US20020182221A1 - Recombinant papillomavirus vaccine and method for production and treatment - Google Patents
Recombinant papillomavirus vaccine and method for production and treatment Download PDFInfo
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- US20020182221A1 US20020182221A1 US10/000,903 US90301A US2002182221A1 US 20020182221 A1 US20020182221 A1 US 20020182221A1 US 90301 A US90301 A US 90301A US 2002182221 A1 US2002182221 A1 US 2002182221A1
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
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- C12N2710/00011—Details
- C12N2710/20011—Papillomaviridae
- C12N2710/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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- C—CHEMISTRY; METALLURGY
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- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/20011—Papillomaviridae
- C12N2710/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
Definitions
- the present invention relates to fusions proteins, comprising a protein or part of a protein that provides T helper epitopes and an antigen from a human-papilloma virus that find utility in the treatment or prophylaxis of human papilloma induced tumours.
- the invention relates to fusion proteins comprising an E6 or E7 protein from HPV strain 16 or 18 linked to protein D from Heamophilius influenza B.
- Papillomaviruses are small naked DNA tumour viruses (7.9 kilobases, double strand), which are highly species-specific. Over 70 individual human papillomavirus (HPV) genotypes have been described. Papillomaviruses are classified on the basis of species of origin (human, bovine etc.) and of the degree of genetic relatedness with other papillomaviruses from the same species. HPVs are generally specific for the skin or mucosal surfaces and have been broadly classified into “low” and “high” risk on the basis of rare and common, respectively, detection in abnormal or tumour tissue. Low risk HPVs usually cause benign lesions (warts or papillomas) that persist for several months or years.
- HPVs High risk HPVs are associated with cancer.
- the strongest positive association between an HPV virus and human cancer is that which exist between HPV 16 and 18 and cervical carcinoma. More than ten other HPV types have also been found in cervical carcinomas including HPV 31 and HPV 33 although at less frequency.
- Genital HPV infection in young sexually active women is common and most individuals either clear the infection, or if lesions develop, these regress. Only a subset of infected individuals has lesions which progress to high grade intraephithelial neoplasia and only a fraction of these progress further to invasive carcinoma.
- HPV bovine papillomavirus type 1
- HPVs genomes described have at least seven early genes, E1 to E7 and two late genes L1 and L2.
- an upstream regulatory region harbors the regulatory sequences which appears to control most transcriptional events of the HPV genome.
- E1 and E2 genes are involved in viral replication and transcriptional control, respectively and tend to be disrupted by viral integration.
- E6 and E7 are involved in viral transformation.
- E5 has also been implicated in this process.
- Carcinoma of the cervix is common in women and develops through a pre-cancerous intermediate stage to the invasive carcinoma which frequently leads to death.
- the intermediate stages of the disease is known as cervical intraepithelial neoplasia and is graded I to III in terms of increasing severity (CIN I-III).
- HPV infection of the female anogenital tract manifests as cervical flat condylomas, the hallmark of which is the koilocytosis affecting predominantly the superficial and intermediate cells of the cervical squamous epithelium.
- Koilocytes which are the consequence of a cytopathic effect of the virus, appear as multinucleated cells with a perinuclear clear haloe.
- the epithelium is thickened with abnormal keratinisation responsible for the warty appearance of the lesion.
- compositions comprising either an E6 or E7 or an E6/E7 fusion protein linked to an immunological fusion partner having T cell epitopes.
- the immunological fusion partner is derived from protein D of Heamophilus influenza B.
- the protein D derivative comprises approximately the first ⁇ fraction (1/3) ⁇ of the protein, in particular approximately the first N-terminal 100-110 amino acids.
- the protein D may be lipidated (Lipo Protein D).
- Other immunological fusion partners include the non-structural protein from influenzae virus, NS1 (hemagglutinin). Typically the N terminal 81 amino acids are utilised, although different fragments may be used provided they include T-helper epitopes.
- the immunological fusion partner is the protein known as LYTA.
- LYTA the protein known as LYTA.
- the C terminal portion of the molecule is used.
- Lyta is derived from Streptococcus pneumoniae which synthesize an N-acetyl-L-alanine amidase, amidase LYTA, (coded by the lytA gen ⁇ Gene, 43 (1986) page 265-272 ⁇ an autolysin that specifically degrades certain bonds in the peptidoglycan backbone.
- the C-terminal domain of the LYTA protein is responsible for the affinity to the choline or to some choline analogues such as DEAE.
- the present invention in preferred embodiment provides fusion proteins comprising Protein D-E6 from HPV 16, Protein D-E7 from HPV 16 Protein D-E7 from HPV 18, Protein D-E6 from HPV 18, and Protein D E6 E7 from both HPV 16 and 18.
- the protein D part preferably comprises the first ⁇ fraction (1/3) ⁇ of protein D. It will be appreciated that other E6 and E7 proteins may be utilised from other HPV subtypes.
- the proteins of the present invention preferably are expressed in E. coli .
- the proteins are expressed with a Histidine tail comprising between 5 to 9 and preferably six Histidine residues. These are advantageous in aiding purification.
- the protein E7 may in a preferred embodiment carry a mutation to reduce the binding for the rb site (retinoblastoma gene product) and hence eliminate any potential transforming capacity.
- Preferred mutations for HPV 16 E7 involve replacing Cys 24 with Glycine, or Glutamic acid 26 with Glutamine.
- the E7 protein contains both these mutations.
- Preferred mutations for the HPV 18 E 7 involve replacing Cys 27 with Glycine and/or Glutamic acid 26 with Glutamine. Again preferably both mutations are present.
- Single or double mutations may also be introduced p53 region of E 6 to eliminate any potential transforming ability.
- E6 E7 fusion protein from HPV linked to an immunological fusion partner is provided and E6 E7 fusion protein from HPV linked to an immunological fusion partner.
- a preferred Immunological fusion partner is Protein D, more preferable the first ⁇ fraction (1/3) ⁇ of protein D.
- the present invention also provides a DNA encoding the proteins of the present invention. Such sequences can be inserted into a suitable expression vector and expressed in a suitable host.
- a DNA sequence encoding the proteins of the present invention can be synthesized using standard DNA synthesis techniques, such as by enzymatic ligation as described by D. M. Roberts et al. in Biochemistry 1985, 24, 5090-5098, by chemical synthesis, by in vitro enzymatic polymerization, or by PCR technology utilising for example a heat stable polymerase, or by a combination of these techniques.
- Enzymatic polymerisation of DNA may be carried out in vitro using a DNA polymerase such as DNA polymerase I (Klenow fragment) in an appropriate buffer containing the nucleoside triphosphates dATP, dCTP, dGTP and dTTP as required at a temperature of 10°-37° C., generally in a volume of 50 ⁇ l or less.
- a DNA polymerase such as DNA polymerase I (Klenow fragment) in an appropriate buffer containing the nucleoside triphosphates dATP, dCTP, dGTP and dTTP as required at a temperature of 10°-37° C., generally in a volume of 50 ⁇ l or less.
- Enzymatic ligation of DNA fragments may be carried out using a DNA ligase such as T4 DNA ligase in an appropriate buffer, such as 0.05M Tris (pH 7.4), 0.01M MgCl 2 , 0.01M dithiothreitol, 1 mM spermidine, 1 mM ATP and 0.1 mg/ml bovine serum albumin, at a temperature of 4° C. to ambient, generally in a volume of 50 ml or less.
- a DNA ligase such as T4 DNA ligase in an appropriate buffer, such as 0.05M Tris (pH 7.4), 0.01M MgCl 2 , 0.01M dithiothreitol, 1 mM spermidine, 1 mM ATP and 0.1 mg/ml bovine serum albumin, at a temperature of 4° C. to ambient, generally in a volume of 50 ml or less.
- the chemical synthesis of the DNA polymer or fragments may be carried out by conventional phosphotriester, phosphite or phosphoramidite chemistry, using solid phase techniques such as those described in ‘Chemical and Enzymatic Synthesis of Gene Fragments—A Laboratory Manual’ (ed. H. G. Gassen and A. Lang), Verlag Chemie, Weinheim (1982), or in other scientific publications, for example M. J. Gait, H. W. D. Matthes, M. Singh, B. S. Sproat, and R. C. Titmas, Nucleic Acids Research, 1982, 10, 6243; B. S. Sproat, and W. Bannwarth, Tetrahedron Letters, 1983, 24, 5771; M. D.
- the process of the invention may be performed by conventional recombinant techniques such as described in Maniatis et al., Molecular Cloning—A Laboratory Manual; Cold Spring Harbor, 1982-1989.
- the process may comprise the steps of:
- transforming is used herein to mean the introduction of foreign DNA into a host cell. This can be achieved for example by transformation, transfection or infection with an appropriate plasmid or viral vector using e.g. conventional techniques as described in Genetic Engineering; Eds. S. M. Kingsman and A. J. Kingsman; Blackwell Scientific Publications; Oxford, England, 1988.
- transformation or transformationant’ will hereafter apply to the resulting host cell containing and expressing the foreign gene of interest.
- recombinant antigen of the invention are expressed in E. coli .
- the expression strategy include fusion of E7, E6 or E6/E7 fusion to the 1/3-N-terminal portion of protein D from Haemophilus influenzae B, an immunological fusion partner providing T cell helper epitopes.
- An affinity polyhistidine tail is engineered at the carboxy terminus of the fusion protein allowing for simplified purification.
- Such recombinant antigen is overexpressed in E. coli as insoluble protein.
- the proteins of the invention are coexpressed with thioredoxin in trans (TIT).
- TIT thioredoxin in trans
- Coexpression of thioredoxin in trans versus in cis is preferred to keep antigen free of thioredoxin without the need for protease.
- Thioredoxin coexpression eases the solubilisation of the proteins of the invention.
- Thioredoxin coexpression has also a significant impact on protein purification yield, on purified-protein solubility and quality.
- the replicable expression vectors may be prepared in accordance with the invention, by cleaving a vector compatible with the host cell to provide a linear DNA segment having an intact replicon, and combining said linear segment with one or more DNA molecules which, together with said linear segment encode the desired product, such as the DNA polymer encoding the protein of the invention, or derivative thereof, under ligating conditions.
- the DNA polymer may be preformed or formed during the construction of the vector, as desired.
- the choice of vector will be determined in part by the host cell, which may be prokaryotic or eukaryotic but preferably is E. coli .
- Suitable vectors include plasmids, bacteriophages, cosmids and recombinant viruses.
- the preparation of the replicable expression vector may be carried out conventionally with appropriate enzymes for restriction, polymerisation and ligation of the DNA, by procedures described in, for example, Maniatis et al. cited above.
- the recombinant host cell is prepared, in accordance with the invention, by transforming a host cell with a replicable expression vector of the invention under transforming conditions.
- Suitable transforming conditions are conventional and are described in, for example, Maniatis et al. cited above, or “DNA Cloning” Vol. II, D. M. Glover ed., IRL Press Ltd, 1985.
- a bacterial host such as E. coli may be treated with a solution of CaCl 2 (Cohen et al., Proc. Nat. Acad. Sci., 1973, 69, 2110) or with a solution comprising a mixture of RbCl, MnCl 2 , potassium acetate and glycerol, and then with 3-[N-morpholino]-propane-sulphonic acid, RbCl and glycerol.
- Mammalian cells in culture may be transformed by calcium co-precipitation of the vector DNA onto the cells.
- the invention also extends to a host cell transformed with a replicable expression vector of the invention.
- Culturing the transformed host cell under conditions permitting expression of the DNA polymer is carried out conventionally, as described in, for example, Maniatis et al. and “DNA Cloning” cited above.
- the cell is supplied with nutrient and cultured at a temperature below 50° C.
- the product is recovered by conventional methods according to the host cell.
- the host cell is bacterial, such as E. coli it may be lysed physically, chemically or enzymatically and the protein product isolated from the resulting lysate.
- the product may generally be isolated from the nutrient medium or from cell free extracts.
- Conventional protein isolation techniques include selective precipitation, adsorption chromatography, and affinity chromatography including a monoclonal antibody affinity column.
- proteins of the present invention are expressed with a hisitidine tail (His tag).
- His tag hisitidine tail
- the proteins can easily be purified by affinity chromatography using an ion metal affinity chromatography column (IMAC) column.
- IMAC ion metal affinity chromatography column
- a second chromatographic step such as Q-sepharose may be utilised either before or after the IMAC column to yield highly purified protein.
- the immunological fusion partner is C-LYTA
- Products containing both C-LYTA and his tags can be easily and efficiently purified in a two step process involving differential affinity chromatography.
- One step involves the affinity of the His tag to IMAC columns, the other involves the affinity of the C-terminal domain of LYTA for choline or DEAE.
- Proteins comprising both a C-LYTA and Hisitidine tag are new and accordingly form one aspect of the invention. These may be purified to high levels (greater than 80% preferably greater than 90%) by a simple two step differential affinity procedure.
- the proteins of the present invention are provided preferably at least 80% pure more preferably 90% pure as visualized by SDS PAGE.
- the protein present a major single band when analysed by SDS PAGE under reducing conditions, and western blot analysis show less than 5% host cell protein contamination.
- the present invention also provides pharmaceutical composition comprising a protein of the present invention in a pharmaceutically acceptable excipient.
- a preferred vaccine composition comprises at least Protein D-E6 from HPV 16 or derivative thereof together with Protein D-E7 from HPV 16.
- the E6 and E7 may be presented in a single molecule, preferably a Protein D E6/E7 fusion.
- Such vaccine may optionally contain either or both E6 and E7 proteins from HPV 18, preferably in the form of a Protein D-E6 or Protein D-E7 fusion protein or Protein D E6/E7 fusion protein.
- the vaccines of the present invention may contain other HPV antigens from HPV 16 or 18. In particular, the vaccine may contain L1 or L2 antigen monomers.
- L1 or L2 antigens may be presented together as a virus like particle or the L1 alone protein may be presented as virus like particle or caposmer structure.
- antigens, virus like particles and capsomer are per se known. See for example WO94/00152, WO94/20137, WO94/05792, & WO93/02184. Additional early proteins may be included such as E2 or preferably E5 for example
- the vaccine of the present invention may additionally comprise antigens from other HPV strains, preferably from strains HPV 6 11, HPV 31 or 33.
- Vaccine preparation is generally described in Vaccine Design—The subunit and adjuvant approach (Ed. Powell and Newman) Pharmaceutical Biotechnology Vol. 6 Plenum Press 1995. Encapsulation within liposomes is described by Fullerton, U.S. Pat. No. 4,235,877.
- the proteins of the present invention are preferably adjuvanted in the vaccine formulation of the invention.
- Suitable adjuvants include an aluminium salt such as aluminium hydroxide gel (alum) or aluminium phosphate, but may also be a salt of calcium, iron or zinc, or may be an insoluble suspension of acylated tyrosine, or acylated sugars, cationically or anionically derivatised polysaccharides, or polyphosphazenes.
- the adjuvant composition induces a preferential TH 1 response.
- Suitable adjuvant systems include, for example, a combination of monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipid A (3D-MPL) together with an aluminium salt.
- An enhanced system involves the combination of a monophosphoryl lipid A and a saponin derivative particularly the combination of QS21 and 3D-MPL as disclosed in WO 94/00153, or a less reactogenic composition where the QS21 is quenched with cholesterol as disclosed in WO 96/33739.
- a particularly potent adjuvant formulation involving QS21, 3D-MPL & tocopherol in an oil in water emulsion is described in WO 95/17210 and is a preferred formulation.
- a vaccine comprising a protein D (or derivative thereof)—E6 or protein D (or derivative thereof)—E7 adjuvanted with a monophosphoryl lipid A or derivative thereof.
- the vaccine additionally comprises a saponin, more preferably QS21.
- the formulation additional comprises an oil in water emulsion and tocopherol.
- the present invention also provides a method for producing a vaccine formulation comprising mixing a protein of the present invention together with a pharmaceutically acceptable excipient, such as 3D-MPL.
- nucleotides sequences corresponding to amino acids 1 ⁇ 98 of E7 protein are amplified from pRIT14462.
- the insert was sequenced to verify that no modification had been generated during the polymerase chain reaction.
- the sequence for the fusion protein-D1/3-E7-His HPV 16 is described in FIG. 1.
- Plasmid pRIT14501 was introduced into E. coli AR58 (Mott et al., 1985, Proc. Natl. Acad. Sci., 82:88) a defective ⁇ lysogen containing a thermosensitive repressor of the ⁇ pL promoter.
- Cells of AR58 transformed with plasmid pRIT14501 were grown in 100 ml of LB medium supplemented with 50 ⁇ gr/ml of Kanamycin at 30° C. During the logarithmic phase of growth bacteria were shifted to 39° C. to inactivate the ⁇ repressor and turn on the synthesis of protein D1/3-E7-His. The incubation at 39° C. was continued for 4 hours. Bacteria were pelleted and stored at ⁇ 20° C.
- Frozen cells are thawed and resuspended in 10 ml of PBS buffer. Cells are broken in a French pressure cell press SLM Aminco at 20.000 psi (three passages). The extract is centrifuged at 16.000 g for 30 minutes at 4° C.
- Proteins are solubilised after 2 hours incubation of the pellet in 30 ml of 8 M urea, 50 mM phosphate pH 7.5 at RT. Cells debris are eliminated by 15 min centrifugation at 17,000 g, 4° C. Protein purification is carried out at RT°, 15 ml of solubilised protein are applied onto a 5 ml Ni2+NTA (Qiagen) resin (Pharmacia column XK 16/20) preequilibrated in 8M urea, 50 mM phosphate pH 7.5 at a flow rate of 0.2 ml/min. The column is washed in the same buffer until the absorbance at 280 nm reaches the base line.
- Ni2+NTA Qiagen
- the protein is eluted with a 0-600 mM Imidazole gradient in 8M urea, 50 mM phosphate pH 7.5. The flow rate of these two last steps is brought to 1 ml/min. Eluted fractions are analysed by SDS polyacrylamide gel electrophoresis and by Western blotting. ProtD1/3-E7-His, visualised by Coomassie blue staining, by a polyclonal anti protein D or by a monoclonal anti E7 antibody, appears as a major single band at about 32 kDalton and is estimated as a 95% pure protein. No E. coli contaminants, traced with a polyclonal anti E. coli proteins antibody, are observed.
- prot-D1/3-E7-His at 0.665 mg/ml is assayed for stability.
- SDS PAGE analysis showed no evolution of the protein after 7 days incubation at ⁇ 20° C., 4° C., RT° or 37 ° C.
- the sequence of Prot-D1/3 is followed by a multiple cloning site (11 residues) and a coding region for a C-terminal histidine tail (6 His). This plasmid is used to express the fusion protein D1/3-E6-his.
- HPV genomic E6 and E7 sequences type HPV 16 were amplified from HPV16 full length genome cloned in pBR322 (obtained from Deutsches Krebsgeberstechnik (DKFZ), Referenztechnik für human pathogen Papillomaviruses—
- nucleotides sequences corresponding to amino acid.
- E6 protein 1 ⁇ 151 of E6 protein were amplified from pRIT 14462.
- the insert was sequenced to verify that no modification had been generated during the polymerase chain reaction.
- the coding sequence for the fusion protein-D1/3-E6-His is described in FIG. 3.
- Plasmid pRIT14497 was introduced into E. coli AR58 (Mott et al., 1985, Proc. Natl. Acad. Sci., 82:88) a defective ⁇ lysogen containing a thermosensitive repressor of the ⁇ pL promoter.
- Cells of AR58 transformed with plasmid pRIT14497 were grown in 100 ml of LB medium supplemented with 50 ⁇ gr/ml of Kanamycin at 30° C. During the logarithmic phase of growth bacteria were shifted to 39° C. to inactivate the ⁇ repressor and turn on the synthesis of protein D1/3-E6-his. The incubation at 39° C. was continued for 4 hours. Bacteria were pelleted and stored at ⁇ 20C.
- Frozen cells are thawed and resuspended in 10 ml of PBS buffer. Cells are broken in a French pressure cell press SLM Aminco at 20.000 psi (three passages). The extract is centrifuged at 16.000 g for 30 minutes at 4° C.
- the level of expression represents about 5% of total protein.
- HPV-16 ProtD1/3 E6 recombinant antigen was expressed in E. coli (AR58). Expression strategy included fusion of E6 to the 1/3-N-terminal portion of protein D from Haemophilus influenzae, an immunological fusion partner providing T cell helper epitopes. An affinity polyhistidine tail was engineered at the carboxy terminus of the fusion protein. The recombinant antigen was overexpressed in E. coli as insoluble proteins.
- Bacterial expressions are conducted in LB media in presence of 0.05 mg/ml of kanamycin at 30° C. plus 0.2 mg/ml of Ampicillin when Thioredoxin is coexpressed. Recombinant protein expression is thermally induced by transferring the cells to 42° C., when cell optical density (OD 600 nm) of 0.4 is reached. Protein expression is maintained for 4 hours. Purification was carried out according to the following protocol. Cell Culture Pellet 60 OD 600 1 mM pefabloc, 2 M NaCl, PBS pH 7.4 (Buffer A) French Press Disruptor Three passes 20,000 psi Centrifugation 17,000 g 30 min, 4° C.
- Starting material is 10 flasks of 400 ml culture each.
- Cell paste is suspended to 60 OD 600 in Buffer A (240 ml of Buffer A in this case), prior cell lysis by three passes through a French press disrupter (20,000 psi). Lysed cells are pelleted 30 min at 15,000 g at 4° C. Bacterial cell pellet containing the recombinant protein is washed once in 240 ml Buffer B, then twice in 240 ml Buffer C.
- Prot D E6-His (TIT) is solubilised by 240 ml Buffer D overnight at 4° C. on a rotating wheel. Cell debris are pelleted 30 min at 15,000 g at 4° C. Supernatant (230 ml) is stored at ⁇ 20° C. The material is then subjected to IMAC chromatography.
- the chelating ligand NTA (nitrilo-tri-acetic-acid) is attached to an Agarose support (Qiagen). NTA ligand is charged with nickel metal ion with which it interacts through 4 of the 6 coordination sites of the nickel. The two remaining coordination sites of nickel interact strongly with histidine residues of the 6 ⁇ His-tagged protein. Elution is achieved by competition with Imidazole which bind to the Ni-NTA and displace the tagged antigen.
- Ni-NTA Agarose Qiagen (catalogue number: 30 250) was used.
- Antigen positive fractions are pooled (30 ml).
- Endotoxin is removed by affiprep chromatography.
- Affi-Prep® Polymyxin support consists of USP Grade Polymyxin B coupled to the Affi-Prep® Matrix. Due to its high affinity to the lipid A moiety of endotoxins, polymixin B binds endotoxin molecules with high capacity and selectivity.
- E 8M Urea, 20 mM PO4 (NaH 2 PO4 (2H 2 O)/K 2 HPO4 (3H 2 O)), pH 7.0 (apyrogenic).
- Affi-Prep® Polymyxin resin is washed in 10 volumes of 0.1 M NaOH, followed by 10 volumes of pyrogen free water.
- Buffers I 4 M Urea, 0.5 M Arginine, 0.15 M NaCl, 10 mM PO4 (NaH 2 PO4 (2H 2 O)/K 2 HPO4 (3H 2 O)) pH 6.8 J: 2 M Urea, 0.5 M Arginine, 0.15 M NaCl, 10 mM PO4 (NaH 2 PO4 (2H 2 O)/K 2 HPO4 (3H 2 O)) pH 6.8 K: 0 M Urea, 0.5 M Arginine, 0.15 M NaCl, 10 mM PO4 (NaH 2 PO4 (2H 2 O)/K 2 HPO4 (3H 2 O)) pH 6.8
- Dialysis tubing is placed in a 2 liters cylinder containing Buffer K (under stirring) at 4° C. O/N. Buffer is changed and dialysis is pursued 2 more hours at 4° C.
- Antigen solution is filtered through a 0.2 ⁇ m filter to prevent any bacterial growth. Antigen is kept at ⁇ 20° C. in Nunc vials.
- Protein D1/3 E6 His is characterised as follows:
- ProteinD1/3-E6-His is a 273 amino acids long peptide with 112 amino acids coming from Protein D part. ProteinD1/3-E6-His has a theoretical Molecular Weight of 32 kD and migrates on SDS-PAGE as a 33 kD protein. ProteinD1/3-E6-His theoretical isoelectric point is 8.17.
- the viral Protein E6 is a basic protein containing 14 cystein residues, eight of them (Cys 30,33,63,66 and Cys 103,106,136,139) are involved in two C-terminal zinc binding motifs.
- Protein D 1/3-E6-His is expressed as insoluble protein, in E. coli -AR 58 strain, with Thioredoxin in Trans, a folding partner. Cell culture is produced in 400 ml flask.
- nucleotides sequences corresponding to amino acids 1 ⁇ 249 of fused E6E7 protein were amplified from pRIT14556.
- the insert was sequenced to verify that no modification had been generated during the polymerase chain reaction.
- the coding sequence for the fusion protein-D1/3-His is described FIG. 6.
- Plasmid pRIT14512 was introduced into E. coli AR58 (Mott et al., 1985, Proc. Natl. Acad. Sci., 82:88) a defective ⁇ lysogen containing a thermosensitive repressor of the ⁇ pL promoter.
- Cells of AR58 transformed with plasmid pRIT14512 were grown in 100 ml of LB medium supplemented with 50 ⁇ gr/ml of Kanamycin at 30° C. During the logarithmic phase of growth bacteria were shifted to 39° C. to inactivate the ⁇ repressor and turn on the synthesis of protein D1/3-E6E7-his. The incubation at 39° C. was continued for 4 hours. Bacteria were pelleted and stored at ⁇ 20C.
- Frozen cells are thawed and resuspended in 10 ml of PBS buffer. Cells are broken in a French pressure cell press SLM Aminco at 20.000 psi (three passages). The extract is centrifuged at 16.000 g for 30 minutes at 4° C.
- the level of expression represents about 1% of total protein.
- the fusion protein of Lipo D 1/3 and E6-E7 from HPV 16 was expressed in E. coli in the presence of thioredoxin.
- the N-terminal of the pre-protein (388 aa) contains MDP residues followed by 16 amino acids of signal peptide of lipoprotein D (from Haemophilus Influenzae) which is cleaved in vivo to give the mature protein (370 aa).
- Lipoprotein portion (aa 1 to 127) is followed by the proteins E6 and E7 in fusion.
- the C terminal of the protein is elongated by TSGHHHHHH.
- the protein was purified by the following protocol:
- Cell paste is suspended to 60 OD 600 in 2 M NaCl, 20 mM Phosphate (NaH 2 PO4/K 2 HPO4) pH 7.5 in presence of 1 mM Pefabloc as protease inhibitor prior cell lysis by three passes through a French press disruptor (20,000 psi). Lysed cells are pelleted 30 min at 15,000 g at 4° C.
- bacterial cell pellet containing the recombinant protein is washed twice in 4 M urea, 2 M NaCl, 20 mM Phosphate pH 7.5, once in 2% Empigen BB, 20 mM Phosphate pH 7.5 and finally twice in 20 mM Phosphate buffer pH 7.0 to eliminate trace of detergent (each wash is performed in the same volume used for cell suspension).
- IMAC Ion Metal Affinity Chromatography
- 0.5 M NaCl-eluted fractions from Q Sepharose step are pooled and dialyzed versus 0.2 M NaCl, 8 M urea, 20 mM Phosphate pH 10 before loading onto a Ni2+-NTA (Qiagen) column (XK 26/20, Pharmacia) preequilibrated in 8 M urea, 20 mM PO4 pH 12 (30 ml resin/61 ml sample) at 5.6 cm/h. Column is washed in 8 M urea, 20 mM PO4 pH 12 until the base line is reached then by 8 M urea, 20 mM PO4 pH 10.
- Antigen is eluted by Imidazole steps (0.025 M, 0.05 M, 0.1 M, 0.15 M, 0.2 M, 0.5 M Imidazole, each step in two column volumes) in 8 M urea, 20 mM PO4 pH 10, at 45 cm/h. 0.05 M Imidazole-eluted fractions are pooled.
- Imac sample is concentrated about 5 times (to 0.407 mg/ml) on a 5 kDa Filtron Omega membrane in a stirred cell from AMICON at RT°.
- Concentrated sample is dialyzed at RT versus decreasing-urea-concentration steps (4 M, 2 M urea) in 0.5 M Arginine, 150 mM NaCl, 10 mM PO4 pH 6.8. Last dialysis against 0.5 M Arginine, 150 mM NaCl, 10 mM PO4 pH 6.8 is achieved at 4° C.
- IMAC step is able to eliminate a 32 kD contaminant at 0.025 M Imidazole which eluted also some antigen.
- 0.05 M Imidazole-eluted Antigen is estimated pure at 90% by Coomassie blue staining of SDS-PAGE. After these two purification steps, sample is free of E. coli contaminants.
- Western blotting analysis using specific antigen-N and/or C terminus antibodies shows a heterogeneous pattern of bands with higher and lower MW than the full length protein. This pattern suggests the presence of aggregates and incompletely processed protein and/or degraded one, copurified with the full length protein.
- Prot-D1/3 is followed by a multiple cloning site (11 residues) and a coding region for a C-terminal histidine tail (6 His)
- Construction of plasmid pRIT 14733( TCA347): a plasmid expressing the fusion Protein-D1/3-E7 mutated (cys24 ⁇ gly, glu26 ⁇ gln) with His tail
- Double mutations cys24 gly (Edmonds and Vousden, J. Virology 63: 2650 (1989) and glu26 gln (Phelps et al, J. Virology 66: 2418-27 (1992) were chosen to impair the binding to the antioncogene product of Retinoblastome gene (pRB).
- Plasmid pRIT 14733 was introduced into E.coli AR58 (Mott et al.,1985, Proc. Natl. Acad. Sci., 82:88) a defective X lysogen containing a thermosensitive repressor of the ⁇ pL promoter, to give strain B1002, by selection for transformants resistant to kanamycine
- Frozen cells were thawed and resuspended in 10 ml of PBS buffer.Cells were broken in a French Pressure cell press SLM Aminco at 20 000 psi (three passages). The extract was centrifuged at 16000 g for 30 minutes at 4° C.
- the level of expression represents about 3 to5% of total protein.
- the P3 fraction is store at ⁇ 20° C. waiting for the next purification step.
- the P3 fraction is thawed and injected in a packed and equilibrated Zn-chelating sepharose FF.
- the column is the washed with EDTA 50 mM and NaOH 0.5M.
- Zn chelating sepharose eluate (Zn-500) is stored between 2-8° c. before the next purification step.
- the Zn-500 fraction is injected in a packed and equilibrated Q-sepharose FF.
- QS-sepharose eluate (QS-500) is stored between 2-8°c before the next purification step.
- the QS-500 fraction is then treated on a 10 kD utrafiltration unit (Ultrasette—Pall Filtron)
- the product is first concentrated to around 1 mg/mL of protein and then diafiltrated against 10 volumes of phosphate buffer.
- the final concentration is between 0.5 and 1.0 ⁇ g/mL.
- the sterile bulk is stored at ⁇ 20° C.
- Lyta is derived from Streptococcus pneumoniae which synthesize an N-acetyl-L-alanine amidase, amidase LYTA, (coded by the lytA gene ⁇ Gene, 43 (1986) pag 265-272 ⁇ an autolysin that specifically degrades certain bonds in the peptidoglycan backbone.
- the C-terminal domain of the LYTA protein is responsible for the affinity to the choline or to some choline analogues such as DEAE.
- the first step was the purification of the large NcoI-AflII restriction fragment from plasmid pRIT14497 and the purification of the small AflII-AflIII restriction fragment from pRIT14661
- Plasmid pRIT14634 was introduced into E. coli AR58 (Mott et al., 1985, Proc. Natl. Acad. Sci., 82:88) a defective ⁇ lysogen containing a thermosensitive repressor of the ⁇ pL promoter.
- Cells of AR58 transformed with plasmid pRIT14634 were grown in 100 ml of LB medium supplemented with 50 ⁇ gr/ml of Kanamycin at 30° C. During the logarithmic phase of growth bacteria were shifted to 39° C. to inactivate the ⁇ repressor and turn on the synthesis of protein clyta-E6-his. The incubation at 39° C. was continued for 4 hours. Bacteria were pelleted and stored at ⁇ 20° C.
- Frozen cells were thawed and resuspended in 10 ml of PBS buffer. Cells were broken in a French pressure cell press SLM Aminco at 20.000 psi (three passages). The extract was centrifuged at 16.000 g for 30 minutes at 4° C. After centrifugation of extracts described above, aliquots of supernatant and pellet were analysed by SDS-polyacrylamide gel electrophoresis and Western blotting.
- the level of expression represents about 3% of total protein.
- the first step was the purification of the large NcoI-AflIII restriction fragment from plasmid pRIT14501 and the purification of the small AflII-AflIII restriction fragment from pRIT14661
- Cells of AR58 transformed with plasmid pRIT14626 were grown in 100 ml of LB medium supplemented with 50 ⁇ gr/ml of Kanamycin at 30° C. During the logarithmic phase of growth bacteria were shifted to 39° C. to inactivate the ⁇ repressor and turn on the synthesis of protein clyta-E7-his. The incubation at 39° C. was continued for 4 hours. Bacteria were pelleted and stored at ⁇ 20° C.
- Frozen cells were thawed and resuspended in 10 ml of PBS buffer. Cells were broken in a French pressure cell press SLM Aminco at 20.000 psi (three passages). The extract was centrifuged at 16.000 g for 30 minutes at 4° C. After centrifugation of extracts described above, aliquots of supernatant and pellet were analysed by SDS-polyacrylamide gel electrophoresis and Western blotting.
- the level of expression represents about 5% of total protein.
- the first step was the purification of the large NcoI-AflII restriction fragment from plasmid pRIT14512 and the purification of the small AflII-AflIII restriction fragment from pRIT14661
- Plasmid pRIT14629 was introduced into E. coli AR58 (Mott et al., 1985, Proc. Natl. Acad. Sci., 82:88) a defective ⁇ lysogen containing a thermosensitive repressor of the ⁇ pL promoter.
- Cells of AR58 transformed with plasmid pRIT14629 were grown in 100 ml of LB medium supplemented with 50 ⁇ gr/ml of Kanamycin at 30° C. During the logarithmic phase of growth bacteria were shifted to 39° C. to inactivate the ⁇ repressor and turn on the synthesis of protein clyta-E6E7-his. The incubation at 39° C. was continued for 4 hours. Bacteria were pelleted and stored at ⁇ 20° C.
- Frozen cells were thawed and resuspended in 10 ml of PBS buffer. Cells were broken in a French pressure cell press SLM Aminco at 20.000 psi (three passages). The extract was centrifuged at 16.000 g for 30 minutes at 4° C.
- the level of expression represents about 1% of total protein.
- Prot-D1/3 The sequence of Prot-D1/3 is followed by a multiple cloning site (11 residues) and a coding region for a C-terminal histidine tail (6 His) (see FIG. 15). This plasmid is used to express the fusion protein D1/3-E7-his.
- the insert was sequenced and a modification versus E7/HPV18 prototype sequence was identified in E7 gene (nucleotide 128 G ⁇ A) generating a substitution of a glycine by a glutamic acid (aa 43 in E7, position 156 in fusion protein).
- the sequence for the fusion protein-D1/3-E7-His/HPV18 is described in FIG. 16.
- Plasmid pRIT14532 was introduced into E. coli AR58 (Mott et al., 1985, Proc. Natl. Acad. Sci., 82:88) a defective ⁇ lysogen containing a thermosensitive repressor of the ⁇ pL promoter, by selection for transformants resistant to kanamycine.
- Plasmid pRIT14532 and pRIT14523 were introduced into E. coli AR58 (Mott et al., 1985, Proc. Natl. Acad. Sci., 82:88) a defective ⁇ lysogen containing a thermosensitive repressor of the ⁇ pL promoter, by double selection for transformants resistant to kanamycin and ampicillin.
- Frozen cells are thawed and resuspended in 10 ml of PBS buffer. Cells are broken in a French pressure cell press SLM Aminco at 20.000 psi (three passages). The extract is centrifuged at 16.000 g for 30 minutes at 4° C.
- the fusion protD1/3-E7-His (about 31 kDa) was visualised by Coomassie stained gels in the pellet fraction for strain B1011 and partially localized (30%) in the supernatant fraction for strain 1012 and was identified in Western blots by rabbit polyclonal anti-protein-D and by Ni-NTA conjugate coupled to calf intestinal alkaline phosphatase (Qiagen cat. n° 34510) which detects accessible histidine tail.
- the level of expression represents about 1-3% of total protein as shown on a Coomassie-stained SDS-polyacrylamide gel.
- Purified antigen is stable one week at ⁇ 20° C. and 4° C. (no degradation) but appears more susceptible to oxidation after incubation at 37° C.
- Protein solubility is pH dependent (see below) with decrease of solubility for pH ⁇ 7.4: PBS pH 7.4 686 ⁇ g/ml 100% PBS pH 7.2 560 ⁇ g/ml 81% PBS pH 7.0 498 ⁇ g/ml 72% PBS pH 6.8 327 ⁇ g/ml 48%
- HPV 18 Prot D1/3 E7 proteiin is composed of 227 amino acids. Its theoretical molecular weight is 25.9 kDa, and a theoretical isoelectric point of 5.83. It migrates at about 31.5 kDa in reducing SDS PAGE.
- Cell paste is suspended to 60 OD 600 in 2 M NaCl, 20 mM Phosphate. (NaH 2 PO4/K 2 HPO4) pH 7.6 prior cell lysis by two passes through a Rannie disruptor. Lysed cells are pelleted 30 min at 9,000 rpm in a JA 10 rotor at 4° C.
- bacterial cell pellet containing the recombinant protein is washed once in 5 mM EDTA, 2 M NaCl, PBS pH 7.4; once in 4 M urea, 20 mM Phosphate pH 7.4 and finally once in PBS pH 7.4 to eliminate trace of EDTA (each wash is performed in twice volume used for cell suspension).
- HPV18Prot.D1/3-E7-His TIT for Thioredoxin In Trans
- Cell debris are pelleted 30 min at 9,000 rpm in a JA 10 rotor at 4° C.
- Supernatant is supplemented with 0.5% Empigen BB and incubated 30 min at RT.
- Antigen is eluted by 0.25 M-Imidazole in 6 M urea, 0.5 M NaCl, 50 mM PO4 pH 7.6, at 2 ml/min (FIG. 1B). IMAC-eluted sample is dialyzed at 4° C. versus PBS pH 7.4
- Purified antigen is stable one week at ⁇ 20° C. and 4° C. (no degradation) but appears more susceptible to oxidation after incubation at 37° C.
- Protein solubility is pH dependent (see below) with decrease of solubility for pH ⁇ 7.4: PBS pH 7.4 686 ⁇ g/ml 100% PBS pH 7.2 560 ⁇ g/ml 81% PBS pH 7.0 498 ⁇ g/ml 72% PBS pH 6.8 327 ⁇ g/ml 48%
- HPV18-ProtD1/3-E7-His protein is composed of 227 amino acids. Its theoretical molecular weight is 25.9 kDa. It migrates at about 31.5 kDa in reducing SDS-PAGE. Theoretical isoelectric point is 5.83.
- Plasmid pRIT 14831 was introduced into E.coli AR58 (Mott et al., 1985, Proc. Natl. Acad. Sci., 82:88) a defective ⁇ lysogen containing a thermosensitive repressor of the ⁇ pL promoter, to give strain B1098, by selection for transformants resistant to kanamycin.
- Frozen cells were thawed and resuspended in 10 ml of PBS buffer. Cells were broken in a French Pressure cell press SLM Aminco at 20 000 psi (three passages). The extract was centrifuged at 16000 g for 30 minutes at 4° C.
- Plasmid pMG MCS prot D1/3 is a derivative of pMG81 (described supra) in which the codons 4-81 of NS1 coding region from Influenza were replaced by the codons corresponding to residues Ser 20 ⁇ Thr 127 of mature protein D of Haemophilus Influenzae strain 772, biotype 2 (H. Janson et al., 1991, Infection and Immunity, Jan. p.119-125).
- the sequence of Prot-D 1/3 is followed by a multiple cloning site (11 residues) and a coding region for a C-terminal histidine tail (6 His). This plasmid is used to express the fusion protein D1/3-E6-his.
- Plasmid pRIT14526 was introduced into E. coli AR58 (Mott et al., 1985, Proc. Natl. Acad. Sci., 82:88) a defective ⁇ lysogen containing a thermosensitive repressor of the ⁇ pL promoter.
- Cells of AR58 transformed with plasmid pRIT14526 were grown in 100 ml of LB medium supplemented with 50 ⁇ gr/ml of Kanamycin at 30° C. During the logarithmic phase of growth bacteria were shifted to 39° C. to inactivate the ⁇ repressor and turn on the synthesis of protein D1/3-E6-his. The incubation at 39° C. was continued for 4 hours. Bacteria were pelleted and stored at ⁇ 20C.
- Frozen cells are thawed and resuspended in 10 ml of PBS buffer. Cells are broken in a French pressure cell press SLM Aminco at 20.000 psi (three passages). The extract is centrifuged at 16.000 g for 30 minutes at 4° C. After centrifugation of extracts described above, aliquots of supernatant and pellet were analysed by SDS-polyacrylamide gel electrophoresis and Western blotting.
- the level of expression represents about 3-5% of total protein.
- Plasmid pRIT14567 was introduced into E. coli AR58 (Mott et al., 1985, Proc. Natl. Acad. Sci., 82:88) a defective ⁇ lysogen containing a thermosensitive repressor of the ⁇ pL promoter.
- Cells of AR58 transformed with plasmid pRIT14512 were grown in 100 ml of LB medium supplemented with 50 ⁇ gr/ml of Kanamycin at 30° C. During the logarithmic phase of growth bacteria were shifted to 39° C. to inactivate the ⁇ repressor and turn on the synthesis of protein D1/3-E6E7-his. The incubation at 39° C. was continued for 4 hours. Bacteria were pelleted and stored at ⁇ 20C.
- Frozen cells are thawed and resuspended in 10 ml of PBS buffer. Cells are broken in a French pressure cell press SLM Aminco at 20.000 psi (three passages). The extract is centrifuged at 16.000 g for 30 minutes at 4° C.
- Vaccines are formulated with a Protein from the above examples expressed in E. coli from the strain AR58, and as adjuvant, the formulation comprising a mixture of 3 de -O-acylated monophosphoryl lipid A (3D-MPL) and aluminium hydroxide or 3D-MPL and/or QS21 optionally in an oil/water emulsion, and optionally formulated with cholesterol.
- 3D-MPL 3 de -O-acylated monophosphoryl lipid A
- QS21 aluminium hydroxide or 3D-MPL and/or QS21 optionally in an oil/water emulsion, and optionally formulated with cholesterol.
- 3D-MPL is a chemically detoxified form of the lipopolysaccharide (LPS) of the Gram-negative bacteria Salmonella minnesota.
- LPS lipopolysaccharide
- QS21 is one saponin purified from a crude extract of the bark of the Quillaja Saponaria Molina tree, which has a strong adjuvant activity: it activates both antigen-specific lymphoproliferation and CTLs to several antigens.
- Vaccine containing an antigen of the invention containing 3D-MPL and alum may be prepared in analogous manner to that described in WO93/19780 or 92/16231.
- the oil/water emulsion is composed of 2 oils (a tocopherol and squalene), and of PBS containing Tween 80 as emulsifier.
- the emulsion comprised 5% squalene 5% tocopherol 0.4% Tween 80 and had an average particle size of 180 nm and is known as SB62 (see WO 95/17210).
- Tween 80 is dissolved in phosphate buffered saline (PBS) to give a 2% solution in the PBS.
- PBS phosphate buffered saline
- To provide 100 ml two fold concentrate emulsion 5 g of DL alpha tocopherol and 5 ml of squalene are vortexed to mix thoroughly.
- 90 ml of PBS/Tween solution is added and mixed thoroughly.
- the resulting emulsion is then passed through a syringe and finally microfluidised by using an M110S microfluidics machine.
- the resulting oil droplets have a size of approximately 180 nm.
- ProtD1/3-E7 (5 ⁇ g) was diluted in 10 fold concentrated PBS pH 6.8 and H 2 O before consecutive addition of SB62, 3 D MPL (5 ⁇ g), QS21 (5 ⁇ g) and 50 ⁇ g/ml thiomersal as preservative at 5 min interval.
- the emulsion volume is equal to 50% of the total volume (50 ⁇ l for a dose of 100 ⁇ l). All incubations were carried out at room temperature with agitation. The adjuvants controls without antigen were prepared by replacing the protein by PBS.
- Protein D is a lipoprotein exposed on the surface of the Gram-negative bacteria Haemophilus influenzae.
- Tumour cell line TC1 In vivo Tumour Regression Experiments Tumour cell line TC1:
- TC 1 cells growing in vitro culture were trypsinised, washed two times in serum-free medium and were injected S.C. in the right flank of the mice.
- TC1 cells were injected at a dose of 3 ⁇ 10e4 cells/mouse.
- mice were vaccinated with 5 ⁇ g in 100 ⁇ l of protD 1/3 E7 His intra foot pad (50 ⁇ l/foot pad) in PBS or in the 3D-MPL, QS21 and SB62 or with PBS or with the adjuvant alone.
- Five C57BL/6 mice (Iffa Credo) were used in each group. Mice were monitored twice a week for tumour growth.
- mice vaccinated with protD 1/3 E7 His in PBS or with PBS or the adjuvant alone developed progressively growing tumours (0-1 tumour-free animal/group).
- protD1/3 E7 His in adjuvant did not develop a tumour, one animal developed a very small and stable tumour at day 40.
- This results indicate that the protein protD1/3 E7 His from HPV 16 formulated in adjuvant is able to induce the regression of small established tumours expressing this antigen.
- Lymphocytes were prepared by crushing the spleen or the popliteal lymph nodes from the vaccinated mice at day 69.
- FIGS. 27 and 28 compares the ability of protD E7 to stimulate the proliferation of splenocytes and lymph node cells primed in vivo either by PBS, 3D-MPL, QS21 SB62, ProtD1/3 E7 His and ProtD1/3 E7 His+the adjuvant of 3D-MPL, QS21, SB62 and shows that high proliferative responses in spleen were detected only in mice immunised with protD1/3 E7 His in adjuvant compared to the other groups.
- biotinylated goat Anti mouse Ig ( ⁇ fraction (1/1000) ⁇ ) or goat anti mouse Ig subclass (total IgG, IgGl, IgG2a, IgG2b) antisera (1/5000) were used as second antibodies, after an incubation of 90 min at 37° C., streptavidin coupled to peroxydase was added and TMB (tetra-methyl-benzidine/peroxide) was used as substrate, after 10 min. the reaction was stopped with H2SO4 0.5 M and the O.D.450 was determined.
- IgG1, IgG2a, and IgG2b concentration of all types of IgG subclass analyzed
- IgG1, IgG2a, and IgG2b concentration of all types of IgG subclass analyzed
- the predominant isotype found was IgG2b which represented more than 80% of the total of IgG), this isotype is generally said to be associated with the induction of a TH1 type immune response.
- mice were immunised 2 times at 14 days interval with either PBS, adjuvant of example 1, 5 ⁇ g of protD1/3 E7 His or 5 ⁇ g of protD1/3 E7 His in the adjuvant of example 1 intra foot pad in a volume of 100 ⁇ l (50 ⁇ l/foot pad).
- mice Four weeks after the latest vaccination mice were challenged with 2 ⁇ 10e5 TC1 cells/mouse S.C. in the flank. TC1 cells growing in vitro culture, were trypsinised and washed two times in serum-free medium and injected. 5 mice used in each group were monitored twice a week for tumour growth.
- FIG. 30 shows that vaccination with the E7 protein in the SB62 QS21, 3D-MPL adjuvant protect the mice against the development of tumour (only one animal/5 has a very small and stable tumour) in all the other groups, that received the E7 protein without the adjuvant or the adjuvant alone developed growing tumours.
- Lymphocytes were prepared as described above from the spleen and from the popliteal draining lymph nodes. An aliquot of 2 ⁇ 10e5 cells was plated in triplicate in 96 well plates with decreasing concentrations (10, 1, 0.1 ⁇ g/ml) of protD 1/3 E7 His coated or not onto latex microbeads (Sigma) to restimulate the cells in vitro (72 Hrs). T cell proliferation was measured by 3H thymidine incorporation.
- FIGS. 31 and 32 show repectively that, both with splenocytes or popliteal lymph node cells, as it was observed in the therapeutic settings, a better lymphoproliferative activity was obtained for the mice that received the E7 protein in the SB62 QS21, 3D-MPL adjuvant antibody response.
- FIG. 33 shows that as in the therapeutic settings, a better antibody response was observed in the serum of mice vaccinated with the ProtD1/3 E7 protein formulated in the 3D-MPL, QS21 O/W adjuvant.
- a mixed antibody response was triggered, as all the IgG subclass tested (IgG 2a, IgG2b, IgG1), in this case also, IgG2b was the predominant isotype found, representing 75% of the total IgG.
- mice were vaccinated twice, 2 weeks apart, with 5 ⁇ g in 100 ⁇ l of protD 1/3 18 E7 His intra foot pad (50 ⁇ l/foot pad) in PBS or QS21, 3D-MPL and SB62, DQ MPL as described in WO96/33739 or DQ alum MPL as described in WO98/15827. Eight 6-8 weeks old Balb/c mice (Iffa Credo) were used in each group.
- lymphocytes were prepared by crushing the spleen or the popliteal lymph nodes from the vaccinated mice at day 28
- FIGS. 34 and 35 compares the ability of protD 1/3 18 E7 to stimulate the proliferation of splenocytes or lymph node cells primed in vivo either by ProtD1/3 18 E7 His or Prot D1/3 18 E7 His+adjuvant and shows that only a basal lymphoproliferation is seen in mice that received the protein alone, on the contrary, high proliferative responses in spleen and very high responses in lymph nodes were detected in mice immunised with protD1/3 18 E7 His in adjuvant.
- cytokines IL-5 and IFNg
- IL-5 and IFNg The cytokines produced in the culture supernatant after a 96 Hrs period of in vitro re-stimulation of spleen or lymph node cells, with medium or with the ProtD1/3 18E7 (1 or 3 ⁇ g/ml) was measured by ELISA as described:
- IL5 Quantitation of IL5 was performed by Elisa using reagents from Pharmingen. Samples and antibody solutions were used at 50 ⁇ l per well. 96-well microtiter plates (Maxisorb Immuno-plate, Nunc, Denmark) were coated overnight at 4° c. with 50 ⁇ l of rat anti-mouse IL5 diluted at 1 ⁇ g/ml in carbonate buffer pH 9.5. Plates were then incubated for lhr at 37° c. with 100 ⁇ l PBS containing 1% bovine serum albumin and 0.1% Tween 20 (saturation buffer).
- lymph node cells a very weak IFNg production was obtained in the group of mice that received the protein alone and a 5-10 fold increase is observed with the adjuvanted protein. IL5 could only be detected in the group of mice receiving the SBAS2 adjuvanted protein.
- FIGS. 36 and 37 compares the ability of ProtD1/3 18 E7 His to stimulate the production of cytokines 'IFNg and IL5) after in vitro re-stimulation of spleen or lymph node cells respectively.
- biotinylated goat Anti mouse Ig ( ⁇ fraction (1/1000) ⁇ ) or goat anti mouse Ig subclass (total IgG, IgGI, IgG2a, IgG2b) antisera ( ⁇ fraction (1/5000) ⁇ ) were used as second antibodies, after an incubation of 90 min at 37° C., streptavidin coupled to peroxydase was added and TMB (tetra-methyl-benzidine/peroxide) was used as substrate, after 10 min. the reaction was stopped with H2SO4 0.5 M and the O.D.450 was determined.
- FIG. 38 The comparison of the midpoint dilution of the serum and relative percentage of the different isotypes elicited by the vaccinations in the different groups of mice are shown.
- ProtD1/3 E7 HPV16 protein in adjuvant is capable of enhancing helper T cell proliferation suggesting that the antitumour immune response induced by this vaccine is at least in part associated with a CD4+ T cell response.
Abstract
The present invention relates to fusions proteins, comprising a protein or part of a protein that provides T helper epitopes and an antigen from a human-papilloma virus. In particular the invention relates to fusion proteins comprising an E6 or E7 protein from HPV strain 16 or 18 linked to protein D from Haemophilus influenza B. The invention also provides vaccine compositions that are useful in the treatment or prophylaxis of human papilloma induced tumors.
Description
- The present invention relates to fusions proteins, comprising a protein or part of a protein that provides T helper epitopes and an antigen from a human-papilloma virus that find utility in the treatment or prophylaxis of human papilloma induced tumours. In particular the invention relates to fusion proteins comprising an E6 or E7 protein from
HPV strain - Papillomaviruses are small naked DNA tumour viruses (7.9 kilobases, double strand), which are highly species-specific. Over 70 individual human papillomavirus (HPV) genotypes have been described. Papillomaviruses are classified on the basis of species of origin (human, bovine etc.) and of the degree of genetic relatedness with other papillomaviruses from the same species. HPVs are generally specific for the skin or mucosal surfaces and have been broadly classified into “low” and “high” risk on the basis of rare and common, respectively, detection in abnormal or tumour tissue. Low risk HPVs usually cause benign lesions (warts or papillomas) that persist for several months or years. High risk HPVs are associated with cancer. The strongest positive association between an HPV virus and human cancer is that which exist between
HPV - Genital HPV infection in young sexually active women is common and most individuals either clear the infection, or if lesions develop, these regress. Only a subset of infected individuals has lesions which progress to high grade intraephithelial neoplasia and only a fraction of these progress further to invasive carcinoma.
- The molecular events leading to HPV infection have not been clearly established. The lack of an adequate in vitro system to propagate human papillomaviruses has hampered the progress to a best information about the viral cycle.
- Today, the different types of HPVs have been isolated and characterised with the help of cloning systems in bacteria and more recently by PCR amplification. The molecular organisation of the HPV genomes has been defined on a comparative basis with that of the well characterised bovine papillomavirus type 1 (BPV 1).
- Although minor variations do occur, all HPVs genomes described have at least seven early genes, E1 to E7 and two late genes L1 and L2. In addition, an upstream regulatory region harbors the regulatory sequences which appears to control most transcriptional events of the HPV genome.
- E1 and E2 genes are involved in viral replication and transcriptional control, respectively and tend to be disrupted by viral integration. E6 and E7 are involved in viral transformation. E5 has also been implicated in this process.
- In the HPVs involved in cervical carcinoma such as
HPV - Carcinoma of the cervix is common in women and develops through a pre-cancerous intermediate stage to the invasive carcinoma which frequently leads to death. The intermediate stages of the disease is known as cervical intraepithelial neoplasia and is graded I to III in terms of increasing severity (CIN I-III).
- Clinically, HPV infection of the female anogenital tract manifests as cervical flat condylomas, the hallmark of which is the koilocytosis affecting predominantly the superficial and intermediate cells of the cervical squamous epithelium.
- Koilocytes which are the consequence of a cytopathic effect of the virus, appear as multinucleated cells with a perinuclear clear haloe. The epithelium is thickened with abnormal keratinisation responsible for the warty appearance of the lesion.
- Such flat condylomas when positive for the
HPV - The natural history of oncogenic HPV infection presents 3 consecutive phases, namely:
- (1) a latent infection phase,
- (2) a phase of intranuclear viral replication with product of complete virions, which corresponds to the occurrence of koilocytes. At this stage, the HPV is producing its full range of proteins including E2, E5, E6, E7, L1 and L2.
- (3) a phase of viral integration into the cellular genome, which triggers the onset of malignant transformation, and corresponds to CIN II and CIN III/CIS with progressive disappearance of koilocytes. At this stage, the expression of E2 is down-regulated, the expression of E6 and E7 is enhanced. Between CIN II/III and CIN III/Cervix carcinoma the viral DNA changes from being episomal in the basal cells to integration of E6 and E7 genes only (tumoral cells). 85% of all cervix carcinomas are squamos cell carcinomas most predominantly related to the HPV16 serotype. 10% and 5% are adenocarcinomas and adenosquamos cell carcinomas respectively, and both types are predominantly related to
HPV 18 serotype. Nevertheless other oncogenic HPV's exist. - International Patent Application No.
WO 96/19496 discloses variants of human papilloma virus E6 and E7 proteins, particularly fusion proteins of E6/E7 with a deletion in both the E6 and E7 proteins. These deletion fusion proteins are said to be immunogenic. - The present invention provides compositions comprising either an E6 or E7 or an E6/E7 fusion protein linked to an immunological fusion partner having T cell epitopes.
- In a preferred form of the invention, the immunological fusion partner is derived from protein D of Heamophilus influenza B. Preferably the protein D derivative comprises approximately the first {fraction (1/3)} of the protein, in particular approximately the first N-terminal 100-110 amino acids. The protein D may be lipidated (Lipo Protein D). Other immunological fusion partners include the non-structural protein from influenzae virus, NS1 (hemagglutinin). Typically the
N terminal 81 amino acids are utilised, although different fragments may be used provided they include T-helper epitopes. - In another embodiment the immunological fusion partner is the protein known as LYTA. Preferably the C terminal portion of the molecule is used. Lyta is derived fromStreptococcus pneumoniae which synthesize an N-acetyl-L-alanine amidase, amidase LYTA, (coded by the lytA gen {Gene, 43 (1986) page 265-272} an autolysin that specifically degrades certain bonds in the peptidoglycan backbone. The C-terminal domain of the LYTA protein is responsible for the affinity to the choline or to some choline analogues such as DEAE. This property has been exploited for the development of E.coli C-LYTA expressing plasmids useful for expression of fusion proteins. Purification of hybrid proteins containing the C-LYTA fragment at its amino terminus has been described {Biotechnology: 10, (1992) page 795-798}. As used herein a preferred embodiment utilises the repeat portion of the Lyta molecule found in the C terminal end starting at residue 178. A particularly preferred form incorporates residues 188-305.
- Accordingly, the present invention in preferred embodiment provides fusion proteins comprising Protein D-E6 from
HPV 16, Protein D-E7 fromHPV 16 Protein D-E7 fromHPV 18, Protein D-E6 fromHPV 18, and Protein D E6 E7 from bothHPV - The proteins of the present invention preferably are expressed inE. coli. In a preferred embodiment the proteins are expressed with a Histidine tail comprising between 5 to 9 and preferably six Histidine residues. These are advantageous in aiding purification.
- The protein E7 may in a preferred embodiment carry a mutation to reduce the binding for the rb site (retinoblastoma gene product) and hence eliminate any potential transforming capacity. Preferred mutations for
HPV 16 E7 involve replacing Cys24 with Glycine, or Glutamic acid26 with Glutamine. In a preferred embodiment the E7 protein contains both these mutations. - Preferred mutations for the HPV 18 E7 involve replacing Cys27 with Glycine and/or Glutamic acid26 with Glutamine. Again preferably both mutations are present.
- Single or double mutations may also be introduced p53 region of E6 to eliminate any potential transforming ability.
- In a further embodiment of the invention there is provided and E6 E7 fusion protein from HPV linked to an immunological fusion partner. A preferred Immunological fusion partner is Protein D, more preferable the first {fraction (1/3)} of protein D.
- The present invention also provides a DNA encoding the proteins of the present invention. Such sequences can be inserted into a suitable expression vector and expressed in a suitable host.
- A DNA sequence encoding the proteins of the present invention can be synthesized using standard DNA synthesis techniques, such as by enzymatic ligation as described by D. M. Roberts et al. in Biochemistry 1985, 24, 5090-5098, by chemical synthesis, by in vitro enzymatic polymerization, or by PCR technology utilising for example a heat stable polymerase, or by a combination of these techniques.
- Enzymatic polymerisation of DNA may be carried out in vitro using a DNA polymerase such as DNA polymerase I (Klenow fragment) in an appropriate buffer containing the nucleoside triphosphates dATP, dCTP, dGTP and dTTP as required at a temperature of 10°-37° C., generally in a volume of 50 μl or less. Enzymatic ligation of DNA fragments may be carried out using a DNA ligase such as T4 DNA ligase in an appropriate buffer, such as 0.05M Tris (pH 7.4), 0.01M MgCl2, 0.01M dithiothreitol, 1 mM spermidine, 1 mM ATP and 0.1 mg/ml bovine serum albumin, at a temperature of 4° C. to ambient, generally in a volume of 50 ml or less. The chemical synthesis of the DNA polymer or fragments may be carried out by conventional phosphotriester, phosphite or phosphoramidite chemistry, using solid phase techniques such as those described in ‘Chemical and Enzymatic Synthesis of Gene Fragments—A Laboratory Manual’ (ed. H. G. Gassen and A. Lang), Verlag Chemie, Weinheim (1982), or in other scientific publications, for example M. J. Gait, H. W. D. Matthes, M. Singh, B. S. Sproat, and R. C. Titmas, Nucleic Acids Research, 1982, 10, 6243; B. S. Sproat, and W. Bannwarth, Tetrahedron Letters, 1983, 24, 5771; M. D. Matteucci and M. H. Caruthers, Tetrahedron Letters, 1980, 21, 719; M. D. Matteucci and M. H. Caruthers, Journal of the American Chemical Society, 1981, 103, 3185; S. P. Adams et al., Journal of the American Chemical Society, 1983, 105, 661; N. D. Sinha, J. Biernat, J. McMannus, and H. Koester, Nucleic Acids Research, 1984, 12, 4539; and H. W. D. Matthes et al., EMBO Journal, 1984, 3, 801.
- The process of the invention may be performed by conventional recombinant techniques such as described in Maniatis et al., Molecular Cloning—A Laboratory Manual; Cold Spring Harbor, 1982-1989.
- In particular, the process may comprise the steps of:
- i) preparing a replicable or integrating expression vector capable, in a host cell, of expressing a DNA polymer comprising a nucleotide sequence that encodes the protein or an immunogenic derivative thereof;
- ii) transforming a host cell with said vector;
- iii) culturing said transformed host cell under conditions permitting expression of said DNA polymer to produce said protein; and
- iv) recovering said protein.
- The term ‘transforming’ is used herein to mean the introduction of foreign DNA into a host cell. This can be achieved for example by transformation, transfection or infection with an appropriate plasmid or viral vector using e.g. conventional techniques as described in Genetic Engineering; Eds. S. M. Kingsman and A. J. Kingsman; Blackwell Scientific Publications; Oxford, England, 1988. The term ‘transformed’ or ‘transformant’ will hereafter apply to the resulting host cell containing and expressing the foreign gene of interest.
- Preferably recombinant antigen of the invention are expressed inE. coli. The expression strategy include fusion of E7, E6 or E6/E7 fusion to the 1/3-N-terminal portion of protein D from Haemophilus influenzae B, an immunological fusion partner providing T cell helper epitopes. An affinity polyhistidine tail is engineered at the carboxy terminus of the fusion protein allowing for simplified purification. Such recombinant antigen is overexpressed in E. coli as insoluble protein.
- Preferably the proteins of the invention are coexpressed with thioredoxin in trans (TIT). Coexpression of thioredoxin in trans versus in cis is preferred to keep antigen free of thioredoxin without the need for protease. Thioredoxin coexpression eases the solubilisation of the proteins of the invention. Thioredoxin coexpression has also a significant impact on protein purification yield, on purified-protein solubility and quality.
- The expression vectors are novel and also form part of the invention.
- The replicable expression vectors may be prepared in accordance with the invention, by cleaving a vector compatible with the host cell to provide a linear DNA segment having an intact replicon, and combining said linear segment with one or more DNA molecules which, together with said linear segment encode the desired product, such as the DNA polymer encoding the protein of the invention, or derivative thereof, under ligating conditions.
- Thus, the DNA polymer may be preformed or formed during the construction of the vector, as desired.
- The choice of vector will be determined in part by the host cell, which may be prokaryotic or eukaryotic but preferably isE. coli. Suitable vectors include plasmids, bacteriophages, cosmids and recombinant viruses.
- The preparation of the replicable expression vector may be carried out conventionally with appropriate enzymes for restriction, polymerisation and ligation of the DNA, by procedures described in, for example, Maniatis et al. cited above.
- The recombinant host cell is prepared, in accordance with the invention, by transforming a host cell with a replicable expression vector of the invention under transforming conditions. Suitable transforming conditions are conventional and are described in, for example, Maniatis et al. cited above, or “DNA Cloning” Vol. II, D. M. Glover ed., IRL Press Ltd, 1985.
- The choice of transforming conditions is determined by the host cell. Thus, a bacterial host such asE. coli may be treated with a solution of CaCl2 (Cohen et al., Proc. Nat. Acad. Sci., 1973, 69, 2110) or with a solution comprising a mixture of RbCl, MnCl2, potassium acetate and glycerol, and then with 3-[N-morpholino]-propane-sulphonic acid, RbCl and glycerol. Mammalian cells in culture may be transformed by calcium co-precipitation of the vector DNA onto the cells. The invention also extends to a host cell transformed with a replicable expression vector of the invention.
- Culturing the transformed host cell under conditions permitting expression of the DNA polymer is carried out conventionally, as described in, for example, Maniatis et al. and “DNA Cloning” cited above. Thus, preferably the cell is supplied with nutrient and cultured at a temperature below 50° C.
- The product is recovered by conventional methods according to the host cell. Thus, where the host cell is bacterial, such asE. coli it may be lysed physically, chemically or enzymatically and the protein product isolated from the resulting lysate. Where the host cell is mammalian, the product may generally be isolated from the nutrient medium or from cell free extracts. Conventional protein isolation techniques include selective precipitation, adsorption chromatography, and affinity chromatography including a monoclonal antibody affinity column.
- When the proteins of the present invention are expressed with a hisitidine tail (His tag). The proteins can easily be purified by affinity chromatography using an ion metal affinity chromatography column (IMAC) column.
- A second chromatographic step, such as Q-sepharose may be utilised either before or after the IMAC column to yield highly purified protein. If the immunological fusion partner is C-LYTA, then it is possible to exploit the affinity of CLYTA for choline and/or DEAE to purify this product. Products containing both C-LYTA and his tags can be easily and efficiently purified in a two step process involving differential affinity chromatography. One step involves the affinity of the His tag to IMAC columns, the other involves the affinity of the C-terminal domain of LYTA for choline or DEAE.
- Proteins comprising both a C-LYTA and Hisitidine tag are new and accordingly form one aspect of the invention. These may be purified to high levels (greater than 80% preferably greater than 90%) by a simple two step differential affinity procedure.
- The proteins of the present invention are provided preferably at least 80% pure more preferably 90% pure as visualized by SDS PAGE. The protein present a major single band when analysed by SDS PAGE under reducing conditions, and western blot analysis show less than 5% host cell protein contamination.
- The present invention also provides pharmaceutical composition comprising a protein of the present invention in a pharmaceutically acceptable excipient. A preferred vaccine composition comprises at least Protein D-E6 from
HPV 16 or derivative thereof together with Protein D-E7 fromHPV 16. Alternatively the E6 and E7 may be presented in a single molecule, preferably a Protein D E6/E7 fusion. Such vaccine may optionally contain either or both E6 and E7 proteins fromHPV 18, preferably in the form of a Protein D-E6 or Protein D-E7 fusion protein or Protein D E6/E7 fusion protein. The vaccines of the present invention may contain other HPV antigens fromHPV HPV 6 11, HPV 31 or 33. - Vaccine preparation is generally described in Vaccine Design—The subunit and adjuvant approach (Ed. Powell and Newman) Pharmaceutical Biotechnology Vol. 6 Plenum Press 1995. Encapsulation within liposomes is described by Fullerton, U.S. Pat. No. 4,235,877.
- The proteins of the present invention are preferably adjuvanted in the vaccine formulation of the invention. Suitable adjuvants include an aluminium salt such as aluminium hydroxide gel (alum) or aluminium phosphate, but may also be a salt of calcium, iron or zinc, or may be an insoluble suspension of acylated tyrosine, or acylated sugars, cationically or anionically derivatised polysaccharides, or polyphosphazenes.
- In the formulation of the inventions it is preferred that the adjuvant composition induces a
preferential TH 1 response. Suitable adjuvant systems include, for example, a combination of monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipid A (3D-MPL) together with an aluminium salt. - An enhanced system involves the combination of a monophosphoryl lipid A and a saponin derivative particularly the combination of QS21 and 3D-MPL as disclosed in WO 94/00153, or a less reactogenic composition where the QS21 is quenched with cholesterol as disclosed in WO 96/33739.
- A particularly potent adjuvant formulation involving QS21, 3D-MPL & tocopherol in an oil in water emulsion is described in WO 95/17210 and is a preferred formulation.
- Accordingly in one embodiment of the present invention there is provided a vaccine comprising a protein D (or derivative thereof)—E6 or protein D (or derivative thereof)—E7 adjuvanted with a monophosphoryl lipid A or derivative thereof.
- Preferably the vaccine additionally comprises a saponin, more preferably QS21.
- Preferably the formulation additional comprises an oil in water emulsion and tocopherol. The present invention also provides a method for producing a vaccine formulation comprising mixing a protein of the present invention together with a pharmaceutically acceptable excipient, such as 3D-MPL.
- The invention will be further described by reference to the following examples:
- 1)—Construction of Expression Plasmid
- a)—Plasmid pMG MCS prot D1/3 (=pRIT14589) is a derivative of pMG81 (described in UK patent application n° 951 3261.9 published as WO97/01640) in which the codons 4-81 of
NS 1 coding region from Influenza were replaced by the codons corresponding to residues Ser 20→Thrl27 of mature protein D of Haemophilus Influenzae strain 772, biotype 2 (H. Janson et al., 1991, Infection and Immunity, Jan. p.119-125). The sequence of Prot-D1/3 is followed by a multiple cloning site (11 residues) and a coding region for a C-terminal histidine tail (6 His). This plasmid is used to express the fusion protein D1/3-E7-His. - b)—HPV genomic E6 and E7 sequences type HPV 16 (See Dorf et al., Virology 1985, 145, p. 181-185) were amplified from
HPV 16 full length genome cloned in pBR322 (obtained from Deutsches Krebsforschungszentrum (DKFZ), Referenzzentrum für human pathogen Papillomaviruses—D 69120—Heidelberg) and were subcloned into pUC19 to give TCA 301 (=pRIT14462). - Construction of Plasmid TCA 308 (=pRIT14501): a Plasmid Expressing the Fusion Protein-D1/3-E7-His
- The nucleotides sequences corresponding to
amino acids 1→98 of E7 protein are amplified from pRIT14462. During the polymerase chain reaction, NcoI and Spel restriction sites were generated at the 5′ and 3′ ends of the E7 sequences allowing insertion into the same sites of plasmid pMGMCS Prot D1/3 to give plasmid TCA308 (=pRIT14501). The insert was sequenced to verify that no modification had been generated during the polymerase chain reaction. The sequence for the fusion protein-D1/3-E7-His (HPV 16) is described in FIG. 1. - 2)—Transformation of AR58 Strain
- Plasmid pRIT14501 was introduced intoE. coli AR58 (Mott et al., 1985, Proc. Natl. Acad. Sci., 82:88) a defective λ lysogen containing a thermosensitive repressor of the λ pL promoter.
- 3)—Growth and Induction of Bacterial Strain—Expression of Prot -D1/3-E7-His
- Cells of AR58 transformed with plasmid pRIT14501 were grown in 100 ml of LB medium supplemented with 50 μgr/ml of Kanamycin at 30° C. During the logarithmic phase of growth bacteria were shifted to 39° C. to inactivate the λ repressor and turn on the synthesis of protein D1/3-E7-His. The incubation at 39° C. was continued for 4 hours. Bacteria were pelleted and stored at −20° C.
- Frozen cells are thawed and resuspended in 10 ml of PBS buffer. Cells are broken in a French pressure cell press SLM Aminco at 20.000 psi (three passages). The extract is centrifuged at 16.000 g for 30 minutes at 4° C.
- After centrifugation of extracts described above, aliquots of supernatant and pellet were analysed by SDS-polyacrylamide gel electrophoresis and Western blotting. A major band of about 33 kDa, localised in the pellet fraction, was visualised by Coomassie stained gels and identified in Western blots by rabbit polyclonal anti-protein-D and by Ni-NTA conjugate coupled to calf intestinal alkaline phosphatase (Qiagen cat. n° 34510) which detects accessible histidine tail. The level of expression represents about 5% of total protein as shown on a Coomassie-stained SDS-polyacrylamide gel.
- One liter culture of bacteria expressing protein-D1/3-E7-His, is centrifuged at 11,300 g for 30 min at 4° C. and cell pellet is kept at −80° C. until further treatment. After resuspension in 75 ml PBS buffer,E. coli cells are broken in a French pressure cell press (SLM Aminco®) at 20,000 psi. Lysed cells are pelleted by centrifugation at 17,000 g for 30 minutes. Pellet, containing the protein-D1/3-E7-His, is washed once in 30 ml of 2M NaCI, 50 mM Phosphate pH 7.5, then twice in 30
ml 50 mM Phosphate pH 7.5. Proteins are solubilised after 2 hours incubation of the pellet in 30 ml of 8 M urea, 50 mM phosphate pH 7.5 at RT. Cells debris are eliminated by 15 min centrifugation at 17,000 g, 4° C. Protein purification is carried out at RT°, 15 ml of solubilised protein are applied onto a 5 ml Ni2+NTA (Qiagen) resin (Pharmacia column XK 16/20) preequilibrated in 8M urea, 50 mM phosphate pH 7.5 at a flow rate of 0.2 ml/min. The column is washed in the same buffer until the absorbance at 280 nm reaches the base line. The protein is eluted with a 0-600 mM Imidazole gradient in 8M urea, 50 mM phosphate pH 7.5. The flow rate of these two last steps is brought to 1 ml/min. Eluted fractions are analysed by SDS polyacrylamide gel electrophoresis and by Western blotting. ProtD1/3-E7-His, visualised by Coomassie blue staining, by a polyclonal anti protein D or by a monoclonal anti E7 antibody, appears as a major single band at about 32 kDalton and is estimated as a 95% pure protein. No E. coli contaminants, traced with a polyclonal anti E. coli proteins antibody, are observed. - In order to eliminate urea, 9 ml of purified antigen, at 1.33 mg/ml (Bradford), is dialysed against 3 liters of PBS buffer overnight at RT° followed by a 4 hours dialysis against a fresh PBS buffer. 80% of urea free protein is recovered as soluble protein. To eliminate contaminating endotoxins, 6 ml of dialysed protein are incubated with 1 ml of Affiprep polymixin gel (Biorad), for 3 hours at 4° C. under gentle stirring. A second incubation with 500 μl of Affiprep polymixin resin is performed to minimise the endotoxin level to 8.8 EU/μg protein. After sterile filtration on a 0.22 μm filter device (Millex 0.22 GV, Millipore), prot-D1/3-E7-His at 0.665 mg/ml is assayed for stability. SDS PAGE analysis showed no evolution of the protein after 7 days incubation at −20° C., 4° C., RT° or37° C.
- 1. Construction of Expression Plasmid
- a) Plasmid pMG MCS prot D1/3 (=pRIT14589) is a derivative of pMG81 (described in WO97/01640 in which the codons 4-81 of NS1 coding region from Influenza were replaced by the codons corresponding to residues Ser 20→Thr 127 of mature protein D of Haemophilus Influenzae strain 772, biotype 2 (H. Janson et al., 1991, Infection and Immunity, Jan. p.119-125). The sequence of Prot-D1/3 is followed by a multiple cloning site (11 residues) and a coding region for a C-terminal histidine tail (6 His). This plasmid is used to express the fusion protein D1/3-E6-his.
- b) HPV genomic E6 and E7 sequences type HPV 16 (Seedorf et al., Virology 1985, 145, p.181-185) were amplified from HPV16 full length genome cloned in pBR322 (obtained from Deutsches Krebsforschungszentrum (DKFZ), Referenzzentrum für human pathogen Papillomaviruses—
- c) D 69120—Heidelberg) and were subcloned into pUC19 to give TCA 301 (=pRIT14462).
- Construction of Plasmid TCA 307 (=pRIT14497): a Plasmid Expressing the Fusion Protein-D1/3-E6-His/HPV16
- The nucleotides sequences corresponding to amino acid.
- 1→151 of E6 protein were amplified from
pRIT 14462. During the polymerase chain reaction, NcoI and Spel restriction sites were generated at the 5′ and 3′ ends of the E6 sequences allowing insertion into the same sites of plasmid pMGMCS Prot D1/3 to give plasmid TCA307 (=pRIT14497) (see FIG. 2). The insert was sequenced to verify that no modification had been generated during the polymerase chain reaction. The coding sequence for the fusion protein-D1/3-E6-His is described in FIG. 3. - 2. Transformation of AR58 Strain
- Plasmid pRIT14497 was introduced intoE. coli AR58 (Mott et al., 1985, Proc. Natl. Acad. Sci., 82:88) a defective λ lysogen containing a thermosensitive repressor of the λ pL promoter.
- 3. Growth and Induction of Bacterial Strain—Expression of Prot-D1/3-E6-His
- Cells of AR58 transformed with plasmid pRIT14497 were grown in 100 ml of LB medium supplemented with 50 μgr/ml of Kanamycin at 30° C. During the logarithmic phase of growth bacteria were shifted to 39° C. to inactivate the λ repressor and turn on the synthesis of protein D1/3-E6-his. The incubation at 39° C. was continued for 4 hours. Bacteria were pelleted and stored at −20C.
- 4. Characterization of Fusion Protein D1/3-E6-his (HPV 16) Preparation of Extracts
- Frozen cells are thawed and resuspended in 10 ml of PBS buffer. Cells are broken in a French pressure cell press SLM Aminco at 20.000 psi (three passages). The extract is centrifuged at 16.000 g for 30 minutes at 4° C.
- Analysis on Coomassie-Stained SDS-Polyacrylamide Gels and Western Blots
- After centrifugation of extracts described above, aliquots of supernatant and pellet were analysed by SDS-polyacrylamide gel electrophoresis and Western blotting.
- A major band of about 32 kDa, localized in the pellet fraction, was visualised by Coomassie stained gels and identified in Western blots by rabbit polyclonal anti-protein-D and by Ni-NTA conjugate coupled to calf intestinal alkaline phosphatase (Qiagen cat. n° 34510) which detects accessible histidine tail. The level of expression represents about 5% of total protein.
- 5. Coexpression with Thioredoxin
- In an analagons fashion to the expression of
prot D 1/3 E7 His from HPV 18 (example XIII) an E.coli strain AR58 was transformed with a plasmid encoding thioredoxin andprotein D 1/3 E7 His (HPV 16). - HPV-16 ProtD1/3 E6 recombinant antigen was expressed inE. coli (AR58). Expression strategy included fusion of E6 to the 1/3-N-terminal portion of protein D from Haemophilus influenzae, an immunological fusion partner providing T cell helper epitopes. An affinity polyhistidine tail was engineered at the carboxy terminus of the fusion protein. The recombinant antigen was overexpressed in E. coli as insoluble proteins.
- Solubilisation of the antigen required denaturing agents. In absence of denaturing agent, ProtD1/3-E6-His precipitated at neutral pH. To circumvent the solubility problems, co-expression of these proteins with Thioredoxin in Trans (TIT), a folding partner was carried out.
- Bacterial expressions are conducted in LB media in presence of 0.05 mg/ml of kanamycin at 30° C. plus 0.2 mg/ml of Ampicillin when Thioredoxin is coexpressed. Recombinant protein expression is thermally induced by transferring the cells to 42° C., when cell optical density (OD 600 nm) of 0.4 is reached. Protein expression is maintained for 4 hours. Purification was carried out according to the following protocol.
Cell Culture Pellet 60 OD 6001 mM pefabloc, 2 M NaCl, PBS pH 7.4 (Buffer A) French Press Disruptor Three passes 20,000 psi Centrifugation 17,000 g 30 min, 4° C.Pellet Washes 2 M NaCl, PBS pH 7.4 (Buffer B) x1 PBS pH 7.4 (Buffer C) x2 Centrifugation 17,000 g 30 min, 4° C.Pellet Solubilisation 6 M Guanidine Chloride, 20 mM PO4, pH 7.0 (Buffer D) Overnight at 4° C. Centrifugation 17,000 g 30 min, 4° C.Supernatant on IMAC Equilibration: 6 M Guanidine Chloride, 20 mM PO4, pH 7.0 (Buffer D) Elution: Imidazole steps (0.025 M, 0.1 M, 0.5 M) in 8 M Urea, 20 mM PO4, pH 7.0 Affiprep Polymyxin 8 M Urea, 20 mM PO4, pH 7.0 (Buffer E) 2 h RT° Dialysis 4 M Urea, 0.5 M Arginine, 150 mM NaCl, 10 mM PO4, pH 6.8 (Buffer I) 2 M Urea, 0.5 M Arginine, 150 mM NaCl, 10 mM PO4 pH 6.8, (Buffer J) 0 M Urea, 0.5 M Arginine, 150 mM NaCl, 10 mM PO4 pH 6.8 (Buffer K) - Cells are efficiently broken by high-pressure homogenisation using a French pressure cell device. Antigen is extracted with high concentration of protein denaturant. This first step breaks open the bacterial cell wall and antigen is extracted from the bacterial insoluble fraction. The following purification was carried out on 4 liter culture.
- Buffers
- A. PBS/2M NaCl/1 mM Pefabloc
- B. PBS/2MNaCl
- C. PBS: 137 mM NaCl, 2.7 mM KCl, 8.1 mM NaH2PO4, 1.47 mM KH2PO4 pH 7.4.
- D. 6 M Guanidium Chloride, 20 mM PO4 (NaH2PO4 (2H2O)/K2HPO4 (3H2O)) pH 7.0
- Starting material is 10 flasks of 400 ml culture each.
- Cell paste is suspended to 60 OD600 in Buffer A (240 ml of Buffer A in this case), prior cell lysis by three passes through a French press disrupter (20,000 psi). Lysed cells are pelleted 30 min at 15,000 g at 4° C. Bacterial cell pellet containing the recombinant protein is washed once in 240 ml Buffer B, then twice in 240 ml Buffer C.
- Prot D E6-His (TIT) is solubilised by 240 ml Buffer D overnight at 4° C. on a rotating wheel. Cell debris are pelleted 30 min at 15,000 g at 4° C. Supernatant (230 ml) is stored at −20° C. The material is then subjected to IMAC chromatography.
- The chelating ligand NTA (nitrilo-tri-acetic-acid) is attached to an Agarose support (Qiagen). NTA ligand is charged with nickel metal ion with which it interacts through 4 of the 6 coordination sites of the nickel. The two remaining coordination sites of nickel interact strongly with histidine residues of the 6× His-tagged protein. Elution is achieved by competition with Imidazole which bind to the Ni-NTA and displace the tagged antigen.
- Ni-NTA Agarose Qiagen (catalogue number: 30 250) was used.
- Solutions
D: 6 M Guanidium Chloride, 20 mM PO4 (NaH2PO4 (2H2O)/K2HPO4 (3H2O)), pH 7.0 E: 8 M Urea, 20 mM PO4 (NaH2PO4 (2H2O)/K2HPO4 (3H2O)), pH 7.0 F: E + 0.025 M Imidazole G: E + 0.1 M Imidazole H: E + 0.5 M Imidazole - 0.5 M NaOH
- Deionized water
- 0.02% NaN3
- Purification
- a) The resin (15 ml resin/230 ml sample) is packed and equilibrated in 10 column volumes (C.V.) of Buffer D at 15 cm h−1.
- b) Supernatant from solubilised fraction is injected onto the column at 15 cm h−1.
- c) Column is washed at 15 cm h−1 with buffer D until OD 280 nm returns to the baseline.
- d) Column is washed with 2 CV of Buffer E at 15 cm h−1. The wash fraction is recovered.
- e) Column is first eluted with 5 CV of Buffer F. Elimination of 25 kD major contaminant.
- f) Column is then eluted with 2 CV of Buffer G.
- g) Column is finally eluted with 3 CV of Buffer H. Elution of the antigen.
- Antigen positive fractions are pooled (30 ml).
- Endotoxin is removed by affiprep chromatography.
- Affi-Prep® Polymyxin support consists of USP Grade Polymyxin B coupled to the Affi-Prep® Matrix. Due to its high affinity to the lipid A moiety of endotoxins, polymixin B binds endotoxin molecules with high capacity and selectivity.
- Solutions
- E: 8M Urea, 20 mM PO4 (NaH2PO4 (2H2O)/K2HPO4 (3H2O)), pH 7.0 (apyrogenic).
- 0.5 M Na OH
- Deionized apyrogenic water
- Procedure
- 1) Affi-Prep® Polymyxin resin is washed in 10 volumes of 0.1 M NaOH, followed by 10 volumes of pyrogen free water.
- 2) Resin is equilibrated in 10 volumes of Buffer E.
- 3) 15 ml (half-pool) of IMAC-eluted sample is incubated with 3 ml of Affi-Prep® Polymyxin resin in a batch mode.
- 4) Incubation is pursued 4 hours at Room Temperature or O/N at 4° C. on a rotating wheel.
- 5) Sample is centrifuged 10 min at 2000 g (Beckman GS-6R).
- 6) Supernatant containing the antigen is collected and submitted to endotoxins and protein assays.
- 7) Resin is discarded.
- Small molecules diffuse through a semi-permeable membrane while large molecules are retained. The process of dialysis is driven by the difference in concentration of the solutes on the two sides of the membrane. New buffer solution is introduced until buffer composition on each side equalises.
- Buffers
I: 4 M Urea, 0.5 M Arginine, 0.15 M NaCl, 10 mM PO4 (NaH2PO4 (2H2O)/K2HPO4 (3H2O)) pH 6.8 J: 2 M Urea, 0.5 M Arginine, 0.15 M NaCl, 10 mM PO4 (NaH2PO4 (2H2O)/K2HPO4 (3H2O)) pH 6.8 K: 0 M Urea, 0.5 M Arginine, 0.15 M NaCl, 10 mM PO4 (NaH2PO4 (2H2O)/K2HPO4 (3H2O)) pH 6.8 - 1) The Sample (15 ml) is introduced into a dialysis tubing (20.4 mm diameter and 6 cm height).
- 2) Dialysis tubing is placed in a 2 liters cylinder containing Buffer I under stirring at 4° C. for 2 hours.
- 3) Dialysis tubing is placed in a 2 liters cylinder (under stirring) containing Buffer J; at 4° C. for 2 hours.
- 4) Dialysis tubing is placed in a 2 liters cylinder containing Buffer K (under stirring) at 4° C. O/N. Buffer is changed and dialysis is pursued 2 more hours at 4° C.
- Millipore Sterile Millex-GV 0.22μ, 13 mm. Catalogue number: SLGV0130S.
- All steps are performed at room temperature (RT≅22° C.), the antigen appears stable.
- Antigen solution is filtered through a 0.2 μm filter to prevent any bacterial growth. Antigen is kept at −20° C. in Nunc vials.
- Characterisation:
- Protein D1/3 E6 His is characterised as follows:
- ProteinD1/3-E6-His is a 273 amino acids long peptide with 112 amino acids coming from Protein D part. ProteinD1/3-E6-His has a theoretical Molecular Weight of 32 kD and migrates on SDS-PAGE as a 33 kD protein. ProteinD1/3-E6-His theoretical isoelectric point is 8.17.
- The viral Protein E6 is a basic protein containing 14 cystein residues, eight of them (
Cys 30,33,63,66 and Cys 103,106,136,139) are involved in two C-terminal zinc binding motifs. -
Protein D 1/3-E6-His is expressed as insoluble protein, in E. coli-AR 58 strain, with Thioredoxin in Trans, a folding partner. Cell culture is produced in 400 ml flask. - 5.4 mg of 95% pure protein is obtained per liter of culture.
- 1. Construction of Expression Plasmid
- a) Plasmid pMG MCS prot D1/3 (=pRIT14589) is a derivative of pMG81 (described Supra) in which the codons 4-81 of
NS 1 coding region from Influenza were replaced by the codons corresponding to residues Ser 20→Thr 127 of mature protein D of Haemophilus Influenzae strain 772, biotype 2 (H. Janson et al., 1991, Infection and Immunity, Jan. p.119-125). The sequence of Prot-D1/3 is followed by a multiple cloning site (11 residues) and a coding region for a C-terminal histidine tail (6 His). This plasmid is used to express the fusion protein D1/3-E6E7-his. - b) HPV genomic E6 and E7 sequences type HPV16 (Seedorf et al., Virology 1985, 145, p.181-185) were amplified from HPV16 full length genome cloned in pBR322 (obtained from Deutsches Krebsforschungszentrum (DKFZ), Referenzzentrum für human pathogen Papillomaviruses—D 69120—Heidelberg) and were subcloned into pUC19 to give TCA 301 (=pRIT14462).
- c) The coding sequences for E6 and E7 in TCA301 (=pRIT 14462) were modified with a synthetic oligonucleotides adaptor (inserted between Afl III and Nsi I sites) introducing a deletion of 5 nucleotides between E6 and E7 genes to remove the stop codon of E6 and create fused E6 and E7 coding sequences in the plasmid TCA309(=pRIT 14556) see FIG. 4.
- Construction of Plasmid TCA 311(=pRIT14512): a Plasmid Expressing the Fusion Protein-D1/3-E6E7-His/HPV16
- The nucleotides sequences corresponding to
amino acids 1→249 of fused E6E7 protein were amplified from pRIT14556. During the polymerase chain reaction, NcoI and Spel restriction sites were generated at the 5′ and 3′ ends of the E6E7 fused sequences allowing insertion into the same sites of plasmid pMGMCS Prot D1/3 to give plasmid TCA311 (=pRIT14512) (see FIG. 5). The insert was sequenced to verify that no modification had been generated during the polymerase chain reaction. The coding sequence for the fusion protein-D1/3-His is described FIG. 6. - 2. Transformation of AR58 Strain
- Plasmid pRIT14512 was introduced intoE. coli AR58 (Mott et al., 1985, Proc. Natl. Acad. Sci., 82:88) a defective λ lysogen containing a thermosensitive repressor of the λ pL promoter.
- 3. Growth and Induction of Bacterial Strain—Expression of Prot-D1/3-E6E7-His
- Cells of AR58 transformed with plasmid pRIT14512 were grown in 100 ml of LB medium supplemented with 50 μgr/ml of Kanamycin at 30° C. During the logarithmic phase of growth bacteria were shifted to 39° C. to inactivate the λ repressor and turn on the synthesis of protein D1/3-E6E7-his. The incubation at 39° C. was continued for 4 hours. Bacteria were pelleted and stored at −20C.
- 4. Characterization of Fusion Protein D1/3-E6E7-his
- Frozen cells are thawed and resuspended in 10 ml of PBS buffer. Cells are broken in a French pressure cell press SLM Aminco at 20.000 psi (three passages). The extract is centrifuged at 16.000 g for 30 minutes at 4° C.
- After centrifugation of extracts described above, aliquots of supernatant and pellet were analysed by SDS-polyacrylamide gel electrophoresis and Western blotting.
- A major band of about 48 kDa, localized in the pellet fraction, was visualised by Coomassie stained gels and identified in Western blots by rabbit polyclonal anti-protein-D and by Ni-NTA conjugate coupled to calf intestinal alkaline phosphatase (Qiagen cat. n° 34510) which detects accessible histidine tail. The level of expression represents about 1% of total protein.
- In an analagous fashion the fusion protein of
Lipo D 1/3 and E6-E7 fromHPV 16 was expressed in E. coli in the presence of thioredoxin. The N-terminal of the pre-protein (388 aa) contains MDP residues followed by 16 amino acids of signal peptide of lipoprotein D (from Haemophilus Influenzae) which is cleaved in vivo to give the mature protein (370 aa). Lipoprotein portion (aa 1 to 127) is followed by the proteins E6 and E7 in fusion. The C terminal of the protein is elongated by TSGHHHHHH. - The protein was purified by the following protocol:
- A) Solubilisation.
- Cell paste is suspended to 60 OD600 in 2 M NaCl, 20 mM Phosphate (NaH2PO4/K2HPO4) pH 7.5 in presence of 1 mM Pefabloc as protease inhibitor prior cell lysis by three passes through a French press disruptor (20,000 psi). Lysed cells are pelleted 30 min at 15,000 g at 4° C. In order to reduce endotoxin level, bacterial cell pellet containing the recombinant protein is washed twice in 4 M urea, 2 M NaCl, 20 mM Phosphate pH 7.5, once in 2% Empigen BB, 20 mM Phosphate pH 7.5 and finally twice in 20 mM Phosphate buffer pH 7.0 to eliminate trace of detergent (each wash is performed in the same volume used for cell suspension). LipoProt.D1/3-E6-E7-His (TIT) is solubilised (in the same volume used for cell suspension) by 8 M urea in 0.2 M βMercaptoEthanol (=βMeOH), 20
mM PO4 pH 12 overnight at 4° C. followed by a two hours incubation at RT° versus the same buffer. Cell debris are pelleted 30 min at 15,000 g at 4° C. Supernatant is kept at −20° C. - B) Purification
- 1) Anion Exchange Chromatography on Q-Sepharose Fast Flow.
- 225 ml of frozen sample is thawed at room temperature in a cold water bath and is applied onto a Q-Sepharose fast flow column (Pharmacia, XK 26/20) preequilibrated in 8 M urea, 0.2 M βMEOH, 20 mM PO4 pH 12 (30 ml resin/225 ml supernatant) at 45 cm/h. Column is washed by 8 M urea, 0.2 M MEOH, 20
mM PO4 pH 12, until OD 280 nm reaches the baseline, followed by a second wash in 8 M urea, 20 mM Phosphate pH 12 (in 2 column volumes) Elution is performed by NaCl steps (0.1 M, 0.25 M, 0.5 M NaCl, each step in about 2 column volumes) in 8 M urea, 20mM Phosphate pH 12, at 45 cm/h. 0.5 M NaCl-eluted fractions are pooled. - 2) Ion Metal Affinity Chromatography (IMAC).
- 0.5 M NaCl-eluted fractions from Q Sepharose step are pooled and dialyzed versus 0.2 M NaCl, 8 M urea, 20
mM Phosphate pH 10 before loading onto a Ni2+-NTA (Qiagen) column (XK 26/20, Pharmacia) preequilibrated in 8 M urea, 20 mM PO4 pH 12 (30 ml resin/61 ml sample) at 5.6 cm/h. Column is washed in 8 M urea, 20mM PO4 pH 12 until the base line is reached then by 8 M urea, 20mM PO4 pH 10. Antigen is eluted by Imidazole steps (0.025 M, 0.05 M, 0.1 M, 0.15 M, 0.2 M, 0.5 M Imidazole, each step in two column volumes) in 8 M urea, 20mM PO4 pH 10, at 45 cm/h. 0.05 M Imidazole-eluted fractions are pooled. - C) Concentration.
- Imac sample is concentrated about 5 times (to 0.407 mg/ml) on a 5 kDa Filtron Omega membrane in a stirred cell from AMICON at RT°.
- D) Dialysis
- Concentrated sample is dialyzed at RT versus decreasing-urea-concentration steps (4 M, 2 M urea) in 0.5 M Arginine, 150 mM NaCl, 10 mM PO4 pH 6.8. Last dialysis against 0.5 M Arginine, 150 mM NaCl, 10 mM PO4 pH 6.8 is achieved at 4° C.
- Results:
- IMAC step is able to eliminate a 32 kD contaminant at 0.025 M Imidazole which eluted also some antigen. 0.05 M Imidazole-eluted Antigen is estimated pure at 90% by Coomassie blue staining of SDS-PAGE. After these two purification steps, sample is free ofE. coli contaminants. Western blotting analysis using specific antigen-N and/or C terminus antibodies shows a heterogeneous pattern of bands with higher and lower MW than the full length protein. This pattern suggests the presence of aggregates and incompletely processed protein and/or degraded one, copurified with the full length protein.
- Mutated (cys24→gly,glu26→gln) type HPV16
- 1)-Construction of Expression Plasmid
- Starting Material:
- a)—Plasmid pRIT 14501 (=TCA 308) which codes for fusion ProtD1/3-E7-His
- b)—Plasmid LITMUS 28 (New England Biolabs cat n° 306-28), a cloning vector pUC-derived
- c)—Plasmid pMG MCS ProtD1/3 (pRIT 14589), a derivative of pMG81 (described Supra) in which the codons 4-81 of NS1 coding region from Influenza were replaced by the codons corresponding to residues Ser 20→Thr 127 of mature protein D of Haemophilus Influenzae strain 772, biotype 2 (H. Janson et al., 1991, Infection and Immunity, Jan. p.119-125). The sequence of Prot-D1/3 is followed by a multiple cloning site (11 residues) and a coding region for a C-terminal histidine tail (6 His) Construction of plasmid pRIT 14733(=TCA347): a plasmid expressing the fusion Protein-D1/3-E7 mutated (cys24→gly, glu26→gln) with His tail
- The NcoI—XbaI fragment from pRIT 14501 (=TCA 308), bearing the coding sequence of E7 gene from HPV16, elongated with an His tail, was subcloned in an
intermediate vector Litmus 28 useful for mutagenesis to give pRIT 14909 (=TCA337) Double mutations cys24gly (Edmonds and Vousden, J. Virology 63: 2650 (1989) and glu26gln (Phelps et al, J. Virology 66: 2418-27 (1992) were chosen to impair the binding to the antioncogene product of Retinoblastome gene (pRB). The introduction of mutations in E7 gene was realized with the kit “Quick Change Site directed Mutagenesis (Stratagene cat n° 200518) to give plasmid pRIT 14681 (=TCA343) After verification of presence of mutations and integrity of the complete E7 gene by sequencing, the mutated E7 gene was introduced into vector pRIT 14589 (=pMG MCS ProtD1/3) to give plasmid pRIT 14733 (=TCA347) (FIG. 7). - The sequence for the fusion protein-D1/3-E 7 mutated (cys24→gly, glu26→gln)-His is described in the FIG. 8.
-
-
Plasmid pRIT 14733 was introduced into E.coli AR58 (Mott et al.,1985, Proc. Natl. Acad. Sci., 82:88) a defective X lysogen containing a thermosensitive repressor of the λ pL promoter, to give strain B1002, by selection for transformants resistant to kanamycine - 3)—Growth and Induction of Bacterial Strain B1002—Expression of ProtD1/3-E7 Mutated (cys 24→gly, glu26→gln)-His/HPV16
- Cells of AR58 transformed with plasmid pRIT 14733 (B1002 strain) were grown at 30° C. in 100 ml of LB medium supplemented with 50 μgr/ml of Kanamycin. During the logarithmic phase of growth bacteria were shifted to 39° C. to inactivate the λ repressor and turn on the synthesis of ProtD1/3-E7 mutated-His/HPV16. The incubation at 39° C. was continued for 4 hours. Bacteria were pelleted and stored at −20° C.
- 4)-Characterization of Fusion ProtD1/3-E7 mut (cys24→gly, glu26→gln)-His type HPV16.
- Frozen cells were thawed and resuspended in 10 ml of PBS buffer.Cells were broken in a French Pressure cell press SLM Aminco at 20 000 psi (three passages). The extract was centrifuged at 16000 g for 30 minutes at 4° C.
- After centrifugation of extracts described above, aliquots of supernatant and pellet were analysed by SDS-polyacrylamide gel electrophoresis and Western blotting.
- A major band of about 33 kDa, localized in the pellet fraction, was visualised by Coomassie stained gels and identified in Western blots by rabbit polyclonal 22
J 70 anti-protein D, by monoclonal anti E7/HPV16 from Zymed and by Ni-NTA conjugate coupled to calf intestinal alkaline phosphatase (Qiagen cat. n° 34510) which detects accessible histidine tail. The level of expression represents about 3 to5% of total protein. - Cells of B1002 were separated from the culture broth by centrifugation. The concentrated cells of B1002 were stored at −65° C.
-
- a) Preparation of Cell Suspension
- The frozen concentrated cells of B1002 were thawed and resuspended in a cell disruption buffer at +4° C. (see table 1) to a final optical density OD650 of 60 (corresponding to a cell concentration of approximately 25 g DCW L−1)
- b) Cell Disruption
- The cells were disrupted by two passes at 1000 bar through a high-pressure homogeniser (Rannie). The broken cell suspension was collected in a flask maintained at 4° C. CELL DISRUPTION BUFFER: Na2 HPO4 (0.02N), NaCl (2M) pH adjusted to 7.5 with HCl 3N (Merck)
- Purification
- 2a) Dynamic Membrane Filtration (DMF®-PALL FILTRON)
- 2 Litres of broken cell suspension (OD 60) is loaded on the DMF®, a dynamic filtration system from PALL, mounted with a 0.2 μm cut-off membrane.
- concentration from 2 Litres to 1L to give sample PCC1
- washing at constant volume with 3 volumes (3L) of empigen-EDTA buffer (concentration EDTA 1.86 g, Empigen (30%) 3.33 mL, PO4 3− 0.5M 40.00 mL 40.00 mL) gave sample PD!
- concentration from 1L to 300 mL gave sample PCC2
- washing at constant volume with 10 volumes (3L) of empigen buffer (concentration L−1:
Empigen 30%, 3.33 mL, PO4 3− 0.5M 40 mL) pH 7.5 gave sample PD2 - solubilisation of the protein in by addition of the same volume (300 mL) of Guanidine hydrochloride 8M buffer (concentration L−1 Gu.HCl 764 g;
- Empigen 30% 3.33 mL, PO4 3− 0.5
M 40 mL) pH 7.5 - recovery of the protein: Collection of the permeate—sample P3 during Concentration to initial volume (300 mL) and
- Diafiltration with 3 volume of Guanidine hydrochloride 4M buffer (concentration L−1: Gu.HCl 328.12 g, Empigen (30%) 3.33 mL Po43-0.5M 40.00 mL) pH 7.5.
- All these steps are made in a cold room (2-8° C.), pH adjusted with 0.5M PO4 3 −.
- The P3 fraction is store at −20° C. waiting for the next purification step.
- 2b) Zn-Chelating Sepharose Chromatography
- The P3 fraction is thawed and injected in a packed and equilibrated Zn-chelating sepharose FF.
- After that, the column is:
- Washed with around 3 volumes of Guanidine hydrochloride 4M buffer (see above)—sample Zn-FT
- Washed with around 5 volumes of Urea 4M buffer (concentration L−1: Urea 240.24 g Empigen 3.33 mL PO4 3− 0.5M 40.00 mL)—sample Zn-W
- Eluted with around 3 volumes of Urea 4M-
Imidazole 20 mM buffer (concentration L−1 Urea 240.24 g, Empigen (30%) 3.33 mL Imidazole (1.36 g) PO4 3− 0.5M 40.00 mL pH 7.5) buffer as above, but concentration L−1 of Imidazole 34.04 g—sample Zn-20 - Eluted with Urea 4M-
Imidazole 500 mM to the end of the UV peak—sample Zn-500 - The column is the washed with
EDTA 50 mM and NaOH 0.5M. Zn chelating sepharose eluate (Zn-500) is stored between 2-8° c. before the next purification step. - The Zn-chelating sepharose chromatography operations are carried out at room temperature.
- 2c) Q-sepharose Chromatography
- The Zn-500 fraction is injected in a packed and equilibrated Q-sepharose FF.
- After that, the column is:
- Washed with around 7 volumes of Urea 4M buffer (see above) - sample QS-FT
- Washed with around 10 volumes of Urea 4M buffer without empigen (concentration L−1 Urea 240.24 g PO4 3− 0.5M 40.00 mL)—sample QS-W1
- Washed with around 10 volumes of Urea 6M buffer without empigen (Urea 360.36 g/L)—sample QS-W2
- Eluted with around 5 volumes of Urea 6M-
NaCl 200 mM buffer (conctration L−1: Urea 360.36 g NaCl 11.69 g, 40.00 MnL PO4 3−. - Eluted with around 3 volumes of Urea 6M-
NaCl 500 mM buffer (as above, but NaCl 29.22 g/L). The exact end of the fraction is determined by the end of the UV peak.—sample QS-500 - Eluted with 4 volumes of Urea 4M-NaCl 1M buffer (conctration L−1 Urea 360.36, NaCl 58.44 g 40.00 mL PO4 3− (0.5)—sample QS-1M
- The column is then washed with NaOH 0.5M
- QS-sepharose eluate (QS-500) is stored between 2-8°c before the next purification step.
- The Q-sepharose chromatography operations are carried out at room temperature.
- 2d) Ultrafiltration
- The QS-500 fraction is then treated on a 10 kD utrafiltration unit (Ultrasette—Pall Filtron)
- The product is first concentrated to around 1 mg/mL of protein and then diafiltrated against 10 volumes of phosphate buffer.
- The permeate (fraction UF-P) is discarded and the retentate (fraction UF-R) is stored at 2-8° c. waiting for final filtration.
- Ultrafiltration operations are carried out at 2-8° C.
- 2e) Final Filtration
- The final bulk (UF-R fraction) is filtered through a 0.22 cm sterile filter (Millipak-Millipore) under laminar flow and in an
aseptic class 100 room. - The final concentration is between 0.5 and 1.0 μg/mL.
- The sterile bulk is stored at −20° C.
- 1. Construction of Expression Plasmid
- a)—Plasmid pRIT14497 (=TCA307), that codes for fusion ProtD1/3-E6-His/HPV16
- b)—Plasmid pRIT14661 (=DVA2), an intermediate vector containing the coding sequence for the 117 C-terminal codons of LytA ofStreptococcus Pneumoniae. Lyta is derived from Streptococcus pneumoniae which synthesize an N-acetyl-L-alanine amidase, amidase LYTA, (coded by the lytA gene {Gene, 43 (1986) pag 265-272} an autolysin that specifically degrades certain bonds in the peptidoglycan backbone. The C-terminal domain of the LYTA protein is responsible for the affinity to the choline or to some choline analogues such as DEAE.
- 1.b Construction of Plasmid pRIT14634 (=TCA332): a Plasmid Expressing the Fusion clyta-E6-His/HPV16
- a) The first step was the purification of the large NcoI-AflII restriction fragment from plasmid pRIT14497 and the purification of the small AflII-AflIII restriction fragment from pRIT14661
- b) The second step was linking of clyta sequences to the E7-His sequences (NcoI and AfilIII are compatible restriction sites) that gave rise to the plasmid pRIT 14634 (=TCA332), coding for the fusion protein clyta-E6-His under the control of the pL promoter. (see FIG. 9) The coding sequence for the fusion protein clyta-E6-His is decribed in FIG. 10.
- Transformation of AR58 Strain
- Plasmid pRIT14634 was introduced intoE. coli AR58 (Mott et al., 1985, Proc. Natl. Acad. Sci., 82:88) a defective λ lysogen containing a thermosensitive repressor of the λ pL promoter.
- Growth and Induction of Bacterial Strain—Expression of clyta-E6-His
- Cells of AR58 transformed with plasmid pRIT14634 were grown in 100 ml of LB medium supplemented with 50 μgr/ml of Kanamycin at 30° C. During the logarithmic phase of growth bacteria were shifted to 39° C. to inactivate the λ repressor and turn on the synthesis of protein clyta-E6-his. The incubation at 39° C. was continued for 4 hours. Bacteria were pelleted and stored at −20° C.
- 4. Characterization of Fusion clyta-E6-his
- Frozen cells were thawed and resuspended in 10 ml of PBS buffer. Cells were broken in a French pressure cell press SLM Aminco at 20.000 psi (three passages). The extract was centrifuged at 16.000 g for 30 minutes at 4° C. After centrifugation of extracts described above, aliquots of supernatant and pellet were analysed by SDS-polyacrylamide gel electrophoresis and Western blotting.
- A major band of about 33 kDa, localized in the pellet fraction, was visualised by Coomassie stained gels and identified in Western blots by rabbit polyclonal anti-clyta antibodies and by Ni-NTA conjugate coupled to calf intestinal alkaline phosphatase (Qiagen cat. n° 34510) which detects accessible histidine tail. The level of expression represents about 3% of total protein.
- 1. Construction of Expression Plasmid
- 1.a Starting Materials
- a)—Plasmid pRIT14501 (=TCA308), that codes for fusion ProtD1/3-E7-His/HPV16
- b)-Plasmid pRIT14661 (=DVA2), an intermediate vector containing the coding sequence for the 117 C-terminal codons of LytA ofStreptococcus Pneumoniae.
- 1.b Construction of Plasmid pRIT14626 (=TCA330): a plasmid expressing the fusion clyta-E7-His/HPV16
- a) The first step was the purification of the large NcoI-AflIII restriction fragment from plasmid pRIT14501 and the purification of the small AflII-AflIII restriction fragment from pRIT14661
- b) The second step was linking of clyta sequences to the E7-His sequences (NcoI and AflIII are compatible restriction sites)that gave rise to the plasmid pRIT 14626 (=TCA330), coding for the fusion protein clyta-E7-His under the control of the pL promoter.
- (FIG. 11)
- The coding sequence for the fusion protein clyta-E7-His is decribed in FIG. 12.
- 2. Transformation of AR58 Strain
- Plasmid pRIT14626 was introduced intoE. coli AR58 (Mott et al., 1985, Proc. Natl. Acad. Sci., 82:88) a defective ? lysogen containing a thermosensitive repressor of the λ pL promoter.
- 3. Growth and Induction of Bacterial Strain—Expression of clyta-E7-His
- Cells of AR58 transformed with plasmid pRIT14626 were grown in 100 ml of LB medium supplemented with 50 μgr/ml of Kanamycin at 30° C. During the logarithmic phase of growth bacteria were shifted to 39° C. to inactivate the λ repressor and turn on the synthesis of protein clyta-E7-his. The incubation at 39° C. was continued for 4 hours. Bacteria were pelleted and stored at −20° C.
- 4. Characterization of Fusion clyta-E7-his
- Frozen cells were thawed and resuspended in 10 ml of PBS buffer. Cells were broken in a French pressure cell press SLM Aminco at 20.000 psi (three passages). The extract was centrifuged at 16.000 g for 30 minutes at 4° C. After centrifugation of extracts described above, aliquots of supernatant and pellet were analysed by SDS-polyacrylamide gel electrophoresis and Western blotting.
- A major band of about 35 kDa, localized in the pellet fraction, was visualised by Coomassie stained gels and identified in Western blots by rabbit polyclonal anti-clyta antibodies and by Ni-NTA conjugate coupled to calf intestinal alkaline phosphatase (Qiagen cat. n° 34510) which detects accessible histidine tail. The level of expression represents about 5% of total protein.
- 1. Construction of Expression Plasmid
- 1.a Starting Materials
- a)—Plasmid pRIT14512 (=TCA311), that codes for fusion ProtD1/3-E6E7-His/HPV16
- b)—Plasmid pRIT14661 (=DVA2), an intermediate vector containing the coding sequence for the 117 C-terminal codons of LytA of Streptococcus Pneumoniae.
- 1.b Construction of Plasmid pRIT14629 (=TCA331): a Plasmid Expressing the Fusion clyta-E6E7-His/HPV16
- a) The first step was the purification of the large NcoI-AflII restriction fragment from plasmid pRIT14512 and the purification of the small AflII-AflIII restriction fragment from pRIT14661
- b) The second step was linking of clyta sequences to the E7-His sequences (NcoI and AflIII are compatible restriction sites)that gave rise to the plasmid pRIT 14629 (=TCA33 1), coding for the fusion protein clyta-E6E7-His under the control of the pL promoter. (see FIG. 13)
- The coding sequence for the fusion protein clyta-E6E7-His is decribed in FIG. 14.
- 2. Transformation of AR58 Strain
- Plasmid pRIT14629 was introduced intoE. coli AR58 (Mott et al., 1985, Proc. Natl. Acad. Sci., 82:88) a defective λ lysogen containing a thermosensitive repressor of the λ pL promoter.
- 3. Growth and Induction of Bacterial Strain—Expression of clyta-E6E7-His
- Cells of AR58 transformed with plasmid pRIT14629 were grown in 100 ml of LB medium supplemented with 50 μgr/ml of Kanamycin at 30° C. During the logarithmic phase of growth bacteria were shifted to 39° C. to inactivate the λ repressor and turn on the synthesis of protein clyta-E6E7-his. The incubation at 39° C. was continued for 4 hours. Bacteria were pelleted and stored at −20° C.
- 4. Characterization of Fusion clyta-E6E7-his
- Frozen cells were thawed and resuspended in 10 ml of PBS buffer. Cells were broken in a French pressure cell press SLM Aminco at 20.000 psi (three passages). The extract was centrifuged at 16.000 g for 30 minutes at 4° C.
- After centrifugation of extracts described above, aliquots of supernatant and pellet were analysed by SDS-polyacrylamide gel electrophoresis and Western blotting.
- A major band of about 48 kDa, localized in the pellet fraction, was visualised by Coomassie stained gels and identified in Western blots by rabbit polyclonal anti-clyta antibodies and by Ni-NTA conjugate coupled to calf intestinal alkaline phosphatase (Qiagen cat. n° 34510) which detects accessible histidine tail. The level of expression represents about 1% of total protein.
- 1)—Construction of Expression Plasmids
- 1).a. Construction of Plasmid TCA316(=pRIT 14532) a Plasmid Expressing the Fusion Protein-D1/3-E7-His IHPV18
- Starting Materials
- a)—Plasmid pMG MCS prot D1/3 (=pRIT14589) is a derivative of pMG81 (described in UK patent application n° 951 3261.9 published as WO97/01640 in which the codons 4-81 of NS1 coding region from Influenza were replaced by the codons corresponding to residues Ser 20→Thr 127 of mature protein D ofHaemophilus Influenzae strain 772, biotype 2 (H. Janson et al., 1991, Infection and Immunity, Jan. p.119-125). The sequence of Prot-D1/3 is followed by a multiple cloning site (11 residues) and a coding region for a C-terminal histidine tail (6 His) (see FIG. 15). This plasmid is used to express the fusion protein D1/3-E7-his.
- b)—HPV genomic E6 and E7 sequences of prototype HPV18 (Cole et al, J. Mol. Biol. (1987) 193,599-608) were amplified from HPV16 full length genome cloned in pBR322 (obtained from Deutsche Krebsforschungszentrum (DKFZ), Referenzzentrum für human pathogen Papillomaviruses—D 69120—Heidelberg) and were subcloned into pUC19 to give TCA 302 (=pRIT14467).
- Construction of Plasmid TCA 316(=pRIT14532)
- The nucleotides sequences corresponding to
amino acids 1→105 of E7 protein were amplified from pRIT14467. During the polymerase chain reaction, NcoI and SpeI restriction sites were generated at the 5′ and 3′ ends of the E7 sequences allowing insertion into the same sites of plasmid pMGMCS Prot D1/3 to give plasmid TCA316 (=pRIT14532). The insert was sequenced and a modification versus E7/HPV18 prototype sequence was identified in E7 gene (nucleotide 128 G→A) generating a substitution of a glycine by a glutamic acid (aa 43 in E7, position 156 in fusion protein). The sequence for the fusion protein-D1/3-E7-His/HPV18 is described in FIG. 16. - 1).b. Construction of Plasmid TCA313 (=pRIT14523): a Plasmid Expressing Thioredoxin
- Starting Materials
- a)—Plasmid pBBR1MCS4(Antoine R. and C. Locht, Mol. Microbiol. 1992, 6, 1785-1799 ; M. E. Kovach et al. Biotechniques 16, (5), 800-802)which is compatible with plasmids containing ColE1 or P15a origins of replication.
- b)—Plasmid pMG42 (described in WO93/04175) containing the sequence of promoter pL of Lambda phage
- c)—Plasmid pTRX (Invitrogen, kit Thiofusion K350-01) bearing the coding sequence for thioredoxin followed by AspA transcription terminator.
- Construction of Plasmid TCA313(=pRIT14523)
- The fragment EcoRI-NdeI fragment from pMG42, bearing pL promoter and the NdeI-HindIII fragment from pTRX, bearing the coding sequence for thioredoxin followed by AspA terminator, were purified and ligated into the EcoRI and HindIII sites of plasmid vector pBBR 1MCS4 to give plasmid TCA313(=pRIT14523) (see FIG. 17).
- The sequence for thioredoxin is described in FIG. 18.
- 2)—Transformation of AR58 Strain
- 2).a. To Obtain Strain B1011 Expressing ProtD1/3-E7-His/HPV18
- Plasmid pRIT14532 was introduced intoE. coli AR58 (Mott et al., 1985, Proc. Natl. Acad. Sci., 82:88) a defective λ lysogen containing a thermosensitive repressor of the λ pL promoter, by selection for transformants resistant to kanamycine.
- 2).b. Construction of Strain B1012 Expressing ProtD1/3-E7-His/HPV18 and Thioredoxin
- Plasmid pRIT14532 and pRIT14523 were introduced intoE. coli AR58 (Mott et al., 1985, Proc. Natl. Acad. Sci., 82:88) a defective λ lysogen containing a thermosensitive repressor of the λ pL promoter, by double selection for transformants resistant to kanamycin and ampicillin.
- 3)—Growth and Induction of Bacterial Strains B1011 and B1012—Expression of Prot-D1/3-E7-His/HPV18 Without and With Thioredoxin in Trans
- Cells of AR58 transformed with plasmids pRIT14532 (B1011 strain) and Cells of AR58 transformed with plasmids pRIT14532 and pRIT14523 (B1012 strain) were grown at 30° c. in 100 ml of LB medium supplemented with 50 μgr/ml of Kanamycin for B1011 strain and supplemented 50 μgr/ml of Kanamycin and 100 μgr/ml of Ampicillin for B1012 strain. During the logarithmic phase of growth bacteria were shifted to 39° C. to inactivate the λ repressor and turn on the synthesis of protein D1/3-E7-his/HPV18 and thioredoxin. The incubation at 39° C. was continued for 4 hours. Bacteria were pelleted and stored at −20° C.
- Characterization of Fusion Protein D1/3-E7-his/HPV18
- Preparation of Extracts
- Frozen cells are thawed and resuspended in 10 ml of PBS buffer. Cells are broken in a French pressure cell press SLM Aminco at 20.000 psi (three passages). The extract is centrifuged at 16.000 g for 30 minutes at 4° C.
- Analysis on Coomassie-Stained SDS-Polyacrylamide Gels and Western Blots
- After centrifugation of extracts described above, aliquots of supernatant and pellet were analysed by SDS-polyacrylamide gel electrophoresis and Western blotting.
- The fusion protD1/3-E7-His (about 31 kDa) was visualised by Coomassie stained gels in the pellet fraction for strain B1011 and partially localized (30%) in the supernatant fraction for strain 1012 and was identified in Western blots by rabbit polyclonal anti-protein-D and by Ni-NTA conjugate coupled to calf intestinal alkaline phosphatase (Qiagen cat. n° 34510) which detects accessible histidine tail. The level of expression represents about 1-3% of total protein as shown on a Coomassie-stained SDS-polyacrylamide gel.
- For the extract of strain B1012 the thioredoxin (about 12 KDa) was visualised by coomassie stained gel in the supernatant and identified in western blots by monoclonal anti thioredoxin (Invitrogen R920-25)
- Purification of Prot D1/3 E7-his/HPV18
- Recombinant HPV 18-ProtD1/3-E7-His is expressed inE. coli (as described above) AR58 strain. All steps are performed at room temperature (RT≅22° C.). Proteins are followed by monitoring OD 280 nm. Between steps, antigens positive fractions are kept at −20° C.
- Purified antigen is stable one week at −20° C. and 4° C. (no degradation) but appears more susceptible to oxidation after incubation at 37° C.
- d)—Solubility
- Protein solubility is pH dependent (see below) with decrease of solubility for pH<7.4:
PBS pH 7.4 686 μg/ ml 100% PBS pH 7.2 560 μg/ ml 81% PBS pH 7.0 498 μg/ ml 72% PBS pH 6.8 327 μg/ ml 48% - e)—The
HPV 18 Prot D1/3 E7 proteiin is composed of 227 amino acids. Its theoretical molecular weight is 25.9 kDa, and a theoretical isoelectric point of 5.83. It migrates at about 31.5 kDa in reducing SDS PAGE. - a)—Solubilisation
- Cell paste is suspended to 60 OD600 in 2 M NaCl, 20 mM Phosphate. (NaH2PO4/K2HPO4) pH 7.6 prior cell lysis by two passes through a Rannie disruptor. Lysed cells are pelleted 30 min at 9,000 rpm in a
JA 10 rotor at 4° C. In order to reduce endotoxin level, bacterial cell pellet containing the recombinant protein is washed once in 5 mM EDTA, 2 M NaCl, PBS pH 7.4; once in 4 M urea, 20 mM Phosphate pH 7.4 and finally once in PBS pH 7.4 to eliminate trace of EDTA (each wash is performed in twice volume used for cell suspension). HPV18Prot.D1/3-E7-His (TIT for Thioredoxin In Trans) is solubilised (in the same volume used for cell suspension) by 6 M Guanidine-Chloride, 50 mM PO4 pH 7.6 overnight at 4° C. Cell debris are pelleted 30 min at 9,000 rpm in aJA 10 rotor at 4° C. Supernatant is supplemented with 0.5% Empigen BB and incubated 30 min at RT. - b)—Purification
- 1).a. Immobilized Metal Affinity Chromatography
- 125 .ml of sample are loaded onto a Zn2+-Chelating Sepharose FF column (XK 26/20, Pharmacia; 50 ml gel/125 ml solubilisation) preequilibrated in 0.5% Empigen BB, 6 M Guanidine-Chloride, 50 mM PO4 pH 7.6 at 4 ml/min. Column is washed by Guanidine Chloride 6M,
PO4 50 mM pH 7.6 until the base line is reached then by 6 M urea, 0.5 M NaCl, 50 mM PO4 pH 7.6. Antigen is eluted by 0.25 M-Imidazole in 6 M urea, 0.5 M NaCl, 50 mM PO4 pH 7.6, at 2 ml/min (FIG. 1B). IMAC-eluted sample is dialyzed at 4° C. versus PBS pH 7.4 - 1).b. Affi-Prep Polymixin (Bio-Rad)
- To reduce endotoxin level, 28 mg (37 ml) of antigen are incubated in batch mode with 2 ml of Affiprep Polymyxin resin prequilibrated in PBS pH 7.4, over night at room temperature. Protein recovery is estimate at 60% and endotoxin content is reduced 6.5 times.
- 1).c. Analysis
- Purified antigen analyzed on reducing-SDS-PAGE presents a major 30 kDa band with a second one at 55 kDa, after Coomassie Blue or Silver Staining. In a non reducing SDS-PAGE, HPV-18-ProtD1/3-E7-His appears mainly like a smear with Molecular Weight≧175 kDa. However this oxidation can be reversed by addition of 5 mM of β-Mercapto-Ethanol. This pattern is confirmed by anti ProtD or by anti His Western Blotting analysis.
- c)—Stability
- Purified antigen is stable one week at −20° C. and 4° C. (no degradation) but appears more susceptible to oxidation after incubation at 37° C.
- d)—Solubility
- Protein solubility is pH dependent (see below) with decrease of solubility for pH <7.4:
PBS pH 7.4 686 μg/ ml 100% PBS pH 7.2 560 μg/ ml 81% PBS pH 7.0 498 μg/ ml 72% PBS pH 6.8 327 μg/ ml 48% - HPV18-ProtD1/3-E7-His protein is composed of 227 amino acids. Its theoretical molecular weight is 25.9 kDa. It migrates at about 31.5 kDa in reducing SDS-PAGE. Theoretical isoelectric point is 5.83.
- Mutated (cys27→gly,glu29→gln) type HPV18
- 1)-Construction of Expression Plasmid
- Starting Material:
- a)—Plasmid pRIT 14532 (=TCA 316) which codes for
fusion ProtD 1/3-E7-His - b)—Plasmid LITMUS 28 (New England Biolabs cat n° 306-28), a cloning vector pUC-derived
- c)—Plasmid pMG MCS ProtD1/3 (pRIT 14589), a derivative of pMG81 (described supra) in which the codons 4-81 of NS1 coding region from Influenza were replaced by the codons corresponding to residues Ser 20→Thr 127 of mature protein D ofHaemophilus Influenzae strain 772, biotype 2 (H. Janson et al., 1991, Infection and Immunity, Jan. p. 119-125). The sequence of Prot-D1/3 is followed by a multiple cloning site (11 residues) and a coding region for a C-terminal histidine tail (6 His)
- Construction of Plasmid pRIT 14831(=TCA355): a Plasmid Expressing the Fusion Protein-D1/3-E7 Mutated (cys27→gly, glu29→gln) With His Tail
- The NcoI-XbaI fragment from pRIT 14532 (—TCA 316), bearing the coding sequence of E7 gene from HPV18, elongated with an His tail, was subcloned in an
intermediate vector Litmus 28 useful for mutagenesis to give pRIT 14910 (=TCA348) By analogy with E7/HPV 16 mutagenesis, double mutations cys27gly and glu29gln were chosen to impair the binding to the antioncogene product of Retinoblastome gene (pRB). - The introduction of mutations in E7 gene was realized with the kit “Quick Change Site directed Mutagenesis (Stratagene cat n° 200518). As the sequencing of pRIT14532 had pointed out the presence of a glutamic acid in
position 43 of E7 instead of a glycine in the prototype sequence ofHPV 18, a second cycle of mutagenesis was realized to introduce a glycine inposition 43. We obtained plasmid pRIT 14829 (=TCA353). After verification of presence of mutations and integrity of the complete E7 gene by sequencing, the mutated E7 gene was introduced into vector pRIT 14589 (=pMG MCS ProtD1/3) to give plasmid pRIT 14831 (=TCA355) (see FIG. 17). - The sequence for the fusion protein-D1/3-E 7 mutated (cys27→gly, glu29→gln)-His is described in the FIG. 18.
-
-
Plasmid pRIT 14831 was introduced into E.coli AR58 (Mott et al., 1985, Proc. Natl. Acad. Sci., 82:88) a defective λ lysogen containing a thermosensitive repressor of the λ pL promoter, to give strain B1098, by selection for transformants resistant to kanamycin. - 3)—Growth and Induction of Bacterial Strain B1098—Expression of ProtD1/3-E7 Mutated (
cys 27→gly, glu29→gln)-His/HPV18 - Cells of AR58 transformed with plasmid pRIT 14831 (B1098 strain) were grown at 30° C. in 100 ml of LB medium supplemented with 50 μgr/ml of Kanamycin. During the logarithmic phase of growth bacteria were shifted to 39° C. to inactivate the λ repressor and turn on the synthesis of ProtD1/3-E7 mutated -His/HPV18. The incubation at 39° C. was continued for 4 hours. Bacteria were pelleted and stored at −20° C.
- 4)—Characterization of Fusion ProtD1/3-E7 mut (cys24→gly, glu26→gln)-His Type HPV16
- Frozen cells were thawed and resuspended in 10 ml of PBS buffer. Cells were broken in a French Pressure cell press SLM Aminco at 20 000 psi (three passages). The extract was centrifuged at 16000 g for 30 minutes at 4° C.
- Analysis on Coomassie Stained SDS-Polyacrylamide Gels and Western Blots
- After centrifugation of extracts described above, aliquots of supernatant and pellet were analysed by SDS-polyacrylamide gel electrophoresis and Western blotting. A major band of about 31 kDa, localized in the pellet fraction, was visualised by Coomassie stained gels and identified in Western blots by rabbit polyclonal 22
J 70 anti-protein D and by monoclonal Penta-His (Qiagen cat. n° 34660) which detects accessible histidine tail. The level of expression represents about 3 to 5% of total protein. - 1. Construction of Expression Plasmid
- a) Plasmid pMG MCS prot D1/3 (=pRIT14589) is a derivative of pMG81 (described supra) in which the codons 4-81 of NS1 coding region from Influenza were replaced by the codons corresponding to residues Ser 20→Thr 127 of mature protein D of Haemophilus Influenzae strain 772, biotype 2 (H. Janson et al., 1991, Infection and Immunity, Jan. p.119-125). The sequence of Prot-
D 1/3 is followed by a multiple cloning site (11 residues) and a coding region for a C-terminal histidine tail (6 His). This plasmid is used to express the fusion protein D1/3-E6-his. - HPV genomic E6 and E7 sequences type HPV18 (Cole et al., J. Mol. Biol. 1987, 193, p.599-608.) were amplified from HPV18 full length genome cloned in pBR322 (obtained from Deutsches Krebsforschungszentrum (DKFZ), Referenzzentrum für human pathogen Papillomaviruses—D 69120—Heidelberg) and were subcloned into pUC19 to give TCA 302 (=pRIT14467).
- Construction of Plasmid TCA 314(=pRIT14526): a Plasmid Expressing the Fusion Protein-D1/3-E6-His/HPV18
- The nucleotides sequences corresponding to amino acids
- 1→158 of E6 protein were amplified from pRIT14467. During the polymerase chain reaction, NcoI and Spel restriction sites were generated at the 5′ and 3′ ends of the E6 sequences allowing insertion into the same sites of plasmid pMGMCS Prot D1/3 to give plasmid TCA314 (=pRIT14526) (see FIG. 21). The insert was sequenced to verify that no modification had been generated during the polymerase chain reaction. The coding sequence for the fusion protein-D1/3-E6-His is described in FIG. 22.
- Transformation of AR58 Strain
- Plasmid pRIT14526 was introduced intoE. coli AR58 (Mott et al., 1985, Proc. Natl. Acad. Sci., 82:88) a defective λ lysogen containing a thermosensitive repressor of the λ pL promoter.
- 3. Growth and Induction of Bacterial Strain—Expression of Prot-D1/3-E6-His
- Cells of AR58 transformed with plasmid pRIT14526 were grown in 100 ml of LB medium supplemented with 50 μgr/ml of Kanamycin at 30° C. During the logarithmic phase of growth bacteria were shifted to 39° C. to inactivate the λ repressor and turn on the synthesis of protein D1/3-E6-his. The incubation at 39° C. was continued for 4 hours. Bacteria were pelleted and stored at −20C.
- 4. Characterization of Fusion Protein D1/3-E6-his
- Frozen cells are thawed and resuspended in 10 ml of PBS buffer. Cells are broken in a French pressure cell press SLM Aminco at 20.000 psi (three passages). The extract is centrifuged at 16.000 g for 30 minutes at 4° C. After centrifugation of extracts described above, aliquots of supernatant and pellet were analysed by SDS-polyacrylamide gel electrophoresis and Western blotting.
- A major band of about 32 kDa, localized in the pellet fraction, was visualised by Coomassie stained gels and identified in Western blots by rabbit polyclonal anti-protein-D and by Ni-NTA conjugate coupled to calf intestinal alkaline phosphatase (Qiagen cat. n° 34510) which detects accessible histidine tail. The level of expression represents about 3-5% of total protein.
- 1. Construction of Expression Plasmid
- a) Plasmid pMG MCS prot D1/3 (=pRIT14589) is a derivative of pMG81 (described supra) in which the codons 4-81 of
NS 1 coding region from Influenza were replaced by the codons corresponding to residues Ser 20→Thr 127 of mature protein D of Haemophilus Influenzae strain 772, biotype 2 (H. Janson et al., 1991, Infection and Immunity, Jan. p.119-125). The sequence of Prot-D1/3 is followed by a multiple cloning site (11 residues) and a coding region for a C-terminal histidine tail (6 His). This plasmid is used to express the fusion protein D1/3-E6E7-his. - b) HPV genomic E6 and E7 sequences type HPV18 (Cole et al., J. Mol. Biol. 1987, 193, 599-608) were amplified from HPV18 full length genome cloned in pBR322 (obtained from Deutsches Krebsforschungszentrum (DKFZ), Referenzzentrum für human pathogen Papillomaviruses—D 69120—Heidelberg) and were subcloned into pUC19 to give TCA 302 (=pRIT14467).
- c) The coding sequences for E6 and E7 in TCA302 (=pRIT 14467) were modified with a synthetic oligonucleotides adaptor (inserted between Hga I and Nsi I sites) introducing a deletion of 11 nucleotides between E6 and E7 genes, removing the stop codon of E6 and creating fused E6 and E7 coding sequences in the plasmid TCA320(=pRIT 14618) see FIG. 23.
- Construction of Plasmid TCA 328(=pRIT14567): a Plasmid Expressing the Fusion Protein-D1/3-E6E7-His/HPV18
- The nucleotides sequences corresponding to amino acids
- 1→263 of fused E6E7 protein were amplified from pRIT14618. During the polymerase chain reaction, NcoI and SpeI restriction sites were generated at the 5′ and 3′ ends of the E6E7 fused sequences allowing insertion into the same sites of plasmid pMGMCS Prot D1/3 to give plasmid TCA328 (=pRIT14567) (see FIG. 24). The insert was sequenced to verify that no modification had been generated during the polymerase chain reaction. The coding sequence for the fusion protein-D1/3-E6E7-His is described in FIG. 25.
- 2. Transformation of AR58 Strain
- Plasmid pRIT14567 was introduced intoE. coli AR58 (Mott et al., 1985, Proc. Natl. Acad. Sci., 82:88) a defective λ lysogen containing a thermosensitive repressor of the λ pL promoter.
- 3. Growth and Induction of Bacterial Strain—Expression of Prot-D1/3-E6E7-His
- Cells of AR58 transformed with plasmid pRIT14512 were grown in 100 ml of LB medium supplemented with 50 μgr/ml of Kanamycin at 30° C. During the logarithmic phase of growth bacteria were shifted to 39° C. to inactivate the λ repressor and turn on the synthesis of protein D1/3-E6E7-his. The incubation at 39° C. was continued for 4 hours. Bacteria were pelleted and stored at −20C.
- 4. Characterization of Fusion Protein D1/3-E6E7-his
- Frozen cells are thawed and resuspended in 10 ml of PBS buffer. Cells are broken in a French pressure cell press SLM Aminco at 20.000 psi (three passages). The extract is centrifuged at 16.000 g for 30 minutes at 4° C.
- After centrifugation of extracts described above, aliquots of supernatant and pellet were analysed by SDS-polyacrylamide gel electrophoresis and Western blotting. A major band of about 48 kDa, localized in the pellet fraction, was visualised by Coomassie stained gels and identified in Western blots by rabbit polyclonal anti-protein-D and by Ni-NTA conjugate coupled to calf intestinal alkaline phosphatase (Qiagen cat. n° 34510) which detects accessible histidine tail. The level of expression represents about 1% of total protein.
- Vaccines are formulated with a Protein from the above examples expressed inE. coli from the strain AR58, and as adjuvant, the formulation comprising a mixture of 3 de -O-acylated monophosphoryl lipid A (3D-MPL) and aluminium hydroxide or 3D-MPL and/or QS21 optionally in an oil/water emulsion, and optionally formulated with cholesterol.
- 3D-MPL: is a chemically detoxified form of the lipopolysaccharide (LPS) of the Gram-negative bacteria Salmonella minnesota.
- Experiments performed at Smith Kline Beecham Biologicals have shown that 3D-MPL combined with various vehicles strongly enhances both the humoral and a THI type of cellular immunity.
- QS21: is one saponin purified from a crude extract of the bark of the Quillaja Saponaria Molina tree, which has a strong adjuvant activity: it activates both antigen-specific lymphoproliferation and CTLs to several antigens.
- Vaccine containing an antigen of the invention containing 3D-MPL and alum may be prepared in analogous manner to that described in WO93/19780 or 92/16231.
- Experiments performed at Smith Kline Beecham Biologicals have demonstrated a clear synergistic effect of combinations of 3D-MPL and QS21 in the induction of both humoral and TH1 type cellular immune responses. Vaccines containing an antigen such antigens are described in U.S. Pat. No. 5,750,110.
- The oil/water emulsion is composed of 2 oils (a tocopherol and squalene), and of
PBS containing Tween 80 as emulsifier. The emulsion comprised 5% squalene 5% tocopherol 0.4% Tween 80 and had an average particle size of 180 nm and is known as SB62 (see WO 95/17210). - Experiments performed at Smith Kline Beecham Biologicals have proven that the adjunction of this O/W emulsion to MPL/QS21 further increases their immunostimulant properties.
- Preparation of Emulsion SB62 (2 Fold Concentrate)
-
Tween 80 is dissolved in phosphate buffered saline (PBS) to give a 2% solution in the PBS. To provide 100 ml two fold concentrate emulsion 5 g of DL alpha tocopherol and 5 ml of squalene are vortexed to mix thoroughly. 90 ml of PBS/Tween solution is added and mixed thoroughly. The resulting emulsion is then passed through a syringe and finally microfluidised by using an M110S microfluidics machine. The resulting oil droplets have a size of approximately 180 nm. - Preparation of Prot.D1/3 E7 QS21/3D MPL oil in Water Formulation
- ProtD1/3-E7 (5 μg) was diluted in 10 fold concentrated PBS pH 6.8 and H2O before consecutive addition of SB62, 3 D MPL (5 μg), QS21 (5 μg) and 50 μg/ml thiomersal as preservative at 5 min interval. The emulsion volume is equal to 50% of the total volume (50 μl for a dose of 100 μl). All incubations were carried out at room temperature with agitation. The adjuvants controls without antigen were prepared by replacing the protein by PBS.
- Tumour Regression Experiments (HPV 16) with PROT D E7
- Vaccine Antigen: Fusion Protein ProtD E7
- Protein D is a lipoprotein exposed on the surface of the Gram-negative bacteria Haemophilus influenzae.
- The inclusion of the 109 first residues of the protein D as fusion partner is incorporated to provide the vaccine antigen with bystander help properties. The antigen was formulated with
QS21 3D-MPL and SB62 as described supra. - Primary lung epithelial cells from C57BL.6 mice were immortalised by
HPV 16 E6 and E7 and then transformed with an activated ras oncogene, producing a tumourigenic cell line expressing E6 and E7 (Lin KY et al. 1996). The E7 expression has been verified by FACS analysis of fixed and permeabilised TC1 cells using the mouse anti-HPV 16 E7 Mab (Triton Corp. Alameda, Calif.) - Tumour Growth:
-
TC 1 cells growing in vitro culture were trypsinised, washed two times in serum-free medium and were injected S.C. in the right flank of the mice. To assess treatment of established tumours, TC1 cells were injected at a dose of 3×10e4 cells/mouse. One and two weeks after the tumour cell injection, mice were vaccinated with 5 μg in 100 μl ofprotD 1/3 E7 His intra foot pad (50 μl/foot pad) in PBS or in the 3D-MPL, QS21 and SB62 or with PBS or with the adjuvant alone. Five C57BL/6 mice (Iffa Credo) were used in each group. Mice were monitored twice a week for tumour growth. The mean tumour mass/group is shown in FIG. 26, the mice vaccinated withprotD 1/3 E7 His in PBS or with PBS or the adjuvant alone developed progressively growing tumours (0-1 tumour-free animal/group). On the contrary, four out of five mice vaccinated with protD1/3 E7 His in adjuvant did not develop a tumour, one animal developed a very small and stable tumour atday 40. This results indicate that the protein protD1/3 E7 His fromHPV 16 formulated in adjuvant is able to induce the regression of small established tumours expressing this antigen. - Immunological Read Out
- Proliferation Assay:
- For in vitro assay, Lymphocytes were prepared by crushing the spleen or the popliteal lymph nodes from the vaccinated mice at day 69.
- An aliquot of 2×10e5 cells was plated in triplicate in 96 well plates with decreasing concentrations (10, 1, 0.1 μg/ml) of
protD 1/3 E7 His coated or not onto latex microbeads (Sigma) to restimulate the cells in vitro (72 Hrs). T cell proliferation was measured by 3H thymidine incorporation. - FIGS. 27 and 28 compares the ability of protD E7 to stimulate the proliferation of splenocytes and lymph node cells primed in vivo either by PBS, 3D-MPL, QS21 SB62, ProtD1/3 E7 His and ProtD1/3 E7 His+the adjuvant of 3D-MPL, QS21, SB62 and shows that high proliferative responses in spleen were detected only in mice immunised with protD1/3 E7 His in adjuvant compared to the other groups.
- Antibody Response
- Individual serum were taken at the same time as the organs were taken and submitted to indirect ELISAs.
- 5 μg/ml of purified E7 protein was used as coated antigen. After saturation in PBS+1%
newborn calf serum 1 Hr at 37° C., the sera were serially diluted (starting at {fraction (1/100)}) in the saturation buffer and incubated O/N at 4° C. or 90 min at 37° C. After washing inPBS Tween 20 0.1%, biotinylated goat Anti mouse Ig ({fraction (1/1000)}) or goat anti mouse Ig subclass (total IgG, IgGl, IgG2a, IgG2b) antisera (1/5000) were used as second antibodies, after an incubation of 90 min at 37° C., streptavidin coupled to peroxydase was added and TMB (tetra-methyl-benzidine/peroxide) was used as substrate, after 10 min. the reaction was stopped with H2SO4 0.5 M and the O.D.450 was determined. - The subclass-specific anti E7 titers elicited by the vaccinations in the different groups of mice are shown in FIG. 29 as a comparison of the relative mean midpoint dilution of the serum.
- These results show that a weak antibody response is triggered with 2 injections of
ProtD 1/3 E7 HPV16 alone. - Much more anti-E7 antibodies are generated when ProtD1/3 E7 was injected in the presence of the adjuvant SB62, QS21+3D-MPL.
- No IgA nor IgM were detected in any of the serum samples even in the serum of the mice that received
ProtD 1/3 E7 in the adjuvant SB62, QS21+3D-MPL (data not shown) On the contrary, the total IgG level was slightly increased by the vaccination of the mice withProtD 1/3 E7 alone and was greatly increased by the addition of the adjuvant SB62, QS21+3D-MPL to the protein. The analysis of the concentrations of the different IgG subclass show that a mixed antibody response has been induced as the concentration of all types of IgG subclass analyzed (IgG1, IgG2a, and IgG2b) were increased in the serum of the mice that received the adjuvanted antigen, compared to the concentration observed in the serum of mice that received the antigen or the adjuvant alone. The predominant isotype found was IgG2b which represented more than 80% of the total of IgG), this isotype is generally said to be associated with the induction of a TH1 type immune response. - Mice were immunised 2 times at 14 days interval with either PBS, adjuvant of example 1, 5 μg of protD1/3 E7 His or 5 μg of protD1/3 E7 His in the adjuvant of example 1 intra foot pad in a volume of 100 μl (50 μl/foot pad).
- Tumour Growth:
- Four weeks after the latest vaccination mice were challenged with 2×10e5 TC1 cells/mouse S.C. in the flank. TC1 cells growing in vitro culture, were trypsinised and washed two times in serum-free medium and injected. 5 mice used in each group were monitored twice a week for tumour growth.
- FIG. 30 shows that vaccination with the E7 protein in the SB62 QS21, 3D-MPL adjuvant protect the mice against the development of tumour (only one animal/5 has a very small and stable tumour) in all the other groups, that received the E7 protein without the adjuvant or the adjuvant alone developed growing tumours.
- Immunological Read Out
- Three weeks after the latest vaccination, before the
tumour challenge 5 mice in each group were sacrified for immunological read out. - Proliferation Assay
- For in vitro assay, Lymphocytes were prepared as described above from the spleen and from the popliteal draining lymph nodes. An aliquot of 2×10e5 cells was plated in triplicate in 96 well plates with decreasing concentrations (10, 1, 0.1 μg/ml) of
protD 1/3 E7 His coated or not onto latex microbeads (Sigma) to restimulate the cells in vitro (72 Hrs). T cell proliferation was measured by 3H thymidine incorporation. - FIGS. 31 and 32 show repectively that, both with splenocytes or popliteal lymph node cells, as it was observed in the therapeutic settings, a better lymphoproliferative activity was obtained for the mice that received the E7 protein in the SB62 QS21, 3D-MPL adjuvant antibody response.
- FIG. 33 shows that as in the therapeutic settings, a better antibody response was observed in the serum of mice vaccinated with the ProtD1/3 E7 protein formulated in the 3D-MPL, QS21 O/W adjuvant. A mixed antibody response was triggered, as all the IgG subclass tested (IgG 2a, IgG2b, IgG1), in this case also, IgG2b was the predominant isotype found, representing 75% of the total IgG.
- Mice were vaccinated twice, 2 weeks apart, with 5 μg in 100 μl of
protD 1/3 18 E7 His intra foot pad (50 μl/foot pad) in PBS or QS21, 3D-MPL and SB62, DQ MPL as described in WO96/33739 or DQ alum MPL as described in WO98/15827. Eight 6-8 weeks old Balb/c mice (Iffa Credo) were used in each group. - 14 days post II, the spleen and lymph nodes were taken for immunological read out and blood sampling for serology.
- Immunological Read Out:
- Proliferation Assay:
- For in vitro assay, lymphocytes were prepared by crushing the spleen or the popliteal lymph nodes from the vaccinated mice at
day 28 - An aliquot of 2×10e5 cells was plated in triplicate in 96 well plates with decreasing concentrations (10, 1, 0.1, 0.01 μg/ml) of
protD 1/3 18 E7 His to restimulate the cells in vitro (72 Hrs). T cell proliferation was measured by 3H thymidine incorporation. The results are expressed as stimulation index (cpm sample/cpm baseline) - FIGS. 34 and 35 compares the ability of
protD 1/3 18 E7 to stimulate the proliferation of splenocytes or lymph node cells primed in vivo either by ProtD1/3 18 E7 His or Prot D1/3 18 E7 His+adjuvant and shows that only a basal lymphoproliferation is seen in mice that received the protein alone, on the contrary, high proliferative responses in spleen and very high responses in lymph nodes were detected in mice immunised with protD1/3 18 E7 His in adjuvant. - Cytokine Production
- The cytokines (IL-5 and IFNg) produced in the culture supernatant after a 96 Hrs period of in vitro re-stimulation of spleen or lymph node cells, with medium or with the ProtD1/3 18E7 (1 or 3 μg/ml) was measured by ELISA as described:
- IFNg (Genzyme)
- Quantitation of IFNγ was performed by Elisa using reagents from Genzyme. Samples and antibody solutions were used at 50 μl per well. 96-well microtiter plates (Maxisorb Immuno-plate, Nunc, Denmark) were coated overnight at 4° c. with 50 μl of hamster anti-mouse IFNg diluted at 1.5 μg/ml in carbonate buffer pH 9.5. Plates were then incubated for lhr at 37° c. with 100 μl of PBS containing 1% bovine serum albumin and 0.1% Tween 20 (saturation buffer). Two-fold dilutions of supernatant from in vitro stimulation (starting at {fraction (1/2)}) in saturation buffer were added to the anti-IFNg-coated plates and incubated for 1
hr 30 at 37° c. The plates were washed 4 times with PBS Tween 0.1% (wash buffer) and biotin-conjugated goat anti-mouse IFNg diluted in saturation buffer at a final concentration of 0.5 g/ml was added to each well and incubated for 1 hr at 37° c. After a washing step, AMDEX conjugate (Amersham) diluted {fraction (1/10000)} in saturation buffer was added for 30 min at 37° c. Plates were washed as above and incubated with 50 μl of TMB (Biorad) for 15 min. The reaction was stopped with H2SO4 0.4N and read at 450 nm. Concentrations were calculated using a standard curve (mouse IFNγ standard) by SoftmaxPro (four parameters equation) and expressed in pg/ml. - IL5 (Pharmingen)
- Quantitation of IL5 was performed by Elisa using reagents from Pharmingen. Samples and antibody solutions were used at 50 μl per well. 96-well microtiter plates (Maxisorb Immuno-plate, Nunc, Denmark) were coated overnight at 4° c. with 50 μl of rat anti-mouse IL5 diluted at 1 μg/ml in carbonate buffer pH 9.5. Plates were then incubated for lhr at 37° c. with 100 μl PBS containing 1% bovine serum albumin and 0.1% Tween 20 (saturation buffer). Two-fold dilutions of supernatant from in vitro stimulation (starting at {fraction (1/2)}) in saturation buffer were added to the anti-IFNg-coated plates and incubated for 1
hr 30 at 37° c. The plates were washed 4 times with PBS Tween 0.1% (wash buffer) and biotin-conjugated rat anti-mouse IL5 diluted in saturation buffer at a final concentration of 1 μg/ml was added to each well and incubated for 1 hr at 37° c. After a washing step, AMDEX conjugate (Amersham) diluted {fraction (1/10000)} in saturation buffer was added for 30 min at 37° c. Plates were washed as above and incubated with 50 μl of TMB (Biorad) for 15 min. The reaction was stopped with H2SO4 0.4N and read at 450 nm. Concentrations were calculated using a standard curve (recombinant mouse IL5) by SoftmaxPro (four parameters equation) and expressed in pg/ml. - Starting with spleen cells, no IL-5 could be detected whatever the group tested, on the contrary, a very high production of IFNg production was observed in all groups, with only a slight increase in the group of mice that received the SBAS1c adjuvanted protein compared to the other groups. This suggest the induction of a TH1 type of immune response.
- Regarding lymph node cells, a very weak IFNg production was obtained in the group of mice that received the protein alone and a 5-10 fold increase is observed with the adjuvanted protein. IL5 could only be detected in the group of mice receiving the SBAS2 adjuvanted protein.
- FIGS. 36 and 37 compares the ability of ProtD1/3 18 E7 His to stimulate the production of cytokines 'IFNg and IL5) after in vitro re-stimulation of spleen or lymph node cells respectively.
- Antibody Response
- Individual serum were taken at the same time as the organs and submitted to indirect ELISAs.
- 2.5 μg/ml of purified of protD1/3
18E7 protein HPV 18 was used as coated antigen. After saturation in PBS+1%newborn calf serum 1 Hr at 37° C., the sera were serially diluted (starting at {fraction (1/100)}) in the saturation buffer and incubated O/N at 4° C. or 90 min at 37° C. After washing inPBS Tween 20 0.1%, biotinylated goat Anti mouse Ig ({fraction (1/1000)}) or goat anti mouse Ig subclass (total IgG, IgGI, IgG2a, IgG2b) antisera ({fraction (1/5000)}) were used as second antibodies, after an incubation of 90 min at 37° C., streptavidin coupled to peroxydase was added and TMB (tetra-methyl-benzidine/peroxide) was used as substrate, after 10 min. the reaction was stopped with H2SO4 0.5 M and the O.D.450 was determined. - A very weak antibody response is triggered with 2 injections of
ProtD 1/3 18 E7 alone. The total IgG level was greatly increased by the addition of adjuvants to the protein vaccine. - The analysis of the concentrations of the different IgG subclass show that when the protein was injected in the presence of adjuvants,
DQS21 3D-MPL or SB62, QS21/3D-MPL, a slight increase of the IgG2a subtype percentage was obtained: 28% IgG1, 48% IgG2a and 43% IgG1, 44% IgG2a respectively, compared to 46% of IgG1, 32% of IgG2a with the non adjuvanted protein. The strongest antibody response is obtained with the protein formulated in DQ alum with a clear shift in the isotype concentration (80% IgG1, 8% IgG2a). As the IgG2a isotype in Balb/c mice is generally conidered to be associated with the induction of a TH1 type of immune response, these results suggested that the DQS21, 3D-MPL and SB62 QS21/3D-MPL adjuvants tend toincrease the THI type profile of the humoral response while SBAS5 induce a clear TH2 type of response. - FIG. 38. The comparison of the midpoint dilution of the serum and relative percentage of the different isotypes elicited by the vaccinations in the different groups of mice are shown.
- Conclusion:
- We have demonstrated that the fused protein: {fraction (1/3)} Prot D and early protein E7 of
HPV 16 induced a potent systemic antitumour immunity and the fusion protein ProtD1/3 and E7 of HPV18 has also been showed to be immunogenic in mice Vaccination with the prot D1/3 E7 HPV16 fusion protein protected the mice from a tumour challenge with E7 expressing tumour cells and eliminated small pre-established tumours expressing the E7 of HPV16 injected at a distant site from the vaccination site. - We have demonstrated that the ProtD1/3 E7 HPV16 protein in adjuvant is capable of enhancing helper T cell proliferation suggesting that the antitumour immune response induced by this vaccine is at least in part associated with a CD4+ T cell response.
- We have also demonstrated that a better antibody response was triggered by the vaccination with the ProtD1/3 E7 in the presence of the 3D-MPL containing adjuvant. The predominant isotype found in the serum of C57BL/6 mice being IgG2b suggesting that a THI type immune response was raised.
-
1 23 1 220 PRT Homo sapien 1 Met Asp Pro Ser Ser His Ser Ser Asn Met Ala Asn Thr Gln Met Lys 1 5 10 15 Ser Asp Lys Ile Ile Ile Ala His Arg Gly Ala Ser Gly Tyr Leu Pro 20 25 30 Glu His Thr Leu Glu Ser Lys Ala Leu Ala Phe Ala Gln Gln Ala Asp 35 40 45 Tyr Leu Glu Gln Asp Leu Ala Met Thr Lys Asp Gly Arg Leu Val Val 50 55 60 Ile His Asp His Phe Leu Asp Gly Leu Thr Asp Val Ala Lys Lys Phe 65 70 75 80 Pro His Arg His Arg Lys Asp Gly Arg Tyr Tyr Val Ile Asp Phe Thr 85 90 95 Leu Lys Glu Ile Gln Ser Leu Glu Met Thr Glu Asn Phe Glu Thr Met 100 105 110 Ala Met His Gly Asp Thr Pro Thr Leu His Glu Tyr Met Leu Asp Leu 115 120 125 Gln Pro Glu Thr Thr Asp Leu Tyr Cys Tyr Glu Gln Leu Asn Asp Ser 130 135 140 Ser Glu Glu Glu Asp Glu Ile Asp Gly Pro Ala Gly Gln Ala Glu Pro 145 150 155 160 Asp Arg Ala His Tyr Asn Ile Val Thr Phe Cys Cys Lys Cys Asp Ser 165 170 175 Thr Leu Arg Leu Cys Val Gln Ser Thr His Val Asp Ile Arg Thr Leu 180 185 190 Glu Asp Leu Leu Met Gly Thr Leu Gly Ile Val Cys Pro Ile Cys Ser 195 200 205 Gln Lys Pro Thr Ser Gly His His His His His His 210 215 220 2 663 DNA Homo sapien 2 atggatccaa gcagccattc atcaaatatg gcgaataccc aaatgaaatc agacaaaatc 60 attattgctc accgtggtgc tagcggttat ttaccagagc atacgttaga atctaaagca 120 cttgcgtttg cacaacaggc tgattattta gagcaagatt tagcaatgac taaggatggt 180 cgtttagtgg ttattcacga tcacttttta gatggcttga ctgatgttgc gaaaaaattc 240 ccacatcgtc atcgtaaaga tggccgttac tatgtcatcg actttacctt aaaagaaatt 300 caaagtttag aaatgacaga aaactttgaa accatggcca tgcatggaga tacacctaca 360 ttgcatgaat atatgttaga tttgcaacca gagacaactg atctctactg ttatgagcaa 420 ttaaatgaca gctcagagga ggaggatgaa atagatggtc cagctggaca agcagaaccg 480 gacagagccc attacaatat tgtaaccttt tgttgcaagt gtgactctac gcttcggttg 540 tgcgtacaaa gcacacacgt agacattcgt actttggaag acctgttaat gggcacacta 600 ggaattgtgt gccccatctg ttctcagaaa ccaactagtg gccaccatca ccatcaccat 660 taa 663 3 822 DNA Homo sapien 3 atggatccaa gcagccattc atcaaatatg gcgaataccc aaatgaaatc agacaaaatc 60 attattgctc accgtggtgc tagcggttat ttaccagagc atacgttaga atctaaagca 120 cttgcgtttg cacaacaggc tgattattta gagcaagatt tagcaatgac taaggatggt 180 cgtttagtgg ttattcacga tcacttttta gatggcttga ctgatgttgc gaaaaaattc 240 ccacatcgtc atcgtaaaga tggccgttac tatgtcatcg actttacctt aaaagaaatt 300 caaagtttag aaatgacaga aaactttgaa accatggcca tgtttcagga cccacaggag 360 cgacccagaa agttaccaca gttatgcaca gagctgcaaa caactataca tgatataata 420 ttagaatgtg tgtactgcaa gcaacagtta ctgcgacgtg aggtatatga ctttgctttt 480 cgggatttat gcatagtata tagagatggg aatccatatg ctgtatgtga taaatgttta 540 aagttttatt ctaaaattag tgagtataga cattattgtt atagtttgta tggaacaaca 600 ttagaacagc aatacaacaa accgttgtgt gatttgttaa ttaggtgtat taactgtcaa 660 aagccactgt gtcctgaaga aaagcaaaga catctggaca aaaagcaaag attccataat 720 ataaggggtc ggtggaccgg tcgatgtatg tcttgttgca gatcatcaag aacacgtaga 780 gaaacccagc tgactagtgg ccaccatcac catcaccatt aa 822 4 273 PRT Homo sapien 4 Met Asp Pro Ser Ser His Ser Ser Asn Met Ala Asn Thr Gln Met Lys 1 5 10 15 Ser Asp Lys Ile Ile Ile Ala His Arg Gly Ala Ser Gly Tyr Leu Pro 20 25 30 Glu His Thr Leu Glu Ser Lys Ala Leu Ala Phe Ala Gln Gln Ala Asp 35 40 45 Tyr Leu Glu Gln Asp Leu Ala Met Thr Lys Asp Gly Arg Leu Val Val 50 55 60 Ile His Asp His Phe Leu Asp Gly Leu Thr Asp Val Ala Lys Lys Phe 65 70 75 80 Pro His Arg His Arg Lys Asp Gly Arg Tyr Tyr Val Ile Asp Phe Thr 85 90 95 Leu Lys Glu Ile Gln Ser Leu Glu Met Thr Glu Asn Phe Glu Thr Met 100 105 110 Ala Met Phe Gln Asp Pro Gln Glu Arg Pro Arg Lys Leu Pro Gln Leu 115 120 125 Cys Thr Glu Leu Gln Thr Thr Ile His Asp Ile Ile Leu Glu Cys Val 130 135 140 Tyr Cys Lys Gln Gln Leu Leu Arg Arg Glu Val Tyr Asp Phe Ala Phe 145 150 155 160 Arg Asp Leu Cys Ile Val Tyr Arg Asp Gly Asn Pro Tyr Ala Val Cys 165 170 175 Asp Lys Cys Leu Lys Phe Tyr Ser Lys Ile Ser Glu Tyr Arg His Tyr 180 185 190 Cys Tyr Ser Leu Tyr Gly Thr Thr Leu Glu Gln Gln Tyr Asn Lys Pro 195 200 205 Leu Cys Asp Leu Leu Ile Arg Cys Ile Asn Cys Gln Lys Pro Leu Cys 210 215 220 Pro Glu Glu Lys Gln Arg His Leu Asp Lys Lys Gln Arg Phe His Asn 225 230 235 240 Ile Arg Gly Arg Trp Thr Gly Arg Cys Met Ser Cys Cys Arg Ser Ser 245 250 255 Arg Thr Arg Arg Glu Thr Gln Leu Thr Ser Gly His His His His His 260 265 270 His 5 1116 DNA Homo sapien 5 atggatccaa gcagccattc atcaaatatg gcgaataccc aaatgaaatc agacaaaatc 60 attattgctc accgtggtgc tagcggttat ttaccagagc atacgttaga atctaaagca 120 cttgcgtttg cacaacaggc tgattattta gagcaagatt tagcaatgac taaggatggt 180 cgtttagtgg ttattcacga tcacttttta gatggcttga ctgatgttgc gaaaaaattc 240 ccacatcgtc atcgtaaaga tggccgttac tatgtcatcg actttacctt aaaagaaatt 300 caaagtttag aaatgacaga aaactttgaa accatggcca tgtttcagga cccacaggag 360 cgacccagaa agttaccaca gttatgcaca gagctgcaaa caactataca tgatataata 420 ttagaatgtg tgtactgcaa gcaacagtta ctgcgacgtg aggtatatga ctttgctttt 480 cgggatttat gcatagtata tagagatggg aatccatatg ctgtatgtga taaatgttta 540 aagttttatt ctaaaattag tgagtataga cattattgtt atagtttgta tggaacaaca 600 ttagaacagc aatacaacaa accgttgtgt gatttgttaa ttaggtgtat taactgtcaa 660 aagccactgt gtcctgaaga aaagcaaaga catctggaca aaaagcaaag attccataat 720 ataaggggtc ggtggaccgg tcgatgtatg tcttgttgca gatcatcaag aacacgtaga 780 gaaacccagc tgatgcatgg agatacacct acattgcatg aatatatgtt agatttgcaa 840 ccagagacaa ctgatctcta ctgttatgag caattaaatg acagctcaga ggaggaggat 900 gaaatagatg gtccagctgg acaagcagaa ccggacagag cccattacaa tattgtaacc 960 ttttgttgca agtgtgactc tacgcttcgg ttgtgcgtac aaagcacaca cgtagacatt 1020 cgtactttgg aagacctgtt aatgggcaca ctaggaattg tgtgccccat ctgttctcag 1080 aaaccaacta gtggccacca tcaccatcac cattaa 1116 6 371 PRT Homo sapien 6 Met Asp Pro Ser Ser His Ser Ser Asn Met Ala Asn Thr Gln Met Lys 1 5 10 15 Ser Asp Lys Ile Ile Ile Ala His Arg Gly Ala Ser Gly Tyr Leu Pro 20 25 30 Glu His Thr Leu Glu Ser Lys Ala Leu Ala Phe Ala Gln Gln Ala Asp 35 40 45 Tyr Leu Glu Gln Asp Leu Ala Met Thr Lys Asp Gly Arg Leu Val Val 50 55 60 Ile His Asp His Phe Leu Asp Gly Leu Thr Asp Val Ala Lys Lys Phe 65 70 75 80 Pro His Arg His Arg Lys Asp Gly Arg Tyr Tyr Val Ile Asp Phe Thr 85 90 95 Leu Lys Glu Ile Gln Ser Leu Glu Met Thr Glu Asn Phe Glu Thr Met 100 105 110 Ala Met Phe Gln Asp Pro Gln Glu Arg Pro Arg Lys Leu Pro Gln Leu 115 120 125 Cys Thr Glu Leu Gln Thr Thr Ile His Asp Ile Ile Leu Glu Cys Val 130 135 140 Tyr Cys Lys Gln Gln Leu Leu Arg Arg Glu Val Tyr Asp Phe Ala Phe 145 150 155 160 Arg Asp Leu Cys Ile Val Tyr Arg Asp Gly Asn Pro Tyr Ala Val Cys 165 170 175 Asp Lys Cys Leu Lys Phe Tyr Ser Lys Ile Ser Glu Tyr Arg His Tyr 180 185 190 Cys Tyr Ser Leu Tyr Gly Thr Thr Leu Glu Gln Gln Tyr Asn Lys Pro 195 200 205 Leu Cys Asp Leu Leu Ile Arg Cys Ile Asn Cys Gln Lys Pro Leu Cys 210 215 220 Pro Glu Glu Lys Gln Arg His Leu Asp Lys Lys Gln Arg Phe His Asn 225 230 235 240 Ile Arg Gly Arg Trp Thr Gly Arg Cys Met Ser Cys Cys Arg Ser Ser 245 250 255 Arg Thr Arg Arg Glu Thr Gln Leu Met His Gly Asp Thr Pro Thr Leu 260 265 270 His Glu Tyr Met Leu Asp Leu Gln Pro Glu Thr Thr Asp Leu Tyr Cys 275 280 285 Tyr Glu Gln Leu Asn Asp Ser Ser Glu Glu Glu Asp Glu Ile Asp Gly 290 295 300 Pro Ala Gly Gln Ala Glu Pro Asp Arg Ala His Tyr Asn Ile Val Thr 305 310 315 320 Phe Cys Cys Lys Cys Asp Ser Thr Leu Arg Leu Cys Val Gln Ser Thr 325 330 335 His Val Asp Ile Arg Thr Leu Glu Asp Leu Leu Met Gly Thr Leu Gly 340 345 350 Ile Val Cys Pro Ile Cys Ser Gln Lys Pro Thr Ser Gly His His His 355 360 365 His His His 370 7 663 DNA Homo sapien 7 atggatccaa gcagccattc atcaaatatg gcgaataccc aaatgaaatc agacaaaatc 60 attattgctc accgtggtgc tagcggttat ttaccagagc atacgttaga atctaaagca 120 cttgcgtttg cacaacaggc tgattattta gagcaagatt tagcaatgac taaggatggt 180 cgtttagtgg ttattcacga tcacttttta gatggcttga ctgatgttgc gaaaaaattc 240 ccacatcgtc atcgtaaaga tggccgttac tatgtcatcg actttacctt aaaagaaatt 300 caaagtttag aaatgacaga aaactttgaa accatggcca tgcatggaga tacacctaca 360 ttgcatgaat atatgttaga tttgcaacca gagacaactg atctctacgg ttatcagcaa 420 ttaaatgaca gctcagagga ggaggatgaa atagatggtc cagctggaca agcagaaccg 480 gacagagccc attacaatat tgtaaccttt tgttgcaagt gtgactctac gcttcggttg 540 tgcgtacaaa gcacacacgt agacattcgt actttggaag acctgttaat gggcacacta 600 ggaattgtgt gccccatctg ttctcagaaa ccaactagtg gccaccatca ccatcaccat 660 taa 663 8 220 PRT Homo sapien 8 Met Asp Pro Ser Ser His Ser Ser Asn Met Ala Asn Thr Gln Met Lys 1 5 10 15 Ser Asp Lys Ile Ile Ile Ala His Arg Gly Ala Ser Gly Tyr Leu Pro 20 25 30 Glu His Thr Leu Glu Ser Lys Ala Leu Ala Phe Ala Gln Gln Ala Asp 35 40 45 Tyr Leu Glu Gln Asp Leu Ala Met Thr Lys Asp Gly Arg Leu Val Val 50 55 60 Ile His Asp His Phe Leu Asp Gly Leu Thr Asp Val Ala Lys Lys Phe 65 70 75 80 Pro His Arg His Arg Lys Asp Gly Arg Tyr Tyr Val Ile Asp Phe Thr 85 90 95 Leu Lys Glu Ile Gln Ser Leu Glu Met Thr Glu Asn Phe Glu Thr Met 100 105 110 Ala Met His Gly Asp Thr Pro Thr Leu His Glu Tyr Met Leu Asp Leu 115 120 125 Gln Pro Glu Thr Thr Asp Leu Tyr Gly Tyr Gln Gln Leu Asn Asp Ser 130 135 140 Ser Glu Glu Glu Asp Glu Ile Asp Gly Pro Ala Gly Gln Ala Glu Pro 145 150 155 160 Asp Arg Ala His Tyr Asn Ile Val Thr Phe Cys Cys Lys Cys Asp Ser 165 170 175 Thr Leu Arg Leu Cys Val Gln Ser Thr His Val Asp Ile Arg Thr Leu 180 185 190 Glu Asp Leu Leu Met Gly Thr Leu Gly Ile Val Cys Pro Ile Cys Ser 195 200 205 Gln Lys Pro Thr Ser Gly His His His His His His 210 215 220 9 879 DNA Homo sapien 9 atgaaagggg gaattgtaca ttcagacggc tcttatccaa aagacaagtt tgagaaaatc 60 aatggcactt ggtactactt tgacagttca ggctatatgc ttgcagaccg ctggaggaag 120 cacacagacg gcaactggta ctggttcgac aactcaggcg aaatggctac aggctggaag 180 aaaatcgctg ataagtggta ctatttcaac gaagaaggtg ccatgaagac aggctgggtc 240 aagtacaagg acacttggta ctacttagac gctaaagaag gcgccatggt atcaaatgcc 300 tttatccagt cagcggacgg aacaggctgg tactacctca aaccagacgg aacactggca 360 gacaggccag aattggccag catgctggac atggccatgt ttcaggaccc acaggagcga 420 cccagaaagt taccacagtt atgcacagag ctgcaaacaa ctatacatga tataatatta 480 gaatgtgtgt actgcaagca acagttactg cgacgtgagg tatatgactt tgcttttcgg 540 gatttatgca tagtatatag agatgggaat ccatatgctg tatgtgataa atgtttaaag 600 ttttattcta aaattagtga gtatagacat tattgttata gtttgtatgg aacaacatta 660 gaacagcaat acaacaaacc gttgtgtgat ttgttaatta ggtgtattaa ctgtcaaaag 720 ccactgtgtc ctgaagaaaa gcaaagacat ctggacaaaa agcaaagatt ccataatata 780 aggggtcggt ggaccggtcg atgtatgtct tgttgcagat catcaagaac acgtagagaa 840 acccagctga ctagtggcca ccatcaccat caccattaa 879 10 292 PRT Homo sapien 10 Met Lys Gly Gly Ile Val His Ser Asp Gly Ser Tyr Pro Lys Asp Lys 1 5 10 15 Phe Glu Lys Ile Asn Gly Thr Trp Tyr Tyr Phe Asp Ser Ser Gly Tyr 20 25 30 Met Leu Ala Asp Arg Trp Arg Lys His Thr Asp Gly Asn Trp Tyr Trp 35 40 45 Phe Asp Asn Ser Gly Glu Met Ala Thr Gly Trp Lys Lys Ile Ala Asp 50 55 60 Lys Trp Tyr Tyr Phe Asn Glu Glu Gly Ala Met Lys Thr Gly Trp Val 65 70 75 80 Lys Tyr Lys Asp Thr Trp Tyr Tyr Leu Asp Ala Lys Glu Gly Ala Met 85 90 95 Val Ser Asn Ala Phe Ile Gln Ser Ala Asp Gly Thr Gly Trp Tyr Tyr 100 105 110 Leu Lys Pro Asp Gly Thr Leu Ala Asp Arg Pro Glu Leu Ala Ser Met 115 120 125 Leu Asp Met Ala Met Phe Gln Asp Pro Gln Glu Arg Pro Arg Lys Leu 130 135 140 Pro Gln Leu Cys Thr Glu Leu Gln Thr Thr Ile His Asp Ile Ile Leu 145 150 155 160 Glu Cys Val Tyr Cys Lys Gln Gln Leu Leu Arg Arg Glu Val Tyr Asp 165 170 175 Phe Ala Phe Arg Asp Leu Cys Ile Val Tyr Arg Asp Gly Asn Pro Tyr 180 185 190 Ala Val Cys Asp Lys Cys Leu Lys Phe Tyr Ser Lys Ile Ser Glu Tyr 195 200 205 Arg His Tyr Cys Tyr Ser Leu Tyr Gly Thr Thr Leu Glu Gln Gln Tyr 210 215 220 Asn Lys Pro Leu Cys Asp Leu Leu Ile Arg Cys Ile Asn Cys Gln Lys 225 230 235 240 Pro Leu Cys Pro Glu Glu Lys Gln Arg His Leu Asp Lys Lys Gln Arg 245 250 255 Phe His Asn Ile Arg Gly Arg Trp Thr Gly Arg Cys Met Ser Cys Cys 260 265 270 Arg Ser Ser Arg Thr Arg Arg Glu Thr Gln Leu Thr Ser Gly His His 275 280 285 His His His His 290 11 720 DNA Homo sapien 11 atgaaagggg gaattgtaca ttcagacggc tcttatccaa aagacaagtt tgagaaaatc 60 aatggcactt ggtactactt tgacagttca ggctatatgc ttgcagaccg ctggaggaag 120 cacacagacg gcaactggta ctggttcgac aactcaggcg aaatggctac aggctggaag 180 aaaatcgctg ataagtggta ctatttcaac gaagaaggtg ccatgaagac aggctgggtc 240 aagtacaagg acacttggta ctacttagac gctaaagaag gcgccatggt atcaaatgcc 300 tttatccagt cagcggacgg aacaggctgg tactacctca aaccagacgg aacactggca 360 gacaggccag aattggccag catgctggac atggccatgc atggagatac acctacattg 420 catgaatata tgttagattt gcaaccagag acaactgatc tctactgtta tgagcaatta 480 aatgacagct cagaggagga ggatgaaata gatggtccag ctggacaagc agaaccggac 540 agagcccatt acaatattgt aaccttttgt tgcaagtgtg actctacgct tcggttgtgc 600 gtacaaagca cacacgtaga cattcgtact ttggaagacc tgttaatggg cacactagga 660 attgtgtgcc ccatctgttc tcagaaacca actagtggcc accatcacca tcaccattaa 720 12 239 PRT Homo sapien 12 Met Lys Gly Gly Ile Val His Ser Asp Gly Ser Tyr Pro Lys Asp Lys 1 5 10 15 Phe Glu Lys Ile Asn Gly Thr Trp Tyr Tyr Phe Asp Ser Ser Gly Tyr 20 25 30 Met Leu Ala Asp Arg Trp Arg Lys His Thr Asp Gly Asn Trp Tyr Trp 35 40 45 Phe Asp Asn Ser Gly Glu Met Ala Thr Gly Trp Lys Lys Ile Ala Asp 50 55 60 Lys Trp Tyr Tyr Phe Asn Glu Glu Gly Ala Met Lys Thr Gly Trp Val 65 70 75 80 Lys Tyr Lys Asp Thr Trp Tyr Tyr Leu Asp Ala Lys Glu Gly Ala Met 85 90 95 Val Ser Asn Ala Phe Ile Gln Ser Ala Asp Gly Thr Gly Trp Tyr Tyr 100 105 110 Leu Lys Pro Asp Gly Thr Leu Ala Asp Arg Pro Glu Leu Ala Ser Met 115 120 125 Leu Asp Met Ala Met His Gly Asp Thr Pro Thr Leu His Glu Tyr Met 130 135 140 Leu Asp Leu Gln Pro Glu Thr Thr Asp Leu Tyr Cys Tyr Glu Gln Leu 145 150 155 160 Asn Asp Ser Ser Glu Glu Glu Asp Glu Ile Asp Gly Pro Ala Gly Gln 165 170 175 Ala Glu Pro Asp Arg Ala His Tyr Asn Ile Val Thr Phe Cys Cys Lys 180 185 190 Cys Asp Ser Thr Leu Arg Leu Cys Val Gln Ser Thr His Val Asp Ile 195 200 205 Arg Thr Leu Glu Asp Leu Leu Met Gly Thr Leu Gly Ile Val Cys Pro 210 215 220 Ile Cys Ser Gln Lys Pro Thr Ser Gly His His His His His His 225 230 235 13 1173 DNA Homo sapien 13 atgaaagggg gaattgtaca ttcagacggc tcttatccaa aagacaagtt tgagaaaatc 60 aatggcactt ggtactactt tgacagttca ggctatatgc ttgcagaccg ctggaggaag 120 cacacagacg gcaactggta ctggttcgac aactcaggcg aaatggctac aggctggaag 180 aaaatcgctg ataagtggta ctatttcaac gaagaaggtg ccatgaagac aggctgggtc 240 aagtacaagg acacttggta ctacttagac gctaaagaag gcgccatggt atcaaatgcc 300 tttatccagt cagcggacgg aacaggctgg tactacctca aaccagacgg aacactggca 360 gacaggccag aattggccag catgctggac atggccatgt ttcaggaccc acaggagcga 420 cccagaaagt taccacagtt atgcacagag ctgcaaacaa ctatacatga tataatatta 480 gaatgtgtgt actgcaagca acagttactg cgacgtgagg tatatgactt tgcttttcgg 540 gatttatgca tagtatatag agatgggaat ccatatgctg tatgtgataa atgtttaaag 600 ttttattcta aaattagtga gtatagacat tattgttata gtttgtatgg aacaacatta 660 gaacagcaat acaacaaacc gttgtgtgat ttgttaatta ggtgtattaa ctgtcaaaag 720 ccactgtgtc ctgaagaaaa gcaaagacat ctggacaaaa agcaaagatt ccataatata 780 aggggtcggt ggaccggtcg atgtatgtct tgttgcagat catcaagaac acgtagagaa 840 acccagctga tgcatggaga tacacctaca ttgcatgaat atatgttaga tttgcaacca 900 gagacaactg atctctactg ttatgagcaa ttaaatgaca gctcagagga ggaggatgaa 960 atagatggtc cagctggaca agcagaaccg gacagagccc attacaatat tgtaaccttt 1020 tgttgcaagt gtgactctac gcttcggttg tgcgtacaaa gcacacacgt agacattcgt 1080 actttggaag acctgttaat gggcacacta ggaattgtgt gccccatctg ttctcagaaa 1140 ccaactagtg gccaccatca ccatcaccat taa 1173 14 390 PRT Homo sapien 14 Met Lys Gly Gly Ile Val His Ser Asp Gly Ser Tyr Pro Lys Asp Lys 1 5 10 15 Phe Glu Lys Ile Asn Gly Thr Trp Tyr Tyr Phe Asp Ser Ser Gly Tyr 20 25 30 Met Leu Ala Asp Arg Trp Arg Lys His Thr Asp Gly Asn Trp Tyr Trp 35 40 45 Phe Asp Asn Ser Gly Glu Met Ala Thr Gly Trp Lys Lys Ile Ala Asp 50 55 60 Lys Trp Tyr Tyr Phe Asn Glu Glu Gly Ala Met Lys Thr Gly Trp Val 65 70 75 80 Lys Tyr Lys Asp Thr Trp Tyr Tyr Leu Asp Ala Lys Glu Gly Ala Met 85 90 95 Val Ser Asn Ala Phe Ile Gln Ser Ala Asp Gly Thr Gly Trp Tyr Tyr 100 105 110 Leu Lys Pro Asp Gly Thr Leu Ala Asp Arg Pro Glu Leu Ala Ser Met 115 120 125 Leu Asp Met Ala Met Phe Gln Asp Pro Gln Glu Arg Pro Arg Lys Leu 130 135 140 Pro Gln Leu Cys Thr Glu Leu Gln Thr Thr Ile His Asp Ile Ile Leu 145 150 155 160 Glu Cys Val Tyr Cys Lys Gln Gln Leu Leu Arg Arg Glu Val Tyr Asp 165 170 175 Phe Ala Phe Arg Asp Leu Cys Ile Val Tyr Arg Asp Gly Asn Pro Tyr 180 185 190 Ala Val Cys Asp Lys Cys Leu Lys Phe Tyr Ser Lys Ile Ser Glu Tyr 195 200 205 Arg His Tyr Cys Tyr Ser Leu Tyr Gly Thr Thr Leu Glu Gln Gln Tyr 210 215 220 Asn Lys Pro Leu Cys Asp Leu Leu Ile Arg Cys Ile Asn Cys Gln Lys 225 230 235 240 Pro Leu Cys Pro Glu Glu Lys Gln Arg His Leu Asp Lys Lys Gln Arg 245 250 255 Phe His Asn Ile Arg Gly Arg Trp Thr Gly Arg Cys Met Ser Cys Cys 260 265 270 Arg Ser Ser Arg Thr Arg Arg Glu Thr Gln Leu Met His Gly Asp Thr 275 280 285 Pro Thr Leu His Glu Tyr Met Leu Asp Leu Gln Pro Glu Thr Thr Asp 290 295 300 Leu Tyr Cys Tyr Glu Gln Leu Asn Asp Ser Ser Glu Glu Glu Asp Glu 305 310 315 320 Ile Asp Gly Pro Ala Gly Gln Ala Glu Pro Asp Arg Ala His Tyr Asn 325 330 335 Ile Val Thr Phe Cys Cys Lys Cys Asp Ser Thr Leu Arg Leu Cys Val 340 345 350 Gln Ser Thr His Val Asp Ile Arg Thr Leu Glu Asp Leu Leu Met Gly 355 360 365 Thr Leu Gly Ile Val Cys Pro Ile Cys Ser Gln Lys Pro Thr Ser Gly 370 375 380 His His His His His His 385 390 15 684 DNA Homo sapien 15 atggatccaa gcagccattc atcaaatatg gcgaataccc aaatgaaatc agacaaaatc 60 attattgctc accgtggtgc tagcggttat ttaccagagc atacgttaga atctaaagca 120 cttgcgtttg cacaacaggc tgattattta gagcaagatt tagcaatgac taaggatggt 180 cgtttagtgg ttattcacga tcacttttta gatggcttga ctgatgttgc gaaaaaattc 240 ccacatcgtc atcgtaaaga tggccgttac tatgtcatcg actttacctt aaaagaaatt 300 caaagtttag aaatgacaga aaactttgaa accatggcca tgcatggacc taaggcaaca 360 ttgcaagaca ttgtattgca tttagagccc caaaatgaaa ttccggttga ccttctatgt 420 cacgagcaat taagcgactc agaggaagaa aacgatgaaa tagatgaagt taatcatcaa 480 catttaccag cccgacgagc cgaaccacaa cgtcacacaa tgttgtgtat gtgttgtaag 540 tgtgaagcca gaattgagct agtagtagaa agctcagcag acgaccttcg agcattccag 600 cagctgtttc tgaacaccct gtcctttgtg tgtccgtggt gtgcatccca gcagactagt 660 ggccaccatc accatcacca ttaa 684 16 227 PRT Homo sapien 16 Met Asp Pro Ser Ser His Ser Ser Asn Met Ala Asn Thr Gln Met Lys 1 5 10 15 Ser Asp Lys Ile Ile Ile Ala His Arg Gly Ala Ser Gly Tyr Leu Pro 20 25 30 Glu His Thr Leu Glu Ser Lys Ala Leu Ala Phe Ala Gln Gln Ala Asp 35 40 45 Tyr Leu Glu Gln Asp Leu Ala Met Thr Lys Asp Gly Arg Leu Val Val 50 55 60 Ile His Asp His Phe Leu Asp Gly Leu Thr Asp Val Ala Lys Lys Phe 65 70 75 80 Pro His Arg His Arg Lys Asp Gly Arg Tyr Tyr Val Ile Asp Phe Thr 85 90 95 Leu Lys Glu Ile Gln Ser Leu Glu Met Thr Glu Asn Phe Glu Thr Met 100 105 110 Ala Met His Gly Pro Lys Ala Thr Leu Gln Asp Ile Val Leu His Leu 115 120 125 Glu Pro Gln Asn Glu Ile Pro Val Asp Leu Leu Cys His Glu Gln Leu 130 135 140 Ser Asp Ser Glu Glu Glu Asn Asp Glu Ile Asp Glu Val Asn His Gln 145 150 155 160 His Leu Pro Ala Arg Arg Ala Glu Pro Gln Arg His Thr Met Leu Cys 165 170 175 Met Cys Cys Lys Cys Glu Ala Arg Ile Glu Leu Val Val Glu Ser Ser 180 185 190 Ala Asp Asp Leu Arg Ala Phe Gln Gln Leu Phe Leu Asn Thr Leu Ser 195 200 205 Phe Val Cys Pro Trp Cys Ala Ser Gln Gln Thr Ser Gly His His His 210 215 220 His His His 225 17 109 PRT Homo sapien 17 Met Ser Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp 1 5 10 15 Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp 20 25 30 Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp 35 40 45 Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn 50 55 60 Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu 65 70 75 80 Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser 85 90 95 Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala 100 105 18 684 DNA Homo sapien 18 atggatccaa gcagccattc atcaaatatg gcgaataccc aaatgaaatc agacaaaatc 60 attattgctc accgtggtgc tagcggttat ttaccagagc atacgttaga atctaaagca 120 cttgcgtttg cacaacaggc tgattattta gagcaagatt tagcaatgac taaggatggt 180 cgtttagtgg ttattcacga tcacttttta gatggcttga ctgatgttgc gaaaaaattc 240 ccacatcgtc atcgtaaaga tggccgttac tatgtcatcg actttacctt aaaagaaatt 300 caaagtttag aaatgacaga aaactttgaa accatggcca tgcatggacc taaggcaaca 360 ttgcaagaca ttgtattgca tttagagccc caaaatgaaa ttccggttga ccttctaggt 420 caccagcaat taagcgactc agaggaagaa aacgatgaaa tagatggagt taatcatcaa 480 catttaccag cccgacgagc cgaaccacaa cgtcacacaa tgttgtgtat gtgttgtaag 540 tgtgaagcca gaattgagct agtagtagaa agctcagcag acgaccttcg agcattccag 600 cagctgtttc tgaacaccct gtcctttgtg tgtccgtggt gtgcatccca gcagactagt 660 ggccaccatc accatcacca ttaa 684 19 227 PRT Homo sapien 19 Met Asp Pro Ser Ser His Ser Ser Asn Met Ala Asn Thr Gln Met Lys 1 5 10 15 Ser Asp Lys Ile Ile Ile Ala His Arg Gly Ala Ser Gly Tyr Leu Pro 20 25 30 Glu His Thr Leu Glu Ser Lys Ala Leu Ala Phe Ala Gln Gln Ala Asp 35 40 45 Tyr Leu Glu Gln Asp Leu Ala Met Thr Lys Asp Gly Arg Leu Val Val 50 55 60 Ile His Asp His Phe Leu Asp Gly Leu Thr Asp Val Ala Lys Lys Phe 65 70 75 80 Pro His Arg His Arg Lys Asp Gly Arg Tyr Tyr Val Ile Asp Phe Thr 85 90 95 Leu Lys Glu Ile Gln Ser Leu Glu Met Thr Glu Asn Phe Glu Thr Met 100 105 110 Ala Met His Gly Pro Lys Ala Thr Leu Gln Asp Ile Val Leu His Leu 115 120 125 Glu Pro Gln Asn Glu Ile Pro Val Asp Leu Leu Gly His Gln Gln Leu 130 135 140 Ser Asp Ser Glu Glu Glu Asn Asp Glu Ile Asp Gly Val Asn His Gln 145 150 155 160 His Leu Pro Ala Arg Arg Ala Glu Pro Gln Arg His Thr Met Leu Cys 165 170 175 Met Cys Cys Lys Cys Glu Ala Arg Ile Glu Leu Val Val Glu Ser Ser 180 185 190 Ala Asp Asp Leu Arg Ala Phe Gln Gln Leu Phe Leu Asn Thr Leu Ser 195 200 205 Phe Val Cys Pro Trp Cys Ala Ser Gln Gln Thr Ser Gly His His His 210 215 220 His His His 225 20 837 DNA Homo sapien 20 atggatccaa gcagccattc atcaaatatg gcgaataccc aaatgaaatc agacaaaatc 60 attattgctc accgtggtgc tagcggttat ttaccagagc atacgttaga atctaaagca 120 cttgcgtttg cacaacaggc tgattattta gagcaagatt tagcaatgac taaggatggt 180 cgtttagtgg ttattcacga tcacttttta gatggcttga ctgatgttgc gaaaaaattc 240 ccacatcgtc atcgtaaaga tggccgttac tatgtcatcg actttacctt aaaagaaatt 300 caaagtttag aaatgacaga aaactttgaa accatggcgc gctttgagga tccaacacgg 360 cgaccctaca agctacctga tctgtgcacg gaactgaaca cttcactgca agacatagaa 420 ataacctgtg tatattgcaa gacagtattg gaacttacag aggtatttga atttgcattt 480 aaagatttat ttgtggtgta tagagacagt ataccgcatg ctgcatgcca taaatgtata 540 gatttttatt ctagaattag agaattaaga cattattcag actctgtgta tggagacaca 600 ttggaaaaac taactaacac tgggttatac aatttattaa taaggtgcct gcggtgccag 660 aaaccgttga atccagcaga aaaacttaga caccttaatg aaaaacgacg atttcacaac 720 atagctgggc actatagagg ccagtgccat tcgtgctgca accgagcacg acaggaacga 780 ctccaacgac gcagagaaac acaagtaact agtggccacc atcaccatca ccattaa 837 21 278 PRT Homo sapien 21 Met Asp Pro Ser Ser His Ser Ser Asn Met Ala Asn Thr Gln Met Lys 1 5 10 15 Ser Asp Lys Ile Ile Ile Ala His Arg Gly Ala Ser Gly Tyr Leu Pro 20 25 30 Glu His Thr Leu Glu Ser Lys Ala Leu Ala Phe Ala Gln Gln Ala Asp 35 40 45 Tyr Leu Glu Gln Asp Leu Ala Met Thr Lys Asp Gly Arg Leu Val Val 50 55 60 Ile His Asp His Phe Leu Asp Gly Leu Thr Asp Val Ala Lys Lys Phe 65 70 75 80 Pro His Arg His Arg Lys Asp Gly Arg Tyr Tyr Val Ile Asp Phe Thr 85 90 95 Leu Lys Glu Ile Gln Ser Leu Glu Met Thr Glu Asn Phe Glu Thr Met 100 105 110 Ala Arg Phe Glu Asp Pro Thr Arg Arg Pro Tyr Lys Leu Pro Asp Leu 115 120 125 Cys Thr Glu Leu Asn Thr Ser Leu Gln Asp Ile Glu Ile Thr Cys Val 130 135 140 Tyr Cys Lys Thr Val Leu Glu Leu Thr Glu Val Phe Glu Phe Ala Phe 145 150 155 160 Lys Asp Leu Phe Val Val Tyr Arg Asp Ser Ile Pro His Ala Ala Cys 165 170 175 His Lys Cys Ile Asp Phe Tyr Ser Arg Ile Arg Glu Leu Arg His Tyr 180 185 190 Ser Asp Ser Val Tyr Gly Asp Thr Leu Glu Lys Leu Thr Asn Thr Gly 195 200 205 Leu Tyr Asn Leu Leu Ile Arg Cys Leu Arg Cys Gln Lys Pro Leu Asn 210 215 220 Pro Ala Glu Lys Leu Arg His Leu Asn Glu Lys Arg Arg Phe His Asn 225 230 235 240 Ile Ala Gly His Tyr Arg Gly Gln Cys His Ser Cys Cys Asn Arg Ala 245 250 255 Arg Gln Glu Arg Leu Gln Arg Arg Arg Glu Thr Gln Val Thr Ser Gly 260 265 270 His His His His His His 275 22 1152 DNA Homo sapien 22 atggatccaa gcagccattc atcaaatatg gcgaataccc aaatgaaatc agacaaaatc 60 attattgctc accgtggtgc tagcggttat ttaccagagc atacgttaga atctaaagca 120 cttgcgtttg cacaacaggc tgattattta gagcaagatt tagcaatgac taaggatggt 180 cgtttagtgg ttattcacga tcacttttta gatggcttga ctgatgttgc gaaaaaattc 240 ccacatcgtc atcgtaaaga tggccgttac tatgtcatcg actttacctt aaaagaaatt 300 caaagtttag aaatgacaga aaactttgaa accatggcgc gctttgagga tccaacacgg 360 cgaccctaca agctacctga tctgtgcacg gaactgaaca cttcactgca agacatagaa 420 ataacctgtg tatattgcaa gacagtattg gaacttacag aggtatttga atttgcattt 480 aaagatttat ttgtggtgta tagagacagt ataccgcatg ctgcatgcca taaatgtata 540 gatttttatt ctagaattag agaattaaga cattattcag actctgtgta tggagacaca 600 ttggaaaaac taactaacac tgggttatac aatttattaa taaggtgcct gcggtgccag 660 aaaccgttga atccagcaga aaaacttaga caccttaatg aaaaacgacg atttcacaac 720 atagctgggc actatagagg ccagtgccat tcgtgctgca accgagcacg acaggaacga 780 ctccaacgac gcagagaaac acaagtaatg catggaccta aggcaacatt gcaagacatt 840 gtattgcatt tagagcccca aaatgaaatt ccggttgacc ttctatgtca cgagcaatta 900 agcgactcag aggaagaaaa cgatgaaata gatggagtta atcatcaaca tttaccagcc 960 cgacgagccg aaccacaacg tcacacaatg ttgtgtatgt gttgtaagtg tgaagccaga 1020 attgagctag tagtagaaag ctcagcagac gaccttcgag cattccagca gctgtttctg 1080 aacaccctgt cctttgtgtg tccgtggtgt gcatcccagc agactagtgg ccaccatcac 1140 catcaccatt aa 1152 23 383 PRT Homo sapien 23 Met Asp Pro Ser Ser His Ser Ser Asn Met Ala Asn Thr Gln Met Lys 1 5 10 15 Ser Asp Lys Ile Ile Ile Ala His Arg Gly Ala Ser Gly Tyr Leu Pro 20 25 30 Glu His Thr Leu Glu Ser Lys Ala Leu Ala Phe Ala Gln Gln Ala Asp 35 40 45 Tyr Leu Glu Gln Asp Leu Ala Met Thr Lys Asp Gly Arg Leu Val Val 50 55 60 Ile His Asp His Phe Leu Asp Gly Leu Thr Asp Val Ala Lys Lys Phe 65 70 75 80 Pro His Arg His Arg Lys Asp Gly Arg Tyr Tyr Val Ile Asp Phe Thr 85 90 95 Leu Lys Glu Ile Gln Ser Leu Glu Met Thr Glu Asn Phe Glu Thr Met 100 105 110 Ala Arg Phe Glu Asp Pro Thr Arg Arg Pro Tyr Lys Leu Pro Asp Leu 115 120 125 Cys Thr Glu Leu Asn Thr Ser Leu Gln Asp Ile Glu Ile Thr Cys Val 130 135 140 Tyr Cys Lys Thr Val Leu Glu Leu Thr Glu Val Phe Glu Phe Ala Phe 145 150 155 160 Lys Asp Leu Phe Val Val Tyr Arg Asp Ser Ile Pro His Ala Ala Cys 165 170 175 His Lys Cys Ile Asp Phe Tyr Ser Arg Ile Arg Glu Leu Arg His Tyr 180 185 190 Ser Asp Ser Val Tyr Gly Asp Thr Leu Glu Lys Leu Thr Asn Thr Gly 195 200 205 Leu Tyr Asn Leu Leu Ile Arg Cys Leu Arg Cys Gln Lys Pro Leu Asn 210 215 220 Pro Ala Glu Lys Leu Arg His Leu Asn Glu Lys Arg Arg Phe His Asn 225 230 235 240 Ile Ala Gly His Tyr Arg Gly Gln Cys His Ser Cys Cys Asn Arg Ala 245 250 255 Arg Gln Glu Arg Leu Gln Arg Arg Arg Glu Thr Gln Val Met His Gly 260 265 270 Pro Lys Ala Thr Leu Gln Asp Ile Val Leu His Leu Glu Pro Gln Asn 275 280 285 Glu Ile Pro Val Asp Leu Leu Cys His Glu Gln Leu Ser Asp Ser Glu 290 295 300 Glu Glu Asn Asp Glu Ile Asp Gly Val Asn His Gln His Leu Pro Ala 305 310 315 320 Arg Arg Ala Glu Pro Gln Arg His Thr Met Leu Cys Met Cys Cys Lys 325 330 335 Cys Glu Ala Arg Ile Glu Leu Val Val Glu Ser Ser Ala Asp Asp Leu 340 345 350 Arg Ala Phe Gln Gln Leu Phe Leu Asn Thr Leu Ser Phe Val Cys Pro 355 360 365 Trp Cys Ala Ser Gln Gln Thr Ser Gly His His His His His His 370 375 380
Claims (23)
1. An E6 or E7 protein or E6/E7 fusion protein from HPV linked to an immunological fusion partner.
2. A protein as claimed in claim 1 wherein the fusion partner is selected from the group; protein D or a fragment thereof from Heamophilius influenzae B, lipoprotein D or fragment thereof from Heamophilius influenzae B, NS1 or fragment thereof from Influenzae Virus, and LYTA or fragment thereof from Streptococcus Pneumoniae.
3. A protein as claimed in claim 1 or 2 wherein the E6 or E7 proteins are derived from HPV16 or HPV18.
4. A protein as claimed in claim 1 , 2 or 3 wherein the E7 protein is mutated.
5. A protein as claimed in claim 1 , 2 or 3 wherein the E6 protein is mutated.
6. A protein as claimed in any of claims 1 to 5 additionally comprising a hisitidine tag of at least 4 hisitidine residues.
7. A fusion protein comprising a heterologous protein, a hisitidine tag and a C-LYTA tag.
8. A DNA sequence encoding a protein as claimed herein.
9. A vaccine containing a protein as claimed in any of claims 1 to 7 and a pharmaceutically acceptable diluent or excipient.
10. A vaccine as claimed in claim 9 additionally comprising an adjuvant.
11. A vaccine as claimed in claim 9 or 10 wherein the protein is presented in an oil in water emulsion vehicle.
12. A vaccine as claimed in claim 10 or 11 wherein the adjuvant comprises 3D-MPL or QS21 or both.
13. A vaccine as claimed herein comprising an additional HPV antigen.
14. A vaccine as claimed herein for use in medicine.
15. Use of a protein as claimed herein for the manufacture of a vaccine for immunotherapeutically treating a patient suffering from HPV induced tumour lesions (benign or malignant).
16. Use of a protein as claimed herein for the manufacture of a vaccine to prevent HPV viral infection.
17. A vector containing a DNA sequence of claim 8 .
18. A vector containing a DNA sequence as claimed in claim 8 and a DNA sequence encoding thioredoxin.
19. A host transformed with a DNA sequence of claim 8 .
20. A host transformed with a vector of claim 17 or 18.
21. A host as claimed in claim 19 additionally transformed with a DNA sequence encoding thioredoxin.
22. A process for the production of a protein as claimed herein comprising transforming a host cell with a DNA sequence of claim 6 , expressing said sequence and isolating the desired product.
23. A process for the production of a vaccine as claimed herein, comprising admixing a protein as claimed herein with a suitable adjuvant, diluent or other pharmaceutically acceptable excipient.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US10/000,903 US20020182221A1 (en) | 1997-08-22 | 2001-11-01 | Recombinant papillomavirus vaccine and method for production and treatment |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GBGB9717953.5A GB9717953D0 (en) | 1997-08-22 | 1997-08-22 | Vaccine |
GB9717953.5 | 1997-08-22 | ||
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MA (1) | MA24638A1 (en) |
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NZ (1) | NZ502632A (en) |
PL (1) | PL339009A1 (en) |
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UY (1) | UY25146A1 (en) |
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ZA (1) | ZA987591B (en) |
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1997
- 1997-08-22 GB GBGB9717953.5A patent/GB9717953D0/en active Pending
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1998
- 1998-08-17 DE DE69824013T patent/DE69824013T2/en not_active Expired - Lifetime
- 1998-08-17 EP EP98945269A patent/EP1007551B1/en not_active Expired - Lifetime
- 1998-08-17 WO PCT/EP1998/005285 patent/WO1999010375A2/en active IP Right Grant
- 1998-08-17 TR TR2000/00480T patent/TR200000480T2/en unknown
- 1998-08-17 ES ES98945269T patent/ES2221198T3/en not_active Expired - Lifetime
- 1998-08-17 US US09/485,885 patent/US6342224B1/en not_active Expired - Lifetime
- 1998-08-17 HU HU0004327A patent/HUP0004327A3/en unknown
- 1998-08-17 BR BR9812139-1A patent/BR9812139A/en not_active IP Right Cessation
- 1998-08-17 AU AU92639/98A patent/AU732946B2/en not_active Ceased
- 1998-08-17 NZ NZ502632A patent/NZ502632A/en unknown
- 1998-08-17 KR KR1020007001821A patent/KR20010023193A/en not_active Application Discontinuation
- 1998-08-17 IL IL13439298A patent/IL134392A0/en unknown
- 1998-08-17 PL PL98339009A patent/PL339009A1/en not_active Application Discontinuation
- 1998-08-17 JP JP2000507701A patent/JP2001513986A/en active Pending
- 1998-08-17 CA CA2301920A patent/CA2301920C/en not_active Expired - Fee Related
- 1998-08-17 AT AT98945269T patent/ATE267211T1/en not_active IP Right Cessation
- 1998-08-17 CN CN98810251A patent/CN1276833A/en active Pending
- 1998-08-19 UY UY25146A patent/UY25146A1/en not_active Application Discontinuation
- 1998-08-20 CO CO98047511A patent/CO4810234A1/en unknown
- 1998-08-21 ZA ZA9807591A patent/ZA987591B/en unknown
- 1998-08-21 MA MA25223A patent/MA24638A1/en unknown
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2000
- 2000-02-21 NO NO20000850A patent/NO20000850L/en not_active Application Discontinuation
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- 2001-11-01 US US10/000,903 patent/US20020182221A1/en not_active Abandoned
Cited By (7)
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WO2008087102A1 (en) * | 2007-01-15 | 2008-07-24 | Glaxosmithkline Biologicals Sa | Vaccine |
US20080187535A1 (en) * | 2007-01-15 | 2008-08-07 | Normand Blais | Vaccine |
EA016326B1 (en) * | 2007-01-15 | 2012-04-30 | Глаксосмитклайн Байолоджикалс С.А. | Vaccine |
WO2011068934A3 (en) * | 2009-12-04 | 2011-11-03 | Consejo Nacional De Investigaciones Cientificas Y Tecnicas | Vaccine against neoplastic or cancerous lesions caused by human papillomavirus (hpv), procedures, uses and methods |
WO2016069774A1 (en) * | 2014-10-28 | 2016-05-06 | Agrivida, Inc. | Methods and compositions for stabilizing trans-splicing intein modified proteases |
US10731143B2 (en) | 2014-10-28 | 2020-08-04 | Agrivida, Inc. | Methods and compositions for stabilizing trans-splicing intein modified proteases |
US11066657B2 (en) | 2014-10-28 | 2021-07-20 | Agrivida, Inc | Methods and compositions for stabilizing trans-splicing intein modified proteases |
Also Published As
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TR200000480T2 (en) | 2000-07-21 |
NZ502632A (en) | 2002-03-28 |
DE69824013D1 (en) | 2004-06-24 |
JP2001513986A (en) | 2001-09-11 |
HUP0004327A1 (en) | 2001-03-28 |
CA2301920A1 (en) | 1999-03-04 |
WO1999010375A3 (en) | 1999-06-10 |
NO20000850D0 (en) | 2000-02-21 |
CA2301920C (en) | 2011-01-04 |
IL134392A0 (en) | 2001-04-30 |
AU9263998A (en) | 1999-03-16 |
CO4810234A1 (en) | 1999-06-30 |
PL339009A1 (en) | 2000-12-04 |
AU732946B2 (en) | 2001-05-03 |
BR9812139A (en) | 2000-07-18 |
MA24638A1 (en) | 1999-04-01 |
ATE267211T1 (en) | 2004-06-15 |
WO1999010375A2 (en) | 1999-03-04 |
GB9717953D0 (en) | 1997-10-29 |
ZA987591B (en) | 2000-02-21 |
KR20010023193A (en) | 2001-03-26 |
NO20000850L (en) | 2000-04-14 |
US6342224B1 (en) | 2002-01-29 |
ES2221198T3 (en) | 2004-12-16 |
EP1007551A2 (en) | 2000-06-14 |
EP1007551B1 (en) | 2004-05-19 |
CN1276833A (en) | 2000-12-13 |
DE69824013T2 (en) | 2005-06-02 |
HUP0004327A3 (en) | 2003-08-28 |
UY25146A1 (en) | 2000-12-29 |
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