HPV antigenic composition and uses thereof

Abstract

Disclosed herein is an immunotherapy for human papillomavirus. More particular, disclosed is a therapeutic composition that includes one or more polypeptides that comprise a plurality of epitopes derived from multiple human papillomavirus antigens, which, when used in immunotherapy is capable of eliciting a protective and durable cell mediated immune response, without being limited thereto. Also disclosed are methods of treating or preventing HPV and / or cancer.

Description

TITLE OF THE INVENTION HPV ANTIGENICCOMPOSITION ANDUSESTHEREOF RELATED APPLICATIONS

[0001] This application claims priority to Australian Provisional Application No.2024902692 entitled “HPV Antigenic Composition And Uses Thereof” filed 28 August 2024, the contents of which are incorporated herein by reference in their entirety. FIELD OF THE INVENTION

[0002] The present invention relates to an immunotherapy for human papillomavirus. In particular, the invention relates to a vaccine composition that includes one or more polypeptides that comprise a plurality of epitopes derived from multiple human papillomavirus antigens, which, when used in immunotherapy is capable of eliciting a protective and durable cell mediated immune response, without being limited thereto. BACKGROUND

[0003] Human papillomaviruses (HPVs) are a family of double strand DNA viruses comprising more than 150 types [1]. These viruses contain double-stranded DNA with 8000 bp arranged in 8 well defined genes. Six early genes are involved in virus replication and two late genes are involved in virus assembly. Selective infection of cutaneous or mucosal epithelia is a classic feature of HPVs, and their replication is closely linked to the differentiation of the epithelium [2, 3]. Most common high-risk HPV serotype 16 (HPV-16) and HPV serotype 11 (HPV-11) are responsible for 90% of genital warts and recurrent respiratory papilloma’s [1]. HPV-16 and HPV-18 account for the majority of cancers of the cervix, anus, vagina, vulva, penis, tongue, larynx, and tonsil [4]. Recent United States population-based studies conducted by the Centre for Disease Control show that 66% of cervical cancers, 55% of vaginal cancers, 79% of anal cancers, and 62% of oropharyngeal cancers are attributable to HPV 16 or 18 [5]. Globally, HPV infection accounts for an estimated 530,000 cervical cancer cases (-270,000 deaths) annually, with the majority (86% of cases, 88% of deaths) occurring in developing countries [4-6]. In total, HPV accounts for 5.2% of the worldwide cancer burden. Each year in the United States, an estimated 26,000 new cancers are attributable to HPV, about 17,000 in women and 9,000 in men [4]. The incidence of HPV-associated cervical cancer is significantly higher in Hispanic, black and American Indian / Alaskan Native women when compared to Caucasians [7]. HPV-associated vaginal cancers are slightly more frequent among black individuals, and vulvar cancers are more frequent among Caucasians [8]. HPV-associated oropharyngeal cancers have been increasing in frequency among both sexes, more among males than females, as wellas among most racial / ethnic groups, with the exception of black individuals [9]. HPV-associated anal cancers have been increasing among males and females across all racial / ethnic groups [4].

[0004] Standard therapeutic options for HPV-associated lesions include physical elimination by cryotherapy (i.e. using extreme cold to destroy tissue), chemical cauterization (i.e. using a chemical to destroy tissue), and laser or surgical removal

[0010] . However, surgical procedures alone are not very effective in treating pre-cancerous lesions, since recurrences occur at rates of 20-30% or more with lesions both at previously treated sites due to failure of the procedure to eliminate the HPV, and at new sites due to new infections

[0010] . When this occurs, radiotherapy and chemotherapy are then used with relative success, however about 50% of the HPV-associated cancer patients still die of the disease [11, 12]. Clearly, new treatment strategies are urgently needed to control the burden of HPV-related cancer. The fact that most HPV infections are cleared spontaneously shows that the immune system can effectively eliminate virus-infected cells. But in the case of persistent infections, the immune system has clearly failed. Uncleared HPV-16 infections are considered to be a major risk factor for the development of epithelium cancers with suboptimal survival in patients having the worst prognosis of advanced, recurrent or metastatic disease, despite the availability of various treatment modalities.

[0005] A study in The Cancer Genome Atlas Program analyzed RNA-Seq data from tumors of head and neck cancer (HNC) patients which suggested that more than 70% of tumors contain HPV16 genome in mixed form (integrated and episomal). This discovery unlocks the possibility of the presence of other HPV antigens apart from E6 and E7 antigens to target particularly the patients with mixed viral genome status [13,14]. Recent studies have demonstrated that HPV-specific T cell responses from HNC patients were not constrained to the E6 and E7 antigens, but also recognized E1, E2, E4, E5 and L1 proteins as dominant targets for virus-specific CD8+and CD4+T cells [15, 16]. SUMMARY OF THE INVENTION

[0006] Provided herein are compositions and methods related to development of HPV- specific prophylactic and / or therapeutic immunotherapy based on T cell epitopes (e.g., HPV epitopes listed in Table 1) that are recognized by cytotoxic T lymphocytes (CTLs) and can be employed in the prevention and / or therapeutic treatment of HPV infection, and / or cancer (e.g., a cancer expressing a HPV antigen provided herein), and / or precancerous legions.

[0007] In one aspect, the present invention provides an isolated polypeptide comprising a plurality of HLA class I-restricted cytotoxic T lymphocyte (CTL) epitopes selected from the T cell epitope amino acid sequence set forth in SEQ ID NOs: 1-35, wherein the epitopes are from two or more antigens from human papillomavirus (HPV).

[0008] In some embodiments, the HPV serotype is HPV-16. In some embodiments of this type, the HPV serotype is only HPV-16.

[0009] In some embodiments, the epitopes are restricted by HLA class I specificities HLA- A1, -A2, -A3, -A11, -A23, -A24, -A26, -A29, -A32, -A68, -B2, -B7, -B8, -B15, -B18, -B27, -B35, -B40, -B44, -B53, -B57, -B62, -B68, -C5, -C7, and / or -C8. In some preferred embodiments, the HLA class I specificities include HLA-A1, -A2, -A24, -B8, and -B35.

[0010] In some embodiments, the CTL epitopes are derived from antigens selected from E1, E2, E4, E5, E6 and / or E7.

[0011] In some embodiments, the polypeptide comprises the CTL epitopes selected from the amino acid sequences set forth in SEQ ID NO: 1-3, 5-20, 35.

[0012] In some embodiments, the isolated polypeptide comprises an intervening amino acid sequence between each of the epitopes, wherein the intervening amino acid sequence comprises a proteasome liberation amino acid sequence.

[0013] In some embodiments, the proteasome liberation amino acids or amino acid sequences comprise AD, K and / or R.

[0014] In another aspect, the present invention provides a polynucleotide that encodes the polypeptide described above and / or elsewhere herein.

[0015] In some embodiments, the polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO: 40.

[0016] In some alternative embodiments, the polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO: 38.

[0017] In yet another aspect, the present invention provides a genetic construct comprising the polynucleotide described above and / or elsewhere herein.

[0018] In still yet another aspect, the invention includes an expression vector comprising the polynucleotide described above and / or elsewhere herein.

[0019] In some embodiments, the expression vector is a viral vector. In some embodiments of this type, the viral vector is an adenovirus-based expression vector.

[0020] In still yet another aspect, the present invention comprises a cell comprising an isolated nucleic acid described above and / or elsewhere herein, or an expression vector as described above and / or elsewhere herein.

[0021] In some embodiments, the cell is a host cell that comprises the isolated nucleic acid, genetic construct, and / or an expression vector as described above or elsewhere herein.

[0022] In some specific embodiments, the host cell is or comprises a T cell.

[0023] In still yet another aspect, the present invention provides a pharmaceutical composition comprising the isolated polypeptides described above and / or elsewhere herein, and a pharmaceutically acceptable carrier, diluent or excipient.

[0024] In some embodiments of this type, the pharmaceutical composition further comprisies an immunostimulatory molecule or adjuvant.

[0025] In another aspect, the invention provides a vaccine that comprises the pharmaceutical composition described above and / or elsewhere herein for eliciting a protective immune response against a human papillomavirus in a subject.

[0026] In some embodiments, the human papillomavirus is HPV-16.

[0027] In some embodiments, the pharmaceutical composition or vaccine is for use in the treatment or prevention of an HPV-associated cancer in subject.

[0028] In some embodiments, the cancer is cervical cancer, vaginal cancer, anal cancer or oropharyngeal cancer. In some embodiments, the cancer is HPV-16 positive.

[0029] In still yet another aspect, the invention provides a polypeptide encoded by the nucleic acid sequence set forth in SEQ ID NO: 40. In a related aspect, the invention provides a cell comprising the polypeptide encoded by the nucleic acid sequence set forth in SEQ ID NO: 40.

[0030] In still another aspect, the invention provides a messenger RNA (mRNA) composition for eliciting an immune response to an HPV antigen, the mRNA comprising an open reading frame (ORF) encoding a plurality of epitopes from at least two HPV antigens.

[0031] In some embodiments, further comprising one or more of a 5’ cap, a 3’ UTR, and a poly(A) tail.

[0032] In some embodiments, the present invention further comprises one or both of an optimised codon and a chemical modification when compared to a corresponding mRNA that does notcomprise the optimised codon and / or chemical modification. In some embodiments of this typie, the chemical modification and / or the optimised codon increases mRNA stability and / or mRNA translation in a mammalian cell when compared to a mRNA without the chemical modification and / or the optimized codon.

[0033] In some embodiments, the HPV serotype is HPV-16. In some embodiments of this type, the HPV serotype is only HPV-16.

[0034] In some embodiments, the epitopes are restricted by HLA class I specificities HLA- A1, -A2, -A3, -A11, -A23, -A24, -A26, -A29, -A32, -A68, -B2, -B7, -B8, -B15, -B18, -B27, -B35, -B40, -B44, -B53, -B57, -B62, -B68, -C5, -C7, and / or -C8. In some preferred embodiments, the HLA class I specificities include HLA-A1, -A2, -A24, -B8, and -B35. BREIF DESCRIPTION OF THE FIGURES

[0035] Figure 1 shows HLA coverage in Europe, United States of America and world-wide of HPV-16 CD8+T cell epitopes included in HPVpoly-1, HPVpoly-2, HPVpoly-3, HPVpoly-4, and HPVpoly- 5.

[0036] Figure 2 shows the agarose gel image showing Pac-1 digest of AdHPVpoly-1, AdHPVpoly-2, AdHPVpoly-3, AdHPVpoly-4, and AdHPVpoly-5.

[0037] Figure 3 is a schematic outline for the construction of AdHPVpoly vectors. Synthetic DNA sequences encoding polyepitope proteins containing HLA class I-restricted T cell epitopes from HPV were cloned into a pShuttle vector and then subcloned into the Ad5F35 expression vector. The recombinant Ad5F35 vector was packaged into infectious adenovirus by transfecting HEK 293 cells, and recombinant adenovirus was harvested from transfected cells by repeated freeze-thawing cycles.

[0038] Figure 4 shows frequency of IFNγ+, TNFα+, IL-2+and CD107a+CD8+T cells following simulation of HLA A*02:01-restricted TIHDIILECV (HPV E6) and TLQDVSLEVYL (HPV E2) and HLA A*01:01-restricted QVDYYGLYY (HPV E2) HPV-specific TCR transgenic T cells with HLA matched fibroblast infected with AdHPVpoly-1, AdHPVpoly-2, AdHPVpoly-3, AdHPVpoly-4, and AdHPVpoly-5.

[0039] Figure 5 illustrates in vitro expansion of HPV-specific T cells from three HPV+HNC patients following stimulation with AdHPVpoly-1. Frequency of IFNγ+and TNF+CD8+T cells were assessed using intracellular cytokine assay.

[0040] Figure 6 shows priming of HPV-specific T cells following immunization with AdHPVpoly-1. (A & B) Ex vivo polyfunctional HPV-specific CD8+T cell responses in HLA A1, A2, A24, B8 and B35 transgenic mice in blood and spleen. (C) In vitro expansion of polyfunctional HPV-specific CD8+T cell following stimulation with HLA matched HPV T cell epitopes. Stacked bar graph showing percentageof HPV-specific CD8+T cell expressing IFNγ, TNF, IL-2 and IFNγ+TNF. T cell specificity was assessed using an intracellular cytokine assay.

[0041] Figure 7 illustrates priming of HPV-specific T cells following immunization with AdHPVpoly-2. (A & B) Ex vivo polyfunctional HPV-specific CD8+T cell responses in HLA A1, A2, A24, B8 and B35 transgenic mice in blood and spleen. (C) In vitro expansion of polyfunctional HPV-specific CD8+T cell following stimulation with HLA matched HPV T cell epitopes. Stacked bar graph showing percentage of HPV-specific CD8+T cell expressing IFNγ, TNF, IL-2 and IFNγ+TNF. T cell specificity was assessed using an intracellular cytokine assay.

[0042] Figure 8 shows priming of HPV-specific T cells following immunization with AdHPVpoly-3. (A & B) Ex vivo polyfunctional HPV-specific CD8+T cell responses in HLA A1, A2, A24, B8 and B35 transgenic mice in blood and spleen. (C) In vitro expansion of polyfunctional HPV-specific CD8+T cell following stimulation with HLA matched HPV T cell epitopes. Stacked bar graph showing percentage of HPV-specific CD8+T cell expressing IFNγ, TNF, IL-2 and IFNγ+TNF. T cell specificity was assessed using an intracellular cytokine assay.

[0043] Figure 9 illustrates priming of HPV-specific T cells following immunization with AdHPVpoly-4. (A & B) Ex vivo polyfunctional HPV-specific CD8+T cell responses in HLA A1, A2, A24, B8 and B35 transgenic mice in blood and spleen. (C) In vitro expansion of polyfunctional HPV-specific CD8+T cell following stimulation with HLA matched HPV T cell epitopes. Stacked bar graph showing percentage of HPV-specific CD8+T cell expressing IFNγ, TNF, IL-2 and IFNγ+TNF. T cell specificity was assessed using an intracellular cytokine assay.

[0044] Figure 10 shows priming of HPV-specific T cells following immunization with AdHPVpoly-5. (A & B) Ex vivo polyfunctional HPV-specific CD8+T cell responses in HLA A1, A2, A24, B8 and B35 transgenic mice in blood and spleen. (C) In vitro expansion of polyfunctional HPV-specific CD8+T cell following stimulation with HLA matched HPV T cell epitopes. Stacked bar graph showing percentage of HPV-specific CD8+T cell expressing IFNγ, TNF, IL-2 and IFNγ+TNF. T cell specificity was assessed using an intracellular cytokine assay.

[0045] Figures 11-13 shows priming of HPV-specific T cells following immunization with mRNA HPVpoly-3 and / or HPVpoly-5. (A & B) Ex vivo polyfunctional HPV-specific CD8+T cell responses in HLA A1, A2, A24, B8 and B35 transgenic mice in blood and spleen. (C) In vitro expansion of polyfunctional HPV-specific CD8+T cell following stimulation with HLA matched HPV T cell epitopes. Stacked bar graph showing percentage of HPV-specific CD8+T cell expressing IFNγ, TNF, IL-2 and IFNγ+TNF. T cell specificity was assessed using an intracellular cytokine assay.DETAILED DESCRIPTION OF THE INVENTION Definitions

[0046] For convenience, certain terms employed in the specification, examples, and appended claims are collected here.

[0047] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0048] As used herein, the term “administering” means providing a pharmaceutical agent or composition to a subject, and includes, but is not limited to, administering by a medical professional and self-administering. Such an agent can contain, for example, peptide described herein, an antigen presenting cell provided herein and / or a CTL provided herein.

[0049] The term “amino acid” is intended to embrace all molecules, whether natural or synthetic, which include both an amino functionality and an acid functionality and capable of being included in a polymer of naturally occurring amino acids. Exemplary amino acids include naturally occurring amino acids; analogs, derivatives and congeners thereof; amino acid analogs having variant side chains; and all stereoisomers of any of the foregoing.

[0050] As used herein, the term “antibody” may refer to both an intact antibody and an antigen binding fragment thereof. Intact antibodies are glycoproteins that include at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain includes a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain includes a light chain variable region (abbreviated herein as VL) and a light chain constant region. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The term “antibody” includes, for example, monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies and antigen-binding antibody fragments.

[0051] The terms “antigen-binding fragment” and “antigen-binding portion” of an antibody, as used herein, refers to one or more fragments of an antibody that retain the ability to bind to an antigen. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibodyinclude Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabodies, single-chain antibodies, camelid antibodies, isolated CDRH3, a Designed Ankyrin Repeat Protein (DARPin) and other antibody fragments that retain at least a portion of the variable region of an intact antibody. These antibody fragments can be obtained using conventional recombinant and / or enzymatic techniques and can be screened for antigen binding in the same manner as intact antibodies.

[0052] The term “binding” or “interacting” refers to an association, which may be a stable association, between two molecules, e.g., between a peptide and a binding partner or agent, e.g., small molecule, due to, for example, electrostatic, hydrophobic, ionic and / or hydrogen-bond interactions under physiological conditions.

[0053] The term “biological sample”, “tissue sample”, or simply “sample” each refers to a collection of cells obtained from a tissue of a subject. The source of the tissue sample may be solid tissue, as from a fresh, frozen and / or preserved organ, tissue sample, biopsy, or aspirate; blood or any blood constituents, serum, blood; bodily fluids such as cerebral spinal fluid, amniotic fluid, peritoneal fluid or interstitial fluid, urine, saliva, stool, tears; or cells from any time in gestation or development of the subject.

[0054] As used herein, the term “cancer” includes, but is not limited to, solid tumors and blood borne tumors. The term cancer includes diseases of the skin, tissues, organs, bone, cartilage, blood, and vessels, including the cervix, anus, vagina, vulva, penis, tongue base, larynx, and tonsil. The term “cancer” further encompasses primary and metastatic cancers.

[0055] The term “precancerous lesions” or “precancerous condition” refers to atypical cells and / or tissues that are associated with an increased risk of cancer. The term “precancerous lesions” may refer, for example, to dysplasia, benign neoplasia, or carcinoma in situ.

[0056] The term “epitope” means a protein determinant capable of specific binding to an antibody. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains. Certain epitopes can be defined by a particular sequence of amino acids to which a T cell receptor or antibody is capable of binding.

[0057] The term “isolated nucleic acid” refers to a polynucleotide of natural or synthetic origin or some combination thereof, which (1) is not associated with the cell in which the “isolated nucleic acid” is found in nature, and / or (2) is operably linked to a polynucleotide to which it is not linked in nature.

[0058] The term “isolated polypeptide” refers to a polypeptide, in certain embodiments prepared from recombinant DNA or RNA, or of synthetic origin, or some combination thereof, which (1) is not associated with proteins that it is normally found with in nature, (2) is isolated from the cell in which it normally occurs, (3) is isolated free of other proteins from the same cellular source, (4) is expressed by a cell from a different species, or (5) does not occur in nature.

[0059] As used herein, the term “lipid nanoparticle” or “LNP” shall be understood to refer to lipid-based particles having at least one dimension in the order of nanometers (e.g., 1-1,000 nm) and which typically comprises a mRNA described herein.

[0060] As used herein, the phrase “pharmaceutically acceptable” refers to those agents, compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0061] As used herein, the phrase “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting an agent from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffered solutions; polyesters, polycarbonates and / or polyanhydrides; and other non-toxic compatible substances employed in pharmaceutical formulations.

[0062] The terms “polynucleotide”, and “nucleic acid” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure and may perform any function. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. A polynucleotide may be further modified, such as by conjugation with a labeling component. In all nucleic acid sequences provided herein, U nucleotides are interchangeable with T nucleotides.

[0063] As used herein, a therapeutic that “prevents” a condition refers to a compound that, when administered to a statistical sample prior to the onset of the disorder or condition, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition relative to the untreated control sample.

[0064] As used herein, “specific binding” refers to the ability of an antibody to bind to a predetermined antigen or the ability of a peptide to bind to its predetermined binding partner. Typically, an antibody or peptide specifically binds to its predetermined antigen or binding partner with an affinity corresponding to a KD of about 107M or less, and binds to the predetermined antigen / binding partner with an affinity (as expressed by KD) that is at least 10 fold less, at least 100 fold less or at least 1000 fold less than its affinity for binding to a non-specific and unrelated antigen / binding partner (e.g., BSA, casein).

[0065] As used herein, the term “subject” means a human or non-human animal selected for treatment or therapy.

[0066] The phrases "therapeutically effective amount" and “effective amount” as used herein means the amount of an agent which is effective for producing the desired prophylactic and / or therapeutic effect in at least a sub-population of cells in a subject at a reasonable benefit / risk ratio applicable to any medical treatment.

[0067] As used herein, “treating” (or “treat” or “treatment”) refers to a therapeutic intervention that ameliorates a sign or symptom of a HPV infection, inclusive of a HPV-associated disease, disorder or condition, after it has begun to develop. The term “ameliorating”, with reference to a HPV-associated disease, disorder or condition, refers to any observable beneficial effect of the treatment. Treatment need not be absolute to be beneficial to the subject. The beneficial effect can be determined using any methods or standards known to the ordinarily skilled artisan.

[0068] The term “vector” refers to the means by which a nucleic acid can be propagated and / or transferred between organisms, cells, or cellular components. Vectors include plasmids, viruses, bacteriophage, pro-viruses, phagemids, transposons, and artificial chromosomes, and the like, that may or may not be able to replicate autonomously or integrate into a chromosome of a host cell. General

[0069] While innate immune responses play an important role in controlling initial HPV infection, long-term protection is dependent on adaptive immune responses including humoral and cell- mediated immunity. In immunocompetent individuals, the majority of HPV infections are cleared within two years of initial infection. Infiltration of CD4+and CD8+T cells is frequently observed in spontaneously regressing lesions.

[0070] A number of immunotherapeutic strategies have been tested for the treatment of HPV-associated diseases. While the HPV prophylactic vaccine is based on L1 protein, this viral antigen is not relevant for the treatment of HPV-associated diseases. This protein is only expressed in the late stages of HPV replication, especially in terminally differentiated keratinocytes. In contrast, other proteins associated with the HPV replicative cycle, i.e., E1, E2, E6 and E7, have been identified as important targets for immunotherapeutic strategies. This is primarily due to the fact that the expression of all these proteins are retained through multiple stages of infection. While much of the emphasis on the design of immunotherapeutic strategies has focused on E6 and E7 antigens, it is important to appreciate that E1 and E2 proteins are implicated in HPV DNA replication and thus the expression of these proteins is retained throughout multiple stages of infection. This highlights the importance of these proteins as potential targets for immunotherapy aimed at eliminating persistently HPV-infected cells regardless of the stage of pathogenesis. Indeed, previous studies using animal models (canine and rabbit) have shown that immunization with a DNA vaccine encoding codon-optimized E1 or E2 genes results in complete regression of papillomas. The primary mode of protection in these animal models is mediated through the induction of an effective T cell response to E1 and E2 antigens. Further clinical studies using a modified vaccinia Ankara vector encoding E2 in human subjects with HPV-induced cervical lesions (C1N1 to C1N3) demonstrated complete elimination of cervical lesions to regression from C1N3 to C1N1 and significant reduction in HPV viral load. Here again, the induction of E2-specific T cell immunity correlated strongly with clinical response.

[0071] Development of anti-vector antibodies resulted in a poor response to booster immunization and some patients showed recurrence of lesions after the completion of the study. Moreover, this therapy required direct injection of the vector into uterine tissue to be effective, thus limiting its wider use in the general population.

[0072] Retrospective clinical studies have been done on patients who had high-grade intraepithelial lesions that regressed to cleared cervical intraepithelial neoplasia lesions, and subsequently exhibited reduced HPV viral load. HLA class II-restricted CD4+T cell epitopes from HPV E7 protein were identified in these patients, and immune profiling of their peripheral blood mononuclear cells revealed that the HPV E7-specific T cells displayed a Th1 bias, characterized by IFNγ and TNF expression. Indeed, ex vivo analysis in patients showing regression of HPV-driven pathology post-intervention without disease recurrence revealed that a strong T cell response was directed towards the E6 and E7 proteins. In contrast, T cells from patients who were diagnosed with recurrent disease did not show this antigen specificity profile. Reconstitution of robust T cell immunity against the E6 and E7 antigens may help confer long-term protection from disease recurrence.

[0073] Provided herein are compositions and methods related to HPV epitopes (e.g., HPV epitopes listed in Table 1) that are recognized by cytotoxic T lymphocytes (CTLs) and that are useful in the prevention and / or treatment of HPV infection, and / or cancer, and / or precancerous lesions. In certainaspects, provided herein are compositions (e.g., prophylactic and / or therapeutic compositions, such as vaccine compositions) containing a polypeptide comprising one or more of the HPV epitopes described herein (e.g., HPV epitopes listed in Table 1), nucleic acids encoding such a polypeptide, CTLs that recognize such a peptide, APCs presenting such peptides and / or antigen-binding molecules that bind specifically to such peptides, as well as methods of treating and / or preventing HPV infection, and / or cancer, and / or precancerous lesions by administering such compositions to a subject. In some embodiments, also provided herein are methods of identifying a subject suitable for treatment according to a method provided herein. Peptides

[0074] Provided herein are polypeptides comprising HPV epitopes that are recognized by CTLs and that are useful in the prevention and / or treatment of HPV infection, and / or cancer (e.g., a cancer expressing an HPV epitope provided herein), and / or precancerous lesions.

[0075] The HPV epitopes of the present invention were selected based on their MHC allele restriction, in order to provide the broadest HLA allele coverage. The polypeptide comprising the multiple epitopes derived from two or more HPV antigens, target 24-48 HLA class I alleles, covering 94-99% of the multi-ethnic population worldwide (see, Figure 1). This broad MHC allele coverage allows the polypeptide therapy to be applicable to whole populations, and thus effective as an “off-the-shelf” allogeneic immunotherapy. In certain embodiments, the HPV epitopes are listed in Table 1. TABLE 1 LIST OFEXEMPLARYHLA CLASSI RESTRICTEDCD8+T CELLEPITOPESGIVCPICSQK HPV-16-E7 A*11:01 19 TLQDVSLEVYL HPV-16-E2 A*02:01 20 HLA B*35:01, HLA C*07:02, HLA MVYDFAFRDLCIVY HPV16-E6 A*24:02 HLA A*26:01 HLA B*53:01 HLA[ ] n some em o mens, e poypep es prov e eren are u eng proeins. In some embodiments, the polypeptides provided herein comprise less than 100, 90, 80, 70, 60, 50, 40, 30, 25, 20, 15 or 10 contiguous amino acids of the HPV viral protein. In some embodiments, the polypeptides provided herein comprise two or more of the HPV epitopes listed in Table 1. For example, in some embodiments, the polypeptide provided herein comprises two or more of the HPV epitopes listed in Table 1 connected by polypeptide linkers. In some embodiments, the polypeptide provided herein comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25 or 30 of the epitopes listed in Table 1.

[0077] In some embodiments, the polypeptide provided herein consists of an epitope listed in Table 1. In some embodiments, the polypeptide provided herein consists essentially of an epitope listed in Table 1. In some embodiments, the polypeptide provided herein comprises no more than 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acids in addition to the epitopes listed in Table 1. The preferred polypeptides of the invention comprising the selection of HPV epitope are listed in Tables 2-6 below (i.e., HPVpoly-1, HPVpoly-2, HPVpoly-3, HPVpoly-4 and HPVpoly-5). TABLE 2 LIST OF HLA CLASS I RESTRICTED CD8+T CELL EPITOPES INCLUDED IN HPVPOLY-1VYDFAFRDL HPV-16-E6 A*24 4 LLIRCINCQK HPV-16-E6 A*03:01 / A*11:01 5 TTLEQQYNK HPV-16-E6 A*03:01 6 I I ILIST OFHLA CLASSI RESTRICTEDCD8+T CELLEPITOPESINCLUDED INHPVPOLY-2 Epitope Sequence Antigen HLA Restriction SEQ ID NO:SAFRCFIVY HPV16-E5 HLA B*35:01 / B*35:43 14 HNIRGRWTGRCM HPV16-E6 HLA B*27:05, HLA A*68 32 TLQDVSLEVYL HPV16-E2 HLA A*02:01 20LIST OF HLA CLASS I RESTRICTED CD8+T CELL EPITOPES INCLUDED IN HPVPOLY-3 Epitope Sequence Antigen HLA Restriction SEQ ID NO: MVYDFAFRDLCIVY HPV16-E6 HLA A*24:02, HLA B*35:01 21 *TABLE 5 LIST OF HLA CLASS I RESTRICTED CD8+T CELL EPITOPES INCLUDED IN HPVPOLY-4 Epitope Sequence Antigen HLA Restriction SEQ ID NO: MVYDFAFRDL HPV16-E6 HLA A*24:02 35YLHNRLVVF HPV16-E1 HLA B*08:01 17 IILECVYCK HPV16-E6 HLA A*11:01 18 GIVCPICSQK HPV16-E7 HLA A*11:01 19LIST OFHLACLASSIRESTRICTEDCD8+TCELL EPITOPES INCLUDED INHPVPOLY-5 Epitope Sequence Antigen HLA Restriction SEQ ID NO: *

[0078] In some embodiments, the sequence of the polypeptides comprise an HPV viral protein sequence except for one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) conservative sequence modifications. As used herein, the term “conservative sequence modifications” is intended to refer to amino acid modifications that do not significantly affect or alter the interaction between a TCR and a peptide containing the amino acid sequence presented on an MHC. Such conservative modifications include amino acid substitutions, additions (e.g., additions of amino acids to the N or C terminus of the peptide) and deletions (e.g., deletions of amino acids from the N or C terminus of the peptide). Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues of the peptides described herein can be replaced with other amino acid residues from the same side chain family and the altered peptide can be tested for retention of TCR binding using methods known in the art. Modifications can be introduced into an antibody by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.

[0079] To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non- identical sequences can be disregarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[0080] Also provided herein are chimeric or fusion proteins. As used herein, a “chimeric protein” or “fusion protein” comprises a peptide(s) provided herein (e.g., those comprising an epitope listed in Table 1) linked to a distinct peptide to which it is not linked in nature. For example, the distinct peptide can be fused to the N-terminus or C-terminus of the peptide either directly, through a peptide bond, or indirectly through a chemical linker. In some embodiments, the peptide provided herein is linked to polypeptides comprising other HPV epitopes. In some embodiments, the peptide provided herein is linked to peptides comprising epitopes from other viral and / or infectious diseases. In some embodiments, the peptide provided herein is linked to a peptide encoding a cancer-associated epitope.

[0081] A chimeric or fusion peptide provided herein can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different peptide sequences are ligated together in-frame in accordance with conventional techniques, for example by employing blunt- ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and re-amplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons: 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety.

[0082] In some aspects, provided herein are cells that present a peptide described herein (e.g., a peptide comprising a plurality of epitopes listed in Table 1). In some embodiments, the cell is a mammalian cell. In some embodiments the cell is an antigen presenting cell (APC) (e.g., an antigen presenting T cell, a dendritic cell, a B cell, a macrophage or an artificial antigen presenting cell, such as a K562 cell). A cell presenting a peptide described herein can be produced by standard techniques known in the art. For example, a cell may be pulsed to encourage peptide uptake. In some embodiments, the cells are transfected with a nucleic acid encoding a peptide provided herein. In some aspects, provided herein are methods of producing antigen presenting cells (APCs), comprising pulsing a cell with the peptides described herein. Exemplary examples of producing antigen presenting cells can be found in International PCT Patent Publication No. WO 2013 / 088114, hereby incorporated in its entirety.

[0083] The peptides provided herein can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques, can be produced by recombinant DNA techniques, and / or can be chemically synthesized using standard peptide synthesis techniques. The peptides described herein can be produced in prokaryotic or eukaryotic host cells by expression of nucleotides encoding a peptide(s) of the present invention. Alternatively, such peptides can be synthesized by chemical methods. Methods for expression of heterologous peptides in recombinant hosts, chemical synthesis of peptides, and in vitro translation are well known in the art and are described further in Maniatis et al., Molecular Cloning: A Laboratory Manual (1989), 2nd Ed., Cold Spring Harbor, N. Y.; Berger and Kimmel, Methods in Enzymology, Volume 152, Guide to Molecular Cloning Techniques (1987), Academic Press, Inc., San Diego, Calif.; Merrifield, J. (1969) J. Am. Chem. Soc.91 :501; Chaiken I. M. (1981) CRC Crit. Rev. Biochem.11 :255; Kaiser et al. (1989) Science 243: 187; Merrifield, B. (1986) Science 232:342; Kent, S. B. H. (1988) Annu. Rev. Biochem. 57:957; and Offord, R. E. (1980) Semisynthetic Proteins, Wiley Publishing, which are incorporated herein by reference.Nucleic Acid Molecules

[0084] Provided herein are nucleic acid molecules that encode the polypeptides described herein. In some aspects, provided herein are methods of treating cancer, precancerous lesions, or HPV by administering to a subject the nucleic acids disclosed herein. The nucleic acids may be present, for example, in whole cells, in a cell lysate, or in a partially purified or substantially pure form.

[0085] In some embodiments, provided herein are vectors (e.g., a viral vector, such as an adenovirus-based expression vector) that contain the nucleic acid molecules described herein. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid” which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication, episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby be replicated along with the host genome.

[0086] Moreover, certain vectors are capable of directing the expression of genes. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”). In some embodiments, provided herein are nucleic acids operable linked to one or more regulatory sequences (e.g., a promoter) in an expression vector. In some embodiments the cell transcribes the nucleic acid provided herein and thereby expresses an antibody, antigen binding fragment thereof or peptide described herein. The nucleic acid molecule can be integrated into the genome of the cell or it can be extrachromosomal.

[0087] In some embodiments, the nucleic acid vectors or recombinant adenoviruses provided herein consist of two or more epitopes from at least two different viral epitopes listed in Table 1. In some embodiments, the nucleic acid vectors or recombinant adenoviruses provided herein encoded for essentially an epitope listed in Table 1. In some embodiments, the nucleic acid vectors or recombinant adenoviruses provided herein encode no more than 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acids in addition to the epitopes listed in Table 1. In some embodiments, the sequence of the T cell epitopes comprise an epitope sequence provided herein except for 1 or more (e.g., 1, 2, 3, 4 or 5) conservative sequence modifications.

[0088] In some embodiments, the nucleic acid vectors or recombinant adenoviruses comprise nucleic acid sequences that have undergone codon optimization. In such embodiments a coding sequence is constructed by varying the codons in each nucleic acid used to assemble the coding sequence. In general, a method to identify a nucleotide sequence that optimizes codon usages for production of apeptide comprises at least the following steps (a) through (e). In step (a), oligomers are provided encoding portions of the polypeptide containing degenerate forms of the codon for an amino acid encoded in the portions, with the oligomers extended to provide flanking coding sequences with overlapping sequences. In step (b), the oligomers are treated to effect assembly of the coding sequence for the peptide. The reassembled peptide is included in an expression system that is operably linked to control sequences to effect its expression. In step (c), the expression system is transfected into a culture of compatible host cells. In step (d), the colonies obtained from the transformed host cells are tested for levels of production of the polypeptide. In step (e), at least one colony with the highest or a satisfactory production of the polypeptide is obtained from the expression system. The sequence of the portion of the expression system that encodes the protein is determined. Further description of codon optimization is provided in U.S. Patent Publication number US2010 / 035768, which is incorporated by reference in its entirety. Exemplary codon-optimized nucleotide sequences are provided for HPVpoly-1, HPVpoly-2, HPVpoly-3, HPVpoly-4, and HPVpoly-5 below: HPVpoly-1 GCTAGCATGTTCGCCTTCCGCGACCTCTGCATCGTGTACTACATGCTGGACCTTCAGCCTGAGACTACTTGGCCCAC TACTCCTCCACGGCCCATCGTGTACGACTTCGCGTTCCGGGACCTCCTGTTGATTAGATGTATCAATTGCCAAAAGA CCACACTGGAGCAGCAGTACAACAAACAAGTGGATTACTACGGCTTGTACTACACCATCCACGACATCATCCTGGAA TGCGTGAGCGAGTACAGGCATTATTGTTACTCCCTGATCCACGATATCATTCTCGAATGCGTGTACATTTTCGTGTA CATCCCGCTGTTCCTGAACCTGGATACCGCATCGACCACGCTGAAGTCAGCCATTGTCACCCTGACCTATTCCGCCT TCCGGTGCTTTATTGTGTACGCTTTTCGCGACCTGTGCATCGTCTACGGAAGATGGACCGGACGGTGCATGTACCTC CACAACCGCCTCGTGGTGTTCATCATTCTTGAGTGCGTGTACTGCAAGGGGATCGTGTGCCCGATCTGTTCCCAAAA GACCCTGCAGGACGTCAGCCTGGAAGTCTACCTGTAATGAGGTACC [SEQ ID NO: 36] HPVpoly-2 GCTAGCATGGTGTACGATTTCGCGTTCCGGGATCTGTGTATCGTGTACTGGCCGACAACTCCGCCTAGACCGATTCA AGTGGACTACTACGGGCTGTATTACGACACCCCCACCCTTCATGAGTACATGCTGGATCTGCAGCCCGAAACCACCG ATCTGTACTGTTACGAGCTGCTCATCCGCTGCATCAACTGTCAGAAGCAGGCCGAACCTGACCGCGCCCACTACAAT ATTGTGACTTTCTGCTACCTCCACAACCGGCTCGTCGTGTTTACGATCCATGACATCATCCTGGAATGCGTGTACTG CAAGCGGACTCTCGAGGACCTCCTGATGGGCACCCTGGGAATTGTGTGCCCCATCTGCTCCCAAAAAGTCTGCGACA AGTGCCTGAAGTTCTACTCCAAAATCAGCGAGTACCGGCACTACTGCTATTCGCTGAAGTCCGCCATTGTGACCCTT ACCTATACCACCCTGGAACAGCAGTACAACAAGATTTTTGTCTACATTCCACTGTTCTTGAACCTCGACACTGCTTC CACCACCCTTGTGCTGCTGCTCTGGATCACTGCCGCATCAGCCTTCCGCTGCTTCATCGTGTACCACAACATCAGGG GCAGATGGACCGGACGCTGTATGACTCTGCAAGACGTCAGCCTGGAGGTGTACTTGCAAGAACGGCCGAGGAAGCTG CCTCAGCTGTGATAAGGTACC [SEQ ID NO: 37] HPVpoly-3GCTAGCATGGTGTACGACTTCGCCTTCCGCGACCTCTGCATCGTGTACTACATGCTGGACCTTCAGCCTGAGACTAC TTGGCCCACTACTCCTCCACGGCCCATCCTGCTGATGGGCACCCTCGGAATTGTGCTGTTGATTAGATGTATCAATT GCCAAAAGACCACACTGGAGCAGCAGTACAACAAACAAGTGGATTACTACGGCTTGTACTACACCATCCACGACATC ATCCTGGAATGCGTGAGCGAGTACAGGCATTATTGTTACTCCCTGATCCACGATATCATTCTCGAATGCGTGTACAT TTTCGTGTACATCCCGCTGTTCCTGAACCTGGATACCGCATCGACCACGCTGAAGTCAGCCATTGTCACCCTGACCT ATTCCGCCTTCCGGTGCTTTATTGTGTACGCTTTTCGCGACCTGTGCATCGTCTACGGAAGATGGACCGGACGGTGC ATGTACCTCCACAACCGCCTCGTGGTGTTCATCATTCTTGAGTGCGTGTACTGCAAGGGGATCGTGTGCCCGATCTG TTCCCAAAAGACCCTGCAGGACGTCAGCCTGGAAGTCTACCTGTAATAAGGTACC [SEQ ID NO: 38] HPVpoly-4 GCTAGCATGGTGTACGACTTCGCGTTTCGGGACCTTTACATGCTGGACCTCCAACCTGAGACTACTTGGCCCACTAC TCCCCCTCGCCCAATCTTCGCCTTCCGGGACCTCTGCATCGTGTACCTGTTGATTAGATGTATCAATTGCCAAAAGA CCACACTGGAGCAGCAGTACAACAAACAAGTGGATTACTACGGCTTGTACTACACCATCCACGACATCATCCTGGAA TGCGTGAGCGAGTACAGGCATTATTGTTACTCCCTGATCCACGATATCATTCTCGAATGCGTGTACATTTTCGTGTA CATCCCGCTGTTCCTGAACCTGGATACCGCATCGACCACGCTGAAGTCAGCCATTGTCACCCTGACCTATTCCGCCT TCCGGTGCTTTATTGTGTACGCTTTTCGCGACCTGTGCATCGTCTACGGAAGATGGACCGGACGGTGCATGTACCTC CACAACCGCCTCGTGGTGTTCATCATTCTTGAGTGCGTGTACTGCAAGGGGATCGTGTGCCCGATCTGTTCCCAAAA GACCCTGCAGGACGTCAGCCTGGAAGTCTACCTGTGATAAGGTACC [SEQ ID NO: 39] HPVpoly-5 GCTAGCATGGTGTACGACTTCGCCTTCCGCGATCTGAAGTACATGCTTGATCTGCAACCCGAAACCACTGCCGATTG GCCAACAACGCCCCCGAGGCCGATTAGATTCGCGTTCCGGGATCTGTGTATCGTGTACGCGGACCTGTTGATCCGGT GCATAAACTGCCAGAAGAGGACCACTCTGGAGCAGCAGTACAACAAGGCCGATCAGGTCGACTACTACGGATTGTAC TACAAGACCATCCATGACATCATTCTCGAATGCGTGCGCTCCGAGTATCGGCACTACTGCTACTCACTGGCTGACAT TCACGACATCATCCTTGAGTGTGTCTACAAGATCTTCGTCTACATCCCTCTGTTTCTGCGCAACCTGGACACCGCAA GCACCACCCTGAAGAAAAGCGCGATTGTCACTCTGACTTACGCCGACTCCGCTTTCCGGTGCTTCATTGTGTATCGG GCCTTTCGGGACCTGTGCATTGTGTACAAGGGTCGCTGGACCGGCCGCTGTATGGCCGACTACCTCCACAATCGGCT CGTGGTGTTCAAGATCATCCTGGAATGCGTGTACTGCAAGAGAGGGATCGTGTGCCCTATCTGCTCCCAAAAGAGAA CCCTGCAAGACGTGTCGCTCGAAGTGTACCTCGCCGACTGATAAGGTACC [SEQ ID NO: 40]

[0089] In some embodiments, the nucleic acid vectors, recombinant adenoviruses, mRNA compositions, or polyepitopes provided herein are part of a vaccine. In some embodiments, the vaccine is delivered to a subject in a vector, including, but not limited to, a bacterial vector and / or a viral vector. Examples of bacterial vectors include, but are not limited to, Mycobacterium bovis (BCG), Salmonella Typhimurium ssp., Salmonella Typhi ssp., Clostridium sp. spores, Escherichia coli Nissle 1917, Escherichia coli K-12 / LLO, Listeria monocytogenes, and Shigella flexneri. Examples of viral vectors include, but are not limited to, vaccinia, adenovirus, RNA viruses (replicons), and replication-defective like avipox, fowlpox, canarypox, MVA, and adenovirus.

[0090] In some embodiments, provided herein are cells that contain nucleic acid vectors or recombinant adenoviruses described herein. The cell can be, for example, prokaryotic, eukaryotic, mammalian, avian, murine and / or human. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell may be HEK 293 cells. In some embodiments, the cell is an APC (e.g., an antigen- presenting T cell, a dendritic cell, a B cell, or an aK562 cell). In the present methods, nucleic acid vectors or recombinant adenoviruses described herein can be administered to the cell, for example, as nucleic acid without delivery vehicle, in combination with a delivery reagent. In some embodiments, any nucleic acid delivery method known in the art can be used in the methods described herein. Suitable delivery reagents include, but are not limited to, e.g., the Minis Transit TKO lipophilic reagent; lipofectin; lipofectamine; cellfectin; polycations (e.g., polylysine), atelocollagen, nanoplexes and liposomes. In some embodiments of the methods described herein, liposomes are used to deliver a nucleic acid to a cell or subject. Liposomes suitable for use in the methods described herein can be formed from standard vesicle-forming lipids, which generally include neutral or negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of factors such as the desired liposome size and half-hie of the liposomes in the blood stream. A variety of methods are known for preparing liposomes, for example, as described in Szoka et al. (1980), Ann. Rev. Biophys. Bioeng 9:467; and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369, the entire disclosures of which are herein incorporated by reference. mRNA compositions

[0091] In some embodiments the present invention is an mRNA molecule that encodes a polypeptide that comprises a plurality of HPV epitopes from at least two antigens. In this regard, in some preferred embodiments, the mRNA molecule comprises, consists, or consists essentially of the nucleotide sequence set forth in any one of SEQ ID NOs: 36-40. In some alternative embodiments, the mRNA molecule comprises, consists, or consists essentially of a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ IDs 36-40.

[0092] In a particularly preferred embodiment, the mRNA molecule comprises, consists, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 38. In an alternative particularly preferred embodiment, the mRNA molecule comprises, consists, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 40.

[0093] In some embodiments, the mRNA composition comprises a coding sequence that encodes the polypeptide sequence set forth, below: HPVpoly-3MVYDFAFRDLCIVYYMLDLQPETTWPTTPPRPILLMGTLGIVLLIRCINCQKTTLEQQYNKQVDYY GLYYTIHDIILECVSEYRHYCYSLIHDIILECVYIFVYIPLFLNLDTASTTLKSAIVTLTYSAFRCFIVYA FRDLCIVYGRWTGRCMYLHNRLVVFIILECVYCKGIVCPICSQKTLQDVSLEVYL or HPVpoly-5 MVYDFAFRDLKYMLDLQPETTADWPTTPPRPIRFAFRDLCIVYADLLIRCINCQKRTTLEQQYNKA DQVDYYGLYYKTIHDIILECVRSEYRHYCYSLADIHDIILECVYKIFVYIPLFLRNLDTASTTLKKSAIV TLTYADSAFRCFIVYRAFRDLCIVYKGRWTGRCMADYLHNRLVVFKIILECVYCKRGIVCPICSQK RTLQDVSLEVYLAD

[0094] As used herein, the terms “poly(A) sequence,” “poly(A) tail,” and “poly(A) region” refer to a sequence of adenosine nucleotides at the 3′ end of the mRNA molecule. The poly(A) tail may confer stability to the mRNA and protect it from exonuclease degradation. The poly(A) tail may enhance translation. In some embodiments, the poly(A) tail is essentially homopolymeric. For example, a poly(A) tail of 100 adenosine nucleotides may have essentially a length of 100 nucleotides. In certain embodiments, the poly(A) tail may be interrupted by at least one nucleotide different from an adenosine nucleotide (e.g., a nucleotide that is not an adenosine nucleotide). For example, a poly(A) tail of 100 adenosine nucleotides may have a length of more than 100 nucleotides (comprising 100 adenosine nucleotides and at least one nucleotide, or a stretch of nucleotides, that are different from an adenosine nucleotide).

[0095] The “poly(A) tail,” as used herein, typically relates to RNA (e.g., mRNA). However, in the context of the disclosure, the term likewise relates to corresponding sequences in a DNA molecule (e.g., a “poly(T) sequence”).

[0096] The poly(A) tail may comprise about 10 to about 500 adenosine nucleotides, about 10 to about 200 adenosine nucleotides, about 40 to about 200 adenosine nucleotides, or about 40 to about 150 adenosine nucleotides. The length of the poly(A) tail may be at least about 10, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or 500 adenosine nucleotides.

[0097] In some embodiments, the poly(A) tail comprises the sequence: AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAA.

[0098] In some embodiments where the nucleic acid is an RNA, the poly(A) tail of the nucleic acid is obtained from a DNA template during RNA in vitro transcription. In certain embodiments, the poly(A)tail is obtained in vitro by common methods of chemical synthesis without being transcribed from a DNA template. In various embodiments, poly(A) tails are generated by enzymatic polyadenylation of the RNA (after RNA in vitro transcription) using commercially available polyadenylation kits and corresponding protocols, or alternatively, by using immobilized poly(A)polymerases (e.g., using methods and means as described in International PCT Patent Publication No. WO2016 / 174271).

[0099] The nucleic acid may comprise a poly(A) tail obtained by enzymatic polyadenylation, wherein the majority of nucleic acid molecules comprise about 100 (+ / -20) to about 500 (+ / -50) or about 250 (+ / -20) adenosine nucleotides.

[0100] In some embodiments, the nucleic acid may comprise a poly(A) tail derived from a template DNA and may additionally comprise at least one additional poly(A) tail generated by enzymatic polyadenylation, e.g., as described in International PCT Patent No. WO2016 / 091391.

[0101] In certain embodiments, the nucleic acid comprises at least one polyadenylation signal. In various embodiments, the nucleic acid may comprise at least one poly(C) sequence. The term “poly(C) sequence,” as used herein, is intended to be a sequence of cytosine nucleotides of up to about 200 cytosine nucleotides. In some embodiments, the poly(C) sequence comprises about 10 to about 200 cytosine nucleotides, about 10 to about 100 cytosine nucleotides, about 20 to about 70 cytosine nucleotides, about 20 to about 60 cytosine nucleotides, or about 10 to about 40 cytosine nucleotides. In some embodiments, the poly(C) sequence comprises about 30 cytosine nucleotides. Viral Vectors

[0102] The nucleic acid that encodes the HPV polyepitope polypeptides described above and / or may be encoded within a recombinant vector. The vectors can be used to deliver the mRNA that encodes the HPC polyepitope polypeptide. The vector may be a mammalian, a viral, or a bacterial expression vector.

[0103] The vectors may be, for example, a plasmid, an artificial chromosome (e.g., a BAG, PAC, or YAC), or a virus or phage vector, and may optionally include a promoter, enhancer, or regulator for the expression of the polynucleotide. The vector may also contain one or more selectable marker genes, for example an ampicillin, neomycin, and / or kanamycin resistance gene in the case of a bacterial plasmid or a resistance gene for a fungal vector. Vectors may be used in vitro, for example, for the production of DNA or RNA or used to transfect or transform a host cell, for example, a mammalian host cell (e.g., for the production of protein encoded by the vector). The vectors may also be adapted to be used in vivo, for example in a method of DNA vaccination, RNA vaccination, or gene therapy.

[0104] Viral genomes provide a rich source of vectors that can be used for the efficient delivery of the mRNA encoding the HPV polyepitope polypeptide into the genome of a cell (e.g., a eukaryoticor prokaryotic cell). Viral genomes are particularly useful vectors for gene delivery because the polynucleotides contained within such genomes are typically incorporated into the genome of a target cell by generalized or specialized transduction. These processes occur as part of the natural viral replication cycle, and do not require added proteins or reagents in order to induce gene integration. Examples of viral vectors that can be used to deliver the mRNA encoding the coronavirus antigen include a retrovirus, adenovirus (e.g., Ad2, Ad5, Ad11 , Ad12, Ad24, Ad26, Ad34, Ad35, Ad40, Ad48, Ad49, Ad50, Ad52 (e.g., a RhAd52), Ad59 (e.g., a RhAd59), and Pan9 (also known as AdC68)), parvovirus (e.g., adeno-associated viruses), negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g. measles and Sendai), positive strand RNA viruses, such as picornavirus and alphavirus, and double stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox and canarypox). Other viruses useful for delivering polynucleotides encoding immunogens (e.g., polypeptides) include Norwalk virus, togavirus, coronavirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example. Examples of retroviruses include: avian leukosis-sarcoma, mammalian C-type, B-type viruses, D-type viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, B. N. Fields, et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996). These adenovirus vectors can be derived from, for example, human, chimpanzee, or rhesus adenoviruses. Other examples include murine leukemia viruses, murine sarcoma viruses, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, Gibbon ape leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus and lentiviruses. Other examples of vectors are described, for example, in McVey et al., (U.S. Patent. No.5,801,030); incorporated herein in its entirety by reference. The nucleic acid material (e.g., including a nucleic acid molecule) of the viral vector may be encapsulated, e.g., in a lipid membrane or by structural proteins (e.g., capsid proteins), that may include one or more viral polypeptides (e.g., a glycoprotein). The viral vector can be used to infect cells of a subject, which, in turn, promotes the translation of the heterologous gene(s) of the viral vector into the immunogens.

[0105] Adenoviral vectors disclosed in International Patent Application Publication Nos. WO 2006 / 040330 and WO 2007 / 104792, each incorporated by reference herein, are particularly useful as vectors. These adenoviral vectors can encode and / or deliver the HPV polyepitope polypeptides to treat a subject having a pathological condition associated with a viral infection (e.g., an HPV infection or a cancer). In some embodiments, one or more recombinant adenovirus vectors can be administered to the subject in order to express more than one type of immunogen (e.g., an HPV infection and / or cancer). Besides adenoviral vectors, other viral vectors and techniques are known in the art that can be used to facilitate delivery and / or expression of one or more of the immunogens in a subject (e.g., a human). These viruses include poxviruses (e.g., vaccinia virus and modified vaccinia virus Ankara (MVA); see, e.g., U.S. Patent Nos.4,603,112 and 5,762,938, each incorporated by reference herein), herpesviruses, togaviruses (e.g.,Venezuelan Equine Encephalitis virus; see, e.g., U.S. Patent No. 5,643,576, incorporated by reference herein), picornaviruses (e.g., poliovirus; see, e.g., U.S. Patent No. 5,639,649, incorporated by reference herein), baculoviruses, and others described by Wattanapitayakul and Bauer (Biomed. Pharmacother. 54:487 (2000), incorporated by reference herein).

[0106] In some embodiments, the mRNA encoding a polyepitope polypeptide. antigen is incorporated into a recombinant AAV (rAAV) vectors and / or virions in order to facilitate their introduction into a cell. rAAV vectors useful in the compositions and methods described herein are recombinant polynucleotide constructs that include (1) a heterologous sequence to be expressed (e.g., a polynucleotide encoding a coronavirus antigen to be expressed) and (2) viral sequences that facilitate stability and expression of the heterologous genes. The viral sequences may include those sequences of AAV that are required in cis for replication and packaging (e.g., functional ITRs) of the DNA into a virion. Such rAAV vectors may also contain marker or reporter genes. Useful rAAV vectors have one or more of the AAV WT genes deleted in whole or in part but retain functional flanking ITR sequences. The AAV ITRs may be of any serotype suitable for a particular application. Methods for using rAAV vectors are described, for example, in Tal et al., J. Biomed. Sci.7:279 (2000), and Monahan and Samulski, Gene Delivery 7:24 (2000), the disclosures of each of which are incorporated herein by reference as they pertain to AAV vectors for gene delivery.

[0107] The mRNA encoding a HPVSobwq antigen can be incorporated into a rAAV virion in order to facilitate introduction of the mRNA encoding a polyepitope polypeptides antigen into a cell. The capsid proteins of an AAV compose the exterior, non-nucleic acid portion of the virion and are encoded by the AAV cap gene. The cap gene encodes three viral coat proteins, VP1 , VP2 and VP3, which are required for virion assembly. The construction of rAAV virions has been described, for instance, in United States Patent NOs 5,173,414; 5,139,941; 5,863,541; 5,869,305; 6,057,152; and 6,376,237; as well as in Rabinowitz et al., J. Virol.76:791 (2002) and Bowles et al., J. Virol.77:423 (2003), the disclosures of each of which are incorporated herein by reference as they pertain to AAV vectors for gene delivery.

[0108] Useful rAAV virions include those derived from a variety of AAV serotypes including AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , AAV12 rh10, rh39, rh43, rfi74, and Anc80. Construction and use of AAV vectors and AAV proteins of different serotypes are described, for instance, in Chao et al., Mol. Ther.2:619 (2000); Davidson et al., Proc. Natl. Acad. Sci. USA 97:3428 (2000); Xiao et al., J. Virol.72:2224 (1998); Halbert et al., J. Virol.74:1524 (2000); Halbert et al., J. Virol.75:6615 (2001); and Auricchio et al., Hum. Molec. Genet.10:3075 (2001), the disclosures of each of which are incorporated herein by reference as they pertain to AAV vectors for gene delivery. AAV vectors may be pseudotyped vectors. Pseudotyped vectors include AAV vectors of a given serotype (e.g., AAV9) pseudotyped with a capsid gene derived from a serotype other than the given serotype (e.g., AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, etc.).

[0109] Techniques involving the construction and use of pseudotyped rAAV virions are known in the art and are described, for instance, in Duan et al., J. Virol.75:7662 (2001); Halbert et al., J. Virol.74:1524 (2000); Zolotukhin et al., Methods, 28:158 (2002); and Auricchio et al., Hum. Molec. Genet. 10:3075 (2001).

[0110] AAV virions that have mutations within the virion capsid may be used to infect particular cell types more effectively than non-mutated capsid virions. For example, suitable AAV mutants may have ligand insertion mutations for the facilitation of targeting an AAV to specific cell types. The construction and characterization of AAV capsid mutants including insertion mutants, alanine screening mutants, and epitope tag mutants is described in Wu et al., J. Virol.74:8635 (2000). Other rAAV virions that can be used in methods described herein include those capsid hybrids that are generated by molecular breeding of viruses as well as by exon shuffling. See, e.g., Soong et al., Nat. Genet., 25:436 (2000) and Kolman and Stemmer, Nat. Biotechnol.19:423 (2001).

[0111] Gene transfer techniques using these viruses are known to those skilled in the art. Retrovirus vectors for example may be used to stably integrate the polynucleotide into the host genome, although such recombination is not preferred. Replication-defective adenovirus vectors by contrast remain episomal and therefore allow transient expression.

[0112] Vectors capable of driving expression in insect cells (for example baculovirus vectors), in human cells, in yeast or in bacteria may be employed in order to produce quantities of HPV polyepitope polypeptide encoded by the mRNA, for example, for use as subunit vaccines or in immunoassays. Liposome and lipoplex delivery

[0113] Suitably, the mRNA compositions described above and / or elsewhere herein could be delivered to the subject in a liposome and / or lipoplex. Liposomes are artificially-prepared vesicles which are primarily be composed of a lipid bilayer and may be used as a delivery vehicle for the administration of nutrients and pharmaceutical formulations. Liposomes can be of different sizes such as, but not limited to, a multilamellar vesicle (MLV) which may be hundreds of nanometers in diameter and may contain a series of concentric bilayers separated by narrow aqueous compartments, a small unicellular vesicle (SUV) which may be smaller than 50 nm in diameter, and a large unilamellar vesicle (LUV) which may be between 50 and 500 nm in diameter.

[0114] Liposomes and / or lipoplexes may include opsonins or ligands in order to improve the attachment of liposomes, lipoplex or lipid nanoparticles to unhealthy tissue or to activate events such as, but not limited to, endocytosis. Lipid nanoparticle delivery

[0115] In some embodiments, the mRNA molecules of the invention are formulated a lipid nanoparticle. LNPs are composed primarily of cationic lipids along with other lipid ingredients. These typically include neutral phospholipid molecules belonging to the phosphatidylcholine (PC) class and sterols, such as cholesterol. Another common lipid ingredient is what is known as a PEGylated phospholipid-a polyethylene glycol (PEG) polymer covalently attached to the head-group of a phospholipid. In some embodiments, lipid nanoparticles (LNPs) are formulated in a composition for delivery of the mRNA to a desired target such as a cell, tissue, or organ. In some preferred embodiments, the LNPs are formulated for delivery of the mRNA to the lungs of a subject. In even more preferred embodiments, the LNPs are formulated for delivery of the mRNA to the macrophages of a subject. The LNPs of the invention may be any lipid composition For example, the LNP may be selected from, but not limited to, liposomes or vesicles in which an aqueous volume is encapsulated by amphipathic lipid bilayers, micelle-like lipid nanoparticles having a non-aqueous core, and solid lipid nanoparticles.

[0116] Whilst liposomes include one or more rings of lipid bilayer surrounding an aqueous pocket, not all lipid nanoparticles have a contiguous bilayer like liposomes. Instead, it is understood that some LNPs assume a micelle-like structure, encapsulating drug molecules in a non-aqueous core.

[0117] Where the use of lipid nanoparticles is contemplated, a lipid nanoparticle generally comprises a cationic lipid, a non-cationic lipid, a PEG lipid and a structural lipid. Suitable cationic lipids may include those described in the cationic lipid may be selected from, but not limited to, a cationic lipid described in International PCT Publication Nos. WO2012 / 040184, WO2011 / 153120, WO2011 / 149733, WO2011 / 090965, WO2011 / 043913, WO2011 / 022460, WO2012 / 061259, WO2012 / 054365, WO2012 / 044638, WO2010 / 080724, WO2010 / 21865, WO2008 / 103276, WO2013 / 086373 and WO2013 / 086354, US Patent Nos. 7,893,302, 7,404,969, 8,283,333, and 8,466,122 and U.S. Patent Publication No. US2010 / 0036115, US2012 / 0202871, US2013 / 0064894, US2013 / 0129785, US2013 / 0150625, 2US013 / 0178541 and US2013 / 0225836. Other suitable cationic lipids, non-cationic lipids, PEG lipids and structural lipids, and suitable ratios thereof include those disclosed in WO 2015 / 164674 and WO 2013 / 090648. For example, mRNA according to the invention may be formulated in a lipid nanoparticle at a 20:1 weight ratio of total lipid to modified mRNA.

[0118] Exemplary lipid nanoparticle compositions and methods of making same that are suitable for use with the present invention are described, for example, in Semple et al. (2010); Jayarama et al. (2012); and Maier et al. (2013). Alternatively, the LNP formulation may be formulated by the methods described in International PCT Publication Nos. WO 2011 / 127255 or WO 2008 / 103276.

[0119] Further, the particle size of the lipid nanoparticle may be increased and / or decreased. The change in particle size may be able to help counter biological reaction such as, but not limited to, inflammation or may increase the biological effect of the modified mRNA delivered to a given subject.

[0120] Lipid nanoparticles suitable for use in the present disclosure will be apparent to the skilled person and / or are described herein. The lipids can have an anionic, cationic or zwitterionic hydrophilic head group. In some embodiments, the lipid nanoparticle comprises a PEG-lipid, a sterol structural lipid, and / or a neutral lipid. In one example, the lipid nanoparticle further comprises a cationic lipid. In one example, the lipid nanoparticle does not comprise a cationic lipid. In one embodiment, the LNP comprises a PEG-lipid. For example, the PEG-lipid is selected from the group consisting of PEG-DSPE, PEG-c-DMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEGDPPC, a lipid and combinations thereof.

[0121] In some embodiments, the LNP comprises a structural lipid. For example, the structural lipid is selected from the group consisting of cholesterol fecosterol, sitosterol, campesterol, stigmasterol, brassicasterol, ergosterol, tomatidine, tomatine, ursolic acid and ^-tocopherol, and combinations thereof.

[0122] In some embodiments, the LNP comprises a neutral lipid. Exemplary phospholipids (anionic or zwitterionic) for use in the present disclosure include, for example, phosphatidylethanolamines, phosphatidylcholines, phosphatidylserines, and phosphatidylglycerols. For example, the neutral lipid is selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn- glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2- dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl- sn-glycero-3 -phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3- phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecylsn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3- phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3- phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn- glycero-3-phosphoethanolamine (DSPE), 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2- dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoylsn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1- glycerol) sodium salt (DOPG), and sphingomyelin and combinations thereof.

[0123] In some embodiments, the LNP comprises a cationic lipid. Exemplary cationic lipids include, but are not limited to, dioleoyl trimethylammonium propane (DOTAP), 1,2-distearyloxy- N,Ndimethyl-3-aminopropane (DSDMA), 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA),1,2- dilinoleyloxy-N,N-dimethyl-3- aminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA), 2,5-bis((9Z,12Z)-octadeca-9,12,dien-1-yloxyl)benzyl-4-(dimethylamino)butnoate (LKY750). Exemplary zwitterionic lipids include, but are not limited to, acyl zwitterionic lipids and ether zwitterionic lipids, such as dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC) and dodecylphosphocholine. The lipids can be saturated or unsaturated.

[0124] By way of an illustrative example, in some embodiments the lipid nanoparticle comprises: (i) hyaluronic acid (HA) to target CD44 proteins overexpressed on the cell surface of macrophages; (ii) 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) (DSPE-PEG) to stabilise the nanoparticle system; and (iii) a targeting moiety of mannose conjugated to DSPE-PEG on the mRNA nanoparticle surface to further dual target glycose transporters overexpressed on the surface of macrophages. Alveolar macrophages are the major cell type known to internalize HA for degradation in normal lung, and CD44 participates in this process (Hajj et al., 2019; and Wang et al., 2012). Polymeric microparticles

[0125] In one example, the pharmaceutical composition further comprises a polymeric microparticle.

[0126] The skilled person will be aware that various polymers can form microparticles to encapsulate or adsorb the mRNA molecules of the present invention. It will be apparent that use of a substantially non-toxic polymer means that particles are safe, and the use of a biodegradable polymer means that the particles can be metabolised after delivery to avoid long-term persistence. Useful polymers are also sterilisable, to assist in the preparation of pharmaceutical grade formulations.

[0127] Exemplary non-toxic and biodegradable polymers include, but are not limited to, poly(^-hydroxy acids), polyhydroxy butyric acids, polylactones (including polycaprolactones), polydioxanones, polyvalerolactone, polyorthoesters, polyanhydrides, polycyanoacrylates, tyrosine-derived polycarbonates, polyvinyl-pyrrolidinones or polyester-amides, and combinations thereof. Cells

[0128] In some aspects, provided herein are APCs that present on an MHC two or more T cell epitopes (e.g., two or more of the T cell epitopes listed in Table 1), wherein the two or more T cell epitopes comprise T cell epitopes from at least two different HPV antigens (e.g., E1, E2, E4, E5, E6, and / or E7). In some particularly preferred embodiments, the MHC is a class I MHC.

[0129] In some embodiments, the MHC is a class II MHC. In some particularly preferred embodiments, the class I MHC has an α chain polypeptide that is HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-g, HLA-K or HLA-L. In some embodiments, the class II MHC has an α chain polypeptide that is HLA- DMA, HLA-DOA, HLA-DPA, HLA-DQA or HLA-DRA. In some embodiments, the class II MHC has a β chain polypeptide that is HLA-DMB, HLA-DOB, HLA-DPB, HLA-DQB or HLA-DRB.

[0130] In some embodiments the APCs are B cells, antigen presenting T-cells, dendritic cells, or artificial antigen-presenting cells (e.g., aK562 cells). Dendritic cells for use in the process may be prepared by taking PBMCs from a patient sample and adhering them to plastic. Generally, the monocytepopulation sticks, and all other cells can be washed off. The adherent population is then differentiated with IL-4 and GM-CSF to produce monocyte derived dendritic cells. These cells may be matured by the addition of IL-Ιβ, IL-6, PGE-1 and TNF-a (which upregulates the important co-stimulatory molecules on the surface of the dendritic cell) and are then contacted with a recombinant adenovirus described herein.

[0131] In some embodiments, the APC is an artificial antigen-presenting cell, such as an aK562 cell. In some embodiments, the artificial antigen-presenting cells are engineered to express CD80, CD83, 41BB-L, and / or CD86. Exemplary artificial antigen-presenting cells, including aK562 cells, are described U.S. Pat. Pub. No.2003 / 0147869, which is hereby incorporated by reference.

[0132] In certain aspects, provided herein are methods of generating APCs that present the two or more of the T cell epitopes described herein comprising contacting an APC with a nucleic acid vector and / or recombinant adenoviruses encoding T cell epitopes described herein and / or with a polyepitope produced by the nucleic acid vectors or recombinant adenoviruses described herein. In some embodiments, the APCs are irradiated.

[0133] In certain aspects, provided herein are T cells (e.g., CD4 T cells and / or CD8 T cells) that express a TCR (e.g., an αβ TCR or a γδ TCR) that recognizes a peptide described herein (a peptide comprising a HPV epitope listed in Table 1) presented on an MHC. In some embodiments, the T cell is a CD8 T cell (a CTL) that expresses a TCR that recognizes a peptide described herein presented on a class I MHC. In some embodiments, the T cell is a CD4 T cell (a helper T cell) that recognizes a peptide described herein presented on a class II MHC.

[0134] In some aspects, provided herein are methods of generating, activating and / or inducing proliferation of T cells (e.g., CTLs) that recognize two or more T cell epitopes from at least two different HPV antigens. In some embodiments, the CTLs are incubated in culture with an APC provided herein (e.g., an APC that presents a peptide comprising a T cell epitope).

[0135] In some embodiments, the sample containing T cells are incubated 2 or more times with APCs provided herein. In some embodiments, the T cells are incubated with the APCs in the presence of at least one cytokine. In some embodiments, the cytokine is IL-4, 1L-7 and / or IL-15. Exemplary methods for inducing proliferation of T cells using APCs are provided, for example, in U.S. Pat. Pub. No. 2015 / 0017723, which is hereby incorporated by reference.

[0136] In some aspects, provided herein is a population of CTLs collectively comprising T cell receptors that recognize two or more T cell epitopes (e.g., two or more of the T cell epitopes listed in Table 1), wherein the two or more T cell epitopes comprise T cell epitopes from at least two different HPV antigens (e.g., E1, E2, E4, E5, E6, and / or E7). In some embodiments, the epitopes are HLA class I- restricted T cell epitopes.

[0137] In some aspects, provided herein are compositions (e.g., therapeutic compositions) comprising the nucleic acid vector described herein, peptides produced by the nucleic acid vector described herein, multivirus-specific CTLs and / or APCs provided herein (e.g., comprising the nucleic acid vector described herein) and a pharmaceutically acceptable carrier. In some embodiments, such compositions are used in adoptive immunotherapy to boost multi-virus-specific immunity in a subject by administering to the subject an effective amount of the composition. In some embodiments, the multivirus-specific CTLs and / or APCs are not autologous to the subject.

[0138] In some embodiments, the T cells and / or APCs are autologous to the subject. In some embodiments, the T cells and / or APCs are stored in a cell bank before they are administered to the subject (i.e., the cells are allogeneic to the subject). Pharmaceutical Compositions

[0139] In some aspects, provided herein is a composition (e.g., a pharmaceutical composition, such as a vaccine composition), containing a peptide, nucleic acid, vector, CTL, or an APC described above and / or elsewhere herein, formulated together with a pharmaceutically acceptable carrier. In some embodiments, the composition includes a combination of multiple (e.g., two or more) agents provided herein. Adjuvants

[0140] In some embodiments, the pharmaceutical composition further comprises an adjuvant. As used herein, the term “adjuvant” broadly refers to an agent that affects an immunological or physiological response in a patient or subject. For example, an adjuvant might increase the presence of an antigen over time or to an area of interest like a tumour, help absorb an antigen-presenting cell antigen, activate macrophages and lymphocytes and support the production of cytokines. By changing an immune response, an adjuvant might permit a smaller dose of an immune interacting agent to increase the effectiveness or safety of a particular dose of the immune interacting agent. For example, an adjuvant might prevent T cell exhaustion and thus increase the effectiveness or safety of a particular immune interacting agent.

[0141] An “adjuvant” as used herein refers to a composition that enhances the immune response to an immunogen. Examples of such adjuvants include, but are not limited to, inorganic adjuvants (e.g., inorganic metal salts such as aluminium phosphate or aluminium hydroxide), organic adjuvants (e.g., saponins, such as QS21, or squalene), oil-based adjuvants (e.g. Freund’s complete adjuvant and Freund’s incomplete adjuvant), cytokines (e.g., IL-1β, IL-2, IL-7, IL-12, IL-18, GM-CSF, and IFN-γ) particulate adjuvants (e.g., immunostimulatory complexes (ISCOMS), liposomes, or biodegradable microspheres), virosomes, bacterial adjuvants (e.g., monophosphoryl lipid A, such as 3-de-O-acylated monophosphoryllipid A (3D-MPL), or muramyl peptides), synthetic adjuvants (e.g., non-ionic block co-polymers, muramyl peptide analogues, or synthetic lipid A), synthetic polynucleotide adjuvants (e.g., polyarginine or polylysine) and immunostimulatory oligonucleotides containing unmethylated CpG dinucleotides (“CpG”).

[0142] One suitable adjuvant is monophosphoryl lipid A (MPL), in particular 3-de-O-acylated monophosphoryl lipid A (3D-MPL). Chemically it is often supplied as a mixture of 3-de-O-acylated monophosphoryl lipid A with either 4, 5, or 6 acylated chains. It can be purified and prepared by the methods taught in GB 2122204B, which reference also discloses the preparation of diphosphoryl lipid A, and 3-O- deacylated variants thereof. Other purified and synthetic lipopolysaccharides have been described (U.S. Pat. No. 6,005,099 and European Patent No. EP 0729473 B1; Hilgers et al., 1986, Int. Arch. Allergy. lmmunol., 79(4):392-6; Hilgers et al., 1987, Immunology, 60(1):141 -6; and European Patent No. EP 0549 074 B1).

[0143] Saponins are also suitable adjuvants (see Lacaille-Dubois, M and Wagner H, A review of the biological and pharmacological activities of saponins. Phytomedicine vol 2 pp 363-386 (1996)). For example, the saponin Quil A (derived from the bark of the South American tree Quillaja Saponaria Molina), and fractions thereof, are described in U.S. Pat. No.5,057,540 and Kensil, Crit. Rev. Ther. Drug Carrier Syst., 1996, 12:1 -55; and European Patent No. EP 0362279 B1. Purified fractions of Quil A are also known as immunostimulants, such as QS21 and QS17; methods of their production is disclosed in U.S. Pat. No.5,057,540 and EP 0362279 B1. Also described in these references is QS7 (a non-haemolytic fraction of Quil-A). Use of QS21 is further described in Kensil et al. (1991, J. Immunology, 146: 431 -437). Combinations of QS21 and polysorbate or cyclodextrin are also known (International Patent Publication No. WO 99 / 10008). Particulate adjuvant systems comprising fractions of QuilA, such as QS21 and QS7 are described in International Patent Publication Nos. WO 96 / 33739 and WO 96 / 11711. CpG

[0144] CpG oligodeoxynucleotides (ODNs) are short synthetic single-stranded DNA molecules containing unmethylated CpG dinucleotides in particular sequence contexts. CpG ODNs possess a partially or completely phosphorothioated (PS) backbone, as opposed to the natural phosphodiester (PO) backbone in DNA molecules. Three major classes of stimulatory CpG ODNs have been identified based on structural characteristics and activity on human peripheral blood mononuclear cells (PBMCs), in particular B cells and plasmacytoid dendritic cells (pDCs). These three classes are Class A (Type D), Class B (Type K), and Class C.

[0145] In some embodiments, the CpG ODN may be a Class A ODN. For example, the Class A ODN may be selected from the group including CpG 1585, having an amino acid sequence of GGGGTCAACGTTGAGGGGGG (SEQ ID NO: 41); CpG 2216, having an amino acid sequence ofGGGGGACGATCGTCGGGGGG (SEQ ID NO: 42); and CpG 2336, having the amino acid sequence of GGGGACGACGTCGTGGGGGGG (SEQ ID NO: 43).

[0146] In some embodiments, the CpG ODN may be a Class B ODN. Class B CpG ODNs contain a full PS backbone with one or more CpG dinucleotides. They strongly activate B cells and TLR9- dependent NF-κB signaling but weakly stimulate IFN-^ secretion. For example, the Class B ODN may be selected from the group including CpG 1668, having the amino acid sequence of TCCATGACGTTCCTGATGCT (SEQ ID NO:44); CpG 7909, also known as CpG 2006, having the amino acid sequence of TCGTCGTTTTGTCGTTTTGTCGTT (SEQ ID NO: 45); CpG 2007, having the amino acid sequence of TCGTCGTTGTCGTTTTGTCGTT (SEQ ID NO: 46); CpG D-SL01, having the amino acid sequence of TCGCGACGTTCGCCCGACGTTCGGTA (SEQ ID NO: 47); CpG 1018, having the amino acid sequence of TGACTGTGAACGTTCGAGATGA (SEQ ID NO: 48); and CpG 1826, having an amino acid sequence of TCCATGACGTTCCTGACGTT (SEQ ID NO: 49). In some embodiments, the CpG ODN is CpG 7909 (SEQ ID NO: 45). In some embodiments, the CpG ODN is CpG 1826 (SEQ ID NO: 49).

[0147] In some embodiments, the CpG ODN may be a Class C ODN. For example, the Class C ODN may be selected from the group including CpG 2395, having the amino acid sequence of TCGTCGTTTTCGGCGCGCGCCG (SEQ ID NO: 50); CpG M362, having the amino acid sequence of having the amino acid sequence of TCGTCGTCGTTCGAACGACGTTGAT (SEQ ID NO: 51); and CpG D-SL03, having the amino acid sequence of TCGCGAACGTTCGCCGCGTTCGAACGCGG (SEQ ID NO: 52).

[0148] In some embodiments, all the internucleoside groups connecting the nucleosides in the CpG sequence are phosphorothionates.

[0149] In some embodiments, an immunogenic composition includes an amphiphilic conjugate. An amphiphilic conjugate refers to a conjugate that includes a CpG ODN covalently linked to an albumin-binding domain (e.g., a lipid). In some embodiments, an amphiphilic conjugate includes a CpG ODN that is covalently linked to an albumin-binding domain (e.g., a lipid) directly. In some embodiments, an amphiphilic conjugate includes a CpG ODN that is covalently linked to an albumin-binding domain (e.g., a lipid) through a linker. For amphiphilic conjugates that include CpG ODN conjugated to an albumin-binding domain either directly or through a linker, the albumin binding domain binds to endogenous albumin, which prevents the CpG-amphiphile from rapidly flushing into the bloodstream and instead re-targets them to lymphatics and draining lymph nodes where they accumulate due to filtering of albumin by antigen presenting cells.

[0150] CpG ODNs may be bonded directly or linked by way of a linker to a lipid to a form an CpG amphiphile. These compounds may be produced using the ordinary phosphoramidite chemistry known in the art. In some examples, the CpG ODN or CpG ODN-GG may be reacted with the following compound:to produce an intermediate, which upon oxidation with (e.g., phosphite oxidation methods known in the art, e.g., a sulfurizing agent, such as 3-((N,N- dimethylaminomethylidene)amino)-3H-1,2,4-dithiazole-5-thione) and hydrolysis of the cyanoethyl group may produce a compound of the invention.

[0151] Reference to CpG molecules herein, as well as amphiphiles including a CpG molecule (AMP-CpG), is to be understood as including pharmaceutically acceptable salts thereof. Lipid

[0152] The CpG-amphiphiles (AMP-CpG) disclosed herein include a hydrophobic lipid, which may be an albumin binding domain. The lipid can be linear, branched, or cyclic. The lipid is preferably at least 17 to 18 carbons in length but may be shorter if it shows good albumin binding and adequate targeting to the lymph nodes. In some embodiments, the activity relies, in part, on the ability of the AMP-CpG to associate with albumin in the blood of the subject. Therefore, lymph node-targeted CpG-amphiphiles typically include a lipid that can bind to albumin under physiological conditions. Lipids suitable for targeting the lymph node can be selected based on the ability of the lipid or a lipid conjugated to a CpG ODN to bind to albumin. Suitable methods for testing the ability of the lipid or lipid conjugated to a CpG ODN to bind to albumin are known in the art.

[0153] Examples of preferred lipids for use in lymph node targeting with CpG-amphiphiles include, but are not limited to fatty acids with aliphatic tails of 8-30 carbons including, but not limited to, linear and unsaturated saturated fatty acids, branched saturated and unsaturated fatty acids, and fatty acids derivatives, such as fatty acid esters, fatty acid amides, and fatty acid thioesters, diacyl lipids, cholesterol, cholesterol derivatives, and steroid acids such as bile acids; Lipid A or combinations thereof.

[0154] In some embodiments, the lipid is a diacyl lipid or two-tailed lipid. In some embodiments, the tails in the diacyl lipid contain from about 8 to about 30 carbons and can be saturated, unsaturated, or combinations thereof. In some embodiments, the diacyl lipid has the following structure: or a salt thereof, wherein X is O or S. The tails of a lipid can be coupled to the head group via ester bond linkages, amide bond linkages, thioester bond linkages, or combinations thereof. In a particular embodiment, the diacyl lipids are phosphate lipids, glycolipids, sphingolipids, or combinations thereof.

[0155] Lymph node-targeting conjugates typically include a lipid that is 8 or more carbon units in length. Increasing the number of lipid units can reduce insertion of the lipid into plasma membrane of cells, allowing the lipid conjugate to remain free to bind albumin and traffic to the lymph node. For example, the lipid can be a diacyl lipid composed of two C18 hydrocarbon tails. In some embodiments, the lipid for use in preparing lymph node targeting lipid conjugates is not a single chain hydrocarbon (e.g., C18), or cholesterol. Cholesterol conjugation has been explored to enhance the immunomodulation of molecular adjuvants such as CpG and immunogenicity of peptides.

[0156] Reference to lipids herein, as well as amphiphiles including the lipid, is to be understood as including pharmaceutically acceptable salts thereof. Linkers

[0157] For the AMP-CpG to be trafficked efficiently to the lymph node, the CpG ODN should remain soluble. Therefore, a polar block linker can be included between the CpG ODN and the lipid to which it is conjugated to increase solubility of the CpG ODN. In some embodiments, the AMP-CpG includes a CpG sequence linked to a lipid by a linker. The linker may reduce or prevent the ability of the lipid to insert into the plasma membrane of cells, such as cells in the tissue adjacent to the injection site. The linker can also reduce or prevent the ability of the CpG ODN from non-specifically associating with extracellular matrix proteins at the site of administration. The linker may increase the solubility of the CpG ODN without preventing its ability to bind to albumin. This combination of characteristics can allow the CpG ODN to bind to albumin present in the serum or interstitial fluid and remain in circulation until the albumin is trafficked to and retained in a lymph node.

[0158] The length and composition of the linker can be adjusted based on the lipid and CpG ODN selected. For example, for some CpG ODNs, the oligonucleotide itself may be polar enough to ensure solubility; for example, oligonucleotides that are 10, 15, 20 or more nucleotides in length. Therefore, in some embodiments, no additional linker is required. However, depending on the amino acid sequence, some lipidated peptides can be essentially insoluble. In these cases, it can be desirable to include a linker that mimics the effect of a polar oligonucleotide. A linker can be used as part of any of lipid conjugates described herein, for example, lipid-oligonucleotide conjugates and lipid-peptide conjugates, which reduce cell membrane insertion / preferential portioning onto albumin.

[0159] Suitable linkers include, but are not limited to, oligonucleotides such as those discussed above, including a string of nucleic acids, a hydrophilic polymer including but not limited to polyethylene glycol) (MW: 500 Da to 20,000 Da), polyacrylamide (MW: 500 Da to 20,000 Da), polyacrylic acid; a string of hydrophilic amino acids such as serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, or combinations thereof; polysaccharides, including but not limited to, dextran (MW: 1,000 Da to 2,000,000 Da), or combinations thereof. The hydrophobic lipid and the linker / CpG ODN are covalently linked. The covalent bond may be a non-cleavable linkage or a cleavable linkage. The non-cleavable linkage can include an amide bond or phosphate bond, and the cleavable linkage can include a disulfide bond, acid-cleavable linkage, ester bond, anhydride bond, biodegradable bond, or enzyme-cleavable linkage.

[0160] In some embodiments, the linker is one or more ethylene glycol (EG) units, more preferably two or more EG units (i.e., polyethylene glycol (PEG)). For example, in some embodiments, theAMP-CpG includes a CpG and a hydrophobic lipid linked by a polyethylene glycol (PEG) molecule or a derivative or analog thereof.

[0161] In some embodiments, AMP-CpG described herein contain a CpG ODN linked to PEG which is in turn linked to a hydrophobic lipid, or lipid-Gn-ON conjugates, either covalently or via formation of protein-oligo conjugates that hybridize to oligo micelles. The precise number of PEG units depends on the lipid and the cargo, however, typically, a linker can have between about 1 and about 100, between about 20 and about 80, between about 30 and about 70, or between about 40 and about 60 PEG units. In some embodiments, the linker has between about 45 and 55 PEG units. For example, in some embodiments, the linker has 48 PEG units.

[0162] As discussed above, in some embodiments, the linker is an oligonucleotide which includes a string of nucleic acids. In some embodiments, the CpG-amphiphiles described above and / or elsewhere herein include a CpG ODN linked to a string of nucleic acids, which is in turn linked to a hydrophobic lipid. The linker can have any sequence, for example, the sequence of the oligonucleotide can be a random sequence, or a sequence specifically chosen for its molecular or biochemical properties (e.g., highly polar). In some embodiments, the linker includes 20 one or more series of consecutive adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), or analog thereof. In some embodiments, the linker consists of a series of consecutive adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), or analog thereof.

[0163] In some embodiments, the string of nucleic acids includes between 1 and 50 nucleic acid residues. In some embodiments, the string of nucleic acids includes between 5 and 30 nucleic acid residues. In some embodiments, the linker includes one or more guanines, for example between 1- 10 guanines. It has been discovered that altering the number of guanines between a CpG ODN and a lipid tail controls micelle stability in the presence of serum proteins. Therefore, the number of guanines in the linker can be selected based on the desired affinity of the CpG ODN for serum proteins such as albumin.

[0164] In some embodiments, the linker is an oligonucleotide that includes a string of amino acids.

[0165] In some embodiments, the AMP-CpG includes a CpG ODN linked to string of amino acids, which is in turn linked to a hydrophobic lipid. The linker can have any amino acid sequence, for example, the sequence of the oligonucleotide can be a random sequence, or a sequence chosen for its molecular or biochemical properties (e.g., high flexibility). In some embodiments, the linker includes a series of glycine residue to form a polyglycine linker. In some embodiments, the linker includes an amino acid sequence of (Gly)n, wherein n may be between 2 and 20 residues. Examples of polyglycine linkers include but are not limited to GGG, GGGA (SEQ ID NO:53), GGGG (SEQ ID NO:54), GGGAG (SEQ ID NO:55), GGGAGG (SEQ ID NO:56), GGGAGGG (SEQ ID NO:57), GGAG (SEQ ID NO:58), GGSG (SEQ ID NO:59),AGGG (SEQ ID NO:60), SGGG (SEQ ID NO:61), GGAGGA (SEQ ID NO:62), GGSGGS (SEQ ID NO:63), GGAGGAGGA (SEQ ID NO:64), GGSGGSGGS (SEQ ID NO:65), GGAGGAGGAGGA (SEQ ID NO:66), GGSGGSGGSGGS (SEQ ID NO:67), GGAGGGAG (SEQ ID NO:68), GGSGGGSG (SEQ ID NO:69), GGAGGGAGGGAG (SEQ ID NO:70), GGSGGGSGGGSG (SEQ ID NO:71), GGGGAGGGGAGGGGA (SEQ ID NO:72), GGGGSGGGGSGGGGS (SEQ ID NO:73), and GGGSGGGS (SEQ ID NO:74). Other adjuvants

[0166] Adjuvants such as those described above may be formulated together with carriers, such as liposomes, oil in water emulsions, and / or metallic salts (including aluminum salts such as aluminum hydroxide). For example, 3D-MPL may be formulated with aluminum hydroxide (European Patent A 0689 454) or oil in water emulsions (International Patent Publication No. WO 95 / 17210); QS21 may be formulated with cholesterol containing liposomes (International Patent Publication No. WO 96 / 33739), oil in water emulsions (International Patent Publication No. WO 95 / 17210) or alum (International Patent Publication No. WO 98 / 15287); CpG may be formulated with alum (Brazolot-Millan, supra) or with other cationic carriers.

[0167] Combinations of adjuvants may be utilized in the present invention, in particular a combination of a monophosphoryl lipid A and a saponin derivative (see, e.g., International Patent Publication Nos. WO 94 / 00153; WO 95 / 17210; WO 96 / 33739; WO 98 / 56414; WO 99 / 12565; WO 99 / 1 1241), more particularly the combination of QS21 and 3D-MPL as disclosed in International Patent Publication No. WO 94 / 00153, or a composition where the QS21 is quenched in cholesterol-containing liposomes (DQ) as disclosed in International Patent Publication No. WO 96 / 33739. Alternatively, a combination of CpG plus a saponin such as QS21 is an adjuvant suitable for use in the present invention. A potent adjuvant formulation involving QS21, 3D-MPL and tocopherol in an oil in water emulsion is described in International Patent Publication No. WO 95 / 17210 and is another formulation for use in the present invention. Saponin adjuvants may be formulated in a liposome and combined with an immunostimulatory oligonucleotide. Thus, suitable adjuvant systems include, for example, a combination of monophosphoryl lipid A, preferably 3D-MPL, together with an aluminium salt (e.g., as described in International Patent Publication No. WO 00 / 23105). A further exemplary adjuvant comprises QS21 and / or MPL and / or CpG. QS21 may be quenched in cholesterol-containing liposomes as disclosed in International Patent Publication No. WO 96 / 33739.

[0168] Other suitable adjuvants include alkyl glucosaminide phosphates (AGPs) such as those disclosed in International Patent Publication No. WO 98 / 50399 or U.S. Pat. No.6,303,347 (processes for preparation of AGPs are also disclosed), or pharmaceutically acceptable salts of AGPs as disclosed in U.S. Pat. No.6,764,840. Some AGPs are TLR4 agonists, and some are TLR4 antagonists. Both are known to be useful as adjuvants.

[0169] It has been found that the fusion of the invariant chain to an antigen which is comprised by an expression system used for vaccination increases the immune response against said antigen, if it is administered with an adenovirus. Accordingly, in one embodiment of the invention, the immunogenic transgene may be co-expressed with invariant chain in a recombinant adenovirus viral vector.

[0170] Examples of adjuvants include, but are not limited to, an immune modulatory protein, Adjuvant 65, α-GalCer, aluminum phosphate, aluminium hydroxide, calcium phosphate, β-Glucan Peptide, GPI-0100, lipid A, lipopolysaccharide, Lipovant, Montanide, N-acetyl-muramyl-L-alanyl-D-isoglutamine, Pam3CSK4, quil A and trehalose dimycolate, and / or combinations thereof. Methods of preparing these formulations or compositions include the step of bringing into association an agent described herein with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association an agent described herein with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0171] Pharmaceutical compositions of this invention suitable for parenteral administration comprise one or more agents described herein in combination with one or more pharmaceutically- acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain sugars, alcohols, antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

[0172] Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0173] Regardless of the route of administration selected, the agents of the present invention, which may be used in a suitable hydrated form, and / or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art. Methods of Inducing an Immune Response and Treatment

[0174] New treatment approaches take advantage of our knowledge of how the immune system can eliminate virus-infected cells, which is done mainly by cytotoxic T cells (also known as “killer” T cells). These new therapies involve the use of anti-cancer vaccines and intra-lesion immune system- based therapy, with the idea to activate T cells so they can then locate and kill HPV-infected cells.

[0175] In some embodiments, the present invention includes a method of eliciting or inducing an immune response to an HPV antigen, the method comprising administering to the subject, or a cell from the subject, a pharmaceutical composition or vaccine as described above and / or elsewhere herein, to thereby elicit or induce an immune response to the HPV antigen.

[0176] In certain embodiments, provided herein are methods of treating or preventing an HPV infection, and / or a cancer, and / or precancerous lesions in a subject comprising administering to the subject a pharmaceutical composition or vaccine as described above and / or elsewhere herein, to thereby treat or prevent an HPV infection, and / or a cancer, and / or precancerous lesions in the subject.

[0177] In some embodiments, provided herein is a method of treating or preventing an HPV infection in a subject. In some embodiments, the subject treated is immunocompromised. For example, in some embodiments, the subject has a T cell deficiency. In some embodiments, the subject has leukemia, lymphoma or multiple myeloma. In some embodiments, the subject is infected with HIV and / or has AIDS. In some embodiments, the subject has undergone a tissue, organ and / or bone marrow transplant. In some embodiments, the subject is being administered immunosuppressive drugs. In some embodiments, the subject has undergone and / or is undergoing chemotherapy. In some embodiments, the subject has undergone and / or is undergoing radiation therapy. In some embodiments the subject has undergone immunotherapy, for example, with a checkpoint inhibitor molecule.

[0178] In some embodiments, the subject is also administered an anti-viral drug that inhibits HPV replication. For example, in some embodiments, the subject is administered one or more of podofilox, imiquimod, sinecatechins, podophyllin resin, trichloroacetic acid, or bichloracetic acid. In some embodiments, the subject is also treated with an intervention that physically affects the HPV infected lesions and / or HPV-associated tumors. For example, in some embodiments, the lesions are treated with surgical excision, chemical ablation, cryotherapy, or cauterization.

[0179] In some embodiments, the subject has cancer or precancerous lesions. In some embodiments, the methods described herein may be used to treat any cancerous or pre-cancerous tumour. In some embodiments, the cancer and / or precancerous lesions expresses one or more of the HPV epitopes provided herein (e.g., the HPV epitopes listed in Table 1). In some embodiments, the precancerous lesions include abnormal cell changes and / or precancerous cell changes. Precancerous lesions that may be treated by methods and compositions provided herein include, but are not limited to, cervical intraepithelial neoplasia (CIN), squamous intraepithelial lesions (SIL), or warts on the cervix. In some embodiments, the cancer includes a solid tumor. Cancers that may be treated by methods and compositions provided herein include, but are not limited to, cancer cells from the cervix, anus, vagina, vulva, penis, tongue base, larynx, tonsil, bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, non-melanoma skin cancer (NMSC), cutaneous squamous cell carcinoma (SCC), stomach, testis, tongue, or uterus. In addition, the cancer mayspecifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; non encapsulating sclerosing carcinoma; adrenal cortical carcinoma endometrioid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; mammary paget's disease; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w / squamous metaplasia; malignant thymoma; malignant ovarian stromal tumor; malignant thecoma; malignant granulosa cell tumor; and malignant roblastoma; sertoli cell carcinoma; malignant leydig cell tumor; malignant lipid cell tumor; malignant paraganglioma; malignant extra-mammary paraganglioma; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; malignant blue nevus; sarcoma; fibrosarcoma; malignant fibrous histiocytoma; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; malignant mixed tumor; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; malignant mesenchymoma; malignant brenner tumor; malignant phyllodes tumor; synovial sarcoma; malignant mesothelioma; dysgerminoma; embryonal carcinoma; malignant teratoma; malignant struma ovarii; choriocarcinoma; malignant mesonephroma; hemangiosarcoma; malignant hemangioendothelioma; kaposi's sarcoma; malignant hemangiopericytoma; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; malignant chondroblastoma; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; malignant odontogenic tumor; ameloblastic odontosarcoma; malignant ameloblastoma; ameloblastic fibrosarcoma; malignant pinealoma; chordoma; malignant glioma; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglion euroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; malignant meningioma; neurofibrosarcoma; malignant neurilemmoma; malignant granular cell tumor; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; small lymphocytic malignant lymphoma; diffuse large cell malignant lymphoma; follicular malignant lymphoma; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

[0180] In some embodiments, the subject is also administered an anti-cancer compound. Exemplary anti-cancer compounds include, but are not limited to, Alemtuzumab (Campath®), Alitretinoin (Panretin®), Anastrozole (Arimidex®), Bevacizumab (Avastin®), Bexarotene (Targretin®), Bortezomib (Velcade®), Bosutinib (Bosulif®), Brentuximab vedotin (Adcetris®), Cabozantinib (Cometriq™), Carfilzomib (Kyprolis™), Cetuximab (Erbitux®), Crizotinib (Xalkori®), Dasatinib (Sprycel®), Denileukin diftitox (Ontak®), Erlotinib hydrochloride (Tarceva®), Everolimus (Afmitor®), Exemestane (Aromasin®), Fulvestrant (Faslodex®), Gefitinib (Iressa®), Ibritumomab tiuxetan (Zevalin®), Imatinib mesylate (Gleevec®), Ipilimumab (Yervoy™), Lapatinib ditosylate (Tykerb®), Letrozole (Femara®), Nilotinib (Tasigna®), Ofatumumab (Arzerra®), Panitumumab (Vectibix®), Pazopanib hydrochloride (Votrient®), Pertuzumab (Peijeta™), Pralatrexate (Folotyn®), Regorafenib (Stivarga®), Rituximab (Rituxan®), Romidepsin (Istodax®), Sorafenib tosylate (Nexavar®), Sunitinib malate (Sutent®), Tamoxifen, Temsirolimus (Torisel®), Toremifene (Fareston®), Tositumomab and 13 1I-tositumomab (Bexxar®), Trastuzumab (Herceptin®), Tretinoin (Vesanoid®), Vandetanib (Caprelsa®), Vemurafenib (Zelboraf®), Vorinostat (Zolinza®), and Ziv-aflibercept (Zaltrap®).

[0181] In some embodiments, the subject is also administered a chemotherapeutic agent. Examples of such chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; cally statin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancrati statin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall; dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomysins, actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5- FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti- adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel and doxetaxel; chlorambucil; gemcitabine; 6- thioguanine; mercaptopurine; methotrexate; platinum coordination complexes such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT- 11); topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0182] In some embodiments, the subject is also administered an immunotherapeutic agent. Immunotherapy refers to a treatment that uses a subject’s immune system to treat cancer, e.g. cancer vaccines, cytokines, use of cancer-specific antibodies, T cell therapy, and dendritic cell therapy.

[0183] In some embodiments, the subject is also administered an immune modulatory protein. Examples of immune modulatory proteins include, but are not limited to, B lymphocyte chemoattractant (“BLC”), C-C motif chemokine 11 (“Eotaxin-1”), Eosinophil chemotactic protein 2 (“Eotaxin- 2”), Granulocyte colony-stimulating factor (“G-CSF”), Granulocyte macrophage colony-stimulating factor (“GM-CSF”), Intercellular Adhesion Molecule 1 (“ICAM-1”), Interferon gamma (“IFN-ɣ”), Interlukin-1 alpha (“IL-1^”), Interleukin-1 beta (“IL-1β”), Interleukin 1 receptor antagonist (“IL-1ra”), Interleukin-2 (“IL-2”), Interleukin-4 (“IL-4”), Interleukin-5 (“IL-5”), Interleukin-6 (“IL-6”), Interleukin-6 soluble receptor (“IL-6sR”), Interleukin-7 (“IL-7”), Interleukin-8 (“IL-8”), Interleukin- 10 (“IL-10”), Interleukin- 11 (“IL-11”), Subunit beta of Interleukin- 12 (“IL-12 p40” or “IL-12 p70”), Interleukin- 13 (“IL-13”), Interleukin- 15 (“IL-15”), Interleukin- 16 (“IL-16”), Interleukin- 17 (“IL-17”), Chemokine (C-C motif) Ligand 2 (“MCP-1”), Macrophage colony-stimulating factor (“M-CSF”), Monokine induced by gamma interferon (“MIG”), Chemokine (C-C motif) ligand 2 (“MIP-1^”), Chemokine (C-C motif) ligand 4 (“MIP-1β”), Macrophage inflammatory protein-1-delta (“MIP-l delta”), Platelet-derived growth factor subunit B (“PDGF-BB”), Chemokine (C-C motif) ligand 5, Regulated on Activation, Normal T cell Expressed and Secreted (“RANTES”), metallopeptidase inhibitor 1 (“TIMP-1”), metallopeptidase inhibitor 2 (“TIMP-2”), Tumor necrosis factor (TNF), Soluble TNF receptor type 1 (“sTNFRI”), sTNFRIIAR, Brain-derived neurotrophic factor (“BDNF”), Basic fibroblast growth factor (“bFGF”), Bone morphogenetic protein 4 (“BMP-4”), Bone morphogenetic protein 5 (“BMP-5”), Bone morphogenetic protein 7 (“BMP-7”), Nerve growth factor (“b-NGF”), Epidermal growth factor (“EGF”), Epidermal growth factor receptor (“EGFR”), Endocrine-gland-derived vascular endothelial growth factor (“EG-VEGF”), Fibroblast growth factor 4 (“FGF-4”), Keratinocyte growth factor (“FGF-7”), Growth differentiation factor 15 (“GDF-15”), Glial cell-derived neurotrophic factor (“GDNF”), Growth Hormone, Heparin-binding EGF-like growth factor (“HB-EGF”), Hepatocyte growth factor (“HGF”), Insulin-like growth factor binding protein 1 (“IGFBP-1”), Insulin-like growth factor binding protein 2 (“IGFBP-2”), Insulin-like growth factor binding protein 3 (“IGFBP-3”), Insulin-like growth factor binding protein 4 (“IGFBP-4”), Insulin- like growth factor binding protein 6 (“IGFBP-6”), Insulin-like growth factor 1 (“IGF-1”), Insulin, Macrophage colony-stimulating factor (“M-CSFR”), Nerve growth factor receptor (“NGFR”), Neurotrophin-3 (“NT-3”), Neurotrophin-4 (“NT-4”), Osteoclastogenesis inhibitory factor (“Osteoprotegerin”), Platelet-derived growth factor receptors (“PDGF-AA”), Phosphatidylinositol-glycan biosynthesis (“PIGF”), Skp, Cullin, F-box containing complex (“SCF”), Stem cell factor receptor (“SCFR”), Transforming growth factor alpha (“TGF^”), Transforming growth factor beta-1 (“TGFβ1”), Transforming growth factor beta-3 (“TGFβ3”), Vascular endothelial growth factor (“VEGF”), Vascular endothelial growth factor receptor 2 (“VEGFR2”), Vascular endothelial growth factor receptor 3 (“VEGFR3”), VEGF-D, 6Ckine, Axl tyrosine-protein kinase receptor (“Axl”), Betacellulin (“BTC”), Mucosae-associated epithelial chemokine (“CCL28”), Chemokine (C- C motif) ligand 27 (“CTACK”), Chemokine (C-X-C motif) ligand 16 (“CXCL16”), C-X-C motif chemokine 5 (“ENA-78”), Chemokine (C-C motif) ligand 26 (“Eotaxin-3”), Granulocyte chemotactic protein 2 (“GCP-2”), GRO, Chemokine (C-C motif) ligand 14 (“HCC-l”), Chemokine (C-C motif) ligand 16 (“HCC-4”), Interleukin-9 (“IL-9”), Interleukin-17 F (“IL-17F”), Interleukin- 18-binding protein (“IL-18 BPa”), Interleukin-28 A (“IL-28A”), Interleukin 29 (“IL-29”), Interleukin 31 (“IL-31”), C-X-C motif chemokine 10 (“IP-10”), Chemokine receptor CXCR3 (“I-TAC”), Leukemia inhibitory factor (“LIF”), Light, Chemokine (C motif) ligand (“Lymphotactin”), Monocyte chemoattractant protein 2 (“MCP-2”), Monocyte chemoattractant protein 3 (“MCP-3”), Monocyte chemoattractant protein 4 (“MCP-4”), Macrophage-derived chemokine (“MDC”), Macrophage migration inhibitory factor (“MIF”), Chemokine (C-C motif) ligand 20 (“MIP-3^”), C-C motif chemokine 19 (“MIP-3β”), Chemokine (C-C motif) ligand 23 (“MPIF-1”), Macrophage stimulating protein alpha chain (“MSP^”), Nucleosome assembly protein l-like 4 (“NAP-2”), Secreted phosphoprotein 1 (“Osteopontin”), Pulmonary and activation-regulated cytokine (“PARC”), Platelet factor 4 (“PF4”), Stroma cell-derived factor-1 alpha (“SDF-1^”), Chemokine (C-C motif) ligand 17 (“TARC”), Thymus-expressed chemokine (“TECK”), Thymic stromal lymphopoietin (“TSLP 4-1BB”), CD166 antigen (“ALCAM”), Cluster of Differentiation 80 (“B7-1”),Tumor necrosis factor receptor superfamily member 17 (“BCMA”), Cluster of Differentiation 14 (“CD14”), Cluster of Differentiation 30 (“CD30”), Cluster of Differentiation 40 (“CD40 ligand”), Carcinoembryonic antigen-related cell adhesion molecule 1 (biliary glycoprotein) (“CEACAM-1“), Death Receptor 6 (“DR6”), Deoxythymidine kinase (“Dtk”), Type 1 membrane glycoprotein (“Endoglin”), Receptor tyrosine-protein kinase ErbB-3 (“ErbB3”), Endothelial-leukocyte adhesion molecule 1 (“E-Selectin”), Apoptosis antigen 1 (“Fas”), Fms-like tyrosine kinase 3 (“Flt-3L”), Tumor necrosis factor receptor superfamily member 1 (“GITR”), Tumor necrosis factor receptor superfamily member 14 (“HVEM”), Intercellular adhesion molecule 3 (“ICAM-3”), IL-1R4, IL1R1, IL-101Rβ, IL-17R, IL-2Rɣ, IL-21R, Lysosome membrane protein 2 (“LIMPH”), Neutrophil gelatinase-associated lipocalin (“Lipocalin-2”), CD62L (“L-Selectin”), Lymphatic endothelium (“LYVE-1), MHC class I polypeptide-related sequence A (“MICA”), MHC class I polypeptide- related sequence B (“MICB”), NRG1-β1, Beta-type platelet-derived growth factor receptor (“PDGFRβ”), Platelet endothelial cell adhesion molecule (“PECAM-1”), RAGE, Hepatitis A virus cellular receptor 1 (“TIM-1”), Tumor necrosis factor receptor superfamily member 3 (“TRAIL-R3”), Trappin protein transglutaminase binding domain (“Trappin-2”), Urokinase receptor (“uPAR”), Vascular cell adhesion protein 1 (“VCAM-1”), XEDAR, Activin A, Agouti -related protein (“AgRP”), Ribonuclease 5 (“Angiogenin”), Angiopoietin 1, Angiostatin, Cathepsin S, CD40, Cryptic family protein 1 (“Cripto-1”), DAN, Dickkopf-related protein 1 (“DKK-1”), E-Cadherin, Epithelial cell adhesion molecule (“EpCAM”), Fas Ligand (FasL or CD95L), FcɣRIIB / C, Follistatin, Galectin-7, Intercellular adhesion molecule 2 (“ICAM-2”), IL-13R1, IL-13R2, IL-17B, IL-2 receptor alpha (“IL2RA”), IL-2 receptor beta (IL2RB), IL-23, LAP, Neuronal cell adhesion molecule (“NRCAM”), Plasminogen activator inhibitor-1 (“PAI-1”), Platelet derived growth factor receptors (“PDGF- AB”), Resistin, stromal cell-derived factor 1 (“SDF-1β”), soluble glycoprotein 130 (“sgp130”), Secreted frizzled-related protein 2 (“SFRP2”), Sialic acid-binding immunoglobulin-type lectin 5 (“Siglec-5”), ST2, Transforming growth factor-beta 2 (“TGFβ2”), Tie-2, Thrombopoietin (“TPO”), Tumor necrosis factor receptor superfamily member 10D (“TRAIL-R4”), Triggering receptor expressed on myeloid cells 1 (“TREM-1”), Vascular endothelial growth factor C (“VEGF-C”), VEGFR1, Adiponectin, Adipsin (“AND”), Alpha-fetoprotein (“AFP”), Angiopoietin-like 4 (“ANGPTL4”), Beta-2-microglobulin (“β2M”), Basal cell adhesion molecule (“BCAM”), Carbohydrate antigen 125 (“CA125”), Cancer Antigen 15-3 (“CA15-3”), Carcinoembryonic antigen (“CEA”), cAMP receptor protein (“CRP”), Human Epidermal Growth Factor Receptor 2 (“ErbB2”), Follicle-stimulating hormone (“FSH”), Chemokine (C-X-C motif) ligand 1 (“GRO^”), human chorionic gonadotropin (“βHCG”), Insulin-like growth factor 1 receptor 1 (“IGF1R1”), IL-2R2, IL-3, IL-18Rβ, IL-21, Leptin, Matrix metalloproteinase- 1 (“MMP-l”), Matrix metalloproteinase-2 (“MMP-2”), Matrix metalloproteinase-3 (“MMP-3”), Matrix metalloproteinase-8 (“MMP-8”), Matrix metalloproteinase-9 (“MMP-9”), Matrix metalloproteinase-10 (“MMP-10”), Matrix metalloproteinase-13 (“MMP-13”), Neural Cell Adhesion Molecule (“NCAM-1”), Entactin-1 (“Nidogen-1”), Neuron specific enolase (“NSE”), Oncostatin M (“OSM”), Procalcitonin, Prolactin, Prostate specific antigen (“PSA”), Sialic acid-binding Ig-like lectin 9 (“Siglec-9”), ADAM 17 endopeptidase (“TACE”), Thyroglobulin, Metalloproteinase inhibitor 4 (“TIMP-4”), TSH2B4, Disintegrin and metalloproteinase domain-containing protein 9 (“ADAM-9”), Angiopoietin 2,Tumor necrosis factor ligand superfamily member 13 / Acidic leucine-rich nuclear phosphoprotein 32 family member B (“APRIL”), Bone morphogenetic protein 2 (“BMP-2”), Bone morphogenetic protein 9 (“BMP-9”), Complement component 5a (“C5a”), Cathepsin L, CD200, CD97, Chemerin, Tumor necrosis factor receptor superfamily member 6B (“DcR3”), Fatty acid-binding protein 2 (“FABP2”), Fibroblast activation protein, alpha (“FAP”), Fibroblast growth factor 19 (“FGF-19”), Galectin-3, Hepatocyte growth factor receptor (“HGF R”), IFN-^ / β R2, Insulin-like growth factor 2 (“IGF-2”), Insulin-like growth factor 2 receptor (“IGF2R”), Interleukin-l receptor 6 (“IL1R6”), Interleukin 24 (“IL-24”), Interleukin 33 (“IL-33”, Kallikrein 14, Asparaginyl endopeptidase (“Legumain”), Oxidized low-density lipoprotein receptor 1 (“LOX-l”), Mannose-binding lectin (“MBL”), Neprilysin (“NEP”), Notch homolog 1, translocation-associated (Drosophila) (“Notch-1”), Nephroblastoma overexpressed (“NOV”), Osteoactivin, Programmed cell death protein 1 (“PD-1”), N- acetylmuramoyl-L-alanine amidase (“PGRP-5”), Serpin A4, Secreted frizzled related protein 3 (“sFRP-3”), Thrombomodulin, Toll-like receptor 2 (“TLR2”), Tumor necrosis factor receptor superfamily member 10A (“TRAIL-R1”), Transferrin (“TRF”), WIF-1, ACE-2, Albumin, AMICA, Angiopoietin 4, B-cell activating factor (“BAFF”), Carbohydrate antigen 19-9 (“CA19-9”), CD163 , Clusterin, CRT AM, Chemokine (C-X-C motif) ligand 14 (“CXCL14”), Cystatin C, Decorin (“DCN”), Dickkopf-related protein 3 (“Dkk-3”), Delta-like protein 1 (“DLL1”), Fetuin A, Heparin-binding growth factor 1 (“aFGF”), Folate receptor alpha (“FOLR1”), Furin, GPCR-associated sorting protein 1 (“GASP-1”), GPCR-associated sorting protein 2 (“GASP-2”), Granulocyte colony-stimulating factor receptor (“GCSFR”), Serine protease hepsin (“HAI-2”), Interleukin-17B Receptor (“IL-17BR”), Interleukin 27 (“IL-27”), Lymphocyte-activation gene 3 (“LAG-3”), Apolipoprotein A-V (“LDLR”), Pepsinogen I, Retinol binding protein 4 (“RBP4”), SOST, Heparan sulfate proteoglycan (“Syndecan-l”), Tumor necrosis factor receptor superfamily member 13B (“TACI”), Tissue factor pathway inhibitor (“TFPI”), TSP-1, Tumor necrosis factor receptor superfamily member 10B (“TRAILR2”), TRANCE, Troponin I, Urokinase Plasminogen Activator (“uPA”), Cadherin 5, type 2 or VE- cadherin (vascular endothelial) also known as CD144 (“VE-Cadherin”), WNT1-inducible-signaling pathway protein 1 (“W1SP-1”), and Receptor Activator of Nuclear Factor κB (“RANK”).

[0184] In some embodiments, the subject is also administered an immune checkpoint inhibitor. Immune Checkpoint inhibition broadly refers to inhibiting the checkpoints that cancer cells can produce to prevent or downregulate an immune response. Examples of immune checkpoint proteins include, but are not limited to, CTLA4, PD-1, PD-L1, PD-L2, A2AR, B7-H3, B7-H4, BTLA, KIR, LAG3, TIM-3 or VISTA. Immune checkpoint inhibitors can be antibodies or antigen binding fragments thereof that bind to and inhibit an immune checkpoint protein. Examples of immune checkpoint inhibitors include, but are not limited to, nivolumab, pembrolizumab, pidilizumab, AMP-224, AMP-514, STI-A1110, TSR-042, RG-7446, BMS-936559, MEDI-4736, MSB-0020718C, AUR-012 and STI-A1010.

[0185] In some embodiments, a composition provided herein (e.g., a vaccine composition described above and / or elsewhere herein) is administered prophylactically to prevent cancer, precancerous lesions, and / or an HPV infection. In some embodiments, the vaccine is administered to inhibit tumor cellexpansion. The vaccine may be administered prior to or after the detection of cancer cells, precancerous lesions, or HPV infected cells in a patient. Inhibition of tumor cell expansion is understood to refer to preventing, stopping, slowing the growth, or killing of tumor cells. In some embodiments, after administration of a vaccine comprising peptides, nucleic acids, antibodies or APCs described herein, a proinflammatory response is induced. The proinflammatory immune response comprises production of proinflammatory cytokines and / or chemokines, for example, interferon gamma (IFN-ɣ) and / or interleukin 2 (IL-2). Proinflammatory cytokines and chemokines are well known in the art.

[0186] In some embodiments, the pharmaceutical compositions described above and / or elsewhere herein may be used for boosting adoptively transferred HPV-specific T cells. In some embodiments, the T cells are genetically modified T cells (e.g., TCR / CAR T cells). Delivery Methods and Dosage

[0187] The vector may be prepared for administration by being suspended or dissolved in a pharmaceutically or physiologically acceptable carrier such as isotonic saline; isotonic salts solution or other formulations that will be apparent to those skilled in the art. The appropriate carrier will be evident to those skilled in the art and will depend in large part upon the route of administration. The compositions described herein may be administered to a mammal in a sustained release formulation using a biodegradable biocompatible polymer, or by on-site delivery using micelles, gels and liposomes.

[0188] In some embodiments, the recombinant viral vectors of the invention may be administered to a subject by intradermal administration, intramuscular injection, subcutaneous injection, intranasal administration, intravenous injection, intravaginal injection, intraperitoneal injection, epicutaneous administration, or oral administration. In some preferred embodiments, the administration is by intradermal injection. In some embodiments, the administration is by intramuscular injection.

[0189] If the therapeutic regimen involves co-administration of one or more viral vectors and a further component, each formulated in different compositions, they are favourably administered co- locationally at or near the same site. For example, the components can be administered (e.g., via an administration route selected from intramuscular, transdermal, intradermal, sub-cutaneous) to the same side or extremity (“co-lateral” administration) or to opposite sides or extremities (“contra-lateral” administration).

[0190] Conjunctive therapy includes sequential, simultaneous and separate, and / or co- administration of the active compounds in such a way that the therapeutic effects of the first agent administered have not entirely disappeared when the subsequent treatment is administered. In some embodiments, the second agent may be co-formulated with the first agent or be formulated in a separate pharmaceutical composition.

[0191] Dosages of the viral vector will depend primarily on factors such as the condition being treated, the age, sex, weight, condition, health, and prior medical history of the patient being treated, and may thus vary among patients. Furthermore, actual dosage levels of the active ingredients in the pharmaceutical compositions provided herein may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of factors including the activity of the particular agent employed, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound being employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compound employed.

[0192] For example, a therapeutically effective adult human or veterinary dosage of the viral vector generally contains 1 x 105to 1 x 1015viral particles, such as from 1 x 108to 1 x 1012(e.g., 1 x 108, 2.5 x 108, 5 x 108,1 x 109, 1.5 x 109, 2.5 x 109, 5 x 109, 1 x 1010, 1.5 x 1010, 2.5 x 1010, 5 x 1010, 1 x 1011, 1.5 x 1011, 2.5 x 1011, 5 x 1011, 1 x 1012particles). Alternatively, a viral vector can be administered at a dose that is typically from 1 x 105to 1 x 1010plaque forming units (PFU), such as 1 x 105PFU, 2.5 x 105PFU, 5 x 105PFU, 1 x 106PFU, 2.5 x 106PFU, 5 x 106PFU,1 x 107PFU, 2.5 x 107PFU, 5 x 107PFU, 1 x 108PFU, 2.5 x 108PFU, 5 x 108PFU, 1 x 109PFU, 2.5 x 109PFU, 5 x 109PFU, or 1 x 1010PFU. Dosages will vary depending upon the size of the animal and the route of administration. For example, a suitable human or veterinary dosage (for about an 80 kg animal) for intramuscular injection is in the range of about 1 x 109to about 5 x 1012particles per mL, for a single site. Optionally, multiple sites of administration may be used. In another example, a suitable human or veterinary dosage may be in the range of about 1 x 1011to about 1 x 1015particles for an oral formulation.

[0193] The viral vector can be quantified by Quantitative PCR Analysis (Q-PCR), for example with primers and probe designed on CMV promoter region using as standard curve serial dilution of plasmid DNA containing the vector genome with expression cassette including HCMV promoter. The copy number in the test sample is determined by the parallel line analysis method. Alternative methods for vector particle quantification can be analytical HPLC or spectrophotometric method based on A260 nm.

[0194] An immunologically effective amount of a nucleic acid may suitably be between 1 ng and 100 mg. For example, a suitable amount can be from 1 µg to 100 mg. An appropriate amount of the particular nucleic acid (e.g., vector) can readily be determined by those of skill in the art. Exemplary effective amounts of a nucleic acid component can be between 1 ng and 100 µg, such as between 1 ng and 1 µg (e.g., 100 ng to 1 µg), or between 1 µg and 100 µg, such as 10 ng, 50 ng, 100 ng, 150 ng, 200 ng, 250 ng, 500 ng, 750 ng, or 1 µg. Effective amounts of a nucleic acid can also include from 1 µg to 500 µg, such as between 1 µg and 200 µg, such as between 10 and 100 µg, for example 1 µg, 2 µg, 5 µg, 10 µg, 20 µg, 50 µg, 75 µg, 100 µg, 150 µg, or 200 µg. Alternatively, an exemplary effective amount of a nucleic acid canbe between 100 µg and 1 mg, such as from 100 µg to 500 µg, for example, 100 µg, 150 µg, 200 µg, 250 µg, 300 µg, 400 µg, 500 µg, 600 µg, 700 µg, 800 µg, 900 µg, or 1 mg.

[0195] Generally, a human dose will be in a volume of between 0.1 mL and 2 mL. Thus, the composition described herein can be formulated in a volume of, for example 0.1, 0.15, 0.2, 0.5, 1.0, 1.5, or 2.0 mL human dose per individual or combined immunogenic components.

[0196] One of skill in the art may adjust these doses, depending on the route of administration and the therapeutic or vaccine application for which the recombinant vector is employed. The levels of expression of the transgene, or for an adjuvant, the level of circulating antibody, can be monitored to determine the frequency of dosage administration.

[0197] If one or more priming and / or boosting steps are used, this step may include a single dose that is administered hourly, daily, weekly or monthly, or yearly. As an example, mammals may receive one or two doses containing between about 10 µg to about 50 µg of plasmid in carrier. The amount or site of delivery is desirably selected based upon the identity and condition of the mammal.

[0198] The therapeutic levels of, or level of immune response against, the protein encoded by the selected transgene can be monitored to determine the need, if any, for boosters. Following an assessment of CD8+ T cell response, or optionally, antibody titers, in the serum, optional booster immunizations may be desired. Optionally, the recombinant viral vectors may be delivered in a single administration or in various combination regimens (e.g., in combination with a regimen or course of treatment involving other active ingredients or in a prime-boost regimen).

[0199] In some embodiments a prime-boost administration regimen of the pharmaceutical compositions of the invention. In some embodiments of this type, the composition used in the first priming dose is not the same as the composition used in the second and subsequent booster dose. By way of an illustrative example, the priming dose could comprise an adenovirus vector composition, whereas the booster dose may be an mRNA composition.

[0200] In some embodiments, the plurality of epitopes is the same in the first priming dose as the second and subsequent booster doses (i.e., only the modality of the pharmaceutical composition has changed, e.g., polypeptide and gene therapy; Adenovirus and mRNA molecule).

[0201] In some specific embodiments, the priming dose is a viral vector that includes a nucleotides sequence as set forth in SEQ ID NO: 38. In some embodiments of this type, the booster does in an mRNA composition. Methods of Detection

[0202] In some aspects, provided herein is a method of identifying a subject suitable for a therapy provided herein (methods of treating an HPV infection, a cancer, and / or precancerous lesions in a subject comprising administering to the subject a pharmaceutical composition provided herein). In some embodiments, the method comprises isolating a sample from the subject (e.g., a blood sample, a tissue sample, a tumor sample) and detecting the presence of an HPV epitope listed in Table 1 in the sample. In some embodiments the epitope is detected using an ELISA assay, a western blot assay, a FACS assay, a fluorescent microscopy assay, an Edman degradation assay and / or a mass spectrometry assay (e.g., protein sequencing). In some embodiments, the presence of the HPV epitope is detected by detecting a nucleic acid encoding the HPV epitope. In some embodiments, the nucleic acid encoding the HPV epitope is detected using a nucleic acid probe, a nucleic acid amplification assay and / or a sequencing assay.

[0203] Examples of nucleic acid amplification assays that can be used in the methods provided herein include, but are not limited to polymerase chain reaction (PCR), LATE-PCR, ligase chain reaction (LCR), strand displacement amplification (SDA), transcription mediated amplification (TMA), self- sustained sequence replication (3 SR), Qβ replicase based amplification, nucleic acid sequence-based amplification (NASBA), repair chain reaction (RCR), boomerang DNA amplification (BDA) and / or rolling circle amplification (RCA).

[0204] In some embodiments the product of the amplification reaction is detected as an indication of the presence and / or identity of the bacteria in the sample. In some embodiments, the amplification product is detected after completion of the amplification reaction (i.e., endpoint detection). Examples of end-point detection methods include gel-electrophoresis based methods, probe-binding based methods (e.g., molecular beacons, HPA probes, lights-on / lights-off probes) and double-stranded DNA binding fluorescent-dye based methods (e.g., ethidium bromide, SYBR-green). In some embodiments, the amplification product is detected as it is produced in the amplification reaction (i.e., real-time detection). Examples of real-time detection methods include probe-binding based methods (e.g., molecular beacons, TaqMan probes, scorpion probes, lights-on / lights-off probes) and double-stranded DNA binding fluorescent-dye based methods (e.g., ethidium bromide, SYBR-green). In some embodiments, the product of the amplification reaction is detected and / or identified by sequencing (e.g., through the use of a sequencing assay described herein).

[0205] In some embodiments, the detection of the nucleic acid sequence comprises contacting the nucleic acid sequence with a nucleic acid probe that hybridizes specifically to the nucleic acid sequence. In some embodiments, the probe is detectably labeled. In some embodiments, the probe is labeled (directly or indirectly) with a fluorescent moiety. Examples of fluorescent moieties useful in the methods provided herein include, but are not limited to Allophycocyanin, Fluorescein, Phycoerythrin, Peridinin-chlorophyll protein complex, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594,Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, GFP, RFP, YFP, EGFP, mPlum, mCherry, mOrange, mKO, EYFP, mCitrine, Venus, YPet, Emerald, Cerulean and CyPet. In some embodiments, the probe is a molecular beacon probe, a molecular torch probe, a TaqMan probe, a SDA probe, a scorpion probe, a HPA probe, or a lights on / lights off probe.

[0206] In some embodiments, the nucleic acid sequence is detected by sequencing (e.g., whole genome sequencing, transcriptome sequence and / or targeted gene sequencing). Examples of sequencing processes that can be used in the methods provided herein include, but are not limited to, chain termination sequencing, massively parallel signature sequencing, ion semiconductor sequencing, polony sequencing, Illumina sequencing, sequencing by ligation, sequencing by synthesis, pyrosequencing, single-molecule real-time sequencing, SOLiD sequencing, DNA nanoball sequencing, heliscope single molecule sequencing, single molecule real time sequencing, 454 sequencing, nanopore sequencing, tunneling currents DNA sequencing or sequencing by hybridization.

[0207] In some embodiments, the methods provided herein further comprise treating the identified subject using a therapeutic method provided herein (e.g., by administering to the subject a pharmaceutical composition provided herein. EXPERIMENTAL Example 1 Construction of Adenovirus HPV Polyepitopes

[0208] To generate recombinant adenovirus encoding multiple HPV CD8+ T cell epitopes as a polyepitope construct, HLA class I restricted CD8+ T cell from multiple HPV antigens were selected. Table 7 lists HPV T cell epitopes included in each HPV polyepitope sequence (referred to as HPVpoly-1, HPVpoly-2, HPVpoly-3, HPVpoly-4, and HPVpoly-5). These polyepitope sequences target 24-48 HLA class I alleles and cover 94-99% of a multi-ethnic population worldwide (see, Figure 1). Codon optimized sequences of each HPV polyepitope are set forth in SEQ ID NOs: 36, 37, 38, 39, 40. TABLE 7 List of HLA class I-restricted CD8+ epitopes Construct E ito e Se uence Anti en HLA Restriction(s)TIHDIILECV HPV-16-E6 A*02:01 SEYRHYCYSL HPV-16-E6 B*40:01 *SEYRHYCYSL HPV16-E6 HLA B*40:01 IHDIILECVY HPV16-E6 HLA B*18:01 *YLHNRLVVFK HPV16-E1 HLA B*08:01 IILECVYCKR HPV16-E6 HLA A*11:01I pathway proteasome liberation amino acids (i.e., AD, K, or R residues) were added at the carboxyl-terminus of each epitope in HPVpoly-5 (see, Table 1, underlined residues). The nucleotide acid sequence encoding the polyepitope with Nhe I and Kpn I restriction sites at 5’ and 3’ respectively was cloned into the pShuttle expression vector. Following amplification. The expression cassette from pShuttle was sub-cloned into an Ad5F35 expression vector (see, Figure 2; referred to as AdHPVpoly-1, AdHPVpoly-2, AdHPVpoly-3, AdHPVpoly-4, and AdHPVpoly-5).

[0210] The recombinant Ad5F35 vector was transfected into human embryonic kidney HEK293 cells and recombinant adenovirus stocks were produced in HEK293 cells (see, Figure 3). Endogenous Processing of HPV CD8+ T cell epitopes from Recombinant Polyepitopes

[0211] To assess the processing and presentation of HPV CD8+T cell epitope encoded by recombinant adenoviral constructs, HLA matched fibroblasts infected with AdHPVpoly-1, AdHPVpoly-2, AdHPVpoly-3, AdHPVpoly-4, or AdHPVpoly-5 were exposed to HPV TCR transgenic T cells. Following incubation, these T cells were assessed for polyfunctional effector function. Data presented in Figure 5 illustrates that HLA A*02:01-restricted TIHDIILECV (HPV E6; SEQ ID NO: 8) and TLQDVSLEVYL (HPV E2; SEQ ID NO: 20) and HLA A*01:01-restricted QVDYYGLYY (HPV E2; SEQ ID NO: 7) epitopes were efficiently processed and presented from all five HPV polyepitope constructs and stimulated HLA matched TCR transgenic T cells. These T cells also showed mobilization of CD107 which is a surrogate marker for T cell mediated cytotoxicity.

[0212] To further characterize immunogenicity of the HPVpoly constructs, PBMCs from six HPV16+HNC patients were stimulated with AdHPVpoly-1 and these cells were cultured for 14 days to expand pre-existing memory HPV-specific CD8+ T cells. The AdHPVpoly-1 construct induced expansion of functional HPV-specific CD8+ T cells against multiple HPV epitopes in three out of six HNC patients (see, Figure 6). These expanded HPV-specific CD8+T cells showed polyfunctional effector function as indicated by section of IFN-γ and TNF. In Vivo Priming of HPV-Specific T cells with AdHPVpoly Constructs

[0213] To evaluate the immunogenicity of recombinant HPV polyepitope vaccine, five groups of HLA transgenic mice expressing HLA A*01:01, HLA A*02:01, HLA B*35:01, HLA A*24:02 or HLA B*08:01 were each vaccinated with recombinant AdHPV poly vaccine (108PFUs) via intramuscular route with threedoses of vaccine on day 0, 21 and 42. Peripheral blood was collected on days 21, 28, 42, and 49 and assessed for ex vivo HPV-specific CD8+T cell responses using intracellular cytokine assays. In addition, HPV-specific T cell responses were also assessed (ex vivo and following in vitro expansion) in spleen on day 49.

[0214] Immunization with AdHPVpoly-1 vaccine induced robust HPV-specific CD8+T cell response in the peripheral blood and spleen of HLA A1, HLA A2, HLA B8 and HLA B35 transgenic mice on day 21, 28, 42 and 49 (see, Figure 6A & 6B). However, no HPV-specific CD8+T cell responses were observed in HLA A24 transgenic mice in blood and spleen (see, Figure 6A). In vitro culture of splenocytes with HLA matched peptide epitopes showed expansion of T cells specific for multiple epitopes in HLA A1 and A2 transgenic mice and individual epitopes in HLA B8 and B35 transgenic mice (see, Figure 6C).

[0215] AdHPVpoly-2 which included 48 overlapping HLA class I-restricted epitopes (see, Table 2), induced robust HPV-specific CD8+T cell response in HLA A24 mice and comparable responses in HLA A1, HLA B8 and HLA B35 transgenic mice in blood and spleen on day 21, 28, 42 and 49 (see, Figure 7A & 7B). Ex vivo T cell responses induced in HLA A24A transgenic mice ranged from 10-20% and these T cell expanded to >80% following in vitro stimulation (see, Figure 7C). AdHPVpoly-2 showed low or undetectable HPV-specific CD8+T cell responses ex vivo in HLA A2 transgenic mice when compared with AdHPVpoly-1 (see, Figure 7A & 7B). However, in vitro stimulation of splenocytes with HLA A2-restricted peptide epitopes showed massive expansion of T cells directed to multiple HLA A2-restricted epitopes (see, Figure 7C). Similar T cell expansions were also observed following in vitro stimulation of splenocytes from HLA A1, HLA B8, HLA B35 and HLA A24 mice (see, Figure 7C).

[0216] AdHPVpoly-3, AdHPVpoly-4, and AdHPVpoly-5 includes 20 HLA class I-restricted HPV CD8+T cell epitopes with minor modifications in the encoding sequences. AdHPVpoly-3 and AdHPVpoly-4 differ in the N-terminal sequence with a longer peptide sequence for the first epitope in AdHPVpoly-3 (see, Table 3 and Table 4). AdHPVpoly-5 includes proteasome liberation amino acids (AD or K or R) to optimize the endogenous processing and presentation of HPV epitopes through HLA class I pathway (see, Table 5). These vectors were tested in HLA A2, HLA A24 and HLA B35 transgenic mice. Ex vivo analysis of T cell responses in these transgenic mice following immunization with AdHPVpoly-4 and AdHPVpoly-5 showed low to medium level T cell responses in peripheral blood and spleen (see, Figure 9A & 9B and Figure 10A & 10B). In vitro stimulation of splenocytes with HLA matching HPV peptide epitopes led to dramatic expansion of T cell responses directed to multiple epitopes in HLA A2, HLA A24 and HLA B35 transgenic mice (see, Figure 9C and 10C). In contrast, ex vivo analysis of T cells in peripheral blood and spleen from HLA transgenic mice immunized with AsHPVpoly-3 showed moderate to high levels of T cells responses in HLA A2, HLA A24 and HLA B35 mice (see, Figure 8A & 8B). In vitro stimulation of splenocytes with HPV peptide epitopes led to 5-10 fold expansion of antigen-specific T cells in all three HLA-transgenic mice (see, Figure 8C).Materials & Methods Antigen Presentation Assay and Intracellular Cytokine Secretion Assay

[0217] To determine the processing and presentation of HPV-specific CD8+T cell epitopes encoded by recombinant adenoviral vectors, primary human fibroblast cells (HLA A*01:01, A*02:01, B*08:01, B*51:01) were infected with AdHPVpoly-1, AdHPVpoly-2, AdHPVpoly-3, AdHPVpoly-4, or AdHPVpoly-5 at the multiplicity of infection (MOI) of 10:1. Prior to infection, these fibroblasts were treated with recombinant IFNγ (100 IU / mL) for 24 hours to enhance surface HLA class I expression. Uninfected or peptide epitope pulsed fibroblasts were used as negative and positive controls respectively. These target cells were then co-cultured with HLA matched CD8+T cells expressing transgenic TCR specific for HLA A*02:01-restricted TIHDIILECV (HPV E6; SEQ ID NO: 8) and TLQDVSLEVYL (HPV E2; SEQ ID NO: 20) and HLA A*01:01-restricted QVDYYGLYY (HPV E2; SEQ ID NO: 7) epitopes. Following incubation, these T cells were assessed for expression of IFNγ, TNF, IL-2 and CD107 mobilization using intracellular cytokine assays. In Vitro Expansion of HPV-Specific T Cells Using Adenoviral Polyepitopes

[0218] Peripheral blood mononuclear cells (PBMC) isolated from HPV+HNC patients were revived from frozen stocks and rested for at least 1 hour at 37°C before using for in vitro T cell expansion. These PBMCs were divided into responder and stimulator cells at a responder to stimulator ratio of 2:1. The stimulator cells were infected with AdHPVpoly-1, AdHPVpoly-2, AdHPVpoly-3, AdHPVpoly-4, or AdHPVpoly-5 at a MOI of 10:1 for 1 hour at 37°C. Unbound virus particles were washed off and the stimulator cells were co-cultured with the responder cells in RPMI-1640 medium supplemented 10% fetal calf serum (growth medium) and recombinant IL-2 (rIL-2; 120 IU / mL). Every 3-4 days the cultures were supplemented with growth medium and rIL-2. HPV-specific T cell expansion was tested on day 14 using an intracellular cytokine assay. Characterization of HPV-Specific CTL by Intracellular Cytokine Assay and Flow Cytometry

[0219] In vitro expanded HPV-specific T cells or TCR transgenic T cells were stimulated with 1 µg / mL HPV peptide epitope(s) corresponding to defined HLA class I-restricted CD8+T cell epitopes derived from HPV antigens and incubated in the presence of a CD107a-antibody, Brefeldin A and Monensin for 5 hours. After surface staining for CD8 and CD4, cells were fixed and permeabilized with cytofix / cytoperm and strain for IFNγ, IL-2, and TNF. Stained cells were resuspended in phosphate buffered saline containing 2% paraformaldehyde and acquired using a LSR Fortessa with FACSDiva software (BD Biosciences) Post acquisition analysis was conducted using FlowJo software (version 10.1r7). AdHPVpoly Immunization in HLA Transgenic Mice

[0220] All animal immunization protocols were conducted in compliance with the QIMR Berghofer Medical Research Institute Animal Ethics Committee. HLA A*01:01, HLA A*02:01, HLA B*35:01,HLA A*24:02 and HLA B*08:01 transgenic mice were maintained in a pathogen free animal facility at QIMR Berghofer. Two groups (placebo and prime-boost) of 6-8 week old mice (for each HLA allele) were injected intramuscularly with 50 µL phosphate buffered saline or AdHPVpoly-1, AdHPVpoly-2, AdHPVpoly-3, AdHPVpoly-4, or AdHPVpoly-5 (1 x 108pfu / mL). Booster doses were given on day 21 and day 42 to the prime boost group. Peripheral blood samples were collected from all animals on day 21, 28, 42, and 49. These blood samples were assessed from HPV epitope-specific T cell response using an intracellular cytokine staining assay. Mice were sacrificed on day 49, splenocytes from all the groups were stimulated in vitro with HLA matched HPV peptide epitopes. Splenocytes were cultured in a 24-well plate for 10 days at 37°C, 10% CO2. On day 3 and day 6, cultures were supplemented with growth medium containing rIL-2. T cell specificity was assessed using an intracellular cytokine staining assay.

[0221] The group difference between mice immunized with placebo and AdHPVpoly constructs was evaluated by a linear mixed-effect model with time, group, and the interaction of time and group as predictors. Example 2 IMMUNOGENICITY EVALUATION OF MRNA BASED HPVPOLY3 AND HPVPOLY5 VACCINE FORMULATIONS IN HLA TRANSGENIC MICE

[0222] To evaluate the immunogenicity of recombinant HPV polyepitope vaccine, HLA transgenic mice expressing HLA A*01:01, HLA B835:01, HLA A*24:02 and HLA B*08:01 were vaccinated with recombinant mRNA poly vaccine (5 µg / dose) via intramuscular route with three doses of vaccine on days 0, 21 and 42. Peripheral blood was collected on Days 21, 28, 42 and 49 and assessed for ex vivo HPV-specific CD8+ T cell responses using intracellular cytokine assays. In addition, HPV-specific T cell responses were also assessed (ex vivo and following in vitro expansion) in spleen on day 49. Data presented in Figure 11A and 11B show that immunization with mRNApoly-3 vaccine induced robust HPV- specific CD8+T cell response in the peripheral blood and spleen of HLA A1, HLA B8, HLA A24 and HLA B35 transgenic mice on Days 21, 28, 42 and 49. Data in Figure 11C show that in vitro culture of splenocytes with HLA matched peptide epitopes showed expansion of HPV-specific CD8+ T cells against multiple epitopes in HLA A1, HLA B8, HLA A24 and HLA B35 transgenic mice. Similarly data in Figure 12A and 12B show that immunization with mRNApoly-5, which includes proteasome liberation amino acids (AD or K or R) to optimise the endogenous processing and presentation of HPV epitopes through HLA class I pathway (Table 2) also induced robust HPV-specific CD8+T cell responses directed to multiple HLA A1, HLA A2, HLA A24 and HLA B8-restricted epitopes. Data presented in Figure 12C shows that in vitro culture of splenocytes with HLA matched peptides epitopes induced robust expansion of HPV-specific CD8+ T cell responses in all HLA transgenic mice. However, in comparison between mRNApoly-3 and mRNApoly-5; exvivo analysis in spleen shows that superior HPV-specific CD8+ T cells responses were observed with mRNApoly-3 vaccine in HLA B8 and B35 mice (Figure 11B and 12B). Materials & Methods Construction of mRNA HPV polyepitopes

[0223] To generate recombinant mRNA encoding multiple HPV CD8+T cell epitopes as a polyepitope, HLA class I restricted CD8+T cell epitopes from multiple HPV antigens were selected. Table 1-2 list HPV T cell epitopes included in each HPV polyepitope sequence (referred to as HPVpoly-m3 and HPVpoly-m5). These polyepitope sequences target 24-48 HLA class I alleles and they cover 94-99% of a multi-ethnic population worldwide (Figure 1). To optimise the processing and presentation of HPV epitopes through HLA class I pathway proteasome liberation amino acids (AD or K or R) were added at the carboxyl- terminus of each epitope in HPVpoly-5 (Table 2). HLA transgenic mice immunisation

[0224] All animal immunization protocols were conducted in compliance with the QIMR Berghofer Medical Research Institute Animal Ethics Committee. HLA A*01:01, HLA B35:01, HLA A*24:02 and HLA B*08:01 transgenic mice were maintained in a pathogen–free animal facility at QIMR Berghofer. Mice were immunised with either the mRNA-based HPVpoly3 or HPVpoly5 vaccine (vaccine group, n = 6) or phosphate-buffered saline (PBS; placebo group, n = 4). Mice received booster doses on days 21 and 42 following the primary immunisation. Blood was collected from the tail vein on days 21, 28, 42, and 49. HPV- specific CD8+ T cell responses were analysed in both the blood and spleen. Splenocytes from all the groups were stimulated in vitro with HLA matched HPV peptide epitopes. Splenocytes were cultured in a 24-well plate for 10 days at 37°C, 10% CO2. On days 3 and 6, cultures were supplemented with growth medium containing recombinant IL-2. T-cell specificity was assessed using an intracellular cytokine staining assay. Example 3 HOMOLOGUES AND HETEROLOGOUS BOOST VACCINATION STRATEGY EVALUATION

[0225] Heterologous booster vaccines, which employ a different vaccine platform for the booster dose compared to the primary vaccination series, may offer advantages such as enhanced CD8+ T cell responses against infections and cancer by combining different immunological properties of various vaccines, as well as reduced toxicity. Thus, we aimed to evaluate heterologous and homologous booster strategies designed to improve the immunogenicity of the HPV vaccine. As stated above, we developed two distinct vaccine formulations: a replication-defective adenoviral vector (AdHPVpoly) and an mRNA– LNP delivery platform (HPVpoly mRNA). Both vaccine formulations express the HPVpoly3 protein. Then, we evaluated immunogenicity in HLA-A1 transgenic mice.

[0226] In order to test the immunogenicity of the HPVpoly3 vaccine in homologous and heterologous settings, the first two groups of HLA-A1 transgenic mice were immunized with AdHPVpoly3 (10^8 viral particles per dose); the next two groups were immunized with the mRNApoly3 vaccine, and the control group was vaccinated with phosphate-buffered saline (PBS). Twenty-one days after the primary dose, mice were immunized with a booster dose administered using either a homologous or heterologous strategy. Mice were sacrificed seven days after the booster dose, and immune responses were analyzed in both the blood and spleen.

[0227] To evaluate the immunogenicity of HPV vaccine in homologous and heterologous manner, two groups (n = 6 / group) of HLA-A1 transgenic mice were immunized with AdHPVpoly3 (108viral particles per dose). The next two groups were immunized with the HPVpoly3 mRNA vaccine, and the control group was vaccinated with phosphate-buffered saline (PBS). Twenty-one days after the primary dose, mice were immunized with a booster dose using either a homologous or heterologous strategy. Priming with AdHPVpoly-3 vaccine and mRNApoly-3 vaccine induced comparable HPV-specific CD8+ T cell response in HLA A1 mice in blood on Day 21 and Day28 (Figure 13A). Compared to homologus boost with AdHPVpoly3, either homologus boost or hetrologous boost with mRNApoly-3 vaccination strategy induced slightly higher the HPV-specific CD8+T cell response in the spleen in HLA A1 transgenic mice spleens on Days 28 (Figure 13B). REFERENCES 1. Burk, R.D., A. Harari, and Z. Chen, Human papillomavirus genome variants. Virology, 2013.445(1- 2): p.232-43. 2. Della Fera, A.N., et al., Persistent Human Papillomavirus Infection. Viruses, 2021.13(2). 3. Doorbar, J., et al., Principles of epithelial homeostasis control during persistent human papillomavirus infection and its deregulation at the cervical transformation zone. Curr Opin Virol, 2021.51: p.96-105. 4. Lin, Y.Y., et al., Trends in the Incidence of Human Papillomavirus-Associated Cancers by County- Level Income and Smoking Prevalence in the United States, 2000-2018. JNCI Cancer Spectr, 2022.6(2). 5. Guo, F., et al., Incidence of human papillomavirus-related cancers among males and females aged 15-34 years in the United States. JNCI Cancer Spectr, 2023.7(2). 6. Gillison, M.L., Flashback Foreword: Human Papillomavirus and Incidence and Survival of Oropharyngeal Cancers. J Clin Oncol, 2023.41(17): p.3079-3080. 7. Villalona, S., et al., Human Papillomavirus (HPV)-Associated Cancers Among Hispanic Males in the United States: Late-Stage Diagnosis by Country of Origin. Cancer Control, 2023. 30: p. 10732748231218088.8. Rincon, N.L., et al., Racial and ethnic disparities in human papillomavirus (HPV) vaccine uptake among United States adults, aged 27-45 years. Hum Vaccin Immunother, 2024.20(1): p.2313249. 9. Wijesekera, A., et al., Surveillance of human papillomavirus through salivary diagnostics - A roadmap to early detection of oropharyngeal cancer men. Tumour Virus Res, 2024.17: p.200278. 10. Wolf, J., et al., Human papillomavirus infection: Epidemiology, biology, host interactions, cancer development, prevention, and therapeutics. Rev Med Virol, 2024.34(3): p. e2537. 11. Alhamlan, F.S., et al., Human Papillomavirus-Associated Cancers. Adv Exp Med Biol, 2021.1313: p.1-14. 12. Bekkers, R.L., et al., Epidemiological and clinical aspects of human papillomavirus detection in the prevention of cervical cancer. Rev Med Virol, 2004.14(2): p.95-105. 13. Arias-Pulido, H., et al., Human papillomavirus type 16 integration in cervical carcinoma in situ and in invasive cervical cancer. J Clin Microbiol, 2006.44(5): p.1755-62. 14. Morgan, I.M., L.J. DiNardo, and B. Windle, Integration of Human Papillomavirus Genomes in Head and Neck Cancer: Is It Time to Consider a Paradigm Shift? Viruses, 2017.9(8): p.208. 15. Bhatt, K.H., et al., Profiling HPV-16-specific T cell responses reveals broad antigen reactivities in oropharyngeal cancer patients. J Exp Med, 2020.217(10). 16. Eberhardt, C.S., et al., Functional HPV-specific PD-1(+) stem-like CD8 T cells in head and neck cancer. Nature, 2021.597(7875): p.279-284.

Claims

1. THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. An isolated polypeptide comprising a plurality of HLA class I-restricted cytotoxic T lymphocyte (CTL) epitopes selected from the T cell epitope amino acid sequence set forth in SEQ ID NOs: 1- 35, wherein the epitopes are from two or more antigens from human papillomavirus (HPV).

2. The isolated polypeptide of claim 1, wherein the HPV is serotype 16 (HPV-16).

3. The isolated polypeptide of any one of the preceding claims, wherein the epitopes are restricted by HLA class I specificities HLA-A1, -A2, -A3, -A11, -A23, -A24, -A26, -A29, -A32, -A68, -B2, -B7, -B8, -B15, -B18, -B27, -B35, -B40, -B44, -B53, -B57, -B62, -B68, -C5, -C7, and / or -C8.

4. The isolated polypeptide of claim 1 or claim 2, wherein the CTL epitopes are derived from E1, E2, E4, E5, E6 and / or E7.

5. The isolated polypeptide of claim 3, wherein polypeptide comprises the CTL epitopes selected from the amino acid sequences set forth in SEQ ID NO: 1-3, 5-20, 35.

6. The isolated polypeptide of claim 6, wherein the isolated polypeptide comprises an intervening amino acid sequence between each of the epitopes, wherein the intervening amino acid sequence comprises a proteasome liberation amino acid sequence.

7. The isolated polypeptide of claim 6, wherein the proteasome liberation amino acids or amino acid sequences comprise AD, K and / or R.

8. An polynucleotide encoding the isolated polypeptide of any one of claims 1 to 7.

9. The polynucleotide of claim 8, wherein the polynucleotide is isolated.

10. The polynucleotide of claim 8 or claim 9, which comprises the nucleotide sequence set forth in SEQ ID NO:

40.

11. The polynucleotide of claim 8 or claim 9, which comprises the nucleotide sequence set forth in SEQ ID NO:

38.

12. A genetic construct comprising the polynucleotide of any one of claims 8 to 11.

13. An expression vector comprising a polynucleotide of any one of claims 8 to 11 14. The expression vector of claim 11, wherein the expression vector is a viral vector.

15. The expression vector of claim 12, wherein the viral vector is an adenovirus-based expression vector.

16. A cell comprising polynucleotide according to any one of claims 8 to 11, or the genetic construct of claim 12, or the expression vector of claim 14 or claim 15.

17. A host cell comprising the polynucleotide according to any one of claims 8 to 11, or the genetic construct of claim 12, or the expression vector of claim 14 or claim 15.

18. The host cell of claim 15, wherein the host cell is or comprises a T cell or a CAR T cell.

19. A pharmaceutical composition comprising one or more of the isolated polypeptides of claim 1, and a pharmaceutically acceptable carrier, diluent or excipient.

20. The pharmaceutical composition of claim 17, further comprising an immunostimulatory molecule or adjuvant.

21. A vaccine that comprises the pharmaceutical composition of claim 17 for eliciting a protective immune response against a human papillomavirus in a subject.

22. The vaccine of claim 19, wherein the human papillomavirus is HPV-16.

23. The pharmaceutical composition of claim 17 or claim 18, for use in the treatment or prevention of a HPV associated cancer in subject.

24. The pharmaceutical composition of claim 21, wherein the cancer is cervical cancer, vaginal cancer, anal cancer or oropharyngeal cancer.

25. The pharmaceutical composition of claim 22, wherein the cancer is HPV-16 positive.

26. A polypeptide encoded by the nucleic acid sequence set forth in SEQ ID NO:

40.

27. A cell comprising the polypeptide encoded by the nucleic acid sequence set forth in SEQ ID NO:

40.

28. A messenger RNA (mRNA) composition for eliciting an immune response to an HPV antigen, the mRNA comprising an open reading frame (ORF) encoding a plurality of epitopes from at least two HPV antigens..

29. The mRNA of claim 28, further comprising one or more of a 5’ cap, a 3’ UTR, and a poly(A) tail.

30. The mRNA of claim 28 or claim 29, further comprising one or both of an optimised codon and a chemical modification when compared to a corresponding mRNA that does not comprise the optimised codon and / or chemical modification.

31. The mRNA of claim 30, wherein the chemical modification and / or the optimised codon increases mRNA stability and / or mRNA translation in a mammalian cell when compared to a mRNA without the chemical modification and / or the optimized codon.

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