CA2155120A1 - Expression of protective antigens - Google Patents

Abstract

The invention provides a method for the expression of an enzyme antigen which in nature is parasite gut membrane bound enzyme or a fragment thereof having similar enzyme and/or antigenic activity, wherein mammalian host cells are transfected with a vector adapted to express said enzyme antigen or fragment thereof, characterised in that prior to transformation the host cells are substantially free from endogenous enzymes having (a) the same function and (b) the same cell membrane integration or lack of integration, as said parasite enzyme antigen or fragment thereof. Such a method is particularly useful for the expression of helminthic integral membrane aminopeptidase antigens.

Description

W094/1~20 ~ ~ 5 5 1 2 0 PCT/GB94/00204 . =

Expression of Protective Antiqens This invention relates to the production of certain protective antigens by recombinant DNA technology.
Parasites are responsible for a wide range of diseases of humans and of domestic animals. These have hitherto been treated by chemotherapy but more recently, immunological methods have been used. Although parasites in most stages of their life cycle are relatively large and are not readily dealt with by the cell-mediated immune defence system of an animal host, they may be susceptible to antibodies which act to inactivate essential functions of the parasite. In particular, it has now been found possible to immunise host animals with antigens which are membrane bound on the gut surface of the parasite so that the antibodies generated bind to the antigens on such internal membranes when body fluids containing the antibodies are ingested by the parasite. Such antigens can be termed 'hidden antigensl since they do not give rise to natural immunity against the parasite.
We have found that a particularly effective 'hidden antigenl is the anthelmintic antigen HllOD derived from Haemonchus contortus as described in W088/00835 and 90/11086. In W093/23542 we have described the further finding that HllOD is a membrane-bound aminopeptidase from the gut of the helminth. This enzyme appears to be essential for conversion of protein nutrients into amino acids for uptake from the gut and when inactivated by the anti-HllOD antibody, causes the parasite to die from impaired nutrient uptake. We have further found that a large number of parasites are susceptible to the same Ihidden antigen' strategy in that gut membrane bound enzymes are essential to the processing of protein nutrients. Such enzymes include aspartyl proteases (as described in PCT/GB93 01521) and thiol proteases such as cathepsin. These are W094/18320 , PCT/GB94/00204 ~,~5~2~

present in the gut of a wide range of parasites such as helminths, eg. various species of the families Haemonchus, ostertaqia, Trichostronq~lus, Nematodirus, DictYocaulus, CooPeria, Ascaris, Dirofilaria, Trichuris, Strongylus and Fasciola; and arthropod species especially members of the arachnid and insect classes and in particular ectoparasites such as the blood-feeding insects (eg. members of the insect divisions exopterygota and endopterygota), flies such as blowfly (Lucilia), myiasis flies and suckers, lice, mites, fleas, keds and bugs.
W093/23542 referred to above also describes the production of antigenic fragments of HllOD by recombinant DNA technology. In this work, the HllOD
material was expressed in E.coli using a pGEX vector and on injection into sheep, raised anti-HllOD antibodies.
In further work using baculovirus in insect host cells (Sf9 cells) we have successfully expressed a 3.5 Kb clone of the HllOD gene. The nucleotide sequence of this is shown in Figure 1 (seq. ID No:l). The corresponding translation is shown in Figure 2 (seq. ID
No:2).
However, it is preferable for production of vaccines for use in animals to express an antigen in mammalian host cells. This provides good reproduction of the native form and protective epitopes of the antigen since a eukaryotic expression system will give rise to more similar glycosylation patterns, disulphide bonding and other post-translational modifications than E.coli which produces an insoluble protein requiring refolding and having poor reproduction of the native form.
In addition mammalian glycosylation is unlikely to induce an immune response which distracts from a protective anti-protein response which can occur with insect cell line derived material because of its very different glycosylation pattern. For protection of humans and domestic animals, it is thus preferable to WO94/18320 2 15 5 ~ 2 ~ PCT/GB94/00204 - 3 - . ;~
use human or animal fibroblast or myeloma cell lines such as HeLa - a human cell line; BHK - baby hamster kidney cells; VERO and COS, a monkey kidney cell line;
FR3T3, Fisher rat fibroblasts; NIH3T3, a mouse fibroblast cell line; Cl27I, a mouse mAmm~ry tumour cell line; CV-l, African green monkey kidney fibroblasts;
3T6, mouse embryo fibroblasts; L cells, a mouse cell line; CHO, a Chinese Hamster ovary cell line; NSO NSI, SP2 and other mouse myeloma cell lines and rat myeloma cell lines such as YB2/0 and Y3.
Since the hidden antigens to be produced are those essential to the processing of nutrients by the parasite, enzymes or other functional proteins having the same activity are common in a wide range of available eukaryotic cell lines and not only hinder the selection of clones producing the desired antigen but render difficult the purification of the desired antigen from the cell products. Additionally, the chosen host cell-line will inevitably be genetically close, in terms of protein sequence, to the animal to be protected, so that contamination of the desired foreign hidden antigen with such endogenous antigen is much more likely to give an undesirable auto-immune host reaction. In addition, quality control assay of the expressed hidden antigen will be made more difficult.
The present invention is based on the concept of carrying out recombinant DNA expression of a desired foreign enzymic "hidden antigen" in a transformed m~mm~lian host cell-line which, when both enzymes are associated with the cell membrane or both are cytoplasmic, thus precluding physico-chemical separation, is substantially free from endogenous antigens having the same enzyme function as the foreign "hidden antigen".
According to the present invention therefore we provide a method for the expression of an enzyme antigen which in nature is a parasite gut membrane bound enzyme or a fragment thereof having similar enzyme and/or WO94/1~20 , PCT/GB94/00204 ~5~2~ - 4 -antigenic activity, wherein mammalian host cells are transfected with a vector adapted to express said enzyme antigen or fragment thereof, characterised in that prior to transformation the host cells are substantially free from endogenous enzymes having (a) the same function and (b) the same cell membrane integration or lack of integration, as said parasite enzyme antigen or fragment thereof.
Thus, if the host cell contains an endogenous enzyme such as an aminopeptidase in the cytoplasm, while the foreign enzyme is expressed with a trans-membrane sequence which becomes located in the membrane of the host cell, there is no difficulty in effecting separation of the endogenous and foreign enzymes by processing the cells to separate out membrane fragments, eg. by centrifugation. On the other hand, the foreign antigen may be modified to produce a fragment lacking the membrane binding region and if the host cell membrane carries an endogenous enzyme having the same activity as the foreign enzyme, it will also be possible to effect separation by removal of cell-membrane material; thus the coding sequence for the transmembrane region of the parasite enzyme may be replaced, in the gene to be expressed, by a signal sequence effecting secretion.
Of particular interest are helminthic aminopeptidase antigens such as HllOD referred to above and fragments thereof. These may be expressed in a wide range of host mammalian cells, using appropriate vectors. For expression of the H.contortus antigen HllOD, which shows predominantly aminopeptidase A-like or M-like activity and thus cleaves predominantly methionine and leucine peptide bonds, we have found COS-l cells to be useable in accordance with the invention in that they lack significant A-like and M-like aminopeptidase activity. They appear to possess an aminopeptidase enzyme cleaving alanine peptide bonds, this being weakly associated with the cell membrane, so ~55120 that there is no difficulty in separating the endogenous enzyme from the expressed HllOD which is located in the cell membrane.
Proteolytic enzymes such as trypsin have been shown to cleave the parasite antigen HllOD from the membrane to produce HllOD-soluble (HllS). Such enzymes may thus be used to cleave differentially a foreign hidden antigen away from an endogenous enzyme with similar activity.
Suitable cell lines for use in accordance with the invention may either be selected from existing strains by screening for their profile of appropriate enzyme activity and/or location of any relevant enzyme in relation to the cell membrane or the cytoplasm. In the latter case, association with cell membrane can be established by extracting the lysed cells firstly with a detergent such as Tween which does not extract integral membrane enzymes and then with a detergent such as Triton which can release such enzymes.
It is also possible to create a ~m~lian cell line low in a particular enzyme. One way in which this could be done is as follows: antibodies are raised to the m~mm~l ian enzyme to be deleted. The cell lines are modified by irradiation or chemicals to induce point mutations. The cells are cultured in media suitably fortified to compensate for the lost enzyme activity.
The cells are then exposed to fluorescently labelled antibodies and passed through a Fluorescence Activated Cell Sorter (FACS). The fluorescence negative cells are cloned and re-selected and stability of enzyme loss is monitored.
The cells may also be modified by gene deletion or rational (directed) mutation techniques to remove or mutate the gene for the relevant endogenous enzyme.
Vectors appropriate for different classes of mammalian cell lines are well known in the art. In general, these will comprise a promoter and/or enhancer operably connected to a gene expressing the enzyme 2~5S~ 6 -antigen or fragment thereof. Thus, in particular, the 3.5 kb fragment of the HllO gene may be connected in frame with an appropriate promoter. Suitable promoters include SV40 early or late promoter, eg. PSVL vector, cytomegalovirus (CMV) promoter, mouse metallothionein I
promoter and mouse mammary tumour virus long terminal repeat. The vector preferably includes a suitable marker such as a gene for dihydrofolate reductase or glutamine synthetase. Vectors of those types are described in W086/05807, W087/04462, W089/01036 and W089/10404.
Transfection of the host cells may be effected using standard techniques, for example using calcium phosphate, DEAE dextran, polybrene, protoplast fusion, liposomes, direct microinjection, gene cannon or electroporation. The latter technique is preferred and methods of transfection of mammalian cell lines using electroporation are described by Andreason G.L. and Evans G.A., Introduction and expression of DNA molecules in eukaryotic cells by electroporation, Biotechniques 6, 650, 1980). In general, linear DNA is introduced more readily than circular DNA. The HllOD antigen shows leucine amino peptidase (M-like) and methionine (A-like) aminopeptidase activity and in general, it is preferred that the host cells are free from at least these types of aminopeptidase activity.
The following Examples are given by way of illustration only. In these Examples, the Figures represent:
Fiqure 1 shows the DNA sequence of 3.5 Kb PCR clone 2 (sequence ID No:l); and Fiqure 2 shows the amino acid translation of 3.5 Kb PCR clone 2 (sequence ID No.2).

W094/1~20 PCT/GB94/00204 ~1S~12.~
~ .! . ,: I

Cos-l Cell Enz~me AssaY

Cos-1 cells were received from the University of Surrey in 5mls of DMEM growth medium, the cells were split into two 200ml culture flasks each containing llmls of media where they were grown until confluent. Cos cells are adherent and were removed from the flask surface by aspiration using a glass pasteur pipette into lOmls of PBS buffer. When all of the cells were in suspension they were transferred to a universal tube and frozen down at -20C. The cells were freeze thawed several times in liquid nitrogen to break open the cells, they were then spun to release the PBS supernatant (PLS).
Subsequent extractions used 500~1 of PBS 0.1% Tween and PBS 2% Triton to give TwLS and TrLS supernatants. All supernatants were concentrated to 200~1 using Millipore micro-concentrators. PBS alone extracts enzymes which are free in the cytoplasm, Tween extracts enzymes which may be weakly bound to the cell membrane, while Triton extracts integral membrane proteins.

The supernatants were assayed against 25mM;
Phenylalanine, ~ Glutamic acid, Leucine, Lysine, Methionine, Alanine, ~ Glutamic acid, Gly-Pro and Aspartic acid pNA substrates at pH 7.0 in HEPES
Bicarbonate buffer. No activity was apparent after 30 minutes incubation at 37C so the assay was allowed to incubate for 18 hours to determine the presence of any enzymatic activity. Specific activities were calculated and the results are shown in Table 1.

Most activity was present in the Tween extraction, where the greatest activity was Alanine pNA specific, some activity was also evident to Phe, ~Ga, Leu, Lys and Met pNA substrates. PLS and TrLS contained little activity with only suggestions of ~GTP and residual Lysine AP and alanine AP.

~ 8 -Table l Specific Activities (OD/min/~q Protein) of Cos Cell Supernatants Preparation p-NA Substrate PLS TwLS TrLS

Phenylalanine 0.0000 0.089 X10-3 0.000 ~ Glutamic Acid 0.0015 X10-3 0.165 X10-3 0. 033 X10-3 Leucine 0.0012 X10-3 0. 066 X10-3 0.000 X10-3 Lysine 0.0029 X10-3 0. 273 X10-3 0.010 X10-3 Methionine 0.00ll X10-3 0. 083 X10-3 0.000 Alanine 0.0270 X10-3 1.110 X103 0.159 X10-3 ~ Glutamic Acid 0.0000 0.000 0.000 Gly-Pro 0.0007 X10-3 0.000 0.000 l0~l of each Cos-l cell supernatant was added to 25mM
p-nitroanilide substrate in 250~1 of HEPES bicarbonate buffer at pH 7.0 and incubated at 37~C for 18 hours.

Chinese Hamster Ovar~ (CHO) and NSO Cell Enz~me Assav Extracts of cultured CHO cells and NSO cells were prepared by the same method as described for COS-l cells above. These extracts were assayed against the following paranitroanilide substrates: Alanine, Arginine, Glycine, ~ Glutamic acid, ~ Glutamic acid, Leucine, Lycine, Methionine, Phenylanine, Proline and Gly-Pro, all at 25mM in HEPES Bicarbonate buffer pH 7Ø
The assay was incubated for 30 minutes at 37C when a final OD reading was taken and the specific activities calculated.

Table 2 shows that the CH0 cell soluble and membrane associated extracts contained low levels of enzyme activity, except for the activity of TwLS against the Gly-Pro substrate. In contrast, the TrLS extract had low activity in all cases except against Arginine substrate. This latter activity being distinct from z~s5~2P
g HllOD aminopeptidase activity.

Table 2 SPecific activities (OD/min/uq Protein) of CHO cell supernatants Preparation p-NA Substrate PLS TwLS TrLS

Leucine 0.070 X10-3 0.147 X10-3 0.106 X10-3 Phenylalanine 0.059 X10-3 0. 052 X10-3 0 Lysine 0.178 X10-3 0.123 X10-3 0.106 X10-3 Methionine 0.130 X10-3 0.118 X10-3 0.106 X10-3 Alanine 0.148 X10-3 0.169 X10-3 0.137 X10-3 Glycine 0 0.052 X10-3 0.068 x10 3 Arginine 0.078 X10-3 0.118 X10-3 0.889 X10-3 Proline 0.024 X10-3 0. 030 X10-3 0. 046 X10-3 Arg-Pro 0.024 X10-3 0. 052 X10-3 0.061 X10-3 Gly-Pro 0.074 X10-3 0.405 X10-3 0. 068 X10-3 Glutamic Acid 0.011 x10-3 0. 052 X10-3 0.144 X10-3 ~ Glutamic Acid 0.030 0 0.053 x10 NSO cells PLS and TwLS extracts both had some aminopeptidase activity particularly against Leucine and Methionine (Table 3). In contrast, the TrLS had low levels of such activity suggesting that a purification schedule involving a Triton extraction, such as that currently used for HllOD, would result in very little contaminating endogenous enzyme activity.

~,~55~%G' . ~

Table 3 SPecific activities (OD/min/uq protein) of NSO cell supernatants Preparation p-NA Substrate PLS TwLS TrLS

Leucine 0.640 X10-31. 840 X10-3 0.137 x10-3 Phenylalanine 0.108 x10-30. 205 x10-3 0. 087 x10-3 Lysine 0.136 X10-30.188 X10-3 0. 093 X10-3 Methionine 0.280 X10-30. 764 x10-3 0.099 x10-3 Alanine 0.047 X10-30.150 X10-3 0. 067 X10-3 Glycine 0.014 X10-30.045 x10 3 0.019 X10-3 Arginine 0.110 X10-30.114 X10-3 0. 081 x10-3 Proline 0.014 X10-30. 034 X10-3 0.019 x10-3 Arg-Pro 0.043 X10-30. 085 x10-3 0. 050 x10-3 Gly-Pro 0.058 x10-30. 063 X10~3 0.050 x10-3 Glutamic Acid 0.017 X10-3 0.270 X10-3 0.019 x103 ~ Glutamic Acid 0.017 x10 3 0.040 X103 0.019 x10-3 Comparative ExamPle BHK cells were prepared by the same method as the Cos-l cells described in Example 1, although in much greater quantity (2xlL roller bottles of confluent cells in 100mls of media). The supernatants were assayed against; phe, Leu, ~GA, Ala, Arg, Asp, Lys, Met, Gly-Pro and aGA pNA substrates at pH 7Ø The assay was incubated at 37C for 30 minutes when a final OD reading was taken.

The BHK cell extracts contained considerable enzyme activity which was present in all supernatants (Table 4). There was negligible activity to ~GA, Asp or ~GA
substrates. All supernatants showed good activity to Lys pNA (which was greatest in PLS), leu, Ala, and Gly-Pro. Activity to Met pNA which was negligible in PLS

WO94/18320 21551 ~ O PCTIGB94/00204 ..

was maximal in the TrLS supernatant. It will be seen that such BHK cells would not be suitable for expression of HllOD since a high level of A-like and M-like aminopeptidase activity is found both in the cytoplasm and integrally in the membrane.

Table 4 SPecific Activities (OD/min/uq protein~ of BHK Cell Supernatants Preparation p-NA Substrate PLS TwLS TrLS

Phenylalanine 0.102 x10 3 0. 080 X10-3 0.113 X10-3 Leucine 0.320 X10-3 0 . 340 X10-3 0. ~33 x10-3 ~ Glutamic Acid 0.006 X10-3 0 . 000 0 . 004 x10-3 Alanine 0.2~0 X10-3 0 . 220 x10-3 0.328 x10-3 Arginine 0.150 X10-3 0 . 200 x10-3 0.344 x10-3 Asparagine 0.017 X10-3 0 . 015 x10-3 0.008 X10-3 Lysine 0.330 x10-3 0.259 x10-3 0.420 x10-3 Methionine 0.020 x10-3 0.185 X10-3 0. 355 x10-3 Gly-Pro 0.312 x10-3 0.318 x10-3 0.225 X10-3 Glutamic Acid 0.014 x10-3 0.008 X10-3 0. 008 x103 10~1 of each BHK cell supernatant was added to 25mM
p-nitroanilide substrate in 250~1 of HEPES bicarbonate buffer at pH 7.0 and incubated at 37C for 30 minutes.

Example 2 Cloninq of HllOD sequence into a mammalian expression vector The DNA to be cloned was the 3.5Kb PCR clone 2 HllOD

gene described in WO 93/23542. The DNA sequence (Sequence ID No: 1) of this insert, obtained by polymerase chain reaction (PCR), is shown in Figure 1 and the amino acid translation (Sequence ID NO:2) in Figure 2. This DNA was excised from the vector pT7Blue-T
Vector (Novagene) by BamHI-digestion and cloned into the W094/l~20 ,~ PCT/GB94/00204 ~55~ 12 -BamHI site of the multiple cloning site of the vector pSPT18 (Boehringer Mannheim) to yield Clone pSPT18-3 . 5-2.
Partial BamHI digestion of the pSPT18-3 . 5-2 clone was performed and the linear DNA purified twice by gel electrophoresis. The 'sticky' ends were blunt ended with dNTPs using Klenow enzyme (DNA polymerase large fragment) and an NcoI linker containing an ATG (Boehringer Mannheim Cat No: 1171 160) ligated into the plasmid. Clones were screened by restriction analysis for those that had this linker at the 5' end of the 3 . 5Kb insert giving them an in-frame ATG initiation site under control of the T7 promotor. The modified 3 . 5Kb insert from one such clone (Clone pSPT18-3 . 5-2N44) was excised and then sub-cloned into the mammalian cell expression vector pRC/CMV using the following strategy:

1. DNA of the clone pSPT18 (T7) -3 . 5-2N44) was digested with the restriction enzyme SmaI.
2. A NotI linker was ligated into this modified SmaI
site to give rise to clones with a NotI site at the 5' end of the insert preceding the NcoI linker containing the ATG start site.
3. Purified DNA from a suitable clone was digested with both NotI and XbaI to release the 3 . 5Kb insert. The enzyme PvuI was added to the incubation in order to cut the pSPT18 vector DNA in half, this was necessary because of the similar size of the vector and insert making purification difficult.
4. The 3 . 5Kb insert was purified on an 0.6-0.7% agarose gel.
5. The mammalian expression vector pRC/CMV was digested with NotI and XbaI and the linear plasmid band was purified on an agarose gel.

6. Ligation was performed between the 3 . 5Kb insert and the linearised vector.

7. Appropriate clones were selected that had a 3 . 5Kb on digestion with NotI and XbaI. The clones were named pRC/CMV-3.5-2.

W094/18320 2 $ 5 5 Y ~G PCT/GB94/00204 Transfection of mammalian COS-l cells DNA of the mammalian expression vector with the HllOD
insert Clone pRC/CMV-3.5-2 is highly purified by centrifugation in a caesium chloride gradient. (Sambrook J., Fritsch E.F. and Maniatis T. Molecular Cloning, A
Laboratory Manual, Second edition. Cold Spring Harbor Press, 1989).

Transient expression of HllOD can be obtained by using this purified DNA of clone pRC/CMV-3.5-2 to transfect COS-1 cells (obtainable from ECACC, Porton). Transfection is performed using DEAE-dextran (Cullen B.R., Use of Eukaryotic expression technology in the functional analysis of cloned genes, Methods in Enzymology: Guide to molecular cloning techniques, Eds S.L. Berger and A.R.
Kimmal, Academic Press, 1987, pp684-704). Cells are cultured in Dulbecco's Modified Eagles Medium (DMEM, Gibco BRL) and 10% foetal calf serum (FCS, Gibco BRL) and expression analysed following 48-72 hours incubation.

Transfection of mammalian Chinese Hamster Ovary fCHO) cells Vector DNA is transfected into CHO cells (obtainable from ECACC, Porton) using the calcium phosphate method (such as that described by Cullen B.R. Use of eukaryotic compression technology in the functional analysis of cloned genes. Methods in enzymology: Guide to Molecular Cloning Techniques, Eds S.L. Berger and A.R. Kimmel, Academic Press, 1987 pp684-704). The transformed cells are cultured in DMEM plus 10% FCS (Gibco BRL). Geneticin (G418) is only the transformed cell lines grow well and untransformed cells do not, this may be up to 800~g/ml.
The transformed cells are then cloned by limiting dilution in microtitre plates.

Analysis of transfected mammalian cells WO94/18320 . PCT/GB94/00204 ~ 14 -Transformed and untransformed CHO cells can be transferred to growth on coverslips for immunofluorescence analyses.
Calls are allowed to grow into small colonies, fixed with methanol and probed with a sheep anti-HllOD antisera followed by a fluorescent dye (e.g. FTTC) conjugated anti-sheep immunoglobulin antiserum. A fluorescence microscope is used to look for positive colonies.

Transformed cell lines are disrupted in RIPA buffer (150mM
sodium chloride, 1~ Nonidet P40, 0.5% deoxycholate, 0.1%
sodium dodecyl sulphate, 50mM Tris-HCl pH 8.0) by removing the growth medium from the cells and then gently swirling the cells for 5 minutes in RIPA buffer. Cells are then transferred to a microfuge tube and spun in the microfuge at full speed for 15 minutes to obtain a clear lysate which is transferred to a fresh tube. Aliquots of this lysate equivalent to 2 x 105 cells are electrophoresed on SDS polyacrylamide gel electrophoresis (SDS-PAGE) and proteins in the gel are transferred to nitrocellulose membrane by Western blotting. The membrane is processed, possibly including a step that involves periodate treatment, and analysed using antisera raised to various forms of the HllOD antigen. The periodate treatment disrupts carbohydrate epitopes; mammalian carbohydrate epitopes may be significantly different from the native helminth carbohydrate. Western blots of transformed cells show the presence of a protein recognised by antisera specific for HllOD.

Extracts of transformed and untransformed cells prepared as described in Example 1 and lysates obtained using RIPA
are assayed for enzyme activity exactly as described in Example 1. Cells transformed with HllOD show higher levels of aminopeptidase activity than untransformed cells.

The presence of transfected vector DNA containing the 3.5Kb PCR clone 2 in Geneticin resistant CHO cell lines is WO94/l~Z0 21~5 12q PCT/GB94/00204 determined by Southern analysis of DNA preparations from these cell lines. DNA is extracted from cells using the method of Sambrook, J., Fritsch, E.F. and Maniatis, T.
Molecular Cloning, A Laboratory Manual, Second Ed. Cold Spring Harbor Press, 1989. 10-20~g of DNA is digested with restriction enzymes and is then electrophoresed on an agarose gel and the DNA transferred to a membrane by Southern blotting (Southern, E. Detection of specific sequences among DNA fragments separated by gel electrophoreses. J Mol. Biol. 98 p503, 1975). This membrane is hybridised with a probe encoding the 3.5Kb PCR
clone 2 and after high stringency washing the membrane is exposed to autoradiography. Bands specific for the 3.5Kb PCR clone 2 are present in transformed cells.

The expression of the 3.5Kb PCR clone 2 at the RNA level in transformed mammalian cells is determined by Northern analysis of RNA isolated from these cells. RNA is extracted from the cells using RNAzol (Cinna/Biotecx, Texas) following the manufacturer's directions and up to 20~g run on an agarose gel and transferred to a membrane by Northern blotting (Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular Cloning, A Laboratory Manual, Second Ed. Cold Spring Harbor Press, 1989). This membrane is then hybridised with a probe encoding the 3.5Kb PCR
clone 2 and after high stringency washing the membrane is exposed to autoradiography. A specific hybridising band is seen in transformed cells expressing the 3.5Kb PCR
clone 2.

W O 94/18320 ' . ~ r PCTIGB94/00204 ~ 16 -SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:
(A) NAME: PITMAN-MOORE, INC., (B) STREET: 421 EAST HAWLEY STREET
(C) CITY: MUNDELEIN
(D) STATE: ILLINOIS
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(B) STREET: BABRAHAM HALL, BABRAHAM
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(A) NAME: EDWARD ALBERT MUNN
(B) STREET: 72 STATION ROAD, FULBOURN
(C) CITY: CAMBRIDGE
(E) COUNTRY: UNITED KINGDOM
(F) POSTAL CODE (ZIP): CBl 5ES

(A) NAME: MARGARET GRAHAM
(B) STREET: 17 BAWTREE CRESCENT, LINTON
(C) CITY: CAMBRIDGE
(E) COUNTRY: UNITED KINGDOM
(F) POSTAL CODE (ZIP): CBl 6XQ

(A) NAME: TREVOR STANLEY SMITH
(B) STREET: 14 THE GROVE, LINTON
(C) CITY: CAMBRIDGE
(E) COUNTRY: UNITED KINGDOM
(F) POSTAL CODE (ZIP): CBl 6UQ

(A) NAME: TIMOTHY PETER ROLPH

W O 94/18320 ~15 5 1 ~ 0 PCTIGB94/00204 (B) STREET: 42 LITTLEWORTH
(C) CITY: WHEATLEY
(D) STATE: OXON
(E) COUNTRY: UNITED KINGDOM
(F) POSTAL CODE (ZIP): OX33 ITR

(A) NAME: SUSAN ELIZABETH NEWTON
(B) STREET: 3 LEBANON STREET
(C) CITY: STRATHMORE
(D) STATE: VICTORIA
(E) COUNTRY: AUSTRALIA
(F) POSTAL CODE (ZIP): 3401 (ii) TITLE OF INVENTION: EXPESSION OF PROTECTIVE ANTIGENS

(iii) NUMBER OF SEQUENCES: 2 (iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC co~patible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (EPO) (vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: GB 9302302.6 (B) FILING DATE: 05-FEB-1993 (2) INFORMATION EOR SEQ ID NO: 1 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3303 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: sin~le (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

~5~ - 18 - -(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

GCTGAATCTA ACTCCAATCC GTCTTATTGT CGCATTATTT CTAGTAGCTG CTGCAGTCGG

CCTCTCTATT GGTCTCACCT ATTACTTTAC TCGCAAAGCG TTCGATACCT CAGAAAAGCC

AGGGAAGGAT GATACTGGTG GCAAGGACAA AGACAATTCT CCCTCTGCGG CGGAACTACT

CCTTCCAAGT AATATAAAAC CATTGTCTTA CGACTTGACG ATCAAAACAT ATCTACCTGG

TTATGTGGAC TTCCCACCGG AGAAAAACCT CACATTCGAT GGGCGTGTGG AAATATCAAT

GGTTGTAATT GAGCCAACAA AGAGTATCGT ACTCAATTCA AAGAAGATCT CTGTAATACC

CCAAGAATGT GAACTGGTAT CGGGCGATAA AAAACTCGAA ATTGAAAGTG TAAAGGAGCA

CCCAAGACTG GAAAAGGTTG AGTTTCTTAT CAAAAGCCAA CTGGAAAAAG ATCAACAAAT

CTTGCTCAAG GTCGGCTACA TCGGTCTCAT CAGCAACAGC TTTGGTGGAA TCTACCAGAC

CACTTATACC ACCCCGGATG GCACCCCTAA GATCGCTGCA GTTTCACAAA ATGAGCCCAT

AGATGCTCGT CGAATGGTAC CATGCATGGA TGAACCGAAA TACAAAGCAA ACTGGACCGT

TACTGTCATT CATCCAAAAG GCACCAAAGC CGTCTCGAAT GGAATCGAAG TGAACGGAGA

215S~2D

TGGAGAGATC AGTGGTGATT GGATCACATC GAAGTTCTTG ACTACTCCAC GGATGTCATC

CTA~Ll~LlG GCAGTTATGG TTTCAGAATT TGAATACATC GAAGGTGAAA CAAAGACGGG

TGTTCGGTTC CGTATATGGT CACGCCCAGA GGCAAAGAAG ATGACACAAT ATGCTCTGCA

ATCTGGTATC AAGTGCATAG AATTCTACGA AGATTTCTTT GATATCAGAT TCCCTCTGAA

GAAACAAGAT ATGATTGCCC TTCCTGATTT CTCTGCCGGT GCCATGGAGA ATTGGGGCCT

CATCACTTAC AGGGAAAACT CTTTGTTGTA CGATGACAGA TTCTATGCAC CGATGAATAA

ACAGCGAATT GCTCGCATTG TTGCTCATGA GCTTGCTCAT CAGTGGTTCG GCGACTTGGT

TACGATGAAG TGGTGGGATA ATTTGTGGTT (;AATGAAGGT TTTGCAAGAT TCACAGAATT

TATTGGAGCT GGTCAGATAA CTCAAGATGA CGCCAGAATG AGGAACTACT TCCTGATTGA

TGTACTTGAA CGCGCTTTGA AAGCTGATTC GGTAGCGTCA AGCCATCCAC TTTCCTTCAG

AATCGACAAA GCTGCAGAAG TTGAAGAAGC CTTTGATGAT ATCACATACG CCAAAGGAGC

~ t~ - 20 -TTCTGTTCTT ACTATGCTGA GAGCCTTGAT TGGAGAAGAA AAACATAAGC ATGCAGTATC

GCAGTACCTC AAGAAGTTCT CGTATAGCAA TGCAGAAGCG ACTGATCTAT GGGCAGTTTT

TGATGAAGTT GTCACTGACG TCGAAGGTCC AGACGGCAAA CCTATGAAAA CCACAGAGTT

TGCAAGTCAG TGGACGACTC AGATGGGCTT CCCAGTTATT TCCGTAGCAG AGTTTAACTC

GACTACTTTG AAATTAACGC AAAGTCGATA TGAGGCGAAT AAAGACGCTG TGGAGAAAGA

GAAGTACCGT CACCCGAAAT ACGGATTTAA ATGGGATATT CCACTGTGGT ATCAGGAAGG

CGATAAGAAG GAGATAAAGC GAACATGGTT GAGAAGAGAT GAACCGCTTT ACTTGCATGT

TAGTGATGCT GGCGCTCCCT TTGTGGTGAA CGCAGACCGC TATGGATTTT ATCGACAAAA

TCATGACGCT AATGGTTGGA AAAAGATAAT CAAGCAGCTC AAGGATAATC ATGAGGTTTA

CAGTCCCCGG ACAAGAAATG CCATCATTAG CGATGCGTTT GCTGCGGCTG CAACTGACGC

AATTGAGTAT GAGACTGTAT TTGAACTTCT GAATTATGCC GAAAAAGAAA CGGAATATCT

ACCATTAGAA ATCGCAATGT CCGGGATCTC TTCGATTTTG AAATACTTCG GTACCGAGCC

~ 2155i20 AGAGGCAAAG CCAGCTCAAA CATACATGAT GAACATATTG AAACCGATGT ATGAAAAAAG

CAGTATCGAC TTCATTGCCA ATAACTACAG AAATGACAAG ~L~llLlLCC AAATCAACCT

CCAAAAAGAT GTCATTGATA TGTTCTGCGC CCTCGGATCG CAAGACTGCA GGAAGAAATA

TAAAAAACTT TTCGATGACG AAGTCATGAA CAAATGCAGG GATGGTCAAG CAGCAACCGA

ATGCGTAAGA ATCGCCGCTC CTCTCCGATC AAGTGTTTAT TGTTATGGTG TGAAGGAAGG

CGGTGATTAT GCTTCCGACA AGGTGATGGA GCTTTATACG GCCGAAACAC TCGCCCTAGA

AAAAGACTTC CTACGCCTAG CATTGGGATG TCATAAAGAT GTTACTGCTT TGAAAGGACT

TCTCTTGCGG GCTCTGGACA GGAATTCGTC GTTCGTACGT ATGCAGGATA TCCCAAGTGC

TTTCAATGAT GTAGCAGCAA ATCCTATCGG CGGAGAATTC ATTTTCAATT TCCTTATTGA

AAGATGGCCA GATATCATTG AAAGTATAGG AACGAAGCAC ACATACGTTG AGAAAGTGAT

ACCAGCCTGC ACTTCAGGAA TCCGCTCACA ACAGCAGATT GACCAGCTGA AGAATCTGCA

GAAAAATGGC ATGAACGCTC GTCAATTCGG TGCATTCGAT AAAGCAATCG AACGAGCACA

..
~ Q - 22 -AAATAGGGTG GATTGGATTA AAAAACATTT CCAAAAATTA GCGGCTTTCT Tr~AA~AAAGc CACCTTGTAA TTCGAATTAC ATTGCCAGTA ATCCAGATCT TAAAGTTCAT GAAGGAATAT

GACAGGGAAC TGACTGTCTG TTGGTCACTG TTCCACTGAA TGGAAGTTTT TACCTACAAA

AATTTTTATC GTTATATTTG CCTTCCGTGA GGGGTCATTG TTGTCACTTG AATAGTAAAC

AAAGCTCAGT ATTGCAACCA GTGAACAATA TTACTTTCGC TTCATCAAAT TGTTATCTTC

CCTATACTCT CTTCCTAACT GAATTCGGAA.ATTTGTTCAT ATTCGTTTGT AGTCTGTTGC

TCAGAACACT TTCTCCTCAA TAGCTTCTTG TTT~ LLL TTTGATTGTA TTGATCGTTT

TACAATTGTA TAGATTAGTT ATCTTATAAA TATTGATGGT TAAAAAAAAA AA~UU~UAAAA

AAA

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 962 amino acids (B) TYPE: a~ino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide W O 94/18320 ~ ~ ~ 5 ~ 2 Q PCT/GB94/00204 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Leu Asn Leu Thr Pro Ile Arg Leu Ile Val Ala Leu Phe Leu Val Ala . 1 5 10 15 Ala Ala Val Gly Leu Ser Ile Gly Leu Thr Tyr Tyr Phe Thr Ar~ Lys Ala Phe Asp Thr Ser Glu Lys Pro Gly Lys Asp Asp Thr Gly Gly Lys Asp Lys Asp Asn Ser Pro Ser Ala Ala Glu Leu Leu Leu Pro Ser Asn Ile Lys Pro Leu Ser Tyr Asp Leu Thr Ile Lys Thr Tyr Leu Pro Gly Tyr Val Asp Phe Pro Pro Glu Lys Asn Leu Thr Phe Asp Gly Arg Val Glu Ile Ser Met Val Val Ile Glu Pro Thr Lys Ser Ile Val Leu Asn Ser Lys Lys Ile Ser Val Ile Pro Gln Glu Cys Glu Leu Val Ser Gly Asp Lys Lys Leu Glu Ile Glu Ser Val Lys Glu His Pro Ar~ Leu Glu Lys Val Glu Phe Leu Ile Lys Ser Gln Leu Glu Lys Asp Gln Gln Ile Leu Leu Lys Val Gly Tyr Ile Gly Leu Ile Ser Asn Ser Phe Gly Gly Ile Tyr Gln Thr Thr Tyr Thr Thr Pro Asp Gly Thr Pro Lys Ile Ala WO 94/l~20 . ~ PCT/GB94/00204 Ala Val Ser Gln Asn Glu Pro Ile Asp Ala Arg Arg Met Val Pro Cys 19~ ~0() 205 Met Asp Glu Pro Lys Tyr Lys Ala Asn Trp Thr Val Thr Val Ile His Pro Lys Gly Thr Lys Ala Val Ser Asn Gly Ile Glu Val Asn Gly Asp Gly Glu Ile Ser Gly Asp Trp Ile Thr Ser Lys Phe Leu Thr Thr Pro Arg Met Ser Ser Tyr Leu Leu Ala Val Met Val Ser Glu Phe Glu Tyr Ile Glu Gly Glu Thr Lys Thr Gly Val Arg Phe Arg Ile Trp Ser Arg Pro Glu Ala Lys Lys Met Thr Gln Tyr Ala Leu Gln Ser Gly Ile Lys Cys Ile Glu Phe Tyr Glu Asp Phe Phe Asp Ile Arg Phe Pro Leu Lys Lys Gln Asp Met Ile Ala Leu Pro Asp Phe Ser Ala Gly Ala Met Glu Asn Trp Gly Leu Ile Thr Tyr Arg Glu Asn Ser Leu Leu Tyr Asp Asp Arg Phe Tyr Ala Pro Met Asn Lys Gln Arg Ile Ala Arg Ile Val Ala His Glu Leu Ala His Gln Trp Phe Gly Asp Leu Val Thr Met Lys Trp Trp Asp Asn Leu Trp Leu Asn Glu Gly Phe Ala Arg Phe Thr Glu Phe W O 94/18320 2 ~ 2 Q PCT/GB94/00204 : .' Ile Gly Ala Gly Gln Ile Thr Gln Asp Asp Ala Arg Met Arg Asn Tyr Phe Leu Ile Asp Val Leu Glu Arg Ala Leu Lys Ala Asp Ser Val Ala Ser Ser His Pro Leu Ser Phe Arg Ile Asp Lys Ala Ala Glu Val Glu Glu Ala Phe Asp Asp Ile Thr Tyr Ala Lys Gly Ala Ser Val Leu Thr Met Leu Arg Ala Leu Ile Gly Glu Glu Lys His Lys His Ala Val Ser Gln Tyr Leu Lys Lys Phe Ser Tyr Ser Asn Ala Glu Ala Thr Asp Leu Trp Ala Val Phe Asp Glu Val Val Thr Asp Val Glu Gly Pro Asp Gly Lys Pro Met Lys Thr Thr Glu Phe Ala Ser Gln Trp Thr Thr Gln Met Gly Phe Pro Val Ile Ser Val Ala Glu Phe Asn Ser Thr Thr Leu Lys Leu Thr Gln Ser Arg Tyr Glu Ala Asn Lys Asp Ala Val Glu Lys Glu Lys Tyr Arg His Pro Lys Tyr Gly Phe Lys Trp Asp Ile Pro Leu Trp Tyr Gln Glu Gly Asp Lys Lys Glu Ile Lys Arg Thr Trp Leu Arg Arg Asp Glu Pro Leu Tyr Leu His Val Ser Asp Ala Gly Ala Pro Phe Val WO 94/l8320 ~ PCT/GB94/00204 2~ 26 -Val Asn Ala Asp Arg Tyr Gly Phe Tyr Arg Gln Asn His Asp Ala Asn Gly Trp Lys Lys Ile Ile Lys Gln Leu Lys Asp Asn His Glu Val Tyr Ser Pro Arg Thr Arg Asn Ala Ile Ile Ser Asp Ala Phe Ala Ala Ala Ala Thr Asp Ala Ile Glu Tyr Glu Thr Val Phe Glu L~u Leu Asn Tyr 66~ 665 670 Ala Glu Lys Glu Thr Glu Tyr Leu Pro Leu Glu Ile Ala Met Ser Gly Ile Ser Ser Ile Leu Lys Tyr Phe Gly Thr Glu Pro Glu Ala Lys Pro Ala Gln Thr Tyr Met Met Asn Ile Leu Lys Pro Met Tyr Glu Lys Ser Ser Ile Asp Phe Ile Ala Asn Asn Tyr Arg Asn Asp Lys Leu Phe Phe Gln Ile Asn Leu Gln Lys Asp Val Ile Asp Met Phe Cys Ala Leu Gly Ser Gln Asp Cys Arg Lys Lys Tyr Lys Lys Leu Phe Asp Asp Glu Val Met Asn Lys Cys Arg Asp Gly Gln Ala Ala Thr Glu Cys Val Arg Ile Ala Ala Pro Leu Arg Ser Ser Val Tyr Cys Tyr Gly Val Lys Glu Gly Gly Asp Tyr Ala Ser Asp Lys Val Met Glu Leu Tyr Thr Ala Glu Thr ' Leu Ala Leu Glu Lys Asp Phe Leu Arg Leu Ala Leu Gly Cys His Lys Asp Val Thr Ala Leu Lys Gly Leu Leu Leu Arg Ala Leu Asp Arg Asn -Ser Ser Phe Val Arg Met Gln Asp Ile Pro Ser Ala Phe Asn Asp Val Ala Ala Asn Pro Ile Gly Gly Glu Phe Ile Phe Asn Phe Leu Ile Glu Arg Trp Pro Asp Ile Ile Glu Ser Ile Gly Thr Lys His Thr Tyr Val Glu Lys Val Ile Pro Ala Cys Thr Ser Gly Ile Arg Ser Gln Gln Gln Ile Asp Gln Leu Lys Asn Leu Gln Lys Asn Gly Met Asn Ala Arg Gln Phe Gly Ala Phe Asp Lys Ala Ile Glu Arg Ala Gln Asn Arg Val Asp Trp Ile Lys Lys His Phe Gln Lys Leu Ala Ala Phe Phe Lys Lys Ala Thr Leu

Claims (8)

1. A method for the expression of an enzyme antigen which in nature is a parasite gut membrane bound enzyme or a fragment thereof having similar enzyme and/or antigenic activity, wherein mammalian host cells are transfected with a vector adapted to express said enzyme antigen or fragment thereof, characterised in that prior to transformation the host cells are substantially free from endogenous enzymes having (a) the same function and (b) the same cell membrane integration or lack of integration, as said parasite enzyme antigen or fragment thereof.

2. A method as claimed in claim 1, wherein said antigen is a helmintic enæyme.

3. A method as claimed in claim 1 or claim 2, wherein said antigen is an aminopeptidase enzyme.

4. A method as claimed in any one of claims 1 to 3, wherein said antigen is H110D, or an antigenic fragment thereof.

5. A method as claimed in claim 3 or claim 4, wherein said host cells lack significant aminopeptidase A-like or aminopeptidase M-like activity.

6. A method as claimed in claim 5, wherein said host cells are COS-1 or CHO cells.

7. A method as claimed in any one of claims 1 to 6 wherein said antigen is expressed integrated within a cell membrane of the host cells.

8. A method as claimed in any one of claims 1 to 6, wherein said antigen is expressed as a soluble cytoplasmic enzyme.