Compositions and methods for treating anal high-grade squamous intraepithelial lesions (HSIL)

Abstract

Use of anti-HPV immunogens and the nucleic acid molecules encoding them for the treatment and prevention of anal high-grade squamous intraepithelial lesions are disclosed. Pharmaceutical compositions, recombinant vaccines including DNA plasmids, and live attenuated vaccines, as well as methods of inducing an immune response to treat or prevent anal high-grade squamous intraepithelial lesions are disclosed.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 270,929, filed October 22, 2021, the disclosure of which is incorporated herein by reference in its entirety.

[0002] The present invention relates to improved vaccines, improved methods for inducing an immune response and for prophylactically and / or therapeutically immunizing individuals against anal high-grade squamous intraepithelial lesions (HSIL). [Background technology]

[0003] Anal squamous intraepithelial lesions include both low-grade squamous intraepithelial lesions (LSIL) and high-grade squamous intraepithelial lesions (HSIL), which are caused by chronic infection with human papillomavirus (HPV).Current management of anal high-grade squamous intraepithelial lesions (HSIL) is challenging, and there is significant evidence that untreated HSIL can progress to squamous cell carcinoma.

[0004] Thus, there is a need in the art for improved compositions and methods for the treatment or prevention of anal HSIL. The present invention fulfills this unmet need. Summary of the Invention

[0005] Aspects of the present invention provide compositions comprising at least one nucleotide sequence comprising an HPV16 E6-E7 fusion antigen, an HPV18 E6-E7 fusion antigen, or a combination thereof, and uses thereof for the treatment or prevention of anal high-grade squamous intraepithelial lesions.

[0006] Another aspect provides compositions comprising one or more nucleotide sequences encoding an HPV16 E6-E7 fusion antigen selected from the group consisting of a nucleotide sequence encoding SEQ ID NO:2, a nucleotide sequence that is at least 95% homologous to a nucleotide sequence encoding SEQ ID NO:2, or a nucleotide sequence that is at least 95% homologous to a fragment of a nucleotide sequence encoding SEQ ID NO:2. In some embodiments, the nucleotide sequence encoding the HPV6 E6-E7 fusion antigen does not have a leader sequence at the 5' end.

[0007] In another aspect of the invention, compositions are provided that include one or more nucleotide sequences encoding an HPV16 E6-E7 fusion antigen selected from the group consisting of: SEQ ID NO:1, a nucleotide sequence that is at least 95% homologous to SEQ ID NO:1, a fragment of SEQ ID NO:1, a nucleotide sequence that is at least 95% homologous to a fragment of SEQ ID NO:1. In some embodiments, the nucleotide sequence encoding the HPV16 E6-E7 fusion antigen does not have a leader sequence at the 5' end.

[0008] Another aspect provides compositions comprising one or more nucleotide sequences encoding an HPV18 E6-E7 fusion antigen selected from the group consisting of a nucleotide sequence encoding SEQ ID NO: 10, a nucleotide sequence that is at least 95% homologous to a nucleotide sequence encoding SEQ ID NO: 10, or a nucleotide sequence that is at least 95% homologous to a fragment of a nucleotide sequence encoding SEQ ID NO: 10. In some embodiments, the nucleotide sequence encoding the HPV6 E6-E7 fusion antigen further comprises nucleotides encoding a leader sequence at the 5' end.

[0009] In another aspect of the invention, compositions are provided comprising one or more nucleotide sequences encoding an HPV18 E6-E7 fusion antigen selected from the group consisting of: SEQ ID NO:9, a nucleotide sequence that is at least 95% homologous to SEQ ID NO:9, a fragment of SEQ ID NO:9, a nucleotide sequence that is at least 95% homologous to a fragment of SEQ ID NO:9. In some embodiments, the nucleotide sequence encoding the HPV16 E6-E7 fusion antigen further comprises nucleotides encoding a leader sequence at the 5' end.

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

[0011] In an additional aspect, pharmaceutical compositions comprising the disclosed nucleotide sequences are provided.

[0012] In some aspects, there are methods of treating or preventing anal high-grade squamous intraepithelial lesions in an individual by inducing an effective immune response in the individual, comprising administering to the individual a composition comprising one or more of the provided nucleotide sequences, preferably comprising the step of introducing the provided nucleotide sequences into the individual by electroporation. [Brief description of the drawings]

[0013] [Figure 1-1] Draw the research design of this study. [Figure 1-2] Draw the research design of this study. [Diagram 2] Data on vaccine efficacy at 36 and 64 weeks will be provided. [Diagram 3] We provide data demonstrating that VGX-3100 induces cellular immune responses to both HPV16 and HPV18 antigens. [Figure 4] We provide data demonstrating that VGX-3100 induces humoral immune responses against HPV16E7 and HPV18E7 antigens. [Diagram 5]Provide data on the safety of the VGX-3100 vaccine. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] definition The disclosed methods may be more readily understood by reference to the following detailed description, which forms a part of this disclosure: It is to be understood that the disclosed methods are not limited to the specific methods described and / or described herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only, and is not intended to be a limitation of the claimed methods.

[0015] Unless specifically stated otherwise, any description of a possible mechanism or mode of action, or reason for improvement, is intended for illustration only, and the disclosed methods are not constrained by the accuracy or inaccuracy of any such proposed mechanism or mode of action, or reason for improvement.

[0016] When values ​​are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. Reference to a particular numerical value includes at least the particular value unless the context clearly dictates otherwise.

[0017] It should be understood that certain features of the disclosed methods that are, for clarity, described herein in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, for brevity, various features of the disclosed methods that are described in the context of a single embodiment can also be provided separately or in any subcombination.

[0018] Throughout the specification and claims, various terms relating to the aspects of the present specification are used. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be interpreted in a manner consistent with the definitions provided herein.

[0019] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0020] For the recitation of numerical ranges herein, each intervening numerical value is expressly contemplated with the same degree of precision. For example, for the range 6 to 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 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 expressly contemplated.

[0021] As used herein, the term "at least one" means "one or more."

[0022] As used herein, the term "subject" or "individual" as used herein refers to any animal, but particularly to humans. Thus, the present methods are applicable to humans and non-human animals, but are most preferably used in humans. "Subject" and "patient" as well as "individual" are used interchangeably herein.

[0023] As used herein, the term "comprising" is intended to include examples encompassed by the terms "consisting essentially of" and "consisting of," and similarly, the term "consisting essentially of" is intended to include examples encompassed by the term "consisting of."

[0024] As used herein, "adjuvant" may refer to any molecule that is added to the DNA plasmid vaccines described herein to enhance the antigenicity of one or more antigens encoded by the DNA plasmid and that encodes a nucleic acid sequence as described below.

[0025] "Antibody" may refer to an antibody of class IgG, IgM, IgA, IgD, or IgE, or a fragment, fragment, or derivative thereof, including Fab, F(ab')2, Fd, and single chain antibodies, diabodies, bispecific antibodies, bifunctional antibodies, and derivatives thereof. The antibody may be an antibody isolated from a mammalian serum sample, a polyclonal antibody, an affinity purified antibody, or a mixture thereof, which exhibits sufficient binding specificity for a desired epitope or a sequence derived therefrom.

[0026] "Antigen" refers to proteins having HPV E6 or HPV E7 domains, and preferably E6 and E7 fusions with an intracellular proteolytic cleavage site between them. Antigens include SEQ ID NO:2 (subtype 16) and SEQ ID NO:4 (subtype 18), fragments thereof of lengths described herein, variants, i.e., proteins having sequences homologous to SEQ ID NO:2 or SEQ ID NO:4 described herein, fragments of variants of lengths described herein, and combinations thereof. Antigens may have the IgE leader sequence of SEQ ID NO:7 or SEQ ID NO:12, or alternatively, may have such sequences removed from the N-terminus. Antigens may optionally include signal peptides, such as those from other proteins.

[0027] "Biosimilar" (of an approved reference product / biological drug, i.e., of a reference-listed drug) refers to a biological product that is highly similar to the reference product, despite minor differences in clinically inactive ingredients, with no clinically meaningful differences between the biosimilar and the reference product in terms of safety, purity, and potency, based on data obtained from (a) analytical studies showing that the biological product is highly similar to the reference product, despite minor differences in clinically inactive ingredients, (b) animal studies (including evaluation of toxicity), and / or (c) clinical studies or studies (including evaluation of immunogenicity and pharmacokinetics or pharmacodynamics) sufficient to demonstrate safety, purity, and potency under one or more appropriate conditions of use for which the reference product is approved and intended to be used, and for which licensure is sought for the biosimilar. A biosimilar may be an interchangeable product that may be substituted for the reference product in a pharmacy without the intervention of a prescribing healthcare professional. To meet the additional criteria of "interchangeability," the biosimilar is expected to produce the same clinical outcome as the reference product in any given patient, and if the biosimilar is administered more than once to an individual, the risks in terms of reduced safety or efficacy of alternating or switching between the use of the biosimilar and the reference product are not greater than the risks of using the reference product without such alternation or switching. The biosimilar utilizes the same mechanism of action for the proposed conditions of use, to the extent that the mechanism is known for the reference product. The conditions of use specified, recommended, or proposed in the proposed label for the biosimilar have previously been approved for the reference product. The route of administration, dosage form, and / or strength of the biosimilar are the same as those of the reference product, and the biosimilar is manufactured, processed, packaged, or held in facilities that meet standards designed to ensure that the biosimilar remains safe, pure, and potent. The biosimilar may contain minor modifications in amino acid sequence, such as, for example, N- or C-terminal truncations, that are not expected to alter the biosimilar performance when compared to the reference product.

[0028] As used herein, "coding sequence" or "encoding nucleic acid" may be meant to refer to a nucleic acid (RNA or DNA molecule) that comprises a nucleotide sequence that encodes an antigen as described in section c above. The coding sequence may further comprise initiation and termination signals operably linked to regulatory elements, including a promoter and a polyadenylation signal, capable of directing expression in cells of an individual or mammal to which the nucleic acid is administered. The coding sequence may further comprise a sequence encoding a signal peptide, e.g., an IgE leader sequence, such as SEQ ID NO: 7 or 12.

[0029] As used herein, "complement" or "complementary" can refer to a nucleic acid and can refer to Watson-Crick (e.g., AT / U and CG) or Hoogsteen base pairing between nucleotides or nucleotide analogs of a nucleic acid molecule.

[0030] "Fragment" may refer to a polypeptide fragment of an antigen capable of eliciting an immune response in a mammal against the antigen. A fragment of an antigen may be 100% identical to the full length, except for the deletion of at least one amino acid from the N- and / or C-terminus, in each case with or without a signal peptide and / or methionine at position 1. A fragment may comprise a percent of 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, 99% or more of the length of a particular full length antigen, excluding any added heterologous signal peptide. A fragment preferably comprises a fragment of a polypeptide that is 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homologous to the antigen, and may additionally include an N-terminal methionine or a heterologous signal peptide that is not included when calculating the percent homology. The fragment may further comprise a signal peptide, such as an N-terminal methionine and / or an immunoglobulin signal peptide, e.g., an IgE or IgG signal peptide. The N-terminal methionine and / or the signal peptide may be linked to the fragment of the antigen.

[0031] Fragments of nucleic acid sequences encoding antigens can be 100% identical to the full length, except for lacking at least one nucleotide from the 5' and / or 3' end, in each case with or without sequences encoding a signal peptide and / or methionine at position 1. Fragments can comprise a percentage of 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, 99% or more of the length of the particular full length coding sequence, excluding any added heterologous signal peptide. Fragments preferably include fragments encoding polypeptides that are 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homologous to the antigen, and may additionally optionally include sequences encoding an N-terminal methionine or a heterologous signal peptide that are not included in calculating the percentage of homology. The fragment may further comprise a coding sequence for an N-terminal methionine and / or a signal peptide, such as an immunoglobulin signal peptide, e.g., an IgE or IgG signal peptide. A coding sequence encoding an N-terminal methionine and / or a signal peptide may be linked to the fragment of the coding sequence.

[0032] "Identical" or "identity" as used herein in the context of two or more nucleic acid or polypeptide sequences may mean that the sequences have a certain percentage of residues that are the same over a particular region. The percentage may be calculated by optimally aligning the two sequences, comparing the two sequences over a particular region, determining the number of positions where identical residues occur in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the particular region, and raising the result to the power of 100 to obtain the percentage of sequence identity. If the two sequences have different lengths or the alignment results in one or more staggered ends and a particular comparison region contains only a single sequence, the residues of the single sequence are included in the denominator rather than the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) may be considered equivalent. Identity may be performed manually or by using a computer sequence algorithm such as BLAST or BLAST 2.0.

[0033] As used herein, "immune response" may refer to activation of a host's immune system, e.g., a mammal's immune system, in response to the introduction of one or more antigens via a provided DNA plasmid vaccine. The immune response may be in the form of a cellular or humoral response, or both.

[0034] As used herein, "nucleic acid" or "oligonucleotide" or "polynucleotide" may refer to at least two nucleotides covalently linked together. The depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the complementary strand of a depicted single strand. Many variants of a nucleic acid may be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also encompasses substantially identical nucleic acids and their complements. A single strand provides a probe that can hybridize to a target sequence under stringent hybridization conditions. Thus, a nucleic acid also encompasses a probe that hybridizes under stringent hybridization conditions.

[0035] Nucleic acids may be single-stranded or double-stranded, or may contain portions of both double-stranded and single-stranded sequences. Nucleic acids may be DNA, both genomic and cDNA, RNA, or hybrids, and may contain combinations of deoxyribonucleotides and ribonucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. Nucleic acids may be obtained by chemical synthesis methods or by recombinant methods.

[0036] As used herein, "operably linked" may mean that the expression of a gene is under the control of a promoter to which it is spatially connected. The promoter may be located 5' (upstream) or 3' (downstream) of the gene under its control. 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 is derived. As is known in the art, variations in this distance may be accommodated without loss of promoter function.

[0037] As used herein, the term "placebo" refers to the administration of a pharmaceutical composition that does not contain VGX-3100.

[0038] As used herein, a "promoter" may refer to a synthetic or naturally derived molecule that can confer, activate, or enhance expression of a nucleic acid in a cell. A promoter may contain one or more specific transcriptional regulatory sequences to further enhance expression and / or modify its spatial and / or temporal expression. A promoter may also contain distal enhancer or repressor elements, which may be located as many as several thousand base pairs from the start site of transcription. Promoters may be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. A promoter may regulate the expression of genetic components constitutively or differentially with respect to the cell, tissue, or organ in which expression occurs, or with respect to the developmental stage in which expression occurs, or in response to external stimuli such as physiological stress, pathogens, metal ions, or inducers. Representative examples of promoters include a bacteriophage T7 promoter, a bacteriophage T3 promoter, an SP6 promoter, a lac operator-promoter, a tac promoter, an SV40 late promoter, an SV40 early promoter, an RSV-LTR promoter, a CMV IE promoter, an SV40 early promoter or an SV40 late promoter, and a CMV IE promoter.

[0039] As used herein, "stringent hybridization conditions" may refer to conditions under which a first nucleic acid sequence (e.g., a probe) hybridizes to a second nucleic acid sequence (e.g., a target) as in a complex mixture of nucleic acids. Stringent conditions are sequence-dependent and will be different in different circumstances. Stringent conditions may 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 may be the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (the target sequence is present in excess, so at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions can be those in which the salt concentration is less than about 1.0 M sodium ion, e.g., about 0.01 to 1.0 M sodium ion concentration (or other salt) at pH 7.0 to 8.3, and the temperature is at least about 30° C. for short probes (e.g., about 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., more than about 50 nucleotides). Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal can be at least 2 to 10 times background hybridization. Exemplary stringent hybridization conditions include: 50% formamide, 5× SSC, and 1% SDS, incubation at 42° C., or 5× SSC, 1% SDS, incubation at 65° C., washing with 0.2× SSC and 0.1% SDS at 65° C.

[0040] As used herein, "substantially complementary" can mean that a first sequence is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the complement of a second sequence over a region 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 stringent hybridization conditions.

[0041] As used herein, "substantially identical" can mean that a first and second sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical over a region 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 to a nucleic acid, where the first sequence is substantially complementary to the complement of the second sequence.

[0042] As used herein, "treating" and similar terms refer to, for example, reducing the severity and / or frequency of anal and / or perianal symptoms, such as human papillomavirus (HPV) type 16 or HPV type 18 associated high-grade anal or anal / perianal intraepithelial lesions (HSIL) and anal or anal / perianal high-grade squamous intraepithelial lesions (HSIL) lesions, eliminating HPV type 16 or HPV type 18 infection symptoms, particularly HSIL lesions, and / or clearing the HPV type 16 or HPV type 18 virus from a subject, and / or resolution of anal or anal / perianal low-grade squamous intraepithelial lesions (LSIL) or normal tissue.

[0043] "Variant," as used herein with respect to a nucleic acid, may mean (i) a portion or fragment of a referenced nucleotide sequence; (ii) the complement of the referenced nucleotide sequence or a portion thereof; (iii) a nucleic acid that is substantially identical to the referenced nucleic acid or its complement; or (iv) a nucleic acid that hybridizes under stringent conditions to the referenced nucleic acid, its complement, or a sequence substantially identical thereto.

[0044] "Variant" refers to a peptide or polypeptide that differs in amino acid sequence by insertion, deletion, or conservative substitution of amino acids, but retains at least one biological activity. Variant can also refer to a protein having an amino acid sequence that is substantially identical to a reference protein having an amino acid sequence that retains at least one biological activity. Conservative substitutions of amino acids, i.e., replacing an amino acid with an amino acid of different similar properties (e.g., hydrophilicity, degree and distribution of charged regions), are recognized in the art as typically involving minor changes. These minor changes can be identified, in part, by considering the hydropathic index of an amino acid, as understood in the art. Kyte et al., J. Mol. Biol. 157:105-132 (1982). The hydropathic index of an amino acid is based on consideration of its hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic indexes can be substituted and still retain protein function. In one aspect, amino acids with hydropathic indexes of ±2 are substituted. The hydrophilicity of an amino acid can also be used to identify substitutions that will result in a protein that retains biological function. Consideration of the hydrophilicity of an amino acid in the context of a peptide allows for the calculation of the greatest local average hydrophilicity of the peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. U.S. Patent No. 4,554,101, fully incorporated herein by reference. Substitution of amino acids with similar hydrophilicity values ​​can result in peptides that retain biological activity, e.g., immunogenicity, as understood in the art. Substitutions can be made with amino acids that have hydrophilicity values ​​within ±2 of each other. Both the hydrophobicity index and hydrophilicity value of an amino acid are influenced by the particular side chain of that amino acid. Consistent with that observation, it is understood that amino acid substitutions that are compatible with biological function depend on the relative similarity of the amino acids, and in particular the side chains of those amino acids, as revealed by hydrophobicity, hydrophilicity, charge, size, and other properties.

[0045] "Vector" as used herein may refer to a nucleic acid sequence that contains a replication origin. A vector may be a plasmid, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. A vector may be a DNA or an RNA vector. A vector may be either a self-replicating extrachromosomal vector or a vector that is integrated into a host genome.

[0046] explanation Improved vaccines are disclosed that result from a multi-phase strategy to enhance the cellular immune response induced by immunogens. Modified consensus sequences have been generated. Genetic modifications, including codon optimization, RNA optimization, and the addition of highly efficient immunoglobulin leader sequences, are also disclosed. The novel constructs are designed to induce stronger and broader cellular immune responses than the corresponding codon-optimized immunogens.

[0047] The improved HPV vaccine is based on proteins and genetic constructs that code for proteins with epitopes that are particularly effective as immunogens to mediate preventative or therapeutic strategies against anal high-grade squamous intraepithelial lesions (HSIL). Thus, the vaccine can induce a therapeutic or preventative immune response. In some embodiments, the means for delivering the immunogen is a DNA vaccine, a recombinant vaccine, a protein subunit vaccine, a composition comprising an immunogen, an attenuated vaccine, or a killed 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 comprising an immunogen, one or more attenuated vaccines, and one or more killed vaccines.

[0048] According to some embodiments, the vaccine is delivered to an individual to modulate the activity of the individual's immune system, thereby enhancing the immune response to HPV to treat anal high-grade squamous intraepithelial lesions. When the nucleic acid molecule encoding the protein is taken up by the cells of the individual, the nucleotide sequence is expressed in the cells, and the protein is thereby delivered to the individual. Methods are provided for delivering the coding sequence of the protein on a nucleic acid molecule, such as a plasmid, as an isolated protein or as a vector, as part of a recombinant vaccine and as part of an attenuated vaccine.

[0049] Compositions and methods are provided that provide prophylactic and / or therapeutic treatment for anal high-grade squamous intraepithelial lesions in an individual.

[0050] The composition for delivering a nucleic acid molecule comprising a nucleotide sequence encoding an immunogen is operably linked to a regulatory element. The composition may comprise a plasmid encoding an immunogen, a recombinant vaccine comprising a nucleotide sequence encoding an immunogen, a live attenuated pathogen encoding and / or comprising a protein of the invention, a killed pathogen comprising a protein of the invention, or a liposomal or subunit vaccine comprising a protein of the invention. The invention further relates to an injectable pharmaceutical composition comprising the composition.

[0051] Aspects of the invention provide compositions comprising at least one nucleotide sequence encoding at least one HPV E6-E7 fusion antigen, e.g., HPV16 E6-E7 fusion antigen or HPV18 E6-E7 fusion antigen. In one embodiment, the composition comprises nucleotide sequences encoding an HPV16 E6-E7 fusion antigen and an HPV18 E6-E7 fusion antigen.

[0052] In one embodiment, the invention includes a method of administering a composition of the invention to a subject in need thereof. In one embodiment, the subject is a subject diagnosed with anal high-grade squamous intraepithelial lesion. In one embodiment, the subject is a subject with anal high-grade squamous intraepithelial lesion. In one embodiment, the subject is a subject at risk of developing anal high-grade squamous intraepithelial lesion.

[0053] HPV16 E6-E7 fusion Another aspect provides a composition comprising one or more nucleotide sequences encoding an HPV16 E6-E7 fusion antigen selected from the group consisting of a nucleotide sequence encoding SEQ ID NO:2, a nucleotide sequence that is at least 95% homologous to a nucleotide sequence encoding SEQ ID NO:2, a fragment of a nucleotide sequence encoding SEQ ID NO:2, a nucleotide sequence that is at least 95% homologous to a fragment of a nucleotide sequence encoding SEQ ID NO:2.

[0054] In some embodiments, the composition comprises an HPV16 E6-E7 fusion antigen selected from the group consisting of a nucleotide sequence encoding SEQ ID NO:2, a nucleotide sequence that is at least 95% homologous to a nucleotide sequence encoding SEQ ID NO:2, a fragment of a nucleotide sequence encoding SEQ ID NO:2, or a nucleotide sequence that is at least 95% homologous to a fragment of a nucleotide sequence encoding SEQ ID NO:2.

[0055] In another aspect of the invention, there is provided a composition comprising one or more nucleotide sequences encoding an HPV16 E6-E7 fusion antigen selected from the group consisting of: SEQ ID NO:1, a nucleotide sequence that is at least 95% homologous to SEQ ID NO:1, a fragment of SEQ ID NO:1, a nucleotide sequence that is at least 95% homologous to a fragment of SEQ ID NO:1.

[0056] In some embodiments, the nucleotide sequences described herein are absent of a leader sequence. In one embodiment, the nucleotide sequence comprising the HPV16 E6-E7 fusion antigen is absent of a leader sequence. In particular, the HPV16 E6-E7 fusion antigen comprising the nucleotide sequence encoding SEQ ID NO:2 is absent of a leader sequence at the 5' end, for example, a nucleotide sequence encoding SEQ ID NO:7. In particular, the HPV6 E6-E7 fusion antigen comprising the nucleotide sequence SEQ ID NO:1 is absent of a leader sequence at the 5' end, for example, a nucleotide sequence encoding SEQ ID NO:7.

[0057] In some embodiments, the nucleotide sequences of the invention may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably 95%, 96%, 97%, 98%, or 99%, or 98% or 99% homologous to the nucleotide sequences provided.

[0058] The nucleotide sequences provided may be included in one of a variety of known vectors or delivery systems, including plasmids, viral vectors, lipid vectors, nanoparticles, preferably plasmids.

[0059] In an additional aspect, pharmaceutical compositions comprising the disclosed nucleotide sequences are provided.

[0060] In some aspects, there are methods of inducing an effective immune response in an individual against more than one subtype of HPV, thereby providing prophylactic or therapeutic treatment against anal high-grade squamous intraepithelial lesions, comprising administering to the individual a composition comprising one or more of the provided nucleotide sequences, preferably the composition has more than one antigen. The method preferably comprises the step of introducing the provided nucleotide sequences into the individual by electroporation.

[0061] SEQ ID NO:1 comprises a nucleotide sequence encoding a consensus immunogen of HPV16 E6 and E7 proteins, which comprises an IgE leader sequence, which is a consensus sequence of HPV E6 linked to the consensus sequence of HPV E7 by a proteolytic cleavage sequence. SEQ ID NO:2 comprises an amino acid sequence of a consensus immunogen of HPV16 E6 and E7 proteins, which comprises an IgE leader sequence, which is a consensus sequence of HPV E6 linked to the consensus sequence of HPV E7 by a proteolytic cleavage sequence. The consensus sequence of HPV16 E6 comprises an immunodominant epitope set forth in SEQ ID NO:3. The consensus sequence of HPV16 E7 comprises an immunodominant epitope set forth in SEQ ID NO:4. The consensus sequence of HPV E6 is SEQ ID NO:5. The consensus sequence of HPV E6 is SEQ ID NO:6. The IgE leader sequence is SEQ ID NO:7. A proteolytic cleavage sequence useful for linking the two consensus sequences is SEQ ID NO:8.

[0062] Further information regarding HPV16 E6-E7 fusion antigens can be found in at least US Pat. No. 8,168,769, which is incorporated by reference in its entirety.

[0063] In some embodiments, the vaccine comprises SEQ ID NO:2, or a nucleic acid molecule encoding SEQ ID NO:2. In some embodiments, the vaccine of the invention comprises a nucleic acid sequence encoding one of SEQ ID NO:3 and / or SEQ ID NO:4, or both. In some embodiments, the vaccine of the invention comprises a nucleic acid sequence encoding SEQ ID NO:5 and / or SEQ ID NO:6, or one or both. In some embodiments, the vaccine of the invention comprises SEQ ID NO:5 linked to SEQ ID NO:6 by a proteolytic cleavage sequence, such as SEQ ID NO:8, or a nucleic acid sequence encoding a fusion protein. In some embodiments, the vaccine of the invention comprises the IgE leader SEQ ID NO:7, or a nucleic acid sequence encoding same. In some embodiments, the vaccine of the invention comprises SEQ ID NO:2, or a nucleic acid sequence in SEQ ID NO:1.

[0064] A fragment of SEQ ID NO:2 may be 100% identical to the full length, except for missing at least one amino acid from the N- and / or C-terminus, in each case with or without a signal peptide and / or methionine at position 1. A fragment of SEQ ID NO:2 may comprise a percent of 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, 99% or more of the length of the full length SEQ ID NO:2, excluding any added heterologous signal peptide. Fragments preferably include fragments of SEQ ID NO:2 that are 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homologous to SEQ ID NO:2, and additionally may include an N-terminal methionine or heterologous signal peptide that is not included when calculating the percent homology. The fragment may further comprise a signal peptide, such as an N-terminal methionine and / or an immunoglobulin signal peptide, e.g., an IgE or IgG signal peptide. The N-terminal methionine and / or the signal peptide may be linked to the fragment.

[0065] A fragment of the nucleic acid sequence SEQ ID NO:1 may be 100% identical to the full length, except for the deletion of at least one nucleotide from the 5' and / or 3' end, in each case with or without a sequence encoding a signal peptide and / or a methionine at position 1. A fragment may comprise a percentage of 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, 99% or more of the length of the full length coding sequence SEQ ID NO:1, excluding any added heterologous signal peptide. Fragments preferably include fragments encoding polypeptides that are 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homologous to the antigen SEQ ID NO:2, and may additionally optionally include a sequence encoding an N-terminal methionine or a heterologous signal peptide that is not included in calculating the percentage of homology. The fragment may further comprise a coding sequence for an N-terminal methionine and / or a signal peptide, such as an immunoglobulin signal peptide, e.g., an IgE or IgG signal peptide. A coding sequence encoding an N-terminal methionine and / or a signal peptide may be linked to the fragment.

[0066] A fragment of SEQ ID NO:1 may preferably comprise 30 or more nucleotides, including sequences encoding immunodominant epitopes. In some embodiments, a fragment of SEQ ID NO:1 may preferably comprise 45 or more nucleotides, including sequences encoding immunodominant epitopes. In some embodiments, a fragment of SEQ ID NO:1 may preferably comprise 60 or more nucleotides, including sequences encoding immunodominant epitopes. In some embodiments, a fragment of SEQ ID NO:1 may preferably comprise 75 or more nucleotides, including sequences encoding immunodominant epitopes. In some embodiments, a fragment of SEQ ID NO:1 may preferably comprise 90 or more nucleotides, including sequences encoding immunodominant epitopes. In some embodiments, a fragment of SEQ ID NO:1 may preferably comprise 120 or more nucleotides, including sequences encoding immunodominant epitopes. In some embodiments, a fragment of SEQ ID NO:1 may preferably comprise 150 or more nucleotides, including sequences encoding immunodominant epitopes. In some embodiments, a fragment of SEQ ID NO:1 may preferably comprise 180 or more nucleotides, including sequences encoding immunodominant epitopes. In some embodiments, a fragment of SEQ ID NO:1 may comprise 210 or more nucleotides, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:1 may comprise 240 or more nucleotides, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:1 may comprise 270 or more nucleotides, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:1 may comprise 300 or more nucleotides, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:1 may comprise 360 ​​or more nucleotides, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:1 may comprise 420 or more nucleotides, preferably including a sequence encoding an immunodominant epitope.In some embodiments, a fragment of SEQ ID NO:1 may comprise 480 or more nucleotides, preferably including sequences encoding immunodominant epitopes. In some embodiments, a fragment of SEQ ID NO:1 may comprise 540 or more nucleotides, preferably including sequences encoding immunodominant epitopes. In some embodiments, a fragment of SEQ ID NO:1 may comprise 600 or more nucleotides, preferably including sequences encoding immunodominant epitopes. In some embodiments, a fragment of SEQ ID NO:1 may comprise 300 or more nucleotides, preferably including sequences encoding immunodominant epitopes. In some embodiments, a fragment of SEQ ID NO:1 may comprise 660 or more nucleotides, preferably including sequences encoding immunodominant epitopes. In some embodiments, a fragment of SEQ ID NO:1 may comprise 720 or more nucleotides, preferably including sequences encoding immunodominant epitopes. In some embodiments, a fragment of SEQ ID NO:1 may comprise 780 or more nucleotides, preferably including sequences encoding immunodominant epitopes. In some embodiments, a fragment of SEQ ID NO:1 may comprise a coding sequence for an IgE leader sequence. In some embodiments, the fragment of SEQ ID NO:1 does not include the coding sequence for the IgE leader sequence.Fragments may comprise fewer than 60 nucleotides, in some embodiments fewer than 75 nucleotides, in some embodiments fewer than 90 nucleotides, in some embodiments fewer than 120 nucleotides, in some embodiments fewer than 150 nucleotides, in some embodiments fewer than 180 nucleotides, in some embodiments fewer than 210 nucleotides, in some embodiments fewer than 240 nucleotides, in some embodiments fewer than 270 nucleotides, in some embodiments fewer than 300 nucleotides, in some embodiments fewer than 360 nucleotides, in some embodiments fewer than 420 nucleotides, in some embodiments fewer than 480 nucleotides, in some embodiments fewer than 540 nucleotides, in some embodiments fewer than 600 nucleotides, in some embodiments fewer than 660 nucleotides, in some embodiments fewer than 720 nucleotides, and in some embodiments fewer than 780 nucleotides.

[0067] A fragment of SEQ ID NO:2 may preferably comprise 15 or more amino acids, including sequences encoding immunodominant epitopes. In some embodiments, a fragment of SEQ ID NO:2 may preferably comprise 18 or more amino acids, including sequences encoding immunodominant epitopes. In some embodiments, a fragment of SEQ ID NO:2 may preferably comprise 21 or more amino acids, including sequences encoding immunodominant epitopes. In some embodiments, a fragment of SEQ ID NO:2 may preferably comprise 24 or more amino acids, including sequences encoding immunodominant epitopes. In some embodiments, a fragment of SEQ ID NO:2 may preferably comprise 30 or more amino acids, including sequences encoding immunodominant epitopes. In some embodiments, a fragment of SEQ ID NO:2 may preferably comprise 36 or more amino acids, including sequences encoding immunodominant epitopes. In some embodiments, a fragment of SEQ ID NO:2 may preferably comprise 42 or more amino acids, including sequences encoding immunodominant epitopes. In some embodiments, a fragment of SEQ ID NO:2 may preferably comprise 48 or more amino acids, including sequences encoding immunodominant epitopes. In some embodiments, a fragment of SEQ ID NO:2 may comprise 54 or more amino acids, preferably including sequences encoding immunodominant epitopes. In some embodiments, a fragment of SEQ ID NO:2 may comprise 60 or more amino acids, preferably including sequences encoding immunodominant epitopes. In some embodiments, a fragment of SEQ ID NO:2 may comprise 18 or more amino acids, preferably including sequences encoding immunodominant epitopes. In some embodiments, a fragment of SEQ ID NO:2 may comprise 72 or more amino acids, preferably including sequences encoding immunodominant epitopes. In some embodiments, a fragment of SEQ ID NO:2 may comprise 90 or more amino acids, preferably including sequences encoding immunodominant epitopes. In some embodiments, a fragment of SEQ ID NO:2 may comprise 120 or more amino acids, preferably including sequences encoding immunodominant epitopes. In some embodiments, a fragment of SEQ ID NO:2 may comprise 150 or more amino acids, preferably including sequences encoding immunodominant epitopes.In some embodiments, a fragment of SEQ ID NO:2 may comprise 180 or more amino acids, preferably including sequences encoding immunodominant epitopes. In some embodiments, a fragment of SEQ ID NO:2 may comprise 210 or more amino acids, preferably including sequences encoding immunodominant epitopes. In some embodiments, a fragment of SEQ ID NO:2 may comprise 240 or more amino acids, preferably including sequences encoding immunodominant epitopes. In some embodiments, a fragment of SEQ ID NO:2 may comprise 260 or more amino acids, preferably including sequences encoding immunodominant epitopes. In some embodiments, a fragment of SEQ ID NO:2 may comprise coding sequences for an IgE leader sequence. In some embodiments, a fragment of SEQ ID NO:2 does not comprise coding sequences for an IgE leader sequence. Fragments may comprise fewer than 24 amino acids, in some embodiments fewer than 30 amino acids, in some embodiments fewer than 36 amino acids, in some embodiments fewer than 42 amino acids, in some embodiments fewer than 48 amino acids, in some embodiments fewer than 54 amino acids, in some embodiments fewer than 60 amino acids, in some embodiments fewer than 72 amino acids, in some embodiments fewer than 90 amino acids, in some embodiments fewer than 120 amino acids, in some embodiments fewer than 150 amino acids, in some embodiments fewer than 180 amino acids, in some embodiments fewer than 210 amino acids, in some embodiments fewer than 240 amino acids, and in some embodiments fewer than 260 amino acids.

[0068] HPV18 E6-E7 fusion Another aspect provides a composition comprising one or more nucleotide sequences encoding an HPV18 E6-E7 fusion antigen selected from the group consisting of a nucleotide sequence encoding SEQ ID NO:10, a nucleotide sequence that is at least 95% homologous to a nucleotide sequence encoding SEQ ID NO:10, a fragment of a nucleotide sequence encoding SEQ ID NO:10, a nucleotide sequence that is at least 95% homologous to a fragment of a nucleotide sequence encoding SEQ ID NO:10.

[0069] In some embodiments, the composition comprises an HPV18 E6-E7 fusion antigen selected from the group consisting of a nucleotide sequence encoding SEQ ID NO:10, a nucleotide sequence that is at least 95% homologous to a nucleotide sequence encoding SEQ ID NO:10, a fragment of a nucleotide sequence encoding SEQ ID NO:10, or a nucleotide sequence that is at least 95% homologous to a fragment of a nucleotide sequence encoding SEQ ID NO:10.

[0070] In another aspect of the invention, there is provided a composition comprising one or more nucleotide sequences encoding an HPV18 E6-E7 fusion antigen selected from the group consisting of: SEQ ID NO:9, a nucleotide sequence that is at least 95% homologous to SEQ ID NO:9, a fragment of SEQ ID NO:9, a nucleotide sequence that is at least 95% homologous to a fragment of SEQ ID NO:9.

[0071] In some embodiments, the nucleotide sequences described herein are absent of a leader sequence. In one embodiment, the nucleotide sequence comprising the HPV18 E6-E7 fusion antigen is absent of a leader sequence. In particular, the HPV18 E6-E7 fusion antigen comprising the nucleotide sequence encoding SEQ ID NO: 10 is absent of a leader sequence at the 5' end, for example, a nucleotide sequence encoding SEQ ID NO: 12. In particular, the HPV16 E6-E7 fusion antigen comprising the nucleotide sequence SEQ ID NO: 9 is absent of a leader sequence at the 5' end, for example, a nucleotide sequence comprising SEQ ID NO: 11.

[0072] In some embodiments, the composition comprises an HPV18 E6-E7 fusion antigen selected from the group consisting of a nucleotide sequence encoding SEQ ID NO: 14, a nucleotide sequence that is at least 95% homologous to a nucleotide sequence encoding SEQ ID NO: 14, a fragment of a nucleotide sequence encoding SEQ ID NO: 14, a nucleotide sequence that is at least 95% homologous to a fragment of a nucleotide sequence encoding SEQ ID NO: 14. SEQ ID NO: 14 comprises the amino acid sequence of the HPV18 E6-E7 fusion antigen of SEQ ID NO: 10, and further comprises an IgE leader sequence.

[0073] In another aspect of the invention, there is provided a composition comprising one or more nucleotide sequences encoding an HPV18 E6-E7 fusion antigen selected from the group consisting of: SEQ ID NO: 13, a nucleotide sequence that is at least 95% homologous to SEQ ID NO: 13, a fragment of SEQ ID NO: 9, a nucleotide sequence that is at least 95% homologous to a fragment of SEQ ID NO: 13. SEQ ID NO: 13 comprises the nucleotide sequence of SEQ ID NO: 9 that encodes the HPV18 E6-E7 fusion antigen and further comprises a nucleotide sequence encoding an IgE leader sequence.

[0074] In some embodiments, the nucleotide sequences of the invention may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably 95%, 96%, 97%, 98%, or 99%, or 98% or 99% homologous to the nucleotide sequences provided.

[0075] The nucleotide sequences provided may be included in one of a variety of known vectors or delivery systems, including plasmids, viral vectors, lipid vectors, nanoparticles, preferably plasmids.

[0076] In an additional aspect, pharmaceutical compositions comprising the disclosed nucleotide sequences are provided.

[0077] In some aspects, there are methods of inducing an effective immune response in an individual against more than one subtype of HPV, thereby providing prophylactic or therapeutic treatment against anal high-grade squamous intraepithelial lesions, comprising administering to the individual a composition comprising one or more of the provided nucleotide sequences, preferably the composition has more than one antigen. The method preferably comprises the step of introducing the provided nucleotide sequences into the individual by electroporation.

[0078] SEQ ID NO:9 comprises a nucleotide sequence encoding a consensus immunogen of HPV18 E6 and E7 proteins. SEQ ID NO:13 comprises SEQ ID NO:9 and further comprises an IgE leader sequence linked to the nucleotide sequence encoding the consensus immunogen of HPV18 E6 and E7 proteins. SEQ ID NO:10 comprises an amino acid sequence of a consensus immunogen of HPV18 E6 and E7 proteins. SEQ ID NO:14 comprises SEQ ID NO:10 and further comprises an IgE leader sequence linked to the consensus immunogen sequence. The IgE leader sequence is SEQ ID NO:12 and can be encoded by SEQ ID NO:11. SEQ ID NO:15 is the nucleic acid sequence of plasmid pGX3002 having SEQ ID NO:13 incorporated therein for expression.

[0079] Further information regarding HPV16 E6-E7 fusion antigens can be found in at least US Pat. No. 8,389,706, which is incorporated by reference in its entirety.

[0080] In some embodiments, the vaccine comprises SEQ ID NO: 10, or a nucleic acid molecule encoding SEQ ID NO: 10. In some embodiments, the vaccine comprises SEQ ID NO: 9 as the nucleic acid molecule encoding SEQ ID NO: 10. In some embodiments, the vaccine comprises SEQ ID NO: 14, or a nucleic acid molecule encoding SEQ ID NO: 14. In some embodiments, the vaccine comprises SEQ ID NO: 13 as the nucleic acid molecule encoding SEQ ID NO: 14. In some embodiments, the vaccine comprises SEQ ID NO: 15.

[0081] A fragment of SEQ ID NO: 10 or 14 may be 100% identical to the full length, except for missing at least one amino acid from the N- and / or C-terminus, in each case with or without a signal peptide and / or methionine at position 1. A fragment of SEQ ID NO: 10 or 15 may comprise a percentage of 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, 99% or more of the length of the full length SEQ ID NO: 10 or 14, excluding any added heterologous signal peptide. Fragments preferably include fragments of SEQ ID NO: 10 or 15 that are 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homologous to SEQ ID NO: 10 or 14, and additionally may include an N-terminal methionine or heterologous signal peptide that is not included when calculating the percentage of homology. The fragment may further comprise a signal peptide, such as an N-terminal methionine and / or an immunoglobulin signal peptide, e.g., an IgE or IgG signal peptide. The N-terminal methionine and / or the signal peptide may be linked to the fragment.

[0082] A fragment of nucleic acid SEQ ID NO: 9 or 13 may be 100% identical to the full length, except for missing at least one nucleotide from the 5' and / or 3' end, in each case with or without a sequence encoding a signal peptide and / or methionine at position 1. A fragment may comprise a percentage of 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, 99% or more of the length of the full length coding SEQ ID NO: 9 or 13, excluding any added heterologous signal peptide. Fragments preferably include fragments encoding polypeptides that are 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homologous to antigen SEQ ID NO: 10 or 14, and may additionally optionally include a sequence encoding an N-terminal methionine or a heterologous signal peptide that is not included in calculating the percentage of homology. The fragment may further comprise a coding sequence for an N-terminal methionine and / or a signal peptide, such as an immunoglobulin signal peptide, e.g., an IgE or IgG signal peptide. A coding sequence encoding an N-terminal methionine and / or a signal peptide may be linked to the fragment.

[0083] A fragment of SEQ ID NO:9 may comprise 90 or more nucleotides. In some embodiments, a fragment of SEQ ID NO:9 may comprise 180 or more nucleotides, in some embodiments 270 or more nucleotides, in some embodiments 360 or more nucleotides, in some embodiments 450 or more nucleotides, in some embodiments 540 or more nucleotides, in some embodiments 630 or more nucleotides, in some embodiments 720 or more nucleotides, and in some embodiments 770 or more nucleotides. In some embodiments, a fragment of SEQ ID NO:9, such as those described herein, may further comprise a coding sequence for an IgE leader sequence. In some embodiments, a fragment of SEQ ID NO:9 does not comprise a coding sequence for an IgE leader sequence. A fragment of SEQ ID NO:9 may comprise fewer than 180 nucleotides, in some embodiments fewer than 270 nucleotides, in some embodiments fewer than 360 nucleotides, in some embodiments fewer than 450 nucleotides, in some embodiments fewer than 540 nucleotides, in some embodiments fewer than 630 nucleotides, in some embodiments fewer than 690 nucleotides, in some embodiments fewer than 760 nucleotides, and in some embodiments fewer than 780 nucleotides.

[0084] A fragment of SEQ ID NO: 10 may comprise 30 or more amino acids. In some embodiments, a fragment of SEQ ID NO: 10 may comprise 60 or more amino acids, in some embodiments 90 or more amino acids, in some embodiments 120 or more amino acids, in some embodiments 150 or more amino acids, in some embodiments 180 or more amino acids, in some embodiments 210 or more amino acids, and in some embodiments 240 or more amino acids. A fragment may comprise fewer than 90 amino acids, in some embodiments fewer than 120 amino acids, in some embodiments fewer than 150 amino acids, in some embodiments fewer than 180 amino acids, in some embodiments fewer than 210 amino acids, and in some embodiments fewer than 240 amino acids.

[0085] All fragments of SEQ ID NO: 13 contain coding sequences encoding HPV sequences, i.e., fragments of SEQ ID NO: 13 must contain sequences in addition to sequences encoding the IgE leader peptide. In some embodiments, fragments of SEQ ID NO: 13 contain 90 or more nucleotides. In some embodiments, fragments of SEQ ID NO: 13 may contain 180 or more nucleotides, in some embodiments 270 or more nucleotides, in some embodiments 360 or more nucleotides, in some embodiments 450 or more nucleotides, in some embodiments 540 or more nucleotides, in some embodiments 630 or more nucleotides, in some embodiments 720 or more nucleotides, in some embodiments 810 or more nucleotides, and in some embodiments 830 or more nucleotides. A fragment of SEQ ID NO:13 may comprise fewer than 180 nucleotides, in some embodiments fewer than 270 nucleotides, in some embodiments fewer than 360 nucleotides, in some embodiments fewer than 450 nucleotides, in some embodiments fewer than 540 nucleotides, in some embodiments fewer than 630 nucleotides, in some embodiments fewer than 690 nucleotides, in some embodiments fewer than 720 nucleotides, in some embodiments fewer than 780 nucleotides, and in some embodiments fewer than 840 nucleotides.

[0086] A fragment of SEQ ID NO: 14 may comprise 30 or more amino acids that include the HPV sequence. In some embodiments, a fragment of SEQ ID NO: 14 may comprise 60 or more amino acids that include the HPV sequence, in some embodiments 90 or more amino acids that include the HPV sequence, in some embodiments 120 or more amino acids that include the HPV sequence, in some embodiments 150 or more amino acids that include the HPV sequence, in some embodiments 180 or more amino acids that include the HPV sequence, in some embodiments 210 or more amino acids that include the HPV sequence, in some embodiments 240 or more amino acids that include the HPV sequence, and in some embodiments 270 or more amino acids that include the HPV sequence. A fragment may comprise less than 90 amino acids that include the HPV sequence, in some embodiments less than 120 amino acids that include the HPV sequence, in some embodiments less than 150 amino acids that include the HPV sequence, in some embodiments less than 180 amino acids that include the HPV sequence, in some embodiments less than 210 amino acids that include the HPV sequence, in some embodiments less than 240 amino acids that include the HPV sequence, and in some embodiments less than 270 amino acids that include the HPV sequence.

[0087] In one embodiment, the HPV16 E6-E7 immunogen, HPV18 E6-E7 immunogen, or a nucleic acid molecule encoding an HPV16 E6-E7 immunogen or HPV16 E6-E7 immunogen is administered in combination with IL-12. In one embodiment, the IL-12 is encoded from a synthetic DNA plasmid.

[0088] Provided herein are methods of treating or preventing anal high-grade squamous intraepithelial lesions in a subject by inducing an immune response in the individual against HPV, comprising administering to the individual a composition comprising a nucleic acid sequence provided herein. In some embodiments, the method also comprises introducing the nucleic acid sequence into the individual by electroporation.

[0089] In some aspects, there are methods of treating or preventing anal high-grade squamous intraepithelial lesions in a subject by inducing an immune response in the individual against HPV, comprising administering to the individual a composition comprising an amino acid sequence provided herein, in some embodiments, the method also comprises introducing the amino acid sequence into the individual by electroporation.

[0090] Improved vaccines include proteins and genetic constructs that code for proteins with epitopes that are particularly effective as immunogens against which anti-HPV immune responses can be induced. Thus, vaccines can be provided to induce therapeutic or prophylactic immune responses. In some embodiments, the means for delivering the immunogen is a DNA vaccine, a recombinant vaccine, a protein subunit vaccine, a composition comprising an immunogen, an attenuated vaccine, or a killed 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 comprising an immunogen, one or more attenuated vaccines, and one or more killed vaccines.

[0091] Aspects of the invention provide methods for delivering coding sequences for proteins on nucleic acid molecules such as plasmids, either as isolated proteins or as part of a vector, as part of a recombinant vaccine and as part of an attenuated vaccine.

[0092] According to some aspects of the present invention, compositions and methods are provided for prophylactically and / or therapeutically immunizing an individual.

[0093] 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 the priority applications cited therein, each of which is incorporated herein by reference. In addition to the delivery protocols described in those applications, alternative methods of delivering DNA are described in U.S. Patent Nos. 4,945,050 and 5,036,006, both of which are incorporated herein by reference.

[0094] The present invention relates to improved live attenuated vaccines, improved killed vaccines, and improved vaccines that use recombinant vectors to deliver foreign genes encoding antigens, as well as subunit and glycoprotein vaccines. Examples of live attenuated vaccines, those that use recombinant vectors to deliver foreign 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, 5,294,548, and the like. Nos. 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, each of which is incorporated herein by reference.

[0095] When taken up by a cell, the genetic construct(s) may remain present in the cell as a functional extrachromosomal molecule and / or may integrate into the chromosomal DNA of the cell. DNA may be introduced into the cell, where it remains as separate genetic material in the form of a plasmid(s). Alternatively, linear DNA that can integrate into a chromosome may be introduced into the cell. When introducing DNA into a cell, reagents may be added that promote DNA integration into the chromosome. DNA sequences useful for promoting integration may also be included in the DNA molecule. Alternatively, RNA may be administered to the cell. It is also contemplated to provide the genetic construct as a linear minichromosome that includes a centromere, telomeres, and an origin of replication. The genetic construct may remain part of the genetic material in an attenuated live microorganism or recombinant microbial vector that survives in the cell. The genetic construct may be part of the genome of a recombinant viral vaccine, where the genetic material is either integrated into the chromosome of the cell or remains extrachromosomal. The genetic construct contains the regulatory elements necessary for gene expression of the nucleic acid molecule. The elements include a promoter, a start codon, a stop codon, and a polyadenylation signal. In addition, enhancers are often required for gene expression of sequences encoding target proteins or immunomodulatory proteins. These elements need to be operably linked to the sequence encoding the desired protein, and the regulatory elements need to be operative in the individual to whom they are administered.

[0096] The start codon and stop codon 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 whom the genetic construct is administered. The start codon and stop codon must be in frame with the coding sequence.

[0097] The promoter and polyadenylation signal used must be functional in the cells of the individual.

[0098] Examples of promoters useful in practicing the present invention, particularly in the production of genetic vaccines for humans, include, but are not limited to, promoters from Simian Virus 40 (SV40), mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus (MV) such as the BIV long terminal repeat (LTR) promoter, Moloney virus, ALV, cytomegalovirus (CMV) such as the CMV immediate early promoter, Epstein-Barr virus (EBV), Rous sarcoma virus (RSV), and human genes such as human actin, human myosin, human hemoglobin, human muscle creatine, and human metallothionein.

[0099] Examples of polyadenylation signals useful for carrying out the present invention, particularly in the production of genetic vaccines for humans, include, but are not limited to, the SV40 polyadenylation signal and the LTR polyadenylation signal. In particular, the SV40 polyadenylation signal found in pCEP4 plasmid (Invitrogen, San Diego CA), referred to as the SV40 polyadenylation signal, is used.

[0100] In addition to the regulatory elements required for DNA expression, other elements can also be included in the DNA molecule.Such additional elements include enhancers.Enhancers can be selected from the group including, but not limited to, human actin, human myosin, human hemoglobin, human muscle creatine, and viral enhancers such as those derived from CMV, RSV, and EBV.

[0101] The genetic construct can be provided with a mammalian origin of replication to maintain the construct extrachromosomally and produce multiple copies of the construct in cells. Plasmids pVAX1, pCEP4, and pREP4 from Invitrogen (San Diego, Calif.) contain the Epstein-Barr virus origin of replication and the nuclear antigen EBNA-1 coding region, which provides high-copy episomal replication without integration.

[0102] In some preferred embodiments related to immunization applications, a nucleic acid molecule(s) is delivered that includes a nucleotide sequence encoding a protein of the invention and, in addition, a gene for a protein that further enhances the immune response against such target protein. Examples of such genes are those that encode other cytokines and lymphokines such as alpha-interferon, gamma-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 the signal sequence deleted and optionally including a signal peptide from IgE. Other genes that may be useful include those encoding: MCP-1, MIP-lα, MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18, CD4 0, CD40L, vascular growth 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 genes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK ligand, Ox40, Ox40 ligand, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2, and functional fragments thereof.

[0103] If for any reason it is desirable to eliminate cells that receive the genetic construct, additional elements can be added that target cells for destruction. An expressible form of the herpes thymidine kinase (tk) gene can be included in the genetic construct. The drug ganciclovir can be administered to an individual, which causes the selective killing of any cells producing tk, thus providing a means for the selective destruction of cells that carry the genetic construct.

[0104] To maximize protein production, regulatory sequences can be selected that are well suited for gene expression in the cells to which the construct is administered. Furthermore, codons that are most efficiently transcribed in the cells can be selected. Those skilled in the art can generate DNA constructs that are functional in cells.

[0105] In some embodiments, genetic constructs may be provided in which the coding sequences for the proteins described herein are linked to an IgE signal peptide. In some embodiments, the proteins described herein are linked to an IgE signal peptide.

[0106] In some embodiments where a protein is used, for example, one of skill in the art can use well-known techniques to produce and isolate the protein of the invention. In some embodiments where a protein is used, for example, one of skill in the art can use well-known techniques to insert a DNA molecule encoding the protein of the invention into a commercially available expression vector for use in a well-known expression system. For example, the commercially available plasmid pSE420 (Invitrogen, San Diego, Calif.) can be used for production of the protein in E. coli. The commercially available plasmid pYES2 (Invitrogen, San Diego, Calif.) can be used for production in, for example, S. cerevisiae strains of yeast. The commercially available MAXBAC™ complete baculovirus expression system (Invitrogen, San Diego, Calif.) can be used for production in, for example, insect cells. The commercially available plasmids pcDNA I or pcDNA3 (Invitrogen, San Diego, Calif.) can be used for production in mammalian cells, such as, for example, Chinese hamster ovary cells. Those skilled in the art can use these commercial expression vectors and expression systems, or others, to produce proteins using routine techniques and readily available starting materials. (See, e.g., Sambrook et al., Molecular Cloning a Laboratory Manual, Second Ed. Cold Spring Harbor Press (1989), incorporated herein by reference.) Thus, the desired protein can be prepared in both prokaryotic and eukaryotic systems, resulting in a spectrum of processed forms of the protein.

[0107] Those skilled in the art may use other commercially available expression vectors and expression systems or may produce vectors using well-known methods and readily available starting materials. Expression systems containing the necessary 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, Second Ed. Cold Spring Harbor Press (1989). The genetic construct comprises a protein coding sequence operably linked to a promoter that is functional in the cell line in which the construct is transfected. 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 easily produce genetic constructs useful for transfecting cells with DNA encoding the protein of the present invention from readily available starting materials. The expression vector containing the DNA encoding the protein is used to transform a compatible host, which is then cultured and maintained under conditions in which expression of the foreign DNA occurs.

[0108] The produced protein is recovered by lysing the cells or from the culture medium as required, as known to those skilled in the art. Those skilled in the art can use well-known techniques to isolate proteins produced using such expression systems. The methods of purifying proteins from natural sources using antibodies that specifically bind to a particular protein as described above can be equally applied to purifying proteins produced by recombinant DNA methodology.

[0109] In addition to producing proteins by recombinant techniques, automated peptide synthesizers can also be used to produce isolated, essentially pure proteins. Such techniques are well known to those of skill in the art and are useful when the protein is a derivative having substitutions not provided for in DNA-encoded protein production.

[0110] Nucleic acid molecules can be delivered using any of a number of well-known techniques, including DNA injection (also called DNA vaccination), recombinant vectors such as recombinant adenoviruses, recombinant adenovirus-associated viruses, and recombinant vaccinia.

[0111] Routes of administration include, but are not limited to, intramuscular, intranasal, intraperitoneal, intradermal, subcutaneous, intravenous, intraarterial, intraocular, and oral, as well as topical, transdermal, by inhalation or suppository, or by lavage into mucosal tissues, such as vaginal, rectal, urethral, ​​buccal, and sublingual tissues. Preferred routes of administration include intramuscular, intraperitoneal, intradermal, and subcutaneous injection. Gene constructs may be administered by means including, but not limited to, electroporation methods and devices, conventional syringes, needleless injection devices, or "microprojectile bombardment gene guns."

[0112] Examples of preferred electroporation devices and methods for enhancing delivery of DNA vaccines include those described in U.S. Patent No. 7,245,963 by Draghia-Akli, et al., and U.S. Patent Publication No. 2005 / 0052630 filed by Smith, et al., the contents of which are incorporated herein by reference in their entireties. Also preferred are electroporation devices and methods for enhancing delivery of DNA vaccines provided in co-pending, co-owned U.S. Patent Application No. 11 / 874,072, filed October 17, 2007, which claims benefit under 35 USC 119(e) to U.S. Provisional Application No. 60 / 852,149, filed October 17, 2006, and U.S. Provisional Application No. 60 / 978,982, filed October 10, 2007, all of which are incorporated herein in their entireties.

[0113] The following are examples of embodiments using electroporation techniques, discussed in more detail in the patent references mentioned above: The electroporation device can be configured to deliver pulses of energy to a desired tissue of a mammal that produces a constant current similar to a current input preset by a user. The electroporation device includes an electroporation component and an electrode assembly or handle assembly. The electroporation component can include and incorporate one or more of the various elements of an electroporation device, including a controller, a current waveform generator, an impedance tester, a waveform logger, an input element, a status reporting element, a communication port, a memory component, a power source, and a power switch. The electroporation component can function as one element of the electroporation device, and other elements are separate elements (or components) that communicate with the electroporation component. In some embodiments, the electroporation component can function as more than one element of the electroporation device, which can communicate with yet other elements of the electroporation device that are separate from the electroporation component. The use of electroporation technology to deliver improved HPV vaccines is not limited by the elements of the electroporation device being part of one electromechanical or mechanical device, as the elements can function as one device or as separate elements in communication with each other. The electroporation component can deliver a pulse of energy that produces a constant current in the desired tissue and includes a feedback mechanism. The electrode assembly includes an electrode array having a plurality of electrodes in a spatial arrangement, the electrode assembly receives a pulse of energy from the electroporation component and delivers it through the electrodes to the desired tissue. At least one of the plurality of electrodes is neutral during delivery of the pulse of energy, measures impedance in the desired tissue, and communicates the impedance to the electroporation component.A feedback mechanism can receive the measured impedance and can adjust the pulses of energy delivered by the electroporation component to maintain a constant current.

[0114] In some embodiments, the multiple electrodes can deliver pulses of energy in a decentralized pattern. In some embodiments, the multiple electrodes can deliver pulses of energy in a decentralized pattern through control of the electrodes under a programmed sequence, the programmed sequence being input by a user into the electroporation component. In some embodiments, the programmed sequence includes multiple pulses delivered sequentially, each pulse of the multiple pulses being delivered by at least two active electrodes with one indifferent electrode that measures impedance, and each subsequent pulse of the multiple pulses being delivered by a different one of the at least two active electrodes with one indifferent electrode that measures impedance.

[0115] In some embodiments, the feedback mechanism is implemented by either hardware or software. Preferably, the feedback mechanism is implemented by an analog closed loop circuit. Preferably, this feedback occurs every 50 μs, 20 μs, 10 μs, or 1 μs, but is preferably real-time feedback or instantaneous (i.e., substantially instantaneous, as determined by available techniques for determining response time). In some embodiments, the indifferent electrode measures the impedance at the desired tissue and communicates the impedance to the feedback mechanism, which responds to the impedance and adjusts the pulse of energy to maintain the constant current at a value similar to the preset current. In some embodiments, the feedback mechanism maintains the constant current continuously and instantaneously during the delivery of the pulse of energy.

[0116] In some embodiments, the nucleic acid molecule is delivered to the cell in conjunction with administration of a polynucleotide function enhancer or genetic vaccine facilitator. Polynucleotide function enhancers are described in U.S. Patent Nos. 5,593,972, 5,962,428, and International Patent Application No. PCT / US94 / 00899, filed January 26, 1994, each of which is incorporated herein by reference. Genetic vaccine facilitator agents are described in U.S. Patent Application No. 021,579, filed April 1, 1994, each of which is incorporated herein by reference. The adjuvant administered in conjunction with the nucleic acid molecule may be administered as a mixture with the nucleic acid molecule, or may be administered separately and simultaneously, before or after administration of the nucleic acid molecule. In addition, other agents that may function as transfection and / or replication and / or inflammatory agents and may be co-administered with GVF include growth factors, cytokines, and lymphocytes, 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, as well as fibroblast growth factors, surfactants, such as immune stimulating complexes (ISCOMS), Freund's incomplete adjuvant, LPS analogs including monophosphoryl lipid A (WL), muramyl peptides, quinone analogs, and vesicles such as squalene and squalene, and hyaluronic acid may also be used and administered in conjunction with the genetic construct. In some embodiments, immunomodulatory proteins may be used as GVFs. In some embodiments, nucleic acid molecules are provided in association with PLG to enhance delivery / uptake.

[0117] The pharmaceutical composition according to the present invention contains about 1 nanogram to about 2000 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition according to the present invention contains about 5 nanograms to about 1000 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition contains about 10 to about 800 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition contains about 0.1 to about 500 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition contains about 1 to about 350 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition contains about 25 to about 250 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition contains about 100 to about 200 micrograms of DNA.

[0118] The pharmaceutical compositions according to the present invention are formulated according to the mode of administration to be used. When the pharmaceutical compositions are injectable pharmaceutical compositions, they are sterile, pyrogen-free, and particulate-free. An isotonic formulation is preferably used. In general, additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol, and lactose. In some cases, an isotonic solution such as phosphate buffered saline is preferred. Stabilizers include gelatin and albumin. In some embodiments, a vasoconstrictor is added to the formulation.

[0119] Provided herein is a method of treating human papillomavirus (HPV) type 16 or HPV type 18 associated anal or anal / perianal high-grade intraepithelial lesion (HSIL) in a subject in need thereof, comprising, consisting of, or consisting essentially of administering to the subject a therapeutically effective amount of a pharmaceutical composition according to the present invention. According to some embodiments, the pharmaceutical composition is VGX-3100 or a biosimilar thereof.

[0120] Also provided herein is VGX-3100 for use in a method of treating human papillomavirus (HPV) type 16 or HPV type 18 associated anal or anal / perianal high-grade intraepithelial lesion (HSIL) in a subject in need thereof, comprising, consisting of, or consisting essentially of administering to the subject a therapeutically effective amount of a pharmaceutical composition according to the present invention. According to some embodiments, the pharmaceutical composition is VGX-3100 or a biosimilar thereof.

[0121] Also provided herein is the use of VGX-3100 in the manufacture of a medicament for treating human papillomavirus (HPV) type 16 or HPV type 18 associated anal or anal / perianal high-grade intraepithelial lesions (HSIL), the method comprising, consisting of, or consisting essentially of administering to a subject a therapeutically effective amount of a pharmaceutical composition according to the invention. According to some embodiments, the pharmaceutical composition is VGX-3100 or a biosimilar thereof.

[0122] Also provided herein is a method of treating anal or anal / perianal HSIL in a subject for which the improvement is to improve histopathological regression, comprising, consisting of, or consisting essentially of administering to the subject a therapeutically effective amount of a pharmaceutical composition according to the present invention. According to some embodiments, the pharmaceutical composition is VGX-3100 or a biosimilar thereof. In certain embodiments, the improvement in histopathological regression of anal or anal / perianal HSIL is relative to no treatment of the subject or population of subjects. In certain embodiments, the improvement in histopathological regression of anal or anal / perianal HSIL is relative to treatment of a subject in a population of subjects with standard of care.

[0123] Also provided herein is a method of improving virologic clearance of HPV-16 and / or HPV-18 in a subject with anal or anal / perianal HSIL, comprising, consisting of, or consisting essentially of administering to the subject a therapeutically effective amount of a pharmaceutical composition according to the present invention. According to some embodiments, the pharmaceutical composition is VGX-3100 or a biosimilar thereof.

[0124] Also provided herein is a method of achieving partial or complete histopathological regression of anal or anal / perianal HSIL to normal or non-hisopathological progression in a subject with HPV type 16 or HPV type 18 associated anal or perianal HSIL, comprising, consisting of, or consisting essentially of administering to the subject a therapeutically effective amount of a pharmaceutical composition according to the invention. According to some embodiments, the pharmaceutical composition is VGX-3100 or a biosimilar thereof. In certain embodiments, the achievement of partial or complete histopathological regression of anal or anal / perianal HSIL to normal or non-hisopathological progression is relative to administration of a placebo to the subject or population of subjects. In certain embodiments, the achievement of partial or complete histopathological regression of anal or anal / perianal HSIL to normal or non-hisopathological progression is relative to no treatment of the subject or population of subjects. In certain embodiments, the achievement of partial or complete histopathological regression or non-hisopathological progression of anal or anal / perianal HSIL is relative to treatment of a subject in a population of subjects with standard of care.

[0125] Also provided herein is a method of improving clearance of HPV-16 and / or HPV-18 infection from non-anal anatomical locations in a subject with HPV type 16 or HPV type 18 associated anal or perianal HSIL, comprising, consisting of, or consisting essentially of administering to the subject a therapeutically effective amount of a pharmaceutical composition according to the present invention. According to some embodiments, the pharmaceutical composition is VGX-3100 or a biosimilar thereof.

[0126] In some embodiments, VGX-3100 is administered to the subject by intramuscular injection followed by electroporation. In certain embodiments, VGX-3100 is administered to the subject at a dose of 6 mg. In further embodiments, VGX-3100 is administered to the subject three times over a 12 week period. In further embodiments, VGX-3100 is administered to the subject four times over a 40 week period. In yet further embodiments, VGX-3100 is formulated at a concentration of 6 mg / ml in 150 mM sodium chloride and 15 mM sodium citrate.

[0127] In certain embodiments, administration of VGX-3100 results in virologic clearance of HPV-16 and / or HPV-18 and histopathological regression of anal HSIL. In further embodiments, administration of VGX-3100 results in histopathological regression of anal HSIL. In yet further embodiments, administration of VGX-3100 results in virologic clearance of HPV-16 and / or HPV-18. In certain embodiments, administration of VGX-3100 results in complete histopathological regression of anal HSIL to normal. In further embodiments, administration of VGX-3100 results in complete histopathological regression of anal HSIL to normal and virologic clearance of HPV-16 and / or HPV-18. In yet further embodiments, administration of VGX-3100 results in histopathological non-progression. In certain embodiments, administration of VGX-3100 results in clearance of HPV-16 and / or HPV-18 infection from non-anal anatomical locations. In further embodiments, administration of VGX-3100 results in improvement in humoral and cellular immune responses to VGX-3100, assessed relative to baseline after the third administration of VGX-3100 and 36 weeks after administration of VGX-3100.

[0128] In a further embodiment, administration of VGX-3100 results in improved humoral and cellular immune responses to VGX-3100, as assessed after the fourth administration of VGX-3100 compared to baseline.

[0129] In certain embodiments, the results of administration of VGX-3100 are evaluated 36 weeks after administration of VGX-3100.

[0130] According to some embodiments of the present invention, a method of inducing an immune response is provided. The vaccine can be a protein-based, live attenuated vaccine, a cellular vaccine, a recombinant vaccine, or a nucleic acid or DNA vaccine. In some embodiments, a method of inducing an immune response in an individual against an immunogen, including a method of inducing a mucosal immune response, comprises administering to the individual one or more of a CTACK protein, a TECK protein, a MEC protein, and functional fragments thereof, or expressible coding sequences thereof, in combination with an isolated nucleic acid molecule encoding a protein of the present invention and / or a recombinant vaccine encoding a protein of the present invention and / or a subunit vaccine encoding a protein of the present invention and / or a live attenuated vaccine and / or a killed vaccine. One or more of a CTACK protein, a TECK protein, a MEC protein, and functional fragments thereof can be administered before, simultaneously with, or after administration of an isolated nucleic acid molecule encoding an immunogen and / or a recombinant vaccine encoding an immunogen and / or a subunit vaccine comprising an immunogen and / or a live attenuated vaccine and / or a killed vaccine. In some embodiments, an isolated nucleic acid molecule encoding one or more proteins selected from the group consisting of CTACK, TECK, MEC, and functional fragments thereof is administered to the individual.

[0131] The present invention is further illustrated in the following examples. It should be understood that the examples show embodiments of the present invention, but are provided by way of illustration only. From the above discussion and the examples, those skilled in the art can ascertain the essential features of the present invention, and can make various changes and modifications to the present invention to accommodate various uses and conditions without departing from the spirit and scope thereof. Thus, in addition to those shown and described herein, various modifications of the present invention will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

[0132] Each of the U.S. patents, U.S. applications, and references cited throughout this disclosure is hereby incorporated by reference in its entirety.

[0133] Illustrative embodiments: Embodiment 1. A method for treating or preventing anal high-grade squamous intraepithelial lesions in an individual, comprising administering to the individual a composition comprising at least one nucleic acid molecule encoding at least one selected from the group consisting of HPV16 antigens and HPV18 antigens. Embodiment 2. The method of embodiment 1, wherein the HPV16 antigen is an HPV16 E6-E7 fusion antigen. Embodiment 3. The method of embodiment 1 or 2, wherein the HPV16 antigen comprises the amino acid sequence of SEQ ID NO:5 and the amino acid sequence of SEQ ID NO:6. Embodiment 4. The method of any preceding embodiment, wherein the HPV 16 antigen is encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:17 and the nucleotide sequence of SEQ ID NO:19. Embodiment 5 The method of any preceding embodiment, wherein the HPV18 antigen is an HPV18 E6-E7 fusion antigen. Embodiment 6 The method of any preceding embodiment, wherein the HPV18 antigen comprises the amino acid sequence of SEQ ID NO:21 and the amino acid sequence of SEQ ID NO:22. Embodiment 7. The method of any preceding embodiment, wherein the composition comprises a nucleotide sequence encoding an HPV 16 antigen and a nucleotide sequence encoding an HPV 18 antigen. Embodiment 8. The nucleic acid molecule comprises: a nucleotide sequence encoding SEQ ID NO:2; a nucleotide sequence that is at least 95% identical to the nucleotide sequence encoding SEQ ID NO:2; a fragment of a nucleotide sequence encoding SEQ ID NO:2;

[0036] The method of any preceding embodiment, comprising one or more nucleotide sequences selected from the group consisting of: a nucleotide sequence that is at least 95% homologous to a fragment of a nucleotide sequence encoding SEQ ID NO:2. Embodiment 9. The method of embodiment 8, wherein the nucleic acid molecule comprises a nucleotide sequence that is at least 98% homologous to a nucleotide sequence encoding SEQ ID NO:2. Embodiment 10. The method of embodiment 8, wherein the nucleic acid molecule comprises a nucleotide sequence that is at least 99% homologous to a nucleotide sequence encoding SEQ ID NO:2. Embodiment 11. The nucleic acid molecule comprises: A nucleotide sequence comprising nucleotides 19 to 795 of SEQ ID NO:1; A nucleotide sequence comprising nucleotides 1 to 795 of SEQ ID NO:1; A nucleotide sequence comprising SEQ ID NO:1; A nucleotide sequence that is at least 95% identical to SEQ ID NO:1; A fragment of SEQ ID NO: 1; A nucleotide sequence that is at least 95% homologous to a fragment of SEQ ID NO:1. Embodiment 12. The method of embodiment 11, wherein the nucleic acid molecule comprises a nucleotide sequence that is at least 98% identical to SEQ ID NO:1. Embodiment 13. The method of embodiment 11, wherein the nucleic acid molecule comprises a nucleotide sequence that is at least 99% identical to SEQ ID NO:1. Embodiment 14 The method of any preceding embodiment, wherein the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:23 and the nucleotide sequence of SEQ ID NO:24. Embodiment 15. The nucleic acid molecule comprises: A nucleotide sequence encoding SEQ ID NO:10; a nucleotide sequence that is at least 95% identical to the nucleotide sequence encoding SEQ ID NO:10; A fragment of a nucleotide sequence encoding SEQ ID NO: 10;

[0036] The method of any preceding embodiment, comprising one or more nucleotide sequences selected from the group consisting of: a nucleotide sequence that is at least 95% homologous to a fragment of a nucleotide sequence encoding SEQ ID NO: 10. Embodiment 16. The method of embodiment 15, wherein the nucleic acid molecule comprises a nucleotide sequence that is at least 98% identical to a nucleotide sequence encoding SEQ ID NO:10. Embodiment 17. The method of embodiment 15, wherein the nucleic acid molecule comprises a nucleotide sequence that is at least 99% homologous to a nucleotide sequence encoding SEQ ID NO:10. Embodiment 18. The method of any one of embodiments 1 to 7 or 14 to 17, wherein the nucleic acid molecule encoding the HPV16 E6-E7 fusion antigen further comprises a nucleotide sequence encoding a leader sequence, the nucleotide sequence encoding the HPV18 E6-E7 fusion antigen further comprises a nucleotide sequence encoding a leader sequence, or the nucleotide sequence encoding the HPV16 E6-E7 fusion antigen further comprises a nucleotide sequence encoding a leader sequence, and the nucleotide sequence encoding the HPV18 E6-E7 fusion antigen further comprises a nucleotide sequence encoding a leader sequence. Embodiment 19. The nucleic acid molecule comprises: A nucleotide sequence comprising nucleotides 1 to 780 of SEQ ID NO:9; A nucleotide sequence comprising SEQ ID NO:9; A nucleotide sequence that is at least 95% identical to SEQ ID NO:9; A fragment of SEQ ID NO: 9, 9. The method of any preceding embodiment, comprising one or more nucleotide sequences selected from the group consisting of: a nucleotide sequence at least 95% homologous to a fragment of SEQ ID NO:9. Embodiment 20. The method of embodiment 19, wherein the nucleic acid molecule comprises a nucleotide sequence that is at least 98% identical to SEQ ID NO:9. Embodiment 21 The method of embodiment 19, wherein the nucleic acid molecule comprises a nucleotide sequence that is at least 99% identical to SEQ ID NO:9. Embodiment 22 The method of any preceding embodiment, wherein the at least one nucleic acid molecule comprises at least one plasmid. Embodiment 23 The method of any preceding embodiment, wherein the composition is a pharmaceutical composition. Embodiment 24 The method of any preceding embodiment, further comprising administering to the individual a composition comprising an adjuvant. Embodiment 25. A method comprising administering to an individual a nucleic acid molecule comprising a nucleotide sequence encoding an HPV16 E6-E7 fusion antigen and a nucleic acid molecule comprising a nucleotide sequence encoding an HPV18 E6-E7 fusion antigen, the nucleotide sequence encoding the HPV16 E6-E7 fusion antigen is selected from the group consisting of a nucleotide sequence encoding SEQ ID NO:2, a nucleotide sequence that is at least 95% identical to a nucleotide sequence encoding SEQ ID NO:2, a fragment of a nucleotide sequence encoding SEQ ID NO:2, and a nucleotide sequence that is at least 95% identical to a fragment of a nucleotide sequence encoding SEQ ID NO:2; 10. The method of any preceding embodiment, wherein the nucleotide sequence encoding the HPV18 E6-E7 fusion antigen is selected from the group consisting of a nucleotide sequence encoding SEQ ID NO:10, a nucleotide sequence that is at least 95% homologous to a nucleotide sequence encoding SEQ ID NO:10, a fragment of a nucleotide sequence encoding SEQ ID NO:10, and a nucleotide sequence that is at least 95% homologous to a fragment of a nucleotide sequence encoding SEQ ID NO:10. Embodiment 26. The method of embodiment 25, wherein one nucleic acid molecule comprises a nucleotide sequence encoding an HPV16 E6-E7 fusion antigen and a nucleotide sequence encoding an HPV18 E6-E7 fusion antigen. Embodiment 27 The method of any preceding embodiment, wherein administering the nucleic acid molecule to the individual comprises electroporation. Embodiment 28 The method of any preceding embodiment, wherein the composition is administered intramuscularly. Embodiment 29. The method of any preceding embodiment, wherein the composition comprises VGX-3100 or a biosimilar thereof. Embodiment 30. The method of any preceding embodiment, wherein the composition is administered in three or four doses. Embodiment 31 The method of embodiment 30, wherein the second dose of the composition is administered about 4 weeks after the first dose. Embodiment 32 The method of embodiment 30, wherein the third dose is administered about 12 weeks after the first dose. Embodiment 33 The method of embodiment 30, wherein the fourth dose is administered about 40 weeks after the first dose. Embodiment 34 The method of any preceding embodiment, wherein the HSIL is anal or anal / perianal HSIL. Embodiment 35 The method of embodiment 34, wherein the leader sequence comprises the amino acid sequence of SEQ ID NO:7. Embodiment 36 The method of embodiment 18, wherein the leader sequence is encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:11. EXAMPLES

[0134] The present invention is further defined in the following examples. It should be understood that these examples, while showing preferred embodiments of the present invention, are provided by way of illustration only. From the above discussion and these examples, those skilled in the art can ascertain the essential features of the present invention, and can make various changes and modifications to the present invention to accommodate various uses and conditions without departing from the spirit and scope thereof. Thus, in addition to those shown and described herein, various modifications of the present invention will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

[0135] Example 1: VGX-3100 delivered intramuscularly (IM) followed by electroporation (EP) for the treatment of HPV-16 and / or HPV-18 associated anal or anal / perianal high-grade squamous intraepithelial lesions (HSIL) in individuals who are seronegative for human immunodeficiency virus (HIV)-1 / 2 (ClinicalTrials_gov Identifier: NCT03499795) This is a Phase 2, open-label efficacy study of VGX-3100 administered by intramuscular (IM) injection followed by electroporation (EP) in adult men and women who are human immunodeficiency virus (HIV)-negative with histologically confirmed anal or anal / perianal high-grade squamous intraepithelial lesions (HSIL) associated with human papillomavirus (HPV)-16 and / or HPV-18.

[0136] Management of anal high-grade squamous intraepithelial lesions (HSIL) remains challenging. VGX-3100, a DNA-based HPV16 / 18-specific immunotherapy encoding HPV16 and 18 E6 / E7 proteins, was evaluated in an open-label Phase 2 study for treating anal HSIL (Figure 1).

[0137] Inclusion Criteria: 18 years of age or older, A negative screening test for HIV-1 / 2 within 30 days of dose 1; Confirmed anal or anal / perianal HPV-16 / 18 infection at screening by polymerase chain reaction (PCR) from HSIL specimens; At least one anal or anal / perianal (AIN2 / 3 and / or PAIN2 / PAIN3) lesion histologically confirmed as HSIL at screening, Suitable candidates for histology collection procedures (i.e., resection or biopsy); Female subjects must be postmenopausal, surgically sterilized, or agree to avoid pregnancy by continuous abstinence or by use of a contraceptive method with a failure rate of less than 1% per year from screening through 1 month (week 12 or week 40) after the last dose of study drug. Men who could father children must agree to use at least one form of contraception during heterosexual intercourse or continuous abstinence from heterosexual intercourse prior to the study, throughout their study participation, and one month after the last dose of study medication.

[0138] Normal screening electrocardiogram (ECG).

[0139] Exclusion criteria: Untreated microinvasive or invasive cancer, Biopsy-proven vaginal intraepithelial neoplasia (VAIN) without prior medical treatment and / or therapy for VAIN; Biopsy-proven vulvar intraepithelial neoplasia (VIN) without prior medical treatment and / or therapy for VIN; Biopsy-proven cervical intraepithelial neoplasia (CIN) 2 / 3 without medical treatment and / or therapy for CIN; Biopsy-proven penile intraepithelial neoplasia (PIN) without prior medical treatment and / or therapy for PIN; Anal or anal / perianal HSIL that is not accessible for sampling with a biopsy device; Intra-anal and / or perianal lesion(s) that cannot be fully visualized at screening, inability to perform complete and satisfactory high-resolution anoscopy (HRA); Any treatment (e.g., surgery) for anal or anal / perianal HSIL within 4 weeks of screening; pregnancy, breastfeeding, or consideration of pregnancy within 1 month after the last dose of study drug; The presence of any abnormal laboratory value greater than Grade 1 according to the Common Toxicity Criteria for Adverse Events (CTCAE) version 4.03 within 45 days prior to Day 0, or less than Grade 1 but deemed clinically significant by the investigator; Immunosuppression as a result of the underlying disease or treatment, History of previous therapeutic HPV vaccination receiving any non-study-related non-live vaccine within 2 weeks of any VGX-3100 dose, receiving any non-study-related non-live vaccine (e.g., measles vaccine) within 4 weeks of any VGX-3100 dose; Significant acute or chronic medical illness that may be adversely affected by electroporation, as determined by the investigator and treated; Any current clinically significant, medically unstable illness or history that, in the investigator's judgment, jeopardizes the subject's safety, interferes with the study evaluation or endpoint assessment, or otherwise affects the validity of the study results; Previous major surgery within 4 weeks of day 0, Participation in an interventional study using an investigational compound or device; Any disease or condition that, in the opinion of the Investigator, may affect the subject's safety or the assessment of any study endpoints.

[0140] Primary outcome measures: Proportion of participants without histologic evidence of anal or anal / perianal HSIL and without evidence of HPV-16 / 18 at week 36 [Timeframe:Week 36] Secondary outcome measures: Number of local and systemic safety events during the 7 days after each dose [time frames: days 0-7 (7 days after the day 0 dose), days 22-29 (7 days after the week 4 dose), and days 78-85 (7 days after the week 12 dose)] Number of Adverse Events [Time Frame: Baseline to Week 88 Visit] Proportion of participants without histologic evidence of anal or anal / perianal HSIL at the Week 36 visit [Time Frame: Week 36] Proportion of participants with no evidence of HPV-16 / 18 in anal and / or perianal tissue by species-specific HPV testing at the week 36 visit [Timeframe: Week 36] Proportion of participants with no evidence of HPV-16 / 18 from anal swabs by specific HPV testing at the 36-, 64-, and 88-week visits [Time Frame: 36, 64, and 88 weeks] Proportion of participants without histologic evidence of anal or anal / perianal low-grade squamous intraepithelial lesions (LSIL) or HSIL at the Week 36 visit [Time Frame: Week 36] Proportion of participants without progression of anal or anal / perianal HSIL from baseline to cancer by histology at the 36-week visit [Time frame: baseline to week 36] Percent reduction from baseline in the number of intra-anal and / or perianal lesion(s) as determined by the investigator at the Week 36, 64, and 88 visits [Time Frame: Baseline to Weeks 36, 64, and 88] Percent reduction from baseline in size of perianal lesion(s) as determined by the investigator at Week 36, 64, and 88 visits [Time Frame: Baseline to Weeks 36, 64, and 88] Magnitude of Flow Cytometry Response [Time Frame: Baseline and Week 15] Proportion of participants with no histologic evidence of anal or anal / perianal HSIL or no evidence of HPV-16 / 18 at Week 36 [Time Frame: Week 36]

[0141] Investigational Drug Generic name: VGX-3100 Chemical name: circular double-stranded deoxyribonucleic acid consisting of 3782 base pairs for the pGX3001 plasmid and 3824 base pairs for the pGX3002 plasmid.

[0142] Identification name: Eukaryotic expression plasmid containing transcription units encoding HPV 16 and 18-E6 and E7 controlled by the synthetic CMV promoter and elements required for replication and selection in E. coli, namely the pUC origin of replication (pUC Ori) and the kanamycin resistance gene (Kan R).

[0143] Detailed Description: VGX-3100, the HPV therapeutic vaccine, is a combination of two plasmids in equal amounts (i.e., a 6 mg dose delivers 3 mg of each pGX3001 and pGX3002 plasmid): a) pGX3001: p16ConE6E7, a plasmid encoding a synthetic HPV16 consensus E6 and E7 fusion gene ("consensus HPV16-6 and 7") into a pVAX1 backbone (Invitrogen, Carlsbad, CA) under control of the cytomegalovirus immediate-early (CMV) promoter, and b) pGX3002: p18ConE6E7, a plasmid encoding an HPV18 consensus E6 and E7 fusion gene ("consensus HPV 18-6 and 7") into a pVAX1 backbone (Invitrogen, Carlsbad, CA) under control of the cytomegalovirus immediate-early (CMV) promoter. VGX-3100 is described in WO2014 / 165291, which is incorporated by reference in its entirety. The nucleic acid and amino acid consensus sequences are provided in Tables 1 and 2, respectively. [Table 1] [Table 2]

[0144] Subjects with HPV16 / 18 positive anal HSIL received 3 or 4 doses of VGX-3100 intramuscularly followed by CELLECTRA® electroporation. Subjects underwent biopsy of HSIL and histology-based clinical endpoints were assessed 36 weeks after initiation of VGX-3100 treatment. Endpoints included resolution of anal HSIL and clearance of HPV16 / 18 in anal tissue (primary), and reduction in lesion count and size (secondary). In addition, samples were collected to demonstrate concordance between baseline anal swabs and excised tissue for HPV, using the SPF10-LiPA25 (line probe assay) version 1 system (tissue) and Roche cobas® HPV test (swab).

[0145] Figure 2 provides data on vaccine efficacy at 36 and 64 weeks.

[0146] FIG. 3 provides data demonstrating that VGX-3100 induces cellular immune responses to both HPV16 and HPV18 antigens.

[0147] FIG. 4 provides data demonstrating that VGX-3100 induces a humoral immune response against HPV16E7 and HPV18E7 antigens.

[0148] Figure 5 provides data on the safety of the VGX-3100 vaccine.

[0149] Given the challenging nature of anal HSIL, an immunotherapeutic approach such as VGX-3100 would represent a significant advance in its management. VGX-3100 is tolerable, immunogenic, and exerts a therapeutic effect on HPV-16 / 18-associated anal HSIL. In addition, detection of HPV-16 / 18 from anal swabs and tissues was highly concordant (data not shown), providing a minimally invasive screening strategy for detecting anal lesions that may simplify screening and surveillance.

[0150] Example 2: Sequences HPV genotype 16 consensus E6-E7 DNA sequence (SEQ ID NO:1) [ka] HPV genotype 16 consensus E6-E7 protein sequence (SEQ ID NO:2) [ka] HPV16 E6 immunodominant epitope (SEQ ID NO:3) [ka] HPV16 E7 immunodominant epitope (SEQ ID NO: 4) [ka] HPV16 E6 consensus sequence (SEQ ID NO:5) [ka] HPV16 E7 consensus sequence (SEQ ID NO:6) [ka] IgE leader sequence (SEQ ID NO:7) [ka] Proteolytic cleavage sequence (SEQ ID NO:8) [ka] HPV18 consensus sequence encoding E6 and E7 (SEQ ID NO:9) [ka] HPV18 consensus HPV peptide sequence with E6 and E7 (SEQ ID NO:10) [ka] IgE leader DNA sequence (SEQ ID NO:11) [ka] IgE leader peptide sequence (SEQ ID NO:12) [ka] Contains the HPV18 consensus sequence encoding E6 and E7, the IgE leader (SEQ ID NO: 13) [ka] HPV18 consensus HPV peptide sequence with E6 and E7, including the IgE leader (SEQ ID NO: 14) [ka] pGX3002 plasmid with consensus and IgE leaders (SEQ ID NO: 15) [ka] [ka] HPV genotype 16 consensus E6-E7 peptide sequence without the IgE leader coding sequence (SEQ ID NO: 16) [ka] HPV genotype 16 consensus E6 nucleotide sequence (SEQ ID NO:17) [ka] Nucleotide sequence encoding the proteolytic cleavage sequence (SEQ ID NO: 18) [ka] HPV genotype 16 consensus E7 nucleotide sequence (SEQ ID NO: 19) [ka] HPV genotype 16 consensus E6-E7 peptide sequence without the IgE leader sequence (SEQ ID NO:20) [ka] HPV18 consensus E6 peptide (SEQ ID NO:21) [ka] HPV18 consensus E7 peptide (SEQ ID NO:22) [ka] HPV18 consensus sequence encoding E6 (SEQ ID NO:23) [ka] HPV18 consensus sequence encoding E7 (SEQ ID NO:24) [ka]

[0151] It is understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes will be suggested to those skilled in the art and are to be included within the spirit and scope of this application and the scope of the appended claims.

Claims

1. A composition comprising at least one nucleic acid molecule encoding at least one antigen selected from the group consisting of human papillomavirus (HPV) 16 antigen and HPV 18 antigen for use in a method for treating or preventing anal high-grade squamous intraepithelial lesions (HSIL) or anal / perianal HSIL.

2. A composition for use as described in claim 1, wherein the anal HSIL or anal / peri-anal HSIL has been histologically confirmed to be associated with human papillomavirus type 16 (HPV16), human papillomavirus type 18 (HPV18), or both.

3. The composition for use according to claim 1, wherein the HPV16 antigen is an HPV16 E6-E7 fusion antigen.

4. The composition for use according to claim 1, wherein the HPV16 antigen comprises the amino acid sequence of SEQ ID NO:5 and the amino acid sequence of SEQ ID NO:

6.

5. 2. The composition for use according to claim 1, wherein the nucleic acid molecule encoding the HPV16 antigen comprises the nucleotide sequence of SEQ ID NO:17 and the nucleotide sequence of SEQ ID NO:

19.

6. The composition for use according to claim 1, wherein the HPV18 antigen is an HPV18 E6-E7 fusion antigen.

7. 2. The composition for use according to claim 1, wherein the HPV18 antigen comprises the amino acid sequence of SEQ ID NO:21 and the amino acid sequence of SEQ ID NO:

22.

8. The composition for use according to claim 1, wherein the composition comprises a nucleic acid molecule comprising a nucleotide sequence encoding an HPV16 antigen and a nucleic acid molecule comprising a nucleotide sequence encoding an HPV18 antigen.

9. The nucleic acid molecule encoding the HPV16 antigen, a nucleotide sequence encoding SEQ ID NO:2, or A nucleotide sequence that is at least 95% identical to the nucleotide sequence encoding SEQ ID NO:2 9. The composition for use according to claim 8, comprising:

10. The composition for use according to claim 9, wherein the nucleic acid molecule encoding the HPV16 antigen comprises a nucleotide sequence that is at least 98% identical to the nucleotide sequence encoding SEQ ID NO:

2.

11. A composition for use as described in claim 9, wherein the nucleic acid molecule encoding the HPV16 antigen comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence encoding SEQ ID NO:

2.

12. The nucleic acid molecule encoding the HPV16 antigen, a nucleotide sequence comprising nucleotides 19 to 795 of SEQ ID NO:1; a nucleotide sequence comprising nucleotides 1 to 795 of SEQ ID NO:1; a nucleotide sequence comprising SEQ ID NO: 1, or A nucleotide sequence that is at least 95% identical to SEQ ID NO:1 9. The composition for use according to claim 8, comprising:

13. The composition for use according to claim 12, wherein the nucleic acid molecule encoding the HPV16 antigen comprises a nucleotide sequence that is at least 98% identical to SEQ ID NO:

1.

14. The composition for use according to claim 12, wherein the nucleic acid molecule encoding the HPV16 antigen comprises a nucleotide sequence that is at least 99% identical to SEQ ID NO:

1.

15. 2. The composition for use according to claim 1, wherein the nucleic acid molecule encoding the HPV18 antigen comprises a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:23 and the nucleotide sequence of SEQ ID NO:

24.

16. The nucleic acid molecule encoding the HPV18 antigen, a nucleotide sequence encoding SEQ ID NO: 10, or A nucleotide sequence that is at least 95% identical to the nucleotide sequence encoding SEQ ID NO: 10 2. The composition for use according to claim 1, comprising:

17. The composition for use according to claim 16, wherein the nucleic acid molecule encoding the HPV18 antigen comprises a nucleotide sequence that is at least 98% identical to a nucleotide sequence encoding SEQ ID NO:

10.

18. The composition for use according to claim 16, wherein the nucleic acid molecule encoding the HPV18 antigen comprises a nucleotide sequence that is at least 99% identical to a nucleotide sequence encoding SEQ ID NO:

10.

19. 2. The composition for use according to claim 1, wherein the nucleic acid molecule encoding the HPV16 E6-E7 fusion antigen further comprises a nucleotide sequence encoding a leader sequence, the nucleotide sequence encoding the HPV18 E6-E7 fusion antigen further comprises a nucleotide sequence encoding a leader sequence, or the nucleotide sequence encoding the HPV16 E6-E7 fusion antigen further comprises a nucleotide sequence encoding a leader sequence and the nucleotide sequence encoding the HPV18 E6-E7 fusion antigen further comprises a nucleotide sequence encoding a leader sequence.

20. The nucleic acid molecule encoding the HPV18 antigen, a nucleotide sequence comprising nucleotides 1 to 780 of SEQ ID NO:9; a nucleotide sequence comprising SEQ ID NO:9, or A nucleotide sequence that is at least 95% identical to SEQ ID NO:9 2. The composition for use according to claim 1, comprising:

21. The composition for use according to claim 1, wherein the nucleic acid molecule encoding the HPV18 antigen comprises a nucleotide sequence that is at least 98% identical to SEQ ID NO:

9.

22. The composition for use according to claim 1, wherein the nucleic acid molecule encoding the HPV18 antigen comprises a nucleotide sequence that is at least 99% identical to SEQ ID NO:

9.

23. The composition for use according to claim 1 , wherein the at least one nucleic acid molecule comprises at least one plasmid.

24. The composition for use according to claim 1, wherein the composition is a pharmaceutical composition.

25. A composition for use according to claim 1, further comprising an adjuvant.

26. a nucleic acid molecule comprising a nucleotide sequence encoding an HPV16 E6-E7 fusion antigen and a nucleic acid molecule comprising a nucleotide sequence encoding an HPV18 E6-E7 fusion antigen; the nucleotide sequence encoding the HPV16 E6-E7 fusion antigen comprises a nucleotide sequence encoding SEQ ID NO:2 or a nucleotide sequence that is at least 95% identical to a nucleotide sequence encoding SEQ ID NO:2; 2. The composition for use according to claim 1, wherein the nucleotide sequence encoding the HPV18 E6-E7 fusion antigen comprises a nucleotide sequence encoding SEQ ID NO:10 or a nucleotide sequence that is at least 95% identical to a nucleotide sequence encoding SEQ ID NO:

10.

27. The composition for use according to claim 1 , wherein the method comprises electroporation.

28. The composition for use according to claim 1 , wherein the method comprises intramuscular administration.

29. 10. The composition for use according to claim 1, wherein the method comprises administering the composition in three or four doses.

30. 30. The composition for use according to claim 29, wherein the second dose of the composition is administered about 4 weeks after the first dose.

31. 30. The composition for use according to claim 29, wherein the third dose is administered about 12 weeks after the first dose.

32. 30. The composition for use according to claim 29, wherein the fourth dose is administered about 40 weeks after the first dose.

33. 20. The composition for use according to claim 19, wherein the leader sequence comprises the amino acid sequence of SEQ ID NO:

7.

34. 20. The method of claim 19, wherein the nucleic acid sequence encoding the leader sequence comprises the nucleotide sequence of SEQ ID NO:11.

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