WO2008145745A1

WO2008145745A1 - Vaccine against hpv

Info

Publication number: WO2008145745A1
Application number: PCT/EP2008/056719
Authority: WO
Inventors: Peter Franz Ertl; Gerald Wayne Gough; Vanita Parmar; Paul Alexander Wilson
Original assignee: Smithkline Beecham Corporation
Priority date: 2007-06-01
Filing date: 2008-05-30
Publication date: 2008-12-04
Also published as: GB0710538D0

Abstract

The present invention relates to methods and compositions useful in the treatment and prevention of human papilloma virus infections. In particular the invention relates to nucleic acid molecules typically encoding a polyprotein based on Early antigens from different HPV strains, and vectors suitable for DNA vaccine delivery, and pharmaceutical compositions containing them. Methods for manufacturing said molecules, vectors and composition are also contemplated, as are their use in medicine.

Description

VACCINE AGAINST HPV

Background to the Invention

Papillomaviruses are small DNA tumour viruses, which are species specific. They infect basal epithelial cells and replicate and complete their full life cycle within the cell nucleus. Viral gene expression is tightly linked to epithelial cell differentiation and capsid assembly and maturation only occurs in fully differentiated epithelial cells in the upper epithelial cell layers.

So far, over 100 individual human papillomavirus (HPV) types have been described. HPVs are generally specific either for the skin (e.g. HPV-1 and -2) or mucosal surfaces (e.g. HPV-6 and -1 1 ) and usually cause benign tumours (warts) that persist for several months or years. Such benign tumours may be distressing for the individuals concerned but tend not to be life threatening, with a few exceptions.

Some HPVs are also associated with cancers, known as oncogenic HPV types. The strongest positive association between an HPV and human cancer is the one existing between HPV-16 and HPV-18 and cervical carcinoma. Cervical cancer is the most common malignancy in developing countries, with about 500,000 new cases occurring in the world each year.

Other HPV types besides HPV-16 and HPV-18 which can cause cancer are types 31 , 33, 35, 39, 45, 51 , 52, 56, 58, 59, 66 and 68 (referred to as "oncogenic-HPV types"). Types 16 and 18 are those which have the highest association with cervical cancer. After HPV types 16 (found in 53.5% of cervical cancer) and 18 (found in 17.2% of cervical cancer), types 45 (6.7%) and 31 (2.9%) are the next most significant in terms of their frequency in cervical cancers. HPV 33 (2.6%) is next, followed by HPV 52 (2.3%). (Munoz N, Bosch FX, de Sanjose S et al. International Agency for Research on Cancer Multicenter Cervical Cancer Study Group. N Engl J Med 2003; 348: 518-27). Papillomaviruses are not naturally very immunogenic and during the course of natural infection antibodies may only occur very late (during or after resolution), and in a fraction of patients whilst some patients may resolve disease without developing detectable antibody at all.

Vaccination using papillomavirus early antigens has been widely studied in several different animal model systems. However there are only a few reports studying therapeutic immunisation. For example, cattle immunised therapeutically with a cocktail of proteins comprising bovine papillomavirus (BPV) proteins E1 , E2, E4 and E7 showed a reduced papilloma disease burden in a proportion of animals compared to controls.

Papilloma virus infections have been observed in a variety of species, including sheep, dogs, rabbits, monkeys, cattle and humans. Human papilloma viruses (HPV) have been classified into more than 80 types [Epidemiology and Biology of Cervical Cancer Seminars in Surgical Oncology 1999 16:203-211. Wolfgang MJ, Schoell MD, Janicek MF and Mirhashemi R.], some of which are further divided into sub-types (e.g. type 6a and 6b), based on the extent of DNA sequence homology. Papilloma viruses generally infect epithelia, but the different HPV types cause distinct diseases. For example, types 1-4, 7, 10 and 26-29 cause benign warts, types 16, 18, 31 , 33, 35, 39, 45, 51 , 52, 56, 58, 59, and 68 are associated with cervical cancers and types 6 and 11 are implicated in genital warts (non-malignant condylomata of the genital tract).

HPV has proven difficult to grow in tissue culture, so there is no traditional live or attenuated viral vaccine. Development of an HPV vaccine has also been slowed by the lack of a suitable animal model in which the human virus can be studied. This is because the viruses are highly species specific, so it is very difficult to infect an animal with a papilloma virus from a host of a different species, as would be required for safety testing before a vaccine was first tried in humans.

Papilloma viruses have a DNA genome which encodes "early" and "late" genes designated E1 to E7, L1 and L2. The early gene sequences have been shown to have functions relating to viral DNA replication and transcription, evasion of host immunity, and alteration of the normal host cell cycle and other processes. For example the E1 protein is an ATP-dependent DNA helicase and is involved in initiation of the viral DNA replication process whilst E2 is a regulatory protein controlling both viral gene expression and DNA replication. Through its ability to bind to both E1 and the viral origin of replication, E2 brings about a local concentration of E1 at the origin, thus stimulating the initiation of viral DNA replication. The E4 protein appears to have a number of poorly defined functions but amongst these may be binding to the host cell cytoskeleton, whilst E5 appears to delay acidification of endosomes resulting in increased expression of EGF receptor at the cell surface and both E6 and E7 are known to bind cell proteins p53 and pRB respectively. The E6 and E7 proteins form HPV types associated with cervical cancer are known oncogenes. L1 and L2 encode the two viral structural (capsid) proteins.

Historically, vaccines have been seen as a way to prevent infection by a pathogen, priming the immune system to recognise the pathogen and neutralise it should an infection occur. The vaccine includes one or more antigens from the pathogen, commonly the entire organism, either killed or in a weakened (attenuated) form, or selected antigenic peptides from the organism. When the immune system is exposed to the antigen(s), cells are generated which retain an immunological "memory" of it for the lifetime of the individual. Subsequent exposure to the same antigen (e.g. upon infection by the pathogen) stimulates a specific immune response which results in elimination or inactivation of the infectious agent.

There are two arms to the immune response: a humoral (antibody) response and a cell-mediated response. Protein antigens derived from pathogens that replicate intracellular^ (viruses and some bacteria) are processed within the infected host cell releasing short peptides which are subsequently displayed on the infected cell surface in association with class I major histocompatability (MHC I) molecules. When this associated complex of MHC I and peptide is contacted by antigen-specific CD8+ T-cells the T-cell is activated, acquiring cytotoxic activity. These cytotoxic T- cells (CTLs) can lyse infected host cells, so limiting the replication and spread of the infecting pathogen. Another important arm of the immune response is controlled by CD4+ T-cells. When antigen derived from pathogens is released into the extracellular milieu they may be taken up by specialised antigen-presenting cells (APCs) and displayed upon the surface of these cells in association with MHC Il molecules. Recognition of antigen in this complex stimulates CD4+ T-cells to secrete soluble factors (cytokines) which regulate the effector mechanisms of other T-cells. Antibody is produced by B-cells. Binding of antigen to secreted antibody may neutralise the infectivity of a pathogen and binding of antigen to membrane-bound antibody on the surface of B-cells stimulates division of the B-cell so amplifying the B-cell response. In general, good antibody responses are required to control bacterial infections and both antibody and cell-mediated immune responses (CD8+ and CD4+) are required to control infections by viruses.

It is believed that it may be possible to harness the immune system by vaccination, even after infection by a pathogen, to control or resolve the infection by inactivation or elimination of the pathogen. Such "therapeutic" vaccines would require a cell- mediated response to be effective, and would ideally invoke both humoral and cell- mediated immune responses.

It has been demonstrated (Benvenisty, N and Reshaf, L. PNAS 83 9551-9555) that inoculation of mice with calcium phosphate precipitated DNA results in expression of the peptides encoded by the DNA. Subsequently, intramuscular injection into mice of plasmid DNA which had not been precipitated was shown to result in uptake of the DNA into the muscle cells and expression of the encoded protein. Because expression of the DNA results in production of the encoded pathogen proteins within the host's cells, as in a natural infection, this mechanism can stimulate the cell- mediated immune response required for therapeutic vaccination. DNA vaccines are described in WO90/1 1092 (Vical, Inc.).

DNA vaccination may be delivered by mechanisms other than intra-muscular injection. For example, delivery into the skin takes advantage of the fact that immune mechanisms are highly active in tissues that are barriers to infection such as skin and mucous membranes. Delivery into skin could be via injection, via jet injector (which forces a liquid into the skin under pressure) or via particle bombardment, in which the DNA may be coated onto particles of sufficient density to penetrate the epithelium (US Patent No. 5371015). Projection of these particles into the skin results in direct transfection of both epidermal cells and epidermal Langerhan cells. Langerhan cells are antigen presenting cells (APC) which take up the DNA, express the encoded peptides, and process these for display on cell surface MHC proteins. Transfected Langerhan cells migrate to the lymph nodes where they present the displayed antigen fragments to lymphocytes, invoking an immune response. Very small amounts of DNA (0.5-1 μg) are required to induce an immune response via particle delivery into skin and this contrasts with the milligram quantities of DNA known to be required to generate immune responses subsequent to direct intramuscular injection.

It has been reported, for example in studies using virus like particles formed from the L1 and L2 capsid proteins or using these proteins alone (1 ), that HPV is poorly immunogenic. Furthermore, HPV genes have proven difficult to express in human or other mammalian cells, leading difficulties in developing protein subunit vaccines. Monocystronic E1 has proven particularly resistant to expression from heterologous promoters in mammalian cells (Remm M, Remm A and Mart Ustav, J.Virol 1999 73, 3062-3070). Human papilloma virus type 18 E1 is translated from polycistronic mRNA by a discontinuous scanning mechanism). Expression of E1 is most often detected using in vitro DNA replication of an HPV origin containing plasmid as a surrogate (Lu, JZJ, Sun et al J.Virol 1993 67, 7131-7139 and Del Vecchio AM et al J.Virol 1992 66, 5949-5958).

International patent application WO02/08435 provides HPV polynucleotides wherein the sequence has been optimised to resemble the usage patterns of a highly expressed human gene. In particular codon optimised HPV6bE1 , and HPV 1 1 E2 are disclosed. International patent application WO2004/031222 describes codon- optimised HPV polynucleotides encoding multiple genes within one expression cassette. Surprisingly we have found that certain combinations of genes encoding early HPV proteins can be combined in one expression cassette without any significant loss of expression, and providing broad HPV type coverage. Furthermore it was found that the combination of two expression cassettes with certain combinations of genes encoding early HPV genes can provide a method for delivering a larger number of genes to a subject.

Additionally it was found that such combinations of one or more expression vectors comprising genes encoding early HPV genes can provide cross-reactivity against other HPV strains which are not present within the construct.

The present invention therefore provides a polynucleotide sequence which encodes a polypeptide sequence comprising at least two HPV Early antigens or fragments thereof, said polypeptide being capable of raising an immune response to HPV types selected from 18, 45, 56, 39, 16, 31 , 35, 33, 58, 52, 51 and 59 when administered in vivo, wherein at least one of the HPV types against which an immune response is capable of being raised is not encoded by the polynucleotide sequence.

In one embodiment the invention provides a polynucleotide sequence which encodes a polypeptide sequence comprising at least two HPV Early antigens or fragments thereof, said polypeptide being capable of raising an immune response to HPV types selected from 18, 45, 56, 39, 16, 31 , 35, 33, 58, 52, 51 , 68, 82, 73 and 59 when administered in vivo, wherein at least one of the HPV types against which an immune response is capable of being raised is not encoded by the polynucleotide sequence.

In a further embodiment, the invention provides a polynucleotide sequence which encodes a polypeptide sequence comprising a first HPV type early antigen or fragment thereof from one HPV cluster and a second HPV type early antigen or fragment thereof from a different HPV cluster said polynucleotide being capable of raising an immune response to more than one HPV type within each of the clusters when administered in vivo, wherein at least one of the HPV types being treated is not encoded by the polynucleotide sequence. For example, wherein the polynucleotide sequence encodes an amino acid sequence comprising HPV 16 and 18 early antigens or fragments thereof, or wherein the polynucleotide sequence encodes an amino acid sequence comprising two or more of HPV 33, HPV 51 , HPV 51 and HPV56 early antigens or fragments thereof.

In one embodiment the invention provides a polynucleotide sequence as set out in SEQ ID NO: 23 or SEQ ID NO: 24.

In a further embodiment the invention provides a polynucleotide sequence encoding the polyprotein which set out in SEQ ID NO 28 or 29. The invention also provides expression vector comprising polynucleotide sequences of the invention operably linked to a control sequence which is capable of providing for the expression of the polynucleotide sequence in a host cell.

Any suitable promoter may be used in the expression vectors of the present invention. One example of a suitable vector is the promoter from the HCMV IE gene, for example wherein the 5' untranslated region of the HCMV IE gene comprising exon 1 is included and for example wherein intron A is partially or completely excluded as described in WO 02/36792.

The expression vector of the present invention may comprise one or more expression cassettes. For example in one embodiment the invention provides an expression vector comprising two expression cassettes, the first expression cassette comprising the polynucleotide sequences set out in SEQ ID NO: 23 and the second expression cassette comprising the polynucleotide sequences set out in SEQ ID NO: 24, each of which are operably linked to a promoter capable of driving expression. In a further embodiment of the present invention these two expression cassettes are present in different expression vectors, for example each expression cassette will be in a different plasmid.

In one embodiment the invention provides an expression vector encoding the polyprotein set out in SEQ ID NO: 28 or SEQ ID NO: 29.

The invention also provides host cells comprising the polynucleotide sequences of the present invention, or expression vectors of the present invention.

The invention further provides pharmaceutical compositions comprising polynucleotide sequences of the present invention, or expression vectors of the present invention.

The pharmaceutical composition of the present invention may be in a format suitable for particle-mediated epidermal delivery, for example it may comprise a plurality of dense particles, for example gold particles, coated with the polynucleotides of the invention. In one embodiment the expression vectors are co-coated onto a plurality of gold beads.

The invention also provides use of the polynucleotide, vector or pharmaceutical composition of the present invention in the treatment or prophylaxis of an HPV infection. Such treatment or prophylaxis may be for cervical dysplasia, cervical intraepithelial neoplasia (CIN), cervical cancer, vulval intraepithelial neoplasia (VIN), vaginal intraepithelial neoplasia (VAIN), anal intraepithelial neoplasia (AIN) or associated cancers. The present invention also provides methods of treating or preventing HPV infections or any symptoms or diseases associated therewith comprising administering an effective amount of a protein, polynucleotide or a vector or a vaccine according to the invention. Administration of a vaccine may take the form of one or more individual doses, for example in a "prime-boost" regime. In certain cases the "prime" vaccination may be via DNA vaccine delivery, in particular via particle mediated DNA delivery of a polynucleotide according to the present invention, for example it may be incorporated into a plasmid-derived vector and the "boost" by administration of a recombinant viral vector comprising the same polynucleotide sequence. Alternatively, a protein adjuvant approach may act as part of the priming or boosting approach, with DNA delivered as the other arm of the prime-boost regime (the protein being the same as, or at least sharing one or more epitopes, for example the majority of epitopes, with the protein encoded by the DNA).

In one embodiment the present invention provides the use of a composition comprising a polynucleotide sequence encoding human HPV early antigen or fragments thereof of at least two different HPV types in the manufacture of a medicament for the treatment of HPV infection by HPV types selected from 18, 45, 56, 39, 16, 31 , 35, 33, 58, 52, 51 and 59, wherein at least one of the HPV types being treated is omitted from the composition.

A further embodiment of the present invention is a method of generating an immune response against more than one HPV type by administration of a composition comprising a polynucleotide encoding at least one HPV early antigen or fragment thereof from each HPV cluster wherein at least one of the HPV types against which an immune response is generated is omitted from the composition, for example wherein the immune response is generated against three or more of the HPV E1 and or E2 types 18, 45, 56, 39, 16, 31 , 35, 33, 58, 52, 51 and 59, for example wherein the immune response is generated against at least HPV16, HPV18 and HPV45.

A yet further embodiment of the present invention is the use of a polynucleotide encoding an HPV Early protein in the preparation of a medicament for the prevention of infection or disease caused by an HPV virus containing a second different HPV Early protein type, wherein the Early protein encoded by the polynucleotide of the medicament has a sequence identity of greater than 80 % in the predicted epitope regions when compared with a sequence from the second HPV type.

It is likely that the epitopes of the HPV Early proteins expressed by the polynucleotides of the present invention will be HLA restricted and distributed throughout the protein, however some regions have been identified as set out in Example 20 as potential key regions for certain HLA types. Therefore in one embodiment the polynucleotide sequence of the invention comprises for example one or more of the key regions in the E1 gene identified in Figures 41 , 43 and 44, for example one or more regions selected from residues 65 to 72 of E1 Cluster 1 ; residues 127 to 134 of E1 Cluster 1 ; residues 153 to 162 of E1 Cluster 1 ; residues, 306 to 320 of E1 Cluster 1 ; residues 420 to 427 of E1 Cluster 1 ; 593 to 6020 of E1 Cluster 1 ; residues 107 to 115 of E1 Cluster 1 ; and 467 to 475 of E1 Cluster 1.

In a further embodiment the polynucleotide sequence of the invention comprises one or more of the equivalent regions identified as set out in Example 20 as potential key regions in the E2 gene identified in Figure 42, for example one or more regions selected from residues 8 to 17 of E2 Cluster 1 ; residues 69 to 78 of E2 Cluster 1 , residues 92 to 103 of E2 Cluster 1 ; residues 138 to 149 of E2 Cluster 1 ; residues 156 to 165 of E2 Cluster 1 ; residues 192 to 201 of E2 Cluster 1 ; residues 229 to 238 of E2 Cluster 1 ; residues 250 to 261 of E2 Cluster 1 ; residues 317 to 327 of E2 Cluster 1.

In yet a further embodiment the polynucleotide sequence of the invention comprises one or more of the key regions in the E1 gene and one or more of the key regions in the E2 gene.

In the polynucleotides of the present invention, the codon usage pattern is altered from that typical of human papilloma viruses to more closely represent the codon bias of highly expressed genes in human. The "codon usage coefficient" is a measure of how closely the codon pattern of a given polynucleotide sequence resembles that of a target species. Codon frequencies can be derived from literature sources for the highly expressed genes of many species (see e.g. Nakamura et.al. Nucleic Acids Research 1996, 24:214-215). The codon frequencies for each of the 61 codons (expressed as the number of occurrences occurrence per 1000 codons of the selected class of genes) are normalised for each of the twenty natural amino acids, so that the value for the most frequently used codon for each amino acid is set to 1 and the frequencies for the less common codons are scaled to lie between zero and 1. Thus each of the 61 codons is assigned a value of 1 or lower for the highly expressed genes of the target species. In order to calculate a codon usage coefficient for a specific polynucleotide, relative to the highly expressed genes of that species, the scaled value for each codon of the specific polynucleotide are noted and the geometric mean of all these values is taken (by dividing the sum of the natural logs of these values by the total number of codons and take the anti-log). The coefficient will have a value between zero and 1 and the higher the coefficient the more codons in the polynucleotide are frequently used codons. If a polynucleotide sequence has a codon usage coefficient of 1 , all of the codons are "most frequent" codons for highly expressed genes of the target species.

Shorter polynucleotide sequences are within the scope of the invention. For example, a polynucleotide of the invention may encode a fragment of a HPV protein. A polynucleotide which encodes a fragment of at least 8, for example 1 to 10 amino acids or up to 20, 50, 60, 70, 80, 100, 150 or 200 amino acids in length is considered to fall within the scope of the invention as long as the polynucleotide encodes a polypeptide that demonstrates HPV antigenicity. In particular, but not exclusively, this aspect of the invention encompasses the situation when the polynucleotide encodes a fragment of a complete HPV protein sequence and may represent one or more discrete epitopes of that protein.

As discussed above, the present invention includes expression vectors that comprise the nucleotide sequences of the invention. Such expression vectors are routinely constructed in the art of molecular biology and may for example involve the use of plasmid DNA and appropriate initiators, promoters, enhancers and other elements, such as for example polyadenylation signals which may be necessary, and which are positioned in the correct orientation, in order to allow for protein expression. Other suitable vectors would be apparent to persons skilled in the art. By way of further example in this regard we refer to Sambrook et al. Molecular Cloning: a Laboratory Manual. 2^nd Edition. CSH Laboratory Press. (1989).

A polynucleotide of the invention or for use in the invention in a vector is operably linked to a control sequence which is capable of providing for the expression of the coding sequence by the host cell, i.e. the vector is an expression vector. The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A regulatory sequence, such as a promoter, "operably linked" to a coding sequence is positioned in such a way that expression of the coding sequence is achieved under conditions compatible with the regulatory sequence.

The vectors may be for example, plasmid, artificial chromosome, virus or phage vectors provided with an origin of replication, they may have a promoter for the expression of the said polynucleotide and they may also have a regulator of the promoter. In one embodiment there may be a polyadenylation signal sequence. The vectors may contain one or more selectable marker genes, for example an ampicillin or kanomycin resistance gene in the case of a bacterial plasmid or a resistance gene for a fungal vector. Vectors may be used in vitro, for example for the production of DNA or RNA or used to transfect or transform a host cell, for example, a mammalian host cell. The vectors may also be adapted to be used in vivo, for example in a method of DNA vaccination or of gene therapy.

Promoters and other expression regulation signals may be selected to be compatible with the host cell for which expression is designed. For example, mammalian promoters include the metallothionein promoter, which can be induced in response to heavy metals such as cadmium, and the β-actin promoter. Viral promoters such as the SV40 large T antigen promoter, human cytomegalovirus (CMV) immediate early (IE) promoter, rous sarcoma virus LTR promoter, adenovirus promoter), or a HPV promoter, particularly the HPV upstream regulatory region (URR) may also be used. All these promoters are readily available in the art.

Examples of suitable viral vectors include herpes simplex viral vectors, vaccinia or alpha-virus vectors and retroviruses, including Antiviruses, adenoviruses and adeno- associated viruses. Gene transfer techniques using these viruses are known to those skilled in the art. Retrovirus vectors for example may be used to stably integrate the polynucleotide of the invention into the host genome, although such recombination is not preferred. Replication-defective adenovirus vectors by contrast remain episomal and therefore allow transient expression. Vectors capable of driving expression in insect cells (for example baculovirus vectors), in human cells or in bacteria may be employed in order to produce quantities of the HPV protein encoded by the polynucleotides of the present invention, for example for use as subunit vaccines. Preferred viral vectors are those derived from non-human primate adenovirus such as C68 chimp adenovirus (US 6, 083, 716) other wise known as Pan 9.

Where the polynucleotides of the present invention find use as therapeutic agents, e.g. in DNA vaccination, the nucleic acid will be administered to the mammal e.g. human to be vaccinated. The nucleic acid, such as RNA or DNA, for example, DNA, is provided in the form of a vector, such as those described above, which may be expressed in the cells of the mammal. The polynucleotides may be administered by any available technique. For example, the nucleic acid may be introduced by needle injection, for example intradermally, subcutaneously or intramuscularly. Alternatively, the nucleic acid may be delivered directly across the skin using a nucleic acid delivery device such as particle-mediated epidermal delivery (PMED). In this method, inert particles (such as gold beads) are coated with a nucleic acid, and are accelerated at speeds sufficient to enable them to penetrate a surface of a recipient (e.g. skin), for example by means of discharge under high pressure from a projecting device. (Particles coated with a nucleic acid molecule of the present invention are within the scope of the present invention, as are devices loaded with such particles).

Suitable techniques for introducing the naked polynucleotide or vector into a patient include topical application with an appropriate vehicle. The nucleic acid may be administered topically to the skin, or to mucosal surfaces for example by intranasal, oral, intravaginal or intrarectal administration. The naked polynucleotide or vector may be present together with a pharmaceutically acceptable excipient, such as phosphate buffered saline (PBS). DNA uptake may be further facilitated by addition of facilitating agents such as bupivacaine to the composition. Other methods of administering the nucleic acid directly to a recipient include ultrasound, electrical stimulation, electroporation and microseeding which is described in US-5,697,901. Uptake of nucleic acid constructs may be enhanced by several known transfection techniques, for example those including the use of transfection agents. Examples of these agents includes cationic agents, for example, calcium phosphate and DEAE- Dextran and lipofectants, for example, lipofectam and transfectam. The dosage of the nucleic acid to be administered can be altered. Typically the nucleic acid is administered in an amount in the range of 1 pg to 1 mg, for example of 1 pg to 10μg nucleic acid for particle mediated gene delivery and 10μg to 1 mg for other routes.

A nucleic acid sequence of the present invention may also be administered by means of specialised delivery vectors useful in gene therapy. Gene therapy approaches are discussed for example by Verme et al, Nature 1997, 389:239-242. Both viral and non-viral systems can be used. Viral based systems include retroviral, lentiviral, adenoviral, adeno-associated viral, herpes viral, Canarypox and vaccinia-viral based systems. Non-viral based systems include direct administration of nucleic acids and liposome-based systems.

A nucleic acid sequence of the present invention may also be administered by means of transformed cells. Such cells include cells harvested from a subject. The naked polynucleotide or vector of the present invention can be introduced into such cells in vitro and the transformed cells can later be returned to the subject. The polynucleotide of the invention may integrate into nucleic acid already present in a cell by homologous recombination events. A transformed cell may, if desired, be grown up in vitro and one or more of the resultant cells may be used in the present invention. Cells can be provided at an appropriate site in a patient by known surgical or microsurgical techniques (e.g. grafting, micro-injection, etc.)

The vaccine compositions of the present invention may include adjuvant compounds which may serve to increase the immune response induced by the protein itself or which is encoded by the plasmid DNA. Alteration of the codon bias to suit the vaccinated species is proposed herein as a means of increasing expression and thereby boosting the immune response, but an adjuvant may never-the-less be desirable because, while DNA vaccines tend to work well in murine models, there is evidence of a somewhat weaker potency in larger species such as non-human primates which is thought to be predictive of the likely potency in humans.

The vaccine composition of the invention may also comprise an adjuvant, such as, in an embodiment, imiquimod, tucaresol, GM-CSF or alum.

In one embodiment of the present invention one or more adjuvants may administered at the same time as the polynucleotide or expression vector of the invention and for example the polynucleotide or expression vector and one or more adjuvants may be formulated together. Such adjuvant agents contemplated by the invention include, but this list is by no means exhaustive and does not preclude other agents: synthetic imidazoquinolines such as imiquimod [S-26308, R-837], (Harrison, et al. 'Reduction of recurrent HSV disease using imiquimod alone or combined with a glycoprotein vaccine', Vaccine 19: 1820-1826, (2001 )); and resiquimod [S-28463, R-848] (Vasilakos, et al. ' Adjuvant activates of immune response modifier R-848: Comparison with CpG ODN', Cellular immunology 204: 64-74 (2000).), Schiff bases of carbonyls and amines that are constitutively expressed on antigen presenting cell and T-cell surfaces, such as tucaresol (Rhodes, J. et al. ' Therapeutic potentiation of the immune system by costimulatory Schiff-base-forming drugs', Nature 377: 71-75 (1995)), cytokine, chemokine and co-stimulatory molecules, Th1 inducers such as interferon gamma, IL-2, IL-12, IL-15 and IL-18, Th2 inducers such as IL-4, IL-5, IL-6, IL-10 and IL-13 and other chemokine and co-stimulatory genes such as MCP-1 , MIP- 1 alpha, MIP-1 beta, RANTES, TCA-3, CD80, CD86 and CD40L, other immunostimulatory targeting ligands such as CTLA-4 and L-selectin, apoptosis stimulating proteins and peptides such as Fas, synthetic lipid based adjuvants, such as vaxfectin, (Reyes et al., Vaxfectin enhances antigen specific antibody titres and maintains Th1 type immune responses to plasmid DNA immunization', Vaccine 19: 3778-3786) squalene, alpha- tocopherol, polysorbate 80, DOPC and cholesterol, endotoxin, [LPS], Beutler, B., 'Endotoxin, Toll-like receptor 4, and the afferent limb of innate immunity', Current Opinion in Microbiology 3: 23-30 (2000)) ; CpG oligo- and di-nucleotides, Sato, Y. et al., 'Immunostimulatory DNA sequences necessary for effective intradermal gene immunization', Science 273 (5273): 352-354 (1996). Hemmi, H. et al., 'A Toll-like receptor recognizes bacterial DNA', Nature 408: 740- 745, (2000) and other potential ligands that trigger Toll receptors to produce Th1- inducing cytokines, such as synthetic Mycobacterial lipoproteins, Mycobacterial protein p19, peptidoglycan, teichoic acid and lipid A.

Certain preferred adjuvants for eliciting a predominantly Th1-type response include, for example, a Lipid A derivative such as monophosphoryl lipid A, or preferably 3-de- O-acylated monophosphoryl lipid A. MPL^® adjuvants are available from Corixa Corporation (Seattle, WA; see, for example, US Patent Nos. 4,436,727; 4,877,61 1 ; 4,866,034 and 4,912,094). CpG-containing oligonucleotides (in which the CpG dinucleotide is unmethylated) also induce a predominantly Th1 response. Such oligonucleotides are well known and are described, for example, in WO 96/02555, WO 99/33488 and U.S. Patent Nos. 6,008,200 and 5,856,462. Immunostimulatory DNA sequences are also described, for example, by Sato et al., Science 273:352, 1996. Another preferred adjuvant comprises a saponin, such as Quil A, or derivatives thereof, including QS21 and QS7 (Aquila Biopharmaceuticals Inc., Framingham, MA); Escin; Digitonin; or Gypsophila or Chenopodium quinoa saponins.

Toll-like receptor (TLR) ligands, that can mimic pathogen-associated molecular patterns and activate immune cells via Toll-like receptors (TLRs), have been demonstrated to act as adjuvants for DNA immunization in animal models (Thomsen 2004). Imiquimod, which is a TLR-7 agonist, has been shown to exert adjuvant effects, enhancing the efficacy of DNA plasmids administered by PMED.

In one embodiment of the invention the pharmaceutical composition will further comprise one or more adjuvants, for example a TLR agonist, for example a TLR-7 agonist such as imiquimod.

In a further embodiment of the invention the pharmaceutical composition will further comprise GM-CSF, or polynucleotides encoding GM-CSF. In one embodiment the pharmaceutical composition of the invention will comprise both GM-CSF, or polynucleotides encoding GM-CSF and imiquimod.

These adjuvants and the combination of these two adjuvant components are described in WO2005025614.

In an embodiment, the adjuvant comprises an immunostimulatory CpG oligonucleotide, such as disclosed in (WO96102555). Typical immunostimulatory oligonucleotides will be between 8-100 bases in length and comprises the general formula X₁ CpGX₂ where X₁ and X₂ are nucleotide bases, and the C and G are unmethylated.

The preferred oligonucleotides for use in adjuvants or vaccines of the present invention preferably contain two or more dinucleotide CpG motifs preferably separated by at least three, more preferably at least six or more nucleotides. The oligonucleotides of the present invention are typically deoxynucleotides. In a preferred embodiment 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, e.g. mixed phosphorothioate/phophodiesters. Other internucleotide bonds which stabilise the oligonucleotide may be used.

Methods for producing phosphorothioate oligonucleotides or phosphorodithioate are described in US5,666,153, US5,278,302 and WO95/26204.

Examples of preferred oligonucleotides have the following sequences. The sequences preferably contain phosphorothioate modified internucleotide linkages.

OLIGO 1 : TCC ATG ACG TTC CTG ACG TT (CpG 1826) (SEQ ID NO 30) OLIGO 2: TCT CCC AGC GTG CGC CAT (CpG 1758) (SEQ ID NO 31 ) OLIGO 3: ACC GAT GAC GTC GCC GGT GAC GGC ACC ACG (SEQ ID NO 32) OLIGO 4: TCG TCG TTT TGT CGT TTT GTC GTT (CpG 2006) (SEQ ID NO 33) OLIGO 5: TCC ATG ACG TTC CTG ATG CT (CpG 1668) (SEQ ID NO 34) Alternative CpG oligonucleotides may comprise the preferred sequences above in that they have inconsequential deletions or additions thereto.

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. An adjuvant formulation containing CpG oligonucleotide can be purchased from Qiagen under the trade name "ImmunEasy".

Throughout the present specification and the accompanying claims the words

"comprise" and "include" and variations such as "comprises", "comprising", "includes" and "including" are to be interpreted inclusively. That is, these words are intended to convey the possible inclusion of other elements or integers not specifically recited, where the context allows.

The term "variant" as used herein refers to a polynucleotide which encodes the same amino acid sequence as another polynucleotide of the present invention but which, through the redundancy of the genetic code, has a different nucleotide sequence whilst maintaining the same codon usage pattern, for example having the same codon usage coefficient or a codon usage coefficient within 0.1 , for example within 0.05 of that of the other polynucleotide.

The term "codon usage pattern" as used herein refers to the average frequencies for all codons in the nucleotide sequence, gene or class of genes under discussion (e.g. highly expressed mammalian genes). Codon usage patterns for mammals, including humans can be found in the literature (see e.g. Nakamura et.al. Nucleic Acids Research 1996, 24:214-215).

The term "cluster" as used herein refers to phlogenetically related HPV types, for example as set out in the phylogenetic trees of figure 32 (HPV E1 ) and figure 33 (HPV E2).

Examples

Example 1

Sequence selection:

Protein sequences for E1 and E2 antigens of HPV 16, 18, 33, 56 and 51 were obtained from Los Alamos HPV database, with the exception of the HPV 18 E2 sequence which was obtained from the SwissProt database. The sequences obtained were codon-optimised (c/o) for human cell expression using the GSK Syngene software (version 4d).

The DNA and protein sequences for these antigens are set out in SEQ ID NO: 1-29.

Example 2

Polyprotein fusion strategy for HPV242 (HPV18 E1 and HPV16 E1 and HPV18 E2 and HPV16 E2)

Codon-optimised HPV18 E1 and HPV16 E1 and HPV18 E2 and HPV16 E2 genes were cloned into vector p7313 backbone. This backbone consists of a bacterial pUC19-based vector that has a rabbit globin poly adenylation signal, an enhanced human CMV immediate early promoter with exon-1 as an expression-enhancing element (ie) and a kanamycin resistance selection marker (p7313ie). The cloning sites are Notl and BamHI. A stop signal was introduced after the C terminal cloning site (BamHI), to enable cloning of multiple components of the polyprotein fusion (7313ies). A plasmid map is shown in Figure 1.

The N terminus of each gene was modified to accommodate the fusion cloning strategy. A BgIII site was engineered after the Not 1 site followed by two Glycine (G) motifs before the start of the sequence. The fusion was between BgIII of the second component e.g. HPV16E1 and BamHI site of the first component e.g. HPV18E1 as shown in Figure 2.

On fusion, a Glycine-Serine-Glycine-Glycine (GSGG) linker sequence was generated between each of the fusion components. Glycine (G) and Serine(S) are not charged and confer flexibility to protein structures. The junctions between each antigen are therefore artificial. Consequently each junction protein sequence between fused antigens was analysed for the introduction of neo-epitopes using bioinformatics tools including GenBank redundant peptide database and, RefSeq human peptide database. No significant neo-epitopes were detected. Example 3

E1 Mutation

To disable the replication function in E1 , a mutation at amino acid position 482 in HPV16E1 was introduced, where Glycine (G) was mutated to Aspartic Acid (D), G482D. This mutation occurred in a highly conserved ATP binding consensus sequence and E1 protein carrying this mutation has been shown to have multiple functional deficits such as in replication, ATP binding and helicase activity.

HPV16E1 , HPV18E1 , HPV56E1 and HPV33E1 genes were mutated at the equivalent position to HPV16E1 (amino acid 482) and cloned into the p7HS7313ies vector and sequence verified and checked for expression using His tag antibodies (see Figure 3). Once expression and sequence had been verified, each of these clones were transferred into 7313ies without the His Tag and checked for correct sequence. Additionally, expression of HPV 16 and HPV18 proteins were checked using antibodies specific to the HPV type.

Example 4

E2 Mutation

A specific mutation was introduced to disable each E2 antigen. Mutation of amino acid position 1 11 from Lysine (K) to Alanine (A) impaired replication function, nuclear localisation and cooperative origin of binding and transcriptional activity of HPV- 16E2. A similar Lysine to Alanine mutation was introduced into HPV-18, HPV-33, HPV-56 and HPV-51 at the equivalent position based on the sequence similarity to HPV-16E2. The mutated HPV-18, HPV-33, HPV-56 and HPV-51 E2 were each cloned into vector p7HS7313ies, DNA sequenced and protein expression confirmed by western blot using using His-tagged antibodies (see Figure 4). Once expression and sequence had been verified, each of these clones were transferred into 7313ies without the His Tag and checked for correct sequence. Additionally, expression of HPV16 and HPV18 proteins were checked using antibodies specific to the HPV type.

Example 5

Construction of a polynucleotide construct encoding polyprotein fusion HPV242 (HPV18 E1 and HPV16 E1 and HPV18 E2 and HPV16 E2)

HPV18E1 was digested Bam HI and Hindlll and HPV16E1 was digested BgIII and Hindi 11 using the fusion strategy set out in Example 2. The HPV18E1 BamHI- Hindlll fragment of 3359bp size was ligated to HPV16E1 BgIII- Hindlll fragment of 4034bp, transformed in JM109 cells and selection was via kanamycin. Minipreps were checked for correct clones by restriction enzymes and verified further via sequencing.

HPV18E2 was digested BamHI, CIaI and Dral and HPV16E2 was digested BgIII and CIaI. The HPV18E2 BamHI-Clal fragment of 2501 bp size was ligated to HPV16E2 BgIII-CIaI fragment of 3608bp, transformed in JM109 cells and selection was via kanamycin. Minipreps were checked for correct clones by restriction enzymes and verified further via sequencing.

The HPV18,16E1 fusion BamHI - MIuI (6954bp) and HPV18,16E2 fusion BgIII-MIuI (2582bp) fragments were then fused together and correct clones were checked via restriction enzymes and sequencing to obtain HPV242.

HPV242 was then checked for protein expression by transfecting 1 ug of DNA into HEK293T cells using Optimem and Lipofectamine 2000, allowing transfection of cells for 24hours and then harvesting for protein. HPV242 was Western blotted and detected using HPV16E2 mAb from OEM Concepts Catalogue No: 286-17261 ; HPV18E2 polyclonal Ab from OEM Concepts Catalogue No: SCB VN20; HPV16E1 mAb (in-house) and HPV18E1 polyclonal Ab (in-house). All primary antibody reactions were carried out overnight.

HPV242 expressed a protein of -245KdA as expected by the HPV18E1 and HPV18E2 Ab Western Blots (see figure 5). HPV16E1 and HPV16E2 gave similar results (data not shown).

Example 6

Construction of a polynucleotide construct encoding polyprotein fusion HPV275 (HPV33 E1 , HPV51 E2, HPV56 E2, HPV33 E2 and HPV56 E1 )

HPV33 E1 was digested with BamHI-HINDIII to give a fragment of 3322bp and was ligated to HPV51 E2 BgIII-HINDIII fragment (3158bp). This ligation was transformed using JM109 cells and selection was via kanamycin. Colonies were minipreped and checked via restriction enzymes. The correct HPV 33E1 , HPV 51 E2 clones were then digested BamHI-HINDIII to give a fragment of 4408bp. HPV56E2 was digested BgIII-HINDIII to give a fragment of 3185bp. These two fragments were ligated and transformed. Colonies minipreped after kanamycin selection gave correct clones of HPV33E1 , HPV51 E2, HPV56E2 using restriction enzymes. HPV33 E2 was digested using BamHI-HINDIII to give a fragment of 2449bp and this was ligated to HPV56 E1 BgIII-HINDIII fragment of 3992bp. The correct clones after transformation and selection via kanamycin were HPV33E2, HPV56E1. HPV33E1 , HPV51 E2, HPV56E2 was digested using BamHI-HINDIII to give a fragment of 5521 bp and it was ligated to HPV33E2, HPV 56E1 BgIII-HINDIII fragment of 3992bp. The correct clones after transformation and selection via kanamycin were referred to as HPV271 and they were checked via restriction enzymes and sequencing. This initial version (called HPV271 ) was histidine-tagged in p7HS7313ies and was subsequently transferred over to p7313ies as a Notl- BamHI fragment of 7142bp to create the plasmid HPV275.

Expression of HPV275 in HEK293 cells was checked via Western Blotting using a mixture of HPV16E1 , 16E2, 18E1 and 18E2 Abs since no specific antibodies were available for HPV33E1/E2, HPV56E1/E2 or HPV51 E2. Polyhistidine antibodies initially confirmed expression of HPV271 which prompted transfer into the non-his- tagged 7313ies vector. HPV275 was checked for expression by transfection on its own as well as in combination with HPV242. HPV275 should give a protein of -285KdA and HPV242 should give a protein of ~245KdA based on the size of the component proteins.

HPV275 gave a protein of the expected size, and HPV275 and HPV242 in combination gave two protein products of the correct size (see figure 6).

Other multivalent constructs encoding polyprotein fusions of HPV33 E1 , HPV33 E2, HPV51 E2, HPV56 E1 and HPV56 E2 were produced using a similar strategy to that outlined for HPV 275. These were HPV273 (HPV51 E2, HPV56 E1 , HPV56 E2, HPV33 E1 , and HPV33 E2), HPV274 (HPV56 E1 HPV33 E1 , HPV51 E2, HPV56 E2, HPV33 E2), and HPV276 (HPV51 E2, HPV56 E2, HPV33 E2, HPV56 E1 , HPV33 E1 ). Expression of the correct size polyprotein from these constructs was demonstrated in the same way as for H PV275.

Example 7 Other multivalent constructs were produced comprising of two plasmids, one with E1 genes and the other with E2 genes. These constructs had E1 genes from different HPV types on a single plasmid (HPV216) and E2 genes from different HPV types on a second plasmid (HPV204 and HPV263).

HPV216 (HPV18 E1 , HPV56 E1 , HPV33 E1 , HPV16E1 ) was constructed using the same strategy as for HPV242 and HPV275 as set out in the previous examples. 56El

The plasmid HPV216 was verified by sequencing the clone and expressed a protein of -312KdA as shown by the HPV16E1 and HPV18E1 Ab Western Blots (see figure 7).

Example 8

HPV204 (HPV18 E2, HPV31 E2, HPV51 E2, HPV39 E2, HPV56E2, HPV33 E2, HPV16E2) was constructed using the same strategy as for HPV242 and HPV275 as set out in the previous examples.

HPV204 was verified by sequencing and expressed a protein ~309KdA as shown by the HPV16E2 and HPV18E2 Ab Western Blots (see figure 8).

Example 9

HPV263 (HPV18 E2, HPV51 E2, HPV56E2, HPV33 E2, HPV16 E2) was constructed by removal of HPV31 E2 and HPV39E2 from HPV204.

HPV263 was verified by sequencing and gave a protein upon expression of -219KdA.

Example 10

Mouse Immunology Data For HPV242

HPV242 was tested for immunogenicity in mice following immunisation using particle mediated epidermal delivery technology (PMED). For comparative purposes vectors expressing HPV16 E1 , HPV16 E2, HPV18 E1 and, HPV18 E2 (single protein expression vectors) were evaluated alongside HPV242. Cellular immune responses were measured using IL-2 and IFN-gamma ELIspot assays. HPV242 was associated with high levels of cellular immune responses. Overall, the level of immunogenicity recorded to individual antigens following immunization of mice with HPV242 was marginally lower compared to that invoked by plasmids expressing each of the single proteins individually. The response to this plasmid was enhanced by topical application of imiquimod 24 hours after immunization.

All of the murine in vivo studies described were carried out using the Balb/c (H2^d) inbred strain of mice. PMED was used for delivery of plasmid DNA.

The interferon-gamma (IFN-gamma ELIspot was selected as the main assay for investigation of specific cellular immune responses, as the secretion of IFN-gamma can be used as a surrogate for cytotoxicity (Michel 2002). Peptides (of 9 amino acids) corresponding to two previously published CD8 epitopes in HPV-16 E1 (Tobery 2001 ) were obtained and their activity confirmed (see Peptide library screen method below). Additional new peptide reagents for use in the evaluation of murine cellular immune responses to other HPV antigens were identified in Balb/c mice using peptide library screening. Reagent activity was confirmed using IFN-gamma and IL-2 ELIspot assays and peptides were further characterized using flow cytometry. CD8 cytotoxic T cell epitopes predominantly induce IFN-gamma secretion from activated cells, whereas CD4 helper T cells tend to induce IL-2 release.

Peptide Library Screen Method: 15-mer peptide sequences overlapping by 1 1 , for each HPV antigen (HPV16E1 , HPV16E2, HPV18E1 , and HPV18E2) were sourced and screened in an interferon- gamma ELISpot against splenocytes from Balb/C mice immunised with the homologous antigen DNA construct. Peptides which produced a cellular immune response were subsequently used as reagents in further studies.

Experiments were carried out to investigate the immunogenicity induced by HPV242 in Balb/c mice. Immunogenicity to the four encoded viral antigens was compared with that of animals immunized with plasmids encoding each of the four individual antigens. During the course of these murine immunology studies three separate experiments were undertaken.

Figure 10 shows the mean responses to HPV peptide library pools by IFN-gamma ELIspot by splenocytes isolated from PMED immunized mice at 1 1 days post-primary immunization. Mice were cluster primed (1 μg doses on each of 3 alternate days) using PMED with the indicated vector and spleens harvested 11 days later. Isolated splenocytes were incubated overnight in an IFN-gamma ELIspot assay. The strongest IFN-gamma response was observed following re-stimulation with HPV-18 E1 peptide 401 , which we have shown by ICS to be a CD8 epitope. Figure 1 1 shows the mean responses to HPV peptide library pools by IL-2 ELIspot by splenocytes isolated from PMED immunized mice at 1 1 days post-primary immunization. Mice were cluster primed (1 μg doses on each of 3 alternate days) using PMED with the indicated vector and spleens harvested 11 days later. Isolated splenocytes were incubated overnight in an IL-2 ELIspot assay. With the exception of HPV-16 E1 CD8 peptide (pep2), the number of splenocytes releasing IL-2 isolated from the HPV242 immunized animals was shown to be greater than, or equal to the number invoked by immunization of mice with plasmids encoding expressing proteins. Peptide 2 has previously been shown to be a H2^d restricted HPV-16 E1 CD8 epitope (Tobery 2003).

The results confirm that HPV242 is immunogenic in Balb/c mice and immune responses to all four antigens can be detected. When the results for HPV242 immunized animals were compared with those for splenocytes isolated from mice immunized with plasmids encoding each of the individual genes, the overall trend was for a reduction in magnitude of responses for the animals immunized with four antigens compared to those receiving a single antigen. In some cases significantly greater or equal numbers of IFN-gamma or IL-2 releasing cells were detected from the HPV242 immunized animals when results were compared with those from mice immunized with plasmids encoding each of the individual genes. However, in a number of cases the responses to individual antigens from the HPV242 immunized animals were lower than those recorded for animals immunized with the corresponding single antigen. Taken together the immunology data in Balb/c mice indicate that level of cellular immune response elicited by immunization with HPV242 is marginally lower compared with the magnitude of the cellular immune response raised against the individually expressed gene products. Interestingly some cross- reactivity was also observed. For example, splenocytes from animals immunized with HPV-16 E2 produced both IFN-gamma and IL-2 following re-stimulation with peptide 524 (which we have shown is a mixed HPV-18 E2 CD4/CD8 epitope) and to a lesser extent with peptide 337 (a CD4 epitope from HPV 16 E2).

Most of the in vivo studies were carried out using a stock of HPV242 that was subsequently found to contain a single point mutation in the plasmid vector backbone. This point mutation was corrected in plasmid HPV242mk2. The location of the point mutation in the backbone of the plasmid was not expected to have any effect on the immunogenicity of the plasmid inserts, but nevertheless a study was carried out to compare the immunogenicity of HPV242 and HPV242mk2. The results indicated that there were no significant differences between the numbers of spots recorded by either the IFN-gamma or IL-2 ELIspots (data not shown). Therefore immunogenicity results from experiments using either plasmid were valid. Example 11

In vivo Cytotoxicity assay

In order to demonstrate the killing efficacy of specific cytotoxic T-cells (CTLs) generated in animals immunised with HPV242 an in vivo CTL assay was undertaken. Naϊve splenocytes from un-immunised animals were isolated and labelled by incubating with either a low or a high concentration of a fluorescent green dye (CFSE). The cells labelled with the high concentration of CFSE were loaded by incubating with Peptide 2. CFSE high + Peptide 2 and CFSE low labelled cells were mixed in equal proportions and adoptively transferred into animals that had previously been primed and boosted with HPV242 using PMED. Twenty hours after the labelled cells had been intravenously injected, blood from the recipient animals was collected and the ratio of high versus low CFSE labelling in the recovered cells was investigated using flow cytometry. If no epitope specific lysis had occurred it would be expected that the ratio of high to low labelled cells would remain constant (i.e. cell numbers would remain approximately equal). However, if one of the subsets of cells had been selectively lysed the ratio would be skewed in favour of the cells that had not been incubated with peptide (low CFSE labelling). The results (see Figure 12) indicate that fewer of the cells that had been incubated with peptide (high CFSE) could be recovered from the immunised animals indicating that these cells had been selectively killed in vivo.

Results indicate that in vivo killing had occurred in the HPV242 immunized animals but not in control (empty vector, p7313ies) immunized mice. Splenocytes were harvested from naϊve Balb/c mice and labelled with either high or low levels of CFSE. The high CFSE labelled population were loaded with Peptide 2. High (+ peptide) and low CFSE labelled cells were mixed in equal proportions and adoptively transferred into mice that had previously been immunized with either HPV242 or p7313ies (empty vector). Twenty hours later, PBMC were isolated and levels of CFSE fluorescence evaluated using flow cytometry. A reduction in the proportion of high CFSE-labelled cells compared with the low labelled cells indicates specific in vivo killing has occurred.

Example 12

Enhancement of the PMED response in mice using imiquimod

Investigations were carried out to determine whether imiquimod could enhance the magnitude of specific cellular responses elicited following immunization of Balb/c mice with HPV242. Mice were immunized using a pulse (2 x 1 μg HPV242 DNA) dosed onto a shaved area of abdomen. On the following day, selected mice were treated with topical imiquimod. A 20 μl volume of imiquimod cream was rubbed into each immunisation site, until the cream was no longer visible. At various times post- primary immunisation, animals were killed and splenocytes were isolated and incubated overnight in IFN-gamma and IL-2 ELIspot assays. Data from one experiment is shown in Figure 13 where mice were immunised using a pulse regimen (2 doses x 1 μg) and HPV242 (research device at 400 p.s.i). 24 hours later one group was dosed by rubbing a 20 μl volume of Imiquimod (5% imiquimod) onto each immunization site. Animals were culled 14 days later, splenocytes isolated and an IL-2 ELIspot assay undertaken.

The responses of the HPV242 Imiquimod treated mice are higher than the responses of the HPV242 treated mice indicating that Imiquimod was exerting an adjuvant effect by increasing the magnitude of the specific cellular immune response.

Example 13 Minipig Immunology Data For HPV242

The immunogenicity of HPV242 was also tested in minipigs (in the presence or absence of imiquimod as adjuvant) and specific interferon-gamma responses were detected at 2 weeks post-primary immunization. One week after a boost (at 9 weeks post-primary) cellular immune responses, detected by ELIspot assay, indicated that the numbers of porcine cells releasing IFN-gamma can be boosted by subsequent immunizations and show a trend consistent with imiquimod exerting adjuvant effects. The results also confirmed that minipigs can mount responses to all four HPV genes coded by HPV242 plasmid. The minipig data also yielded evidence that HPV242 immunized animals may exhibit a level of cross-reactivity with at least one other high risk HPV HPV type (HPV-33).

Vector HPV242 was evaluated for immunogenicity in the Gottingen minipig, an outbred large animal species (5 - 40 kg) with skin that is structurally similar to that of humans. Gottingen minipigs were obtained from Ellegaard, Denmark, and were allowed to acclimatise for at least 2 weeks before the start of the study.

Twelve minipigs each received 8 doses of 0.5 μg DNA delivered to the ventral abdomen using a Research Device at 500 p.s.i. helium. Twenty four hours later, six minipigs had 20μl of imiquimod cream rubbed into each immunization site, until the cream was no longer visible. Blood samples were collected from each animal at 2 weeks post-primary and peripheral blood mononuclear cells (PBMC) isolated. The immune response to the antigens encoded by HPV242 was evaluated using an IFN- gamma ELIspot assay. The results (see Figure 14) indicate that specific responses to HPV16 E1 , HPV18 E1 and HPV18 E2 antigens could be detected at 2 weeks postprimary. The results also show a trend (that did not reach statistical significance) for the animals receiving imiquimod to have higher responses than those which were not treated with imiquimod. This observation is consistent with imiquimod acting as an adjuvant to PMED immunization.

The results indicate responses (above background) to the three amino terminal antigens coded by HPV242.

Animals were boosted 8 weeks after the primary immunization. IFN-gamma ELIspot data at this time point indicate that all responses had returned to resting levels, with a mean of less than 50 spots. Animals were boosted using the Research device and imiquimod was applied 24 hours later as described above. One week later PBMC were isolated and IFN-gamma ELIspot assays undertaken (results are shown in figure 15).

The results shown above indicate that the magnitude of the cellular immune responses had boosted at 1 week after the second immunization and, were at least 3-fold higher than the levels recorded at 2 weeks post-primary. The responses for several animals were too numerous to count accurately as some of the spots had merged, so these were assigned an arbitrary value of 750 spots/million PBMC. The trend for enhanced responses in the imiquimod treated group that had been observed 2 weeks after primary immunization was still evident 1 week post-boost. HPV242 only encodes HPV-16 and 18 E1 and E2 genes. However, a response to HPV-33 E1 above background levels was also recorded, suggesting that a level of cross-reactivity with a related HPV type was occurring.

Example 14

Murine data comparing multivalent DNA constructs

An experiment was set up to compare the immune responses in groups of 6 Balb/C mice immunized by PMED with the following plasmids:

• HPV273 (51 E2/56 E 1/56 E2/33 E 1/33 E2)

• HPV274 (56 E1/33 E1/51 E2/56 E2/33 E2)

• HPV275 (33 E1/51 E2/56 E2/33 E2/56 E1 )

• HPV276 (51 E2/56 E2/33 E2/56 E 1/33 E1 )

• HPV242 (18 E1/16 E1/18 E2/16 E2)

• p7313 (Empty vector)

Mice received a cluster primary immunization consisting of 3 doses of -1 μg plasmid each, administered by PMED (Research Device, 400 p.s.i. helium) on days 0, 2 and 4. A twenty microlitre volume of Imiquimod was rubbed into each immunization site at 24 hours post- each PMED immunization. Splenocytes were isolated 6 days post final immunization dose (day 4), and restimulated overnight in the presence of specific HPV peptide pools (the entire sequence of each antigen was represented as a pool of 15-mer peptides, overlapping by 11 amino acids) and subsequently evaluated using IFN-gamma and IL-2 ELIspot assays (Figures 16 and 17).

Results demonstrated that constructs HPV273 and HPV276 produced lower overall interferon-g responses than HPV274 and HPV275 (Figure 8). The same trend was observed for IL-2 responses where the lower cellular responses were to HPV273 and HPV276 (Figure 9). The results from the interferon-gamma ELISpot (Figure 16) demonstrated that

HPV274 and HPV275 both produced equivalent cellular responses. However, in the IL-2 ELISpot (Figure 17), HPV275 demonstrated slightly higher responses to 33E1 , 33E2 and 56E2.

Example 15 lmmunogenicity of Multivalent vector HPV242mk2 and HPV275.

Multivalent vectors HPV242mk2 and HPV275 (multivalent V2) were evaluated in a murine study to demonstrate immunogenicity following primary immunisation. Balb/C mice were immunised using a cluster protocol (1.0 μg co-coated DNA delivered to the abdomen using a PowderMed Research Device at 400 p.s.i. helium on days 0, 2, 4) and spleens harvested 11 days after the first dose.

Results from the interferon-gamma ELISpot (Figure 18) demonstrated good multivalent V2 cellular immune responses (predominantly CD8 T cells) to HPV18E1 ,

56E1 and 51 E2 antigens. The IL-2 ELISpot assay (Figure 19) demonstrated that following administration of HPV multivalent V2, good cellular immune responses

(predominantly CD4 T cells) to all 5 antigens present in HPV275 were detected. In the HPV242mk2 alone group there were no responses to HPV33E2, 56E1 , 56E2 and 51 E2 antigens. However, a cross-reactive immune response was observed to

HPV33E1 antigen (which was not represented in the HPV242mk2 plasmid).

Example 16 Evidence of cross-reactive immune responses to HPV types not represented in HPV242mk2 and HPV275.

Possible cross-reactive immunity was investigated following primary immunisation of mice with the multivalent V2 vector, by restimulating splenocytes from immunized animals in the presence of HPV45E1 and 45E2 antigens (HPV types not represented in the multivalent V2) in IFN-g and IL-2 ELISpot assays. Balb/C mice were immunised using a cluster protocol (1.0 μg co-coated multivalent DNA delivered to the abdomen using a PowderMed Research Device at 400 p.s.i. helium on days 0, 2, 4) and spleens harvested 1 1 days after the first dose. The strong cellular immune responses to HPV 45 E1 detected indicate that cross reactive cellular immune responses (against other HPV types) can be demonstrated in mice (Figures 20 and 21 ). Weaker responses to HPV 33E1 were detected in animals immunized with HPV242mk2 (Figures 20 and 21 ). A construct that only encodes HPV16 and HPV18 E1 and E2 genes gave a response to HPV33 E1 above background levels indicating that a level of cross-reactivity with a related HPV type was occurring.

Example 17

In vivo Cytotoxicity assay

A therapeutic vaccination would need to elicit cells that had functional capacity to kill virally infected cells. In order to demonstrate the killing efficacy of specific cytotoxic T-cells (CTLs) generated in animals immunised with the HPV multivalent V2, an in vivo CTL assay was undertaken. Naϊve splenocytes from unimmunised animals were isolated and labelled by incubating with either a low or a high concentration of a fluorescent green dye (CFSE). The cells labelled with the high concentration of CFSE were loaded by incubating with Peptide 2, a 9-mer HPV 16E1 peptide (Tobery 2001 ). CFSE high + Peptide 2 and CFSE low labelled cells were mixed in equal proportions and adoptively transferred into animals that had previously been primed and boosted with HPV multivalent V2 using PMED. Twenty hours after the labelled cells had been intravenously injected, blood from the recipient animals was collected and the ratio of high versus low CFSE labelling in the recovered cells was investigated using flow cytometry. If no epitope specific lysis had occurred it would be expected that the ratio of high to low labelled cells would remain constant (i.e. cell numbers would remain approximately equal). However, if the subset of cells that had been incubated with the peptide (high CFSE) had been selectively lysed the ratio would be skewed in favour of the cells that had not been incubated with peptide (low CFSE labelling). The results (illustrated in Figure 22 below) indicate that fewer of the cells that had been incubated with peptide (high CFSE) could be recovered from the immunised animals, demonstrating an average of 24% of specific HPV16E1 labelled cells had been selectively killed in vivo (values calculated by subtracting the non-specific percentage lysis from the specific percentage lysis).

Results indicate that in vivo killing had occurred in the HPV multivalent immunized animals but not in control (empty vector, p7313ies) immunized mice. Splenocytes were harvested from naϊve Balb/c mice and labelled with either high or low levels of CFSE. The high CFSE labelled population were loaded with Peptide 2. High (+ peptide) and low CFSE labelled cells were mixed in equal proportions and adoptively transferred into mice that had previously been immunized with either HPV242mk2 or p7313ies (empty vector). Twenty hours later, PBMC were isolated and levels of CFSE fluorescence evaluated using flow cytometry. A reduction in the proportion of high CFSE-labelled cells compared with the low labelled cells indicated that specific in vivo killing had occurred. The HPV multivalent V2 dosed individual shown in Figure 15, demonstrated a specific killing value of 30% compared with the control individual where no specific cell killing had occurred. This demonstrates functional activity of the cells elicited by immunization with HPV multivalent V2.

Example 18

Investigation of immunogenicity of two versions of HPV multivalent construct in minipigs

An experiment was set up to compare the immune responses in groups of 5 minipigs immunized by PMED with the following plasmids:

HPV Bivalent plasmid

(HPV242: 18 E1/16 E1/18 E2/16 E2)

Multivalent Version 1 : HPV Multivalent (1 : 1 ratio) (HPV216: 18 E1/56 E1/33 E1/16 E1 + HPV263: 18 E2/51 E2/56 E2/33 E2/16 E2)

Multivalent Version 2: HPV bivalent plasmid + partner (1 : 1 ratio)

(HPV242: 18 E1/16 E1/18 E2/16 E2 + HPV275: 33 E1/51 E2/56 E2/33 E2/56 E1 ).

Minipigs received a prime and a boost immunization with a 4 week interval. Each immunization consisted of 4 doses of -1 μg plasmid administered by PMED (Research Device, 500 p.s.i. helium). A twenty microlitre volume of Imiquimod was rubbed into each immunization site at 24 hours post-PMED. Blood samples were obtained from each animal on the day of each immunization, at 2 weeks post-primary and at 1 and 2 weeks post-boost. The peripheral blood mononuclear cells (PBMC) were isolated and restimulated overnight in the presence of specific HPV peptide pools in IFN-g ELIspot assays.

Two weeks after the primary immunization low level responses were detected by IFN-gamma ELIspot for the Bivalent (HPV242) and the Bivalent + partner (HPV242 + HPV275) groups. No responses were detected above background for Multivalent Version 1 (HPV216 + HPV263) group. (The results for the Bivalent and Multivalent Version 1 were of similar levels to those detected in a previous experiment (see Figure 14)). Mean responses to HPV16 E1 peptide library pools over time by IFN- gamma ELIspot are shown in Fig 23. Mean responses to HPV56 E2 peptide library pools over time by IFN-gamma ELIspot are shown in Fig 24. Figure 25 shows mean responses to HPV peptide library pools at 6 weeks post-primary immunization (2 weeks post-boost). The responses in all groups were enhanced by the boost immunization (carried out at Week 4) and mean spot forming cell numbers were roughly equivalent between the two groups immunized with the different Versions of the multivalent plasmids. However, the individual results indicated that the five minipigs immunized with the Bivalent + partner combination (HPV242 + HPV275) gave an unusually consistent response across the group. By comparison, the

Multivalent Version 1 group had one very high responder and four low responders. Both versions of the multivalent plasmid give responses to the antigens that are not present in the bivalent plasmid (results for HPV 56 E2 are shown). A level of cross reactivity with HPV types not present in the plasmids used to immunize the animals was also detected as responses to HPV 45 E1 and E2 were detected (data for shown in the individual graph at 1 week post boost).

Example 19

Adjuvant Effects of Imiquimod

Groups of 6 minipigs (3 females + 3 males) were immunized with either Multivalent V1 (HPV216 + HPV263) or Bivalent (HPV242mk2) constructs. (The results of the previous experiment had indicated that after a boost the responses from animals immunized with Multivalent V2 were equivalent or better to those from animals immunized with Multivalent V1 ). The immunizations were carried out using a PowderMed Research Device (500 p.s.i. helium) at 8 week intervals. Animals received a total of 5 immunizations (one prime and four boosts). At 24 hours after each immunization selected groups of animals had a 20μl volume of Imiquimod (5% Imiquimod cream) rubbed into each immunization site, until the cream was no longer visible. Blood samples were collected from each animal at various times pre- and post-immunization and peripheral blood mononuclear cells (PBMC) isolated. The cellular immune response to the antigens encoded by the vectors (the entire sequence of each antigen was represented as a pool of 15-mer peptides, overlapping by 1 1 amino acids) was evaluated using IFN-gamma ELIspot assays. The results indicate that there is an increase in the magnitude of the cellular responses recorded for the minipigs treated with Imiquimod 24 hours after each dosing occasion compared with the animals that received PMED alone (Figure 26). The increases were statistically significant on Weeks 33 and 34 (1 and 2 weeks post- fourth boost) for all groups with Imiquimod, compared to those without. These results indicate that Imiquimod was acting as an adjuvant and enhancing the magnitude of the specific cellular immune responses.

Some of the remaining animals were given an additional PMED boost and have their responses on the day of boost and 1 week post-boost were investigated. Three groups of 3 female animals were boosted: Bivalent 4 wk, Bivalent 4 wk + Imiquimod and the Multivalent Version 1 8 wk + Imiquimod. The Bivalent groups received p242 (18 E1-16E1-18E2-16E2) and the multivalent version 1 received a mixture of two plasmids coding for 16 E1 + E2, 18 E1 + E2, 33 E1 + E2, 51 E2 and 56 E1 + E2. The animals had already received a prime and four boosts at either 4 (Bivalent) or 8 (Multivalent V1 ) intervals. The minipigs were rested for 19 weeks before being boosted (Week 55). The Week 55 and Week 56 (1 week post-boost) ELIspot data are shown in Figs 28-31.

Owing to the size of the animals they had to be sedated prior to bleeding and this may have contributed to higher backgrounds than usual. The peptide libraries for HPV31 E1 and HPV31 E2 were tested at the time of the boost and high numbers of IFN-g secreting cells were recorded in several animals.

Cross-reactive Cellular Immune Responses:

The strong cellular immune responses to HPV45E1 (detected in all immunized groups), indicates that cross reactive cellular immune responses (against other HPV types) can be demonstrated in the minipig. The HPV45 type was not present in any of the vaccine constructs (Figure 27). Weaker responses to HPV33E1 were detected in animals immunized with HPV242mk2, a construct that only encodes HPV16E1 , HPV18E1 , HPV16E2 and HPV18E2 genes. The response to HPV33 E1 (above background levels) indicated that a level of cross-reactivity with a related HPV type was occurring.

Data was also generated using the Bivalent construct (HPV242mk2) to show that there was no statistical difference between the results after the final boost, for the 4 week and 8 week schedules of dosing .

Example 20

Sequence alignments of the HPV types within each of the clusters were put together. These are set out in Figures 34 to 40.

Predictions of various HLA epitopes were identified by deriving a consensus prediction from four epitope programs Syfpeithi, HLS peptide binding, Predep and Rankpep. These are set out in figures 41 to 44. Brief Description of Figures:

Figure 1 : Vector map of p7313ies. Genes can be cloned between the Notl (N- terminal) and BamHI (C-terminal) restriction sites.

Figure 2: Cloning strategy for fusing several E1 and E2 genes together using BgIII and BamHI sites.

Figure 3: Western blot of protein expression of 7HS7313ies HPV16E1 ; HPV18E1 ; HPV33E1 and HPV56E1 using His-tagged antibodies.

Figure 4: Western blot of protein expression of 7HS7313ies HPV16E2; HPV18E2; HPV33E2; HPV31 E2; HPV39E2 and HPV56E2 using His-tagged antibodies.

Figure 5: Western blot of protein expression of HPV242 using antibodies against HPV18E2 (Fig 5a) and HPV18E1 (Fig 5b).

Figure 6: Western blot of protein expression of HPV275, HPV273, HOV274, HPV276, HPV242, and the mixtures of HPV275 + HPV242, HPV273 + HPV242, HPV274 + HPV242, HPV276 + HPV242 using antibodies against HPV16E1 , HPV18E1 , HPV16E2 and HPV18E2.

Figure 7: Western blot of protein expression of HPV216 and HPV16E1 and HPV18E1 using antibodies against HPV16E1 and HPV18E1 respectively.

Figure 8: Western blot of protein expression of HPV204 and HPV16 E2 and HPV18 E2 using antibodies against HPV16 E2 (Fig 8a) and HPV18 E2 (Fig 8b).

Figure 9: Western blot of protein expression of HPV263 and HPV204 using antibodies against HPV16E2 (Fig 9a) and HPV18E2 (Fig 9b).

Figure 10: Mean responses to HPV peptide library pools by INF-gamma ELIspot by splenocytes isolated from PMED immunized mice at 1 1 days post-primary immunization with HPV p242.

Figure 1 1 : Mean responses to HPV peptide library pools by IL-2 ELIspot by splenocytes isolated from PMED immunized mice at 1 1 days post- immunization with HPV p242.

Figure 12: Flow cytometry histograms showing representative populations of CSFE- labelled cells recovered from mice after 20 hours. Fig 12a sets out the CFSE fluorescence of PBMC harvested at 20 hours after transfer of peptide-pulsed donor splenocytes from mice immunised with HPV242 and Fig. 12b sets out the CFSE fluorescence of PBMC harvested at 20 hours after transfer of peptide-pulsed donor splenocytes from empty vector- immunised mice Figure 13: Mean responses of Balb/c mice to HPV specific peptides by IL-2 ELIspot at 14 days post-primary immunization with HPV242

Figure 14: Mean responses to HPV peptide library pools by IFN-gamma ELIspot at 2 weeks post primary immunisation with HPV242.

Figure 15: Mean responses to peptide library pools (15-mers overlapping by 11 ) covering the sequence of each of six different HPV antigens at 9 weeks post-primary (1 week post-boost) by IFN-gamma ELIspot. (Note that the scale of the graph for the post-boost results is higher than for the post-primary results set out in figure 14 - 700 compared with 100).

Figure 16: Results from HPV242mk2 partner selection study. Balb/C mice were immunised using a cluster protocol and splenocytes were isolated 11 days post first prime dose. Cellular immune responses determined by interferon-gamma ELISpot.

Figure 17: Results from HPV242mk2 partner selection study. Balb/C mice were immunised using a cluster protocol and splenocytes were isolated 11 days post first prime dose. Cellular immune responses were determined by IL-2 ELISpot.

Figure 18: Results from an HPV Multivalent V2 administration study, where Balb/C mice were immunised using a cluster protocol. Splenocytes were isolated 1 1 days post first prime dose and cellular immune responses determined by interferon- gamma ELISpot. Results demonstrated that HPV275 induced good interferon- gamma responses (predominantly CD8 T cell responses) to HPV51 E2 and HPV56E1 antigens expressed by the vector. HPV242mk2 alone did not induce an interferon- gamma immune response to any of the antigens expressed by HPV275 vector.

Figure 19: Results from the HPV Multivalent V2 administration study, where Balb/C mice were immunised using a cluster protocol. Splenocytes were isolated 1 1 days post first prime dose and cellular immune responses determined by IL-2 ELISpot. Results demonstrated that HPV275 induced good IL-2 responses (predominantly CD4 T cell responses) to all 5 antigens expressed by the vector. HPV242mk2 alone did not induce an immune response to HPV33E2, HPV56E1 , HPV56E2 and HPV51 E2. However, a cross-reactive response to HPV33E1 was observed in the HPV242mk2 immunized mice. (This antigen is not present in the plasmid).

Figure 21 : Results from the HPV Multivalent V2 administration study. Balb/C mice were immunised using a cluster protocol, splenocytes were isolated 11 days post first prime dose and cellular immune responses determined by IL-2 ELISpot. Similar to the results observed for interferon-gamma responses, results demonstrated that mice immunised with either HPV242mk2 or multivalent V2 (HPV242mk2+HPV275) produced good cross-reactive cellular responses to HPV45E1 , an antigen from an HPV type not represented in either vector.

Figure 22: Flow cytometry histograms showing representative populations of CSFE- labelled cells recovered from mice after 20 hours. Fig 22a sets out the CFSE fluorescence of PBMC harvested at 20 hours after transfer of peptide-pulsed donor splenocytes from mice immunised with HPV Multivalent V2 and Fig. 22b sets out the CFSE fluorescence of PBMC harvested at 20 hours after transfer of peptide-pulsed donor splenocytes from empty vector- immunised mice

Figure 23: Mean responses to HPV16 E1 peptide library pools over time by IFN- gamma ELIspot.

Figure 24: Mean responses to HPV56 E2 peptide library pools over time by IFN- gamma ELIspot.

Figure 25: Mean responses to HPV peptide library pools at 6 weeks post-primary (2 weeks post-boost. Each bar represents Mean of 5 samples (each tested in triplicate) +/- 1 Standard Error.

Figure 26: Mean responses to HPV peptide library pools by IFN-gamma ELIspot at 34 weeks post primary immunisation (2 weeks post-4^th boost).

Figure 27: Individual responses to HPV 45 peptide library pools by IFN-gamma ELIspot at 33 weeks post primary immunisation (1 week post 4^th boost).

Figure 28: Mean responses to HPV E1 peptide library pools by IFN-gamma ELIspot 55 weeks post primary immunisation (day of 5^th boost).

Figure 29: Mean responses to HPV E2 peptide library pools by IFN-gamma ELIspot

55 weeks post primary immunisation (day of 5^th boost).

Figure 30: Mean responses to HPV E1 peptide library pools by IFN-gamma ELIspot

56 weeks post primary immunisation (1 week post-5^th boost).

Figure 31 : Mean responses to HPV E2 peptide library pools by IFN-gamma ELIspot 56 weeks post primary immunisation (1 week post-5^th boost).

Figure 32: Phylogenetic tree for HPV E1 genes.

Figure 33: Phylogenetic tree for HPV E2 genes.

Figure 34: HPV E1 - cluster 1. HPV31 and HPV35 E1 sequences demonstrate 75 and 72% sequence identity with HPV16 E1 , respectively. Figure 35: HPV E1 - cluster 2. HPV39, HPV45 and HPV59 E1 sequences demonstrate 68, 83 and 71% sequence identity with HPV18 E1 , respectively. Figure 36: HPV E1 - cluster 3. HPV52 & HPV58 E1 sequences demonstrate 72 & 86% sequence identity with HPV33 E1 , respectively

Figure 37: HPV E1 - cluster 4. HPV51 E1 sequence demonstrates 58% identity with HPV56 E1

Figure 38: HPV E2 - cluster 1. HPV31 and HPV35 E2 sequences demonstrate 61 and 59% sequence identity with HPV16 E2, respectively

Figure 39: HPV E2 - cluster. HPV39, HPV45 & HPV59 E2sequences demonstrate 50, 72 & 46% sequence identity with HPV18 E2, respectively

Figure 40: HPV E2 - cluster 3. HPV52 & HPV58 E2 sequences demonstrate 56 & 72% sequence identity with HPV33 E2, respectively Figure 41 : HPV16 E1 regions with predicted HLA-A0201 binding affinity Figure 42: HPV16 E2 regions with predicted HLA-A0201 binding affinity

Figure 43: HPV16 E1 regions with predicted HLA-B7 binding affinity

Figure 44: HPV16 E1 regions with predicted HLA-A1 binding affinity

Figure 45: Sequence Listings (see Table 1 )

Table 1:

Claims

1. A polynucleotide sequence which encodes a polypeptide sequence comprising at least two HPV Early antigens or fragments thereof, said polypeptide being capable of raising an immune response to HPV types selected from 18, 45, 56, 39, 16, 31 , 35, 33, 58, 52, 51 , 68, 82, 73 and 59 when administered in vivo, wherein at least one of the HPV types against which an immune response is capable of being raised is not encoded by the polynucleotide sequence.

2. A polynucleotide sequence which encodes a polypeptide sequence comprising a first HPV type early antigen or fragment thereof from one HPV cluster and a second HPV type early antigen or fragment thereof from a different HPV cluster said polynucleotide being capable of raising an immune response to more than one HPV type within each of the clusters when administered in vivo, wherein at least one of the HPV types being treated is not encoded by the polynucleotide sequence.

3. A polynucleotide sequence according to claim 1 or 2 wherein the polynucleotide sequence encodes an amino acid sequence comprising HPV 16 and 18 early antigens or fragments thereof.

4. A polynucleotide sequence according to claim 1 or 2 wherein the polynucleotide sequence encodes an amino acid sequence comprising two or more of HPV 33, HPV 51 , HPV 51 and HPV56 early antigens or fragments thereof.

5. A polynucleotide sequence as set out in SEQ ID NO: 23.

6. A polynucleotide sequence encoding the polyprotein set out in SEQ ID NO 28 or 29.

7. An expression vector comprising a polynucleotide sequence according to any preceding claim operably linked to a control sequence which is capable of providing for the expression of the polynucleotide sequence in a host cell.

8. An expression vector according to claim 7 wherein the control sequence is a CMV promoter.

9. An expression vector according to claim 7 or 8 comprising one or more expression cassettes.

10. An expression vector according to any one of claim 7 to 9 encoding the polyprotein encoded by the polynucleotide set out in SEQ ID NO: 23.

11. An expression vector according to any one of claim 7 to 9 encoding the polyprotein encoded by the polynucleotide set out in SEQ ID NO: 24.

12. An expression vector according to claim 1 1 comprising two expression cassettes, the first expression cassette comprising the polynucleotide sequences set out in SEQ

ID NO: 23 and the second expression cassette comprising the polynucleotide sequences set out in SEQ ID NO: 24, each of which are operably linked to a promoter capable of driving expression.

13. A host cell comprising a polynucleotide sequence according to any one of claims 1 to 6, or an expression vector according to any one of claims 7 to 12.

14. A pharmaceutical composition comprising a polynucleotide sequence according to any one of claims 1 to 6.

15. A pharmaceutical composition comprising one or more expression vectors according to any one of claims 7 to 12.

16. A pharmaceutical composition according to claim 14 or 15 which further comprises one or more adjuvants.

17. A pharmaceutical composition according to claim 16 wherein the adjuvant is a TLR 7 agonist.

18. A pharmaceutical composition according to claim 17 wherein the adjuvant is imiquimod.

19. A pharmaceutical composition according to claim 16 in which the adjuvant is encoded as a fusion with the HPV polypeptide encoded by the polynucleotide.

20. A pharmaceutical composition according to claim 19 wherein the adjuvant is GM- CSF.

21. A pharmaceutical composition according to any one of claims 14 to 20 comprising a plurality, gold particles, coated with the polynucleotides set out in any one of claims 1 to 6.

22. A pharmaceutical composition according to claim 21 wherein the expression vectors of claims 10 and 1 1 are co-coated onto a plurality of gold beads.

23. The use of a polynucleotide according to any one of claims 1 to 6 in the treatment or prophylaxis of an HPV infection.

24. The use of a vector according to any one of claims 7 to 12 in the treatment or prophylaxis of a HPV infection.

25. The use of a composition according to any one of claims 13 to 22 in the treatment or prophylaxis of an HPV infection.

26. The use of a polynucleotide according to any one of claims 1 to 6, a vector according to any one of claims 7 to 12 or a pharmaceutical composition according to any one of claims 13 to 22 in the treatment or prophylaxis of cervical dysplasia, cervical intraepithelial neoplasia (CIN), cervical cancer, vulval intraepithelial neoplasia (VIN), vaginal intraepithelial neoplasia (VAIN), anal intraepithelial neoplasia (AIN) or associated cancers.

27. Use of a composition comprising a polynucleotide sequence encoding human HPV early antigen or fragments thereof of at least two different HPV types in the manufacture of a medicament for the treatment of HPV infection by HPV types selected from 18, 45, 56, 39, 16, 31 , 35, 33, 58, 52, 51 and 59, wherein at least one of the HPV types being treated is omitted from the composition.

28. Method of generating an immune response against more than one HPV type by administration of a composition comprising a polynucleotide encoding at least one HPV early antigen or fragment thereof from each HPV cluster wherein at least one of the HPV types against which an immune response is generated is omitted from the composition.

29. Method according to claim 24 wherein the immune response is generated against three or more of the HPV E1 and or E2 types 18, 45, 56, 39, 16, 31 , 35, 33, 58, 52,

51 and 59.

30. Method according to claim 29 wherein the immune response is generated against at least HPV16, HPV18 and HPV45.

31. Use of a polynucleotide encoding an HPV Early protein in the preparation of a medicament for the prevention of infection or disease caused by an HPV virus containing a second different HPV Early protein type, wherein the Early protein encoded by the polynucleotide of the medicament has a sequence identity of greater than 80 % in the predicted epitope regions when compared with a sequence from the second HPV type.