EP1399182A2 - Papillomavirus vaccines - Google Patents

Abstract

The invention relates to immunogens and vaccines and to their preparation and use. In particular, the invention extends to combination immunogens comprising a primer and a booster immunogen directed against papillomavirus protein antigens, and their use to stimulate an immune response against papillomaviruses, such as a prophylactic or therapeutic immune response against human papillomavirus (HPV) infection.

Description

Immunogens and Vaccines and their Preparation and Use

Field of the invention:

This invention relates to immunogens and vaccines and to their preparation and use. In particular the invention relates to combination immunogens and vaccines. In certain embodiments 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.

Background:

It is known to administer vaccines in successive doses, often designated 'primer' and 'booster' doses. Commonly, 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.

For example, 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.

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.

Also, WO 98/56919 (SC Gilbert et al) 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. An aim of the present invention is to provide further combination immunogens and vaccines with particularly useful immunogenicity.

Description and summary of the present invention

According to an aspect of the present invention, there are provided combination immunogens directed against papillomavirus protein antigen(s). These 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, and 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.

Suitable examples of forms of the primer and booster are described further below:

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.

The booster can in certain useful examples be an immunogen or vaccine as described in WO 92/16636 and US 5,719,054 (Cantab Pharmaceuticals: ME

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.

It has surprisingly been found that a combination as described above can induce an immune response which is more intense than if two similar compositions are used but in the reciprocal order, i.e. with a viral vector immunogen or vaccine used first.

The antigens contained in or encoded by the primer and booster respectively can correspond to each other in whole or in part. For example, in an embodiment described below, 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.

Further features of the primer immunogen can be as mentioned in documents cited herein.

The virus vector used to encode papillomavirus antigen(s) in the booster can be a poxvirus vector, e.g. vaccinia. Alternative and generally equivalent poxvirus vectors include per-se known avipox vectors such as canarypox virus, and genetically attenuated or disabled poxvirus vectors. Alternatively, the virus vector used in the booster can be based on a virus (vector) of another type, e.g. a per-se known herpesvirus or adenovirus or adenoassociated virus vector, or a herpesvirus amplicon, carrying inserted heterologous sequences encoding a papillomavirus antigen and suitably placed and under control of a suitable promoter to express the papillomavirus antigen when the virus vector infects a host cell of a subject of vaccine treatment. In further alternative embodiments the booster can be a DNA vaccine encoding the desired antigen, or a non-virus vector carrying such a DNA vaccine. In a further preferred embodiment the primer can be a DNA vaccine encoding the desired antigen, or a non-virus vector carrying such a DNA vaccine.

For example, 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, e.g. for use as a therapeutic or prophylactic vaccine in humans or non-human animals, 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. 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.

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. as described in US 4,912,094 (Ribi Immunochem Research: KR Myers and AT Truchot: describing adjuvants based on modified lipopoly-saccharide, de-3-O-acyl monophosphoryl lipid A), which can be applied for example as described in US 4,912,094 or as in specification WO 94/21292 (Smithkline Beecham: P Hauser et al: Vaccine compositions containing 3-O- deacylated monophosphoryl Lipid A). Where both alum and MPL are used, the protein is preferably adsorbed first to alum and MPL added afterwards. Also usable are 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. Also usable as 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). An example of 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). Alternatively liposomes of these or other kinds can be used alongside the polypeptides, as adjuvants.

Further accessory or immunostimulatory molecules useful in this connection include cytokines, such as interleukins, including but not limited to GM-CSF, IL-12, IL-2, IL-3 and IL-7, also useful are 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 1-2000μg of the protein, preferably 10-300μg, e.g. 10-250μg. 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.

Equally, 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^Λ4-10^Λ8 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. Example:

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. from a W12 cell line (as described in MA Stanley et al, Int J Cancer 1989, 43, 672-676), derived from HPV-infected keratinocytes from CIN1 cervical biopsy material, but it is not essential to use this or any other particular source, many clinical sources are suitable, and the resulting sequences can be compared in routine manner for verification or otherwise with sequences of L2, E7 and E6 entered in the EMBL (European Molecular Biology Laboratory) database e.g. at PPH16: accession number K02718. It is not essential that the sequences used be either complete, or identical with the EMBL reference sequences.

In order to optimise expression and production of recombinant full-length fusion protein in an E.coli host, it is currently preferred to introduce 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. It is also known and preferred, on general considerations of safety in use, so as to avoid a possible connection with oncogenic conditions, to use a non-transforming point mutation in the early protein sequence, as in WO 92/16636. In E7 two preferred mutations are at residues 70 and 77 in the E7 coding sequence to change cys and glu to gly in each case, shown to eliminate binding of E7 to RB protein in accordance with the teaching of C Edmonds & KH Vousden, J Virol (1989) 63, 2650-2656, which discloses HPV E7 mutant sequences lacking in the transforming ability encoded by the normal gene sequences.

In 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.

Mouse experiments with such a primer and booster show, as described below, pre- clinical safety and efficacy of a recombinant HPV16 L2E6E7 fusion protein vaccine, in heterologous prime-boost regimens.

Thus, an example of the applicability of the present invention, including this example, 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. 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. These immune responses have effectively prevented outgrowth of HPV-specific tumour cells in a prophylactic setting as well as in a minimal residual disease setting. Most vigorous CTL immunity was induced when TA-CIN was employed in heterologous prime-boost regimens in combination with TA-HPV, a clinical grade vaccinia-based vaccine.

These results tend to support the use of the vaccine for HPV induced cervical lesions.

The accompanying drawings, Figures 1-4, indicate features of results obtained using the hereinbelow-described vaccination regimes including vaccination regimes according to the invention, (which are given without intent to limit the scope of the invention thereby). The Figures have the following significance:-

Figure 1 shows prevention of TC-1 tumour development in mice (n=10 per group) vaccinated (a-c): twice at three week interval with adjuvant only (a), 32μg TA-CIN (b) or 200μg TA-CIN (b) or, (d-f): once at the day of TC-1 tumour challenge. 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. 3 groups of mice (n=3) were vaccinated twice at three weeks interval with adjuvant only (left), 32μg TA- CIN (middle) or 200 g TA-CIN (right), (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 E7₄₉.₅₇: RAHYNIVTF peptide. The specific cytotoxicity, calculated by subtracting the RMA-specific lysis from the lysis of RMA+ E7₄₉.₅₇ 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 IFNγ upon stimulation as detected by ELISPOT. (c) The percentage of IFNγ-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 IFNγ production by CD8+ splenocytes upon stimulation with E7₄₉.₅₇: RAHYNIVTF peptide. Splenocytes derived from a control mouse (M #3), a mouse injected with 32 g TA-CIN (M #5) or injected with 200μg 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 IFNγ staining. The values indicate the percentage of double positive, IFNγ producing CD8+ T-cells.

Figure 4 shows an analysis of the percentage of E7₄₉.₅₇-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. Indicated are one example of each group: control mouse (M #1 ), TA-CIN / TA-CIN (M #10), TA-CIN / TA-HPV (M #15), TA-HPV / TA-CIN (M#18) or TA-HPV / TA-HPV (M #21 ). The percentage of double positive, H-2D^b-RAHYNIVTF tetramer positive CD8+ T-cells is depicted. Background to Example:

In the following description, numerals in square brackets refer to the list of references at the foot.

Cervical intra-epithelial neoplasia (CIN) 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].

The majority of cervical cancers express the HPV16 derived E6 and E7 proteins [2].

Therefore, these proteins are excellent target antigens for immunological intervention and hence to prevent cervical cancer.

Evidence for the involvement of the immune system in the protection against HPV- induced disease comes from a number of findings. Firstly, the increased prevalence of CIN and cervical carcinoma in immunodeficient patients [3, 4]. Secondly, the spontaneous regression of HPV-induced lesions in a manner characteristic of a cell- mediated immune response [5, 6]. Thirdly, the association between lymphocytic infiltrate and improved clinical outcome in cervical cancer patients [7]. There may be a number of reasons for the failure of the immune system to respond adequately in a normal infection. For instance, HPV infection is confined and usually does not result in inflammation. As such, it is likely to result in the generation of only weak immune responses or even immunological ignorance (reviewed in [8]). The close association of HPV infection and disease, however, provides a clear opportunity to develop strategies that are based on harnessing the power of the human immune system to treat cervical lesions.

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. Furthermore, such peptide vaccines are often restricted to patients with certain HLA-types [9-11]. In contrast, 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. Previously, a recombinant 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.

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. Furthermore, TA-CIN has been shown to prevent outgrowth of HPV16+ tumours both prophylactically as well as therapeutically in a minimal residual disease setting.

Material and Methods

Antigens and Vaccine formulations. 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 μm 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 0.2-5.0μm. The Novasomes adjuvant was formulated in 5.9mM phosphate, 3.5mM glycine buffer (pH7.5) containing 0.63mM cysteine. 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.

Mice, Vaccination, TC-1 protection and therapy experiments.

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 32μg or 200μg TA-CIN in 200μl adjuvants or intraperitoneal (subcutaneously when indicated) with TA-HPV (5x10⁶ 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 250μl 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.

Antibody analysis. 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.

Plates were washed and developed with o-phenylene diamine (OPD)/H₂0₂ for 30min at room temperature and the absorbance was measured at 490nm. Titres are recorded as the logio of the reciprocal of the dilution at an absorbance of 1.0. CTL analysis: Spleen cells were tested either freshly isolated (ELISPOT and FACS analysis with H2-D^b E7₄₉.₅₇ (RAHYNIVTF)-containing tetramers) or following 7-day in vitro expansion (cytotoxicity, intracellular IFNγ-staining and tetramer staining) using the 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 E7₄₉.₅₇ CTL epitope, as stimulator cells.

ELISPOT: The number of peptide-specific IFNγ-producing CTL in freshly isolated spleen cells using ELISPOT were measured as follows. 5x10⁶ spleen cells were stimulated overnight with or without 1μg/ml of E7₄₉.₅₇-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. The next day the cells were harvested, washed and plated at a concentration of 250,000 cells/well in a Multiscreen 96-well plate (Millipore, Etten- Leur, The Netherlands) coated with an IFNγ capture antibody (rat-anti-mouse IFNγ, 5μg/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, 100μl of biotin-labelled rat-anti-mouse IFNγ (5μg/ml in PBS Tween 0.05%, Pharmingen, Cat. 554410) was added and incubated overnight at 4°C. The next day plates were washed six times with PBS/Tween 0.05% and 100μl of extravidin alkaline phosphatase conjugate (1 :2000 in PBSTween0.05%/BSA1%, Sigma) was added. Following incubation for 1 h at room temperature, the plates were washed 3 times with PBS/TweenO.05% and 3 times with PBS. Colour was developed by adding BCIP/NBT (Sigma, B-5655) substrate in 100 l/well. When spots were well developed the reaction was stopped by extensively washing with tap water. Plates were dried and transferred to Millipore adhesive tissue. The number of spots were analysed with a fully automated computer assisted video imaging analysis system (Carl Zeiss Vision).

Intracellular cytokine staining. The percentage of CD8+ IFNγ-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 1x10⁶ cells/ml. Two hundred microliters of responding spleen cells were added to 200 l ISCOVE's/BSA 0.1% with + 10 g/ml E7₄₉.₅₇- peptide (STIMULATED) or without (NON-STIMULATED). Following 1 h of incubation at 37°C 1600μl of ISCOVE's + 10% FCS + 12.5μg/ml Brefeldin A (Sigma) was added and cells were incubated for another 5 h at 37°C. Then the cells were washed twice with ice-cold PBS and fixed with 1 ml paraformaldehyde 4% for 4 minutes on ice. Following fixation, the cells were washed twice with cold PBS and incubated in 1 ml PBS/NaAz 0.2%/BSA 0.5%/Saponin 0.1%/FCS 10% for 10 minutes on ice. Cells were washed twice with PBS/NaAz 0.2%/BSA 0.5%/Saponin 0.1% and transferred to a 96- well V-bottom plate (Costar). Cells were spun down and supernatant was removed before 25 l of PBS/NaAz 0.2%/BSA 0.5%/Saponin 0.1% containing 1μl PE-labelled rat-anti-mouse IFNγ (0.5μg/ml, Pharmingen, Cat. 554412) and 2μl FITC-labelled anti- CD8a (2.5 g/ml, Pharmingen, Cat. 01044D) was added. Following 30 minutes of (delete of) incubation at 4°C the cells were washed, suspended in 100 l paraformaldehyde and analysed on a FACScan.

Tetramers. H2-D^b E7₄₉.₅₇ (RAHYNIVTF)-containing tetramers were constructed and used for the analysis of the number of peptide-specific CTL as described earlier [19]. Both freshly isolated as well as 7 day expanded spleen cells were used.

Cytotoxicity. Cell mediated E7₄₉.₅₇-speciflc cytotoxicity was measured in a standard ⁵¹CR-release assay. RMA (H-2Db+ tumour cells) were radioactively labelled and pulsed with or without E7 ₉.₅ -peptide at a concentration of 10μg/ml. Varying numbers of in vitro expanded effector cells were added to 2000 Na₂ ⁵¹CrO₄ (51 Cr)-labelled target cells and incubated for 5 h at 37°C. The percentage of specific lysis was calculated as follows: %specific lysis=[(cpm experimental release - cpm spontaneous release)/(cpm maximum, 2% Triton X-100, release - cpm spontaneous release)] x100. Peptide-specific lysis was calculated by subtracting the specific lysis of RMA cells from the specific lysis of peptide-pulsed RMA cells.

Results

Prophylactic vaccination with TA-CIN

B6 mice are protected against HPV16+ tumour cells via the H-2Db restricted HPV16 E7₄₉.₅₇ CD8+ CTL epitope, RAHYNIVTF [14, 20]. The HPV-16 E6 and E7-positive tumour cell line TC-1 [21], which is of B6-origin, dominantly expresses this CTL epitope and represents an appropriate model to establish the efficacy of new vaccines against HPV induced tumours [22, 23]. First, we analysed the efficacy of 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. 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 μg TA-CIN were completely protected against tumour outgrowth (Figure 1c).

Therapeutic vaccination with TA-CIN.

The protective capacity of TA-CIN was established. It was further investigated whether TA-CIN could also be used in a therapeutic setting. Upon challenge with 50,000 TC-1 cells, mice develop palpable, rapidly growing tumours within 4-7 days, that are lethal to the mice within 14-days (Figure 1a). As such, 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 μg 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). The protective effect of 32μg TA-CIN was considerably weaker (Figure 1e). Taken together, the data show that TA-CIN, when administered at a dose of 200μg per injection in adjuvant, constitutes a potent vaccine formulation, which is highly effective in prophylactic as well as therapeutic settings.

TA-CIN induces E7-specific CTL in a dose dependent fashion.

The CTL response to the H2-Db restricted E7₄₉.₅₇ 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 E7₄₉.₅₇-specific CTL was assessed. Mice were vaccinated and boosted at three-week intervals with either 32μg or 200 μg TA-CIN in adjuvant, or with adjuvant alone. Three weeks after the last vaccination spleen cells were tested directly in an ELISPOT IFNγ assay or put into culture for one week to expand effector cells for measurement of E7 ₉.₅₇ specific cytotoxicity and IFNγ production by CD8+ CTL. In addition, serum was isolated for the detection of TA-CIN specific IgG antibodies. As shown in Figure 2, injection of the 200μg dose of TA-CIN elicited the highest number of E7₄₉.₅₇-specific CTL. A dose-dependent increase of peptide- specific cytotoxicity (Figure 2a) as well as the number of spots in the IFNγ-ELISPOT (Figure 2b) was measured in 32 μg TA-CIN versus 200 μg TA-CIN injected mice. Analysis of E7-specific CTL by intracellular staining of IFNγ production by CD8+ T- cells in splenocyte cultures, which had been stimulated for 1 week in vitro, confirmed these results (Figure 2c and Figure 3).

TA-CIN induces specific antibodies and Th-cells.

Since whole antigen vaccination is expected to induce humoral immunity, sera from all mice were analysed for TA-CIN specific IgG antibodies. Indeed, both groups injected with TA-CIN displayed high titers of TA-CIN-specific antibodies (Figure 2d). More detailed analysis of the antibody responses detected in a separate experiment following immunization with TA-CIN in adjuvants, revealed the presence of both lgG1 and lgG2b type antibodies indicating a mixed T-helper type 2 and type 1 cytokine response (see Table 1 below).

The appearance of demonstrable levels of anti-TA-CIN IgG antibodies indirectly points to the existence of TA-CIN specific CD4+ Th-cells, since isotype switching to IgG is Th-cell dependent [24, 25]. Analysis of the T-helper immunity of TA-CIN vaccinated mice through IFNγ ELISPOT (Table 2), shows that certain spleen cell cultures react to TA-CIN but not to the control protein TA-GW. Mice #4-6, #10-12 and #16-18, which were all injected with TA-CIN, showed high numbers of TA-CIN- specific IFNγ-spots but lacked a response to the CTL epitope, indicating a concomitant TA-CIN specific Th-type 1 response (see Table 2 below) [24, 25].

Heterologous prime-boost regimens employing TA-CIN and TA-HPV result in optimal induction of the most vigorous CTL immunity. In several studies with murine virus infections, 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]). We made use of the availability of a clinical grade recombinant vaccinia vector expressing modified versions of HPV16/18 E6 and E7 (TA-HPV) [12, 13] and evaluated the efficacy of this vaccine and TA-CIN in expanding E7-specific CTL using several vaccination schemes.

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 IFNγ ELISPOT and H2-Db E7₄₉.₅₇ (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. Interestingly, priming with TA-CIN followed by boosting with TA-HPV resulted in particularly high levels of antigen-specific CTL (Table 2 and Figure 4). An independent experiment confirmed that TA-CIN followed by TA-HPV was by far the most effective vaccination regimen in these experiements (Table 3). In addition, the experiments demonstrated that this heterologous regimen works equally well when TA-HPV is administered subcutaneously or intravenously. The efficacy also did not depend on whether TA-CIN was injected with or without adjuvants (Table 3 and data not shown). In all cases, this heterologous protocol resulted in optimal induction of the E7₄₉.₅₇ CTL response.

These experiments provide evaluation of a clinical-grade vaccine against HPV16 in a pre-clinical mouse model. The data show that prophylactic vaccination with TA-CIN results in complete protection against HPV16+ tumour cells. The majority of mice were protected against tumour outgrowth when TA-CIN was therapeutically injected in a setting of minimal residual disease. Vaccination with TA-CIN resulted in the induction of both TA-CIN-specific cellular (CTL and Th) and humoral immunity. These results support the use of this candidate vaccine for both prophylactic and therapeutic intervention against HPV16 induced disease.

The availability of the vaccinia virus-based vaccine TA-HPV, which has undergone pre-clinical mouse studies [12] and has been tested in cervical cancer patients [13], provided an opportunity to employ TA-CIN and TA-HPV in different prime-boost regimens. Although both vaccines, when used in a homologous prime-boost strategy, were capable of inducing substantial numbers of specific CTL, it was found that priming with TA-CIN and boosting with TA-HPV was significantly more efficient than all other combinations. It is possible that TA-CIN primes and focuses the immune system towards the E7₄₉.₅₇ CTL epitope. As a consequence of this channelling of CTL immunity, 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 E7₄₉.₅ directed CTL response only. Furthermore, 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]).

Therapeutic vaccination of HPV16+ individuals with high grade CIN lesions or cervical carcinoma using peptide-based vaccines or TA-HPV led to minor responses in cervical cancer patients and to more pronounced responses in patients diagnosed with CIN [9-11 , 13]. The combination of TA-CIN and TA-HPV, however, has now been shown to constitute a powerful blend for the induction of humoral and cellular immunity. Therapeutic vaccination of, in particular, non-immunocompromised

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.

The present invention can be modified and varied as will be appreciated by the skilled reader: this disclosure extends to modifications and variations including all combinations and subcombinations of the features mentioned or described herein and in the documents cited herein, each of which is incorporated by reference in its entirety for all purposes.

References

1. zur Hausen H, Papillomavirus infections - a major cause of human cancers. Biochimica et Biophysica Acta 1996; 1288F55-F78.

2. Bosch FX, Manos MM, Munoz N, Sherman M, Jansen AM, Peto J, et al., Prevalence of human papillomavirus in cervical cancer: a worldwide perspective. International biological study on cervical cancer (IBSCC) Study Group [see comments]. J Natl Cancer Inst 1995; 87(1 1 ):796-802.

3. Benton C, Shahidullah H, Hunter JAA, Human papillomavirus in the immunosupressed. Papillomavirus Rep 1992; 323-26. 4. Serraino D, Carrieri P, Pradier C, Bidoli E, Dorrucci M, Ghetti E, et al., Risk of invasive cervical cancer among women with, or at risk for, HIV infection. Int J Cancer 1999; 82(3):334-337.

5. Aiba S, Rokugo M.Tagami H, Immunohistologic analysis of the phenomenon of spontaneous regression of numerous flat warts. Cancer 1986; 58(6): 1246- 1251.

6. Coleman N, Birley HD, Renton AM, Hanna NF, Ryait BK, Byrne M, et al., Immunological events in regressing genital warts. Am J Clin Pathol 1994;

102(6):768-774.

7. Bethwaite PB, Holloway LJ, Thornton A.Delahunt B, Infiltration by immunocompetent cells in early stage invasive carcinoma of the uterine cervix: a prognostic study. Pathology 1996; 28(4):321-327.

8. Van der Burg SH, Offringa R.Melief CJM. In: Rosenberg SA. Principles and Practice of Biologic Therapy of Cancer. Philadelphia, Lippinscott Williams & Wilkins, 2000: 514 - 526.

9. Muderspach L, Wilczynski S, Roman L, Bade L, Felix J, Small LA, et al., A phase I trial of a human papillomavirus (HPV) peptide vaccine for women with high-grade cervical and vulvar intraepithelial neoplasia who are HPV 16 positive. Clin Cancer Res 2000; 6(9):3406-3416.

10. Steller MA, Gurski KJ, Murakami M, Daniel RW, Shah KV, Celis E, et al., Cell- mediated immunological responses in cervical and vaginal cancer patients immunized with a lipidated epitope of human papillomavirus type 16 E7. Clin Cancer Res 1998; 4(9):2103-2109.

11. Ressing ME, van Driel WJ, Brandt RM, Kenter GG, de Jong JH, Bauknecht T, et al., Detection of T helper responses, but not of human papillomavirus- specific cytotoxic T lymphocyte responses, after peptide vaccination of patients with cervical carcinoma. J Immunother 2000; 23(2):255-266.

12. Boursnell ME, Rutherford E, Hickling JK, Rollinson EA, Munro AJ, Rolley N, et al., Construction and characterisation of a recombinant vaccinia virus expressing human papillomavirus proteins for immunotherapy of cervical cancer. Vaccine 1996; 14(16):1485-1494. 13. Borysiewicz LK, Fiander A, Nimako M, Man S, Wilkinson GWG, Westmoreland D, et al., A recombinant vaccinia virus encoding human papillomavirus types 16 and 18, E6 and E7 proteins as immunotherapy for cervical cancer. Lancet 1996; 3471523-1527.

14. 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.

15. Lin YL, Borenstein LA, Selvakumar R, Ahmed R.Wettstein FO, Effective vaccination against papilloma development by immunization with L1 or L2 structural protein of cottontail rabbit papillomavirus. Virology 1992; 187(2):612- 619.

16. Campo MS, O'Neil BW, Grindlay GJ, Curtis F, Knowles G.Chandrachud L, A peptide encoding a B-cell epitope from the N-terminus of the capsid protein L2 of bovine papillomavirus-4 prevents disease. Virology 1997; 234(2):261-266.

17. Han R, Cladel NM, Reed CA, Peng X.Christensen ND, Protection of rabbits from viral challenge by gene gun-based intracutaneous vaccination with a combination of cottontail rabbit papillomavirus E1 , E2, E6, and E7 genes. J Virol 1999; 73(8):7039-7043.

18. Thompson HS, Davies ML, Holding FP, Fallon RE, Mann AE, O'Neill T, et al., Phase I safety and antigenicity of TA-GW: a recombinant HPV6 L2E7 vaccine for the treatment of genital warts. Vaccine 1999; 17(1):40-49.

19. Diehl L, den Boer AT, Schoenberger SP, van der Voort El, Schumacher TN, Melief CJ, et al., CD40 activation in vivo overcomes peptide-induced peripheral cytotoxic T-lymphocyte tolerance and augments anti-tumor vaccine efficacy . Nat Med 1999; 5(7):774-779.

20. Feltkamp MC, Vreugdenhil GR, Vierboom MP, Ras E, van der Burg SH, ter Schegget J, et al., Cytotoxic T lymphocytes raised against a subdominant epitope offered as a synthetic peptide eradicate human papillomavirus type 16-induced tumors. Eur J Immunol 1995; 25(9):2638-2642.

21. Lin KY, Guarnieri FG, Staveley-O'Carroll KF, Levitsky HI, August JT, Pardoll DM, et al., Treatment of established tumors with a novel vaccine that enhances major histocompatibility class II presentation of tumor antigen. Cancer Res 1996; 56(1 ):21-26.

22. Greenstone HL, Nieland JD, de Visser KE, De Bruijn ML, Kirnbauer R, Roden RB, et al., Chimeric papillomavirus virus-like particles elicit antitumor immunity against the E7 oncoprotein in an HPV16 tumor model. Proc Natl Acad Sci USA 1998; 95(4):1800-1805.

23. Ji H, Chang EY, Lin KY, Kurman RJ, Pardoll DM.Wu TC, Antigen-specific immunotherapy for murine lung metastatic tumors expressing human papillomavirus type 16 E7 oncoprotein. Int J Cancer 1998; 78(1 ):41-45.

24. Coffman RL.Mosmann TR, Isotype regulation by helper T cells and lymphokines. Monogr Allergy 1988; 2496-103.

25. Bergstedt-Lindqvist S, Sideras P, MacDonald HR.Severinson E, Regulation of Ig class secretion by soluble products of certain T-cell lines. Immunol Rev 1984; 7825-50.

26. Fu TM, Guan L, Friedman A, Schofield TL, Ulmer JB, Liu MA, et al., Dose dependence of CTL precursor frequency induced by a DNA vaccine and correlation with protective immunity against influenza virus challenge. J Immunol 1999; 162(7):4163-4170.

27. Sedlik C, Dadaglio G, Saron MF, Deriaud E, Rojas M, Casal SI, et al., In vivo induction of a high-avidity, high-frequency cytotoxic T-lymphocyte response is associated with antiviral protective immunity. J Virol 2000; 74(13):5769-5775.

28. Schneider J, Gilbert SC, Hannan CM, Degano P, Prieur E, Sheu EG, et al., Induction of CD8+ T cells using heterologous prime-boost immunisation strategies. Immunol Rev 1999; 17029-38. Table 1 TA-CIN specific lgG1 and lgG2b antibody responses in mice vaccinated with TA-CIN, TA-HPV or both. lgG1^a{tc \I3 "lgG1^a} lgG2b{tc \I3 "lgG2b}

Vaccine M1 M2 M3 M1 M2 M3 (-) 0.132* 0.067* 0.085^* 0.065^* 0.075^* 0.067^*

(-) / TA-CIN 3.934 3.840 3.881 0.590^* 3.302 2.224

(-) / TA-HPV 0.105* 0.118^* 0.101^* 0.063* 0.089^* 0.059^*

TA-CIN / TA-CIN NT 4.610 4.559 2.378 0.749* 2.487

TA-CIN / TA-HPV 3.805 4.424 3.876 0.885* 0.524^* 1.906

TA-HPV / TA-CIN 3.694 3.902 3.917 0.787^* 0.727* 2.476

TA-HPV / TA-HPV 0.081^* 0.057^* 0.004* 0.066* 0.697^* 0.091^{* a}TA-CIN specific antibody titers. Titers are expressed as the log of the reciprocal serum dilution that results in an OD value of 1. The values marked with an asterisk (*) are the OD values of sera that displayed an OD value <1 at the lowest dilution (100x). NT: Not Tested

Table 2. Analysis of E7₄₉.₅₇ CTL frequencies by ELISPOT and H-2D^b-

RAHYNIVTF tetramers in several prime-boost regimens.

ELISPOT^a{tc \I3 "ELISPOT³} TETRAMER STAINING°{TC \L3 "TETRAMER STAINING⁰}

Vaccine Mouse CTL TA-CIN TA-GW Fresh Following epitope stimulation

(-) #1 1 9 4 0.02 % 0.35% #2 <1 <1 <1 0.04 % - #3 <1 6 2 0.04 % -

(-) / TA-CIN #4 <1 76 2 0.04 % 1.99 % #5 <1 101 3 0.07 % 1.66 % #6 <1 43 4 0.07 % 1.61 %

(-) / TA-HPV #7 <1 <1 <1 0.06 % 0.30 % #8 <1 16 <1 0.05 % 0.73% #9 1 <1 6 0.04 % 3.62 %

TA-CIN / TA-CIN #10 <1 80 1 0.10 % 4.91 % #11 <1 134 <1 0.02 % 0.60 % #12 1 21 <1 0.06 % 1.81 %

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 %

TA-HPV / TA-CIN #16 <1 24 <1 0.03 % 4.06 % #17 <1 82 <1 0.07 % 5.28 % #18 <1 43 <1 0.06 % 3.78 %

TA-HPV / TA-HPV #19 2 3 <1 0.04 % 4.27 % #20 <1 <1 2 0.04 % 4.35 % #21 <1 <1 <1 0.11 % 9.24 % ^a Shown are the number of spots per 250,000 splenocytes following stimulation with indicated antigens. CTL epitope: RAHYNIVTF peptide. The percentage of H-2D^b-RAHYNIVTF tetramer positive CD8+ T-cells in freshly isolated splenocytes and in 7-day stimulated splenocyte cultures are shown.

Table 3 Analysis of E7₄₉.₅₇ CTL frequencies by H-2D -RAHYNIVTF tetramers following intraperitoneal or subcutaneous administration of T HPV.

Vaccine{tc \I3 Mouse{tc TETRAMERSTAINING{

"Vaccine} \I3 TC \L3

"Mouse} "TETRAMERSTAINING

}

(-) #22 0.22 %^a

#23 0.00 %

#24 0.22 %

TA-CIN / TA-CIN #25 0.25%

#26 0.66%

#27 0.87%

TA-CIN / TA-HPV #28 5.89 % intraperitoneal

#29 12.42 %

#30 5.47 %

TA-CIN / TA-HPV #31 10.79 % subcutaneous

#32 3.35 %

#33 2.01 %

TA-CIN without #34 6.08%

Novasomes / TA-HPV

#35 1.02%

#36 9.31%

TA-HPV / TA-CIN #37 0.92%

#38 0.38%

#39 1.09%

TA-HPV / TA-HPV #40 0.73%

#41 0.86%

#42 0.87% age of H-2D^b-RAHYNIVTF tetramer positive CD8+ T-cells in 7-day stimulated splenocyte cultures.

Claims

CLAIMS: 1 : A combination immunogen, for sequential administration of the components, to evoke an immune response against papillomavirus comprising (i) a primer for first administration, and (ii) a booster for second administration, wherein the primer comprises a polypeptide with antigenic determinants derived from papillomavirus protein, or DNA encoding said polypeptide, and the booster comprises a virus vector encoding papillomavirus antigen and which can express said papillomavirus antigen when it infects a host cell.

2: An immunogen according to claim 1 , wherein the primer and/or the booster comprises a polypeptide antigen, or DNA encoding said polypeptide, and which polypeptide is from a human papillomavirus selected from types 6, 11 , 16 and 18.

3: An immunogen according to any one of claims 1 or 2, wherein the papillomavirus antigens contained in, or encoded by, the primer and booster correspond to each other in whole or in part.

4: An immunogen according to any one of claims 1 to 3, wherein the primer is a fusion polypeptide comprising sequences from each of at least two different papillomavirus proteins. 5: An immunogen according to claim 4, wherein the fusion polypeptide comprises antigenic determinants from at least two papillomavirus proteins selected from L2, L1 , E1 , E2, E4, E5, E6 and E7 papillomavirus proteins.

6: An immunogen according to claim 5, wherein the papillomavirus proteins are from different papillomavirus types.

7: An immunogen according to claim 5 or 6, wherein the fusion polypeptide comprises antigenic determinants from papillomavirus L2 protein. 8: An immunogen according to any one of the preceding claims, wherein the booster is a virus selected from a poxvirus, herpesvirus, adenovirus and adenoassociated virus.

9: An immunogen according to claim 8, wherein the booster virus is a vaccinia virus which carries inserted heterologous sequences encoding and expressing a fusion protein based on E6 and E7 sequences of human papillomavirus of types 16 and 18.

10: An immunogen according to claim 9, wherein the primer is an L2E7E6 fusion protein containing sequences based on those of human papillomavirus type 16.

11 : An immunogen according to any one of the preceding claims, which further comprises an adjuvant and/or another accessory substance such as a further immunostimulatory molecule. 12: A pharmaceutical which comprises an immunogen according to any one of the preceding claims in combination with a pharmaceutically acceptable excipient.

13: A pharmaceutical according to claim 12, for use as a medicament. 14: Use of a pharmaceutical according to claim 12, in the manufacture of a medicament to evoke an immune response against papillomavirus. 15: A method of evoking an immune response against papillomavirus in a subject, which comprises administering to said subject (i) a primer, followed by (ii) a booster, wherein said primer comprises a polypeptide with antigenic determinants derived from papillomavirus protein, or DNA encoding said polypeptide, and said booster comprises a virus vector encoding papillomavirus antigen and which can express said antigen when it infects a host cell.

16: A method according to claim 15, wherein an adjuvant and/or another accessory substance such as a further immunostimulatory molecule is also administered to said subject.