WO2002002142A1

WO2002002142A1 - Papillomavirus vaccine

Info

Publication number: WO2002002142A1
Application number: PCT/SE2001/001501
Authority: WO
Inventors: Stefan Schwartz
Original assignee: Stefan Schwartz
Priority date: 2000-07-03
Filing date: 2001-06-29
Publication date: 2002-01-10
Also published as: AU2001266534A1; SE0002498D0; WO2002002142B1

Abstract

The present invention within the field of papillomavirus vaccines and is related to nucleic acid vaccines comprising nucleic acid sequences encoding papillomavirus proteins, such as L1 and L2. More closely, the invention relates to a human papilloma virus (HPV) vaccine comprising a HPV nucleic acid which encodes HPV protein which is effectively expressed in human cells and which leads to an effective immune response. This is achieved by inactivating the negative regulatory elements of the HPV nucleic acid.

Description

Title: Papillomavirus vaccine

Field of the invention

The present invention within the field of papillomavirus vaccines and is related to nucleic acid vaccines comprising nucleic acid sequences encoding papillomavirus proteins, such as LI and L2.

Background of the invention

Human papillomavirus (HPVs) are epitheliotropic small DNA tumor viruses (1). There are approximately 100 different HPV types known to date, some of which cause common warts while others are associated with cancer. Genital human papillomavirus infections are the virally sexually transmitted diseases most frequently diagnosed and include anogenital condylomas and squamous intrapithelial lesions. These are precursors of invasive carcinoma of the uterine cervix (1). At least 50% of the sexually active adults have had a genital HPV infection. A subset of HPVs, the so called high risk HPV types are associated with high grade cervical lesions and carcinomas but also with a proportion of carcinomas of the upper aerodigestive tract, penis, vulva and anus, thereby substantially contributing to the cancer burden world wide. For example, cancer of the uterine cervix is one of the most common cancer forms in women, especially in developing countries, and is a leading cause of cancer death (2). In developing countries it accounts for the highest cancer mortality. Up to 98 % of these cancers have been reported to contain HPV DNA and approximately 50% of them are HPV- 16, which is the most common cancer associated HPV type, HPV- 16, -18, -31 and -45 account for 80% and HPV types -16, - 18, -31, -33, -45, -52, and -58 account for more than 90% of all cervical carcinomas (2). Although cervical screening programs for dysplastic precursors exists and have been shown to reduce incidence and mortality from cervical cancer in some Western countries, such programs will not eliminate HPV due to the occurrence of silent infections. The medical importance of the HPVs prompted research on vaccine development against HPV infection.

The early HPV gene products have various roles in transcription and replication of the genomic HPV DNA and in host cell transformation. The late gene products LI and L2 assemble into an icosahedral capsid with a diameter of -60 nm that contains the circular HPV DNA genome (1). The HPV replication cycle starts as the virus enters the cells in the basal layer of the squamous epithelium. The completion of the viral replication cycle is dependent on terminal differentiation of the infected epithelial cell (1) and LI and L2 proteins are produced only in terminally differentiated cells. The various HPV types have also been found to produce different amounts of progeny virus in vivo and may be classified as productive, weakly productive or non-productive HPV types (1).

Development of antiviral substances and vaccines against HPVs have been hampered by the lack of an in vitro cell culture system for propagation of infectious virus. One reason for the lack of virus production in vitro is the strict coupling between the cell differentiation stage and expression of viral late genes LI and L2 that encode the virus structural proteins, the major and the minor capsid proteins, respectively (3). With other words, production of LI and L2 and infectious virus is blocked in proliferating, non-terminally differentiated cells. In vivo, this is evidenced by lack of LI and L2 production in the lower and middle layers of the epithelium, while high levels are detected in the uppermost layer. Production of LI and L2 is inhibited in dividing cells that are not terminally differentiated. Differentiation activates late mRNA synthesis but production of LI and L2 proteins is delayed to the upper layers of the epithelium by posttranscriptional regulatory events. It was previously found that the HPV- 16 LI and L2 coding regions contain RNA elements with inhibitory activity (3, 4, 5, 6). These elements act by reducing mRNA half-life and translation resulting in efficient inhibition of HPV- 16 LI and L2 production. The location of these elements is not known.

The HPV- 16 LI and L2 are not expressed in animal cells but are efficiently expressed in insect cells and in yeast cells. Analysis of HPV LI produced in insect cells from recombinant insect viruses or in yeast cells revealed that the HPV LI protein from several different HPV types could assemble into virus-like particles (VLPs). Coexpression with L2 resulted in incorporation of L2 and the production of empty capsids that are virtually identical to virus particles, but lack viral genomic DNA. VLPs have been produced also in human cells from replicating viral vectors such as vaccinia virus and semliki forest virus carrying the HPV LI gene.

There is no animal model for HPV infection and HPVs infect only human cells as papillomaviruses are strictly species-specific (1). VLPs of LI and L2 of the animal papillomaviruses have been produced and used in vaccination studies (10). In contrast to denatured LI and L2 proteins, LI and L2 proteins that have assembled into VLPs induce antibodies that neutralise the virus particles and prevent virus infection (8, 9, 10). VLPs assemble only when the LI and L2 proteins are produced in eukaryotic cells and not when the proteins are produced in bacteria. Using the VLPs in animal systems it has been shown that vaccination with VLPs protect animals from experimental infection (10).

An alternative to using VLPs produced in insect cells as vaccine, is to use nucleic acid vaccination (7). This is a relatively recent vaccination method in which plasmid DNA containing a promoter that efficiently directs transcription initiation in human cells, the gene producing the protein to which an immune response is desired and a polyA signal for termination of transcription. The plasmids also contain sequences that encode antibiotic resistance for selection when maintained in bacteria and sequences for replication of the plasmid DNA in bacteria. When such plasmids are generated and purified from bacteria and for example intramuscularly injected into an individual, the plasmid is taken up by the cells, transcribed and translated into protein, to which an immune response is elicited. The injected plasmids are maintained in an extrachromosomal state and are continuously expressed resulting in a sustained immune response. Persistent expression have been reported for a number of experimentally infected animals. The proteins produced in vivo in the human cells assume the correct conformation and contain the correct posttranslational modifications. The use of nucleic acid vaccination also offers the advantage of the generation of cell mediated immune responses as a result of the intracellular production of the proteins.

Nucleic acid vaccination can also bee performed using in vitro synthesised, capped and polyadenylated RNA that is injected intramuscularly or intradermally. When these RNAs enter the cells, they direct production of the encoded proteins. This approach has been further developed by using "self-replicating" RNA. In this case the RNA encodes the replicase proteins of and RNA virus such as Sindbis virus, Semliki forest virus or Venezuelan equine encephalitis virus (7, 13). The replicase multiplies the injected RNA in the cell which results in a stronger immune response against the protein of interest. The replicase unit could also be inserted in a DNA plasmid encoding an antigen. This results in multiplication of the mRNA that is produced from the plasmid after introduction in cells (7, 13).

Vaccination of animals with DNA sequences encoding the LI and /or the L2 gene has been performed with LI genes of the cottontail rabbit papillomavirus. In this animal model systems, the LI and L2 genes were inserted between a promoter and a polyadenylation signal in an eukaryotic expression plasmid. The animals received two doses of the naked DNA vaccine and were subsequently challenged with infectious virus. Sundaram et al reported that nearly all the animals that received the LI expression plasmid were protected against experimental infection whereas none of the unvaccinated animals were protected (11). All protected animals had antibodies that reacted with intact virions and neutralised virus. Donnely et al reported that vaccination of cottontail rabbits with naked DNA expressing the LI, but not the L2 gene, induced the production of virus neutralising antibodies and almost complete protection against papillomavirus infection when the animals were challenged with virus (12). It would be desirable with nucleic acid vaccination against HPV infection in humans but this has hitherto been not been possible.

Summary of the invention

Production of HPV- 16 LI and L2 in mammalian cells from non- replicating, plasmid expression vectors has hitherto been unsuccessful. The present inventor found that this is due to the presence of the negative regulatory elements in the LI and L2 coding sequences.

The present invention provides a human papilloma virus (HPV) vaccine comprising a HPV nucleic acid which encodes HPV protein which is effectively expressed in human cells and which leads to an effective immune response. In one embodiment, the vaccine comprises a HPV nucleic acid in which negative regulatory element(s) have been inactivated, deleted or substituted with HPV protein encoding sequences lacking said element(s). In a further embodiment, the vaccine comprises a synthetic HPV nucleic acid in which negative regulatory element(s) have been^' inactivated by genetically altering said nucleic acid without changing the coding sequence of its corresponding protein. The nucleic acid may be DNA or RNA.

The synthetic HPV sequence is derived from oncogenic HPV , such as HPV

6, 1 1, 16, 18, 31, 33 and/or 45. Preferably HPV sequences are HPV late sequence(s).

In a further embodiment, the vaccine comprises a combination of HPV sequences from one or more HPV types. Such a vaccine may also include HPV early sequences and immune response enhancer(s).

The present inventor has developed an assay for identification and characterisation of inhibitory RNA elements located in the coding region of mRNAs. By using this assay, deletion analysis combined with mutational analysis mapped the location of the negative elements in HPV- 16 LI and L2. It was found that the 5' part of the LI coding sequence displayed strong inhibitory activity that decreased gradually as further deletions were introduced from the 5' or 3' ends whereas the HPV- 16 L2 coding region contained negative elements in the 5' end and in the 3' end. The location of negative RNA elements in the HPV- 16 LI and L2 coding regions have prevented production of HPV- 16 LI and L2 from plasmids in transfected mammalian cells.

In a preferred embodiment, the invention comprises DNA constructs that encode genetically altered human papillomavirus type 16 LI and L2 genes in which the negative regulatory elements have been inactivated in order to accomplish high LI and L2 expression levels when introduced into human cells or animal tissue. The produced proteins could provide immune protection against infection with papillomavirus.

Detailed description of the invention

The invention will be described more closely below in association with the accompanying drawings, in which: Fig. 1 shows testing of the HPV- 16 LI sequence in the transfection assay for negative elements. Plasmids encoding the capsid protein of HPV- 16 or EIAV were transfected into cells and RNA was extracted and analysed by Northern RNA blotting. The results demonstrate that the HPV- 16 LI encoding plasmid produced undetectable levels of LI mRNA due to the negative elements in HPV- 16 LI whereas the EIAV sequence directed the synthesis of high RNA levels.

Fig. 2 shows the sequences of the long oligonucleotides encoding the synthetic HPV- 16 LI gene are shown. These sequences were used in PCR reactions with the PCR primers shown in Fig. 3 to generate the fragments that are schematically shown in Fig. 5.

Fig. 3 shows the sequences of the PCR primers used for PCR amplification of the long oligonucleotides encoding the synthetic HPV- 16 LI gene that are shown in Fig. 2.

Fig. 4 shows schematic drawing of the 9 PCR fragments that were generated by PCR amplifying the sequences shown in Fig. 2 with the PCR primers shown in Fig. 3. The restriction sites at the ends of the fragments that were used to fuse the nine fragments are indicated.

Fig. 5 shows schematic drawing of the wt HPV- 16 LI coding region, the synthetic HPV- 16 LI coding region and one hybrid encoding synthetic HPV- 16 LI sequences upto the BamHI site and wt sequences between the BamHI and the Xhol sites; and

Fig. 6 shows an immunoblot on extracts from cells transfected with the expression plasmids encoding the HPV- 16 LI hybrid gene shown in Fig. 5 and the HPV- 16 LI wt gene. The results show specific detection of HPV- 16 LI protein in the cells that had been transfected with the hybrid gene that contains a genetically altered HPV- 16 LI sequence, but not in the cells transfected with the wt HPV- 16 LI gene. In order to generate plasmids that can produce HPV- 16 LI and L2 in human cells, the negative regulatory elements in the HPV- 16 LI and L2 coding regions described above must be inactivated.

The invention provides HPV- 16 LI and L2 genes that have been genetically altered to inactivate the negative elements in the LI and L2 coding regions, without affecting the protein coding sequence of LI and L2, and therefore direct the synthesis of high levels of LI and L2 when inserted into expression plasmids that are introduced into human cells. When these nucleic acid sequences are introduced into cells of a vertebrate animal such as humans in the form of plasmid DNA, RNA or self replicating RNA, the LI and L2 proteins are processed and could induce a cellular immune response. In addition, LI can self assemble into VLPs in human cells and incorporate HPV- 16 L2. These VLPs could elicit an immune response with virus neutralising antibodies. The HPV- 16 LI ands L2 expression plasmids in which the negative elements have been inactivated to obtain high expression levels are highly suitable for use as nucleic acid vaccines.

AN ASSAY FOR TESTING IF CODING SEQUENCES CONTAIN NEGATIVE ELEMENTS

This assay can be used to test any coding sequence for the presence of negative elements. The coding region to be tested is PCR amplified with a sense prime that contains a BssHII site and an anti sense primer that contains an Xhol site. The PCR fragment is cleaved with the two en2ym.es and subcloned into the expression plasmid pC, generated by the inventor. The resulting plasmid with the coding region to be tested is transfected into human cells, RNA is extracted and analysed on Northern RNA blots with a probe that hybridises to the first 76 nucleotides that are derived from the CMV promoter and thus are present on all the pC-derived plasmid. An example is shown in Fig. 1. The high RNA levels seen in cells transfected with pC containing the EIAV (an equine retrovirus) gag capsid coding sequence that lacks negative elements and produces high RNA levels are shown. The pCHPV16Ll plasmid that contains the wild type HPV- 16 LI sequence produces undetectable RNA levels as a result of the negative elements (Fig. 1). This assay could be used to determine if the LI and L2 sequences of other HPV types contain negative elements. Similar results are obtained with the LI and L2 sequences. In order to map the negative elements, hybrids between the EIAV gag sequence, which lacks negative elements, and the gene that contains negative elements (for example HPV- 16 LI) can be generated and analysed in the assay.

CONSTRUCTION OF A SYNTHETIC HPV- 16 LI GENE THAT LACKS NEGATIVE ELEMENTS

In order to generate a synthetic HPV- 16 LI gene in which the negative elements were inactivated, 9 large consecutive oligonucleotides comprising the entire LI coding sequence were synthesised. These oligonucleotides contained sequence alterations that inactivated the negative elements. The sequences are shown in Fig. 2. Due to the degenerate nature of the genetic code, most amino acids are represented by multiple codon triplets. By changing the DNA sequence in such a way that it results in the usage of an alternative codon for the same amino acid, it is possible to alter the DNA sequence of the LI gene without affecting the sequence of the LI protein that the LI gene is coding for. This strategy was used and the 9 large oligonucleotides were altered in that manner, throughout the LI gene. In order to generate a complete LI sequence containing the sequence alterations, 9 PCR primer pairs (one pair for each large oligonucleotide) were used. The sequences of the PCR primers are shown (Fig. 3). These primers were used for PCR amplification of each of the long oligonucleotides and the PCR reactions resulted in the nine fragments shown schematically in Fig. 4. The restriction endonuclease cleavage site that are introduced in the PCR primers are indicated. Each primer contained a restriction enzyme site that allowed the nine PCR fragments comprising the HPV- 16 LI gene to be fused in frame to generate a complete HPV- 16 LI coding sequence. The nine large PCR fragments with the restriction sites are shown schematically in Fig. 4. Subcloning of the nine PCR fragments after each other using the restriction sites shown in Fig. 4 resulted in the synthetic HPV- 16 LI gene named 16Lls.

By using the assay for identification and characterisation of negative RNA elements described in the text above, the major negative element in the HPV- 16 LI coding sequence was mapped to the first 25% of the HPV- 16 LI gene. By generating a hybrid gene consisting of the first 25% of the synthetic LI gene and the remaining 75% of the wild type sequence, the major negative element should be inactivated. A number of hybrids between the wt and the synthetic sequence could be envisaged and could be generated with the help of the various restriction sites introduced into the synthetic gene. The restriction sites that are introduced in the 16Lls sequence are shown schematically in Fig. 5. These sites can also be used for generation of hybrids with the wt LI sequence or with other sequences such as for example early HPV proteins. An example of a hybrid between synthetic and wild type LI sequence is shown schematically in Fig. 5. By using the restriction sites in the synthetic part it is possible to generate hybrids with less than 25% if the synthetic sequence and more than 75% of the wt sequence. These hybrids may have sufficient capacity to inactivate the negative regulatory elements in HPV 16 LI.

HPV- 16 LI EXPRESSION PLASMIDS

In order to express the synthetic LI genes and derivatives thereof in human cells, mammalian expression plasmids were generated. These plasmids are based on the commercially available pUC8 plasmid in which the immediate early promoter of human cytomegalovirus was inserted. The promoter sequence consists of human cytomegalovirus sequences from -671 to + 76 with respect to the transcriptional start site. This sequence is followed by a leader sequence consisting of exon 1 sequence between nucleotide 257 and 289 in exon 1 of the HIV-1 infectious molecular clone HXB2R and exon 4 sequence from 5324 to 5332 from the same HIV-1 clone. This sequence is followed by a Sail, Clal, Hindlll and BssHII restriction sites, a four nucleotide CAAG "Kozak sequence" upstream of the translational start codon and the entire HPV- 16 LI coding sequence. After the translational stop codon of HPV- 16 LI, an Xhol site has been introduced followed by the HPV- 16 late 3' UTR from 7290 up to nucleotide position 7453. The HPV- 16 late 3' UTR sequence contains the HPV- 16 late polyA signal. The EcoRI is followed by the pUC8 sequence. This plasmid was named pC16Ll and contains the wild type HPV- 16 LI region. In this plasmid we replaced the wt HPV- 16 LI with the synthetic HPV- 16 LI gene or the hybrid gene consisting of the first 25% of the synthetic LI sequence and the last 75% of the wt sequence. In order to do so, the synthetic LI gene and the hybrid between the synthetic and the wild type LI gene were separately PCR amplified with primers that introduced and upstream BssHII site and a downstream Xhol site for cloning. The resulting PCR fragments were digested with BssHII and Xhol and inserted into the BssHII and Xhol digested pC16Ll plasmid thereby replacing the wild type HPV- 16 LI sequence. When the pC16Ll plasmid lacks insert and instead contains a poly linker sequence it is referred to as pC and can be used for insertion of any sequence that one wishes to test in the assay for identification of negative elements in the coding regions.

This is one example of a vector that can be used for expression of HPV- 16 LI from the synthetic HPV- 16 LI gene and derivatives thereof. Different vectors with various promoters and polyA signals, recombinant viruses, expression vectors for other species such as for example yeast, fungi, insect cells, plants and bacteria could be used. Nucleic acid vaccination can also be performed using in vitro synthesised, capped and polyadenylated RNA or by using "self- replicating" RNA. In the latter case the RNA encodes the replicase proteins of and RNA virus such as Sindbis virus, Semliki forest virus or Venezuelan equine encephalitis virus that multiplies the injected RNA, which results in a stronger immune response against the protein of interest.

EXPRESSION OF THE SYNTHETIC HPV- 16 LI GENE FROM EXPRESSION PLASMIDS IN HUMAN CELLS

In order to determine the expression levels of the various plasmids that are aimed at the development of nucleic acid vaccines against HPV infection, the plasmids are transfected into human cells by one of various techniques, for example fugene transfection, calcium phosphate coprecipitation or electroporation. The cells are harvested at 20-24 hrs posttransfection and a cell lysate is prepared by lysis of the cells in for example 100 mM Tris pH 7.8 and 1% triton X-100. The cell lysate is freeze thawed and centrifuged to remove debris, and the supernatant is analysed by Western blotting with antibodies specifically reacting with HPV- 16 LI. The three generated plasmids (pC16Ll, pC16Lls and pC1625%L175%EIAVgag) were tested and the results revealed that the wt HPV- 16 LI gene failed to produce detectable levels of HPV- 16 LI as expected, due to the presence of the negative elements. In contrast, the synthetic HPV- 16 LI gene and the hybrid consisting of the first 25% of the synthetic gene and the remaining 75% of the wt gene produced high levels of LI protein. High levels of LI protein are seen in the cells transfected with the synthetic gene and with the hybrid gene whereas LI protein was undetectable in cells transfected with the wt HPV- 16 LI gene. An example of the results with the 25% synthetic and 75% wt hybrid and the wt HPV- 16 LI gene are seen in Fig. 6.

The process according to the invention enables development of expression plasmids that can be used to produce HPV- 16 LI and L2 in mammalian cells and therefore can be used for development of nucleic acid vaccines against HPV- 16 infection. It is also possible to test if the LI and L2 coding sequences of other HPV types for example HPV-18, - 31 , -33 and -45, contain negative elements, map them and inactivate them to generate plasmid that efficiently express LI and L2 as described here for HPV- 16 LI . These plasmids could be used in combination with the HPV- 16 LI expression plasmid in a multivalent vaccine against HPV infection.

Alternatively, the LI and L2 encoding plasmids may be used in combination with plasmids expressing HPV early proteins and/ or genes that enhance the immune response against HPV.

REFERENCES

1. Howley, P. M. 1996. Papillomaviridae: The viruses and their replication, p. 2045-2076. In B. N. Fields and D. M. Knipe and P. M. Howley (ed.), Fields Virology, 3rd ed, vol. 2. Lippincott - Raven publishers, Philadelphia.

2. zur Hausen, H. 1996. Papillomavirus infections - a major cause of human cancers. Biochem Biophys Acta. 1288:55-78.

3. Schwartz, S. 1997. Cis-acting negative RNA elements on papillomavirus late mRNAs. Sem Virol. 8:291-300.

4. Schwartz, S., S. Sokolowski, B. Collier, A. Carlsson, and L. Goobar- Larsson. 1999. Cis-acting regulatory sequences on human papillomavirus late mRNAs. Recent Res Devel Virol. 1 :53-74.

5. Sokolowski, M., W. Tan, M. Jellne, and S. Schwartz. 1998. mRNA instability elements in the human papillomavirus type 16 L2 coding region. J Virol. 72: 1504-1515.

6. Tan, W., B. K. Felber, A. S. Zolotukhin, G. N. Pavlakis, and S. Schwartz. 1995. Efficient expression of human papillomavirus typelό LI protein in epithelial cells by using rev and the rev-responsive element of human immunodeficiency virus or the cis-acting transactivation element of simian retrovirus type 1. J Virol. 69:5607-5620.

7. Leitner, W W., Ying, H., Restifo, N P. 2000. DNA and RNA-based vaccines: principles, progress and prospects. Vaccine. 18: 765-777.

8. Kirnbauer, R., F. Booy, N. Cheng, D. R. Lowy, and J. T. Schiller. 1992. Papillomavirus LI major capsid protein self-assembles into virus-like particles that are highly immunogenic. Proc Natl Acad Sci USA. 89: 12180- 12184. 9. Roden, R. B. S., H. L. Greenstone, R. Kirnbauer, F. P. Booy, J. Jessie, D. R. Lowy, and J. T. Schiller. 1996. In vitro generation of type-specific neutralization of a human papillomavirus type 16 virion pseudotype. J Virol. 70:5875-5883.

10. Schiller, J. T., and D. R. Lowy. 1996. Papillomavirus-like particles and HPV vaccine development. Sem Cancer Biol. 7:373-382.

11. Sundaram, P., Tigelaar, R E., Brandsma, L. 1997. Intracutaneous vaccination of rabbits with the cottontail rabbit papillomavirus (CRPV) LI gene protects against virus challenge. Vaccine. 15:664-671.

12. Donnely, J J., Martinez, D., Jansen., K U, Ellis, R W., Montgomery, D L., Liu, M A. 1996. Protection against papillomavirus with a polynucelotide vaccine. J. Infect. Dis.,713: 314-320.

13. Restifo, NP, Yiang, H, Hwang, L, Leitner, WW. 2000. The promise of nucleic acid vaccines. Gene Therapy, 7: 89-92. ,

Claims

1. A human papilloma virus (HPV) vaccine comprising a HPV nucleic acid which encodes HPV protein which is effectively expressed in human cells and which leads to an effective immune response.

2. A vaccine according to claim 1 , comprising a HPV nucleic acid in which negative regulatory element(s) have been inactivated, deleted or substituted with HPV protein encoding sequences lacking said element(s) .

3. A vaccine according to claims 1 or 2, comprising a synthetic HPV nucleic acid in which negative regulatory element(s) have been inactivated by genetically altering said nucleic acid without changing the coding sequence of its corresponding protein.

4. A vaccine according to claims 1, 2 or 3, wherein the nucleic acid is DNA or RNA.

5. A vaccine according to claim 4, comprising:

- a nucleic acid vector;

- a synthetic HPV DNA sequence; and

- elements necessary for expressing said sequence when the vaccine has been introduced into a human subject.

6. A vaccine according to claim 4, wherein the HPV nucleic acid is in vitro synthesised, capped and polyadenylated RNA.

7. A vaccine according to claim 6, wherein the RNA is self-replicating.

8. A vaccine according to one or more of the above claims, wherein the synthetic HPV sequence is/ are derived from oncogenic HPV.

9. A vaccine according to claim 8, wherein the synthetic HPV sequence is/ are derived from HPV late sequence(s).

10. A vaccine according to claim 9, wherein the HPV late sequence is HPV 16 LI .

11. A vaccine according to claim 10, wherein the first 25% of the nucleic acids in the sequence have been replaced with other nucleic acids in a way not changing the amino acid sequence of the gene product.

12. A vaccine according to one or more of the above claims, comprising a combination of HPV sequences derived from one or more HPV types.

13. A vaccine according to claim 12, wherein the sequences are derived from HPV 6, 11, 16, 18, 31, 33 and/or 45.

14. A vaccine according to claim 12, further comprising HPV early sequences.

15. A vaccine according to claim 12, 13 or 14, comprising immune response enhancer(s).