MXPA06010983A - Optimized expression of hpv 52 l1 in yeast - Google Patents

Abstract

Synthetic DNA molecules encoding the HPV 52 L1 protein are provided. Specifically, the present invention provides polynucleotides encoding HPV 52 L1 protein, wherein said polynucleotides are codon-optimized for high level expression in a yeast cell. In alternative embodiments of the invention, the nucleotide sequence of the synthetic molecule is altered to eliminate transcription termination signals that are recognized by yeast. The synthetic molecules may be used to produce HPV 52 virus-like particles (VLPs), and to produce vaccines and pharmaceutical compositions comprising the HPV 52 VLPs. The vaccines of the present invention provide effective immunoprophylaxis against papillomavirus infection through neutralizing antibody and cell-mediated immunity and may also be useful for treatment of existing HPV infections.

Description

OPTIMIZED EXPRESSION OF HPV 52 L1 IN YEASTS FIELD OF THE INVENTION In general, the present invention relates to the prevention and / or therapy of human papillomavirus (HPV) infection. More specifically, the present invention relates to synthetic polynucleotides encoding the HPV 52 L1 protein, and to recombinant vectors and hosts comprising said polynucleotides. This invention also relates to HPV virus-like particles (VLPs), where VLPs are produced by the recombinant expression of HPV 52 L1 or L1 + L2 in yeast cells and their uses in vaccines and vaccines. in pharmaceutical compositions for preventing and treating HPV infections.

BACKGROUND OF THE INVENTION There are more than 80 types of human papillomavirus (HPV), much of which has been associated with a wide variety of biological phenotypes, from benign proliferative warts to malignant carcinomas (for consultation, see McMurray et al., Int. J. Exp. Pathol. 82 (1): 15-33 (2001)). The types most commonly associated with benign warts, non-malignant condyloma acuminatum and / or low-grade dysplasia of the genital or respiratory mucosa are HPV6 and HPV1. Types HPV16 and HPV18 are the highest risk types most frequently associated with in situ and invasive carcinomas of the cervix, vagina, vulva, and anal canal. More than 90% of cervical carcinomas are associated with infections of HPV16, HPV18 or with the less prevalent oncogenic types HPV31, -33, -45, -52 and -58 (Schiffman et al., J. Nati. Cancer Inst. (12): 958-64 (1993)). The observation that HPV DNA is detected in 90-100% of cervical cancers is a strong epidemiological evidence that cervical carcinoma is caused by HPVs (see Bosch et al., J. Clin. Pathol. 244-265 (2002)). Papillomaviruses are small icosahedral DNA viruses (50-60 nm), uncoated that encode up to eight early and two late genes. The open reading frames (ORFs) of the viral genomes are designated from E1 to E7, and L1 and L2, where "E" denotes early and "L" denotes late. L1 and L2 code for virus capsid proteins, while E genes are associated with functions such as viral replication and cell transformation. The L1 protein is the major capsid protein and has a molecular weight of 55-60 kDa. The L2 protein is the minor capsid protein. Immunological data suggest that most of the L2 protein is internal to the L1 protein of the viral capsid. Both the L1 and L2 proteins are highly conserved in the different papillomaviruses. The expression of the L1 protein or a combination of the L1 and L2 proteins in yeast, insect cells, mammalian cells or bacteria produce self-assembly of virus-like particles (VLPs) (see Schiller and Roden, in Papillomavirus Reviews: Current Research on Papillomaviruses; Lacey, ed. Leeds, UK: Leeds Medical Information, pp 101-12 (1996)). VLPs are morphologically similar to authentic virions and are capable of inducing high titers of antibodies by neutralization when administered to animals or humans. Because VLPs do not contain the potentially oncogenic viral genome, they present a safe alternative to the use of live viruses in the development of the HPV vaccine (see Schiller and Hidesheim, J. Clin. Virol. 19: 67-74 (2000) ). For this reason, the L1 and L2 genes have been identified as immunological targets for the development of prophylactic and therapeutic vaccines for HPV infection and disease. The development of the HPV vaccine and commercialization have been impeded by the difficulties associated with obtaining high levels of expression of capsid proteins in successfully transformed host organisms, limiting the production of purified protein. Thus, despite the identification of wild-type nucleotide sequences encoding HPV L1 proteins such as the HPV 52 L1 protein, it would be highly desirable to develop an easily renewable source of crude HPV L1 protein using HPV 52 L1 coding nucleotide sequences that are optimized for expression in the intended host cell. Additionally, it may be useful to produce large quantities of HPV 52 L1 VLPs that have the conferred immunity properties of the native proteins for use in the development of vaccines.

BRIEF DESCRIPTION OF THE INVENTION The present invention relates to compositions and methods for obtaining or improving the immunity of protein products expressed by the HPV 52L1 genes. Specifically, the present invention provides polynucleotides encoding the HPV 52 L1 protein, where the polynucleotides have been optimized at the codon for high level expression in a yeast cell. In alternative embodiments of the invention, the nucleotide sequence of the polynucleotide is altered to eliminate transcription of the termination signals that are recognized by the yeast. The present invention also provides HPV 52 virus-like particles (VLPs), wherein said VLPs are produced by the recombinant expression of HPV 52 L1 or L1 + L2 in yeast cells, and describes the use of HPV 52 VLPs in immunogenic compositions and Vaccines for the Prevention and / or Treatment of HPV Disease and HPV-Associated Cancer The present invention relates to synthetic DNA molecules that encode the HPV 52 L1 protein. The codons of the synthetic molecules are designed to use the codons preferably by a yeast cell. In an alternative embodiment of the invention, the nucleotide sequence of the synthetic molecule is altered to eliminate the transcription of termination signals that are recognized by the yeast. The synthetic molecules can be used as a source of the HPV 52 L1 protein, which can self-assemble into VLPs. These VLPs can be used in vaccines based on VLP. An embodiment example of the present invention comprises a synthetic nucleic acid molecule which encodes the HPV 52 L1 protein as indicated in SEQ ID NO: 2, said nucleic acid molecule comprises a nucleotide sequence that is optimized at the codon for express a high level in a yeast cell.

Recombinant vectors and recombinant host cells are also provided, both prokaryotes and eukaryotes, which contain nucleic acid molecules described in this specification. In a preferred embodiment of the present invention, the host cell is a yeast cell. The present invention also relates to a method for expressing an HPV 52 L1 protein in a recombinant host cell, comprising: (a) introducing a vector comprising a nucleic acid encoding an HPV 52 L1 protein in a yeast host cell; and (b) culture of the yeast host cell under conditions which allow the expression of said HPV 52 L1 protein. The present invention further relates to a method for the expression of an HPV 52 L1 protein in a recombinant host cell, comprising: (a) introducing a vector comprising a nucleic acid molecule encoding an HPV 52 L1 protein in a cell yeast host, wherein the nucleic acid molecule is optimized at the codon for optimal expression in the yeast host cell and; (b) culture of the host yeast cell under conditions that allow the expression of said HPV 52 L1 protein. In preferred embodiments, the nucleic acid molecule comprises a nucleotide sequence as set forth in SEQ ID NO: 1 (designated here "sequence 52 L1 R"). This invention also relates to HPV 52 virus-like particles (VLPs) which are produced in yeast cells, the production methods of HPV 52 VLPs, and the methods of using HPV 52 VLPs.

In a preferred embodiment of this invention, the yeast is selected from the group consisting of: Saccharomyces cerevisiae, Hansenula polymorpha, Pichia pastoris, Kluyvermyces fragilis, Kluveromyces lactis, and Schizosaccharomyces pombe. Another aspect of this invention is a HPV 52 VLP, where VLP is produced by recombinant expression of HPV 52 L1 or HPV 52 L1 + L2 in a yeast cell. Yet another aspect of this invention is a HPV 52 VLP which comprises an HPV 52 L1 protein produced by HPV 52 L1 gene with codon optimized. In an embodiment example of this aspect of the invention, the optimized codon of the HPV 52 L1 gene comprises a nucleotide sequence as indicated in SEQ ID NO: 1. This invention also provides a method for inducing an immune response in an animal comprising administering to the animal HPV virus-like particles 52. In a preferred embodiment, HPV 52 VLPs are produced by an optimized codon gene. In yet another aspect of this embodiment is a method of prevention or treatment of cervical cancer associated with HPV which comprises the administration to a mammal of a vaccine comprising HPV 52 VLPs. In a preferred embodiment of this aspect of the invention, the HPV VLPs 52 are produced in yeast.

This invention also relates to a vaccine comprising virus-like particles (VLPs) HPV 52, where HPV 52 VLPs are produced in yeast. In an embodiment of this aspect of the invention, the vaccine further comprises VLPs of at least one additional type of HPV. Another last type of HPV may be some type of HPV of interest including some type of HPV described in the state of the art, or these subsequently identified. In a preferred embodiment, the HPV type is a type that is associated with a clinical phenotype such as warts or cervical cancer. In another preferred embodiment, at least one additional type of HPV is selected from the group consisting of: HPV6, HPV1, HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV55, HPV56, HPV58, HPV59, and HPV68 . This invention also relates to pharmaceutical compositions comprising particles similar to HPV 52 viruses, where HPV 52 VLPs are produced in yeast. In addition, this invention relates to pharmaceutical compositions comprising HPV 52 VLPs and VLPs of at least one additional type of HPV. In a preferred embodiment, at least one additional type of HPV is selected from the group consisting of: HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV55, HPV56, HPV58, HPV59, and HPV68 . As used throughout the specification and in the claims, the singular forms "a" and "the" include the plural reference unless the context clearly dictates otherwise.

As used throughout the specification and in the claims, the following definitions and abbreviations refer: The term "promoter" refers to the recognition site on a strand of DNA to which the RNA polymerase is attached. The promoter forms an initiation complex with the RNA polymerase to initiate and conduct the transcription activity. The complex can be modified by activating sequences called "enhancers" or "upstream activated sequences" or inhibitors of sequences called "silencers". The term "vector" refers to some meaning by which DNA fragments can be introduced into an organism or a host tissue. There are several types of vectors that include plasmids, viruses (including adenoviruses), bacteriophages and cosmids. The term "cassette" refers to a nucleotide or a sequence of a gene that is expressed from a vector, for example, the nucleotide or the sequence of the gene encoding the HPV 52 L1 protein. In general, a cassette comprises a gene sequence inserted into a vector, which, in some of the embodiments, provides regulatory sequences for the expression of the nucleotide or the sequence of the gene. In other embodiments, the nucleotide or sequence of the gene provides the regulatory sequences for expression. In other embodiments, the vector provides some regulatory sequences and the nucleotide or genetic sequence provides other regulatory sequences. For example, the vector can provide a promoter for the transcription of the nucleotide or the sequence of the gene and the nucleotide or the genetic sequence provides a transcription termination sequence. Regulatory sequences can be provided by the included vector, but are not limited to, enhancers, transcription termination sequences, splice donor and acceptor sequences, introns, ribosome binding sequences, and poly (A) addition sequences. The designations "wild-type sequence 52 L1" and "sequence 52 L1 wt" refer to the HPV sequence 52 L1 described herein as SEQ ID NO: 3. Although the HPV 52 L1 wild-type sequence has been previously described, it is common to find minor sequence variations between the DNAs obtained in clinical isolates. Therefore, a wild-type sequence representative of HPV 52 L1 was isolated from clinical samples previously shown to contain HPV 52 DNA (see Example 1). The HPV 52 L1 wild-type sequence was used as a reference when comparing the HPV 52 L1 optimized codon sequences described here (see Figures 1A-1C). The designations "HPV 52 L1 R" and "52 L1 R" refer to an example synthetic nucleotide sequence of HPV52 L1, described herein, where the sequence was reconstructed so that it comprises codons that are preferred by the high level of expression in yeast cells. The term "effective amount" means that the vaccine composition sufficient to produce the appropriate levels of the polypeptide is introduced, resulting in an immune response. One skilled in the art recognizes that this level may vary. A "conservative substitution of an amino acid" refers to the change from one amino acid residue to another, chemically similar. Examples of these conservative substitutions are: substitution of a hydrophobic residue (isoleucine, leucine, valine, or methionine) for another; substitution of a polar residue for another polar residue of the same charge (eg, arginine for lysine, glutamic acid for aspartic acid). The term "mammal" refers to any mammal, including a human. "VLP" or "VLPs" means particle resembling viruses or virus-like particles. "Synthetic" means that the HPV 52 L1 gene is created so it contains a nucleotide sequence that is not the same as the nucleotide sequence present in the natural sequence designated as the wild-type HPV 52 L1 gene (52 L1 wt, SEQ ID NO: 3). As found above, the synthetic molecules that are provided herein comprise a sequence of nucleotides comprising codons that are preferentially for expression by yeast cells. The synthetic molecules provided herein encode the same amino acid sequences as the wild-type HPV 52 L1 gene (SEQ ID NO: 2).

BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A-1C show an alignment sequence comparing nucleotides that are altered in the HPV 52 L1 synthetic gene of the present invention (SEQ ID NO: 1, indicated as "52 L1 R") (See Example 2). The reference sequence is the wild-type sequence 52 L1 (SEQ ID NO: 3, indicated as "52 L1 wt", see example 1). The altered nucleotides are indicated in their corresponding locations. The nucleotide number is in parentheses. Identical nucleotides in the reconstructed sequence 52 L1 are indicated with points. Figures 2A-2C show the reconstructed synthetic HPV 52 L1 double-stranded nucleic acid (SEQ ID NOs: 1 and 7) and the single encoding amino acid sequence (SEQ ID NO: 2). The nucleotide number is indicated on the left. Figure 3 shows a Northern analysis of the HPV 52 L1 wt and HPV 52 L1 R transcripts (see example 4). Northern analysis was tested with a mixture of DNA tests generated against both the 52 L1 wt and the 52 L1 R sequences. The arrow to the right indicates the predicted position of a full length 52 L1 transcript. No transcript of any length was detected in the lanes of 5 and 10 μg of 52 L1 wt RNA. The full-length transcripts are shown in 52 L1 R, in both lanes of 5 and 10 μg. Figure 4 shows a Western analysis of HPV 52 L1 wt proteins (52 wt), and 52 L1 R (52R). HPV 16 L1 was included as reference (16). Ten, five and two and a half micrograms of the extracted total yeast protein were denatured and applied to the 10% SDS-PAGE gel. The protein was transferred to a Western. The HPV 52 L1 protein was detected in the test result using a yeast-absorbed anti-trpE-HPV 31 L1 goat polyclonal antiserum which cross-reacted with HPV 52 L1 and HPV 16 L1. The molecular weight of the markers is indicated in kDa on the left. The arrow indicates the position of the protein ~ 55 kDa HPV 52 L1. Figure 5 represents a representative sample of HPV VLPs 52 composed of the HPV 52 L1 R protein molecules, described herein, as visualized by transmission electron microscopy (see example 7). The diameter of the spherical particles in this crude test is in the range between 40 and 70 nm with some particles exposing a regular matrix of capsomeres. The bar represents approximately 0.1 μm.

DETAILED DESCRIPTION OF THE INVENTION Most cervical carcinomas are associated with infections of specific oncogenic types of human papillomavirus (HPV). The present invention relates to compositions and methods for obtaining or improving the immunity of the products of the proteins expressed by genes of the oncogenic HPV types. Specifically, the present invention provides polynucleotides encoding HPV 52 L1, where the polynucleotides are codon-optimized for a high level of expression in yeast. The present invention also provides virus-like particles (VLPs), which are produced in yeast, and describes the use of said polynucleotides and VLPs in immunogenic compositions and vaccines for the prevention and / or treatment of cancer associated with HPV. A nucleotide sequence of wild-type HPV52 L1 has been described (Genbank Accession # NC 001592). The present invention provides synthetic DNA molecules encoding the HPV 52 L1 protein. In one aspect of the invention, the synthetic molecules comprise a sequence of codons, wherein at least some of the codons have been altered to use the preferred codons by a yeast cell for high levels of expression. In an alternative aspect of the invention, the nucleotide sequence of the synthetic molecule is altered to eliminate the transcription termination signals that are recognized by yeast. The synthetic molecules can be used as a coding sequence for the expression of the HPV 52 L1 protein, which can self-assemble into VLPs. Said VLPs can be used in VLP-based vaccines to provide effective immunoprophylaxis against papillomavirus infection through neutralizing antibodies and cell-mediated immunity. Such VLP-based vaccines may also be useful for the treatment or for established HPV infections. The expression of HPV VLPs in yeast cells offers the advantages of an effective cost and that can be easily adapted to the large-scale growth in fermenters. In addition, the yeast genome can be easily altered to ensure selection of recombination, transformed yeast with increased growth and potential expression. However, many HPV L1 proteins, including HPV 52 L1 are expressed in yeast cells at levels lower than the desirable commercial scale (see example 2). Accordingly, the present invention relates to HPV 52 L1 gene sequences that are "optimized" for high level expression in a yeast cell environment. A "triplet" codon of four possible nucleotide bases can exist in more than 60 variant forms. Because these codons provide the message for only 20 different amino acids (as well as the initiation of transcription and termination), some of the amino acids can be encoded by more than one codon, a phenomenon known as codon redundancy. For reasons not completely understood, alternative codons are not uniformly present in the endogenous DNA of different cell types. Even, there seems to be a variable natural hierarchy or "preference" for certain codons in certain cell types. As an example, the amino acid leucine is specified by one of the six DNA codons including CTA, CTC, CTG, CTT, TTA, and TTG. The exhaustive analysis of the frequencies used of the codons of the genome for microorganisms has revealed that the most common endogenous DNA of E. coli contains the codon CTG that specifies leucine, whereas the DNA of yeasts and molds more commonly include a codon TTA that specifies Leucine In view of this hierarchy, it is generally believed that the probability of obtaining high levels of expression of a leucine-rich polypeptide by an E. coli host will depend to some extent on the frequency of the codon used. For example, it is as if a gene rich in TTA codons would be poorly expressed in E. coli, where a gene rich in CTG would probably be more highly expressed in this host. Similarly, a preferred codon for the expression of a leucine-rich polypeptide in yeast host cells would be TTA. The implication of the phenomenon of codon preference in recombinant DNA techniques are manifest, and the phenomenon may serve to explain many of the previous failures to develop high expression levels of exogenous genes in host organisms successfully transformed to a codon minus " "preferred" may be present repeatedly in the inserted gene and the machinery of the host cell for expression may not operate as efficiently. This phenomenon suggests that synthetic genes which have been designated to include preferred codons of projected host cells provide an optimal form of foreign genetic material for the practice of expression of recombinant proteins. Although, one aspect of this invention is an HPV 52 L1 gene which is codon optimized for the expression of high levels in a yeast cell. In a preferred embodiment of this invention, it has been found that the use of alternative codons encoding the same protein sequence can eliminate the restriction on expression of HPV 52 L1 proteins by yeast cells. According to the invention, the HPV 52 L1 gene segments were converted to sequences having identical translated sequences but with alternative codon usage as described by Sharp and Cowe (Synonymous Codon Usage in Saccharomyces cerevisiae, Yeast 7: 657-678 ( 1991)), which is incorporated here as a reference. The methodology generally consists in the identification of codons in the wild-type sequence that are not commonly associated with highly expressed yeast genes and replace it with optimal codons for high expression in yeast cells. The new gene sequence is then examined in unwanted sequences generated by these codon replacements (eg, "ATTTA" sequences, inadvertent creation of intron splice recognition sites, unwanted restriction enzyme sites, high GC content , presence of transcription termination signals that are recognized by the yeast, etc.). Undesired sequences are eliminated by substitution of existing codons with different codons encoded for the same amino acid. The synthetic gene segments are then tested to improve expression.

The methods described above were used to create the synthetic gene segments for HPV 52 L1, resulting in a gene comprising the codons optimized for the high level of expression. While the above procedure provides a summary of sample methodology for the design of genes with codons optimized for use in HPV vaccines, it is understood by a specialist in the art that vaccines of similar efficacies or of increased expression of genes can be developed. with minor variations in the procedure or with minor variations in the sequence. Accordingly, the present invention relates to a synthetic polynucleotide comprising a nucleotide sequence encoding an HPV 52 L1 protein, or a biologically active fragment or a mutant form of an HPV 52 L1 protein, the polynucleotide sequence comprises codons optimized for expression in a yeast host cell. Said mutant forms of the HPV 52 L1 protein include, but are not limited to: conservative amino acid substitutions, amino-terminal truncations, carboxy-terminal truncations, deletions, or additions. Any biologically active fragment and / or mutant will encode both a protein and a protein fragment which at least substantially mimic the immunological properties of the HPV 52 L1 protein as shown in SEQ ID NO: 2. The synthetic polynucleotides of the present invention encode mRNA molecules that express a functional HPV 52 L1 protein for use in the development of a therapeutic or prophylactic HPV vaccine.

One aspect of this invention is a codon-optimized nucleic acid molecule which encodes the HPV 52 L1 protein as shown in SEQ ID NO: 2, said nucleic acid molecule comprises a sequence of nucleotides that are codon optimized for the expression of high levels in yeast cells. In a preferred embodiment of this aspect of the invention, the nucleic acid molecule comprises a nucleotide sequence as described in SEQ ID NO: 1. The present invention also relates to recombinant vectors and recombinant host cells, both prokaryotes and eukaryotes, which contain the nucleic acid molecules described through this specification. In a preferred embodiment of this invention, the host cell is a yeast host cell. Synthetic HPV 52 L1 DNA, and functional equivalents thereof, and fragments thereof, constructed through the methods described herein can be recombinantly expressed by molecular cloning in an expression vector containing a suitable promoter and other transcriptional regulatory elements. adequate. Said expression vector can be transferred into prokaryotic and eukaryotic host cells to produce the recombinant protein HPV 52 L1. Techniques for manipulations are fully described in the art (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, (1989), Current Protocols in Molecular Biology, Ausubel et al., Green Pub. Associates and Wiley-lnterscience, New York (1988), Yeast Genetics: A Laboratory Course Manual, Rose et al., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, (1990), which are incorporated herein. integrity by reference) Although, the present invention relates to a method for expressing the HPV 52 L1 protein in a recombinant host cell, it comprises: (a) introducing a vector comprising a nucleic acid encoding a protein HPV 52 L1 in a yeast host cell; and (b) culturing a yeast host cell under conditions that allow the expression of said HPV 52 L1 protein. The present invention further relates to a method for expressing an HPV 52 L1 protein in a recombinant host cell, comprising: (a) introducing a vector comprising a nucleic acid encoding an HPV 52 L1 protein in a yeast host cell; wherein the nucleic acid molecule is a codon optimized for optimal expression in a yeast host cell and; (b) culture of the yeast host cell under conditions that allow the expression of said HPV 52 L1 protein. The invention further relates to a method for expressing a HPV 52 L1 protein in a recombinant host cell, comprising: (a) introducing a vector comprising a nucleic acid as described in SEQ ID NO: 1 into a host cell of yeast; and, (b) culturing in a yeast host cell under conditions that allow the expression of said HPV 52 L1 protein. The synthetic genes of the present invention can be assembled into an expression cassette comprising the sequences designated to give efficient expression of the HPV 52 L1 protein in a host cell. The cassette preferably contains the synthetic gene, with the transcriptional and translational control sequences operably linked thereto, such as the promoter, and the termination sequences. In a preferred embodiment, the promoter is the S. cerevisiae GAL1 promoter, therefore those skilled in the art recognize that any of the other yeast promoters known as the GAL10, GAL7, ADH1, TDH3 or PGK promoters, or other gene promoters. Eukaryotes can be used. A preferred transcriptional terminator is the terminator S. cerevisiae ADH1, although other transcriptional terminators may be used. The combination of the GAL1-terminator ADH1 promoter is particularly preferred. Another aspect of this invention is a virus-like particle (VLP) HPV 52 produced by recombinant expression of the HPV 52 L1 or L1 + L2 genes in a yeast cell, the production methods of the HPV 52 VLPs, and the methods of Use of HPV VLPs 52. VLPs can self-assemble when L1, the papillomavirus major capsid protein of animals and humans, is expressed in yeast, insect cells, mammalian cells or bacteria (for review, see Schiller and Roden, in Papillomavirus Reviews: Current Research on Papillomaviruses; Lacey, ed. Leeds, UK: Leeds Medical Information, pp 101-12 (1996)). Morphologically indistinct HPV VLPs can be produced by expressing a combination of capsid proteins L1 and L2. The VLPs are composed of 72 pentamers of L1 in a icosahedral structure T = 7 (Baker et al., Biophys., J. 60 (6): 1445-56 (1991)). VLPs are morphologically similar to authentic virions and are capable of inducing high titers of antibody neutralization upon administration in an animal. Immunization of rabbits (Breitburd et al., J. Virol. 69 (6): 3959-63 (1995)) and dogs (Suzich et al., Proc. Nati. Acad. Sci. USA 92 (25): 1553 -57 (1995)) with VLPs shows that both induce neutralizing antibodies and protect against experimental infection of papillomaviruses. Additionally, immunization of adult women with HPV 16 VLPs protects against HPV 16 infection and HPV 16 cervical intraepithelial neoplasia (Koutsky et al., Engl. J. Med. 347: 1645-51 (2002)). Because VLPs contain no potentially oncogenic viral genome and can self-assemble when expressed from a single gene, they present a safe alternative for the use of live viruses in the development of HPV vaccines (for review, see Schiller and Hidesheim, J. Clin. Virol. 19: 67-74 (2000)). Accordingly, the present invention relates to virus-like particles comprising a recombinant L1 protein or recombinant proteins L1 + L2 of HPV 52, where the recombinant protein is expressed in a yeast cell. As stated above, in a preferred embodiment of the invention, HPV 52 VLPs are produced in yeast. In another embodiment, the yeast is selected from the group consisting of: Saccharomyces cerevisiae, Hansenula polymorpha, Pichia pastoris, Kluyveromyces fragilis, Kluyveromyces lactis, and Schizosaccharomyces pombe. Another aspect of this invention is a HPV 52 VLP which comprises an HPV 52 L1 protein produced by a codon-optimized HPV 52 L1 gene. In a preferred embodiment of this aspect of the invention, the codon-optimized HPV 52 L1 gene comprises a nucleotide sequence as described in SEQ ID NO: 1. In yet another aspect of this invention is a method of producing HPV 52 VLPs, comprising: (a) yeast transformed with a recombinant DNA molecule encoding the HPV 52 L1 protein or the HPV 52 L1 + L2 proteins; (b) culture of the transformed yeast under conditions that allow the expression of the recombinant DNA molecule to produce the recombinant protein HPV 52; and (c) isolation of the recombinant protein HPV 52 to produce HPV52 VLPs. In a preferred embodiment of this aspect of the invention, the protein is transformed with an optimized codon gene HPV 52 L1 to produce VLPs HPV 52. In a preferred preferred embodiment, the HPV 52 L1 codon-optimized gene comprises a nucleotide sequence as described in SEQ ID NO: 1. This invention also provides a method for inducing an immune response in an animal comprising the administration of virus-like particles.

HPV 52 to the animal. In a preferred embodiment, HPV 52 VLPs are produced by recombinant expression of a codon-optimized gene encoding HPV 52 L1 or HPV 52 L1 + L2. In still another aspect of the invention is a method for the prevention and / or treatment of cervical cancer associated with HPV comprising the administration to a mammal of a vaccine comprising HPV 52 VLPs. In a preferred embodiment of this invention, the VLPs HPV 52 are produced in yeast. This invention also relates to a vaccine comprising HPV 52 virus-like particles (VLPs).

In an alternative embodiment of this aspect, the vaccine further comprises VLPs of at least one additional type of HPV. In a preferred embodiment, at least one additional HPV type is selected from the group consisting of: HPV 6, HPV 11, HPV 16, HPV 18, HPV 31, HPV 33, HPV 35, HPV 39, HPV 45, HPV 51, HPV 55, HPV 56, HPV 58, HPV 59, and HPV 68. In a preferred embodiment of this aspect of the invention, the vaccine further comprises HPV VLPs 16. In another preferred embodiment, the vaccine further comprises HPV 16 VLPs and HPV 18 VLPs. In still another preferred embodiment of the invention, the vaccine further comprises HPV 6 VLPs, VLPs HPV 11, HPV 16 VLPs and HPV VLPs 18. This invention also relates to pharmaceutical compositions comprising HPV 52 virus-like particles. In addition, this invention relates to pharmaceutical compositions comprising HPV 52 VLPs and VLPs of at least one additional type of HPV. In a preferred embodiment, at least one additional type of HPV is selected from the group consisting of: HPV 6, HPV 11, HPV 16, HPV 18, HPV 31, HPV 33, HPV 35, HPV 39, HPV 45, HPV 51, HPV 55, HPV 56, HPV 58, HPV 59, and HPV 68. The vaccine compositions of The present invention can be used alone in appropriate doses which allow optimal inhibition of HPV 52 infection with minimal potential toxicity. In additional, co-administration or sequential administration of other agents may be desirable.

The amount of virus-like particles that are introduced into a vaccine container will depend on the immunogenicity of the product of the expressed gene. In general, an immunologically or prophylactically effective dose of from about 10 μg to 100 μg, and preferably from about 20 μ to 60 μg of VLPs are administered directly to the muscle tissue. Subcutaneous injection, intradermal introduction, impression through the skin, and other modes of administration such as intraperitoneal, intravenous, or inhalation are also contemplated. It is also contemplated that vaccination amplification can be provided. It is also an advantage, parental administration, such as intravenous, subcutaneous intramuscular or others such as administration with adjuvants such as alum or Merck alum adjuvant, concurrently with or subsequent to the parenteral introduction of the vaccine of this invention. All publications mentioned herein are incorporated by reference for the purpose of describing and disclosing methodologies and materials that may be used in connection with the present invention. Nothing here is interpreted as a request for the invention if it is not entirely novel as claimed by the virtue of previous inventions. Having described the preferred embodiments of the invention with reference to the accompanying drawings, it is understood that the invention is not limited to those precise embodiments, and that various changes and modifications can be made by an expert in the material without departing from the scope or scope of the invention. spirit of the invention as defined in the claims. The following examples illustrate, but do not limit the invention.

EXAMPLE 1 Determination of a representative HPV 52 L1 sequence The HPV 52 L1 sequence has been previously described (Genbank Accession # NC 001592). It is common, however, to find smaller variations in DNA sequences obtained from clinical isolates. To determine a representative wild-type HPV 52 L1 sequence, the DNA was isolated from three clinical examples previously shown to contain HPV 52 DNA. HPV 52 L1 sequences were amplified in a polymerase chain reaction (PCR) using Taq DNA polymerase and the following primers: 5'L15'-ATGTCCGTGTGGCGG CCTAGT-3 '(SEQ ID NO: 4) and 3"52BgMI5'-GAGATCTCAATTACACAA AGTG-3' (SEQ ID NO: 5) The amplified products were subjected to a gel electrophoresis of agarose and were visualized by staining with ethidium bromide.The bands ~ 1500 bp L1 were cut and the DNA was purified using the Geneclean Spin kit (Q-Bio Gene, Carlsbad, CA) .The DNA was then ligated to a cloned vector TA, pCR2.1 (Invitrogen) The TOP10F 'E. coli cells were transformed with the ligation mixture and placed on an LB agar with kanamicin plus IPTG and X-gal for the selection of blue / white colonies. and incubated for 16 hours at 37 ° C. The PCR colony was developed in five white colonies originating each of the three amplified clinical isolates. The 5 'L1 and 3' 52 Bgl II primers were used in a two-step PCR in which the first step comprised 10 cycles of 96 ° C for 15 seconds (denaturation), 55 ° C for 30 seconds (alignment) and 68 cycles. ° C for 2 minutes (extension), and the second step comprised 35 cycles of an essentially similar program, except that the alignment step was developed at 50 ° C for 30 seconds. The PCR products were electrophoresed on agarose gels and visualized with ethidium bromide staining. Several colonies of each clinical isolate contained amplified products with bands of -1500 bp. The colonies were cultivated in LB medium with canamicin, shaking at 37 ° C for 16 hours. Minipreps were developed to extract the plasmid DNAs, which were digested with restriction endonuclease to demonstrate the presence of the L1 gene in the plasmid. The resulting restriction fragments were visualized, by electrophoresis in agarose gel and staining with ethidium bromide. DNA sequencing was developed on plasmids containing L1 inserts cloned from each of the three clinical isolates. DNA and sequencing was developed on plasmids containing L1 inserts cloned from each of the three clinical isolates. DNA and translated amino acid sequences were compared with each other and with the previously published Genbank HPV 52 L1 sequences. Sequence analyzes of the three clinical isolates revealed that no sequence was identical to the Genbank sequence (Accession No. NC 001592). The pCR2.1 HPV 52L1 # 2C clone was chosen as the HPV 52 L1 representative sequence and is referred to herein as the "wild type 52 L1 sequence" (SEQ ID NO: 3, see Figures 1A-1C). The sequence chosen as 52 L1 wild-type (wt) contains a mutation site when compared to the Genbank sequence, which consisted of a silent mutation at nucleotide 1308 (guanine adenine). The amino acid sequence of the HPV 52 L1 wt sequence was identical to the 52 L1 Genbank sequence.

Wild-type HPV 52 L1 sequence was amplified using the 5 'L1 Bgl II primer (5'-GAGATCTCACAAAACAAAATG TCCGTGTGGC-3' (SEQ ID NO: 6)) and the 3 '52 Bgl II primer described above to add the Bgl II extensions . The PCR was developed using Taq polymerase. The PCR product was subjected to electrophoresis on an agarose gel and visualized with an ethidium bromide stain. The ~ 1500 bp band was cut and DNA purified using the Geneclean Spin kit (Q-Bio Gene, Carlsbad, CA). The PCR product was then ligated to the vector pCR2.1 and the TOP10F 'cells were transformed with a ligation mixture. The white colonies were cultivated in LB medium with canamicin, were stirred at 37 ° C for 16 hours. Minipreps were developed to extract plasmid DNA. The HPV 52 L1 gene was freed from the vector sequences with the digestion of the Bgl II restriction endonucleases. The digested DNA was identified with agarose gel electrophoresis and visualized with ethidium bromide staining. The L1 band was purified using the Geneclean kit and ligated to a dephosphorylated, Sa / nH I-digested vector pGAL110. TOP10F E. coli cells were transformed with the ligation mixture. To project the HPV 52 L1 insert in the correct orientation, the plasmid DNA of the colonies was amplified in PCR. DNA sequencing led to confirming the sequence and orientation of the inserts. The selected clone was named pGAL1 10-HPV 52L1 # 5. Maxiprep DNA was prepared from the selected clone. The Saccharomyces cerevisiae cells were made competent by transformation into spheroplasts with glusulase and transformed with pGAL110-HPV 52L1 # 5. The yeast transformation mixture was placed in Leu sorbitol top agar plates and incubated inverted for 3-5 days at 30 ° C. Colonies were collected and streaked for isolation in Leu sorbitol dishes. The isolated colonies were subsequently grown in 5 ml of 5 X Leu-Adepo-sorbitol with 1.6% glucose and 4% galactose in rotary culture tubes at 30 ° C to induce transcription of HPV 52 L1 and the expression of the protein.

EXAMPLE 2 Optimization of the yeast codon The preferred codons of yeast have been described (Sharp, Paul M and Cowe, Elizabeth.) Synonymous Codon Usage in Saccharomyces cerevisiae YEAST 7: 657-678 (1991)). The expression of the HPV 52 L1 wt protein was detected; however, the level of transcription was very low and was not detected by Northern analysis. It was postulated that the termination of premature transcription may be responsible for the low levels of expression of the HPV 52 L1 gene. To increase the transcription of this gene and ensure that the full length of the transcriptionists would be produced, the HPV 52 L1 gene was reconstructed using the preferred yeast codons. The sequence was inspected to present yeast transcription termination signals that are recognized by yeast, and these sequences were removed by substitution with alternative codons, while preserving the same amino acid sequence. The reconstructed HPV 52 L1 sequence, which comprises optimized codon sequences of yeast, contains 379 nucleotide alterations compared to the HPV 52 L1 wt sequence. The resulting sequence is referred to herein as "52 L1 R" (R = reconstructed, see Figures 1A-1 C). Nucleotide alterations between the 52 L1 wt (SEQ ID NO: 3) and the 52 L1 R sequences (SEQ ID NO: 1) are shown in Figures 1A-1C. The translated amino acid sequence of 58L1R is not altered (SEQ ID NO: 2, see figures 2A-2C). The reconstructed sequence provides an expression of the increased HPV 52 L1 protein, which is a significant advance over the wild type for use in the development of vaccines. The strategy employed to produce the optimized gene was to design long overlapping oligomers with sense and antisense that span the gene, replacing the nucleotides with codon sequences preferably from yeast while maintaining the amino acid sequence. These oligomers were used in the positioning of the DNA template in a PCR reaction with Pfu DNA polymerase. The additional amplification of the primers was designated and used to amplify the reconstructed sequences from template oligomers. The optimal conditions for the amplification were section specific; however, most reactions used a program at 94 ° C for 5 minutes (denaturation) followed by 25 cycles of 95 ° C for 30 sec. (denaturation), 50-55 ° C for 30 sec. (alignment), 72 ° C for 1.5 minutes (extension), followed by a final extension at 72 ° C for 7 minutes and maintenance at 4 ° C. The PCR products were examined by agarose gel electrophoresis. Bands of appropriate size were examined by agarose gel electrophoresis. Bands of appropriate size were cut and the DNA was purified from the gel. The amplified fragments were then used as templates to join the reconstructed 1512 nt HPV 52 L1 gene.

The next reconstruction, the 1512 nt band was gel purified, and ligated to a pCR4 Blunt vector (Invitrogen, Carlsbad, CA). The next ligation, competent TOP10 E. coli cells were transformed with ligation mixture. The colonies grew in 4 ml LB with ampicillin and the plasmid DNA was extracted from colonies by miniprep techniques. The plasmid DNA was sequenced to confirm the presence of the desired changes in the reconstructed HPV 52 L1. To add the SamHI extensions to both terminations, the 52 L1 R (reconstructed) was re-amplified from pCR4Blunt-52 L1 R. The amplified fragment was cloned as above and the resulting plasmid DNA was sequenced. The plasmid, pCR4 Blunt-52 L1 R (Bam) was digested with SamHI and the inserts of the resulting DNA fragment were electrophoresed on an agarose gel. The -1530 bp HPV 52 L1 R (Bam) fragment was purified and ligated to a SamHI-digested pGAL1 10. The TOP10F 'E.coli cells (Invitrogen) were transformed with the ligation mixture. The resulting colonies were projected by PCR for the HPV 52 L1 R insert in the correct orientation. The sequence and orientation was confirmed by DNA sequencing. Maxiprep of plasmid DNA was prepared. The cells of S. cerevisiae were made competent by spheroplasty and transformation. The yeast transformation was placed on Leu sorbitol top agar in Lys sorbitol agar dishes and incubated inverted for 7 days. The colonies were collected and streaked, for clonal isolation in Lys sorbitol agar dishes. The isolated colonies subsequently grew in 5 ml of sorbitol 5 X Leu-Ade- with 1.6% glucose and 4% galactose in rotating tube culture at 30 ° C to induce transcription of L1 and protein expression. After 48 and / or 72 hours, an equivalent volume of culture at OD ODOO = 10 was agglomerated, and supernatant was removed and the agglomerates were frozen and stored at -70 ° C.

EXAMPLE 3 Preparation of RNA Cell pellets of transformed yeast induced to express HPV 52 L1 by galactose were thawed on ice, suspended in 0.8 ml of Trizol reagent (Life Technologies, Gibco BRL) and incubated at room temperature for 5 minutes. One fifth of the volume of chloroform was added to the vial. Then it was stirred vigorously for 15 seconds to mix and incubate at room temperature for 3 minutes. After centrifuging 5 minutes at 13 k rpms, the upper phase was collected and transferred to a new vial. The mixture was incubated at room temperature for 10 minutes. The supernatant was decanted, the RNA pellet was washed with 75% EtOH and the centrifugation step was repeated. The supernatant was decanted and the RNA pellet was air dried for 15 minutes followed by suspension in RNase-free water. Spectrophotometry was developed to determine the concentration of RNA in the sample using the assumption that an A260 reading of RNA 1 = 40 Dg / ml when the A260 / 280 is 1.7-2.0.

EXAMPLE 4 Northern Analysis A 1.1% formaldehyde agarose gel was spread. Five and ten micrograms of RNA were combined with denatured buffer (final concentrations: 6% formaldehyde, 50% formamide and 0.1 x MOPS) and heated at 65 ° C for 10 minutes. One-tenth volume of the gel loading buffer was added and the sample loaded on the gel. Electrophoresis was developed at 75 volts in 1 x MOPS buffer for - 3 hours. The gel was washed for 60 minutes in 10 x SSC. The RNA was transferred to a Hybond-N + nylon membrane (Amersham Biosciences, Piscataway, NJ) by capillary action for 16 hours in 10 x SSC. The RNA was then fixed to the nylon membrane by cross-links using the Stratagene UV Stratalinker with self-cross functions (Stratagene, LA Jolla, CA). After fixation, the nylon membrane was left in dry air. The Roche DIG High Prime DNA detection and marking kit I (Hoffmann-La Roche Ltd., Basel, Switzerland) was used to label 52 L1 wt DNA sequences and 52 L1 R with DIG to be used as a test to detect RNA 52 L1 wt and 52 L1 R RNA in Northern analysis. The prehybridization, hybridization, and immunological development using an alkaline phosphatase-conjugated anti-DIG antibody were developed as recommended by the producers. In brief, the analysis was prehybridized by shaking at 37 ° C for 30 minutes. The test was denatured by heat at 95 ° C for 5 minutes and subsequently the reaction was stopped on ice. The test was added to the hybridization solution and applied to the membrane for 4 hours at 44.6 ° C with considerable stirring. The hybridization solution was removed and the analysis was washed 2 x for 5 minutes in 2 x SSC with 0.1% SDS at room temperature, followed by an additional wash at 65 ° C with 0.5 x SSC and 0.1% SDS. The analysis was then blocked for 30 minutes and the anti-DIG alkaline phosphatase-conjugated antibody was applied at a 1: 5000 dilution for 30 minutes. The analysis washed the presence of an RNA bound to the test was determined by detecting NBT / BCIP substrate of the antibody-bound anti-DIG conjugated alkaline phosphatase. Initial analyzes of the expression of yeast HPV 52 L1 wt suggested that the HPV 52 L1 protein was expressed; however, the level was low. Northern RNA analysis of yeast extracts from cultures to express HPV 52 L1 wt did not reveal any detectable HPV 52 L1 RNA. Since some protein of appropriate size was detected, it was clear that full-length RNA transcripts were made. The HPV 52 L1 gene was reconstructed with codon sequences preferentially from yeast and modified to omit any site of premature termination of transcription to ensure robust transcription. Northern analsis of the HPV 52 L1 R transcriptor revealed that the full-length transcripts were generated and detected by Northern analysis (figure 3).

EXAMPLE 5 Expression of the HPV 52 L1 protein Agglomerates of frozen yeast cells from galactose-induced cultures equivalent to OD600 = 10 were thawed on ice and suspended in 300 μl of PC buffer (100 mM Na2HP? 4 and 0.5 M NaCl, pH 7.0) with 2mM PMSF. Acid washes of 0.5mm glass beads at a concentration of - 0.5g / tube were added. The tubes were placed in a vortex in 3 cycles of 5 minutes at 4 ° C with 1 min rest. 7.5 μl of 20% TritonX100 was added and the vortex passage was repeated for 5 minutes at 4 ° C. The tubes were placed on ice for 15 minutes, followed by centrifugation for 10 minutes at 4 ° C. The supernatant was transferred to a microcentrifuge tube, which was labeled as a yeast protein extract, stained, and stored at -70 ° C.

EXAMPLE 6 Western Analysis The total yeast protein extract of twenty yeast colonies isolated from each HPV 52 L1 construct was analyzed by Western analysis to confirm the expression of the HPV 52 L1 protein after galactose induction. Ten, five, and two and a half micrograms of the total yeast protein extract were combined with the SDS-PAGE loaded buffer and heated at 95 ° C for minutes. The HPV 16 L1 protein, which is approximately 55 kDa, was included as a positive control, together with total protein extract HPV L1-free as negative control (data not shown). Proteins were loaded on 10% SDS-PAGE gel and electrophoresed in Tris-Glycine buffer. After separation of the protein, the proteins were transferred to Western from the gel to nitrocellulose and the resulting analysis was blocked in 1 x buffer dilution (Kirkegaard and Perry Laboratories, Gaithersburg, MD) for 1 hour at room temperature with one movement of oscillation. The analysis was washed three times and the yeast was absorbed in goat anti-trpE-HPV 31 L1 serum, which cross-reacted with the proteins HPV 16 and HPV 52 L1, was applied at room temperature for 16 hours. Then the blot was washed three times and incubated with a 1: 2500 dilution of anti-goat conjugate antibody-HRP for 1 hr. The analysis was then washed three times and the detection of the substrate NBT / BCIP (Kirkegaard and Perry Laboratories) was applied. The immunoreactive proteins were detected in violet bands in the analysis. In all cases, the HPV 52 L1 protein was detected as a distinct immunoreactive band in the nitrocellulose corresponds to approximately 55 kDa. (Figure 4) The intensity of the HPV 52 L1 R band (2.5 μg line) appeared to be more significant than the HPV 52 L1 wt band (10 μg). It is clear that on reconstruction, the level of expression of the codon-optimized HPV 52 L1 R was increased more than four times, which is the limit of the direct comparison of the Western analysis.

EXAMPLE 7 Transmission electron microscope To demonstrate that 52 L1 protein was indeed self-assembling to form L1 pentameric capsomers, which self-assemble into virus-like particles, the partially purified protein extract was subjected to transmission electron microscopy (TEM).

The yeast grew in a small-scale burner and was grouped. The resulting groupings were subjected to purification treatments. Grouping and clarified yeast extracts were analyzed by immunoblot to demonstrate the expression of the HPV 52 L1 protein and retention through the purification procedure. The clarified yeast extracts were centrifuged on a 45% -sucrose band and the resulting agglomerate was suspended in buffer for the analysis of HPV 52 L1 VLPs by TEM. A representative sample of the produced HPV 52 L1 R VLPs is shown in Figure 5. The diameter of the spherical particles in this crude sample is in a range between 40 and 70 nm with some particles developing a regular matrix of capsomeres.

Claims (28)

NOVELTY OF THE INVENTION CLAIMS

1. - A nucleic acid molecule comprising a sequence of nucleotides encoding an HPV52 L1 protein as shown in SEQ ID NO: 2, the codon-optimized nucleic acid sequence being for a high level of expression in a yeast cell .

2. A vector comprising the nucleic acid molecule of claim 1.

3.- A host cell comprising the vector of claim 2.

4. The host cell according to claim 3, further characterized in that the cell guest is a yeast cell.

5. The host cell according to claim 4, further characterized in that the yeast cell is selected from the group consisting of: Saccharomyces cerevisiae, Hansenula polymorpha, Pichia pastoris, Kluyveromyces fragilis, Kluyveromyces lactis, and Schizosaccharomyces pombe.

6. The host cell according to claim 4, further characterized in that the host cell is Saccharomyces cerevisiae.

7. The nucleic acid molecule according to claim 1, further characterized in that the nucleotide sequence comprises a nucleotide sequence as indicated in SEQ ID NO: 1.

8. Virus-like particles (VLPs) comprising the recombinant protein L1 or the recombinant proteins L1 + L2 of HPV52, where the recombinant protein L1 or the recombinant proteins L1 + L2 are produced in yeast.

9. The VLPs according to claim 8, further characterized in that the recombinant protein L1 or the recombinant proteins L1 + L2 are encoded by the codon optimized nucleic acid molecule HPV52 L1.

10. The VLPs according to claim 9, further characterized in that the codon-optimized nucleic acid molecule comprises a nucleotide sequence as described in SEQ ID NO: 1.

11. A method for producing the VLPs of claim 9, comprising: (a) transforming the yeast with a codon-optimized DNA molecule encoding the HPV52 L1 protein or the HPV52 L1 + L2 proteins; (b) culture of the transformed yeast under conditions that allow the expression of the codon-optimized DNA molecule to produce the recombinant papillomavirus protein; and (c) isolation of the recombinant papillomavirus protein to produce the VLPs of claim 9.

12. A vaccine comprising the VLPs of claim 9.

13. The pharmaceutical compositions comprising the VLPs of claim 9.

14 The use of the VLPs claimed in claim 9 for manufacturing a vaccine for the prevention of HPV infection in a mammal.

15. The use of the VLPs claimed in claim 11 for the manufacture of a medicament for inducing an immune response in an animal.

16. - The virus-like particles according to claim 9, further characterized in that the yeast is selected from the group consisting of Saccharomyces cerevisiae, Hansenula polymorpha, Pichia pastoris, Kluyveromyces fragilis, Kluyveromyces lactis, and Schizosaccharomyces pombe.

17. The virus-like particles according to claim 16, further characterized in that the yeast is Saccharomyces cerevisiae.

18. The vaccine according to claim 12, further characterized in that it also comprises the VLPs of at least one type an additional HPV type.

19. The vaccine according to claim 18 further characterized in that at least one additional HPV type is selected from the group consisting of: HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV39, HPV45, HPV51, HPV55, HPV56 , HPV58, HPV59, and HPV68.

20. The vaccine according to claim 19, further characterized in that at least one type of HPV comprising HPV16.

21. The vaccine according to claim 20, further characterized in that it also comprises HPV18 VLPs.

22. The vaccine according to claim 21, further characterized in that it also comprises HPV6 and HPV11 VLPs.

23. The vaccine according to claim 22, further characterized in that it also comprises HPV31 VLPs.

24. - The vaccine according to claim 21, further characterized in that it also comprises HPV31 VLPs.

25. The vaccine according to claim 23, further characterized in that it also comprises HPV45 VLPs.

26. The vaccine according to claim 24, further characterized in that it also comprises HPV45 VLPs.

27. The vaccine according to claim 26, further characterized in that it also comprises HPV58 VLPs.

28. The vaccine according to claim 25, further characterized in that it also comprises HPV58 VLPs.