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  • A61K39/00—Medicinal preparations containing antigens or antibodies
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  • A61K2039/585—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation wherein the target is cancer
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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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  • 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
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  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• the present invention relates to vaccine compositions, comprising an E6 or/ and E7 or E6, E7 fusion protein from an HPV strain optionally linked with an immunological fusion partner and formulated with a CpG containing oligonucleotide into vaccines that find utility in the treatment or prophylaxis of human papilloma virus induced tumours or lesions.
• the present invention relates to vaccines comprising fusions proteins, comprising a protein or part of a protein that provides T helper epitopes (such as protein D from Haemophilus influenzae B) and an antigen from a human-papilloma virus (eg comprising an E6 or E7 protein from HPV 16 or 18 strain associated with cancer) that find utility in the treatment or prophylaxis of human papilloma induced tumours, wherein the vaccine is formulated with a CpG containing oligonucleotide as an adjuvant.
• T helper epitopes such as protein D from Haemophilus influenzae B
• an antigen from a human-papilloma virus eg comprising an E6 or E7 protein from HPV 16 or 18 strain associated with cancer
• 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 viruses.
• High risk HPVs usually cause benign lesions (warts or papillomas) that persist for several months or years.
• High risk HPVs are associated with pre-neoplastic lesions and 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. 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.
• HPV1 bovine papillomavirus type 1
• HPVs genomes described have at least seven early genes, El to E7 and two late genes LI and L2.
• an upstream regulatory region harbors the regulatory sequences which appears to control most transcriptional events of the HPV genome.
• El 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-HI).
• 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 16 or 18 serotypes, are high- risk factors for the evolution toward cervical intraepithelial neoplasia (CIN) and carcinoma in situ (CIS) which are themselves regarded as precursor lesions of invasive cervix carcinoma.
• CIN cervical intraepithelial neoplasia
• CIS carcinoma in situ
• 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. Immunomodulatory oligonucleotides contain unmethylated CpG dinucleotides
• CpG CpG
• cytosine-guanosine dinucleotide motifs present in DNA.
• synthetic oligonucleotides derived from BCG gene sequences were shown to be capable of inducing immunostimulatory effects (both in vitro and in vivo).
• the authors of these studies concluded that certain palindromic sequences, including a central CG motif, carried this activity.
• the central role of the CG motif in immunostimulation was later elucidated in a publication by Krieg, Nature 374, p546 1995.
• Purine Purine CG pyrimidine pyrimidine and where the CG motif is not methylated In certain combinations of the six nucleotides a palindromic sequence is present. Several of these motifs, either as repeats of one motif or a combination of different motifs, can be present in the same oligonucleotide. The presence of one or more of these immunostimulatory sequence containing oligonucleotides can activate various immune subsets, including natural killer cells (which produce interferon ⁇ and have cytolytic activity) and macrophages (Wooldrige et al Vol 89 (no. 8), 1977). Although other unmethylated CpG containing sequences not having this consensus sequence have now been shown to be immunomodulatory.
• compositions comprising either an E6 or/and E7 or an E6/E7 fusion protein optionally linked to an immunological fusion partner having T cell epitopes, and adjuvanted with an immunomodulatory CpG containing oligonucleotide.
• the immunological fusion partner is derived from protein D of Heamophilus influenza B.
• the protein D derivative comprises approximately the first 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.
• 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. This property has been exploited for the development of E.coli C-LYTA expressing plasmids useful for expression of fusion proteins.
• compositions comprising an immunomodulatory CpG oligonucleotide and a 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 1/3 of protein D. It will be appreciated that other E6 and E7 proteins may be utilised from other HPV subtypes.
• the proteins utilised in 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 one or several mutations in the binding site for the rb (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.
• HPV 18 E 7 involves replacing Cys-,- with Glycine and/or Glutamic acid- 9 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 and a CpG immunomodulatory oligonucleotide.
• the vaccine of the present invention preferentially induce a TH1 immune response.
• TH1 and TH2 Two main types of Helper T cells have been characterized TH1 and TH2, which differ in the type of cytokines they secrete. These cytokines can be considered as the driving force behind the development of 2 different types of immune response : THl-type of immune response is associated with cell mediated effector mechanisms such as production of the INF- ⁇ and IL-2 cytokines by T-lymphocytes.
• INF- ⁇ which in turn can activate other cells and induce them to secrete other important cytokines and mediators
• INF- ⁇ - activated NK cells produce IL12
• IL2-activated NK cells are transformed into lymphokine activated killer cell ( LAK)
• INF- ⁇ -activated macrophages secrete inflamatory mediators like TNF a, IL1, IL6 and release nitric oxyde
• IL2 can provide help for the differentiation of antigen specific, haplotype restricted cytotoxic T lymphocytes (CTL).
• CTL antigen specific, haplotype restricted cytotoxic T lymphocytes
• Thl-type of immune response is also associated with the generation of antibodies of the IgG2 isotype (IgG2a in Balb/c mice and IgG2b in C57BL/6 mice) .
• the Th2-type of immune response is associated with a humoral immune response to the antigen, with the production of cytokines like IL4, IL5, IL6, IL 10 and by the generation of a broad range of immunoglobulin isotypes including in mice IgGl, IgA, and IgM.
• Thl and Th2-type immune responses are not absolute. An individual will support an immune response which is predominantly Thl or predominantly Th2.
• mice In the human TH1 type of response is also associated with the presence of cytokine (IFNg and IL2) eventually with the presence of CTl and IgG2 isotypes in mice correspond to IgGl type antibodies This type 1 phenotype is of particular importance in protecting against viral and intracellular bacterial infections as well as in the treatment of cancer.
• IFNg and IL2 cytokine
• an expression strategy can be used which involves 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 my be 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 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.
• vector 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 : (Cohen et al., Proc. Nat. Acad. Sci., 1973, 69, 21 10) 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.
• Culruring 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.
• the 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. 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.
• 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.
• the vaccine may contain LI or L2 antigen monomers.
• LI or L2 antigens may be presented together as a virus like particle or the LI alone protein may be presented as virus like particle or caposmer structure.
• Such antigens, virus like particles and capsomer are per se known. See for example WO94/00152, WO94/20137, WO94/05792, and 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, 1 1, 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, US Patent 4,235,877.
• the preferrred oligonucleotides preferably contain two or more CpG motifs separated by six or more nucleotides.
• the oligonucleotides of the present invention are typically deoxynucleotides.
• the internucleotide in the oligonucleotide is phosphorodithioate, or more preferably a phosphorothioate bond, although phosphodiester and other internucleotide bonds are within the scope of the invention including oligonucleotides with mixed internucleotide linkages.
• Preferred oligonucleotides have the following sequences: The sequences preferably contain all phosphorothioate modified internucleotide linkages.
• OLIGO 1 TCC ATG ACG TTC CTG ACG TT
• OLIGO 2 TCT CCC AGC GTG CGC CAT
• OLIGO 3 ACC GAT GAC GTC GCC GGT GAC GGC ACC ACG
• the CpG oligonucleotides utilised in the present invention may be synthesized by any method known in the art (eg EP 468520). Conveniently, such oligonucleotides may be synthesized utilising an automated synthesizer. Methods for producing phosphorothioate oligonucleotides or phosphorodithioate are described in US5,666,153, US5,278,302 and WO95/26204.
• 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.
• 3) - Growth and induction of bacterial strain - Expression of Prot -Dl/3-E7-His Cells of AR58 transformed with plasmid pRIT14501 were grown in 100 ml of
• the protein and coding sequence for the fusion protein-D l/3-E6-His is described in sequence ID No.3 and 4.
• Cells of AR58 transformed with plasmid pRIT 14497 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 Dl/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.
• E.coli strain AR58 was transformed with a plasmid encoding thioredoxin and protein D 1/3 E7 His (HPV 16).
• EXAMPLE III Construction of an E. coli strain expressing fusion Protein-Dl/3-
• the sequence of Prot-Dl/3 is followed by a multiple cloning site (11 residues) and a coding region for a C-terminal histidine tail (6 His).
• pBR322 obtained from Deutsches Krebsforschungstechnik (DKFZ), Referenz scholar fur human pathogen Papillomaviruses - D 69120 - Heidelberg
• the protein and coding sequence for the fusion protein-D E6/E7 1/3 -His is described sequence ID No. 5 and 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.
• 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 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.
• EXAMPLE V Construction of E.coli strain B1002 expressing fusion ProtDl/3- E7 Mutated (cys24->gly,glu26->gln ) type HPV16 l)-Construction of expression plasmid
• b) - Plasmid LITMUS 28 ( New England Biolabs cat n° 306-28 ) a cloning vector pUC-derived c) - Plasmid pMG MCS ProtDl/3 ( pRIT 14589) , a derivative of pMG81 (described Supra) in which the codons 4-81 of NS l 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.
• 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 ).
• sequence for the fusion protein-D 1/3 -E 7 mutated ( cys24->gly, glu26- >gln ) -His is described in sequence ID No. 7 and 8.
• Cells of AR58 transformed with plasmid pRIT 14733 ( B1002 strain ) were grown at 30°C in 100 ml of LB medium supplemented with 50 ⁇ gr /ml of Kanamycin. During the logarithmic phase of growth bacteria were shifted to 39°C to inactivate the ⁇ repressor and turn on the synthesis of ProtDl/3-E7 mutated -His /HPV16 . 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 16000 g for 30 minutes at 4°C.
• the C-terminal domain of the LYTA protein is responsible for the affinity to the choline or to some choline analogues such as DEAE.
• 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 die ⁇ 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.
• the first step was the purification of the large Ncol-Aflll restriction fragment from plasmid pRIT14501 and d e purification of the small Aflll-Afllll restriction fragment from pRIT 14661 b)
• Plasmid pRIT14626 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. 3. Growth and induction of bacterial strain - Expression of clyta-E7-His
• Cells of AR58 transformed with plasmid pRIT 14626 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. 4. Characterization of fusion clyta-E7-his Frozen cells were thawed and resuspended in 10 ml of PBS buffer.
• 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 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).
• 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.
• the coding sequence for diioredoxin is described in ID No. 17.
• 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.
• Cells of AR58 transformed with plasmids pRIT14532 ( B101 1 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 ⁇ gr/ml of Kanamycin for B1011 strain and supplemented 50 ⁇ gr/ml of Kanamycin and 100 ⁇ gr/ml of Ampicillin for Bl 012 strain .
• During the logarithmic phase of growth bacteria were shifted to 39°C to inactivate the ⁇ repressor and turn on the synthesis of protein Dl/3-E7-his/ ⁇ PVl 8 and diioredoxin. The incubation at 39°C was continued for 4 hours.
• 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 protDl/3-E7-His (about 31 kDa) was visualised by Coomassie stained gels in the pellet fraction for strain BlOl 1 and partially localized (30%) in the supernatant fraction for strain BlOl 2 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.
• EXAMPLE X Construction of E.coli strain B1098 expressing fusion ProtDl/3- E7
• 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.
• Cells of AR58 transformed with plasmid pRIT 14831 ( B 1098 strain ) were grown at 30°C in 100 ml of LB medium supplemented with 50 ⁇ gr /ml of Kanamycin. During the logarithmic phase of growth bacteria were shifted to 39°C to inactivate the ⁇ repressor and turn on the synthesis of ProtDl/3-E7 mutated -His /HPV18 . The incubation at 39°C was continued for 4 hours. Bacteria were pelleted and stored at - 20°C.
• the level of expression represents about 3 to5 % of total protein.
• EXAMPLE XI Construction of an E. coli strain expressing fusion Protein-Dl/3- E6-his / HPV18
• This plasmid is used to express the fusion protein Dl/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.
• 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.
• the protein and coding sequence for the fusion protein-D 1/3 -E6E7- His is described in sequence ID No. 22 and 23.
• 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 Dl/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 Dl/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.
• Protocol 10e6 TCI cells, E7 expressing tumour cells were injected subcutaneously
• mice (200 ⁇ l) in the flank of C57BL/6 immunocompetent mice. Mice were vaccinated 7 and 14 days after the tumour challenge, with 5 ⁇ g ProtD 1/3 E7 HPV 16 injected intra- footpad (lOO ⁇ l : 50 ⁇ l / footpad) in the presence of different adjuvants:
• mice/group were killed and spleens or popliteal lymph nodes were taken and analyzed for immune response.
• Tumour Growth was monitored by measuring individual tumours twice a week.
• tumour growth As shown in figure 1 and 2, in the groups of mice that received the antigen with a CpG oligonucleotide the mean tumour growth remained very low and very similar between groups, reflecting that the tumour growth either was slowed down or that several tumours were completely rejected.
• tumour growth/group of mice vaccinated with PD1/3 E7+ the CpG oligos are quite similar and analysis of the individual tumour growth showed that the CpG oligos induce prolonged complete tumour rejection.
• Lymphoproliferative response was analysed by in vitro restimulation of spleen and lymph nodes cells for 72 hrs with either PD1/3E7, the protein E7(Bollen) and PD (whole) PD1/3 (coated or not on latex ⁇ beads) (10, 1, 0.1 ⁇ g/ml) 2 and 4 weeks post II. • Positive controls (ConA stimulaltion) were positive. • Surprisingly, no E7 specific and no PD specific proliferative response could be observed starting with spleen cells 2 or 4 weeks post II (probably due to a technical problem: data not shown).
• lymph node cells from mice that received ProtD 1/3 E7 in CpG oligos 1 and 2 showed a very good E7 specific proliferative response although almost no PD (whole) specific response could be observed even at the -lightest concentration of lOO ⁇ g/ml no PD1/3 specific responses was observed even when coated on latex ⁇ beads.
• IgG tot and isotypes (IgGl, IgG2a, IgG2b, IgGTot) were measured by ELISA using the E7 protein as coating antigen as described in the Materials and Methods.
• Figures 3 and 4 show the relative percentage of the different IgG isotypes in the total of IgGs, 2 and 4 weeks post II respectively.
• the Oligos affect only weakly (oligo 2) or not at all (Oligo 1) the weak antibody response observed when PD1/3E7 alone was injected.
• a CTL response could be detected when measured 2 weeks after the latest vaccination, when cells were re-stimulated in vitro with irradiated TCI when TCI or peptide E7 pulsed EL4, were used as target cells, when mice immunised with PD1/3 E7 + CpG oligo 2> 1 (25-40% specific lysis) and not with oligos alone.
• Formulations containing oligo alone without other adjuvant were prepared by addition of CpG to the diluted PrtDl/3-E7 in PBS pH 7.4.
• the adjuvant controls without antigen were prepared by replacing the protein by PBS.
• TCI obtained from the John Hopkin's University
• EL4 cells were grown in RPMI 1640 (Bio Whittaker) containig 10% FCS and additives: 2mM L-Glutamine .
• antibiotics lOOOOU/ml penicilin. lOOOO ⁇ g/ml streptomycin
• non essential amino acid lOOx 1% sodium pyruvate (Gibco)
• sodium pyruvate Gibco
• 5 10e-5 M 2- mercaptoethanol Before injection TCI cells were trypsynized and washed in serum free medium.
• lymphoproliferation was performed on individual spleens and on lymph node pools. 200000 spleen cells or popliteal lymph node cells were plated in triplicate, in 96 well microplate, in RPMI medium containing 1% normal mice serum and additives . After 72 hrs of in vitro re-stimulation with different amounts of PD1/3 E7 (1, 0.1, 0.01 ⁇ g/ml) or E7 ( 10-1-0.1 ⁇ g/ml ) After 72hrs, 100 ⁇ l of culture supernatant were removed and replaced by fresh medium containing l ⁇ Ci 3H thymidine (Amersham 5Ci/mmol) . After 16 hrs, cells were harvested onto filter plates. Incorporated radioactivity was counted in a ⁇ counter. Results are expressed in CPM (mean of triplicate wells) or as stimulation indexes (mean CPM in cultures with antigen / mean CPM in cultures without antigen).
• Target cells used to assess cytotoxicity were either Cr51 ( DuPont NEN
• E7as coating antigen Quantitation of anti E7 antibody was performed by Elisa using E7as coating antigen. Antigen and antibody solutions were used at 50 ⁇ l per well. Antigen was diluted at a final concentration of 3 ⁇ g/ml in carbonate buffer ph9.5 and was adsorbed overnight at 4°c to the wells of 96 wells microtiter plates (Maxisorb Immuno-plate, Nunc, Denmark). The plates were then incubated for lhr at 37°c with PBS containing 1% bovine serum albumin and 0.1 % Tween 20 (saturation buffer).
• the tumor growth was monitored by measuring individual tumors twice a week and the mean tumor growth/ ' group of 5 animals is depicted in figure 5 and show the phosphorothioate modified oligonucleotides are effective in bringing about tumour regression.
• the first concentration at which this therapeutic effect on tumor regression is not fully obtained is E7+ l ⁇ g CpG oligo 1001.
• the vaccines ofthe invention were evaluated in transgenic mice expressing E7 protein.
• the transgenic mouse strain has been generated by M. Parmentier and C. Ledent at the IRIBHN (ULB). (Ref: PNAS (USA) 1990, 87; 6176-6180).
• transgenic mice live with the E7 HPV 16 gene from birth, they are considered “tolerant” to this gene: E7 from HPV 16, in this situation is considered as a "self antigen”. • The expression of the transgene is driven by the thyroglobulin promoter. As Thyroglobulin is constitutively expressed only In the Thyroid, E7 is expressed in the thyroid.
• mice were vaccinated 7 and 14 days after the tumor challenge, with 5 ⁇ g ProtD 1/3 E7 HPV16 injected intra- footpad (100 ⁇ l : 50 ⁇ l / footpad) in the 2 presence of CpG oligonucleotide TCT CCC AGC GTG CGC CAT and two control adjuvants:,
• the vaccines ofthe invention are effective in bringing about tumour regression in HPV induced tumours.

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