Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Parasitic organism or component thereof or substance...
Reexamination Certificate
1999-10-04
2001-03-20
Minnifield, Nita (Department: 1645)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Parasitic organism or component thereof or substance...
C424S265100, C424S266100, C424S271100, C424S093100, C424S093200, C424S184100, C424S191100, C424S185100, C424S269100, C514S04400A, C536S023500, C536S023700, C435S069100, C435S069300, C435S252300, C435S320100, C435S328000, C435S173300
Reexamination Certificate
active
06203801
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to Eimeria polypeptides, DNA-fragments encoding those peptides, recombinant DNA molecules comprising such fragments, live recombinant carriers comprising such fragments or molecules, host cells comprising such fragments, molecules or carriers, antibodies against the polypeptide and coccidiosis vaccines. The invention also relates to methods for the preparation of such antibodies and vaccines, and to methods for the detection of Eimeria parasites and antibodies against Eimeria parasites.
BACKGROUND OF THE INVENTION
Parasitic protozoa belonging to the genus Eimeria are the causative agents of intestinal coccidiosis, an enteritis which affects birds. This causes significant economic loss, especially to the poultry industry. (For the purposes of the present application, the term “poultry” is taken to mean birds that serve as sources of eggs or meat. It includes, inter alia, chickens, turkeys, ducks, geese, guinea fowl, pheasants, pigeons and pea fowl). Nowadays, coccidiosis is mainly controlled by the use of antibiotic drugs in the feed. The rapid emergence of drug resistant strains (Chapman H. D. Parasitology Today 9, 159-162 (1993)) and the prohibitive costs of development and registration of a novel drug have led to increased interest in the development of an alternative method of control. The development of effective vaccines has therefore been desirable for many years. However only partial success has been obtained.
Currently available vaccination strategies consist of controlled infections with either virulent or live attenuated parasites (Shirley M. W. In: Proceedings of the Vlth. International Coccidiosis Conference (Eds.: J. R. Barta and M. A. Fernando) Moffitt Print Craft Ltd., Guelph. pp. 61-72 (1993)). For reasons of safety and cost, the most desirable method of immunoprophylaxis against coccidiosis appears to be the use of a subunit vaccine. Although many attempts have been made to immunise chickens against coccidiosis with fractions of parasite material (Murray P. K., Bhogal B. S., Crane M. S. J. & MacDonald T. T. In: Research in Avian Coccidiosis. Proceedings of the Georgia Coccidiosis Conference (Eds.: L. R. McDougald, Joyner L. P. and P. L. Long) Athens, University of Georgia. pp. 564-573 (1986), McKenzie M. E. & Long P. L. Poultry Science 65, 892-897 (1986)) or recombinant Eimeria polypeptides (Danforth H. D., Augustine P. C., Ruff M. D., McCandliss R., Strausberg R. L. & Likel M. Poultry Science 68, 1643-1652 (1989), Jenkins M. C., Augustine P. C., Danforth H. D. & Barta J. R. Infection and Immunity 59, 4042-4048 (1991)) only limited protection against challenge infection could be achieved. The parasite stages responsible for the induction of protective immunity are generally thought to be early asexual developmental stages (Jenkins et al. 1991). Initially, selection of candidate antigens was performed using antibodies from immune chickens but, in view of the fundamental role of cell mediated responses in protective immunity (reviewed in Lillehoj H. S. & Trout J. M. Avian Pathology 22, 3-31 (1993), Rose M. E. In: Poultry Immunology (Ed.: T. F. Davison, T. R. Morris and L. N. Payne), Carfax Publishing Company, Oxfordshire, U. K. pp. 265-299 (1996), attention has now focused, next to B-cell inducing antigens, on screening antigens for their ability to stimulate specific T-cell responses (Dunn P. P. J., Billington K., Bumstead J. M. & Tomley F. M. Molecular and Biochemical Parasitology 70, 211-215 (1995)).
It is an objective of the present invention to provide polypeptides that are capable of inducing protection against the pathogenic effects of Eimeria infection in poultry.
SUMMARY OF THE INVENTION
It was now surprisingly found that 6 different polypeptides could be specifically identified and isolated, essentially free from other Eimeria polypeptides, from a hydrophilic fraction of Eimeria polypeptides, each of these different polypeptides being capable of inducing an immune response against Eimeria parasites. The inventors have found that these polypeptides can be used, either alone or in combination with each other, to provide a vaccine which gives a significant degree of protection to birds (preferably poultry). For example, protection against the formation of cecal lesions can be achieved in birds immunised with such a vaccine, when subjected to subsequent challenge with Eimeria parasites.
DETAILED DESCRIPTION OF THE INVENTION
A first embodiment of the invention relates to a hydrophilic polypeptide of Eimeria that in its full-length form has a molecular weight of 25 kD and comprises an amino acid sequence that shares at least 70% homology with the amino acid sequence MPFELPPLPYPMDALEPYISKETLEYHYGKHHAAYVNNLNRLVEGKPEASKSLEEIIKTSSGSVLNNAGQAWNHTFYWKSMRPASAGGPPGAPGGGPPGAPGAPLREELESAFGGVEKFREAFAAAAAAHFGSGWAWLCFCKKSRSLFLLQTHDGATPFRDNPNCAPLLTCDLWEHAYYIDRRNDRKSYLDAWWSVVNWDFANENLKKAMQGSD (further referred to as SEQ ID NO: 1:) and immunogenic fragments of that polypeptide capable of inducing an immune response against said polypeptide. The polypeptide is functionally related to a Superoxide Dismutase (SOD) found in non-Eimeria parasites and is therefore characterised as SOD-like.
Also, this embodiment relates to a hydrophilic polypeptide of Eimeria that is a peroxidoxin-like polypeptide, in its full-length form has a molecular weight of 22 kD and comprises an amino acid sequence that shares at least 70% homology with the amino acid sequence LGPLALPLLADVR (further referred to as SEQ ID NO: 2:), and immunogenic fragments of the polypeptide capable of inducing an immune response against that polypeptide.
A hydrophilic polypeptide of Eimeria that is a peroxidoxin-like polypeptide, in its full-length form has a molecular weight of 25 kD and comprises an amino acid sequence that shares at least 70% homology with the amino acid sequence MPLNLGDSFPDFQAEALGAEHFRLHEYLGDSWGVMFSHPNDFTPVCTTELAEAVKLQDSFTKKNCKLVGFSCNDLQSHREWAKDIMAYAGRSGNLPFPLVCDPNRELAASLGIMDPAEKDKKGLPLTCRCVFFISPEKKLAASILYPATTGRNFAEILRVLDSLQLTAKFPVATPVDWTAGAKCCVVPNLAAEEAQRLLPKGHEALQLPSGKPYLRLTPDPRG (further referred to as SEQ ID NO: 3:), as well as immunogenic fragments of the polypeptide capable of inducing an immune response against that polypeptide are also part of this embodiment.
Also part of this embodiment is a hydrophilic polypeptide of Eimeria that in its full-length form has a molecular weight of 22 kD and comprises an amino acid sequence that shares at least 70% homology with the amino acid sequence MSPSPAGVAEYLASL (further referred to as SEQ ID NO: 4:), or an immunogenic fragment of that polypeptide capable of inducing an immune response against said polypeptide.
This embodiment also includes a triosephosphate isomerase-like hydrophilic polypeptide of Eimeria that in its full-length form has a molecular weight of 28 kD and comprises an amino acid sequence that shares at least 70% homology with the amino acid sequence NHAEFDPSQTEVVVFP (further referred to as SEQ ID NO: 5:), or an immunogenic fragment of that polypeptide capable of inducing an immune response against said polypeptide.
Finally, this embodiment relates to a hydrophilic polypeptide of Eimeria that in its full-length form has a molecular weight of 28 kD and comprises an amino acid sequence that shares at least 70% homology with the amino acid sequence VDSFTPSVGCVFAGMPADFR (further referred to as SEQ ID NO: 6:), or an immunogenic fragment of that polypeptide capable of inducing an immune response against said polypeptide.
Although various groups have disclosed Eimeria derived proteins which might, by chance, have molecular masses within the 26-30 kDa±5 kDa range disclosed above, these proteins are quite different from the polypeptides of the present invention.
For example, in EP-A-0231537 (Newman et al) a 25 kDa surface protein is disclosed. However under reducing conditions this splits to form two bands on SDS-PAGE of about 17 and about 8 kDa, whereas the polypeptides of the present invention had relative molecular masses of at least 21 kDa when separated under reducing conditions.
Bouv
Kuiper Catharina Maria
Schaap Theodorus Cornelis
Vermeulen Arnoldus Nicolaas
Akzo Nobel N.V.
Baskar Padma
Blackstone William M.
Minnifield Nita
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