Vaccine for prevention and treatment of human papilloma virus related cancer

Abstract

The present disclosure provides novel combinations of nucleic acid sequences encoding regions of E6 and E7 proteins of human papilloma virus (HPV) strains, as nucleic acid vaccines, to treat HPV and / or HPV-related cancers, prevent HPV and / or HPV-related cancers, or a combination thereof.

Description

VACCINE FOR PREVENTION AND TREATMENT OF HUMAN PAPILLOMA VIRUSRELATED CANCERRELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Application No. 63 / 622,436, filed January 18, 2024. The entire content of this related application is expressly incorporated herein by reference in its entirety.REFERENCE TO SEQUENCE LISTING

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 88LW-374178-WO_SequenceListing, created January 15, 2025, which is 296 kilobytes in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.BACKGROUND

[0003] Human papilloma viruses (HPV) are known to be the main cause of various types of cancers. Prophylactic vaccines have been commercialized to prevent infections of the high-risk variants of HPV (especially HP VI 6 and 18) in humans, which reduces the possibility of developing HPV related cancer (especially cervical cancer) among the receivers of these prophylactic vaccines. Current prophylactic vaccines protect populations from age 9 to 45, with the idea to establish immunity against high-risk HPV variants before contracting the virus. However, the prophylactic vaccines do not protect patients with prior HPV infections and those patients developed precancerous lesions and cancers. In addition, after nearly two decades after their commercialization, the worldwide vaccination rate of HPV prophylactic vaccines is still relatively low, even among the appropriate age groups. Further, cost wise, HPV prophylactic vaccines are the most expensive preventative vaccine in history. There is a significant need for therapeutic vaccines to prevent and treat HPV related cancer.

[0004] E6 and E7 proteins of high-risk HPV are the major oncoproteins that may immortalize normal cells and cause tumor in permissive cells. Therefore, E6 and E7 proteins have been major therapeutic targets in efforts to develop treatments against HPV related cancers.

[0005] Intact E6 and E7 proteins are oncogenic. Mutations have been introduced in E6 and E7 to disable their interaction with pRb and p53, respectively, to block their major mechanisms of oncogenesis. However, blocking E7-p53 and E6-pRb interactions would reduce the risk of cancer caused by introducing high-risk HPV E6 and E7 proteins into normal cells, but areunlikely to completely eliminate the oncogenic risks, since E6 and E7 proteins have complicated binding partner networks in cells.

[0006] In addition to previous modifications that disrupt pRB-E6 and p53-E7 interactions, the present disclosure disrupted structurally important Zinc-finger elements of E6 and E7 proteins to separate E6 and E7 proteins into smaller peptide fragments. Moreover, the present disclosure rearranged such fragments in orders unrelated to the natural arrangement in E6 and E7 proteins, to further reduce the oncogenic risks.

[0007] The present disclosure employed the structurally disrupted E6 and E7 proteins of high-risk HPV type 16 and 18, to prevent and treat tumor caused by high-risk HPV viruses.SUMMARY

[0008] One aspect of the present disclosure relates to a polypeptide comprising, or consisting of, one or more amino acid sequences that is the same as, or differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from, or shares at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater sequence identity with the amino acid sequences set forth in Table 1 or Table 2 or Table 3 or Table 4. In certain embodiments, there is provided a polynucleotide that may comprise, or consist of, one or more nucleic acid sequences that encode an amino acid sequence that is the same as, or differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from, or shares at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater sequence identity with the amino acid sequences set forth in Table 1 or Table 2 or Table 3 or Table 4. In certain embodiments, the polynucleotide may be circular or linear. In certain embodiments, the polynucleotide may comprise DNA, RNA, or both.

[0009] Another aspect of the present disclosure relates to the use of a polynucleotide comprising, or consisting of, one or more nucleic acid sequences that encode an amino acid sequence that is the same as, or differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from, or shares at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater sequence identity with the amino acid sequences set forth in Table 1 or Table 2 or Table 3 or Table 4, compositions, or pharmaceutical formulations provided herein in the manufacture of a medicament for the prevention or treatment of a condition caused by infection of HPV variants or contracting HPV virus components.

[0010] Disclosed herein include polynucleotides. In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a structurally disrupted (SD) human papilloma virus (HPV) E6 protein or SD HPV E7 protein.

[0011] In some embodiments, the SD HPV E6 protein or the SD HPV E7 protein is derived from a wild type HPV E6 protein or a wild type HPV E7 protein. In some embodiments, the wild type HPV E6 protein or the wild type HPV E7 protein comprises HP VI 6 E6 protein, HP VI 6 E7 protein, HP VI 8 E6 protein, HP VI 8 E7 protein, or any combination thereof. In some embodiments, the SD HPV E6 protein exhibits reduced binding affinity to pRB and / or the SD HPV E7 protein exhibits reduced binding affinity to p53, e.g., as compared to the wild type HPV E6 protein or the wild type HPV E7 protein. In some embodiments, the SD HPV E6 protein is not capable of binding to pRB and / or the SD HPV E7 protein is not capable of binding to p53. In some embodiments, the SD HPV E6 protein and / or the SD HPV E7 protein comprises one or more disrupted or deleted zinc-finger elements, e.g., as compared to the wild type HPV E6 protein or the wild type HPV E7 protein.

[0012] In some embodiments, the SD HPV E6 protein comprises a fragment of the wild type HPV E6 protein, the SD HPV E7 comprises a fragment of the wild type HPV E7 protein, or both. In some embodiments, the fragment of the wild type HPV E6 protein and / or the fragment of the wild type HPV E7 protein is less than 40 amino acids in length. In some embodiments, the fragment of the wild type HPV E6 protein comprises the sequence of any one of SEQ ID NOs: 5-8 and 12-15 or a sequence having one, two, or three mismatches relative to any one of SEQ ID NOs: 5-8 and 12-15. In some embodiments, the fragment of the wild type HPV E7 protein comprises the sequence of any one of SEQ ID NOs: 9-11 and 16-18 or a sequence having one, two, or three mismatches relative to any one of SEQ ID NOs: 9-11 and 16-18.

[0013] In some embodiments, the wild type HPV E6 protein or the wild type HPV E7 protein comprises an amino acid sequence of any one of SEQ ID NOs: 1-4.

[0014] The polynucleotide can comprise a nucleotide sequence encoding a polypeptide comprising two or more SD HPV E6 proteins, two or more SD HPV E7 proteins, or any combination thereof. In some embodiments, the polypeptide comprises four SD HPV E6 proteins and three SD HPV E7 proteins. In some embodiments, the polypeptide comprises, from N-terminus to C-terminus: a first SD HPV E6 protein, a first SD HPV E7 protein, a second SD HPV E6 protein, a second SD HPV E7 protein, a third SD HPV E6 protein, a third SD HPV E7 protein, and a fourth SD HPV E6 protein.

[0015] In some embodiments, the polypeptide further comprises an N-terminal signal peptide, a C-terminal signal peptide, or both. In some embodiments, the N-terminal signal peptide, the C- terminal signal peptide, or both, is a signal peptide of a human leukocyte antigen (HLA). In some embodiments, the HLA is HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G. In some embodiments, the polypeptide comprises an N-terminal signal peptide of HLA-F, a C-terminalsignal peptide ofHLA-G, or both. In some embodiments, the N-terminal signal peptide ofHLA- F comprises the sequence of SEQ ID NO: 301 or a sequence have one, two, or three mismatches relative to the sequence of SEQ ID NO: 301. In some embodiments, the C-terminal signal peptide ofHLA-G comprises the sequence of SEQ ID NO: 302 or a sequence have one, two, or three mismatches relative to the sequence of SEQ ID NO: 302.

[0016] In some embodiments, the polynucleotide encodes a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of any one of SEQ ID NOs: 5-180. In some embodiments, the polynucleotide encodes a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 5-180 or an amino acid sequence having one, two, three, four, five, six, seven, eight, nine, or ten mismatches relative to any one of SEQ ID NOs: 5-180.

[0017] Disclosed herein include polypeptides. In some embodiments, the polypeptide comprises an SD HPV E6 protein or an SD HPV E7 protein.

[0018] In some embodiments, the SD HPV E6 protein or the SD HPV E7 protein is derived from a wild type HPV E6 protein or a wild type HPV E7 protein. In some embodiments, the wild type HPV E6 or the wild type HPV E7 protein comprises HPV16 E6 protein, HPV16 E7 protein, HPV18 E6 protein, HPV18 E7 protein, or any combination thereof. In some embodiments, the SD HPV E6 protein exhibits reduced binding affinity to pRB and / or the SD HPV E7 protein exhibits reduced binding affinity to p53, e.g., as compared to the wild type HPV E6 protein or the wild type HPV E7 protein. In some embodiments, the SD HPV E6 protein is not capable of binding to pRB and / or the SD HPV E7 protein is not capable of binding to p53. In some embodiments, the SD HPV E6 protein and / or the SD HPV E7 protein comprises one or more disrupted or deleted zinc-finger elements, e.g., as compared to the wild type HPV E6 protein or the wild type HPV E7 protein.

[0019] In some embodiments, the SD HPV E6 protein comprises a fragment of the wild type HPV E6 protein, the SD HPV E7 comprises a fragment of the wild type HPV E7 protein, or both. In some embodiments, the fragment of the wild type HPV E6 protein and / or the fragment of the wild type HPV E7 protein is less than 40 amino acids in length. In some embodiments, the fragment of the wild type HPV E6 protein comprises the sequence of any one of SEQ ID NOs: 5-8 and 12-15 or a sequence having one, two, or three mismatches relative to any one of SEQ ID NOs: 5-8 and 12-15. In some embodiments, the fragment of the wild type HPV E7 protein comprises the sequence of any one of SEQ ID NOs: 9-11 and 16-18 or a sequence having one, two, or three mismatches relative to any one of SEQ ID NOs: 9-11 and 16-18.

[0020] In some embodiments, the wild type HPV E6 protein or the wild type HPV E7 protein comprises an amino acid sequence of any one of SEQ ID NOs: 1-4.

[0021] The polypeptide can comprise an amino acid sequence of two or more SD HPV E6 proteins, two or more SD HPV E7 proteins, or any combination thereof. In some embodiments, the polypeptide comprises four SD HPV E6 proteins and three SD HPV E7 proteins. In some embodiments, the polypeptide comprises, from N-terminus to C-terminus: a first SD HPV E6 protein, a first SD HPV E7 protein, a second SD HPV E6 protein, a second SD HPV E7 protein, a third SD HPV E6 protein, a third SD HPV E7 protein, and a fourth SD HPV E6 protein.

[0022] In some embodiments, the polypeptide further comprises an N-terminal signal peptide, a C-terminal signal peptide, or both. In some embodiments, the N-terminal signal peptide, the C- terminal signal peptide, or both, is a signal peptide of a human leukocyte antigen (HLA). In some embodiments, the HLA is HLA- A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G. In some embodiments, the polypeptide comprises an N-terminal signal peptide of HLA-F, a C-terminal signal peptide of HLA-G, or both. In some embodiments, the N-terminal signal peptide ofHLA- F comprises the sequence of SEQ ID NO: 301 or a sequence have one, two, or three mismatches relative to the sequence of SEQ ID NO: 301. In some embodiments, the C-terminal signal peptide of HLA-G comprises the sequence of SEQ ID NO: 302 or a sequence have one, two, or three mismatches relative to the sequence of SEQ ID NO: 302.

[0023] The polypeptide can comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of any one of SEQ ID NOs: 5- 180. The polypeptide can comprise an amino sequence of any one of SEQ ID NOs: 5-180 or an amino acid sequence having one, two, three, four, five, six, seven, eight, nine, or ten mismatches relative to any one of SEQ ID NOs: 5-180.

[0024] Disclosed herein include compositions. In some embodiments, the composition comprises: a nucleic acid composition comprising one or more polynucleotides of the disclosure. In some embodiments, the composition comprises: one or more polypeptides disclosed herein.

[0025] In some embodiments, the nucleic acid composition is complexed or associated with one or more lipids or lipid-based carriers, thereby forming liposomes, lipid nanoparticles (LNPs), lipoplexes, and / or nanoliposomes, e.g., encapsulating the nucleic acid composition. In some embodiments, the one or more polynucleotides are situated on the same nucleic acid and / or different nucleic acids. In some embodiments, the nucleic acid composition is, comprises, or further comprises, one or more vectors. In some embodiments, at least one of the one or more vectors is a viral vector, a plasmid, a naked DNA vector, a lipid nanoparticle (LNP), or anycombination thereof. In some embodiments, the viral vector is an AAV vector, a lentivirus vector, a retrovirus vector, an adenovirus vector, a herpesvirus vector, a herpes simplex virus vector, a cytomegalovirus vector, a vaccinia virus vector, a MVA vector, a baculovirus vector, a vesicular stomatitis virus vector, a human papillomavirus vector, an avipox virus vector, a Sindbis virus vector, a VEE vector, a Measles virus vector, an influenza virus vector, a hepatitis B virus vector, an integration-deficient lentivirus (IDLV) vector, or any combination thereof. In some embodiments, the one or more polynucleotides are comprised in the one or more vectors. In some embodiments, the one or more polynucleotides are comprised in the same vector and / or different vectors. In some embodiments, the one or more vectors is a DNA vaccine. In some embodiments, the one or more polynucleotides are operably linked to one or more promoters capable of inducing transcription of the one or more polynucleotides. In some embodiments, the DNA vaccine is a plasmid-based DNA vaccine, a minicircle-based DNA vaccine, a bacmid- based DNA vaccine, a minigene-based DNA vaccine, a ministring DNA (linear covalently closed DNA vector) vaccine, a closed-ended linear duplex DNA (CELiD or ceDNA) vaccine, a doggybone™ DNA vaccine, a dumbbell shaped DNA vaccine, or a minimalistic immunological- defined gene expression (MIDGE)-vector DNA vaccine. In some embodiments, the one or more promoters comprise a ubiquitous promoter, an inducible promoter, a tissue-specific promoter and / or a lineage-specific promoter. In some embodiments, the ubiquitous promoter is selected from the group comprising a cytomegalovirus (CMV) immediate early promoter, a CMV promoter, a viral simian virus 40 (SV40) (e.g., early or late), a Moloney murine leukemia virus (MoMLV) LTR promoter, a Rous sarcoma virus (RS V) LTR, an RSV promoter, a herpes simplex virus (HSV) (thymidine kinase) promoter, H5, P7.5, and Pl 1 promoters from vaccinia virus, an elongation factor 1 -alpha (EFla) promoter, early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock 70 kDa protein 5 (HSPA5), heat shock protein 90 kDa beta, member 1 (HSP90B1), heat shock protein 70 kDa (HSP70), P-kinesin (P- KIN), the human ROSA 26 locus, a Ubiquitin C promoter (UBC), a phosphoglycerate kinase-1 (PGK) promoter, 3 -phosphoglycerate kinase promoter, a cytomegalovirus enhancer, human P- actin (HBA) promoter, chicken P-actin (CB A) promoter, a CAG promoter, a CASI promoter, a CBH promoter, or any combination thereof. In some embodiments, the one or more polynucleotides are operably linked to a tandem gene expression element. In some embodiments, the tandem gene expression element is an internal ribosomal entry site (IRES), foot-and-mouth disease virus 2A peptide (F2A), equine rhinitis A virus 2A peptide (E2A), porcine teschovirus 2A peptide (P2A) or Thosea asigna virus 2A peptide (T2A), or any combination thereof. In some embodiments, the one or more polynucleotides comprise atranscript stabilization element. In some embodiments, the transcript stabilization element comprises woodchuck hepatitis post-translational regulatory element (WPRE), bovine growth hormone polyadenylation (bGH-polyA) signal sequence, human growth hormone polyadenylation (hGH-polyA) signal sequence, or any combination thereof.

[0026] In some embodiments, the nucleic acid composition is or comprises mRNA. In some embodiments, the mRNA is formulated in a lipid nanoparticle (LNP). In some embodiments, the mRNA comprises a 5' untranslated region (UTR), a 3' UTR, and / or a cap. In some embodiments, the mRNA comprises one or more modified nucleotides selected from the group comprising pseudouridine, N-l-methyl-pseudouridine, 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo- pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7- deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, and 2-thiocytidine. In some embodiments, the mRNA comprises a modified nucleotide in place of one or more uridines. In some embodiments, the modified nucleotide is selected from pseudouridine (y), N 1-methyl-pseudouridine (m IT), and 5-methyl-uridine (m5U).

[0027] The composition can comprise particles comprising the one or more polypeptides. In some embodiments, the particles are iron oxide particles, liposomes, micelles, polymer complexes, cationic peptide nanoemulsions, virus-like particles (VLPs), lipid nanoparticles (LNP) and / or lipoplex (LPX) particles. In some embodiments, the LNP comprises one or more of an ionizable cationic lipid, a non-cationic lipid, a sterol, and a PEG-modified lipid. In some embodiments, the non-cationic lipid is a neutral lipid. In some embodiments, the LNP comprises 0.5-15 mol% PEG-modified lipid, 5-25 mol% non-cationic lipid, 25-55 mol% sterol, and 20-60 mol% ionizable cationic lipid. In some embodiments, the LNP comprises: 40-55 mol% ionizable cationic lipid, 5-15 mol% neutral lipid, 35-45 mol% sterol, and 1-5 mol% PEG-modified lipid. In some embodiments, the LNP comprises: 47 mol% ionizable cationic lipid, 11.5 mol% neutral lipid, 38.5 mol% sterol, and 3.0 mol% PEG-modified lipid; 48 mol% ionizable cationic lipid, 11 mol% neutral lipid, 38.5 mol% sterol, and 2.5 mol% PEG-modified lipid; 49 mol% ionizable cationic lipid, 10.5 mol% neutral lipid, 38.5 mol% sterol, and 2.0 mol% PEG-modified lipid; 50 mol% ionizable cationic lipid, 10 mol% neutral lipid, 38.5 mol% sterol, and 1.5 mol% PEG- modified lipid; or 51 mol% ionizable cationic lipid, 9.5 mol% neutral lipid, 38.5 mol% sterol, and 1.0 mol% PEG-modified lipid. In some embodiments: the ionizable cationic lipid is heptadecan-9-yl 8 ((2 hydroxyethyl)(6 oxo 6-(undecyloxy)hexyl)amino)octanoate; the neutral lipid is l,2-distearoyl-sn-glycero-3 phosphocholine (DSPC); the sterol is cholesterol; and / or thePEG-modified lipid is l-monomethoxypolyethyleneglycol-2,3-dimyristylglycerol with polyethylene glycol of average molecular weight 2000 (PEG2000 DMG). In some embodiments, the wt / wt ratio of lipid to mRNA is from about 1 : 100 to about 100: 1.

[0028] In some embodiments, the composition comprises one or more polynucleotides encoding immunostimulatory agents. In some embodiments, the immunostimulatory agents are selected from the group comprising toll-like receptor (TLR) agonists, cytokine receptor agonists, CD40 agonists, Fc receptor agonists, CpG-containing nucleic acids, complement receptor agonists, or any combination thereof. In some embodiments, the TLR agonist is a TLR-1 agonist, TLR-2 agonist, TLR-3 agonist, TLR-4 agonist, TLR-5 agonist, TLR-6 agonist, TLR-7 agonist, TLR-8 agonist, TLR-9 agonist, and / or TLR- 10 agonist. In some embodiments, the Fc receptor agonist is a Fc-gamma receptor agonist. In some embodiments, the complement receptor agonist binds to CD21 or CD35. In some embodiments, the cytokine receptor agonist is a cytokine. In some embodiments, the cytokine receptor agonist is a small molecule, antibody, fusion protein, or aptamer. The composition can comprise an adjuvant. In some embodiments, the adjuvant comprises aluminum hydroxide, alhydrogel, AddaVax, MF59, AS03, Freund’s adjuvant, Montanide ISA51, CpG, Poly EC, glucopyranosyl lipid A, flagellin, resiquimod, or any combination thereof.

[0029] In some embodiments, the composition further comprises Tris buffer, sucrose, and / or sodium acetate. In some embodiments, the composition is a lyophilized composition. In some embodiments, the lyophilized composition has a water content of less than about 10%. In some embodiments, the composition is formulated or is to be formulated: as a liquid, a solid, or a combination thereof; for injection; and / or for intramuscular administration, intranasal administration, transdermal administration, aerosol delivery, nasal delivery, vaginal delivery, rectal delivery, buccal delivery, ocular delivery, local delivery, topical delivery, intracistemal delivery, intraperitoneal delivery, oral delivery, intramuscular injection, intravenous injection, subcutaneous injection, intranodal injection, intratumoral injection, intraperitoneal injection, or intradermal injection. In some embodiments, the composition is a pharmaceutical composition, wherein the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents and / or excipients.

[0030] The composition can comprise instructions for use of the composition for: stimulating an immune response in a subject in need thereof; treating or preventing HPV-related disease or disorder in a subject in need thereof; and / or treating or preventing an HPV-related cancer in a subject in need thereof. Disclosed herein include kits. The kit can comprise any composition ofthe disclosure. Also disclosed are cells comprising a nucleic acid composition described herein. The compositions disclosed herein can be used as a medicament.

[0031] Disclosed herein include methods of stimulating an immune response in a subject in need thereof. In some embodiments, the method comprises: administering to the subject a pharmaceutically effective amount of a polynucleotide, a polypeptide, or a composition of the disclosure, thereby stimulating the immune response in the subject. Disclosed herein include methods of treating or preventing a disease or disorder in a subject in need thereof. In some embodiments, the method comprises: administering to the subject a pharmaceutically effective amount of a polynucleotide, a polypeptide, or a composition of the disclosure, thereby treating or preventing the disease or disorder in the subject. In some embodiments, the disease or disorder is an HPV-related disease or disorder. In some embodiments, the HPV-related disease or disorder is cancer. In some embodiments, the cancer is anal cancer, cervical cancer, oropharyngeal cancer, penile cancer, vaginal cancer, or vulvar cancer.

[0032] In some embodiments, immunogenic levels of the SD HPV E6 protein, the SD HPV E7 protein, or both, are produced in serum of the subject at about 1 hour to about 6 months post administration of the composition. In some embodiments, a neutralizing antibody titer of about 50 to about 100000 half-maximal inhibitory dilutions (ID50s values) is produced in the serum of the subject at about 1 hour to about 6 months post administration of the composition. In some embodiments, the method comprises administering to the subject one or more doses of the composition. In some embodiments, a second dose of the composition is administered to the subject at least 14 days after a first dose of the composition is administered to the subject. In some embodiments, the second dose of the composition is administered to the subject at least 28 days after the first dose of the composition is administered to the subject. In some embodiments, the first dose is a prime, and the second dose is a boost. In some embodiments, each of the one or more doses of the composition comprises about 2 pg to about 5 pg of the nucleic acid composition.

[0033] In some embodiments, administering the composition induces neutralizing responses against HPV, cancer cells, or both. In some embodiments, the HPV is HPV16, HPV18, or both. In some embodiments, the administration of the composition elicits protective and long-lasting immunity against HPV, cancer, or both. In some embodiments, the polynucleotide, the polypeptide, or the composition is administered in an effective amount to: induce a robust antibody response against the HPV, cancer cells, or both. In some embodiments, a robust antibody response comprises a neutralizing antibody response. In some embodiments, a robust antibody response comprises Fc domain effector functions that recruit immune cells to infectedcells. In some embodiments, said immune cells are macrophages, neutrophils, and / or natural killer cells. In some embodiments, said recruitment induces antibody-dependent cellular cytotoxicity (ADCC) and / or antibody-dependent cellular phagocytosis (ADCP); elicit a robust CD4 and / or CD8 T cell response against the HPV, the cancer cells, or both in the subject; and / or elicit a balanced Thl / Th2 response against the HPV, the cancer cells, or both in the subject. In some embodiments, the polynucleotide, the polypeptide, or the composition is administered in an effective amount to induce the production of one or more cytokines in the subject. In some embodiments, the one or more cytokines comprise IL-2, IFN-y, TNF-a, IL-1, IL-12, IL-17, IL- 18, or any combination thereof. In some embodiments, at least a portion of the cancer cells express the wild type HPV E6 protein or variants thereof, the wild type HPV E7 protein or variants thereof, or any combination thereof.

[0034] In some embodiments, treating or preventing the disease or disorder in the subject comprises inhibiting the onset or progression of the cancer. In some embodiments, the inhibition of progression of the cancer is measured by tumor growth inhibition (TGI) and wherein the TGI resulted by the administration is at least 30% higher as compared to an untreated subject. In some embodiments, a tumor volume after the administration decreases at least 50% relative to the tumor volume with no treatment or the tumor volume prior to the administration. In some embodiments, the subject achieves a complete response. In some embodiments, the subject does not develop HPV-related cancer following the administration.

[0035] In some embodiments, the subject is a human subject. In some embodiments, the human subject is at least 9 years old. In some embodiments, the subject has had a prior HPV infection. In some embodiments, the composition is co-administered with an adjuvant. In some embodiments, the composition is not co-administered with an adjuvant. In some embodiments, the composition is administered intramuscularly, e.g., into a deltoid region of an arm. In some embodiments, the administering comprises aerosol delivery, nasal delivery, vaginal delivery, rectal delivery, buccal delivery, ocular delivery, local delivery, topical delivery, intracistemal delivery, intraperitoneal delivery, oral delivery, intramuscular injection, intravenous injection, subcutaneous injection, intranodal injection, intratumoral injection, intraperitoneal injection, intradermal injection, or any combination thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The accompanying drawings are intended to provide illustrative, and schematic rather than comprehensive, examples of certain aspects and embodiments of the present disclosure. The drawings are not intended to be limiting or binding to any particular theory or model, and are not necessarily to scale.

[0037] FIGs. 1A-1I depict growth curves of tumors from individual animals (mice) in the tumor growth blocking study. X-axis shows the time in unit of the number of days after the starting date (Day 0) of the IxlO6C3.43 tumor cell challenge. The Y-axis shows the size of the tumors (in unit of mm3), calculated based on the measurement of three dimensions of the tumors: volume = length x width x depth. Mice were vaccinated on Day -21 (21 days before the tumor cell challenge) and Day -7 (7 days before the tumor cell challenge) and received C3.43 tumor cell challenge on Day 0.

[0038] FIG. 2 depicts a survival (*all-cause mortality) curve of mice groups in the tumor growth blocking study. The X-axis shows the time in unit of the number of days after the starting date (Day 0) of IxlO6C3.43 tumor cell challenge. The Y-axis shows the percentage of animals surviving before death or euthanasia due to tumor volumes exceeding 1500 mm3or development of severe ulcerations. Mice were vaccinated on Day -21 (21 days before tumor cell challenge) and Day -7 (7 days before tumor cell challenge), and received C3.43 tumor cell challenge on Day 0.

[0039] FIG. 3A depicts growth curves of tumors from animals (mice) in treatment of preinoculated tumor study. The X-axis shows the time in unit of the number of days after the starting date (Day 0) of 5xl05TC-1 tumor cell challenge. The Y-axis shows the size of the tumors (in unit of mm3), calculated based on the measurement of three dimensions of the tumors: volume = length x width x depth. Mice received TC-1 tumor cell challenge on Day 0, and then received three vaccine doses on Days 7, 28, and 58.

[0040] FIG. 3B depicts growth curves of tumors from animals (mice) with repeating doses of AOV CFR treatments in pre-inoculated tumor study. The X-axis shows the time in unit of the number of days after the starting date (Day 0) of 5* 105TC-1 tumor cell challenge. The Y-axis shows the size of the tumors (in unit of mm3), calculated based on the measurement of three dimensions of the tumors: volume = length x width x depth. Mice received TC-1 tumor cell challenge on Day 0, and then received three 5 pg of AOV CFR encapsulated in lipid nanoparticle (LNP) on Days 7, 28, and 58.

[0041] FIG. 4 depicts growth curves of pre-inoculated tumors from animals (mice) untreated (FIG. 4A) or treated with depicted dosage of treatments (FIG. 4B, 4C). X-axis shows the time in unit of the number of days after the starting date (Day 0) of the 5xl05TC-1 tumor cell challenge. The Y-axis shows the size of the tumors (in unit of mm3), calculated based on the measurement of three dimensions of the tumors: volume = length x width x depth. Mice received TC-1 tumor cell challenge on Day 0 and were treated with single dose of vaccine on Day 3 (3 days after the tumor cell challenge).

[0042] FIGS. 5A-5B depicts the specific cellular immune responses induced by the treatments. Animals were separated into four groups (5 animals / group): Group 1. no tumor cells inoculated followed by empty LNP particle treatment on Day 3; Group 2. 5xl05TC-1 tumor cells / animal challenge on Day 0, followed by empty LNP particle treatment on Day 3; Group 3. 5xl05TC-1 tumor cells / animal challenge on Day 0, followed by LNP containing 5|ig AOV-SD1 (Table 3) treatment on Day 3; Group 4. 5xl05TC-1 tumor cells / animal challenge on Day 0, followed by LNP particle enclosed 30|ig AOV-SD1 treatment on Day 3. All animals were euthanized on Day 14, spleen lymphocytes were collected. Elispot Assays were performed to evaluate HPV16 / 18 E6 / E7 specific cellular immune responses by measuring the level of IL-2 (FIG. 5B) and IFN-y (FIG. 5 A) after stimulated by HPV16 / 18 E6 / E7 peptides fragments. The X-axis shows Groups 1-4, and the Y-axis shows IL-2 (FIG. 5B) or IFN-y (FIG. 5A) level in Elispot (spots per million cells).

[0043] FIG. 6 depicts in vitro cellular expression level, as well as the specific cellular immune responses induced by constructs with various fragment combinations. For in vitro cellular expression, I pg of LNP encapsulated mRNA of individual constructs AOV-16SD001 to AOV- 16SD144 was incubated with 3 x 105viable 293T cells for 24 hours, respectively. Expression levels of each individual construct was measured by relative dot blot signal against construct AOV-16SD017. For cellular immune response, Animals were separated into 144 groups (3 animals / group) treated with LNPs encapsulate AOV-16SD001 to AOV-16SD144, respectively, and an additional group of three (3) animals treated with empty LNP particle treatment on Day 0. All animals were euthanized on Day 21, spleen lymphocytes were collected. Elispot Assays were performed to evaluate HP VI 6 E7 specific cellular immune responses by measuring the level of IFN-y after stimulated by HPV16 E7 peptides fragments pool (Table 5). The X-axis shows groups 1-144, and the left Y-axis scales the relative expression level, and right Y-axis scales the IFN-y level in Elispot (spots per million cells).DETAILED DESCRIPTION

[0044] The following description provides specific details for a thorough understanding of, and enabling description for, embodiments of the disclosure. However, one skilled in the art will understand that the disclosure may be practiced without these details. In other instances, well- known structures and functions have not been shown or described in detail to avoid unnecessarily obscuring the description of the embodiments of the disclosure.

[0045] Disclosed herein include polynucleotides. In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a structurally disrupted (SD) human papilloma virus(HPV) E6 protein (e.g., a fragment of HPV E6 full-length protein) or SD HPV E7 protein (e.g., a fragment of HPV E7 full-length protein). Disclosed herein include compositions. In some embodiments, the composition comprises: a nucleic acid composition comprising one or more polynucleotides of the disclosure. In some embodiments, the composition comprises one or more polypeptides disclosed herein.

[0046] Disclosed herein include kits. The kit can comprise any composition of the disclosure. Also disclosed are cells comprising a nucleic acid composition described herein. The compositions disclosed herein can be used as a medicament. Disclosed herein include methods of stimulating an immune response in a subject in need thereof. In some embodiments, the method comprises: administering to the subject a pharmaceutically effective amount of a polynucleotide, a polypeptide, or a composition of the disclosure, thereby stimulating the immune response in the subject. Disclosed herein include methods of treating or preventing a disease or disorder in a subject in need thereof. In some embodiments, the method comprises: administering to the subject a pharmaceutically effective amount of a polynucleotide, a polypeptide, or a composition of the disclosure, thereby treating or preventing the disease or disorder in the subject.Definitions

[0047] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. See, e.g. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, NY 1989). For purposes of the present disclosure, the following terms are defined below.

[0048] “Immune response” as used herein can refer to a response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus. In one embodiment, the response is specific for a particular antigen (an “antigen-specific response”). In one embodiment, an immune response is a T cell response, such as a CD4+ response or a CD8+ response. In another embodiment, the response is a B cell response and results in the production of specific antibodies.

[0049] As used herein, the term “isolated” shall be given its ordinary meaning and shall also refer to an “isolated” biological component (such as a protein, for example, a disclosed antigen or nucleic acid encoding such an antigen) that has been substantially separated or purified away from other biological components in which the component naturally occurs, such as otherchromosomal and extrachromosomal DNA, RNA, and proteins. Proteins, peptides, and nucleic acids that have been “isolated” include proteins purified by standard purification methods. The term also embraces proteins or peptides prepared by recombinant expression in a host cell as well as chemically synthesized proteins, peptides, and nucleic acid molecules. Isolated (or purified) does not require absolute purity, and can include protein, peptide, or nucleic acid molecules that are at least 50% isolated, such as at least 75%, 80%, 90%, 95%, 98%, 99%, or even 99.9% isolated.

[0050] As used herein, “sequence identity” or “identity” in the context of two nucleic acid or polypeptide sequences makes reference to the nucleotide bases or residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5: 151-3, 1989; Corpet et al., Nuc. Acids Res. 16: 10881-90, 1988; Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; Pearson et al., Meth. Mol. Bio. 24:307-31, 1994; and Altschul et al., J. Mol. Biol. 215:403-10, 1990 (the content of each of these references is incorporated herein in its entirety).

[0051] When percentage of sequence identity or similarity is used in reference to proteins, it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted with a functionally equivalent residue of the amino acid residues with similar physiochemical properties and therefore do not change the functional properties of the molecule. A functionally equivalent residue of an amino acid used herein typically can refer to other amino acid residues having physiochemical and stereochemical characteristics substantially similar to the original amino acid. The physiochemical properties include water solubility (hydrophobicity or hydrophilicity), dielectric and electrochemical properties, physiological pH, partial charge of side chains (positive, negative or neutral) and other properties identifiable to one of skill in the art. The stereochemical characteristics include spatial and conformational arrangement of the amino acids and their chirality. For example, glutamic acid is considered to be a functionally equivalent residue to aspartic acid in the sense of the current disclosure. Tyrosine and tryptophan are considered as functionally equivalent residues to phenylalanine. Arginine and lysine are considered as functionally equivalent residues to histidine.

[0052] As used herein, an “antibody” or “antigen-binding polypeptide” can refer to a polypeptide or a polypeptide complex that specifically recognizes and binds to an antigen (e.g., a spike protein receptor binding domain). An antibody can be a whole antibody and any antigen binding fragment or a single chain thereof. Thus, the term “antibody” includes any protein or peptide-containing molecule that comprises at least a portion of an immunoglobulin molecule having biological activity of binding to the antigen. Examples of such include, but are not limited to, a complementarity determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework (FR) region, or any portion thereof, or at least one portion of a binding protein.

[0053] Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques can be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures can be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference for any purpose. Unless specific definitions are provided, the nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those commonly known and used in the art. Standard techniques can be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0054] Certain embodiments herein relate to the discovery in the examples below that demonstrates that certain combinations of fragments of the high-risk variant HPV16 and HPV18 E6 and E7 oncoproteins prevent tumor progression before and / or after challenge with tumor cells.Sequences

[0055] Disclosed herein include polynucleotides. In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a structurally disrupted (SD) human papilloma virus (HPV) E6 protein (e.g., a fragment of HPV E6 full-length protein) or SD HPV E7 protein (e.g., a fragment of HPV E7 full-length protein).

[0056] In some embodiments, the SD HPV E6 protein or the SD HPV E7 protein is derived from a wild type HPV E6 protein or a wild type HPV E7 protein. The wild type HPV E6 protein or the wild type HPV E7 protein can comprise HPV16 E6 protein, HPV16 E7 protein, HPV18 E6 protein, HPV18 E7 protein, or any combination thereof. In some embodiments, the SD HPV E6 protein exhibits reduced binding affinity to pRB and / or the SD HPV E7 protein exhibits reduced binding affinity to p53, e.g., as compared to the wild type HPV E6 protein or the wild type HPV E7 protein. The reduction can be 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%,35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%,51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 100%, or a number or a range between any two of these values, e.g., as compared to the wild type HPV E6 protein or the wild type HPV E7 protein. In some embodiments, the SD HPV E6 protein is not capable of binding to pRB and / or the SD HPV E7 protein is not capable of binding to p53. The SD HPV E6 protein and / or the SD HPV E7 protein can comprise one or more disrupted or deleted zinc-finger elements, e.g., as compared to the wild type HPV E6 protein or the wild type HPV E7 protein.

[0057] In some embodiments, the SD HPV E6 protein comprises a fragment of the wild type HPV E6 protein, the SD HPV E7 comprises a fragment of the wild type HPV E7 protein, or both. The fragment of the wild type HPV E6 protein and / or the fragment of the wild type HPV E7 protein can be less than 40 amino acids in length (e.g., 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 amino acids in length).

[0058] The fragment of the wild type HPV E6 protein can comprise the sequence of any one of SEQ ID NOs: 5-8 and 12-15 or a sequence having one, two, or three mismatches relative to any one of SEQ ID NOs: 5-8 and 12-15. The fragment of the wild type HPV E7 protein can comprise the sequence of any one of SEQ ID NOs: 9-11 and 16-18 or a sequence having one, two, or three mismatches relative to any one of SEQ ID NOs: 9-11 and 16-18.

[0059] The wild type HPV E6 protein or the wild type HPV E7 protein can comprise an amino acid sequence of any one of SEQ ID NOs: 1-4.

[0060] The polynucleotide can comprise a nucleotide sequence encoding a polypeptide comprising two or more SD E6 HPV proteins and / or SD HPV E7 proteins (e.g., fragments of HPV E6 full-length protein and / or fragments of HPV E7 full-length protein). In someembodiments, the polynucleotide can comprise a nucleotide sequence encoding a polypeptide comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 SD E6 HPV proteins and / or SD HPV E7 proteins (e.g., fragments of wild type HPV E6 and / or fragments of HPV E7). The polynucleotide can comprise a nucleotide sequence encoding a polypeptide comprising two or more SD HPV E6 proteins, two or more SD HPV E7 proteins, or any combination thereof. The polypeptide can comprise four SD HPV E6 proteins and three SD HPV E7 proteins. The polypeptide can comprise, for example from N-terminus to C-terminus: a first SD HPV E6 protein, a first SD HPV E7 protein, a second SD HPV E6 protein, a second SD HPV E7 protein, a third SD HPV E6 protein, a third SD HPV E7 protein, and a fourth SD HPV E6 protein. A SD HPV E6 protein can be, or comprise, a fragment of HPV E6 full length protein. A SD HPV E6 protein can be, or comprise, a fragment of HPV E7 full length protein.

[0061] In some embodiments, the polypeptide further comprises an N-terminal signal peptide, a C-terminal signal peptide, or both. In some embodiments, the N-terminal signal peptide, the C- terminal signal peptide, or both, is a signal peptide of a human leukocyte antigen (HLA). The HLA can be HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G. The polypeptide can comprise an N-terminal signal peptide of HLA-F, a C-terminal signal peptide of HLA-G, or both. The N-terminal signal peptide of HLA-F can comprise the sequence of SEQ ID NO: 301 or a sequence have one, two, or three mismatches relative to the sequence of SEQ ID NO: 301. The C-terminal signal peptide of HLA-G can comprise the sequence of SEQ ID NO: 302 or a sequence have one, two, or three mismatches relative to the sequence of SEQ ID NO: 302.

[0062] In some embodiments, the polynucleotide encodes a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values) identical to the sequence of any one of SEQ ID NOs: 5-180. In some embodiments, the polynucleotide encodes a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 5-180 or an amino acid sequence having one, two, three, four, five, six, seven, eight, nine, or ten mismatches relative to any one of SEQ ID NOs: 5-180.

[0063] Disclosed herein include polypeptides. In some embodiments, the polypeptide comprises an SD HPV E6 protein (e.g., a fragment of HPV E6 full-length protein) or an SD HPV E7 protein (e.g., a fragment of HPV E7 full-length protein). In some embodiments, the SD HPV E6 protein or the SD HPV E7 protein is derived from a wild type HPV E6 protein or a wild type HPV E7 protein. The wild type HPV E6 or the wild type HPV E7 protein can comprise HP VI 6E6 protein, HP VI 6 E7 protein, HP VI 8 E6 protein, HP VI 8 E7 protein, or any combination thereof.

[0064] In some embodiments, the SD HPV E6 protein exhibits reduced binding affinity to pRB and / or the SD HPV E7 protein exhibits reduced binding affinity to p53, e.g., as compared to the wild type HPV E6 protein or the wild type HPV E7 protein. The reduction can be 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%,28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%,44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values, e.g., as compared to the wild type HPV E6 protein or the wild type HPV E7 protein. In some embodiments, the SD HPV E6 protein is not capable of binding to pRB and / or the SD HPV E7 protein is not capable of binding to p53. The SD HPV E6 protein and / or the SD HPV E7 protein can comprise one or more disrupted or deleted zinc-finger elements, e.g., as compared to the wild type HPV E6 protein or the wild type HPV E7 protein.

[0065] In some embodiments, the SD HPV E6 protein comprises a fragment of the wild type HPV E6 protein, the SD HPV E7 comprises a fragment of the wild type HPV E7 protein, or both. In some embodiments, the fragment of the wild type HPV E6 protein and / or the fragment of the wild type HPV E7 protein is less than 40 amino acids in length (e.g., 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 amino acids in length). The fragment of the wild type HPV E6 protein can comprise the sequence of any one of SEQ ID NOs: 5-8 and 12-15 or a sequence having one, two, or three mismatches relative to any one of SEQ ID NOs: 5-8 and 12-15. The fragment of the wild type HPV E7 protein can comprise the sequence of any one of SEQ ID NOs: 9-11 and 16-18 or a sequence having one, two, or three mismatches relative to any one of SEQ ID NOs: 9-11 and 16- 18.

[0066] The wild type HPV E6 protein or the wild type HPV E7 protein can comprise an amino acid sequence of any one of SEQ ID NOs: 1-4.

[0067] The polypeptide can comprise two or more SD E6 HPV proteins and / or SD HPV E7 proteins (e.g., fragments of HPV E6 full length protein and / or fragments of HPV E7 full-length protein). In some embodiments, the polypeptide can comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 SD E6 HPV proteins and / or SD HPV E7 proteins (e.g., fragments of wild type HPV E6 and / or fragments of HPV E7). The polypeptide can comprise an amino acid sequence of twoor more SD HPV E6 proteins, two or more SD HPV E7 proteins, or any combination thereof. The polypeptide can comprise four SD HPV E6 proteins and three SD HPV E7 proteins. The polypeptide can comprise, for example from N-terminus to C-terminus: a first SD HPV E6 protein, a first SD HPV E7 protein, a second SD HPV E6 protein, a second SD HPV E7 protein, a third SD HPV E6 protein, a third SD HPV E7 protein, and a fourth SD HPV E6 protein. A SD HPV E6 protein can be, or comprise, a fragment of HPV E6 full length protein. A SD HPV E6 protein can be, or comprise, a fragment of HPV E7 full length protein.

[0068] In some embodiments, the polypeptide further comprises an N-terminal signal peptide, a C-terminal signal peptide, or both. In some embodiments, the N-terminal signal peptide, the C- terminal signal peptide, or both, is a signal peptide of a human leukocyte antigen (HLA). In some embodiments, the HLA is HLA- A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G. The polypeptide can comprise an N-terminal signal peptide of HLA-F, a C-terminal signal peptide of HLA-G, or both.

[0069] The N-terminal signal peptide of HLA-F can comprise the sequence of SEQ ID NO: 301 or a sequence have one, two, or three mismatches relative to the sequence of SEQ ID NO: 301. The C-terminal signal peptide of HLA-G can comprise the sequence of SEQ ID NO: 302 or a sequence have one, two, or three mismatches relative to the sequence of SEQ ID NO: 302.

[0070] The polypeptide can comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values) identical to the sequence of any one of SEQ ID NOs: 5-180.

[0071] The polypeptide can comprise an amino sequence of any one of SEQ ID NOs: 5-180 or an amino acid sequence having one, two, three, four, five, six, seven, eight, nine, or ten mismatches relative to any one of SEQ ID NOs: 5-180.

[0072] Provided below are the amino acid sequences for the high-risk variant HPV16 and HP VI 8 E6 and E7 oncoproteins:

[0073] HPV16 E6 Sequence:MHQKRTAMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDLCI VYRDGNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLC PEEKQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRETQL (SEQ ID NO: 1)

[0074] HPV16 E7 Sequence:MHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCC KCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP (SEQ ID NO: 2)

[0075] HPV18 E6 Sequence: MARFEDPTRRPYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDLFVVYRDSIP HAACHKCIDFYSRIRELRHYSDSVYGDTLEKLTNTGLYNLLIRCLRCQKPLNPAEKLRH LNEKRRFHNIAGHYRGQCHSCCNRARQERLQRRRETQV (SEQ ID NO: 3)

[0076] HPV18 E7 Sequence: MHGPKATLQDIVLHLEPQNEIPVDLLCHEQLSDSEEENDEIDGVNHQHLPARRAEPQRH TMLCMCCKCEARIKLVVESSADDLRAFQQLFLNTLSFVCPWCASQQ (SEQ ID NO: 4)

[0077] Set forth in Table 1 below are amino acid sequences of structurally disrupted (SD) fragments of the HPV16 and HPV18 E6 and E7 oncoproteins. Skilled artisans will be aware that there are multiple nucleic acid sequences that can encode the amino acid sequences set forth in Table 1.Table 1. HPV16 and HPV18 E6 and E7 Fragments

[0078] Different combinations of the HPV16 and HPV18 E6 and E7 fragments from Table 1 are set forth below in Table 2. Skilled artisans will be aware that there are multiple nucleic acid sequences that can encode the amino acid sequences set forth in Table 2.Table 2. HPV16 and HPV18 E6 and E7 Fragment Combinations

[0079] Different combinations of the HPV16 and HPV18 E6 and E7 fragments from Table 1 are set forth below in Table 3. The amino acid sequences set forth in Table 3 are exemplary.Skilled artisans will be aware that there are multiple nucleic acid sequences that can encode the amino acid sequences set forth in Table 3.Table 3. HPV16 and HPV18 E6 and E7 Fragment Combinations

[0080] Different combinations of the HPV16 and HP VI 8 E6 and E7 fragments from Table 1 are set forth below in Table 4. The amino acid sequences set forth in Table 4 are exemplary.Skilled artisans will be aware that there are multiple nucleic acid sequences that can encode the amino acid sequences set forth in Table 4.Table 4. HPV16 and HPV18 E6 and E7 Fragment Combinations

[0081] Different overlapping peptide pool compositions are set forth below in Table 5-Table 8.Each peptide pool contains equal mass of all peptides listed in corresponding column.Table 5: HPV16 E7 overlapping peptide poolTable 6: HPV16 E6 overlapping peptide poolTable 7: HPV18 E7 overlapping peptide poolTable 8: HPV18 E6 overlapping peptide pool

[0082] In certain embodiments, provided herein are polynucleotides comprising, or consisting of, one or more nucleic acid sequences that encode an amino acid sequence that is the same as, or differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from, or shares at least 80%,85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater sequence identity with the amino acid sequences set forth in Table 1 or Table 2 or Table 3 or Table 4.

[0083] In certain embodiments, a polynucleotide may comprise one or more nucleic acid sequences that encode one or more amino acid sequences set forth in Table 1 or Table 2 or Table 3 or Table 4.Compositions

[0084] Disclosed herein include compositions. In some embodiments, the composition comprises: a nucleic acid composition comprising one or more polynucleotides of the disclosure, or the composition comprises one or more polypeptides disclosed herein.

[0085] The nucleic acid composition can be complexed or associated with one or more lipids or lipid-based carriers, thereby forming liposomes, lipid nanoparticles (LNPs), lipoplexes, and / or nanoliposomes, e.g., encapsulating the nucleic acid composition.

[0086] The one or more polynucleotides can be situated on the same nucleic acid and / or different nucleic acids. In some embodiments, the nucleic acid composition is, comprises, or further comprises, one or more vectors. At least one of the one or more vectors can be a viral vector, a plasmid, a naked DNA vector, a lipid nanoparticle (LNP), or any combination thereof. The viral vector can be an AAV vector, a lentivirus vector, a retrovirus vector, an adenovirus vector, a herpesvirus vector, a herpes simplex virus vector, a cytomegalovirus vector, a vaccinia virus vector, a MVA vector, a baculovirus vector, a vesicular stomatitis virus vector, a human papillomavirus vector, an avipox virus vector, a Sindbis virus vector, a VEE vector, a Measles virus vector, an influenza virus vector, a hepatitis B virus vector, an integration-deficient lentivirus (IDLV) vector, or any combination thereof. The one or more polynucleotides can be comprised in the one or more vectors. The one or more polynucleotides can be comprised in the same vector and / or different vectors. The one or more vectors can be a DNA vaccine. The one or more polynucleotides can be operably linked to one or more promoters capable of inducing transcription of the one or more polynucleotides. The DNA vaccine can be a plasmid-based DNA vaccine, a minicircle-based DNA vaccine, a bacmid-based DNA vaccine, a minigenebased DNA vaccine, a ministring DNA (linear covalently closed DNA vector) vaccine, a closed- ended linear duplex DNA (CELiD or ceDNA) vaccine, a doggybone™ DNA vaccine, a dumbbell shaped DNA vaccine, or a minimalistic immunological-defmed gene expression (MIDGE)-vector DNA vaccine.

[0087] The one or more promoters can comprise a ubiquitous promoter, an inducible promoter, a tissue-specific promoter and / or a lineage-specific promoter. In some embodiments, theubiquitous promoter is selected from the group comprising a cytomegalovirus (CMV) immediate early promoter, a CMV promoter, a viral simian virus 40 (SV40) (e.g., early or late), a Moloney murine leukemia virus (MoMLV) LTR promoter, a Rous sarcoma virus (RSV) LTR, an RSV promoter, a herpes simplex virus (HSV) (thymidine kinase) promoter, H5, P7.5, and Pl 1 promoters from vaccinia virus, an elongation factor 1 -alpha (EFla) promoter, early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock 70 kDa protein 5 (HSPA5), heat shock protein 90 kDa beta, member 1 (HSP90B1), heat shock protein 70 kDa (HSP70), P-kinesin (P-KIN), the human ROSA 26 locus, a Ubiquitin C promoter (UBC), a phosphoglycerate kinase- 1 (PGK) promoter, 3 -phosphoglycerate kinase promoter, a cytomegalovirus enhancer, human P-actin (HBA) promoter, chicken P-actin (CBA) promoter, a CAG promoter, a CASI promoter, a CBH promoter, or any combination thereof.

[0088] As used herein, the term “promoter” is a nucleotide sequence that permits binding of RNA polymerase and directs the transcription of a gene. Typically, a promoter is located in the 5’ non-coding region of a gene, proximal to the transcriptional start site of the gene. Sequence elements within promoters that function in the initiation of transcription are often characterized by consensus nucleotide sequences. Examples of promoters include, but are not limited to, promoters from bacteria, yeast, plants, viruses, and mammals (including humans). A promoter can be inducible, repressible, and / or constitutive. Inducible promoters initiate increased levels of transcription from DNA under their control in response to some change in culture conditions, such as a change in temperature.

[0089] As used herein, the term “operably linked” is used to describe the connection between regulatory elements and a gene or its coding region. Typically, gene expression is placed under the control of one or more regulatory elements, for example, without limitation, constitutive or inducible promoters, tissue-specific regulatory elements, and enhancers. A gene or coding region is said to be “operably linked to” or “operatively linked to” or “operably associated with” the regulatory elements, meaning that the gene or coding region is controlled or influenced by the regulatory element. For instance, a promoter is operably linked to a coding sequence if the promoter effects transcription or expression of the coding sequence.

[0090] The one or more polynucleotides can be operably linked to a tandem gene expression element. The tandem gene expression element can be an internal ribosomal entry site (IRES), foot-and-mouth disease virus 2A peptide (F2A), equine rhinitis A virus 2A peptide (E2A), porcine teschovirus 2A peptide (P2A) or Thosea asigna virus 2A peptide (T2A), or any combination thereof. The one or more polynucleotides can comprise a transcript stabilizationelement. The transcript stabilization element can comprise woodchuck hepatitis post- translational regulatory element (WPRE), bovine growth hormone polyadenylation (bGH- polyA) signal sequence, human growth hormone polyadenylation (hGH-polyA) signal sequence, or any combination thereof.

[0091] In some embodiments, the nucleic acid composition is or comprises mRNA. The composition (e.g., nucleic acid composition) can be an mRNA vaccine. The mRNA can be formulated in a lipid nanoparticle (LNP). The term “lipid nanoparticle”, also referred to as LNP, refers to a particle having at least one dimension on the order of nanometers (e.g., 1-1,000 nm) which includes one or more lipids. In some embodiments, such lipid nanoparticles comprise a cationic lipid and one or more excipient selected from neutral lipids, charged lipids, steroids and polymer conjugated lipids (e.g., a pegylated lipid). In some embodiments, the mRNA, or a portion thereof, is encapsulated in the lipid portion of the lipid nanoparticle or an aqueous space enveloped by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cells e.g. an adverse immune response. In some embodiments, the mRNA or a portion thereof is associated with the lipid nanoparticles. An LNP may comprise any lipid capable of forming a particle to which the one or more nucleic acid molecules are attached, or in which the one or more nucleic acid molecules are encapsulated. The term “lipid” refers to a group of organic compounds that are derivatives of fatty acids (e.g., esters) and are generally characterized by being insoluble in water but soluble in many organic solvents. Lipids are usually divided in at least three classes: (1) “simple lipids” which include fats and oils as well as waxes; (2) “compound lipids” which include phospholipids and glycolipids; and (3) “derived lipids” such as steroids.

[0092] The LNP can comprise one or more of an ionizable cationic lipid, a non- cationic lipid (e.g., a neutral lipid), a sterol, and a PEG-modified lipid. The LNP can comprise 0.5-15 mol% PEG-modified lipid, 5-25 mol% non-cationic lipid, 25-55 mol% sterol, and 20-60 mol% ionizable cationic lipid. The LNP can comprise 40-55 mol% ionizable cationic lipid, 5-15 mol% neutral lipid, 35-45 mol% sterol, and 1-5 mol% PEG-modified lipid. In some embodiments, the RNA (e.g., mRNA) of the disclosure is formulated in a lipid nanoparticle (LNP). Lipid nanoparticles typically comprise ionizable cationic lipid, non-cationic lipid, sterol and PEG lipid components along with the nucleic acid cargo of interest. The lipid nanoparticles of the disclosure can be generated using components, compositions, and methods as are generally known in the art, see for example PCT / US2016 / 052352; PCT / US2016 / 068300;PCT / US2017 / 037551; PCT / US2015 / 027400; PCT / US2016 / 047406; PCT / US2016 / 000129;PCT / US2016 / 014280; PCT / US2016 / 014280; PCT / US2017 / 038426; PCT / US2014 / 027077; PCT / US2014 / 055394; PCT / US2016 / 052117; PCT / US2012 / 069610; PCT / US2017 / 027492; PCT / US2016 / 059575 and PCT / US2016 / 069491 all of which are incorporated by reference herein in their entirety.

[0093] In some embodiments, the LNP comprises: 47 mol% ionizable cationic lipid, 11.5 mol% neutral lipid, 38.5 mol% sterol, and 3.0 mol% PEG-modified lipid; 48 mol% ionizable cationic lipid, 11 mol% neutral lipid, 38.5 mol% sterol, and 2.5 mol% PEG-modified lipid; 49 mol% ionizable cationic lipid, 10.5 mol% neutral lipid, 38.5 mol% sterol, and 2.0 mol% PEG-modified lipid; 50 mol% ionizable cationic lipid, 10 mol% neutral lipid, 38.5 mol% sterol, and 1.5 mol% PEG-modified lipid; or 51 mol% ionizable cationic lipid, 9.5 mol% neutral lipid, 38.5 mol% sterol, and 1.0 mol% PEG-modified lipid.

[0094] The ionizable cationic lipid can be heptadecan-9-yl 8 ((2 hydroxyethyl)(6 oxo 6- (undecyloxy)hexyl)amino)octanoate. The neutral lipid can be 1,2 distearoyl sn glycero-3 phosphocholine (DSPC). The sterol can be cholesterol. The PEG-modified lipid can be 1- monomethoxypolyethyleneglycol-2,3-dimyristylglycerol with polyethylene glycol of average molecular weight 2000 (PEG2000 DMG).

[0095] The wt / wt ratio of lipid to mRNA can be from about 1:100 to about 100:1 (e.g., 1:1, 1:1.1, 1:1.2, 1: 1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.5, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, E33, 1:34, 1:35, 1:36, 1:37, E38, 1:39, 1:40, 1:41, 1:42,1:43, 1:44, 1:45, 1:46, 1:47, 1:48, 1:49, 1:50, 1:51, 1:52, 1:53, 1:54, 1:55, 1:56, 1:57, 1:58, 1:59,1:60, 1:61, 1:62, 1:63, 1:64, 1:65, 1:66, 1:67, 1:68, 1:69, 1:70, 1:71, 1:72, 1:73, 1:74, 1:75, 1:76,1:77, 1:78, 1:79, 1:80, 1:81, 1:82, 1:83, 1:84, 1:85, 1:86, 1:87, 1:88, 1:89, 1:90, 1:91, 1:92, 1:93,1:94, 1:95, 1:96, 1:97, 1:98, 1:99, 1:100 to 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.5:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1,34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1,51:1, 52:1, 53:1, 54:1, 55:1, 56:1, 57:1, 58:1, 59:1, 60:1, 61:1, 62:1, 63:1, 64:1, 65:1, 66:1, 67:1,68:1, 69:1, 70:1, 71:1, 72:1, 73:1, 74:1, 75:1, 76:1, 77:1, 78:1, 79:1, 80:1, 81:1, 82:1, 83:1, 84:1,85:1, 86:1, 87:1, 88:1, 89:1, 90:1, 91:1, 92:1, 93:1, 94:1, 95:1, 96:1, 97:1, 98:1, 99:1, 100: 1, ora number or a range between any of these values).

[0096] The LNP can comprise a cationic lipid. The cationic lipid is can be cationisable, i.e. it becomes protonated as the pH is lowered below the pKa of the ionizable group of the lipid, but is progressively more neutral at higher pH values. When positively charged, the lipid is then ableto associate with negatively charged nucleic acids. In some embodiments, the cationic lipid comprises a zwitterionic lipid that assumes a positive charge on pH decrease. The LNP may comprise any lipid capable of forming a particle to which the one or more nucleic acid molecules are attached, or in which the one or more nucleic acid molecules are encapsulated. In some embodiments, the LNP may comprise any further cationic or cationisable lipid, i.e. any of a number of lipid species which carry a net positive charge at a selective pH, such as physiological pH. Such lipids include, but are not limited to, N,N-dioleyl- N,N- dimethylammonium chloride (DODAC); N-(2,3-dioleyloxy)propyl)-N,N,N- trimethylammonium chloride (DOTMA); N,N-distearyl-N,N-dimethylammonium bromide (DDAB); N-(2,3dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); 3-(N — (N’,N’dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(l-(2,3- dioleoyloxy)propyl)N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoracetate (DOSPA), dioctadecylamidoglycyl carboxyspermine (DOGS), l,2-dioleoyl-3- dimethylammonium propane (DODAP), N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), and N-(l,2dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (DMRLE).

[0097] Additionally, a number of commercial preparations of cationic lipids are available which can be used in embodiments provided herein. These include, for example, LIPOFECTIN® (commercially available cationic liposomes comprising DOTMA and 1,2- dioleoyl-sn- 3phosphoethanolamine (DOPE), from GIBCO / BRL, Grand Island, N.Y ); LIPOFECT AMINE® (commercially available cationic liposomes comprising N-(l- (2,3dioleyloxy)propyl)-N-(2- (sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA) and (DOPE), from GIBCO / BRL); and TRANSFECTAM® (commercially available cationic lipids comprising dioctadecylamidoglycyl carboxyspermine (DOGS) in ethanol from Promega Corp., Madison, Wis.). The following lipids are cationic and have a positive charge at below physiological pH: DODAP, DODMA, DMDMA, 1,2- dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1 ,2-dilinolenyloxy-N,N- dimethylaminopropane (DLenDMA).

[0098] Exemplary neutral lipids include, for example, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4- (N-maleimidomethyl)-cyclohexane-lcarboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-0-monom ethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1- stearioyl-2-oleoylphosphatidy ethanol amine (SOPE), and l,2-dielaidoyl-sn-glycero-3- phophoethanolamine (transDOPE). In some embodiments, the neutral lipid is 1,2-distearoyl-sn- glycero-3 phosphocholine (DSPC).

[0099] In some embodiments, the cationic lipid is an amino lipid. Suitable amino lipids useful include those described in W02012 / 016184, incorporated herein by reference in its entirety. Representative amino lipids include, but are not limited to, 1, 2-dilinoley oxy-3 - (dimethylamino)acetoxypropane (DLin-DAC), l,2-dilinoleyoxy-3morpholinopropane (DLin- MA), l,2-dilinoleoyl-3 -dimethylaminopropane (DLinDAP), l,2-dilinoleylthio-3- dimethylaminopropane (DLin-S-DMA), l-linoleoyl-2-linoleyloxy-3 dimethylaminopropane (DLin-2-DMAP), l,2-dilinoleyloxy-3 -trimethylaminopropane chloride salt (DLin-TMA.Cl),

[0100] 1.2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2- dilinoleyloxy-3-(N- methylpiperazino)propane (DLin-MPZ), 3 -(N,Ndilinoleylamino)- 1,2- propanediol (DLinAP), 3- (N,N-di oleylamino)- 1 ,2-propanediol (DO AP), 1 ,2-dilinoleyloxo-3 - (2-N,N- dimethylamino)ethoxypropane (DLin-EG-DMA), and 2,2-dilinoleyl-4- dimethylaminomethyl- [l,3]-dioxolane (DLin-K-DMA).

[0101] In some embodiments, a non-cationic lipid comprises 1,2-distearoyl-sn- glycero-3- phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1.2- dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-gly cero- phosphocholine (DMPC), l,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), l,2-di-0-octadecenyl-sn-glycero-3- phosphocholine (18:0 Diether PC), l-oleoyl-2 cholesterylhemisuccinoyl-sn-glycero-3- phosphocholine (OChemsPC), l-hexadecyl-sn-glycero-3 -phosphocholine (Cl 6 Lyso PC), 1,2- dilinolenoyl-sn-glycero-3 -phosphocholine, l,2-diarachidonoyl-sn-glycero-3 -phosphocholine, 1,2- didocosahexaenoyl-sn-glycero-3 -phosphocholine, l,2-diphytanoyl-sn-glycero-3- phosphoethanolamine (ME 16.0 PE), l,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2- dilinoleoyl-sn-glycero-3 -phosphoethanolamine, l,2-dilinolenoyl-sn-glycero-3- phosphoethanolamine, 1 ,2-diarachidonoyl-sn-glycero-3 -phosphoethanolamine, 1 ,2- didocosahexaenoyl-sn-glycero-3 -phosphoethanolamine, l,2-dioleoyl-sn-glycero-3-phospho-rac- (1 -glycerol) sodium salt (DOPG), sphingomyelin, and mixtures thereof.

[0102] In some embodiments, a PEG modified lipid comprises a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol,and mixtures thereof. In some embodiments, the PEG-modified lipid is DMG-PEG, PEG-c- DOMG (also referred to as PEG-DOMG), PEG-DSG and / or PEG-DPG. In some embodiments, a sterol comprises cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha- tocopherol, and mixtures thereof.

[0103] The LNP can comprise one or more of an ionizable cationic lipid, a non-cationic lipid, a sterol, and a PEG-modified lipid. The non-cationic lipid can be a neutral lipid. The LNP can comprise 0.5-15 mol% PEG-modified lipid, 5-25 mol% non-cationic lipid, 25-55 mol% sterol, and 20-60 mol% ionizable cationic lipid. The LNP can comprise: 40-55 mol% ionizable cationic lipid, 5-15 mol% neutral lipid, 35-45 mol% sterol, and 1-5 mol% PEG-modified lipid.

[0104] The LNP can comprise: 47 mol% ionizable cationic lipid, 11.5 mol% neutral lipid, 38.5 mol% sterol, and 3.0 mol% PEG-modified lipid; 48 mol% ionizable cationic lipid, 11 mol% neutral lipid, 38.5 mol% sterol, and 2.5 mol% PEG-modified lipid; 49 mol% ionizable cationic lipid, 10.5 mol% neutral lipid, 38.5 mol% sterol, and 2.0 mol% PEG-modified lipid; 50 mol% ionizable cationic lipid, 10 mol% neutral lipid, 38.5 mol% sterol, and 1.5 mol% PEG-modified lipid; or 51 mol% ionizable cationic lipid, 9.5 mol% neutral lipid, 38.5 mol% sterol, and 1.0 mol% PEG-modified lipid. In some embodiments: the ionizable cationic lipid is heptadecan-9-yl 8 ((2 hydroxy ethyl)(6 oxo 6-(undecyloxy)hexyl)amino)octanoate; the neutral lipid is 1,2- distearoyl-sn-glycero-3 phosphocholine (DSPC); the sterol is cholesterol; and / or the PEG- modified lipid is l-monomethoxypolyethyleneglycol-2,3-dimyristylglycerol with polyethylene glycol of average molecular weight 2000 (PEG2000 DMG).

[0105] The composition can comprise particles comprising the one or more polypeptides. In some embodiments, the particles are iron oxide particles, liposomes, micelles, polymer complexes, cationic peptide nanoemulsions, virus-like particles (VLPs), lipid nanoparticles (LNP) and / or lipoplex (LPX) particles.

[0106] The mRNA can comprise a 5' untranslated region (UTR), a 3' UTR, and / or a cap. In some embodiments, the mRNA comprises one or more modified nucleotides selected from the group comprising pseudouridine, N-l-methyl-pseudouridine, 2-aminoadenosine, 2- thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5- fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, and 2-thiocytidine. The mRNA can comprise a modified nucleotide in place of one or more uridines. In some embodiments, the modified nucleotide is selected from pseudouridine (y), N 1-methyl-pseudouridine (m IT), and 5-methyl-uridine (m5U).

[0107] In some embodiments, the nucleic acid composition comprising an mRNA sequence is a modified mRNA sequence. In this context, a modification as defined herein can lead to a stabilization of the mRNA sequence provided herein. In some embodiments, there is thus provided a stabilized mRNA sequence comprising at least one coding region as defined herein (e.g. polynucleotide(s) encoding fusion protein(s)). In some embodiments, the nucleic acid composition comprising an mRNA sequence may thus be provided as a “stabilized mRNA sequence”, that is to say as an mRNA that is essentially resistant to in vivo degradation (e.g. by an exo- or endo-nuclease). Such stabilization can be effected, for example, by a modified phosphate backbone of an mRNA provided herein. A backbone modification can be a modification in which phosphates of the backbone of the nucleotides contained in the mRNA are chemically modified. Nucleotides that can be used in this connection contain e.g. a phosphorothioate-modified phosphate backbone, such as at least one of the phosphate oxygens contained in the phosphate backbone being replaced by a sulfur atom. Stabilized mRNAs may further include, for example: non-ionic phosphate analogues, such as, for example, alkyl and aryl phosphonates, in which the charged phosphonate oxygen is replaced by an alkyl or aryl group, or phosphodiesters and alkylphosphotriesters, in which the charged oxygen residue is present in alkylated form. Such backbone modifications typically include, without implying any limitation, modifications from the group consisting of methylphosphonates, phosphoramidates and phosphorothioates (e.g. cytidine-5’-0-(l -thiophosphate)). The term “mRNA modification” as used herein may refer to chemical modifications comprising backbone modifications as well as sugar modifications or base modifications. In this context, a modified mRNA (sequence) as defined herein may contain nucleotide analogues / modifications, e.g. backbone modifications, sugar modifications or base modifications. A backbone modification can be a modification, in which phosphates of the backbone of the nucleotides contained in an mRNA compound comprising an mRNA sequence as defined herein are chemically modified. A sugar modification can be a chemical modification of the sugar of the nucleotides of the mRNA compound comprising an mRNA sequence as defined herein. Furthermore, a base modification can be a chemical modification of the base moiety of the nucleotides of the mRNA compound comprising an mRNA sequence. In this context, nucleotide analogues or modifications can be selected from nucleotide analogues, which are applicable for transcription and / or translation.

[0108] The mRNA provided herein can comprise a 5' untranslated region (UTR), a 3' UTR, and / or a cap (e.g., a CAP analogue). A modified mRNA sequence as defined herein, can be modified by the addition of a so-called “5’ -CAP structure”, which can stabilize the mRNA as described herein. A 5’ -CAP is an entity, typically a modified nucleotide entity, which generally “caps” the 5’ -end of a mature mRNA. A 5’ -CAP may typically be formed by a modifiednucleotide, particularly by a derivative of a guanine nucleotide. In some embodiments, the 5’- CAP is linked to the 5’ -terminus via a 5 ’ -5 ’ -triphosphate linkage. A 5 ’-CAP may be methylated, e.g. m7GpppN, wherein N is the terminal 5’ nucleotide of the nucleic acid carrying the 5 ’-CAP, typically the 5’-end of an mRNA. m7GpppN is the 5’-CAP structure, which naturally occurs in mRNA transcribed by polymerase II and is therefore in some embodiments is not considered as modification comprised in a modified mRNA in this context. Accordingly, a modified mRNA sequence may comprise a m7GpppN as 5’-cap, but additionally the modified mRNA sequence typically comprises at least one further modification as defined herein. A CAP analogue refers to a non-polymerizable di-nucleotide that has CAP functionality in that it facilitates translation or localization, and / or prevents degradation of the RNA molecule when incorporated at the 5 ’-end of the RNA molecule. Non-polymerizable means that the CAP analogue will be incorporated only at the 5 ’-terminus because it does not have a 5’ triphosphate and therefore cannot be extended in the 3 ’-direction by a template-dependent RNA polymerase. CAP analogues include, but are not limited to, a chemical structure selected from the group consisting of m7GpppG, m7GpppA, m7GpppC; unmethylated CAP analogues (e.g., GpppG); dimethylated CAP analogue (e.g., m2,7GpppG), trimethylated CAP analogue (e.g., m2,2,7GpppG), dimethylated symmetrical CAP analogues (e.g., m7Gpppm7G), or anti reverse CAP analogues (e.g., ARC A; m7,2’OmeGpppG, m7,2’dGpppG, m7,3’OmeGpppG, m7,3’dGpppG and their tetraphosphate derivatives) (Stepinski et al., 2001. RNA 7(10): 1486- 95). Further CAP analogues have been described previously (U.S. Pat. No. 7,074,596, W02008 / 016473, W02008 / 157688, WO2009 / 149253, WO2011 / 015347, and WO2013 / 059475).

[0109] The mRNA can comprise one or more modified nucleotides selected from the group comprising pseudouridine, N-l -methyl-pseudouridine, 2-aminoadenosine, 2- thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5- fluorouridine, C5-iodouridine, C5- propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7- deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, and 2-thiocytidine. The mRNA can comprise a modified nucleotide in place of one or more uridines. The modified nucleoside can be selected from pseudouridine (y), N 1 -methyl-pseudouridine (m 1 Y), and 5 -methyl -uridine (m5U). In some embodiments, a non- naturally occurring modified nucleotide or nucleoside of the disclosure is one as is generally known or recognized in the art. Non-limiting examples of such non-naturally occurring modified nucleotides and nucleosides can be found, inter alia, in published application Nos. PCT / US2012 / 058519;PCT / US2013 / 075177; PCT / US2014 / 058897; PCT / US2014 / 058891; PCT / US2014 / 070413;PCT / US2015 / 36773; PCT / US2015 / 36759; PCT / US2015 / 36771; or PCT / IB 2017 / 051367 all of which are incorporated by reference herein.

[0110] The composition can comprise one or more polynucleotides encoding immunostimulatory agents. In some embodiments, the immunostimulatory agents are selected from the group comprising toll-like receptor (TLR) agonists, cytokine receptor agonists, CD40 agonists, Fc receptor agonists, CpG-containing nucleic acids, complement receptor agonists, or any combination thereof. The TLR agonist can be a TLR-1 agonist, TLR-2 agonist, TLR-3 agonist, TLR-4 agonist, TLR-5 agonist, TLR-6 agonist, TLR-7 agonist, TLR-8 agonist, TLR-9 agonist, and / or TLR- 10 agonist. The Fc receptor agonist can be a Fc-gamma receptor agonist. The complement receptor agonist binds to CD21 or CD35. The cytokine receptor agonist can be a cytokine. In some embodiments, the cytokine receptor agonist can be a small molecule, antibody, fusion protein, or aptamer. The composition can comprise an adjuvant. In some embodiments, the adjuvant comprises aluminum hydroxide, alhydrogel, AddaVax, MF59, AS03, Freund’s adjuvant, Montanide ISA51, CpG, Poly LC, glucopyranosyl lipid A, flagellin, resiquimod, or any combination thereof.

[0111] The composition can be formulated for a variety of modes of administration. Techniques for formulation and administration can be found, for example, in “Remington's Pharmaceutical Sciences”, 18thed., 1990, Mack Publishing Co., Easton, Pa. In some embodiments, the compositions of the present disclosure may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes; (2) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension: (3) topical application, for example, as a cream, ointment or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; or (5) aerosol, for example, as an aqueous aerosol, liposomal preparation or solid particles containing the hydrogel composition. The pharmaceutical compositions can comprise one or more pharmaceutically-acceptable carriers.

[0112] Formulations useful in the methods of the present disclosure include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol and / or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a pharmaceutically acceptable carrier to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. Theamount of active ingredient, which can be combined with a pharmaceutically acceptable carrier to produce a single dosage form will generally be that amount of the composition (e.g., a nucleic acid composition, a population of ENPs) which produces a therapeutic effect or an immune response. Generally, out of one hundred percent, this amount will range from about 1% to about 99% of active ingredient, optionally from about 5% to about 70%, optionally from about 10% to about 30%.

[0113] The composition can be formulated for parenteral administration by injection, e.g. by bolus injection or continuous infusion. Formulations for injection can be presented in a unit dosage form, e.g. in ampoules or in multi-dose containers, with an optionally added preservative. The pharmaceutical compositions can further be formulated as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain other agents including suspending, stabilizing and / or dispersing agents.

[0114] In some embodiments, the composition further comprises Tris buffer, sucrose, and / or sodium acetate. The composition can be a lyophilized composition. In some embodiments, the lyophilized composition has a water content of less than about 10%. In some embodiments, the composition is formulated or is to be formulated: as a liquid, a solid, or a combination thereof; for injection; and / or for intramuscular administration, intranasal administration, transdermal administration, aerosol delivery, nasal delivery, vaginal delivery, rectal delivery, buccal delivery, ocular delivery, local delivery, topical delivery, intraci sternal delivery, intraperitoneal delivery, oral delivery, intramuscular injection, intravenous injection, subcutaneous injection, intranodal injection, intratumoral injection, intraperitoneal injection, or intradermal injection.

[0115] In some embodiments, the composition is a pharmaceutical composition, wherein the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents and / or excipients. The composition can comprise instructions for use of the composition for: stimulating an immune response in a subject in need thereof; treating or preventing HPV- related disease or disorder in a subject in need thereof; and / or treating or preventing an HPV- related cancer in a subject in need thereof. Disclosed herein include kits. The kit can comprise any composition of the disclosure. Also disclosed are cells comprising a nucleic acid composition described herein. The compositions disclosed herein can be used as a medicament (e.g., for treating an HPV infection, cancer, or both).

[0116] In some embodiments, provided herein are compositions comprising a polynucleotide comprising one or more nucleic acids described herein. In some embodiments, the compositions are formulated for delivery to a subject. In some embodiments, the compositions may further comprise one or more pharmaceutically acceptable carriers, excipients, preservatives, or acombination thereof. A “pharmaceutically acceptable carrier” refers to a pharmaceutically- acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting an active ingredient from one location, body fluid, tissue, organ (interior or exterior), or portion of the body, to another location, body fluid, tissue, organ, or portion of the body. Each carrier is “pharmaceutically acceptable” in the sense of being compatible with the other ingredients, e.g., the nucleic acids described herein or other ingredients, of the formulation and suitable for use in contact with the tissue or organ of a biological subject without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0117] Pharmaceutically acceptable carriers are well known in the art and include, without limitation, (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) alcohol, such as ethyl alcohol and propane alcohol; (20) phosphate buffer solutions; (21) lipid nanoparticles; and (22) other non-toxic compatible substances employed in pharmaceutical formulations.

[0118] The pharmaceutical formulations disclosed herein may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like.Methods of Treatment

[0119] In some aspects, provided herein are methods for preventing and / or treating a disease in a subject in need thereof. In certain embodiment, the disease may be HPV infection. In some embodiments, the HPV may be caused by HPV types or variants. In certain embodiments, the HPV variants may be HPV16, HPV 18, or a combination thereof. In some embodiments, the HPV is a persistent infection of HPV. In certain embodiments, the methods prevent carcinogenesis after HPV infections. In certain embodiments, the methods treat early-stage carcinogenesis caused by high-risk HPV strains, such as HPV16 and / or HPV18.

[0120] In certain embodiments, methods of preventing or treating a disease in a subject may comprise administering to the subject a polynucleotide comprising, or consisting of, one or more nucleic acid sequences that encodes an amino acid sequence that is the same as, or differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from, or shares at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater sequence identity with the amino acid sequences set forth in Table 1 or Table 2 or Table 3 or Table 4.

[0121] As used herein, the term “treatment” or “treat” refers to an intervention made in response to a disease, disorder or physiological condition (e.g., an HPV infection and / or cancer) manifested by a patient. The aim of treatment may include, but is not limited to, one or more of the alleviation or prevention of symptoms, slowing or stopping the progression or worsening of a disease, disorder, or condition and the remission of the disease, disorder or condition. The term “treat” and “treatment” includes, for example, therapeutic treatments, prophylactic treatments, and applications in which one reduces the risk that a subject will develop a disorder or other risk factor. Treatment does not require the complete curing of a disorder and encompasses embodiments in which one reduces symptoms or underlying risk factors. In some embodiments, “treatment” refers to both therapeutic treatment and prophylactic or preventative measures.Those in need of treatment include those already affected by a disease or disorder or undesired physiological condition as well as those in which the disease or disorder or undesired physiological condition is to be prevented. As used herein, the term “prevention” refers to any activity that reduces the burden of the individual later expressing those symptoms. This can take place at primary, secondary and / or tertiary prevention levels, wherein: a) primary prevention avoids the development of symptom s / disorder / conditi on; b) secondary prevention activities are aimed at early stages of the condition / disorder / symptom treatment, thereby increasing opportunities for interventions to prevent progression of the condition / disorder / symptom and emergence of symptoms; and c) tertiary prevention reduces the negative impact of an already established condition / disorder / symptom by, for example, restoring function and / or reducing any condition / disorder / symptom or related complications. The term “prevent” does not require the 100% elimination of the possibility of an event. Rather, it denotes that the likelihood of the occurrence of the event has been reduced in the presence of the compound or method.

[0122] The term “condition” as used herein indicates a physical status of the body of an individual (as a whole or as one or more of its parts), that does not conform to a standard physical status associated with a state of complete physical, mental and social well-being for the individual. Conditions herein described include but are not limited disorders and diseases wherein the term “disorder” indicates a condition of the living individual that is associated to afunctional abnormality of the body or of any of its parts, and the term “disease” indicates a condition of the living individual that impairs normal functioning of the body or of any of its parts and is typically manifested by distinguishing signs and symptoms.

[0123] The terms “subject”, “subject in need”, and “individual” as used herein refer to an animal and in particular higher animals and in particular vertebrates such as mammals and more particularly human beings. In some embodiments, the subject or individual has been exposed to HPV. The term “exposed” indicates the subject has come in contact with a person or an animal that is known to be infected with HPV. In some embodiments, a subject in need can be a healthy subject exposed to or at risk of being exposed to HPV. In some embodiments, subjects in need include those already suffering from the disease or disorder caused by an HPV infection and / or cancer or those diagnosed with an HPV infection and / or cancer.

[0124] Accordingly, the compositions can be administered in advance of any symptom, for example, in advance of an HPV infection and / or cancer. The compositions can also be administered at or after the onset of a symptom of disease or infection, for example, after development of a symptom of infection or after diagnosis of the infection.

[0125] The phrase “therapeutically effective amount” as used herein means that amount of carriers disclosed herein which is effective for producing some desired therapeutic effect and / or generating a desired response, such as reduce or eliminate a sign or symptom of a condition or disease, such as pneumonia, at a reasonable benefit / risk ratio. The therapeutically effective amount also varies depending on the structure and antigens of the carrier, the route of administration utilized, and the specific diseases or disorders to be treated as will be understood to a person skilled in the art. For example, if a given clinical treatment is considered effective when there is at least a 20% reduction in a measurable parameter associated with a disease or disorder, a therapeutically effective amount of the carriers for the treatment of that disease or disorder is the amount necessary to achieve at least a 20% reduction in that measurable parameter.

[0126] Disclosed herein include methods of stimulating an immune response in a subject in need thereof. In some embodiments, the method comprises: administering to the subject a pharmaceutically effective amount of a polynucleotide, a polypeptide, or a composition of the disclosure, thereby stimulating the immune response in the subject.

[0127] Disclosed herein include methods of treating or preventing a disease or disorder in a subject in need thereof. In some embodiments, the method comprises: administering to thesubject a pharmaceutically effective amount of a polynucleotide, a polypeptide, or a composition of the disclosure, thereby treating or preventing the disease or disorder in the subject.

[0128] In some embodiments, the disease or disorder is an HPV-related disease or disorder. The HPV-related disease or disorder can be cancer. The cancer can be anal cancer, cervical cancer, oropharyngeal cancer, penile cancer, vaginal cancer, or vulvar cancer.

[0129] In some embodiments, immunogenic levels of the SD HPV E6 protein, the SD HPV E7 protein, or both, are produced in serum of the subject at about 1 hour to about 6 months (e.g., 1 hour, 2, hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours,12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 8 days, 9 days,10 days, 11 days, 12 days, 13 days, 2 weeks, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 3 weeks, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or a number or a range between any of these values) post administration of the composition. In some embodiments, a neutralizing antibody titer of about 50 to about 100000 half-maximal inhibitory dilutions (ID50s values) is produced in the serum of the subject at about 1 hour to about 6 months (e.g., 1 hour, 2, hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours,13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 8 days, 9 days, 10 days,11 days, 12 days, 13 days, 2 weeks, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 3 weeks, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or a number or a range between any of these values) post administration of the composition.

[0130] The method can comprise administering to the subject one or more doses of the composition (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or a number or a range between any of these values). In some embodiments, a second dose of the composition is administered to the subject at least 14 days (e.g., 2 weeks, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 3 weeks, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 2 years or more) after a first dose of the composition is administered to the subject. In some embodiments, the second dose of the composition is administered to the subject at least 28 days (e.g., 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9months, 10 months, 11 months, 1 year, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 2 years or more) after the first dose of the composition is administered to the subject. In some embodiments, the first dose is a prime, and the second dose is a boost.

[0131] Each of the one or more doses of the composition can comprise about 2 pg to about 5 pg (e.g., 2 pg, 2.1 pg, 2.2 pg, 2.3 pg, 2.4 pg, 2.5 pg, 2.6 pg, 2.7 pg, 2.8 pg, 2.9 pg, 2.1 pg, 2.2 pg, 2.3 pg, 2.4 pg, 2.5 pg, 2.6 pg, 2.7 pg, 2.8 pg, 2.9 pg, 3 pg, 3.1 pg, 3.2 pg, 3.3 pg, 3.4 pg, 3.5 pg, 3.6 pg, 3.7 pg, 3.8 pg, 3.9 pg, 4 pg, 4.1 pg, 4.2 pg, 4.3 pg, 4.4 pg, 4.5 pg, 4.6 pg, 4.7 pg, 4.8 pg, 4.9 pg, 5 pg, or a number or a range between any of these values) of the nucleic acid composition. In some embodiments, each of the one or more doses of the composition can comprise less than 2 pg of the nucleic acid composition. In some embodiments, each of the one or more doses of the composition can comprise more than 5 pg of the nucleic acid composition.

[0132] In some embodiments, administering the composition induces neutralizing responses against HPV, cancer cells, or both. The HPV can be HPV16, HPV18, or both. In some embodiments, the administration of the composition elicits protective and long-lasting immunity against HPV, cancer, or both. The polynucleotide, the polypeptide, or the composition can be administered in an effective amount to induce a robust antibody response against the HPV, cancer cells, or both. A robust antibody response can comprise a neutralizing antibody response. In some embodiments, a robust antibody response comprises Fc domain effector functions that recruit immune cells to infected cells. In some embodiments, said immune cells are macrophages, neutrophils, and / or natural killer cells. In some embodiments, said recruitment induces antibody-dependent cellular cytotoxicity (ADCC) and / or antibody-dependent cellular phagocytosis (ADCP). The polynucleotide, the polypeptide, or the composition can be administered in an effective amount to elicit a robust CD4 and / or CD8 T cell response against the HPV, the cancer cells, or both in the subject. The polynucleotide, the polypeptide, or the composition can be administered in an effective amount to elicit a balanced Thl / Th2 response against the HPV, the cancer cells, or both in the subject. The polynucleotide, the polypeptide, or the composition can be administered in an effective amount to polynucleotide, the polypeptide, or the composition can be administered in an effective amount to induce the production of one or more cytokines in the subject. The one or more cytokines can comprise IL-2, IFN-y, TNF-a, IL-1, IL- 12, IL- 17, IL- 18, or any combination thereof. In some embodiments, at least a portion of the cancer cells express the wild type HPV E6 protein or variants thereof, the wild type HPV E7 protein or variants thereof, or any combination thereof.

[0133] Treating or preventing the disease or disorder in the subject can comprise inhibiting the onset or progression of the cancer. The inhibition of progression of the cancer can be measured by tumor growth inhibition (TGI) and wherein the TGI resulted by the administration is at least 30% higher (e.g., 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%,43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% higher, or a number or a range between any two of these values) as compared to an untreated subject. In some embodiments, a tumor volume after the administration decreases at least 50% (e.g., 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values) relative to the tumor volume with no treatment or the tumor volume prior to the administration. In some embodiments, the subject achieves a complete response. In some embodiments, the subject does not develop HPV-related cancer following the administration.

[0134] The subject can be a human subject. The human subject can be at least 9 years old. In some embodiments, the subject has had a prior HPV infection. The compositions herein described can be administered using techniques well known to those skilled in the art, such as injection, inhalation or insulation or by oral, parenteral or rectal administration. The composition can be administered by means including, but not limited to, traditional syringes and needleless injection devices. Suitable routes of administration include, but are not limited to, parenteral delivery, such as intramuscular, intradermal, subcutaneous, intramedullary injections, as well as, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections. For injection, the compositions described herein described can be formulated in aqueous solutions, optionally in physiologically compatible buffers such as Hanks’ solution, Ringer's solution, or physiological saline buffer. In some embodiments, administering can comprise aerosol delivery, nasal delivery, vaginal delivery, rectal delivery, buccal delivery, ocular delivery, local delivery, topical delivery, intracistemal delivery, intraperitoneal delivery, oral delivery, intramuscular injection, intravenous injection, subcutaneous injection, intranodal injection, intratumoral injection, intraperitoneal injection, intradermal injection, or any combination thereof. The composition can be administered intramuscularly (e.g., into a deltoid region of an arm). The composition can be co-administered with an adjuvant. In some embodiments, the composition is not co-administered with an adjuvant.

[0135] In some embodiments, the compositions provided herein can be administered to a subject systematically. The wording “systemic administration” as used herein indicates any route of administration by which a composition is brought in contact with the body of the individual, so that the resulting composition location in the body is systemic (i.e. non limited to a specific tissue, organ or other body part where the vaccine is administered). Systemic administration includes enteral and parenteral administration. Enteral administration is a systemic route of administration where the substance is given via the digestive tract, and includes but is not limited to oral administration, administration by gastric feeding tube, administration by duodenal feeding tube, gastrostomy, enteral nutrition, and rectal administration. Parenteral administration is a systemic route of administration where the substance is given by route other than the digestive tract and includes but is not limited to intravenous administration, intra-arterial administration, intramuscular administration, subcutaneous administration, intradermal, administration, intraperitoneal administration, and intravesical infusion.

[0136] The following examples are intended to illustrate various embodiments of the disclosure. As such, the specific embodiments discussed are not to be construed as limitations on the scope of the disclosure. It will be apparent to one skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of disclosure, and it is understood that such equivalent embodiments are to be included herein. Further, all references cited in the disclosure are hereby incorporated by reference in their entirety, as if fully set forth herein.EXAMPLESExample 1 : AOV Vaccines Block Tumor Growth in Mice When Administered Before Tumor Challenge

[0137] To test whether vaccines comprising different combinations of HPV16 and HPV18 E6 and E7 fragments (AOV C2, AOV C6, AOV C8, AOV C9, AOV CC, AOV CD, AOV CE, or AOV CF (Table 2)) were able to prevent tumor growth in mice when administered prior to challenge with tumor cells, mice were administered the vaccines and then challenged with tumor cells. As shown herein, five AOV vaccines (pAOV_C2, pAOV_CC, pAOV_CD, pAOV_CE, and pAOV CF) completely prevented tumor growth in mice.

[0138] Cell Lines. C3.43, a progressive subclone of the HPV16-transformed B6 mouse embryo cell line C3, was used for the tumor challenge studies described below. C3.43 cellswere grown and expanded in vitro with Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 10% fetal bovine serum (FBS).

[0139] Gene Gun Immunization. Pathogen-free 6-8-week-old C57BL / 6 female mice were anesthetized with isoflurane (4% induction, 2% maintenance) in an induction chamber and then individually placed onto a nose cone apparatus for procedures. The abdomen area around the administration site was shaved with animal clippers to expose skin. After fur removal was completed, the area was cleansed of loose fur and disinfected using an isopropanol wipe. While still under anesthesia, DNA gold microcarriers loaded with plasmid DNA that encodes AOV_C2, AOV_C6, AOV C8, AOV C9, AOV CC, AOV CD, AOV CE, or AOV CF (Table 2) were delivered to the abdomen subcutaneously by helium-pressure-based gene gun bombardment. Mice were placed into clean cages post immunization and were monitored until they woke up from anesthesia. Mice were monitored the day after vaccination for postvaccination adverse events.

[0140] Immunization - Tumor Challenge Schedule. Mice were separated into nine groups of 10 mice each. Mice were either untreated or dosed with one of eight different AOV vaccines comprising plasmid DNA that encoded AOV_C2, AOV_C6, AOV_C8, AOV_C9, AOV_CC, AOV CD, AOV CE, or AOV CF. The amino acid sequences of each of the AOV constructs are set forth in Table 2. Each immunization dose contained DNA gold microcarriers loaded with 2 pg of the plasmid DNA constructs (Table 2) mixed with 2 pg of the pBOOST2sahIRF3 (Invitrogen) cloning vector. Each group of mice (N=10 mice) received two doses of the same vaccine. The first dose was administrated 21 days before the C3.43 tumor cell challenge (see below). Fourteen days later, the mice received a second vaccination with the same DNA vaccine construct as the first dose. Seven days later, all mice were challenged with C3.43 tumor cells as described below.

[0141] Tumor Challenge. The nine groups of mice were challenged subcutaneously with IxlO5C3.43 tumor cells in 100 pl Hanks’ Balanced Salt Solution (HBSS). Tumor growth was measured two times per week with manual calipers by measuring tumor length, height, and depth to generate a tumor volume (volume = 1 x w x d). Tumor volumes exceeding 1500 mm3resulted in euthanasia. Mildly ulcerated tumors less than 1500 mm3were subjected to a veterinary-approved wound care regimen (flushing with sterile saline, treating with Vetericyn spray, and covering wound with liquid bandage every 2-3 days). Mice that developed severe ulcerations (actively bleeding, or ulceration >50% of tumor surface area) were euthanized regardless of tumor size. No mice had tumor volumes exceeding 1500 mm3or severe ulcerations.

[0142] Study Period. Tumor growth was monitored for up to 60 days post tumor challenge. Mice with tumors exceeding 10% body weight or tumors ulcerating through the skin surface were euthanized. Tumor dimensions were measured twice weekly in three dimensions (1 x w x d) to calculate tumor volume. Mice that did not develop tumors were euthanized after 60 days or 1 week after all other tumor-bearing mice were euthanized.

[0143] Results. Out of the eight vaccines tested, five of them (pAOV_C2, pAOV CC, pAOV CD, pAOV CE, and pAOV CF) completely blocked tumor growth when challenged by C3.43 cells (FIGs. IB, IF, 1G, 1H, and II, respectively). All mice immunized with these five vaccines were 100% tumor free and survived the whole length of the study period (FIGs. IB, IF, 1G, 1H, II, and FIG. 2). In contrast, all mice in the untreated group failed in the blocking tumor challenge (FIG. 1A), with a median survival of 32.5 days (FIG. 2). The other three AOV DNA vaccine constructs (pAOV_C6, pAOV_C8, pAOV_C9) also failed in the blocking tumor challenge (FIGs. 1C, ID, and IE, respectively), with modest survival extension (pAOV_C6: median survival 35 days; pAOV_C8: medium survival 35 days; pAOV_C9: medium survival 37.5 days) (FIG. 2).Example 2: AOV-CFR (Table 3) Block Tumor Growth in Mice When Administered after Tumor Challenge

[0144] To test whether vaccines comprising different combinations of HPV16 and HPV18 E6 and E7 fragments (AOV CF (Table 2)) were able to prevent tumor growth in mice when administered prior to challenge with tumor cells, mice were administered the vaccines and then challenged with tumor cells. As shown herein, five AOV vaccines (pAOV_C2, pAOV_CC, pAOV CD, pAOV CE, and pAOV CF) completely prevented tumor growth in mice.

[0145] Structurally disrupted fragment combination AOV CF (Table 2) was engineered into a proper open reading frame of a typical IVT vector to generate corresponding plasmids. In vitro transcriptions were performed on linearized plasmids to generate mRNA encode AOV CF. The mRNA contains a T7 promoter, a 5’UTR, an open reading frame encodes AOV CF, a 3’UTR, and a poly- A sequence of 110 A. Purified AOV CF mRNA was enzymatically capped, purified and quantified following standard procedures and encapsulated in lipid nanoparticles (LNP) with standard procedures to generate AOV-CFR (Table 3).

[0146] Cell Lines. TC-1, a progressive subclone of the HPV16 E6 and E7 proteins- transformed lung epithelial cells, was used for the tumor challenge studies described below. TC-1 cells were grown and expanded in vitro with Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 10% fetal bovine serum (FBS).

[0147] Tumor Challenge. Pathogen-free 6-8-week-old C57BL / 6 female mice each were challenged subcutaneously with 5xl05TC-1 tumor cells in 100 pl Hanks’ Balanced Salt Solution (HBSS). Tumor growth was measured two times per week with manual calipers by measuring tumor length, height, and depth to generate a tumor volume (volume = 1 x w x d). Tumor volumes exceeding 1500 mm3resulted in euthanasia. Mildly ulcerated tumors less than 1500 mm3were subjected to a veterinary-approved wound care regimen (flushing with sterile saline, treating with Vetericyn spray, and covering wound with liquid bandage every 2-3 days). Mice that developed severe ulcerations (actively bleeding, or ulceration >50% of tumor surface area) were euthanized regardless of tumor size.

[0148] Intramuscular Injection. Pathogen-free 6-8-week-old C57BL / 6 female mice were anesthetized with isoflurane (4% induction, 2% maintenance). The right hind limb area around the administration site was shaved with animal clippers to expose skin. After fur removal was completed, the area was cleansed of loose fur and disinfected using an isopropanol wipe. One hundred microliter ( lOOpl) of empty LNP or LNP loaded with 5pg AOV-CFR mRNA (Table 3) mixture were injected into the thigh muscle of the hind limb with 1ml syringe. Mice were placed into clean cages post immunization and were monitored until they woke up from anesthesia. Mice were monitored the day after vaccination for post-vaccination adverse events.

[0149] Tumor Challenge - Treatment Schedule. The mice were separated into two groups of 10 mice each. Group 1 mice were challenged with 5xl05TC-1 cells on Day 0 and were treated with empty LNP on Day 7, Day 28, and Day 58. Group 2 mice were challenged with 5xl05TC- 1 cells on Day 0 and were treated with LNP contains 5pg AOV CFR (Table 3) on Day 7, Day 28, and Day 58.

[0150] Study Period. Tumor growth was monitored for up to 60 days post tumor challenge. Mice with tumors exceeding 10% body weight or tumors ulcerating through the skin surface were euthanized. Tumor dimensions were measured twice weekly in three dimensions (1 x w x d) to calculate tumor volume. Mice that did not develop tumors were euthanized after 60 days or 1 week after all other tumor-bearing mice were euthanized.

[0151] Results. In group 1 mice, tumor volumes increased with time, and all mice were euthanized on or before day 46 when tumor volume exceeded 1500mm3or severe ulcerations (FIG. 3A). In group 2 mice, tumor size peaked around Day 21, then diminished over time and completely disappeared around Day 33 (FIG. 3B). All mice in group 2 were 100% tumor free afterward and survived the whole length of the study period (FIG. 3B).Example 3: AOV-SD1 Block Tumor Growth in Mice When Administered after TumorChallenge

[0152] Each structurally disrupted E6 and E7 proteins of HPV16 and 18 were individually engineered into a proper open reading frame of a typical IVT vector to generate corresponding plasmids. In vitro transcriptions were performed on linearized plasmids to generate mRNA encoding individual structurally disrupted E6 and E7 proteins of HPV16 and 18. Each type of mRNA contains a T7 promoter, a 5’UTR, an open reading frame encodes one of the structurally disrupted E6 and E7 proteins of HPV16 and 18, a 3’UTR, and a poly-A sequence. Each type of mRNA was capped, purified and quantified following standard procedures. Equal mass of the four types of mRNAs encoding structurally disrupted HPV16E6, HPV16E7, HPV18E6, and HPV18E7 (Table 3) were mixed together at equal molar ratio and encapsulated in lipid nanoparticles (LNP) with standard procedures to generate AOV-SD1.

[0153] Cell Lines. TC-1, a progressive subclone of the HPV16 E6 and E7 proteins- transformed lung epithelial cells, was used for the tumor challenge studies described below. TC-1 cells were grown and expanded in vitro with Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 10% fetal bovine serum (FBS).

[0154] Tumor Challenge. Pathogen-free 6-8-week-old C57BL / 6 female mice each were challenged subcutaneously with 5xl05TC-1 tumor cells in 100 pl Hanks’ Balanced Salt Solution (HBSS). Tumor growth was measured two times per week with manual calipers by measuring tumor length, height, and depth to generate a tumor volume (volume = 1 * w2* 1 / 2). Tumor volumes exceeding 1500 mm3resulted in euthanasia. Mildly ulcerated tumors less than 1500 mm3were subjected to a veterinary-approved wound care regimen (flushing with sterile saline, treating with Vetericyn spray, and covering wound with liquid bandage every 2-3 days). Mice that developed severe ulcerations (actively bleeding, or ulceration >50% of tumor surface area) were euthanized regardless of tumor size.

[0155] Intramuscular Injection. Pathogen-free 6-8-week-old C57BL / 6 female mice were anesthetized with isoflurane (4% induction, 2% maintenance). The right hind limb area around the administration site was shaved with animal clippers to expose skin. After fur removal was completed, the area was cleansed of loose fur and disinfected using an isopropanol wipe. One hundred microliter (lOOpl) of empty LNP or LNP loaded with 5pg or 30pg AOV-SD1 mRNA mixture were injected into the thigh muscle of the hind limb with 1ml syringe. Mice were placed into clean cages post immunization and were monitored until they woke up from anesthesia. Mice were monitored the day after vaccination for post-vaccination adverse events.

[0156] Tumor Challenge - Treatment Schedule. The mice were separated into four groups of 15 mice each. Group 1 mice were not challenged with TC-1 cells on Day 0 and were treated with empty LNP on Day 3. Group 2 mice were challenged with 5xl05TC-1 cells on Day 0 and were treated with empty LNP on Day 3. Group 3 mice were challenged with 5xl05TC-1 cells on Day 0 and were treated with LNP containing 5 pg of AOV-SD1 mRNA mixture on Day 3. Group 4 were challenged with 5xl05TC-1 cells on Day 0 and were treated with LNP containing 30pg of AOV-SD1 mRNA mixture on Day 3. On Day 14, 5 mice from each group were euthanized and spleen lymphocytes were collected for Elispot Assay.

[0157] Elispot Assay. Five mice in each group were euthanized on Day 14, spleen lymphocytes were collected. Elispot Assays were performed to evaluate specific cellular immune responses by measuring the number of cells (per million cells) expressing IL-2 and IFN-y after stimulated with HPV16 E7, HPV16 E6, HPV18 E6 and HPV18 E7 peptides fragments pools (Table 5- 8). Briefly, 6 x 10A4 viable spleen lymphocytes were plated in

[0158] Elispot Assay. Five mice in each group were euthanized on Day 14, spleen lymphocytes were collected, and counted for viable cells. Elispot Assays were performed to evaluate HPV16 / HPV18 E6ZE7 specific cellular immune responses by measuring the number of cells (per million cells) expressing IFN-y or IL-2 after stimulated with HPV16 / HPV18 E6ZE7 peptide mix contains 200ng of each peptide in the HP VI 6 E7, HP VI 6 E6, HPV18 E7 and HP VI 8 E6 overlapping peptide pools (Table 5-8). Elispot assays were performed using Mabtech Mouse IFN-y (ALP) kit, or Mabtech Mouse IL-2 (ALP) kit according to manufacture recommendation. Briefly, the assay was carried out in 96 well plates precoated with IFN-y or IL-2 antibody at bottom. For each animal, 6 x 10A4 of viable spleen lymphocytes (diluted in final volume of lOOpl) were mixed with equal volume of 5ng PMA / Ionomycin for 20 hours as positive control well, 2 x 10A5 of spleen lymphocytes (diluted in final volume of 1 OOpl) were mixed with equal volume of 1 x PBS contains 1% DMSO for 20 hours as negative control well, and 2 x 10A5 of spleen lymphocytes (diluted in final volume of 1 OOpil) were mixed with equal volume of HP VI 6 E7 peptide pool (Table 5) dissolved in 1 x PBS contains 1% DMSO for 20 hours as stimulation well. Then the wells were thoroughly washed, then incubated with R4-6A2 -biotin or 5H6-biotin in PBS-0.5% FCS for IFN-y and IL-2, respectively, for 2 hours, washed and incubated with Streptavidin- ALP in PBS-0.5% FCS for Ihour, washed and developed with BCIP / NBT-plus, the color development was then stopped with extensive wash with water. The plates were read with CTL6 Elispot plate reader. Cellular immune of IFN-y or IL-2 was calculated as stimulated reading minus negative control reading per one million spleen lymphocytes. For each group,intensity of cellular immune was reported as mathematical average of Elispot readings of all animals in the same group, and illustrated in FIG. 5A (IFN-y) and FIG. 5B (IL-2).

[0159] Study Period. Tumor growth was monitored for up to 42 days post tumor challenge. Mice with tumors exceeding 10% body weight or tumors ulcerating through the skin surface were euthanized. Tumor dimensions were measured twice weekly in three dimensions (1 x w2x 1 / 2) to calculate tumor volume. Mice that did not develop tumors were euthanized after 42 days or 1 week after all other tumor-bearing mice were euthanized.

[0160] Results. Tumors were not grown in group 1 mice without TC-1 tumor cell challenge, while in group 2, average tumor size increased over time until end of the study (42 days post tumor challenge). In Group 3 and group 4, tumor size peaked on Day 21, then diminished over time. Two weeks after tumor challenge (11 days after treatment), mice in group 3 and group 4 exhibited significantly higher levels of specific cellular immune responses against HPV16 / 18 E6ZE7 peptides fragments stimuli, while tumor challenge alone only generated minor levels of HPV16 / 18 E6ZE7 specific cellular immune responses. Higher levels of AOV-SD1 mRNA (30|ig) treatment resulted in higher levels of specific cellular immune responses, indicating that on Day 14, the specific cellular immune response against HPV16 / 18 E6 / E7 is dose dependent on AOV-SD1 mRNA treatment. AOV-SD1 was able to eliminate pre-inoculated tumor in mice when administered after challenge with tumor cells (FIG. 4). As shown herein, a single treatment of AOV-SD1 almost eliminates pre-inoculated tumors in mice.

[0161] Example 4: AQV-16SD001 to AOV-16SD144 possessed various ability to induce cellular immune response.

[0162] The AOV-16SD001 to AOV16-SD144 core sequences follows a basic fragmentation arrangement pattern of F1-F2-F3-F4-F5-F6-F7, with Fl to F7 selected from the fragmentation pool of F1661, F1662, F1663, F1664, F1671, F1672, and F1673 in Table 1.

[0163] More specifically, F1 / F3 / F5 / F7 of such arrangement pattern were selected from the fragmentation pool of Fl 661, Fl 662, Fl 663, Fl 664; and F2 / F4 / F6 were selected from the fragmentation pool of Fl 671, Fl 672, Fl 673.

[0164] To clarify: AOV-16SD001 core sequence is composed of F1661-F1671-F1662-F1672- F 1663 -Fl 673 -Fl 664, with the C-terminus of the prior fragment directly connected with N- terminus of the following fragment to form a complete polypeptide sequence.

[0165] The full core sequence of AOV-16SD001-16SD144 are described in Table 4.

[0166] The N-terminal signal peptide of human leukocyte antigen HLA-F (MAPRSLLLLLSGALALTDTWAGS, SEQ ID NO: 301) was added to N-terminus of each core sequence, and C-terminal signal peptide of HLA-G (SSLPTIPIMGIVAGLVVLAAVVTGAAVAAVLWRKKSSD, SEQ ID NO: 302) was added to C-terminus of each core sequence to generate the full peptide sequence of AOV-16SD001 to AOV-16SD144.

[0167] Corresponding typical coding DNA sequences of AOV-16SD001 to AOV-16SD144 core peptides constructs, were cloned into the open reading frame (ORF) of a circular in vitro transcription (IVT) vector. The IVT vector contained a T7 promoter, a 5’ UTR (untranslated region), and a Kozak (GCCACC) sequence, a nucleotide sequence that encodes N-terminal signal peptide of human leukocyte antigen HLA-F (MAPRSLLLLLSGALALTDTWAGS, SEQ ID NO: 301) upstream of the ORF of the inserted construct, and a nucleotide sequence that encodes a C-terminal signal peptide of HLA-G (SSLPTIPIMGIVAGLVVLAAVVTGAAVAAVLWRKKSSD, SEQ ID NO: 302), followed by two (2) stop codons, a 3’ UTR, and a poly(A) tail of total 110 A, and a BspQI restriction site downstream of the ORF.

[0168] The resulting plasmids were sequenced to ensure the nucleotide sequence correctness of each construct.

[0169] Each plasmid was purified and digested with BspQI restriction enzymes to generate corresponding linearized template for in vitro transcription (IVT).

[0170] IVT: Each linearized template was purified and quantified. One (1) micro-gram (pg) of each particular template was added in a typical IVT reaction mixture containing proper buffer, and reagents (T7 RNA polymerase, ATP, GTP, CTP and Nl-me-UTP, a Capl analogs, RNase inhibitor, inorganic pyrophosphatase) with proper concentrations, to generate mRNA corresponding to each construct. Resulting mRNA was purified with Thermofisher Dynabeads MyOne Carboxylic Acid according to manufacture recommendation, and dissolved in RNase- Free water.

[0171] LNP production: Each mRNA was individually encapsulated in 8mM SM102 LNP formulation (SM102: 50%, DMG-PEG2000: 1.5%, DSPC: 10%, Cholesterol: 38.5%), with calculated N / P ratio 6, FRR=3, total flow rate=20 ml / min, using Micro&Nano’s INano E microfluidic system, according to manufacture recommendation. The resulting mRNA containing LNP was buffer exchanged to 5% sucrose in IxPBS (pH=7.4), and stored at -20°C.The encapsulation RNA was quantified with Thermofisher RiboGreen RNA reagent, according to manufacture recommendations.

[0172] Cellular Expression'. An amino acid sequence of YKDDDDK (SEQ ID NO: 303) was engineered and cloned to the C-terminus of each individual construct to generate a Flag tag (D YKDDDDK, SEQ ID NO: 304), which allowed binding and quantification of expression levels of the constructs. In vitro cellular expressions were performed using I pg of LNP encapsulated mRNA of individual constructs AOV-16SD001 to AOV-16SD144, each LNP- mRNA complex was incubated with 3 x 10A5 viable 293T cells in standard 12-well tissue culture plate for 24 hours, respectively. The cells were then harvested, lysed, electrophoresed through a denaturing polyacrylamide gel, electro-transferred to a PVDF membrane, and standard western blot procedure with an anti-flag mouse antibody were performed to detect the expression of Flag tag signal at corresponding position of each construct. Expression of each construct was compared to the Flag tag signal generated by construct AOV-16SD017 to calculate a relative expression level. The results were illustrated in FIG. 6.

[0173] Expression levels of each individual construct was measured by relative dot blot signal against construct AOV-16SD017.

[0174] Intramuscular Injection. Pathogen-free 6-8-week-old C57BL / 6 female mice were randomly grouped into 145 groups, with 3 mice in each group. Animals were anesthetized with isoflurane (4% induction, 2% maintenance). The right hind limb area around the administration site was shaved with animal clippers to expose skin. After fur removal was completed, the area was cleansed of loose fur and disinfected using an isopropanol wipe. Each animal group was coded with a particular construct in Table 4, and one hundred microliter (100|il) of LNP loaded with 5 pg mRNA of a particular construct or empty LNP in Table 4 were injected into the thigh muscle of the hind limb with 1ml syringe to each individual animal in the group. Mice were placed into clean cages post immunization and were monitored until they woke up from anesthesia. Mice were monitored the day after vaccination for post-vaccination adverse events.

[0175] Elispot Assay. Three mice in each group were euthanized on Day 21, spleen lymphocytes were collected, and counted for viable cells. Elispot Assays were performed to evaluate HPV16 E7 specific cellular immune responses by measuring the number of cells (per million cells) expressing IFN-y after stimulated with HPV16 E7 peptide mix contains 200ng of each peptide in the HPV16 E7 overlapping peptides pool (Table 5). Elispot assays were performed using Mabtech Mouse IFN-y (ALP) kit, according to manufacture recommendation. Briefly, the assay was carried out in 96 well plates precoated with IFN-y antibody at bottom. Foreach animal, 6 x 10A4 of viable spleen lymphocytes (diluted in final volume of lOOpl) were mixed with equal volume of 5ng PMA / Ionomycin for 20 hours as positive control well, 2 x 10A5 of spleen lymphocytes (diluted in final volume of 1 OOpil) were mixed with equal volume of 1 x PBS contains 1% DMSO for 20 hours as negative control well, and 2 x 10A5 of spleen lymphocytes (diluted in final volume of 1 OOpil) were mixed with equal volume of HP VI 6 E7 peptide pool (Table 5) dissolved in 1 x PBS contains 1% DMSO for 20 hours as stimulation well. Then the wells were thoroughly washed, then incubated with R4-6A2 -biotin in PBS-0.5% FCS for 2 hours, washed and incubated with Streptavidin-ALP in PBS-0.5% FCS for Ihour, washed and developed with BCIP / NBT-plus, the color development was then stopped with extensive wash with water. The plates were read with CTL6 Elispot plate reader. Cellular immune of IFN-y was calculated as stimulated reading minus negative control reading per one million spleen lymphocytes. For each group, intensity of cellular immune response was reported as mathematical average of Elispot readings of three animals in the same group, and illustrated in FIG. 6

[0176] In at least some of the previously described embodiments, one or more elements used in an embodiment can interchangeably be used in another embodiment unless such a replacement is not technically feasible. It will be appreciated by those skilled in the art that various other omissions, additions and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter, as defined by the appended claims.

[0177] With respect to the use of substantially any plural and / or singular terms herein, those having skill in the art can translate from the plural to the singular and / or from the singular to the plural as is appropriate to the context and / or application. The various singular / plural permutations may be expressly set forth herein for sake of clarity. As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Any reference to “or” herein is intended to encompass “and / or” unless otherwise stated.

[0178] It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent willbe explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and / or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms.

[0179] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0180] As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each rangediscussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

[0181] While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.

Claims

WHAT IS CLAIMED IS:

1. A polynucleotide comprising a nucleotide sequence encoding a structurally disrupted (SD) human papilloma virus (HPV) E6 protein or SD HPV E7 protein.

2. The polynucleotide of claim 1, wherein the SD HPV E6 protein or the SD HPV E7 protein is derived from a wild type HPV E6 protein or a wild type HPV E7 protein, optionally the wild type HPV E6 protein or the wild type HPV E7 protein comprises HPV 16 E6 protein, HPV 16 E7 protein, HPV18 E6 protein, HPV18 E7 protein, or any combination thereof.

3. The polynucleotide of any one of claims 1-2, wherein the SD HPV E6 protein exhibits reduced binding affinity to pRB and / or the SD HPV E7 protein exhibits reduced binding affinity to p53, optionally as compared to the wild type HPV E6 protein or the wild type HPV E7 protein.

4. The polynucleotide of any one of claims 1-3, wherein the SD HPV E6 protein is not capable of binding to pRB and / or the SD HPV E7 protein is not capable of binding to p53.

5. The polynucleotide of any one of claims 1-4, wherein the SD HPV E6 protein and / or the SD HPV E7 protein comprises one or more disrupted or deleted zinc-finger elements, optionally as compared to the wild type HPV E6 protein or the wild type HPV E7 protein.

6. The polynucleotide of any one of claims 2-5, wherein the SD HPV E6 protein comprises a fragment of the wild type HPV E6 protein, the SD HPV E7 comprises a fragment of the wild type HPV E7 protein, or both, optionally the fragment of the wild type HPV E6 protein and / or the fragment of the wild type HPV E7 protein is less than 40 amino acids in length.

7. The polynucleotide of claim 6, wherein: the fragment of the wild type HPV E6 protein comprises the sequence of any one of SEQ ID NOs: 5-8 and 12-15 or a sequence having one, two, or three mismatches relative to any one of SEQ ID NOs: 5-8 and 12-15; and / or the fragment of the wild type HPV E7 protein comprises the sequence of any one of SEQ ID NOs: 9-11 and 16-18 or a sequence having one, two, or three mismatches relative to any one of SEQ ID NOs: 9-11 and 16-18.

8. The polynucleotide of any one of claims 2-7, wherein the wild type HPV E6 protein or the wild type HPV E7 protein comprises an amino acid sequence of any one of SEQ ID NOs: 1-4.

9. The polynucleotide of any one of claims 1-8, comprising a nucleotide sequence encoding a polypeptide comprising two or more SD HPV E6 proteins, two or more SD HPV E7 proteins, or any combination thereof.

10. The polynucleotide of claim 9, wherein the polypeptide comprises four SD HPV E6 proteins and three SD HPV E7 proteins.-n-11. The polynucleotide of claim 10, wherein the polypeptide comprises, from N- terminus to C-terminus: a first SD HPV E6 protein, a first SD HPV E7 protein, a second SD HPV E6 protein, a second SD HPV E7 protein, a third SD HPV E6 protein, a third SD HPV E7 protein, and a fourth SD HPV E6 protein.

12. The polynucleotide of any one of claims 9-11, wherein the polypeptide further comprises an N-terminal signal peptide, a C-terminal signal peptide, or both.

13. The polynucleotide of claim 11, wherein the N-terminal signal peptide, the C- terminal signal peptide, or both, is a signal peptide of a human leukocyte antigen (HLA), optionally the HLA is HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G.

14. The polynucleotide of claim 13, wherein the polypeptide comprises an N-terminal signal peptide of HLA-F, a C-terminal signal peptide of HLA-G, or both.

15. The polynucleotide of claim 14, wherein: the N-terminal signal peptide of HLA-F comprises the sequence of SEQ ID NO:301 or a sequence have one, two, or three mismatches relative to the sequence of SEQ ID NO: 301; and the C-terminal signal peptide of HLA-G comprises the sequence of SEQ ID NO:302 or a sequence have one, two, or three mismatches relative to the sequence of SEQ ID NO: 302.

16. The polynucleotide of any one of claims 1-15, wherein the polynucleotide encodes a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of any one of SEQ ID NOs: 5-180.

17. The polynucleotide of any one of claims 1-16, wherein the polynucleotide encodes a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 5-180 or an amino acid sequence having one, two, three, four, five, six, seven, eight, nine, or ten mismatches relative to any one of SEQ ID NOs: 5-180.

18. A polypeptide comprising an SD HPV E6 protein or an SD HPV E7 protein.

19. The polypeptide of claim 18, wherein the SD HPV E6 protein or the SD HPV E7 protein is derived from a wild type HPV E6 protein or a wild type HPV E7 protein, optionally the wild type HPV E6 or the wild type HPV E7 protein comprises HPV16 E6 protein, HPV16 E7 protein, HPV18 E6 protein, HPV18 E7 protein, or any combination thereof.

20. The polypeptide of any one of claims 18-19, wherein the SD HPV E6 protein exhibits reduced binding affinity to pRB and / or the SD HPV E7 protein exhibits reduced binding affinity to p53, optionally as compared to the wild type HPV E6 protein or the wild type HPV E7 protein.

21. The polypeptide of any one of claims 18-20, wherein the SD HPV E6 protein is not capable of binding to pRB and / or the SD HPV E7 protein is not capable of binding to p53.

22. The polypeptide of any one of claims 18-21, wherein the SD HPV E6 protein and / or the SD HPV E7 protein comprises one or more disrupted or deleted zinc-finger elements, optionally as compared to the wild type HPV E6 protein or the wild type HPV E7 protein.

23. The polypeptide of any one of claims 19-22, wherein the SD HPV E6 protein comprises a fragment of the wild type HPV E6 protein, the SD HPV E7 comprises a fragment of the wild type HPV E7 protein, or both, optionally the fragment of the wild type HPV E6 protein and / or the fragment of the wild type HPV E7 protein is less than 40 amino acids in length.

24. The polypeptide of claim 22, wherein: the fragment of the wild type HPV E6 protein comprises the sequence of any one of SEQ ID NOs: 5-8 and 12-15 or a sequence having one, two, or three mismatches relative to any one of SEQ ID NOs: 5-8 and 12-15; and / or the fragment of the wild type HPV E7 protein comprises the sequence of any one of SEQ ID NOs: 9-11 and 16-18 or a sequence having one, two, or three mismatches relative to any one of SEQ ID NOs: 9-11 and 16-18.

25. The polypeptide of any one of claims 19-24, wherein the wild type HPV E6 protein or the wild type HPV E7 protein comprises an amino acid sequence of any one of SEQ ID NOs: 1-4.

26. The polypeptide of any one of claims 18-25, comprising an amino acid sequence of two or more SD HPV E6 proteins, two or more SD HPV E7 proteins, or any combination thereof.

27. The polynucleotide of claim 26, wherein the polypeptide comprises four SD HPV E6 proteins and three SD HPV E7 proteins.

28. The polynucleotide of claim 27, wherein the polypeptide comprises, from N- terminus to C-terminus: a first SD HPV E6 protein, a first SD HPV E7 protein, a second SD HPV E6 protein, a second SD HPV E7 protein, a third SD HPV E6 protein, a third SD HPV E7 protein, and a fourth SD HPV E6 protein.

29. The polypeptide of any one of claims 26-28, wherein the polypeptide further comprises an N-terminal signal peptide, a C-terminal signal peptide, or both.

30. The polypeptide of claim 29, wherein the N-terminal signal peptide, the C-terminal signal peptide, or both, is a signal peptide of a human leukocyte antigen (HLA), optionally the HLA is HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G.

31. The polypeptide of claim 30, wherein the polypeptide comprises an N-terminal signal peptide of HLA-F, a C-terminal signal peptide of HLA-G, or both.

32. The polypeptide of claim 31, wherein: the N-terminal signal peptide of HLA-F comprises the sequence of SEQ ID NO:301 or a sequence have one, two, or three mismatches relative to the sequence of SEQ ID NO: 301; and the C-terminal signal peptide of HLA-G comprises the sequence of SEQ ID NO:302 or a sequence have one, two, or three mismatches relative to the sequence of SEQ ID NO: 302.

33. The polypeptide of any one of claims 18-32, comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of any one of SEQ ID NOs: 5-180.

34. The polypeptide of any one of claims 18-33, comprising an amino sequence of any one of SEQ ID NOs: 5-180 or an amino acid sequence having one, two, three, four, five, six, seven, eight, nine, or ten mismatches relative to any one of SEQ ID NOs: 5-180.

35. A composition comprising: a nucleic acid composition comprising one or more polynucleotides of any one of claims 1-17, or one or more polypeptides of any one of claims 18-34.

36. The composition of claim 35, wherein the nucleic acid composition is complexed or associated with one or more lipids or lipid-based carriers, thereby forming liposomes, lipid nanoparticles (LNPs), lipoplexes, and / or nanoliposomes, optionally encapsulating the nucleic acid composition.

37. The composition of any one of claims 35-36, wherein the one or more polynucleotides are situated on the same nucleic acid and / or different nucleic acids.

38. The composition of any one of claims 35-37, wherein the nucleic acid composition is, comprises, or further comprises, one or more vectors, optionally at least one of the one or more vectors is a viral vector, a plasmid, a naked DNA vector, a lipid nanoparticle (LNP), or any combination thereof, and optionally the viral vector is an AAV vector, a lentivirus vector, a retrovirus vector, an adenovirus vector, a herpesvirus vector, a herpes simplex virus vector, a cytomegalovirus vector, a vaccinia virus vector, a MVA vector, a baculovirus vector, a vesicular stomatitis virus vector, a human papillomavirus vector, an avipox virus vector, a Sindbis virus vector, a VEE vector, a Measles virus vector, an influenza virus vector, a hepatitis B virus vector, an integration-deficient lentivirus (IDLV) vector, or any combination thereof.

39. The composition of claim 38, wherein the one or more polynucleotides are comprised in the one or more vectors,-SO-optionally the one or more polynucleotides are comprised in the same vector and / or different vectors.

40. The composition of any one of claims 38-39, wherein the one or more vectors is a DNA vaccine, optionally the one or more polynucleotides are operably linked to one or more promoters capable of inducing transcription of the one or more polynucleotides, optionally the DNA vaccine is a plasmid-based DNA vaccine, a minicircle-based DNA vaccine, a bacmid-based DNA vaccine, a minigene-based DNA vaccine, a ministring DNA (linear covalently closed DNA vector) vaccine, a closed-ended linear duplex DNA (CELiD or ceDNA) vaccine, a doggybone™ DNA vaccine, a dumbbell shaped DNA vaccine, or a minimalistic immunological-defmed gene expression (MIDGE)-vector DNA vaccine.

41. The composition of claim 40, wherein the one or more promoters comprise a ubiquitous promoter, an inducible promoter, a tissue-specific promoter and / or a lineage-specific promoter, optionally the ubiquitous promoter is selected from the group comprising a cytomegalovirus (CMV) immediate early promoter, a CMV promoter, a viral simian virus 40 (SV40) (e.g., early or late), a Moloney murine leukemia virus (MoMLV) LTR promoter, a Rous sarcoma virus (RSV) LTR, an RSV promoter, a herpes simplex virus (HSV) (thymidine kinase) promoter, H5, P7.5, and Pl l promoters from vaccinia virus, an elongation factor 1 -alpha (EFla) promoter, early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock 70 kDa protein 5 (HSPA5), heat shock protein 90 kDa beta, member 1 (HSP90B1), heat shock protein 70 kDa (HSP70), P-kinesin (P-KIN), the human ROSA 26 locus, a Ubiquitin C promoter (UBC), a phosphoglycerate kinase- 1 (PGK) promoter, 3 -phosphoglycerate kinase promoter, a cytomegalovirus enhancer, human P-actin (HBA) promoter, chicken P-actin (CBA) promoter, a CAG promoter, a CASI promoter, a CBH promoter, or any combination thereof.

42. The composition of any one of claims 35-41, wherein the one or more polynucleotides are operably linked to a tandem gene expression element, optionally the tandem gene expression element is an internal ribosomal entry site (IRES), foot-and-mouth disease virus 2A peptide (F2A), equine rhinitis A virus 2A peptide (E2A), porcine teschovirus 2A peptide (P2A) or Thosea asigna virus 2A peptide (T2A), or any combination thereof.

43. The composition of any one of claims 35-42, wherein the one or more polynucleotides comprise a transcript stabilization element, optionally the transcript stabilization element comprises woodchuck hepatitis post-translational regulatory element (WPRE), bovine growth hormone polyadenylation (bGH-polyA) signal sequence, human growth hormone polyadenylation (hGH-polyA) signal sequence, or any combination thereof.

44. The composition of any one of claims 35-39, wherein the nucleic acid composition is or comprises mRNA, optionally the mRNA is formulated in a lipid nanoparticle (LNP).

45. The composition of claim 44, wherein the mRNA comprises a 5' untranslated region (UTR), a 3' UTR, and / or a cap.

46. The composition of any one of claims 44-45, wherein the mRNA comprises one or more modified nucleotides selected from the group comprising pseudouridine, N-l -methylpseudouridine, 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8- oxoguanosine, 0(6)-methylguanine, and 2-thiocytidine.

47. The composition of any one of claims 44-46, wherein the mRNA comprises a modified nucleotide in place of one or more uridines, optionally the modified nucleotide is selected from pseudouridine (y), N 1-methyl-pseudouridine (m IT), and 5-methyl-uridine (m5U).

48. The composition of any one of claims 35-47, comprising particles comprising the one or more polypeptides, optionally the particles are iron oxide particles, liposomes, micelles, polymer complexes, cationic peptide nanoemulsions, virus-like particles (VLPs), lipid nanoparticles (LNP) and / or lipoplex (LPX) particles.

49. The composition of any one of claims 36-48, wherein the LNP comprises one or more of an ionizable cationic lipid, a non-cationic lipid, a sterol, and a PEG-modified lipid, optionally the non-cationic lipid is a neutral lipid.

50. The composition of any one of claims 36-49, wherein the LNP comprises 0.5-15 mol% PEG-modified lipid, 5-25 mol% non-cationic lipid, 25-55 mol% sterol, and 20-60 mol% ionizable cationic lipid.

51. The composition of any one of claims 36-50, wherein the LNP comprises: 40-55 mol% ionizable cationic lipid, 5-15 mol% neutral lipid, 35-45 mol% sterol, and 1-5 mol% PEG- modified lipid.

52. The composition of any one of claims 36-51, wherein the LNP comprises:47 mol% ionizable cationic lipid, 11.5 mol% neutral lipid, 38.5 mol% sterol, and 3.0 mol% PEG-modified lipid;48 mol% ionizable cationic lipid, 11 mol% neutral lipid, 38.5 mol% sterol, and 2.5 mol% PEG-modified lipid;49 mol% ionizable cationic lipid, 10.5 mol% neutral lipid, 38.5 mol% sterol, and 2.0 mol% PEG-modified lipid;50 mol% ionizable cationic lipid, 10 mol% neutral lipid, 38.5 mol% sterol, and 1.5 mol% PEG-modified lipid; or51 mol% ionizable cationic lipid, 9.5 mol% neutral lipid, 38.5 mol% sterol, and 1.0 mol% PEG-modified lipid.

53. The composition of any one of claims 49-52, wherein: the ionizable cationic lipid is heptadecan-9-yl 8 ((2 hydroxyethyl)(6 oxo 6- (undecyloxy)hexyl)amino)octanoate; the neutral lipid is l,2-distearoyl-sn-glycero-3 phosphocholine (DSPC); the sterol is cholesterol; and / or the PEG-modified lipid is l-monomethoxypolyethyleneglycol-2,3-dimyristylglycerol with polyethylene glycol of average molecular weight 2000 (PEG2000 DMG).

54. The composition of any one of claims 44-53, wherein the wt / wt ratio of lipid to mRNA is from about 1 : 100 to about 100: 1.

55. The composition of any one of claims 35-54, wherein the composition comprises one or more polynucleotides encoding immunostimulatory agents, optionally the immunostimulatory agents are selected from the group comprising toll-like receptor (TLR) agonists, cytokine receptor agonists, CD40 agonists, Fc receptor agonists, CpG- containing nucleic acids, complement receptor agonists, or any combination thereof.

56. The composition of claim 55, wherein: the TLR agonist is a TLR-1 agonist, TLR-2 agonist, TLR-3 agonist, TLR-4 agonist, TLR- 5 agonist, TLR-6 agonist, TLR-7 agonist, TLR-8 agonist, TLR-9 agonist, and / or TLR- 10 agonist; the Fc receptor agonist is a Fc-gamma receptor agonist; the complement receptor agonist binds to CD21 or CD35; the cytokine receptor agonist is a cytokine; and / or the cytokine receptor agonist is a small molecule, antibody, fusion protein, or aptamer.

57. The composition of any one of claims 35-56, further comprising an adjuvant, optionally the adjuvant comprises aluminum hydroxide, alhydrogel, AddaVax, MF59, AS03, Freund’s adjuvant, Montanide ISA51, CpG, Poly EC, glucopyranosyl lipid A, flagellin, resiquimod, or any combination thereof.

58. The composition of any one of claims 35-57, wherein the composition further comprises Tris buffer, sucrose, and / or sodium acetate.

59. The composition of any one of claims 35-58, wherein the composition is a lyophilized composition, optionally the lyophilized composition has a water content of less than about 10%.

60. The composition of any one of claims 35-59, wherein the composition is formulated or is to be formulated: as a liquid, a solid, or a combination thereof; for injection; and / or for intramuscular administration, intranasal administration, transdermal administration, aerosol delivery, nasal delivery, vaginal delivery, rectal delivery, buccal delivery, ocular delivery, local delivery, topical delivery, intraci sternal delivery, intraperitoneal delivery, oral delivery, intramuscular injection, intravenous injection, subcutaneous injection, intranodal injection, intratumoral injection, intraperitoneal injection, or intradermal injection.

61. The composition of any one of claims 35-60, wherein the composition is a pharmaceutical composition, wherein the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents and / or excipients.

62. The composition of any one of claims 35-61, further comprising instructions for use of the composition for: stimulating an immune response in a subject in need thereof; treating or preventing HPV-related disease or disorder in a subject in need thereof; and / or treating or preventing an HPV-related cancer in a subject in need thereof.

63. A kit, comprising the composition of any one of claims 35-62.

64. A cell comprising the nucleic acid composition of any one of claims 35-62.

65. The composition of any one of claims 35-62 for use as a medicament.

66. A method of stimulating an immune response in a subject in need thereof, comprising: administering to the subject a pharmaceutically effective amount of the polynucleotide of any one of claims 1-17, the polypeptide of any one of claims 18-34, or the composition of any one of claims 35-62, thereby stimulating the immune response in the subject.

67. A method of treating or preventing a disease or disorder in a subj ect in need thereof, comprising: administering to the subject a pharmaceutically effective amount of the polynucleotide of any one of claims 1-17, the polypeptide of any one of claims 18-34, or the composition of any one of claims 35-62, thereby treating or preventing the disease or disorder in the subject, optionally the disease or disorder is an HPV-related disease or disorder.

68. The method of claim 67, wherein the HPV-related disease or disorder is cancer, optionally the cancer is anal cancer, cervical cancer, oropharyngeal cancer, penile cancer, vaginal cancer, or vulvar cancer.

69. The method of any one of claims 66-68, wherein immunogenic levels of the SD HPV E6 protein, the SD HPV E7 protein, or both, are produced in serum of the subject at about 1 hour to about 6 months post administration of the composition.

70. The method of any one of claims 66-69, wherein a neutralizing antibody titer of about 50 to about 100000 half-maximal inhibitory dilutions (ID50s values) is produced in the serum of the subject at about 1 hour to about 6 months post administration of the composition.

71. The method of any one of claims 66-70, wherein the method comprises administering to the subject one or more doses of the composition, optionally a second dose of the composition is administered to the subject at least 14 days after a first dose of the composition is administered to the subject.

72. The method of claim 71, wherein the second dose of the composition is administered to the subject at least 28 days after the first dose of the composition is administered to the subj ect.

73. The method of any one of claims 71-72, wherein the first dose is a prime, and the second dose is a boost.

74. The method of any one of claims 71-73, wherein each of the one or more doses of the composition comprises about 2 pg to about 5 pg of the nucleic acid composition.

75. The method of any one of claims 66-74, wherein administering the composition induces neutralizing responses against HPV, cancer cells, or both.

76. The method of claim 75, wherein the HPV is HPV16, HPV18, or both.

77. The method of any one of claims 66-76, wherein the administration of the composition elicits protective and long-lasting immunity against HPV, cancer, or both.

78. The method of any one of claims 66-77, wherein the polynucleotide, the polypeptide, or the composition is administered in an effective amount to: induce a robust antibody response against the HPV, cancer cells, or both; optionally a robust antibody response comprises a neutralizing antibody response, further optionally a robust antibody response comprises Fc domain effector functions that recruit immune cells to infected cells, optionally said immune cells are macrophages, neutrophils, and / or natural killer cells, further optionally said recruitment induces antibody-dependent cellular cytotoxicity (ADCC) and / or antibody-dependent cellular phagocytosis (ADCP); elicit a robust CD4 and / or CD8 T cell response against the HPV, the cancer cells, or both in the subject; and / or elicit a balanced Thl / Th2 response against the HPV, the cancer cells, or both in the subject.

79. The method of any one of claims 66-78, wherein the polynucleotide, the polypeptide, or the composition is administered in an effective amount to induce the productionof one or more cytokines in the subject, optionally the one or more cytokines comprise IL-2, IFN- y, TNF-a, IL-1, IL-12, IL-17, IL-18, or any combination thereof.

80. The method of any one of claims 75-79, wherein at least a portion of the cancer cells express the wild type HPV E6 protein or variants thereof, the wild type HPV E7 protein or variants thereof, or any combination thereof.

81. The method of any one of claims 67-80, wherein treating or preventing the disease or disorder in the subject comprises inhibiting the onset or progression of the cancer.

82. The method of claim 81, wherein the inhibition of progression of the cancer is measured by tumor growth inhibition (TGI) and wherein the TGI resulted by the administration is at least 30% higher as compared to an untreated subject.

83. The method of any one of claims 81-82, wherein a tumor volume after the administration decreases at least 50% relative to the tumor volume with no treatment or the tumor volume prior to the administration.

84. The method of any one of claims 67-83, wherein the subject achieves a complete response.

85. The method of any one of claims 67-84, wherein the subject does not develop HPV-related cancer following the administration.

86. The method of any one of claims 66-85, wherein the subject is a human subject, optionally the human subject is at least 9 years old.

87. The method of any one of claims 66-86, wherein the subject has had a prior HPV infection.

88. The method of any one of claims 66-87, wherein the composition is coadministered with an adjuvant.

89. The method of any one of claims 66-87, wherein the composition is not coadministered with an adjuvant.

90. The method of any one of claims 66-89, wherein the composition is administered intramuscularly, optionally into a deltoid region of an arm.

91. The method of any one of claims 66-90, wherein the administering comprises aerosol delivery, nasal delivery, vaginal delivery, rectal delivery, buccal delivery, ocular delivery, local delivery, topical delivery, intraci sternal delivery, intraperitoneal delivery, oral delivery, intramuscular injection, intravenous injection, subcutaneous injection, intranodal injection, intratumoral injection, intraperitoneal injection, intradermal injection, or any combination thereof.

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