EP1399182A2 - Immunogenes et vaccins ainsi que leur preparation et leur utilisation - Google Patents

Immunogenes et vaccins ainsi que leur preparation et leur utilisation

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Publication number
EP1399182A2
EP1399182A2 EP02702541A EP02702541A EP1399182A2 EP 1399182 A2 EP1399182 A2 EP 1399182A2 EP 02702541 A EP02702541 A EP 02702541A EP 02702541 A EP02702541 A EP 02702541A EP 1399182 A2 EP1399182 A2 EP 1399182A2
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European Patent Office
Prior art keywords
papillomavirus
cin
booster
primer
polypeptide
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP02702541A
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German (de)
English (en)
Inventor
Julian Kingdon Hickling
Terry O'neill
Rienk Leiden Univ. Med. Center OFFRINGA
Sjoerd Leiden Univ. Med. Center VAN DER BURG
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Xenova Research Ltd
Leids Universitair Medisch Centrum LUMC
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Xenova Research Ltd
Leids Universitair Medisch Centrum LUMC
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Publication of EP1399182A2 publication Critical patent/EP1399182A2/fr
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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/20011—Rhabdoviridae
    • C12N2760/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/20011—Rhabdoviridae
    • C12N2760/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • This invention relates to immunogens and vaccines and to their preparation and use.
  • the invention relates to combination immunogens and vaccines.
  • the invention relates to vaccines for use in prophylactic or therapeutic treatment of papillomavirus infection, especially for example human papillomavirus (HPV) infection, e.g. chronic infection, and of the tumours or other lesions produced by such infection.
  • HPV human papillomavirus
  • booster doses are administered after a chosen interval during which a vaccine dose that was previously given may be expected to have evoked an immune response, e.g. after an interval of a few weeks.
  • Combination vaccines have also previously been proposed, in which the primer and booster vaccines are of different formulation.
  • US 5,686,078 (Connaught Laboratories: RS Becker et al) proposes a primary and secondary immunization procedure using different physico-chemical forms of a "viral bacterial antigen" in which a particulate highly-immunogenic form of an antigen, e.g. inactivated or attenuated "whole cell virus", e.g. influenza virus, is first administered to an animal, and later a weakly-immunogenic form of the antigen, e.g. isolated and purified HA(p) antigen from influenza virus, is given with the intent to achieve a booster immune reaction.
  • a particulate highly-immunogenic form of an antigen e.g. inactivated or attenuated "whole cell virus", e.g. influenza virus
  • a weakly-immunogenic form of the antigen e.g. isolated and purified HA(p) antigen from influenza virus
  • J W Hodge et al (in Vaccine, 15(6/7) (1997) pp 759-768) described inter alia 'prime and boost' immunization of mice based on priming immunization with a vaccinia virus vector encoding carcinoembryonic antigen CEA, followed by boosting of the immune response with a non-replicating recombinant avipox virus vector also encoding carcinoembryonic antigen. This pair of immunizations was reported to give increased CEA-specific T-cell response compared with successive immunizations using the recombinant vaccinia virus on both occasions.
  • WO 98/56919 propose combinations for generating CD8+ T- cell immune responses, e.g. against Plasmodium falciparum (malaria) or HIV, based on a priming vaccine which can be a viral vector or DNA or RNA or a target antigen, and a boosting vaccine which is a non-replicating or replication-impaired poxvirus vector.
  • a priming vaccine which can be a viral vector or DNA or RNA or a target antigen
  • a boosting vaccine which is a non-replicating or replication-impaired poxvirus vector.
  • An aim of the present invention is to provide further combination immunogens and vaccines with particularly useful immunogenicity.
  • combination immunogens directed against papillomavirus protein antigen(s). can comprise (i) a primer immunogen and (ii) a booster immunogen.
  • the primer (i) can comprise a polypeptide having antigenic determinants derived from papillomavirus protein, or it can comprise DNA encoding such antigenic determinants derived from papillomavirus protein
  • the booster (ii) can comprise a virus vector encoding papillomavirus antigen and able to express said antigen when it infects a host cell of a human or non-human animal treated with the vector.
  • the booster is formulated separately and is intended for administration to a subject previously treated with the primer, after a chosen interval during which it may be expected that an immune response can develop against the antigens presented by the primer. More than one dose of each component can be given: commonly a primer is followed by a booster or two boosters at intervals but other combinations and numbers of doses are possible within the scope of the invention.
  • the primer can for example be an immunogen or vaccine as described in WO 96/26277 and US 6,123,948 (Cantab Pharmaceuticals: NR Whittle et al) (hereby incorporated by reference in its entirety for all purposes). In certain useful examples it can comprise a polypeptide vaccine based on antigens from human papillomavirus type 16.
  • Boursnell et al (hereby incorporated by reference in its entirety for all purposes), e.g. a vaccinia virus vector carrying heterologous DNA inserts which encode and can express fusion proteins based on E6 and E7 protein sequences of human papillomavirus types 16 and 18.
  • both the primer and booster can contain or encode (inter alia) the full length or substantially the full length aminoacid sequence of the E6 and/or E7 protein of human papillomavirus type 16.
  • the primer immunogen preferably is based on fusion polypeptides that combine papilloma-virus-derived antigens, e.g. from each of at least two different papillomavirus proteins, e.g. comprising (a) preferably at least an antigenic determinant of a papillomavirus L2 protein and/or L1 protein, and (b) at least an antigenic determinant selected from E1 , E2, E4, E5, E6 and E7 papillomavirus proteins and L2 papillomavirus proteins of different papillomavirus type than in (a).
  • Further fusion polypeptides provided hereby comprise antigenic determinants from at least two papillomavirus proteins selected from E1 , E2, E4, E5, E6 and E7 papillomavirus proteins e.g. where the said proteins are from different papillomavirus types.
  • Particularly preferred polypeptides and compositions comprise antigenic determinants of human papillomavirus proteins, e.g. of HPV type 6, 11 , 16, 18, though antigens of other human papillomavirus types and papillomavirus from other species are also susceptible to application in the present invention.
  • Antigenic determinants of proteins from other HPV types and proteins of non-human animal papillomaviruses can also be made and used. Also useful are synthetic peptide sequences which encode such useful antigenic determinants of papillomavirus proteins.
  • the polypeptide may comprise a fusion molecule or can be derived from individual polypeptides coupled or aggregated together. Soluble or solubilised forms of the polypeptide can be used according to the invention.
  • the virus vector used to encode papillomavirus antigen(s) in the booster can be a poxvirus vector, e.g. vaccinia.
  • poxvirus vectors include per-se known avipox vectors such as canarypox virus, and genetically attenuated or disabled poxvirus vectors.
  • the virus vector used in the booster can be based on a virus (vector) of another type, e.g.
  • the booster can be a DNA vaccine encoding the desired antigen, or a non-virus vector carrying such a DNA vaccine.
  • the primer can be a DNA vaccine encoding the desired antigen, or a non-virus vector carrying such a DNA vaccine.
  • papillomavirus sequences can be inserted in a genetically-disabled herpesvirus as described in WO 92/05263 and US 5,665,362 (Cantab Pharmaceuticals/Immunology Ltd: Inglis et al)(hereby incorporated by reference in their entirety for all purposes), for example in place of the SIV antigen described in an example therein, and such a resulting virus vector encoding papillomavirus protein antigen can for example be used as an alternative form of booster in connection with the present invention.
  • a primer component of immunogenic compositions according to examples of the present invention can comprise a polypeptide antigen forming a component of an adsorption complex comprising "alum" (i.e. aluminium hydroxide usually Alhydrogel (TM) or Rehydrogel (TM) as conventionally used as vaccine adjuvant) having adsorbed thereon a polypeptide obtainable as mentioned above.
  • alum i.e. aluminium hydroxide usually Alhydrogel (TM) or Rehydrogel (TM) as conventionally used as vaccine adjuvant
  • the adsorption complex can be a binary complex consisting of the alum and the polypeptide, or there may be further constituents, e.g. MPL as described below, making for example a ternary complex of MPL, alum and polypeptide.
  • Polypeptide immunogens can be formulated with an adjuvant or other accessory substance such as an immunostimulatory molecule in order to enhance its effect as a therapeutic antigen, and also to stimulate a preferred type of immune response in the recipient patient.
  • an adjuvant or other accessory substance such as an immunostimulatory molecule
  • Useful adjuvants include, but are not limited to; aluminium hydroxide ("alum”), e.g. in the form of Alhydrogel(TM) or Rehydrogel(TM); 3D-MPL (3- deacylated monophosphoryl lipid A) e.g.
  • trehalose diesters such as trehalose dimycolate; saponins and their derivatives such as Quil A or QS-21 , as for example described in specifications WO 88/09336 (Cambridge Bioscience: CA Kensil et al: Saponin adjuvant) and WO 93/05789 (Cambridge Biotech: CA Kensil et al: Saponin-antigen conjugates); ISCOMS or ISCOM matrices, as for example described in specifications WO 90/03184 (B Morein et al: Iscom matrix with immunomodulating activity, comprising lipid and optionally also adjuvants) and WO 92/21331 (Kabi Pharmacia AB: B Morein et al: Pharmaceutical carriers comprising sterol and saponin); or muramyl dipeptide, or cholera toxin B.
  • adjuvants are oligodeoxynucleotides containing CpG motifs (AM Krieg & HL Davis, Curr. Opin. Mol. Ther., 3 (1), 2001 , pp 15-24) and these adjuvants can be especially preferred when the primer comprises polypeptides.
  • the polypeptide vaccine can be encapsulated, e.g. by encapsulation in biodegradable microparticles or liposomes or nonionic surfactant vesicles: for these techniques see respectively e.g. specifications WO 94/27718 (DT O'Hagan et al: microparticles containing entrapped antigens and their use in immunization) and WO 93/19781 (PCT/GB93/00716) (Proteus Molecular Design: J Alexander et al: Vaccines containing non-ionic surfactant vesicles with entrapped antigen).
  • microparticles which can usefully be used to encapsulate polypeptide vaccines are PLG microspheres (C Berkland et al., J Control Release 73 (1), 2001 , pp 59-74).
  • liposomes of these or other kinds can be used alongside the polypeptides, as adjuvants.
  • cytokines such as interleukins, including but not limited to GM-CSF, IL-12, IL-2, IL-3 and IL-7
  • molecules which activate CD40 e.g. agonistic anti-CD40 antibodies or CD40L.
  • Such adjuvants and/or other accessory substances can be used separately or in combinations as desired.
  • the amount of polypeptide administered can be chosen according to the formulation and the condition to be treated. Generally it is expected that doses will be between 1-2000 ⁇ g of the protein, preferably 10-300 ⁇ g, e.g. 10-250 ⁇ g. Optimal amounts can readily be determined in subjects. One or more doses of the vaccine may be administered at intervals (see e.g. Example 13). This regime can readily be optimised in subjects.
  • doses of booster based on virus vector vaccine can be chosen and optimised according to per-se well-known methods in the art: for example doses in the range 10 ⁇ 4-10 ⁇ 8 plaque-forming units (pfu) can be used: in the case of disabled virus vectors, pfu are measured on test cell cultures of a host cell type or recombinant cell type chosen for its ability to support production and multiplication of new virus particles.
  • pfu plaque-forming units
  • a non-limitative example of the present invention has been made using, as a polypeptide priming immunogen, a L2E7E6 fusion protein containing sequences based on those of human papillomavirus type 16, in accordance with the general teaching of US 6,123,948 (Cantab Pharmaceuticals: NR Whittle et al), by per-se routine adaptation of its specific examples changed so as to use an alternate combination of source antigens.
  • This fusion protein is used along with a liposome adjuvant. The adjuvant and its nature are not critical.
  • the booster immunogen in this example of the present invention is a vaccinia virus vector as described in US 5,719,054 (Cantab Pharmaceuticals: ME Boursnell et al) carrying heterologous DNA inserts which encode and can express fusion proteins based on E6 and E7 protein sequences of human papillomavirus of both types 16 and 18.
  • a suitable L2E7E6 fusion protein antigen for use in the present example can be made using HPV protein-encoding nucleic acid sequences, obtained using standard PCR techniques from clinical isolates, and modifying the sequences so obtained to form a fusion-protein sequence for expression in E.coli, e.g. as exemplified in US 6,123,948 (Cantab Pharmaceuticals: NR Whittle et al).
  • the disclosure referred to can be used with per-se known routine modifications and adaptations chosen to suit the system to genetic source materials of different source and sequence. Genetic material of HPV type 16 has been obtained e.g.
  • substitution mutations into the sequence as follows: to replace TTT codons by TTC; to replace codons rarely used in the E.coli host by other codons more frequently used in that host, so as to encode the same amino acids (this applies particularly to the first 100 nucleic acid residues of the E6 gene coding sequence); and to replace a pro-rich sequence in L2 (pro val pro ser val pro) by a substitute sequence (ala-6) including alanine instead.
  • E6 two preferred mutations are at residues 66 and 106 in the E6 coding sequence to change cys to gly in each case, here to make on a similar basis a substitution of residues known to be involved in complex formation between E6 and the p53 tumour suppressor gene product, in accordance with the teaching of T Kanda et al (Virology (1991 ) 185 pp 536-543), which discloses HPV16 E6 protein sequence mutants lacking in the capacity shown by the native protein to enhance HPV16 E7 transformation.
  • an example of the applicability of the present invention is in the targetting of human papillomavirus (HPV) E6 and E7 oncoproteins, e.g. in the T-cell-based immunotherapy of cervical intraepithelial neoplasia (CIN) (or vaginal intraepithelial neoplasia VIN) and cancer.
  • HPV human papillomavirus
  • E6 and E7 oncoproteins e.g. in the T-cell-based immunotherapy of cervical intraepithelial neoplasia (CIN) (or vaginal intraepithelial neoplasia VIN) and cancer.
  • a vaccine comprising HPV16 L2, E6 and E7 as a single fusion protein (designated herein TA-CIN), is shown to elicit HPV16- specific CTL, T-helper cells and antibodies in a pre-clinical mouse model.
  • Each line represents the volume of TC-1 tumours of an individual mouse measured at the indicated days in the follow-up.
  • Figure 2 shows monitoring of vaccine induced immune responses.
  • (a) Splenocytes were stimulated in vitro for 7 days with the HPV16 E7+ cell line 13.2 and then tested for cytotoxicity against HLA- D°+ RMA cells with or without E7 49 .
  • 57 RAHYNIVTF peptide. The specific cytotoxicity, calculated by subtracting the RMA-specific lysis from the lysis of RMA+ E7 49 .
  • 57 peptide, of all three individual mice are depicted, (b) The number of T-cells per 250,000 splenocytes that spontaneously (white bars) or following stimulation with E7 9 . 57 peptide (black bars) produce IFN ⁇ upon stimulation as detected by ELISPOT. (c) The percentage of IFN ⁇ -producing CD8+ E7-specific CTL present in 7-day in vitro 13.2 stimulated splenocyte cultures as detected by intracellular cytokine staining upon stimulation without (white bars) or with (black bars) the E7 9 . 57 CTL epitope. (d) TA- CIN specific IgG antibodies present in the sera of all three individual mice.
  • Figure 3 shows intracellular cytokine staining FACS analysis of IFN ⁇ production by CD8+ splenocytes upon stimulation with E7 49 . 57 : RAHYNIVTF peptide.
  • Splenocytes derived from a control mouse (M #3), a mouse injected with 32 g TA-CIN (M #5) or injected with 200 ⁇ g TA-CIN (M #9) are shown.
  • Plots show the cells that were gated on CD8+ staining.
  • the horizontal axis of plots shows CD8 staining and the vertical axis shows IFN ⁇ staining. The values indicate the percentage of double positive, IFN ⁇ producing CD8+ T-cells.
  • Figure 4 shows an analysis of the percentage of E7 49 . 57 -specific CTL in splenocytes of mice after vaccination with indicated prime-boost combinations of TA-CIN and/or TA-HPV. FACS analysis plots (gated on CD8+ T-cells) of tetramer positive T-cells in freshly isolated splenocytes (top row) or in 7 day stimulated splenocytes (bottom row) are shown.
  • control mouse M #1
  • TA-CIN / TA-CIN M #10
  • TA-CIN / TA-HPV M #15
  • TA-HPV / TA-CIN M#18
  • TA-HPV / TA-HPV M #21
  • the percentage of double positive, H-2D b -RAHYNIVTF tetramer positive CD8+ T-cells is depicted.
  • Cervical intra-epithelial neoplasia is a condition in which the epithelial cells of the cervix proliferate abnormally. In a significant proportion of patients this condition progresses to cervical cancer, which is one of the main causes of cancer-related death for women under the age of 40 worldwide. Strong epidemiological and molecular biological evidence indicates that the origin of cervical cancer is closely linked to genital infection with oncogenic types of human papilloma viruses (HPV) [1].
  • these proteins are excellent target antigens for immunological intervention and hence to prevent cervical cancer.
  • Vaccines designed to induce or boost T-cell activity against HPV16-induced neoplastic lesions can come in various formulations.
  • Peptide-based vaccines comprising minimal T cell epitopes, are well defined but trigger only a small T-cell repertoire which implies that the restricted breadth of the response may limit the efficacy of such vaccines.
  • such peptide vaccines are often restricted to patients with certain HLA-types [9-11].
  • recombinant protein, DNA or virus-based vectors that comprise or encode entire antigens contain all possible CTL and T-helper (Th) epitopes and thus enable the immune system to choose the most appropriate CTL and Th-epitopes by itself.
  • Th T-helper
  • vaccinia-based vaccine expressing modified forms of HPV16 and 18 E6 and E7 genes (designated TA-HPV) was tested in a clinical trial for therapeutic treatment of cervical cancer patients [12, 13]. Although this vaccine was shown to induce HPV-specific T cell immunity in such patients, the use of vaccinia can have some limitations regarding its use, for instance in immunocompromised individuals.
  • TA-CIN A protein-based vaccine, (designated TA-CIN) has been developed on the basis of the teaching given in published patent application WO 96/26277, cited above.
  • TA- CIN is a fusion protein that, as described above, is made up of aminoacid sequences derived from the HPV16 L2, E6 and E7 antigens. The choice of these antigens was based on vaccination studies in animal models using HPV, bovine papillomavirus or cottontail rabbit papillomavirus [14-17].
  • Analysis of the immunogenicity of TA-CIN in a C57/BL6 pre-clinical mouse model (TC-1) demonstrated that TA-CIN effectively induces HPV16-specific CTL, Th-cells and antibodies.
  • TA-CIN has been shown to prevent outgrowth of HPV16+ tumours both prophylactically as well as therapeutically in a minimal residual disease setting.
  • TA-CIN consists of recombinant HPV16 L2E7E6 that was isolated from solubilised E. coli inclusion bodies under reducing conditions and purified by chromatography, as described in references cited herein.
  • the 80kD L2E7E6 monomer comprised 725 amino acids.
  • the final product was a discrete, 0.22 ⁇ m filterable, stable protein aggregate formulated in 5mM phosphate, 5mM glycine buffer (pH8.0) containing 0.9mM cysteine. The protein was stored at -70°C until use.
  • the adjuvant used in this study (designated as Novasome, acknowledged herein as a trade name of Novavax Inc.) consisted of amphiphile-based non-phospholipid vesicular membrane structures with particle sizes in the range 0.2-5.0 ⁇ m.
  • the Novasomes adjuvant was formulated in 5.9mM phosphate, 3.5mM glycine buffer (pH7.5) containing 0.63mM cysteine.
  • TA-CIN protein Prior to administration of the vaccine TA-CIN protein was added to the adjuvant at a ratio of 7:3.
  • the resultant adjuvanted TA-CIN vaccine was a white homogeneous liquid.
  • the construction and characterisation of a closely related fusion protein composition derived from HPV6, designated TA-GW [18], and the recombinant vaccinia-virus designated TA-HPV [12] have been described in detail previously.
  • C57BL/6 (B6, H-2 b ) mice were obtained from the Netherlands Cancer Institute and held under specific pathogen-free conditions.
  • TC-1 which was derived from primary epithelial cells of C57BL/6 mice co transformed with HPV-16 E6 and E7 and c-Ha-ras oncogenes (a kind gift of dr. T.C. Wu), were cultured in IMDM + 10% FCS.
  • B6 mice were vaccinated subcutaneously with 32 ⁇ g or 200 ⁇ g TA-CIN in 200 ⁇ l adjuvants or intraperitoneal (subcutaneously when indicated) with TA-HPV (5x10 6 pfu;) in 200/J PBS at day 0 and day 21 (prime-boost experiments).
  • mice were either offered at day 42, for the analysis of HPV-specific cellular immunity or challenged with 50,000 TC-1 cells in 250 ⁇ l of PBS (TA-CIN vaccination experiments only). Following TC-1 challenge, tumour development in mice was monitored for 70-days during follow-up. In the therapeutic experiments B6 mice were challenged with 50,000 TC-1 cells and then received the vaccine 4 hours later. Tumour development was monitored during a 90-day follow-up.
  • L2E7E6 specific serum antibodies were measured by ELISA. 96 well plates (Nunc Maxisorp) were coated with L2E7E6 in 100mM carbonate buffer, pH9.6, overnight at 4°C. Wells were blocked with 2% bovine serum albumin in PBS for 1h at 37°C. Titrations, either from 1/100 or 1/500, of serum samples and a known positive sample diluted in 2% BSA / PBS were added to triplicate wells and incubated for 1 h at 37°C.
  • IgG and lgG2b After washing with PBS/0.05%Tween-20 the detection reagent, either goat anti-mouse IgG horseradish peroxidase (Biorad) or bovine anti-mouse lgG2b horseradish peroxidase (Serotec), were added and incubated for 1 h at 37°C.
  • lgG1 After washing with PBS/0.05%Tween-20 the detection reagent, monoclonal rat anti-mouse lgG1 (Pharmingen), was added and incubated for 1 h at 37°C. Plates were washed and incubated with goat anti rat- horseradish peroxidase conjugate (Southern Biotechnology Associates) for 1 h at 37°C.
  • tumour cell line 13.2 which was derived from mouse embryo cells transformed with adenovirus type 5 derived E1 protein in which the H-2D b E1A epitope was replaced with the HPV16 E7 49 .
  • 57 CTL epitope as stimulator cells.
  • ELISPOT The number of peptide-specific IFN ⁇ -producing CTL in freshly isolated spleen cells using ELISPOT were measured as follows. 5x10 6 spleen cells were stimulated overnight with or without 1 ⁇ g/ml of E7 49 . 57 -peptide and 5IU rlL-2/ml in a 24- well plate (Costar, Cambridge, MA) in 1 ml of ISCOVE's medium (Gibco) enriched with 10% FCS at 37 °C.
  • rat-anti-mouse IFN ⁇ 5 ⁇ g/ml in PBS, Pharmingen, Cat. 554431 . Plates were incubated for 24 h at 37°C. Then plates were washed five times with PBS/Tween 0.5% and five times with tap water. To each well, 100 ⁇ l of biotin-labelled rat-anti-mouse IFN ⁇ (5 ⁇ g/ml in PBS Tween 0.05%, Pharmingen, Cat.
  • Intracellular cytokine staining The percentage of CD8+ IFN ⁇ -producing T-cells in 7- day 13.2 stimulated spleen cultures was measured by intracellular cytokine staining as follows. The responding spleen cells were harvested, counted and suspended in ISCOVE'S/BSA 0.1% at 1x10 6 cells/ml. Two hundred microliters of responding spleen cells were added to 200 l ISCOVE's/BSA 0.1% with + 10 g/ml E7 49 . 57 - peptide (STIMULATED) or without (NON-STIMULATED).
  • Cytotoxicity Cell mediated E7 49 . 57 -speciflc cytotoxicity was measured in a standard 51 CR-release assay.
  • RMA H-2Db+ tumour cells
  • Varying numbers of in vitro expanded effector cells were added to 2000 Na 2 51 CrO 4 (51 Cr)-labelled target cells and incubated for 5 h at 37°C.
  • Peptide-specific lysis was calculated by subtracting the specific lysis of RMA cells from the specific lysis of peptide-pulsed RMA cells.
  • B6 mice are protected against HPV16+ tumour cells via the H-2Db restricted HPV16 E7 49 . 57 CD8+ CTL epitope, RAHYNIVTF [14, 20].
  • TA-CIN when used as prophylactic vaccine.
  • Groups of 10 mice were vaccinated and boosted at 3 weeks interval with TA-CIN mixed with adjuvant at two different doses.
  • mice Three weeks after the booster injection mice were challenged with a lethal dose of 50,000 TC-1 tumour cells. As shown in Figure 1a, control mice that were injected with adjuvant only developed large aggressive tumours within 7 days. Mice injected with 32 g of TA-CIN were partially protected ( Figure 1 b). Importantly, mice injected with 200 ⁇ g TA-CIN were completely protected against tumour outgrowth (Figure 1c).
  • mice develop palpable, rapidly growing tumours within 4-7 days, that are lethal to the mice within 14-days (Figure 1a).
  • Figure 1a newly challenged mice can be regarded as a proper model for immune-intervention against minimal residual disease. Therefore, mice challenged with 50,000 TC-1 cells were vaccinated at the day of challenge and monitored for the development of tumours. All control mice quickly developed tumours ( Figure 1d).
  • Therapeutic vaccination with 200 ⁇ g TA-CIN protected the majority of mice against tumour outgrowth. The onset of tumour growth in the 3 tumour-positive mice was delayed (28-50 days after challenge).
  • TA-CIN induces E7-specific CTL in a dose dependent fashion.
  • 57 CD8+ CTL epitope [21] was shown to be a key feature in the protective immune response against TC-1 , the capacity of TA-CIN to induce E7 49 .
  • 57 -specific CTL was assessed.
  • Mice were vaccinated and boosted at three-week intervals with either 32 ⁇ g or 200 ⁇ g TA-CIN in adjuvant, or with adjuvant alone. Three weeks after the last vaccination spleen cells were tested directly in an ELISPOT IFN ⁇ assay or put into culture for one week to expand effector cells for measurement of E7 9 .
  • 57 specific cytotoxicity and IFN ⁇ production by CD8+ CTL were tested directly in an ELISPOT IFN ⁇ assay or put into culture for one week to expand effector cells for measurement of E7 9 .
  • TA-CIN induces specific antibodies and Th-cells.
  • Heterologous prime-boost regimens employing TA-CIN and TA-HPV result in optimal induction of the most vigorous CTL immunity.
  • TA-CIN and TA-HPV result in optimal induction of the most vigorous CTL immunity.
  • a positive correlation was detected between the frequency of CTL precursors and protective immunity [26, 27].
  • Certain heterologous prime-boost immunization regimens, in which two different types of vaccines sharing the antigen of choice are used, have been reported more effective in stimulating the T-cell response than homologous (prime-boost using one type of vaccine) immunization regimens (reviewed in [28]).
  • mice were injected according to seven different vaccination regimens and three weeks after the last immunization, spleens were taken out and the specific CTL response was measured by IFN ⁇ ELISPOT and H2-Db E7 49 .
  • 57 (RAHYNIVTF)- containing tetramers directly and following in vitro expansion (tetramers only). All vaccination regimens resulted in the priming of E7 9 .
  • 57 -specific CTL and those mice that were vaccinated twice generally showed higher numbers of tetramer-positive CD8+ CTL.
  • priming with TA-CIN followed by boosting with TA-HPV resulted in particularly high levels of antigen-specific CTL (Table 2 and Figure 4).
  • the response towards the TA-HPV recombinant vaccinia virus is focused at the E7 gene product and less to the virus particle itself, resulting in strong amplification of the E7 49 . 5 directed CTL response only.
  • poxviruses can be effective in boosting CD8+ T-cell responses possibly due to the broad host range of these viruses and the strong inflammatory response they evoke (reviewed in [28]).
  • HPV16+ individuals diagnosed with either high grade CIN or primary cervical cancer, using the prime-boost regimen of this or related examples, is expected to result in an effective immune response against papillomavirus, here HPV16.
  • Feltkamp MC Smits HL, Vierboom MP, Minnaar RP, de Jongh BM, Drijfhout JW, et al., Vaccination with cytotoxic T lymphocyte epitope-containing peptide protects against a tumor induced by human papillomavirus type 16- transformed cells. Eur J Immunol 1993; 23(9):2242-2249.
  • TA-CIN / TA-HPV #13 6 33 3 0.16 % 13.65 % #14 51 46 5 0.36 % 27.29 % #15 61 37 ⁇ 1 0.61 % 41.89 %

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Abstract

L'invention concerne des immunogènes et des vaccins ainsi que leur préparation et leur utilisation. L'invention concerne, en particulier, des immunogènes de combinaison comprenant un immunogène d'activation et de rappel dirigé contre les antigènes protéiques de papillomavirus, et leur utilisation en vue de stimuler une réponse immunitaire contre ces papillomavirus, notamment une réponse immunitaire prophylactique ou thérapeutique contre une infection due au papillomavirus humain (HPV).
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