Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1998-08-05
2003-03-18
Minnifield, Nita (Department: 1645)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023200, C536S023700, C536S023400, C435S069100, C435S069300, C435S069700, C435S071200, C435S071100, C435S320100, C435S243000, C435S252300
Reexamination Certificate
active
06534638
ABSTRACT:
The present invention relates to the preparation of protective antigens by recombinant DNA technology for use as anthelmintic agents and as protective immunogens in the control of diseases caused by helminth parasites.
Helminth parasites are responsible for a wide range of diseases and infestations of domestic animals which, leading as they do to loss of production and even animal mortality, are of considerable economic importance. Thus for example, the blood feeding nematode Haemonchus infects the lining of the gastrointestinal tract of ruminants, causing anaemia and weight loss and if untreated frequently leads to death. Animals infected with the related non-blood feeding nematode Ostertagia similarly fail to thrive and may die if untreated. Other genera of helminths of economic importance include Trichostrongylus and Nematodirus which cause enteritis in various animals, and trematodes.
Problems are also caused by nematodes such as hookworms (eg. Necator, Ancylostoma, Uncinaria and Bunostomum spp) and flukes (eg. Fasciola, Paramphistomum and Dicrocoelium) and their relatives which in addition to ruminants and domestic pets, also infect humans, frequently with fatal results.
Control of nelminth parasites presently relies primarily on the use of anthelmintic drugs combined with pasture management. Such techniques have a number of drawbacks however—frequent administration of drugs and pasture management are often not practical, and drug-resistant helminth strains are becoming increasingly widespread.
There is therefore a need in this field for an effective anti-helminth vaccine and many efforts have been concentrated in this area in recent years. However, as yet there are no commercially available molecular or sub-unit vaccines for the major helminth species, particularly for the gastrointestinal nematodes of ruminants, such as Haemonchus and Ostertagia.
Most promising results to data have been obtained with novel proteins isolated from Haemonchus, which have potential as protective antigens not only against Haemonchus but also against a range of other helminths. In particular the protein doublet H11OD, found at the luminal surface of the intestine of
H.contortus
has been shown to confer protective immunity against haemonchosis in sheep.
H11OD from
H.contortus
has an approximate molecular weight of 110 kilodaltons (kd) under reducing and non-reducing conditions, as defined by SDS-PAGE, and is described in WO88/00835 and WO90/11086. The term “H11OD” as used herein refers to the protein doublet H11OD as defined in WO88/00835 and WO90/11086. Corresponding proteins have also recently been shown in other helminth species, eg.
Necator americanus.
A number of methods for the purification of H11OD have been described in WO88/00835 which suffice for the characterisation of the protein, and may be scaled up to permit production of the protein in experimentally and commercially useful quantities. There is however a need for an improved and convenient source from which to prepare not only H11OD but also related antigenic proteins, especially for a process based on recombinant DNA technology and expression of the proteins in suitably transformed prokaryotic or eukaryotic organisms.
The present invention seeks to provide such an improved procedure. Sequence determination of cDNAs for H11OD from
Haemonchus contortus
has been performed and the predicted amino acid sequences have been found to display homology with a family of integral membrane aminopeptidases (systematic name: &agr;-amino acyl peptide hydrolase (microsomal)).
The mammalian integral membrane aminopeptidases are located in several tissues, eg. on the microvillar brush border of intestines, and kidney. Their role in the kidney is unclear, but in the intestine their functional is to cleave the small peptides which are the final products of digestion (for reviews, see Kenny & Maroux, 1982; Kenny & Turner, 1987; Noren et al, 1986; Semenza, 1986).
In one aspect the present invention thus provides nucleic acid molecules comprising one or more nucleotide sequences which encode helminth aminopeptidase enzymes or antigenic portions thereof substantially corresponding to all or a portion of the nucleotide sequences as shown in
FIGS. 2
,
3
,
4
or
5
(SEQ ID NOS: 1 to 15) or sequences coding for helminth aminopeptidase enzymes which are substantially homologous with or which hybridise with any of said sequences.
A nucleic acid according to the invention may thus be a single or double stranded DNA, cDNA and RNA.
Variations in the aminopeptidase-encoding nucleotide sequences may occur between different strains of helminth within a species, between different stages of a helminth life cycle (e.g. between larval and adult stages), between similar strains of different geographical origin, and also within the same helminth. Such variations are included within the scope of this invention.
“Substantially homologous” as used herein includes those sequences having a sequence identity of approximately 50% or more, eg. 60% or more, and also functionally-equivalent allelic variants and related sequences modified by single or multiple base substitution, addition and/or deletion. By “functionally equivalent” is meant nucleic acid sequences which encode polypeptides having aminopeptidase activities which are similarly immunoreactive ie. which raise host protective antibodies against helminths.
Nucleic acid molecules which hybridise with the sequences shown in
FIGS. 2
,
3
,
4
or
5
(composed of SEQ ID NOS: 1 to 15) or any substantially homologous or functionally equivalent sequences as defined above are also included within the scope of the invention. “Hybridisation” as used herein defines those sequences binding under non-stringent conditions (6×SSC/50% formamide at room temperature) and washed under conditions of low stringency (2×SSC, room temperature, more preferably 2×SCC, 42° C.) or conditions of higher stringency eg. 2×SSC, 65° C. (where SSC=0.15M NaCl, 0.015M sodium citrate, pH 7.2).
Methods for producing such derivative related sequences, for example by site-directed mutagenesis, random mutagenesis, or enzymatic cleavage and/or ligation of nucleic acids are well known in the art, as are methods for determining whether the thus-modified nucleic acid has significant homology to the subject sequence, for example by hybridisation.
Provision of a nucleic acid molecule according to the invention thus enables recombinant aminopeptidase enzymes, or immunogenic fragments thereof, to be obtained in quantities heretofore unavailable, thereby permitting the development of anti-helminth vaccines.
In another aspect the present invention thus provides nucleic acid molecules comprising one or more nucleotide sequences encoding one or more polypeptides capable of raising protective antibodies against helminth parasites, which sequences incorporate one or more antigenic determinant-encoding regions from the aminopeptidase-encoding sequences as shown in
FIGS. 2
,
3
,
4
or
5
(composed from SEQ ID NOS: 1 to 15).
The present invention also extends to synthetic polypeptides comprising one or more amino acid sequences constituting an aminopeptidase enzyme or antigenic portions thereof, substantially corresponding to all or a portion of the nucleotide sequences as shown in
FIG. 2
,
3
,
4
or
5
(SEQ ID NOS: 1 to 15), or a functionally-equivalent variant thereof other than a synthetic polypeptide corresponding to the protein doublet H11OD, or a synthetic polypeptide corresponding to any of the individual polypeptide sequences disclosed in WO90/11086.
Alternatively viewed, the invention also provides synthetic polypeptides comprising an amino acid sequence constituting an aminopeptidase enzyme or an antigenic portion thereof, substantially corresponding to all or a portion of the nucleotide sequences as shown in
FIG. 2
,
3
,
4
or
5
(SEQ ID NOS: 1 to 15) or a functionally-equivalent variant thereof, substantially free from other
Haemonchus contortus
components.
The invention further extends to vaccine composit
Graham Margaret
Knox David Patrick
Munn Edward Albert
Newton Susan Elizabeth
Oliver Joanna Jane
Barbraham Institute
Minnifield Nita
Rothwell Figg Ernst & Manbeck
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