AU682488B2 - Expression of protective antigens - Google Patents

Description

Expression of Protective Antigens

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 antigens' since they do not give rise to natural immunity against the parasite.

We have found that a particularly effective 'hidden antigen' is the anthelmintic antigen H110D derived from Haemonchus contortus as described in W088/00835 and 90/11086. In W093/23542 we have described the further finding that H110D 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 'hidden 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 present in the gut of a wide range of parasites such as helminths, eg. various species of the families Haemonchus, Ostertacria, Trichostrongylus. 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 H110D by recombinant DNA technology. In this work, the H110D 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 H110D gene. The nucleotide seguence of this is shown in Figure 1 (seg. ID No:l) . The corresponding translation is shown in Figure 2 (seg. 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 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; C127I, a mouse mammary tumour cell line; CV-1, 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 mammalian 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 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 H110D 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 H110D, which shows predominantly aminopeptidase A-like or M-like activity and thus cleaves predominantly methionine and leucine peptide bonds, we have found COS-1 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 that there is no difficulty in separating the endogenous enzyme from the expressed H110D which is located in the cell membrane.

Proteolytic enzymes such as trypsin have been shown to cleave the parasite antigen H110D from the membrane to produce HllOD-soluble (H11S) . 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 mammalian cell line low in a particular enzyme. One way in which this could be done is as follows: antibodies are raised to the mammalian 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 antigen or fragment thereof. Thus, in particular, the 3.5 kb fragment of the H110 gene may be connected in frame with an appropriate promoter. Suitable promoters include SV40 early or late promoter, eg. PSVL vector, cytomegaloviruε (CMV) promoter, mouse metallofchionein 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 _5, 650, 1980) . In general, linear DNA is introduced more readily than circular DNA. The H110D 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:

Figure 1 shows the DNA sequence of 3.5 Kb PCR clone 2 (sequence ID No:l) ; and

Figure 2 shows the amino acid translation of 3.5 Kb PCR clone 2 (sequence ID No.2) . EXAMPLE 1

Cos-1 Cell Enzyme 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 lO ls of PBS buffer. When all of the cells were in suspension they were transferred to a universal tube and frozen down at -20°C. 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) . Subseguent extractions used 500μl of PBS 0.1% Tween and PBS 2% Triton to give TwLS and TrLS supernatants. All supernatants were concentrated to 200μl 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 37°C 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 ₇GTP and residual Lysine AP and alanine AP. Table 1

Specific Activities (OD/min/μg protein) of Cos Cell

Supernatants

Preparation p-NA Substrate PLS TwLS TrLS

Phenylalanine 0.0000 0.089 X10^"3 0.000

₇ Glutamic Acid 0.0015 xlO^"3 0.165 XlO^"3 0.033 XlO^"3

Leucine 0.0012 XlO^"3 0.066 XlO^"3 0.000 XlO^"3

Lysine 0.0029 XlO^'3 0.273 XlO^"3 0.010 XlO^"3

Methionine 0.0011 XlO^"3 0.083 XlO^"3 0.000

Alanine 0.0270 XlO^"3 1.110 lO^"3 0.159 XlO^'3 α Glutamic Acid 0.0000 0.000 0.000

Gly-Pro 0.0007 XlO^"3 0.000 0.000

lOμl of each Cos-1 cell supernatant was added to 25mM p-nitroanilide substrate in 250μl of HEPES bicarbonate buffer at pH 7.0 and incubated at 37"C for 18 hours.

Chinese Hamster Ovary (CHO) and NSO Cell Enzyme Assay

Extracts of cultured CHO cells and NSO cells were prepared by the same method as described for COS-1 cells above. These extracts were assayed against the following paranitroanilide substrates: Alanine, Arginine, Glycine, Glutamic acid, _t Glutamic acid, Leucine, Lycine, Methionine, Phenylanine, Proline and Gly-Pro, all at 25mM in HEPES Bicarbonate buffer pH 7.0. The assay was incubated for 30 minutes at 37 °C when a final OD reading was taken and the specific activities calculated.

Table 2 shows that the CHO 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 HllOD aminopeptidase activity.

Table 2

Specific activities (OD/min/μg protein) of CHO cell supernatants

Preparation p-NA Substrate PLS TwLS TrLS

Leucine 0.070 XlO^" 0.147 XlO^"3 0.106 XlO^"3

Phenylalanine 0.059 lO -3 0.052 XlO^"3 0

Lysine 0.178 XlO^" 0.123 XlO^"3 0.106 XlO^"3

Methionine 0.130 xlO -3 0.118 XlO^"3 0.106 XlO^"3

Alanine 0.148 XlO^" 0.169 xlO^"3 0.137 xlO^"3

Glycine 0 0.052 XlO^"3 0.068 XlO^'3

Arginine 0.078 XlO^" 0.118 XlO^"3 0.889 XlO^"3

Proline 0.024 XlO^" 0.030 XlO^"3 0.046 XlO^*3

Arg-Pro 0.024 XlO -3 0.052 xlO^"3 0.061 XlO^"3

Gly-Pro 0.074 XlO^' 0.405 XlO^"3 0.068 xlO^"3

₇ Glutamic Acid 0.011 XlO^" 0.052 XlO^"3 0.144 XlO -3 a Glutamic Acid 0.030 0 0.053 XlO

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. Table 3

Specific activities (OD/min/μg protein) of NSO cell supernatants

p-NA Substrate PLS TrLS

xlO^" xlO -3

XlO^" xlO xlO^" xlO^"3

XlO -3 xlO^" xlO^" xlO^" xlO^" xlO -3

XlO^" xlO^"

XlO -3 lO^"

XlO^" XlO -3

XlO^" xlO^" lO^' XlO^" XlO^" XlO -3

Comparative Example

BHK cells were prepared by the same method as the Cos-1 cells described in Example 1, although in much greater quantity (2xlL roller bottles of confluent cells in lOOmls of media) . The supernatants were assayed against; phe, Leu, ₇GA, Ala, Arg, Asp, Lys, Met, Gly-Pro and αGA pNA substrates at pH 7.0. The assay was incubated at 37"C 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 ₇GA, Asp or αGA 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 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/μg protein) of BHK Cell

Supernatants

Preparation p-NA Substrate PLS TwLS TrLS

XlO^'

XlO -3

XlO^"

XlO^"

XlO -3

XlO^'

XlO^"

XlO -3 xlO^" XlO -3

lOμl of each BHK cell supernatant was added to 25mM p-nitroanilide substrate in 250μl of HEPES bicarbonate buffer at pH 7.0 and incubated at 37°C for 30 minutes.

Example 2

Cloning of HllOD seguence 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 Fiqure 2. This DNA was excised from the vector pT7Blue-T Vector (Novagene) by BamHI-digestion and cloned into the 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 Ncol 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 Smal.

2. A NotI linker was ligated into this modified Smal site to give rise to clones with a NotI site at the 5' end of the insert preceding the Ncol linker containing the ATG start site.

3. Purified DNA from a suitable clone was digested with both NotI and Xbal to release the 3.5Kb insert. The enzyme Pvul was added to the incubation in order to cut the pSPTlδ 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 Xbal 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 Xbal . The clones were named pRC/CMV-3.5-2. Transfection of mammalian COS-1 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 (CHO) 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 800μg/ml. The transformed cells are then cloned by limiting dilution in microtitre plates.

Analysis of transfected mammalian cells 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 polyacryla ide 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 determined by Southern analysis of DNA preparations from these cell lines. DNA is extracted from cells using the method of Sa brook, J., Fritsch, E.F. and Ma iatis, T. Molecular Cloning, A Laboratory Manual, Second Ed. Cold Spring Harbor Press, 1989. 10-20μg 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 20μg 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. SEQUENCE LISTING

L INFORMATION :

PPLICANT:

(A NAME: PITMAN-MOORE, INC., (B STREET: 421 EAST HAWLEY STREET (C CITY: MUNDELEIN (D STATE: ILLINOIS (E COUNTRY: USA (F POSTAL CODE (ZIP): 60062

(A NAME: THE AGRICULTURAL AND FOOD RESEARCH COUNCIL (B STREET: BABRAHAM HALL, BABRAHAM (C CITY: CAMBRIDGE (E COUNTRY: UNITED KINGDOM (F POSTAL CODE (ZIP): CB2 4AT

(A NAME: EDWARD ALBERT MUNN (B STREET: 72 STATION ROAD, FULBOURN (C CITY: CAMBRIDGE (E COUNTRY: UNITED KINGDOM (F POSTAL CODE (ZIP) : CB1 5ES

(A NAME: MARGARET GRAHAM (B STREET: 17 BAWTREE CRESCENT, LINTON (C CITY: CAMBRIDGE (E COUNTRY: UNITED KINGDOM (F POSTAL CODE (ZIP): CB1 6XQ

(A NAME: TREVOR STANLEY SMITH (B STREET: 14 THE GROVE, LINTON (C CITY: CAMBRIDGE (E COUNTRY: UNITED KINGDOM (F POSTAL CODE (ZIP) : CB1 6UQ

(A) NAME: TIMOTHY PETER ROLPH (B) STREET: 42 LITTLEWORTH

(C) CITY: WHEATLEY

(D) STATE: OXON

(E) COUNTRY: UNITED KINGDOM

(F) POSTAL CODE (ZIP): 0X33 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 compatible

(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 FOR SEQ ID NO^* 1

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3303 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

GCTGAATCTA ACTCCAATCC GTCTTATTGT CGCATTATTT CTAGTAGCTG CTGCAGTCGG 60

CCTCTCTATT GGTCTCACCT ATTACTTTAC TCGCAAAGCG TTCGATACCT CAGAAAAGCC 120

AGGGAAGGAT GATACTGGTG GCAAGGACAA AGACAATTCT CCCTCTGCGG CGGAACTACT 180

CCTTCCAAGT AATATAAAAC CATTGTCTTA CGACTTGACG ATCAAAACAT ATCTACCTGG 240

TTATGTGGAC TTCCCACCGG AGAAAAACCT CACATTCGAT GGGCGTGTGG AAATATCAAT 300

GGTTGTAATT GAGCCAACAA AGAGTATCGT ACTCAATTCA AAGAAGATCT CTGTAATACC 360

CCAAGAATGT GAACTGGTAT CGGGCGATAA AAAACTCGAA ATTGAAAGTG TAAAGGAGCA 420

CCCAAGACTG GAAAAGGTTG AGTTTCTTAT CAAAAGCCAA CTGGAAAAAG ATCAACAAAT 480

CTTGCTCAAG GTCGGCTACA TCGGTCTCAT CAGCAACAGC TTTGGTGGAA TCTACCAGAC 540

CACTTATACC ACCCCGGATG GCACCCCTAA GATCGCTGCA GTTTCACAAA ATGAGCCCAT 600

AGATGCTCGT CGAATGGTAC CATGCATGGA TGAACCGAAA TACAAAGCAA ACTGGACCGT 660

TACTGTCATT CATCCAAAAG GCACCAAAGC CGTCTCGAAT GGAATCGAAG TGAACGGAGA 720 TGGAGAGATC AGTGGTGATT GGATCACATC GAAGTTCTTG ACTACTCCAC GGATGTCATC 780

CTACTTGTTG GCAGTTATGG TTTCAGAATT TGAATACATC GAAGGTGAAA CAAAGACGGG 840

TGTTCGGTTC CGTATATGGT CACGCCCAGA GGCAAAGAAG ATGACACAAT ATGCTCTGCA 900

ATCTGGTATC AAGTGCATAG AATTCTACGA AGATTTCTTT GATATCAGAT TCCCTCTGAA 960

GAAACAAGAT ATGATTGCCC TTCCTGATTT CTCTGCCGGT GCCATGGAGA ATTGGGGCCT 1020

CATCACTTAC AGGGAAAACT CTTTGTTGTA CGATGACAGA TTCTATGCAC CGATGAATAA 1080

ACAGCGAATT GCTCGCATTG TTGCTCATGA GCTTGCTCAT CAGTGGTTCG GCGACTTGGT 1140

TACGATGAAG TGGTGGGATA ATTTGTGGTT GAATGAAGGT TTTGCAAGAT TCACAGAATT 1200

TATTGGAGCT GGTCAGATAA CTCAAGATGA CGCCAGAATG AGGAACTACT TCCTGATTGA 1260

TGTACTTGAA CGCGCTTTGA AAGCTGATTC GGTAGCGTCA AGCCATCCAC TTTCCTTCAG 1320

AATCGACAAA GCTGCAGAAG TTGAAGAAGC CTTTGATGAT ATCACATACG CCAAAGGAGC 1380 TTCTGTTCTT ACTATGCTGA GAGCCTTGAT TGGAGAAGAA AAACATAAGC ATGCAGTATC 1440

GCAGTACCTC AAGAAGTTCT CGTATAGCAA TGCAGAAGCG ACTGATCTAT GGGCAGTTTT 1500

TGATGAAGTT GTCACTGACG TCGAAGGTCC AGACGGCAAA CCTATGAAAA CCACAGAGTT 1560

TGCAAGTCAG TGGACGACTC AGATGGGCTT CCCAGTTATT TCCGTAGCAG AGTTTAACTC 1620

GACTACTTTG AAATTAACGC AAAGTCGATA TGAGGCGAAT AAAGACGCTG TGGAGAAAGA 1680

GAAGTACCGT CACCCGAAAT ACGGATTTAA ATGGGATATT CCACTGTGGT ATCAGGAAGG 1740

CGATAAGAAG GAGATAAAGC GAACATGGTT GAGAAGAGAT GAACCGCTTT ACTTGCATGT 1800

TAGTGATGCT GGCGCTCCCT TTGTGGTGAA CGCAGACCGC TATGGATTTT ATCGACAAAA 1860

TCATGACGCT AATGGTTGGA AAAAGATAAT CAAGCAGCTC AAGGATAATC ATGAGGTTTA 1920

CAGTCCCCGG ACAAGAAATG CCATCATTAG CGATGCGTTT GCTGCGGCTG CAACTGACGC 1980

AATTGAGTAT GAGACTGTAT TTGAACTTCT GAATTATGCC GAAAAAGAAA CGGAATATCT 2040

ACCATTAGAA ATCGCAATGT CCGGGATCTC TTCGATTTTG AAATACTTCG GTACCGAGCC 2100 AGAGGCAAAG CCAGCTCAAA CATACATGAT GAACATATTG AAACCGATGT ATGAAAAAAG 2160

CAGTATCGAC TTCATTGCCA ATAACTACAG AAATGACAAG CTGTTTTTCC AAATCAACCT 2220

CCAAAAAGAT GTCATTGATA TGTTCTGCGC CCTCGGATCG CAAGACTGCA GGAAGAAATA 2280

TAAAAAACTT TTCGATGACG AAGTCATGAA CAAATGCAGG GATGGTCAAG CAGCAACCGA 2340

ATGCGTAAGA ATCGCCGCTC CTCTCCGATC AAGTGTTTAT TGTTATGGTG TGAAGGAAGG 2400

CGGTGATTAT GCTTCCGACA AGGTGATGGA GCTTTATACG GCCGAAACAC TCGCCCTAGA 2460

AAAAGACTTC CTACGCCTAG CATTGGGATG TCATAAAGAT GTTACTGCTT TGAAAGGACT 2520

TCTCTTGCGG GCTCTGGACA GGAATTCGTC GTTCGTACGT ATGCAGGATA TCCCAAGTGC 2580

TTTCAATGAT GTAGCAGCAA ATCCTATCGG CGGAGAATTC ATTTTCAATT TCCTTATTGA 2640

AAGATGGCCA GATATCATTG AAAGTATAGG AACGAAGCAC ACATACGTTG AGAAAGTGAT 2700

ACCAGCCTGC ACTTCAGGAA TCCGCTCACA ACAGCAGATT GACCAGCTGA AGAATCTGCA 2760

GAAAAATGGC ATGAACGCTC GTCAATTCGG TGCATTCGAT AAAGCAATCG AACGAGCACA 2820 AAATAGGGTG GATTGGATTA AAAAACATTT CCAAAAATTA GCGGCTTTCT TCAAGAAAGC 2880

CACCTTGTAA TTCGAATTAC ATTGCCAGTA ATCCAGATCT TAAAGTTCAT GAAGGAATAT 2940

GACAGGGAAC TGACTGTCTG TTGGTCACTG TTCCACTGAA TGGAAGTTTT TACCTACAAA 3000

AATTTTTATC GTTATATTTG CCTTCCGTGA GGGGTCATTG TTGTCACTTG AATAGTAAAC 3060

AAAGCTCAGT ATTGCAACCA GTGAACAATA TTACTTTCGC TTCATCAAAT TGTTATCTTC 3120

CCTATACTCT CTTCCTAACT GAATTCGGAA ATTTGTTCAT ATTCGTTTGT AGTCTGTTGC 3180

TCAGAACACT TTCTCCTCAA TAGCTTCTTG TTTGTTTTTT TTTGATTGTA TTGATCGTTT 3240

TACAATTGTA TAGATTAGTT ATCTTATAAA TATTGATGGT TAAAAAAAAA AAAAAAAAAA 3300

AAA 3303

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 962 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Leu Asn Leu Thr Pro He Arg Leu He Val Ala Leu Phe Leu Val Ala 1 5 10 15

Ala Ala Val Gly Leu Ser He Gly Leu Thr Tyr Tyr Phe Thr Arg Lys 20 25 30

Ala Phe Asp Thr Ser Glu Lys Pro Gly Lys Asp Asp Thr Gly Gly Lys 35 40 45

Asp Lys Asp Asn Ser Pro Ser Ala Ala Glu Leu Leu Leu Pro Ser Asn 50 55 60

He Lys Pro Leu Ser Tyr Asp Leu Thr He Lys Thr Tyr Leu Pro Gly 65 70 75 80

Tyr Val Asp Phe Pro Pro Glu Lys Asn Leu Thr Phe Asp Gly Arg Val 85 90 95

Glu He Ser Met Val Val He Glu Pro Thr Lys Ser He Val Leu Asn 100 105 110

Ser Lys Lys He Ser Val He Pro Gin Glu Cys Glu Leu Val Ser Gly 115 120 125

Asp Lys Lys Leu Glu He Glu Ser Val Lys Glu His Pro Arg Leu Glu 130 135 140

Lys Val Glu Phe Leu He Lys Ser Gin Leu Glu Lys Asp Gin Gin He 145 150 155 160

Leu Leu Lys Val Gly Tyr He Gly Leu He Ser Asn Ser Phe Gly Gly 165 170 175

He Tyr Gin Thr Thr Tyr Thr Thr Pro Asp Gly Thr Pro Lys He Ala 180 185 190 Ala Val Ser Gin Asn Glu Pro He Asp Ala Arg Arg Met Val Pro Cys

19b 200 205

Met Asp Glu Pro Lys Tyr Lys Ala Asn Trp Thr Val Thr Val He His 210 215 220

Pro Lys Gly Thr Lys Ala Val Ser Asn Gly He Glu Val Asn Gly Asp 225 230 235 240

Gly Glu He Ser Gly Asp Trp He Thr Ser Lys Phe Leu Thr Thr Pro 245 250 255

Arg Met Ser Ser Tyr Leu Leu Ala Val Met Val Ser Glu Phe Glu Tyr 260 265 270

He Glu Gly Glu Thr Lys Thr Gly Val Arg Phe Arg He Trp Ser Arg 275 280 285

Pro Glu Ala Lys Lys Met Thr Gin Tyr Ala Leu Gin Ser Gly He Lys 290 295 300

Cys He Glu Phe Tyr Glu Asp Phe Phe Asp He Arg Phe Pro Leu Lys 305 310 315 320

Lys Gin Asp Met He Ala Leu Pro Asp Phe Ser Ala Gly Ala Met Glu 325 330 335

Asn Trp Gly Leu He Thr Tyr Arg Glu Asn Ser Leu Leu Tyr Asp Asp 340 345 350

Arg Phe Tyr Ala Pro Met Asn Lys Gin Arg He Ala Arg He Val Ala 355 360 365

His Glu Leu Ala His Gin Trp Phe Gly Asp Leu Val Thr Met Lys Trp 370 375 380

Trp Asp Asn Leu Trp Leu Asn Glu Gly Phe Ala Arg Phe Thr Glu Phe 385 390 395 400 He Gly Ala Gly Gin He Thr Gin Asp Asp Ala Arg Met Arg Asn Tyr 405 410 415

Phe Leu He Asp Val Leu Glu Arg Ala Leu Lys Ala Asp Ser Val Ala 420 425 430

Ser Ser His Pro Leu Ser Phe Arg He Asp Lys Ala Ala Glu Val Glu 435 440 445

Glu Ala Phe Asp Asp He Thr Tyr Ala Lys Gly Ala Ser Val Leu Thr 450 455 460

Met Leu Arg Ala Leu He Gly Glu Glu Lys His Lys His Ala Val Ser 465 470 475 480

Gin Tyr Leu Lys Lys Phe Ser Tyr Ser Asn Ala Glu Ala Thr Asp Leu 485 490 495

Trp Ala Val Phe Asp Glu Val Val Thr Asp Val Glu Gly Pro Asp Gly 500 505 510

Lys Pro Met Lys Thr Thr Glu Phe Ala Ser Gin Trp Thr Thr Gin Met 515 520 525

Gly Phe Pro Val He Ser Val Ala Glu Phe Asn Ser Thr Thr Leu Lys 530 535 540

Leu Thr Gin Ser Arg Tyr Glu Ala Asn Lys Asp Ala Val Glu Lys Glu 545 550 555 560

Lys Tyr Arg His Pro Lys Tyr Gly Phe Lys Trp Asp He Pro Leu Trp 565 570 575

Tyr Gin Glu Gly Asp Lys Lys Glu He Lys Arg Thr Trp Leu Arg Arg 580 585 590

Asp Glu Pro Leu Tyr Leu His Val Ser Asp Ala Gly Ala Pro Phe Val 595 600 605 Val Asn Ala Asp Arg Tyr Gly Phe Tyr Arg Gin Asn His Asp Ala Asn 610 615 620

Gly Trp Lys Lys He He Lys Gin Leu Lys Asp Asn His Glu Val Tyr 625 630 635 640

Ser Pro Arg Thr Arg Asn Ala He He Ser Asp Ala Phe Ala Ala Ala 645 650 655

Ala Thr Asp Ala He Glu Tyr Glu Thr Val Phe Glu L^u Leu Asn Tyr 660 665 670

Ala Glu Lys Glu Thr Glu Tyr Leu Pro Leu Glu He Ala Met Ser Gly 675 680 685

He Ser Ser He Leu Lys Tyr Phe Gly Thr Glu Pro Glu Ala Lys Pro 690 695 700

Ala Gin Thr Tyr Met Met Asn He Leu Lys Pro Met Tyr Glu Lys Ser 705 710 715 720

Ser He Asp Phe He Ala Asn Asn Tyr Arg Asn Asp Lys Leu Phe Phe 725 730 735

Gin He Asn Leu Gin Lys Asp Val He Asp Met Phe Cys Ala Leu Gly 740 745 750

Ser Gin Asp Cys Arg Lys Lys Tyr Lys Lys Leu Phe Asp Asp Glu Val 755 760 765

Met Asn Lys Cys Arg Asp Gly Gin Ala Ala Thr Glu Cys Val Arg He 770 775 780

Ala Ala Pro Leu Arg Ser Ser Val Tyr Cys Tyr Gly Val Lys Glu Gly 785 790 795 800

Gly Asp Tyr Ala Ser Asp Lys Val Met Glu Leu Tyr Thr Ala Glu Thr 805 810 815 Leu Ala Leu Glu Lys Asp Phe Leu Arg Leu Ala Leu Gly Cys His Lys 820 825 830

Asp Val Thr Ala Leu Lys Gly Leu Leu Leu Arg Ala Leu Asp Arg Asn 835 840 845

Ser Ser Phe Val Arg Met Gin Asp He Pro Ser Ala Phe Asn Asp Val 850 855 860

Ala Ala Asn Pro He Gly Gly Glu Phe He Phe Asn Phe Leu He Glu 865 870 875 880

Arg Trp Pro Asp He He Glu Ser He Gly Thr Lys His Thr Tyr Val 885 890 895

Glu Lys Val He Pro Ala Cys Thr Ser Gly He Arg Ser Gin Gin Gin 900 905 910

He Asp Gin Leu Lys Asn Leu Gin Lys Asn Gly Met Asn Ala Arg Gin 915 920 925

Phe Gly Ala Phe Asp Lys Ala He Glu Arg Ala Gin Asn Arg Val Asp 930 935 940

Trp He Lys Lys His Phe Gin Lys Leu Ala Ala Phe Phe Lys Lys Ala 945 950 955 960

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 enzyme.

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 HllOD, 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.