DNA encoded nanoparticle vaccines against human papillomavirus, and methods of use thereof

Abstract

Disclosed herein are nanoparticles comprising one or more Human papillomavirus (HPV) antigen and nucleic acid molecules encoding the same. Also disclosed herein is a method of treating a HPV infection or treating or preventing a disease or disorder associated therewith in a subject in need thereof, by administering the nanoparticles, or encoding nucleic acid molecules, to the subject.

Description

DNA ENCODED NANOPARTICLE VACCINES AGAINST HUMAN PAPILLOMAVIRUS, AND METHODS OF USE THEREOFCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 727,909, filed December 4, 2024 which is hereby incorporated by reference herein in its entirety.REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] This application contains a Sequence Listing, which is submitted electronically via EFS-Web as an XML Document formatted sequence listing with a file name “206193-0149- OOWO_SequenceListing.xml,” having a creation date of December 1, 2025, and having a size of 150,822 bytes. The sequence listing submitted via EFS-Web is part of the specification and is herein incorporated by reference in its entirety.BACKGROUND

[0003] Papillomavirus are small DNA viruses that comprise up to seven early genes and two late genes. Generally, papilloma virus early genes are designated E1-E7, and papilloma virus late genes are designated LI and L2. Several species of animals can be infected by members of the papillomavirus family.

[0004] Human Papillomavirus (HPV) infection is common and can be transmitted sexually. HPV have been differentiated into 56 or more types based upon DNA sequence homology. HPV type 16, which causes epithelial dysplasia and other lesions, is often associated with an increased risk of cancer, particularly in situ and invasive carcinomas of the cervix, vagina, vulva and anal canal. Nearly 88% of cervical cancers worldwide are the result of HPV subtypes 16, 18, 45, 31, 33, 52 and 58.

[0005] There remains a need for improved vaccines and methods for preventing and treating HPV infection, and diseases or disorders associated with HPV infections such as cervical cancer and / or head and neck carcinomas.BRIEF DESCRIPTION OF THE DRAWINGS

[0006] Figure 1A through Figure IF depict data demonstrating the stabilized nanoparticle design workflow. Figure 1 A provides a flow diagram showing that the desired protein sequence is run through structural prediction algorithms such as AlphaFold2 and RoseTTafold2. Confidence metrics associated with the structural prediction help determine ordered regions from disordered regions, along with visual inspection of the predicted structure. This results in the design of a minimized ‘foldable’ domain. Additional design stabilization can be engineered in; here, disulfide scanning was used to introduce pairs of disulfide bonds to stabilize local folds. Finally, the resultant candidate designs are scaffolded onto a self-assembling 60-mer nanoparticle scaffold. Figure IB shows an example of structural prediction. The top ranked model of wild type (WT) E7 from RoseTTaFold2, colored by per-residue RMS-error. Red residues are lower confidence while blue residues are higher confidence. Figure 1C shows an example of a confidence metric graph. Predicted local distance difference test (pLDDT) scores from the top ranked Alphafold2 prediction score of WT E7 is depicted. Scores above 80 are considered high confidence and guide selection of structurally intact domains. Residues that comprise the selected foldable domain are shown in yellow. Figure ID shows an example of a selected foldable domain. Using confidence metric scores and visual inspection, residues that comprise a purported foldable domain were selected. The sequence was then re-predicted to confirm purported structural integrity, and the top ranked AlphaFold2 prediction is shown. Figure IE shows an example of an iterative foldable domain design. WT E7 can form a dimer. This guided creation of a covalent dimer of the truncated domain design in Figure ID). The top ranked Alphafold2 prediction of the designed dimeric sequence is shown. Figure IF shows an example of addition design stabilization. A disulfide scanning algorithm was used to select pairs of residues that could be mutated to cysteine to form a new disulfide bridge. Here, the scanner was applied to find disulfide pairs that bridge the dimeric interface to create additional stabilization of the fold. The engineered disulfide bridge is depicted in stick representation and colored red.

[0007] Figure 2A through Figure 2E depicts the structural prediction and confidence metrics for E7 and associated designs. Figure 2A and Figure 2B provide additional structural prediction outputs for E7 FL, colored by confidence metrics. Red residues are lower confidence while blue residues are higher confidence. Figure 2A) The top ranked model from AlphaFold2,colored by per-residue pLDDT score. All AlphaFold2 models predicted an unstructured N- terminal domain. Figure 2B) The fourth ranked model from RoseTTaFold2, colored by per- residue RMS-error. This model was included because it suggested an alternative conformation for the N-terminal domain. Figure 2C and Figure 2D depict examples of confidence metric graphs. Per residue RMS-error for E7 FL from Figure 2C) the top ranked RoseTTaFold2 model or Figure 2D) the fourth ranked RoseTTaFold2 model. Figure 2E) Comparison of E7_Tr to partial structure 2F8B. The partial NMR structure of HPV45 E7 (individual subunits shown in dark red and pink) is aligned to the designed foldable domain HPV16 E7_Tr (tan). Cartoon representation was used. E7_Tr has very similar folds to a single subunit of the HPV45 E7 dimer.

[0008] Figure 3 depicts the sequence alignment for E7 designs. All designs were aligned relative to the WT HPV16 E7 sequence (E7 FL). Dots represent identical amino acids.

[0009] Figure 4A through Figure 4G depict data demonstrating the in vitro characterization of the E7 designs. Figure 4A shows the purified transfection yield for designed E7 nanoparticles. Nanoparticles are grouped by their base design: full length, truncated, or truncated dimer. Transfection volume was lOOmL and constructs were purified by lectin affinity chromatography and size exclusion chromatography before yield was determined. Figure 4B shows the binding of designed E7 nanoparticles to an HPV16 E7 polyclonal antibody. Plates were coated with 15 pg / mL of each nanoparticle followed by incubation with the serially diluted polyclonal antibody at the concentrations indicated. Size exclusion chromatography traces (215nm) of E7 nanoparticles with models in the inset for E7_FL_Dsl_nano (Figure 4C) E7 Tr nano (Figure 4D) E7 Tr Dim nano (Figure 4E) and E7 FL ApRb nano (Figure 4F). Figure 4G) Binding of E7_FL_ApRb_nano to an HPV16 E7 polyclonal antibody. Plates were coated with 15 pg / mL of each nanoparticle followed by incubation with the serially diluted polyclonal antibody at the concentrations indicated.

[0010] Figure 5 A through Figure 5C depicts the immunogenicity of first-pass E7 and E6 nanoparticle designs. Figure 5A provides an immunization overview. C57 / BL6 were immunized with 10 pg of DNA (IM-EP) at week 0. Animals were boosted with an equivalent dose at week 2, and their spleens were harvested at week 3. Splenocytes were isolated for downstream ELISpot analysis. Figure 5B provides an ELISpot of splenocytes stimulated with pooled overlapping peptides of WT HPV16 E7. n=5 mice / group. Mean ± SEM shown. One-wayANOVA was conducted. ** p<0.005. Figure 5C provides an ELISpot of splenocytes stimulated with pooled overlapping peptides (peptides 1-11) of WT HPV16 E6. n=5 mice / group. Mean ± SEM shown. One-way ANOVA was conducted. * p<0.05, *** p<0.0005.

[0011] Figure 6A through Figure 6E depicts the characterization of designs for non- ocogenic E7 and E6 nanoparticles. Figure 6A) Alignment of top ranked Alphafold2 model of E7_FL_ pRb (orange) to top ranked Alphafold2 model of E7 FL (gray). Cartoon representation was used. The N terminal domain is predicted to be unstructured in either case, and introduction of the pRb mutations does not perturb folding of the well-structured C-terminal domain. Figure 6B) Purified transfection yield for E7_FL_ pRb nano. Transfection volume was lOOmL or 300mL and constructs were purified by lectin affinity chromatography and size exclusion chromatography before yield was determined. Figure 6C) Alignment of top ranked Alphafold2 model of E6_FL_ Sol_ p53 (light blue) to E6 from PDB: 6SJA (gray). Cartoon representation was used. E6_FL_ Sol_ p53 shows similar shape to the crystal structure. Figure 6D) Alignment of top ranked Alphafold2 model of E6_Tr_ Sol_ p53 (dark blue) to E6 from PDB: 6SJA (gray), truncated to similar residues. Cartoon representation was used. E6_Tr_ Sol_ p53 shows similar shape to the truncated crystal structure, with main differences in the position of the C-terminal alpha helix. Figure 6E) Purified transfection yield for E6_FL_ Sol_ p53_nano and E6_Tr_ Sol_ p53_nano. Transfection volume was lOOmL and constructs were purified by lectin affinity chromatography and size exclusion chromatography before yield was determined.

[0012] Figure 7A through Figure 7D depicts negative stain electron microscopy of lectin and SEC purified E6 and E7 lead nanoparticle design. Left image is raw micrograph with scale bar and a corresponding zoomed in view on right to show representative nanoparticles. Figure 7A) E7_2Tr_nano. Figure 7B) E7_FL_ApRb_nano. Figure 7C) E6_FL_Sol_Ap53_nano. Figure 7D) E6_Tr_Sol_Ap53_nano.

[0013] Figure 8 A and Figure 8B depicts structural prediction and confidence metrics for E6 FL. Figure 8A) Alignment of E6 crystal structure 6SJA (gray; contains Sol muts) to top ranked Alphafold2 model of WT E6 FL (colored by confidence metrics). Red residues are lower confidence while blue residues are higher confidence. Cartoon representation was used. Figure 8B) Confidence metric graph. pLDDT for E6 FL from top ranked Alphafold2 model; pLDDT above 80 is considered an indication of strong model confidence.

[0014] Figure 9 depicts the sequence alignment for E6 designs. All designs were aligned relative to the WT HPV16 E6 sequence (E6 FL). Dots represent identical amino acids.

[0015] Figure 10A through Figure 10G depict data demonstrating the in vitro characterization of the E6 designs. Figure 10A depicts the purified transfection yield transfection for designed E6 nanoparticles. Nanoparticles are grouped by their base design: full length or truncated. Transfection volume was 100 mL and constructs were purified by lectin affinity chromatography and size exclusion chromatography before yield was determined. Figure 10B depicts the binding of designed E6 nanoparticles to an HPV16 E6 antibody. Plates were coated with 15 pg / mL of each nanoparticle followed by incubation with the serially diluted antibody at the concentrations indicated. Size exclusion chromatography traces (215 nm) of E6 nanoparticles with models in the inset for E6_FL_Sol_nano (Figure 10C) E6_Tr_Sol_nano (Figure 10D) E6_FL_Sol_Ap53_nano (Figure 10E) and E6_Tr_Sol_Ap53_nano (Figure 10F). Figure 10G) Binding of Ap53 E6 nanoparticle to an HPV16 E6 antibody. Plates were coated with 15 pg / mL of each nanoparticle followed by incubation with the serial diluted antibody at the concentrations indicated.

[0016] Figure HA through Figure 11G depict data demonstrating the T-cell responses of designed E7 nanoparticles. Figure 11 A) Immunizations overview. Either C57 / BL6 or CD-I mice were immunized with lOpg of DNA (IM-EP) at week 0. Animals were boosted with an equivalent dose at week 2, and their spleens were harvested at week 3. Splenocytes were isolated for downstream use in ELISpot and flow cytometry experiments. Figure 1 IB) ELISpot responses in C57 / BL6 mice. Splenocytes from immunized mice were stimulated with overlapping peptide pools that span WT E7. IFNy spot forming units per million splenocytes are quantified. (n=5 mice / group). Mean ± SEM. Figure 11C) Positive responders in CD-I mice. ELISpot responses were quantified as above, with additional normalization to the average positive SFUs in the naive group. Any mouse retaining strong responses after normalization to naive was counted as a responder. The number of responders per pool per immunization group was determined. (n=5 mice / group). Black = naive, gray = E7_FL_ApRb_monomer, orange = E7_FL_ApRb_nano, pink = E7_2Tr_nano, purple = E6 / E7 nano cocktail. Figure 1 ID) Top immunogenic constructs per pool in CD-I mice. The pool number is indicated per wedge, and colored according to the immunization group that had the highest number of mice responding for that pool. In cases where multiple constructs had the same number of responders, the wedge color is splitaccordingly. Color scheme is identical to Figure 11 C). Figure 1 IE) IFNy+ and TNFa+ CD8+ T- cell responses in C57 / BL6 mice. Splenocytes were stimulated with E7 peptides and CD8+ T cell responses were quantified by flow cytometry. Mean ± SEM shown. One way ANOVA was conducted. **p<0.005, ***p<0.0005. Figure 1 IF) IFNv+ and TNFa+ CD8+ T-cell responses in CD-I mice. Splenoctyes were analyzed as above. Mean ± SEM shown. One sided T-test was conducted. Figure 11G) Nanoparticle T-cell bias. CD8:CD4 T-cell ratios were determined as the average of (%)IFNy+ and TNFa+ CD8+ T-cells / (%)FFNy+ and TNFa+ CD4+ T-cells per group. These were normalized to the average CD8:CD4 ratio for the E7 monomer to determine the fold CD8:CD4 stimulation bias for nanoparticle groups over monomer. Anything above the dotted line is superior to monomer.

[0017] Figure 12A through Figure 12D depicts ELISpot responses (outbred mice) and CD8+:CD4+ T-cell ratios for designed nanoparticles. Figure 12A) E7 ELISpot responses in outbred CD-I mice. Splenocytes from immunized mice were stimulated with overlapping peptide pools that span WT E7. IFNy spot forming units per million splenocytes were quantified. (n=5 mice / group). Mean ± SEM. One mouse in the naive group responded strongly to all peptide pools and was excluded from analysis due to this nonspecific response. Figure 12B) E6 ELISpot responses in outbred CD-I mice. Splenocytes from immunized mice were stimulated with overlapping peptide pools that span WT E6. IFNy SFU spot forming units per million splenocytes were quantified. (n=5 mice / group). Mean ± SEM. Figure 12C and Figure 12D show CD8+:CD4+ T-cell ratios. Ratios were determined as the ratio of average IFNY+ Or TNFa+ CD8+ T-cell responses to average IFNy+ or TNFa+ CD4+ T-cell responses for both C57 / BL6 (dark green) and CD-I (light green) mice. Figure 12C) CD8+:CD4+ ratios for mice immunized with E7 groups. Figure 12D) CD8+:CD4+ ratios for mice immunized with E6 groups.

[0018] Figure 13 A through Figure 13F depict data demonstrating the T-cell responses of designed E6 nanoparticles. Figure 13 A) ELISpot responses in C57 / BL6 mice. Splenocytes from immunized mice were stimulated with overlapping peptide pools that span WT E6. IFNy spot forming units per million splenocytes are quantified. (n=5 mice / group). Mean ± SEM. Figure 13B) Positive responders in CD-I mice. ELISpot responses were quantified as above, with additional normalization to the average positive SFUs in the naive group. Any mouse retaining strong responses after normalization to naive was counted as a responder. The number of responders per pool per immunization group was determined. (n=5 mice / group). Black = naive,gray = E6_FL_Ap53_monomer, light blue = E6_FL_Sol_Ap53_nano, dark blue = E6_Tr_Sol_Ap53_nano, purple = E6 / E7 nano cocktail. Figure 13C) Top immunogenic constructs per pool in CD-I mice. The pool number is indicated per wedge, and colored according to the immunization group that had the highest number of mice responding for that pool. In cases where multiple constructs had the same number of responders, the wedge color is split accordingly. Color scheme is identical to Figure 13B). Figure 13D) IFNy+ and TNFa+ CD8+ T- cell responses in C57 / BL6 mice. Splenocytes were stimulated with E6 peptides and CD8+ T cell responses were quantified by flow cytometry. Mean ± SEM shown. One sided T-test was conducted. Figure 13E) IFNy+ and TNFa+ CD8+ T-cell responses in CD-I mice. Splenoctyes were analyzed as above. Mean ± SEM shown. One sided T-test was conducted. Figure 13F) Nanoparticle T-cell bias. CD8:CD4 T-cell ratios were determined as the average of (%)IFNy+ and TNFa+ CD8+ T-cells / (%)ZFNy+ and TNFa+ CD4+ T-cells per group. These were normalized to the average CD8:CD4 ratio for the E6 monomer to determine the fold CD8:CD4 stimulation bias for nanoparticle groups over monomer. Anything above the dotted line is superior to monomer.

[0019] Figure 14A and Figure 14B depicts predicted immunogenic epitopes in humans for designed nanoparticles. Peptides predicted to bind human HLA alleles were determined using NetMHCpan using the ‘HLA supertype representative’ set of loci. The epitope corresponding to a strong HLA binder is specified, or shown as NA if no strong binders were predicted. If that sequence is present in the designed nanoparticle, it is highlighted green to show predicted immunogenicity. Binding peptides (9-mers) were predicted from the E7_FL_ pRb sequence (Figure 14A) or E6 FL p53 sequence (Figure 14A). Italics indicate residues that are mutated in the designed nanoparticle.

[0020] Figure 15A and Figure 15B depict data demonstrating expanded epitope targeting of nanoparticle vaccines. Figure 15 A) Predicted binding of common human alleles to epitopes from the indicated nanoparticles. Peptides predicted to have strong binding to each supertype representative allele were predicted using NetMHCPan for both E7 and E6. If the epitope for the peptide was present in the indicated nanoparticle, then the allele is graphed by its frequency in the global human population. E7 nanoparticles were predicted against E7_FL_ pRb ; E6 nanoparticles were predicted against E6_FL_ p53; the cocktail was predicted against both. Figure 15B) Overview of expanded epitope nanoparticle vaccine workflow. This approach usescomputational tools to design stabilized, more-full length cancer antigens scaffolded onto nanoparticles with the aim of providing maximal immunogenic epitopes to the immune system. This allows for greater diversity of T-cell responses and subsequently should allow a greater portion of the population to generate CTL responses to the vaccine.DETAILED DESCRIPTION OF THE INVENTION

[0021] The present invention relates to an immunogenic composition or a vaccine comprising a self-assembling nanoparticle comprising one or more human papillomavirus (HPV) antigen. The HPV antigen can be the HPV E7 protein or a variant or fragment thereof. The HPV antigen can be the HPV E6 protein, or a variant or fragment thereof.

[0022] In one embodiment, the invention relates to a nucleic acid molecule encoding a nanoparticle subunit comprising one or more Human papillomavirus (HPV) antigen. In one embodiment, the nucleic acid molecule encodes a subunit of a self-assembling nanoparticle comprising the HPV E7 protein, or a variant or fragment thereof. In one embodiment, the nucleic acid molecule encodes a subunit of a self-assembling nanoparticle comprising the HPV E6 protein, or a variant or fragment thereof.

[0023] The vaccine can be used treat HPV infection or to prevent or treat a disease or disorder associated with HPV infection. In one embodiment, the disease or disorder associated with HPV infection include, but is not limited to, cervical cancer, anogenital cancers (e.g., penile, anal, vulvar, and vaginal cancers) and head and neck cancers (e.g., oropharyngeal cancer). The vaccine can elicit both humoral and cellular immune responses that target at least one HPV antigen. The vaccine can elicit neutralizing antibodies and immunoglobulin G (IgG) antibodies that are reactive with at least one HPV antigen. The vaccine can also elicit CD8+ and CD4+ T cell responses that are reactive to at least one HPV antigen and produce interferongamma (IFN-y), tumor necrosis factor alpha (TNF-ot), and interleukin-2 (IL-2).Definitions

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described hereincan be used in practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0025] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of’ and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0026] “Adjuvant” as used herein means any molecule added to the vaccine described herein to enhance the immunogenicity of the antigen.

[0027] “Antibody” as used herein means an antibody of classes IgG, IgM, IgA, IgD or IgE, or fragments, fragments or derivatives thereof, including Fab, F(ab')2, Fd, and single chain antibodies, diabodies, bispecific antibodies, bifunctional antibodies and derivatives thereof. The antibody can be an antibody isolated from the serum sample of mammal, a polyclonal antibody, affinity purified antibody, or mixtures thereof which exhibits sufficient binding specificity to a desired epitope or a sequence derived therefrom.

[0028] “Coding sequence” or “encoding nucleic acid” as used herein means the nucleic acids (RNA or DNA molecule) that comprise a nucleotide sequence which encodes a protein. The coding sequence can further include initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of an individual or mammal to which the nucleic acid is administered.

[0029] “Complement” or “complementary” as used herein means Watson-Crick (e.g., A- T / U and C-G) or Hoogsteen base pairing between nucleotides or nucleotide analogs of nucleic acid molecules.

[0030] “Consensus” or “Consensus Sequence” as used herein may mean a synthetic nucleic acid sequence, or corresponding polypeptide sequence, constructed based on analysis of an alignment of multiple subtypes of a particular antigen. The sequence may be used to induce broad immunity against multiple subtypes, serotypes, or strains of a particular antigen. Syntheticantigens, such as fusion proteins, may be manipulated to generate consensus sequences (or consensus antigens).

[0031] “Electroporation,” “electro-permeabilization,” or “electro-kinetic enhancement” (“EP”) as used interchangeably herein means the use of a transmembrane electric field pulse to induce microscopic pathways (pores) in a bio-membrane; their presence allows biomolecules such as plasmids, oligonucleotides, siRNA, drugs, ions, and water to pass from one side of the cellular membrane to the other.

[0032] “Fragment” as used herein means a nucleic acid sequence or a portion thereof that encodes a polypeptide capable of eliciting an immune response in a mammal. The fragments can be DNA fragments selected from at least one of the various nucleotide sequences that encode protein fragments set forth below.

[0033] “Fragment” or “immunogenic fragment” with respect to polypeptide sequences means a polypeptide capable of eliciting an immune response in a mammal that cross reacts with a full length wild type strain HPV antigen. Fragments of consensus proteins can comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% of a consensus protein. In some embodiments, fragments of consensus proteins can comprise at least 20 amino acids or more, at least 30 amino acids or more, at least 40 amino acids or more, at least 50 amino acids or more, at least 60 amino acids or more, at least 70 amino acids or more, at least 80 amino acids or more, at least 90 amino acids or more, at least 100 amino acids or more, at least 110 amino acids or more, at least 120 amino acids or more, at least 130 amino acids or more, at least 140 amino acids or more, at least 150 amino acids or more, at least 160 amino acids or more, at least 170 amino acids or more, at least 180 amino acids or more, at least 190 amino acids or more, at least 200 amino acids or more, at least 210 amino acids or more, at least 220 amino acids or more, at least 230 amino acids or more, or at least 240 amino acids or more of a consensus protein.

[0034] As used herein, the term “genetic construct” refers to the DNA or RNA molecules that comprise a nucleotide sequence which encodes a protein. The coding sequence includes initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of the individual to whom the nucleic acid molecule is administered. As used herein, the term “expressible form” refers to gene constructs that contain the necessary regulatory elements operable linked to a codingsequence that encodes a protein such that when present in the cell of the individual, the coding sequence will be expressed.

[0035] “Identical” or “identity” as used herein in the context of two or more nucleic acids or polypeptide sequences, means that the sequences have a specified percentage of residues that are the same over a specified region. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity. In cases where the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified region of comparison includes only a single sequence, the residues of single sequence are included in the denominator but not the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identity can be performed manually or by using a computer sequence algorithm such as BLAST or BLAST 2.0.

[0036] “Immune response” as used herein means the activation of a host’s immune system, e.g., that of a mammal, in response to the introduction of antigen. The immune response can be in the form of a cellular or humoral response, or both.

[0037] “Nucleic acid” or “oligonucleotide” or “polynucleotide” as used herein means at least two nucleotides covalently linked together. The depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the complementary strand of a depicted single strand. Many variants of a nucleic acid can be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also encompasses substantially identical nucleic acids and complements thereof. A single strand provides a probe that can hybridize to a target sequence under stringent hybridization conditions. Thus, a nucleic acid also encompasses a probe that hybridizes under stringent hybridization conditions.

[0038] Nucleic acids can be single stranded or double stranded, or can contain portions of both double stranded and single stranded sequence. The nucleic acid can be DNA, both genomic and cDNA, RNA, or a hybrid, where the nucleic acid can contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine,guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine. Nucleic acids can be obtained by chemical synthesis methods or by recombinant methods.

[0039] “Operably linked” as used herein means that expression of a gene is under the control of a promoter with which it is spatially connected. A promoter can be positioned 5' (upstream) or 3' (downstream) of a gene under its control. The distance between the promoter and a gene can be approximately the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variation in this distance can be accommodated without loss of promoter function.

[0040] A “peptide,” “protein,” or “polypeptide” as used herein can mean a linked sequence of amino acids and can be natural, synthetic, or a modification or combination of natural and synthetic.

[0041] “Promoter” as used herein means a synthetic or naturally-derived molecule which is capable of conferring, activating or enhancing expression of a nucleic acid in a cell. A promoter can comprise one or more specific transcriptional regulatory sequences to further enhance expression and / or to alter the spatial expression and / or temporal expression of same. A promoter can also comprise distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. A promoter can be derived from sources including viral, bacterial, fungal, plants, insects, and animals. A promoter can regulate the expression of a gene component constitutively or differentially with respect to cell, the tissue or organ in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, pathogens, metal ions, or inducing agents. Representative examples of promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV40 late promoter and the CMV IE promoter.

[0042] “Signal peptide” and “leader sequence” are used interchangeably herein and refer to an amino acid sequence that can be linked at the amino terminus of a HPV protein set forth herein. Signal peptides / leader sequences typically direct localization of a protein. Signal peptides / leader sequences used herein preferably facilitate secretion of the protein from the cell in which it is produced. Signal peptides / leader sequences are often cleaved from the remainder ofthe protein, often referred to as the mature protein, upon secretion from the cell. Signal peptides / leader sequences are linked at the N terminus of the protein.

[0043] “Subject” as used herein can mean a mammal that wants to or is in need of being immunized with the herein described vaccine. The mammal can be a human, chimpanzee, dog, cat, horse, cow, mouse, or rat.

[0044] “Substantially identical” as used herein can mean that a first and second amino acid sequence are at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% over a region of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100 or more amino acids. Substantially identical can also mean that a first nucleic acid sequence and a second nucleic acid sequence are at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% over a region of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100 or more nucleotides.

[0045] “Treatment” or “treating,” as used herein can mean protecting of an animal from a disease through means of preventing, suppressing, repressing, or completely eliminating the disease. Preventing the disease involves administering a vaccine of the present invention to an animal prior to onset of the disease. Suppressing the disease involves administering a vaccine of the present invention to an animal after induction of the disease but before its clinical appearance. Repressing the disease involves administering a vaccine of the present invention to an animal after clinical appearance of the disease.

[0046] “Variant” used herein with respect to a nucleic acid means (i) a portion or fragment of a referenced nucleotide sequence; (ii) the complement of a referenced nucleotide sequence or portion thereof; (iii) a nucleic acid that is substantially identical to a referenced nucleic acid or the complement thereof; or (iv) a nucleic acid that hybridizes under stringent conditions to the referenced nucleic acid, complement thereof, or a sequences substantially identical thereto.

[0047] Variant can further be defined as a peptide or polypeptide that differs in amino acid sequence by the insertion, deletion, or conservative substitution of amino acids, but retain atleast one biological activity. Representative examples of “biological activity” include the ability to be bound by a specific antibody or to promote an immune response. Variant can also mean a protein with an amino acid sequence that is substantially identical to a referenced protein with an amino acid sequence that retains at least one biological activity. A conservative substitution of an amino acid, i.e., replacing an amino acid with a different amino acid of similar properties (e.g., hydrophilicity, degree and distribution of charged regions) is recognized in the art as typically involving a minor change. These minor changes can be identified, in part, by considering the hydropathic index of amino acids, as understood in the art. Kyte et al., J. Mol. Biol. 157:105-132 (1982). The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic indexes can be substituted and still retain protein function. In one aspect, amino acids having hydropathic indexes of ±2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that would result in proteins retaining biological function. A consideration of the hydrophilicity of amino acids in the context of a peptide permits calculation of the greatest local average hydrophilicity of that peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. Substitution of amino acids having similar hydrophilicity values can result in peptides retaining biological activity, for example immunogenicity, as is understood in the art. Substitutions can be performed with amino acids having hydrophilicity values within ±2 of each other. Both the hydrophobicity index and the hydrophilicity value of amino acids are influenced by the particular side chain of that amino acid. Consistent with that observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and particularly the side chains of those amino acids, as revealed by the hydrophobicity, hydrophilicity, charge, size, and other properties.

[0048] A variant may be a nucleic acid sequence that is substantially identical over the full length of the full gene sequence or a fragment thereof. The nucleic acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the full length of the gene sequence or a fragment thereof. A variant may be an amino acid sequence that is substantially identical over the full length of the amino acid sequence or fragment thereof. The amino acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,^ / o, 98%, 99%, or 100% identical over the full length of the amino acid sequence or a fragment thereof.

[0049] “Vector” as used herein means a nucleic acid sequence containing an origin of replication. A vector can be a viral vector, bacteriophage, bacterial artificial chromosome or yeast artificial chromosome. A vector can be a DNA or RNA vector. A vector can be a selfreplicating extrachromosomal vector, and preferably, is a DNA plasmid.

[0050] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.Vaccine

[0051] Provided herein are immunogenic compositions, such as vaccines, comprising a Human papillomavirus (HPV) antigen, a fragment thereof, a variant thereof, or a combination thereof. The vaccine can be used to treat HPV infection, thereby treating, preventing, and / or protecting against HPV based pathologies. In one embodiment, the HPV based pathology is cervical cancer, an anogenital cancer (e.g., penile, anal, vulvar, or vaginal cancer) or head and neck cancer (e.g., oropharyngeal cancer). The vaccine can significantly induce an immune response of a subject administered the vaccine, thereby protecting against and treating HPV infection, or a disease or disorder associated with HPV infection.

[0052] In one embodiment, the immunogenic composition of the invention comprises an HPV antigen. In some embodiments, the HPV antigen is operably linked to an aggregation domain for self-assembly into a nanoparticle, referred to herein as an HPV antigen fusion molecule. In one embodiment, the HPV antigen comprises an HPV E6 or E7 antigen, or a variant or fragment thereof. In some embodiments, the HPV antigen is from HPV16.

[0053] In one embodiment, the antigen comprises SEQ ID NO:5, SEQ ID NO:7, SEQ ID NOV, SEQ ID NO: 11, SEQ ID N0: 13, SEQ ID NO: 15, SEQ ID NO:17, SEQ ID NO 19, SEQ ID N0:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO: 27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, or SEQ ID NO:45, or a fragment or variant thereof. In one embodiment, the HPV antigen fusion molecule comprises SEQ ID NO:5, SEQ ID NO:7, SEQ ID NOV, SEQ ID NO: 11, SEQID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ TD NO:21 , SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, or SEQ ID NO:45, or a fragment or variant thereof operably linked to a nucleotide sequence encoding a nanoparticle core domain. In one embodiment, the nanoparticle core domain comprises SEQ ID NO:3. In one embodiment, the HPV antigen fusion molecule comprises SEQ ID NO:47, SEQ ID NO:49, SEQ ID N0:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85 or SEQ ID NO:87 or a fragment or variant thereof.

[0054] In one embodiment, the immunogenic composition of the invention comprises an isolated nucleic acid molecule that encodes an HPV antigen. In one embodiment, the immunogenic composition of the invention comprises an isolated nucleic acid molecule that encodes an HPV antigen fusion molecule of the invention. Some embodiments comprise an isolated nucleic acid molecule encodes an HPV E6 or E7 antigen, or a variant or fragment thereof. In some embodiments, the HPV antigen is from HPV16.

[0055] In one embodiment, the nucleotide sequence encoding the antigen comprises SEQ ID NO:6, SEQ ID NO 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO 22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44 or SEQ ID NO:46, or a fragment or variant thereof. In one embodiment, the nucleotide sequence encoding the HPV antigen fusion molecule comprises SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO:14, SEQ ID NO 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44 or SEQ ID NO:46, or a fragment or variant thereof operably linked to a nucleotide sequence encoding a nanoparticle core domain. In one embodiment, the nucleotide sequence encoding a nanoparticle core domain comprises SEQ ID NON. In one embodiment, the HPV antigen fusion molecule comprises SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO 52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO 66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72,SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86 or SEQ ID NO:88 or a fragment or variant thereof.

[0056] The vaccine can be a DNA vaccine, an RNA vaccine, or a peptide vaccine, or a combination thereof. The vaccine can include a nucleic acid sequence encoding the HPV antigen fusion molecule. The nucleic acid sequence can be DNA, RNA, cDNA, a variant thereof, a fragment thereof, or a combination thereof. The nucleic acid sequence can also include additional sequences that encode linker, leader, or tag sequences that are linked to the HPV antigen fusion molecule. In one embodiment, the nucleic acid sequence encodes SEQ ID NO:47, SEQ ID NO:49, SEQ ID N0:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85 or SEQ ID NO:87. In one embodiment, the nucleic acid sequence comprises SEQ ID NO:6, SEQ ID NO:8, SEQ ID NOTO, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NOTO, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NOTO, SEQ ID NO:42, SEQ ID NO:44 or SEQ ID NO:46.

[0057] In one embodiment, one or more HPV antigen fusion molecule comprising an HPV antigen domain and an aggregation domain forms a self-assembling nanoparticle upon expression. Self-assembling protein nanoparticles (SAPN) may be formed by the assembly of one or more polypeptide chains comprising at least one antigen and at least one protein oligomerization domain. Without limitation, the SAPN of the invention may self-assemble into a tetrahedron, a cube, an octahedron, a dodecahedron, or an icosahedron. In one embodiment, the SAPN of the invention is generated by the aggregation of at least 60 HPV fusion molecules. The SAPN of the invention may be used as an efficient means for presenting one or more HPV antigen.

[0058] The vaccine can induce a humoral immune response in the subject administered the vaccine. The induced humoral immune response can be specific for the HPV antigen. The induced humoral immune response can be reactive with the HPV antigen. The humoral immune response can be induced in the subject administered the vaccine by about 1.5-fold to about 16- fold, about 2-fold to about 12-fold, or about 3 -fold to about 10-fold. The humoral immune response can be induced in the subject administered the vaccine by at least about 1.5-fold, atleast about 2.0-fold, at least about 2.5-fold, at least about 3.0-fold, at least about 3.5-fold, at least about 4.0-fold, at least about 4.5-fold, at least about 5.0-fold, at least about 5.5-fold, at least about 6.0-fold, at least about 6.5-fold, at least about 7.0-fold, at least about 7.5-fold, at least about 8.0-fold, at least about 8.5-fold, at least about 9.0-fold, at least about 9.5-fold, at least about 10.0-fold, at least about 10.5-fold, at least about 11.0-fold, at least about 11.5-fold, at least about 12.0-fold, at least about 12.5-fold, at least about 13.0-fold, at least about 13.5-fold, at least about 14.0-fold, at least about 14.5-fold, at least about 15.0-fold, at least about 15.5-fold, or at least about 16.0-fold.

[0059] The humoral immune response induced by the vaccine can include an increased level of neutralizing antibodies associated with the subject administered the vaccine as compared to a subject not administered the vaccine. The neutralizing antibodies can be specific for the HPV antigen. The neutralizing antibodies can be reactive with the HPV antigen. The neutralizing antibodies can provide protection against and / or treatment of HPV infection and its associated pathologies in the subject administered the vaccine.

[0060] The humoral immune response induced by the vaccine can include an increased level of IgG antibodies associated with the subject administered the vaccine as compared to a subject not administered the vaccine. These IgG antibodies can be specific for the HPV antigen. These IgG antibodies can be reactive with the HPV antigen. Preferably, the humoral response is cross-reactive against two or more strains of the HPV. The level of IgG antibody associated with the subject administered the vaccine can be increased by about 1.5-fold to about 16-fold, about 2-fold to about 12-fold, or about 3 -fold to about 10-fold as compared to the subject not administered the vaccine. The level of IgG antibody associated with the subject administered the vaccine can be increased by at least about 1.5-fold, at least about 2.0-fold, at least about 2.5-fold, at least about 3.0-fold, at least about 3.5-fold, at least about 4.0-fold, at least about 4.5-fold, at least about 5.0-fold, at least about 5.5-fold, at least about 6.0-fold, at least about 6.5-fold, at least about 7.0-fold, at least about 7.5-fold, at least about 8.0-fold, at least about 8.5-fold, at least about 9.0-fold, at least about 9.5-fold, at least about 10.0-fold, at least about 10.5-fold, at least about 11.0-fold, at least about 11.5-fold, at least about 12.0-fold, at least about 12.5-fold, at least about 13.0-fold, at least about 13.5-fold, at least about 14.0-fold, at least about 14.5-fold, at least about 15.0-fold, at least about 15.5-fold, or at least about 16.0-fold as compared to the subject not administered the vaccine.

[0061] The vaccine can induce a cellular immune response in the subject administered the vaccine. The induced cellular immune response can be specific for the HPV antigen. The induced cellular immune response can be reactive to the HPV antigen. In some embodiments, the cellular response is cross-reactive against two or more strains of HPV. The induced cellular immune response can include eliciting a CD8 T cell response. The elicited CD8+T cell response can be reactive with the HPV antigen. The elicited CD8+T cell response can be polyfunctional. The induced cellular immune response can include eliciting a CD8+T cell response, in which the CD8+T cells produce interferon-gamma (IFN-y), tumor necrosis factor alpha (TNF-a), interleukin-2 (IL-2), or a combination of IFN-y and TNF-a.

[0062] The induced cellular immune response can include an increased CD8+T cell response associated with the subject administered the vaccine as compared to the subject not administered the vaccine. The CD8+T cell response associated with the subject administered the vaccine can be increased by about 2-fold to about 30-fold, about 3-fold to about 25-fold, or about4-fold to about 20-fold as compared to the subject not administered the vaccine. The CD81T cell response associated with the subject administered the vaccine can be increased by at least about 1.5-fold, at least about 2.0-fold, at least about 3.0-fold, at least about 4.0-fold, at least about 5.0- fold, at least about 6.0-fold, at least about 6.5-fold, at least about 7.0-fold, at least about 7.5-fold, at least about 8.0-fold, at least about 8.5-fold, at least about 9.0-fold, at least about 9.5-fold, at least about 10.0-fold, at least about 10.5-fold, at least about 11.0-fold, at least about 11.5-fold, at least about 12.0-fold, at least about 12.5-fold, at least about 13.0-fold, at least about 13.5-fold, at least about 14.0-fold, at least about 14.5-fold, at least about 15.0-fold, at least about 16.0-fold, at least about 17.0-fold, at least about 18.0-fold, at least about 19.0-fold, at least about 20.0-fold, at least about 21.0-fold, at least about 22.0-fold, at least about 23.0-fold, at least about 24.0-fold, at least about 25.0-fold, at least about 26.0-fold, at least about 27.0-fold, at least about 28.0-fold, at least about 29.0-fold, or at least about 30.0-fold as compared to the subject not administered the vaccine.

[0063] The induced cellular immune response can include an increased frequency of CD3+CD8+T cells that produce IFN-y. The frequency of CD3 CD8 IFN-y T cells associated with the subject administered the vaccine can be increased by at least about 2-fold, 3-fold, 4-fold,5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, or 20-fold as compared to the subject not administered the vaccine.

[0064] The induced cellular immune response can include an increased frequency of CD3+CD8+T cells that produce TNF-a. The frequency of CD3+CD8+TNF-a+T cells associated with the subject administered the vaccine can be increased by at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, or 14-fold as compared to the subject not administered the vaccine.

[0065] The induced cellular immune response can include an increased frequency of CD3+CD8+T cells that produce IL-2. The frequency of CD3+CD8+IL-2+T cells associated with the subject administered the vaccine can be increased by at least about 0.5-fold, 1.0-fold, 1.5- fold, 2.0-fold, 2.5-fold, 3.0-fold, 3.5-fold, 4.0-fold, 4.5-fold, or 5.0-fold as compared to the subject not administered the vaccine.

[0066] The induced cellular immune response can include an increased frequency of CD3+CD8+T cells that produce both IFN-y and TNF-a. The frequency of CD3+CD8+IFN- y+TNF-a+T cells associated with the subject administered the vaccine can be increased by at least about 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold, 110-fold, 120-fold, 130-fold, 140-fold, 150- fold, 160-fold, 170-fold, or 180-fold as compared to the subject not administered the vaccine.

[0067] The cellular immune response induced by the vaccine can include eliciting a CD4+T cell response. The elicited CD4+T cell response can be reactive with the HPV antigen. The elicited CD4+T cell response can be polyfunctional. The induced cellular immune response can include eliciting a CD4 T cell response, in which the CD4+T cells produce IFN-y, TNF-a, IL-2, or a combination of IFN-y and TNF-a.

[0068] The induced cellular immune response can include an increased frequency of CD3+CD4+T cells that produce IFN-y. The frequency of CD3 CD4 IFN-y T cells associated with the subject administered the vaccine can be increased by at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, or 20-fold as compared to the subject not administered the vaccine.

[0069] The induced cellular immune response can include an increased frequency of CD3+CD4+T cells that produce TNF-a. The frequency of CD3+CD4+TNF-a+T cells associated with the subject administered the vaccine can be increased by at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold,17-fold, 18-fold, 19-fold, 20-fold, 21 -fold, or 22-fold as compared to the subject not administered the vaccine.

[0070] The induced cellular immune response can include an increased frequency of CD3+CD4+T cells that produce IL-2. The frequency of CD3+CD4+IL-2+T cells associated with the subject administered the vaccine can be increased by at least about 2-fold, 3-fold, 4-fold, 5- fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30-fold, 31-fold, 32-fold, 33-fold, 34-fold, 35-fold, 36-fold, 37-fold, 38-fold, 39-fold, 40-fold, 45-fold, 50-fold, 55-fold, or 60-fold as compared to the subject not administered the vaccine.

[0071] The induced cellular immune response can include an increased frequency of CD3+CD4+T cells that produce both IFN-y and TNF-a. The frequency of CD3+CD4+IFN- y+TNF-a+associated with the subject administered the vaccine can be increased by at least about 2-fold, 2.5-fold, 3.0-fold, 3.5-fold, 4.0-fold, 4.5-fold, 5.0-fold, 5.5-fold, 6.0-fold, 6.5-fold, 7.0- fold, 7.5-fold, 8.0-fold, 8.5-fold, 9.0-fold, 9.5-fold, 10.0-fold, 10.5-fold, 11.0-fold, 11.5-fold, 12.0-fold, 12.5-fold, 13.0-fold, 13.5-fold, 14.0-fold, 14.5-fold, 15.0-fold, 15.5-fold, 16.0-fold, 16.5-fold, 17.0-fold, 17.5-fold, 18.0-fold, 18.5-fold, 19.0-fold, 19.5-fold, 20.0-fold, 21 -fold, 22- fold, 23-fold 24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30-fold, 31-fold, 32-fold, 33- fold, 34-fold, or 35-fold as compared to the subject not administered the vaccine.

[0072] The vaccine of the present invention can have features required of effective vaccines such as being safe so the vaccine itself does not cause illness or death; is protective against illness resulting from exposure to live pathogens such as viruses or bacteria; induces neutralizing antibody to prevent invention of cells; induces protective T cells against intracellular pathogens; and provides ease of administration, few side effects, biological stability, and low cost per dose.

[0073] The vaccine can further induce an immune response when administered to different tissues such as the muscle or skin. The vaccine can further induce an immune response when administered via electroporation, or injection, or subcutaneously, or intramuscularly.Human papillomavirus (HPV) Antigen

[0074] As described above, in one embodiment, the invention relates to a vaccine comprising an HPV16 E6 or E7 antigen, a fragment thereof, a variant thereof, or a combination thereof.

[0075] In one embodiment, the composition of the invention is capable of eliciting an immune response in a mammal against one or more HPV strains. The HPV antigen can comprise an epitope(s) that makes it particularly effective as an immunogen against which an anti-HPV immune response can be induced.

[0076] The HPV antigen can be a consensus sequence derived from two or more strains of HPV. The nucleic acid molecule encoding the HPV antigen can comprise a consensus sequence and / or modification(s) for improved expression. Modification can include codon optimization, RNA optimization, addition of a kozak sequence for increased translation initiation, and / or the addition of an immunoglobulin leader sequence to increase the immunogenicity of the one or more HPV antigen, or fragment thereof. The one or more HPV antigen can comprise a signal peptide such as an immunoglobulin signal peptide, for example, but not limited to, an immunoglobulin E (IgE) or immunoglobulin (IgG) signal peptide.Self-Assembling Nanoparticles

[0077] In one embodiment, the invention relates to a vaccine comprising a selfassembling nanoparticle further comprising a HPV antigen, a fragment thereof, a variant thereof, or a combination thereof. In one embodiment, the nanoparticle functions as a scaffold for presentation of the HPV antigen. Therefore, in one embodiment, the invention relates to polypeptide subunits for self-assembly into a spherical nanoparticle, comprising an aggregation or scaffold domain and an HPV antigen domain. In one embodiment, the aggregation or scaffold domain comprises lumazine synthase, or a fragment or variant thereof. In one embodiment, the aggregation or scaffold domain comprises lumazine synthase and GT60.

[0078] In one embodiment, the invention relates to a nucleic acid molecule encoding a subunit of a self-assembling nanoparticle comprising an oligomerization domain and further comprising a HPV antigen, a fragment thereof, a variant thereof, or a combination thereof. In some embodiments, the nucleic acid molecule encoding the oligomerization domain can comprise a nucleotide sequence that encodes the amino acid sequence having at least about 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over an entire length of the amino acid sequence set forth in SEQ ID NO:3. In some embodiments, the nucleic acid molecule encoding the oligomerization domain can comprise a nucleotide sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over an entire length of the nucleic acid sequence set forth in SEQ ID NO:4. In some embodiments, the nucleotide sequence encoding the oligomerization domain can be operably linked to a sequence encoding at least one linker sequence, such as an LS3 or GGS linker sequence.

[0079] Immunogenic fragments of SEQ ID NO:3 can be provided. Immunogenic fragments can comprise at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of SEQ ID NO:3.

[0080] Some embodiments relate to immunogenic fragments of SEQ ID NO:4. Immunogenic fragments can be at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the full length of SEQ ID NO:4. Immunogenic fragments can comprise at least 95%, at least 96%, at least 97% at least 98% or at least 99% identity to fragments of SEQ ID NO:4.Leader Sequence

[0081] In some embodiments, the amino acid sequence for a subunit of a self-assembling nanoparticle of the invention is operably linked to at least one leader sequence or a pharmaceutically acceptable salt thereof. In some embodiments, the nucleic acid molecules of the invention encoding a subunit of a self-assembling nanoparticle of the invention is operably linked to at least one nucleotide sequence encoding a leader sequence or a pharmaceutically acceptable salt thereof. "Signal peptide" and "leader sequence" are used interchangeably herein and refer to an amino acid sequence that can be linked at the amino terminus of a protein set forth herein. Signal peptides / leader sequences typically direct localization of a protein. Signal peptides / leader sequences used herein preferably facilitate secretion of the protein from the cell in which it is produced. Signal peptides / leader sequences are often cleaved from the remainder ofthe protein, often referred to as the mature protein, upon secretion from the cell. Signal peptides / leader sequences are linked at the N terminus of the protein.

[0082] In some embodiments, the fusion construct comprising the HPV antigen can be operably linked to an IgE leader sequence (SEQ ID NO: 1). In some embodiments, the nucleic acid molecule encoding the fusion construct comprising the HPV antigen comprises a sequence encoding an IgE leader sequence (SEQ ID NO:2).Linker Sequence

[0083] In some embodiments, the amino acid sequence for a subunit of a self-assembling nanoparticle of the invention is operably linked to at least one linker sequence. For example, in some embodiments the sequence for the subunit of a self-assembling nanoparticle sequences comprise a linker between one or more scaffold sequences and at least one downstream domain, such as an HPV domain sequence. In some embodiments the sequence for the subunit of a selfassembling nanoparticle sequences comprises two or more linkers (e.g., a first linker between a lumazine synthase domain and a GT60 domain and a second linker between a GT60 domain and an HPV domain.) A linker can be either flexible or rigid or a combination thereof. In one embodiment, the linker is a (GGS)n repeat wherein, the GGS is repeated at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more than 10 times.

[0084] The HPV antigen can have an amino acid sequence of SEQ ID NO:5, SEQ ID NOV, SEQ ID NOV, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NOV 1, SEQ ID NO:33, SEQ ID NO 35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, or SEQ ID NO:45. In some embodiments, the HPV antigen can be an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%>, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over an entire length of the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:7, SEQ ID NOV, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO:15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO: 29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, or SEQ ID NO:45.

[0085] The fusion construct comprising the HPV antigen can have an amino acid sequence of SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO 83, SEQ ID NO:85 or SEQ ID NO:87. In some embodiments, the fusion construct can be an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over an entire length of the amino acid sequence set forth in SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85 or SEQ ID NO:87.

[0086] The nucleic acid molecule encoding the HPV antigen can comprise a nucleotide sequence that encodes the amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over an entire length of the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NOT, SEQ ID NOV, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO: 25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, or SEQ ID NO:45. In some embodiments, the nucleic acid molecule encoding the HPV antigen can comprise a nucleotide sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over an entire length of the nucleic acid sequence set forth in SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NOTO, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NOTO, SEQ ID NO 42, SEQ ID NO:44 or SEQ ID NO:46.

[0087] The nucleic acid molecule encoding the HPV antigen can comprise the nucleic acid sequence of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NOTO, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO 28, SEQ ID NOTO, SEQ ID NO:32, SEQ ID NO 34, SEQ ID NO:36, SEQID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44 or SEQ ID NO:46, which encodes SEQ ID N0:5, SEQ ID N0:7, SEQ ID N0:9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO 43, or SEQ ID NO:45 respectively.

[0088] The nucleic acid molecule encoding the fusion construct comprising the HPV antigen can comprise a nucleotide sequence that encodes the amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over an entire length of the amino acid sequence set forth in SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO 69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO 83, SEQ ID NO:85 or SEQ ID NO:87. In some embodiments, the nucleic acid molecule encoding the fusion construct comprising the HPV antigen can comprise a nucleotide sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over an entire length of the nucleic acid sequence set forth in SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO 66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO 86 or SEQ ID NO:88.

[0089] The nucleic acid molecule encoding the fusion construct comprising the HPV antigen fusion construct can comprise the nucleic acid sequence of SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO 52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO 86 or SEQ ID NO:88, which encodes SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO 75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85 or SEQ ID NO:87 respectively.Vector

[0090] The vaccine can comprise one or more vectors that include a nucleic acid encoding a subunit of a self-assembling nanoparticle of the invention. The one or more vectors can be capable of expressing the encoded subunit polypeptides. The vector can have a nucleic acid sequence containing an origin of replication. The vector can be a plasmid, bacteriophage, bacterial artificial chromosome or yeast artificial chromosome. The vector can be either a selfreplicating extrachromosomal vector or a vector which integrates into a host genome.

[0091] The one or more vectors can be an expression construct, which is generally a plasmid that is used to introduce a specific gene into a target cell. Once the expression vector is inside the cell, the protein that is encoded by the gene is produced by the cellular-transcription and translation machinery ribosomal complexes. The plasmid is frequently engineered to contain regulatory sequences that act as enhancer and promoter regions and lead to efficient transcription of the gene carried on the expression vector. The vectors of the present invention express large amounts of stable messenger RNA, and therefore proteins.

[0092] The vectors may have expression signals such as a strong promoter, a strong termination codon, adjustment of the distance between the promoter and the cloned gene, and the insertion of a transcription termination sequence and a PTIS (portable translation initiation sequence).Expression Vectors

[0093] The vector can be a circular plasmid or a linear nucleic acid. The circular plasmid and linear nucleic acid are capable of directing expression of a particular nucleotide sequence in an appropriate subject cell. The vector can have a promoter operably linked to the antigenencoding nucleotide sequence, which may be operably linked to termination signals. The vector can also contain sequences required for proper translation of the nucleotide sequence. The vector comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. The expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or of an inducible promoter, which initiates transcription only when the host cell isexposed to some particular external stimulus. In the case of a multicellular organism, the promoter can also be specific to a particular tissue or organ or stage of development.Circular and Linear Vectors

[0094] The vector may be a circular plasmid, which may transform a target cell by integration into the cellular genome or exist extrachromosomally (e.g., autonomous replicating plasmid with an origin of replication).

[0095] The vector can be pVAX, pcDNA3.0, or provax, or any other expression vector capable of expressing DNA encoding the antigen and enabling a cell to translate the sequence to an antigen that is recognized by the immune system.

[0096] Also provided herein is a linear nucleic acid vaccine, or linear expression cassette (“LEC”), that is capable of being efficiently delivered to a subject via electroporation and expressing one or more desired antigens. The LEC may be any linear DNA devoid of any phosphate backbone. The DNA may encode one or more antigens. The LEC may contain a promoter, an intron, a stop codon, and / or a polyadenylation signal. The expression of the antigen may be controlled by the promoter. The LEC may not contain any antibiotic resistance genes and / or a phosphate backbone. The LEC may not contain other nucleic acid sequences unrelated to the desired antigen gene expression.Promoter, Intron, Stop Codon, and Polyadenylation Signal

[0097] The vector may have a promoter. A promoter may be any promoter that is capable of driving gene expression and regulating expression of the isolated nucleic acid. Such a promoter is a cis-acting sequence element required for transcription via a DNA dependent RNA polymerase, which transcribes the antigen sequence described herein. Selection of the promoter used to direct expression of a heterologous nucleic acid depends on the particular application. The promoter may be positioned about the same distance from the transcription start in the vector as it is from the transcription start site in its natural setting. However, variation in this distance may be accommodated without loss of promoter function.

[0098] The promoter may be operably linked to the nucleic acid sequence encoding the antigen and signals required for efficient polyadenylation of the transcript, ribosome binding sites, and translation termination. The promoter may be a CMV promoter, SV40 early promoter,SV40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or another promoter shown effective for expression in eukaryotic cells.

[0099] The vector may include an enhancer and an intron with functional splice donor and acceptor sites. The vector may contain a transcription termination region downstream of the structural gene to provide for efficient termination. The termination region may be obtained from the same gene as the promoter sequence or may be obtained from different genes.Excipients and other Components of the Vaccine

[0100] The vaccine may further comprise a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient can be functional molecules such as vehicles, carriers, or diluents. The pharmaceutically acceptable excipient can be a transfection facilitating agent, which can include surface active agents, such as immune-stimulating complexes (ISCOMS), Freunds incomplete adjuvant, LPS analog including monophosphoryl lipid A, muramyl peptides, quinone analogs, vesicles such as squalene and squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection facilitating agents.

[0101] The transfection facilitating agent is a polyanion, polycation, including poly-L- glutamate (LGS), or lipid. The transfection facilitating agent is poly-L-glutamate, and the poly- L-glutamate may be present in the vaccine at a concentration less than 6 mg / ml. The transfection facilitating agent may also include surface active agents such as immune-stimulating complexes (ISCOMS), Freunds incomplete adjuvant, LPS analog including monophosphoryl lipid A, muramyl peptides, quinone analogs and vesicles such as squalene and squalene, and hyaluronic acid may also be used administered in conjunction with the genetic construct. The DNA plasmid vaccines may also include a transfection facilitating agent such as lipids, liposomes, including lecithin liposomes or other liposomes known in the art, as a DNA-liposome mixture (see for example W09324640), calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection facilitating agents. The transfection facilitating agent is a polyanion, polycation, including poly-L-glutamate (LGS), or lipid. Concentration of the transfection agent in the vaccine is less than 4 mg / ml, less than 2 mg / ml, less than 1 mg / ml, less than 0.750 mg / ml,less than 0.500 mg / ml, less than 0.250 mg / ml, less than 0.100 mg / ml, less than 0.050 mg / ml, or less than 0.010 mg / ml.

[0102] The pharmaceutically acceptable excipient can be an adjuvant. The adjuvant can be other genes that are expressed in an alternative plasmid or are delivered as proteins in combination with the plasmid above in the vaccine. The adjuvant may be selected from the group consisting of: a-interferon(IFN- a), P-interferon (IFN-P), y-interferon. platelet derived growth factor (PDGF), TNFa, TNFp, GM-CSF, epidermal growth factor (EGF), cutaneous T cellattracting chemokine (CTACK), epithelial thymus-expressed chemokine (TECK), mucosae- associated epithelial chemokine (MEC), IL-12, IL-15, MHC, CD80, CD86 including IL-15 having the signal sequence deleted and optionally including the signal peptide from IgE. The adjuvant can be IL-12, IL-15, IL-28, CTACK, TECK, platelet derived growth factor (PDGF), TNFa, TNFp, GM-CSF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL- 12, IL- 18, or a combination thereof.

[0103] Other genes that can be useful as adjuvants include those encoding: MCP-1, MIP- la, MIP-lp, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18, CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7, IL-22, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Fit, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, Caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, Inactive NIK, SAP K, SAP-1, INK, interferon response genes, NFkB, Bax, TRAIL, TRAlLrec, TRAlLrecDRC5, TRA1L-R3, TRA1L-R4, RANK, RANK LIGAND, 0x40, 0x40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAPI, TAP2 and functional fragments thereof.

[0104] The vaccine may further comprise a genetic vaccine facilitator agent as described in U.S. Serial No. 021,579 filed April 1, 1994, which is fully incorporated by reference.

[0105] The vaccine can be formulated according to the mode of administration to be used. An injectable vaccine pharmaceutical composition can be sterile, pyrogen free and particulate free. An isotonic formulation or solution can be used. Additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol, and lactose. The vaccine can comprise a vasoconstriction agent. The isotonic solutions can include phosphate buffered saline. Vaccinecan further comprise stabilizers including gelatin and albumin. The stabilizers can allow the formulation to be stable at room or ambient temperature for extended periods of time, including LGS or polycations or polyanions.Methods of Vaccination

[0106] Also provided herein is a method of treating, protecting against, and / or preventing disease in a subject in need thereof by administering the vaccine to the subject. Administration of the vaccine to the subject can induce or elicit an immune response in the subject. The induced immune response can be used to treat, prevent, and / or protect against disease, for example, pathologies relating to HPV infection. In one embodiment, the pathology relating to HPV infection is cervical cancer or a carcinoma of the lung, tonsil, or larynx.

[0107] The induced immune response can include an induced humoral immune response and / or an induced cellular immune response. The humoral immune response can be induced by about 1.5-fold to about 16-fold, about 2-fold to about 12-fold, or about 3-fold to about 10-fold. The induced humoral immune response can include IgG antibodies and / or neutralizing antibodies that are reactive to the HPV E7 protein. The induced cellular immune response can include a CD8+ T cell response, which is induced by about 2-fold to about 30-fold, about 3-fold to about25-fold, or about 4-fold to about 20-fold.

[0108] The vaccine dose can be between 1 pg to 10 mg active component / kg body weight / time, and can be 20 pg to 10 mg component / kg body weight / time. The vaccine can be administered every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. The number of vaccine doses for effective treatment can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.Administration

[0109] The vaccine can be formulated in accordance with standard techniques well known to those skilled in the pharmaceutical art. Such compositions can be administered in dosages and by techniques well known to those skilled in the medical arts taking into consideration such factors as the age, sex, weight, and condition of the particular subject, and the route of administration. The subject can be a mammal, such as a human, a horse, a cow, a pig, a sheep, a cat, a dog, a rat, or a mouse.

[0110] The vaccine can be administered prophylactically or therapeutically. In prophylactic administration, the vaccines can be administered in an amount sufficient to induce an immune response. In therapeutic applications, the vaccines are administered to a subject in need thereof in an amount sufficient to elicit a therapeutic effect. An amount adequate to accomplish this is defined as “therapeutically effective dose.” Amounts effective for this use will depend on, e.g., the particular composition of the vaccine regimen administered, the manner of administration, the stage and severity of the disease, the general state of health of the patient, and the judgment of the prescribing physician.

[0111] The vaccine can be administered by methods well known in the art as described in Donnelly et al. (Ann. Rev. Immunol. 15:617-648 (1997)); Feigner et al. (U.S. Pat. No. 5,580,859, issued Dec. 3, 1996); Feigner (U.S. Pat. No. 5,703,055, issued Dec. 30, 1997); and Carson et al. (U.S. Pat. No. 5,679,647, issued Oct. 21, 1997), the contents of all of which are incorporated herein by reference in their entirety. The DNA of the vaccine can be complexed to particles or beads that can be administered to an individual, for example, using a vaccine gun. One skilled in the art would know that the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable compound, depends, for example, on the route of administration of the expression vector.

[0112] The vaccine can be delivered via a variety of routes. Typical delivery routes include parenteral administration, e.g., intradermal, intramuscular or subcutaneous delivery. Other routes include oral administration, intranasal, and intravaginal routes. For the DNA of the vaccine in particular, the vaccine can be delivered to the interstitial spaces of tissues of an individual (Feigner et al., U.S. Pat. Nos. 5,580,859 and 5,703,055, the contents of all of which are incorporated herein by reference in their entirety). The vaccine can also be administered to muscle, or can be administered via intradermal or subcutaneous injections, or transdermally, such as by iontophoresis. Epidermal administration of the vaccine can also be employed. Epidermal administration can involve mechanically or chemically irritating the outermost layer of epidermis to stimulate an immune response to the irritant (Carson et al., U.S. Pat. No. 5,679,647, the contents of which are incorporated herein by reference in its entirety).

[0113] The vaccine can also be formulated for administration via the nasal passages. Formulations suitable for nasal administration, wherein the carrier is a solid, can include a coarse powder having a particle size, for example, in the range of about 10 to about 500 microns whichis administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. The formulation can be a nasal spray, nasal drops, or by aerosol administration by nebulizer. The formulation can include aqueous or oily solutions of the vaccine.

[0114] The vaccine can be a liquid preparation such as a suspension, syrup or elixir. The vaccine can also be a preparation for parenteral, subcutaneous, intradermal, intramuscular or intravenous administration (e.g., injectable administration), such as a sterile suspension or emulsion.

[0115] The vaccine can be incorporated into liposomes, microspheres or other polymer matrices (Feigner et al., U.S. Pat. No. 5,703,055; Gregoriadis, Liposome Technology, Vols. Ito III (2nd ed. 1993), the contents of which are incorporated herein by reference in their entirety). Liposomes can consist of phospholipids or other lipids, and can be nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.Kit

[0116] Provided herein is a kit, which can be used for treating a subject using the method of vaccination described above. In one embodiment, the kit can comprise the vaccine. In one embodiment, the kit can comprise a nucleic acid molecule encoding a subunit of a selfassembling HPV nanoparticle of the invention.

[0117] The kit can also comprise instructions for carrying out the vaccination method described above and / or how to use the kit. Instructions included in the kit can be affixed to packaging material or can be included as a package insert. While instructions are typically written or printed materials, they are not limited to such. Any medium capable of storing instructions and communicating them to an end user is contemplated by this disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges), optical media (e.g., CD ROM), and the like. As used herein, the term “instructions” can include the address of an internet site which provides instructions.EXPERIMENTAL EXAMPLES

[0118] The present invention has multiple aspects, illustrated by the following nonlimiting examples.Example 1 : Structural Engineering of Stabilized, Expanded Epitope Nanoparticle Vaccines for HPV

[0119] The data presented here demonstrate the development of DNA encoded nanoparticle vaccines for HPV with improved potency. Further, the data demonstrate the development of a generalizable pipeline to create stabilized, more full-length nanoparticle vaccines, including safer versions with oncogenic properties removed for the HPV16 E7 and E6 antigens. The use of Al structural prediction algorithms as well as computational tools increase the power and novelty of this design platform. Designs created using Al-driven techniques successfully form in vitro and retain their structure. In vivo immunogenicity experiments in both inbred and a more translationally relevant outbred mouse model showed that designed nanoparticles were immunogenic and capable of eliciting strong CD8+ T-cell responses. In particular, designed nanoparticles elicited a strongly biased CD8+ T-cell response over monomer. The combination immunization group of both E6 and E7 nanoparticles, the E6 / E7 nano cocktail, was able to induce the most positive responders in outbred mouse models and likewise would be predicted to elicit immunogenicity for several common HLA alleles.

[0120] One question this study brings up is the link between structure, expression, and immunogenicity. E7_FL_DpRb_nano had lower expression levels, in concordance with its partially unfolded structural prediction. In general, both E7 and E6 full-length nanoparticles and their derivatives generally had poorer expression in vitro than their corresponding truncated antigen counterparts. However, despite this poorer in vitro expression, in vivo E7_FL_DpRb_nano elicited the strongest response for E7, and E6_FL_Dp53_monomer elicited the strongest ELISpot response for E6 in BL6 mice. One possible explanation for these findings could be that an unfolded antigen is actually advantageous for more efficient MHC-I presentation. Proteins that don’t achieve native folds will be sent for degradation; these degraded proteins are a huge source of peptides that are eventually loaded onto MHC-I for T-cellsurveillance (Reits et al., 2000, Nature, 404, 774-8; Schubert et al., 2000, Nature, 404, 770-4). If, for example, the E6 monomer and E7_FL_DpRb_nano express poorly and are often misfolded, they may be frequently targeted for degradation and end up being presented on MHC- I more often. Indeed, there has been some suggestion that a different HPV antigen, El, which is unstructured, could make an attractive new vaccine target for this very reason (Boilesen et al., 2021, Vaccines (Basel), 9).

[0121] However, at odds with this idea is that E7_2Tr_nano and the E6 / E7 cocktail elicited stronger immunity than the E7 monomer, despite expressing far better in vitro and presumably having much higher stability. Further, in CD-I mice, the E6 / E7 cocktail composed of two truncated nanoparticles, E7_2Tr_nano and E6_Tr_Sol_Dp53_nano, outperformed monomer and full-length nanoparticles for both E7 and E6. This brings up an interesting role that dendritic cells may play in eliciting CTL immunity in DNA-delivery platforms. Antigens that have been properly assembled and secreted, such as the presumably well-structured nanoparticles truncated nanoparticles, can be taken up by antigen-presenting cells such as dendritic cells for cross-presentation on MHC-I (Kutzler and Weiner, 2008, Nat Rev Genet, 9, 776-88; Gary and Weiner, 2020, Curr Opin Immunol, 65, 21-27; Pandya et al., 2023, Med Oncol, 40, 200). Therefore, the overall ability to prime immunogenicity may be driven by a balance of both degraded antigens and well-folded secreted antigens. Finding a good balance, if one exists, may inform future vaccine design.

[0122] One critical consideration for full-length nanoparticle vaccine design is also a balance of the actual ability to prime immunity balanced with the theoretical ability to have more epitopes recognized by a global suite of HLA molecules. While full-length nanoparticles contain more theoretical epitopes, as was observed in the more translationally relevant CD-I mouse model, they were inferior at eliciting immunity in comparison to their truncated, stabilized counterparts. Thus, vaccine decisions need to balance these possibilities or weigh allele frequencies in target populations. In cases where there are known human CTL epitopes, these could also be appended via flexible linkers to the stabilized truncated domain, possibly allowing a balance between good immunogenicity and higher theoretical coverage.

[0123] Because this platform is adaptable, an ideal vaccine may also cover targets beyond simply E7 and E6. A study investigating T-cell responses in 66 cervical cancer patients found that over half of them had El-specific T-cells (Ma et al., 2018, Infect Agent Cancer, 13,35). Another study demonstrated that some patients with higher E2-directed T-cell responses had less cervical dysplasia and responses were correlated with disease regression (Dillon et al., 2007, J Gen Virol, 88, 803-813). The implication of other HPV proteins E4 and E5 is less clear in cervical cancer, but in patients with oropharyngeal squamous cell carcinoma, T-cell responses were detected against E4 and E5, in addition to E1-E2 and E6-E7 (Bhatt et al., 2020, J Exp Med, 217). Though E6 and E7 have remained the most thoroughly investigated antigens, there is potential for the creation of additional nanoparticle vaccines with these other specificities to augment potency, using the design platform presented here.

[0124] The use of this platform is generally tailorable for desired antigens and desired specificities. All told, these findings offer both a guideline for general vaccine development and point to a strong starting ground for improvement on existing HPV vaccine efforts. The computationally driven E6 and E7 nanoparticle designs are safe and immunogenic and are easily translatable. The pathway presented here serves as a model for the design efforts on the path to a globally effective therapeutic HPV vaccine.The Materials and Methods are now describedSequences

[0125] All starting sequences used for both E6 and E7 were from the HPV16 subtype. The full-length WT sequence for E7 was obtained from UniProt (Accession number: P03129). Any AprB constructs contained mutations and deletions in regions that bind pRb as previously described (Yan et al., 2009, Vaccine, 27, 431-40).

[0126] The full-length WT sequence for E6 was obtained from UniProt (Accession number: P03126). Any constructs designated with ‘Sol’ were based on PDB:6SJA and contained mutations that interrupt the E6 homodimerization domain (F47R) and prevent disulfide-mediated aggregation (4C / 4S) as previously described (Nomine et al., 2006, Mol Cell, 21, 665-78; Zanier et al., 2012, Structure, 20, 604-17). Any Ap53 constructs contained mutations and deletions in regions that bind p53 as previously described (Yan et al., 2009, Vaccine, 27, 431-40).Structural Prediction

[0127] Full-length antigen structures were predicted using either Alphafold only (E6) or Alphafold and RoseTTAFold (E7). In all cases, five structures were predicted per input sequence. Alphafold structures were predicted using non-templated ColabFold, an online accessible version of AlphaFold2 (Mirdita et al., 2022, Nat Methods, 19, 679-682; Jumper et al., 2021, Nature, 596, 583-589). A local installation of RoseTTAFold (Baek et al., 2021, Science, 373, 871-876) was downloaded from (github.com / RosettaCommons / RoseTTAFold) and also used to run predictions. All structures were loaded into Pymol 2.0 for visualization. The topranked structure, or in some cases structures with unique folds, was then used for further domain engineering. Per-residue pLDDT scores (AlphaFold) or RMS-error scores (RoseTTAFold) were also extracted from models of interest. A crystal structure of E6_FL_Sol with only an additional N-terminal glycine was available (PDB:6SJA) and so no structural prediction was needed for domains based off this full-length sequence, unless they contained the additional Dp53 mutations.Domain Minimization

[0128] Determination of purported foldable domains was determined through a variety of methods. This included the use of pLDDT / RMS-error. For example, pLDDT scores generally predict stable domains and regions of disorder quite well (Necci et al., 2021, Nat Methods, 18, 472-481; Piovesan et al., 2022, Protein Sci, 31, e4466; Tunyasuvunakool et al., 2021, Nature, 596, 590-596), thus pLDDT scores can be used to determine regions of disorder. These per residue scores were used to down-select to structured regions of interest, which was then accompanied by visual inspection in Pymol of the new domains. Some partial structures of E7 for a different subtype (HPV45) were available in the PDB (2F8B, 2EWL) and were aligned to the AlphaFol d2 and RosettaFold2 predictions to refine domain minimization. Once sequences of minimized domains were selected, the structures were re-predicted using Alphafold2 to ensure that the truncations were predicted to maintain the desired geometries.After initial rounds of characterization of minimized designs, the most successful E6 minimizations were engineered to incorporate the Ap53 mutations and E7 minimizations were engineered to incorporate the ApRb mutations.Disulfide Engineering

[0129] For additional stability, the engineering of disulfide bonds to stabilize local protein folds was considered. Using an MSL library (Kulp et al., 2012, J Comput Chem, 33, 1645-61) a disulfide scanning tool called FindDisulfides was created. It takes an input PDB file of the protein to be designed, then scans for candidate pairs of residues to create a disulfide bridge. The backbone geometries of each pair of residues are compared against the PDB to determine how many other structures in the PDB contain disulfide pairs with similar geometries. New disulfide bridges under consideration were then filtered so that mutated residues had to be >20 residues apart and have >500 matching PDB geometries. The disulfides that met these criteria were used to model disulfides in the input starting structure. Purported disulfide bridges were manually inspected in PyMOL and combinations of disulfides were determined from there (1-2 disulfide pairs per structure). If the structure contained more than one pair of disulfides, efforts were made to have them located on distal sides in order to avoid the mispairing of cysteines.Nanoparticle Design

[0130] Any construct with the designation ‘nano’ was scaffolded onto a stabilized, engineered lumazine synthase scaffold (previously described as DLnano_LS_GT8) (Xu et al., 2020, Cancer Immunol Res, 8, 1354-1364). All constructs were cloned into the pVax vector and contained the IgE leader sequence. Constructs were codon optimized for homo sapiens and / or mus musculus,' all E6 and E7 nanoparticle constructs and their derivatives were codon optimized identically to the monomer.Nanoparticle Production and Characterization

[0131] Each nanoparticle was transfected into ExpiF293 cells following the manufacturer’s guidelines ((ExpiFectamine™ 293 Transfection Kit(Gibco)) with a transfection size of lOOmL. Supernatants were harvested 7 days post-transfection and purified using an inhouse column packed with Galnthus Nivalis Lectin Beads (Vector Lab) on an AKTA Pure system. Following lectin purification, fractions were pooled, concentrated, and dialyzed into IX PBS. Size exclusion chromatography (SEC) was then run using a Superose 6 10 / 300 GL Increase column (Cytiva), again using an AKTA Pure system. In some cases, two rounds of size exclusion chromatography were run. The relevant fractions were then pooled and concentrated.Concentrations of the nanoparticles were then determined using a NanoDrop™ One spectrophotometer (Thermo Scientific) to calculate the final transfection yields of the purified nanoparticles.ELISAs

[0132] All ELISAs were performed using polystyrene high binding, 96-well Flat-Bottom, Half-Area Microplates (Corning). Plates were coated at 15pg / mL with the relevant E6 or E7 nanoparticle overnight at 4°C, washed with IX PBS / 0.05% Tween-20, then blocked for Ihr at RT with 5% milk / lX PBS / 0.01% Tween-20. Following the wash, dilutions of the relevant antibody were performed. For E6 ELISAs, anti-E6 monoclonal antibody (MAB874, Millipore Sigma) was prepared in duplicate (either 50pg / mL or 70pg / mL starting concentration, 3X dilution series). For E7 ELISAs, anti-E7 polyclonal antibody (PA5-117383, Fisher Scientific) was prepared in duplicate (70pg / mL starting concentration, 3X dilution series). Plates were then incubated for 1 hour at RT, washed, then detected for 45min at RT with 1 : 10,000 of the relevant secondary antibody. For E6 ELISAs, anti-mouse H+L-HRP (Bethyl, A90-116P) was used and for E7 ELISAs, anti-rabbit H+L-HRP was used (Bethyl, A120-201P). Following the wash, plates were incubated with 1-StepTM Ultra TMB-ELISA Substrate Solution (Thermo Scientific) for 1 min before being quenched with 1 M H2SO4. The absorbance of plates was then read at 450nm and 570nm using a Biotek Synergy 2 plate reader. Absorbance was 450nm-570nm normalized and the background of blank wells was subtracted. All data was exported to Microsoft Excel and analyzed in GraphPad Prism 10.Negative Stain Electron Microscopy

[0133] Purified nanoparticles in PBS (3uL) at 0.03-0.05 mg / mL were adsorbed onto glow discharged carbon-coated Cu400 EM grids. Grids were rinsed several times with TBS. The grids were then stained with 3 pL of 2% uranyl formate, blotted, and stained again with 3 pL of the stain for 90 seconds followed by a final blot. A FEI Tecnai T12 microscope equipped with Oneview Gatan camera at 52000x magnification was used for data collection. Data is at a 2.356 A / pixel ratio.Immunizations

[0134] Six to eight week old female C57BL / 6J (The Jackson Laboratory) or CD-I IGS mice were obtained. To obtain cellular responses, mice were immunized with lOpg of the relevant E6 or E7 DNA vaccine, or 20 pg total in the case of combination groups, in their tibialus anterior muscle. To promote plasmid uptake use of a CELLECTRA EP (Inovio Pharmaceuticals) delivery device was employed. Two sets of 0.2 A pulses with a 3-second interval were delivered; each pulse consisted of 52 ms pulses with 198 ms delay. Mice received an identical vaccination two weeks post-initial immunization. At week 3, terminal bleeds were collected and mice were euthanized under CO2. Spleens were collected into RPMI media supplemented with 10% HI FBS and 1% P / S, then processed using a Seward Stomacher 80 (Seward) followed by filtration through 40 pm cell strainers. Red blood cells were lysed using ACK lysis buffer (Thermo Fisher Scientific).ELISpot

[0135] Cellular responses were quantified using ELISpot assays. Briefly, 200,000 splenocytes were plated onto mouse IFNy ELI-SpotPLUS plates (MabTech) and stimulated with 5 pg / mL peptides. Overlapping peptide pools were constructed so that the length of E6 or E7 was spanned by overlapping peptides (15AA long, 8AA overlaps). This method of epitope determination has been previously described (Fiore-Gartland et al., 2016, PLoS One, 11, e0147812). Peptides spanning E6 were pooled into 10 different pools, while those spanning E7 were pooled into 8 different pools. Splenocytes from the relevant mice were incubated with each of the relevant peptide pools for 20 hours at 37°C, then developed in accordance with the manufacturer’s instructions. Concanavalin A or R10 were used as positive or negative controls, respectively. Spots were quantified using MabTech IRIS Fluorospot / ELIspot reader, normalized to an unstimulated control (R10).For additional normalization in CD-I mice, ‘responders’ were defined from ELISpot data. The average of positive spots was determined for naive mice, and this value was subtracted from all ELISpot data. If a mouse still had >300 positive spots post-normalization, it was counted as a positive responder to that pool. Some mice had such high responses the plate reader could not quantify the spots. While these mice were excluded in Figure 12 because no numeric value can be assigned as ‘above the limit of detection for responses’, they were included in the responders analysis as a positive responder.Intracellular Staining and Flow Cytometry

[0136] Splenocytes (IM cells / well) were isolated as described in the Immunizations section, then stimulated with peptide pools for full-length E6 or E7 in the presence of protein transport inhibitor for 5 hours at 37°C. Cells were then stained with anti-mouse CD3-PE-Cy5, CD4-BV510, CD8-APC-Cy7, IFNy-APC, IL-2-PE-Cy7 and TNFa-BV605. All antibodies were purchased from Biolegend. To assess cellular viability, cells were also stained with Live / Dead violet (Invitrogen). Samples were then run on an 18-color LSRII flow cytometer (BD Biosciences), gated relative to naive mice, and analyzed by FlowJo software.HLA Allele Binding Specificities and Frequencies

[0137] Peptides predicted to bind human HLA alleles were determined using NetMHCpan (Reynisson et al., 2020, Nucleic Acids Res, 48, W449-W454). Due to the extreme diversity of the human HLA repertoire, predictions were generated using the ‘HLA supertype representative’ set of loci, which are HLA proteins clustered according to similar binding specificities (Lund et al., 2004, Immunogenetics, 55, 797-810; Nielsen et al., 2007, PLoS One, 2, e796; Sette and Sidney, 1999, Immunogenetics, 50, 201-12). These consist of HLA-A*02:01, HLA-A*01:01, HLA-A*03:01, HLA-A*24:02, HLA-A*26:01, HLA-B*07:02, HLA-B*08:01, HLA-B*27:05, HLA-B*39:01, HLA-B*40:01, HLA-B*58:01, and HLA-B*15:01. Binding peptides (9-mers) were determined from E7_FL_ DpRb or E6_FL_Dp53 sequences for E7 and E6, respectively. The threshold for strong binders was specified as 0.5% rank.

[0138] Population frequencies of the HLA supertype alleles were determined using the HLA allele report from the Allele Frequency Net Database (Gonzalez-Galarza et al., 2020, Nucleic Acids Res, 48, D783-D788). Percentages were determined as 100*allele count / number of people typed for a given allele and is based on their gold datasets.The Experimental Results are now DescribedStabilized Nanoparticle Design Pipeline

[0139] To create next generation vaccines that are not limited by MHC restriction, it is imperative to design antigens that display maximal epitopes. An ideal vaccine candidate wouldscaffold these more full-length antigens on a DNA-launched, 60-mer nanoparticle scaffold, as this has been shown to drive superior immune responses (Tursi et al., 2023, Front Immunol, 14, 1072810; Xu et al., 2020, Cancer Immunol Res, 8, 1354-1364; Xu et al., 2020, Adv Sci (Weinh), 7, 1902802). With this in mind, it was sought to develop a cancer antigen design pipeline, utilizing the power of new Al tools (Figure 1A). The developed workflow consists of 5 steps: determination of gene sequence, structural prediction, design of a ‘foldable’ domain, additional design stabilization, and formulation as a nanoparticle.

[0140] Once the gene sequence for the desired antigen is determined, a predicted structure for the full length antigen is generated through prediction tools such as RoseTTafold2, AlphaFold2 or the newer AlphaFold3 (Baek et al., 2021, Science, 373, 871-876; Jumper et al., 2021, Nature, 596, 583-589; Abramson et al., 2024, Nature, 630, 493-500). In some cases, the full-length structure may not have well-folded domains which could interfere with display. Confidence metrics from structural prediction algorithms, such as Alphafold’s pLDDT score have been shown to correlate with well-folded regions (Piovesan et al., 2022, Protein Sci, 31, e4466; Tunyasuvunakool et al., 2021, Nature, 596, 590-596). Loading the predicted structures into structure visualization tools like Pymol, coupled with confidence metric scores, allows for the selection of residues in a truncated, purported ‘foldable’ domain in the event that the full- length antigen cannot be displayed. Additional stabilization can be engineered into these constructs as desired. One way to do so is through the use of disulfide engineering to mutate pairs of residues to cysteines to create a covalent disulfide bridge and stabilize local folds. ProteinMPNN (Dauparas et al., 2022, Science, 378, 49-56) could also be used at this stage to change residues with the hope of achieving display, though there is the tradeoff that less nativelike sequences will be inferior at achieving robust anti-cancer T-cell responses. Finally, candidate designs are scaffolded onto a stabilized, lumazine-synthase domain that creates selfassembling 60-mer nanoparticles in vivo, as previously described (Xu et al., 2020, Cancer Immunol Res, 8, 1354-1364). For simplicity, this will be referred to as ‘nano’ throughout.

[0141] Though these steps can be applied to any desired cancer antigen, HPV16 E7 was used as a model antigen for a proof-of-concept study, as it is a well-described, important target for HPV vaccine efforts (Pal and Kundu, 2019, Front Microbiol, 10, 3116). Example outputs from structural prediction algorithms and examples of per residue confidence metrics are shown for full-length E7 (E7_FL) (Figure IB, 1C, Figure 2A-D). Comparison of the structure with per-residue pLDDT scores showed high confidence for the C-terminal domain folds, which was used as the basis for creating a truncated, foldable domain. A cutoff of pLDDT > 80 was used as this falls into the confident-very confident prediction score range to down-select residues (Guo et al., 2022, Scientific Reports, 12; Jumper et al., 2021, Nature, 596, 583-589). Though residues 43-46 have pLDDT scores lower than this cutoff, they were preserved in the truncated structure to create a more native-like linker when scaffolded onto the nanoparticle core. The resultant structure that forms the basis of the E7, truncated foldable domain (E7_Tr) is shown in Figure ID. An NMR partial structure of E7 for a different strain (HPV45) showed E7 in a dimeric form (PDB: 2F8B). Single subunits of the resolved dimer have a highly similar structure to the designed E7_Tr (Figure 2E). It was reasoned that E7_Tr was likely to be stable as a dimer but with the potential to be more immunogenic, so a dimeric version (E7_2Tr) was formulated with a flexible GS linker between individual subunits (Figure IE). In order to further stabilize the dimeric form, a disulfide scanner was used to select pairs of residues amenable to mutation to cysteine in order to staple the dimeric interface together (Figure IF). This method was also used to create pairs of disulfides that might stabilize local folds in the full-length structure (E7 FL) to promote expression on a nanoparticle, as well as to E7_Tr in the event domain minimization was not sufficient to achieve display. Three disulfide modified full-length versions (E7_FL_Dsl, E7_FL_Ds2, E7_FL_Ds3), and one each for the truncated and truncated dimer versions (E7_Tr_Dsl, E7_2Tr_Dsl) were generated. Sequences for designs can be found in Figure 3. All E7 designs were then formulated onto the self-assembling 60-mer scaffold to create nanoparticles.In vitro characterization of E7 nanoparticle designs

[0142] The success of E7 nanoparticle designs was first assessed in vitro. All nanoparticle designs were easily able to form in vitro in contrast to E7_monomer (Ogg yield), showcasing the power of the nanoparticle platform to aid in design expression and stabilization (Figure 4A). The full length nanoparticle designs had the lowest average purified yields, ranging from 311.5-490.1 pg from a lOOmL transfection. This was expected as all E7 FL nanos contained a large unstructured portion according to the structural prediction algorithms, which would likely reduce their ability to stably form. In this case, introduction of disulfide bonds through E7_FL_Dsl, Ds2 or Ds3 nanos did not have a large effect on the yield. In contrast,designs based on E7_Tr or E7_2Tr both had higher average yields than designs based on E7_FL, and disulfides further increased the yield, showcasing the importance of introducing structure- guided truncations. The average yield of E7 Tr nano was 1012.3pg and 1128.3pg for E7_Tr_Dsl_nano; E7_2Tr_nano had an average yield of 586.5pg and E7_2Tr_Dsl_nano had an average yield of 862pg.

[0143] To assess structural integrity of the E7 antigens displayed on nanoparticles, an ELISA was performed to assess binding to a polyclonal anti-E7 antibody. Most designs bound well, showing proper antigen formation (Figure 4B). Though both E7_Tr_Dsl_nano and E7_2Tr_Dsl_nano formed well in vitro, they maintained only partial binding suggesting that some of the disulfides introduced might cause partial antigen misfolding.

[0144] In vitro yield as well as antigen structural integrity were weighed to decide which nanoparticles to use in a first-pass immunogenicity experiment. First, one nanoparticle from each base construct category was chosen; SEC traces confirmed their formation as nanoparticles and models of each 60-mer are shown in the insets (Figure 4C-E). E7_FL_Dsl_nano was chosen in the full-length category as it had similar yields to E7_FL_nano but slightly better ELISA binding. E7_Tr_nano and E7_2Tr_nano were both selected over their disulfide-modified counterparts due to the better structural integrity of these antigens. Ultimately E7_2Tr_nano was chosen over E7_Tr_nano as they both contain the same truncated domains but E7_2Tr_nano has twice as many of these domains, meaning that it might generate an even more potent immune response.

[0145] To assess whether E7_FL_Dsl_nano and E7_2Tr_nano could elicit immunity, mice were immunized and both nanoparticle groups mounted a significant T-cell response as determined by ELISpot (Figure 5A-B).Formulation of safer E7 nanoparticles

[0146] Encouraged by this immunogenicity experiment, it was decided to iterate on the initial designs to create safer nanoparticles. E7 has oncogenic properties, conferred by its ability to bind pRb and initiate pro-tumorigenic downstream effects (Pal and Kundu, 2019, Front Microbiol, 10, 3116). A safer vaccine candidate would be pRb binding null (and indeed, this mirrors the sequence found in the VGX-3100 clinical trial) (Yan et al., 2009, Vaccine, 27, 431- 40). The location of the truncation found in E7_2Tr_nano is such that this design naturally lackspRb binding, and so no changes had to be made for this design. However, pRb binding knockout mutations (hereafter DpRb) were introduced into E7_FL_nano over E7_FL_Dsl to maximize native-like epitopes. This became E7 FL DpRb nano (Figure 3).

[0147] Alphafold2 predictions showed no major changes to structure introduced by the ApRb mutations confirming this was a viable new design (Figure 6A). In vitro formation of the design as well as the assembly of E7_FL_DpRb_nano into a 60-mer was confirmed via size exclusion chromatography (Figure 4F, Figure 6B). A model of the resultant nanoparticle is shown in the inset. The structural integrity of E7 FL DpRb nano was also assessed by ELISA binding to the polyclonal anti-E7 antibody; antigen integrity was preserved (Figure 4G). This provided two final E7 nanoparticle candidates of interest for more extensive in vivo immunogenicity experiments: E7_FL_DpRb_nano and E7_2Tr_nano; both of which lack binding to pRb for safety considerations. Their formation as nanoparticles was further confirmed by negative stain electron microscopy (nsEM) (Figure 7A-B).In vitro characterization of E6 nanoparticle designs

[0148] Both E6 and E7 have been the targets of many vaccination efforts since they both are oncogenic and are constitutively expressed (Pal and Kundu, 2019, Front Microbiol, 10, 3116; Trimble et al., 2015, Lancet, 386, 2078-2088; Choi et al., 2020, Clin Cancer Res, 26, 1616-1623; Yang et al., 2016, Expert Rev Vaccines, 15, 989-1007). Furthermore, there have been suggestions that immune responses synergize to create a more potent response when both E6 and E7 antigens are present (Yan et al., 2009, Vaccine, 27, 431-40).

[0149] As the nanoparticle pipeline is generalizable, E6-targeting nanoparticles were also engineered. In the case of HPV16 E6, a resolved crystal structure (PDB: 6SJA) is available, though it contains mutations relative to WT that enhance solubility and reduce aggregation (hereafter referred to as ‘Sol’ mutations). Structural prediction algorithms were used to confirm that the WT sequence of full-length HPV16 E6 (E6 FL) was predicted to fold in a similar manner to the crystal structure (Figure 8A); they are essentially identical and fold into an N- terminal and C-terminal domain separated by an alpha helix.

[0150] Ordinarily, the next step in the nanoparticle design pipeline would be to select a truncated domain solely composed of regions predicted to be well folded. However, E6 has very high per residue pLDDT scores across the full protein (Figure 8B), reflecting its well-foldednature. A truncated domain was generated based on residues 7-87 in the event that the entire full- length protein was too large to display properly as a nanoparticle. This design became E6_Tr. It was reasoned that the Sol mutations might be necessary for stability and formation as they were integral to crystal structure resolution, so versions of E6 FL and E6_Tr with Sol muts (E6 FL S0I and E6_Tr_Sol) were engineered. Both full-length and truncated E6 designs with additional disulfides engineered in were also created, resulting in 3 disulfide engineered full- length variants (E6_FL_Dsl, E6_FL_Ds2, E6_FL_Sol_Dsl) and 2 disulfide engineered truncated variants (E6_Tr_Dsl, E6_Tr_Ds2). Sequences for all designs can be found in Figure 9. Finally, all designs were formulated onto the self-assembling 60-mer scaffold to create nanoparticles.

[0151] This suite of designs was assessed for in vitro expression. All formed well in vitro in contrast to E6_monomer (Opg yield), once again demonstrating the power of the nanoparticle platform to aid in design expression and stabilization (Figure 10A). Interestingly, the Sol muts and disulfide modifications made little difference in overall yield; yield instead changed according to full-length constructs (lower yield) or truncated constructs (higher yield). Designs based on the full-length E6 sequence had average purified yields of 443.2-805.5 pg while designs based on the truncated domain had average purified yields of 1429.6pg-2656.6pg. ELISA binding to an HPV16 anti-E6 antibody was used as a proxy to assess the structural integrity of each nanoparticle antigen (Figure 10B). In this case, Sol mutations were key to maintaining structural integrity. Only E6_FL_Sol_nano and E6_Tr_Sol_nano displayed meaningful binding to the anti-E6 antibody. SEC traces of these two constructs further demonstrate their formation as nanoparticles and models of each 60-mer are shown in the insets (Figure 10C-D).

[0152] To assess whether E6_FL_Sol_nano and E6_Tr_Sol_nano were not just structurally intact, but also capable of mounting an immune response, mice were immunized and responses were assessed. Good immune responses were generated by both nanoparticles, with higher responses in mice immunized with E6_Tr_Sol_nano (Figure 5A, 4C).Formulation of safer E6 nanoparticles

[0153] Akin to E7, the oncogenic properties of E6 are driven by its ability to bind another protein, in this case, tumor suppressor p53. In order to create safer versions of E6, leadnanoparticles were formulated without safety risks but by introducing mutations and deletions in the p53 binding region. This also corresponds with mutations to E6 found in clinical trials (Yan et al., 2009, Vaccine, 27, 431-40). The set of changes to knock out p53 binding will be referred to as Dp53. Two new nanoparticles were engineered without oncogenic properties, E6_FL_Sol_Dp53_nano and E6_Tr_Sol_Dp53_nano (Figure 9). Alphafold2 models of the new designs showed that designs incorporating Dp53 mutations are predicted to maintain similar folds to the crystal structure (Figure 6C-D).

[0154] In vitro formation of the designs as well as the proper assembly of both nanoparticles were confirmed (Figure 10E-F, Figure 6E). Models of the new nanoparticles demonstrate similarities between E6_FL_Sol_nano and E6_FL_Sol_Dp53_nano, as well as between E6_Tr_Sol_nano and E6_Tr_Sol_Dp53_nano, as predicted. Finally, the structural integrity of E6_FL_Sol_Dp53 and E6_Tr_Sol_Dp53_nano was assessed by ELISA binding to an anti-E6 monoclonal antibody. Binding was preserved with identical binding patterns to the E6 designs with intact p53 binding; the truncated antigen displayed superior binding to the full length(Figure 10G). These results provided E6_FL_Sol_Dp53_nano and E6_Tr_Sol_Dp53_nano as designs to move forward for more extensive in vivo immunogenicity experiments, and their formation as nanoparticles was further confirmed by nsEM (Figure 7C-D).T-cell responses to designed E7 nanoparticles

[0155] Mice were immunized with lOpg of DNA in a prime / boost immunization scheme (Figure 11 A). Spleens were harvested one week post boost and used to determine the immunogenicity of the DNA-launched nanoparticle designs via interferon-y ELISpot and intracellular cytokine staining by flow cytometry. E7_FL_DpRb_nano and E7_2Tr_nano were compared to a monomeric construct, E7_FL_DpRb_monomer, in order to ascertain if nanoparticle responses were superior to monomer. Further, because of the suggestion that E6 and E7 responses can synergize, a group consisting of E6_Tr_Sol_Dp53_nano and E7_2Tr_nano was also assessed (hereafter ‘E6 / E7 nano cocktail’).

[0156] Immunogenicity was first analyzed for inbred C57 / BL6 mice (Figure 1 IB). All monomeric and nanoparticle groups displayed E7-specific T-cell responses. As expected, all nanoparticle groups were also able to elicit stronger E7-directed immunogenicity than the monomer. The monomer had an average of 6,666 spots for the highest-responding E7 peptidepool 2, compared to average spots of 9,752, 10,030, or 12,957 for E7_2Tr_nano, E6 / E7 nano cocktail, and E7_FL_DpRb_nano respectively. Across all groups, the strongest responses were directed at pools 2, 7 and 8, reflecting the genetically identical nature of inbred mice. These pools contain the known MHC Db-restricted immunodominant peptide, RAHYNIVTF (SEQ ID NO:95), for BL6 mice (Feltkamp et al., 1993, Eur J Immunol, 23, 2242-9). Interestingly, though E7_FL_DpRb_nano had the lowest in vitro expression, the highest magnitude of E7-directed immunogenicity was observed. It may be that the partially unfolded nature of the full-length protein is in fact advantageous in the case of intracellular processing onto MHCI though this may depend on the exact mechanisms contributing to DNA-primed cellular immunity.

[0157] Importantly, experiments were designed to assess immunogenicity in a genetically heterogeneous population as this would be more reflective of the variable nature of responses in a human population. Outbred CD-I mice were immunized according to the same immunization scheme (Figure 11 A). As expected, responses in outbred mice are much more variable since each mouse has different MHC alleles to bind different epitopes to prime immunity (Figure 12A). In order to better compare overall responses and determine if the designed nanoparticles were eliciting responses to multiple different E7 epitopes, each CD1 mouse was characterized as a responder or non-responder on a per-pool basis. Responders were normalized to naive background and were required to have >300 spots after normalization to be considered a positive response. Both the monomer and nanoparticle groups were able to achieve responses to a variety of epitopes, as shown by the diversity of pools with positive responders (Figure 11C). The group that elicited the highest number of responders on a per-pool basis was determined (Figure 1 ID); each pool is colored according to the group with the most responders. The E6 / E7 nano cocktail group had the highest responders in pools 1,2, and 6 and was tied for pool 8. This group was able to achieve the highest number of responders across diverse pools, suggesting synergy of immune responses in the cocktail group. The E7_2Tr_nano group was a close second with the highest responses in pools 3 and 7 and tied for pools 5 and 8. Together, these demonstrate the ability of the designed stabilized nanoparticles to elicit immune responses in a genetically diverse background.

[0158] To more directly assess whether the nanoparticle groups were priming a CTL response, the degree of IFNy+ or TNFa+ CD8+ T-cell responses (Figure 1 IE) was determined. In inbred BL6 mice, a strong CD8+ T-cell response was observed, especially in theE7_FL_DpRb_nano group where an average of 5.16% of the CD8+ cells were IFNy+ or TNFa+. This corresponds with the ELISpot response data. This strong response was significantly higher than the monomer group, and not statistically different than either E7 2Tr nano or the E6 / E7 nano cocktail groups, showcasing the CTL priming abilities of the designed nanoparticles. In outbred CD-I mice, the E6 / E7 nano cocktail has the highest levels of CD8+ cells (Figure 1 IF) with an average of 1.93% IFNy+ or TNFa+ CD8+ T-cells, reflecting the high responder numbers observed in the ELISpot data.

[0159] Experiments were designed to determine to what degree the observed nanoparticles bias a CTL over T-cell help response, i.e. to what degree responses were CD8+ biased over CD4+ biased. Strongly enhanced CD8+:CD4+ responses were observed in both inbred and outbred mice for the nanoparticle groups (Figure 12C) and enhanced abilities to prime CD8+ cells over monomer (Figure 11G). Average CD8+ bias ranges from 1.8-3.3X over monomer for inbred mice, and 2.3-3.4X over monomer in outbred mice. These showcase the strong CTL priming ability of the designed E7 nanoparticle constructs, key to potent vaccine responses.T-cell responses to designed E6 nanoparticles

[0160] Next, the immunogenicity of the designed E6 nanoparticles was determined as compared to monomer following the immunization scheme in Figure 11 A. Groups assessed were E6_FL_Sol_Ap53_nano, E6_Tr_Sol_Ap53_nano and the E6 / E7 cocktail group to determine if there would be E6-directed synergy. These were compared to E6_FL_Ap53_monomer. Unexpectedly, E6_FL_Dp53 monomer had the highest responses in inbred mice via ELISpot, though all nanoparticle groups also were able to elicit E6-directed responses (Figure 13A). The monomer does not contain Sol mutations which perhaps contributes to the differences observed. Overall, the strongest responses across all groups are to pool 2 and pool 9 in the genetically identical BL6 mice background. These pools overlap with the MHC Db-restricted E6 peptide YRDGNPYAV (SEQ ID NO: 105) (Feltkamp et al., 1993, Eur J Immunol, 23, 2242-9).

[0161] To better compare the ability of the nanoparticles to elicit diverse immune responses, the E6 groups were also assessed in an outbred CD-I mouse mode. Responses were observed to a much greater variety of pools in the outbred mice for both the monomer and nanoparticle groups (Figure 12B). For more effective comparisons, the number of positiveresponders in each group was characterized against each E6 pool; this demonstrates the diversity of responses (Figure 13B).

[0162] Akin to E7, the E6 group that elicited the highest number of responders on a perpool basis was determined (Figure 13C). The combination E6 / E7 nano cocktail group clearly elicits the highest number of responders across pools, with the most responders in pools 1, 4-7, 9 and a tie for pool 2. This suggests that the presence of E7-directed nanoparticles in the cocktail may help cross-prime a stronger E6-directed responses as well.

[0163] Though the strongest ELISpot responses for inbred BL6 mice were detected in the monomer group, it is interesting that the monomer does not have the highest average IFNy+ or TNFa+. CD8+ cells for BL6 mice (Figure 13D). In fact, though the cocktail group had the weakest ELISpot responses this group has the highest lFNy+ or TNFa+ CD8+ responses for BL6 mice. For outbred CD1 mice, the cocktail group similarly had the highest IFNY+ or TNFa+ CD8+ responses (Figure 13E); in this case the trend matches with the highest number of responders detected for the cocktail group. One possible reason for this phenomenon could be that the monomer elicits a more CD4+ biased response. The monomer has the lowest CD8+:CD4+ T-cell ratio for both BL6 and CD1 mice out of all groups (Figure 12D) suggesting poor elicitation of CTLs; strong ELISpot responses may be due solely to T-cell help CD4+ responses.

[0164] All of the nanoparticle groups elicit stronger CD8+ biased responses than the monomer, ranging from 1.6-2.2X or 1.5-2. IX more CD8+ bias than monomer for BL6 and CD1 mice respectively (Figure 13F). All told, potent CTL-like responses were seen across all designed nanoparticle groups, and in a genetically diverse model more reflective of population MHC diversity, the highest degree of responses was observe in the E6 / E7 cocktail group.Expanded epitope targeting of nanoparticle vaccines

[0165] It was desired to determine how the designed nanoparticles might translate to a human system, as the ultimate goal is making a translationally useful vaccine. It was demonstrated that the nanoparticles can target expanded epitopes in a mouse system, but mouse and human MHC alleles present peptides differently. To determine how the designed nanoparticles might function, NetMHCPan(Nielsen et al., 2007) was used to predict what E6 and E7 peptides would bind different HLA alleles in humans (Figure 15A, Figure 14). RepresentativeHLA supertype alleles were used to determine how some of the more common HLA alleles would bind peptides and compared these predicted epitopes against the sequences of the designed nanoparticles. Because a vaccine targeted not just for the United States, but for a global population was desired, the frequency at which a given allele is present in the worldwide population was also graphed.

[0166] For E7, both E7_FL_ApRb_nano and E7_2Tr_nano cover more alleles and a greater portion of the global population than a peptide only approach. For E6, E6_FL_Sol_Ap53_nano and E6_Tr_Sol_Ap53_nano also cover more human alleles and a greater portion of the global population than a peptide only approach. In addition, the E6 / E7 cocktail can elicit immunity in a greater number of alleles than either E7_2Tr_nano or E6_Tr_Sol_Ap53_nano alone. While both full length nanoparticles (E7_FL_ApRb_nano and E6_FL_Sol_Ap53_nano) have the largest theoretical population coverage, it is important to note that maximum theoretical coverage can only be achieved if the vaccine is in fact immunogenic. In the in vivo immunogenicity experiments, in the CD-I mouse model with more similarities to human population diversity, both full length constructs did not perform as strongly as the nanoparticle cocktail and would therefore be unlikely to elicit maximum epitope coverage. Thus, the ideal vaccine candidate will weigh determined immunogenicity with theoretical population coverage; in this case the E6 / E7 nano cocktail achieves the best balance of both.Earlier approaches use a single peptide or epitope; even scaffolded as a nanoparticle the epitope will only be able to prime responses to a single 9-mer, resulting in low global protection. In contrast, when stabilized, more full length antigen nanoparticles are used, as designed here, CTLs reacting to a more diverse array of peptides can be elicited, providing greater population coverage and a more effective vaccine.

[0167] Altogether, these expanded epitope, full length antigen DNA-launched nanoparticles represent the next step in eliciting a more potent immune response (Figure 15C). In sequential order, it has been shown that monomeric DNA-launched antigens demonstrate superior response over peptides. Peptide-formulated nanoparticles demonstrate superior response monomeric DNA-launched antigens; stabilized antigen nanoparticles increase potential immunity over peptide-formulated nanoparticles, and finally here were present superior immune responses of DNA-launched stabilized antigen nanoparticle cocktail. The findings presented here can aid in reducing MHC-class restriction of current vaccine formulations while simultaneouslyincreasing potency and safety. The developed pipeline may also be easily applied to other desired antigens, aiding in development of new and potent vaccine candidates.Example 2: SequencesHPV16 E7 nanoparticle designsAll contain Kozak leader sequence and are in the pVaxl vector.IgE leader sequence(SEQ ID NO: 1)MDWTWILFLVAAATRVHS(SEQ ID NO:2)ATGGACTGGACCTGGATTCTGTTCCTGGTGGCCGCCGCCACAAGGGTGCACAGCNanoparticle core(SEQ ID N0:3)MQIYEGKLTAEGLRFGIVASRANHALVDRL VEGAID AIVRHGGREEDITLVRVCGSWEIP VAAGELARKEDIDAVIAIGVLCRGATPSFDYIASEVSKGLADLSLELRKPITFGVITADTL EQAIEAAGTCHGNKGWEAALCAIEMANLFKSLRGGSGGSGGSGGSGGGDTITLPCRPAP PPNCTSNITGLILTRQGGYSNDNTVIFRPSGGNWSDIARCNITGTVVSTQLFLNGSLAGNG TVIRSEDWRDNAKSICVQLNTSVEINCTGNGTCNISRAKWDNTLKQIASKLREQYGNKTI IFKPSSGGDPEFVNHSFNCGNVTFYCDSTQLFDSTWFDSTGGSGSGGSG(SEQ ID NO:4)ATGCAGATCTACGAAGGAAAACTGACCGCTGAGGGACTGAGGTTCGGAATTGTCGC AAGCCGCGCGAATCACGCACTGGTGGATAGGCTGGTGGAAGGCGCTATCGACGCAA TTGTCCGGCACGGCGGGAGAGAGGAAGACATCACACTGGTGAGAGTCTGCGGCAGC TGGGAGATTCCCGTGGCAGCTGGAGAACTGGCTCGAAAGGAGGACATCGATGCCGT GATCGCTATTGGGGTCCTGTGCCGAGGAGCAACTCCCAGCTTCGACTACATCGCCTC AGAAGTGAGCAAGGGGCTGGCTGATCTGTCCCTGGAGCTGAGGAAACCTATCACTT TTGGCGTGATTACTGCCGACACCCTGGAACAGGCAATCGAGGCGGCCGGCACCTGC CATGGAAACAAAGGCTGGGAAGCAGCCCTGTGCGCTATTGAGATGGCAAATCTGTTCAAATCTCTGCGAGGAGGCTCCGGAGGATCTGGAGGGAGTGGAGGCTCAGGAGGAG GCGACACCATCACACTGCCATGCCGCCCTGCACCACCTCCAAACTGTACCTCCAACA TCACCGGCCTGATTCTGACAAGACAGGGGGGATATAGTAACGATAATACCGTGATTT TCAGGCCCTCAGGAGGGAATTGGAGTGACATCGCACGATGCAATATTACTGGAACA GTGGTCTCTACTCAGCTGTTTCTGAACGGCAGTCTGGCTGGAAACGGGACAGTCATC CGATCTGAAGACTGGCGGGATAATGCAAAGTCAATTTGTGTGCAGCTGAACACAAG CGTCGAGATCAATTGCACTGGCAATGGGACCTGTAACATTTCTCGGGCCAAATGGGATAATACCCTGAAGCAGATCGCCAGTAAACTGAGAGAGCAGTACGGCAATAAGACAATCATCTTCAAGCCTTCTAGTGGAGGCGACCCAGAGTTCGTGAACCATAGCTTTAATTGCGGGAACGTCACCTTTTATTGTGATTCCACACAGCTGTTCGATAGCACTTGGTTTGATTCCACCGGTGGGAGCGGAAGTGGCGGTTCCGGA(SEQ ID N0:5)MHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCD STLRLC VQ STHVDIRTLEDLLMGTLGI VCPIC SQKP * *(SEQ ID N0:6)ATGCACGGCGACACCCCTACACTGCACGAGTACATGCTGGACCTGCAGCCCGAGACCACAGATCTGTACTGCTATGAGCAGCTGAACGATAGCTCCGAGGAGGAGGACGAGATCGATGGACCAGCAGGACAGGCAGAGCCTGACAGGGCCCACTATAATATCGTGACCTTCTGCTGTAAGTGCGATTCTACACTGAGGCTGTGCGTGCAGAGCACCCACGTGGACATCAGAACACTGGAGGATCTGCTGATGGGCACCCTGGGCATCGTGTGCCCAATCTGTAGCCAGAAGCCCTGATAAE7_FL_Ds I nano(SEQ ID NO:7)MHGDTPTCHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQCTHVDIRTLEDLLMGTLGIVCPICSQKP**(SEQ ID NO: 8)ATGCACGGCGACACCCCTACATGCCACGAGTACATGCTGGACCTGCAGCCCGAGACCACAGATCTGTACTGCTATGAGCAGCTGAACGATAGCTCCGAGGAGGAGGACGAGATCGATGGACCAGCAGGACAGGCAGAGCCTGACAGGGCCCACTATAATATCGTGACCTTCTGCTGTAAGTGCGATTCTACACTGAGGCTGTGCGTGCAGTGCACCCACGTGGACATCAGAACACTGGAGGATCTGCTGATGGGCACCCTGGGCATCGTGTGCCCAATCTGTAGCCAGAAGCCCTGATAAE7_FL_Ds2_nano(SEQ ID NO:9)MHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDCAHYNIVTFCCKCDSTLRLCVQSCHVDIRTLEDLLMGTLGIVCPICSQKP**(SEQ ID NO: 10)ATGCACGGCGACACCCCTACACTGCACGAGTACATGCTGGACCTGCAGCCCGAGAC CACAGATCTGTACTGCTATGAGCAGCTGAACGATAGCTCCGAGGAGGAGGACGAGA TCGATGGACCAGCAGGACAGGCAGAGCCTGACTGCGCCCACTATAATATCGTGACC TTCTGCTGTAAGTGCGATTCTACACTGAGGCTGTGCGTGCAGAGCTGCCACGTGGACATCAGAACACTGGAGGATCTGCTGATGGGCACCCTGGGCATCGTGTGCCCAATCTGT AGCCAGAAGCCCTGATAAE7_FL_Ds3_nano(SEQ ID NO:11)MHGDTCTLHEYMCDLQPETTDLYCYEQLNDSSEEEDEICGPAGQAEPDRAHYNIVCFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP**(SEQ ID N0:12)ATGCACGGCGACACCTGCACACTGCACGAGTACATGTGCGACCTGCAGCCCGAGAC CACAGATCTGTACTGCTATGAGCAGCTGAACGATAGCTCCGAGGAGGAGGACGAGA TCTGCGGACCAGCAGGACAGGCAGAGCCTGACAGGGCCCACTATAATATCGTGTGC TTCTGCTGTAAGTGCGATTCTACACTGAGGCTGTGCGTGCAGAGCACCCACGTGGACATCAGAACACTGGAGGATCTGCTGATGGGCACCCTGGGCATCGTGTGCCCAATCTGT AGCCAGAAGCCCTGATAAE7_Tr_nano(SEQ ID N0: 13)MHGDTPTLHEYMLDLOPETTDLYCYEQLNDSSEEEDEIDGPAGOAEPDRAHYNIVTFCCKCD STLRLC VQ STHVDTRTLEDLLMGTLGI VCPIC SQKP * *(SEQ ID NO:14)ATGCACGGCGACACCCCTACACTGCACGAGTACATGCTGGACCTGCAGCCCGAGACCACAGATCTGTACTGCTATGAGCAGCTGAACGATAGCTCCGAGGAGGAGGACGAGATCGATGGACCAGCAGGACAGGCAGAGCCTGACAGGGCCCACTATAATATCGTGACCTTCTGCTGTAAGTGCGATTCTACACTGAGGCTGTGCGTGCAGAGCACCCACGTGGACATCAGAACACTGGAGGATCTGCTGATGGGCACCCTGGGCATCGTGTGCCCAATCTGTAGCCAGAAGCCCTGATAAE7_Tr_Dsl_nano(SEQ ID NO:15)MHGDTPTLHEYMLDLOPETTDLYCYEQLNDSSEEEDEIDGPAGOAEPDRAHYNIVTFCCKCDSTLRECVQSCHVDIRTLEDLLMGTCGIVCPICSQKP**(SEQ ID NO:16)ATGCACGGCGACACCCCTACACTGCACGAGTACATGCTGGACCTGCAGCCCGAGAC CACAGATCTGTACTGCTATGAGCAGCTGAACGATAGCTCCGAGGAGGAGGACGAGA TCGATGGACCAGCAGGACAGGCAGAGCCTGACAGGGCCCACTATAATATCGTGACC TTCTGCTGTAAGTGCGATTCTACACTGAGGCTGTGCGTGCAGAGCTGCCACGTGGACATCAGAACACTGGAGGATCTGCTGATGGGCACCTGCGGCATCGTGTGCCCAATCTG TAGCCAGAAGCCCTGATAAE7_2Tr_nano(SEQ ID NO: 17)MHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGOAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSOKPGGSGSGSRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP**(SEQ ID NO: 18)ATGCACGGCGACACCCCTACACTGCACGAGTACATGCTGGACCTGCAGCCCGAGACCACAGATCTGTACTGCTATGAGCAGCTGAACGATAGCTCCGAGGAGGAGGACGAGATCGATGGACCAGCAGGACAGGCAGAGCCTGACAGGGCCCACTATAATATCGTGACCTTCTGCTGTAAGTGCGATTCTACACTGAGGCTGTGCGTGCAGAGCACCCACGTGGACATCAGAACACTGGAGGATCTGCTGATGGGCACCCTGGGCATCGTGTGCCCAATCTGTAGCCAGAAGCCCGGAGGCAGCGGCTCCGGCTCTAGGGCCCACTATAATATCGTGACCTTCTGCTGTAAGTGCGATTCTACACTGAGGCTGTGCGTGCAGAGCACCCACGTGGACATCAGAACACTGGAGGATCTGCTGATGGGCACCCTGGGCATCGTGTGCCCAATCTGTAGCCAGAAGCCCTGATAAE7 2Tr Dsl(SEQ ID NO: 19)MHGDTPTLHEYMLDLOPETTDLYCYEQLNDSSEEEDEIDGPAGOAEPDRAHYNIVTFCCKCDSTLRLCVOCTHVDIRTLEDLLMGTLGIVCPICSOKPGGSGSGSRAHYNTVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGCLGIVCPICSQKP**(SEQ ID NO:20)ATGCACGGCGACACCCCTACACTGCACGAGTACATGCTGGACCTGCAGCCCGAGACCACAGATCTGTACTGCTATGAGCAGCTGAACGATAGCTCCGAGGAGGAGGACGAGATCGATGGACCAGCAGGACAGGCAGAGCCTGACAGGGCCCACTATAATATCGTGACCTTCTGCTGTAAGTGCGATTCTACACTGAGGCTGTGCGTGCAGTGCACCCACGTGGACATCAGAACACTGGAGGATCTGCTGATGGGCACCCTGGGCATCGTGTGCCCAATCTGTAGCCAGAAGCCCGGAGGCAGCGGCTCCGGCTCTAGGGCCCACTATAATATCGTGACCTTCTGCTGTAAGTGCGATTCTACACTGAGGCTGTGCGTGCAGAGCACCCACGTGGACATCAGAACACTGGAGGATCTGCTGATGGGCTGCCTGGGCATCGTGTGCCCAATCTGTAGCCAGAAGCCCTGATAAE7_FL_ApRb_nano(SEQ ID NO:21)MHGDTPTLHEYMLDLQPETTDLYGYGQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP**(SEQ ID NO:22)ATGCACGGCGACACCCCTACACTGCACGAGTACATGCTGGACCTGCAGCCCGAGACCACAGATCTGTACGGCTATGGCCAGCTGAACGATAGCTCCGAGGAGGAGGACGAG ATCGATGGACCAGCAGGACAGGCAGAGCCTGACAGGGCCCACTATAATATCGTGAC CTTCTGCTGTAAGTGCGATTCTACACTGAGGCTGTGCGTGCAGAGCACCCACGTGGACATCAGAACACTGGAGGATCTGCTGATGGGCACCCTGGGCATCGTGTGCCCAATCTGTAGCCAGAAGCCCTGATAAHPV16 E6 nanoparticle designsAll contain Kozak leader sequence and are in the pVaxl vectorAntigenMutations from WTDeletions from WTE6_FL_nano- changes in this section are denoted relative to this construct(SEQ ID NO:23)MHQKRTAMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDLCIVYRDGNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLCPEEKQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRETQL**(SEQ ID NO:24)ATGCACCAGAAGAGGACCGCCATGTTCCAGGACCCACAGGAGAGGCCCAGAAAGCTGCCTCAGCTGTGCACAGAGCTGCAGACCACAATCCACGATATCATCCTGGAGTGCGTGTACTGTAAGCAGCAGCTGCTGAGGAGAGAGGTGTATGACTTCGCCTTTCGGGATCTGTGCATCGTGTACCGCGACGGCAACCCATATGCCGTGTGCGATAAGTGTCTGAAGTT CTACTCTAAGATCAGCGAGTATCGGCACTACTGTTATTCCCTGTACGGCACCACACT GGAGCAGCAGTATAACAAGCCCCTGTGCGACCTGCTGATCCGCTGCATCAATTGTCA GAAGCCCCTGTGCCCTGAGGAGAAGCAGAGGCACCTGGATAAGAAGCAGAGATTTCACAATATCAGGGGCCGCTGGACCGGCCGGTGTATGTCTTGCTGTAGGAGCTCCAGA ACCCGGCGCGAGACACAGCTGTGATAAE6_FL_Dsl_nano(SEQ ID NO:25)MHQKRTAMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDLCIVYRDGNPYAVCDKCLKFYSKISEYRHYCYSLCGTTLEQQYNKPLCDLLCRCINCQKPLCP EEKQRHLDKKQCFHNIRGRCTGRCMSCCRS SRTRRETQL* *(SEQ ID NO:26)ATGCACCAGAAGAGGACCGCCATGTTCCAGGACCCACAGGAGAGGCCCAGAAAGCTGCCTCAGCTGTGCACAGAGCTGCAGACCACAATCCACGATATCATCCTGGAGTGCGTGTACTGTAAGCAGCAGCTGCTGAGGAGAGAGGTGTATGACTTCGCCTTTCGGGATCTGTGCATCGTGTACCGCGACGGCAACCCATATGCCGTGTGCGATAAGEGTCTGAAGTTCTACTCTAAGATCAGCGAGTATCGGCACTACTGTTATTCCCTGTGCGGCACCACACTGGAGCAGCAGTATAACAAGCCCCTGTGCGACCTGCTGTGCCGCTGCATCAATTGTCAGAAGCCCCTGTGCCCTGAGGAGAAGCAGAGGCACCTGGATAAGAAGCAGTGCTTTCACAATATCAGGGGCCGCTGCACCGGCCGGTGTATGTCTTGCTGTAGGAGCTCCAGAACCCGGCGCGAGACACAGCTGTGATAAE6_FL_Ds2_nano(SEQ ID NO:27)MHQKRTAMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDLCIVYRDGNPYAVCDKCLKFYSKISEYRCYCYSLYCTTLEQQYNKPLCDLLIRCINCQKPLCPE EKQRHCDKKQRFHNCRGRWTGRCMSCCRS SRTRRETQL * *(SEQ ID NO:28)ATGCACCAGAAGAGGACCGCCATGTTCCAGGACCCACAGGAGAGGCCCAGAAAGCTGCCTCAGCTGTGCACAGAGCTGCAGACCACAATCCACGATATCATCCTGGAGTGCGTGTACTGTAAGCAGCAGCTGCTGAGGAGAGAGGTGTATGACTTCGCCTTTCGGGATCTGTGCATCGTGTACCGCGACGGCAACCCATATGCCGTGTGCGATAAGTGTCTGAAGTTCTACTCTAAGATCAGCGAGTATCGGTGCTACTGTTATTCCCTGTACTGCACCACACT GGAGCAGCAGTATAACAAGCCCCTGTGCGACCTGCTGATCCGCTGCATCAATTGTCA GAAGCCCCTGTGCCCTGAGGAGAAGCAGAGGCACTGCGATAAGAAGCAGAGATTT CACAATTGCAGGGGCCGCTGGACCGGCCGGTGTATGTCTTGCTGTAGGAGCTCCAGAACCCGGCGCGAGACACAGCTGTGATAAE6_FL_Sol_nano(SEQ ID NO:29)MHQKRTAMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFARRDLCIVYRDGNPYAVCDKCLKFYSKISEYRHYSYSLYGTTLEQQYNKPLSDLLIRCINCQKPLSP EEKQRHLDKKQRFHNIRGRWTGRCMSCSRS SRTRRETQL * *(SEQ ID NO:30)ATGCACCAGAAGAGGACCGCCATGTTCCAGGACCCACAGGAGAGGCCCAGAAAGCTGCCTCAGCTGTGCACAGAGCTGCAGACCACAATCCACGATATCATCCTGGAGTGCGT GTACTGTAAGCAGCAGCTGCTGAGGAGAGAGGTGTATGACTTCGCCCGGCGGGATC TGTGCATCGTGTACCGCGACGGCAACCCATATGCCGTGTGCGATAAGTGTCTGAAGT TCTACTCTAAGATCAGCGAGTATCGGCACTACTCCTATTCCCTGTACGGCACCACACTGGAGCAGCAGTATAACAAGCCCCTGTCCGACCTGCTGATCCGCTGCATCAATTGTC AGAAGCCCCTGTCCCCTGAGGAGAAGCAGAGGCACCTGGATAAGAAGCAGAGATTT CACAATATCAGGGGCCGCTGGACCGGCCGGTGTATGTCTTGCAGCAGGAGCTCCAG AACCCGGCGCGAGACACAGCTGTGATAAE6_FL_Sol_Ds I nano(SEQ 1D NO:31)MHQKRTAMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFARRDLCI VYRDGNPYAVCDKCLKFYSKISEYRHCSYSLYGTTLEQQYNKPLSDLLIRCINCQKPLSP EEKQRHLDKKQRFHNCRGRWTGRCMSCSRS SRTRRETQL* *(SEQ ID NO:32)ATGCACCAGAAGAGGACCGCCATGTTCCAGGACCCACAGGAGAGGCCCAGAAAGCTGCCTCAGCTGTGCACAGAGCTGCAGACCACAATCCACGATATCATCCTGGAGTGCGT GTACTGTAAGCAGCAGCTGCTGAGGAGAGAGGTGTATGACTTCGCCCGGCGGGATC TGTGCATCGTGTACCGCGACGGCAACCCATATGCCGTGTGCGATAAGTGTCTGAAGT TCTACTCTAAGATCAGCGAGTATCGGCACTGCTCCTATTCCCTGTACGGCACCACACTGGAGCAGCAGTATAACAAGCCCCTGTCCGACCTGCTGATCCGCTGCATCAATTGTC AGAAGCCCCTGTCCCCTGAGGAGAAGCAGAGGCACCTGGATAAGAAGCAGAGATTT CACAATTGCAGGGGCCGCTGGACCGGCCGGTGTATGTCTTGCAGCAGGAGCTCCAG AACCCGGCGCGAGACACAGCTGTGATAAE6_Tr_nano(SEQ ID NO:33)MHOKRTAMFODPOERPRKLPOLCTELOTTTHDITLECVYCKOQLLRREVYDFAFRDLCIV YRDGNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEOQYNKPLCDLLIRCINCOKPLCPE EKORHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRETQL**(SEQ ID NO:34)ATGCACCAGAAGAGGACCGCCATGTTCCAGGACCCACAGGAGAGGCCCAGAAAGCTGCCTCAGCTGTGCACAGAGCTGCAGACCACAATCCACGATATCATCCTGGAGTGCGTGTACTGTAAGCAGCAGCTGCTGAGGAGAGAGGTGTATGACTTCGCCTTTCGGGATCTGTGCATCGTGTACCGCGACGGCAACCCATATGCCGTGTGCGATAAGTGTCTGAAGTT CTACTCTAAGATCAGCGAGTATCGGCACTACTGTTATTCCCTGTACGGCACCACACT GGAGCAGCAGTATAACAAGCCCCTGTGCGACCTGCTGATCCGCTGCATCAATTGTCA GAAGCCCCTGTGCCCTGAGGAGAAGCAGAGGCACCTGGATAAGAAGCAGAGATTTCACAATATCAGGGGCCGCTGGACCGGCCGGTGTATGTCTTGCTGTAGGAGCTCCAGA ACCCGGCGCGAGACACAGCTGTGATAAE6_Tr_Dsl_nano(SEQ ID NO:35)MHOKRTAMFODPOERPRKLPOLCTELOTTIHDIILECCYCKOOLLRREVYDFAFRDLCIVYRDGNPCAVCDKCLKFYSKISEYRHYCYSLYGTTLEOQYNKPLCDLLIRCINCOKPLCPEEKORHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRETQL**(SEQ ID NO:36)ATGCACCAGAAGAGGACCGCCATGTTCCAGGACCCACAGGAGAGGCCCAGAAAGCTGCCTCAGCTGTGCACAGAGCTGCAGACCACAATCCACGATATCATCCTGGAGTGCTGCTACTGTAAGCAGCAGCTGCTGAGGAGAGAGGTGTATGACTTCGCCTTTCGGGATCTGTGCATCGTGTACCGCGACGGCAACCCATGCGCCGTGTGCGATAAGTGTCTGAAGTTCTACTCTAAGATCAGCGAGTATCGGCACTACTGTTATTCCCTGTACGGCACCACACTGGAGCAGCAGTATAACAAGCCCCTGTGCGACCTGCTGATCCGCTGCATCAATTGTCAGAAGCCCCTGTGCCCTGAGGAGAAGCAGAGGCACCTGGATAAGAAGCAGAGATTTCACAATATCAGGGGCCGCTGGACCGGCCGGTGTATGTCTTGCTGTAGGAGCTCCAGAACCCGGCGCGAGACACAGCTGTGATAAE6_Tr_Ds2_nano(SEQ ID NO:37)MHQKRTCMFODPOERPRKLPOLCTELOTTIHDIICECVYCKOQLLRREVYDFAFRDLCI VYRDGNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEOQYNKPLCDLLIRCINCOKPLC PEEKQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRETOL**(SEQ ID NO:38)ATGCACCAGAAGAGGACCTGCATGTTCCAGGACCCACAGGAGAGGCCCAGAAAGCTGCCTCAGCTGTGCACAGAGCTGCAGACCACAATCCACGATATCATCTGCGAGTGC GTGTACTGTAAGCAGCAGCTGCTGAGGAGAGAGGTGTATGACTTCGCCTTTCGGGAT CTGTGCATCGTGTACCGCGACGGCAACCCATATGCCGTGTGCGATAAGTGTCTGAAG TTCTACTCTAAGATCAGCGAGTATCGGCACTACTGTTATTCCCTGTACGGCACCACACTGGAGCAGCAGTATAACAAGCCCCTGTGCGACCTGCTGATCCGCTGCATCAATTGT CAGAAGCCCCTGTGCCCTGAGGAGAAGCAGAGGCACCTGGATAAGAAGCAGAGATT TCACAATATCAGGGGCCGCTGGACCGGCCGGTGTATGTCTTGCTGTAGGAGCTCCAG AACCCGGCGCGAGACACAGCTGTGATAAE6_T r Sol nano(SEQ ID NO:39)MHQKRTAMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFARRDLCIVYRDGNPYAVCDKCLKFYSKISEYRHYSYSLYGTTLEOQYNKPLCDLLIRCINCOKPLCPEEKORHLDKKQRFHNIRGRWTGRCMSCCRS SRTRRETQL * *(SEQ ID NO:40)ATGCACCAGAAGAGGACCGCCATGTTCCAGGACCCACAGGAGAGGCCCAGAAAGCTGCCTCAGCTGTGCACAGAGCTGCAGACCACAATCCACGATATCATCCTGGAGTGCGTGTACTGTAAGCAGCAGCTGCTGAGGAGAGAGGTGTATGACTTCGCCCGGCGGGATCTGTGCATCGTGTACCGCGACGGCAACCCATATGCCGTGTGCGATAAGTGTCTGAAGTTCTACTCTAAGATCAGCGAGTATCGGCACTACTCCTATTCCCTGTACGGCACCACACTGGAGCAGCAGTATAACAAGCCCCTGTGCGACCTGCTGATCCGCTGCATCAATTGTCAGAAGCCCCTGTGCCCTGAGGAGAAGCAGAGGCACCTGGATAAGAAGCAGAGATTTCACAATATCAGGGGCCGCTGGACCGGCCGGTGTATGTCTTGCTGTAGGAGCTCCAGAACCCGGCGCGAGACACAGCTGTGATAAE6_FL_Ap53_nano(SEQ ID N0:41)MHOKRTAMFODPQESGRKLPQLCTELQTTIHDIILECVYCKQOLLRREVYDFAFRDLCIVYRDGNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLCPEEKQRHLDKKQRFHN1RGRWTGRCMSCCRSSRTRRETQL**(SEQ ID NO:42)ATGCACCAGAAGAGGACCGCCATGTTCCAGGACCCACAGGAGAGCGGCAGAAAGC TGCCTCAGCTGTGCACAGAGCTGCAGACCACAATCCACGATATCATCCTGGAGTGCG TGTACTGTAAGCAGCAGCTGCTGAGGAGAGAGGTGTATGACTTCGCCTTTCGGGATC TGTGCATCGTGTACCGCGACGGCAACCCATATGCCGTGTGCGATAAGTGTCTGAAGTTCTACTCTAAGATCAGCGAGTATCGGCACTACTGTTATTCCCTGTACGGCACCACAC TGGAGCAGCAGTATAACAAGCCCCTGTGCGACCTGCTGATCCGCTGCATCAATTGTC AGAAGCCCCTGTGCCCTGAGGAGAAGCAGAGGCACCTGGATAAGAAGCAGAGATTT CACAATATCAGGGGCCGCTGGACCGGCCGGTGTATGTCTTGCTGTAGGAGCTCCAGAACCCGGCGCGAGACACAGCTGTGATAAE6_FL_Sol_Ap53_nano(SEQ ID NO:43)MHOKRTAMFODPOESGRKLPOLCTELOTTIHDIILECVYCKOOLLRREVYDFAFRDLCIVYRDGNPYAVCDKCLKFYSKISEYRHYSYSLYGTTLEQQYNKPLSDLLIRCINCQKPLCPEEKQRHLDKKQRFHNIRGRWTGRCMSCSRS SRTRRETQL**(SEQ ID NO:44)ATGCACCAGAAGAGGACCGCCATGTTCCAGGACCCACAGGAGAGCGGCAGAAAGCTGCCTCAGCTGTGCACAGAGCTGCAGACCACAATCCACGATATCATCCTGGAGTGCGTGTACTGTAAGCAGCAGCTGCTGAGGAGAGAGGTGTATGACTTCGCCTTTCGGGATCTGTGCATCGTGTACCGCGACGGCAACCCATATGCCGTGTGCGATAAGTGTCTGAAGTTCTACTCTAAGATCAGCGAGTATCGGCACTACTCCTATTCCCTGTACGGCACCACACTGGAGCAGCAGTATAACAAGCCCCTGTCCGACCTGCTGATCCGCTGCATCAATTGTCAGAAGCCCCTGTGCCCTGAGGAGAAGCAGAGGCACCTGGATAAGAAGCAGAGATTTCACAATATCAGGGGCCGCTGGACCGGCCGGTGTATGTCTTGCAGCAGGAGCTCCAGAACCCGGCGCGAGACACAGCTGTGATAAE6_Tr_Sol_Ap53 nano(SEQ ID NO:45)MHQKRTAMFODPOESGRKLPOLCTELQTTIHDIILECVYCKOOLLRREVYDFAFRDLCIVYRDGNPYAVCDKCLKFYSKISEYRHYSYSLYGTTLEOQYNKPLCDLLIRCINCOKPLCPEEKQRHLDKKQRFHNIRGRWTGRCMSCCRS SRTRRETQL * *(SEQ ID NO:46)ATGCACCAGAAGAGGACCGCCATGTTCCAGGACCCACAGGAGAGCGGCAGAAAGC TGCCTCAGCTGTGCACAGAGCTGCAGACCACAATCCACGATATCATCCTGGAGTGCG TGTACTGTAAGCAGCAGCTGCTGAGGAGAGAGGTGTATGACTTCGCCTTTCGGGATC TGTGCATCGTGTACCGCGACGGCAACCCATATGCCGTGTGCGATAAGTGTCTGAAGTTCTACTCTAAGATCAGCGAGTATCGGCACTACTCCTATTCCCTGTACGGCACCACAC TGGAGCAGCAGTATAACAAGCCCCTGTCCGACCTGCTGATCCGCTGCATCAATTGTC AGAAGCCCCTGTGCCCTGAGGAGAAGCAGAGGCACCTGGATAAGAAGCAGAGATTT CACAATATCAGGGGCCGCTGGACCGGCCGGTGTATGTCTTGCAGCAGGAGCTCCAGAACCCGGCGCGAGACACAGCTGTGATAAAntigenMutations from WTDeletions from WTInsertions from WTE7_FL_nano - changes in this section are denoted relative to this construct(SEQ ID NO:47)MDWTWILFLVAAATRVHSMQIYEGKLTAEGLRFGIVASRANHALVDRLVEGAIDAIVR HGGREEDITLVRVCGSWEIPVAAGELARKEDIDAVIAIGVLCRGATPSFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAIEAAGTCHGNKGWEAALCAIEMANLFKSLRGGSGGS GGSGGSGGGDTITLPCRPAPPPNCTSNITGLILTRQGGYSNDNTVIFRPSGGNWSDIARCN ITGTVVSTQLFLNGSLAGNGTVIRSEDWRDNAKSICVQLNTSVEINCTGNGTCNISRAKW DNTLKQIASKLREQYGNKTIIFKPSSGGDPEFVNHSFNCGNVTFYCDSTQLFDSTWFDSTGG GSGGSGMHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHY NIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP**(SEQ ID NO:48)ATGGACTGGACCTGGATTCTGTTCCTGGTGGCCGCCGCCACAAGGGTGCACAGCATG CAGATCTACGAAGGAAAACTGACCGCTGAGGGACTGAGGTTCGGAATTGTCGCAAG CCGCGCGAATCACGCACTGGTGGATAGGCTGGTGGAAGGCGCTATCGACGCAATTGTCCGGCACGGCGGGAGAGAGGAAGACATCACACTGGTGAGAGTCTGCGGCAGCTGG GAGATTCCCGTGGCAGCTGGAGAACTGGCTCGAAAGGAGGACATCGATGCCGTGAT CGCTATTGGGGTCCTGTGCCGAGGAGCAACTCCCAGCTTCGACTACATCGCCTCAGA AGTGAGCAAGGGGCTGGCTGATCTGTCCCTGGAGCTGAGGAAACCTATCACTTTTGG CGTGATTACTGCCGACACCCTGGAACAGGCAATCGAGGCGGCCGGCACCTGCCATG GAAACAAAGGCTGGGAAGCAGCCCTGTGCGCTATTGAGATGGCAAATCTGTTCAAA TCTCTGCGAGGAGGCTCCGGAGGATCTGGAGGGAGTGGAGGCTCAGGAGGAGGCGA CACCATCACACTGCCATGCCGCCCTGCACCACCTCCAAACTGTACCTCCAACATCACCGGCCTGATTCTGACAAGACAGGGGGGATATAGTAACGATAATACCGTGATTTTCA GGCCCTCAGGAGGGAATTGGAGTGACATCGCACGATGCAATATTACTGGAACAGTG GTCTCTACTCAGCTGTTTCTGAACGGCAGTCTGGCTGGAAACGGGACAGTCATCCGA TCTGAAGACTGGCGGGATAATGCAAAGTCAATTTGTGTGCAGCTGAACACAAGCGT CGAGATCAATTGCACTGGCAATGGGACCTGTAACATTTCTCGGGCCAAATGGGATA ATACCCTGAAGCAGATCGCCAGTAAACTGAGAGAGCAGTACGGCAATAAGACAATCATCTTCAAGCCTTCTAGTGGAGGCGACCCAGAGTTCGTGAACCATAGCTTTAATTGC GGGAACGTCACCTTTTATTGTGATTCCACACAGCTGTTCGATAGCACTTGGTTTGATTCCACCGGTGGGAGCGGAAGTGGCGGTTCCGGA^7GG4CGGCG4G4CCCC 4C4C7UCACGAGTACATGCTGGACCTGCAGCCCGAGACCACAGATCTGTACTGCTATGAGCAGCTGA ACGATAGCTCCGAGGAGGAGGACGAGATCGATGGACCAGCAGGACAGGCAGAGCCTGA CAGGGCCCACTATAATATCGTGACCTTCTGCTGTAAGTGCGATTCTACACTGAGGCTGTGC GTGCAGAGCACCCACGTGGACA TCAGAACACTGGAGGA TCTGCTGA TGGGCACCCTGGG CA TCGTGTGCCCAA TCTGTAGCCAGAAGCCCTGATAAE7_FL_Dsl_nano(SEQ ID NO:49)MDWTWILFLVAAATRVHSMQIYEGKLTAEGLRFGIVASRANHALVDRL VEGAID AIVR HGGREEDITLVRVCGSWEIPVAAGELARKEDIDAVIAIGVLCRGATPSFDYIASEVSKGL ADLSLELRKPITFGVITADTLEQAIEAAGTCHGNKGWEAALCAIEMANLFKSLRGGSGGS GGSGGSGGGDTITLPCRPAPPPNCTSNITGLILTRQGGYSNDNTVIFRPSGGNWSDIARCN ITGTVVSTQLFLNGSLAGNGTVIRSEDWRDNAKSICVQLNTSVEINCTGNGTCNISRAKW DNTLKQIASKLREQYGNKTIIFKPSSGGDPEFVNHSFNCGNVTFYCDSTQLFDSTWFDST GGSGSGGSGMHGDTPTCHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAH YNIVTFCCKCDSTLRLCVQCTHVDIRTLEDLLMGTLGIVCPICSQKP**(SEQ ID NO: 50)ATGGACTGGACCTGGATTCTGTTCCTGGTGGCCGCCGCCACAAGGGTGCACAGCATG CAGATCTACGAAGGAAAACTGACCGCTGAGGGACTGAGGTTCGGAATTGTCGCAAG CCGCGCGAATCACGCACTGGTGGATAGGCTGGTGGAAGGCGCTATCGACGCAATTG TCCGGCACGGCGGGAGAGAGGAAGACATCACACTGGTGAGAGTCTGCGGCAGCTGG GAGATTCCCGTGGCAGCTGGAGAACTGGCTCGAAAGGAGGACATCGATGCCGTGAT CGCTATTGGGGTCCTGTGCCGAGGAGCAACTCCCAGCTTCGACTACATCGCCTCAGA AGTGAGCAAGGGGCTGGCTGATCTGTCCCTGGAGCTGAGGAAACCTATCACTTTTGG CGTGATTACTGCCGACACCCTGGAACAGGCAATCGAGGCGGCCGGCACCTGCCATG GAAACAAAGGCTGGGAAGCAGCCCTGTGCGCTATTGAGATGGCAAATCTGTTCAAA TCTCTGCGAGGAGGCTCCGGAGGATCTGGAGGGAGTGGAGGCTCAGGAGGAGGCGA CACCATCACACTGCCATGCCGCCCTGCACCACCTCCAAACTGTACCTCCAACATCACCGGCCTGATTCTGACAAGACAGGGGGGATATAGTAACGATAATACCGTGATTTTCA GGCCCTCAGGAGGGAATTGGAGTGACATCGCACGATGCAATATTACTGGAACAGTG GTCTCTACTCAGCTGTTTCTGAACGGCAGTCTGGCTGGAAACGGGACAGTCATCCGA TCTGAAGACTGGCGGGATAATGCAAAGTCAATTTGTGTGCAGCTGAACACAAGCGT CGAGATCAATTGCACTGGCAATGGGACCTGTAACATTTCTCGGGCCAAATGGGATA ATACCCTGAAGCAGATCGCCAGTAAACTGAGAGAGCAGTACGGCAATAAGACAATC ATCTTCAAGCCTTCTAGTGGAGGCGACCCAGAGTTCGTGAACCATAGCTTTAATTGC GGGAACGTCACCTTTTATTGTGATTCCACACAGCTGTTCGATAGCACTTGGTTTGATTCC ACCGGTGGG AGCGG A AGTGGCGGTTCCGG AA TGCACGGCGACACCCCTACA TGCC ACGAGTACATGCTGGACCTGCAGCCCGAGACCACAGATCTGTACTGCTATGAGCAGCTGA ACGATAGCTCCGAGGAGGAGGACGAGATCGATGGACCAGCAGGACAGGCAGAGCCTGA CAGGGCCCACTATAATATCGTGACCTTCTGCTGTAAGTGCGATTCTACACTGAGGCTGTGC GTGCAGTGCACCCACGTGGACA TCAGAACACTGGAGGA TCTGCTGA TGGGCACCCTGGG CA TCGTGTGCCCAA TC TG TA GCCAGAAGCCCTG AT A A206193-0149-00WQE7_FL_Ds2_nano(SEQ ID N0:51)MDWTW1LFLVAAATRVHSMQ1YEGKLTAEGLRFG1VASRANHALVDRL VEGAID A1VR HGGREEDITLVRVCGSWEIPVAAGELARKEDIDAVIAIGVLCRGATPSFDYIASEVSKGL ADLSLELRKPITFGVITADTLEQAIEAAGTCHGNKGWEAALCAIEMANLFKSLRGGSGGS GGSGGSGGGDTITLPCRPAPPPNCTSNITGLILTRQGGYSNDNTVIFRPSGGNWSDIARCN ITGTVVSTQLFLNGSLAGNGTVIRSEDWRDNAKSICVQLNTSVEINCTGNGTCNISRAKW DNTLKQIASKLREQYGNKTIIFKPSSGGDPEFVNHSFNCGNVTFYCDSTQLFDSTWFDST GGSGSGGSGMHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDCAHY NIVTFCCKCDSTLRLCVQSCHVDIRTLEDLLMGTLGIVCPICSQKP^(SEQ ID NO:52)ATGGACTGGACCTGGATTCTGTTCCTGGTGGCCGCCGCCACAAGGGTGCACAGCATG CAGATCTACGAAGGAAAACTGACCGCTGAGGGACTGAGGTTCGGAATTGTCGCAAG CCGCGCGAATCACGCACTGGTGGATAGGCTGGTGGAAGGCGCTATCGACGCAATTG TCCGGCACGGCGGGAGAGAGGAAGACATCACACTGGTGAGAGTCTGCGGCAGCTGG GAGATTCCCGTGGCAGCTGGAGAACTGGCTCGAAAGGAGGACATCGATGCCGTGAT CGCTATTGGGGTCCTGTGCCGAGGAGCAACTCCCAGCTTCGACTACATCGCCTCAGA AGTGAGCAAGGGGCTGGCTGATCTGTCCCTGGAGCTGAGGAAACCTATCACTTTTGG CGTGATTACTGCCGACACCCTGGAACAGGCAATCGAGGCGGCCGGCACCTGCCATG GAAACAAAGGCTGGGAAGCAGCCCTGTGCGCTATTGAGATGGCAAATCTGTTCAAA TCTCTGCGAGGAGGCTCCGGAGGATCTGGAGGGAGTGGAGGCTCAGGAGGAGGCGA CACCATCACACTGCCATGCCGCCCTGCACCACCTCCAAACTGTACCTCCAACATCAC CGGCCTGATTCTGACAAGACAGGGGGGATATAGTAACGATAATACCGTGATTTTCA GGCCCTCAGGAGGGAATTGGAGTGACATCGCACGATGCAATATTACTGGAACAGTG GTCTCTACTCAGCTGTTTCTGAACGGCAGTCTGGCTGGAAACGGGACAGTCATCCGA TCTGAAGACTGGCGGGATAATGCAAAGTCAATTTGTGTGCAGCTGAACACAAGCGT CGAGATCAATTGCACTGGCAATGGGACCTGTAACATTTCTCGGGCCAAATGGGATA ATACCCTGAAGCAGATCGCCAGTAAACTGAGAGAGCAGTACGGCAATAAGACAATC ATCTTCAAGCCTTCTAGTGGAGGCGACCCAGAGTTCGTGAACCATAGCTTTAATTGC GGGAACGTCACCTTTTATTGTGATTCCACACAGCTGTTCGATAGCACTTGGTTTGATT CCACCGGTGGGAGCGGAAGTGGCGGTTCCGGA^TGCACGGCGACACCCCTACACTUCACGAGTACATGCTGGACCTGCAGCCCGAGACCACAGATCTGTACTGCTATGAGCAGCTGA ACGATAGCTCCGAGGAGGAGGACGAGATCGATGGACCAGCAGGACAGGCAGAGCCTGA CTGCGCCCACTA TAA TA TCGTGACCTTCTGCTGTAAGTGCGATTCTACACTGAGGCTGTGC GTGCAGAGCTGCCACGTGGACA TCAGAACACTGGAGGA TCTGCTGA TGGGCACCCTGGG CA TCGTGTGCCCAA TCTGTAGCCAGAAGCCCTGATAAE7_FL_Ds3_nano(SEQ ID NO:53)206193-0149-00WQMDWTWTLFLVAAATRVHSMQIYEGKLTAEGLRFGTVASRANHALVDRLVEGAIDAIVR HGGREEDITLVRVCGSWEIPVAAGELARKEDIDAVIAIGVLCRGATPSFDYIASEVSKGL ADLSLELRKPITFGVITADTLEQAIEAAGTCHGNKGWEAALCAIEMANLFKSLRGGSGGS GGSGGSGGGDTITLPCRPAPPPNCTSNITGLILTRQGGYSNDNTVIFRPSGGNWSDIARCN ITGTVVSTQLFLNGSLAGNGTVIRSEDWRDNAKSICVQLNTSVEINCTGNGTCNISRAKW DNTLKQ1ASKLREQYGNKT11FKPSSGGDPEFVNHSFNCGNVTFYCDSTQLFDSTWFDST GGSGSGGSGMHGDTCTLHEYMCDLQPETTDLYCYEQLNDSSEEEDEICGPAGQAEPDRAH YNIVCFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP**(SEQ ID NO:54)ATGGACTGGACCTGGATTCTGTTCCTGGTGGCCGCCGCCACAAGGGTGCACAGCATG CAGATCTACGAAGGAAAACTGACCGCTGAGGGACTGAGGTTCGGAATTGTCGCAAG CCGCGCGAATCACGCACTGGTGGATAGGCTGGTGGAAGGCGCTATCGACGCAATTG TCCGGCACGGCGGGAGAGAGGAAGACATCACACTGGTGAGAGTCTGCGGCAGCTGG GAGATTCCCGTGGCAGCTGGAGAACTGGCTCGAAAGGAGGACATCGATGCCGTGAT CGCTATTGGGGTCCTGTGCCGAGGAGCAACTCCCAGCTTCGACTACATCGCCTCAGA AGTGAGCAAGGGGCTGGCTGATCTGTCCCTGGAGCTGAGGAAACCTATCACTTTTGG CGTGATTACTGCCGACACCCTGGAACAGGCAATCGAGGCGGCCGGCACCTGCCATG GAAACAAAGGCTGGGAAGCAGCCCTGTGCGCTATTGAGATGGCAAATCTGTTCAAA TCTCTGCGAGGAGGCTCCGGAGGATCTGGAGGGAGTGGAGGCTCAGGAGGAGGCGA CACCATCACACTGCCATGCCGCCCTGCACCACCTCCAAACTGTACCTCCAACATCAC CGGCCTGATTCTGACAAGACAGGGGGGATATAGTAACGATAATACCGTGATTTTCA GGCCCTCAGGAGGGAATTGGAGTGACATCGCACGATGCAATATTACTGGAACAGTG GTCTCTACTCAGCTGTTTCTGAACGGCAGTCTGGCTGGAAACGGGACAGTCATCCGA TCTGAAGACTGGCGGGATAATGCAAAGTCAATTTGTGTGCAGCTGAACACAAGCGT CGAGATCAATTGCACTGGCAATGGGACCTGTAACATTTCTCGGGCCAAATGGGATA ATACCCTGAAGCAGATCGCCAGTAAACTGAGAGAGCAGTACGGCAATAAGACAATC ATCTTCAAGCCTTCTAGTGGAGGCGACCCAGAGTTCGTGAACCATAGCTTTAATTGC GGGAACGTCACCTTTTATTGTGATTCCACACAGCTGTTCGATAGCACTTGGTTTGATT CCACCGGTGGGAGCGGAAGTGGCGGTTCCGGA^7GG4CGGCG4G4CCrGC4G4C7UC ACGAGTACATGTGCGACCTGCAGCCCGAGACCACAGATCTGTACTGCTATGAGCAGCTGA ACGATAGCTCCGAGGAGGAGGACGAGATCTGCGGACCAGCAGGACAGGCAGAGCCTGA CAGGGCCCACTATAATATCGTGTGCTTCTGCTGTAAGTGCGATTCTACACTGAGGCTGTGC GTGCAGAGCACCCACGTGGACATCAGAACACTGGAGGATCTGCTGATGGGCACCCTGGG CA TCGTGTGCCCAA TCTGTAGCCAGAAGCCCTGATAAE7_Tr_nano(SEQ ID NO:55)MDWTWILFLVAAATRVHSMQIYEGKLTAEGLRFGIVASRANHALVDRL VEGAID AIVR HGGREEDITLVRVCGSWEIPVAAGELARKEDIDAVIAIGVLCRGATPSFDYIASEVSKGL ADLSLELRKPITFGVITADTLEQAIEAAGTCHGNKGWEAALCAIEMANLFKSLRGGSGGS GGSGGSGGGDT1TLPCRPAPPPNCTSN1TGL1LTRQGGYSNDNTV1FRPSGGNWSD1ARCN ITGTVVSTQLFLNGSLAGNGTVIRSEDWRDNAKSICVQLNTSVEINCTGNGTCNISRAKW DNTLKQIASKLREQYGNKTIIFKPSSGGDPEFVNHSFNCGNVTFYCDSTQLFDSTWFDSTGG G GGSGMHGDTPTJ.HEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHY NIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP**(SEQ ID NO:56)ATGGACTGGACCTGGATTCTGTTCCTGGTGGCCGCCGCCACAAGGGTGCACAGCATG CAGATCTACGAAGGAAAACTGACCGCTGAGGGACTGAGGTTCGGAATTGTCGCAAG CCGCGCGAATCACGCACTGGTGGATAGGCTGGTGGAAGGCGCTATCGACGCAATTG TCCGGCACGGCGGGAGAGAGGAAGACATCACACTGGTGAGAGTCTGCGGCAGCTGG GAGATTCCCGTGGCAGCTGGAGAACTGGCTCGAAAGGAGGACATCGATGCCGTGAT CGCTATTGGGGTCCTGTGCCGAGGAGCAACTCCCAGCTTCGACTACATCGCCTCAGA AGTGAGCAAGGGGCTGGCTGATCTGTCCCTGGAGCTGAGGAAACCTATCACTTTTGG CGTGATTACTGCCGACACCCTGGAACAGGCAATCGAGGCGGCCGGCACCTGCCATG GAAACAAAGGCTGGGAAGCAGCCCTGTGCGCTATTGAGATGGCAAATCTGTTCAAA TCTCTGCGAGGAGGCTCCGGAGGATCTGGAGGGAGTGGAGGCTCAGGAGGAGGCGA CACCATCACACTGCCATGCCGCCCTGCACCACCTCCAAACTGTACCTCCAACATCAC CGGCCTGATTCTGACAAGACAGGGGGGATATAGTAACGATAATACCGTGATTTTCA GGCCCTCAGGAGGGAATTGGAGTGACATCGCACGATGCAATATTACTGGAACAGTG GTCTCTACTCAGCTGTTTCTGAACGGCAGTCTGGCTGGAAACGGGACAGTCATCCGA TCTGAAGACTGGCGGGATAATGCAAAGTCAATTTGTGTGCAGCTGAACACAAGCGT CGAGATCAATTGCACTGGCAATGGGACCTGTAACATTTCTCGGGCCAAATGGGATA ATACCCTGAAGCAGATCGCCAGTAAACTGAGAGAGCAGTACGGCAATAAGACAATC ATCTTCAAGCCTTCTAGTGGAGGCGACCCAGAGTTCGTGAACCATAGCTTTAATTGC GGGAACGTCACCTTTTATTGTGATTCCACACAGCTGTTCGATAGCACTTGGTTTGATT CCACCGGYGGGAGCGGAAGYGGCGGTYCCGGAATGCACGGCGACACCCCTACACTGC ACGAGTACATGCTGGACCTGCAGCCCGAGACCACAGATCTGTACTGCTATGAGCAGCTGAACGATAGCTCCGAGGAGGAGGACGAGATCGATGGACCAGCAGGACAGGCAGAGCCTGA CAGGGCCCACTATAATATCGTGACCTTCTGCTGTAAGTGCGATTCTACACTGAGGCTGTGC GTGCAGAGCACCCACGTGGACA TCAGAACACTGGAGGA TCTGCTGA TGGGCACCCTGGG CA TCGTGTGCCCAA TCTGTAGCCAGAAGCCCTGATAAE7_Tr_Dsl_nano(SEQ ID NO:57)MDWTWILFLVAAATRVHSMQIYEGKLTAEGLRFGIVASRANHALVDRL VEGAID AIVR HGGREEDITLVRVCGSWEIPVAAGELARKEDIDAVIAIGVLCRGATPSFDYIASEVSKGL ADLSLELRKPITFGVITADTLEQAIEAAGTCHGNKGWEAALCAIEMANLFKSLRGGSGGS GGSGGSGGGDTITLPCRPAPPPNCTSNITGLILTRQGGYSNDNTVIFRPSGGNWSDIARCN ITGTVVSTQLFLNGSLAGNGTVIRSEDWRDNAKSICVQLNTSVEINCTGNGTCNISRAKW DNTLKQIASKLREQYGNKTIIFKPSSGGDPEFVNHSFNCGNVTFYCDSTQLFDSTWFDST GGSGSGGSGMHGDTPTLHEYMLDLOPEITDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHY NIVTFCCKCDSTLRLCVQSCHVDIRTLEDLLMGTCGIVCPICSQKP^(SEQ ID NO:58)ATGGACTGGACCTGGATTCTGTTCCTGGTGGCCGCCGCCACAAGGGTGCACAGCATGCAGATCTACGAAGGAAAACTGACCGCTGAGGGACTGAGGTTCGGAATTGTCGCAAG206193-0149-00WQCCGCGCGAATCACGCACTGGTGGATAGGCTGGTGGAAGGCGCTATCGACGCAATTG TCCGGCACGGCGGGAGAGAGGAAGACATCACACTGGTGAGAGTCTGCGGCAGCTGG GAGATTCCCGTGGCAGCTGGAGAACTGGCTCGAAAGGAGGACATCGATGCCGTGAT CGCTATTGGGGTCCTGTGCCGAGGAGCAACTCCCAGCTTCGACTACATCGCCTCAGA AGTGAGCAAGGGGCTGGCTGATCTGTCCCTGGAGCTGAGGAAACCTATCACTTTTGG CGTGATTACTGCCGACACCCTGGAACAGGCAATCGAGGCGGCCGGCACCTGCCATG GAAACAAAGGCTGGGAAGCAGCCCTGTGCGCTATTGAGATGGCAAATCTGTTCAAA TCTCTGCGAGGAGGCTCCGGAGGATCTGGAGGGAGTGGAGGCTCAGGAGGAGGCGA CACCATCACACTGCCATGCCGCCCTGCACCACCTCCAAACTGTACCTCCAACATCAC CGGCCTGATTCTGACAAGACAGGGGGGATATAGTAACGATAATACCGTGATTTTCA GGCCCTCAGGAGGGAATTGGAGTGACATCGCACGATGCAATATTACTGGAACAGTG GTCTCTACTCAGCTGTTTCTGAACGGCAGTCTGGCTGGAAACGGGACAGTCATCCGA TCTGAAGACTGGCGGGATAATGCAAAGTCAATTTGTGTGCAGCTGAACACAAGCGT CGAGATCAATTGCACTGGCAATGGGACCTGTAACATTTCTCGGGCCAAATGGGATA ATACCCTGAAGCAGATCGCCAGTAAACTGAGAGAGCAGTACGGCAATAAGACAATC ATCTTCAAGCCTTCTAGTGGAGGCGACCCAGAGTTCGTGAACCATAGCTTTAATTGC GGGAACGTCACCTTTTATTGTGATTCCACACAGCTGTTCGATAGCACTTGGTTTGATTCCACCGGTGGGAGCGGAAGTGGCGGTTCCGGA^7GG4CGGCG4C4CCCC7^C^C7GC ACGAGTACATGCTGGACCTGCAGCCCGAGACCACAGATCTGTACTGCTATGAGCAGCTGA ACGA TAGCTCCGAGGAGGAGGACGAGA TCGA TGGACCAGCAGGACAGGCAGAGCCTGA CAGGGCCCACTATAATATCGTGACCTTCTGCTGTAAGTGCGATTCTACACTGAGGCTGTGC GTGCAGAGCTGCCACGTGGACATCAGAACACTGGAGGATCTGCTGATGGGCACCTGCGG CA TCGTGTGCCCAA TCTGTAGCCAGAAGCCCTGATAAE7_2Tr_nano(SEQ ID NO:59)MDWTWILFLVAAATRVHSMQIYEGKLTAEGLRFGIVASRANHALVDRL VEGAID AIVR HGGREEDITLVRVCGSWEIPVAAGELARKEDIDAVIAIGVLCRGATPSFDYIASEVSKGL ADLSLELRKPITFGVITADTLEQAIEAAGTCHGNKGWEAALCAIEMANLFKSLRGGSGGS GGSGGSGGGDTITLPCRPAPPPNCTSNITGLILTRQGGYSNDNTVIFRPSGGNWSDIARCN ITGTVVSTQLFLNGSLAGNGTVIRSEDWRDNAKSICVQLNTSVEINCTGNGTCNISRAKW DNTLKQIASKLREQYGNKTIIFKPSSGGDPEFVNHSFNCGNVTFYCDSTQLFDSTWFDST GGSGSGGSGMHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHY NIVTFCCKCDSTLRLCVOSTHVDIRTLEDLLMGTLGIVCPICSQKPGGSGSGSRAHYNIVTFC CKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP**(SEQ ID NO:60)ATGGACTGGACCTGGATTCTGTTCCTGGTGGCCGCCGCCACAAGGGTGCACAGCATG CAGATCTACGAAGGAAAACTGACCGCTGAGGGACTGAGGTTCGGAATTGTCGCAAG CCGCGCGAATCACGCACTGGTGGATAGGCTGGTGGAAGGCGCTATCGACGCAATTG TCCGGCACGGCGGGAGAGAGGAAGACATCACACTGGTGAGAGTCTGCGGCAGCTGG GAGATTCCCGTGGCAGCTGGAGAACTGGCTCGAAAGGAGGACATCGATGCCGTGAT CGCTATTGGGGTCCTGTGCCGAGGAGCAACTCCCAGCTTCGACTACATCGCCTCAGAAGTGAGCAAGGGGCTGGCTGATCTGTCCCTGGAGCTGAGGAAACCTATCACTTTTGG CGTGATTACTGCCGACACCCTGGAACAGGCAATCGAGGCGGCCGGCACCTGCCATG GAAACAAAGGCTGGGAAGCAGCCCTGTGCGCTATTGAGATGGCAAATCTGTTCAAA TCTCTGCGAGGAGGCTCCGGAGGATCTGGAGGGAGTGGAGGCTCAGGAGGAGGCGA CACCATCACACTGCCATGCCGCCCTGCACCACCTCCAAACTGTACCTCCAACATCAC CGGCCTGATTCTGACAAGACAGGGGGGATATAGTAACGATAATACCGTGATTTTCA GGCCCTCAGGAGGGAATTGGAGTGACATCGCACGATGCAATATTACTGGAACAGTG GTCTCTACTCAGCTGTTTCTGAACGGCAGTCTGGCTGGAAACGGGACAGTCATCCGA TCTGAAGACTGGCGGGATAATGCAAAGTCAATTTGTGTGCAGCTGAACACAAGCGT CGAGATCAATTGCACTGGCAATGGGACCTGTAACATTTCTCGGGCCAAATGGGATA ATACCCTGAAGCAGATCGCCAGTAAACTGAGAGAGCAGTACGGCAATAAGACAATC ATCTTCAAGCCTTCTAGTGGAGGCGACCCAGAGTTCGTGAACCATAGCTTTAATTGC GGGAACGTCACCTTTTATTGTGATTCCACACAGCTGTTCGATAGCACTTGGTTTGATT CCACCGGYGGGAGCGGAAGYGGCGGYYCCGGAATGCACGGCGACACCCCTACACTGC ACGAGTACATGCTGGACCTGCAGCCCGAGACCACAGATCTGTACTGCTATGAGCAGCTGA ACGATAGCTCCGAGGAGGAGGACGAGATCGATGGACCAGCAGGACAGGCAGAGCCTGA CAGGGCCCACTATAATATCGTGACCTTCTGCTGTAAGTGCGATTCTACACTGAGGCTGTGC GTGCAGAGCACCCACGTGGACATCAGAACACTGGAGGATCTGCTGATGGGCACCCTGGG CA TCGTGTGCCCAA TCTGTAGCCAGAAGCCCGGAGGCAGCGGCTCCGGCTCTAGGGCC CA CT A TA A TA TCGTGA CCTTCTGCTGTAA GTGCGA TTCTA CA CTGA GGCTGTGCGTGC A GA GCA CCCA CGTGGA CA TCA GAA CA CTGGA GGA TCTGCTGA TGGGCA CCCTGGGCA TCGTGTGCCCAA TCTGTA GCCA GAA GCCCTGAYAAE7 2Tr Dsl(SEQ ID N0:61)MDWTWILFLVAAATRVHSMQIYEGKLTAEGLRFGIVASRANHALVDRL VEGAID AIVRHGGREEDITLVRVCGSWEIPVAAGELARKEDIDAVIAIGVLCRGATPSFDYIASEVSKGL ADLSLELRKPITFGVITADTLEQAIEAAGTCHGNKGWEAALCAIEMANLFKSLRGGSGGSGGSGGSGGGDTITLPCRPAPPPNCTSNITGLILTRQGGYSNDNTVIFRPSGGNWSDIARCN ITGTVVSTQLFLNGSLAGNGTVIRSEDWRDNAKSICVQLNTSVEINCTGNGTCNISRAKW DNTLKQIASKLREQYGNKTIIFKPSSGGDPEFVNHSFNCGNVTFYCDSTQLFDSTWFDSTGGSGSGGSGMHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGOAEPDRAHY NIVTFCCKCDSTLRLCVOCTHVDIRTLEDLLMGTLGIVCPICSQKPGGSGSGSRAH NIVTFC CKCDSTLRLCVOSTHVDIRTLEDLLMGCLGIVCPICSOKP^*(SEQ ID NO:62)AEGGACTGGACCTGGATECTGTTCCEGGTGGCCGCCGCCACAAGGGEGCACAGCATG CAGATCTACGAAGGAAAACTGACCGCTGAGGGACTGAGGTTCGGAATTGTCGCAAG CCGCGCGAATCACGCACTGGTGGATAGGCTGGTGGAAGGCGCTATCGACGCAATTGTCCGGCACGGCGGGAGAGAGGAAGACATCACACTGGTGAGAGTCTGCGGCAGCTGG GAGATTCCCGTGGCAGCTGGAGAACTGGCTCGAAAGGAGGACATCGATGCCGTGATCGCTATTGGGGTCCTGTGCCGAGGAGCAACFCCCAGCTTCGACTACATCGCCTCAGA AGTGAGCAAGGGGCTGGCTGATCTGTCCCTGGAGCTGAGGAAACCTATCACTTTTGG CGTGATTACTGCCGACACCCTGGAACAGGCAAECGAGGCGGCCGGCACCTGCCATG GAAACAAAGGCTGGGAAGCAGCCCTGTGCGCTATTGAGATGGCAAATCTGTTCAAA TCTCTGCGAGGAGGCTCCGGAGGATCTGGAGGGAGTGGAGGCTCAGGAGGAGGCGACACCATCACACTGCCATGCCGCCCTGCACCACCTCCAAACTGTACCTCCAACATCAC CGGCCTGATTCTGACAAGACAGGGGGGATATAGTAACGATAATACCGTGATTTTCA GGCCCTCAGGAGGGAATTGGAGTGACATCGCACGATGCAATATTACTGGAACAGTG GTCTCTACTCAGCTGTTTCTGAACGGCAGTCTGGCTGGAAACGGGACAGTCATCCGA TCTGAAGACTGGCGGGATAATGCAAAGTCAATTTGTGTGCAGCTGAACACAAGCGT CGAGATCAATTGCACTGGCAATGGGACCTGTAACATTTCTCGGGCCAAATGGGATA ATACCCTGAAGCAGATCGCCAGTAAACTGAGAGAGCAGTACGGCAATAAGACAATC ATCTTCAAGCCTTCTAGTGGAGGCGACCCAGAGTTCGTGAACCATAGCTTTAATTGC GGGAACGTCACCTTTTATTGTGATTCCACACAGCTGTTCGATAGCACTTGGTTTGATT CCACCGGTGGGAGCGGAAGTGGCGGTTCCGGA47GC4CGGCG4G4CCCC 4C4C7UC ACGAGTACA TGCTGGACCTGCAGCCCGAGACCACAGA TCTGTACTGCTA TGAGCAGCTGA ACGATAGCTCCGAGGAGGAGGACGAGATCGATGGACCAGCAGGACAGGCAGAGCCTGA CAGGGCCCACTATAATATCGTGACCTTCTGCTGTAAGTGCGATTCTACACTGAGGCTGTGC GTGCAGTGCACCCACGTGGACA TCAGAACACTGGAGGA TCTGCTGA TGGGCACCCTGGG CA TCGTGTGCCCAA TCTGTAGCCAGAAGCCCGGAGGCAGCGGCTCCGGCTCTAGGGCC CA CTA TAA TA TCGTGA CCTTCTGCTGTAA GTGCGA TTCTA CA CTGA GGCTGTGCGTGC A GA GCA CCCA CGTGGA CA TCA GAA CA CTGGA GGA TCTGCTGA TGGGCTGCCTGGGCA TCGTGTGCCCAA TCTGTA GCCA GAA GCCCTGAT AAE7_FL_ApRb_nano(SEQ ID NO: 63)MDWTWILFLVAAATRVHSMQIYEGKLTAEGLRFGIVASRANHALVDRL VEGAID AIVR HGGREEDITLVRVCGSWEIPVAAGELARKEDIDAVIAIGVLCRGATPSFDYIASEVSKGL ADLSLELRKPITFGVITADTLEQAIEAAGTCHGNKGWEAALCAIEMANLFKSLRGGSGGS GGSGGSGGGDTITLPCRPAPPPNCTSNITGLILTRQGGYSNDNTVIFRPSGGNWSDIARCN ITGTVVSTQLFLNGSLAGNGTVIRSEDWRDNAKSICVQLNTSVEINCTGNGTCNISRAKW DNTLKQIASKLREQYGNKTIIFKPSSGGDPEFVNHSFNCGNVTFYCDSTQLFDSTWFDST GGSG^GGSGMHGDTPTLHEYMLDLQPETTDLYGYGQLNDSSEEEDEIDGPAGQAEPDRAH YNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP^(SEQ ID NO: 64)ATGGACTGGACCTGGATTCTGTTCCTGGTGGCCGCCGCCACAAGGGTGCACAGCATG CAGATCTACGAAGGAAAACTGACCGCTGAGGGACTGAGGTTCGGAATTGTCGCAAG CCGCGCGAATCACGCACTGGTGGATAGGCTGGTGGAAGGCGCTATCGACGCAATTG TCCGGCACGGCGGGAGAGAGGAAGACATCACACTGGTGAGAGTCTGCGGCAGCTGG GAGATTCCCGTGGCAGCTGGAGAACTGGCTCGAAAGGAGGACATCGATGCCGTGAT CGCTATTGGGGTCCTGTGCCGAGGAGCAACTCCCAGCTTCGACTACATCGCCTCAGA AGTGAGCAAGGGGCTGGCTGATCTGTCCCTGGAGCTGAGGAAACCTATCACTTTTGG CGTGATTACTGCCGACACCCTGGAACAGGCAATCGAGGCGGCCGGCACCTGCCATG GAAACAAAGGCTGGGAAGCAGCCCTGTGCGCTATTGAGATGGCAAATCTGTTCAAA TCTCTGCGAGGAGGCTCCGGAGGATCTGGAGGGAGTGGAGGCTCAGGAGGAGGCGA CACCATCACACTGCCATGCCGCCCTGCACCACCTCCAAACTGTACCTCCAACATCAC CGGCCTGATTCTGACAAGACAGGGGGGATATAGTAACGATAATACCGTGATTTTCAGGCCCTCAGGAGGGAATTGGAGTGACATCGCACGATGCAATATTACTGGAACAGTG GTCTCTACTCAGCTGTTTCTGAACGGCAGTCTGGCTGGAAACGGGACAGTCATCCGA TCTGAAGACTGGCGGGATAATGCAAAGTCAATTTGTGTGCAGCTGAACACAAGCGT CGAGATCAATTGCACTGGCAATGGGACCTGTAACATTTCTCGGGCCAAATGGGATAATACCCTGAAGCAGATCGCCAGTAAACTGAGAGAGCAGTACGGCAATAAGACAATC ATCTTCAAGCCTTCTAGTGGAGGCGACCCAGAGTTCGTGAACCATAGCTTTAATTGC GGGAACGTCACCTTTTATTGTGATTCCACACAGCTGTTCGATAGCACTTGGTTTGATT CCACCGGTGGGAGCGGAAGTGGCGGTTCCGGA47UCT4CGGCGAC^CCCCL4C^CZGCACGAGTACATGCTGGACCTGCAGCCCGAGACCACAGATCTGTACGGCTATGGCCAGCTG AACGATAGCTCCGAGGAGGAGGACGAGATCGATGGACCAGCAGGACAGGCAGAGCCTGA CAGGGCCCACTATAATATCGTGACCTTCTGCTGTAAGTGCGATTCTACACTGAGGCTGTGC GTGCAGAGCACCCACGTGGACA TCAGAACACTGGAGGA TCTGCTGA TGGGCACCCTGGGCA TCGTGTGCCCAA TCTGTAGCCAGAAGCCCTGATAAHPV16 E6 nanoparticle designsAll contain Kozak leader sequence and are in the pVaxl vectorAntigenMutations from WTDeletions from WTE6_FL_nano- changes in this section are denoted relative to this construct(SEQ ID NO: 65)MDWTWILFLVAAATRVHSMQIYEGKLTAEGLRFGIVASRANHALVDRL VEGAID AIVRHGGREEDITLVRVCGSWEIPVAAGELARKEDIDAVIAIGVLCRGATPSFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAIEAAGTCHGNKGWEAALCAIEMANLFKSLRGGSGGSGGSGGSGGGDTITLPCRPAPPPNCTSNITGLILTRQGGYSNDNTVIFRPSGGNWSDIARCNITGTVVSTQLFLNGSLAGNGTVIRSEDWRDNAKSICVQLNTSVEINCTGNGTCNISRAKWDNTLKQIASKLREQYGNKTIIFKPSSGGDPEFVNHSFNCGNVTFYCDSTQLFDSTWFDSTGGSGSGGSGMHQKRTAMFQDPQERPRKLPQLCTELQiriHDIILECVYCKQQLLRREVYDF AFRDLCIVYRDGNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLCPEEKQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRETQL**(SEQ ID NO: 66)ATGGACTGGACCTGGATTCTGTTCCTGGTGGCCGCCGCCACAAGGGTGCACAGCATGCAGATCTACGAAGGAAAACTGACCGCTGAGGGACTGAGGTTCGGAATTGTCGCAAGCCGCGCGAATCACGCACTGGTGGATAGGCTGGTGGAAGGCGCTATCGACGCAATTGTCCGGCACGGCGGGAGAGAGGAAGACATCACACTGGTGAGAGTCTGCGGCAGCTGGGAGATTCCCGTGGCAGCTGGAGAACTGGCTCGAAAGGAGGACATCGATGCCGTGATCGCTATTGGGGTCCTGTGCCGAGGAGCAACTCCCAGCTTCGACTACATCGCCTCAGA AGTGAGCAAGGGGCTGGCTGATCTGTCCCTGGAGCTGAGGAAACCTATCACTTTTGG CGTGATTACTGCCGACACCCTGGAACAGGCAATCGAGGCGGCCGGCACCTGCCATGGAAACAAAGGCTGGGAAGCAGCCCTGTGCGCTATTGAGATGGCAAATCTGTTCAAA TCTCTGCGAGGAGGCTCCGGAGGATCTGGAGGGAGTGGAGGCTCAGGAGGAGGCGA CACCATCACACTGCCATGCCGCCCTGCACCACCTCCAAACTGTACCTCCAACATCAC CGGCCTGATTCTGACAAGACAGGGGGGATATAGTAACGATAATACCGTGATTTTCAGGCCCTCAGGAGGGAATTGGAGTGACATCGCACGATGCAATATTACTGGAACAGTG GTCTCTACTCAGCTGTTTCTGAACGGCAGTCTGGCTGGAAACGGGACAGTCATCCGATCTGAAGACTGGCGGGATAATGCAAAGTCAATTTGTGTGCAGCTGAACACAAGCGT CGAGATCAATTGCACTGGCAATGGGACCTGTAACATTTCTCGGGCCAAATGGGATA ATACCCTGAAGCAGATCGCCAGTAAACTGAGAGAGCAGTACGGCAATAAGACAATCATCTTCAAGCCTTCTAGTGGAGGCGACCCAGAGTTCGTGAACCATAGCTTTAATTGC GGGAACGTCACCTTTTATTGTGATTCCACACAGCTGTTCGATAGCACTTGGTTTGATTCCACCGGYGGGAGCGGAAGYGGCGGYYCCGGAATGCACCAGAAGAGGACCGCCATGT TCCAGGACCCACAGGAGAGGCCCAGAAAGCTGCCTCAGCTGTGCACAGAGCTGCAGACC ACAA TCCACGA TATCA TCCTGGAGTGCGTGTACTGTAAGCAGCAGCTGCTGAGGAGAGAG GTGTA TGACTTCGCCTTTCGGGA TCTGTGCA TCGTGTACCGCGACGGCAACCCA TA TGCCGTGTGCGATAAGTGTCTGAAGTTCTACTCTAAGATCAGCGAGTATCGGCACTACTGTTATTCCCTGTACGGCACCACACTGGAGCAGCAGTATAACAAGCCCCTGTGCGACCTGCTGATCC GCTGCATCAATTGTCAGAAGCCCCTGTGCCCTGAGGAGAAGCAGAGGCACCTGGATAAGA AGCAGAGATTTCACAATATCAGGGGCCGCTGGACCGGCCGGTGTATGTCTTGCTGTAGGA GCTCCAGAA CCCGGCGCGAGA CA CAGCTGTGATA AE6_FL_Dsl_nano(SEQ ID NO: 67)MDWTWILFLVAAATRVHSMQIYEGKLTAEGLRFGIVASRANHALVDRL VEGAID AIVR HGGREEDITLVRVCGSWEIPVAAGELARKEDIDAVIAIGVLCRGATPSFDYIASEVSKGL ADLSLELRKPITFGVITADTLEQAIEAAGTCHGNKGWEAALCAIEMANLFKSLRGGSGGS GGSGGSGGGDTITLPCRPAPPPNCTSNITGLILTRQGGYSNDNTVIFRPSGGNWSDIARCN ITGTVVSTQLFLNGSLAGNGTVIRSEDWRDNAKSICVQLNTSVEINCTGNGTCNISRAKW DNTLKQIASKLREQYGNKTIIFKPSSGGDPEFVNHSFNCGNVTFYCDSTQLFDSTWFDST GGSGSGGSGMHQKRTAMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKOOLLRREVYDF AFRDLCFFRDGNPYAVCDKCLKFYSKISEYRHYCYSLCGTTLEQQYNKPLCDLLCRCINCQK PLCPEEKQRHLDKKQCFHNIRGRCTGRCMSCCRSSRTRRETQL**(SEQ ID NO: 68)ATGGACTGGACCTGGATTCTGTTCCTGGTGGCCGCCGCCACAAGGGTGCACAGCATG CAGATCTACGAAGGAAAACTGACCGCTGAGGGACTGAGGTTCGGAATTGTCGCAAG CCGCGCGAATCACGCACTGGTGGATAGGCTGGTGGAAGGCGCTATCGACGCAATTG TCCGGCACGGCGGGAGAGAGGAAGACATCACACTGGTGAGAGTCTGCGGCAGCTGG GAGATTCCCGTGGCAGCTGGAGAACTGGCTCGAAAGGAGGACATCGATGCCGTGAT CGCTATTGGGGTCCTGTGCCGAGGAGCAACFCCCAGCTTCGACTACATCGCCTCAGA AGTGAGCAAGGGGCTGGCTGATCTGTCCCTGGAGCTGAGGAAACCTATCACTTTTGG CGTGATTACTGCCGACACCCTGGAACAGGCAAECGAGGCGGCCGGCACCTGCCATG GAAACAAAGGCTGGGAAGCAGCCCTGTGCGCTATTGAGATGGCAAATCTGTTCAAA TCTCTGCGAGGAGGCTCCGGAGGATCTGGAGGGAGTGGAGGCTCAGGAGGAGGCGACACCATCACACTGCCATGCCGCCCTGCACCACCTCCAAACTGTACCTCCAACATCAC CGGCCTGATTCTGACAAGACAGGGGGGATATAGTAACGATAATACCGTGATTTTCA GGCCCTCAGGAGGGAATTGGAGTGACATCGCACGATGCAATATTACTGGAACAGTG GTCTCTACTCAGCTGTTTCTGAACGGCAGTCTGGCTGGAAACGGGACAGTCATCCGA TCTGAAGACTGGCGGGATAATGCAAAGTCAATTTGTGTGCAGCTGAACACAAGCGT CGAGATCAATTGCACTGGCAATGGGACCTGTAACATTTCTCGGGCCAAATGGGATA ATACCCTGAAGCAGATCGCCAGTAAACTGAGAGAGCAGTACGGCAATAAGACAATC ATCTTCAAGCCTTCTAGTGGAGGCGACCCAGAGTTCGTGAACCATAGCTTTAATTGC GGGAACGTCACCTTTTATTGTGATTCCACACAGCTGTTCGATAGCACTTGGTTTGATT CCACCGGTGGGAGCGGAAGTGGCGGTTCCGGA^7GCz4CC^G^4G^GG^CCGCC^ZGZ TCCAGGACCCACAGGAGAGGCCCAGAAAGCTGCCTCAGCTGTGCACAGAGCTGCAGACC ACAA TCCACGA TATCA TCCTGGAGTGCGTGTACTGTAAGCAGCAGCTGCTGAGGAGAGAG GTGTA TGACTTCGCCTTTCGGGA TCTGTGCA TCGTGTACCGCGACGGCAACCCA TA TGCC GTGTGCGATAAGTGTCTGAAGTTCTACTCTAAGATCAGCGAGTATCGGCACTACTGTTATT CCCTGTGCGGCACCACACTGGAGCAGCAGTATAACAAGCCCCTGTGCGACCTGCTGTGC CGCTGCATCAATTGTCAGAAGCCCCTGTGCCCTGAGGAGAAGCAGAGGCACCTGGATAAGAAGCAGTGCTTTCACAATATCAGGGGCCGCTGCACCGGCCGGTGTATGTCTTGCTGTAG GAGCTCCAGAACCCGGCGCGAGACACAGCTGTGATAAE6_FL_Ds2_nano(SEQ ID NO: 69)MDWTWILFLVAAATRVHSMQIYEGKLTAEGLRFGIVASRANHALVDRL VEGAID AIVR HGGREEDITLVRVCGSWEIPVAAGELARKEDIDAVIAIGVLCRGATPSFDYIASEVSKGL ADLSLELRKPITFGVITADTLEQAIEAAGTCHGNKGWEAALCAIEMANLFKSLRGGSGGS GGSGGSGGGDTITLPCRPAPPPNCTSNITGLILTRQGGYSNDNTVIFRPSGGNWSDIARCN ITGTVVSTQLFLNGSLAGNGTVIRSEDWRDNAKSICVQLNTSVEINCTGNGTCNISRAKW DNTLKQIASKLREQYGNKTIIFKPSSGGDPEFVNHSFNCGNVTFYCDSTQLFDSTWFDST GGSGSGGSGMHQKRTAMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDF AFRDLCP^RDGNPYAVCDKCLKFYSKISEYRCYCYSLYCTTLEQQYNKPLCDLLIRCINCQKP LCPEEKQRHCDKKQRFHNCRGRWTGRCMSCCRSSRTRRETQL^(SEQ ID NO: 70)ATGGACTGGACCTGGATTCTGTTCCTGGTGGCCGCCGCCACAAGGGTGCACAGCATG CAGATCTACGAAGGAAAACTGACCGCTGAGGGACTGAGGTTCGGAATTGTCGCAAG CCGCGCGAATCACGCACTGGTGGATAGGCTGGTGGAAGGCGCTATCGACGCAATTG TCCGGCACGGCGGGAGAGAGGAAGACATCACACTGGTGAGAGTCTGCGGCAGCTGG GAGATTCCCGTGGCAGCTGGAGAACTGGCTCGAAAGGAGGACATCGATGCCGTGAT CGCTATTGGGGTCCTGTGCCGAGGAGCAACTCCCAGCTTCGACTACATCGCCTCAGA AGTGAGCAAGGGGCTGGCTGATCTGTCCCTGGAGCTGAGGAAACCTATCACTTTTGG CGTGATTACTGCCGACACCCTGGAACAGGCAATCGAGGCGGCCGGCACCTGCCATG GAAACAAAGGCTGGGAAGCAGCCCTGTGCGCTATTGAGATGGCAAATCTGTTCAAA TCTCTGCGAGGAGGCTCCGGAGGATCTGGAGGGAGTGGAGGCTCAGGAGGAGGCGA CACCATCACACTGCCATGCCGCCCTGCACCACCTCCAAACTGTACCTCCAACATCAC CGGCCTGATTCTGACAAGACAGGGGGGATATAGTAACGATAATACCGTGATTTTCAGGCCCTCAGGAGGGAATTGGAGTGACATCGCACGATGCAATATTACTGGAACAGTG GTCTCTACTCAGCTGTTTCTGAACGGCAGTCTGGCTGGAAACGGGACAGTCATCCGA TCTGAAGACTGGCGGGATAATGCAAAGTCAATTTGTGTGCAGCTGAACACAAGCGT CGAGATCAATTGCACTGGCAATGGGACCTGTAACATTTCTCGGGCCAAATGGGATA ATACCCTGAAGCAGATCGCCAGTAAACTGAGAGAGCAGTACGGCAATAAGACAATC ATCTTCAAGCCTTCTAGTGGAGGCGACCCAGAGTTCGTGAACCATAGCTTTAATTGC GGGAACGTCACCTTTTATTGTGATTCCACACAGCTGTTCGATAGCACTTGGTTTGATT CCACCGGTGGGAGCGGAAGTGGCGGTTCCGGAATGCACCAGAAGAGGACCGCCATGTTCCAGGACCCACAGGAGAGGCCCAGAAAGCTGCCTCAGCTGTGCACAGAGCTGCAGACC ACAA TCCACGA TA TCA TCCTGGAGTGCGTGTACTGTAAGCAGCAGCTGCTGAGGAGAGAG GTGTA TGACTTCGCCTTTCGGGA TCTGTGCA TCGTGTACCGCGACGGCAACCCA TA TGCC GTGTGCGATAAGTGTCTGAAGTTCTACTCTAAGATCAGCGAGTATCGGTGCTACTGTTATT CCCTGTACTGCACCACACTGGAGCAGCAGTATAACAAGCCCCTGTGCGACCTGCTGATCC GCTGCATCAATTGTCAGAAGCCCCTGTGCCCTGAGGAGAAGCAGAGGCACTGCGATAAG AAGCAGAGA TTTCACAA TTGCAGGGGCCGCTGGACCGGCCGGTGTA TGTCTTGCTGTAG GAGCTCCAGAACCCGGCGCGAGACACAGCTGTGATAAE6_FL_Sol_nano(SEQ ID NO: 71)MDWTWTLFLVAAATRVHSMQIYEGKLTAEGLRFGTVASRANHALVDRLVEGAIDATVR HGGREEDITLVRVCGSWEIPVAAGELARKEDIDAVIAIGVLCRGATPSFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAIEAAGTCHGNKGWEAALCAIEMANLFKSLRGGSGGS GGSGGSGGGDTITLPCRPAPPPNCTSNITGLILTRQGGYSNDNTVIFRPSGGNWSDIARCN ITGTVVSTQLFLNGSLAGNGTVIRSEDWRDNAKSICVQLNTSVEINCTGNGTCNISRAKW DNTLKQIASKLREQYGNKTIIFKPSSGGDPEFVNHSFNCGNVTFYCDSTQLFDSTWFDSTGGSGSGGSGMHQKRTAMFQDPQERPRKLPQLCTELQTTIHDIILEC^CKQQLLRREVYDF ARIUTLCIVYRDGNPYAVCDKCLKFYSKISEYRHYSYSLYGTTLEQOYNKPLSDLLIRCTNCQKP LSPEEKQRHLDKKQRFHNIRGRWTGRCMSCSRSSRTRRETQL**(SEQ ID NO: 72)ATGGACTGGACCTGGATTCTGTTCCTGGTGGCCGCCGCCACAAGGGTGCACAGCATG CAGATCTACGAAGGAAAACTGACCGCTGAGGGACTGAGGTTCGGAATTGTCGCAAG CCGCGCGAATCACGCACTGGTGGATAGGCTGGTGGAAGGCGCTATCGACGCAATTG TCCGGCACGGCGGGAGAGAGGAAGACATCACACTGGTGAGAGTCTGCGGCAGCTGG GAGATTCCCGTGGCAGCTGGAGAACTGGCTCGAAAGGAGGACATCGATGCCGTGAT CGCTATTGGGGTCCTGTGCCGAGGAGCAACTCCCAGCTTCGACTACATCGCCTCAGA AGTGAGCAAGGGGCTGGCTGATCTGTCCCTGGAGCTGAGGAAACCTATCACTTTTGG CGTGATTACTGCCGACACCCTGGAACAGGCAATCGAGGCGGCCGGCACCTGCCATG GAAACAAAGGCTGGGAAGCAGCCCTGTGCGCTATTGAGATGGCAAATCTGTTCAAATCTCTGCGAGGAGGCTCCGGAGGATCTGGAGGGAGTGGAGGCTCAGGAGGAGGCGA CACCATCACACTGCCATGCCGCCCTGCACCACCTCCAAACTGTACCTCCAACATCAC CGGCCTGATTCTGACAAGACAGGGGGGATATAGTAACGATAATACCGTGATTTTCA GGCCCTCAGGAGGGAATTGGAGTGACATCGCACGATGCAATATTACTGGAACAGTG GTCTCTACTCAGCTGTTTCTGAACGGCAGTCTGGCTGGAAACGGGACAGTCATCCGA TCTGAAGACTGGCGGGATAATGCAAAGTCAATTTGTGTGCAGCTGAACACAAGCGT CGAGATCAATTGCACTGGCAATGGGACCTGTAACATTTCTCGGGCCAAATGGGATAATACCCTGAAGCAGATCGCCAGTAAACTGAGAGAGCAGTACGGCAATAAGACAATC ATCTTCAAGCCTTCTAGTGGAGGCGACCCAGAGTTCGTGAACCATAGCTTTAATTGC GGGAACGTCACCTTTTATTGTGATTCCACACAGCTGTTCGATAGCACTTGGTTTGATT CC ACCGGTGGGAGCGGA AGTGGCGGTTCCGGA^ TGCACCAGAAGAGGACCGCCA TGT TCCAGGACCCACAGGAGAGGCCCAGAAAGCTGCCTCAGCTGTGCACAGAGCTGCAGACC ACAA TCCACGA TA TCA TCCTGGAGTGCGTGTACTGTAAGCAGCAGCTGCTGAGGAGAGAG GTGTA TGACTTCGCCCGGCGGGA TCTGTGCA TCGTGTACCGCGACGGCAACCCA TA TGC CGTGTGCGA TAAGTGTCTGAAGTTCTACTCTAAGA TCAGCGAGTA TCGGCACTACTCCTA T TCCCTGTACGGCACCACACTGGAGCAGCAGTATAACAAGCCCCTGTCCGACCTGCTGATC CGCTGCA TCAA TTGTCAGAAGCCCCTGTCCCCTGAGGAGAAGCAGAGGCACCTGGA TAA GAAGCAGAGATTTCACAATATCAGGGGCCGCTGGACCGGCCGGTGTATGTCTTGCAGCA GGAGCTCCAGAACCCGGCGCGAGACACAGCTGTGATAAE6_FL_Sol_Ds I nano(SEQ ID NO: 73)MDWTWILFLVAAATRVHSMQIYEGKLTAEGLRFGIVASRANHALVDRL VEGAID AIVR HGGREEDITLVRVCGSWEIPVAAGELARKEDIDAVIAIGVLCRGATPSFDYIASEVSKGL ADLSLELRKPITFGVITADTLEQAIEAAGTCHGNKGWEAALCAIEMANLFKSLRGGSGGS GGSGGSGGGDTITLPCRPAPPPNCTSNITGLILTRQGGYSNDNTVIFRPSGGNWSDIARCN ITGTVVSTQLFLNGSLAGNGTVIRSEDWRDNAKSICVQLNTSVEINCTGNGTCNISRAKW DNTLKQIASKLREQYGNKTIIFKPSSGGDPEFVNHSFNCGNVTFYCDSTQLFDSTWFDST GG G GG GMHOKRTAMFODPOERPRKLPOLCTELQTTIHDIILECVYCKOOLLRREVYDF ARRDLC1VYRDGNPYAVCDKCLKFYSK1SEYRHCSYSLYGTTLEQQYNKPLSDLL1RC1NCQKP LSPEEKQRHTDKKQRFHNCRGRWTGRCMSCSRSSRTRRETQT**(SEQ ID NO: 74)ATGGACTGGACCTGGATTCTGTTCCTGGTGGCCGCCGCCACAAGGGTGCACAGCATG CAGATCTACGAAGGAAAACTGACCGCTGAGGGACTGAGGTTCGGAATTGTCGCAAG CCGCGCGAATCACGCACTGGTGGATAGGCTGGTGGAAGGCGCTATCGACGCAATTG TCCGGCACGGCGGGAGAGAGGAAGACATCACACTGGTGAGAGTCTGCGGCAGCTGG GAGATTCCCGTGGCAGCTGGAGAACTGGCTCGAAAGGAGGACATCGATGCCGTGAT CGCTATTGGGGTCCTGTGCCGAGGAGCAACTCCCAGCTTCGACTACATCGCCTCAGA AGTGAGCAAGGGGCTGGCTGATCTGTCCCTGGAGCTGAGGAAACCTATCACTTTTGG CGTGATTACTGCCGACACCCTGGAACAGGCAATCGAGGCGGCCGGCACCTGCCATG GAAACAAAGGCTGGGAAGCAGCCCTGTGCGCTATTGAGATGGCAAATCTGTTCAAA TCTCTGCGAGGAGGCTCCGGAGGATCTGGAGGGAGTGGAGGCTCAGGAGGAGGCGA CACCATCACACTGCCATGCCGCCCTGCACCACCTCCAAACTGTACCTCCAACATCAC CGGCCTGATTCTGACAAGACAGGGGGGATATAGTAACGATAATACCGTGATTTTCA GGCCCTCAGGAGGGAATTGGAGTGACATCGCACGATGCAATATTACTGGAACAGTG GTCTCTACTCAGCTGTTTCTGAACGGCAGTCTGGCTGGAAACGGGACAGTCATCCGA TCTGAAGACTGGCGGGATAATGCAAAGTCAATTTGTGTGCAGCTGAACACAAGCGT CGAGATCAATTGCACTGGCAATGGGACCTGTAACATTTCTCGGGCCAAATGGGATA ATACCCTGAAGCAGATCGCCAGTAAACTGAGAGAGCAGTACGGCAATAAGACAATC ATCTTCAAGCCTTCTAGTGGAGGCGACCCAGAGTTCGTGAACCATAGCTTTAATTGCGGGAACGTCACCTTTTATTGTGATTCCACACAGCTGTTCGATAGCACTTGGTTTGATT CCACCGGTGGGAGCGGAAGTGGCGG YCCGGAATGCACCAGAAGAGGACCGCCATGT TCCAGGACCCACAGGAGAGGCCCAGAAAGCTGCCTCAGCTGTGCACAGAGCTGCAGACC ACAA TCCACGA TATCA TCCTGGAGTGCGTGTACTGTAAGCAGCAGCTGCTGAGGAGAGAG GTGTA TGACTTCGCCCGGCGGGA TCTGTGCA TCGTGTACCGCGACGGCAACCCA TA TGC CGTGTGCGA TAAGTGTCTGAAGTTCTACTCTAAGA TCAGCGAGTA TCGGCACTGCTCCTA T TCCCTGTACGGCACCACACTGGAGCAGCAGTA TAACAAGCCCCTGTCCGACCTGCTGA TC CGCTGCATCAATTGTCAGAAGCCCCTGTCCCCTGAGGAGAAGCAGAGGCACCTGGATAA GAAGCAGAGATTTCACAATTGCAGGGGCCGCTGGACCGGCCGGTGTATGTCTTGCAGCAGGAGCTCCAGAACCCGGCGCGAGACACAGCTGTGATAAE6_Tr_nano(SEQ ID NO: 75) MDWTWILFLVAAATRVHSMQIYEGKLTAEGLRFGIVASRANHALVDRL VEGAID AIVR HGGREEDITLVRVCGSWEIPVAAGELARKEDIDAVIAIGVLCRGATPSFDYIASEVSKGL ADLSLELRKPITFGVITADTLEQAIEAAGTCHGNKGWEAALCAIEMANLFKSLRGGSGGS GGSGGSGGGDTITLPCRPAPPPNCTSNITGLILTRQGGYSNDNTVIFRPSGGNWSDIARCN ITGTVVSTQLFLNGSLAGNGTVIRSEDWRDNAKSICVQLNTSVEINCTGNGTCNISRAKW DNTLKQIASKLREQYGNKTIIFKPSSGGDPEFVNHSFNCGNVTFYCDSTQLFDSTWFDST GG G GGSGMHOKRTAMFODPOERPRKLPOLCTELQTTIHDIILECVYCKOOLLRREVYDF AFRDLCIVFRDGNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEOQYNKPLCDLLIRCINCOKP LCPEEKORHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRETQL**(SEQ ID NO: 76)ATGGACTGGACCTGGATTCTGTTCCTGGTGGCCGCCGCCACAAGGGTGCACAGCATG CAGATCTACGAAGGAAAACTGACCGCTGAGGGACTGAGGTTCGGAATTGTCGCAAG CCGCGCGAATCACGCACTGGTGGATAGGCTGGTGGAAGGCGCTATCGACGCAATTG TCCGGCACGGCGGGAGAGAGGAAGACATCACACTGGTGAGAGTCTGCGGCAGCTGG GAGATTCCCGTGGCAGCTGGAGAACTGGCTCGAAAGGAGGACATCGATGCCGTGAT CGCTATTGGGGTCCTGTGCCGAGGAGCAACTCCCAGCTTCGACTACATCGCCTCAGA AGTGAGCAAGGGGCTGGCTGATCTGTCCCTGGAGCTGAGGAAACCTATCACTTTTGG CGTGATTACTGCCGACACCCTGGAACAGGCAATCGAGGCGGCCGGCACCTGCCATG GAAACAAAGGCTGGGAAGCAGCCCTGTGCGCTATTGAGATGGCAAATCTGTTCAAA TCTCTGCGAGGAGGCTCCGGAGGATCTGGAGGGAGTGGAGGCTCAGGAGGAGGCGA CACCATCACACTGCCATGCCGCCCTGCACCACCTCCAAACTGTACCTCCAACATCAC CGGCCTGATTCTGACAAGACAGGGGGGATATAGTAACGATAATACCGTGATTTTCA GGCCCTCAGGAGGGAATTGGAGTGACATCGCACGATGCAATATTACTGGAACAGTG GTCTCTACTCAGCTGTTTCTGAACGGCAGTCTGGCTGGAAACGGGACAGTCATCCGA TCTGAAGACTGGCGGGATAATGCAAAGTCAATTTGTGTGCAGCTGAACACAAGCGTCGAGATCAATTGCACTGGCAATGGGACCTGTAACATTTCTCGGGCCAAATGGGATA ATACCCTGAAGCAGATCGCCAGTAAACTGAGAGAGCAGTACGGCAATAAGACAATC ATCTTCAAGCCTTCTAGTGGAGGCGACCCAGAGTTCGTGAACCATAGCTTTAATTGC GGGAACGTCACCTTTTATTGTGATTCCACACAGCTGTTCGATAGCACTTGGTTTGATT CCACCGGTGGGAGCGGAAGTGGCGG YCCGGAATGCACCAGAAGAGGACCGCCATGTTCCA GGA COCA CA GGA GA GGCCCA GAAA GCTGCCTCA GCTG TGC A CA GA GC TGC A GA CC ACAA TCCACGA TATCA TCCTGGAGTGCGTGTACTGTAAGCAGCAGCTGCTGAGGAGAGAG GTGTA TGACTTCGCCTTTCGGGA TCTGTGCATCGTGTACCGCGACGGCAACCCA TA TGCC GTGTGCGA TAAGTGTCTGAAGTTCTACTCTAAGATCAGCGAGTA TCGGCACTACTGTTA TT CCCTGTACGGCACCACACTGGAGCAGCAGTATAACAAGCCCCTGTGCGACCTGCTGATCC GCTGCATCAATTGTCAGAAGCCCCTGTGCCCTGAGGAGAAGCAGAGGCACCTGGATAAGA AGCAGAGATTTCACAATATCAGGGGCCGCTGGACCGGCCGGTGTATGTCTTGCTGTAGGA GCTCCAGAA CCCGGCGCGAGA CA CAGCTGTGATAAE6_Tr_Dsl_nano(SEQ ID NO: 77)MDWTWILFLVAAATRVHSMQIYEGKLTAEGLRFGIVASRANHALVDRL VEGAID AIVR HGGREEDITLVRVCGSWEIPVAAGELARKEDIDAVIAIGVLCRGATPSFDYIASEVSKGL ADLSLELRKPITFGVITADTLEQAIEAAGTCHGNKGWEAALCAIEMANLFKSLRGGSGGS GGSGGSGGGDTITLPCRPAPPPNCTSNITGLILTRQGGYSNDNTVIFRPSGGNWSDIARCN ITGTVVSTQLFLNGSLAGNGTVIRSEDWRDNAKSICVQLNTSVEINCTGNGTCNISRAKW DNTLKQIASKLREQYGNKTIIFKPSSGGDPEFVNHSFNCGNVTFYCDSTQLFDSTWFDST GG G GG GMHQKRTAMFQDPQERPRKLPQLCTELQTTIHDin.ECCYCKQQLLRREVYDF AFRDLCIVYRDGNPCAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKP LCPEEKORHLDKKORFHNIRGRWTGRCMSCCRSSRTRRETQL**(SEQ ID NO: 78)ATGGACTGGACCTGGATTCTGTTCCTGGTGGCCGCCGCCACAAGGGTGCACAGCATGCAGATCTACGAAGGAAAACTGACCGCTGAGGGACTGAGGTTCGGAATTGTCGCAAGCCGCGCGAATCACGCACTGGTGGATAGGCTGGTGGAAGGCGCTATCGACGCAATTGTCCGGCACGGCGGGAGAGAGGAAGACATCACACTGGTGAGAGTCTGCGGCAGCTGGGAGATTCCCGTGGCAGCTGGAGAACTGGCTCGAAAGGAGGACATCGATGCCGTGATCGCTATTGGGGTCCTGTGCCGAGGAGCAACTCCCAGCTTCGACTACATCGCCTCAGAAGTGAGCAAGGGGCTGGCTGATCTGTCCCTGGAGCTGAGGAAACCTATCACTTTTGGCGTGATTACTGCCGACACCCTGGAACAGGCAATCGAGGCGGCCGGCACCTGCCATGGAAACAAAGGCTGGGAAGCAGCCCTGTGCGCTATTGAGATGGCAAATCTGTTCAAATCTCTGCGAGGAGGCTCCGGAGGATCTGGAGGGAGTGGAGGCTCAGGAGGAGGCGACACCATCACACTGCCATGCCGCCCTGCACCACCTCCAAACTGTACCTCCAACATCACCGGCCTGATTCTGACAAGACAGGGGGGATATAGTAACGATAATACCGTGATTTTCAGGCCCTCAGGAGGGAATTGGAGTGACATCGCACGATGCAATATTACTGGAACAGTGGTCTCTACTCAGCTGTTTCTGAACGGCAGTCTGGCTGGAAACGGGACAGTCATCCGATCTGAAGACTGGCGGGATAATGCAAAGTCAATTTGTGTGCAGCTGAACACAAGCGTCGAGATCAATTGCACTGGCAATGGGACCTGTAACATTTCTCGGGCCAAATGGGATAATACCCTGAAGCAGATCGCCAGTAAACTGAGAGAGCAGTACGGCAATAAGACAATCATCTTCAAGCCTTCTAGTGGAGGCGACCCAGAGTTCGTGAACCATAGCTTTAATTGCGGGAACGTCACCTTTTATTGTGATTCCACACAGCTGTTCGATAGCACTTGGTTTGATTCCECCGGYGGGAGCGGAAGYGGCGGTYCCGGAATGCACCAGAAGAGGACCGCCATGTTCCAGGACCCACAGGAGAGGCCCAGAAAGCTGCCTCAGCTGTGCACAGAGCTGCAGACCACAA TCCACGA TA TCA TCCTGGAGTGCTGCTACTGTAAGCAGCAGCTGCTGAGGAGAGAG GTGTATGACTTCGCCTTTCGGGATCTGTGCATCGTGTACCGCGACGGCAACCCATGCGCC GTGTGCGATAAGTGTCTGAAGTTCTACTCTAAGATCAGCGAGTATCGGCACTACTGTTATT CCCTGTACGGCACCACACTGGAGCAGCAGTATAACAAGCCCCTGTGCGACCTGCTGATCC GCTGCATCAATTGTCAGAAGCCCCTGTGCCCTGAGGAGAAGCAGAGGCACCTGGATAAGA AGCAGAGA TTTCACAA TA TCAGGGGCCGCTGGACCGGCCGGTGTA TGTCTTGCTGTAGGAGCTCCAGAACCCGGCGCGAGACACAGCTGTGATAAE6_Tr_Ds2_nano(SEQ ID NO: 79) MDWTWILFLVAAATRVHSMQIYEGKLTAEGLRFGIVASRANHALVDRL VEGAID AIVR HGGREEDITLVRVCGSWEIPVAAGELARKEDIDAVIAIGVLCRGATPSFDYIASEVSKGL ADLSLELRKPITFGVITADTLEQAIEAAGTCHGNKGWEAALCAIEMANLFKSLRGGSGGS GGSGGSGGGDTITLPCRPAPPPNCTSNITGLILTRQGGYSNDNTVIFRPSGGNWSDIARCN ITGTVVSTQLFLNGSLAGNGTVIRSEDWRDNAKSICVQLNTSVEINCTGNGTCNISRAKW DNTLKQIASKLREQYGNKTIIFKPSSGGDPEFVNHSFNCGNVTFYCDSTQLFDSTWFDST GG G GG GMHQKRTCMFODPQERPRKLPOLCTELOTTIHDIICECVYCKOOLLRRE^DF AFRDLCIVYRDGNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEOOYNKPLCDIJJRCINCOKP LCPEEKQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRETQL**(SEQ ID NO: 80)ATGGACTGGACCTGGATTCTGTTCCTGGTGGCCGCCGCCACAAGGGTGCACAGCATG CAGATCTACGAAGGAAAACTGACCGCTGAGGGACTGAGGTTCGGAATTGTCGCAAG CCGCGCGAATCACGCACTGGTGGATAGGCTGGTGGAAGGCGCTATCGACGCAATTG TCCGGCACGGCGGGAGAGAGGAAGACATCACACTGGTGAGAGTCTGCGGCAGCTGG GAGATTCCCGTGGCAGCTGGAGAACTGGCTCGAAAGGAGGACATCGATGCCGTGAT CGCTATTGGGGTCCTGTGCCGAGGAGCAACTCCCAGCTTCGACTACATCGCCTCAGA AGTGAGCAAGGGGCTGGCTGATCTGTCCCTGGAGCTGAGGAAACCTATCACTTTTGG CGTGATTACTGCCGACACCCTGGAACAGGCAATCGAGGCGGCCGGCACCTGCCATG GAAACAAAGGCTGGGAAGCAGCCCTGTGCGCTATTGAGATGGCAAATCTGTTCAAA TCTCTGCGAGGAGGCTCCGGAGGATCTGGAGGGAGTGGAGGCTCAGGAGGAGGCGA CACCATCACACTGCCATGCCGCCCTGCACCACCTCCAAACTGTACCTCCAACATCAC CGGCCTGATTCTGACAAGACAGGGGGGATATAGTAACGATAATACCGTGATTTTCA GGCCCTCAGGAGGGAATTGGAGTGACATCGCACGATGCAATATTACTGGAACAGTG GTCTCTACTCAGCTGTTTCTGAACGGCAGTCTGGCTGGAAACGGGACAGTCATCCGA TCTGAAGACTGGCGGGATAATGCAAAGTCAATTTGTGTGCAGCTGAACACAAGCGT CGAGATCAATTGCACTGGCAATGGGACCTGTAACATTTCTCGGGCCAAATGGGATA ATACCCTGAAGCAGATCGCCAGTAAACTGAGAGAGCAGTACGGCAATAAGACAATC ATCTTCAAGCCTTCTAGTGGAGGCGACCCAGAGTTCGTGAACCATAGCTTTAATTGC GGGAACGTCACCTTTTATTGTGATTCCACACAGCTGTTCGATAGCACTTGGTTTGATT CC ACCGGTGGGAGCGG A AGTGGCGGTTCCGGA4 TGCACCAGAAGAGGACCTGCA TGT TCCAGGACCCACAGGAGAGGCCCAGAAAGCTGCCTCAGCTGTGCACAGAGCTGCAGACC ACAA TCCACGA TATCA TCTGCGAGTGCGTGTACTGTAAGCAGCAGCTGCTGAGGAGAGAG GTGTA TGA CTTCGCCTTTCGGGA TCTGTGCA TCGTGTA CCGCGACGGCAA CCCA TA TGCC206193-0149-00WQGTGTGCGA TAAGTGTCTGAAGTTCTACTCTAAGA TCAGCGAGTA TCGGCACTACTGTTA TTCCCTGTACGGCACCACACTGGAGCAGCAGTATAACAAGCCCCTGTGCGACCTGCTGATCCGCTGCATCAATTGTCAGAAGCCCCTGTGCCCTGAGGAGAAGCAGAGGCACCTGGATAAGA AGCAGAGA TTTCACAA TA TCAGGGGCCGCTGGACCGGCCGGTGTA TGTCTTGCTGTAGGAGCTCCAGAACCCGGCGCGAGACACAGCTGTGATAAE6_T r Sol nano(SEQ ID NO: 81)MDWTWILFLVAAATRVHSMQIYEGKLTAEGLRFGIVASRANHALVDRL VEGAID AIVR HGGREEDITLVRVCGSWEIPVAAGELARKEDIDAVIAIGVLCRGATPSFDYIASEVSKGL ADLSLELRKPITFGVITADTLEQAIEAAGTCHGNKGWEAALCAIEMANLFKSLRGGSGGS GGSGGSGGGDTITLPCRPAPPPNCTSNITGLILTRQGGYSNDNTVIFRPSGGNWSDIARCN ITGTVVSTQLFLNGSLAGNGTVIRSEDWRDNAKSICVQLNTSVEINCTGNGTCNISRAKW DNTLKQIASKLREQYGNKTIIFKPSSGGDPEFVNHSFNCGNVTFYCDSTQLFDSTWFDST GG G GG GMHQKRTAMFODPOERPRKLPOLCTELQTTIHDIILECVYCKOOLLRREVYDF ARRDLCIVYRDGNPYAVCDKCLKFYSKISEYRHYSYSLYGTTLEOQYNKPLCDLLIRCINCOKP LCPEEKQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRETQL**(SEQ ID NO: 82)ATGGACTGGACCTGGATTCTGTTCCTGGTGGCCGCCGCCACAAGGGTGCACAGCATG CAGATCTACGAAGGAAAACTGACCGCTGAGGGACTGAGGTTCGGAATTGTCGCAAG CCGCGCGAATCACGCACTGGTGGATAGGCTGGTGGAAGGCGCTATCGACGCAATTG TCCGGCACGGCGGGAGAGAGGAAGACATCACACTGGTGAGAGTCTGCGGCAGCTGG GAGATTCCCGTGGCAGCTGGAGAACTGGCTCGAAAGGAGGACATCGATGCCGTGAT CGCTATTGGGGTCCTGTGCCGAGGAGCAACTCCCAGCTTCGACTACATCGCCTCAGA AGTGAGCAAGGGGCTGGCTGATCTGTCCCTGGAGCTGAGGAAACCTATCACTTTTGG CGTGATTACTGCCGACACCCTGGAACAGGCAATCGAGGCGGCCGGCACCTGCCATG GAAACAAAGGCTGGGAAGCAGCCCTGTGCGCTATTGAGATGGCAAATCTGTTCAAA TCTCTGCGAGGAGGCTCCGGAGGATCTGGAGGGAGTGGAGGCTCAGGAGGAGGCGA CACCATCACACTGCCATGCCGCCCTGCACCACCTCCAAACTGTACCTCCAACATCAC CGGCCTGATTCTGACAAGACAGGGGGGATATAGTAACGATAATACCGTGATTTTCA GGCCCTCAGGAGGGAATTGGAGTGACATCGCACGATGCAATATTACTGGAACAGTG GTCTCTACTCAGCTGTTTCTGAACGGCAGTCTGGCTGGAAACGGGACAGTCATCCGA TCTGAAGACTGGCGGGATAATGCAAAGTCAATTTGTGTGCAGCTGAACACAAGCGT CGAGATCAATTGCACTGGCAATGGGACCTGTAACATTTCTCGGGCCAAATGGGATA ATACCCTGAAGCAGATCGCCAGTAAACTGAGAGAGCAGTACGGCAATAAGACAATC ATCTTCAAGCCTTCTAGTGGAGGCGACCCAGAGTTCGTGAACCATAGCTTTAATTGC GGGAACGTCACCTTTTATTGTGATTCCACACAGCTGTTCGATAGCACTTGGTTTGATT CCACCGGTGGGAGCGGAAGTGGCGGT CCGGAATGCACCAGAAGAGGACCGCCATGT TCCAGGACCCACAGGAGAGGCCCAGAAAGCTGCCTCAGCTGTGCACAGAGCTGCAGACC ACAA TCCACGA TA TCA TCCTGGAGTGCGTGTACTGTAAGCAGCAGCTGCTGAGGAGAGAGGTGTA TGACTTCGCCCGGCGGGA TCTGTGCA TCGTGTACCGCGACGGCAACCCA TA TGC CGTGTGCGATAAGTGTCTGAAGTTCTACTCTAAGATCAGCGAGTATCGGCACTACTCCTATTCCC TG TA CGGCA CCA CA C TGGA GCA GCA GT A T A A CA A GCCCCTG TGC GA CC TGC TGA TC CGCTGCATCAATTGTCAGAAGCCCCTGTGCCCTGAGGAGAAGCAGAGGCACCTGGATAA GAAGCAGAGA TTTCACAA TA TCAGGGGCCGCTGGACCGGCCGGTGTA TGTCTTGCTGTAG GAGCTCCAGAACCCGGCGCGAGACACAGCTGTGATAAE6_FL_Ap53_nano(SEQ ID NO: 83)MDWTWILFLVAAATRVHSMQIYEGKLTAEGLRFGIVASRANHALVDRL VEGAID AIVR HGGREEDITLVRVCGSWEIPVAAGELARKEDIDAVIAIGVLCRGATPSFDYIASEVSKGL ADLSLELRKPITFGVITADTLEQAIEAAGTCHGNKGWEAALCAIEMANLFKSLRGGSGGS GGSGGSGGGDTITLPCRPAPPPNCTSNITGLILTRQGGYSNDNTVIFRPSGGNWSDIARCN ITGTVVSTQLFLNGSLAGNGTVIRSEDWRDNAKSICVQLNTSVEINCTGNGTCNISRAKW DNTLKQIASKLREQYGNKTIIFKPSSGGDPEFVNHSFNCGNVTFYCDSTQLFDSTWFDST GG G GGSGMHOKRTAMFODPOESGRKLPOLCTELQTTIHDIILECVYCKOOLLRREVYDF AFRDLCnrYRDGNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKP LCPEEKORHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRETOL**(SEQ ID NO: 84)ATGGACTGGACCTGGATTCTGTTCCTGGTGGCCGCCGCCACAAGGGTGCACAGCATG CAGATCTACGAAGGAAAACTGACCGCTGAGGGACTGAGGTTCGGAATTGTCGCAAG CCGCGCGAATCACGCACTGGTGGATAGGCTGGTGGAAGGCGCTATCGACGCAATTG TCCGGCACGGCGGGAGAGAGGAAGACATCACACTGGTGAGAGTCTGCGGCAGCTGG GAGATTCCCGTGGCAGCTGGAGAACTGGCTCGAAAGGAGGACATCGATGCCGTGAT CGCTATTGGGGTCCTGTGCCGAGGAGCAACTCCCAGCTTCGACTACATCGCCTCAGA AGTGAGCAAGGGGCTGGCTGATCTGTCCCTGGAGCTGAGGAAACCTATCACTTTTGG CGTGATTACTGCCGACACCCTGGAACAGGCAATCGAGGCGGCCGGCACCTGCCATG GAAACAAAGGCTGGGAAGCAGCCCTGTGCGCTATTGAGATGGCAAATCTGTTCAAA TCTCTGCGAGGAGGCTCCGGAGGATCTGGAGGGAGTGGAGGCTCAGGAGGAGGCGA CACCATCACACTGCCATGCCGCCCTGCACCACCTCCAAACTGTACCTCCAACATCAC CGGCCTGATTCTGACAAGACAGGGGGGATATAGTAACGATAATACCGTGATTTTCA GGCCCTCAGGAGGGAATTGGAGTGACATCGCACGATGCAATATTACTGGAACAGTG GTCTCTACTCAGCTGTTTCTGAACGGCAGTCTGGCTGGAAACGGGACAGTCATCCGA TCTGAAGACTGGCGGGATAATGCAAAGTCAATTTGTGTGCAGCTGAACACAAGCGT CGAGATCAATTGCACTGGCAATGGGACCTGTAACATTTCTCGGGCCAAATGGGATA ATACCCTGAAGCAGATCGCCAGTAAACTGAGAGAGCAGTACGGCAATAAGACAATC ATCTTCAAGCCTTCTAGTGGAGGCGACCCAGAGTTCGTGAACCATAGCTTTAATTGCGGGAACGTCACCTTTTATTGTGATTCCACACAGCTGTTCGATAGCACTTGGTTTGATT CCACCGGYGGGAGCGGAAGYGGCGG YCCGGAATGCACCAGAAGAGGACCGCCATGT TCCAGGACCCACAGGAGAGCGGCAGAAAGCTGCCTCAGCTGTGCACAGAGCTGCAGACC ACAA TCCACGA TATCA TCCTGGAGTGCGTGTACTGTAAGCAGCAGCTGCTGAGGAGAGAG GTGTA TGACTTCGCCTTTCGGGA TCTGTGCA TCGTGTACCGCGACGGCAACCCA TA TGCC GTGTGCGA TAAGTGTCTGAAGTTCTACTCTAAGA TCAGCGAGTA TCGGCACTACTGTTA TT CCCTGTACGGCACCACACTGGAGCAGCAGTATAACAAGCCCCTGTGCGACCTGCTGATCC GCTGCATCAATTGTCAGAAGCCCCTGTGCCCTGAGGAGAAGCAGAGGCACCTGGATAAGAAGCAGAGA TTTCACAA TA TCAGGGGCCGCTGGACCGGCCGGTGTA TGTCTTGCTGTAGGAGCTCCAGAACCCGGCGCGAGACACAGCTGTGATAAE6_FL_Sol_Ap53_nano(SEQ ID NO: 85)MDWTWILFLVAAATRVHSMQIYEGKLTAEGLRFGIVASRANHALVDRL VEGAID AIVR HGGREEDITLVRVCGSWEIPVAAGELARKEDIDAVIAIGVLCRGATPSFDYIASEVSKGL ADLSLELRKPITFGVITADTLEQAIEAAGTCHGNKGWEAALCAIEMANLFKSLRGGSGGS GGSGGSGGGDTITLPCRPAPPPNCTSNITGLILTRQGGYSNDNTVIFRPSGGNWSDIARCN ITGTVVSTQLFLNGSLAGNGTVIRSEDWRDNAKSICVQLNTSVEINCTGNGTCNISRAKW DNTLKQIASKLREQYGNKTIIFKPSSGGDPEFVNHSFNCGNVTFYCDSTQLFDSTWFDST GG G GGSGMHQKRTAMFODPQESGRKLPOLCTELOTTIHDIILECVYCKOOLLRREVYDF AFRDLCFFRDGNPYAVCDKCLKFYSKISEYRHYSYSLYGTTLEQQYNKPLSDLLIRCINCQKP LCPEEKQRHLDKKQRFHNIRGRWTGRCMSCSRSSRTRRETQL* *~(SEQ ID NO: 86)ATGGACTGGACCTGGATTCTGTTCCTGGTGGCCGCCGCCACAAGGGTGCACAGCATG CAGATCTACGAAGGAAAACTGACCGCTGAGGGACTGAGGTTCGGAATTGTCGCAAG CCGCGCGAATCACGCACTGGTGGATAGGCTGGTGGAAGGCGCTATCGACGCAATTG TCCGGCACGGCGGGAGAGAGGAAGACATCACACTGGTGAGAGTCTGCGGCAGCTGG GAGATTCCCGTGGCAGCTGGAGAACTGGCTCGAAAGGAGGACATCGATGCCGTGAT CGCTATTGGGGTCCTGTGCCGAGGAGCAACTCCCAGCTTCGACTACATCGCCTCAGA AGTGAGCAAGGGGCTGGCTGATCTGTCCCTGGAGCTGAGGAAACCTATCACTTTTGG CGTGATTACTGCCGACACCCTGGAACAGGCAATCGAGGCGGCCGGCACCTGCCATG GAAACAAAGGCTGGGAAGCAGCCCTGTGCGCTATTGAGATGGCAAATCTGTTCAAA TCTCTGCGAGGAGGCTCCGGAGGATCTGGAGGGAGTGGAGGCTCAGGAGGAGGCGA CACCATCACACTGCCATGCCGCCCTGCACCACCTCCAAACTGTACCTCCAACATCAC CGGCCTGATTCTGACAAGACAGGGGGGATATAGTAACGATAATACCGTGATTTTCA GGCCCTCAGGAGGGAATTGGAGTGACATCGCACGATGCAATATTACTGGAACAGTG GTCTCTACTCAGCTGTTTCTGAACGGCAGTCTGGCTGGAAACGGGACAGTCATCCGA TCTGAAGACTGGCGGGATAATGCAAAGTCAATTTGTGTGCAGCTGAACACAAGCGT CGAGATCAATTGCACTGGCAATGGGACCTGTAACATTTCTCGGGCCAAATGGGATA ATACCCTGAAGCAGATCGCCAGTAAACTGAGAGAGCAGTACGGCAATAAGACAATC ATCTTCAAGCCTTCTAGTGGAGGCGACCCAGAGTTCGTGAACCATAGCTTTAATTGCGGGAACGTCACCTTTTATTGTGATTCCACACAGCTGTTCGATAGCACTTGGTTTGATT CCACCGGYGGGAGCGGAAGYGGCGG TCCGGAATGCACCAGAAGAGGACCGCCATGT TCCAGGACCCACAGGAGAGCGGCAGAAAGCTGCCTCAGCTGTGCACAGAGCTGCAGACC ACAA TCCACGA TATCA TCCTGGAGTGCGTGTACTGTAAGCAGCAGCTGCTGAGGAGAGAG GTGTA TGACTTCGCCTTTCGGGA TCTGTGCA TCGTGTACCGCGACGGCAACCCA TA TGCC GTGTGCGA TAAGTGTCTGAAGTTCTACTCTAAGA TCAGCGAGTA TCGGCACTACTCCTA TT CCCTGTACGGCACCACACTGGAGCAGCAGTA TAACAAGCCCCTGTCCGACCTGCTGA TGC GCTGCATCAATTGTCAGAAGCCCCTGTGCCCTGAGGAGAAGCAGAGGCACCTGGATAAGAAGCAGAGA TTTCACAA TA TCAGGGGCCGCTGGACCGGCCGGTGTA TGTCTTGCAGCAGG AGCTCCAGAACCCGGCGCGAGACACAGCTGTGATAAE6_Tr_Sol_Ap53 nano(SEQ ID NO: 87)MDWTWILFLVAAATRVHSMQIYEGKLTAEGLRFGIVASRANHALVDRL VEGAID AIVR HGGREEDITLVRVCGSWEIPVAAGELARKEDIDAVIAIGVLCRGATPSFDYIASEVSKGL ADLSLELRKPITFGVITADTLEQAIEAAGTCHGNKGWEAALCAIEMANLFKSLRGGSGGS GGSGGSGGGDTITLPCRPAPPPNCTSNITGLILTRQGGYSNDNTVIFRPSGGNWSDIARCN ITGTVVSTQLFLNGSLAGNGTVIRSEDWRDNAKSICVQLNTSVEINCTGNGTCNISRAKW DNTLKQIASKLREQYGNKTIIFKPSSGGDPEFVNHSFNCGNVTFYCDSTQLFDSTWFDST GGSGSGGSGMHOKRTAMFODPOESGRKLPOLCTELQTTIHDIILECVYCKOOLLRREVYDF AFRDLCIVYRDGNPYAVCDKCLKFYSKISEYRHYSYSLYGTTLEOOYNKPLCDLLIRCINCQKP LGPEEKQRHLDKKQRFHNIRGRWTGRGMSCCRSSRTRRETQL**(SEQ ID NO: 88)ATGGACTGGACCTGGATTCTGTTCCTGGTGGCCGCCGCCACAAGGGTGCACAGCATG CAGATCTACGAAGGAAAACTGACCGCTGAGGGACTGAGGTTCGGAATTGTCGCAAG CCGCGCGAATCACGCACTGGTGGATAGGCTGGTGGAAGGCGCTATCGACGCAATTG TCCGGCACGGCGGGAGAGAGGAAGACATCACACTGGTGAGAGTCTGCGGCAGCTGG GAGATTCCCGTGGCAGCTGGAGAACTGGCTCGAAAGGAGGACATCGATGCCGTGAT CGCTATTGGGGTCCTGTGCCGAGGAGCAACTCCCAGCTTCGACTACATCGCCTCAGA AGTGAGCAAGGGGCTGGCTGATCTGTCCCTGGAGCTGAGGAAACCTATCACTTTTGG CGTGATTACTGCCGACACCCTGGAACAGGCAATCGAGGCGGCCGGCACCTGCCATG GAAACAAAGGCTGGGAAGCAGCCCTGTGCGCTATTGAGATGGCAAATCTGTTCAAA TCTCTGCGAGGAGGCTCCGGAGGATCTGGAGGGAGTGGAGGCTCAGGAGGAGGCGA CACCATCACACTGCCATGCCGCCCTGCACCACCTCCAAACTGTACCTCCAACATCAC CGGCCTGATTCTGACAAGACAGGGGGGATATAGTAACGATAATACCGTGATTTTCA GGCCCTCAGGAGGGAATTGGAGTGACATCGCACGATGCAATATTACTGGAACAGTG GTCTCTACTCAGCTGTTTCTGAACGGCAGTCTGGCTGGAAACGGGACAGTCATCCGA TCTGAAGACTGGCGGGATAATGCAAAGTCAATTTGTGTGCAGCTGAACACAAGCGT CGAGATCAATTGCACTGGCAATGGGACCTGTAACATTTCTCGGGCCAAATGGGATA ATACCCTGAAGCAGATCGCCAGTAAACTGAGAGAGCAGTACGGCAATAAGACAATC ATCTTCAAGCCTTCTAGTGGAGGCGACCCAGAGTTCGTGAACCATAGCTTTAATTGC GGGAACGTCACCTTTTATTGTGATTCCACACAGCTGTTCGATAGCACTTGGTTTGATT CCACCGGYGGGAGCGGAAGYGGCGG TCCGGAATGCACCAGAAGAGGACCGCCATGT TGC A GGA CCCA GA GGA GA GCGGCA GA A A GCTGCCTCA GCTGTGCA GAGA GGTGCA GA GG ACAA TCCACGA TATCA TCCTGGAGTGCGTGTACTGTAAGCAGCAGCTGCTGAGGAGAGAG GTGTA TGAGTTCGCCTTTCGGGA TGTGTGCATCGTGTACCGCGACGGCAACCCA TA TGCC GTGTGGGA TAAGTGTCTGAAGTTCTACTCTAAGATCAGCGAGTA TCGGCACTACTCCTATTCCCTGTACGGCAGGACACTGGAGGAGCAGTATAACAAGGCCCTGTCCGACCTGCTGATCC GCTGCATCAATTGTCAGAAGGGCCTGTGCGGTGAGGAGAAGGAGAGGCACCTGGATAAGA AGCAGAGATTTCACAATATCAGGGGCCGCTGGACCGGCCGGTGTATGTCTTGCAGCAGG AGCTGGAGAACCCGGCGGGAGACACAGGTGTGATAA

[0001] It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be taken as limitations upon the scope of the invention, which is defined solely by the appended claims and their equivalents.

[0002] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including without limitation those relating to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations, or methods of use of the invention, may be made without departing from the spirit and scope thereof.

Claims

CLAIMSWhat is claimed is:

1. A self-assembling nanoparticle subunit polypeptide comprising: a) a scaffold domain for nanoparticle aggregation; and b) an HPV16 antigen domain, wherein the HPV16 antigen domain comprises an E7 or E6 protein, or a fragment or variant thereof, wherein the HPV 16 antigen domain comprises: i) an amino acid sequence having at least about 90% identity over an entire length of SEQ ID NO:5, SEQIDNO:7, SEQIDNO:9, SEQIDNO:11, SEQIDNO:13, SEQ ID NO:15, SEQ1DNO:17, SEQ1DNO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, SEQIDNO:29, SEQIDNO:31, SEQIDNO:33, SEQIDNO:35, SEQIDNO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, or SEQ ID NO:45; or ii) the amino acid sequence as set forth in SEQ ID NO:5, SEQ ID NO:7, SEQ IDNO:9, SEQIDNO:11, SEQIDNO:13, SEQIDNO:15, SEQIDNO:17, SEQIDNO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQIDNO:33, SEQIDNO:35, SEQ ID NO:37, SEQIDNO:39, SEQIDNO:41, SEQ ID NO:43, or SEQ ID NO:45.

2. The self-assembling nanoparticle subunit polypeptide of claim 1, wherein the self-assembling nanoparticle subunit polypeptide comprises:(a) an amino acid sequence having at least about 90% identity over an entire length of SEQ ID NO :47, SEQIDNO:49, SEQIDNO:51, SEQIDNO:53, SEQIDNO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO 69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85 or SEQ ID NO:87; or(b) the amino acid sequence as set forth in SEQ ID NO:47, SEQ ID NO:49, SEQ IDNO:51, SEQIDNO:53, SEQIDNO 55, SEQIDNO:57, SEQIDNO:59, SEQ IDNO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ IDNO:73, SEQ ID NO 75, SEQ ID NO:77, SEQ TD NO:79, SEQ ID N0 81, SEQ ID NO:83, SEQ ID NO:85 or SEQ ID NO:87.

3. A nucleic acid molecule encoding a self-assembling nanoparticle subunit polypeptide of claim 1, wherein the nucleotide sequence encoding the HPV antigen is selected from the group consisting of: a) a nucleotide sequence having at least about 90% identity over an entire length of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO: 22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO 40, SEQ ID NO:42, SEQ ID NO:44 or SEQ ID NO:46; or b) the nucleotide sequence of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44 or SEQ ID NO: 46.

4. The nucleic acid molecule of claim 3, wherein the nucleotide sequence comprises:(a) a nucleotide sequence having at least about 90% identity over an entire length of SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO 74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86 or SEQ ID NO:88; or(b) the nucleotide sequence of SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO 66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86 or SEQ ID NO:88.

5. The nucleic acid molecule of claim 3, wherein the nucleic acid molecule comprises an expression vector.

6. An immunogenic composition of comprising at least one self-assembling nanoparticle subunit polypeptide of claim 1.

7. The immunogenic composition of claim 6, wherein the composition comprises a nanoparticle.

8. The immunogenic composition of claim 6, further comprising a pharmaceutically acceptable excipient.

9. The immunogenic composition of claim 6, further comprising an adjuvant.

10. An immunogenic composition of comprising at least one nucleic acid molecule encoding a self-assembling nanoparticle subunit polypeptide of claim 3.

11. The immunogenic composition of claim 10, wherein the composition comprises a nanoparticle.

12. The immunogenic composition of claim 10, further comprising a pharmaceutically acceptable excipient.

13. The immunogenic composition of claim 10, further comprising an adjuvant.

14. A method of inducing an immune response against human papillomavirus (HPV) in a subject in need thereof, the method comprising administering a self-assembling nanoparticle subunit polypeptide of any one of claims 1-2, a nucleic acid molecule of claims 3-5, or an immunogenic composition of any one of claims 6-13 to the subject.

15. A method of protecting a subject in need thereof from a disease or disorder associate with HPV infection, the method comprising administering a self-assembling nanoparticle subunit polypeptide of any one of claims 1-2, a nucleic acid molecule of claims 3-5, or an immunogenic composition of any one of claims 6-13 to the subject.

16. The method of claim 15, wherein the disease or disorder is cervical cancer, an anogenital cancer or head and neck cancer.

17. A method of treating a subject in need thereof for an HPV infection, the method comprising administering a self-assembling nanoparticle subunit polypeptide of any one of claims 1-2, a nucleic acid molecule of claims 3-5, or an immunogenic composition of any one of claims 6-13 to the subject.

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