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Definitions

• 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.
• H110D is the anthelmintic antigen derived from Haemonchus contortus as described in W088/00835 and 90/11086.
• 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.
• 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.
• helminths eg. various species of the families Haemonchus,
• W093/23542 referred to above also describes the production of antigenic fragments of H110D by recombinant DNA technology.
• the H110D material was expressed in E.coli using a pGEX vector and on injection into sheep, raised anti-HllOD antibodies.
• 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) .
• 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.
• 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,
• 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” .
• 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.
• 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.
• an endogenous enzyme such as an aminopeptidase in the cytoplasm
• the foreign enzyme is expressed with a trans-membrane sequence which becomes located in the membrane of the host cell
• 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.
• H110D helminthic aminopeptidase antigens
• H110D which shows predominantly aminopeptidase A-like or M-like activity and thus cleaves predominantly methionine and leucine peptide bonds
• COS-1 cells 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.
• 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.
• a mammalian cell line low in a particular enzyme 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.
• FACS Fluorescence Activated Cell Sorter
• 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, cytomegaloviru ⁇ (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) .
• 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.
• Figure 1 shows the DNA sequence of 3.5 Kb PCR clone 2 (sequence ID No:l) ;
• Figure 2 shows the amino acid translation of 3.5 Kb PCR clone 2 (sequence ID No.2) .
• 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) .
• PBS PBS supernatant
• Subseguent extractions used 500 ⁇ l of PBS 0.1% Tween and PBS 2% Triton to give TwLS and TrLS supernatants. All supernatants were concentrated to 200 ⁇ l 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.
• 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.
• 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, 7 GA, Ala, Arg, Asp, Lys, Met, Gly-Pro and ⁇ GA 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 7 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 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.
• 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:
• DNA of the clone pSPT18 (T7) -3.5-2N44) was digested with the restriction enzyme Smal.
• 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.
• the 3.5Kb insert was purified on an 0.6-0.7% agarose gel .
• the mammalian expression vector pRC/CMV was digested with NotI and Xbal and the linear plasmid band was purified on an agarose gel.
• Ligation was performed between the 3.5Kb insert and the linearised vector.
• 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.
• DMEM Dulbecco's Modified Eagles Medium
• FCS foetal calf serum
• 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.
• 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.
• FTTC fluorescent dye conjugated anti- sheep immunoglobulin antiserum
• 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 10 5 cells are electrophoresed on SDS polyacryla ide gel electrophoresis (SDS-PAGE) and proteins in the gel are transferred to nitrocellulose membrane by Western blotting.
• RIPA buffer 150mM sodium chloride, 1% Nonidet P40, 0.5% deoxycholate, 0.1% sodium dodecyl sulphate, 50mM Tris-HCl pH 8.0
• 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.
• 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. 10-20 ⁇ 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.
• 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.
• CATCACTTAC AGGGAAAACT CTTTGTTGTA CGATGACAGA TTCTATGCAC CGATGAATAA 1080

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