Improved vaccines for human papillomavirus and methods of use thereof

Abstract

Improved vaccines for human papillomavirus and methods of use thereof are disclosed. Specifically disclosed are improved anti-HPV immunogens and nucleic acid molecules encoding the same. The disclosed immunogens include those having consensus HPV6 E6E7, HPV11 E6E7, HPV16 E6E7, HPV18 E6E7, HPV31 E6E7, HPV33 E6E7, HPV39 E6E7, HPV45 E6E7, HPV52 E6E7, and HPV58 E6E7. Pharmaceutical compositions, recombinant vaccines comprising DNA plasmids, and live attenuated vaccines are disclosed, as are methods of inducing an immune response against HPV in an individual.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201480013180.5 entitled “An improved vaccine for human papillomavirus and a method of administration thereof”, filed on March 12, 2014. Invention Field

[0002] This invention relates to an improved human papillomavirus (HPV) vaccine, an improved method for inducing an immune response, and an improved method for prophylactic and / or therapeutic immunization of individuals against HPV. Background of the Invention

[0003] Papillomaviruses are small DNA viruses containing up to seven early genes and two late genes. Typically, early papillomavirus genes are denoted as E1-E7, and late papillomavirus genes are denoted as L1 and L2. Animals of several species can be infected by members of the papillomavirus family.

[0004] Human papillomavirus (HPV) infection is common and sexually transmitted. Based on DNA sequence homology, HPV has been classified into 56 or more types. Types 16 and 18 of HPV (which cause epithelial dysplasia and other lesions) are often associated with an increased risk of cancer, particularly in situ and invasive carcinomas of the cervix, vagina, vulva, and anal canal. Nearly 88% of cervical cancers worldwide are the result of HPV subtypes 16, 18, 45, 31, 33, 52, and 58. Furthermore, various studies have revealed the presence of HPV 6 and HPV 11 in most cases of recurrent respiratory papillomatosis. Although the association of HPV 6 and HPV 11 with genital warts is known and they have been found in a small percentage of cervical cancer cases, HPV 6 and HPV 11 have been found in 2.6% to 5.2% of all cases of low-grade cervical lesions. Furthermore, HPV6 and HPV11 have now been found to be associated with approximately 20% of low-grade squamous intraepithelial lesions currently considered precursors to cervical cancer, including grade 2 and 3 cervical intraepithelial neoplasia and mild cervical dysplasia. Further research has revealed associations between these two serotypes and various forms of otolaryngological diseases, including genital warts, recurrent respiratory papillomatosis, lung cancer, tonsil cancer, laryngeal cancer, and other malignant transformations and dysplasia of otherwise benign head and neck tumors. The findings of this study reveal a promising future for using shared sequence DNA vaccines to combat HPV6 and HPV11.

[0005] DNA vaccines offer several conceptual advantages over more traditional vaccination methods, such as live attenuated viruses and recombinant protein-based vaccines. DNA vaccines are safe, stable, easy to produce, and well-tolerated in humans, with preclinical studies indicating minimal plasmid integration [Martin, T. et al., Plasmid DNA malaria vaccine: the potential for genomic integration after intramuscular injection. Hum Gene Ther, 1999.10(5): 759-68; Nichols, W.W. et al., Potential DNA vaccine integration into host cell genome. Ann NY Acad Sci, 1995.772: 30-9]. Furthermore, DNA vaccines are well-suited for repeated administration because their effectiveness is virtually unaffected by pre-existing antibody titers on the vector [Chattergoon, M., J. Boyer, and D.B. Weiner, Genetic immunization: a new era in vaccines and immunetherapeutics. FASEB J, 1997.11(10): 753-63]. However, a major obstacle to the clinical use of DNA vaccines in larger animals is the reduced platform immunogenicity [Liu, MA and JBUlmer, Human clinicaltrials of plasmid DNA vaccines. Adv Genet, 2005, 55: 25-40]. Recent technological advances in the engineering of DNA vaccine immunogens, such as codon optimization, RNA optimization, and the addition of immunoglobulin leader sequences, have improved the expression and immunogenicity of DNA vaccines [Andre, S. et al., Increased immune response elicited by DNA vaccination with a synthetic gp120 sequence with optimized codon usage. J Virol, 1998, 72(2): 1497-503; Deml, L.].[References: Multiple effects of codon usage optimization on expression and immunogenicity of DNA candidate vaccines encoding the human immunodeficiency virus type 1 Gag protein. J Virol, 2001, 75(22): 10991-1001; Laddy, DJ et al., Immunogenicity of novel consensus-based DNA vaccines against avian influenza. Vaccine, 2007, 25(16): 2984-9; Frelin, L. et al., Codon optimization and mRNA amplification effectively enhances the immunogenicity of the hepatitis C virus nonstructural 3 / 4A gene. Gene Ther, 2004, 11(6): 522-33], and recent developments in plasmid delivery systems such as electroporation [Hirao, LA et al., Intradermal / subcutaneous immunization by electroporation improves splasmid vaccine delivery and potency in pigs and rhesus], macaques.Vaccine, 2008.26(3): pp. 440-8; Luckay, A. et al., Effect of plasmid DNA vaccine design and invivo electroporation on the resulting vaccine-specific immune responses inrhesus macaques.J Virol, 2007.81(10): pp. 5257-69; Ahlen, G.[Yan, J. et al., In vivo electrophoresis enhances the immunogenicity of hepatitis C virus nonstructural 3 / 4A DNA by increased local DNA uptake, protein expression, inflammation, and infiltration of CD3+ T cells. J Immunol, 2007, 179(7): 4741-53]. Furthermore, studies have shown that the use of shared immunogens can increase the breadth of cellular immune responses compared to natural antigens alone [Yan, J. et al., Enhanced cellular immune responses elicited by an engineered HIV-1 subtype B consensus-based envelope DNA vaccine. Mol Ther, 2007, 15(2): 411-21; Rolland, M. et al., Reconstruction and function of ancestral center-of-tree human immunodeficiency virus type 1 proteins. J Virol, 2007, 81(16): 8507-14].

[0006] There remains a need for improved vaccines and methods for the prevention and treatment of HPV infection, particularly that that causes cervical and / or lung cancer, tonsil cancer, and laryngeal cancer. Invention Overview

[0007] An aspect of the present invention provides a composition comprising at least one nucleotide sequence comprising an HPV E6-E7 fusion antigen selected from the group consisting of: HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV39, HPV45, HPV52 and HPV58.

[0008] On the other hand, a composition comprising one or more nucleotide sequences is provided, said nucleotide sequences encoding HPV E6-E7 fusion antigen selected from the group consisting of: a nucleotide sequence encoding SEQ ID NO:2; a nucleotide sequence encoding SEQ ID NO:4; a nucleotide sequence encoding SEQ ID NO:6; a nucleotide sequence encoding SEQ ID NO:8; a nucleotide sequence encoding SEQ ID NO:18; a nucleotide sequence encoding SEQ ID NO:20; a nucleotide sequence encoding SEQ ID NO:22; a nucleotide sequence encoding SEQ ID NO:24; a nucleotide sequence encoding SEQ ID NO:25; a nucleotide sequence encoding SEQ ID NO:26; a nucleic acid sequence having at least 95% homology with the nucleotide sequence encoding SEQ ID NO:2; a nucleic acid sequence having at least 95% homology with the nucleotide sequence encoding SEQ ID NO:4; a nucleic acid sequence having at least 95% homology with the nucleotide sequence encoding SEQ ID NO:6; a nucleic acid sequence having at least 95% homology with the nucleotide sequence encoding SEQ ID NO:8; a nucleic acid sequence having at least 95% homology with the nucleotide sequence encoding SEQ ID NO:18; a nucleic acid sequence having at least 95% homology with the nucleotide sequence encoding SEQ ID NO:20; a nucleic acid sequence encoding SEQ ID NO:22; a nucleotide sequence encoding SEQ ID NO:24; a nucleotide sequence encoding SEQ ID NO:25; a nucleotide sequence encoding SEQ ID NO:26; a nucleic acid sequence having at least 95% homology with the nucleotide sequence encoding SEQ ID NO:20 ... Nucleic acid sequences having at least 95% homology to the nucleotide sequence encoding SEQ ID NO:20; nucleotide sequences having at least 95% homology to the nucleotide sequence encoding SEQ ID NO:22; nucleotide sequences having at least 95% homology to the nucleotide sequence encoding SEQ ID NO:24; nucleotide sequences having at least 95% homology to the nucleotide sequence encoding SEQ ID NO:25; nucleotide sequences having at least 95% homology to the nucleotide sequence encoding SEQ ID NO:26; fragments encoding the nucleotide sequence of SEQ ID NO:2; fragments encoding the nucleotide sequence of SEQ ID NO:4; fragments encoding the nucleotide sequence of SEQ ID NO:6; fragments encoding the nucleotide sequence of SEQ ID NO:8; fragments encoding the nucleotide sequence of SEQ ID NO:18; fragments encoding the nucleotide sequence of SEQ ID NO:20; fragments encoding the nucleotide sequence of SEQ ID NO:22; fragments encoding the nucleotide sequence of SEQ ID NO:24; fragments encoding the nucleotide sequence of SEQ ID NO:25; fragments encoding the nucleotide sequence of SEQ ID NO:26; and fragments encoding the nucleotide sequence of SEQ ID NO:20. The nucleotide sequence fragment encoding NO:2 has at least 95% homology with the nucleic acid sequence encoding SEQ ID NO:4; and the nucleotide sequence encoding SEQ ID NO:6 has at least 95% homology with the nucleic acid sequence encoding SEQ ID NO:6.The nucleic acid sequence having at least 95% homology with the fragment encoding the nucleotide sequence of SEQ ID NO: 8; the nucleic acid sequence having at least 95% homology with the fragment encoding the nucleotide sequence of SEQ ID NO: 18; the nucleic acid sequence having at least 95% homology with the fragment encoding the nucleotide sequence of SEQ ID NO: 20; the nucleic acid sequence having at least 95% homology with the fragment encoding the nucleotide sequence of SEQ ID NO: 22; the nucleic acid sequence having at least 95% homology with the fragment encoding the nucleotide sequence of SEQ ID NO: 24; the nucleic acid sequence having at least 95% homology with the fragment encoding the nucleotide sequence of SEQ ID NO: 25; and the nucleic acid sequence having at least 95% homology with the fragment encoding the nucleotide sequence of SEQ ID NO: 26. In some embodiments, the nucleotide sequence encoding the HPV E6-E7 fusion antigen does not have a leader sequence encoding the nucleotide sequence of SEQ ID NO: 10 at its 5' end.

[0009] In another aspect of the invention, a composition comprising one or more nucleotide sequences is provided, said nucleotide sequences encoding an HPV E6-E7 fusion antigen selected from the group consisting of: SEQ ID NO:1; SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:17; SEQ ID NO:19; SEQ ID NO:21; SEQ ID NO:23; a nucleic acid sequence having at least 95% homology with SEQ ID NO:1; a nucleic acid sequence having at least 95% homology with SEQ ID NO:3; a nucleic acid sequence having at least 95% homology with SEQ ID NO:5; a nucleic acid sequence having at least 95% homology with SEQ ID NO:7; a nucleic acid sequence having at least 95% homology with SEQ ID NO:17; a nucleic acid sequence having at least 95% homology with SEQ ID NO:19; a nucleic acid sequence having at least 95% homology with SEQ ID NO:21; a nucleic acid sequence having at least 95% homology with SEQ ID NO:23; a fragment of SEQ ID NO:1; a fragment of SEQ ID NO:3; SEQ ID NO:1; a fragment of SEQ ID NO:3; a fragment of SEQ ID NO:23; a fragment of ... Fragment NO:5; Fragment NO:7; Fragment NO:17; Fragment NO:19; Fragment NO:21; Fragment NO:23; Nucleic acid sequence having at least 95% homology with the fragment NO:1; Nucleic acid sequence having at least 95% homology with the fragment NO:3; Nucleic acid sequence having at least 95% homology with the fragment NO:5; Nucleic acid sequence having at least 95% homology with the fragment NO:7; Nucleic acid sequence having at least 95% homology with the fragment NO:17; Nucleic acid sequence having at least 95% homology with the fragment NO:19; Nucleic acid sequence having at least 95% homology with the fragment NO:21; and Nucleic acid sequence having at least 95% homology with the fragment NO:23. In some embodiments, the nucleotide sequence encoding the HPV E6-E7 fusion antigen does not have a leader sequence having the nucleotide sequence SEQ ID NO:9 at its 5' end.

[0010] The provided nucleotide sequence can be a plasmid.

[0011] In another aspect, pharmaceutical compositions comprising the disclosed nucleotide sequences are provided; preferably having a plurality of antigens.

[0012] In some aspects, there are methods for inducing an effective immune response against more than one subtype of HPV in an individual, comprising administering to the individual a composition comprising one or more provided nucleotide sequences; preferably, the composition has more than one antigen. The method preferably includes the step of introducing the provided nucleotide sequences into the individual via electroporation. Brief description of the attached diagram

[0013] Figures 1A-1B Phylogenetic trees evaluated based on adjacent-joint alignments from E6 and E7, respectively. An asterisk indicates the location of a common sequence on each tree. Figure 1C The diagram shows several modifications made after generating the shared E6 / E7 fusion sequence.

[0014] Figures 2A-2B In vivo expression of p6E6E7 and p11E6E7. Gene products were isolated from lysed transfected 293-T cells, passed through SDS-PAGE gels, and detected using autoradiography. Both HPV 6 and HPV 11E6 / E7 proteins were approximately 32 kDa each. Figure 2A Human rhabdomyosarcoma (RD) cells were also transfected with p6E6E7 and p11E6E7 and subsequently fixed after immunofluorescence staining. FITC fluorescence was used to determine the expression of p6E6E7 and p11E6E7. Figure 2B DAPI fluorescence determined nuclear localization after Hoescht staining.

[0015] Figures 3A-3B IFN-γ ELISApot assay showed p6E6E7 ( Figure 3A ) and p11E6E7 ( Figure 3B Inducing a robust cell-mediated response in C57BL / 6 mice. Assays were performed using spleen cells isolated from mice in each corresponding group (n=5 per group) following three immunizations every two weeks. Each immunization consisted of 20 μg of each construct. Mice in the combination groups received both p6E6E7 and p11E6E7 at 20 μg each, for a total of 40 μg DNA per immunization. DNA was administered via IM injection followed by electroporation (*indicating p<0.0001;). Indicate p = 0.0001).

[0016] Figures 4A-4B Additional IFN-γ ELISpot assays were performed using individual peptides to characterize dominant peptides. Spleen cells isolated from vaccinated mice and negative controls were analyzed using a assay covering the entire HPV 6E6 / E7 fusion protein (…). Figure 4A ) or HPV 11E6 / E7 fusion protein ( Figure 4B Overlapping peptide stimulation.

[0017] Figures 5A-5B Cytokines produced by antigen-specific T cells, characterized by intracellular cytokine staining. These cytokines will be generated from cells inoculated with p6E6E7 (…). Figure 5A ) and p11E6E7 ( Figure 5B Spleen cells isolated from mice were stimulated for 4 hours with R10 growth medium, PMA, or a shared gene peptide, followed by surface labeling and intracellular staining. The above dot plot shows the difference in the percentage of total CD4+ or CD8+ cells producing IFN-γ, IL-2, and TNF-α minus the background. Because the mean of the negative control group was zero, the p-values ​​of the points marked with an asterisk could not be determined.

[0018] Figure 6 The figure shows the amino acid sequence of HPV31 E6 / E7 (SEQ ID NO:18) with annotations illustrating the IgE leader sequence (IgEL), endonuclease cleavage sites, and sites where the E6 and E7 domains are mutated to enhance expression and immunogenicity. In the figure, the IgE leader sequence is underlined. Endonuclease cleavage sites are indicated by boxes. ▲ indicates sites where the deletion of the domain required for p53 binding / degradation occurs. Mutations at the binding sites of E6 protein with p53 and E7 protein with retinoblastoma (Rb) protein are indicated by *.

[0019] Figure 7 The figure shows the amino acid sequence of HPV52 E6 / E7 (SEQ ID NO:20) with annotations illustrating the IgE leader sequence (IgEL), endonuclease cleavage sites, and sites where the E6 and E7 domains are mutated to enhance expression and immunogenicity. In the figure, the IgE leader sequence is underlined. Endonuclease cleavage sites are indicated by boxes. ▲ indicates sites where the deletion of the domain required for p53 binding / degradation occurs. Mutations at the binding sites of E6 protein with p53 and E7 protein with retinoblastoma (Rb) protein are indicated by *.

[0020] Detailed description of the preferred implementation scheme

[0021] definition.

[0022] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Unless the context clearly requires otherwise, the singular forms “a,” “an,” and “the” as used in this specification and the appended claims include the plural indicators.

[0023] For the description of numerical ranges in this article, each intermediate number with the same precision is explicitly included. For example, for the range of 6 to 9, the numbers 7 and 8 are included in addition to 6 and 9, and for the range of 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly included.

[0024] adjuvant

[0025] As used herein, “adjuvant” can mean any molecule added to the DNA plasmid vaccine described herein to enhance the immunogenicity of one or more antigens encoded by the DNA plasmid and the nucleic acid sequence described below.

[0026] b. Antibodies

[0027] "Antibody" can refer to an antibody, fragment, or derivative thereof of class IgG, IgM, IgA, IgD, or IgE, including Fab, F(ab')2, Fd, as well as single-chain antibodies, bispecific antibodies, bifunctional antibodies, and their derivatives. The antibody may be an antibody isolated from a mammalian serum sample, a polyclonal antibody, an affinity-purified antibody, or a mixture thereof, exhibiting sufficient binding specificity to a desired epitope or a sequence derived therefrom.

[0028] c. Antigen

[0029] "Antigen" refers to a protein having an HPV E6 or HPV E7 domain, preferably fused to an endonuclease cleavage site between E6 and E7. Antigens include SEQ ID NO:2 (subtype 6), 4 (subtype 11), 6 (subtype 33), 8 (subtype 58), 18 (subtype 31), 20 (subtype 52), 22 (subtype 16), and 24 (subtype 18); fragments thereof, variants thereof, having the lengths described herein, i.e., proteins having sequences homologous to SEQ ID NO:2, 4, 6, 8, 18, 20, 22, and 24 as described herein, fragments of variants thereof, and combinations thereof. Antigens may have the IgE leader sequence of SEQ ID NO:10 or alternatively, the sequence may be removed from the N-terminus. Antigens may optionally include a signal peptide, such as those derived from other proteins.

[0030] d. Encoded sequence

[0031] As used herein, "coding sequence" or "coding nucleic acid" can mean a nucleic acid (RNA or DNA molecule) comprising a nucleotide sequence encoding an antigen as described in segment c above. The coding sequence may further include start and stop signals operatively linked to regulatory elements, including promoters and polyadenylation signals capable of directing expression in the cells of an individual or mammal to which the nucleic acid is administered. The coding sequence may also include a sequence encoding a signal peptide, such as an IgE leader sequence like SEQ ID NO:9.

[0032] e. Complementary sequences

[0033] As used herein, “complementary sequence” or “complementary” can refer to nucleic acids and can mean Watson-Crick (e.g., AT / U and CG) or Hoogsteen base pairings between nucleotides or nucleotide analogs of a nucleic acid molecule.

[0034] f. fragment

[0035] "Fragment" can refer to a polypeptide fragment of an antigen capable of evoking an immune response against the antigen in mammals. The fragment of an antigen may be 100% identical to the full-length antigen except for the deletion of at least one amino acid at the N and / or C-terminus, and in each case, may or may not contain a signal peptide and / or methionine at position 1. The fragment may comprise 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more of the length of a specific full-length antigen excluding any added heterologous signal peptides. The fragment may preferably comprise a fragment of a polypeptide having 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher homology to the antigen, and additionally includes an N-terminal methionine or heterologous signal peptide not included when calculating the percentage of homology. The fragment may also include an N-terminal methionine and / or a signal peptide such as an immunoglobulin signal peptide, for example, an IgE or IgG signal peptide. The N-terminal methionine and / or signal peptide may be linked to the fragment of the antigen.

[0036] The fragment encoding the nucleic acid sequence of the antigen may be 100% identical to the full-length sequence, except for the deletion of at least one nucleotide at the 5' and / or 3' ends, and in each case, may or may not contain a sequence encoding a signal peptide and / or methionine at position 1. The fragment may comprise 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more of the length of a specific full-length coding sequence excluding any added heterologous signal peptides. The fragment may preferably comprise a fragment encoding a polypeptide having 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology to the antigen, and optionally additionally comprise a sequence encoding an N-terminal methionine or heterologous signal peptide not included when calculating the percentage of homology. The fragment may also include the coding sequence for an N-terminal methionine and / or a signal peptide, such as an immunoglobulin signal peptide, for example, IgE or IgG signal peptide. The coding sequence encoding the N-terminal methionine and / or signal peptide may be linked to the fragment of the coding sequence.

[0037] g. the same

[0038] As used herein in the context of two or more nucleic acid or polypeptide sequences, the term "identical" or "identical" can mean that the sequences have a specified percentage of identical residues in a specified region. The percentage can be calculated as follows: optimally align the two sequences, compare the two sequences in a specified region, determine the number of positions in both sequences where the identical residues appear to obtain the number of matching positions, divide the number of matching positions by the total number of positions in the specified region, and multiply the result by 100 to obtain the percentage of sequence identity. In cases where the two sequences have different lengths or the alignment produces one or more staggered ends and the specified region being compared includes only a single sequence, the residues of the single sequence are included in the denominator, not the numerator, of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identification can be performed manually or using computer sequencing algorithms such as BLAST or BLAST 2.0.

[0039] h. Immune response

[0040] As used herein, “immune response” can refer to the activation of the host’s immune system, such as that of a mammal, in response to the introduction of one or more antigens via a provided DNA plasmid vaccine. An immune response can take the form of a cellular response, a humoral response, or both.

[0041] i. Nucleic acid

[0042] As used herein, “nucleic acid,” “oligonucleotide,” or “polynucleotide” can mean at least two nucleotides covalently linked together. The depiction of a single strand also defines the sequence of the complementary strand. Therefore, nucleic acid also encompasses the complementary strand of the depicted single strand. Many variants of nucleic acids can be used for the same purpose as a given nucleic acid. Therefore, nucleic acid also encompasses substantially the same nucleic acid and its complementary sequence. Single strands provide probes that can hybridize with the target sequence under strict hybridization conditions. Therefore, nucleic acid also encompasses probes that hybridize under strict hybridization conditions.

[0043] Nucleic acids can be single-stranded or double-stranded, or may contain portions of both double-stranded and single-stranded sequences. Nucleic acids can be DNA, genome and cDNA, RNA, or hybrids, wherein they may contain combinations of deoxyribonucleotides and ribonucleotides, as well as combinations of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. Nucleic acids can be obtained through chemical synthesis or recombinant methods.

[0044] j. Operable connection

[0045] As used herein, "operably linked" may mean that gene expression is controlled by a promoter to which it is spatially linked. The promoter may be located at the 5' (upstream) or 3' (downstream) of the gene it controls. The distance between the promoter and the gene may be approximately the same as the distance between the promoter and the gene it controls in the gene from which the promoter originates. As is known in the art, variations in this distance can be tolerated without loss of promoter function.

[0046] k. Promoter

[0047] As used herein, "promoter" can refer to a synthetic or naturally derived molecule capable of conferring, activating, or enhancing the expression of nucleic acids in a cell. A promoter may include one or more specific transcriptional regulatory sequences to further enhance the expression of the gene and / or alter its spatial and / or temporal expression. A promoter may also include distal enhancer or repressor elements located up to several thousand base pairs from the transcription start site. Promoters can be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. A promoter can constitutively or differentially regulate the expression of a genomic molecule relative to the cell, tissue, or organ in which expression occurs, relative to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stress, pathogens, metal ions, or inducers. Representative examples of promoters include the phage T7 promoter, phage T3 promoter, SP6 promoter, lac operon-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV40 late promoter, and CMV IE promoter.

[0048] l. Strict hybridization conditions

[0049] As used herein, “strict hybridization conditions” can mean conditions under which a first nucleic acid sequence (e.g., a probe) will hybridize with a second nucleic acid sequence (e.g., a target) as in a complex mixture of nucleic acids. Strict conditions are sequence-dependent and will vary under different conditions. Strict conditions can be selected to be about 5-10 °C lower than the thermal melting point (Tm) of a particular sequence at a defined ionic strength pH. Tm can be the temperature at which 50% of the probe complementary to the target hybridizes to the target sequence at equilibrium (because the target sequence is in excess, so at Tm, 50% of the probe is occupied at equilibrium). Strict conditions can be conditions where the salt concentration is below about 1.0 M sodium ions at pH 7.0 to 8.3, such as about 0.01-1.0 M sodium ion concentration (or other salts), and the temperature is at least about 30 °C (for short probes, e.g., about 10-50 nucleotides) and at least about 60 °C (for long probes, e.g., greater than about 50 nucleotides). Strict conditions can also be achieved by adding a destabilizing agent such as formamide. For selective or specific hybridization, the positive signal can be at least 2 to 10 times that of the background hybridization. Exemplary strict hybridization conditions include the following: 50% formamide, 5×SSC and 1% SDS, incubated at 42°C; or 5×SSC, 1% SDS, incubated at 65°C, wherein the mixture is washed at 65°C with 0.2×SSC and 0.1% SDS.

[0050] m. largely complementary

[0051] As used herein, “largely complementary” may mean that the complementary sequences of the first and second sequences have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity in regions of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleotides or amino acids, or that the two sequences hybridize under strict hybridization conditions.

[0052] n. roughly the same

[0053] As used herein, “substantially identical” may mean that the first and second sequences are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical in regions of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleotides or amino acids, or, in terms of nucleic acids, if the complementary sequences of the first and second sequences are substantially complementary, then the first and second sequences are substantially identical.

[0054] o. variant

[0055] The term “variant” as used in this article may mean (i) a portion or fragment of a reference nucleotide sequence; (ii) a complementary sequence of a reference nucleotide sequence or a portion thereof; (iii) a nucleic acid substantially identical to a reference nucleic acid or its complementary sequence; or (iv) a nucleic acid that hybridizes under stringent conditions with a reference nucleic acid, its complementary sequence, or a sequence substantially identical to it.

[0056] A “variant” of a peptide or polypeptide differs in its amino acid sequence through the insertion, deletion, or conserved substitution of amino acids, but retains at least one biological activity. A variant can also refer to a protein whose amino acid sequence is substantially the same as that of a reference protein having a certain amino acid sequence, while retaining at least one biological activity. Conserved substitution of amino acids, i.e., replacing an amino acid with a different amino acid having similar properties (e.g., hydrophilicity, degree and distribution of charged regions), is generally considered in the art to involve minor variations. These minor variations can be identified in part by taking into account the hydrophilicity index of the amino acid, as understood in the art. Kyte et al., J. Mol. Biol. 157:105-132 (1982). The hydrophilicity index of an amino acid is based on considerations of its hydrophobicity and charge. It is known in the art that amino acids with similar hydrophilicity indices can be substituted and still retain protein function. In one aspect, amino acids with a hydrophilicity index of ±2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that will produce proteins that retain biological function. Consideration of the hydrophilicity of amino acids in the context of peptides allows for the calculation of the maximum local average hydrophilicity of the peptide, a useful measurement reported to be well correlated with antigenicity and immunogenicity. U.S. Patent No. 4,554,101, incorporated herein by reference in its entirety. Substitution of amino acids with similar hydrophilicity values ​​can produce peptides that retain biological activity, such as immunogenicity, as understood in the art. Substitution of amino acids with hydrophilicity values ​​within ±2 can be made with each other. Both the hydrophobicity index and the hydrophilicity value of an amino acid are influenced by the specific side chain of that amino acid. Consistent with that observation, biocompatible amino acid substitutions are understood to depend on the relative similarity of the amino acids, and in particular the relative similarity of the side chains of those amino acids, as revealed by hydrophobicity, hydrophilicity, charge, size, and other properties.

[0057] p. carrier

[0058] The term "vector" as used in this article can refer to a nucleic acid sequence containing the origin of replication. Vectors can be plasmids, bacteriophages, bacterial artificial chromosomes, or yeast artificial chromosomes. Vectors can be DNA vectors or RNA vectors. Vectors can be autonomously replicating extrachromosomal vectors or vectors integrated into the host genome.

[0059] An improved vaccine derived from a multi-stage strategy to enhance immunogen-induced cellular immune responses is disclosed. A modified common sequence was generated. Genetic modifications, including codon optimization, RNA optimization, and the addition of highly efficient immunoglobulin leader sequences, are also disclosed. The novel construct has been designed to elicit a stronger and broader cellular immune response than the corresponding codon-optimized immunogen.

[0060] Improved HPV vaccines are based on proteins and gene constructs encoding proteins with epitopes that make them particularly effective as immunogens against HPV. Therefore, vaccines can induce therapeutic or prophylactic immune responses. In some embodiments, the immunogen is delivered as a DNA vaccine, a recombinant vaccine, a protein subunit vaccine, a composition containing an immunogen, a live attenuated vaccine, or an inactivated vaccine. In some embodiments, the vaccine comprises a combination selected from the group consisting of one or more DNA vaccines, one or more recombinant vaccines, one or more protein subunit vaccines, one or more compositions containing an immunogen, one or more live attenuated vaccines, and one or more inactivated vaccines.

[0061] According to some implementation schemes, vaccines are delivered to individuals to modulate the activity of the individual's immune system and thereby enhance the immune response against HPV. When nucleic acid molecules encoding proteins are taken up by the individual's cells, the nucleotide sequence is expressed in the cells and the protein is thus delivered to the individual. Methods for delivering protein-coding sequences on nucleic acid molecules such as plasmids (as part of recombinant vaccines and as part of attenuated vaccines, as isolated proteins, or as protein portions of vectors) are provided.

[0062] Compositions and methods are provided for individuals with prophylactic and / or therapeutic HPV immunity.

[0063] Compositions for delivering nucleic acid molecules comprising a nucleotide sequence encoding an immunogen are operatively linked to regulatory elements. The compositions may comprise: plasmids encoding an immunogen, recombinant vaccines comprising a nucleotide sequence encoding an immunogen, live attenuated pathogens encoding and / or comprising proteins of the present invention, inactivated pathogens comprising proteins of the present invention, or compositions comprising proteins of the present invention such as liposomes or subunit vaccines. The present invention also relates to injectable pharmaceutical compositions comprising the compositions.

[0064] This invention provides compositions comprising at least one nucleotide sequence comprising an HPV E6-E7 fusion antigen selected from the group consisting of HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV39, HPV45, HPV52, and HPV58. In some embodiments, the composition may comprise HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV52, and HPV58. In some embodiments, the composition may comprise HPV16, HPV18, HPV31, HPV33, HPV52, and HPV58. In some embodiments, the composition may comprise HPV6, HPV11, HPV16, and HPV18. In some embodiments, the composition may comprise HPV16, HPV31, and HPV52.

[0065] On the other hand, a composition comprising one or more nucleotide sequences is provided, said nucleotide sequences encoding HPV E6-E7 fusion antigen selected from the group consisting of: a nucleotide sequence encoding SEQ ID NO:2; a nucleotide sequence encoding SEQ ID NO:4; a nucleotide sequence encoding SEQ ID NO:6; a nucleotide sequence encoding SEQ ID NO:8; a nucleotide sequence encoding SEQ ID NO:18; a nucleotide sequence encoding SEQ ID NO:20; a nucleotide sequence encoding SEQ ID NO:22; a nucleotide sequence encoding SEQ ID NO:24; a nucleotide sequence encoding SEQ ID NO:25; a nucleotide sequence encoding SEQ ID NO:26; a nucleic acid sequence having at least 95% homology with the nucleotide sequence encoding SEQ ID NO:2; a nucleic acid sequence having at least 95% homology with the nucleotide sequence encoding SEQ ID NO:4; a nucleic acid sequence having at least 95% homology with the nucleotide sequence encoding SEQ ID NO:6; a nucleic acid sequence having at least 95% homology with the nucleotide sequence encoding SEQ ID NO:8; a nucleic acid sequence having at least 95% homology with the nucleotide sequence encoding SEQ ID NO:18; a nucleic acid sequence having at least 95% homology with the nucleotide sequence encoding SEQ ID NO:20; a nucleic acid sequence encoding SEQ ID NO:22; a nucleotide sequence encoding SEQ ID NO:24; a nucleotide sequence encoding SEQ ID NO:25; a nucleotide sequence encoding SEQ ID NO:26; a nucleic acid sequence having at least 95% homology with the nucleotide sequence encoding SEQ ID NO:20 ... Nucleic acid sequences having at least 95% homology to the nucleotide sequence encoding SEQ ID NO:20; nucleotide sequences having at least 95% homology to the nucleotide sequence encoding SEQ ID NO:22; nucleotide sequences having at least 95% homology to the nucleotide sequence encoding SEQ ID NO:24; nucleotide sequences having at least 95% homology to the nucleotide sequence encoding SEQ ID NO:25; nucleotide sequences having at least 95% homology to the nucleotide sequence encoding SEQ ID NO:26; fragments encoding the nucleotide sequence of SEQ ID NO:2; fragments encoding the nucleotide sequence of SEQ ID NO:4; fragments encoding the nucleotide sequence of SEQ ID NO:6; fragments encoding the nucleotide sequence of SEQ ID NO:8; fragments encoding the nucleotide sequence of SEQ ID NO:18; fragments encoding the nucleotide sequence of SEQ ID NO:20; fragments encoding the nucleotide sequence of SEQ ID NO:22; fragments encoding the nucleotide sequence of SEQ ID NO:24; fragments encoding the nucleotide sequence of SEQ ID NO:25; fragments encoding the nucleotide sequence of SEQ ID NO:26; and fragments encoding the nucleotide sequence of SEQ ID NO:20. The nucleotide sequence fragment encoding NO:2 has at least 95% homology with the nucleic acid sequence encoding SEQ ID NO:4; and the nucleotide sequence encoding SEQ ID NO:6 has at least 95% homology with the nucleic acid sequence encoding SEQ ID NO:6.Nucleic acid sequences having at least 95% homology to the fragment encoding the nucleotide sequence of SEQ ID NO: 8; nucleic acid sequences having at least 95% homology to the fragment encoding the nucleotide sequence of SEQ ID NO: 18; nucleic acid sequences having at least 95% homology to the fragment encoding the nucleotide sequence of SEQ ID NO: 20; nucleic acid sequences having at least 95% homology to the fragment encoding the nucleotide sequence of SEQ ID NO: 22; nucleic acid sequences having at least 95% homology to the fragment encoding the nucleotide sequence of SEQ ID NO: 24; nucleic acid sequences having at least 95% homology to the fragment encoding the nucleotide sequence of SEQ ID NO: 25; and nucleic acid sequences having at least 95% homology to the fragment encoding the nucleotide sequence of SEQ ID NO: 26.

[0066] In some embodiments, the composition comprises an HPV E6-E7 fusion antigen selected from the group consisting of: a nucleotide sequence encoding SEQ ID NO:2; a nucleotide sequence encoding SEQ ID NO:4; a nucleotide sequence encoding SEQ ID NO:6; a nucleotide sequence encoding SEQ ID NO:8; a nucleotide sequence encoding SEQ ID NO:18; a nucleotide sequence encoding SEQ ID NO:20; a nucleotide sequence encoding SEQ ID NO:22; a nucleotide sequence encoding SEQ ID NO:24; a nucleic acid sequence having at least 95% homology with the nucleotide sequence encoding SEQ ID NO:2; a nucleic acid sequence having at least 95% homology with the nucleotide sequence encoding SEQ ID NO:4; a nucleic acid sequence having at least 95% homology with the nucleotide sequence encoding SEQ ID NO:6; a nucleic acid sequence having at least 95% homology with the nucleotide sequence encoding SEQ ID NO:8; a nucleic acid sequence having at least 95% homology with the nucleotide sequence encoding SEQ ID NO:18; a nucleic acid sequence having at least 95% homology with the nucleotide sequence encoding SEQ ID NO:20; and a nucleic acid sequence having at least 95% homology with the nucleotide sequence encoding SEQ ID NO:24. Nucleic acid sequences having at least 95% homology to the nucleotide sequence encoding SEQ ID NO:22; nucleotide sequences having at least 95% homology to the nucleotide sequence encoding SEQ ID NO:24; fragments encoding the nucleotide sequence encoding SEQ ID NO:2; fragments encoding the nucleotide sequence encoding SEQ ID NO:4; fragments encoding the nucleotide sequence encoding SEQ ID NO:6; fragments encoding the nucleotide sequence encoding SEQ ID NO:8; fragments encoding the nucleotide sequence encoding SEQ ID NO:18; fragments encoding the nucleotide sequence encoding SEQ ID NO:20; fragments encoding the nucleotide sequence encoding SEQ ID NO:22; fragments encoding the nucleotide sequence encoding SEQ ID NO:24; nucleotide sequences having at least 95% homology to the fragments encoding the nucleotide sequence encoding SEQ ID NO:2; nucleotide sequences having at least 95% homology to the fragments encoding the nucleotide sequence encoding SEQ ID NO:4; nucleotide sequences having at least 95% homology to the fragments encoding the nucleotide sequence encoding SEQ ID NO:6; nucleotide sequences having at least 95% homology to the fragments encoding the nucleotide sequence encoding SEQ ID NO:8; fragments encoding the nucleotide sequence encoding SEQ ID NO:22; fragments encoding the nucleotide sequence encoding SEQ ID NO:24 ...5; fragments encoding the nucleotide sequence encoding SEQ ID NO:26; fragments encoding the nucleotide sequence encoding SEQ ID NO:27; fragments encoding the nucleotide sequence encoding SEQ ID NO:28; fragments encoding the nucleotide sequence encoding SEQ ID NO:29; fragments encoding the nucleotide sequence encoding SEQ ID NO:20; fragments encoding the nucleotide sequence encoding SEQ ID NO:22; fragments encoding the nucleotide sequence encoding The fragment of nucleotide sequence NO:18 has at least 95% homology with the nucleic acid sequence encoding SEQ ID NO:20; the fragment of nucleotide sequence encoding SEQ ID NO:22 has at least 95% homology with the nucleic acid sequence encoding SEQ ID NO:24; and the fragment of nucleotide sequence encoding SEQ ID NO:24 has at least 95% homology with the nucleic acid sequence encoding SEQ ID NO:24.

[0067] In some embodiments, the composition comprises an HPV E6-E7 fusion antigen selected from the group consisting of: a nucleotide sequence encoding SEQ ID NO:6; a nucleotide sequence encoding SEQ ID NO:8; a nucleotide sequence encoding SEQ ID NO:18; a nucleotide sequence encoding SEQ ID NO:20; a nucleotide sequence encoding SEQ ID NO:22; a nucleotide sequence encoding SEQ ID NO:24; a nucleic acid sequence having at least 95% homology with the nucleotide sequence encoding SEQ ID NO:6; a nucleic acid sequence having at least 95% homology with the nucleotide sequence encoding SEQ ID NO:8; a nucleic acid sequence having at least 95% homology with the nucleotide sequence encoding SEQ ID NO:18; a nucleic acid sequence having at least 95% homology with the nucleotide sequence encoding SEQ ID NO:20; a nucleic acid sequence having at least 95% homology with the nucleotide sequence encoding SEQ ID NO:22; a nucleic acid sequence having at least 95% homology with the nucleotide sequence encoding SEQ ID NO:24; a fragment encoding the nucleotide sequence of SEQ ID NO:6; a fragment encoding the nucleotide sequence of SEQ ID NO:8; a fragment encoding SEQ ID NO:6; a fragment encoding the nucleotide sequence of SEQ ID NO:24; a fragment encoding the nucleotide sequence of SEQ ID NO:6; a fragment encoding the nucleotide sequence of SEQ ID NO:8 ...24; a fragment encoding the nucleotide sequence of SEQ ID NO:25; a fragment encoding the nucleotide sequence of SEQ ID NO:26; a fragment encoding the nucleotide sequence of SEQ ID NO:27; a fragment encoding the nucleotide sequence of SEQ ID NO:28; a fragment encoding the nucleotide sequence Fragment encoding the nucleotide sequence of SEQ ID NO:18; fragment encoding the nucleotide sequence of SEQ ID NO:20; fragment encoding the nucleotide sequence of SEQ ID NO:22; fragment encoding the nucleotide sequence of SEQ ID NO:24; nucleic acid sequence having at least 95% homology with the fragment encoding the nucleotide sequence of SEQ ID NO:6; nucleic acid sequence having at least 95% homology with the fragment encoding the nucleotide sequence of SEQ ID NO:8; nucleic acid sequence having at least 95% homology with the fragment encoding the nucleotide sequence of SEQ ID NO:18; nucleic acid sequence having at least 95% homology with the fragment encoding the nucleotide sequence of SEQ ID NO:20; nucleic acid sequence having at least 95% homology with the fragment encoding the nucleotide sequence of SEQ ID NO:22; and nucleic acid sequence having at least 95% homology with the fragment encoding the nucleotide sequence of SEQ ID NO:24.

[0068] In some embodiments, the composition comprises an HPV E6-E7 fusion antigen selected from the group consisting of: a nucleotide sequence encoding SEQ ID NO:2; a nucleotide sequence encoding SEQ ID NO:4; a nucleotide sequence encoding SEQ ID NO:22; a nucleotide sequence encoding SEQ ID NO:24; a nucleic acid sequence having at least 95% homology to the nucleotide sequence encoding SEQ ID NO:2; a nucleic acid sequence having at least 95% homology to the nucleotide sequence encoding SEQ ID NO:4; a nucleic acid sequence having at least 95% homology to the nucleotide sequence encoding SEQ ID NO:22; a nucleic acid sequence having at least 95% homology to the nucleotide sequence encoding SEQ ID NO:24; a fragment encoding the nucleotide sequence of SEQ ID NO:2; a fragment encoding the nucleotide sequence of SEQ ID NO:4; a fragment encoding the nucleotide sequence of SEQ ID NO:22; a fragment encoding the nucleotide sequence of SEQ ID NO:24; a nucleic acid sequence having at least 95% homology to the fragment encoding the nucleotide sequence of SEQ ID NO:2; a fragment encoding the nucleotide sequence of SEQ ID NO:24; a fragment encoding the nucleotide sequence of SEQ ID NO:2 ... The fragment of nucleotide sequence NO:4 has at least 95% homology with the nucleic acid sequence encoding SEQ ID NO:22; and the fragment of nucleotide sequence encoding SEQ ID NO:24 has at least 95% homology with the nucleic acid sequence encoding SEQ ID NO:24.

[0069] In some embodiments, the composition comprises an HPV E6-E7 fusion antigen selected from the group consisting of: a nucleotide sequence encoding SEQ ID NO:18; a nucleotide sequence encoding SEQ ID NO:20; a nucleotide sequence encoding SEQ ID NO:22; a nucleic acid sequence having at least 95% homology to the nucleotide sequence encoding SEQ ID NO:18; a nucleic acid sequence having at least 95% homology to the nucleotide sequence encoding SEQ ID NO:20; a nucleic acid sequence having at least 95% homology to the nucleotide sequence encoding SEQ ID NO:22; a fragment encoding the nucleotide sequence of SEQ ID NO:18; a fragment encoding the nucleotide sequence of SEQ ID NO:20; a fragment encoding the nucleotide sequence of SEQ ID NO:22; a nucleic acid sequence having at least 95% homology to the fragment encoding the nucleotide sequence of SEQ ID NO:18; a nucleic acid sequence having at least 95% homology to the fragment encoding the nucleotide sequence of SEQ ID NO:20; and a nucleic acid sequence having at least 95% homology to the fragment encoding the nucleotide sequence of SEQ ID NO:22.

[0070] In another aspect of the invention, a composition comprising one or more nucleotide sequences is provided, said nucleotide sequences encoding an HPV E6-E7 fusion antigen selected from the group consisting of: SEQ ID NO:1; SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:17; SEQ ID NO:19; SEQ ID NO:21; SEQ ID NO:23; a nucleic acid sequence having at least 95% homology with SEQ ID NO:1; a nucleic acid sequence having at least 95% homology with SEQ ID NO:3; a nucleic acid sequence having at least 95% homology with SEQ ID NO:5; a nucleic acid sequence having at least 95% homology with SEQ ID NO:7; a nucleic acid sequence having at least 95% homology with SEQ ID NO:17; a nucleic acid sequence having at least 95% homology with SEQ ID NO:19; a nucleic acid sequence having at least 95% homology with SEQ ID NO:21; a nucleic acid sequence having at least 95% homology with SEQ ID NO:23; a fragment of SEQ ID NO:1; a fragment of SEQ ID NO:3; SEQ ID NO:1; a fragment of SEQ ID NO:3; a fragment of SEQ ID NO:23; a fragment of ... Fragment NO:5; Fragment NO:7; Fragment NO:17; Fragment NO:19; Fragment NO:21; Fragment NO:23; Nucleic acid sequence having at least 95% homology with the fragment NO:1; Nucleic acid sequence having at least 95% homology with the fragment NO:3; Nucleic acid sequence having at least 95% homology with the fragment NO:5; Nucleic acid sequence having at least 95% homology with the fragment NO:7; Nucleic acid sequence having at least 95% homology with the fragment NO:17; Nucleic acid sequence having at least 95% homology with the fragment NO:19; Nucleic acid sequence having at least 95% homology with the fragment NO:21; and Nucleic acid sequence having at least 95% homology with the fragment NO:23.

[0071] In some embodiments, the composition comprises an HPV E6-E7 fusion antigen selected from the group consisting of: SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:17; SEQ ID NO:19; SEQ ID NO:21; SEQ ID NO:23; a nucleic acid sequence having at least 95% homology with SEQ ID NO:5; a nucleic acid sequence having at least 95% homology with SEQ ID NO:7; a nucleic acid sequence having at least 95% homology with SEQ ID NO:17; a nucleic acid sequence having at least 95% homology with SEQ ID NO:19; a nucleic acid sequence having at least 95% homology with SEQ ID NO:21; a nucleic acid sequence having at least 95% homology with SEQ ID NO:23; a fragment of SEQ ID NO:5; a fragment of SEQ ID NO:7; a fragment of SEQ ID NO:17; a fragment of SEQ ID NO:19; a fragment of SEQ ID NO:21; a fragment of SEQ ID NO:23; a nucleic acid sequence having at least 95% homology with the fragment of SEQ ID NO:5; a nucleic acid sequence having at least 95% homology with SEQ ID NO:5; a nucleic acid sequence having at least 95% homology with SEQ ID NO:21; a nucleic acid sequence having at least 95% homology with SEQ ID NO:23; a fragment of SEQ ID NO:5; a fragment of SEQ ID NO:7; a fragment of SEQ ID NO:17; a fragment of SEQ ID NO:19; a fragment of SEQ ID NO:21; a fragment of SEQ ID NO:23; a nucleic acid sequence having at least 95% homology with the fragment of SEQ ID NO:5 ... The fragment NO:7 has a nucleic acid sequence with at least 95% homology; the fragment NO:17 has a nucleic acid sequence with at least 95% homology; the fragment NO:19 has a nucleic acid sequence with at least 95% homology; the fragment NO:21 has a nucleic acid sequence with at least 95% homology; and the fragment NO:23 has a nucleic acid sequence with at least 95% homology.

[0072] In some embodiments, the composition comprises an HPV E6-E7 fusion antigen selected from the group consisting of: SEQ ID NO:1; SEQ ID NO:3; SEQ ID NO:21; SEQ ID NO:23; a nucleic acid sequence having at least 95% homology with SEQ ID NO:1; a nucleic acid sequence having at least 95% homology with SEQ ID NO:3; a nucleic acid sequence having at least 95% homology with SEQ ID NO:21; a nucleic acid sequence having at least 95% homology with SEQ ID NO:23; a fragment of SEQ ID NO:1; a fragment of SEQ ID NO:3; a fragment of SEQ ID NO:21; a fragment of SEQ ID NO:23; a nucleic acid sequence having at least 95% homology with the fragment of SEQ ID NO:1; a nucleic acid sequence having at least 95% homology with the fragment of SEQ ID NO:3; a nucleic acid sequence having at least 95% homology with the fragment of SEQ ID NO:21; and a nucleic acid sequence having at least 95% homology with the fragment of SEQ ID NO:23.

[0073] In some embodiments, the composition comprises an HPV E6-E7 fusion antigen selected from the group consisting of: SEQ ID NO:17; SEQ ID NO:19; SEQ ID NO:21; a nucleic acid sequence having at least 95% homology with SEQ ID NO:17; a nucleic acid sequence having at least 95% homology with SEQ ID NO:19; a nucleic acid sequence having at least 95% homology with SEQ ID NO:21; a fragment of SEQ ID NO:17; a fragment of SEQ ID NO:19; a fragment of SEQ ID NO:21; a nucleic acid sequence having at least 95% homology with the fragment of SEQ ID NO:17; a nucleic acid sequence having at least 95% homology with the fragment of SEQ ID NO:19; and a nucleic acid sequence having at least 95% homology with the fragment of SEQ ID NO:21.

[0074] In some embodiments, the nucleotide sequences described herein do not contain a leader sequence. Nucleotide sequences comprising HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV39, HPV45, HPV52, and HPV58 do not contain a leader sequence. Specifically, HPV E6-E7 fusion antigens including the nucleotide sequence encoding SEQ ID NO:2; the nucleotide sequence encoding SEQ ID NO:4; the nucleotide sequence encoding SEQ ID NO:6; the nucleotide sequence encoding SEQ ID NO:8; the nucleotide sequence encoding SEQ ID NO:18; the nucleotide sequence encoding SEQ ID NO:20; the nucleotide sequence encoding SEQ ID NO:22; the nucleotide sequence encoding SEQ ID NO:24; and the nucleotide sequence encoding SEQ ID NO:25 do not contain a leader sequence at the 5' end, for example, the nucleotide sequence encoding SEQ ID NO.10. Specifically, HPV E6-E7 fusion antigens including nucleotide sequences SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, and SEQ ID NO:23 do not have a leader sequence at the 5' end, such as nucleotide sequence SEQ ID NO.9.

[0075] In some embodiments, the nucleotide sequence of the present invention may have 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology with the provided nucleotide sequence; preferably 95%, 96%, 97%, 98%, or 99%; or 98% or 99%.

[0076] The provided nucleotide sequence may be included in one of a variety of known vectors or delivery systems, including plasmids, viral vectors, lipid vectors, and nanoparticles; preferably plasmids.

[0077] In another aspect, pharmaceutical compositions comprising the disclosed nucleotide sequences are provided; preferably having a plurality of antigens.

[0078] In some aspects, there are methods for inducing an effective immune response against more than one subtype of HPV in an individual, comprising administering to the individual a composition comprising one or more provided nucleotide sequences; preferably, the composition has more than one antigen. The method preferably includes the step of introducing the provided nucleotide sequences into the individual via electroporation.

[0079] SEQ ID NO:1 contains the nucleotide sequence encoding a common immunogen of HPV 6E6 and E7 proteins. SEQ ID NO:1 includes an IgE leader sequence SEQ ID NO:9, which is a nucleotide sequence linked to the 5' end of SEQ ID NO:1. SEQ ID NO:2 contains the amino acid sequence of a common immunogen of HPV 6E6 and E7 proteins. SEQ ID NO:2 includes an IgE leader sequence SEQ ID NO:10 located at the N-terminus of the common immunogen sequence. The IgE leader sequence is SEQ ID NO:10 and may be encoded by SEQ ID NO:9.

[0080] In some implementations, the vaccine includes SEQ ID NO:2 or a nucleic acid molecule encoding SEQ ID NO:2.

[0081] The fragment of SEQ ID NO:2 may be 100% identical to the full length except for the deletion of at least one amino acid at the N and / or C terminus, and in each case, may or may not contain a signal peptide and / or methionine at position 1. The fragment of SEQ ID NO:2 may comprise 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more of the length of the full length of SEQ ID NO:2 excluding any added heterologous signal peptides. The fragment may preferably comprise a fragment of SEQ ID NO:2 having 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher homology with SEQ ID NO:2, and additionally comprises an N-terminal methionine or heterologous signal peptide not included when calculating the percentage of homology. The fragment may also comprise an N-terminal methionine and / or a signal peptide such as an immunoglobulin signal peptide, for example, an IgE or IgG signal peptide. The N-terminal methionine and / or signal peptide may be linked to the fragment.

[0082] The fragment of the nucleic acid sequence SEQ ID NO:1 may be 100% identical to the full-length sequence except for the deletion of at least one nucleotide at the 5' and / or 3' ends, and in each case, may or may not contain a sequence encoding a signal peptide and / or methionine at position 1. The fragment may comprise 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more of the length of the full-length coding sequence SEQ ID NO:1 excluding any added heterologous signal peptides. The fragment may preferably comprise a fragment encoding a polypeptide having 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher homology with antigen SEQ ID NO:2, and optionally additionally comprise a sequence encoding an N-terminal methionine or a heterologous signal peptide not included when calculating the percentage of homology. The fragment may also comprise sequences encoding N-terminal methionines and / or signal peptides such as immunoglobulin signal peptides, for example, IgE or IgG signal peptides. The coding sequences encoding N-terminal methionines and / or signal peptides may be linked to the fragment.

[0083] In some embodiments, the fragment of SEQ ID NO:1 may contain 786 or more nucleotides; in some embodiments, it may contain 830 or more nucleotides; in some embodiments, it may contain 856 or more nucleotides; and in some embodiments, it may contain 865 or more nucleotides. In some embodiments, the fragment of SEQ ID NO:1, as those listed herein, may also contain the coding sequence of the IgE leader sequence. In some embodiments, the fragment of SEQ ID NO:1 does not contain the coding sequence of the IgE leader sequence.

[0084] In some embodiments, the fragment of SEQ ID NO:2 may contain 252 or more amino acids; in some embodiments, it may contain 266 or more amino acids; in some embodiments, it may contain 275 or more amino acids; and in some embodiments, it may contain 278 or more amino acids.

[0085] SEQ ID NO:3 contains the nucleotide sequence encoding a common immunogen of HPV 11E6 and E7 proteins. SEQ ID NO:3 includes an IgE leader sequence SEQ ID NO:9, which is a nucleotide sequence linked to the 5' end of SEQ ID NO:3. SEQ ID NO:4 contains the amino acid sequence of a common immunogen of HPV 11E6 and E7 proteins. SEQ ID NO:4 includes an IgE leader sequence SEQ ID NO:10 located at the N-terminus of the common immunogen sequence. The IgE leader sequence is SEQ ID NO:10 and may be encoded by SEQ ID NO:9.

[0086] In some implementations, the vaccine includes SEQ ID NO:4 or a nucleic acid molecule encoding SEQ ID NO:4.

[0087] The fragment of SEQ ID NO:4 may be 100% identical to the full length except for the deletion of at least one amino acid at the N and / or C terminus, and in each case, may or may not contain a signal peptide and / or methionine at position 1. The fragment of SEQ ID NO:4 may comprise 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more of the length of the full length of SEQ ID NO:4 excluding any added heterologous signal peptides. The fragment may preferably comprise a fragment of SEQ ID NO:4 having 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher homology with SEQ ID NO:4, and additionally comprises an N-terminal methionine or heterologous signal peptide not included when calculating the percentage of homology. The fragment may also comprise an N-terminal methionine and / or a signal peptide such as an immunoglobulin signal peptide, for example, an IgE or IgG signal peptide. The N-terminal methionine and / or signal peptide may be linked to the fragment.

[0088] The fragment of the nucleic acid sequence SEQ ID NO:3 may be 100% identical to the full-length sequence except for the deletion of at least one nucleotide at the 5' and / or 3' ends, and in each case, may or may not contain a sequence encoding a signal peptide and / or methionine at position 1. The fragment may comprise 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more of the length of the full-length coding sequence SEQ ID NO:3 excluding any added heterologous signal peptides. The fragment may preferably comprise a fragment encoding a polypeptide having 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher homology with antigen SEQ ID NO:4, and optionally additionally comprise a sequence encoding an N-terminal methionine or a heterologous signal peptide not included when calculating the percentage of homology. The fragment may also comprise sequences encoding N-terminal methionines and / or signal peptides such as immunoglobulin signal peptides, for example, IgE or IgG signal peptides. The coding sequences encoding N-terminal methionines and / or signal peptides may be linked to the fragment.

[0089] In some embodiments, the fragment of SEQ ID NO:3 may contain 786 or more nucleotides; in some embodiments, it may contain 830 or more nucleotides; in some embodiments, it may contain 856 or more nucleotides; and in some embodiments, it may contain 865 or more nucleotides. In some embodiments, the fragment of SEQ ID NO:3, as those listed herein, may also contain the coding sequence of the IgE leader sequence. In some embodiments, the fragment of SEQ ID NO:3 does not contain the coding sequence of the IgE leader sequence.

[0090] In some embodiments, the fragment of SEQ ID NO:4 may contain 252 or more amino acids; in some embodiments, it may contain 266 or more amino acids; in some embodiments, it may contain 275 or more amino acids; and in some embodiments, it may contain 278 or more amino acids.

[0091] SEQ ID NO:5 contains the nucleotide sequence encoding the common immunogen of HPV 33E6 and E7 proteins. SEQ ID NO:5 contains the IgE leader sequence SEQ ID NO:9, which is the nucleotide sequence linked to the 5' end of SEQ ID NO:5. SEQ ID NO:6 contains the amino acid sequence of the common immunogen of HPV 33E6 and E7 proteins. SEQ ID NO:6 includes the IgE leader sequence SEQ ID NO:10, located at the N-terminus of the common immunogen sequence. The IgE leader sequence is SEQ ID NO:10 and may be encoded by SEQ ID NO:9.

[0092] In some implementations, the vaccine includes SEQ ID NO:6 or a nucleic acid molecule encoding SEQ ID NO:6.

[0093] The fragment of SEQ ID NO:6 may be 100% identical to the full length except for the deletion of at least one amino acid at the N and / or C terminus, and in each case, may or may not contain a signal peptide and / or methionine at position 1. The fragment of SEQ ID NO:6 may comprise 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more of the length of the full length of SEQ ID NO:6 excluding any added heterologous signal peptides. The fragment may preferably comprise a fragment of SEQ ID NO:6 having 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher homology with SEQ ID NO:6, and additionally comprises an N-terminal methionine or heterologous signal peptide not included when calculating the percentage of homology. The fragment may also comprise an N-terminal methionine and / or a signal peptide such as an immunoglobulin signal peptide, for example, an IgE or IgG signal peptide. The N-terminal methionine and / or signal peptide may be linked to the fragment.

[0094] The fragment of the nucleic acid sequence SEQ ID NO:5 may be 100% identical to the full length except for the deletion of at least one nucleotide at the 5' and / or 3' ends, and in each case, may or may not have a sequence encoding a signal peptide and / or methionine at position 1. The fragment may comprise 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more of the length of the full-length coding sequence SEQ ID NO:5 excluding any added heterologous signal peptides. The fragment may preferably comprise a fragment encoding a polypeptide having 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher homology with antigen SEQ ID NO:6, and optionally additionally comprise a sequence encoding an N-terminal methionine or a heterologous signal peptide not included when calculating the percentage of homology. The fragment may also comprise coding sequences for N-terminal methionines and / or signal peptides such as immunoglobulin signal peptides, for example, IgE or IgG signal peptides. The coding sequences for N-terminal methionines and / or signal peptides may be linked to the fragment.

[0095] In some embodiments, the fragment of SEQ ID NO:5 may contain 746 or more nucleotides; in some embodiments, it may contain 787 or more nucleotides; in some embodiments, it may contain 812 or more nucleotides; and in some embodiments, it may contain 820 or more nucleotides. In some embodiments, the fragment of SEQ ID NO:5, as those listed herein, may also contain the coding sequence of the IgE leader sequence. In some embodiments, the fragment of SEQ ID NO:5 does not contain the coding sequence of the IgE leader sequence.

[0096] In some embodiments, the fragment of SEQ ID NO:6 may contain 235 or more amino acids; in some embodiments, it may contain 248 or more amino acids; in some embodiments, it may contain 256 or more amino acids; and in some embodiments, it may contain 259 or more amino acids.

[0097] SEQ ID NO:7 contains the nucleotide sequence encoding the common immunogen of HPV 58E6 and E7 proteins. SEQ ID NO:7 includes an IgE leader sequence SEQ ID NO:9, which is the nucleotide sequence linked to the 5' end of SEQ ID NO:7. SEQ ID NO:8 contains the amino acid sequence of the common immunogen of HPV 58E6 and E7 proteins. SEQ ID NO:8 includes an IgE leader sequence SEQ ID NO:10 located at the N-terminus of the common immunogen sequence. The IgE leader sequence is SEQ ID NO:10 and may be encoded by SEQ ID NO:9.

[0098] In some implementations, the vaccine includes SEQ ID NO:8 or a nucleic acid molecule encoding SEQ ID NO:8.

[0099] The fragment of SEQ ID NO:8 may be 100% identical to the full length except for the deletion of at least one amino acid at the N and / or C terminus, and in each case, may or may not contain a signal peptide and / or methionine at position 1. The fragment of SEQ ID NO:8 may comprise 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more of the length of the full-length SEQ ID NO:8 excluding any added heterologous signal peptides. The fragment may preferably comprise a fragment of SEQ ID NO:8 having 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher homology with SEQ ID NO:8, and additionally comprises an N-terminal methionine or heterologous signal peptide not included when calculating the percentage of homology. The fragment may also comprise an N-terminal methionine and / or a signal peptide such as an immunoglobulin signal peptide, for example, an IgE or IgG signal peptide. The N-terminal methionine and / or signal peptide may be linked to the fragment.

[0100] The fragment of the nucleic acid sequence SEQ ID NO:7 may be 100% identical to the full-length sequence except for the deletion of at least one nucleotide at the 5' and / or 3' ends, and in each case, may or may not contain a sequence encoding a signal peptide and / or methionine at position 1. The fragment may comprise 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more of the length of the full-length coding sequence SEQ ID NO:7 excluding any added heterologous signal peptides. The fragment may preferably comprise a fragment encoding a polypeptide having 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher homology with antigen SEQ ID NO:8, and optionally additionally comprise a sequence encoding an N-terminal methionine or a heterologous signal peptide not included when calculating the percentage of homology. The fragment may also comprise encoding sequences of N-terminal methionines and / or signal peptides such as immunoglobulin signal peptides, for example, IgE or IgG signal peptides. The encoding sequences of N-terminal methionines and / or signal peptides may be linked to the fragment.

[0101] In some embodiments, the fragment of SEQ ID NO:7 may contain 752 or more nucleotides; in some embodiments, it may contain 794 or more nucleotides; in some embodiments, it may contain 819 or more nucleotides; and in some embodiments, it may contain 827 or more nucleotides. In some embodiments, the fragment of SEQ ID NO:7, as those listed herein, may also contain the coding sequence of the IgE leader sequence. In some embodiments, the fragment of SEQ ID NO:7 does not contain the coding sequence of the IgE leader sequence.

[0102] In some embodiments, the fragment of SEQ ID NO:8 may contain 235 or more amino acids; in some embodiments, it may contain 249 or more amino acids; in some embodiments, it may contain 257 or more amino acids; and in some embodiments, it may contain 260 or more amino acids.

[0103] SEQ ID NO:17 contains the nucleotide sequence encoding a common immunogen of HPV 31E6 and E7 proteins. SEQ ID NO:17 includes an IgE leader sequence SEQ ID NO:9, which is a nucleotide sequence linked to the 5' end of SEQ ID NO:17. SEQ ID NO:18 contains the amino acid sequence of a common immunogen of HPV 31E6 and E7 proteins. SEQ ID NO:18 includes an IgE leader sequence SEQ ID NO:10 located at the N-terminus of the common immunogen sequence. The IgE leader sequence is SEQ ID NO:10 and may be encoded by SEQ ID NO:9.

[0104] In some implementations, the vaccine includes SEQ ID NO:18 or a nucleic acid molecule encoding SEQ ID NO:18.

[0105] The fragment of SEQ ID NO:18 may be 100% identical to the full length except for the deletion of at least one amino acid at the N and / or C terminus, and in each case, may or may not contain a signal peptide and / or methionine at position 1. The fragment of SEQ ID NO:18 may comprise 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more of the length of the full-length SEQ ID NO:18 excluding any added heterologous signal peptides. The fragment may preferably comprise a fragment of SEQ ID NO:18 having 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher homology with SEQ ID NO:18, and additionally comprises an N-terminal methionine or heterologous signal peptide not included when calculating the percentage of homology. The fragment may also comprise an N-terminal methionine and / or a signal peptide such as an immunoglobulin signal peptide, for example, an IgE or IgG signal peptide. The N-terminal methionine and / or signal peptide may be linked to the fragment.

[0106] The fragment of the nucleic acid sequence SEQ ID NO:17 may be 100% identical to the full length except for the deletion of at least one nucleotide at the 5' and / or 3' ends, and in each case, may or may not have a sequence encoding a signal peptide and / or methionine at position 1. The fragment may comprise 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more of the length of the full-length coding sequence SEQ ID NO:17 excluding any added heterologous signal peptides. The fragment may preferably comprise a fragment encoding a polypeptide having 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher homology with the antigen SEQ ID NO:18, and optionally additionally comprise a sequence encoding an N-terminal methionine or a heterologous signal peptide not included when calculating the percentage of homology. The fragment may also comprise sequences encoding N-terminal methionines and / or signal peptides such as immunoglobulin signal peptides, for example, IgE or IgG signal peptides. The coding sequences encoding N-terminal methionines and / or signal peptides may be linked to the fragment.

[0107] In some embodiments, the fragment of SEQ ID NO:17 may contain 713 or more nucleotides; in some embodiments, it may contain 752 or more nucleotides; in some embodiments, it may contain 776 or more nucleotides; and in some embodiments, it may contain 784 or more nucleotides. In some embodiments, the fragment of SEQ ID NO:17, as those listed herein, may also contain the coding sequence of the IgE leader sequence. In some embodiments, the fragment of SEQ ID NO:17 does not contain the coding sequence of the IgE leader sequence.

[0108] In some embodiments, the fragment of SEQ ID NO:18 may contain 236 or more amino acids; in some embodiments, it may contain 249 or more amino acids; in some embodiments, it may contain 257 or more amino acids; and in some embodiments, it may contain 259 or more amino acids.

[0109] SEQ ID NO:19 contains the nucleotide sequence encoding a common immunogen of HPV 52E6 and E7 proteins. SEQ ID NO:19 includes an IgE leader sequence SEQ ID NO:9, which is a nucleotide sequence linked to the 5' end of SEQ ID NO:19. SEQ ID NO:20 contains the amino acid sequence of a common immunogen of HPV 52E6 and E7 proteins. SEQ ID NO:20 includes an IgE leader sequence SEQ ID NO:10 located at the N-terminus of the common immunogen sequence. The IgE leader sequence is SEQ ID NO:10 and may be encoded by SEQ ID NO:9.

[0110] In some implementations, the vaccine includes SEQ ID NO:20 or a nucleic acid molecule encoding SEQ ID NO:20.

[0111] The fragment of SEQ ID NO:20 may be 100% identical to the full length except for the deletion of at least one amino acid at the N and / or C terminus, and in each case, may or may not contain a signal peptide and / or methionine at position 1. The fragment of SEQ ID NO:20 may comprise 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more of the length of the full length of SEQ ID NO:20 excluding any added heterologous signal peptides. The fragment may preferably comprise a fragment of SEQ ID NO:20 having 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher homology with SEQ ID NO:20, and additionally comprises an N-terminal methionine or heterologous signal peptide not included when calculating the percentage of homology. The fragment may also comprise an N-terminal methionine and / or a signal peptide such as an immunoglobulin signal peptide, for example, an IgE or IgG signal peptide. The N-terminal methionine and / or signal peptide may be linked to the fragment.

[0112] The fragment of the nucleic acid sequence SEQ ID NO:19 may be 100% identical to the full-length sequence except for the deletion of at least one nucleotide at the 5' and / or 3' ends, and in each case, may or may not contain a sequence encoding a signal peptide and / or methionine at position 1. The fragment may comprise 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more of the length of the full-length coding sequence SEQ ID NO:19 excluding any added heterologous signal peptides. The fragment may preferably comprise a fragment encoding a polypeptide having 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher homology to the antigen SEQ ID NO:20, and optionally additionally comprise a sequence encoding an N-terminal methionine or a heterologous signal peptide not included when calculating the percentage of homology. The fragment may also comprise coding sequences for N-terminal methionines and / or signal peptides such as immunoglobulin signal peptides, for example, IgE or IgG signal peptides. The coding sequences for N-terminal methionines and / or signal peptides may be linked to the fragment.

[0113] In some embodiments, the fragment of SEQ ID NO:19 may contain 713 or more nucleotides; in some embodiments, it may contain 752 or more nucleotides; in some embodiments, it may contain 776 or more nucleotides; and in some embodiments, it may contain 784 or more nucleotides. In some embodiments, the fragment of SEQ ID NO:19, as those listed herein, may also contain the coding sequence of the IgE leader sequence. In some embodiments, the fragment of SEQ ID NO:19 does not contain the coding sequence of the IgE leader sequence.

[0114] In some embodiments, the fragment of SEQ ID NO:20 may contain 236 or more amino acids; in some embodiments, it may contain 249 or more amino acids; in some embodiments, it may contain 257 or more amino acids; and in some embodiments, it may contain 259 or more amino acids.

[0115] SEQ ID NO:21 contains the nucleotide sequence encoding a common immunogen of HPV16 E6 and E7 proteins. SEQ ID NO:21 includes an IgE leader sequence SEQ ID NO:9, which is a nucleotide sequence linked to the 5' end of SEQ ID NO:21. SEQ ID NO:22 contains the amino acid sequence of a common immunogen of HPV16 E6 and E7 proteins. SEQ ID NO:22 includes an IgE leader sequence SEQ ID NO:10 located at the N-terminus of the common immunogen sequence. The IgE leader sequence is SEQ ID NO:10 and may be encoded by SEQ ID NO:9.

[0116] In some implementations, the vaccine includes SEQ ID NO:22 or a nucleic acid molecule encoding SEQ ID NO:22.

[0117] The fragment of SEQ ID NO:22 may be 100% identical to the full length except for the deletion of at least one amino acid at the N and / or C terminus, and in each case, may or may not contain a signal peptide and / or methionine at position 1. The fragment of SEQ ID NO:22 may comprise 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more of the length of the full length of SEQ ID NO:22 excluding any added heterologous signal peptides. The fragment may preferably comprise a fragment of SEQ ID NO:22 having 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher homology with SEQ ID NO:22, and additionally comprises an N-terminal methionine or heterologous signal peptide not included when calculating the percentage of homology. The fragment may also comprise an N-terminal methionine and / or a signal peptide such as an immunoglobulin signal peptide, for example, an IgE or IgG signal peptide. The N-terminal methionine and / or signal peptide may be linked to the fragment.

[0118] The fragment of the nucleic acid sequence SEQ ID NO:21 may be 100% identical to the full length except for the deletion of at least one nucleotide at the 5' and / or 3' ends, and in each case, may or may not have a sequence encoding a signal peptide and / or methionine at position 1. The fragment may comprise 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more of the length of the full-length coding sequence SEQ ID NO:21 excluding any added heterologous signal peptides. The fragment may preferably comprise a fragment encoding a polypeptide having 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher homology with the antigen SEQ ID NO:22, and optionally additionally comprise a sequence encoding an N-terminal methionine or a heterologous signal peptide not included when calculating the percentage of homology. The fragment may also comprise coding sequences for N-terminal methionines and / or signal peptides such as immunoglobulin signal peptides, for example, IgE or IgG signal peptides. The coding sequences for N-terminal methionines and / or signal peptides may be linked to the fragment.

[0119] In some embodiments, the fragment of SEQ ID NO:21 may contain 736 or more nucleotides; in some embodiments, it may contain 777 or more nucleotides; in some embodiments, it may contain 802 or more nucleotides; and in some embodiments, it may contain 810 or more nucleotides. In some embodiments, the fragment of SEQ ID NO:21, as those listed herein, may also contain the coding sequence of the IgE leader sequence. In some embodiments, the fragment of SEQ ID NO:21 does not contain the coding sequence of the IgE leader sequence.

[0120] In some embodiments, the fragment of SEQ ID NO:22 may contain 238 or more amino acids; in some embodiments, it may contain 251 or more amino acids; in some embodiments, it may contain 259 or more amino acids; and in some embodiments, it may contain 261 or more amino acids.

[0121] SEQ ID NO:23 contains the nucleotide sequence encoding a common immunogen of HPV18 E6 and E7 proteins. SEQ ID NO:23 includes an IgE leader sequence SEQ ID NO:9, which is a nucleotide sequence linked to the 5' end of SEQ ID NO:23. SEQ ID NO:24 contains the amino acid sequence of a common immunogen of HPV18 E6 and E7 proteins. SEQ ID NO:24 includes an IgE leader sequence SEQ ID NO:10 located at the N-terminus of the common immunogen sequence. The IgE leader sequence is SEQ ID NO:10 and may be encoded by SEQ ID NO:9.

[0122] In some implementations, the vaccine includes SEQ ID NO:24 or a nucleic acid molecule encoding SEQ ID NO:24.

[0123] The fragment of SEQ ID NO:24 may be 100% identical to the full length except for the deletion of at least one amino acid at the N and / or C terminus, and in each case, may or may not contain a signal peptide and / or methionine at position 1. The fragment of SEQ ID NO:24 may comprise 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more of the length of the full length of SEQ ID NO:24 excluding any added heterologous signal peptides. The fragment may preferably comprise a fragment of SEQ ID NO:24 having 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher homology with SEQ ID NO:24, and additionally comprises an N-terminal methionine or heterologous signal peptide not included when calculating the percentage of homology. The fragment may also comprise an N-terminal methionine and / or a signal peptide such as an immunoglobulin signal peptide, for example, an IgE or IgG signal peptide. The N-terminal methionine and / or signal peptide may be linked to the fragment.

[0124] The fragment of the nucleic acid sequence SEQ ID NO:23 may be 100% identical to the full length except for the deletion of at least one nucleotide at the 5' and / or 3' ends, and in each case, may or may not have a sequence encoding a signal peptide and / or methionine at position 1. The fragment may comprise 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more of the length of the full-length coding sequence SEQ ID NO:23 excluding any added heterologous signal peptides. The fragment may preferably comprise a fragment encoding a polypeptide having 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher homology with antigen SEQ ID NO:24, and optionally additionally comprise a sequence encoding an N-terminal methionine or a heterologous signal peptide not included when calculating the percentage of homology. The fragment may also comprise encoding sequences of N-terminal methionines and / or signal peptides such as immunoglobulin signal peptides, for example, IgE or IgG signal peptides. The encoding sequences of N-terminal methionines and / or signal peptides may be linked to the fragment.

[0125] In some embodiments, the fragment of SEQ ID NO:23 may contain 705 or more nucleotides; in some embodiments, it may contain 744 or more nucleotides; in some embodiments, it may contain 767 or more nucleotides; and in some embodiments, it may contain 775 or more nucleotides. In some embodiments, the fragment of SEQ ID NO:23, as those listed herein, may also contain the coding sequence of the IgE leader sequence. In some embodiments, the fragment of SEQ ID NO:23 does not contain the coding sequence of the IgE leader sequence.

[0126] In some embodiments, the fragment of SEQ ID NO:24 may contain 234 or more amino acids; in some embodiments, it may contain 247 or more amino acids; in some embodiments, it may contain 255 or more amino acids; and in some embodiments, it may contain 257 or more amino acids.

[0127] SEQ ID NO:25 contains the amino acid sequence of the common immunogen of HPV45 E6 and E7 proteins. SEQ ID NO:25 includes the IgE leader sequence SEQ ID NO:10 located at the N-terminus of the common immunogen sequence. The IgE leader sequence is SEQ ID NO:10 and may be encoded by SEQ ID NO:9.

[0128] SEQ ID NO:26 contains the amino acid sequence of the common immunogen of HPV39 E6 and E7 proteins. SEQ ID NO:26 includes the IgE leader sequence SEQ ID NO:10 located at the N-terminus of the common immunogen sequence. The IgE leader sequence is SEQ ID NO:10 and may be encoded by SEQ ID NO:9.

[0129] Compositions comprising amino acid sequences selected from SEQ ID NO:2; SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8; SEQ ID NO:18; SEQ ID NO:20; SEQ ID NO:22; SEQ ID NO:24; SEQ ID NO:25; or SEQ ID NO:26; or fragments thereof having at least 95% homology; or combinations thereof. In some embodiments, the composition comprises SEQ ID NO:2; SEQ ID NO:4; SEQ ID NO:6; SEQ ID NO:8; SEQ ID NO:18; SEQ ID NO:20; SEQ ID NO:22; and SEQ ID NO:24. In some embodiments, the composition comprises SEQ ID NO:6; SEQ ID NO:8; SEQ ID NO:18; SEQ ID NO:20; SEQ ID NO:22; and SEQ ID NO:24. In some embodiments, the composition comprises SEQ ID NO:2; SEQ ID NO:4; SEQ ID NO:22; and SEQ ID NO:24. In some embodiments, the composition comprises SEQ ID NO:18; SEQ ID NO:20; and SEQ ID NO:22.

[0130] The amino acid sequence may be a fragment having at least 95% homology with any one of the amino acid sequences. In some embodiments, the amino acid sequence may be a fragment having at least 98% homology with any one of the amino acid sequences. In some embodiments, the amino acid sequence may be a fragment having at least 99% homology with any one of the amino acid sequences.

[0131] According to some implementation schemes, a method for inducing an immune response against an immunogen in an individual includes administering to the individual the amino acid sequence of a common immunogen selected from the group consisting of: HPV 6E6 and E7, HPV 11E6 and E7, HPV 16E6 and E7, HPV 18E6 and E7, HPV 31E6 and E7, HPV 33E6 and E7, HPV 52E6 and E7, HPV58E6 and E7, HPV 45E6 and E7, and HPV 39E6 and E7, their functional fragments, and their expressible coding sequences. Preferably, the immunogen is common HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV52, and HPV58. Preferably, the immunogen is common HPV16, HPV18, HPV31, HPV33, HPV52, and HPV58. Preferably, the immunogens include HPV6, HPV11, HPV16, and HPV18. Preferably, the immunogens include HPV16, HPV31, and HPV52.

[0132] Some implementations include isolated nucleic acid molecules encoding amino acid sequences of a common immunogen selected from the group consisting of: HPV 6E6 and E7, HPV11E6 and E7, HPV 16E6 and E7, HPV 18E6 and E7, HPV31E6 and E7, HPV 33E6 and E7, HPV 52E6 and E7, HPV 58E6 and E7, HPV 45E6 and E7, and HPV 39E6 and E7, and fragments thereof. Some implementations include recombinant vaccines encoding amino acid sequences of a common immunogen selected from the group consisting of: HPV 6E6 and E7, HPV 11E6 and E7, HPV 16E6 and E7, HPV 18E6 and E7, HPV 31E6 and E7, HPV 33E6 and E7, HPV 52E6 and E7, HPV 58E6 and E7, HPV 45E6 and E7, and HPV 39E6 and E7, and fragments thereof. Some implementations include subunit vaccines containing an amino acid sequence of a common immunogen selected from the group consisting of: HPV 6E6 and E7, HPV 11E6 and E7, HPV 16E6 and E7, HPV 18E6 and E7, HPV 31E6 and E7, HPV 33E6 and E7, HPV 52E6 and E7, HPV 58E6 and E7, HPV 45E6 and E7, and HPV 39E6 and E7, and fragments thereof. Some implementations include live attenuated vaccines and / or inactivated vaccines containing the amino acid sequence of a common immunogen selected from the group consisting of: HPV 6E6 and E7, HPV 11E6 and E7, HPV 16E6 and E7, HPV18E6 and E7, HPV 31E6 and E7, HPV 33E6 and E7, HPV52E6 and E7, HPV 58E6 and E7, HPV 45E6 and E7, and HPV 39E6 and E7.

[0133] There are methods for inducing an immune response against HPV in an individual, the methods comprising administering to the individual a composition containing the nucleic acid sequence provided herein. In some embodiments, the method further includes introducing the nucleic acid sequence into the individual via electroporation.

[0134] In some aspects, there are methods for inducing an immune response against HPV in an individual, the methods comprising administering to the individual a composition containing the amino acid sequence provided herein. In some embodiments, the method further includes introducing the amino acid sequence into the individual via electroporation.

[0135] On the one hand, the vaccines described herein are those that elicit an immune response against HPV subtypes that are primarily found to be associated with various forms of head and neck cancer and other forms of ear, nose and throat diseases, particularly those containing HPV 6E6 and E7 and HPV11E6 and E7, and preferably both.

[0136] On the other hand, the vaccines described herein are those that elicit an immune response against various HPV subtypes associated with cervical cancer, primarily found in patients outside the United States and more specifically in Asia, particularly those containing HPV 33E6 and E7 and HPV 58E6 and E7, and preferably both.

[0137] Preferred combinations exist for inducing an immune response against HPV subtypes associated with cervical cancer, including precancerous lesions, including: HPV subtypes 16, 18, 31, 33, 52, 58, 6, 11, 39, and 45 or HPV subtypes 16, 18, 31, 33, 52, 58, 6, and 11. Other sub-combinations for this cervical cancer include:

[0138] 16 and 18; 16, 18 and 6; 16, 18 and 11; 16, 18 and 31; 16, 18 and 33; 16, 18 and 52; 16, 18 and 58 ;16, 18, 6 and 11; 16, 18, 6 and 31; 16, 18, 6 and 33; 16, 18, 6 and 52, 16, 18, 6 and 58; 16, 18, 11 and 31; 16, 18, 11 and 33, 16, 18, 11 and 52; 16, 18, 11 and 58; 16, 18, 31 and 33; 1 6, 18, 31 and 52; 16, 18, 31 and 58; 16, 18, 33 and 52; 16, 18, 33 and 58; 16, 18, 52 and 58;

[0139] 6, 11 and 16; 6, 11 and 18; 6, 11 and 31; 6, 11 and 33; 6, 11 and 52; 6, 11 and 58; 6, 11, 16 and 31; 6, 11, 16 and 33; 6, 11, 16 and 52; 6, 11, 16 and 58; 6, 11, 18 and 3 1; 6, 11, 18 and 33; 6, 11, 18 and 52; 6, 11, 18 and 58; 6, 11, 31 and 33; 6, 11, 31 and 52; 6, 11, 31 and 58; 6, 11, 33 and 52; 6, 11, 33 and 58; 6, 11, 52 and 58;

[0140] 6, 16 and 31; 6, 16 and 33; 6, 16 and 52; 6, 16 and 58; 6, 16, 31 and 33; 6, 16, 31 and 52; 6, 16, 31 and 58; 6, 16, 33 and 52; 6, 16, 33 and 58; 6, 16, 52 and 58;

[0141] 6, 18 and 31; 6, 18 and 33; 6, 18 and 52; 6, 18 and 58; 6, 18, 31 and 33; 6, 18, 31 and 52; 6, 18, 31 and 58; 6, 18, 33 and 52; 6, 18, 33 and 58;

[0142] 6, 31 and 33; 6, 31 and 52; 6, 31 and 58; 6, 31, and 33 and 52; 6, 31, and 33 and 58;

[0143] 6, 33 + 52; 6, 33 + 58; 6, 33, 52 + 58;

[0144] 6, 52 sum 58;

[0145] 11, 31 + 33; 11, 31 + 52; 11, 31 + 58; 11, 31, 33 + 52; 11, 31, 33 + 58; 11, 31, 52 + 58;

[0146] 11, 31 + 52; 11, 31 + 58; 11, 31, 52 + 58;

[0147] 11, 52 sum 58;

[0148] 16, 31 + 33; 16, 31 + 52; 16, 31 + 58; 16, 31, 33 + 52; 16, 31, 52 + 58;

[0149] 16, 33 + 52; 16, 33 + 58; 16, 33, 52 + 58;

[0150] 18, 31 + 33; 18, 31 + 52; 18, 31 + 58; 18, 31, 33 + 52; 18, 31, 33 + 58;

[0151] 31, 33 sum 52; 31, 33 sum 58; 31, 33, 52 sum 58;

[0152] 31, 52 sum 58.

[0153] The improved vaccine comprises proteins and gene constructs encoding proteins having epitopes that make them particularly effective as immunogens for inducing anti-HPV immune responses. Therefore, vaccines can be provided to induce therapeutic or prophylactic immune responses. In some embodiments, the immunogen is delivered in the form of a DNA vaccine, a recombinant vaccine, a protein subunit vaccine, a composition containing an immunogen, a live attenuated vaccine, or an inactivated vaccine. In some embodiments, the vaccine comprises a combination selected from the group consisting of one or more DNA vaccines, one or more recombinant vaccines, one or more protein subunit vaccines, one or more compositions containing an immunogen, one or more live attenuated vaccines, and one or more inactivated vaccines.

[0154] Aspects of the present invention provide a method for delivering protein coding sequences on nucleic acid molecules such as plasmids (as part of a recombinant vaccine and as part of an attenuated vaccine, as a protein portion of an isolated protein or a vector).

[0155] According to some aspects of the present invention, compositions and methods for prophylactic and / or therapeutic immunization of individuals are provided.

[0156] DNA vaccines are described in U.S. Patent Nos. 5,593,972, 5,739,118, 5,817,637, 5,830,876, 5,962,428, 5,981,505, 5,580,859, 5,703,055, and 5,676,594, and priority applications are cited in those applications, which are incorporated herein by reference. Alternative methods for delivering DNA, in addition to the delivery schemes described in those applications, are described in U.S. Patent Nos. 4,945,050 and 5,036,006, both of which are incorporated herein by reference.

[0157] This invention relates to improved live attenuated vaccines, improved inactivated vaccines, and improved vaccines using recombinant vectors to deliver exogenous genes encoding antigens, as well as subunit vaccines and glycoprotein vaccines. Examples of live attenuated vaccines, vaccines using recombinant vectors to deliver exogenous antigens, subunit vaccines, and glycoprotein vaccines are described in U.S. Patent Nos. 4,510,245; 4,797,368; 4,722,848; 4,790,987; 4,920,209; 5,017,487; 5,077,044; 5,110,587; 5,112,749; 5,174,993; 5,223,424; 5,225,336; 5,240,703; 5,242,829; 5,294,441; and 5,294,54 The following entries are incorporated herein by reference: 8; 5,310,668; 5,387,744; 5,389,368; 5,424,065; 5,451,499; 5,453,364; 5,462,734; 5,470,734; 5,474,935; 5,482,713; 5,591,439; 5,643,579; 5,650,309; 5,698,202; 5,955,088; 6,034,298; 6,042,836; 6,156,319; and 6,589,529.

[0158] When taken up by cells, gene constructs can remain present in the cell as functional extrachromosomal molecules and / or integrated into the cell's chromosomal DNA. DNA can be introduced into the cell, where it remains as separate genetic material in the form of one or more plasmids. Alternatively, linear DNA that can integrate into chromosomes can be introduced into the cell. When DNA is introduced into the cell, agents that promote DNA integration into chromosomes can be added. DNA sequences suitable for promoting integration can also be included in the DNA molecule. Alternatively, RNA can be administered to the cell. Also covered are gene constructs providing the form of linear miniature chromosomes, including centromeres, telomeres, and origins of replication. Gene constructs can be maintained as part of the genetic material in attenuated live microorganisms or in recombinant microbial vectors that survive in cells. Gene constructs can be part of the genome of a recombinant viral vaccine, where the genetic material is integrated into the cell's chromosome or maintained extrachromosomally. Gene constructs include regulatory elements necessary for gene expression of nucleic acid molecules. Elements include: promoters, start codons, stop codons, and polyadenylation signals. Additionally, enhancers are often required for gene expression of sequences encoding target proteins or immunomodulatory proteins. These elements need to be operablely linked to the sequence encoding the desired protein, and the regulatory elements need to be operable in the individual to which they are applied.

[0159] Start and stop codons are generally considered to be part of the nucleotide sequence encoding the desired protein. However, these elements need to be functional in the individual to which the gene construct is administered. The start and stop codons must be present in the coding sequence.

[0160] The promoters and polyadenylation signals used must be functional within the individual's cells.

[0161] Examples of promoters suitable for practicing this invention, particularly in the manufacture of human gene vaccines, include, but are not limited to: promoters from simian virus 40 (SV40), mouse mammary tumor virus (MMTV) promoters, human immunodeficiency virus (MV) promoters such as BIV long terminal repeat (LTR) promoters, Moroni virus, ALV, cytomegalovirus (CMV) promoters such as CMV immediate early promoters, Epstein-Barr virus (EBV), Raul's sarcoma virus (RSV), and promoters from human genes such as human actin, human myosin, human hemoglobin, human muscle creatine, and human metallothionein.

[0162] Examples of polyadenylation signals applicable to the practice of this invention, particularly in the manufacture of human gene vaccines, include, but are not limited to, the SV40 polyadenylation signal and the LTR polyadenylation signal. Specifically, the SV40 polyadenylation signal used in the pCEP4 plasmid (Invitrogen, San Diego, CA) is referred to as the SV40 polyadenylation signal.

[0163] In addition to the regulatory elements required for DNA expression, other elements may also be included in the DNA molecule. These additional elements include enhancers. Enhancers can be selected from the group consisting of, but not limited to, human actin, human myosin, human hemoglobin, human muscle creatine, and viral enhancers, such as those from CMV, RSV, and EBV.

[0164] Gene constructs with mammalian origin of replication can be provided to maintain the construct outside the chromosome and generate multiple copies of the construct in the cell. Plasmids pVAX1, pCEP4, and pREP4 from Invitrogen (San Diego, CA) contain the EB virus origin of replication and the coding region for the nuclear antigen EBNA-1, which generates high-copy additivity replication without integration.

[0165] In some preferred embodiments relating to immune applications, a nucleic acid molecule is delivered, the nucleic acid molecule comprising a nucleotide sequence encoding the protein of the present invention, and additionally comprising a gene for a protein that further enhances the immune response against such target proteins. Examples of such genes are those encoding other cytokines and lymphokines, such as α-interferon, γ-interferon, platelet-derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-18, MHC, CD80, CD86, and IL-15 (including IL-15 with a deleted signal sequence and optionally containing a signal peptide from IgE). Other potentially useful genes include those encoding the following: MCP-1, MIP-1α, MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, p150.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18, CD40, CD40L. Angiogenic factor, IL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, Killer, Trail-R2, Trick2, DR6, caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactive NIK, SAP K, SAP-1, JNK, interferon response gene, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2 and their functional fragments

[0166] Additional elements may be added, which act as targets for cell destruction should, for any reason, wish to eliminate cells receiving the gene construct. An expressible form of the herpesvirus thymidine kinase (tk) gene may be included in the gene construct. The drug ganciclovir may be administered to an individual, and the drug will cause the selective killing of any tk-producing cells, thereby providing a means of selectively destroying cells containing the gene construct.

[0167] To maximize protein production, regulatory sequences can be selected that are highly suitable for gene expression in the cells in which the construct is applied. Furthermore, codons that are most efficiently transcribed in the cell can be selected. Those skilled in the art can prepare DNA constructs that function in cells.

[0168] In some embodiments, a gene construct may be provided in which the coding sequence of the protein described herein is linked to an IgE signal peptide.

[0169] In some embodiments using proteins, for example, those skilled in the art can use well-known techniques to prepare and isolate the proteins of the present invention. In some embodiments using proteins, for example, those skilled in the art can use well-known techniques to insert DNA molecules encoding the proteins of the present invention into commercially available expression vectors used in well-known expression systems. For example, the commercially available plasmid pSE420 (Invitrogen, San Diego, Calif.) can be used for the preparation of proteins from Escherichia coli (E. coli). The commercially available plasmid pYES2 (Invitrogen, San Diego, Calif.) can be used, for example, for the preparation of proteins from the yeast strain Saccharomyces cerevisiae. Commercially available MAXBAC... TM A complete baculovirus expression system (Invitrogen, San Diego, Calif.) can be used, for example, for preparation in insect cells. Commercially available plasmids pcDNA I or pcDNA3 (Invitrogen, San Diego, Calif.) can be used, for example, for preparation in mammalian cells such as Chinese hamster ovary cells. Those skilled in the art can use these commercial expression vectors and systems, or other vectors and systems, to prepare proteins using conventional techniques and readily available starting materials. (See, for example, Sambrook et al., Molecular Cloning a Laboratory Manual, 2nd ed., Cold Spring Harbor Press (1989), which is incorporated herein by reference). Thus, desired proteins can be prepared in both prokaryotic and eukaryotic systems, resulting in a range of processed forms of the protein.

[0170] Those skilled in the art can prepare vectors using other commercially available expression vectors and systems or using well-known methods and readily available starting materials. Expression systems containing desired control sequences (such as promoters and polyadenylation signals, and preferably enhancers) are readily available and known in the art for a variety of hosts. See, for example, Sambrook et al., Molecular Cloning a Laboratory Manual, 2nd ed., Cold Spring Harbor Press (1989). Gene constructs include protein-coding sequences operatively linked to promoters that function in the cell lines transfected with the construct. Examples of constitutive promoters include promoters from cytomegalovirus or SV40. Examples of inducible promoters include mouse mammary leukemia virus or metallothionein promoters. Those skilled in the art can readily prepare gene constructs suitable for transfecting cells with DNA encoding the proteins of the present invention from readily available starting materials. A compatible host is transformed using an expression vector containing DNA encoding the protein, and the host is then cultured and maintained under conditions in which exogenous DNA expression occurs.

[0171] The prepared protein is recovered from the culture by cell lysis or from a culture medium suitable and known to those skilled in the art. Those skilled in the art can use well-known techniques to isolate proteins prepared using such expression systems. The method for purifying proteins from natural sources using antibodies that specifically bind to a particular protein, as described above, can also be applied to the purification of proteins prepared by recombinant DNA methods.

[0172] In addition to preparing proteins via recombinant technology, automated peptide synthesizers can also be used to prepare isolated, substantially pure proteins. These techniques are well known to those skilled in the art and are applicable to substituted derivatives not provided in the preparation of DNA-encoded proteins.

[0173] Nucleic acid molecules can be delivered using any of several well-known techniques, including DNA injection (also known as DNA vaccination), recombinant vectors such as recombinant adenovirus, recombinant adenovirus-associated virus, and recombinant vaccinia virus.

[0174] Routes of administration include, but are not limited to, intramuscular, intranasal, intraperitoneal, intradermal, subcutaneous, intravenous, intraarterial, intraocular, and oral administration, as well as local, transdermal, inhalation, or suppository administration, or administration to mucosal tissues, such as through vaginal irrigation, rectal administration, urethral administration, oral cavity, and sublingual administration. Preferred routes of administration include intramuscular, intraperitoneal, intradermal, and subcutaneous injection. Gene constructs can be administered in ways including, but not limited to, electroporation methods and devices, conventional syringes, needle-free injection devices, or "micro-projectile gene gun" methods.

[0175] Examples of preferred electroporation devices and methods for facilitating the delivery of DNA vaccines include those described by Drghia-Akli et al. in U.S. Patent No. 7,245,963 and U.S. Patent Publication 2005 / 0052630 filed by Smith et al., the contents of which are incorporated herein by reference in their entirety. Also preferred are the electroporation devices and methods for facilitating the delivery of DNA vaccines provided in co-pending and co-owned U.S. Patent Application Serial No. 11 / 874072, filed October 17, 2007, which claims priority under 35 USC 119(e) to U.S. Provisional Application Serial No. 60 / 852,149, filed October 17, 2006, and U.S. Provisional Application Serial No. 60 / 978,982, filed October 10, 2007, both of which are incorporated herein by reference in their entirety.

[0176] The following are examples of implementations using electroporation technology, discussed in more detail in the aforementioned patent references: An electroporation device can be configured to deliver energy pulses that generate a constant current similar to a preset current input by a user to desired tissue in a mammal. The electroporation device includes an electroporation component and an electrode assembly or handle assembly. The electroporation component may include one or more of a variety of elements of the electroporation device, including: a controller, a current waveform generator, an impedance tester, a waveform recorder, an input element, a status reporting element, a communication port, a memory component, a power supply, and a power switch. The electroporation component may act as one element of the electroporation device, and other elements are separate elements (or components) communicating with said electroporation component. In some embodiments, the electroporation component may act as more than one element of the electroporation device, which may communicate with other elements of the electroporation device independently of said electroporation component. The use of electroporation technology to deliver improved HPV vaccines is not limited to the elements of existing electroporation devices that are part of an electromechanical or mechanical device, because said elements may act as a device or separate element communicating with each other. The electroporation component is capable of delivering energy pulses that generate a constant current in desired tissue and includes a feedback mechanism. The electrode assembly includes an electrode array having multiple electrodes arranged in a spatial configuration, wherein the electrode assembly receives energy pulses from the electroporation component and delivers the energy pulses to the desired tissue via the electrodes. At least one of the multiple electrodes is neutral during the energy pulse process and measures the resistance in the desired tissue and notifies the electroporation component of the resistance. The feedback mechanism can receive the measured resistance and can adjust the energy pulses delivered by the electroporation component to maintain a constant current.

[0177] In some embodiments, multiple electrodes may be distributed to deliver energy pulses. In some embodiments, multiple electrodes may be distributed to deliver energy pulses via a programmed sequence controlled by control electrodes, and the programming sequence is input by a user to the electroporation component. In some embodiments, the programming sequence comprises multiple pulses delivered sequentially, wherein each pulse of the multiple pulses is delivered through at least two active electrodes (one of which is a neutral electrode measuring resistance), and wherein subsequent pulses of the multiple pulses are delivered through a different one of the at least two active electrodes (one of which is a neutral electrode measuring resistance).

[0178] In some embodiments, the feedback mechanism is implemented via hardware or software. Preferably, the feedback mechanism is implemented via an analog closed-loop circuit. Preferably, the feedback occurs every 50 μs, 20 μs, 10 μs, or 1 μs, but is preferably real-time or instantaneous (i.e., substantially instantaneous, as determined by available techniques for measuring response time). In some embodiments, a neutral electrode measures the resistance in the desired tissue and informs the feedback mechanism of the resistance, and the feedback mechanism responds to the resistance and adjusts the energy pulse to maintain a constant current at a value similar to a preset current. In some embodiments, the feedback mechanism maintains a constant current continuously and instantaneously during energy pulse delivery.

[0179] In some implementations, nucleic acid molecules are delivered to cells by administration of a polynucleotide enhancer or a gene vaccine promoter. Polynucleotide enhancers are described in U.S. Serial Nos. 5,593,972, 5,962,428 and International Application Serial No. PCT / US94 / 00899, filed January 26, 1994, all of which are incorporated herein by reference. Gene vaccine promoters are described in U.S. Serial No. 021,579, filed April 1, 1994, which is also incorporated herein by reference. Adjuvants for the administration of nucleic acid molecules may be administered as a mixture with the nucleic acid molecules, or separately, concurrently with, before, or after the administration of the nucleic acid molecules. In addition, other agents that can be used as transfection agents and / or replication agents and / or inflammatory agents and can be co-administered with GVF include: growth factors, cytokines and lymphokines such as alpha-interferon, gamma-interferon, GM-CSF, platelet-derived growth factor (PDGF), TNF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-6, IL-10, IL-12 and IL-15 and fibroblast growth factor, surfactants such as immunostimulatory complexes (ISCOMS), Freund's incomplete adjuvants, LPS analogs (including monophosphoryl lipid A (WL)), cell wall peptides, quinone analogs and vesicles such as triacontadecene and squalene, and hyaluronic acid may also be used in combination with gene constructs for administration. In some embodiments, immunomodulatory proteins may also be used as GVF. In some embodiments, nucleic acid molecules are provided in combination with PLG to enhance delivery / uptake.

[0180] The pharmaceutical composition according to the invention comprises about 1 nanogram to about 2000 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition according to the invention comprises about 5 nanograms to about 1000 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition comprises about 10 nanograms to about 800 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition comprises about 0.1 to about 500 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition comprises about 1 to about 350 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition comprises about 25 to about 250 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition comprises about 100 to about 200 micrograms of DNA.

[0181] The pharmaceutical compositions according to the invention are formulated according to the method of administration used. In the case of injectable pharmaceutical compositions, they are sterile, pyrogen-free, and particulate-free. Isotonic formulations are preferred. Typically, additives for isotonicity may include sodium chloride, dextran, mannitol, sorbitol, and lactose. In some cases, isotonic solutions such as phosphate-buffered saline are preferred. Stabilizers include gelatin and albumin. In some embodiments, a vasoconstrictor is added to the formulation.

[0182] According to some embodiments of the present invention, methods for inducing an immune response are provided. The vaccine may be protein-based, live attenuated, cell-based, recombinant, or nucleic acid or DNA-based. In some embodiments, methods for inducing an immune response against an immunogen in an individual (including methods for inducing a mucosal immune response) include administering to the individual one or more of the following: CTACK protein, TECK protein, MEC protein and functional fragments thereof, or their expressible coding sequences, in combination with isolated nucleic acid molecules encoding proteins of the present invention, and / or recombinant vaccines encoding proteins of the present invention, and / or subunit vaccines containing proteins of the present invention, and / or live attenuated vaccines and / or inactivated vaccines. One or more of CTACK protein, TECK protein, MEC protein and functional fragments thereof may be administered before, simultaneously with, or after the administration of: isolated nucleic acid molecules encoding immunogens, and / or recombinant vaccines encoding immunogens, and / or subunit vaccines containing immunogens, and / or live attenuated vaccines and / or inactivated vaccines. In some embodiments, administering to the individual an isolated nucleic acid molecule encoding one or more proteins selected from the group consisting of: CTACK, TECK, MEC and functional fragments thereof.

[0183] The invention is further illustrated in the following embodiments. It should be understood that while these embodiments illustrate implementations of the invention, they are given by way of illustration only. Based on the foregoing discussion and these embodiments, those skilled in the art will determine the essential characteristics of the invention, and various changes and modifications can be made to adapt it to various uses and conditions without departing from the spirit and scope of the invention. Therefore, various modifications to the invention will be apparent to those skilled in the art from the preceding description, in addition to the modifications shown and described herein. Such modifications are also intended to fall within the scope of the appended claims.

[0184] All U.S. patents, U.S. applications, and cited references cited throughout this disclosure are incorporated herein by reference in their entirety.

[0185] Example

[0186] The invention is further defined in the following embodiments. It should be understood that these embodiments, while indicating preferred embodiments of the invention, are given by way of illustration only. Based on the foregoing discussion and these embodiments, those skilled in the art will determine the essential characteristics of the invention, and various changes and modifications can be made to adapt it to various uses and conditions without departing from the spirit and scope of the invention. Therefore, various modifications to the invention will be apparent to those skilled in the art from the preceding description, in addition to those modifications shown and described herein. Such modifications are also intended to fall within the scope of the appended claims.

[0187] Example 1

[0188] A novel engineered HPV-16 DNA vaccine encoding the E6 / E7 fusion protein

[0189] The immunogen has been engineered to express a polyprotein, whereby the E6 and E7 sequences are separated by proteolytic cleavage sites. The polyprotein can also be expressed with an IgE leader sequence. Polyprotein design includes deletions or mutations in the E6 sequence, which are important for p53 binding and degradation at the ribosome-binding site on the E7 protein, as well as for mutations in this design.

[0190] The coding sequence of HPV16 E6 / E7 (SEQ ID NO:21) encoding a polyprotein (SEQ ID NO:22) was inserted into the vector pVAX to generate an expression plasmid. The optimized human papillomavirus 16-6&7 antigen (HPV16 E6&7) was used with a pVAX backbone including the kanamycin resistance gene (Kan) and the plasmid replication origin (pUC ori), driven by the CMV promoter (PCMV) and featuring a bovine growth hormone 3'-terminal polyadenylation signal (bGHpA).

[0191] HPV18 E6 / E7 vaccine design and expression

[0192] An optimized concordant sequence for human papillomavirus (HPV) viral protein 18E6&7 was prepared. The sequence was designed for high-level expression. This sequence is useful in our gene immunotherapy technique. Results from experiments using the concordant sequence were positive. The plasmid construct includes the nucleic acid sequence of the concordant HPV 18-6&7 (SEQ ID NO:23). The nucleic acid sequence of the concordant HPV 18-6&7 incorporated into the plasmid includes the coding sequence of an IgE leader peptide linked to the coding sequence of the concordant HPV 18-6&7.

[0193] An expression plasmid pGX3002 was generated to express the human papillomavirus 18-6&7 antigen (HPVI8 E6&7) (SEQ ID NO:24) with optimized expression. It uses a pVAX backbone that includes the kanamycin resistance gene (Kan) and the plasmid replication origin (pUC ori), is driven by the CMV promoter (PCMV), and has a bovine growth hormone 3' end and a polyadenylation signal (bGHpA).

[0194] HPV6 and HPV11 E6 / E7 vaccine design and expression

[0195] Construction of HPV6 and 11E6 / E7 shared sequence fusion immunogen

[0196] HPV types 6 and 11 E6 and E7 gene sequences were collected from a gene bank, and common E6 and E7 nucleotide sequences were obtained after multiple alignment. The common sequences for HPV 6E6 or E7 proteins were generated from 98 or 20 sequences, respectively, while the common sequences for HPV 11E6 or E7 proteins were generated from 76 or 13 sequences, respectively. Multiple alignment programs used in phylogenetic studies included Clustal X (version 2.0). Figure 1A and Figure 1B As shown, there is approximately 0-2% sequence divergence in HPV strains whose E6 and E7 proteins belong to the same type. However, the genetic distance in the E6 protein is as high as 19.3% between HPV 6 and 11, and in the E7 protein, it is as high as 16.3%. Based on these results from phylogenetic analysis, we developed two type-specific E6 / E7 co-DNA vaccines.

[0197] After generating the shared E6 / E7 fusion sequence, several modifications are performed. Figure 1C A highly efficient leader sequence was fused upstream of the start codon to promote expression. Codon and RNA optimization were also performed as previously described (J. Yan et al., Cellular immunity induced by a novel HPV18 DNA vaccine encoding an E6 / E7 fusion consensus protein in mice and rhesus macaques, Vaccine. 26(2008) 5210-5215; and J. Yan et al., Induction of antitumor immunity in vivo following delivery of a novel HPV-16 DNA vaccine encoding an E6 / E7 fusion antigen, Vaccine. 27(2009) 431-440). An endonucleolytic cleavage site was introduced between the E6 and E7 proteins for proper protein folding and better CTL processing. The synthesized engineered 6E6E7 and 11E6E7 genes were both 840 bp in length. The sequence-validated synthesized genes were subcloned into the BamHI and XhoI sites of the pVAX expression vector for further studies. The common amino acid sequence is obtained by translating the common nucleotide sequence.

[0198] After obtaining the shared E6 and E7 sequences for HPV types 6 and 11, codon and RNA optimization were performed as previously described (J. Yan et al., Vaccine. 26 (2008) 5210-5215; and J. Yan et al., Induction, Vaccine. 27 (2009) 431-440). Fusion genes encoding the shared E6 / E7 fusion protein of HPV types 6 or 11 (6E6E7 or 11E6E7) were synthesized and their sequences were validated. The synthesized 6E6E7 or 11E6E7 was digested with BamHI and XhoI, cloned into the expression vector pVAX (Invitrogen) under the control of the cytomegalovirus immediate early promoter, and these constructs were named p6E6E7 or p11E6E7.

[0199] 293-T cells were cultured in 6-well plates and transfected with pVAX, p6E6E7, or p11E6E7 using FuGENE6 transfection reagent (Roche Applied Science, Indianapolis, IN). Two days after transfection, cells were lysed using modified RIPA cell lysis buffer, and cell lysates were collected. Western blot analysis was performed using anti-HA monoclonal antibody (Sigma-Aldrich, St. Louis, MO), and observations were made using an ECL™ Western blot analysis system (Amersham, Piscataway, NJ) with horseradish peroxidase-conjugated goat anti-mouse IgG (Sigma-Aldrich, St. Louis, MO).

[0200] Indirect immunofluorescence assays were performed using human rhabdomyosarcoma cells (RD cells) to verify the expression of p6E6E7 and p11E6E7. RD cells cultured in slides were transfected with pVAX, p6E6E7, or p11E6E7 using Turbofectin 8.0 (Origene, Rockville, MD). Cells were then fixed with PFA and infiltrated with 0.1% Triton-X in PBS. Cells were incubated with primary and secondary antibodies for 1–2 hours, followed by washing with PBS supplemented with glycine and BSA between incubations. The primary and secondary antibodies used were monoclonal mouse anti-HA (Sigma-Aldrich, St. Louis, MO) and FITC-conjugated anti-mouse IgG (Abcam, Cambridge, MA), respectively. Hoechst staining was also performed to identify the nuclei and localize RD cells. After all incubations, cells were placed on slides containing Fluoromount-G (Southern Biotech, Birmingham, AL). The samples were observed and imaged using a confocal microscope (CDB Microscopy Core, University of Pennsylvania Cell and Developmental Biology, Philadelphia, PA).

[0201] HPV antigens 31, 33, 39, 45, 52 and 58E6E7

[0202] HPV E6 and E7 gene sequences for types 31, 33, 39, 45, 52, and 58 were collected individually from a gene bank, and shared E6 and E7 nucleotide sequences were obtained after multiple alignment (type 31 - SEQ ID NO: 18; type 33 - SEQ ID NO: 6; type 52 - SEQ ID NO: 20; and type 58 - SEQ ID NO: 8). The multiple alignment procedure used in the phylogenetic study included Clustal X (version 2.0). Based on the results from the phylogenetic analysis, we developed a type-specific E6 / E7 shared DNA vaccine.

[0203] Several modifications were performed after generating the shared E6 / E7 fusion sequence. A highly efficient leader sequence was fused in-frame upstream of the start codon to promote expression. Codon and RNA optimizations were also performed as previously described (J. Yan et al., Cellular immunity induced by a novel HPV18 DNA vaccine encoding an E6 / E7 fusion consensus protein in mice and rhesus macaques, Vaccine. 26(2008) 5210-5215; and J. Yan et al., Induction of antitumor immunity in vivo following delivery of a novel HPV-16 DNA vaccine encoding an E6 / E7 fusion antigen, Vaccine. 27(2009) 431-440). An endonucleolytic cleavage site was introduced between the E6 and E7 proteins for proper protein folding and better CTL processing. The sequence-validated synthetic gene was subcloned into the BamHI and XhoI sites of the pVAX expression vector for further investigation. The shared amino acid sequence was obtained by translating the shared nucleotide sequence.

[0204] Expression plasmids were generated that expressed optimized human papillomavirus 31, 33, 52, and 58-E6&7 antigens (HPV31 E6&7-SEQ ID NO:17, HPV33 E6&7-SEQ ID NO:5, HPV52 E6&7-SEQ ID NO:19, and HPV58 E6&7-SEQ ID NO:7) using a pVAX backbone including the kanamycin resistance gene (Kan) and plasmid replication origin (pUC ori), driven by the CMV promoter (PCMV) with bovine growth hormone 3'-terminal polyadenylation signal (bGHpA). Similarly, expression plasmids were generated that included inserts for optimized human papillomavirus 39 and 45-E6&E7 antigens (HPV39 E6&E7 sequence encoding SEQ ID NO:26; and HPV45 E6&E7 sequence encoding SEQ ID NO:25).

[0205] Example 2

[0206] Mice and treatment groups vaccinated

[0207] Female C57BL / 6 mice aged 6 to 8 weeks were used in this experiment. The mice were obtained from the Jackson Laboratory (BarHarbor, ME). The mice were bred and maintained in captivity by the University Laboratory Animal Resources at the University of Pennsylvania (Philadelphia, PA) in compliance with the policies of the National Institutes of Health and the University of Pennsylvania Committee on Laboratory Animal Management and Use (IACUC). Mice used in these experiments were divided into four groups for immunization. Mice were immunized with p6E6E7, p11E6E7, or either of these constructs, and the pVAX group served as a negative control.

[0208] DNA vaccination and electroporation

[0209] Each mouse received three doses of 20 μg of each DNA plasmid at 14-day intervals. Mice in the groups receiving both p6E6E7 and p11E6E7 received 20 μg of each plasmid, for a total of 40 μg of DNA per vaccination. The DNA constructs were administered via intramuscular injection into the right quadriceps muscle, followed by administration via CELLECTRA. TM An electroporator (Inovio Pharmaceuticals, Blue Bell, PA) generates square wave pulses. The electroporator is configured to deliver two electrical pulses at 0.2 Amps at a rate of 52 ms / pulse with a 1-second delay. The electroporation procedure is performed as previously described [12, 13].

[0210] IFN-γELISpot assay

[0211] Mice in both the treatment and control groups were sacrificed one week after the third immunization. Spleens were collected from each mouse and transferred to RPMI-1640 medium (R10) containing 10% FBS and antibiotics. The spleens were homogenized using a stomacher (Seward Laboratory Systems, Bohemia, NY) and subsequently transferred through a cell filter and suspended in ACK lysis buffer. After erythrocyte removal, spleen cells were isolated and suspended in R10 medium. High-protein IP 96-well Multiscreen TMPlates (Millipore, Bedford, MA) were pre-coated with monoclonal mouse IFN-γ capture antibody (R&D Systems, Minneapolis, MN) and incubated overnight at 4°C. After washing three times with 1X PBS, the plates were blocked for 2 hours at ambient temperature with 1×PBS solution of 1% BSA and 5% sucrose. Splenocytes isolated from R10 medium were counted and added to wells in triplicate at 2×10⁵ cells per well. Two peptides containing the common E6 / E7 sequence of HPV6 and HPV11 were reconstituted in DMSO (GenScript USA, Piscataway, NJ). The peptides contain 15 amino acid sequences, with 8 residues overlapping each consecutive peptide. The HPV6 and HPV11 peptides were each divided into two pools at a concentration of 2 μg / mL in DMSO—one pool for E6 and the other for E7. Wells reserved for positive and negative controls were treated with concavalin A (Sigma-Aldrich, St. Louis, MO) and R10 medium, respectively, instead of peptide. The plates were then placed in a 5% CO2 incubator. After incubation at 37°C for 24 hours, the wells were washed with 1×PBS. Biotinylated anti-mouse IFN-γ detection antibody (R&D Systems, Minneapolis, MN) was added to each well, followed by overnight incubation at 4°C. The plates were then washed and treated with streptavidin-AP and BCIP / NBT Plus (R&D Systems, Minneapolis, MN) according to the chromogenic protocol provided by R&D Systems. The wells were air-dried overnight and then passed through a plate containing... and The software's ELISpot plate reading system (Cellular Technology Ltd., ShakerHeights, OH) scans and counts the spots within the wells. An algorithm is used to convert the reported spot-forming cell counts to represent spot-forming units per 1 × 10⁶ spleen cells.

[0212] Given the sensitivity and ability of IFN-γ ELISpot assays to indicate T cell activity, they were used to determine the number of antigen-specific IFN-γ secreting cells in response to stimulation by HPV 6 or 11E6 and E7 peptides.

[0213] like Figure 3A and Figure 3BAs shown, the mean SFU / 10⁶ spleen cell count in mice inoculated with p6E6E7 was 1442.8, while the mean SFU / 10⁶ count in mice inoculated with p11E6E7 was 2845, both significantly higher than the negative control group. Therefore, both p6E6E7 and p11E6E7 are effective in inducing stable type-specific E6 and E7-specific immune responses in mice.

[0214] Interestingly, vaccination with p6E6E7 or p11E6E7 also induced cross-reactive cellular immune responses. In mice immunized with p11E6E7, the summative frequency of SFU / 10⁶ splenocytes against the HPV 6E6 / E7 peptide was 552.8 SFU / 10⁶ splenocytes, while the HPV 11E6 / E7-specific immune response in mice immunized with p6E6E7 was 888.3 SFU / 10⁶ splenocytes. The E6 protein of HPV 6 or 11 shares approximately 80% identity, while the E7 protein of HPV 6 or 11 shares approximately 84% identity. There may be some shared immunoepotopes between HPV 6 or 11E6 and E7 antigens. The cross-reactivity observed in IFN-γELISpot assays suggests the existence of shared immunoepotopes between HPV 6 and 11E6 and E7 antigens.

[0215] Spleen cells from mice receiving both p6E6E7 and p11E6E7 (combination group) were also subjected to the above ELISpot assay to examine whether there was any immune interference when the two constructs were co-inoculated. Figure 3A and Figure 3B The combined group exhibited an average of 1670 SFU / 10⁶ splenocytes against HPV6 E6 and E7 peptides (σ = 55.7, p < 0.001). Splenocytes in the same group produced 2010 SFU / 10⁶ splenocytes against HPV11 E6 and E7 peptides (σ = 247.8, p = 0.002). The data indicate that parallel inoculation with these two constructs elicits statistically significant E6 and E7-specific cellular responses against HPV6 and HPV11, and these responses are independent of each other.

[0216] Epitope plotting

[0217] Epitope mapping studies were conducted to identify dominant epitopes within the peptide pool, in order to determine the immunodominant peptides within the E6 / E7 common antigen. Figure 4A and Figure 4B The study was conducted similarly to the previously mentioned IFN-γ ELISpot assay. Individual peptides, rather than pools, were used to stimulate spleen cells.

[0218] Each peptide used in this single-peptide analysis represents a partially overlapping fragment of the E6 and E7 antigens of HPV6 or HPV11. Plotting data indicate that peptide 7 (TAEIYSYAYKQLKVL) SEQ ID NO:11 is the dominant epitope of both the HPV6 E6 and E7 immunogens. Figure 4A TAEIYSYAYKQLKVL SEQ ID NO:11 contains 8, 9, and 10-mer amino acid epitopes, which have been verified as H2-Kb-restricted epitopes by NIH BIMAS HLA-binding prediction software. To further describe the HPV11 E6 and E7 specific T-cell immune responses (… Figure 4B Peptide analysis was also performed using overlapping fragments of HPV11 E6 and E7. Peptide mapping revealed that the dominant epitopes of the HPV11 E6 and E7 antigens were peptides 7 (TAEIYAYAYKNLKVV) SEQ ID NO:12 and 27 (HCYEQLEDSSEDEVD) SEQ ID NO:13. Similar to peptide 7 in the HPV6 epitope mapping assay, TAEIYAYAYKNLKVV SEQ ID NO:12 was identified as an H2-Kb-restricted epitope by BIMAS HLA-binding prediction software. Another HLA-binding peptide database, the Immune Epitope Database and Analysis Resource provided by NIH NIAID, identified HCYEQLEDSSEDEVD SEQ ID NO:13 as an H2-Kb-restricted epitope. This epitope mapping study identified three immune subdominant peptides: No. 6 (FCKNALTTAEIYSYA) SEQ ID NO:14, No. 9 (LFRGGYPYAACACCL) SEQ ID NO:15, and No. 13 (YAGYATTVEEETKQD) SEQ ID NO:16.

[0219] Intracellular cytokine staining

[0220] Intracellular cytokine staining assays were performed to provide a more comprehensive overview of the cellular responses induced by p6E6E7 and p11E6E7, based on the hyperimmune response characterized by IFN-γ ELISpot assays. Spleen cells were isolated from vaccinated and untreated mice and stimulated for 4 hours at 37°C in a 5% CO2 environment with peptides covering the E6 and E7 regions of HPV6 and HPV11. Positive and negative controls were used in the assays by placing cells in phorbol 12-myristate 13-acetate (PMA) and R10 cell culture media, respectively. After incubation, cells were first stained with ViViD dye (Invitrogen, Carlsbad, CA) to distinguish between live and dead cells, followed by staining with the following surface marker antibodies: APC-Cy7 hamster anti-mouse CD3e, PerCP-Cy5.5 rat anti-mouse CD4, and APC rat anti-mouse CD8a (BD Biosciences, San Diego, CA). Cells were then fixed using the Cytofix / Cytoperm kit (BD Biosciences, San Diego, CA). After fixation according to the manufacturer's protocol, cells were stained with the following intracellular marker antibodies: Alexa Fluor 700 rat anti-mouse IFN-γ, PE-Cy7 rat anti-mouse TNF clone, and PE rat anti-mouse IL-2 (BD Biosciences, San Diego, CA). Following staining, cells were fixed with PBS containing 2% paraformaldehyde. Cells were prepared and acquired using an LSR II flow cytometer equipped with BD FACSDiva software (BD Biosciences, San Jose, CA). Data were analyzed using the latest version of FlowJo software (Tree Star, Ashland, OR). The following gating sequences were used to separate CD4+ and CD8+ events: singlet cells from FSC-A versus FSC-H, all spleen cells from FSC-A versus SSC-A, live cells from ViViD dye (Pacific Blue) versus SSC-A, CD3+ cells from CD3 (APC-Cy7) versus SSC-A, and CD4+ or CD8+ cells from CD4 (PerCP-Cy5.5: positive – CD4+, negative – CD8+) versus SSC-A. The latter two populations were gated against Alexa Fluor700, PE-Cy7, and PE, respectively, to observe changes in IL-2, IFN-γ, and TNF-α production.

[0221] Cell gating was employed to differentiate intracellular cytokine staining data based on CD4+ and CD8+ cellular responses. Mice inoculated with p6E6E7 showed mean values ​​of 0.163% (σ = 0.09), 0.003% (σ = 0.07), and 0.188% (σ = 0.20) of total CD4+ cells producing IFN-γ, TNF-α, and IL-2, respectively, upon stimulation with HPV6 E6 and E7-specific peptides. Figure 5A Mice in the same group had mean total CD8+ cells producing IFN-γ, TNF-α, and IL-2 at 3.323% (σ = 1.39), 0.838% (σ = 0.32), and 1.172% (σ = 1.81), respectively. The same intracellular cytokine data were collected using spleen cells from mice inoculated with p11E6E7 after incubation with HPV11 E6 and E7 antigens. Figure 5B In all CD4+ cells of p11E6E7-inoculated mice, the mean percentages were 0.051% (σ = 0.04) producing IFN-γ, 0.068% (σ = 0.09) producing TNF-α, and 0.026% (σ = 0.037) producing IL-2. In addition, in all CD8+ cells of p11E6E7-inoculated mice, the mean percentages of IFN-γ, TNF-α, and IL-2 production were 4.52% (σ = 2.53), 2.08% (σ = 1.56), and 0.21% (σ = 0.22), respectively. With the exception of a few groups, the percentages of cells producing cytokines in the treated groups were statistically significant compared to their corresponding untreated groups at a confidence level of 89% or higher. A comparison of the levels of cytokine production by CD4+ cells and CD8+ T cells led to the conclusion that the immune responses induced by p6E6E7 and p11E6E7 are highly inclined to drive CD8+ lymphocytes, and these immune responses are associated with their cell clearance rates in all models.

[0222] Statistical analysis

[0223] Student's t-tests were performed to analyze the statistical significance of all quantitative data generated in this study. Unless otherwise specified, p-values ​​were calculated to determine statistical significance at different confidence levels.

[0224] This study has provided strong evidence that DNA vaccines can achieve the immunogenicity levels observed in experiments using other popular and conventional vaccine platforms. High levels of cellular responses measured in other similar E6 and E7-specific HPV DNA vaccine studies have been associated with data indicating prophylactic and therapeutic antitumor efficacy. Most HPV-related studies and disease challenge models focus on cervical cancer. Given that HPV6 and HPV11 are not associated with cervical cancer as other HPV serotypes, the therapeutic efficacy of p6E6E7 and p11E6E7 cannot be fully assessed using conventional HPV disease challenge models. It is foreseeable that the protective and therapeutic potential of p6E6E7 and p11E6E7 will be determined when appropriate disease challenge models are available. Therefore, the immunogenicity of p6E6E7 and p11E6E7 can only be inferred by observing high levels of IFN-γ and cytokine production as measured by ELISpot assays and intracellular cytokine staining. However, the significantly higher levels observed indicate that both constructs will elicit high levels of cellular immune responses. Since HPV-related malignancies often result from concurrent infections with multiple serotypes, further research is urgently needed to investigate the feasibility of combining vaccines targeting different viral serotypes. Future studies investigating T-cell dynamics and vaccine competition will further characterize the efficacy of parallel vaccination with two plasmids.

[0225] Intracellular cytokine staining showed that inoculation with p6E6E7 and p11E6E7 elicited a significant percentage of T cells producing IFN-γ, TNF-α, and IL-2. Given the currently known functions of IFN-γ in the immune system, it has historically been used as an indicator of cellular immune responses. Some of these important roles include the ability to regulate and stimulate innate and adaptive immunity. Furthermore, it is widely accepted that T cells are the primary producers of IFN-γ, making IFN-γ production an acceptable modality for measuring cellular immunogenicity following exposure to a given vaccine antigen. TNF-α is another cytokine involved in the regulation of the immune system. TNF-α's known ability to induce apoptosis and regulate tumor proliferation makes it an important parameter to consider when characterizing immune responses after vaccination. TNF-α production may be of particular interest given the potential tumor-proliferating properties of HPV6 and HPV11. IL-2 is another signaling molecule that has been observed to play a central role in the proliferation and differentiation of T cells in the immune system. As a result, IL-2 is often examined along with other cytokines to gain further overall insight into the quantity and quality of specific immune responses. As described above, the significant percentage of CD4+ and CD8+ cells producing IFN-γ, TNF-α, and IL-2 after p6E6E7 vaccination indicates that the vaccine successfully induced an effective immune response. The same trend applied to cells isolated from p11E6E7-vaccinated mice, except for IL-2-secreting CD4+ cells. Furthermore, it is noteworthy that CD8+ cells were observed driving a strong immune response in vaccinated mice, a crucial feature in assessing the antitumor efficacy of both plasmids.

[0226] Example 3

[0227] Mice and treatment groups vaccinated

[0228] Female C57BL / 6 mice aged 6 to 8 weeks can be used. Mice can be obtained from the Jackson Laboratory (Bar Harbor, ME). Mice can be divided into groups of four for immunization. Mice can be immunized with one of the following combinations: a) plasmids encoding HPV16E6E7, HPV18E6E7, HPV6E6E7, HPV11E6E7, HPV31E6E7, HPV33E6E7, HPV52E6E7, and HPV58E6E7; b) plasmids encoding HPV16E6E7, HPV18E6E7, and HPV31E6E7. The plasmids encoding HPV33E6E7, HPV52E6E7, and HPV58E6E7; c) the plasmids encoding HPV16E6E7, HPV18E6E7, HPV31E6E7, and HPV33E6E7; d) the plasmids encoding HPV16E6E7, HPV18E6E7, HPV6E6E7, and HPV11E6E7; and the pVAX group used as a negative control.

[0229] DNA vaccination and electroporation

[0230] Each mouse was administered a total of three doses of 20 μg of each DNA plasmid at 14-day intervals. The DNA constructs were administered via intramuscular injection into the right quadriceps, followed by square-wave pulses generated by a CELLECTRA electroporator (Inovio Pharmaceuticals, BlueBell, PA). The electroporator was configured to deliver two pulses at 0.2 Amps at a rate of 52 ms / pulse with a 1-second delay. The electroporation procedure was performed as previously described in the CELLECTRA information platform of Inovio Pharmaceuticals (information courtesy).

[0231] IFN-γELISpot assay

[0232] Mice in both the treatment and control groups were sacrificed one week after the third immunization. Spleens were collected from each mouse and transferred to RPMI-1640 medium (R10) containing 10% FBS and antibiotics. The spleens were homogenized using a homogenizing bag (Seward Laboratory Systems, Bohemia, NY) and then transferred through a cell filter and resuspended in ACK lysis buffer. After erythrocyte removal, spleen cells were isolated and resuspended in R10 medium. High-protein IP 96-well Multiscreen plates (Millipore, Bedford, MA) were pre-coated with monoclonal mouse IFN-γ capture antibody (R&D Systems, Minneapolis, MN) and incubated overnight at 4°C. After washing three times with 1X PBS, the plates were blocked for 2 hours at ambient temperature with 1×PBS solution of 1% BSA and 5% sucrose. The isolated spleen cells in R10 medium were counted and added to the wells in triplicate at 2 × 10⁵ cells per well. Two sets of peptides, encompassing the common E6 / E7 sequence of each HPV plasmid, were reconstituted in DMSO (GenScript USA, Piscataway, NJ). The peptides contain 15 amino acid sequences, with 8 residues overlapping each consecutive peptide. Each HPV plasmid peptide was divided into two pools at a concentration of 2 μg / mL in DMSO—one pool for E6 and the other for E7. Wells reserved for positive and negative controls were filled with concanavalin A (Sigma-Aldrich, St. Louis, MO) and R10 medium, respectively, instead of the peptides. The plates were then placed in a 5% CO2 incubator. After incubation at 37°C for 24 hours, the wells were washed with 1×PBS. Biotinylated anti-mouse IFN-γ detection antibody (R&D Systems, Minneapolis, MN) was added to each well, followed by overnight incubation at 4°C. The plates were then washed and treated with streptomycin-AP and BCIP / NBT Plus (R&D Systems, Minneapolis, MN) according to the colorimetric protocol provided by R&D Systems. The wells were air-dried overnight and then passed through a container... and The software's ELISpot plate reading system (Cellular Technology Ltd., ShakerHeights, OH) scans and counts the spots within the wells. An algorithm is used to convert the reported spot-forming cell counts to represent spot-forming units per 1 × 10⁶ spleen cells.

[0233] Given the sensitivity and ability of IFN-γ ELISpot assays to indicate T cell activity, they were used to determine the number of antigen-specific IFN-γ secreting cells in response to stimulation by HPV 6 or 11E6 and E7 peptides.

[0234] Example 4: Human Results from Phase 1 of VGX-3100

[0235] (A combination of HPV16 E6E7 and HPV18 E6E7)

[0236] Patients with previously treated CIN 2 / 3

[0237] use VGX-3100 combination DNA vaccine delivered intramuscularly (IM) via a constant current EP device.

[0238] See Bagarazzi et al., Sci Transl Med 4,155ra138 (2012), which is incorporated herein in its entirety.

[0239] 1 6 0.3 x 2 plasmids (HPV16 / HPV18) 2 6 1x2 plasmids (HPV16 / HPV18) 3 6 3x2 plasmids (HPV16 / HPV18)

[0240] Antibody response:

[0241] Antibodies against all four antigens with high titers of 15 / 18 (83%) and confirmed protein blots persisted for up to 9 months.

[0242] Antigen-specific cellular responses to HPV16, 18, E6, and E7:

[0243] - 14 / 18 (78%) POS (>50 SFU / 106 PBMC) via IFN-γELISpot

[0244] - For 1Ag, increase the w / dose to >2500 SFU / 106 PBMC; for all 4 antigens, >5670 SFU / 106 PBMC.

[0245] - Five subjects responded to all four antigens.

[0246] - The response lasted up to 9 months after the initial series.

[0247] - The fourth dose induced T-cell responses lasting for >2 years.

[0248] -HLA DR+ / CD38+CD8+ T cells release granzyme B / perforin for cell killing. sequence list <110> University of Pennsylvania Board of Trustees Aino Pharmaceutical Co., Ltd. <120> Improved vaccines for human papillomavirus and their administration <130> UPVG0025 WO3 <150> 61777198 <151> 2013-03-12 <160> 26 <170> PatentIn version 3.5 <210> 1 <211> 873 <212> DNA <213> Artificial sequence <220> <223> HPV6 E6E7 DNA sequence <400> 1 ggtaccggat ccgccaccat ggactggacc tggattctgt tcctggtggc cgctgccaca 60 cgggtgcaca gcgaaagcgc caatgccagc accagcgcta caaccatcga ccagctgtgc 120 aagaccttca acctgagcat gcacaccctg cagatcaact gcgtgttctg caagaacgcc 180 ctgaccaccg ccgagatcta cagctacgcc tacaagcagc tgaaggtgct gttcagaggc 240 ggctacccct atgccgcctg cgcctgctgc ctggaatttc acggcaagat caaccagtac 300 cggcacttcg actacgccgg ctacgccacc accgtggaag aggaaacaaa gcaggacatc 360 ctggacgtgc tgatccggtg ctacctgtgc cacaagcccc tgtgcgaggt ggaaaaagtg 420 aagcacatcc tgaccaaggc ccggttcatc aagctgaact gcacctggaa gggccggtgc 480 ctgcactgct ggaccacctg tatggaagat atgctgccca gaggccggaa gcggcggagc 540 catggcagac acgtgaccct gaaggacatc gtgctggacc tgcagccccc cgatcctgtg 600 ggcctgcact gttacgagca gctggtggac agcagcgagg acgaggtgga cgaagtggac 660 ggccaggaca gccagcccct gaagcagcac ttccagatcg tgacctgctg ctgcggctgc 720 gacagcaacg tgcggctggt ggtgcagtgc accgagacag acatcagaga ggtccagcag 780 ctcctgctgg gcaccctgaa catcgtgtgc cccatctgcg cccccaagac ctacccttac 840 gacgtgcccg actacgcctg atgactcgag ctc 873 <210> 2 <211> 280 <212> PRT <213> Artificial sequence <220> <223> HPV6 E6E7 protein sequence <400> 2 Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val 1 5 10 15 His Ser Glu Ser Ala Asn Ala Ser Thr Ser Ala Thr Thr Ile Asp Gln 20 25 30 Leu Cys Lys Thr Phe Asn Leu Ser Met His Thr Leu Gln Ile Asn Cys 35 40 45 Val Phe Cys Lys Asn Ala Leu Thr Thr Ala Glu Ile Tyr Ser Tyr Ala 50 55 60 Tyr Lys Gln Leu Lys Val Leu Phe Arg Gly Gly Tyr Pro Tyr Ala Ala 65 70 75 80 Cys Ala Cys Cys Leu Glu Phe His Gly Lys Ile Asn Gln Tyr Arg His 85 90 95 Phe Asp Tyr Ala Gly Tyr Ala Thr Thr Val Glu Glu Glu Thr Lys Gln 100 105 110 Asp Ile Leu Asp Val Leu Ile Arg Cys Tyr Leu Cys His Lys Pro Leu 115 120 125 Cys Glu Val Glu Lys Val Lys His Ile Leu Thr Lys Ala Arg Phe Ile 130 135 140 Lys Leu Asn Cys Thr Trp Lys Gly Arg Cys Leu His Cys Trp Thr Thr 145 150 155 160 Cys Met Glu Asp Met Leu Pro Arg Gly Arg Lys Arg Arg Ser His Gly 165 170 175 Arg His Val Thr Leu Lys Asp Ile Val Leu Asp Leu Gln Pro Pro Asp 180 185 190 Pro Val Gly Leu His Cys Tyr Glu Gln Leu Val Asp Ser Ser Glu Asp 195 200 205 Glu Val Asp Glu Val Asp Gly Gln Asp Ser Gln Pro Leu Lys Gln His 210 215 220 Phe Gln Ile Val Thr Cys Cys Cys Gly Cys Asp Ser Asn Val Arg Leu 225 230 235 240 Val Val Gln Cys Thr Glu Thr Asp Ile Arg Glu Val Gln Gln Leu Leu 245 250 255 Leu Gly Thr Leu Asn Ile Val Cys Pro Ile Cys Ala Pro Lys Thr Tyr 260 265 270 Pro Tyr Asp Val Pro Asp Tyr Ala 275 280 <210> 3 <211> 873 <212> DNA <213> Artificial sequence <220> <223> HPV11 E6E7 DNA sequence <400> 3 ggtaccggat ccgccaccat ggactggacc tggattctgt tcctggtggc cgctgccaca 60 agagtgcaca gcgagagcaa ggatgccagc accagcgcca ccagcatcga ccagctgtgc 120 aagaccttca acctgagcct gcacaccctg cagatccagt gcgtgttctg ccggaacgcc 180 ctgaccaccg ccgagatcta cgcctacgcc tacaagaacc tgaaggtcgt gtggcgggac 240 aacttcccct tcgccgcctg cgcctgctgc ctggaactgc agggcaagat caaccagtac 300 cggcacttca actacgccgc ttacgccccc accgtggaag aggaaacaaa cgaggacatc 360 ctgaaggtgc tgatccggtg ctacctgtgc cacaagcccc tgtgcgagat cgagaagctg 420 aagcacatcc tgggcaaggc ccggttcatc aagctgaaca accagtggaa gggccggtgc 480 ctgcactgct ggaccacctg tatggaagat ctgctgccca gaggccggaa gcggagaagc 540 cacggcagac tggtcaccct gaaggacatc gtgctggacc tgcagccccc cgatcctgtg 600 ggcctgcact gttacgagca gctggaagat agcagcgagg acgaggtgga caaagtggac 660 aagcaggacg cccagcccct gacccagcac taccagatcc tgacctgctg ctgcggctgc 720 gacagcaacg tgcggctggt ggtggaatgc accgacggcg acatccggca gctccaggat 780 ctgctgctgg gcaccctgaa catcgtgtgc cccatctgcg cccccaagcc ctacccctac 840 gacgtgcccg actacgcctg atgactcgag ctc 873 <210> 4 <211> 280 <212> PRT <213> Artificial sequence <220> <223> HPV11 E6E7 protein sequence <400> 4 Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val 1 5 10 15 His Ser Glu Ser Lys Asp Ala Ser Thr Ser Ala Thr Ser Ile Asp Gln 20 25 30 Leu Cys Lys Thr Phe Asn Leu Ser Leu His Thr Leu Gln Ile Gln Cys 35 40 45 Val Phe Cys Arg Asn Ala Leu Thr Thr Ala Glu Ile Tyr Ala Tyr Ala 50 55 60 Tyr Lys Asn Leu Lys Val Val Trp Arg Asp Asn Phe Pro Phe Ala Ala 65 70 75 80 Cys Ala Cys Cys Leu Glu Leu Gln Gly Lys Ile Asn Gln Tyr Arg His 85 90 95 Phe Asn Tyr Ala Ala Tyr Ala Pro Thr Val Glu Glu Glu Thr Asn Glu 100 105 110 Asp Ile Leu Lys Val Leu Ile Arg Cys Tyr Leu Cys His Lys Pro Leu 115 120 125 Cys Glu Ile Glu Lys Leu Lys His Ile Leu Gly Lys Ala Arg Phe Ile 130 135 140 Lys Leu Asn Asn Gln Trp Lys Gly Arg Cys Leu His Cys Trp Thr Thr 145 150 155 160 Cys Met Glu Asp Leu Leu Pro Arg Gly Arg Lys Arg Arg Ser His Gly 165 170 175 Arg Leu Val Thr Leu Lys Asp Ile Val Leu Asp Leu Gln Pro Pro Asp 180 185 190 Pro Val Gly Leu His Cys Tyr Glu Gln Leu Glu Asp Ser Ser Glu Asp 195 200 205 Glu Val Asp Lys Val Asp Lys Gln Asp Ala Gln Pro Leu Thr Gln His 210 215 220 Tyr Gln Ile Leu Thr Cys Cys Cys Gly Cys Asp Ser Asn Val Arg Leu 225 230 235 240 Val Val Glu Cys Thr Asp Gly Asp Ile Arg Gln Leu Gln Asp Leu Leu 245 250 255 Leu Gly Thr Leu Asn Ile Val Cys Pro Ile Cys Ala Pro Lys Pro Tyr 260 265 270 Pro Tyr Asp Val Pro Asp Tyr Ala 275 280 <210> 5 <211> 828 <212> DNA <213> Artificial sequence <220> <223> HPV33 E6E7 DNA sequence <400> 5 ggtaccgtcg acggatccgc caccatggac tggacctgga ttctgttcct ggtggccgct 60 gctacccggg tgcacagctt ccaggacacc gaggaaagcg gccggaccct gcacgatctg 120 tgccaggccc tggaaaccac catccacaac atcgagctgc agtgcgtgga atgcaagaag 180 cccctgcagc ggagcgaggt gtacgacgcc gacctgaccg tggtgtacag agagggcaac 240 cccttcggca tctgcaagct gtgcctgcgg ttcctgagca agatcagcga gtaccggcac 300 tacaactaca gcgtgtacgg caacaccctg gaacagaccg tgaagaagcc tctgaacgag 360 atcctgatcc ggtgcatcat ctgccagcgg cccctgaagc ggcacgtgga cctgaacaag 420 cggttccaca atatcagcgg cagatgggcc ggcagatgcg ccgcctgttg gagaagccgg 480 cggagagaga cagccctgcg gggcagaaag cggcggagca gaggccacaa gcccaccctg 540 aaagaatacg tgctggacct gtaccccgag cccaccgacc tgtacggcta cggccagctg 600 agcgacagca gcgacgagga cgagggcctg gacagacctg atggacaggc ccagcccgcc 660 accgccgact actacatcgt gacctgctgc cacacctgta acaccaccgt gcggctgtgc 720 gtgaacagca ccgccagcga cctgcggaca atccagcagc tcctgatggg caccgtgaac 780 atcgtgtgcc ctacctgcgc ccagctgtga ctcgagacgc gtgagctc 828 <210> 6 <211> 261 <212> PRT <213> Artificial sequence <220> <223> HPV33 E6E7 protein sequence <400> 6 Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val 1 5 10 15 His Ser Phe Gln Asp Thr Glu Glu Ser Gly Arg Thr Leu His Asp Leu 20 25 30 Cys Gln Ala Leu Glu Thr Thr Ile His Asn Ile Glu Leu Gln Cys Val 35 40 45 Glu Cys Lys Lys Pro Leu Gln Arg Ser Glu Val Tyr Asp Ala Asp Leu 50 55 60 Thr Val Val Tyr Arg Glu Gly Asn Pro Phe Gly Ile Cys Lys Leu Cys 65 70 75 80 Leu Arg Phe Leu Ser Lys Ile Ser Glu Tyr Arg His Tyr Asn Tyr Ser 85 90 95 Val Tyr Gly Asn Thr Leu Glu Gln Thr Val Lys Lys Pro Leu Asn Glu 100 105 110 Ile Leu Ile Arg Cys Ile Ile Cys Gln Arg Pro Leu Lys Arg His Val 115 120 125 Asp Leu Asn Lys Arg Phe His Asn Ile Ser Gly Arg Trp Ala Gly Arg 130 135 140 Cys Ala Ala Cys Trp Arg Ser Arg Arg Arg Glu Thr Ala Leu Arg Gly 145 150 155 160 Arg Lys Arg Arg Ser Arg Gly His Lys Pro Thr Leu Lys Glu Tyr Val 165 170 175 Leu Asp Leu Tyr Pro Glu Pro Thr Asp Leu Tyr Gly Tyr Gly Gln Leu 180 185 190 Ser Asp Ser Ser Asp Glu Asp Glu Gly Leu Asp Arg Pro Asp Gly Gln 195 200 205 Ala Gln Pro Ala Thr Ala Asp Tyr Tyr Ile Val Thr Cys Cys His Thr 210 215 220 Cys Asn Thr Thr Val Arg Leu Cys Val Asn Ser Thr Ala Ser Asp Leu 225 230 235 240 Arg Thr Ile Gln Gln Leu Leu Met Gly Thr Val Asn Ile Val Cys Pro 245 250 255 Thr Cys Ala Gln Leu 260 <210> 7 <211> 835 <212> DNA <213> Artificial sequence <220> <223> HPV58 E6E7 DNA sequence <400> 7 ggtaccgttt aaacggatcc gccaccatgg actggacctg gattctgttc ctggtggccg 60 ctgctacccg ggtgcacagc ttccaggatg ccgaggaaag cggccggacc ctgcacgatc 120 tgtgccaggc cctggaaacc agcgtgcacg agatcgagct gaagtgcgtg gaatgcaaga 180 aaaccctgca gcggagcgag gtgtacgacg ccgacctgcg gatcgtgtac cgggacggca 240 accccttcgc cgtgtgcaaa gtgtgcctgc ggctgctgag caagatcagc gagtaccggc 300 actacaacta cagcctgtac ggcgacaccc tggaacagac cctgaagaag tgcctgaacg 360 agatcctgat ccggtgcatc atctgccagc ggcccctgaa gcggcacgtg gacctgaaca 420 agcggttcca caacatcagc ggccggtgga ccggcagatg cgccgtgtgt tggaggccca 480 ggcggagaca gacccaggtg cgcggcagaa agcggcggag cagaggcaac aaccccaccc 540 tgagagagta catcctggac ctgcaccccg agcccaccga cctgtttggc tacggccagc 600 tgtgcgacag cagcgacgag gacgagatcg gcctggacgg ccctgatgga caggcccagc 660 ccgccaccgc caactactac atcgtgacct gctgctacac ctgtggcacc accgtgcggc 720 tgtgcatcaa cagcaccacc accgacgtgc gcaccctgca gcagctcctg atgggcacct 780 gtaccatcgt gtgccccagc tgcgcccagc agtgactcga ggcggccgcg agctc 835 <210> 8 <211> 262 <212> PRT <213> Artificial sequence <220> <223> HPV58 E6E7 protein sequence <400> 8 Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val 1 5 10 15 His Ser Phe Gln Asp Ala Glu Glu Ser Gly Arg Thr Leu His Asp Leu 20 25 30 Cys Gln Ala Leu Glu Thr Ser Val His Glu Ile Glu Leu Lys Cys Val 35 40 45 Glu Cys Lys Lys Thr Leu Gln Arg Ser Glu Val Tyr Asp Ala Asp Leu 50 55 60 Arg Ile Val Tyr Arg Asp Gly Asn Pro Phe Ala Val Cys Lys Val Cys 65 70 75 80 Leu Arg Leu Leu Ser Lys Ile Ser Glu Tyr Arg His Tyr Asn Tyr Ser 85 90 95 Leu Tyr Gly Asp Thr Leu Glu Gln Thr Leu Lys Lys Cys Leu Asn Glu 100 105 110 Ile Leu Ile Arg Cys Ile Ile Cys Gln Arg Pro Leu Lys Arg His Val 115 120 125 Asp Leu Asn Lys Arg Phe His Asn Ile Ser Gly Arg Trp Thr Gly Arg 130 135 140 Cys Ala Val Cys Trp Arg Pro Arg Arg Arg Gln Thr Gln Val Arg Gly 145 150 155 160 Arg Lys Arg Arg Ser Arg Gly Asn Asn Pro Thr Leu Arg Glu Tyr Ile 165 170 175 Leu Asp Leu His Pro Glu Pro Thr Asp Leu Phe Gly Tyr Gly Gln Leu 180 185 190 Cys Asp Ser Ser Asp Glu Asp Glu Ile Gly Leu Asp Gly Pro Asp Gly 195 200 205 Gln Ala Gln Pro Ala Thr Ala Asn Tyr Tyr Ile Val Thr Cys Cys Tyr 210 215 220 Thr Cys Gly Thr Thr Val Arg Leu Cys Ile Asn Ser Thr Thr Thr Asp 225 230 235 240 Val Arg Thr Leu Gln Gln Leu Leu Met Gly Thr Cys Thr Ile Val Cys 245 250 255 Pro Ser Cys Ala Gln Gln 260 <210> 9 <211> 54 <212> DNA <213> Artificial sequence <220> <223> IgE leader DNA sequence <400> 9 atggactgga cctggatcct gttcctggtg gccgctgcca cacgggtgca cagc 54 <210> 10 <211> 18 <212> PRT <213> Artificial sequence <220> <223> IgE precursor protein <400> 10 Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val 1 5 10 15 His Ser <210> 11 <211> 15 <212> PRT <213> Artificial sequence <220> <223> HPV6 peptide 7 <400> 11 Thr Ala Glu Ile Tyr Ser Tyr Ala Tyr Lys Gln Leu Lys Val Leu 1 5 10 15 <210> 12 <211> 15 <212> PRT <213> Artificial sequence <220> <223> HPV11 peptide 7 <400> 12 Thr Ala Glu Ile Tyr Ala Tyr Ala Tyr Lys Asn Leu Lys Val Val 1 5 10 15 <210> 13 <211> 15 <212> PRT <213> Artificial sequence <220> <223> HPV11 peptide 27 <400> 13 His Cys Tyr Glu Gln Leu Glu Asp Ser Ser Glu Asp Glu Val Asp 1 5 10 15 <210> 14 <211> 15 <212> PRT <213> Artificial sequence <220> <223> HPV subdominant peptide 6 <400> 14 Phe Cys Lys Asn Ala Leu Thr Thr Ala Glu Ile Tyr Ser Tyr Ala 1 5 10 15 <210> 15 <211> 15 <212> PRT <213> Artificial sequence <220> <223> HPV subdominant peptide 9 <400> 15 Leu Phe Arg Gly Gly Tyr Pro Tyr Ala Ala Cys Ala Cys Cys Leu 1 5 10 15 <210> 16 <211> 15 <212> PRT <213> Artificial sequence <220> <223> HPV testing13 <400> 16 Tyr Ala Gly Tyr Ala Thr Thr Val Glu Glu Glu Thr Lys Gln Asp 1 5 10 15 <210> 17 <211> 792 <212> DNA <213> The snowstorm <220> <223> pGX3005 harboring HPV31 E6 / E7 DNA <400> 17 atggactgga catggattct gttcctggtc gccgccgca ctaggtgca ttcattcaaa aaccccgccg agtctgggag aaaactgcac gagctgagct ccgcactgga gatcccctac 180. gatgaactga ggctgaactg cgtgtattgt aaaggacagc tgactgagac cgaagtgctg 240. gacactgatc tgaccattgt ctaccgagac gatacacctt atggcgtgtg cactaaatgt ctgcgcttct actcaaaggt gagcgagttt agatggtaca ggtatagcgt ctatggcacc acactggaa aactgacaaa caaggggatc tgcgacctgc tgatcaggtg cattacttgt cagcgccctc tgcagcgaca cctggataag aagaagcggt tccataatat cggcgggcgg 420 tggaccggga gatgcatcgt gtgctggcgg agaccacgga cagagactca ggtccgaggg 480 cggaaaaggc gctctagagg agaaacccca acactgcagg actacgtgct ggatctgcag 540 cccgaggcca cagacctgca tggatatggc cagctgcccg actctagtga tgaggaagac 600 gtgatcgata gtcccgccgg ccaggctaag cctgatacct ccaactacaa tattgtcaca 660 ttttgctgtc agtgcgaatc cactctgagg ctgtgcgtgc agtctaccca ggtcgacatc 720 cgcattctgc aggaactgct gatgggatct ttcggaatcg tgtgccctaa ttgctccacc 780 agactgtgat aa 792 <210> 18 <211> 262 <212> PRT <213> Artificial sequence <220> <223> pGX3005 insert HPV31 E6 / E7 protein sequence <400> 18 Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val 1 5 10 15 His Ser Phe Lys Asn Pro Ala Glu Ser Gly Arg Lys Leu His Glu Leu 20 25 30 Ser Ser Ala Leu Glu Ile Pro Tyr Asp Glu Leu Arg Leu Asn Cys Val 35 40 45 Tyr Cys Lys Gly Gln Leu Thr Glu Thr Glu Val Leu Asp Thr Asp Leu 50 55 60 Thr Ile Val Tyr Arg Asp Asp Thr Pro Tyr Gly Val Cys Thr Lys Cys 65 70 75 80 Leu Arg Phe Tyr Ser Lys Val Ser Glu Phe Arg Trp Tyr Arg Tyr Ser 85 90 95 Val Tyr Gly Thr Thr Leu Glu Lys Leu Thr Asn Lys Gly Ile Cys Asp 100 105 110 Leu Leu Ile Arg Cys Ile Thr Cys Gln Arg Pro Leu Gln Arg His Leu 115 120 125 Asp Lys Lys Lys Arg Phe His Asn Ile Gly Gly Arg Trp Thr Gly Arg 130 135 140 Cys Ile Val Cys Trp Arg Arg Pro Arg Thr Glu Thr Gln Val Arg Gly 145 150 155 160 Arg Lys Arg Arg Ser Arg Gly Glu Thr Pro Thr Leu Gln Asp Tyr Val 165 170 175 Leu Asp Leu Gln Pro Glu Ala Thr Asp Leu His Gly Tyr Gly Gln Leu 180 185 190 Pro Asp Ser Ser Asp Glu Glu Asp Val Ile Asp Ser Pro Ala Gly Gln 195 200 205 Ala Lys Pro Asp Thr Ser Asn Tyr Asn Ile Val Thr Phe Cys Cys Gln 210 215 220 Cys Glu Ser Thr Leu Arg Leu Cys Val Gln Ser Thr Gln Val Asp Ile 225 230 235 240 Arg Ile Leu Gln Glu Leu Leu Met Gly Ser Phe Gly Ile Val Cys Pro 245 250 255 Asn Cys Ser Thr Arg Leu 260 <210> 19 <211> 792 <212> DNA <213> Artificial sequence <220> <223> HPV52 E6 / E7 DNA sequence of pGX3007 insert <400> 19 atggactgga cctggattct gttcctggtg gctgctgcta cacgggtgca ttcatttgaa 60 gaccctgcta catctggacg aactctgcac gagctgtgcg aagtgctgga ggaatctgtc 120 catgagatca ggctgcagtg cgtgcagtgt aagaaagagc tgcagcggag agaagtgtac 180 aagaccgacc tgcggattgt ctacagagat aacaatccct atggagtgtg catcatgtgc 240 ctgcggttcc tgtctaaaat tagtgaatac agacactacc agtattccct gtatggcaag 300 acactggagg aacgagtgcg gaaaccactg tctgagatca ccattcgctg catcatttgt 360 cagacacccc tggaacgaca cgtcaacgca aacaagcggt tccataacat catgggaagg 420 tggactggcc gctgcagtga gtgttggaga ccaaggcctg tgacccaggt ccgcggacga 480 aaacggcgga gccggggcga caaggcaacc atcaaagact acattctgga tctgcagcct 540 gagaccacag atctgcatgg ctatgggcag ctgggggaca gctccgatga ggaagacaca 600 gatggggtgg acagaccaga tggacaggcc gaacaggcta caagcaatta ctatatcgtc 660 acttattgcc actcatgtga cagcactctg aggctgtgca ttcattccac tgctaccgat 720 ctgcgcacac tgcagcagat gctgctggga acactgcagg tggtctgtcc aggatgtgcc 780 cgactgtgat aa 792 <210> 20 <211> 262 <212> PRT <213> Artificial sequence <220> <223> pGX3007 insert HPV52 E6 / E7 protein sequence <400> 20 Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val 1 5 10 15 His Ser Phe Glu Asp Pro Ala Thr Ser Gly Arg Thr Leu His Glu Leu 20 25 30 Cys Glu Val Leu Glu Glu Ser Val His Glu Ile Arg Leu Gln Cys Val 35 40 45 Gln Cys Lys Lys Glu Leu Gln Arg Arg Glu Val Tyr Lys Thr Asp Leu 50 55 60 Arg Ile Val Tyr Arg Asp Asn Asn Pro Tyr Gly Val Cys Ile Met Cys 65 70 75 80 Leu Arg Phe Leu Ser Lys Ile Ser Glu Tyr Arg His Tyr Gln Tyr Ser 85 90 95 Leu Tyr Gly Lys Thr Leu Glu Glu Arg Val Arg Lys Pro Leu Ser Glu 100 105 110 Ile Thr Ile Arg Cys Ile Ile Cys Gln Thr Pro Leu Glu Arg His Val 115 120 125 Asn Ala Asn Lys Arg Phe His Asn Ile Met Gly Arg Trp Thr Gly Arg 130 135 140 Cys Ser Glu Cys Trp Arg Pro Arg Pro Val Thr Gln Val Arg Gly Arg 145 150 155 160 Lys Arg Arg Ser Arg Gly Asp Lys Ala Thr Ile Lys Asp Tyr Ile Leu 165 170 175 Asp Leu Gln Pro Glu Thr Thr Asp Leu His Gly Tyr Gly Gln Leu Gly 180 185 190 Asp Ser Ser Asp Glu Glu Asp Thr Asp Gly Val Asp Arg Pro Asp Gly 195 200 205 Gln Ala Glu Gln Ala Thr Ser Asn Tyr Tyr Ile Val Thr Tyr Cys His 210 215 220 Ser Cys Asp Ser Thr Leu Arg Leu Cys Ile His Ser Thr Ala Thr Asp 225 230 235 240 Leu Arg Thr Leu Gln Gln Met Leu Leu Gly Thr Leu Gln Val Val Cys 245 250 255 Pro Gly Cys Ala Arg Leu 260 <210> 21 <211> 818 <212> DNA <213> Artificial sequence <220> <223> HPV genotype 16 E6-E7 DNA sequence <400> 21 gaattcgcca ccatggactg gacctggatc ctgttcctgg tggccgccgc cacacgggtg 60 cacagcttcc aggaccccca ggagagcggc agaaagctgc ctcagctgtg taccgagctg 120 cagaccacca tccacgacat catcctggag tgtgtgtact gtaagcagca gctgctgagg 180 agagaggtgt acgaccggga cctgtgtatc gtgtacaggg acggcaatcc ctacgccgtg 240 tgtgacaagt gcctgaagtt ctacagcaag atcagcgagt accggcacta ctgctacagc 300 ctgtacggca ccaccctgga gcagcagtac aacaagcccc tgtgtgacct gctgatccgg 360 tgtatcaact gccagaagcc cctgcagaga cacctggaca agaagcagcg gttccacaac atcaggggca gatggaccgg cagatgtatg agctgctgcc ggagcagcag aaccagaagg 480 gagacccagc tgagaggccg gagagaga agccacggcg atacccccac cctgcacgag 540 600. tacatgctgg acctgcagcc tgagaccacc gatctgtacg gctacggcca gctgaatgac agcagcgagg agcagcgagcgacggc cctgccggcc cgacagagcc 660 cactacaaca tcgtgacctt ttgctgtaag tgtgacagca ccctgagact gtgcgtgcag 720 agcacccacg tggacatcag aaccctggag gatctgctga tgggcaccct gggcatcgtg tgtcccatct gctcccagaa accctgatga gcggccgc 818 <210> 22 <211> 264 <212> PRT <213> The snowstorm <220> <223> HPV genes with 16 E6-E7 sequences <400> 22 Met Asp Trp Thr Trp Free Mp3 Download 1 5 10 15 His Ser Phe Gln Asp Pro Gln Glu Ser Gly Arg Lys Leu Pro Gln Leu 20 25 30 Cys Thr Glu Leu Gln Thr Thr Ile His Asp Ile Ile Leu Glu Cys Val 35 40 45 Tyr Cys Lys Gln Gln Leu Leu Arg Arg Glu Val Tyr Asp Arg Asp Leu 50 55 60 Cys Ile Val Tyr Arg Asp Gly Asn Pro Tyr Ala Val Cys Asp Lys Cys 65 70 75 80 Leu Lys Phe Tyr Ser Lys Ile Ser Glu Tyr Arg His Tyr Cys Tyr Ser 85 90 95 Leu Tyr Gly Thr Thr Leu Glu Gln Gln Tyr Asn Lys Pro Leu Cys Asp 100 105 110 Leu Leu Ile Arg Cys Ile Asn Cys Gln Lys Pro Leu Gln Arg His Leu 115 120 125 Asp Lys Lys Gln Arg Phe His Asn Ile Arg Gly Arg Trp Thr Gly Arg 130 135 140 Cys Met Ser Cys Cys Arg Ser Ser Arg Thr Arg Arg Glu Thr Gln Leu 145 150 155 160 Arg Gly Arg Lys Arg Arg Ser His Gly Asp Thr Pro Thr Leu His Glu 165 170 175 Tyr Met Leu Asp Leu Gln Pro Glu Thr Thr Asp Leu Tyr Gly Tyr Gly 180 185 190 Gln Leu Asn Asp Ser Ser Glu Glu Glu Asp Glu Ile Asp Gly Pro Ala 195 200 205 Gly Gln Ala Glu Pro Asp Arg Ala His Tyr Asn Ile Val Thr Phe Cys 210 215 220 Cys Lys Cys Asp Ser Thr Leu Arg Leu Cys Val Gln Ser Thr His Val 225 230 235 240 Asp Ile Arg Thr Leu Glu Asp Leu Leu Met Gly Thr Leu Gly Ile Val 245 250 255 Cys Pro Ile Cys Ser Gln Lys Pro 260 <210> 23 <211> 837 <212> DNA <213> Artificial sequence <220> <223> HPV genotype 18 E6-E7 DNA sequence <400> 23 atggactgga cctggatcct gttcctggtg gccgctgcca cacgggtgca cagcgccaga 60 ttcgaggacc ccaccaggag cggctacaag ctgcccgatc tgtgtaccga gctgaacacc 120 agcctgcagg acatcgagat cacctgtgtg tactgtaaga ccgtgctgga gctgaccgag 180 gtgttcgaga aggacctgtt cgtggtgtac agggacagca tcccccacgc cgcctgccac 240 aagtgtatcg acttctacag ccggatccgg gagctgagac actacagcga cagcgtgtac 300 ggcgataccc tggagaagct gaccaacacc ggcctgtaca acctgctgat ccggtgcctg 360 agatgccaga agcccctgct gagacacctg aacgagaagc ggcggttcca caacatcgcc 420 ggccactaca gaggccagtg ccacagctgc tgtaacaggg ccaggcagga gagactgcag 480 cggagaagag agacccaggt gaggggcagg aagagaagaa gccacggccc caaggccacc 540 ctgcaggata tcgtgctgca cctggagccc cagaatgaga tccccgtgga tctgctgggc 600 cacggccagc tgtccgacag cgaggaggag aacgacgaga tcgacggcgt gaatcaccag 660 cacctgcctg ccagaagagc cgagcctcag aggcacacca tgctgtgtat gtgctgtaag 720 tgtgaggccc ggatcgaact ggtggtggag agcagcgccg acgacctgag agccttccag 780 cagctgttcc tgaacaccct gagcttcgtg tgtccttggt gtgccagcca gcagtga 837 <210> 24 <211> 278 <212> PRT <213> Artificial sequence <220> <223> HPV genotype 18 E6-E7 protein sequence <400> 24 Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val 1 5 10 15 His Ser Ala Arg Phe Glu Asp Pro Thr Arg Ser Gly Tyr Lys Leu Pro 20 25 30 Asp Leu Cys Thr Glu Leu Asn Thr Ser Leu Gln Asp Ile Glu Ile Thr 35 40 45 Cys Val Tyr Cys Lys Thr Val Leu Glu Leu Thr Glu Val Phe Glu Lys 50 55 60 Asp Leu Phe Val Val Tyr Arg Asp Ser Ile Pro His Ala Ala Cys His 65 70 75 80 Lys Cys Ile Asp Phe Tyr Ser Arg Ile Arg Glu Leu Arg His Tyr Ser 85 90 95 Asp Ser Val Tyr Gly Asp Thr Leu Glu Lys Leu Thr Asn Thr Gly Leu 100 105 110 Tyr Asn Leu Leu Ile Arg Cys Leu Arg Cys Gln Lys Pro Leu Leu Arg 115 120 125 His Leu Asn Glu Lys Arg Arg Phe His Asn Ile Ala Gly His Tyr Arg 130 135 140 Gly Gln Cys His Ser Cys Cys Asn Arg Ala Arg Gln Glu Arg Leu Gln 145 150 155 160 Arg Arg Arg Glu Thr Gln Val Arg Gly Arg Lys Arg Arg Ser His Gly 165 170 175 Pro Lys Ala Thr Leu Gln Asp Ile Val Leu His Leu Glu Pro Gln Asn 180 185 190 Glu Ile Pro Val Asp Leu Leu Gly His Gly Gln Leu Ser Asp Ser Glu 195 200 205 Glu Glu Asn Asp Glu Ile Asp Gly Val Asn His Gln His Leu Pro Ala 210 215 220 Arg Arg Ala Glu Pro Gln Arg His Thr Met Leu Cys Met Cys Cys Lys 225 230 235 240 Cys Glu Ala Arg Ile Glu Leu Val Val Glu Ser Ser Ala Asp Asp Leu 245 250 255 Arg Ala Phe Gln Gln Leu Phe Leu Asn Thr Leu Ser Phe Val Cys Pro 260 265 270 Trp Cys Ala Ser Gln Gln 275 <210> 25 <211> 279 <212> PRT <213> Artificial sequence <220> <223> HPV45 E6 / E7 protein sequence <400> 25 Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val 1 5 10 15 His Ser Ala Arg Phe Asp Asp Pro Lys Gln Ser Gly Tyr Lys Leu Pro 20 25 30 Asp Leu Cys Thr Glu Leu Leu Asn Thr Ser Leu Gln Asp Val Ser Ile Ala 35 40 45 Cys Will Tyr Cys Lys Only Thr Lead Glu Arg Thr Glu Will Tyr Gln Lys 50 55 60 Asp Leu Cys Ile Val Tyr Arg Asp Cys Ile Ala Tyr Ala Ala Cys His 65 70 75 80 Lys Cys Ile Asp Phe Tyr Ser Arg Ile Arg Glu Leu Arg Tyr Tyr Ser 85 90 95 Asn Ser Val Tyr Gly Glu Thr Leu Glu Lys Ile Thr Asn Thr Glu Leu 100 105 110 Tyr Asn Leu Ile Arg Cys Leu Cys Gln Lys Pro Leu Iron Arg 115 120 125 His Lys Asp Lys Arg is also known as Gly Gln Tyr Arg. 130 135 140 Gly Gln Cys Asn Thr Cys Asp Gln Ala Arg Gln Gln Arg Leu Arg 145 150 155 160 Arg Arg Arg Glu Thr Gln Val Arg Gly Arg Lys Arg Arg Ser His Gly 165 170 175 Pro Arg Ala Thr Leu Gln Glu Ile Val Leu His Leu Glu Pro Gln Asn 180 185 190 Glu Leu Asp Pro Val Asp Leu Leu Gly Tyr Gly Gln Leu Ser Glu Ser 195 200 205 Glu Glu Glu Asn Asp Glu Ala Asp Gly Val Ser His Ala Gln Leu Pro 210 215 220 Ala Arg Arg Ala Glu Pro Gln Arg His Lys Ile Leu Cys Val Cys Cys 225 230 235 240 Lys Cys Asp Gly Arg Ile Glu Leu Thr Val Glu Ile Ser Ala Glu Asp 245 250 255 Leu Arg Thr Leu Gln Gln Leu Phe Leu Ser Thr Leu Ser Phe Val Cys 260 265 270 Pro Trp Cys Ala Thr Asn Gln 275 <210> 26 <211> 282 <212> PRT <213> artificial sequence <220> <223> HPV39 E6 / E7 protein sequence <400> 26 Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val 1 5 10 15 His Ser Ala Arg Phe His Asn Pro Ala Glu Ser Gly Tyr Lys Leu Pro 20 25 30 Asp Leu Cys Thr Thr Leu Asp Thr Thr Leu Gln Asp Ile Thr Ile Ala 35 40 45 Cys Val Tyr Cys Arg Arg Pro Leu Gln Gln Thr Glu Val Tyr Glu Ser 50 55 60 Asp Leu Tyr Val Val Tyr Arg Asp Gly Glu Pro Leu Ala Ala Cys Gln 65 70 75 80 Ser Cys Ile Lys Phe Tyr Ala Lys Ile Arg Glu Leu Arg Tyr Tyr Ser 85 90 95 Asp Ser Val Tyr Ala Thr Thr Leu Glu Asn Ile Thr Asn Thr Lys Leu 100 105 110 Tyr Asn Leu Leu Ile Arg Cys Met Cys Cys Leu Lys Pro Leu Leu Arg 115 120 125 His Leu Asn Ser Lys Arg Arg Phe His Lys Ile Ala Gly Ser Tyr Thr 130 135 140 Gly Gln Cys Arg Arg Cys Trp Thr Thr Lys Arg Glu Asp Arg Arg Leu 145 150 155 160 Thr Arg Arg Glu Thr Gln Val Arg Gly Arg Lys Arg Arg Ser Arg Gly 165 170 175 Pro Lys Pro Thr Leu Gln Glu Ile Val Leu Asp Leu Cys Pro Tyr Asn 180 185 190 Glu Ile Gln Pro Val Asp Leu Val Gly His Gly Gln Leu Gly Glu Ser 195 200 205 Glu Asp Glu Ile Asp Glu Pro Asp His Ala Val Asn His Gln His Gln 210 215 220 Leu Leu Ala Arg Arg Asp Glu Pro Gln Arg His Thr Ile Gln Cys Ser 225 230 235 240 Cys Cys Lys Cys Asn Asn Thr Leu Gln Leu Val Val Glu Ala Ser Arg 245 250 255 Asp Thr Leu Arg Gln Leu Gln Gln Leu Phe Met Asp Ser Leu Gly Phe 260 265 270 Val Cys Pro Trp Cys Ala Thr Ala Asn Gln 275 280

Claims

1. A nucleic acid molecule having a nucleotide sequence encoding the HPV31 E6-E7 fusion antigen. The nucleotide sequence encoding the HPV 31 E6-E7 fusion antigen encodes SEQ ID NO: 18 or encodes SEQ ID NO: 18 without the leader sequence of SEQ ID NO: 10 at the 5' end.

2. The nucleic acid molecule according to claim 1, wherein the at least one nucleotide sequence is: SEQ ID NO:17; or SEQ ID NO:17 does not have the leading sequence of SEQ ID NO:9 at the 5' end.

3. The nucleic acid molecule according to claim 1, wherein the nucleic acid sequence is incorporated into a plasmid.

4. A pharmaceutical composition having the nucleic acid molecule of claim 1.

5. Use of the composition according to claim 4 in the preparation of a medicament for inducing an effective immune response against HPV in an individual.

6. The use according to claim 5, further wherein the nucleotide sequence is introduced into the individual via electroporation.

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