenlist, et al, Cell 20: 269 (1980)).
In addition to prokaryotes, eukaryotic microbes, such as yeast may also be used. Saccharomyces cerevisiae, or common baker's yeast is the most commonly used among eukaryotic microorganisms, although a number of other strains are commonly available. For expression in
Saccharomyces, the plasmid YRp7, for example, (Stinchcomb et al.,
Nature 282, 39 (1979); Kingsman et al., Gene 7, 141 (1979); Tschemper et al., Gene 10, 157 (1980)) is commonly used. This plasmid already contains the trpl gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example
ATCC No. 44076 or PEP4-1 (Jones, Genetics 85, 12 (1977)).The presence of the trPl lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.
Suitable promoting sequences in yeast vectors include the promoters for 3-phosphoglycerate kinase (Hitzeman et al., J. Biol.
Chem. 255, 2073 (1980)) or other glycolytic enzymes (Hess et al., J.
Adv. Enzyme Re. 7: 149 (1968); Holland et al., Biochemistry 17 4900 (1978). Other promoters, which have the additional advantage of transcription controlled by growth conditions, are the promoter region for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and the aforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Any plasmid vector containinga yEast-compatible prcroter,origin of replication and termination sequences is suitable.
In addition to microorganisms, cultures of cells derived from multicellular organisms such as mammals and insects may also be used as hosts. In principle, any such cell culture is workable, whether from vertebrate or invertebrate culture. However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure in recent years (Tissue Culture, Academic Press, Kruse and Patterson, editors (1973)).
Examples of such useful host cell lines are VERO and HeLa cells and
Chinese hamster ovary (CHO; cells. Expression vectors for such cells ordinarily include (if necessary) an origin of replication, a promoter located upstream from the gene to be expressed, along with any necessary ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences.
For use in mammalian cells, the control functions on the expression vectors are often provided by viral material. For example, commonly used promoters are derived from polyoma, Adenovirus 2, and most frequently Simian Virus 40(SV40). The early and late promoters of
SV40 virus are particularly useful because both are obtained easily from the virus as a fragment which also contains the SV40 viral origin of replication (Fiers et al., Nature 273, 113, (1978)). Smaller or larger SV40 fragments may also be used, provided there is included the approximately 250 bp sequence extending from the HindIII site toward the BglI site located in the viral origin of replication. Further, it is also possible, and often desirable, to utilize promoter or control sequences normally associated with the desired gene sequence, provided such control sequences are compatible with the host cell systems.
An origin of replication may be provided either by construction of the vector to include an exogenous origin, such as may be derived from
SV40 or other viral (e.g Polyoma, Adeno, VSV, BPV) source, or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient.
Upon transformation of the selected host with an appropriate vector, the antigenic peptide encoded can be produced often in the form of a fusion protein by culturing the host cells. The fusion protein is then recovered and purified as necessary.
Where the protein is produced in a soluble form, recovery and purification can be achieved using any of those procedures known in the art, for example by adsorption onto and elution from an anion exchange resin. As will be apparent from the'specific examples provided, where the peptide of the invention is produced as a fusion protein, the carrier portion of the fusion protein can prove useful in this regard.
However, it has been the applicants' experience that only a minor proportion of the protein is expressed in a soluble form, with the major proportion being expressed in a non-soluble form contained with inclusion bodies within the transformed host. In such cases, a solubilisation technique can be employed to extract the product from the inclusion bodies and to render it soluble for subsequent processing and use.
Once again, any of those conventional techniques known in the art for this purpose can be employed. For a general review of these techniques, reference can be made to the following publications:
Marston, F.A.O., "The purification of eukaryotic polypeptides synthesized in Escherichia coli"; Biochem J 240 1-12 (1986); and Sharma
S.K., "On the recovery of Genetically Engineered Proteins from
Escherichia coli", Separation Science and Technology 21 (8), pp 701-726 (1986). Of such techniques, urea solubilisation as described in
Example 4 is preferred.
Following recovery of the antigenic peptide it is purified as desired. The purification procedure adopted will of course depend upon the degree of purity required for the use to which the peptide is to be put. For most vaccination purposes, separation of the fusion protein from most of the remaining components of the cell culture is sufficient as the antigen can be incorporated into a vaccine in a relatively crude form. However, in cases where a greater degree of purity is desired, the carrier component of the fusion protein can be cleaved from the antigenic component. As will again be apparent from the specific examples provided, this can be easily achieved through the provision of an appropriate enzyme cleavage site between the carrier component and the antigen.
Where,as is preferred, recombinant techniques are used to produce the antigenic peptide, the first step is to obtain DNA encoding the desired product. Such DNA molecules comprise still a further aspect of this invention.
The DNA-molecule of the invention preferably encodes the nucleotide sequence of Figure 1. This DNA sequence can be obtained contained within a DNA molecule isolated from an appropriate natural source or can be produced as intron-free cDNA using conventional techniques such as those used in the specific description set out hereinafter.
However, as indicated above, the invention also contemplates variants of the peptide which differ from the native amino acid sequences by the insertion, substitution or deletion of one or more amino acids. Where such a variant is desired, the nucleotide sequence of the native DNA molecule is altered appropriately. This alteration can be made through elective synthesis of the DNA using an appropriate synthesizer such as the Applied Biosystems DNA Synthesizer or by modification of the native DNA by, for example, site specific or cassette mutagenesis.
Once obtained, the DNA molecule is treated to be suitable for insertion together with the selected control sequence into the appropriate cloning and! our expression vector. To this end the DNA is cleaved, tailored and religated as required.
Cleavage is performed by treatment with restriction enzyme(s) in a suitable buffer. Any of the large number of commercially available restriction enzymes can be used as specified by the manufacturer.
After cleavage, the nucleic acid is recovered by, for example, precipitation with ethanol.
Tailoring of the cleaved DNA is performed using conventional techniques. For example, if blunt ends are required, the DNA may be treated with DNA polymerase I (Klenow), phenol and chloroform extracted, and precipitated by ethanol.
Re-ligation can be performed by providing approximately equimolar amounts of the desired components, appropriately tailored for correct matching, and treatment with an appropriate ligase (eg T4 DNA ligase).
In addition to the protective stable antigens of the invention and the method of producing these, a further and most important aspect of the present invention relates to the use of the protective antigen as the active agent in a ruminant vaccine against T.ovis infection. In this aspect, the protective antigen of the invention can be administered either alone or in the form of a composition comprising the protective antigen of the invention as the active ingredient together with a pharmaceutically acceptable diluent carrier or adjuvant.
Examples of suitable adjuvants known to those skilled in the art are saponins (or derivative or related material), muramyl dipeptide, trehalose dimycollate, Freunds's complete adjuvant, Freund's incomplete adjuvant, other water-in-oil emulsions, double emulsions, dextran, diethylaminoethyl-dextran, potassium alum, aluminium phosphate, aluminium hydroxide, bentonite, zymosan, polyelectrolytes, retinol, calcium phosphate, protamine, sarcosine, glycerol, sorbitol, propylene glycol, fixed oils and synthetic esters of higher fatty acids.
Saponins have been found to be particularly effective adjuvants.
The protective antigen of the invention may also be treated in any conventional way to still further enhance its stability or to conserve or potentiate its immunogenic efficacy. For example, the antigen may be treated with a suitable inhibitor, modifier, crosslinker or denaturant in such a way as to enhance its immunogenicity.
In addition, the protective antigen can be administered in combination with other therapeutic agents such as anthelmintics, for example levamisole, or other vaccines.
The vaccine can be administered to the ruminant by any of those methods known in the art. However, the preferred mode of administration of the vaccine is parenteral. The term "parenteral" is used herein to mean intravenous, intramuscular, intradermal and subcutaneous injection. Most conveniently, the administration is by subcutaneous injection.
The amount of the vaccine administered to the ruminant to be treated will depend on the type, size and body weight of the ruminant as well as on the immunogenicity of the vaccine preparation.
Conveniently, the vaccine is formulated such that relatively small dosages of vaccine (1 to 5ml) are sufficient to protect the ruminant.
The immunogenicity of the antigenic peptide of the invention and its effectiveness as the active agent of a ruminant vaccine will be appreciated from the immunogenicity trials detailed in the Examples 4 and 5.
Specific- non-limiting examples of the invention will now be described.
Example 1: Identification and initial cloning of the stable antigen of the invention
cDNA encoding the 45W antigen is contained in E. coli 45W which was deposited at the American Type Culture Collection, Rockville, Md 20852, USA, cn August 27, 1987), under accession number 67507. The details of derivation of E. coli 45W are fully set out in USSN 07/349,723.
The entire 45W complementary cDNA was cloned into the Eco RI site of the vector pUc18 (Yanisch-Perron Gene 33, 103-119 (1985)) prior to cloning into the plasmid pGEX-2T (Smith D.B. and Johnson K.S. Gene 67, 31-40 (1988)). This plasmid pUC18-45W was digested with the restriction endonucleases Bam HI and Xho II. The DNA was electrophoresed on agarose gels and the Bam HIlXho II fragment containing 45W DNA was purified. This fragment was then ligated with pGEX-2T DNA digested with Bam HI and transformed into the E. coli strain JM101 (Yanisch-Perron et al., 1985 supra).
The appropriate clones were selected on the basis of their DNA insert size and molecular weight of their affinity purified GST fusion proteins (Smith and Johnson Gene (1988) supra). The approximate molecular weight of the T.ovis portion of the pGEX-2T-45W (Bam Hl/Xho II) fusion protein is 23841, calculated from the DNA sequence of the molecular. This fusion protein when electrophoresed on a 13Z SDS gel and stained with Coomassie blue was found to be stable.
The T.ovis portion of the 45W DNA sequence contained in the clone pGEX-2T-45W (Bam HI/Xho II) is set out in Figure 1. The predicted amino acid sequence for this T.ovis antigen fragment is also set out in this Figure.
It will however be appreciated that the non-GST portion of cloned fusion protein GST 45W(BlX) includes a small number of additional amino acids in addition to those encoding the fragment of the T.ovis polypeptide. These additional amino acids are included as a consequence of the cloning and expression of the T.ovis fragment as described above.
The non-GST portion of the fusion protein is hereinafter called "45W(BIX)".
Example 2 : Demonstration of antigenicity of the GST-45W(B/X) fusion protein
Western blot analysis was performed with the fusion proteins prepared as described in USSN 071349,723 from pGEX-2T-45W (Bam HIlXho II), pSjlOtBam7Stop7-45S and pGEX-2T-45W. These fusion proteins were electrophoresed on SDS-polyacrylamide gels, transferred to nitrocellulose and probed with the 45W specific monoclonal antibody 3F10F6 prior to incubation with 125I-labeled protein A.
Antibody 3F10F6 was prepared as follows; BALTIC mice were immunised with GST 45W fusion protein and hybridomas were prepared according to the method of Fazekas de St.
Groth and Scheidegger (J. Immunological Methods 35 1-21, Production of
Monoclonal Antibodies; Strategy and tactics (1980)). Monoclonal antibody 3F10F6 was selected by ELISA analysis of hybridoma supernatants against GST 45W, and was purified by the method of Reik et al (1987, J. Immunological Methods 100, 123-130).
The results are shown in Figure 2. As can be seen by reference to this Figure, a positive reaction was obtained with antibody 3F10F6 in relation to the antigen expressed by host cells transformed with pGEX-2T-45W (Bam HIiXho II). This indicated that the stable peptide was likely to be immunogenic.
Example 3 : Preparation of fusion protein for immunogenicity trials
An equivalent procedure to that set out in Example 1 was adopted to prepare fusion protein for the immunogenicity trials. The host cell chosen to express the fusion protein was in this case E.coli DH5 transformed as follows.
The entire 45W cDNA was cloned into the Eco RI site of the vector pUC 18 (Yanisch-Perron, Gene 33, 103-119, 1985) prior to cloning into the plasmid pGEX-2T (Smith and Johnson, Gene (1988) supra). This plasmid pUC 18 was digested with the restriction endonucleases Bam HI and Xho II. The DNA was electrophoresed on agarose gels and the Bam
HIIXho II (658 bp) fragments containining 45W DNA were purified. These fragments were then ligated with pGEX-2T DNA digested with Bam HI and transformed into the E. coli strain DH5.
An overnight 100 ml culture of transformed E. coli DH5 was added to a shaker flask containing 800 ml SOB medium containing 0.1 mglml ampicillin. After 30 minutes, fusion protein expression was induced by adding IPTG to 0.021 mglml and culturing continued for a further 6 hours. The bacteria were pelleted by centrifugation at 5000 x g for 30 minutes and the cell pellet (net weight " 4.7 g) was resuspended in 22 ml PBS pH7.4. Lysozyme and triton-X-100 were added to 0.25 mglml and 0.5Z final concentration respectively and the suspension was mixed for 10 minutes at ambient temperature. The bacteria were disrupted by sonication and the lysate was centrifuged at 4500 x 6 for 40 minutes.
Soluble fusion protein GST-45W(BlX) was purified from the supernatant by affinity chromatography as follows.
The supernatant was mixed at room temperature with 50 ml washed glutathione-agarose beads (sulphur linkage, Sigma) for 30 minutes.
After absorption, the unbound supernatant was eluted and the beads washed on a sintered glass filter with 5 x 200 ml volumes of PBS.
Fusion protein was eluted in 2 x 25 ml volumes (5 minutes each volume) of 50 mM Tris-HCl pH 8.0 containing 5 mM reduced glutathione (Sigma) freshly prepared.
To solubilise the non-soluble fusion protein, the pellet was resuspended in 15 ml of 50 mM borate buffer pH 9.0 containing 2 mM EDTA and 1 tnM DTT. A 5 ml aliquot was removed and stored frozen at -180C.
The remaining 10 ml suspension was made up to 25 ml by addition of borate buffer and dry urea to a final concentration of 7.0 M and was mixed for 2 days at ambient temperature. Triton X-100 was added to 0.4Z final concentration and the solution was dialysed initially against borate buffer as above followed by PBS pH7.2 over 3 days. The dialysed protein solution was centrifuged at 35000 x g for 30 minutes at 40C and the clear supernatant retained.
Part of the urea solubilised pellet was kept for vaccination trials and part was further processed by affinity chromatography on glutathione-agarose as described previously. Amount of fusion protein recovered at each step is shown in Table 1. The proportion of fusion protein GST 45W(BIX) relative to E.coli protein was calculated by densitometry of Coomassie Blue stained SDS-PAGE gels (Andrews A.T., 1986, Electrophoresis, Oxford University Press).
TABLE 1
Amount of GST 45W(BlX) recovered during processing
Total X Fusion Fusion
Protein Protein Protein soluble affinity purified 8.5 mg 80 6.8 mg urea solubilised pellet 42.5 mg 37 15.7 mg urea solubilised, affinity purified 3.0 mg 71 2.1 mg
Example 4 : Vaccination trials with fusion protein extracts
In an initial experiment 5 Romney sheep aged 10 months were immunised with 2 subcutaneous injections of 5 ug of GST 45W(B(X) plus 1 mg saponin in 1 ml saline, four weeks apart. A booster inoculation of 10 ug fusion protein plus saponin was given three weeks later, and the sheep were infected 4 weeks later with 1200 T.ovis eggs. Three control sheep were given injections of GST carrier protein in the same manner.
In a second experiment groups of sheep were immunised as follows:
Group Antigen Total dose of fusion protein* Adjuvant 1 Saline control - saponin, 1 mg 2 Affinity purified 58 ug 3 Urea extract 132 ug 4 Urea extract + affinity
purified 90 ug
* given in three injections
Four weeks after the final immunisation all sheep were infected with 2400 T.ovis eggs.
Six weeks after infection sheep were humanely slaughtered and carcassexamined for cysts.
Results are shown in Table 2.
TABLE 2 : Protection data from vaccination trials
Group Antigen Number of cysts* Mean Z Reduction
Trial A 1 GST 15 28 63 35.3 2 GST 45W(B/X) 0 0 0 0 20 4.0 89
affinity purified
Trial B 1 Saline 3 12 14 15 16 17 40 16.7 2 GST 45W(BIX) 0 0 0 0 1 0.2 99
affinity purified 3 Urea extract 0 0 0 1 0.25 9n 4 Urea extract + 0 0 0 2 0.5 97
affinity purified *Trial A: numbers of cysts in whole carcass
Trial B: numbers of cysts in masseters, heart, diaphragm and external
carcass
It can therefore be seen that immunisation with the fusion protein GST-45W(BIX) resulted in a highly significant reduction in cyst numbers compared with the controls used.
Example 5 : Antibody response of sheep immunised with GST 45W(BlX) Sera from sheep immunised with the extracts described above were analysed by immunoblotting and ELISA.
Immunoblotting was performed as described (Johnson et al Nature (1989) supra and showed that sheep immunised with the fusion protein GST 45W(B/X) made antibodies which recognised oncosphere antigens with relative mobilities of 47-52000 as described for antibody responses to the parent antigen GST 45W (Johnson Set al, Nature 1989, supra). These results are shown in Figure 3.
The GST portion of GST 45W(B/X) was removed by thrombin cleavage and the 45W(B/X) protein obtained free from GST by depletion on glutathione-agarose as described (Smith and Johnson Gene 1988, supra).
The 45WtB/X) protein was used as antigen to coat microtitre plates for a standard ELISA as described in "Practice and Theory of Enzyme Immunoassays", P. Tijssen, Laboratory Techniques in Biochemistry and
Molecular Biology; Volume 15, Elsevier Science Publishers (1985).
Sheep antibody was detected with peroxidase-conjugated rabbit anti-sheep lgG (Cappel, Cooper Biomedical Inc.).
The results are shown in Figure 4. As can be seen from this
Figure, immunisation of sheep with affinity purified fusion protein or urea extracted fusion protein stimulated strong antibody responses in all groups of sheep.
In accordance with the present invention there is provided a stable peptide comprising a fragment of a T.ovis polypeptide which is effective in generating a protective immunological response against T.ovis infection in ruminants. It has been established that vaccination with this peptide stimulates almost complete immunity against challenge infection with T.ovis eggs. Insofar as the applicants are aware, it is the highest level of protection thus far achieved in a natural host-parasite system by injection of a single antigen. The invention also provides a recombinant method for expression of the antigen by which commercial quantities can be obtained.
It will be appreciated that the above description is provided by way of example only and that variations in both the materials and the techniques used which are known to those persons skilled in the art are contemplated.
Claims (27)
1. A purified stable antigenic peptide comprising a fragment of a
T.ovis polypeptide, which fragment
(a) has a molecular weight of from about 23 kD to about 24 kD; and
(b) is capable of generating a protective immunological response to
T.ovis infection in a ruminant;
or a subfragment or variant thereof having substantially equivalent stability and immunological activity thereto.
2. A peptide as claimed in claim 1 comprising the amino acid sequence:
1 CG GAC TAC GAA CAA CCC ATC GAC AGA ACA GTG GTA GAA
Asp Tyr Glu Gln Pro Ile Glu Arg Thr Val Val Glu 12
39 TAT CCA TCA CTA CGT GAC ATC TTT GCT TGG GAA CCT CCG ACT TCT 27
Tyr Pro Ser Leu Arg Asp Ile Phe Ala Trp Glu Pro Pro Thr Ser
84 AAC TCC ATT GGC CTA ACT TGG CAA AGG CAC GCA TTT CCT GGT GTG
Agn Ser Ile Gly Leu Thr Trp Gln Arg His Ala Phe Pro Gly Val 42
129 GAA CGT GAA GTG CTC ACA TTG AAG GCA GTG CCG ACT TCT GAA CCC
Glu Arg Glu Val Leu Thr Leu Lys Alz Val Pro Thr Ser Glu Pro 57
174 AAT AAC ACC AAG ACA GCA TAT GCA AAG CTC GGC AGC GGA AAA GTC
Asn Asn Thr Lys Thr Ala Tyr Ala Lys Leu Gly Ser Gly Lys Val 72
219 ACT CTT GAT GGA CTG AAG CCC AAT GCC ACA TAT CTT GTG ACT GCG
Thr Leu Asp Gly Leu Lys Pro Asn Ala Thr Tyr Leu Val Thr Alz 87
264 ACG GCA AAT ATA AGT GGA GAC ACA ATT CTG GTA TTG AGC AAT ACT
TO Ala Asn Ile Ser Gly Asp Thr Ile Leu Val Leu Ser Asn Thr 102
309 TTT CAT ACA CTG GCC AAT GGC ACA AAT ATT ATA AAT AAC ATC TTC
Phe His Thr Leu Ala Asn Gly Thr Asn Ile Ile Asn Asn Ile Phe 117
354 CAT TGG GGT CCT GTG ACT AAT CAA TCA ATT CAA GTA AGA TGG GAT
His Trp Gly Pro Val Thr Asn Gln Ser Ile Gln Val Arg Trp Asp 132
399 CAG ATA AAA CCG GAG CAA ACA AGC GCT CTG ATA GTC ACA CTG ACG
Gln Ile Lys Pro Glu Glu Thr Ser Ala Leu Ile Val Thr Leu Thr 147
444 GCA GAG ATG GCT TCT GAC CCC GGA GTG GAA AGA TCG GAG TCT GCA
Ala Glu Met Ala Ser Asp Pro Gly Val Glu Arg Ser Glu Ser Ala 162
489 CTC TTC GGT AAA GGA AAG GTC ACT GTT GAC GGA CTG GAG TCC GAC
Leu Phe Gly Lys Gly Lys Val Thr Val Asp Gly Leu Glu Ser Asp 177
534 ACA CTA TAT ATT GCG ACT GTG ATG GTA TTT AGA AAT GGA AGG CAA
Thr Leu Try Ile Ala Thr Val Met Val Phe Arg Asn Gly Arg Gln 192
579 TAC TTC AAT TCC ACC AGA GAT ATT CGA ACA CTC AAA TCT GGC CAT
Tyr Phe Asn Ser Thr Arg Asp Ile Arg Thr Leu Lys Ser Gly His 207
624 AAC GAG GTA ACA CTC GTA ACA ACT ACT GGA TC
Lys Glu Val Thr Val Val Thr Thr Ser Gly
or a subfragment or variant thereof having substantially equivalent stability and immunological activity thereto.
3. A peptide as claimed in claim 1 or claim 2 which is the product of expression of a nucleotide sequence coding therefor in a host cell.
4. A peptide as claimed in claim 3 which is expressed in the host cell as a fusion protein.
5. A peptide as claimed in claim 4 which is expressed as a fusion protein with the enzyme glutathione s-transferase (E.C 2.5.18).
6. A composition of matter capable of generating a protective immunological response to -T.ovis infection in a ruminant which essentially consists of a component selected from the group comprising;
(a) the peptide of claim 2;
(b) a stable immunologically active subfragment of (a); and
(c) a variant of (a) or (b) which has been modified by the insertion, substitution or deletion of one or more amino acids and which has equivalent stability and immunological activity thereto.
7. A DNA molecule comprising a nucleotide sequence which encodes a purified stable antigenic peptide comprising a fragment of a T.ovis polypeptide, which fragment:
(a) has a molecular weight of from about 23 kD to about 24 kD; and
(b) is capable of generating a protective immunological response to
T.ovis infection in a ruminant;
or which encodes a sub fragment or variant of said fragment having substantially equivalent stability and immunological activity thereto.
8. A DNA molecule which encodes an immunologically active peptide comprising a fragment of a T.ovis polypeptide, which fragment is coded for by the nucleotide sequence
1 CGG ACTACGAACA ACCCATCGAG AGAACAGTGG TAGAATATCC ATCACTACGT
54 GACATCTTTG CTTGGGAACC TCCGACTTCT AACTCCATTG GCCTAACTTG GCAAAGGCAT 114 GCATTTCCTG GTGTGGAACG TGAAGTGCTC ACATTGAAGG CAGTGCCSAC TTCTGAACCC 174 AATAACACCA AGACAGCATA TGCAAAGCTC GGCAGCGGAA AAGTCACTCT TGATGGACTG 234 AAGCCCAATG CCACATATCT TGTGACTGCG ACGGCAAATA TAAGTGGAGA CACAATTCTG 294 GTATTGAGCA ATACTTTTCA TACACTGGCC AATGGtACM ATATTATAAA TAACATCTTC 354 CATTGGGGTC CTGTGACTAA TCAATCAATT CAAGTAAGAT GGGATCAGAT AAAACCGGAG 414 GAAACAAGCG CTCTGATAGT CACACTGACG GCAGAGATGG CTTCTGACCC CGGAGTGGAA 474 AGATCGGAGT CTGCACTCTT CGGTAAAGGA AAGGTCACTG TTGACGGACT GGAGTCCGAC 534 ACACTATATA TTGCGACTGT GATGGTATTT AGAAATGGAA GGCAATACTT CAATTCCACC 594 AGAGATATTC GAACACTCAA ATCTGGCCAT AAGGAGGTAA CAGTCGTAAC AACTAGTGGA 654 TC
9. A DNA molecule which is selected from the group comprising:
(a) a nucleotide sequence encoding the immunogenic peptide of claim 2;
(b) a nucleotide sequence encoding a stable immunologically active subfragment of (a); and
(c) a nucleotide sequence encoding a stable immunologically active variant of peptide (a) or subfragment (b) in which the amino acid sequence has been modified by the insertion, substitution or deletion of one or more amino acids.
10. A DNA molecule comprising a 655 bp fragment of DNA contained within
E.coli 45W (ATCC 67507) encoding a stable immunogenic peptide comprising a fragment of a polypeptide of T.ovis.
11. A DNA molecule as claimed in claim 7 which has-been isolated from a natural source.
12. A DNA molecule as claimed in claim 7 which is intron-free.
13. A recombinant expression vector which contains a DNA molecule as claimed in any one of claims 7 to 12.
14. A vector as claimed in claim 13 which is pGEX-2T-45W (Bam HI-Xho
II).
15. A host cell transformed with a vector as claimed in claim 13 or claim 14 and capable of expressing the T.ovis peptide or fragment or variant thereof which is encoded.
16. A host cell as claimed in claim 15 which is a prokaryote.
17. A host cell as claimed in claim 16 wherein the prokaryote host is
E.coli.
18. A host cell as claimed in claim 17 wherein the prokaryote host is
E.coli DH5
19. A host cell as claimed in claim 15 which is a eukaryote.
20. A method of producing a stable antigenic peptide comprising a fragment of T.ovis polypeptide according to claim 1 which comprises culturing a cell as claimed in any one of claims 15 to 19 and recovering the expressed peptide.
21. A method as claimed in claim 20 wherein the antigenic peptide is recovered in a soluble form.
22. A method as claimed in claim 20 wherein the antigenic peptide is recovered in an insoluble form and is subsequently solubilised.
23. A stable antigenic peptide of comprising a fragment of a T.ovis polypeptide produced by the method of claim 20.
24. A vaccine comprising an immunologically-effective amount of a stable peptide as claimed in any one of claims 1 to 5 and 23 in combination with a pharmaceutically acceptable adjuvant, carrier or diluent therefor.
25. A method of protecting a ruminant against T.ovis infection comprising administering to a said ruminant an immunologically effective amount of a stable peptide as claimed in any one of claims 1 to 5 and 23.
26. A method of protecting a ruminant against T.ovis infection comprising administering to a said ruminant an immunologically effective amount of a composition as claimed in claim 6.
27. A method of protecting a ruminant against T.ovis infection comprising administering to a said ruminant an immunologically effective amount of a vaccine as claimed in claim 24.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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US34972389A | 1989-05-09 | 1989-05-09 | |
US47069190A | 1990-01-26 | 1990-01-26 |
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GB9010357D0 GB9010357D0 (en) | 1990-06-27 |
GB2232675A true GB2232675A (en) | 1990-12-19 |
GB2232675B GB2232675B (en) | 1993-05-12 |
Family
ID=26996317
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GB9010357A Expired - Fee Related GB2232675B (en) | 1989-05-09 | 1990-05-09 | Stable forms of antigenic taenia ovis polypeptides |
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---|---|
AU (1) | AU642874B2 (en) |
GB (1) | GB2232675B (en) |
NZ (1) | NZ233621A (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5366728A (en) * | 1988-01-06 | 1994-11-22 | Pitman-Moore New Zealand Limited | Stable forms of antigenic Taenia ovis polypeptides |
EP0629131A1 (en) * | 1992-02-21 | 1994-12-21 | New Zealand Pastoral Agriculture Research Institute Limited | ANTIGENS PROTECTIVE AGAINST $i(ECHINOCOCCUS GRANULOSUS) INFECTION AND VACCINES CONTAINING SUCH ANTIGENS |
US5980911A (en) * | 1994-05-04 | 1999-11-09 | Commonwealth Scientific And Industrial Research Organisation | Adjuvant |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1994022912A1 (en) * | 1993-04-07 | 1994-10-13 | Mallinckrodt Veterinary Limited | Antigenic polypeptides of t. ovis and vaccines containing such polypeptides |
EP0707597A4 (en) * | 1993-04-07 | 1998-08-26 | Mallinckrodt Veterinary Ltd | ANTIGENIC POLYPEPTIDES OF -i(T. OVIS) AND VACCINES CONTAINING SUCH POLYPEPTIDES |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1988001277A1 (en) * | 1986-08-18 | 1988-02-25 | The Australian National University | Helminth parasite vaccine |
GB2219590A (en) * | 1988-05-12 | 1989-12-13 | Nz Ministry Agriculture & Fisheries | Antigenic polypeptides of taena ovis |
-
1990
- 1990-05-08 AU AU54848/90A patent/AU642874B2/en not_active Ceased
- 1990-05-09 NZ NZ233621A patent/NZ233621A/en unknown
- 1990-05-09 GB GB9010357A patent/GB2232675B/en not_active Expired - Fee Related
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1988001277A1 (en) * | 1986-08-18 | 1988-02-25 | The Australian National University | Helminth parasite vaccine |
GB2219590A (en) * | 1988-05-12 | 1989-12-13 | Nz Ministry Agriculture & Fisheries | Antigenic polypeptides of taena ovis |
Non-Patent Citations (3)
Title |
---|
Int. J. Parasitol. 1989, * |
Mol. Biochem. Parasitol. * |
Nature 1989, 338(6216), * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5366728A (en) * | 1988-01-06 | 1994-11-22 | Pitman-Moore New Zealand Limited | Stable forms of antigenic Taenia ovis polypeptides |
EP0629131A1 (en) * | 1992-02-21 | 1994-12-21 | New Zealand Pastoral Agriculture Research Institute Limited | ANTIGENS PROTECTIVE AGAINST $i(ECHINOCOCCUS GRANULOSUS) INFECTION AND VACCINES CONTAINING SUCH ANTIGENS |
EP0629131A4 (en) * | 1992-02-21 | 1995-11-29 | Pastoral Agric Res Inst Nz Ltd | ANTIGENS PROTECTIVE AGAINST -i(ECHINOCOCCUS GRANULOSUS) INFECTION AND VACCINES CONTAINING SUCH ANTIGENS. |
US5980911A (en) * | 1994-05-04 | 1999-11-09 | Commonwealth Scientific And Industrial Research Organisation | Adjuvant |
Also Published As
Publication number | Publication date |
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AU5484890A (en) | 1990-11-15 |
GB9010357D0 (en) | 1990-06-27 |
AU642874B2 (en) | 1993-11-04 |
NZ233621A (en) | 1993-04-28 |
GB2232675B (en) | 1993-05-12 |
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