Therapeutic human papillomavirus mRNA vaccine and adjuvant for same

Abstract

The present disclosure relates to isolated mRNA encoding human papillomavirus (HPV) oncoproteins and another early viral protein. The present disclosure also relates to compositions for expression of a cyclic GMP-AMP synthase and one or more HPV early proteins in cells of a mammalian subject. The compositions of the present disclosure are useful for stimulating an immune response against HPV.

Description

Docket No.: 16553-20011.40 THERAPEUTIC HUMAN PAPILLOMA VIRUS MRNA VACCINE AND ADJUVANT FOR SAMECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and benefit of U.S. Provisional Patent Application No. 63 / 773,323, filed March 17, 2025, U.S. Provisional Patent Application No. 63 / 829,652, filed June 24, 2025, and U.S. Provisional Patent Application No. 63 / 873,010, filed August 29, 2025, each of which is incorporated by reference herein in its entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The content of the electronic sequence listing (165532001140seqlist.xml; Size: 200,742 bytes; and Date of Creation: March 5, 2026) is herein incorporated by reference in its entirety.FIELD

[0003] The present disclosure relates to isolated mRNA encoding human papillomavirus (HPV) oncoproteins and another early viral protein. The present disclosure also relates to compositions for expression of a cyclic GMP-AMP synthase and one or more HPV early proteins in cells of a mammalian subject. The compositions of the present disclosure are useful for stimulating an immune response against HPV.BACKGROUND

[0004] Despite the availability of prophylactic vaccines against human papillomavirus (HPV) in developed countries, HPV infection results in a significant disease burden globally.Prophylactic HPV vaccination is not adopted widely in all parts of the world, and no therapies currently exist to clear infection from individuals who are already infected. Most incident infections resolve within two years, but a subset of high-risk HPV infections progress to persistent infections that generate clonal populations of precancerous cells that over time may evolve into invasive cancer. HPV infection can result in cervical cancer, other anogenital cancers, and oropharyngeal cancers. Notably, more than half of cervical cancers are caused by HPV type 16 (HPV16) (Bruni et al., ICO / IARC Information Centre on HPV and Cancer (HPV Information Centre). Human Papillomavirus and Related Diseases in the World. Summary Report 10 March 2023), and cervical cancer is the fourth most common cancer in women worldwide with nearly 350,000 deaths in 2022 (World Health Organization, Cervical cancer fact sheet, 2024). Prophylactic vaccines have not been demonstrated to clear persistent infections, likely because1MF-367038161Docket No.: 16553-20011.40 they target the viral protein LI that is no longer expressed at the precancerous and cancerous stages of disease. Thus, identifying therapeutic strategies to control persistent HPV16 infections is highly desired.

[0005] In order to treat persistent HPV16 infections, E6 and E7 viral proteins have been targeted because they drive oncogenicity. E6 and E7 are highly expressed at the late precancerous and cancerous stages of disease, and their expression is required to drive cell proliferation. Although numerous therapeutic HPV vaccines have been tested, none have received approval by the U. S. Food and Drug Administration. In developed countries, the standard of care for high grade cervical dysplasia is the loop electrosurgical excision procedure (LEEP), also known as the large loop excision of the transformation zone (LLETZ). While generally efficacious, LEEP has a number of significant drawbacks including surgical complications, potential for negative impact on reproductive health, and failure to treat the underlying HPV infection. As such, non-invasive therapies for treating pre-cancerous and cancerous HPV lesions are needed in the art.

[0006] Specifically, a therapeutic HPV vaccine for reducing prevalence of HPV infection and / or treating HPV lesions is desirable.BRIEF SUMMARY

[0007] The present disclosure relates to isolated mRNA encoding human papillomavirus (HPV) oncoproteins and another early viral protein. The present disclosure also relates to compositions for expression of a cyclic GMP-AMP synthase and one or more HPV early proteins in cells of a mammalian subject. The compositions of the present disclosure are useful for stimulating an immune response against HPV.BRIEF DESCRIPTION OF THE DRAWINGS

[0008] FIG. 1A is an alignment of amino acid sequences of primate cGASΔN proteins including: human (Homo sapiens, SEQ ID NO:1); chimpanzee Pan troglodytes, SEQ ID NO:7); and western gorilla (Gorilla gorilla, SEQ ID NO:8). FIG. 1B is a cGASΔN consensus sequence (SEQ ID NO:9) derived from the alignment.

[0009] FIG.2 is a cartoon depicting activation of the stimulator of interferon genes (STING) innate immune signaling pathway by a cyclic GMP-AMP synthase (cGAS) that has been engineered to be constitutively-active by removal of its amino-terminal phosphoinositide-binding domain. cGASΔN binds to DNA resulting in the production of cGAMP, the cyclic dinucleotide that activates STING, initiating a signaling cascade that includes the phosphorylation of kinases such as TBK1 and the phosphorylation of transcription factors such as IRF3. IRF3 activation results in the transcription of2MF-367038161Docket No.: 16553-20011.40 inflammatory genes such as interferons, cytokines, chemokines, MHC molecules, and costimulatory molecules.

[0010] FIG.3 depicts mRNA constructs designed for expression of HPV16 antigens. Ten mRNA transcripts were designed to express HPV16 E6 and E7 viral proteins, alone or in combination with El and E2 viral proteins. E6 and E7 were mutated using two different strategies (S and H) to disrupt viral protein activities in immune evasion and cell cycling. El was also mutated with an amino acid substitution (G482D) to ablate enzymatic activity. As strategies to improve expression of the viral proteins, some constructs utilized an IgE leader sequence at the N-terminus and furin cleavable linker sequences in between viral proteins. When furin cleavable linker sequences were not added, viral proteins were directly fused together.

[0011] FIG.4 shows a western blot in which cell lysates were assayed for expression of HPV16 E7 protein, with vinculin detection used as a loading control. Transfection of a human monocytic cell line (THP-1 cells) with HPV16-1 mRNA resulted in detectable E7 expression.

[0012] FIG.5A-5C show that both HPV16-1 and HPV16-2 mRNA constructs induce antigen-specific T cell responses in a dose-dependent manner. Mice were immunized intramuscularly on days 0 and 14 with the indicated doses of either HPV16-1 or HPV16-2. On day 28, (FIG. 5A) frequencies of CD8+ T cells and (FIG.5B) frequencies of E7-specific CD8+ T-cells were quantified by tetramer staining. The sequence of the HPV16 E749-57 peptide is RAHYNIVTF (SEQ ID NO:28). Column graphs show mean and SD. Each symbol represents one mouse, n=5 mice per group. In FIG.5C, the mean percentages of tetramer-specific T-cells in three groups are shown.

[0013] FIG.6A-6B show that cGASΔN enhances antigen-specific T cell responses against HPV E6 and E7. Mice were immunized intramuscularly on days 0 and 14 with the indicated doses of HPV16-1 alone or 0.2 mcg HPV16-1 combined with cGASΔN or green fluorescent protein (GFP) control. On day 35, (FIG.6A) E7-specific and (FIG.6B) E6-specific T-cell responses were assessed by IFNγ ELISPOT. Column graphs show mean and SD. Each symbol represents one mouse, n=6 mice per group.

[0014] FIG.7A-7B show that cGASΔN drives a robust T-cell response against the weakly immunogenic E6 antigen. Mice were immunized intramuscularly on days 0 and 14 with the indicated doses of HPV16-1 alone or 0.5 mcg HPV16-1 combined with cGASΔN or GFP control. On day 42, (FIG.7A) E7-specific and (FIG.7B) E6-specific T-cell responses were assessed by IFNγ ELISPOT. Column graphs show mean and SD. Each symbol represents one mouse, n=5 mice per group.3MF-367038161Docket No.: 16553-20011.40

[0015] FIG.8A-8D show that cGASΔN treatment of human monocyte-derived dendritic cells (moDCs) enhances the antigen-specific CD8+ T-cell IFNγ response. FIG.8A is a cartoon depicting the co-culture assay. In brief, moDCs from an HLA-A02+ donor were treated with stimulators of the STING signaling pathway (cGASΔN LNP or diABZI) or control treatments. HPV16 E711-19peptide was added as an antigen. The sequence of the HPV16 E711-19peptide is YMLDLQPET (SEQ ID NO:53). After 3-4 hours, CD8+ T-cells specific for HPV16 E711-19presented on HLA-A02 were added to the cultures. After an overnight incubation, culture supernatant was collected to measure IFNβ and IFNγ, cytokines released by moDCs and T cells respectively. FIG. 8B shows IFNβ release in the presence of a STING agonist in both the presence and absence of antigen, albeit to much higher levels in the presence of cGASΔN than in the presence of diABZI. FIG.8C shows that antigen-specific IFNγ release was significantly higher in the presence of cGASΔN. FIG.8D shows IFNγ release in supernatants of T-cell monocultures. IFN release was measured using a luminescent immunoassay. Columns indicate mean values, symbols represent technical replicates, and error bars indicate standard deviation. Statistical comparisons were made using 2-way ANOVA. * denotes p<0.05 and **** denotes p<0.0001.

[0016] FIG. 9A-9B show that diABZI treatment directly interferes with CD8+ T-cell IFNγ response. FIG. 9A is a cartoon depicting the activation of a clonal population of human CD8+ T-cells specific for the MART-1 melanoma tumor antigen using anti-CD3 and anti-CD28 antibodies. To test if STING agonists inhibit the IFNγ response, T-cells were treated with diABZI or ENPs containing cGASΔN mRNA. FIG. 9B shows IFNγ release in supernatants of T-cell monocultures the day after culture setup. Columns indicate mean values, symbols represent technical replicates, and error bars indicate standard deviation. Statistical comparisons were made using 2-way ANOVA. *** denotes p<0.001.

[0017] FIG. 10A-10H show HPV-16 antigen-specific IFNγ levels in supernatants after a 72-hour culture of splenocytes from BALB / c or C57BL / 6 mice immunized with one of 10 different HPV mRNA constructs in LNPs or a PBS control. Splenocytes were stimulated with media alone (unstimulated) or media containing 1 μg / mL of Pepmix peptide pools for HPV-16 antigens: (FIG. 10A-10B) El antigen; (FIG. 10C-10D) E2 antigen; (FIG. 10E-10F) E6 antigen; or (FIG. 10G-10H) E7 antigen. Data is shown as means and SD of signal after subtraction of background (unstimulated). Boxes indicate antigen inclusion in each construct. Each symbol represents one mouse, n=5 mice per group.

[0018] FIG. 11A shows that all 10 HPV mRNA constructs in LNPs tested in the presence of cGASΔN mRNA LNPs did not cause unacceptable drops (greater than 25% loss) in THP-1 cell viability. Relative viability measurements were normalized to maximum LDH release control of THP-1 cells after treatment with HPV16-1 through HPV16-10 in the presence of cGASΔN or media (untreated). Column4MF-367038161Docket No.: 16553-20011.40 graphs show mean and SD. Each symbol represents one experimental replicate within a group. FIG. 11B shows that THP-1 cells treated with cGASΔN mRNA LNPs alone or in the presence of HPV mRNA LNPs secrete measurable IFNβ responses detectable by IFNβ lumit. Column graphs show mean and SD. Each symbol represents an experimental replicate per treatment group. FIG. 11C confirms expression of HPV antigens and cGASΔN in THP-1 cells after treatment with HPV16 mRNA LNPs in combination with cGASΔN mRNA LNPs. Untreated THP-1 cells did not express HPV antigens but did express endogenous full-length cGAS. The western blot shows that antigens containing HPV16 E2 (with the exception of HPV16-8) can be detected by western blot with an anti-E2 antibody. cGASΔN was detected with the same anti-cGAS antibody that was used for full length cGAS. B-actin was used as a loading control for HPV 16 antigens and Vinculin was used as a loading control for cGASΔN. Lysates from cells treated with HPV16 mRNA constructs showed expected bands at around 150kDA with the exception of cells treated with HPV 16-6. Since HPV16-6 does not encode El, the expected band size was around 75kDA.

[0019] FIG. 12A shows an assessment of cell viability 1 day after LNP treatments of moDCs. All 4 HPV mRNA constructs in LNPs tested did not cause unacceptable decreases (greater than 25% loss) in viability among moDCs from two different donors (donors: HD149 and HD150). Relative viability was assessed based on LDH release and normalized to the maximum LDH release control. moDCs were treated with HPV16-1, HPV16-3, HPV 16-4 and HPV16-6 in the presence or absence of cGASΔN. Bar graphs show mean and SD. FIG. 12B shows IFNβ responses measured by IFNβ lumit immunoassay after treatment of moDCs with HPV mRNA LNPs alone or in the presence of cGASΔN mRNA LNPs. moDCs from two donors (donor: HD149 and HD150) were treated with HPV16-1, HPV16-3, HPV16-4 and HPV16-6 in the presence or absence of cGASΔN for 24h. IFNP secretion was only detectable when cells were treated with cGASΔN. Bar graphs show mean and SD. FIG. 12C is a confirmation of HPV16 antigen expression visualized by western blot by blotting cell lysates with an anti-HPV16 E2 antibody. Vinculin was used as a loading control. HPV16 antigens were detectable in moDCs from two donors (donors: HD149 and HD150) after treatment with HPV16-3, HPV16-4 or HPV16-6 mRNA LNPs for 24h, alone or in the presence of cGASΔN mRNA LNPs. HPV16-1 does not encode E2. HPV16 mRNA constructs encoding El, E2, E6, and E7 had an expected band at 150 kDa. Since HPV16-6 does not encode El, the expected band size was approximately 75 kDa. FIG. 12D shows an enumeration of HPV epitopes presented by moDCs treated with the indicated mRNAs in LNPs. moDCs from human donors with different MHC haplotypes (donors HD149 and HD150) were treated with HPV16-1, HPV 16-3, HPV 16-4 and HPV16-6 with and without cGASΔN for 24h. After treatment, cell pellets were collected for MHC -I immunoprecipitation and subsequent LC-MS / MS analysis. Detectable5MF-367038161Docket No.: 16553-20011.40 HPV16 MHC-I epitopes were tabulated and mapped to the HPV16 antigen from which they were derived. HPV16-3 treatment of moDCs led to the highest number of HPV16 epitopes in groups that did not receive cGASΔN treatment, while moDCs treated with cGASΔN and HPV16-6 led to the highest number HPV16 epitopes presented on MHC-I.

[0020] FIG. 13A-13B show that LNPs containing cGASΔN mRNA increase CD4+ and CD8+ T-cell immunogenicity of HPV E6 and E7 antigens, respectively. The HPV antigens were expressed from LNPs containing HPV mRNA, which were co-administered with LNPs containing cGASΔN mRNA or Moderna-like LNPs containing GFP mRNA.

[0021] FIG. 14 shows that LNPs containing cGASΔN mRNA induce greater numbers of HPV-specific CD8+ T-cells that secrete TNF-a and / or IL-2 in addition to IFNγ+ than Moderna-like LNPs containing GFP mRNA. In brief, splenocytes were recovered on day 35 and restimulated with an HPV16 E7 peptide library. Modema condition represents an antigen and irrelevant protein only immunization approach using HPV16-1 mRNAand GFP mRNA. In the cGASΔN condition, HPV16-1 mRNALNPs were co-injected with LNPs containing cGASΔN mRNA to increase immunogenicity.

[0022] FIG. 15A-15B depict IFNγ responses as determined from an ELISpot assay in which human moDCs were cocultured with T-cells specific for HPV16 E7 antigen under the indicated control or treatment conditions. moDCs from human donor 136 were cultured with T-cells at a 4:1 ratio, while moDCs from human donor 172 were cultured with T-cells at a 1:1 ratio.

[0023] FIG. 16A-16D show numbers of IFNγ producing cells of CD-I outbred mice immunized with LNPs containing HPV16-6 mRNA or LNPs containing HPV16-6 and cGASΔN mRNAs. Splenocytes were stimulated with media alone (unstimulated) or with 1 μg / mL of Pepmix peptide poolsfor HPV16-6 antigens E2, E6 and E7. After 20 hours of stimulation, IFNγ-i- cells were quantified via ELISPOT. In FIG. 16A each symbol represents one mouse and in FIG. 16B each column represents one mouse. After 6 hours of stimulation, IFNγ+ CD4+ T-cells (FIG. 16C) and IFNγ-i- CD8+ T-cells (FIG. 16D) were quantified by intracellular cytokine staining. n=14-15 mice per group.DETAILED DESCRIPTION

[0024] The present disclosure relates to isolated mRNA encoding human papillomavirus (HPV) oncoproteins and another early viral protein. The present disclosure also relates to compositions for expression of a cyclic GMP-AMP synthase and one or more HPV early proteins in cells of a mammalian subject. The compositions of the present disclosure are useful for stimulating an immune response against HPV.6MF-367038161Docket No.: 16553-20011.40

[0025] Inducing an inflammatory response can be desirable, such as for an immunotherapy or vaccination. STING signaling is of particular interest out of all possible innate immune signaling pathways because, unlike some other signaling pathways, cGAS-STING activation does not induce translation inhibition.

[0026] In order to induce cGAS-STING signaling, a cGAS mutant lacking a portion of its N-terminus was designed such that it is constitutively active (cGASΔN). mRNA encoding the cGAS mutant was packaged in an LNP for cellular uptake and protein expression. The LNP-packaged mRNA encoding cGASΔN has the ability to adjuvant an immune response via the cGAS-STING pathway. Importantly, inclusion of mRNA encoding a constitutively active cGAS, such as cGASΔN, increases the potency of LNPs loaded with mRNA encoding an antigen by increasing inflammatory signals.General Techniques and Definitions

[0027] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art.

[0028] As used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural references unless indicated otherwise. For example, “an” excipient includes one or more excipients.

[0029] The phrase “comprising” as used herein is open-ended, indicating that such embodiments may include additional elements. In contrast, the phrase “consisting of” is closed, indicating that such embodiments do not include additional elements (except for trace impurities). The phrase “consisting essentially of’ is partially closed, indicating that such embodiments may further comprise elements that do not materially change the basic characteristics of such embodiments.

[0030] The term “about” as used herein in reference to a value, encompasses from 90% to 110% of that value and includes 100% of that value (e.g., a molecular weight of about 900 daltons, refers to a molecular weight of from 810 daltons to 990 daltons and includes 900 daltons).

[0031] An “effective amount” or a “sufficient amount” of a substance is that amount sufficient to effect beneficial or desired results, including clinical results, and, as such, an “effective amount” depends upon the context in which it is being applied. For instance, in the context of administering an immunogenic composition comprising one or more mRNAs encoding an antigen and a constitutively -active cGAS, an effective amount contains sufficient mRNA, to stimulate an immune response against the antigen (e.g., antigen-reactive antibody and / or cellular immune response).7MF-367038161Docket No.: 16553-20011.40

[0032] The terms “individual” and “subject” refer to mammals. “Mammals” include, but are not limited to, humans, non-human primates (e.g., monkeys), rodents (e.g., mice and rats), and other animals. In some embodiments, the subject is a human patient, such as a human patient suffering from cancer and / or an infectious disease.

[0033] The term “dose” as used herein in reference to a composition refers to a measured portion of the composition taken by (administered to or received by) a subject at any one time.

[0034] The terms “isolated” and “purified” as used herein refers to a material that is removed from at least one component with which it is naturally associated (e.g., removed from its original environment). As an example, when used in reference to a phospholipid, an isolated phospholipid is at least 90%, 95%, 96%, 97%, 98% or 99% pure as determined by thin layer chromatography, or gas chromatography. As a further example, when used in reference to a recombinant protein, an isolated protein refers to a protein that has been removed from the culture medium of the host cell that produced the protein.

[0035] The terms “pharmaceutical formulation” and “pharmaceutical composition” refer to preparations that are in such form as to permit the biological activity of the active ingredient to be effective, and that contain no additional components that are unacceptably toxic to an individual to which the formulation or composition would be administered. Such formulations or compositions are intended to be sterile.

[0036] “Excipients” as used herein include pharmaceutically acceptable excipients, carriers, vehicles or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable excipient is an aqueous pH buffered solution.

[0037] The term “antigen” refers to a substance that is recognized and bound specifically by an antibody or by a T cell antigen receptor. Antigens can include peptides, polypeptides, proteins, glycoproteins, polysaccharides, complex carbohydrates, sugars, gangliosides, lipids and phospholipids; portions thereof and combinations thereof. In the context of the present disclosure, the term “antigen” typically refers to a polypeptide encoded by a nucleic acid sequence of a mRNA or a DNA. Polypeptide antigens are preferably at least eight amino acid residues in length, and may comprise one or more post-translational modifications.

[0038] The term “agonist” is used in the broadest sense and includes any molecule that activates signaling through a receptor. In some embodiments, the agonist binds to the receptor. For instance, a TLR8 agonist binds to a TLR8 and activates a TLR8-signaling pathway.8MF-367038161Docket No.: 16553-20011.40

[0039] “Alkyl” refers to monovalent saturated aliphatic hydrocarbyl groups. Cx alkyl refers to an alkyl group having x number of carbon atoms. Cx-Cy alkyl or Cx-y alkyl refers to an alkyl group having between x number and y number of carbon atoms, inclusive.

[0040] “Alkylene” refers to divalent saturated aliphatic hydrocarbyl groups.

[0041] “Alkenyl” refers to monovalent hydrocarbyl groups having at least one double bond (> C=C<). Cx alkenyl refers to an alkenyl group having x number of carbon atoms. Cx-Cy alkenyl or Cx-y alkenyl refers to an alkenyl group having between x number and y number of carbon atoms, inclusive.

[0042] “Stimulation” of a response or parameter includes eliciting and / or enhancing that response or parameter when compared to otherwise same conditions except for a parameter of interest, or alternatively, as compared to another condition (e.g., increase in TLR-signaling in the presence of a TLR agonist as compared to the absence of the TLR agonist). For example, “stimulation” of an immune response means an increase in the response. Depending upon the parameter measured, the increase may be from 2-fold to 2,000-fold, or from 5-fold to 500-fold or over, or from 2, 5, 10, 50, or 100-fold to 500, 1,000, 2,000, 5,000, or 10,000-fold.

[0043] Conversely, “inhibition” of a response or parameter includes reducing and / or repressing that response or parameter when compared to otherwise same conditions except for a parameter of interest, or alternatively, as compared to another condition (e.g., decrease in abnormal cell proliferation after administration of a composition of the present disclosure as compared to the administration of a placebo composition or no treatment). For example, “inhibition” of an immune response means a decrease in the response. Depending upon the parameter measured, the decrease may be from 2-fold to 2,000-fold, or from 5-fold to 500-fold or over, or from 2, 5, 10, 50, or 100-fold to 500, 1,000, 2,000, 5,000, or 10,000-fold.

[0044] The relative terms “higher” and “lower” refer to a measurable increase or decrease, respectively, in a response or parameter when compared to otherwise same conditions except for a parameter of interest, or alternatively, as compared to another condition. For instance, a “higher level of DC hyperactivation” refers to a level of DC hyperactivation as a consequence of a treatment condition that is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold above a level of DC hyperactivation as a consequence of a control condition. Likewise, a “lower level of DC hyperactivation” refers to a level of DC hyperactivation as a consequence of a treatment condition that is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold below a level of DC hyperactivation as a consequence of a control condition.9MF-367038161Docket No.: 16553-20011.40

[0045] As used herein the term “immunization” refers to a process that increases a mammalian subject’s immune response to an antigen and therefore improves its ability to resist or overcome infection and / or resist disease.

[0046] The term “vaccination” as used herein refers to the introduction of vaccine into a body of a mammalian subject.

[0047] “Adjuvant” refers to a substance which, when added to a composition comprising an antigen or a nucleic acid encoding an antigen, enhances or potentiates an immune response to the antigen in the mammalian recipient upon exposure.

[0048] The terms “treating” or “treatment” of a disease refer to executing a protocol, which may include administering one or more therapeutic agents to an individual (human or otherwise), in an effort to obtain beneficial or desired results in the individual, including clinical results. Beneficial or desired clinical results include, but are not limited to, alleviation or amelioration of one or more signs or symptoms of a disease, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total). “Treatment” also can mean prolonging survival as compared to expected survival of an individual not receiving treatment. Further, “treating” and “treatment” may occur by administration of one dose of a therapeutic agent or therapeutic agents, or may occur upon administration of a series of doses of a therapeutic agent or therapeutic agents. “Treating” or “treatment” does not require complete alleviation of signs or symptoms, and does not require a cure, and specifically includes protocols that have only a palliative effect on the individual. “Palliating” a disease or disorder means that the extent and / or undesirable clinical manifestations of the disease or disorder are lessened and / or time course of progression of the disease or disorder is slowed, as compared to the expected untreated outcome.

[0049] The term “constitutively-active” as used herein in reference to cGAS refers to a cGAS variant that displays self-DNA reactivity, leading to cGAMP synthesis under conditions in which native cGAS (e.g., full length human cGAS) has little to no enzymatic activity. In some preferred embodiments, the “constitutively-active cGAS” is a “truncated cGAS”, such as cGASΔN comprising a C-terminal DNA-binding, enzymatic domain in the absence of a N-terminal disordered domain. That is, cGASΔN is a constitutively-active cGAS devoid of regulation of enzymatic activity imparted by the N-terminal disordered domain of full length cGAS.

[0050] “Percent (%) sequence identity” with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino10MF-367038161Docket No.: 16553-20011.40 acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, the % sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y, where X is the number of amino acid residues scored as identical matches by the sequence in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % sequence identity of A to B will not equal the % sequence identity of B to A.I. Cyclic GMP-AMP Synthase

[0051] Compositions and methods of the present disclosure may comprise an mRNA encoding a cyclic GMP-AMP synthase (cGAS), also referred to as cGAMP synthase. cGAS, is an enzymatic sensor of cytosolic DNA, which recognizes double-stranded DNA independent of its sequence resulting in dimerization, formation of liquid-like droplets and production of the secondary messenger 2’3’cyclic GMP-AMP (cGAMP), which binds to and activates STING resulting in expression of interferons and other inflammatory mediators. Specifically, STING activation results in a signaling cascade that ultimately activates IRF3 and NF-kB transcription factors. IRF3 activation leads to the expression of genes such as IP-10 and Type I interferons that induce antiviral immune responses. NF-kB activation induces the expression of inflammatory cytokines such as IL-6 that enhance inflammatory immune responses.

[0052] Human cGAS is 522 amino acids in length, including a N-terminal phosphoinositidebinding domain (residues 1-59) and a C-terminal DNA-binding and enzymatic domain (residues 160-522) (Barnett et al., Cell, 176:1432-1446, 2019). Importantly, expression of cGASΔN in a human leukemia monocytic cell line was found to result in higher levels of expression of interferon and interferon-stimulated genes (Barnett, supra, 2019).

[0053] The amino acid sequence of human cGAS (GenBank No. NP_612450.2) is:MQPWHGKAMQRASEAGATAPKASARNARGAPMDPTESPAAPEAALPKAGKFGPARKSGSRQKKSAPDTQE11MF-367038161Docket No.: 16553-20011.40 RPPVRATGARAKKAPQRAQDTQPSDATSAPGAEGLEPPAAREPALSRAGSCRQRGARCSTKPRPPPGPWD VPSPGLPVSAPILVRRDAAPGASKLRAVLEKLKLSRDDISTAAGMVKGWDHLLLRLKCDSAFRGVGLLN TGSYYEHVKISAPNEFDVMFKLEVPRIQLEEYSNTRAYYFVKFKRNPKENPLSQFLEGEILSASKMLSKF RKIIKEEINDIKDTDVIMKRKRGGSPAVTLLISEKISVDITLALESKSSWPASTQEGLRIQNWLSAKVRK QLRLKPFYLVPKHAKEGNGFQEETWRLSFSHIEKEILNNHGKSKTCCENKEEKCCRKDCLKLMKYLLEQL KERFKDKKHLDKFSSYHVKTAFFHVCTQNPQDSQWDRKDLGLCFDNCVTYFLQCLRTEKLENYFIPEFNL FSSNLIDKRSKEFLTKQIEYERNNEFPVFDEF (SEQ ID NO: 10).

[0054] The amino acid sequence of the N-terminal domain of cGAS is:MQPWHGKAMQRASEAGATAPKASARNARGAPMDPTESPAAPEAALPKAGKFGPARKSGSRQKKSAPDTQE RPPVRATGARAKKAPQRAQDTQPSDATSAPGAEGLEPPAAREPALSRAGSCRQRGARCSTKPRPPPGPWD VP SPGLPVSAP ILVRRDAA (SEQ ID NO: 11 ).

[0055] The amino acid sequence of the C-terminal domain of cGAS (cGASΔN) is:PGASKLRAVLEKLKLSRDDISTAAGMVKGVVDHLLLRLKCDSAFRGVGLLNTGSYYEHVKISAPNEFDVM FKLEVPRIQLEEYSNTRAYYFVKFKRNPKENPLSQFLEGEILSASKMLSKFRKIIKEEINDIKDTDVIMK RKRGGSPAVTLLISEKISVDITLALESKSSWPASTQEGLRIQNWLSAKVRKQLRLKPFYLVPKHAKEGNG FQEETWRLSFSHIEKEILNNHGKSKTCCENKEEKCCRKDCLKLMKYLLEQLKERFKDKKHLDKFSSYHVK TAFFHVCTQNPQDSQWDRKDLGLCFDNCVTYFLQCLRTEKLENYFIPEFNLFSSNLIDKRSKEFLTKQIE YERNNEFPVFDEF (SEQ ID NO: 1 ).

[0056] The nucleotide sequence encoding human cGASΔN, which was codon-optimized for expression in mouse cells is set forth as SEQ ID NO: 17. A DNA template with this nucleotide sequence was used to prepare mRNA encoding human cGASΔN, which was subsequently loaded into LNPs. The nucleotide sequence encoding human cGASΔN, which was codon-optimized for expression in human cells is set forth as SEQ ID NQ:40.

[0057] Compositions and methods of the present disclosure comprise a nucleic acid encoding a constitutively-active cGAS as a catalytic adjuvant for improving adaptive immune responses elicited by mRNA vaccines. In some preferred embodiments, the constitutively-active cGAS is a truncated cGAS devoid of the N-terminal phosphoinositide-binding domain (cGASΔN). In some preferred embodiments, the constitutively-active cGAS is a truncated cGAS comprising the C-terminal DNA-binding and enzymatic domain (cGASΔN).

[0058] Homologs of cGAS are expressed in species across the animal kingdom, and cGAS amino acid sequences are conserved in higher primates. An alignment of the amino acid sequence of the c-terminal domain of human cGAS with the amino acid sequences of the c-terminal domains of chimp12MF-367038161Docket No.: 16553-20011.40 and gorilla cGAS proteins is shown in FIG. 1. A percent identity matrix for primate cGASΔN proteins is shown in Table I.Table I. Percent Identity Matrix Created by Clustal 12.1A# Species 1 2 3 4 5 6 7 8 1 M. mulatto 100.00 96.42 87.53 85.95 88.71 87.26 86.78 86.50 2 P. anubis 96.42 100.00 87.53 86.23 88.98 88.09 87.88 87.33 3 N. leucogenys 87.53 87.53 100.00 96.14 93.37 89.97 90.03 90.30 4 H. lar 85.95 86.23 96.14 100.00 92.31 88.92 88.98 88.71 5 P. abelii 88.71 88.98 93.37 92.31 100.00 93.07 93.39 92.84 6 P. troglodytes 87.26 88.09 89.97 88.92 93.07 100.00 97.51 96.95 7 H. sapiens 86.78 87.88 90.03 88.98 93.39 97.51 100.00 98.358 G. gorilla 86.50 87.33 90.30 88.71 92.84 96.95 98.35 100.00 ^Numbers in this table are not SEQ ID NOS.

[0059] In some preferred embodiments, the constitutively-active cGAS is a truncated human cGAS devoid of the N-terminal domain (SEQ ID NO:11). In some preferred embodiments, the constitutively-active cGAS is a truncated human cGAS comprising the C-terminal domain (SEQ ID NO:1). In some preferred embodiments, cGASΔN comprises the amino acid sequence of SEQ ID NO:1 or the amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:1. In some preferred embodiments, the truncated human cGAS comprises the C-terminal domain (SEQ ID NO:1) with one, two, three or four conservative substitutions, or up to 18, or up to 36 conservative substitutions. In some preferred embodiments, cGASΔN comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, or the amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8. In some preferred embodiments, cGASΔN comprises the consensus amino acid sequence of SEQ ID NO:9. For expression in transfected cells, the nucleic acid encoding cGASΔN is in operable combination with a start codon (e.g., ATG, CUG, GUG or ACG, preferably ATG).II. Human Papillomavirus Antigen(s)

[0060] Compositions and methods of the present disclosure comprise an mRNA encoding human papillomavirus (HPV) antigen(s).

[0061] The HPV antigen(s) of the present disclosure comprise one or both of HPV E6 and E7 proteins or fragments thereof. In some embodiments, the HPV antigen comprises E6. In some embodiments, the HPV antigen comprises E7. In some embodiments, the HPV antigen comprises both E613MF-367038161Docket No.: 16553-20011.40 and E7. In some embodiments, the HPV antigen(s) comprises E6, E7 and one or both of El and E2. In some embodiments, the HPV antigen(s) comprises E6, E7, El and E2.

[0062] The terms “polypeptide” and “protein” are used interchangeably herein in reference to antigens that comprise peptide chains that are at least 8, 9 or 10 amino acids in length. In some embodiments, the HPV antigen(s) are together more than about 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150 or 1200 amino acids in length, and less than about 1500, 1400, 1300, 1200, 1100, 1000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300 or 250 amino acids in length. Unless the HPV antigen is specified as being a full length antigen or as comprising a SEQ ID NO of a full length protein, the term HPV antigen encompasses fragments thereof.

[0063] In some embodiments, the HPV antigen(s) are of a high-risk type of HPV (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73 and 82 types). In some embodiments, the HPV antigen(s) are of one or both of HPV 16 and HPV18 antigens. In some embodiments, the HPV antigen(s) are HPV16 antigens. The polypeptide may be post-translationally modified such as by phosphorylation, hydroxylation, sulfonation, palmitoylation, and / or glycosylation.

[0064] HPV E6 and E7 are drivers of oncogenicity in persistent infection. HPV El and E2 are larger proteins that increase the number of available immunogenic epitopes. El, E6, and E7 have been identified to inhibit innate immune signaling by multiple mechanisms (Castro-Munoz et al., Sci Rep. 9(l):13620, 2019; Lo Cigno et al., J Med Virol., 96(6):e29685, 2024). Mutations may be introduced into one or more of these three proteins to ablate their immune evasion activities. Desirable mutations in E6 and E7 are present in “S” versions of E6 and E7 sequences of SEQ ID NO:18 and SEQ ID NO:21, respectively. Other desirable mutations in E6 and E7 are present in “H” versions of E6 and E7 sequences of SEQ ID NO:19 and SEQ ID NO:22, respectively.

[0065] Compositions and methods of the present disclosure comprise a nucleic acid encoding an HPV E6 antigen or fragment thereof. The amino acid sequence of exemplary HPV16 E6 antigens are set forth as SEQ ID NO:18 and SEQ ID NO:19, with a consensus amino acid sequence set forth as SEQ ID NQ:20. The amino acid sequence of an exemplary HPV18 E6 antigen is set forth as SEQ ID NO:43. In some embodiments, the amino acid sequence of the E6 antigen is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:18. In some embodiments, the amino acid sequence of the E6 antigen comprises SEQ ID NO:18. In some embodiments, the amino acid sequence of the E6 antigen is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:19. In some embodiments, the amino acid sequence of the E6 antigen comprises SEQ ID NO: 19. In some embodiments, the amino acid sequence of the E6 antigen is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NQ:20. In 14MF-367038161Docket No.: 16553-20011.40 some embodiments, the amino acid sequence of the E6 antigen comprises SEQ ID NO:20. In some embodiments, the amino acid sequence of the E6 antigen is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:43. In some embodiments, the amino acid sequence of the E6 antigen comprises SEQ ID NO: 43. In some embodiments, the HPV E6 antigen is from about 100 to about 160 amino acids in length, optionally from about 100, 110, 120, 130, 140 or 150 amino acids in length, and less than about 160, 150, 140, 130, 120 or 110 amino acids in length.

[0066] Compositions and methods of the present disclosure comprise a nucleic acid encoding an HPV E7 antigen or fragment thereof. The amino acid sequence of exemplary HPV16 E7 antigens are set forth as SEQ ID NO:21 and SEQ ID NO:22, with a consensus amino acid sequence set forth as SEQ ID NO:23. The amino acid sequence of an exemplary HPV18 E7 antigen is set forth as SEQ ID NO:44. In some embodiments, the amino acid sequence of the E7 antigen is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:21. In some embodiments, the amino acid sequence of the E7 antigen comprises SEQ ID NO:21. In some embodiments, the amino acid sequence of the E7 antigen is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:22. In some embodiments, the amino acid sequence of the E7 antigen comprises SEQ ID NO:22. In some embodiments, the amino acid sequence of the E7 antigen is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:23. In some embodiments, the amino acid sequence of the E7 antigen comprises SEQ ID NO:23. In some embodiments, the amino acid sequence of the E7 antigen is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:44. In some embodiments, the amino acid sequence of the E7 antigen comprises SEQ ID NO:44. In some embodiments, the HPV E7 antigen is from about 50 to about 110 amino acids in length, optionally from about 50, 60, 70, 80, 90 or 100 amino acids in length, and less than about 110, 100, 90, 80, 70 or 60 amino acids in length.

[0067] Some compositions and methods of the present disclosure comprise a nucleic acid encoding an HPV E6 antigen and an HPV E7 antigen. In some embodiments, the E6 and E7 antigens are expressed as an E6-E7 fusion protein or an E7-E6 fusion protein. The amino acid sequence of exemplary E7-E6 fusion protein is set forth as SEQ ID NO: 31. In some embodiments, the amino acid sequence of the E7-E6 fusion protein is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:31. In some embodiments, the amino acid sequence of the E7-E6 fusion protein comprises SEQ ID NO:31. In other embodiments, the E6 and E7 antigens are expressed as separate antigens by virtue of their coding regions being separated by an intervening sequence. In an exemplary embodiment, the coding region of the E6 antigen is separated from the coding region of the E7 antigen by a furin cleavable linker (SEQ ID NO:27) as the intervening sequence. In an exemplary embodiment, the amino acid sequence of the E6-linker-E7 is set forth as SEQ ID NO:45 and as residues 19-264 of SEQ ID NQ:30. Residues 1-18 of SEQ15MF-367038161Docket No.: 16553-20011.40 ID NO:30 correspond to an IgE leader sequence, which is also set forth as SEQ ID NO:26. In some embodiments, the amino acid sequence of the E6-linker-E7 is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:45 or comprises SEQ ID NO:45. In some embodiments, the amino acid sequence of the E7 antigen comprises SEQ ID NO:22.

[0068] Some compositions and methods of the present disclosure comprise a nucleic acid further encoding an HPV El antigen or fragment thereof. In some embodiments, the amino acid sequence of the El antigen is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:24. In some embodiments, the amino acid sequence of the El antigen comprises SEQ ID NO:24. In some embodiments, the amino acid sequence of the El antigen is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:41. In some embodiments, the amino acid sequence of the El antigen comprises SEQ ID NO:41. In some embodiments, the El antigen comprises a G482D substitution.

[0069] Additionally, some compositions and methods of the present disclosure comprise a nucleic acid further encoding an HPV E2 antigen or fragment thereof. In some embodiments, the amino acid sequence of the E2 antigen is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:25. In some embodiments, the amino acid sequence of the E2 antigen comprises SEQ ID NO:25. In some embodiments, the amino acid sequence of the E2 antigen is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:42. In some embodiments, the amino acid sequence of the E2 antigen comprises SEQ ID NO:42.

[0070] In some preferred embodiments, the HPV antigen comprises two, three or four HPV antigens (E6, E7 and one or both of El and E2). In some embodiments, the coding regions of the HPV antigens are separated by an intervening sequence. Intervening sequences for use in the constructs of the present disclosure include but are not limited to 2A-like peptide sequences, protease cleavage sites, and an internal ribosome entry sites (IRES). In some embodiments, the coding regions of the HPV antigens are separated by a 2A-like peptide sequence, including but not limited to T2A (SEQ ID NO: 12), P2A (SEQ ID NO:13), E2A (SEQ ID NO:14), and F2A (SEQ ID NO:15) peptide sequences. Additional 2A-like peptide sequences are known in the art (see, e.g., Luke et al., J. Gen. Virol, 89:1036-1042, 2008, 2AL sequences of Figure 2 are incorporated herein by reference). In some embodiments, the intervening sequence is a protease cleavage site, such as a furin cleavage site (SEQ ID NO:27). In some embodiments, intervening sequence is an IRES. In some embodiments, the coding region of the HPV antigens is preceded by a mammalian leader or signal peptide sequence, such as an IgE leader sequence (SEQ ID NO:26).

[0071] Exemplary HPV 16 mRNA constructs are illustrated in FIG.3, with their amino acid sequences set forth as SEQ ID NQs:30-39. In some embodiments, the HPV antigen does not comprise an 16MF-367038161Docket No.: 16553-20011.40 LI antigen and / or an L2 antigen. In some embodiments, the nucleic acids of the present disclosure do not comprise a full HPV genome.

[0072] In some embodiments, the nucleic acid encodes an IgE leader, an E6 antigen, and an E7 antigen. In some embodiments, the IgE leader is fused to the E6 antigen, which is linked by a furin cleavable linker to the E7 antigen. In some embodiments, the E6 antigen is E6 (S). In some embodiments, the E7 antigen is E7 (S). In some preferred embodiments, the amino acid sequence of the HPV antigen comprises SEQ ID NO:30, or the amino acid sequence of at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:30. In some preferred embodiments, the amino acid sequence of the HPV antigen comprises SEQ ID NQ:30. In some preferred embodiments, the amino acid sequence of the HPV antigen is SEQ ID NQ:30.

[0073] In some embodiments, the nucleic acid encodes an E6 antigen and an E7 antigen. In some embodiments, the E6 antigen is fused to the E7 antigen. In some embodiments, the E6 antigen is E6 (H). In embodiments, the E7 antigen is E7 (H). In some preferred embodiments, the amino acid sequence of the HPV antigen comprises SEQ ID NO:31, or the amino acid sequence of at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:31. In some preferred embodiments, the amino acid sequence of the HPV antigen comprises SEQ ID NO:31. In some preferred embodiments, the amino acid sequence of the HPV antigen is SEQ ID NO:31.

[0074] In some embodiments, the nucleic acid encodes an IgE leader, an E6 antigen, an E7 antigen, an El antigen, and an E2 antigen. In some embodiments, the IgE leader is fused to the E6 antigen, which is linked by a furin cleavable linker to the E7 antigen. The E7 antigen is linked by a furin cleavable linker to the El antigen. The El antigen is linked by a furin cleavable linker to the E2 antigen. In some embodiments, the E6 antigen is E6 (S). In some embodiments, the E7 antigen is E7 (S). In some embodiments, the El antigen is El (G482D). In some preferred embodiments, the amino acid sequence of the HPV antigen comprises SEQ ID NO:32, or the amino acid sequence of at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:32. In some preferred embodiments, the amino acid sequence of the HPV antigen comprises SEQ ID NO:32. In some preferred embodiments, the amino acid sequence of the HPV antigen is SEQ ID NO:32.

[0075] In some embodiments, the nucleic acid encodes an IgE leader, an E6 antigen, an E7 antigen, an El antigen, and an E2 antigen. In some embodiments, the IgE leader is fused to the E6 antigen, which is linked by a furin cleavable linker to the E7 antigen. The E7 antigen is linked by a furin cleavable linker to the E2 antigen. The E2 antigen is linked by a furin cleavable linker to the El antigen. In some embodiments, the E6 antigen is E6 (S). In some embodiments, the E7 antigen is E7 (S). In some embodiments, the El antigen is El (G482D). In some preferred embodiments, the amino acid sequence of 17MF-367038161Docket No.: 16553-20011.40 the HPV antigen comprises SEQ ID NO:33, or the amino acid sequence of at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:33. In some preferred embodiments, the amino acid sequence of the HPV antigen comprises SEQ ID NO:33. In some preferred embodiments, the amino acid sequence of the HPV antigen is SEQ ID NO:33.

[0076] In some embodiments, the nucleic acid encodes an IgE leader, an E6 antigen, an E7 antigen, and an El antigen. In some embodiments, the IgE leader is fused to the E6 antigen, which is linked by a furin cleavable linker to the E7 antigen. The E7 antigen is linked by a furin cleavable linker to the El antigen. In some embodiments, the E6 antigen is E6 (S). In some embodiments, the E7 antigen is E7 (S). In some embodiments, the El antigen is El (G482D). In some preferred embodiments, the amino acid sequence of the HPV antigen comprises SEQ ID NO:34, or the amino acid sequence of at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:34. In some preferred embodiments, the amino acid sequence of the HPV antigen comprises SEQ ID NO:34. In some preferred embodiments, the amino acid sequence of the HPV antigen is SEQ ID NO:34.

[0077] In some embodiments, the nucleic acid encodes an IgE leader, an E6 antigen, an E7 antigen, and an E2 antigen. In some embodiments, the IgE leader is fused to the E6 antigen, which is linked by a furin cleavable linker to the E7 antigen. The E7 antigen is linked by a furin cleavable linker to the E2 antigen. In some embodiments, the E6 antigen is E6 (S). In some embodiments, the E7 antigen is E7 (S). In some preferred embodiments, the amino acid sequence of the HPV antigen comprises SEQ ID NO:35, or the amino acid sequence of at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:35. In some preferred embodiments, the amino acid sequence of the HPV antigen comprises SEQ ID NO:35. In some preferred embodiments, the amino acid sequence of the HPV antigen is SEQ ID NO:35.

[0078] In some embodiments, the nucleic acid encodes an IgE leader, an E6 antigen, an E7 antigen, an El antigen, and an E2 antigen. In some embodiments, the IgE leader is fused to the E6 antigen, which is fused to the E7 antigen, which is further fused to the El antigen, which is further fused to the E2 antigen. In some embodiments, the E6 antigen is E6 (S). In some embodiments, the E7 antigen is E7 (S). In some embodiments, the El antigen is El (G482D). In some preferred embodiments, the amino acid sequence of the HPV antigen comprises SEQ ID NO:36, or the amino acid sequence of at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:36. In some preferred embodiments, the amino acid sequence of the HPV antigen comprises SEQ ID NO:36. In some preferred embodiments, the amino acid sequence of the HPV antigen is SEQ ID NO:36.

[0079] In some embodiments, the nucleic acid encodes an E6 antigen, an E7 antigen, an El antigen, and an E2 antigen. In some embodiments, the E6 antigen is linked by a furin cleavable linker to the E7 antigen. In some embodiments, the E7 antigen is linked by a furin cleavable linker to the El 18MF-367038161Docket No.: 16553-20011.40 antigen. In some embodiments, the El antigen is linked by a furin cleavable linker to the E2 antigen. In some embodiments, the E6 antigen is E6 (S). In some embodiments, the E7 antigen is E7 (S). In some embodiments, the El antigen is El (G482D). In some preferred embodiments, the amino acid sequence of the HPV antigen comprises SEQ ID NO:37, or the amino acid sequence of at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:37. In some preferred embodiments, the amino acid sequence of the HPV antigen comprises SEQ ID NO:37. In some preferred embodiments, the amino acid sequence of the HPV antigen is SEQ ID NO:37.

[0080] In some embodiments, the nucleic acid encodes an IgE leader, an E6 antigen, an E7 antigen, an El antigen, and an E2 antigen. In some embodiments, the IgE leader is fused to the E7 antigen, which is linked by a furin cleavable linker to the E6 antigen. The E6 antigen is linked by a furin cleavable linker to the El antigen. The El antigen is linked by a furin cleavable linker to the E2 antigen. In some embodiments, the E6 antigen is E6 (H). In some embodiments, the E7 antigen is E7 (H). In some embodiments, the El antigen is El (G482D). In some preferred embodiments, the amino acid sequence of the HPV antigen comprises SEQ ID NO:38, or the amino acid sequence of at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:38. In some preferred embodiments, the amino acid sequence of the HPV antigen comprises SEQ ID NO:38. In some preferred embodiments, the amino acid sequence of the HPV antigen is SEQ ID NO:38.

[0081] In some embodiments, the nucleic acid encodes an E6 antigen, an E7 antigen, an El antigen, and an E2 antigen. In some embodiments, the E6 antigen fused to the E7 antigen, which is further fused to the El antigen, which is further fused to the E2 antigen. In some embodiments, the E6 antigen is E6 (H). In some embodiments, the E7 antigen is E7 (H). In some embodiments, the El antigen is El (G482D). In some preferred embodiments, the amino acid sequence of the HPV antigen comprises SEQ ID NO:39, or the amino acid sequence of at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:39. In some preferred embodiments, the amino acid sequence of the HPV antigen comprises SEQ ID NO:39. In some preferred embodiments, the amino acid sequence of the HPV antigen is SEQ ID NO:39.III. mRNA Components

[0082] The mRNA of the present disclosure may comprise elements in addition to a coding region of at least one HPV antigen, alone or in combination with a coding region of a cGAS. In some embodiments, the mRNA comprises a 5’ untranslated region (5’UTR) at the 5’ end of the coding region and a 3’ untranslated region (3’UTR) at the 3’ end of the coding region. In some preferred embodiments, the mRNA comprises one or both of a 5 ’ cap structure and a polyA tail.19MF-367038161Docket No.: 16553-20011.40

[0083] Preferably, the mRNA of the present disclosure comprises a modified nucleoside. Modified nucleosides for use in connection with the mRNAs of the present disclosure include but are not limited to modified uridine, modified cytidine, modified adenine and modified guanine. In some embodiments, the modified nucleoside comprises a modified uridine. In some embodiments, the modified uridine comprises Nl-methyl-pseudouridine. In some embodiments, the modified nucleoside comprises one or more of the group consisting of Nl-methyl-pseudouridine, 5 -methylcytidine (m5C), 5-methyluridine (m5U), N6-methyladenosine (m6A), 2-thiouridine (s2U), pseudouridine, 2’-O-methyluridine (um), and 5-methoxyuridine (mo5U). Modified nucleosides for use in the mRNAs of the present disclosure are known in the art (see, e.g., paragraphs

[0049] and

[0050] of US 2025-0019719-Al of Weissman and Kariko, which are incorporated herein by reference).

[0084] In some embodiments, the mRNA comprises a coding region of both a cyclic GMP-AMP synthase (cGAS) and a coding region of a human papillomavirus (HPV) antigen, which are separated by an intervening sequence. In some embodiments, the mRNA comprises a coding regions of two, three or four HPV antigens, wherein an intervening sequence is located between one or more of the HPV antigens. Intervening sequences for use in the constructs of the present disclosure encode a 2A-like peptide sequences, encode a protease cleavage sites, or comprises an internal ribosome entry sites (IRES). In some embodiments, the intervening sequence encodes a T2A (SEQ ID NO:12), P2A (SEQ ID NO:13), E2A (SEQ ID NO: 14), or F2A (SEQ ID NO: 15) peptide sequence. Additional 2A-like peptide sequences are known in the art (see, e.g., Luke et al., J. Gen. Virol, 89:1036-1042, 2008, 2AL sequences of Figure 2 are incorporated herein by reference). In some embodiments, the intervening sequence encodes a protease cleavage site, such as a furin cleavage site (SEQ ID NO:27). In some embodiments, intervening sequence is an IRES.IV. Delivery Vehicles

[0085] Compositions and methods of the present disclosure may comprise mRNA encapsulated in a nanoparticle or as part of a complex. For instance, the compositions and methods of the present disclosure may comprise a lipid-based delivery vehicle for an mRNA vaccine. In some embodiments, the vehicle is a lipid nanoparticle (LNP). In other embodiments, the vehicle is a lipid that forms a complex with the mRNA (RNA-Lipoplex). In some embodiments, the nanoparticle is polymeric nanoparticle (PNP) of approximately 1 nanometer to 1000 nanometers in diameter formed from one or more polymers. Polymeric nanoparticles can be in the form of micelles, metal-organic frameworks, dendrimeric assemblies, hydrogels, liposomes, or polymerosomes (see, e.g., Xiao et al., Front Bioeng Biotechnol, 10:1024143, 2022). In some embodiments, the polymeric nanoparticle comprises a non-lipid polymer. In some embodiments, the nanoparticle is a non-lipid polymeric nanoparticle formed primarily20MF-367038161Docket No.: 16553-20011.40 or exclusively from non-lipid polymers. In some embodiments, the nanoparticle is a protein nanoparticle. In some embodiments, the nanoparticle is a viral particle. In other embodiments, the nanoparticle is a virus-like particle. In some embodiments, the nanoparticle is in the form of a cationic nanoemulsion.

[0086] In some embodiments, the LNP comprises at least one lipid selected from the group consisting of an ionizable lipid, a cationic lipid, a phospholipid, a pegylated lipid, a structural lipid, and mixtures thereof. In some embodiments, the at least one lipid comprises an ionizable lipid. In some embodiments, the at least one lipid comprises a cationic lipid. In some embodiments, the at least one lipid comprises a phospholipid. In some embodiments, the at least one lipid comprises a pegylated lipid. In some embodiments, the at least one lipid comprises a structural lipid. In some embodiments, the at least one lipid comprise an ionizable lipid, a phospholipid, a pegylated lipid, and a structural lipid.

[0087] In some embodiments, the lipid component of RNA-Lipoplex comprises one or more lipids. In some preferred embodiments, the one or more lipids comprise a first lipid and a second lipid, wherein the first lipid is distinct from the second lipid. In some embodiments, the first lipid is a cationic lipid and the second lipid is a neutral or anionic lipid.

[0088] Structures of lipids suitable for use in the lipid-based mRNA delivery vehicles of the present disclosure are known in the art (see, Figure 2 of Hou et al., Nature Review Materials, 6:1078-1094, 2021, herein incorporated by reference).

[0089] A wide variety of polymers can be used to form non-lipid polymeric nanoparticles (PNPs), such as a poly(β-amino ester) (PBAE), a cyclodextrin (CD), a polyethyleneimine (PEI), a poly(lactic-co-glycolic acid) (PLGA), a poly(lactic acid) (PLA), a polyamidoamine (PAMAM) dendrimer or an ionizable amphiphilic Janus dendrimer. Poly(beta-amino ester) polymers have a reversible positive charge, which promotes binding to nucleic acids and imparts high buffering capacity (Karlsson et al., Expert Opin Drug Deliv, 17(10): 1395-1410, 2020). Cyclodextrins are cyclic oligosaccharides composed of glucose units connected by alpha-1,4 linkages. Cyclodextrin nanoparticles (Gadade et al., Adv Pharm Bull, 10(2): 166-183, 2020) have been used for encapsulation of a wide variety of therapeutics, including nucleic acids. Polyethylenimine (PEI) is a cationic polymer, which condenses with nucleic acids to form nanoparticles. Nanoparticles formed from polyesters include but are not limited to poly(lactic-co-glycolic acid) (PLGA) and poly (lactic acid) (PLA). PNPs formed from dendrimers are called dendrimersomes or dendrimeric nanoparticles. Exemplary dendrimers include but are not limited to PAMAM dendrimers (Tarach et al., Int J Mol Sci, 13;22(6):2912, 2021) and ionizable amphiphilic Janus dendrimers (Lu et al., J Am Chem Soc, 145(34):18760-18766, 2023).21MF-367038161Docket No.: 16553-20011.40 V. Pharmaceutical Formulations

[0090] Some compositions of the present disclosure are pharmaceutical formulations comprising a pharmaceutically acceptable excipient. Pharmaceutical formulations of the present disclosure may be in the form of a solution or a suspension. Alternatively, the pharmaceutical formulations may be a dehydrated solid (e.g., freeze dried or spray dried solid). The pharmaceutical formulations of the present disclosure are preferably sterile, and preferably essentially endotoxin-free. The term “pharmaceutical formulations” is used interchangeably herein with the terms “medicinal product” and “medicament”. In some embodiments, the pharmaceutical formation comprises specific ratios of the various components based on the intended purpose of the formulation.

[0091] Pharmaceutically acceptable excipients of the present disclosure include for instance, solvents, buffering agents, tonicity adjusting agents, bulking agents, and preservatives (See, e.g., Pramanick et al., Pharma Times, 45:65-77, 2013). In some embodiments, the pharmaceutical formulations may comprise an excipient that functions as one or more of a solvent, a buffering agent, a tonicity adjusting agent, and a bulking agent (e.g., sodium chloride in saline may serve as both an aqueous vehicle and a tonicity adjusting agent).

[0092] In some embodiments, the pharmaceutical formulations comprise an aqueous vehicle as a solvent. Suitable vehicles include for instance sterile water, saline solution, phosphate buffered saline, and Ringer's solution. In some embodiments, the composition is isotonic.

[0093] The pharmaceutical formulations may comprise a buffering agent. Buffering agents control pH to inhibit degradation of the active agent during processing, storage and optionally reconstitution. Suitable buffers include for instance salts comprising acetate, citrate, phosphate or sulfate. Other suitable buffers include for instance amino acids such as arginine, glycine, histidine, and lysine. The buffering agent may further comprise hydrochloric acid or sodium hydroxide. In some embodiments, the buffering agent maintains the pH of the composition within a range of 6 to 9. In some embodiments, the pH is greater than (lower limit) 6, 7 or 8. In some embodiments, the pH is less than (upper limit) 9, 8, or 7. That is, the pH is in the range of from about 6 to 9 in which the lower limit is less than the upper limit.

[0094] The pharmaceutical compositions may comprise a tonicity adjusting agent. Suitable tonicity adjusting agents include for instance dextrose, glycerol, sodium chloride, glycerin and mannitol.

[0095] The pharmaceutical formulations may comprise a bulking agent. Bulking agents are particularly useful when the pharmaceutical composition is to be lyophilized before administration. In some embodiments, the bulking agent is a protectant that aids in the stabilization and prevention of22MF-367038161Docket No.: 16553-20011.40 degradation of the active agents during freeze or spray drying and / or during storage. Suitable bulking agents are sugars (mono-, di- and polysaccharides) such as sucrose, lactose, trehalose, mannitol, sorbital, glucose and raffinose.

[0096] The pharmaceutical formulations may comprise a preservative. Suitable preservatives include for instance antioxidants and antimicrobial agents. However, in preferred embodiments, the pharmaceutical formulation is prepared under sterile conditions and is in a single use container, and thus does not necessitate inclusion of a preservative.

[0097] The pharmaceutical formulations of the present disclosure are suitable for parenteral administration. That is the pharmaceutical formulations of the present disclosure are not intended for enteral administration (e.g., not by orally, gastrically, or rectally).VI. Methods of Use or Manufacture

[0098] In some aspects, the present disclosure relates to methods of use of any one of the compositions or formulations described herein. The methods of use are suitable for a plurality of uses involving stimulating an immune response. In some embodiments, the methods of use comprise methods of treating cancer. In some embodiments, the methods of use comprise methods of inhibiting abnormal cell proliferation. In some embodiments, the methods of use comprise methods of treating or preventing an infectious disease. The methods comprise administering an effective amount of a formulation or a composition described herein to an individual in need thereof to achieve a specific outcome. The individual is a mammalian subject, such as a human patient. In other embodiments, the individual is a non-human subject. That is in some embodiments, the methods of use involve clinical uses, while in other embodiments the methods of use involve pre-clinical uses. For preclinical uses, the mammalian subject may be a non-human primate (e.g., monkey or ape) or a rodent (e.g., mouse or rat).A. Stimulation of an HPV-Specific Immune Response

[0099] In brief, the present disclosure provides methods of stimulating an immune response in an individual, comprising administering to the individual a composition or formulation described herein in an amount sufficient to stimulate an immune response in the individual. “Stimulating” an immune response (used interchangeably with “eliciting” and immune response), means increasing the immune response, which can arise from eliciting a de novo immune response (e.g., as a consequence of an initial vaccination regimen) or enhancing an existing immune response (e.g., as a consequence of a booster vaccination regimen). In some embodiments, stimulating an immune response comprises one or more of the group consisting of: stimulating cytokine production; stimulating B lymphocyte proliferation; stimulating interferon pathway-associated gene expression; stimulating chemoattractant-associated gene23MF-367038161Docket No.: 16553-20011.40 expression; and stimulating dendritic cell DC maturation. Methods for measuring stimulation of an immune response are known in the art.

[0100] For instance, the present disclosure provides methods of stimulating an HPV antigenspecific immune response in an individual by administering to the individual a composition or formulation described herein in an amount sufficient to stimulate the HPV antigen-specific immune response in the individual. In some embodiments, the composition or formulation is administered to a tissue of the individual comprising the antigen. The immune response may comprise one or more of an antigen-specific antibody response, an antigen-specific cytotoxic T lymphocyte (CTL) response, and an antigen-specific helper T (Th) cell response. The terms “stimulating” and “inducing” in reference to an antigen-specific antibody response means increasing titer of the antigen-specific antibodies above a threshold level such as a pre-administration baseline titer or a seroprotective level. Likewise, “stimulating” or “inducing” an antigen-specific CTL response means increasing frequency of antigenspecific CTL found in peripheral blood above a pre-administration baseline frequency. Similarly, “stimulating” or “inducing” an antigen-specific Th cell response means increasing frequency of antigenspecific Th cells found in peripheral blood above a pre-administration baseline frequency. In some embodiments, the individual is a human subject infected with HPV. In some embodiments, the HPV is HPV-16. In some embodiments, the HPV is HPV-18.

[0101] Analysis (both qualitative and quantitative) of the immune response can be by any method known in the art, including, but not limited to, measuring antigen-specific antibody production (including measuring specific antibody subclasses), activation of specific populations of lymphocytes such as B cells and helper T cells, production of cytokines such as IFN-alpha, IFN-gamma, IL-6, IL- 12 and / or release of histamine. Methods for measuring antigen-specific antibody responses include enzyme-linked immunosorbent assay (ELISA). Activation of specific populations of lymphocytes can be measured by proliferation assays, and with fluorescence-activated cell sorting (FACS). Production of cytokines can also be measured by ELISA. In some embodiments, methods of stimulating an immune response comprise stimulation of interleukin-1beta (IL-1β) secretion, interferon-gamma (IFN-y) secretion, and / or tumor necrosis factor-alpha (TNF-a) secretion by monocyte-derived dendritic cells or peripheral blood mononuclear cells. In some preferred embodiments, at least 50%, 55%, 60%, 65%, 70% or 75% of the cells contacted with a composition of the present disclosure remain viable at 40-56 hours (or about 48 hours) post-contact.B. Treating or Preventing HPV Disease

[0102] The present disclosure further provides methods of treating or preventing an HPV-associated disease in an individual, such as a human subject infected with HPV. Specifically, the present 24MF-367038161Docket No.: 16553-20011.40 disclosure provides methods of treating precancerous genital dysplasia, methods of treating cancer, and methods of treating or preventing other HPV-associated diseases (e.g., genital warts). The methods comprise administering to the human subject a composition or formulation described herein in an amount sufficient (effective amount) to treat or prevent the HPV-associated disease in the individual.

[0103] In some embodiments, the methods involve treating a precancerous lesion in a human subject infected with HPV. In some embodiments, the precancerous lesion is precancerous genital dysplasia. In some embodiments, the precancerous genital dysplasia is cervical dysplasia. In some embodiments, the precancerous genital dysplasia is anal dysplasia. In some embodiments, the precancerous genital dysplasia is both cervical dysplasia and anal dysplasia.

[0104] In some embodiments, the methods involve treating cancer in a human subject or treating a human subject with cancer. In some embodiments, the cancer is a genital cancer. In some embodiments, the genital cancer is a cervical cancer. In some embodiments, the genital cancer is a vulvar cancer. In some embodiments, the genital cancer is a penile cancer. In some embodiments, the genital cancer is a vaginal cancer. In some embodiments, the cancer is an oropharyngeal cancer. In other embodiments, the cancer is an anal cancer.

[0105] In preferred embodiments, the human subject to which a composition or formulation described herein is administered is infected HPV. In some embodiments, the HPV is a high-risk type. In some embodiments, the HPV type is HPV-16. In some embodiments, the HPV type is HPV-18. In some embodiments, the human subject is female (e.g., girl or woman). In other embodiments, the human subject is male (e.g., boy or man).C. Production of Hyperactivated Dendritic Cells

[0106] The present disclosure further provides methods for production of hyperactivated dendritic cells. In some embodiments, production of hyperactivated dendritic cells comprises contacting dendritic cells with an effective amount of a composition or formulation described herein to produce hyperactivated dendritic cells. In some embodiments, the hyperactivated dendritic cells contain apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC) speck(s). The presence of ASC speck(s) can be detected by immunohistochemistry or immunofluorescence using an antibody specific for ASC. In some embodiments, the hyperactivated dendritic cells secrete IL-1 beta without undergoing cell death within about 48 hours of exposure or contact with the composition. In some preferred embodiments, at least 50%, 55%, 60%, 65%, 70% or 75% of the cells contacted with a composition of the present disclosure remain viable at 40-56 hours (or about 48 hours) post-contact.25MF-367038161Docket No.: 16553-20011.40

[0107] In some embodiments, the dendritic cells are contacted in vivo with the composition. In other embodiments, the dendritic cells are contacted ex vivo with the composition. In some embodiments, the hyperactivated dendritic cells secrete higher levels of one or more of RANTES, IP-10 and IFNα than unstimulated dendritic cells or dendritic cells contacted with empty LNPs. In some embodiments, the hyperactivated dendritic cells express higher levels of at least one cell surface marker selected from the group consisting of CD40, CD86, CD69, MHC class II, MHC class I, CCR7, and combinations thereof. In some embodiments, the hyperactivated dendritic cells secrete higher levels of one or more of RANTES, IP-10 and IFNα than unstimulated dendritic cells or dendritic cells contacted with empty LNPs and express higher levels of at least one cell surface marker selected from the group consisting of CD40, CD86, CD69, MHC class II, MHC class I, CCR7, and combinations thereof.Enumerated Embodiments1. A composition comprising a first mRNA and a second mRNA encapsulated in a nanoparticle, wherein the first mRNA comprises a coding region of a cyclic GMP-AMP synthase (cGAS), the second mRNA comprises a coding region of a human papillomavirus (HPV) antigen.2. A composition comprising a first mRNA encapsulated in a first nanoparticle and a second mRNA encapsulated in a second nanoparticle, wherein the first mRNA comprises a coding region of a cyclic GMP-AMP synthase (cGAS), and the second mRNA comprises a coding region of a human papillomavirus (HPV) antigen.3. A composition comprising an mRNA encapsulated in a nanoparticle, wherein the mRNA comprises a first coding region and a second coding region separated by an intervening sequence, wherein the first coding region is a coding region of a cyclic GMP-AMP synthase (cGAS) and the second coding region is a coding region of a human papillomavirus (HPV) antigen or the first coding region is a coding region of a human papillomavirus (HPV) antigen and the second coding region is a coding region of a cyclic GMP-AMP synthase (cGAS), wherein the intervening sequence encodes a 2A-like peptide sequence, encodes a protease cleavage site, or is an internal ribosome entry site (IRES).4. The composition of any one of embodiments 1-3, wherein the nanoparticle is a lipid nanoparticle (LNP) comprising an ionizable lipid, a pegylated lipid, a structural lipid, a phospholipid, or a combination thereof, optionally wherein the LNP comprises an ionizable lipid.5. The composition of embodiment 4, wherein the LNP comprises the ionizable lipid, the pegylated lipid, the structural lipid and the phospholipid.26MF-367038161Docket No.: 16553-20011.40 6. The composition of any one of embodiments 1-5, wherein the ionizable lipid comprises: i) 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1 -octylnonyl ester (SM-102) or analogs or derivatives thereof; and / orii) 6-((2-hexyldecanoyl)oxy)-N-(6-((2-hexyldecanoyl)oxy)hexyl)-N-(4-hydroxybutyl)hexan-l-aminium (ALC-0315) or analogs or derivatives thereof; and / oriii) (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA) or analogs or derivatives thereof.7. The composition of any one of embodiments 1-6, wherein the pegylated lipid is selected from the group consisting of a PEG-modified phosphatidyiethanolamine, a PEG-modified phosphatide acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, and combinations thereof.8. The composition of any one of embodiments 1-6, wherein the pegylated lipid comprises polyethylene glycol [PEG] 2000 dimyristoyl glycerol [DMG].9. The composition of any one of embodiments 1-8, wherein the structural lipid is selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha- tocopherol, and combinations thereof.10. The composition of embodiment 9, wherein the structural lipid comprises cholesterol. 11. The composition of any one of embodiments 1-10, wherein the phospholipid comprises: i) a hydrophilic head moiety selected from the group consisting of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and sphingomyelin; andii) one or more fatty acid tail moieties selected from the group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, arachidic acid, arachidonic acid, phytanoic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.12. The composition of any one of embodiments 1-10, wherein the phospholipid is selected from the group consisting of1.2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC),1.2-dimyristoyl-sn-glycero-phosphocholine (DMPC),1.2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1.2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),27MF-367038161Docket No.: 16553-20011.40 1.2-distearoyl-sn-glycero-3-phosphocholine (DSPC),1.2-diundecanoyl-sn-glycero-phosphocholine (DUPC),1 -palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),1.2-di-O-octadecenyl-sn-glycero-3-phosphocholine,l-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine,1.2-dilinolenoyl-sn-glycero-3 -phosphocholine,1.2-diarachidonoyl-sn-glycero-3 -phosphocholine,1.2-didocosahexaenoyl-sn-glycero-3-phosphocholine,1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),1.2-diphytanoyl-sn-glycero-3-phosphoethanolamine,1.2-distearoyl-sn-glycero-3-phosphoethanolamine,1.2-dilinoleoyl-sn-glycero-3-phosphoethanolamine,1.2-dilinolenoyl-sn-glycero-3 -phosphoethanolamine,1.2-diarachidonoyl-sn-glycero-3 -phosphoethanolamine,1.2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine,1.2-dioleoyl-sn-glycero-3-phospho-rac-(l -glycerol) sodium salt (DOPG),sphingomyelin, andcombinations thereof, optionally wherein the phospholipid comprises l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).13. The composition of any one of embodiments 1-3, wherein the nanoparticle is a polymeric nanoparticle comprising a non-lipid polymer.14. The composition of embodiment 13, wherein the non-lipid polymer comprises a poly(β-amino ester) (PBAE), a cyclodextrin (CD), a polyethyleneimine (PEI), a poly(lactic-co-glycolic acid) (PLGA), a polyamidoamine (PAMAM) dendrimer or an ionizable amphiphilic Janus dendrimer.15. The composition of any one of embodiments 1-3, wherein the nanoparticle is a protein nanoparticle, a viral particle, a virus-like particle (VLP) or a cationic nanoemulsion.16. A composition comprising a first mRNA and a second mRNA complexed with one or more lipids (RNA-Lipoplex), wherein the first mRNA comprises a coding region of a constitutively active cyclic GMP-AMP synthase (cGAS), and the second mRNA comprises a coding region of a human papillomavirus (HPV) antigen, and the one or more lipids comprise a first lipid and a second lipid.17. A composition comprising an mRNA complexed with one or more lipids (RNA-Lipoplex), wherein the mRNA comprises a first coding region and a second coding region separated by28MF-367038161Docket No.: 16553-20011.40 an intervening sequence, the first coding region is a coding region of a constitutively active cyclic GMP-AMP synthase (cGAS) and the second coding region is a coding region of a human papillomavirus (HPV) antigen or the first coding region is a coding region of a human papillomavirus (HPV) antigen and the second coding region is a coding region of a constitutively active cyclic GMP-AMP synthase (cGAS), and the one or more lipids comprise a first lipid and a second lipid, wherein the intervening sequence encodes a 2A-like peptide sequence, encodes a protease cleavage site, or is an internal ribosome entry site (IRES).18. The composition of embodiment 16 or embodiment 17, wherein the first lipid is a cationic lipid, and the second lipid is a neutral or anionic lipid.19. The composition of embodiment 18, wherein the cationic lipid comprises one or both of: i) 1,2-di-O-octadecenyl-3 -trimethylammonium propane (DOTMA) or analogs or derivatives thereof; andii) l,2-dioleoyl-3-trimethylammonium propane (DOTAP) or analogs or derivatives thereof.20. The composition of embodiment 18 or embodiment 19, wherein the neutral or anionic lipid comprises:i) l,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE) or analogs or derivatives thereof; and / orii) cholesterol or analogs or derivatives thereof; and / oriii) l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or analogs or derivatives thereof.21. The composition of any one of embodiments 1-20, wherein the composition does not comprise a TLR7 / 8 agonist.22. The composition of any one of embodiments 1-20, further comprising a TLR7 / 8 agonist, wherein the TLR7 / 8 agonist is a small molecule with a molecule weight of 900 daltons or less.23. The composition of embodiment 22, wherein the TLR7 / 8 agonist comprises an imidazoquinoline compound.24. The composition of embodiment 23, wherein the TLR7 / 8 agonist comprises resiquimod (R848).25. The composition of any one of embodiments 1-24, wherein the mRNA or the first mRNA and the second mRNA comprises a 5’ untranslated region (5’UTR) and a 3’ untranslated region (3’UTR).29MF-367038161Docket No.: 16553-20011.40 26. The composition of any one of embodiments 1-25, wherein the mRNA comprises one or both of a 5' cap structure and a poly A tail.27. The composition of any one of embodiments 1-26, wherein the mRNA comprises a modified nucleoside, optionally wherein the modified nucleoside comprises one or more of N1 -methylpseudouridine, 5-methyluridine, 2-thiouridine, pseudouridine and 5 -methylcytidine, optionally wherein the modified nucleoside comprises N1 -methyl -pseudouridine.28. The composition of any one of embodiments 1-27, wherein the composition further comprises at least one excipient, optionally wherein the excipient comprises sucrose.29. The composition of any one of embodiments 1-28, wherein the constitutively-active cGAS has a greater propensity to self DNA reactivity than its wild-type counterpart.30. The composition of any one of embodiments 1-29, wherein the cGAS is a truncated cGAS devoid of an amino-terminal phosphoinositide-binding domain (cGASΔN).31. The composition of embodiment 30, wherein the cGASΔN comprises the amino acid sequence of SEQ ID NO:1 or the amino acid sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:1, optionally wherein the cGASΔN has one, two, three or four conservative amino acid substitutions.32. The composition of embodiment 31, wherein the cGASΔN comprises the consensus amino acid sequence of SEQ ID NO:9.33. The composition of embodiment 31, wherein the cGASΔN is encoded by the nucleotide sequence of SEQ ID NO:41.34. The composition of any one of embodiments 30-33, wherein the coding region of the cGASΔN is in operable combination with a start codon, optionally wherein the start codon is ATG, CUG, GUG or ACG, optionally wherein the start codon is ATG.35. The composition of any one of embodiments 1-34, wherein the HPV antigen comprises: a) an E6 antigen, an E7 antigen and an E2 antigen of an HPV16 type, optionally wherein:(i) the E6 antigen comprises the amino acid sequence of SEQ ID NO: 18 or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:18; and(ii) the E7 antigen comprises the amino acid sequence of SEQ ID NO:21, or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:21; and(iii) the E2 antigen comprises the amino acid sequence of SEQ ID NO:25 or the amino acid sequence30MF-367038161Docket No.: 16553-20011.40 at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25; orb) an E6 antigen, an E7 antigen and an E2 antigen of an HPV18 type, optionally wherein:(i) the E6 antigen comprises the amino acid sequence of SEQ ID NO: 149 or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:43 or SEQ ID NO:149; and(ii) the E7 antigen comprises the amino acid sequence of SEQ ID NO:150, or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:44 or SEQ ID NO:150; and (iii) the E2 antigen comprises the amino acid sequence of SEQ ID NO: 151 or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:42 or SEQ ID NO:151; or c) both a) and b).36. The composition of any one of embodiments 1-34, wherein the HPV antigen comprises one or both of an E6 antigen and an E7 antigen.37. The composition of embodiment 36, wherein the HPV antigen comprises an E6 antigen and an E7 antigen, and wherein the E6 antigen and the E7 antigen are expressed as a fusion protein.38. The composition of embodiment 36, wherein the HPV antigen comprises an E6 antigen and an E7 antigen, and wherein the coding regions of the E6 antigen and the E7 antigen are separated by an intervening sequence, wherein the intervening sequence encodes a 2A-like peptide sequence, encodes a protease cleavage site, or is an internal ribosome entry site (IRES), optionally wherein the E6 antigen and the E7 antigen are expressed as separate antigens, optionally wherein the intervening sequence encodes a protease cleavage site, optionally wherein the protease cleavage site is a furin cleavable linker, optionally wherein the furin cleavable linker comprises the amino acid sequence of SEQ ID NO:28.39. The composition of any one of embodiments 36-38, wherein the E6 antigen comprises: (i) the amino acid sequence of SEQ ID NO:18 or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:18;(ii) the amino acid sequence of SEQ ID NO:19, or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:19;(iii) the consensus amino acid sequence of SEQ ID NO: 20;(iv) the amino acid sequence of SEQ ID NO:43, or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:43; or(v) the amino acid sequence of SEQ ID NO:149, or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 149.40. The composition of any one of embodiments 36-39, wherein the E7 antigen comprises: (i) the amino acid sequence of SEQ ID NO:21, or the amino acid sequence at least 90%, 95%, 96%,31MF-367038161Docket No.: 16553-20011.40 97%, 98% or 99% identical to SEQ ID NO:21;(ii) the amino acid sequence of SEQ ID NO:22, or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22;(iii) the consensus amino acid sequence of SEQ ID NO: 23;(iv) the amino acid sequence of SEQ ID NO:44, or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:44; or(v) the amino acid sequence of SEQ ID NO:150, or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 150.41. The composition of any one of embodiments 1-40, wherein the HPV antigen further comprises one or both of an El antigen and an E2 antigen.42. The composition of embodiment 41, wherein the HPV antigen comprises an El antigen and an E2 antigen, and wherein the El antigen and the E2 antigen are expressed as a fusion protein.43. The composition of embodiment 41, wherein the HPV antigen comprises an El antigen and an E2 antigen, and wherein the coding regions of the El antigen and the E2 antigen are separated by an intervening sequence, wherein the intervening sequence encodes a 2A-like peptide sequence, encodes a protease cleavage site, or is an internal ribosome entry site (IRES), optionally wherein the El antigen and the E2 antigen are expressed as separate antigens, optionally wherein the intervening sequence encodes a protease cleavage site, optionally wherein the protease cleavage site is a furin cleavable linker, optionally wherein the furin cleavable linker comprises the amino acid sequence of SEQ ID NO:28.44. The composition of any one of embodiments 41-43, wherein the El antigen comprises: (i) the amino acid sequence of SEQ ID NO:24 or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24; or(ii) the amino acid sequence of SEQ ID NO:41 or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:41.45. The composition of any one of embodiments 41-44, wherein the E2 antigen comprises: (i) the amino acid sequence of SEQ ID NO:25 or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25; or(ii) the amino acid sequence of SEQ ID NO:42 or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:42; or(iii) the amino acid sequence of SEQ ID NO:151 or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:151.32MF-367038161Docket No.: 16553-20011.40 46. The composition of any one of embodiments 36-45, wherein the amino acid sequence of the E6 antigen or the E7 antigen is preceded by a methionine or the amino acid sequence of a signal peptide.47. The composition of embodiment 46, wherein the signal peptide is an IgE leader comprising the amino acid sequence of SEQ ID NO:26.48. The composition of any one of embodiments 1-47, wherein the HPV antigen and if present the IgE leader comprises:(i) the amino acid sequence of SEQ ID NO:35, or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:35; or(ii) the amino acid sequence of one of the group consisting of SEQ ID NOs:30-39, or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to one of the group consisting of SEQ ID NOs:30-39; and / or(iii) the amino acid sequence of SEQ ID NO:148, or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 148.49. A pharmaceutical formulation comprising the composition of any one of embodiments 1-48, and a pharmaceutically acceptable excipient.50. A method for production of hyperactivated dendritic cells, the method comprising contacting the dendritic cells with an effective amount of the composition of any one of embodiments 1-48, or the formulation of embodiment 49 to produce hyperactivated dendritic cells, wherein the hyperactivated dendritic cells contain apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC) speck(s), optionally wherein the hyperactivated dendritic cells secrete IL-lbeta without undergoing cell death within about 48 hours of exposure.51. The method of embodiment 50, wherein the dendritic cells are:(i) contacted in vivo with the composition; or(ii) contacted ex vivo with the composition.52. The method of embodiment 50 or embodiment 51, wherein the hyperactivated dendritic cells:(i) secrete higher levels of one or more of IFNβ, RANTES, IP-10 and IFNα than unstimulated dendritic cells or dendritic cells contacted with empty LNPs; and / or(ii) express higher levels of at least one cell surface marker selected from the group consisting of33MF-367038161Docket No.: 16553-20011.40 CD40, CD86, CD69, MHC class II, MHC class I, CCR7, and combinations thereof, than unstimulated dendritic cells or dendritic cells contacted with empty LNPs.53. A pharmaceutical formulation comprising at least 103, 104, 105or 106of the hyperactivated dendritic cells produced by the method of any one of embodiments 50-52, and a pharmaceutically acceptable excipient.54. A method of stimulating an immune response against an HPV antigen, comprising administering an effective amount of the pharmaceutical formulation of embodiment 49 to an individual in need thereof to stimulate the immune response against the HPV antigen.55. The method of embodiment 54, wherein the individual is a human subject infected with HPV.56. The method of embodiment 54 or embodiment 55, wherein the human subject has precancerous genital dysplasia.57. The method of embodiment 54 or embodiment 55, wherein the human subject has a cervical cancer, oropharyngeal cancer, vulvar cancer or anal cancer.58. A method of treating precancerous genital dysplasia, comprising administering an effective amount of the pharmaceutical formulation of embodiment 49 to a human subject in need thereof to treat the genital dysplasia, wherein the human subject is infected with HPV.59. The method of embodiment 58, wherein the precancerous genital dysplasia is one or both of cervical dysplasia and anal dysplasia.60. A method of treating cancer, comprising administering an effective amount of the pharmaceutical formulation of embodiment 49 to a human subject in need thereof to treat the cancer, wherein the human subject is infected with HPV.61. The method of embodiment 60, wherein the cancer is selected from the group consisting of cervical cancer, oropharyngeal cancer, vulvar cancer or anal cancer.62. A method of treating or preventing an HPV -associated disease, comprising administering an effective amount of the pharmaceutical formulation of embodiment 49 to a human subject in need thereof to treat or prevent the HPV-associated disease.63. The method of any one of embodiments 54-62, wherein the HPV is a high-risk type, optionally wherein the HPV is HPV-16 and / or HPV-18, optionally wherein the HPV is HPV-16.34MF-367038161Docket No.: 16553-20011.40 64. A method of preparing the composition of any one of embodiments 1-16, comprising encapsulating the mRNA or the first mRNA and the second mRNA in the particle.65. A method of preparing the composition of any one of embodiments 17-21, comprising forming a complex between the mRNA or the first mRNA and the second mRNA and the one or more lipids.66. An isolated mRNA encoding a human papillomavirus (HPV) antigen, wherein the HPV antigen comprises an E6 antigen and an E7 antigen, and one or both of an El antigen and an E2 antigen.67. The mRNA of embodiment 66, wherein the mRNA encodes the E6 antigen and the E7 antigen as a fusion protein.68. The mRNA of embodiment 66, wherein the coding regions of the E6 antigen and the E7 antigen are separated by an intervening sequence, wherein the intervening sequence encodes a 2A-like peptide sequence, encodes a protease cleavage site, or is an internal ribosome entry site (IRES), optionally wherein the E6 antigen and the E7 antigen are expressed as separate antigens, optionally wherein the intervening sequence encodes a protease cleavage site, optionally wherein the protease cleavage site is a furin cleavable linker, optionally wherein the furin cleavable linker comprises the amino acid sequence of SEQ ID NO:28.69. The mRNA of any one of embodiments 66-68, wherein the E6 antigen comprises:(i) the amino acid sequence of SEQ ID NO:18 or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:18;(ii) the amino acid sequence of SEQ ID NO:19, or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:19;(iii) the consensus amino acid sequence of SEQ ID NO: 20;(iv) the amino acid sequence of SEQ ID NO:43, or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:43; or(v) the amino acid sequence of SEQ ID NO:149, or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 149.70. The mRNA of any one of embodiments 66-69, wherein the E7 antigen comprises:(i) the amino acid sequence of SEQ ID NO:21, or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:21;(ii) the amino acid sequence of SEQ ID NO:22, or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22;35MF-367038161Docket No.: 16553-20011.40 (iii) the consensus amino acid sequence of SEQ ID NO: 23;(iv) the amino acid sequence of SEQ ID NO:44, or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:44; or(v) the amino acid sequence of SEQ ID NO:150, or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 150.71. The mRNA of any one of embodiments 66-70, wherein the El antigen comprises:(i) the amino acid sequence of SEQ ID NO:24 or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24; or(ii) the amino acid sequence of SEQ ID NO:41 or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:41.72. The mRNA of any one of embodiments 66-71, wherein the E2 antigen comprises:(i) the amino acid sequence of SEQ ID NO:25 or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25;(ii) the amino acid sequence of SEQ ID NO:42 or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:42; or(iii) the amino acid sequence of SEQ ID NO:151 or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:151.73. The mRNA of any one of embodiments 66-72, wherein the HPV antigen comprises the E6 antigen, the E7 antigen, and the El antigen.74. The mRNA of any one of embodiments 66-72, wherein the HPV antigen comprises the E6 antigen, the E7 antigen, and the E2 antigen, optionally wherein:a) the E6 antigen, the E7 antigen and the E2 antigen are of an HPV16 type, optionally wherein:(i) the E6 antigen comprises the amino acid sequence of SEQ ID NO: 18 or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:18; and(ii) the E7 antigen comprises the amino acid sequence of SEQ ID NO:21, or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:21; and(iii) the E2 antigen comprises the amino acid sequence of SEQ ID NO:25 or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25; orb) the E6 antigen, the E7 antigen and the E2 antigen are of an HPV 18 type, optionally wherein:(i) the E6 antigen comprises the amino acid sequence of SEQ ID NO: 149 or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:43 or SEQ ID NO:149; and(ii) the E7 antigen comprises the amino acid sequence of SEQ ID NO:150, or the amino acid sequence36MF-367038161Docket No.: 16553-20011.40 at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:44 or SEQ ID NO:150; and (iii) the E2 antigen comprises the amino acid sequence of SEQ ID NO: 151 or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:42 or SEQ ID NO:151; or c) both a) and b).75. The mRNA of any one of embodiments 66-74, wherein the HPV antigen comprises the E6 antigen, the E7 antigen, the El antigen and the E2 antigen.76. The mRNA of embodiment 75, wherein the El antigen and the E2 antigen are expressed as a fusion protein.77. The mRNA of embodiment 75, wherein the coding regions of the El antigen and the E2 antigen are separated by an intervening sequence, wherein the intervening sequence encodes a 2A-like peptide sequence, encodes a protease cleavage site, or is an internal ribosome entry site (IRES), optionally wherein the El antigen and the E2 antigen are expressed as separate antigens, optionally wherein the intervening sequence encodes a protease cleavage site, optionally wherein the protease cleavage site is a furin cleavable linker, optionally wherein the furin cleavable linker comprises the amino acid sequence of SEQ ID NO:28.78. The mRNA of any one of embodiments 66-77, wherein the amino acid sequence of the E6 antigen, the E7 antigen, the El antigen or the E2 antigen is preceded by a methionine or the amino acid sequence of a signal peptide.79. The mRNA of embodiment 78, wherein the signal peptide is an IgE leader comprising the amino acid sequence of SEQ ID NO:26.80. The mRNA of any one of embodiments 66-79, wherein the HPV antigen, and if present the IgE leader, comprises:(i) the amino acid sequence of SEQ ID NO:35, or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:35; or(ii) the amino acid sequence of one of the group consisting of SEQ ID NOs:30-39, or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to one of the group consisting of SEQ ID NOs:30-39; and / or(iii) the amino acid sequence of SEQ ID NO:148, or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 148.81. The mRNA of any one of embodiments 66-80, wherein the mRNA comprises a 5’ untranslated region (5’UTR) and a 3’ untranslated region (3’UTR).37MF-367038161Docket No.: 16553-20011.40 82. The mRNA of any one of embodiments 66-81, wherein the mRNA further comprises one or both of a 5' cap structure and a polyA tail.83. The mRNA of any one of embodiments 66-82, wherein the mRNA comprises a modified nucleoside, optionally wherein the modified nucleoside comprises one or more of N1-methylpseudouridine, 5-methyluridine, 2-thiouridine, pseudouridine and 5-methylcytidine, optionally wherein the modified nucleoside comprises N1-methyl-pseudouridine.84. The mRNA of any one of embodiments 66-83, wherein the HPV antigen does not comprise an LI antigen and / or an L2 antigen, or wherein the mRNA does not comprise a complete HPV genome.85. The method of any one of embodiments 54-57, wherein stimulating the immune response against the HPV antigen comprises increasing HPV E2-specific secretion of IFNγ+ by immune cells of the subject.86. The method of embodiment 85, wherein stimulating the immune response against the HPV antigen comprises increasing frequency of HPV E6-specific IFNγ+, CD4+ T-cells of the subject, and / or increasing frequency of HPV E7-specific IFNγ+, CD8+ T-cells of the subject.87. The method of embodiment 85 or embodiment 86, wherein stimulating the immune response against the HPV antigen comprises increasing frequency of HPV E7-specific IFNγ+, TNFα+ CD8+ T-cells and / or IFNγ+, TNFα+, IL-2+ CD8+ T-cells of the subject.88. The method of any one of embodiments 85-87, wherein stimulating the immune response against the HPV antigen comprises inducing presentation of MHC class I epitope(s) of the HPV antigen on antigen presenting cells of the subject.89. The method of embodiment 88, wherein the subject expresses at least one HLA molecule of the group consisting of A*02:01, A*24:02, B*15:11, B*35:01, and C*03:03 alleles, and the MHC class I epitope(s) comprise one or more of the amino acid sequences of SEQ ID NOs:47-53.90. The method of embodiment 89, wherein the subject expresses at least one HLA molecule of the group consisting of A*02:01, A*24:02, B*15:11, B*35:01, and C*03:03 alleles, and the MHC class I epitope(s) comprise one or more of SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:51 and SEQ ID NO:53.38MF-367038161Docket No.: 16553-20011.40 91. The method of embodiment 88, wherein the subject expresses at least one HLA molecule of the group consisting of A*03:01, A*30:02, B*15:01, B*27:05, C*03:03 and C*05:01 alleles, and the MHC class I epitope(s) comprise one or more of the amino acid sequences of SEQ ID NOs:54-93.92. The method of embodiment 88, wherein the subject expresses at least one HLA molecule of the group consisting of A*02:01, A*24:02, B*15:01, B*35:12, C*03:03 and C*04:01 alleles, and the MHC class I epitope(s) comprise one or more of the amino acid sequences of SEQ ID NOs:94-136.93. The method of any one of embodiments 85-92, wherein stimulating the immune response against the HPV antigen comprises increasing one or more of the group consisting of HPV antigen expression, HPV antigen presentation (e.g., presentation of MHC -I restricted HPV epitopes), CD80 / CD86 / CD40 co-stimulation, CCR7 expression, secretion of Type I interferons, and secretion of T-cell chemokines.94. The method of any one of embodiments 85-93, wherein the increase is relative to preadministration or control administration (e.g., PBS or LNPs devoid of mRNA) values.95. The method of any one of embodiments 85-93, wherein the increase is relative to administration of the mRNA comprising a coding region of a human papillomavirus (HPV) antigen in the absence of the mRNA comprising a coding region of a cyclic GMP-AMP synthase (cGAS).96. The composition of any one of embodiments 1-48, wherein the composition does not comprise a lysophosphatidylcholine (LPC), optionally wherein the LPC has a single C13-C24 acyl chain.97. The composition of embodiment 48, wherein the HPV antigen and if present the IgE leader is encoded by:(i) the nucleic acid sequence of SEQ ID NO: 142; or(ii) the nucleic acid sequence at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:142; or (iii) the nucleic acid sequence of one of the group consisting of SEQ ID NOs:137-147; or(iv) the nucleic acid sequence at least 95%, 96%, 97%, 98% or 99% identical to one of the group consisting of SEQ ID NOs:137-147.98. The mRNA of embodiment 80, wherein the HPV antigen and if present the IgE leader is encoded by:(i) the nucleic acid sequence of SEQ ID NO: 142; or(ii) the nucleic acid sequence at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:142; or (iii) the nucleic acid sequence of one of the group consisting of SEQ ID NOs:137-147; or39MF-367038161Docket No.: 16553-20011.40 (iv) the nucleic acid sequence at least 95%, 96%, 97%, 98% or 99% identical to one of the group consisting of SEQ ID NOs:137-147.99. An isolated DNA comprising:(i) the nucleic acid sequence of SEQ ID NO: 142; or(ii) the nucleic acid sequence at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:142; or (iii) the nucleic acid sequence of one of the group consisting of SEQ ID NOs:137-147; or(iv) the nucleic acid sequence at least 95%, 96%, 97%, 98% or 99% identical to one of the group consisting of SEQ ID NOs:137-147.100. An expression vector comprising the isolated DNA of embodiment 99 in operable combination with a promoter.101. The expression vector of embodiment 100, wherein the vector is a plasmid.102. Use of the composition of any one of embodiments 1-48, the formulation of embodiment 49 or the mRNA of any one of embodiments 66-84 in the manufacture of a medicament for inducing an immune response against the HPV antigen in an individual in need thereof.103. The use of embodiment 102, wherein the individual is a human subject infected with HPV.104. The use of embodiment 102 or embodiment 103, wherein the human subject has precancerous genital dysplasia.105. The use of embodiment 102 or embodiment 103, wherein the human subject has cervical cancer, oropharyngeal cancer, vulvar cancer or anal cancer.106. Use of the composition of any one of embodiments 1-48, the formulation of embodiment 49 or the mRNA of any one of embodiments 66-84 in the manufacture of a medicament for treating precancerous genital dysplasia in human subject infected with HPV.107. The use of embodiment 106, wherein the precancerous genital dysplasia is one or both of cervical dysplasia and anal dysplasia.108. Use of the composition of any one of embodiments 1-48, the formulation of embodiment 49 or the mRNA of any one of embodiments 66-84 in the manufacture of a medicament for treating cancer in a human subject in need thereof, wherein the human subject is infected with HPV.40MF-367038161Docket No.: 16553-20011.40 109. The use of embodiment 108, wherein the cancer is selected from the group consisting of cervical cancer, oropharyngeal cancer, vulvar cancer or anal cancer.110. Use of the composition of any one of embodiments 1-48, the formulation of embodiment 49 or the mRNA of any one of embodiments 66-84 in the manufacture of a medicament for treating or preventing an HPV-associated disease in a human subject in need thereof, wherein the human subject is infected with HPV.111. The use of any one of embodiments 102-110, wherein the HPV is a high-risk type, optionally wherein the HPV is HPV-16 and / or HPV-18, optionally wherein the HPV is HPV-16.112. The composition of any one of embodiments 1-48 or the formulation of embodiment 49 or the method of use comprising the composition or the formulation, wherein the first mRNA comprising the coding region of a cyclic GMP-AMP synthase (cGAS) and the second mRNA comprising a coding region of a human papillomavirus (HPV) antigen are present ati) a weight / weight ratio of from about 0.4: 1 to about 25: 1, optionally from about 1: 1 to about 10:1, further optionally from about 1:1 to about 2:1; orii) a molar ratio of from 1: 1 to 2: 1 or about 1: 1.41MF-367038161Docket No.: 16553-20011.40EXAMPLES

[0108] Abbreviations: BMDC (bone marrow-derived dendritic cell); cyclic GMP-AMP synthase (cGAS); DAMP (damage-associated molecular pattern); DC (dendritic cell); diABZI (amidobenzimidazole compound 3); dLN (draining lymph node); DLS (dynamic light scattering); DMG-PEG-2000 (polyethylene glycol [PEG] 2000 dimyristoyl glycerol [DMG]; DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine); GFP (green fluorescent protein); HPV (human papillomavirus); IFNβ (interferon-beta); IFNγ (interferon-gamma); LNP (lipid nanoparticle); LPC / Lyso PC (lysophosphatidylcholine); Lyso PC(22:0) (1-behenoyl-2-hydroxy-sn-glycero-3-phosphocholine); MC3 ((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, also referred to as DLin-MC3-DMA); mcg or pg (microgram); moDC (monocyte-derived dendritic cell); MW (molecular weight); PAMP (pathogen-associated molecular pattern); PBMCs (peripheral blood mononuclear cells); PRR (pathogen recognition receptor); PBS (phosphate buffered saline); R848 (resiquimod); SFC (spotforming cells); STING (stimulator of interferon genes); TNFa (tumor necrosis factor-alpha); and TLR (toll-like receptor).

[0109] Although the present disclosure has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent to those skilled in the art that certain changes and modifications may be practiced. Therefore, the following examples should not be construed as limiting the scope of the present disclosure, which is delineated by the appended claims.Example 1: LNP loading and ValidationMaterials & Methods

[0110] Antigen Design: HPV16 viral antigens can be challenging to produce as recombinant proteins for use as immunogens. To circumvent this challenge and achieve robust viral protein production with the intent of immunization, mRNA constructs were designed to be delivered by LNPs to express viral antigens in vivo (FIG.3).

[0111] HPV 16 mRNA antigen constructs were designed. An IgE leader sequence was used to improve expression, and a furin cleavable linker sequence was used to allow the antigens to be cut into separate proteins. To test their utility, some constructs were designed with or without the IgE leader sequence and with or without the cleavage sequence in between proteins. Finally, to identify a stable antigen construct that can be successfully expressed, the order of the genes was changed or some genes were excluded.42MF-367038161Docket No.: 16553-20011.40

[0112] Given the many permutations possible from the variables listed above, an initial set of mRNA antigens were constructed. HPV16-1 expresses E6 and E7 with the (S) mutations and has an IgE leader sequence (SEQ ID NO:26), and a furin cleavable linker (SEQ ID NO:27) in between E6 and E7. HPV16-2 utilizes E6 and E7 with (H) mutations without the IgE leader and linker. Additionally, the order of E6 and E7 were reversed. HPV16-3 and HPV16-4 include El and E2 as additional antigens in a design similar to HPV16-1, and they differ in the order of El and E2. For HPV16-5 and HPV16-6 either El or E2 were excluded from the constructs. HPV16-7 was constructed similar to HPV16-3 but the cleavable linkers were removed. HPV16-8 shares similarity to HPV16-3 except the IgE leader sequence was removed. HPV16-9 was also similar to HPV16-3 but the mutations used for E6 and E7 were swapped from (S) to (H) and the order of E6 and E7 were reversed. Finally, HPV16-10 followed a similar design to HPV16-2 with El and E2 added. The HPV16 amino acid sequences encoded by the constructs are set forth as SEQ ID NOs:30-39.

[0113] LNP Synthesis and Characterization: Lipids for LNP synthesis were acquired from Caymen Chemicals {8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester(SM-102)} and Avanti (distearoyl-phosphatidylcholine[DSPC]; l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 [DMG-PEG2000]). Cholesterol was purchased from Sigma. mRNAs for LNP synthesis were purchased from TriLink BioTechnologies. CleanCap mRNA encoding enhanced green fluorescent protein (GFP) modified with 5-methoxyuridine (5moU) was purchased from TriLink. cGASΔN, cGASΔN mutant (containing E225A and D227A amino acid mutations), and HPV16 antigen mRNAs were custom-ordered from TriLink and synthesized via in vitro transcription from linearized template DNA. The mRNA sequences were capped using Trilink’s CleanCap mRNA technology, with an N1 -methylpseudouridine base modification, and a 120 residue polyA tail.

[0114] The lipid mix was prepared with 50% SM-102 as ionizable lipid, 10% DSPC, 38.5% cholesterol, and 1.5% DMG-PEG2000. The total lipid concentration used for synthesis was 12.5 mM in ethanol. mRNAs were each prepared in pH 4 sodium citrate buffer, and mixed with the lipids at an N / P ratio of 4 or 6. LNPs were synthesized using the NanoAssemblr Ignite instrument (Precision Nanosystems). Lipids in ethanol were combined with the mRNA solutions individually at a 1:3 volumetric ratio, using a flow rate of 12 mL / min. LNPs were washed in 10 volumes of Tris buffer to remove residual ethanol, and then concentrated using Amicon 50K MWCO centrifugal filters. LNPs were filtered through a 0.2-pm filter before use.

[0115] Loading of mRNA into LNPs was quantified using a RiboGreen assay (ThermoFisher) following the manufacturer’s protocol. Samples were diluted to fall within the range of the standard curve. LNPs were lysed using Triton X-100 to assess encapsulation of mRNA into LNPs.43MF-367038161Docket No.: 16553-20011.40 Both total mRNA and encapsulated mRNA were quantified. The size of the LNPs was assessed using dynamic light scattering (DLS) on the NanoBrook Omni (Brookhaven). LNPs were diluted 1:10 in PBS before running on the DLS. Four 90 second measurements were recorded for each sample.

[0116] Western Blotting: THP1 -Null2 cells (Invivogen) were grown in Roswell Park Memorial Institute 1640 (RPMI) medium containing 100 U / mL penicillin / streptomycin, 10% heat inactivated FBS, 25mM N-2-hydroxyethylpiperazine-N-2 -ethane sulfonic acid (HEPES), and lOOpg / mL normocin. Cells were harvested and plated at 500,000 cells per well in a flat-bottom 96-well tissue culture-treated plate in the same growth medium minus the addition of normocin. Cells were treated with LNPs containing mRNA constructs at Ipg / mL encapsulated mRNA. After 24 hours, cell culture supernatant was removed and cells were lysed using radioimmunoprecipitation assay (RIP A) buffer containing 50mM Tris-HCl, pH8.0, 150mM NaCl, 0.1% Triton X-100, 0.5% sodium dodecyl sulphate (SDS), ImM sodium orthovanadate, IrnM NaF, and protease inhibitors tablet (Roche). Protein content was measured via the Pierce Dilution-Free Rapid Gold BCA Protein Assay Kit (ThermoScientific) using the manufacturer’s protocol. Lysates were mixed with 3X concentrated sample loading buffer (Cell Signaling Technology) and boiled for 5 minutes at 100°C. Ten pg protein was loaded per lane of a Mini-PROTEAN TGX precast gel (Bio-Rad) for electrophoresis. Separated proteins were transferred to polyvinylidene fluoride (PVDF) membranes using the Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were blocked using 3% bovine serum albumin in Tris-buffered saline with Tween 20 (TBST) which contained 20mM Tris, pH 7.5, 150mM NaCl, and 0.1% Tween 20. Rabbit anti-HPV16 E7 antibody (Bioss 10202) was diluted 500-fold in blocking buffer. Rabbit anti-vinculin (Cell Signaling Technology 13901) was diluted 1,000-fold in blocking buffer. Membranes were incubated overnight at 4°C on a rocker for the primary antibody incubation. Anti-rabbit HRP secondary antibody (Cell Signaling Technology 7074S) was diluted 5,000-fold in blocking buffer. Samples were incubated at room temperature on a shaker for one hour with the secondary antibody. Three, five-minute wash incubations using TBST were done after primary antibody and secondary antibody incubations. The Clarity Western ECL substrate (Bio-Rad) was used to detect luminescing protein bands on the ChemiDoc Go Imaging System (Bio-Rad).Results

[0117] Multiple batches of LNPs containing HPV16-1 or HPV16-2 mRNA were prepared and stored frozen at -80°C.

[0118] To test if the target mRNAs could be translated by treated human cells, THP-1 cells were incubated for 1 day with LNPs at 1 pg mRNA / mL. Cells were harvested and protein lysates were used for SDS-PAGE and western blotting (FIG.4). Of the 6 batches of LNPs containing HPV16-1, 544MF-367038161Docket No.: 16553-20011.40 samples had HPV16 E7-specific bands between 25 and 37 kDa in size that were not present in the media treatment control condition. This sizing suggested that E6 and E7 were translated as a polyprotein and were not cleaved into separate proteins. Bands at the expected size of 11kDa for E7 were not detected.

[0119] One batch of HPV16-1 LNP did not produce an E7 -specific band. This batch was used for a murine in vivo immunogenicity study before it was used for western blotting, and it elicited an E7-specific immune response. Given this observed immunogenicity in vivo, the western blot suggests that the LNP batch may have lost stability during storage at -80°C.

[0120] Three batches of LNPs containing HPV 16-2 were also tested on THP-1 cells, but HPV16 E7-specific bands were not detected. In murine vaccinations with HPV16-2 LNPs, immunogenicity against E7 was also observed, suggesting that the HPV16-2 mRNA sequence may require further optimization for translation and detection in human cells.

[0121] In all lysate samples, vinculin bands were detectable and were of similar intensity, indicating equal sample integrity and loading.Example 2: Immunogenicity of LNP-HPV mRNAMaterials & Methods

[0122] Immunization and Study Design: C57BL / 6 mice were used to assess the immunogenicity of HPV16-1 and HPV16-2 mRNA containing LNPs. Previous studies with mRNA-LNPs have shown that two doses administered intramuscularly (IM) spaced two weeks apart induce robust antigen-specific T-cell responses when assessed on day 28 or later. To this end, mice were injected intramuscularly with 5 pg, 1 pg, 0.2 pg or 0.05 pg of HPV16-1 or HPV16-2 on days 0 and 14. On day 28, spleens were harvested and processed to single cell suspension for T cell readouts.

[0123] Tetramer Staining: Splenocytes were plated into a 96-well clear round bottom plate and centrifuged at 400xg for 4 minutes. Supernatant was discarded and cells were resuspended in 100 pL PBS containing Live / Dead Aqua at a 1:1000 dilution and incubated for 20 minutes at 4°C. Following incubation, cells were washed with 100 pL of FACS buffer (0.5% Bovine serum albumin in PBS), centrifuged at 400xg for 4 minutes and supernatant was discarded. Cells were resuspended in 100 pL FACS buffer containing mouse Fc Block at a 1:100 dilution and incubated for 10 minutes at 4°C.Following incubation, cells were washed with 100 pL of FACS buffer, centrifuged at 400x g for 4 minutes and supernatant was discarded. Cells were then resuspended in 100 pL FACS buffer containing H2-Db HPV16 E7 Tetramer (MBL) presenting RAHYNIVTF (SEQ ID NO:28) epitope at a 1:20 dilution45MF-367038161Docket No.: 16553-20011.40 and incubated for 30 minutes at 37°C. Following incubation, cells were washed with 100 pL of FACS buffer, centrifuged at 400x g for 4 minutes and supernatant was discarded. Cells were resuspended in 100 pL FACS buffer containing anti-mouse CD3, CD4, CD8, CD62L and CD44 each at 1:200 dilution and incubated for 30 minutes at 4°C. Following incubation, cells were washed with 100 pL of FACS buffer, centrifuged at 400x g for 4 minutes and supernatant was discarded. Cells were resuspended in 150 pL FACS buffer and samples were acquired on a BD FACSymphony A3.Results

[0124] To test the immunogenicity of HPV16-1 (SEQ ID NO:30) and HPV16-2 (SEQ ID NO:31) mRNA constructs, C57BL / 6 mice were injected intramuscularly on Days 0 and 14 with different doses of either construct based on mRNA mass. On day 28, spleens from these mice were processed to single cell suspensions and E7-specific CD8+ T cells in the blood were quantified via H2Db-E7 tetramer staining. Frequencies of total CD8+ T cells in the blood were similar across all groups (FIG.5A). Both HPV16-1 and HPV16-2 expanded E7-specific CD8+ T cells in a dose-dependent manner while mice treated with PBS did not have detectable levels of E7-specific CD8 T cells (FIG.5B and FIG. 5C). The observed immune responses suggest that both mRNA constructs were successfully expressed as protein antigens in the mice and are sufficiently immunogenic to induce T cell responses. HPV16-1 showed higher immunogenicity and was chosen for subsequent studies.Example 3: cGASΔN Enhances Antigen-Specific T-Cell Responses In VivoMaterials & Methods

[0125] Immunization and Study Design: C57BL / 6 mice were used to assess the capacity of different doses of cGASΔN to enhance antigen-specific T cell responses when paired with HPV16-1. To this end, mice were immunized intramuscularly with 0.2 pg / mouse (low dose) or 0.5 pg / mouse (high dose) HPV16-1 alone or combined with 0.2 pg, 1 pg, or 5 pg cGASΔN as detailed in Table 3-1 and Table 3-2 below. For control groups, mice were immunized with HPV16-1 combined with 0.2 pg, 1 pg, or 5 pg GFP or with 5 pg HPV16-1 alone. Mice were boosted with the same doses 14 days after immunization. At the indicated time point spleens were harvested and processed to single cell suspension for T-cell readouts.46MF-367038161Docket No.: 16553-20011.40 Table 3-1. Low Dose HPV16-1 Study GroupsGroup Antigen Adjuvant Schedule1 PBS - Intramuscular injection: 2 HPV16-1 (0.2 pg) cGASΔN (0.2 pg) Day 0, Day 143 HPV16-1 (0.2 pg) GFP (0.2 pg)4 Harvest Spleen HPV16-1 (0.2 pg) cGASΔN (1 pg)Day 355 HPV16-1 (0.2 pg) GFP (1 pg)6 HPV16-1 (0.2 pg) cGASΔN (5 pg)7 HPV16-1 (0.2 pg) GFP (5 pg)8 HPV16-1 (0.2 pg) -9 HPV16-1 (5 pg) -Table 3-2. High Dose HPV16-1 Study GroupsGroup Antigen Adjuvant Schedule1 PBS - Intramuscular injection: 2 HPV16-1 (0.5 pg) cGASΔN (0.2 pg) Day 0, Day 143 HPV16-1 (0.5 pg) GFP (0.2 pg)4 Harvest Spleen HPV16-1 (0.5 pg) cGASΔN (1 pg)Day 425 HPV16-1 (0.5 pg) GFP (1 pg)6 HPV16-1 (0.5 pg) cGASΔN (5 pg)7 HPV16-1 (0.5 pg) GFP (5 pg)8 HPV16-1 (0.5 pg) -

[0126] ELISPOT: IFNγ ELISPOT plates (R& D Systems) were blocked with 200 pL RIO media (RPMI-1640 media supplemented with 10% FBS, 100 U / mL Penicillin, 100 pg / mL Streptomycin, 2 mM L-Glutamine, 1 mM Sodium Pyruvate, and 54 pM P-Mercaptoethanol) for 45 minutes. At the end of blocking, media was discarded and 100 pL of either R10 media alone, R10 media containing 1 pg / mL of HPV16 E6 Pepmix (JPT) or R10 media containing 1 pg / mL of HPV16 E7 Pepmix (JPT) were added to respective wells. Splenocytes were seeded at 100,000 cells per well in 100 pL of R10 media and plates were incubated at 37°C for 20 hours. After incubation, cells were discarded and ELISPOTs were developed according to the manufacturer’s instructions. Plates were left to dry overnight at room temperature. The next day, plates were read on the S6 Universal M2 ELISPOT plate analyzer and spots were quantified using the Smart Count™ function.

[0127] Statistical Analysis: Data were analyzed and graphed using Microsoft Excel and GraphPad Prism software. Data are reported in bar graphs as means with standard deviation. On bar47MF-367038161Docket No.: 16553-20011.40 graphs, each data point represents an individual mouse. Statistical significance was determined using One-way ANOVA with multiple comparisons to HPV alone.Results

[0128] In order to identify a dose of cGASΔN that enhances antigen-specific T cell responses induced by HPV16-1, C57BL / 6 mice were injected intramuscularly (IM) on Days 0 and 14 with 0.2 pg / mouse of HPV16-1 mRNA alone or in combination with different doses of cGASΔN mRNA (Table 3-1). Due to the inherent immunogenicity of mRNA-LNPs, control groups were immunized with HPV16-1 combined with mRNA encoding green fluorescent protein (GFP). The addition of cGASΔN mRNA to 0.2 pg HPV16-1 mRNA enhanced the E7-specific response compared to HPV16-1 alone, but only at the 1 pg dose level (FIG.6A). Moreover, 1 pg of cGASΔN mRNA outperformed 1 pg of GFP mRNA in inducing E7-specific T cell responses (FIG.6A). Interestingly, the addition of 1 pg cGASΔN mRNA to 0.2 pg HPV16-1 mRNA was the only condition to induce robust an E6-specific T-cell response (FIG.6B).

[0129] Due to the weaker immunogenicity of HPV E6, a subsequent study was performed with 0.5 pg / mouse of HPV16-1 mRNA combined with various doses of cGASΔN mRNA or GFP mRNA control (Table 3-2). A high dose of HPV16-1 mRNA alone induced a robust E7-specific T cell response, which was only marginally increased by the addition of cGASΔN mRNA (FIG.7A). In contrast, a robust E6 response was observed when HPV16-1 mRNA was combined with cGASΔN mRNA at all dose levels tested (FIG. 7B), while the addition of GFP mRNA did not enhance E6-specific responses over HPV16-1 mRNA alone. This data demonstrates the weaker immunogenicity of E6 compared to E7 in C57BL / 6 mice. Together these results demonstrate that the immunogenicity of HPV16-1 can be enhanced by the addition of cGASΔN at 1 pg, and that cGASΔN is particularly beneficial for enhancing T cell responses against weakly immunogenic antigens such as HPV 16 E6.

[0130] Another perspective is to consider HPV mRNA LNPs (viral antigen) combined with GFP mRNA LNPs akin to Modema, Inc’s COVID-19 mRNA vaccine. In Modema’s vaccine, mRNA encodes the SARS-CoV-2 spike protein (viral antigen), which is formulated in LNPs for administration. For these studies, LNPs containing the same lipid components as Modema’s vaccine were used. HPV mRNA LNPs administered alone or in combination with GFP mRNA LNPs conferred a low level of immunogenicity. In comparison, HPV mRNA LNPs administered in combination with cGASΔN mRNA LNPs conferred significantly higher levels of immunogenicity (FIG. 13A-13B). Specifically, E6-specific CD4+ T-cell responses were improved when cGASΔN mRNA LNPs were added to a Modema-like formulation (FIG.7B and re-graphed in FIG. 13A). Additionally, E7-specific CD8+ T-cell responses48MF-367038161Docket No.: 16553-20011.40 were improved when reduced amounts of HPV mRNA (0.2 mcg) were administered in combination with cGASΔN mRNA (FIG.6A and re-graphed in FIG. 13B). In short, the addition of cGASΔN mRNA LNPs to a Moderna-like vaccination strategy (viral antigen mRNA LNPs) was found to improve T-cell responses in the context of an HPV16 mRNA vaccine.Example 4: cGASΔN mRNA Enhances T-Cell Responses to Peptide AntigenMaterials & Methods

[0131] Production of monocyte derived dendritic cells (moDCs): Human leukapheresis blood products were freshly obtained from Miltenyi via same day or overnight shipping at 4°C. Miltenyi StraightFrom Leukopak CD14 MicroBead isolation kits were used according to manufacturer instructions to isolate CD14+ monocytes. Briefly, leukopaks ranging in quantity from 5E9 to 1E10 total nucleated cells were evenly aliquoted into 50mL conical tubes. Cells were incubated with CD14 microbeads and then loaded on the MultiMACS using the program “Possel2”. After washing out the negative population, CD 14+ cells were eluted from the columns and counted. An aliquot of monocytes was used to stain for purity of cells to ensure that isolated live cells were >80% CD11c+CD14+ by flow cytometry. Cells were cultured using R10++ media which consisted of RPMI, 10% fetal bovine serum, 100U / mL penicillin / streptomycin, ImM sodium pyruvate, 2mM L-glutamine, non-essential amino acids (Gibco), lOmM HEPES, and 55pM beta-mercaptoethanol. These monocytes were plated in T75 culture flasks at 4–6x107cells per flask in a total of 20mL R10++ media containing a final concentration of 50ng / mL GM-CSF (Miltenyi) and 25ng / mL IL-4 (Miltenyi) to carry out moDC differentiation. Three days after the start of culture, an additional lOmL R10++ containing 50ng / mL GM-CSF and 25ng / mL IL-4 was added to each flask. Six days after start of differentiation, R10++ media containing cells were placed into 50mL conical tubes. Flasks were washed with an additional lOmL PBS and also collected. Cells were centrifuged at 400xg for 5 minutes and supernatant was aspirated. Cells were resuspended in R10++ media and counted. Cells were then adjusted to 4xlOA5 cells / mL in R10++ media.

[0132] Cocultures: Human moDCs obtained from HLA-A02:01+ donors were plated at 2xlOA4 cells / well in 96-well U-bottom tissue culture plates in R10++ media by adding 50 pL of cells per well. To each well, 6xlOA4 CD8 T cells derived from an HLA-A02:01+ donor and reactive to the HPV16 E711-19epitope (Charles River) were added. For control conditions where only moDC or only CD8+ T cells were plated, the missing cells were replaced with an equivalent volume of R10++ media.

[0133] STING Agonists: cGASΔN, cGASΔN mutant and GFP mRNAs were encapsulated in LNPs for use at a final concentration of 1 mg mRNA / mL per well. The cell permeable STING agonist49MF-367038161Docket No.: 16553-20011.40 diABZI (Invivogen, Catalog No. trl-diabzi-2, CAS No. 2138299-34-8) was used at a final concentration 0.5 pM per well. Cell cultures received 50 pL of LNPs or diABZI depending on the desired treatment. For wells not receiving LNP or diABZI treatment, an equivalent volume of R10++ media was added to the wells.

[0134] To activate HPV16 E711-19-reactive CD8+ T-cells in the coculture, HPV16 E711-19peptide YMLDLQPET (SEQ ID NO:53) was used to induce T cell receptor signaling. HPV16 E711-19peptide was diluted in R10++ media and added to wells for a final concentration of 100ng / mL. For control conditions not receiving HPV16 E711-19peptide, an equivalent volume of R10++ media was added to wells. After adding all treatments, cell cultures had a total volume of 200 pL / well.

[0135] Plates were sealed with Breathe-Easy sealing membranes (Sigma- Aldrich) and placed in a 37°C, 5% CO2 incubator overnight. For study readouts, plates were centrifuged at 400x g for 4 minutes and cell culture supernatant was collected in 96-well U-bottom plates.

[0136] Lumit IFNβ and IFNγ Immunoassays: To determine the concentrations of IFNβ and IFNγ secreted into the cell culture supernatant, Lumit Immunoassays were used. Conceptually, two antibodies are used to bind to the target cytokine in the sample, and each antibody is tethered to a subunit of an enzyme. Binding of the two antibodies to the target cytokine enables the subunits to be in close enough proximity to bind and function. A substrate for the enzyme is then added to create a luminescent signal that indicates the cytokine concentration in the supernatant.

[0137] The IFNβ and IFNγ kit standards were prepared in cell culture medium (R10++) starting at 25,000 pg / mL with two-fold dilutions to 24.4pg / mL. Blank controls were included for all standards. Sample supernatants were diluted 2-fold in R10++ for the IFNβ and IFNγ assay.

[0138] To prepare antibodies for each assay, recommended protocols from the manufacturer were followed to dilute antibodies in R10++ media to reach a 2-fold concentrate. Antibodies were combined with sample supernatant or standards (12.5 pL of each) by pipetting into white opaque 384-well plates. Samples were then incubated at 37°C for 1 hour. Samples were then allowed to cool to room temperature in the dark while preparing substrate. For every 3,040 pL of Detection Buffer B used, 175 pL of Detection Substrate B was added. Substrate was mixed by briefly vortexing and was added at 6.25 pL / well. On the GloMax Explorer microplate reader (Promega), sample luminescence was recorded at 500 milliseconds / well. Prism software was used to construct a standard curve using 2nd order polynomial logistics analysis. Interpolated results were corrected for sample dilution.

[0139] T-Cell Assay: To activate T cells, anti-CD3 (OKT3, Biolegend) and anti-CD28 (CD28.2, Biolegend) antibodies were diluted in PBS to a final concentration of 10 pg / mL and plated in a 50MF-367038161Docket No.: 16553-20011.40 96-well plate. For control conditions not receiving anti-CD3 and anti-CD28 antibodies, an equivalent volume of PBS was added to wells. The plate was incubated for 3 hours at 37°C. After incubation, the plate was washed 3 times with PBS before addition of cells.

[0140] To each well, 25 pL of HLA-A02+ CD8+ T-cells specific for MART-1 antigen were added at 2.4x106cells / mL. cGASΔN mRNA was encapsulated in LNPs to be used at a final concentration of 1 μg / mL per well. The cell permeable STING agonist diABZI was used at a final concentration 0.5 pM per well. Cell cultures received 50 pL of the cGASΔN LNP or diABZI depending on the desired treatment. For wells not receiving LNP or diABZI treatment, an equivalent volume of R10++ media was added to the wells. After adding all treatments, cell cultures had a total volume of 200 pL / well.

[0141] Plates were sealed with Breathe-Easy Membranes plate sealers and placed in a 37°C, 5% CO2 incubator overnight. For study readouts, plates were centrifuged at 400x g for 4 minutes and cell culture supernatant was collected in 96-well U-bottom plates.Results

[0142] To investigate how cGASΔN stimulation affects interactions between moDCs and CD8+ T-cells, cocultures were set up as illustrated in FIG. 8A. Human moDCs were treated with LNPs delivering cGASΔN, cGASΔN mutant, or GFP mRNA. The cGASΔN mutant construct is similar to cGASΔN but contains a mutation that prevents its binding to DNA. LNP-mediated STING signaling was compared to treatment with the small molecule STING agonist diABZI. For antigen-specific stimulation, HPV16 E711-19peptide was added to the cultures. Three hours later HPV16 E7-specific CD8+ T-cells were added to the cocultures. After overnight incubation, supernatants were harvested, and IFNβ levels were measured to assess activation of STING signaling. Significantly increased secretion of IFNP was observed with cGASΔN treatment, and this effect was independent of HPV E7 peptide treatment (FIG.8B). In contrast no expression of IFNP was observed when cells received cGASΔN mutant LNP, GFP LNP, or were left unstimulated. Treatment with diABZI also induced IFNP secretion, but the amount of IFNP was significantly less compared to cGASΔN treatment (FIG. 8B). IFNP was produced by moDCs because monocultures of moDC and T-cells in isolation produced IFNP only in moDC cultures treated with cGASΔN or diABZI (not shown). These data suggest that CD8+ T-cells are inefficient at taking-up LNPs. Thus, LNPs primarily target moDCs in the cocultures.

[0143] Given that cGASΔN treatment induced IFNP from moDC, IFNγ was measured to assess the CD8+ T-cell response. Without addition of E7 peptide, minimal IFNγ responses were observed51MF-367038161Docket No.: 16553-20011.40 from the various LNP treatments and diABZI treatment (FIG.8C). With addition of HPV E7 peptide, cGASΔN treatment led to significantly increased production of IFNγ by HPV16 E7-specific T-cells compared to negative control treatments (FIG.8C). Surprisingly, diABZI induced almost no IFNγ production from the HPV E7 T cells despite inducing IFNP secretion from moDCs (FIG. 8B and FIG. 8C). In monoculture conditions, moDCs did not produce IFNγ in any treatment conditions (not shown), and T-cells in monoculture were not able to elevate IFNγ production with cGASΔN treatment (FIG.8D).Thus, in moDC-T cell cocultures, cGASΔN treatment of moDCs enhances the T cell IFNγ response.

[0144] Treatment of moDC with diABZI induced STING signaling, similar to cGASΔN treatment as indicated by the IFNP response (FIG. 8B). However, diABZI treatment did not result in an enhanced IFNγ response from the T cells in coculture (FIG. 8C). One substantial difference between the two STING pathway agonists, cGASΔN and diABZI, is that diABZI is a cell permeable small molecule that can potentially target both moDC and T cells. To understand if the cell permeable agonist diABZI acts directly on T cells, antigen-specific CD8+ T-cells were treated in a monoculture as illustrated in FIG. 9A.To activate the T cell receptor (TCR), the culture plate was coated with anti-CD3 and anti-CD28 antibodies. After washing away unbound antibodies, clonal human CD8+ T-cells specific for the melanoma antigen MART-1 were plated and stimulated with diABZI, cGASΔN LNP or no agonist. Control T-cells were plated with no TCR stimulation and no STING pathway agonist treatment. Engagement of the TCR triggered IFNγ production (FIG. 9B). cGASΔN LNP treatment also permitted IFNγ production, but diABZI treatment significantly reduced IFNγ production, suggesting that diABZI has a directly inhibitory effect on TCR signaling (FIG. 9B). These data suggest that selective cell targeting by cGASΔN LNPs can have distinct mechanistic consequences compared to other STING agonist technologies (e.g., cell permeable, small molecules) that do not have cell selectivity.Example 5: Assessment of Expression and Immunogenicity of HPV 16 mRNA Constructs Materials & Methods

[0145] Immunization and Study Design: C57BL / 6 and BALB / c mice were used to assess the immunogenicity of LNPs containing mRNA encoding for 10 different HPV-16 antigens. Mice were injected intramuscularly with LNPs at 2 pg / mouse of each mRNA construct on days 0 and 14. On day 35, spleens were harvested and processed to single cell suspension for T cell readouts.

[0146] Splenocyte Restimulation: Splenocytes were seeded at 2x105cells per well and stimulated for 72 hours with either RIO media alone or RIO media containing 1 pg / mL of either HPV-1652MF-367038161Docket No.: 16553-20011.40 El Pepmix (JPT), HPV-16 E2 Pepmix (JPT), HPV-16 E6 Pepmix (JPT) or HPV-16 E7 Pepmix (JPT). After 72 hours, supernatants were collected and IFNγ was detected by Lumit (Promega) according to manufacturer’s instructions.Results

[0147] In order to assess the immunogenicity of LNPs containing 10 different HPV-16 antigen constructs, two mouse strains with different MHC backgrounds were used. C57BL / 6 and BALB / c mice were injected intramuscularly on Days 0 and 14 with 2 pg / mouse of each construct. Antigenspecific T cell responses were assessed on day 35 by culturing splenocytes for 72 hours with peptide pools corresponding to 4 different HPV-16 proteins encoded on the constructs: El, E2, E6 and E7. IFNγ in cell culture supernatants was quantified as a measure of antigen-specific T cell activation.

[0148] Responses against El were detected at varying levels in both mouse strains for every construct that encoded El (FIG. 10A-FIG. 10B), demonstrating that El is a strong immunogen in both mouse backgrounds. In BALB / c mice, every construct that encoded E2 induced a response against E2 (FIG. 10C), with HPV16-6 inducing the strongest response by 6-fold. Interestingly, HPV16-6 was the only construct that induced a response against E2 in C57BL / 6 mice (FIG. 10D) despite multiple constructs encoding for E2. Similarly, while all constructs encoded E6 only HPV16-1, HPV16-2, HPV16-5 and HPV16-6 induced a T cell response against E6 in BALB / c mice (FIG. 10E) while only HPV16-1, HPV16-2, and HPV16-6 induced an E6 response in C57BL / 6 mice (FIG. 10F). Lastly, although all constructs encoded E7, no responses against E7 were detected against in BALB / c mice (FIG. 10G), suggesting a lack of E7 epitopes in the BALB / c background. Additionally, only HPV16-1, HPV16-2, and HPV16-6 induced an E7 response in C57BL / 6 mice (FIG. 10H).

[0149] This data demonstrates that all constructs were expressed and capable of inducing a T cell response against at least one antigen. In C57BL / 6 mice, but not BALB / c mice, constructs containing El were unable to induce T cell responses against any other antigens encoded on the same mRNA, suggesting strain-specific immunodominance of the El antigen. In both mouse strains, HPV16-6 induced the strongest E2 response while also inducing a response against E6 and E7 (C57BL / 6 only).Example 6: Polyfunctionality of T-Cells Reactive with HPV E6 or E7Materials & Methods

[0150] Immunization and Study Design: C57BL / 6 mice were used to assess the capacity of cGASΔN to enhance the polyfunctionality of antigen-specific T cells in vivo. In one set of experiments,53MF-367038161Docket No.: 16553-20011.40 mice were injected intramuscularly on days 0 and 14 with 0.5 pg / mouse of HPV-16-1 LNPs combined with either 1 pg / mouse of GFP LNPs or 1 pg / mouse of cGASΔN LNPs. On day 35 and day 70, splenocytes were harvested from mice and processed to single cell suspension for T-cell readouts (Table 6-1).

[0151] In another experiment, mice were injected intramuscularly with LNPs containing mRNA for HPV16-1 or HPV16-6 antigens alone or co-formulated with mRNA for cGASΔN. Mice were immunized with an equimolar dose of antigen equating to 0.5 pg / mouse HPV16-1 co-formulated with 1 pg / mouse cGASΔN or 1 pg / mouse HPV16-6 co-formulated with 1 pg / mouse cGASΔN. Mice were immunized on days 0 and 14 and splenocytes were harvested from mice on day 35 for T-cell readouts (Table 6-2).

[0152] The LNPs of these experiments included the ionizable lipid SM-102 and other lipids used in Modema Inc.’s COVID- 19 mRNA vaccine. As such, the HPV mRNA LNP plus GFP mRNA LNP group was considered to be comparable to Modema’ s vaccination approach.

[0153] Intracellular Cytokine Staining: Splenocytes were seeded at 5x105cells per well and stimulated with R10 media alone or R10 media containing 1 ug / mL of HPV16 E6 Pepmix (JPT) or HPV16 E7 Pepmix (JPT). All stimulation conditions also included anti-mouse CD107a antibody. Cells were incubated at 37°C for 1.5 hours, after which Golgi Plug (BD Biosciences) and Golgi Stop (BD Biosciences) was added to each well. Cells were incubated at 37°C for 5 hours and then moved to 4°C overnight. The following day, cells were washed and stained with Fixable Live / DeadAqua in PBS for 20 minutes at 4°C. Cells were washed and incubated with Fc block for 20 minutes at 4°C. Cells were washed and stained for anti-mouse CD4 and CD8 for 45 minutes at 4°C. Cells were washed and fixed with BD Cytofix / Cytoperm according to manufacturer’s instructions. Cells were washed and stained with antimouse CD3, TNFa, IFNγ, and IL-2 for 1 hour at 4°C. Cells were washed and resuspended in FACS buffer and samples were acquired on a BD FACS Symphony A3 cytometer.

[0154] Statistical Analysis: Data were analyzed and graphed using Microsoft Excel and GraphPad Prism software and reported in tables as means. Statistical significance was determined using Student’s T-test or using repeated measures two-way ANOVA with the Geisser-Greenhouse correction. Mouse data were matched, and Sidak’s multiple comparisons test was run with individual variances computed for each comparison.Results

[0155] Polyfunctional T-cells can secrete multiple cytokines in addition to IFNγ, namely TNFa and IL-2. Compared to T-cells that only secrete IFNγ, polyfunctional T-cells are considered54MF-367038161Docket No.: 16553-20011.40 functionally superior and have been associated with better control of chronic infections, as well as better anti-tumor immunity (Donia et al., Cancer Research, 23(19):5779-5788, 2017; and Makedonas and Betts, Springer Seminars in Immunopathology, 28(3):209-219, 2006). In order to assess how cGASΔN influences the polyfunctionality of antigen-specific CD4+ and CD8+ T cells, C57BL / 6 mice were injected intramuscularly on days 0 and 14 with LNPs containing HPV16-1 mRNA combined with LNPs containing GFP or cGASΔN mRNA. On days 35 and day 70, splenocytes were harvested and stimulated with peptide pools corresponding to HPV16 proteins E6 or E7. T-cells were subsequently assessed for production of IFNγ, TNFa, and IL-2 via intracellular cytokine staining (ICS).

[0156] At both early and late time points, the cytokine responses induced by restimulation with E6 peptides were too low to be detected by ICS (data not shown). In contrast, strong IFNγ signal was detected specifically in the CD8+ T cell compartment when cells were stimulated with E7 peptides. At both early and late time points the frequencies of IFNγ+ CD8+ T-cells were comparable when mice were immunized with HPV16-1 mRNA combined with GFP or with cGASΔN mRNA (data not shown).Despite no difference in the frequency of IFNγ-responsive cells, the IFNγ+ CD8+ T cell populations were further analyzed for polyfunctionality. Significantly more IFNγ positive CD8+ T-cells from mice that received cGASΔN mRNA also produced TNFa or TNFa and IL-2 on day 35 (Table 6-1). On day 70 significantly more IFNγ positive CD8+ T-cells additionally produced TNFa from mice receiving cGASΔN mRNA (Table 6-1). Commensurate with these increased frequencies of polyfunctional T-cells, the proportion of cells only expressing IFNγ when treated with cGASΔN mRNA significantly decreased (Table 6-1). Together these data show that while the percentage of IFNγ+ CD8+ T-cells was not increased by the addition of cGASΔN mRNA, the frequency of polyfunctional CD8+ T-cells within this population was significantly increased. This indicates that cGASΔN mRNA improves the quality of E7-specific CD8+ T-cells and that this improvement is durable as it was observed out to 70 days postimmunization.55MF-367038161Docket No.: 16553-20011.40 Table 6-1: Combination of cGASΔN LNPs and HPV16-1 LNPs Enhances Poly functionality of E7-specific CD8+ T-cells0.5 pg HPV16-1 0.5 pg HPV16-l p-value+ 1 pg GFP + 1 pg cGASΔNDay 35IFNγ+ 59.2% 35.0% <0.0001 IFNγ+ / TNFa+ 34.0% 49.4% 0.0002 IFNγ+ / TNFa+ / IL-2+ 5.8% 14.2% 0.0227Day 70IFNγ+ 78.7% 56.4% 0.0003 IFNγ+ / TNFa+ 18.8% 41.3% 0.0003 IFNγ+ / TNFa+ / IL-2+ 2.4% 8.7% 0.2729Table shows mean percentages of IFNγ+ CD8+ T cells expressing the indicated cytokine(s) at day 35 or 70 post-immunization. Statistical significance determined by two-way ANOVA or mixed-effects analysis biological replicates were excluded due to insufficient event counts.

[0157] Next, the ability of cGASΔN mRNA to induce polyfunctional CD8+ T-cells was assessed when cGASΔN mRNA was co-formulated into the same LNP with either HPV16-1 mRNA or HPV16-6 mRNA. Mice were immunized on days 0 and 14 with 0.5 pg HPV16-1 mRNA or 1 pg HPV16-6 mRNA alone or combined with 1 pg cGASΔN mRNA in the same LNP. On day 35, splenocytes were stimulated with peptide pools corresponding to HP V 16 proteins E2, E6 or E7. T-cells were subsequently assessed for production of IFNγ, TNFa, and IL-2 via intracellular cytokine staining (ICS).

[0158] Similar to above, cytokine production upon stimulation with E2 or E6 peptides was too low to be quantified by ICS, however strong IFNγ+ signal was observed in the CD8+ T-cell compartment when cells were restimulated with E7 peptides. Again, the frequencies of IFNγ+ CD8+ T cells was similar when mice were immunized with HPV mRNA alone or combined with cGASΔN mRNA (data not shown). However, the addition of cGASΔN mRNA significantly enhanced the number of polyfunctional IFNγ+TNFa-i- T-cells when co-formulated with HPV16-1 mRNA (Table 6-2). When coformulated with HPV16-6 mRNA, the frequencies of polyfunctional IFNγ+TNFa-i- T-cells were also significantly increased (Table 6-2). For comparisons of both HPV antigen constructs, the proportion of cells only expressing IFNγ significantly decreased when co-formulated with cGASΔN mRNA (Table 6-2). These data show that cGASΔN mRNA enhances the polyfunctionality of E7-specific CD8+ T-cells when combined with HPV16-1 mRNA or HPV16-6 mRNA and this adjuvant effect is seen when cGASΔN mRNA is delivered as a separate LNP or when co-formulated with HPV mRNA in the same LNP.56MF-367038161Docket No.: 16553-20011.40 Table 6-2: Co-formulation of cGASΔN and HPV16 mRNAs in LNPs Leads to Enhanced Polyfunctionality of E7-specific CD8+ T-cellsHPV16-1 0.5 pg HPV16-1 0.5 pg HPV16-1 p-value+ 1 pg cGASΔNIFNγ+ 72.2% 54.2% <0.0001 IFNγ+ / TNFa+ 19.2% 32.9% <0.0001 IFNγ+ / TNFa+ / IL-2+ 5.5% 10.5% 0.2611HPV16-6 1 pg HPV16-6 1 pg HPV16-6 p-value+ 1 pg cGASΔNIFNγ+ 70.5% 48.5% <0.0001 IFNγ+ / TNFa+ 23.6% 41.7% 0.0004 IFNγ+ / TNFa+ / IL-2+ 4.7% 9.0% 0.6828Table shows mean percentages of IFNγ+ CD8+ T-cells expressing the indicated cytokine(s) at day 35 post-immunization. Statistical significance determined by two-way ANOVA or mixed-effects analysis biological replicates were excluded due to insufficient event counts.

[0159] In another study, similar frequencies of IFNγ-i- CD8+ T-cells were observed in splenocytes from mice that received either GFP mRNA (3.8%) or cGASΔN mRNA (4.0%) co-formulated with HPV16-1 mRNA (data not shown, not statistically significant). To determine if there were differences in the polyfunctionality between immunization groups, IFNγ+ CD8+ T-cells were analyzed for their expression of TNFa and / or IL-2. Representative histograms of the IFNγ-i- CD8+ T-cell populations demonstrated a clear increase in expression of TNFa and IL-2 when mice were immunized with cGASΔN mRNA compared to GFP mRNA in a Modema-like formulation (FIG. 14). Moreover, the addition of cGASΔN mRNA resulted in significantly more polyfunctional CD8+ T-cells and a concomitant decrease in monofunctional IFNγ-only T-cells (Table 6-3). In mice that received GFP mRNA, 73.9% of E7 -responsive CD8+ T-cells only produced IFNγ, while 19.0% also produced TNFa (Table 6-3). In contrast, mice that received cGASΔN mRNA had 43.4% of E7 -responsive CD8+ T-cells that produced IFNγ only, while 44.1% also produced TNFa (Table 6-3). The frequency of T-cells that also produced IL-2 was comparable between the two groups, however mice that received cGASΔN mRNA had significantly higher percentage of T-cells that produced IFNγ, TNFa and IL-2 (11.2%) compared to mice that received GFP mRNA (4.9%) (Table 6-3). These data further confirm that including cGASΔN mRNA in an immunogenic composition increases the polyfunctionality of HPV16 E7-specific CD8+ T-cells.57MF-367038161Docket No.: 16553-20011.40 Table 6-3: Co-formulation of cGASΔN and HPV16 mRNAs in LNPs Leads to Enhanced Polyfunctionality of E7-specific CD8+ T-cellsIFNγ+ CD8+ T-cells 0.5 pg HPV16-1 0.5 pg HPV16-l Adjusted + 1 pg GFP + 1 pg cGASΔN p-value IFNγ+ 73.92% 43.40% 0.0005 IFNγ+ / IL-2+ 2.108% 1.252% 0.3360 IFNγ+ / TNFa+ 19.04% 44.12% 0.0001 IFNγ+ / TNFa+ / IL-2+ 4.936% 11.23% 0.0304Table shows mean percentages of CD8+ T cells expressing the indicated cytokine(s) at day 35 postimmunization (N = 5 mice / group). Statistical comparisons were tested using two-way ANOVA followed by Sidak’s multiple comparisons test.

[0160] The adjuvant capacity of cGASΔN mRNA LNPs works through the STING signaling pathway in antigen presenting cells. In in vitro studies, expression of cGASΔN resulted in increased expression of MHC molecules (e.g., antigen presentation) and costimulatory molecules (e.g., CD40), as well as increased production of cytokines (e.g., type I interferons) and chemokines (e.g., the T-cell chemoattractant IP-10). Dendritic cells (DCs) were also observed to increase the expression of CCR7 after cGASΔN mRNA treatment. This is important because CCR7 is a chemokine receptor required for DC trafficking to lymph nodes. These attributes within an antigen presenting cell contribute to durable T-cell responses (Table 6-4). In contrast, immunization with only HPV mRNA (antigen only) in a Modema-like lipid formulation (comprising SM-102), which lacks cGASΔN mRNA and hence STING signaling, results in a less immunogenic composition and induction of less durable T-cell responses.Table 6-4: Addition of cGASΔN mRNA Increases Immunogenicity of HPV mRNA Vaccine HPV LNP + GFP LNPImmune Function(Modema-like) HPV LNP + cGASΔN-LNP Antigen Expression High High Antigen Presentation High High CD80 / 86 / 40 Costimulation Low HighCCR7 Expression Low HighType I IFN Low HighT-cell Chemokines Low HighDurable T cells Low High58MF-367038161Docket No.: 16553-20011.40 Example 7: MHC Immunopeptidomics Analyses in THP-1 CellsMaterials & Methods

[0161] Antigens: mRNA constructs as illustrated in FIG.3 were compared to assess which results in the best antigen presentation of viral epitopes on MHC Class I molecules (MHC-I).

[0162] Treatment of THP-1 cells for Immunopeptidomics: THP-1-Null2 cells were purchased from Invivogen. Twenty-four T75 flasks were seeded at 0.4x106cells / mL in 15mL / flask and were cultured for 3 days. After counting, 20x106cells per 10 mL were seeded in T75 flasks with RPMI media. One pg / mL antigen LNP in combination with 1 pg / mL cGASΔN LNP were added for 24h. After 24h, cells were collected. Twenty-three million cells per treatment group were collected in 15mL tubes and washed twice with PBS. After the last wash the supernatant was removed and cell pellets by themselves were spun down. Residual liquid was removed by using gel loading tips. Cell pellets were stored at -80°C until they were transferred to the Translational Immunogenomics Lab and Blais Proteomics core facilities at Dana Farber Cancer Institute for MHC-I immunoprecipitation, peptide elution, and liquid chromatography-tandem mass spectrometry analysis.

[0163] LDH Release Assay: The CyQUANT cytotoxicity assay (Thermo Fisher) measures cell death by quantifying lactate dehydrogenase (LDH) enzyme released into the culture medium from damaged or lysed cells, serving as an indicator of cell membrane integrity and cytotoxicity. To perform the LDH assay, 35uL of supernatant were transferred to a 96-well ELISA plate and 35uL of Reaction Mix to the same 96-well ELISA plate. Afterwards the plate was mixed briefly and incubated for 30 min.Viability in samples was calculated based on absorbance readings at 490nm and 680 nm wavelengths relative to a lysed cell sample (maximum LDH release control).

[0164] Western Blotting: THP1 cells were cultured in RPMI- 1640 supplemented with 10% heat-inactivated FBS, penicillin / streptomycin, HEPES, and normocin. Cells were plated in 96-well plates and treated with LNPs containing mRNA constructs and cGASΔN constructs. After 24 hours, supernatants were removed and cells lysed in RIPA buffer containing protease and phosphatase inhibitors.

[0165] Protein concentration was determined using a BCA assay. Lysates were mixed with sample buffer, boiled, and 10 pg of protein per sample was resolved by SDS-PAGE. Proteins were transferred to PVDF membranes, blocked in BSA-TBST, and probed overnight at 4°C with a mouse anti-E2 antibody (Abeam, clone TVG 261). After washing, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody and developed using chemiluminescent substrate. Signal detection was performed with a digital imaging system.59MF-367038161Docket No.: 16553-20011.40

[0166] Lumit IFN: To measure secreted IFNβ levels, IFNP lumit immunoassay (Promega) was used according to manufacturer’s instructions. In this assay, two antibodies specific to the target cytokine are each linked to two portions of a luciferase enzyme. When both antibodies bind to the same cytokine molecule, the enzyme subunits are brought into proximity, allowing them to assemble into a complete and active luciferase enzyme. Upon addition of a substrate, the active enzyme produces a luminescent signal, which correlates with the amount of cytokine present in the supernatant. IFNP in cell supernatant samples were determined by comparing to a standard with predetermined IFNP concentrations.Results

[0167] Ten HPV mRNA (viral antigen) constructs were evaluated for their ability to be translated, processed, and presented on MHC-I molecules by THP-1 cells in the context of cGASΔN mRNA treatment. THP-1 cells are derived from a human acute monocytic leukemia, and express HLA-A*02:01; A*24:02; B*15:ll; B*35:01; and C*03:03 alleles. Twenty-four hours after treatment with LNPs, cell culture supernatant and a small proportion of cells were collected to verify the intended activity of the LNP treatments. The majority of THP-1 cells were used to immuno-precipitate MHC-I molecules to elute bound peptides. Epitope sequences were then identified using liquid chromatographytandem mass spectrometry.

[0168] To assess cell viability after 24 hours of treatment, cell supernatant was measured for LDH release as a measure of cell viability. All groups treated with HPV mRNA and cGASΔN mRNA were approximately 80% viable. None of the samples exhibited viability below a 75% threshold for unacceptable cytotoxicity (FIG. 11A).

[0169] To confirm that cGASΔN mRNA treatment induced innate immune signaling in THP-1 cells, IFN secretion was measured using an IFN immunoassay. Cells not treated with cGASΔN mRNA had no detectable IFNβ (FIG. 11B). In contrast, all cGASΔN mRNA treated groups had detectable IFNβ levels in the supernatant, indicative of adjuvant activity (FIG. 11B).

[0170] To confirm the functionality of HPV mRNA constructs, protein expression of viral antigen constructs was validated via western blot (FIG. 11C). β-actin and vinculin were blotted as loading controls, and demonstrated equal protein loading across all treatment conditions (FIG. 11C). An E2 antibody was found to be the most reliable out of all commercially available antibodies tested for detection of relevant HPV16 antigens. For conditions where cells were untreated or treated with HPV16-1, HPV16-2, or HPV16-5 mRNA, only non-specific bands were observed because these constructs do not encode E2. However, E2-specific bands were observed at mass sizes indicative of a lack of cleavage at60MF-367038161Docket No.: 16553-20011.40 furin-cleavable linker sites. In particular, E2-specific bands were detected at the expected molecular weight of -150 kDa for constructs expressing El, E2, E6 and E7 with varying signal intensities. Among the constructs encoding El, E2, E6 and E7, HPV16-4 showed the strongest antigen expression, followed by HPV16-3. HPV16-7 and HPV16-9 showed weaker expression, while HPV16-8 had no detectable antigen expression. HPV16-6, which lacks El, had an observed band at 75 kDa, indicative of antigen expression in THP-1 cells. This band was the strongest among all E2-specific bands.

[0171] In addition to validating antigen expression, cGASΔN expression was also validated by western blot. Since cGASΔN lacks the N-terminal domain, its expected molecular weight is 42 kDa in contrast to the 62 kDa expected size for the full-length, endogenous protein. Following treatment of THP-1 cells with cGASΔN mRNA, both the 42 kDa cGASΔN and 62 kDa wild-type bands were detected (FIG. 11C). In untreated cells, only the endogenous cGAS band at 62 kDa appeared (FIG. 11C).

[0172] After verifying that mRNA-containing LNP treatments resulted in antigen expression and cGASΔN activity, HPV16 epitopes presented by THP-1 cells on MHC-I molecules were identified. To investigate which antigen components influence efficiency of epitope presentation, THP-1 cells were treated with mRNA constructs (HPV16-1 to HPV16-10), which differed in the presence / absence of an IgE leader sequence, presence / absence of furin linkers, mutations in E6 and E7 (present in “H” versions of E6 and E7 sequences of SEQ ID NO: 19 and SEQ ID NO:22 vs. present in “S” versions of E6 and E7 sequences of SEQ ID NO:18 and SEQ ID NO:21), presence / absence of El and E2 genes, and the order of El and E2 genes within the mRNA constructs. On average 10,000 epitopes presented on MHC-I molecules were identified using LC-MS / MS, and among these epitopes, 8 were specific to the HPV antigens encoded by the mRNA constructs. Table 7-1 lists detectable HPV16 MHC-I epitopes (epitope presence is indicated by check mark). All antigens led to robust presentation of the same E7 epitope (YMLDLQPET set forth as SEQ ID NO:53) and variable presentation of El, E2 and E6 epitopes.Characteristics of mRNA constructs are listed in the first 4 rows of Table 7-1.

[0173] When considering the effect of including El and / or E2 in the mRNA constructs, the number of epitopes identified in each condition did not increase with the addition of El or E2 genes, suggesting no correlation. Similarly, no trend was observed when comparing (H) mutations (HPV16-9) versus (S) mutations (HPV16-3). Thus, addition of El and E2 to antigen constructs and the use of either (H) or (S) mutations did not affect the number of unique HPV epitopes that could be presented on MHC-I molecules (Table 7-1). In contrast, removal of the IgE leader or furin linkers resulted in the fewest number of unique epitopes presented (Table 7-1). Constructs containing both the IgE leader and furin linker showed greater epitope presentation (HPV16-3, HPV16-4, HPV16-5, HPV16-6, HPV16-9) compared to those lacking one or both elements (HPV16-7, HPV16-8, HPV16-10). When comparing61MF-367038161Docket No.: 16553-20011.40 HPV16-3 and HPV16-4, the constructs are nearly identical except that the order of El and E2 are reversed. Surprisingly when El was placed at the C-terminus (HPV16-4), half as many epitopes were detected as when E2 was placed at the C-terminus (HPV16-3) (Table 7-1). These data indicate that inclusion of an IgE leader sequence, inclusion of furin linker sequences between viral genes, and the order of viral genes on the mRNA construct affect the efficiency of viral epitope presentation on MHC-I molecules.Table 7-1: MHC-I Epitopes in HPV16 Antigens Presented by THP-1 Cells+ cGASΔN(H) or (S)H H H mutation S s s s s s sIgE leader + - + + + + + - + - Furin linker + - + + + + - + + - E1 / E2 - - E1 / E2 E2 / E1 El E2 E1 / E2 E1 / E2 E1 / E2 E1 / E2 HPV mRNA 16-1 16-2 16-3 16-4 16-5 16-6 16-7 16-8 16-9 16-10 Epitope AgFPFDENGNPVY El z zHMRLECAIY E2 z zTLQDVSLEV E2 zYVHEGIRTY E2 z zALQAIELQL E2 ✓ THDIILECV E6 z z z z z HDIILECVY E6 zzYMLDLQPET E7 z z z z z z z z z Ag = antigen. HPV MHC-I epitope sequences identified include: FPFDENGNPVY (SEQ ID NO:46), HMRLECAIY (SEQ ID NO:47), TLQDVSLEV (SEQ ID NO:48), YVHEGIRTY (SEQ ID NO:49), ALQAIELQL (SEQ ID NO:50), THDIILECV (SEQ ID NO:51), HDIILECVY (SEQ ID NO:52), and YMLDLQPET (SEQ ID NO:53).Example 8: MHC Immunopeptidomics Analyses of Human moDCsMaterials & Methods

[0174] Antigens: HPV16-1, HPV16-3, HPV16-4, and HPV16-6 mRNA constructs as illustrated in FIG.3 were compared to assess which results in the best antigen presentation of viral epitopes on MHC-I.

[0175] Monocyte to moDC Differentiation: Fresh leukapheresis products were obtained from AllCells and shipped at 4°C. CD14+ monocytes were isolated using Miltenyi's StraightFrom Leukopak62MF-367038161Docket No.: 16553-20011.40 CD14 MicroBead kit and the MultiMACS system (“Possel2” program). Purity of >80% CD11c+CD14+ was confirmed by flow cytometry. Monocytes were cultured in R10++ medium (RPMI supplemented with 10% FBS, penicillin / streptomycin, sodium pyruvate, L-glutamine, NEAAs, HEPES, and β-mercaptoethanol) at 4–6x107cells per T75 flask in 20 mL medium containing 50 ng / mL GM-CSF and 25 ng / mL IL-4. On day 3, 10 mL of fresh cytokine-supplemented media was added. On day 6, cells were collected, washed with PBS, centrifuged, resuspended in R10++, counted, and used for experimental treatment.

[0176] Treatment of Cells for Immunop eptidomics: moDCs were plated as 5x106cells / mL in a 6-well plate and treated with either 3 pmol / mL of HPV16-1, HPV16-3, HPV16-4 and HPV16-6 LNP alone or in combination with 1 pg / mL cGASΔN LNP. Cells were incubated for 24 h and then plates were spun down at 400xg for 5 min. Afterwards supernatant was collected for viability analysis by an LDH release assay (Invitrogen) and to determine IFN production by IFNβ Lumit immunoassay (Promega). Assays were performed with the same methods as the previous immunopeptidomics example using THP-1 cells and supernatant. After supernatant was harvested, 1 mL of media was added to each well and cells were washed to collect loosely attached moDCs. Afterwards 2 mL of cold PBS+EDTA were added to residual cells on each well of the 6- well plate and the plates were incubated for 15 min at 4°C to detach strongly adherent cells. Cells from each treatment group were transferred to 50 mL canonical tubes and spun down at 400xg for 5 min. Afterwards cells were resuspended in 10 mL of PBS and counted. Nine million cells per treatment group were distributed among 15 mL canonical tubes and cells were spun down at 400xg for 5 min. Supernatant was removed, another 10 mL DPBS were added and cells were spun down again. Afterwards supernatant was removed and cells were spun down again without addition of DPBS. Residual supernatant was removed and cells were frozen at -80°C prior to shipment to TIGIL and Blais Proteomics Center core facilities at Dana Farber Cancer Institute for elution of MHC-I epitopes and liquid chromatography tandem mass spectrometry (LC-MS / MS).

[0177] Western Blotting: moDCs Cells were plated in 96-well plates and treated with LNPs containing mRNA constructs and cGASΔN constructs. After 24 hours, supernatants were removed and cells lysed in RIPA buffer containing protease and phosphatase inhibitors.

[0178] Protein concentration was determined using a BCA assay. Lysates were mixed with sample buffer, boiled, and 10 pg of protein per sample was resolved by SDS-PAGE. Proteins were transferred to PVDF membranes, blocked in BSA-TBST, and probed overnight at 4°C with a mouse anti-E2 antibody. After washing, membranes were incubated with HRP-conjugated secondary antibody and developed using chemiluminescent substrate. Signal detection was performed with a digital imaging system.63MF-367038161Docket No.: 16553-20011.40 Results

[0179] Four mRNA constructs (HPV16-1, HPV16-3, HPV16-4, and HPV16-6) encoding HPV antigens were evaluated for their ability to induce MHC-I epitope presentation in monocyte-derived dendritic cells (moDCs) isolated from two human donors with distinct HLA haplotypes (Table 8-1). Human donors HD149 and HD150 were used for all treatments and were chosen randomly. HPV16-1 was used as a control antigen, while HPV16-3, HPV16-4, and HPV16-6 were selected based on their ability to generate the highest number of MHC-I epitopes in THP-1 cells in Example 7. To investigate the effect of adjuvant treatment, epitope presentation was assessed in the presence and absence of cGASΔN mRNA. moDCs were treated for 24 hours, after which cells were harvested for MHC-I immunoprecipitation and epitope identification by LC-MS / MS. Supernatants were collected to assess cell viability and adjuvant-induced immune activation. An additional portion of the cell pellet was preserved for confirmation of intracellular antigen expression via western blot.Table 8-1: HLA Haplotypes of Human DonorsHuman Donor HLA alleleHD149 A*03:01 A*30:02 B*15:01 B*27:05 C*03:03 C*05:01HD150 A*02:01 A*24:02 B*15:01 B*35:12 C*03:03 C*04:01

[0180] Viability of moDCs was assessed by measuring LDH release 24 hours post-treatment with HPV mRNA constructs in the presence or absence of cGASΔN mRNA. All experimental groups showed approximately 80% viability, with no sample falling below the 75% threshold. The largest decrease was observed in donor HD149 treated with HPV16-3 mRNA + cGASΔN mRNA, resulting in 76% viability (FIG. 12A). Viability results were consistent across both donors.

[0181] To confirm functionality of cGASΔN mRNA, IFNβ levels were measured in cell supernatants using an IFNβ Lumit immunoassay. moDCs treated with HPV mRNA constructs alone did not secrete detectable IFNβ, while cells treated with HPV mRNA and cGASΔN mRNA produced measurable levels of IFNβ, validating the functionality of the adjuvant (FIG. 12B). These results were consistent across donors.

[0182] Intracellular antigen expression was verified by western blot using an anti-E2 antibody (FIG. 12C). Detectable bands were observed in lysates from cells treated with HPV16-3, HPV16-4, and HPV16-6 mRNA, which are constructs encoding E2. HPV16-3 and HPV16-4 mRNA yielded bands around 150 kDa, while HPV16-6 mRNA showed a band near 75 kDa, consistent with the expected molecular weights. No antigen was detected in HPV16-1 mRNA treated cells, as this construct does not encode E2. Notably, HPV16-6 mRNA treatment led to the strongest antigen expression,64MF-367038161Docket No.: 16553-20011.40 followed by HPV16-4 mRNA. Across both donors, intracellular antigen levels were reduced in the presence of cGASΔN mRNA, suggesting adjuvant treatment may influence expression of protein antigens.

[0183] After confirming moDC viability, antigen expression, and adjuvant functionality, HPV mRNA constructs were evaluated for their induction of MHC-I epitopes in the presence or absence of cGASΔN mRNA treatment. Across both donors, approximately 11,000 unique MHC-I epitopes were identified, of which 2-30 (depending on donor and treatment) were derived from HPV16 antigens.

[0184] Table 8-2 and Table 8-3 show the relative abundance of HPV 16 MHC-I epitopes presented on moDCs after treatment with HPV mRNA LNPs. Results from the two donors, HD149 and HD150, are shown separately. For each specific epitope, the treatment condition that led to the highest abundance (e.g., HPV16-6 for E7 epitope GQAEPDRAHY set forth as SEQ ID NO:54 of donor HD149) was set to 100%. Epitope quantities in all other treatment groups were normalized relative to this maximum. “0” indicates the absence of a specific epitope for a treatment group. “X” indicates epitopes that are not expected to be detected.

[0185] Importantly, all treatment groups led to detectable numbers of presented MHC-I epitopes. In general, cGASΔN mRNA treatment decreased the quantity of E7, E6, El and E2 epitopes, although the extent of reduction varied by donor and epitope sequence. Treatment with HPV16-1 consistently yielded the fewest number of antigen-derived epitopes, in both the presence and absence of cGASΔN (FIG. 12D). In both donors, HPV16-3 treatment in the absence of cGASΔN led to the highest number of presented antigen-derived epitopes. Most of these were El -derived, followed by E2-derived epitopes. In the presence of cGASΔN, HPV16-6 led to the highest number of antigen-derived MHC-I epitopes, outperforming HPV16-3 (FIG. 12D). HPV16-6 treatment resulted primarily in E2-derived epitopes. El epitopes were not detectable for this construct since HPV16-6 does not encode El.Table 8-2: MHC-I Epitopes in HPV16 Antigens Presented by moDCs of Human Donor 149 HPV mRNA LNP 16-1 16-1 16-3 16-3 16-4 16-4 16-6 16-6 cGASΔN mRNA LNP - + - + - + - + Ag Epitope (SEQ ID NO)GQAEPDRAHY (54) 43 47 89 50 45 0 100 60 RAHYNIVTF (55) 32 32 43 21 62 0 100 47 E7GTLGIVCPICSQK (56) 0 0 82 0 69 0 100 93 AEPDRAHY (57) 0 0 0 0 0 0 100 0 FYSKISEY (58) 18 0 100 0 0 0 0 0 E6 QQLLRREVY (59) 94 0 0 0 0 0 100 0KFYSKISEY (60) 0 52 0 22 0 0 100 6765MF-367038161Docket No.: 16553-20011.40 HPV mRNA LNP 16-1 16-1 16-3 16-3 16-4 16-4 16-6 16-6 cGASΔN mRNA LNP - + - + - + - + Ag Epitope (SEQ ID NO)SLYGTTLEQQY (61) 0 0 100 0 0 0 74 0 YRHYCYSLY (62) 0 0 100 0 84 0 0 85 FESEDSGY (63) X X 100 48 59 0 X X RLFESEDSGY (64) X X 100 33 92 21 X X IVDDSEIAY (65) X X 100 35 0 0 X X SIKTLLQQY (66) X X 100 0 66 0 X X MLAKFKELY (67) X X 100 0 0 0 X X FLQGSVICF (68) X X 100 22 73 48 X X ADSIKTLL (69) X X 100 0 0 0 X X GISNISEVY (70) X X 100 38 0 31 X X ElIADSIKTLL (71) X X 100 45 67 0 X X RQTVLQHSF (72) X X 100 69 0 0 X X FLRYQGVEF (73) X X 100 0 0 0 X X SLFGMSLMK (74) X X 100 0 0 25 X X LRYQGVEFMSF (75) X X 100 65 58 0 X X KQIVMFLRY (76) X X 100 44 49 0 X X SRWPYLHNRL (77) X X 100 0 0 0 X X YQGVEFMSF (78) X X 100 59 54 0 X X KSAIVTLTY (79) X X 52 0 0 0 100 70 SVDSAPIL (80) X X 48 0 0 0 100 58 TLQDVSLEVY (81) X X 100 0 77 0 0 96 QFKDDAEKY (82) X X 100 0 0 0 0 0 TIYNSQYSNEK (83) X X 78 0 51 0 100 56 YVHEGIRTY (84) X X 46 26 68 0 100 47 KTITVSTGF (85) X X 74 0 40 0 0 100 E2 GQVDYYGLY (86) X X 47 22 63 26 100 51 VQFKDDAEKY (87) X X 72 0 34 0 100 0 TVVEGQVDY (88) X X 0 0 67 0 100 0 HEGIRTY (89) X X 0 0 100 0 0 0 LQDVSLEVY (90) X X 0 0 55 0 100 53 HMRLECAIYY (91) X X 0 0 57 0 100 54 GQVILCPTSVF (92) X X 0 0 0 0 100 75VQFDGDICNTM (93) X X 0 0 0 0 100 0 Table 8-3: MHC-I Epitopes in HPV16 Antigens Presented by moDCs of Human Donor 150 HPV mRNA LNP 16-1 16-1 16-3 16-3 16-4 16-4 16-6 16-6 cGASΔN mRNA LNP + + + + Ag Epitope (SEQ ID NO)E7 YMLDLQPETTDL (94) 100 0 0 0 0 0 43 066MF-367038161Docket No.: 16553-20011.40 HPV mRNA LNP 16-1 16-1 16-3 16-3 16-4 16-4 16-6 16-6 cGASΔN mRNA LNP + + + + Ag Epitope (SEQ ID NO)TPTLHEYML (95) 79 30 54 16 0 15 100 30 TLGIVCPIC (96) 100 0 0 0 0 0 0 0 TLGIVCPI (97) 100 0 0 0 0 0 0 0 YMLDLQPETTDLYG (98) 100 0 0 0 0 0 42 0 AEPDRAHY (99) 100 0 0 0 30 0 0 0 TLGIVCPICSQKP (100) 100 0 0 0 0 0 0 0 GQAEPDRAHY (101) 76 12 62 8 18 8 100 13 YMLDLQPET (102) 100 34 65 0 48 25 99 35 RAHYNIVTF (103) 76 0 34 0 56 0 100 0 QAEPDRAHY (104) 0 0 0 0 0 0 100 0 QQLLRREVY (105) 100 0 0 0 0 0 0 0 TIHDIILECV (106) 100 0 0 0 0 0 61 0 E6 KFYSKISEY (107) 100 0 0 0 0 0 24 0 PTLHEYML (108) 100 0 0 0 0 0 0 0 SLYGTTLEQQY (109) 84 0 55 0 0 0 100 0 KLLSKLLCV (110) X X 100 9 41 0 X X ISNISEVY (111) X X 100 0 79 0 X X MLAKFKELY (112) X X 100 0 0 0 X X FLQGSVICF (113) X X 100 0 53 0 X X El RYQGVEFMSF (114) X X 100 50 65 46 X X QPLADAKIGM (115) X X 100 0 0 0 X X SQYSGGSGGGCSQY (116) X X 100 0 0 0 X X LYGVSFSEL (117) X X 100 30 38 0 X X FPFDENGNPVYEL (118) X X 100 0 28 0 X X YQGVEFMSF (119) X X 100 14 47 25 X X QFDGDICNTM (120) X X 52 0 0 0 100 56 GQVILCPTSVF (121) X X 76 0 27 0 100 24 LYTAVSSTW (122) X X 43 14 43 11 100 30 KSAIVTLTY (123) X X 100 0 0 0 0 0 TLQDVSLEVY (124) X X 59 0 0 0 100 0 E2VQFDGDICNTM (125) X X 57 0 0 0 100 0 HMRLECAIY (126) X X 57 0 39 0 100 27 YVHEGIRTY (127) X X 41 11 46 12 100 20 VQFKDDAEKY (128) X X 59 0 53 0 100 35 HMRLECAIYY (129) X X 100 0 0 0 0 0LQDVSLEVY (130) X X 0 0 58 0 100 4167MF-367038161Docket No.: 16553-20011.40 HPV mRNA LNP 16-1 16-1 16-3 16-3 16-4 16-4 16-6 16-6 cGASΔN mRNA LNP + + + + Ag Epitope (SEQ ID NO)GQVDYYGLY (131) X X 0 0 45 0 100 20 DSVDSAPIL (132) X X 0 0 0 0 100 0 IYYKAREMGF (133) X X 0 0 0 0 100 0 HYTNWTHIYI (134) X X 0 0 0 0 100 0 YYVHEGIRTYF (135) X X 0 0 0 0 100 0YVHEGIRTYF (136) X X 0 0 0 0 100 0

[0186] HPV16-3 and HPV16-4 are similar constructs encoding four HPV16 viral antigens, El, E2, E6, and E7 (FIG.3). They differ in the order which antigens are arranged. Surprisingly, HPV16-3 outperformed HPV16-4 in the number of epitopes presented in 3 of 4 comparisons (Table 8-2 and Table 8-3). These data indicate that the order in which coding regions of antigens are arranged on the construct affects the efficiency of epitope presentation on MHC-I molecules.

[0187] Altogether, these findings show that HPV 16-3 mRNA treatment led to highest number of MHC-I epitopes without adjuvant, while HPV16-6 mRNA treatment provided the most HPV epitopes, in comparison to the other HPV mRNA constructs tested, when co-administered with cGASΔN. A general reduction in epitope number was observed with cGASΔN addition, although this effect was less pronounced with HPV 16-6 mRNA.

[0188] In summary, HPV16-3 mRNA treatment in the absence of cGASΔN resulted in the highest overall number of HPV MHC-I epitopes, but not the highest abundance of individual El, E2, E6 and E7 epitopes. HPV16-6 mRNA led to the highest abundance of HPV epitopes. Additionally, in the context of cGASΔN, HPV16-6 mRNA treatment induced the highest quantity of specific epitopes within the HPV16 E2, E6, and E7 groups. These results suggest that HPV16-6 mRNA is the most suitable candidate for co-administration with cGASΔN mRNA.Example 9: cGASΔN Enhances T-Cell Responses to mRNA-Encoded AntigenMaterials & Methods

[0189] Cocultures: To model T-cell interactions with antigen presenting cells, human dendritic cells were cocultured with a clonal population of HPV16 E7-specific T-cells. Human monocytes were isolated from leukapheresis blood products using StraightFrom Leukopak CD14 microbead isolation kits (Miltenyi) as directed by the manufacturer. Monocyte derived dendritic cells (moDCs) were68MF-367038161Docket No.: 16553-20011.40 differentiated from two human monocyte donors by culturing monocytes for six days in RPMI media containing 10% fetal bovine serum, 100U / mL penicillin / streptomycin, ImM sodium pyruvate, 2mM L-glutamine, 1% non-essential amino acids (v / v, Gibco), lOmM HEPES, 55pM beta-mercaptoethanol, 50ng / mL GM-CSF, and 25ng / mL IL-4. HPV16 E7-specific T cells were sourced from Charles River Laboratories. T-cells and moDC were cultured in human IFNγ ELISpot plates (Mabtech) overnight with various treatment conditions in triplicate. As positive (E7 peptide) and negative (media) controls, cells were cultured overnight in media in the presence or absence of 100ng / mL HPV16 E711-19peptide YMLDLQPET (SEQ ID NO:53) obtained from Charles River Laboratories. As treatments, cells were incubated with LNPs containing firefly luciferase, HPV16-6, cGASΔN, or a combination of HPV16-6 and cGASΔN mRNA. The next day, culture supernatant was saved for additional cytokine analyses, and the plates were processed according to manufacturer instructions to enumerate spot-forming cells (SFC).Results

[0190] In Example 4, cGASΔN was demonstrated to enhance T-cell responses to peptide antigen. Whether T-cell responses could be enhanced by cGASΔN if antigens were delivered via mRNA containing LNPs had not been shown. In order to determine if cGASΔN could augment T cell-mediated immune responses when antigen, as well as cGASΔN, are delivered as mRNAs, in vitro moDC-T cell cocultures were utilized. Cells were treated with LNPs containing 2pg / mL HPV16-6 and 1.26pg / mL cGASΔN mRNA at 1:1 molar ratio (3.26ug / mL total mRNA). As control conditions, cells were treated with either 2pg / mL HPV16-6 or 1.26pg / mL cGASΔN, thus lacking one of the two mRNA components found in the combination LNP. As another control, cells were treated with 3.26pg / mL LNP containing firefly luciferase mRNA to match the same concentration of mRNA found in the combination group. To confirm the responsivity of the coculture system, cells were also treated with 100ng / mL E711-19peptide, which is the minimal epitope to which the E7-specific T-cells respond. After an overnight incubation, T-cell responses were measured using an IFNγ ELISpot assay.

[0191] Two moDC donors were tested with a single lot of HPV16 E7-specific T-cells. In the first experimental run, moDCs from donor HD136 were combined with T-cells at a 4:1 ratio (200,000 moDCs:50,000 T-cells). In the second run using moDCs from donor HD172, the ratio was adjusted to 1:1 maintaining the same number of T-cells. Treatment of co-cultures with media alone resulted in few spots (FIG. 15A and FIG. 15B). When 100ng / mL E711-19peptide was added to cocultures, more than 300 SFC were detected in each well (FIG. 15A and FIG. 15B). When cells were treated with HPV16-6 mRNA LNPs, minimal IFNγ responses were observed, comparable to the luciferase negative control mRNA-LNP treatment (FIG. 15A and FIG. 15B), suggesting that antigen alone when delivered as mRNA-LNP was minimally immunogenic. Responses observed to treatment with cGASΔN mRNA LNP lacking antigen69MF-367038161Docket No.: 16553-20011.40 mRNA was indicative of non-specific IFNγ responses. In contrast, LNPs containing both HPV16-6 mRNA and cGASΔN mRNA significantly increased the IFNγ response compared to all other LNP treatments (FIG. 15A and FIG. 15B). IFNγ was not detected in the supernatants of the other treatment conditions (moDCs alone or T-cells alone).

[0192] Altogether, these results indicate that a minimally immunogenic mRNA-encoded antigen could elicit T-cell responses when combined with cGASΔN mRNA. Other studies had demonstrated that the LNPs were primarily taken up and expressed by moDCs with minimal delivery to T-cells (data not shown). In the repeat study with HD172, monocultures of moDCs or T-cells were treated with LNPs containing both HPV16-6 and cGASΔN mRNAs. The monoculture control treatments indicated that neither cell type alone was sufficient for producing an IFNγ response since the LNPs need to be delivered to moDCs for antigen presentation and the T-cells need to respond to antigen presented by the APCs in order to produce IFNγ (FIG. 15B). Thus, using moDC from two human donors, cGASΔN mRNA was demonstrated to enhance T-cell responses to mRNA-encoded antigen delivered via LNPs.Example 10: Immunogenicity of HPV 16-6 With or Without cGASΔN in Outbred Mice Materials & Methods

[0193] Immunization and Study Design: T-cell responses to LNPs containing HPV16-6 mRNA alone or in combination with cGASΔN mRNA were assessed in CD-I outbred mice. As shown in Table 10-1, Mice were injected intramuscularly with PBS, or LNPs on days 0 and 14. LNPs containing both HPV16-6 and cGASΔN mRNA were formulated to have a 1:1 molar ratio of the two mRNAs. On day 35, spleens were harvested and processed to single cell suspensions for T-cell readouts.Table 10-1. HPV 16-6 Study GroupsGroup Antigen Adjuvant Schedule1 PBS - Intramuscular injection: 2 HPV16-6 (0.5 pg) - Day 0, Day 143 HPV16-6 (2.0 pg) - 4 HPV16-6 (0.5 pg) cGASΔN (0.3 pg) Harvest Spleens Day 355 HPV16-6 (2.0 pg) cGASΔN (1.26 pg)

[0194] ELISPOT: Splenocytes were seeded into IFNγELISPOT plates (R& D Systems) at 2.5e5 cells per well and stimulated with R10 media alone or R10 media containing 1 pg / mL of Pepmix (JPT) for HPV16 E2, E6, or E7. Plates were incubated at 37°C for 20 hours. After incubation, cells were discarded and ELISPOTs were developed according to manufacturer’s instructions. Plates were left to dry70MF-367038161Docket No.: 16553-20011.40 overnight at room temperature. Plates were then read and spots counted on the S6 Universal M2 ELISPOT plate analyzer (ImmunoSpot® analyzer by Cellular Technology Limited).

[0195] Intracellular Cytokine Staining. Splenocytes were seeded at 5e5 cells per well and stimulated with R10 media alone or R10 media containing 1 µg / mL of Pepmix (JPT) for HPV16 E2, E6, or E7. All stimulation conditions also included anti-mouse CD107a antibody. Cells were incubated at 37°C for 1.5 hours, after which Golgi Plug (BD Biosciences) and Golgi Stop (BD Biosciences) was added to each well. Cells were incubated at 37°C for 5 hours and then moved to 4°C overnight. The following day, cells were washed and stained with Fixable Live / Dead Aqua in PBS for 20 minutes at 4°C, washed and incubated with Fc block for 20 minutes at 4°C, washed and stained for anti-mouse CD4 and CD8 for 45 minutes at 4°C, and washed and fixed with BD Cytofix / Cytoperm according to manufacturer’s instructions. Cells were then washed and stained with anti-mouse CD3, TNFa, IFNγ, and IL-2 for 1 hour at 4°C. Lastly, cells were washed and resuspended for acquisition on a BD FACS Symphony A3 cytometer.

[0196] Statistical Analysis: Data were analyzed and graphed using Microsoft Excel, FlowJo, and Graphpad prism software. Data are reported as bar graphs with each symbol representing one mouse, as heat maps with each column representing one mouse, or as contingency plots.Results

[0197] In order to assess the breadth of T-cell responses elicited by LNPs containing HPV16-6 mRNA alone or in combination with cGASΔN mRNA, CD-I outbred mice were used. Mice were immunized intramuscularly on Days 0 and 14 with a low (0.5 pg) or high (2.0 pg) dose of HPV16-6 mRNA alone or combined with cGASΔN mRNA in LNPs as shown in Table 10-1. Antigen-specific T-cell responses to HPV16 E2, E6 and E7 were assessed in spleens on day 35 via IFNγ ELISPOT and intracellular cytokine staining. T-cell responses as measured by IFNγ ELISPOT were detected against all three antigens (FIG. 16A-B). As expected, the strength of T-cell responses to each antigen was variable among mice within the same experimental group due to the outbred (e.g., non-identical) nature of CD-I mice. To address this, responses to each antigen were stratified into four groups based on SFC per le6 cells: high responders (>1000 SFCs); medium responders (500-1000 SFCs); low responders (100-499 SFCs); and very low responders (< 100 SFCs).

[0198] Mice immunized with HPV16-6 mRNA + cGASΔN mRNA had twice as many high responders to all three antigens compared to mice that received HPV16-6 mRNA alone (FIG. 16A). Moreover, HPV16-6 mRNA + cGASΔN mRNA induced more medium responders to all three antigens compared to HPV16-6 mRNA alone (FIG. 16A), while the number of low and very low responders were 71MF-367038161Docket No.: 16553-20011.40 similar between mice that received HPV16-6 mRNA alone or combined with cGASΔN mRNA (FIG. 16).IFNγ ELISPOT responses to each antigen were also visualized on a per-mouse basis using a heat map (FIG. 16B). Mice that received HPV16-6 mRNA + cGASΔN mRNA had more high responders than HPV16-6 mRNA alone at both the low (0.5 pg) and high (2.0 pg) dose levels (FIG. 16B). Moreover, mice that received HPV16-6 mRNA + cGASΔN mRNA were more likely have high responses against two antigens compared to mice that received only HPV16-6 mRNA (FIG. 16B). These data demonstrate that the addition of cGASΔN mRNA enhanced the antigen-specific T cell responses induced by HPV16-6 mRNA.

[0199] T-cell responses against each antigen were further resolved to CD4+ versus CD8+ T-cell responses using intracellular cytokine staining. E2 responses were largely driven by CD4+ T-cells, while E6 and E7 responses were largely driven by CD8+ T-cells (data not shown). Similar to ELISPOT results, responses to each antigen were variable within the same experimental group. As such, CD4+ and CD8+ T-cell responses were divided into four groups based on the frequency of IFNγ+ cells: high responders (>1.0 % IFNγ+); medium responders (0.5-1.0 % IFNγ+); low responders (0.1-0.49 % IFNγ+); and undetected response (<0.1% IFNγ+).

[0200] Within the CD4+ T-cell compartment, high and medium responders were only observed for E2-specific responses (FIG. 16C). Mice that received HPV16-6 mRNA combined with cGASΔN mRNA had more high responders than mice that received HPV16-6 mRNA alone (FIG. 16C), and the number of medium responders was increased when cGASΔN mRNA was combined with low dose (0.5 pg) HPV16-6 mRNA. Within the CD8+ T-cell compartment, high responders were observed for all three antigens (FIG. 16D). Mice that received HPV16-6 mRNA + cGASΔN mRNA had more high responders to E2 and E6 than mice that received HPV16-6 mRNA alone (FIG. 16D). For E7 responses, high responders were comparable among all groups, with the number of medium responders increased by the addition of cGASΔN mRNA at both dose levels. Together these data demonstrate that HPV16-6 mRNA elicited both CD4+ and CD8+ T-cell responses against E2, E6 and E7 in CD-I outbred mice and that the addition of cGASΔN mRNA enhanced these antigen-specific T cell responses.SEQUENCES SEQ ID NO: 1> H. SAPIENSPGASKLRAVLEKLKLSRDDISTAAGMVKGWDHLLLRLKCDSAFRGVGLLN TGSYYEHVKISAPNEFDVMFKLEVPRIQLEEYSNTRAYYFVKFKRNPKENPLSQFLEGEILSASKMLSKF RKIIKEEINDIKDTDVIMKRKRGGSPAVTLLISEKISVDITLALESKSSWPASTQEGLRIQNWLSAKVRK QLRLKPFYLVPKHAKEGNGFQEETWRLSFSHIEKEILNNHGKSKTCCENKEEKCCRKDCLKLMKYLLEQL72MF-367038161Docket No.: 16553-20011.40 KERFKDKKHLDKFSSYHVKTAFFHVCTQNPQDSQWDRKDLGLCFDNCVTYFLQCLRTEKLENYFIPEFNL FSSNLIDKRSKEFLTKQIEYERNNEFPVFDEF SEQ ID NO: 2> M._MU LATTAPGARKLRAVLEKLRLSHQDISKAAKWNGWGHLLRRLKCEPEFGGVEQLH TGSYYEHVKISAPNEFDVMFKLQVPRIQLEEYSDTGAYYFVKFKRNPEGNPLSLFLEDEILSASKMLSKF RKI IKEEINNIKDVQPDADVIMKRKRPGSPAVTLLINKEISVDITLALELKTSWPASTQEGLAIKNWLSA KVRQQLRLKPFYLVPKHAKEGNGFQEETWRLSFSHIEKEILSNHGKSKTCCENKEMKCCRKDCLKLMKYL LEQLKEKFKDKKHLDKFSSYHVKTAFFHLCTENPQDSQWDPKDLGLCFDNCVTYFLECLRTEQLKNYFIP GFNLFSSNLIDKTSKEFLSKQIEYERNNEFPVFGEF SEQ ID NO: 3> P. _ANUBISPGARKLRAVLEKLRLSHQDISKAAKWNGWGHLLHRLKCESEFEGVEQLH TGSYYEHVKISAPNEFDVMFKLQVPRIQLEEYSDTGAYYFVKFKRNPKGNPLSQFLEDEILSASKMLSQF RKI IKEEINNIKDTDVIMKRKRRGSPAVTLLINKEISVDITLALELKTSWPASTQEGLAIKNWLSAKVRQ QLRLKPFYLVPKHAKEGNGFQEETWRLSFSHIEKEILSNHGKSKTCCENKEMKCCRKDCLKLMKYLLEQL KEKFKDKKHLDKFSSYHVKTAFFHLCTENPQDSQWDPKDLGLCFDNCVTHFLQCLRTEKLANYFIPGFNL FSSNLIDKSSKEFLSKQIEYERNNEFPVFGEF SEQ ID NO: 4> N._LEUCOGENYS PGARKLRAVLEKLKLSRQEISEAAEWNGWDHLLRRLQKCDSEFRGVGLLRTGSYYEH VKISAPNEFDVMFKLEVPRIQLEEYSNTGAYYFVKFKRNPKENPLSQFLEGEILSASKMLSKFRKI IKEE INNIKDTDVIMKRKRGGSPAVTLLISEKISVDITLALESNSNSWPASTQEGLPIKDWLSGKVRRQLRLKP FYLVPKHAKEGNGFQEETWRLSFSHIEKEILNNHGKTKTCCENKNVKCCRKDCLKLMKYLLEQLKERFKD KKHLDKFSSYHVKTAFFHVCTENPQDSQWDPKNLGFCFDKCVIYFLKCLRTEQLGNYFIPGFNLFSSNLI DKRSKEFLSKQIEYERNNEYPVFG SEQ ID NO: 5> H_LARPGARKLPAVLEKLKLSRQ EICEAAEWNGWDHLLRRLQKCDSEFRGVGLLRTGSYYEHVKISAPNEFDVMFKLEVPRIQLEEYSNTG AYYFVKFKRNPKENPLCQFLEGEILSASKMLSKFRKI IKEEINNIKDTDVILKRKRGGSPAVTLLISEKI SVDITLALESKSSSWPASTQEGLPIKDWLSGKVRTQLRLKPFYLVPKHAKEGNGFQEETWRLSFSHIEKE ILNNHGETKTCCENKKVKCCRKDCLKLMKYLLEQLKERFKDKKHLDKFSSYHVKTAFFHVCTENPKDSQW DPKDLGLCFDKCVIYFLQCLSTEQLGNYFIPGFNLFSSNLIDKRSKEFLSKQIEYERNNEYPVFGKC SEQ ID NO: 6> P,_ABELIIPGASKLRAVLEKLKLSRLEISKAAEWNRWDHLLRRLQKYNSEFRGVGLLRT GSYYEHVKISAPNEFDVMFKLEVPRIQLEEYSNTGAYYFVKFKRNPKENPLSQFLEGEILSASKMLSKFR KI IKEEINNIKDTDVIMKRKRGGSPAVTLLISEEISVDITLALESKSSWPASTQEGLPIKNWLSAKVRKQ LRLKPFYLVPKHAKEGNGFQEETWRLSFSHIEKEILNNHGKSKTCCEDKEVKCCRKDCLKLMKYLLEQLK ERFKDKKHLDKFSSYHVKTAFFHVCTQNPQDSQWDPKDLGLCFDNCVTYFLQCLRTEQLGNYFIPGFNLF SSNLIDKRSKEFLTKQIEYERNNEFPVFDEF SEQ ID NO: 7> P._TROGLODYTESPGASKLRAVLEKLKLSRDDISTAAGMVKGWDHLLLRLKCDSAFRGVGLLN73MF-367038161Docket No.: 16553-20011.40 TGSYYEHVKISAPNEFDVMFKLEVPRIQLEEYSNTRAYYFVKFKRNPKENPLSQFLEGEILSASKMLSKF RKIIKEEINNIKDVIMKRKRGGSPAVTLLISEKISVDITLALESKSSWPASTQEGLPIKNWLSAKVRKQL RLKPFYLVPKHAKEGNGFQEETWRLSFSHIEKEILNNHGKSKTCCEIKEAKCCRKDCLKLMKYLLEQLKE KFKDKKHLDKFSSYHVKTAFFHVCTENPQDSQWDRKDLGLCFDNCVTYFLQCLRIEKLENYFIPEFNLFS SDLIDKRSKEFLTKQIEYERNNEFPVFDEF SEQ ID NO: 8> G. _GORILLAPGASKLRAVLEKLKLSRDDISTAAGMVKGWDHLLLRLKCDSAFRGVGLLN TGSYYEHVKISAPNEFDVMFKLEVPRIQLEEYSNTRAYYFVKFKRNPKENPLSQFLEGEILSASKMLSKF RKIIKEEINNIKDTDVIMKRKRGGSPAVTLLISEKISVDITLALESKSSWPASTQEGLRIQNWLSAKVRR QLRLKPFYLVPKHAKEGNGFQEETWRLSFSHIEKEILNNHGKSKTCCENKEAKCCRKDCLKLMKYLLEQL KERFKDKKHLDKFSSYHVKTAFFHVCTQNPQDSQWDRKQLGLCFDNCVTYFLQCLRTERLENYFIPEFNL FSRNLIDKRSKEFLTKQIEYERNNEFPVFDEF SEQ ID NO: 9> SYNTHETIC_CGASAN_CONSENSUS PGASKLRAVLEKLKLSRDDISTAAGMVKGWDHLLLRLKCDSAFRGVGLLN TGSYYEHVKISAPNEFDVMFKLEVPRIQLEEYSNTRAYYFVKFKRNPKENPLSQFLEGEILSASKMLSKF RKIIKEEINX1IKDX2X3VIMKRKRGGSPAVTLLISEKISVDITLALESKSSWPASTQEGLX4IX5NWLSAK VRXsQLRLKPFYLVPKHAKEGNGFQEETWRLSFSHIEKEILNNHGKSKTCCEXvKEXsKCCRKDCLKLMKY LLEQLKEX9FKDKKHLDKFSSYHVKTAFFHVCTX10NPQDSQWDRKX11LGLCFDNCVTYFLQCLRX12EX13L ENYFIPEFNLFSX14X15LIDKRSKEFLTKQIEYERNNEFPVFDEF, WhereinXi = N or D; X2= T or absent; X3= D or absent; X4= R or P; X5= Q or K; X6= K or R; X7= N or I; X8= E or A; X9= R or K; X10= Q or E; X11= D or Q; X12 = T or I; X13= K or R; X14= S or R; and X15= N or D SEQ ID NO: 10> HUMAN-CGAS_FULL-LENGTH MQPWHGKAMQRASEAGATAPKASARNARGAPMDPTESPAAPEAALPKAGKFGPARKSGSRQKKSAPDTQE RPPVRATGARAKKAPQRAQDTQPSDATSAPGAEGLEPPAAREPALSRAGSCRQRGARCSTKPRPPPGPWD VPSPGLPVSAPILVRRDAAPGASKLRAVLEKLKLSRDDISTAAGMVKGWDHLLLRLKCDSAFRGVGLLN TGSYYEHVKISAPNEFDVMFKLEVPRIQLEEYSNTRAYYFVKFKRNPKENPLSQFLEGEILSASKMLSKF RKIIKEEINDIKDTDVIMKRKRGGSPAVTLLISEKISVDITLALESKSSWPASTQEGLRIQNWLSAKVRK QLRLKPFYLVPKHAKEGNGFQEETWRLSFSHIEKEILNNHGKSKTCCENKEEKCCRKDCLKLMKYLLEQL KERFKDKKHLDKFSSYHVKTAFFHVCTQNPQDSQWDRKDLGLCFDNCVTYFLQCLRTEKLENYFIPEFNL FSSNLIDKRSKEFLTKQIEYERNNEFPVFDEF SEQ ID NO: 11> HUMAN-CGAS_N-TERMINUS MQPWHGKAMQRASEAGATAPKASARNARGAPMDPTESPAAPEAALPKAGKFGPARKSGSRQKKSAPDTQE RPPVRATGARAKKAPQRAQDTQPSDATSAPGAEGLEPPAAREPALSRAGSCRQRGARCSTKPRPPPGPWD VP SP GLP VSAP I LVRRD AA SEQ ID NO: 12> THOSEA ASIGNA VIRUS T2A RIBOSOME-SKIPPING PEPTIDE GSGEGRGSLLTCGDVEENPGP SEQ ID NO: 13> PORCINE TESCHOVIRUS-1 P2A RIBOSOME-SKIPPING PEPTIDE GSGATNFSLLKQAGDVEENPGP74MF-367038161Docket No.: 16553-20011.40SEQ ID NO: 14> EQUINE RHINITIS A VIRUS E2A RIBOSOME-SKIPPING PEPTIDEGS GQCTNYALLKLAGDVE SNP GP SEQ ID NO: 15> FOOT-AND-MOUTH DISEASE VIRUS F2A RIBOSOME-SKIPPING PEPTIDE GSGVKQTLNFDLLKLAGDVESNPGP SEQ ID NO: 16> GALLUS GALLUS OVALBUMIN PEPTIDESI INFEKL SEQ ID NO: 17> SYNTHETIC CGASAN MOUSE CODON-OPTIMIZED ATGCCCGGCGCCAGCAAGCTGAGGGCCGTGCTGGAGAAGCTGAAGCTGAGCAGGGACGACATCAGCACCG CCGCCGGCATGGTGAAGGGCGTGGTGGACCACCTGCTGCTGAGGCTGAAGTGCGACAGCGCCTTCAGGGG CGTGGGCCTGCTGAACACCGGCAGCTACTACGAGCACGTGAAGATCAGCGCCCCCAACGAGTTCGACGTG ATGTTCAAGCTGGAGGTGCCCAGGATCCAGCTGGAGGAGTACAGCAACACCAGGGCCTACTACTTCGTGA AGTTCAAGAGGAACCCCAAGGAGAACCCCCTGAGCCAGTTCCTGGAGGGCGAGATCCTGAGCGCCAGCAA GAT G C T G AGC AAG T T C AG G AAG AT C AT C AAGG AG GAG AT C AAC G AC AT C AAG G AC AC C G AC G T G AT C AT G AAGAGGAAGAGGGGCGGCAGCCCCGCCGTGACCCTGCTGATCAGCGAGAAGATCAGCGTGGACATCACCC TGGCCCTGGAGAGCAAGAGCAGCTGGCCCGCCAGCACCCAGGAGGGCCTGAGGATCCAGAACTGGCTGAG CGCCAAGGTGAGGAAGCAGCTGAGGCTGAAGCCCTTCTACCTGGTGCCCAAGCACGCCAAGGAGGGCAAC GGCTTCCAGGAGGAGACCTGGAGGCTGAGCTTCAGCCACATCGAGAAGGAGATCCTGAACAACCACGGCA AG AG C AAG AC C T G C T GC G AGAAC AAG GAG G AG AAGT G C T G C AGG AAG G AC T G C C T G AAG C T G AT GAAG TA C C T G C T G G AG C AG C T GAAG GAG AGG T T C AAGG AC AAG AAG C AC C T GG AC AAG T T C AG C AG C T AC C AC G T G AAGACCGCCTTCTTCCACGTGTGCACCCAGAACCCCCAGGACAGCCAGTGGGACAGGAAGGACCTGGGCC TGTGCTTCGACAACTGCGTGACCTACTTCCTGCAGTGCCTGAGGACCGAGAAGCTGGAGAACTACTTCAT CCCCGAGTT C AAC C T GT T C AG C AGC AAC C T GAT C GAC AAG AG GAG C AAG GAGTTCCTGAC C AAG C AG AT C GAG T AC G AGAG GAAC AAC G AG T T C C C C G T G T T C G AC G AGT T C T AAT G A SEQ ID NO: 18> HPV16_E6S (142aa)FQDPQESGRKLPQLCTELQTTIHDI ILECVYCKQQLLRREVYDRDLCIVYRDGNPYAVCDKCLKFYSKIS EYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRET QL SEQ ID NO: 19> HPV16_E6H (157aa)HQKRTAMFQDPQERPRKLPQLCTELQTTIHDI ILECVYCKQQLLRREVYDFAFRDLCIVYRDGNPYAVGD KCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINGQKPLCPEEKQRHLDKKQRFHNIRGRWTG RCMSCCRSSRTRRETQL SEQ ID NO: 20> SYNTHETIC_HPV16_E 6 CONSENSUS FQDPQEX1X2RKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDX3X4X5RDLCIVYRDGNPYAVX6DK CLKFYSKI SEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINX7QKPLX8X9X10X11X12QRHLDKKQRFH NIRGRWTGRCMSCCRSSRTRRETQL, wherein75MF-367038161Docket No.: 16553-20011.40 XI = S or R; X2 = G or P; X3 = ABSENT OR F; X4 = ABSENT OR A; X5 = ABSENT OR F; X6 = C OR G; X7 = C OR G; X8 = ABSENT OR C; X9 = ABSENT OR P; XI 0 = ABSENT OR E; XI 1 = ABSENT OR E; AND X12 = ABSENT OR K SEQ ID NO: 21> HPV16_E7S (97aa) HGDTPTLHEYMLDLQPETTDLYGYGQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQS THVDIRTLEDLLMGTLGIVCPICSQKP SEQ ID NO: 22> HPV16_E7H (97aa) HGDTPTLHEYMLDLQPETTDLYGYGQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQS THVDIRTLEDLLMGTLGIVGPICSQKP SEQ ID NO: 23> SYNTHETIC_HPV16_E7 CONSENSUS HGDTPTLHEYMLDLQPETTDLYGYGQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQS THVDIRTLEDLLMGTLGIVX1PICSQKP, whereinXI = C OR GSEQ ID NO: 24> HPV16_E1 ( 648aa)ADPAGTNGEEGTGCNGWFYVEAWEKKTGDAI SDDENENDSDTGEDLVDFIVNDNDYLTQAETETAHALF TAQEAKQHRDAVQVLKRKYLGSPLSDISGCVDNNISPRLKAICIEKQSRAAKRRLFESEDSGYGNTEVET QQMLQVEGRHETETPCSQYSGGSGGGCSQYSSGSGGEGVSERHTICQTPLTNILNVLKTSNAKAAMLAKF KELYGVSFSELVRPFKSNKSTCCDWCIAAFGLTPSIADSIKTLLQQYCLYLHIQSLACSWGMWLLLVRY KCGKNRETIEKLLSKLLCVSPMCMMIEPPKLRSTAAALYWYKTGI SNI SEVYGDTPEWIQRQTVLQHSFN DCTFELSQMVQWAYDNDIVDDSEIAYKYAQLADTNSNASAFLKSNSQAKIVKDCATMCRHYKRAEKKQMS MSQWIKYRCDRVDDGGDWKQIVMFLRYQGVEFMSFLTALKRFLQGIPKKNCILLYGAANTDKSLFGMSLM KF LQGS VI CF VNSKS HF WLQP LADAK I GMLDD ATVP CWNY I DDNLRNALDGNLVSMD VKHRP LVQLKCPP LLITSNINAGTDSRWPYLHNRLWFTFPNEFPFDENGNPVYELNDKNWKSFFSRTWSRLSLHEDEDKEND GDSLPTFKCVSGQNTNTL SEQ ID NO: 25> HPV16_E2 (364aa) ETLCQRLNVCQDKILTHYENDSTDLRDHIDYWKHMRLECAIYYKAREMGFKHINHQWPTLAVSKNKALQ AIELQLTLETIYNSQYSNEKWTLQDVSLEVYLTAPTGCIKKHGYTVEVQFDGDICNTMHYTNWTHIYICE EASVTWEGQVDYYGLYYVHEGIRTYFVQFKDDAEKYSKNKVWEVHAGGQVILCPTSVFSSNEVSSPEII RQHLANHPAATHTKAVALGTEETQTTIQRPRSEPDTGNPCHTTKLLHRDSVDSAPILTAFNSSHKGRINC NSNTTP IVHLKGDANTLKCLRYRFKKHCTLYTAVSSTWHWTGHNVKHKSAIVTLTYDSEWQRDQFLSQVK IPKTITVSTGFMSI SEQ ID NO: 26> IGE LEADERMDWTWILFLVAAATRVHS SEQ ID NO: 27>furin cleavable linkerRGRKRRS SEQ ID NO: 2876MF-367038161Docket No.: 16553-20011.40 > HPV E7 EPITOPERAHYNIVTF SEQ ID NO: 29>synthetic CGASAN Negative Control Sequence (single mutant) MPGASKLRAVLEKLKLSRDDI STAAGMVKGWDHLLLRLKCDSAFRGVGLLNTGSYYEHVKI SAPNEFDV MFKLEVPRIQLEEYSNTRAYYFVKFKRNPKENPLSQFLEGEILSASKMLSKFRKI IKEE IND IKDTDVIM KRKRGGSPAVTLLISEKISVDITLALESKSSWPASTQEGLRIQNWLSAKVRKQLRLKPFYLVPKHAKEGN GFQEETWRLSFSHIEKEILNNHGKSKTAAENKEEKCCRKDCLKLMKYLLEQLKERFKDKKHLDKFSSYHV KTAFFHVCTQNPQDSQWDRKDLGLCFDNCVTYFLQCLRTEKLENYFIPEFNLFSSNLIDKRSKEFLTKQI EYERNNEFPVFDEF SEQ ID NO: 30>synthetic HPV16-1 (264aa) MDWTWILFLVAAATRVHSFQDPQESGRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDRDLCIVYRD GNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLQRHLDKKQRFHNIRGR WTGRCMSCCRSSRTRRETQLRGRKRRSHGDTPTLHEYMLDLQPETTDLYGYGQLNDSSEEEDEIDGPAGQ AEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP SEQ ID NO: 31>synthetic HPV16-2 (255aa) MHGDTPTLHEYMLDLQPETTDLYGYGQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQ STHVDIRTLEDLLMGTLGIVGPICSQKPHQKRTAMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQ LLRREVYDFAFRDLCIVYRDGNPYAVGDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCING QKPLCPEEKQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRETQL SEQ ID NO: 32>synthetic HPV16-3 (1290aa) MDWTWILFLVAAATRVHSFQDPQESGRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDRDLCIVYRD GNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLQRHLDKKQRFHNIRGR WTGRCMSCCRSSRTRRETQLRGRKRRSHGDTPTLHEYMLDLQPETTDLYGYGQLNDSSEEEDEIDGPAGQ AEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPRGRKRRSADPAGTNGE EGTGCNGWFYVEAWEKKTGDAISDDENENDSDTGEDLVDFIVNDNDYLTQAETETAHALFTAQEAKQHR DAVQVLKRKYLGSPLSDISGCVDNNISPRLKAICIEKQSRAAKRRLFESEDSGYGNTEVETQQMLQVEGR HETETPCSQYSGGSGGGCSQYSSGSGGEGVSERHTICQTPLTNILNVLKTSNAKAAMLAKFKELYGVSFS ELVRPFKSNKSTCCDWCIAAFGLTPSIADSIKTLLQQYCLYLHIQSLACSWGMWLLLVRYKCGKNRETI EKLLSKLLCVSPMCMMIEPPKLRSTAAALYWYKTGISNISEVYGDTPEWIQRQTVLQHSFNDCTFELSQM VQWAYDNDIVDDSEIAYKYAQLADTNSNASAFLKSNSQAKIVKDCATMCRHYKRAEKKQMSMSQWIKYRC DRVDDGGDWKQIVMFLRYQGVEFMSFLTALKRFLQGIPKKNCILLYGAANTDKSLFGMSLMKFLQGSVIC FVNSKSHFWLQPLADAKIGMLDDATVPCWNYIDDNLRNALDGNLVSMDVKHRPLVQLKCPPLLITSNINA GTDSRWPYLHNRLWFTFPNEFPFDENGNPVYELNDKNWKSFFSRTWSRLSLHEDEDKENDGDSLPTFKC VSGQNTNTLRGRKRRSETLCQRLNVCQDKILTHYENDSTDLRDHIDYWKHMRLECAIYYKAREMGFKHIN HQWPTLAVSKNKALQAIELQLTLETIYNSQYSNEKWTLQDVSLEVYLTAPTGCIKKHGYTVEVQFDGDI CNTMHYTNWTHIYICEEASVTWEGQVDYYGLYYVHEGIRTYFVQFKDDAEKYSKNKVWEVHAGGQVILC PTSVFSSNEVSSPEI IRQHLANHPAATHTKAVALGTEETQTTIQRPRSEPDTGNPCHTTKLLHRDSVDSA PILTAFNSSHKGRINCNSNTTPIVHLKGDANTLKCLRYRFKKHCTLYTAVSSTWHWTGHNVKHKSAIVTL TYDSEWQRDQFLSQVKIPKTITVSTGFMSI SEQ ID NO: 33>synthetic HPV16-4 (1290aa)77MF-367038161Docket No.: 16553-20011.40 MDWTWILFLVAAATRVHSFQDPQESGRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDRDLCIVYRD GNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLQRHLDKKQRFHNIRGR WTGRCMSCCRSSRTRRETQLRGRKRRSHGDTPTLHEYMLDLQPETTDLYGYGQLNDSSEEEDEIDGPAGQ AEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPRGRKRRSETLCQRLNV CQDKILTHYENDSTDLRDHIDYWKHMRLECAI YYKAREMGFKHINHQWPTLAVSKNKALQAIELQLTLE TIYNSQYSNEKWTLQDVSLEVYLTAPTGCIKKHGYTVEVQFDGDICNTMHYTNWTHIYICEEASVTWEG QVDYYGLYYVHEGIRTYFVQFKDDAEKYSKNKVWEVHAGGQVILCPTSVFSSNEVSSPEI IRQHLANHPA ATHTKAVALGTEETQTTIQRPRSEPDTGNPCHTTKLLHRDSVDSAPILTAFNSSHKGRINCNSNTTPIVH LKGDANTLKCLRYRFKKHCTLYTAVSSTWHWTGHNVKHKSAIVTLTYDSEWQRDQFLSQVKIPKTITVST GFMSIRGRKRRSADPAGTNGEEGTGCNGWFYVEAWEKKTGDAISDDENENDSDTGEDLVDFIVNDNDYL TQAETETAHALFTAQEAKQHRDAVQVLKRKYLGSPLSDISGCVDNNISPRLKAICIEKQSRAAKRRLFES EDSGYGNTEVETQQMLQVEGRHETETPCSQYSGGSGGGCSQYSSGSGGEGVSERHTICQTPLTNILNVLK TSNAKAAMLAKFKELYGVSFSELVRPFKSNKSTCCDWCIAAFGLTPSIADSIKTLLQQYCLYLHIQSLAC SWGMWLLLVRYKCGKNRETIEKLLSKLLCVSPMCMMIEPPKLRSTAAALYWYKTGI SNI SEVYGDTPEW IQRQTVLQHSFNDCTFELSQMVQWAYDNDIVDDSEIAYKYAQLADTNSNASAFLKSNSQAKIVKDCATMC RHYKRAEKKQMSMSQWIKYRCDRVDDGGDWKQIVMFLRYQGVEFMSFLTALKRFLQGIPKKNCILLYGAA NTDKSLFGMSLMKFLQGSVICFVNSKSHFWLQPLADAKIGMLDDATVPCWNYIDDNLRNALDGNLVSMDV KHRPLVQLKCPPLLITSNINAGTDSRWPYLHNRLWFTFPNEFPFDENGNPVYELNDKNWKSFFSRTWSR LSLHEDEDKENDGDSLPTFKCVSGQNTNTL SEQ ID NO: 34>synthetic HPV16-5 (919aa) MDWTWILFLVAAATRVHSFQDPQESGRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDRDLCIVYRD GNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLQRHLDKKQRFHNIRGR WTGRCMSCCRSSRTRRETQLRGRKRRSHGDTPTLHEYMLDLQPETTDLYGYGQLNDSSEEEDEIDGPAGQ AEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPRGRKRRSADPAGTNGE EGTGCNGWFYVEAWEKKTGDAISDDENENDSDTGEDLVDFIVNDNDYLTQAETETAHALFTAQEAKQHR DAVQVLKRKYLGSPLSDISGCVDNNISPRLKAICIEKQSRAAKRRLFESEDSGYGNTEVETQQMLQVEGR HETETPCSQYSGGSGGGCSQYSSGSGGEGVSERHTICQTPLTNILNVLKTSNAKAAMLAKFKELYGVSFS ELVRPFKSNKSTCCDWCIAAFGLTPSIADSIKTLLQQYCLYLHIQSLACSWGMWLLLVRYKCGKNRETI EKLLSKLLCVSPMCMMIEPPKLRSTAAALYWYKTGISNISEVYGDTPEWIQRQTVLQHSFNDCTFELSQM VQWAYDNDIVDDSEIAYKYAQLADTNSNASAFLKSNSQAKIVKDCATMCRHYKRAEKKQMSMSQWIKYRC DRVDDGGDWKQIVMFLRYQGVEFMSFLTALKRFLQGIPKKNCILLYGAANTDKSLFGMSLMKFLQGSVIC FVNSKSHFWLQPLADAKIGMLDDATVPCWNYIDDNLRNALDGNLVSMDVKHRPLVQLKCPPLLITSNINA GTDSRWPYLHNRLWFTFPNEFPFDENGNPVYELNDKNWKSFFSRTWSRLSLHEDEDKENDGDSLPTFKC VSGQNTNTL SEQ ID NO: 35>synthetic HPV16-6 (635aa) MDWTWILFLVAAATRVHSFQDPQESGRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDRDLCIVYRD GNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLQRHLDKKQRFHNIRGR WTGRCMSCCRSSRTRRETQLRGRKRRSHGDTPTLHEYMLDLQPETTDLYGYGQLNDSSEEEDEIDGPAGQ AEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPRGRKRRSETLCQRLNV CQDKILTHYENDSTDLRDHIDYWKHMRLECAI YYKAREMGFKHINHQWPTLAVSKNKALQAIELQLTLE TIYNSQYSNEKWTLQDVSLEVYLTAPTGCIKKHGYTVEVQFDGDICNTMHYTNWTHIYICEEASVTWEG QVDYYGLYYVHEGIRTYFVQFKDDAEKYSKNKVWEVHAGGQVILCPTSVFSSNEVSSPE I IRQHLANHPA ATHTKAVALGTEETQTTIQRPRSEPDTGNPCHTTKLLHRDSVDSAPILTAFNSSHKGRINCNSNTTPIVH LKGDANTLKCLRYRFKKHCTLYTAVSSTWHWTGHNVKHKSAIVTLTYDSEWQRDQFLSQVKIPKTITVST GEMS I SEQ ID NO: 3678MF-367038161Docket No.: 16553-20011.40 >synthetic HPV16-7 (1269aa) MDWTWILFLVAAATRVHSFQDPQESGRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDRDLCIVYRD GNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLQRHLDKKQRFHNIRGR WTGRCMSCCRSSRTRRETQLHGDTPTLHEYMLDLQPETTDLYGYGQLNDSSEEEDEIDGPAGQAEPDRAH YNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPADPAGTNGEEGTGCNGWFYVEAV VEKKTGDAISDDENENDSDTGEDLVDFIVNDNDYLTQAETETAHALFTAQEAKQHRDAVQVLKRKYLGSP LSDISGCVDNNISPRLKAICIEKQSRAAKRRLFESEDSGYGNTEVETQQMLQVEGRHETETPCSQYSGGS GGGCSQYSSGSGGEGVSERHTICQTPLTNILNVLKTSNAKAAMLAKFKELYGVSFSELVRPFKSNKSTCC DWCIAAFGLTPSIADSIKTLLQQYCLYLHIQSLACSWGMWLLLVRYKCGKNRETIEKLLSKLLCVSPMC MMIEPPKLRSTAAALYWYKTGISNISEVYGDTPEWIQRQTVLQHSFNDCTFELSQMVQWAYDNDIVDDSE IAYKYAQLADTNSNASAFLKSNSQAKIVKDCATMCRHYKRAEKKQMSMSQWIKYRCDRVDDGGDWKQIVM FLRYQGVEFMSFLTALKRFLQGIPKKNCILLYGAANTDKSLFGMSLMKFLQGSVICFVNSKSHFWLQPLA DAKIGMLDDATVPCWNYIDDNLRNALDGNLVSMDVKHRPLVQLKCPPLLITSNINAGTDSRWPYLHNRLV VFTFPNEFPFDENGNPVYELNDKNWKSFFSRTWSRLSLHEDEDKENDGDSLPTFKCVSGQNTNTLETLCQ RLNVCQDKILTHYENDSTDLRDHIDYWKHMRLECAI YYKAREMGFKHINHQWPTLAVSKNKALQAIELQ LTLETIYNSQYSNEKWTLQDVSLEVYLTAPTGCIKKHGYTVEVQFDGDICNTMHYTNWTHIYICEEASVT WEGQVDYYGLYYVHEGIRTYFVQFKDDAEKYSKNKVWEVHAGGQVILCPTSVFSSNEVSSPEI IRQHLA NHPAATHTKAVALGTEETQTTIQRPRSEPDTGNPCHTTKLLHRDSVDSAPILTAFNSSHKGRINCNSNTT PIVHLKGDANTLKCLRYRFKKHCTLYTAVSSTWHWTGHNVKHKSAIVTLTYDSEWQRDQFLSQVKIPKTI TVSTGFMSI SEQ ID NO: 37>synthetic HPV16-8 (1273aa)MFQDPQESGRKLPQLCTELQTTIHDI ILECVYCKQQLLRREVYDRDLCIVYRDGNPYAVCDKCLKFYSKI SEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRE TQLRGRKRRSHGDTPTLHEYMLDLQPETTDLYGYGQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKC DSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPRGRKRRSADPAGTNGEEGTGCNGWFYVEAWEK KTGDAI SDDENENDSDTGEDLVDFIVNDNDYLTQAETETAHALFTAQEAKQHRDAVQVLKRKYLGSPLSD ISGCVDNNISPRLKAICIEKQSRAAKRRLFESEDSGYGNTEVETQQMLQVEGRHETETPCSQYSGGSGGG CSQYSSGSGGEGVSERHTICQTPLTNILNVLKTSNAKAAMLAKFKELYGVSFSELVRPFKSNKSTCCDWC IAAFGLTPSIADSIKTLLQQYCLYLHIQSLACSWGMWLLLVRYKCGKNRETIEKLLSKLLCVSPMCMMI EPPKLRSTAAALYWYKTGISNISEVYGDTPEWIQRQTVLQHSFNDCTFELSQMVQWAYDNDIVDDSEIAY KYAQLADTNSNASAFLKSNSQAKIVKDCATMCRHYKRAEKKQMSMSQWIKYRCDRVDDGGDWKQIVMFLR YQGVEFMSFLTALKRFLQGIPKKNCILLYGAANTDKSLFGMSLMKFLQGSVICFVNSKSHFWLQPLADAK IGMLDDATVPCWNYIDDNLRNALDGNLVSMDVKHRPLVQLKCPPLLITSNINAGTDSRWPYLHNRLWFT FPNEFPFDENGNPVYELNDKNWKSFFSRTWSRLSLHEDEDKENDGDSLPTFKCVSGQNTNTLRGRKRRSE TLCQRLNVCQDKILTHYENDSTDLRDHIDYWKHMRLECAIYYKAREMGFKHINHQWPTLAVSKNKALQA IELQLTLETIYNSQYSNEKWTLQDVSLEVYLTAPTGCIKKHGYTVEVQFDGDICNTMHYTNWTHIYICEE ASVTWEGQVDYYGLYYVHEGIRTYFVQFKDDAEKYSKNKVWEVHAGGQVILCPTSVFSSNEVSSPEIIR QHLANHPAATHTKAVALGTEETQTTIQRPRSEPDTGNPCHTTKLLHRDSVDSAPILTAFNSSHKGRINCN SNTTPIVHLKGDANTLKCLRYRFKKHCTLYTAVSSTWHWTGHNVKHKSAIVTLTYDSEWQRDQFLSQVKI PKTITVSTGFMSI SEQ ID NO: 38>synthetic HPV16-9 (1305aa) MDWTWILFLVAAATRVHSHGDTPTLHEYMLDLQPETTDLYGYGQLNDSSEEEDEIDGPAGQAEPDRAHYN IVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVGPICSQKPRGRKRRSHQKRTAMFQDPQERPRKL PQLCTELQTTIHDI I LECVYCKQQLLRREVYDFAFRDLCI VYRDGNP YAVGDKCLKFYSKI SEYRHYCYS LYGTTLEQQYNKPLCDLLIRCINGQKPLCPEEKQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRETQLR GRKRRSADPAGTNGEEGTGCNGWFYVEAWEKKTGDAISDDENENDSDTGEDLVDFIVNDNDYLTQAETE TAHALFTAQEAKQHRDAVQVLKRKYLGSPLSDISGCVDNNISPRLKAICIEKQSRAAKRRLFESEDSGYG79MF-367038161Docket No.: 16553-20011.40 NTEVETQQMLQVEGRHETETPCSQYSGGSGGGCSQYSSGSGGEGVSERHTICQTPLTNILNVLKTSNAKA AMLAKFKELYGVSFSELVRPFKSNKSTCCDWCIAAFGLTPSIADSIKTLLQQYCLYLHIQSLACSWGMW LLLVRYKCGKNRETIEKLLSKLLCVSPMCMMIEPPKLRSTAAALYWYKTGISNISEVYGDTPEWIQRQTV LQHSFNDCTFELSQMVQWAYDNDIVDDSEIAYKYAQLADTNSNASAFLKSNSQAKIVKDCATMCRHYKRA EKKQMSMSQWIKYRCDRVDDGGDWKQIVMFLRYQGVEFMSFLTALKRFLQGIPKKNCILLYGAANTDKSL FGMSLMKFLQGSVICFVNSKSHFWLQPLADAKIGMLDDATVPCWNYIDDNLRNALDGNLVSMDVKHRPLV QLKCPPLLITSNINAGTDSRWPYLHNRLWFTFPNEFPFDENGNPVYELNDKNWKSFFSRTWSRLSLHED EDKENDGDSLPTFKCVSGQNTNTLRGRKRRSETLCQRLNVCQDKILTHYENDSTDLRDHIDYWKHMRLEC AIYYKAREMGFKHINHQWPTLAVSKNKALQAIELQLTLETIYNSQYSNEKWTLQDVSLEVYLTAPTGCI KKHGYTVEVQFDGDICNTMHYTNWTHIYICEEASVTWEGQVDYYGLYYVHEGIRTYFVQFKDDAEKYSK NKVWEVHAGGQVILCPTSVFSSNEVSSPEI IRQHLANHPAATHTKAVALGTEETQTTIQRPRSEPDTGNP CHTTKLLHRDSVDSAPILTAFNSSHKGRINCNSNTTPIVHLKGDANTLKCLRYRFKKHCTLYTAVSSTWH WTGHNVKHKSAIVTLTYDSEWQRDQFLSQVKIPKTITVSTGFMSI SEQ ID NO: 39>synthetic HPV16-10 ( 1267aa) MHGDTPTLHEYMLDLQPETTDLYGYGQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQ STHVDIRTLEDLLMGTLGIVGPICSQKPHQKRTAMFQDPQERPRKLPQLCTELQTTIHDI ILECVYCKQQ LLRREVYDFAFRDLCIVYRDGNPYAVGDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCING QKPLCPEEKQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRETQLADPAGTNGEEGTGCNGWFYVEAWE KKTGDAISDDENENDSDTGEDLVDFIVNDNDYLTQAETETAHALFTAQEAKQHRDAVQVLKRKYLGSPLS DISGCVDNNISPRLKAICIEKQSRAAKRRLFESEDSGYGNTEVETQQMLQVEGRHETETPCSQYSGGSGG GCSQYSSGSGGEGVSERHTICQTPLTNILNVLKTSNAKAAMLAKFKELYGVSFSELVRPFKSNKSTCCDW CIAAFGLTPSIADSIKTLLQQYCLYLHIQSLACSWGMWLLLVRYKCGKNRETIEKLLSKLLCVSPMCMM IEPPKLRSTAAALYWYKTGISNISEVYGDTPEWIQRQTVLQHSFNDCTFELSQMVQWAYDNDIVDDSEIA YKYAQLADTNSNASAFLKSNSQAKIVKDCATMCRHYKRAEKKQMSMSQWIKYRCDRVDDGGDWKQIVMFL RYQGVEFMSFLTALKRFLQGIPKKNCILLYGAANTDKSLFGMSLMKFLQGSVICFVNSKSHFWLQPLADA KIGMLDDATVPCWNYIDDNLRNALDGNLVSMDVKHRPLVQLKCPPLLITSNINAGTDSRWPYLHNRLWF TFPNEFPFDENGNPVYELNDKNWKSFFSRTWSRLSLHEDEDKENDGDSLPTFKCVSGQNTNTLETLCQRL NVCQDKILTHYENDSTDLRDHIDYWKHMRLECAIYYKAREMGFKHINHQWPTLAVSKNKALQAIELQLT LETIYNSQYSNEKWTLQDVSLEVYLTAPTGCIKKHGYTVEVQFDGDICNTMHYTNWTHIYICEEASVTW EGQVDYYGLYYVHEGIRTYFVQFKDDAEKYSKNKVWEVHAGGQVILCPTSVFSSNEVSSPEI IRQHLANH PAATHTKAVALGTEETQTTIQRPRSEPDTGNPCHTTKLLHRDSVDSAPILTAFNSSHKGRINCNSNTTPI VHLKGDANTLKCLRYRFKKHCTLYTAVSSTWHWTGHNVKHKSAIVTLTYDSEWQRDQFLSQVKIPKTITV STGFMSI SEQ ID NO: 40>synthetic CGASAN_human_codon-optimized ATGCCCGGCGCCAGCAAGCTCCGGGCCGTGCTGGAGAAGCTGAAGCTCAGCAGGGACGACATCTCCACCG CCGCCGGCATGGTGAAGGGCGTGGTGGACCACCTGCTGCTCAGACTGAAGTGCGACTCCGCCTTCAGAGG CGTCGGCCTGCTGAACACCGGCAGCTACTACGAGCACGTGAAGATCAGCGCCCCCAATGAGTTCGACGTC ATGTTCAAGCTGGAGGTCCCCAGAATCCAGCTCGAGGAGTACTCCAACACTAGGGCCTACTACTTCGTGA AG T T C AAG AG AAAT C C G AAGG AG AAT C C C C T G AG C C AG T T C C T G G AG G G T G AG AT AC T C T C AG C C AG C AA GAT G C T G T C AAAG T T C AG G AAG AT C AT C AAGG AG GAG AT C AAC G AC AT C AAG G AC AC C G AC G T C AT C AT G AAG AGG AAGAG AG G AGG C AGC C C C G C C G T G AC C C T G C T GAT C AG C GAG AAG AT AAG C GT G GAC AT AAC C C TGGCCCTGGAGTCAAAGAGCAGCTGGCCCGCCAGCACCCAGGAGGGCCTGAGGATCCAGAACTGGCTGTC AGCCAAGGTGAGGAAGCAGCTGCGACTGAAGCCATTCTACCTGGTGCCCAAGCACGCCAAGGAGGGAAAT GGTTTCCAGGAGGAGACCTGGCGGCTGTCCTTCAGCCACATCGAGAAGGAGATCCTGAACAATCACGGAA AG AG C AAG AC C T G C T GT G AGAAC AAG GAG G AG AAGT G T T G C AGG AAG G AC T G T C T G AAG C T G AT GAAG TA C C T G C T G G AG C AG C T GAAG GAG AGG T T C AAGG AC AAG AAG C AC C T GG AC AAG T T C AG C AG C T AC C AC G T G AAGACTGCCTTCTTCCACGTGTGTACCCAGAACCCCCAGGACAGCCAGTGGGACAGGAAGGACCTGGGCC80MF-367038161Docket No.: 16553-20011.40 TCTGCTTCGACAACTGCGTGACCTACTTCCTGCAGTGCCTCAGGACCGAGAAGCTGGAGAATTACTTCAT CCCCGAGTT C AAT C T GT T C AG C AGC AAC C T C AT C GAC AAG AG AAG C AAG GAGTTCCTGAC C AAG C AG AT C GAG T AC G AGAG AAAC AAT GAG TTCCCAGTGTTC GAC G AGT T C T AAT GA SEQ ID NO: 41> HPV18_E1 ( 657aa)MADPEGTDGEGTGCNGWFYVQAIVDKKTGDVI SDDEDENATDTGSDMVDFIDTQGTFCEQAELETAQALF HAQEVHNDAQVLHVLKRKFAGGSTENSPLGERLEVDTELSPRLQEISLNSGQKKAKRRLFTISDSGYGCS EVEATQIQVTTNGEHGGNVCSGGSTEAIDNGGTEGNNSSVDGTSDNSNIENVNPQCTIAQLKDLLKVNNK QGAMLAVFKDTYGLSFTDLVRNFKSDKTTCTDWVTAIFGVNPTIAEGFKTLIQPFILYAHIQCLDCKWGV LILALLRYKCGKSRLTVAKGLSTLLHVPETCMLIQPPKLRSSVAALYWYRTGISNISEVMGDTPEWIQRL TI IQHGIDDSNFDLSEMVQWAFDNELTDESDMAFEYALLADSNSNAAAFLKSNCQAKYLKDCATMCKHYR RAQKRQMNMSQWIRFRCSKIDEGGDWRPIVQFLRYQQIEFITFLGALKSFLKGTPKKNCLVFCGPANTGK SYFGMSFIHFIQGAVISFVNSTSHFWLEPLTDTKVAMLDDATTTCWTYFDTYMRNALDGNPISIDRKHKP LIQLKCPPILLTTNIHPAKDNRWPYLESRITVFEFPNAFPFDKNGNPVYEINDKNWKCFFERTWSRLDLH EEEEDADTEGNPFGTFKLRAGQNHRPL SEQ ID NO: 42> HPV18_E2 (365aa) MQTPKETLSERLSCVQDKI IDHYENDSKDIDSQIQYWQLIRWENAIFFAAREHGIQTLNHQWPAYNISK SKAHKAIELQMALQGLAQSRYKTEDWTLQDTCEELWNTEPTHCFKKGGQTVQVYFDGNKDNCMTYVAWDS VYYMTDAGTWDKTATCVSHRGLYYVKEGYNTFYIEFKSECEKYGNTGTWEVHFGNNVIDCNDSMCSTSDD TVSATQLVKQLQHTPSPYSSTVSVGTAKTYGQTSAATRPGHCGLAEKQHCGPVNPLLGAATPTGNNKRRK LCSGNTTPIIHLKGDRNSLKCLRYRLRKHSDHYRDISSTWHWTGAGNEKTGILTVTYHSETQRTKFLNTVAl P D S VQ I LVG YMTMSEQ ID NO: 43> HPV18_E6 ( 158aa) MARFEDPTRRPYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDLFWYRDSIPHAACHKCID FYSRIRELRHYSDSVYGDTLEKLTNTGLYNLLIRCLRCQKPLNPAEKLRHLNEKRRFHNIAGHYRGQCHS CCNRARQERLQRRRETQV SEQ ID NO: 44> HPV18_E7 ( 105aa) MHGPKATLQDIVLHLEPQNEIPVDLLCHEQLSDSEEENDEIDGVNHQHLPARRAEPQRHTMLCMCCKCEA RIELWESSADDLRAFQQLFLNTLSFVCPWCASQQ SEQ ID NO: 45>synthetic HPV16-1 w / o signal peptide (246aa) FQDPQESGRKLPQLCTELQTTIHDI ILECVYCKQQLLRREVYDRDLCIVYRDGNPYAVCDKCLKFYSKIS EYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRET QLRGRKRRSHGDTPTLHEYMLDLQPETTDLYGYGQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCD STLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP SEQ ID NOs: 46-53> HPV16 epitopes of Table 7-1SEQ ID NOs: 54-93> HPV16 epitopes of Table 8-2SEQ ID NOs: 94-13681MF-367038161Docket No.: 16553-20011.40 > HPV16 epitopes of Table 8-3SEQ ID NOs: 137-147Synthetic human codon-optimized HPV constructsSEQ ID NO: 137> SYNTHETIC_HPV16— 1 (Sewell ) ATGGACTGGACCTGGATCCTGTTCCTGGTGGCCGCCGCCACAAGGGTGCACAGCTTCCAGGACCCCCAGG AGAGCGGCAGAAAGCTGCCTCAGCTGTGCACCGAGCTGCAGACCACCATCCACGACATCATCCTGGAGTG CGTGTACTGCAAGCAGCAGCTGCTGAGGAGAGAGGTGTACGACAGGGACCTGTGCATCGTGTACAGGGAC GGCAATCCCTACGCCGTGTGCGACAAGTGCCTGAAGTTCTACAGCAAGATCAGCGAGTACAGGCACTACT GCTACAGCCTGTACGGCACCACCCTGGAGCAGCAGTACAACAAGCCCCTGTGCGACCTGCTGATCAGGTG CATCAACTGCCAGAAGCCCCTGCAGAGACACCTGGACAAGAAGCAGAGGTTCCACAACATCAGGGGCAGA T G G AC C G G C AG AT GCATGAGCTGCTGCAG GAG C AGC AG AAC C AG AAG G GAG AC C C AG C T GAG AG GC AG GA AG AG AAG AAG CCACGGCGATACCCCCACCCTGCACGAGTACATGCTGGACCTGCAGCCT GAG AC C AC C GA TCTGTACGGCTACGGCCAGCTGAATGACAGCAGCGAGGAGGAGGATGAGATCGACGGCCCTGCCGGCCAG GCCGAGCCCGACAGAGCCCACTACAACATCGTGACCTTTTGCTGCAAGTGCGACAGCACCCTGAGACTGT GCGTGCAGAGCACCCACGTGGACATCAGAACCCTGGAGGATCTGCTGATGGGCACCCTGGGCATCGTGTG CCCCATCTGCTCCCAGAAACCCTAATGA SEQ ID NO: 138> SYNTHETIC_HPV16— 2 (Hookipa) ATGCACGGCGACACCCCCACCCTGCACGAGTACATGCTGGACCTGCAGCCCGAGACAACCGACCTGTACG GCTACGGCCAGCTGAACGACAGCAGCGAGGAAGAGGACGAGATCGACGGCCCCGCTGGACAGGCCGAACC CGACAGAGCCCACTACAACATCGTGACATTCTGCTGCAAGTGCGACAGCACCCTGAGACTGTGCGTGCAG AGCACCCACGTGGACATCAGAACCCTGGAAGATCTGCTGATGGGCACCCTGGGCATCGTGGGCCCCATCT GCTCTCAGAAGCCCCACCAGAAAAGAACCGCCATGTTCCAGGACCCCCAGGAAAGACCCAGAAAGCTGCC CCAGCTGTGCACCGAGCTGCAGACCACCATCCACGACATCATCCTGGAATGCGTGTACTGCAAGCAGCAG CTGCTGAGAAGAGAGGTGTACGACTTCGCCTTCCGGGACCTGTGCATCGTGTACAGGGACGGCAACCCCT ACGCCGTGGGCGACAAGTGCCTGAAGTTCTACAGCAAGATCAGCGAGTACCGGCACTACTGCTACAGCCT GTACGGAACCACCCTGGAACAGCAGTACAACAAGCCCCTGTGCGACCTGCTGATCAGATGCATCAACGGC C AG AAAC C C C T GT G C C C C G AG G AAAAGC AG AG AC AC C T GG AC AAG AAG C AG C G GT T C C AC AAC AT C AG AG GCAGATGGACCGGCAGATGCATGAGCTGTTGCAGAAGCAGCAGAACCAGACGCGAGACTCAGCTGTAATG A SEQ ID NO: 139> SYNTHETIC_HPV16-3 ATGGACTGGACCTGGATCCTGTTCCTGGTGGCCGCCGCCACCCGGGTGCACAGCTTCCAGGACCCCCAGG AGAGCGGCAGAAAGCTGCCTCAGCTGTGCACCGAGCTGCAGACCACCATCCACGACATCATCCTGGAGTG CGTGTACTGCAAGCAGCAGCTGCTGAGGAGAGAGGTGTACGACCGGGACCTGTGCATCGTGTACAGGGAC GGCAACCCCTACGCCGTGTGCGACAAGTGCCTGAAGTTCTACAGCAAGATCAGCGAGTACCGGCACTACT GCTACAGCCTGTACGGCACCACCCTGGAGCAGCAGTACAACAAGCCCCTGTGCGACCTGCTGATCCGGTG CATCAACTGCCAGAAGCCCCTGCAGAGACACCTGGACAAGAAGCAGCGGTTCCACAACATCAGGGGCAGA T G G AC C G G C AG AT GCATGAGCTGCTGCCG GAG C AGC AG AAC C AG AAG G GAG AC C C AG C T G AG AG GC C G C A AGAGACGGTCTCACGGCGACACCCCCACCCTGCACGAGTACATGCTGGACCTGCAGCCTGAGACCACCGA CCTGTACGGCTACGGCCAGCTGAACGACAGCAGCGAGGAGGAGGACGAGATCGACGGCCCTGCCGGCCAG GCCGAGCCCGACAGAGCCCACTACAACATCGTGACCTTCTGCTGCAAGTGCGACAGCACCCTGAGACTGT GCGTGCAGAGCACCCACGTGGACATCAGAACCCTGGAGGACCTGCTGATGGGCACCCTGGGCATCGTGTG CCCCATCTGCTCCCAGAAGCCCAGAGGCCGGAAAAGAAGAAGCGCTGACCCTGCCGGCACCAACGGGGAG GAGGGCACGGGCTGCAACGGCTGGTTCTACGTGGAGGCTGTGGTGGAGAAAAAGACCGGGGACGCTATCA GC G AC G AC GAG AAC GAG AAC GACAGCGACACCGGCGAGGACCTGGTGGACTTCATCGT G AAC G AC AAC GA82MF-367038161Docket No.: 16553-20011.40 CTACCTGACCCAGGCCGAGACCGAGACCGCCCACGCGCTGTTCACTGCCCAGGAGGCCAAGCAGCACAGA GACGCCGTGCAGGTTCTGAAGCGAAAGTACCTGGGCAGCCCCCTGAGCGACATTAGCGGCTGCGTGGACA ACAACATTAGCCCTAGACTGAAGGCTATCTGCATCGAGAAGCAGAGCAGAGCTGCCAAGAGGAGACTGTT CGAGAGCGAGGACAGCGGGTACGGCAACACTGAGGTGGAGACTCAGCAGATGCTGCAGGTGGAGGGGCGC CACGAGACTGAGACCCCCTGCAGCCAGTACTCTGGCGGAAGCGGGGGCGGCTGCTCCCAGTACAGCAGCG GCAGCGGGGGCGAGGGCGTTAGCGAGAGACACACTATCTGCCAGACCCCCCTGACCAACATTCTGAACGT GCTGAAAACTAGCAACGCCAAGGCCGCCATGCTGGCCAAGTTCAAGGAGCTGTACGGGGTGAGCTTCAGC GAGCTGGTGAGACCCTTCAAGAGCAACAAGAGCACGTGCTGCGACTGGTGCATTGCTGCCTTCGGCCTGA CCCCCTCTATCGCTGACAGCATCAAGACCCTGCTCCAGCAGTACTGTCTCTACCTGCACATTCAGAGCCT GGCCTGCAGCTGGGGCATGGTTGTGCTGCTGCTCGTGAGATACAAGTGCGGCAAGAACAGAGAGACCATT GAGAAGCTGCTGTCTAAGCTGCTGTGCGTGTCTCCCATGTGCATGATGATCGAGCCTCCCAAGCTGAGAA GCACCGCCGCCGCCCTGTACTGGTACAAGACCGGCATCAGCAACATTAGCGAGGTGTACGGCGACACGCC CGAGTGGATCCAGAGACAGACCGTGCTGCAGCACAGCTTCAACGACTGCACCTTCGAGCTGAGCCAGATG GTGCAGTGGGCCTACGACAACGACATCGTGGACGACAGCGAGATTGCCTACAAGTACGCCCAGCTGGCCG ACACTAACAGCAACGCCAGCGCCTTCCTGAAAAGCAACAGCCAGGCCAAGATTGTGAAGGACTGCGCCAC CATGTGCAGACACTACAAGCGAGCCGAGAAGAAGCAGATGAGCATGAGCCAGTGGATCAAGTACAGATGC GACAGGGTGGACGACGGCGGCGACTGGAAGCAGATTGTTATGTTCCTGAGGTACCAGGGCGTGGAGTTCA TGAGCTTCCTGACTGCCCTGAAGAGATTCCTCCAGGGCATCCCTAAGAAGAACTGCATCCTCCTGTACGG CGCCGCTAACACCGACAAGAGCCTGTTCGGCATGAGCCTGATGAAGTTCCTGCAAGGGTCTGTGATCTGC TTCGTGAACTCTAAGAGCCACTTCTGGCTGCAGCCCCTGGCCGACGCCAAGATCGGCATGCTGGACGACG CTACCGTGCCCTGCTGGAACTACATCGACGACAACCTGAGAAACGCCCTGGACGGCAACCTCGTTTCTAT GGACGTGAAGCACAGACCCCTGGTGCAGCTGAAGTGCCCTCCCCTGCTGATTACCTCTAACATTAACGCT GGCACCGACTCTAGGTGGCCTTACCTGCACAACAGACTGGTGGTGTTCACCTTCCCTAACGAGTTCCCCT TCGACGAGAACGGCAACCCCGTGTACGAGCTGAACGATAAGAACTGGAAGTCCTTCTTCAGCAGGACGTG GTCCAGACTCAGCCTGCACGAGGACGAGGACAAGGAGAACGACGGCGACTCTCTGCCCACGTTCAAGTGC GTGAGCGGC C AGAAC AC T AAC AC C C T GAG AGG C C GG AAGAG AAG AAG C GAG AC T C T G T G C C AG AGAC T GA ACGTGTGCCAG GAC AAG AT C C T G AC C C AC T AC G AGAAC GATAGCACCGACCT C AG AG AC C AC AT C G AC T A C T G G AAG C AC AT G C G C C T C GAG T GC G C T AT C T AC T AC AAG G C C AG AG AG AT G G GC T T C AAGC AC AT T AAC CACCAGGTGGTGCCCACCCTGGCTGTGTCCAAGAACAAGGCCCTCCAGGCCATTGAGCTGCAGCTGACGC TGGAGACCATCTACAACAGCCAGTACAGCAACGAGAAGTGGACCCTGCAGGACGTTAGCCTGGAGGTGTA CCTCACTGCCCCCACCGGCTGCATCAAGAAGCACGGCTACACCGTGGAGGTGCAGTTCGACGGCGACATC TGCAACACCATGCACTACACCAACTGGACCCACATCTACATTTGCGAGGAGGCCAGCGTGACTGTGGTGG AGGGCCAGGTTGACTACTACGGCCTGTACTACGTTCACGAGGGCATCCGAACCTACTTCGTGCAGTTCAA GGACGACGCCGAGAAGTACAGCAAGAACAAGGTGTGGGAGGTTCACGCGGGCGGCCAGGTGATCCTGTGC CCTACCTCTGTGTTCAGCAGCAACGAGGTGTCCTCTCCTGAGATTATTAGGCAGCACCTGGCCAACCACC CCGCCGCGACCCACACCAAGGCCGTCGCCCTGGGCACCGAGGAGACCCAGACGACTATCCAGCGACCCAG AAGCGAGCCCGACACCGGCAACCCCTGCCACACCACTAAGCTGCTGCACAGAGACAGCGTGGACAGCGCT CCCATCCTCACTGCCTTCAACAGCAGCCACAAGGGCCGGATTAACTGCAACAGCAACACTACCCCCATCG T G C AC C T G AAG GG G G AC G C T AAC AC T C T G AAG T G C C T G AG AT AC AGAT T C AAG AAG C AC T GC AC C C T G T A CACTGCCGTGTCCTCTACCTGGCACTGGACCGGCCACAACGTGAAACACAAGAGCGCCATTGTTACCCTG ACCTACGACAGCGAGTGGCAGAGAGACCAGTTCCTGTCTCAGGTTAAGATCCCCAAGACTATTACCGTGT CTACTGGCTTCATGTCTATCTAATGA SEQ ID NO: 140> SYNTHETIC_HPV16-4 ATGGACTGGACCTGGATCCTGTTCCTGGTGGCCGCCGCCACCCGGGTGCACAGCTTCCAGGACCCCCAGG AGAGCGGCAGAAAGCTGCCTCAGCTGTGCACCGAGCTGCAGACCACCATCCACGACATCATCCTGGAGTG CGTGTACTGCAAGCAGCAGCTGCTGAGGAGAGAGGTGTACGACCGGGACCTGTGCATCGTGTACAGGGAC GGCAACCCCTACGCCGTGTGCGACAAGTGCCTGAAGTTCTACAGCAAGATCAGCGAGTACCGGCACTACT GCTACAGCCTGTACGGCACCACCCTGGAGCAGCAGTACAACAAGCCCCTGTGCGACCTGCTGATCCGGTG CATCAACTGCCAGAAGCCCCTGCAGAGACACCTGGACAAGAAGCAGCGGTTCCACAACATCAGGGGCAGA83MF-367038161Docket No.: 16553-20011.40 T G G AC C G G C AG AT GCATGAGCTGCTGCCG GAG C AGC AG AAC C AG AAG G GAG AC C C AG C T G AG AG GC C G GA AAAG AAG AAG C C AC G GC G AC AC C C C C AC C C T G C AC G AG T AC AT G C T G G AC C T G C AG C C T G AG AC C AC C GA CCTGTACGGCTACGGCCAGCTGAACGACAGCAGCGAGGAGGAGGACGAGATCGACGGCCCTGCCGGCCAG GCCGAGCCCGACAGAGCCCACTACAACATCGTGACCTTCTGCTGCAAGTGCGACAGCACCCTGAGACTGT GCGTGCAGAGCACCCACGTGGACATCAGAACCCTGGAGGACCTGCTGATGGGCACCCTGGGCATCGTGTG CCCCATCTGCTCCCAGAAGCCCAGAGGCCGCAAGCGGAGATCTGAGACTCTGTGCCAGCGACTGAACGTG T G C C AG G AC AAGAT CCTGACCCACTAC GAG AAC GAT AG C AC C GAC C T G AGG G AC C AC AT C GAC T AC T G GA AG C AC AT G C G C C T G G AG T G C G C T AT C T AC T AC AAGG C C AG AG AG AT G G G C T T C AAG C AC AT T AAC C AC C A GGTGGTGCCCACCCTGGCTGTGAGCAAGAACAAGGCCCTGCAGGCCATTGAGCTGCAGCTGACGCTGGAG ACCATCTACAACAGCCAGTACAGCAACGAGAAGTGGACCCTGCAGGACGTTAGCCTGGAGGTGTACCTGA CTGCCCCCACCGGCTGCATCAAGAAGCACGGCTACACCGTGGAGGTGCAGTTCGACGGCGACATCTGCAA CACCATGCACTACACCAACTGGACCCACATCTACATTTGCGAGGAGGCCAGCGTGACTGTGGTGGAGGGC CAGGTTGACTACTACGGCCTCTACTACGTTCACGAGGGCATCCGAACCTACTTCGTGCAGTTCAAGGACG ACGCCGAGAAGTACAGCAAGAATAAGGTGTGGGAGGTTCACGCGGGCGGCCAGGTGATCCTCTGCCCTAC CTCTGTGTTCAGCAGCAACGAGGTGTCCTCTCCTGAGATTATTAGGCAGCACCTGGCCAACCACCCCGCC GCGACCCACACCAAGGCCGTCGCCCTGGGCACCGAGGAGACCCAGACGACTATCCAGCGACCCAGAAGCG AGCCCGACACCGGCAACCCCTGCCACACCACTAAGCTGCTGCACAGAGACAGCGTGGACAGCGCTCCCAT C C T C AC T G C C T T C AAC AG C AG C C AC AAG G G C C G G AT T AAC T G C AAC AG C AAC AC T AC C C C C AT C GT G C AC C T G AAG G G C G AC G C T AAC AC T C T GAAGT G C C T G AGAT AC AG AT T C AAG AAG C AC T G C AC C C T G T AC AC T G CCGTGAGCTCTACCTGGCACTGGACCGGCCACAACGTGAAACACAAGAGCGCCATTGTTACCCTGACCTA CGACAGCGAGTGGCAGCGAGACCAGTTCCTGTCTCAGGTTAAGATCCCCAAGACTATTACCGTGTCTACT GGCTTCATGTCTATCAGAGGCCGGAAGAGAAGAAGCGCTGACCCTGCCGGCACCAACGGGGAGGAGGGCA CGGGCTGCAACGGCTGGTTCTACGTGGAGGCTGTGGTGGAGAAGAAAACCGGGGACGCTATCAGCGACGA C GAG AAC GAG AAC GACAGCGACACCGGCGAGGACCTGGTGGACTTCAT C GT G AAC GAC AAC G AC T AC C T G ACCCAGGCCGAGACCGAGACCGCCCACGCGCTGTTCACTGCCCAGGAGGCCAAGCAGCACAGAGACGCCG TGCAGGTTCTCAAGCGAAAGTACCTGGGCAGCCCCCTGAGCGACATTAGCGGCTGCGTGGACAACAACAT TAGCCCTAGACTGAAGGCTATCTGCATCGAGAAGCAGAGCAGAGCTGCCAAGAGGAGACTGTTCGAGAGC GAGGATAGCGGGTACGGCAACACTGAGGTGGAGACTCAGCAGATGCTGCAGGTGGAGGGGCGCCACGAGA CTGAGACCCCCTGTAGCCAGTACTCTGGCGGCTCCGGGGGCGGCTGCAGCCAGTACTCCAGCGGAAGCGG GGGCGAGGGCGTTAGCGAGAGACACACTATCTGCCAGACCCCCCTGACCAACATTCTGAACGTGCTGAAA ACTAGCAACGCCAAGGCCGCCATGCTGGCCAAGTTCAAGGAGCTGTACGGGGTGAGCTTCAGCGAGCTCG TGAGACCCTTCAAGAGCAACAAGAGCACGTGCTGCGACTGGTGCATTGCTGCCTTCGGCCTGACCCCCAG CATTGCTGACAGCATCAAGACCCTGCTGCAACAGTACTGCCTGTACCTGCACATTCAGAGCCTGGCCTGC AGCTGGGGCATGGTTGTGCTGCTGCTCGTGAGATACAAGTGCGGCAAGAACAGAGAGACCATTGAGAAGC TGCTGTCTAAGCTGCTGTGCGTGTCTCCCATGTGCATGATGATCGAGCCTCCCAAGCTGCGCAGCACCGC CGCCGCCCTGTACTGGTACAAGACCGGCATCAGCAACATTAGCGAGGTGTACGGCGACACGCCCGAGTGG ATCCAGAGACAGACCGTCCTCCAGCACAGCTTCAACGACTGCACCTTCGAGCTGAGCCAGATGGTGCAGT GGGCCTACGACAACGACATCGTGGACGACAGCGAGATTGCCTACAAGTACGCCCAGCTGGCCGACACTAA CAGCAACGCCAGCGCCTTCCTCAAGAGCAACTCCCAGGCCAAGATTGTGAAGGACTGCGCCACCATGTGC AGACACTACAAGCGAGCCGAGAAGAAGCAGATGAGCATGAGCCAGTGGATCAAGTACAGATGCGACAGGG TGGACGACGGCGGCGACTGGAAGCAGATTGTTATGTTCCTGAGGTACCAGGGCGTGGAGTTCATGAGCTT CCTGACTGCCCTCAAGAGATTCCTGCAGGGCATCCCTAAGAAGAACTGCATCCTCCTCTACGGCGCCGCT AACACCGACAAGAGCCTCTTCGGCATGAGCCTGATGAAGTTCCTGCAGGGGTCTGTGATCTGCTTCGTGA ACTCTAAGAGCCACTTCTGGCTCCAGCCCCTGGCCGACGCCAAGATCGGCATGCTCGACGACGCTACCGT GCCCTGCTGGAACTACATCGACGACAACCTGAGAAACGCCCTGGACGGCAACCTGGTTTCTATGGACGTG AAGCACAGACCCCTCGTGCAGCTGAAGTGCCCTCCCCTGCTGATTACCTCTAACATTAACGCTGGCACCG ACTCTAGGTGGCCTTACCTGCACAACAGACTGGTGGTGTTCACCTTCCCTAACGAGTTCCCCTTCGACGA GAACGGCAACCCCGTGTACGAGCTGAACGATAAGAACTGGAAGTCCTTCTTCAGCAGGACGTGGTCCAGA CTCAGCCTGCACGAGGACGAGGACAAGGAGAACGACGGCGACTCTCTGCCCACGTTCAAGTGCGTGAGCG GC C AGAAC AC TAACACCCTGTAATGA84MF-367038161Docket No.: 16553-20011.40 SEQ ID NO: 141> SYNTHETIC_HPV16-5 ATGGACTGGACCTGGATCCTGTTCCTGGTGGCCGCCGCCACCCGGGTGCACAGCTTCCAGGACCCCCAGG AGAGCGGCAGAAAGCTGCCCCAGCTGTGCACCGAGCTGCAGACCACCATCCACGACATCATCCTGGAGTG CGTGTACTGCAAGCAGCAGCTGCTGAGGAGAGAGGTGTACGACCGGGACCTGTGCATCGTGTACAGGGAC GGCAACCCCTACGCCGTGTGCGACAAGTGCCTGAAGTTCTACAGCAAGATCAGCGAGTACCGGCACTACT GCTACAGCCTGTACGGCACCACCCTGGAGCAGCAGTACAACAAGCCCCTGTGCGACCTGCTGATCCGGTG CATCAACTGCCAGAAGCCCCTGCAGAGACACCTGGACAAGAAGCAGCGGTTCCACAACATCAGGGGCAGA T G G AC C G G C AG AT GCATGAGCTGCTGCCG GAG C AGC AG AAC C AG AAG G GAG AC C C AG C T G AG AG GC C G GA AAAG AAG AAG C C AC G GC G AC AC C C C C AC C C T G C AC G AG T AC AT G C T G G AC C T G C AG C C C G AG AC C AC C GA CCTGTACGGCTACGGCCAGCTGAACGACAGCAGCGAGGAGGAGGACGAGATCGACGGCCCCGCCGGCCAG GCCGAGCCCGACAGAGCCCACTACAACATCGTGACCTTCTGCTGCAAGTGCGACAGCACCCTGAGACTGT GCGTGCAGAGCACCCACGTGGACATCAGAACCCTGGAGGACCTGCTGATGGGCACCCTGGGCATCGTGTG CCCCATCTGCTCCCAGAAGCCCAGAGGCCGGAAGAGAAGAAGCGCTGACCCCGCCGGCACCAACGGGGAG GAGGGCACGGGCTGCAACGGCTGGTTCTACGTGGAGGCTGTGGTGGAGAAAAAGACCGGGGACGCTATCT C AG AC G AC GAG AAC GAG AAC GACAGCGACACCGGCGAGGATCTGGTGGACTTCATCGT G AAC G AC AAC GA CTACCTGACCCAGGCCGAGACCGAGACCGCCCATGCGCTGTTCACTGCCCAGGAGGCCAAGCAGCATAGA GACGCCGTGCAGGTTCTGAAGCGAAAGTACCTGGGCAGCCCCCTGAGCGACATTAGCGGCTGCGTGGACA ACAACATTAGCCCCAGACTGAAGGCTATCTGCATCGAAAAGCAGAGCAGAGCTGCCAAGAGGAGACTGTT CGAGAGCGAGGATAGCGGGTACGGCAACACTGAGGTGGAGACTCAGCAGATGCTCCAGGTGGAGGGGCGC CATGAGACTGAGACCCCCTGCAGCCAGTACTCTGGCGGCTCCGGGGGCGGCTGCTCTCAGTACAGCAGCG GCAGCGGGGGCGAGGGCGTTAGCGAGAGACACACTATCTGCCAGACCCCCCTGACCAACATTCTGAACGT GCTCAAGACTAGCAACGCCAAGGCCGCCATGCTGGCCAAGTTCAAGGAGCTGTACGGGGTGAGCTTCTCA GAGCTGGTGAGACCCTTCAAGAGCAACAAGAGCACGTGCTGCGACTGGTGCATTGCTGCTTTCGGCCTGA CCCCCTCTATCGCTGACAGCATCAAGACCCTCCTGCAGCAGTACTGCCTGTACCTGCACATTCAGAGCCT CGCCTGCTCATGGGGCATGGTTGTGCTGCTGCTCGTGAGATACAAGTGCGGCAAGAACAGAGAGACCATT GAGAAGCTGCTGTCTAAGCTGCTGTGCGTGTCTCCCATGTGCATGATGATCGAGCCCCCCAAGCTGCGGA GCACCGCCGCCGCCCTCTACTGGTACAAGACCGGCATCTCAAACATTAGCGAGGTGTACGGCGACACGCC CGAGTGGATCCAGAGACAGACCGTGCTGCAGCATAGCTTCAACGACTGCACCTTCGAGCTGTCACAGATG GTGCAGTGGGCCTACGACAACGACATCGTGGACGACAGCGAGATTGCCTACAAGTACGCCCAGCTGGCCG ACACTAACAGCAACGCCAGCGCCTTCCTGAAAAGCAACTCACAGGCCAAGATTGTGAAGGACTGCGCCAC CATGTGCAGACATTACAAGCGAGCCGAGAAGAAGCAGATGAGCATGAGCCAGTGGATCAAGTACAGATGC GACAGGGTGGACGACGGCGGCGACTGGAAGCAGATTGTTATGTTCCTCAGGTACCAGGGCGTGGAGTTCA TGTCATTCCTGACTGCCCTGAAGAGATTCCTGCAGGGCATCCCCAAGAAGAACTGCATCCTGCTGTACGG CGCCGCTAACACCGACAAGAGCCTCTTCGGCATGAGCCTGATGAAGTTCCTGCAGGGGTCTGTGATCTGC TTCGTGAACTCTAAGAGCCATTTCTGGCTGCAGCCCCTGGCCGACGCCAAGATCGGCATGCTGGACGACG CTACCGTGCCCTGCTGGAACTACATCGACGACAACCTGAGAAACGCCCTGGACGGCAACCTCGTTTCTAT GGACGTGAAGCATAGACCCCTGGTGCAGCTGAAGTGCCCCCCCCTGCTGATTACCTCTAACATTAACGCT GGCACCGACTCTAGGTGGCCCTACCTGCATAACAGACTGGTGGTGTTCACCTTCCCCAACGAGTTCCCCT TCGACGAGAACGGCAACCCCGTGTACGAGCTCAACGACAAGAACTGGAAGTCCTTCTTCTCAAGGACGTG GTCCAGACTGAGCCTGCACGAGGACGAGGACAAGGAGAACGACGGCGACTCTCTGCCCACGTTCAAGTGC GTGTCAGGCCAGAACACTAACACCCTGTAATGA SEQ ID NO: 142> SYNTHETIC_HPV16-6 ATGGACTGGACCTGGATCCTGTTCCTGGTGGCCGCCGCCACCAGGGTGCACAGCTTCCAGGACCCCCAGG AGAGCGGCAGAAAGCTGCCTCAGCTGTGCACCGAGCTGCAGACCACCATCCACGACATCATCCTGGAGTG CGTGTACTGCAAGCAGCAGCTGCTGAGGAGAGAGGTGTACGACAGGGACCTGTGCATCGTGTACAGGGAC GGCAATCCCTACGCCGTGTGCGACAAGTGCCTGAAGTTCTACAGCAAGATCAGCGAGTACAGGCACTACT GCTACAGCCTGTACGGCACCACCCTGGAGCAGCAGTACAACAAGCCCCTGTGCGACCTGCTGATCAGGTG CATCAACTGCCAGAAGCCCCTGCAGAGACACCTGGACAAGAAGCAGAGGTTCCACAACATCAGGGGCAGA85MF-367038161Docket No.: 16553-20011.40 T G G AC C G G C AG AT GCATGAGCTGCTGCAG GAG C AGC AG AAC C AG AAG G GAG AC C C AG C T GAG AG GC AG GA AAAG AAG AAG C C AC G GC G AT AC C C C C AC C C T G C AC G AG T AC AT G C T G G AC C T G C AG C C T G AG AC C AC C GA TCTGTACGGCTACGGCCAGCTGAATGACAGCAGCGAGGAGGAGGATGAGATCGACGGCCCTGCCGGCCAG GCCGAGCCCGACAGAGCCCACTACAACATCGTGACCTTTTGCTGCAAGTGCGACAGCACCCTGAGACTGT GCGTGCAGAGCACCCACGTGGACATCAGAACCCTGGAGGATCTGCTGATGGGCACCCTGGGCATCGTGTG CCCCATCTGCTCCCAGAAGCCCAGAGGCAGGAAGAGAAGAAGCGAGACTCTTTGCCAAAGACTGAATGTG T G C C AG G AC AAGAT C C T C AC C C AC T AC G AG AAT G AT AG C AC C GAC C T T C GC G AC C AC AT C GAC T AC T G GA AG C AC AT G C G C C T T G AG T G C G C T AT C T AC T AC AAGG C C AG AG AG AT G G G C T T C AAG C AC AT T AAC C AC C A GGTGGTGCCAACCCTGGCTGTGTCAAAGAATAAGGCCCTTCAAGCCATTGAGCTGCAACTGACGCTGGAG AC C AT C T AC AAC T C AC AAT AC AG C AAT G AG AAG T GG AC C C T C C AAGAC G T T AG C C T T GAG GT G T AC C T GA CTGCCCCAACCGGCTGCATCAAGAAGCACGGCTACACCGTGGAGGTGCAGTTTGATGGCGACATCTGCAA TACCATGCACTACACCAACTGGACCCACATCTACATTTGCGAGGAGGCCTCAGTGACTGTGGTGGAGGGC CAAGTTGACTACTACGGCCTCTACTACGTTCACGAGGGCATCCGAACCTACTTTGTGCAGTTTAAGGATG ATGCCGAGAAGTACAGCAAGAATAAGGTGTGGGAGGTTCACGCGGGCGGCCAGGTGATCCTCTGCCCTAC CTCTGTGTTTAGCAGCAACGAGGTGTCCTCTCCTGAGATTATTAGGCAGCACCTGGCCAACCACCCCGCC GCGACCCACACCAAGGCCGTCGCCCTGGGCACCGAGGAGACCCAGACGACTATCCAGCGACCAAGATCAG AGCCAGACACCGGCAACCCCTGCCACACCACTAAGCTGCTGCACAGAGACTCAGTGGACAGCGCTCCAAT C C T C AC T G C C T T C AAC AG C T C AC AC AAG G G C AG G AT T AAC T G C AAT AG C AAC AC T AC C C C C AT C GT G C AC C T G AAG G G GG AT G C T AAT AC T C T GAAGT G C C T C AGAT AC AG AT T C AAG AAG C AC T G C AC C C T G T AC AC T G CCGTGAGCTCTACCTGGCACTGGACCGGCCACAATGTGAAGCACAAGAGCGCCATTGTTACCCTTACCTA CGATAGCGAGTGGCAAAGAGACCAATTTCTGTCTCAAGTTAAGATCCCAAAGACTATTACCGTGTCTACT GGCTTCATGTCTATCTAATGA SEQ ID NO: 143> SYNTHETIC_HPV16-7 ATGGACTGGACCTGGATCCTGTTCCTGGTGGCCGCCGCCACCCGGGTGCACAGCTTCCAGGACCCCCAGG AGAGCGGCAGAAAGCTGCCTCAGCTGTGCACCGAGCTGCAGACCACCATCCACGACATCATCCTGGAGTG CGTGTACTGCAAGCAGCAGCTGCTGAGGAGAGAGGTGTACGACCGGGACCTGTGCATCGTGTACAGGGAC GGCAACCCCTACGCCGTGTGCGACAAGTGCCTGAAGTTCTACAGCAAGATCAGCGAGTACCGGCACTACT GCTACAGCCTGTACGGCACCACCCTGGAGCAGCAGTACAACAAGCCCCTGTGCGACCTGCTGATCCGGTG CATCAACTGCCAGAAGCCCCTGCAGAGACACCTGGACAAGAAGCAGCGGTTCCACAACATCAGGGGCAGA T G G AC C G G C AG AT GCATGAGCTGCTGCCG GAG C AGC AG AAC C AG AAG G GAG AC CCAGCTGCACGGCGACA CCCCCACCCTGCACGAGTACATGCTGGACCTGCAGCCTGAGACCACCGACCTGTACGGCTACGGCCAGCT GAACGACAGCAGCGAGGAGGAGGACGAGATCGACGGCCCTGCCGGCCAGGCCGAGCCCGACAGAGCCCAC TACAACATCGTGACCTTCTGCTGCAAGTGCGACAGCACCCTGAGACTGTGCGTGCAGAGCACCCACGTGG ACATCAGAACCCTGGAGGACCTGCTGATGGGCACCCTGGGCATCGTGTGCCCCATCTGCTCCCAGAAGCC CGCTGACCCTGCCGGCACCAACGGGGAGGAGGGCACGGGCTGCAACGGCTGGTTCTACGTGGAGGCTGTG GT G G AG AAGAAAAC C GG G GAC GCTATCTCCGAC GAC G AGAAC GAG AAC GAC AG C G AC AC C GG C GAG G AT C TGGTGGACTTCATCGTGAACGACAACGACTACCTGACCCAGGCCGAGACCGAGACCGCCCACGCGCTGTT CACTGCCCAGGAGGCCAAGCAGCACAGAGACGCCGTGCAGGTTCTGAAGCGAAAGTACCTGGGCAGCCCC CTGAGCGACATTAGCGGCTGCGTGGACAACAACATTAGCCCTAGACTGAAGGCTATCTGCATCGAGAAGC AGAGCAGAGCTGCCAAGAGGAGACTGTTCGAGAGCGAGGATAGCGGGTACGGCAACACTGAGGTGGAGAC TCAGCAGATGCTGCAGGTGGAGGGGCGCCACGAGACTGAGACCCCCTGCTCCCAGTACAGCGGCGGCAGC GGGGGCGGATGCAGCCAGTACAGCAGCGGCTCTGGGGGCGAGGGCGTTAGCGAGAGACACACTATCTGCC AGACCCCCCTGACCAACATTCTGAACGTGCTGAAAACTAGCAACGCCAAGGCCGCCATGCTGGCCAAGTT CAAGGAGCTGTACGGGGTGAGCTTCAGCGAGCTGGTGAGACCCTTCAAGAGCAACAAGAGCACGTGCTGC GACTGGTGCATTGCTGCCTTCGGCCTGACCCCCTCTATCGCTGACAGCATCAAGACCCTCCTGCAGCAGT ACTGCCTGTACCTGCACATTCAGAGCCTCGCCTGCAGCTGGGGCATGGTTGTGCTGCTGCTGGTGAGATA CAAGTGCGGCAAGAACAGAGAGACCATTGAGAAGCTGCTGTCTAAGCTCCTGTGCGTGTCTCCCATGTGC ATGATGATCGAGCCTCCCAAGCTGAGAAGCACCGCCGCCGCCCTGTACTGGTACAAGACCGGCATCAGCA ACATTAGCGAGGTGTACGGCGACACGCCC GAG T G GAT C C AG AGAC AG AC CGTGCTGCAGCACAGCTTCAA86MF-367038161Docket No.: 16553-20011.40 CGACTGCACCTTCGAGCTCAGCCAGATGGTGCAGTGGGCCTACGACAACGACATCGTGGACGACAGCGAG ATTGCCTACAAGTACGCCCAGCTGGCCGACACTAACAGCAACGCCAGCGCCTTCCTGAAGTCCAACAGCC AG G C C AAG AT T GT G AAG GACTGCGCCACCATGTGCAGACAC T AC AAG C GAG C C GAG AAG AAG C AGAT GAG CATGAGCCAGTGGATAAAGTACAGATGCGACAGGGTGGACGACGGCGGCGACTGGAAGCAGATTGTTATG TTCCTCAGGTACCAGGGCGTGGAGTTCATGAGCTTCCTGACTGCCCTGAAGAGATTCCTGCAGGGCATCC CTAAGAAGAACTGCATCCTGCTCTACGGCGCCGCTAACACCGACAAGAGCCTCTTCGGCATGAGCCTGAT GAAGTTCCTGCAGGGGTCTGTGATCTGCTTCGTGAACTCTAAGAGCCACTTCTGGCTGCAGCCCCTGGCC GACGCCAAGATCGGCATGCTGGACGACGCTACCGTGCCCTGCTGGAACTACATCGACGACAACCTGAGAA ACGCCCTGGACGGCAACCTGGTTTCTATGGACGTCAAGCACAGACCCCTCGTGCAGCTGAAGTGCCCTCC CCTGCTGATTACCTCTAACATTAACGCTGGCACCGACTCTAGGTGGCCTTACCTGCACAACAGACTGGTG GTGTTCACCTTCCCTAACGAGTTCCCCTTCGACGAGAACGGCAACCCCGTGTACGAGCTCAACGACAAGA AC T G GAAG T C C T T C T T C AG C AG G AC G T G G T C C AG AC T G AG C C T G C AC G AGG AC GAG G AC AAG G AGAAC GA CGGCGACTCTCTGCCCACGTTCAAGTGCGTGAGCGGCCAGAACACTAACACCCTCGAGACTCTGTGCCAG AGGCTCAACGTGTGCCAGGACAAGATCCTGACCCACTACGAGAACGATAGCACCGACCTGCGAGACCACA TCGACTACTGGAAGCACATGCGCCTGGAGTGCGCTATCTACTACAAGGCCAGAGAGATGGGCTTCAAGCA CATTAACCACCAGGTGGTGCCCACCCTGGCTGTGTCCAAGAACAAGGCCCTGCAGGCCATTGAGCTGCAA CTCACGCTGGAGACCATCTACAACAGCCAATACAGCAACGAGAAGTGGACCCTCCAGGACGTTAGCCTGG AGGTGTACCTCACTGCCCCCACCGGCTGCATCAAGAAGCACGGCTACACCGTGGAGGTGCAGTTCGACGG CGACATCTGCAACACCATGCACTACACCAACTGGACCCACATCTACATTTGCGAGGAGGCCAGCGTGACT GTGGTGGAGGGCCAGGTTGACTACTACGGCCTGTACTACGTTCACGAGGGCATCCGAACCTACTTCGTGC AGTTCAAGGACGACGCCGAGAAGTACAGCAAGAACAAGGTGTGGGAGGTTCACGCGGGCGGCCAGGTGAT CCTGTGCCCTACCTCTGTGTTCAGCAGCAACGAGGTGTCCTCTCCTGAGATTATTAGGCAGCACCTGGCC AACCACCCCGCCGCGACCCACACCAAGGCCGTCGCCCTGGGCACCGAGGAGACCCAGACGACTATCCAGC GAC C C AG AAG C GAG C C C G AC AC C GG C AAC C C C T G C C AC AC C AC T AAG C T GC T G C AC AGAG AC AG C G T G GA CAGCGCTCCCATCCTCACTGCCTTCAACAGCAGCCACAAGGGCCGGATTAACTGCAACAGCAACACTACC C C C AT C G T GC AC C T C AAG G GG G AC G C T AAC AC T C T C AAGT G C C T C AG AT AC AG AT T C AAG AAG C AC T G C A CCCTCTACACTGCCGTGAGCTCTACCTGGCACTGGACCGGCCACAACGTGAAGCACAAGAGCGCCATTGT TACCCTGACCTACGACAGCGAGTGGCAGAGAGACCAGTTCCTGTCTCAGGTTAAGATCCCCAAGACTATT ACCGTGTCTACTGGCTTCATGTCTATCTAATGA SEQ ID NO: 144> SYNTHETIC_HPV16-8 ATGTTCCAGGACCCCCAGGAGAGCGGCAGAAAGCTGCCTCAGCTGTGCACCGAGCTGCAGACCACCATCC ACGACATCATCCTGGAGTGCGTGTACTGCAAGCAGCAGCTGCTGAGGAGAGAGGTGTACGACCGGGACCT GTGCATCGTGTACAGGGACGGCAACCCCTACGCCGTGTGCGACAAGTGCCTGAAGTTCTACAGCAAGATC AGCGAGTACCGGCACTACTGCTACAGCCTGTACGGCACCACCCTGGAGCAGCAGTACAACAAGCCCCTGT GCGACCTGCTGATCCGGTGCATCAACTGCCAGAAGCCCCTGCAGAGACACCTGGACAAGAAGCAGCGGTT CCACAACATCAGGGGCAGATGGACCGGCAGATGCATGAGCTGCTGCCGGAGCAGCAGAACCAGAAGGGAG AC C C AG C T GAG AG G C C G G AAAAG AAG AAG C C AC G GC G AC AC C C C C AC C C T G C AC G AG T AC AT G C T G G AC C TGCAGCCT GAG AC CACCGACCTGTACGGCTACGGCCAGCT G AAT GAC AG C AG C GAG GAG GAG GAC GAG AT CGACGGCCCTGCCGGCCAGGCCGAGCCCGACAGAGCCCACTACAACATCGTGACCTTCTGCTGCAAGTGC GACAGCACCCTGAGACTGTGCGTGCAGAGCACCCACGTGGACATCAGAACCCTGGAGGACCTGCTGATGG GCACCCTGGGCATCGTGTGCCCCATCTGCTCCCAGAAGCCCAGAGGCCGCAAGAGAAGATCCGCTGACCC TGCCGGCACCAACGGGGAGGAGGGCACGGGCTGCAACGGCTGGTTCTACGTGGAGGCTGTGGTGGAGAAG AAAAC C G G GG AC G C T AT C AGC G AC G AC G AG AAC G AG AAC G AC AG C GAC AC C G G C G AG GAT C T G G T G G AC T TCATCGTGAACGACAACGACTACCTGACCCAGGCCGAGACCGAGACCGCCCACGCGCTGTTCACTGCCCA GGAGGCCAAGCAGCACAGAGACGCCGTGCAGGTTCTGAAGCGAAAGTACCTGGGCAGCCCCCTGAGCGAC ATTAGCGGCTGCGTGGACAACAACATTAGCCCTAGACTGAAGGCTATCTGCATCGAGAAGCAGAGCAGAG C T G C C AAG AG GAG AC T G T T C GAG AG C GAG GAT AG C G G G T AC G GC AAC AC T G AG GT G GAG AC T C AGC AG AT GCTGCAGGTGGAGGGGCGCCACGAGACTGAGACCCCTTGCAGCCAGTATAGCGGCGGCAGCGGCGGCGGC TGCTCTCAGTACAGCAGCGGCTCTGGGGGCGAGGGCGTTAGCGAGAGACACACTATCTGCCAGACCCCCC87MF-367038161Docket No.: 16553-20011.40 TGACCAACATTCTGAACGTGCTGAAAACTAGCAACGCCAAGGCCGCCATGCTGGCCAAGTTCAAGGAGCT GTACGGGGTGAGCTTCAGCGAGCTGGTGAGACCCTTCAAGAGCAACAAGAGCACGTGCTGCGACTGGTGC ATTGCTGCTTTCGGCCTGACCCCCAGCATTGCTGACAGCATCAAGACCCTGCTCCAGCAGTACTGCCTGT ACCTCCACATTCAGAGCCTGGCCTGCAGCTGGGGCATGGTTGTGCTGCTGCTGGTGAGATACAAGTGCGG CAAGAACAGAGAGACCATTGAGAAGCTGCTGTCTAAGCTGCTGTGCGTGTCTCCCATGTGCATGATGATC GAGCCTCCCAAGCTGAGAAGCACCGCCGCCGCCCTGTACTGGTACAAGACCGGCATCAGCAACATTAGCG AGGTGTACGGCGACACGCCCGAGTGGATCCAGAGACAGACCGTCCTCCAGCACAGCTTCAACGACTGCAC CTTCGAGCTCAGCCAGATGGTGCAGTGGGCCTACGACAACGACATCGTGGACGACAGCGAGATTGCCTAC AAGTACGCCCAGCTGGCCGACACTAACAGCAACGCCAGCGCCTTCCTGAAGTCCAACAGCCAGGCCAAGA T T G T GAAG GACTGCGCCACCATGTG C AG AC AC T AC AAG C G AG C C G AG AAGAAG C AG AT G AGC AT GAG C C A GTGGATCAAGTACAGATGCGACAGGGTGGACGACGGCGGCGACTGGAAGCAGATTGTTATGTTCCTGAGG TACCAGGGCGTGGAGTTCATGAGCTTCCTCACTGCCCTCAAGAGATTCCTGCAGGGCATCCCTAAGAAGA ACTGCATCCTCCTGTACGGCGCCGCTAACACCGACAAGAGCCTGTTCGGCATGAGCCTCATGAAGTTCCT GCAGGGGTCTGTCATCTGCTTCGTGAACTCTAAGAGCCACTTCTGGCTGCAGCCCCTCGCCGACGCCAAG ATCGGCATGCTGGACGACGCTACCGTGCCCTGCTGGAACTACATCGACGACAACCTGAGAAACGCCCTGG ACGGCAACCTCGTTTCTATGGACGTCAAGCACAGACCCCTGGTGCAGCTGAAGTGCCCTCCCCTGCTCAT TACCTCTAACATTAACGCTGGCACCGACTCTAGGTGGCCTTACCTGCACAACAGACTGGTGGTGTTCACC TTCCCTAACGAGTTCCCCTTCGACGAGAACGGCAACCCCGTGTACGAGCTGAACGACAAGAACTGGAAGT CCTTCTTCAGCAGGACGTGGTC C AG AC TGAGCCTGCACGAG G AC GAG G AC AAG GAG AAC G AC G G C G AC AG CCTGCCCACGTTCAAGTGCGTGAGCGGCCAGAACACTAACACCCTCAGAGGCCGGAAGAGAAGAAGCGAG ACTCTGTGCCAGCGGCTGAACGTGTGCCAGGACAAGATCCTGACCCACTACGAGAATGACAGCACCGACC T G AG AG AC C AC AT C G AC T AC T G G AAG C AC AT G C G C C T G GAG T GC G C T AT C T AC T AC AAG G C C AG AG AG AT GGGCTTCAAGCACATTAACCACCAGGTGGTGCCCACCCTGGCTGTGAGCAAGAACAAGGCCCTCCAGGCC AT T G AG C T GC AGC T G AC G C T G G AGAC C AT C T AC AAC AG C C AG T AC AG C AAC G AAAAG T G G AC C C T C C AGG ACGTTAGCCTGGAGGTGTACCTGACTGCCCCCACCGGCTGCATCAAGAAGCACGGCTACACCGTGGAGGT GCAGTTCGACGGCGACATCTGCAACACCATGCACTACACCAACTGGACCCACATCTACATTTGCGAGGAG GCCAGCGTGACTGTGGTGGAGGGCCAGGTTGACTACTACGGCCTGTACTACGTTCACGAGGGCATCCGAA CCTACTTCGTGCAGTTCAAGGACGACGCCGAGAAGTACTCCAAGAACAAGGTGTGGGAGGTTCACGCGGG CGGCCAGGTGATTCTGTGCCCTACCTCTGTGTTCAGCAGCAACGAGGTGTCCTCTCCTGAGATTATTAGG CAGCACCTGGCCAACCACCCCGCCGCGACCCACACCAAGGCCGTCGCCCTGGGCACCGAGGAGACCCAGA CGACTATCCAGCGACCCAGAAGCGAGCCCGACACCGGCAACCCCTGCCACACCACTAAGCTGCTGCACAG AGACAGCGTGGACAGCGCTCCCATCCTCACTGCCTTCAACAGCAGCCACAAGGGCCGGATTAACTGCAAC AG C AAC AC T AC C C C C AT C G T G C AC C T GAAG GG G G AC G C T AAC AC T C T G AAG T G C C T G AG AT AC AGAT T C A AGAAGCACTGCACCCTGTACACTGCCGTGAGCTCTACCTGGCACTGGACCGGCCACAACGTGAAGCACAA GAGCGCCATTGTTACCCTGACCTACGACAGCGAGTGGCAGCGAGACCAGTTCCTGTCTCAGGTTAAGATC CCCAAGACTATTACCGTGTCTACTGGCTTCATGTCTATCTAATGA SEQ ID NO: 145> SYNTHETIC_HPV16-9 ATGGACTGGACCTGGATCCTGTTCCTGGTGGCCGCCGCCACACGGGTGCACAGCCACGGCGACACCCCCA CCCTGCACGAGTACATGCTGGACCTGCAGCCCGAGACCACCGACCTGTACGGCTACGGCCAGCTGAACGA C AG C AG C GAG GAG G AGG AC GAG AT C G AC GGCCCCGCTGGCCAGGCCGAGCCC G AC AG AG C C C AC T AC AAC ATCGTGACCTTCTGCTGCAAGTGCGACAGCACCCTGAGACTGTGCGTGCAGAGCACCCACGTGGACATCA GAACCCTGGAGGACCTGCTGATGGGCACCCTGGGCATCGTGGGCCCCATCTGCTCTCAGAAGCCCAGAGG C C G G AAAAGAAGAAG C C AC C AG AAG AGAAC CGCCATGTTCCAGGACCCCCAG GAG AG AC C C AG AAAG C T G CCCCAGCTGTGCACCGAGCTGCAGACCACCATCCACGACATCATCCTGGAGTGCGTGTACTGCAAGCAGC AGCTGCTGAGAAGAGAGGTGTACGACTTCGCCTTCCGGGACCTGTGCATCGTGTACAGGGACGGCAACCC CTACGCCGTGGGCGACAAGTGCCTGAAGTTCTACAGCAAGATCAGCGAGTACCGGCACTACTGCTACAGC CTGTACGGCACCACCCTGGAGCAGCAGTACAACAAGCCCCTGTGCGACCTGCTGATCAGATGCATCAACG GC C AGAAAC C C C T G T GC C C C G AG GAG AAG C AG AG AC AC C T G G AC AAG AAGC AG C G G T T C C AC AAC AT C AG AGGCAGATGGACCGGCAGATGCATGAGCTGCTGCAGAAGCAGCAGAACCAGACGCGAGACTCAGCTGAGA88MF-367038161Docket No.: 16553-20011.40 GGCCGCAAGAGACGGTCTGCTGACCCCGCCGGCACCAACGGGGAGGAGGGCACGGGCTGCAACGGCTGGT T C T AC G T G GAG GC T G T G G T GG AG AAG AAAAC C G G GG AC GC T AT C T C C G AC G AC GAG AAC G AG AAC G AC AG C G AC AC C G GC G AG GATCTGGTG G AC TTCATCGT G AAC G AC AAC GACTACCTGACCCAGGCC GAG AC C GAG ACCGCCCACGCGCTGTTCACTGCCCAGGAGGCCAAGCAGCACAGAGACGCCGTGCAGGTTCTGAAGCGAA AGTACCTGGGCAGCCCCCTGAGCGACATTAGCGGCTGCGTGGACAACAACATTAGCCCCAGACTGAAGGC TATCTGCATC G AAAAGC AG AG CAGAGCTGC C AAG AG G AGAC T GT T C GAG AG C GAG GATAGCGGGTACGGC AACACTGAGGTGGAGACTCAGCAGATGCTGCAGGTGGAGGGGCGCCACGAGACTGAGACCCCCTGCAGCC AGTACTCTGGGGGCAGCGGGGGCGGCTGCTCCCAGTACAGCAGCGGCAGCGGGGGAGAGGGCGTTAGCGA GAG AC AC AC T AT C T G C C AG AC C C C C C T G AC C AAC AT T C T G AAC G T GC T G AAAAC T AG C AAC G C C AAG G C C GCCATGCTGGCCAAGTTCAAGGAGCTGTACGGGGTGAGCTTCAGCGAGCTGGTGAGACCCTTCAAGAGCA ACAAGAGCACGTGCTGCGACTGGTGCATTGCTGCCTTCGGCCTGACCCCCTCTATCGCTGACAGCATCAA GACCCTGCTCCAGCAGTACTGCCTCTACCTGCACATTCAGAGCCTGGCCTGCAGCTGGGGCATGGTTGTG CTGCTGCTCGTGAGATACAAGTGCGGCAAGAACAGAGAGACCATTGAGAAGCTGCTGTCTAAGCTGCTGT GCGTGTCTCCCATGTGCATGATGATCGAGCCCCCCAAGCTGAGAAGCACCGCCGCCGCCCTGTACTGGTA CAAGACCGGCATCAGCAACATTAGCGAGGTGTACGGCGACACGCCCGAGTGGATCCAGAGACAGACCGTG CTGCAGCACAGCTTCAACGACTGCACCTTCGAGCTGAGCCAGATGGTGCAGTGGGCCTACGACAACGACA TCGTGGACGACAGCGAGATTGCCTACAAGTACGCCCAGCTGGCCGACACTAACAGCAACGCCAGCGCCTT C C T G AAAAGC AAC AG C C AG GC C AAG AT T G T GAAG GAC T GC G C C AC C AT G T G C AGAC AC T AC AAG C G AG C C GAGAAGAAGCAGATGAGCATGAGCCAGTGGATCAAGTACAGATGCGACAGGGTGGACGACGGCGGCGACT GGAAGCAGATTGTTATGTTCCTGAGGTACCAGGGCGTGGAGTTCATGAGCTTCCTGACTGCCCTGAAGAG ATTCCTCCAGGGCATCCCCAAGAAGAACTGCATCCTCCTGTACGGCGCCGCTAACACCGACAAGAGCCTG TTCGGCATGAGCCTGATGAAGTTCCTGCAGGGGTCTGTCATCTGCTTCGTGAACTCTAAGAGCCACTTCT GGCTGCAGCCCCTGGCCGACGCCAAGATCGGCATGCTGGACGACGCTACCGTGCCCTGCTGGAACTACAT CGACGACAACCTGAGAAACGCCCTGGACGGCAACCTCGTTTCTATGGACGTGAAGCACAGACCCCTGGTG CAGCTGAAGTGCCCCCCCCTGCTGATTACCTCTAACATTAACGCTGGCACCGACTCTAGGTGGCCCTACC TGCACAACAGACTGGTGGTGTTCACCTTCCCCAACGAGTTCCCCTTCGACGAGAACGGCAACCCCGTGTA CGAGCTGAATGACAAGAACTGGAAGTCCTTCTTCAGCAGGACGTGGTCCAGACTCAGCCTGCACGAGGAC GAGGACAAGGAGAACGACGGCGACTCTCTGCCCACGTTCAAGTGCGTGAGCGGCCAGAACACTAACACCC T GAG AG G C C G GAAG AGAAG AAG C GAG AC T C T G T G C C AG AG AC T G AAC GTGTGCCAG GAC AAG AT C C T GAC CCACTACGAGAACGATAGCACCGACCTCAGAGACCACATCGACTACTGGAAGCACATGCGCCTCGAGTGC GCTATCTACTACAAGGCCAGAGAGATGGGCTTCAAGCACATTAACCACCAGGTGGTGCCCACCCTGGCTG T G AG C AAG AAC AAAG C C C T C C AG GC C AT T G AG C T GC AG C T G AC G C T G G AGAC C AT C T AC AAC AG C C AG T A CAGCAACGAGAAGTGGACCCTGCAGGACGTTAGCCTGGAGGTGTACCTCACTGCCCCCACCGGCTGCATC AAG AAG C AC G G C T AC AC C G T G G AGG T GC AG T T C G AC G G C G AC AT C T G C AAC AC C AT G C AC T AC AC C AAC T GGACCCACATCTACATTTGCGAGGAGGCCAGCGTGACTGTGGTGGAGGGCCAGGTTGACTACTACGGCCT GTACTACGTTCACGAGGGCATCCGAACCTACTTCGTGCAGTTCAAGGACGACGCCGAGAAGTACAGCAAG AACAAGGTGTGGGAGGTTCACGCGGGCGGCCAGGTGATCCTGTGCCCCACCTCTGTGTTCAGCAGCAACG AGGTGTCCTCTCCCGAGATTATTAGGCAGCACCTGGCCAACCACCCCGCCGCGACCCACACCAAGGCCGT CGCCCTGGGCACCGAGGAGACCCAGACGACTATCCAGCGACCCAGAAGCGAGCCCGACACCGGCAACCCC TGCCACACCACTAAGCTGCTGCACAGAGACAGCGTGGACAGCGCTCCCATCCTCACTGCCTTCAACAGCA GC C AC AAG GG C C G G AT T AAC T G C AAC AG C AAC AC T AC C C C C AT C G T G C AC C T G AAG G GC G AC G C T AAC AC TCTGAAGTGCCTGAGATACAGATTCAAGAAGCACTGCACCCTGTACACTGCCGTGTCCTCTACCTGGCAC T G G AC C G G C C AC AAT GT G AAG C AC AAGAG C GC C AT T G T T AC C C T G AC C T AC G AC AG C GAG T G G C AG AG AG ACCAGTTCCTGTCTCAGGTTAAGATCCCCAAGACTATTACCGTGTCTACTGGCTTCATGTCTATCTAATG A SEQ ID NO: 146> SYNTHETIC_HPV16-10 ATGCACGGCGACACCCCCACCCTGCACGAGTACATGCTGGACCTGCAGCCCGAGACCACCGACCTGTACG GCTACGGCCAGCTGAACGACAGCAGCGAGGAGGAGGACGAGATCGACGGCCCCGCTGGCCAGGCCGAGCC CGACAGAGCCCACTACAACATCGTGACCTTCTGCTGCAAGTGCGACAGCACCCTGAGACTGTGCGTGCAG89MF-367038161Docket No.: 16553-20011.40 AGCACCCACGTGGACATCAGAACCCTGGAGGACCTGCTGATGGGCACCCTGGGCATCGTGGGCCCCATCT GC T C T C AG AAG C C C C AC C AGAAG AG AAC C G C C AT GT T C C AG G AC C C C C AGG AG AG AC C C AGAAAGC T G C C CCAGCTGTGCACCGAGCTGCAGACCACCATCCACGACATCATCCTGGAGTGCGTGTACTGCAAGCAGCAG CTGCTGAGAAGAGAGGTGTACGACTTCGCCTTCCGGGACCTGTGCATCGTGTACAGGGACGGCAACCCCT ACGCCGTGGGCGACAAGTGCCTGAAGTTCTACAGCAAGATCAGCGAGTACCGGCACTACTGCTACAGCCT GTACGGCACCACCCTGGAGCAGCAGTACAACAAGCCCCTGTGCGACCTGCTGATCAGATGCATCAACGGC C AG AAAC C C C T GT G C C C C G AG G AGAAGC AG AG AC AC C T GG AC AAG AAG C AG C G GT T C C AC AAC AT C AG AG GCAGATGGACCGGCAGATGCATGAGCTGCTGCAGAAGCAGCAGAACCAGACGCGAGACTCAGCTGGCTGA CCCCGCCGGCACCAACGGGGAGGAGGGCACGGGCTGCAACGGCTGGTTCTACGTGGAGGCTGTGGTGGAG AAG AAAAC C G G GG AC GC T AT C AG C G AC G AC GAG AAC G AGAAC GAC AG C G AC AC C G G C GAG GAT C T G G T GG ACTTCATCGTGAACGACAACGACTACCTGACCCAGGCCGAGACCGAGACCGCCCACGCGCTGTTCACTGC CCAGGAGGCCAAGCAGCACAGAGACGCCGTGCAGGTTCTGAAGCGAAAGTACCTGGGCAGCCCCCTGAGC GAC AT T AG C G G C T G C GT G G AC AAC AAC AT T AG C C C C AG AC T G AAG GC T AT C T G C AT C GAAAAG C AG AG C A GAG C T G C C AAG AG G AGAC T GT T C GAG AG C GAG GATAGCGGGTACGGCAACACTGAGGTG GAG AC T C AG C A GATGCTGCAGGTGGAGGGGCGCCACGAGACTGAGACCCCCTGCAGCCAGTATAGCGGCGGCTCTGGCGGC GGATGCTCCCAGTACAGCAGCGGCAGCGGGGGCGAGGGCGTTAGCGAGAGACACACTATCTGCCAGACCC CCCTGACCAACATTCTGAACGTGCTCAAGACTAGCAACGCCAAGGCCGCCATGCTGGCCAAGTTCAAGGA GCTGTACGGGGTGAGCTTCAGCGAGCTGGTGAGACCCTTCAAGAGCAACAAGAGCACGTGCTGCGACTGG TGTATTGCTGCCTTCGGCCTGACCCCCAGCATTGCTGACAGCATCAAGACCCTGCTGCAGCAGTACTGCC TGTACCTGCACATTCAGAGCCTGGCCTGCAGCTGGGGCATGGTTGTGCTGCTCCTGGTGAGATACAAGTG CGGCAAGAACAGAGAGACCATTGAGAAGCTGCTGTCTAAGCTGCTGTGCGTGTCTCCCATGTGCATGATG ATCGAGCCCCCCAAGCTGCGGAGCACCGCCGCCGCCCTGTACTGGTACAAGACCGGCATCAGCAACATTA GCGAGGTGTACGGCGACACGCCCGAGTGGATCCAGAGACAGACCGTGCTGCAGCACAGCTTCAACGACTG CACCTTCGAGCTCAGCCAGATGGTGCAGTGGGCCTACGACAACGACATCGTGGACGACAGCGAGATTGCC TACAAGTACGCCCAGCTGGCCGACACTAACAGCAACGCCAGCGCCTTCCTGAAGTCCAACAGCCAGGCCA AG AT T G T G AAG GACTGCGCCACCATGTGCAGACACTACAAGCGAGCC G AGAAG AAG C AG AT GAG CAT GAG CCAGTGGATCAAGTACAGATGCGACAGGGTGGACGACGGCGGCGACTGGAAGCAGATTGTTATGTTCCTG AGGTACCAGGGCGTGGAGTTCATGAGCTTCCTGACTGCCCTGAAGAGATTCCTGCAGGGCATCCCCAAGA AGAACTGCATCCTCCTGTACGGCGCCGCTAACACCGACAAGAGCCTGTTCGGCATGAGCCTGATGAAGTT CCTGCAGGGGTCTGTGATCTGCTTCGTGAACTCTAAGAGCCACTTCTGGCTGCAGCCCCTCGCCGACGCC AAGATCGGCATGCTGGACGACGCTACCGTGCCCTGCTGGAACTACATCGACGACAACCTGAGAAACGCCC TGGACGGCAACCTGGTTTCTATGGACGTGAAGCATAGACCCCTGGTGCAGCTCAAGTGCCCCCCCCTCCT GATTACCTCTAACATTAACGCTGGCACCGACTCTAGGTGGCCCTACCTCCACAACAGACTGGTGGTGTTC ACCTTCCCCAACGAGTTCCCCTTCGACGAGAACGGCAACCCCGTGTACGAGCTCAACGACAAGAACTGGA AGTCCTTCTTCAGCAGGACGTGGTCCAGACTGAGCCTGCACGAGGACGAGGACAAGGAGAACGACGGCGA CTCTCTGCCCACGTTCAAGTGCGTGAGCGGCCAGAACACTAACACCCTGGAGACTCTGTGCCAGAGACTG AAC G T G T G C C AGG AC AAG AT C C T GAC C C AC T AC G AG AAC G AT AG C AC C G AC C T GAG G GAC C AC AT C G AC T ACTGGAAGCACATGCGCCTGGAGTGCGCTATCTACTACAAGGCCAGAGAGATGGGCTTCAAGCACATTAA CCACCAGGTGGTGCCCACCCTGGCTGTGAGCAAGAACAAGGCCCTGCAGGCCATTGAGCTGCAGCTGACG CTGGAGACCATCTACAACAGCCAGTACAGCAACGAGAAGTGGACCCTGCAGGACGTTAGCCTGGAGGTGT ACCTGACTGCCCCCACCGGCTGCATCAAGAAGCACGGCTACACCGTGGAGGTGCAGTTCGACGGCGACAT CTGCAACACCATGCACTACACCAACTGGACCCACATCTACATTTGCGAGGAGGCCAGCGTGACTGTGGTG GAGGGCCAGGTTGACTACTACGGCCTGTACTACGTTCACGAGGGCATCCGAACCTACTTCGTGCAGTTCA AGGACGACGCCGAGAAGTACAGCAAAAACAAGGTGTGGGAGGTTCACGCGGGCGGCCAGGTGATCCTGTG CCCCACCTCTGTGTTCAGCAGCAACGAGGTGTCCTCTCCCGAGATTATTAGGCAGCACCTGGCCAACCAC CCCGCCGCGACCCACACCAAGGCCGTCGCCCTGGGCACCGAGGAGACCCAGACGACTATCCAGCGACCCA GAAGCGAGCCCGACACCGGCAACCCCTGCCACACCACTAAGCTGCTGCACAGAGACAGCGTGGACAGCGC TCCCATCCTCACTGCCTTCAACAGCAGCCACAAGGGCCGGATTAACTGCAACAGCAACACTACCCCCATC GTGCACCTGAAGGGCGACGCTAACACTCTGAAGTGCCTGAGATACAGATTCAAGAAGCACTGCACCCTGT ACACTGCCGTGTCCTCTACCTGGCACTGGACCGGCCACAACGTGAAGCACAAGAGCGCCATTGTTACCCT90MF-367038161Docket No.: 16553-20011.40 GACCTACGACAGCGAGTGGCAGCGCGACCAATTCCTGTCTCAGGTTAAGATCCCCAAGACTATTACCGTG TCTACTGGCTTCATGTCTATCTAATGA SEQ ID NO: 147> SYNTHETIC_HPV18— 6 ATGGACTGGACCTGGATCCTGTTCCTGGTGGCCGCCGCCACCCGGGTGCACAGCTTCGAGGATCCCACCC GGAGCGGCTACAAGCTGCCCGATCTGTGCACCGAGCTGAACACTAGCCTGCAGGATATCGAGATCACCTG CGTGTACTGCAAGACCGTGCTGGAGCTGACCGAGGTGTTCGAGAAGGATCTCTTCGTGGTGTACAGAGAC AGCATCCCCCACGCTGCCTGCCACAAGTGCATCGATTTCTACTCTAGAATTAGAGAGCTGAGACACTACA GCGACTCTGTGTACGGCGACACCCTGGAGAAGCTGACTAACACTGGGCTGTACAATCTGCTCATCAGGTG CCTGCGGTGCCAGAAGCCCCTGCTGAGACACCTGAATGAGAAGAGGAGGTTCCACAACATCGCTGGGCAC TACAGAGGCCAGTGCCACAGCTGCTGCAACAGGGCCAGGCAGGAGAGGCTCCAGAGGCGCAGAGAGACCC AGGTCAGAGGCCGGAAAAGAAGAAGCCACGGCCCCAAGGCCACCCTCCAGGACATTGTGCTGCACCTGGA GCCCCAGAATGAGATTCCCGTTGACCTGCTGGGCCACGGCCAGCTGAGCGACAGCGAGGAGGAGAACGAT GAGATCGATGGCGTTAATCACCAGCACCTGCCCGCCAGGAGGGCCGAGCCCCAGCGGCACACCATGCTGT GCATGTGCTGCAAGTGCGAGGCCAGAATTGAGCTGGTGGTGGAGAGCAGCGCCGACGACCTGAGGGCCTT CCAGCAGCTGTTCCTGAACACCCTGTCCTTCGTGTGCCCCTGGTGCGCCTCCCAGCAGAGAGGCCGGAAG AGAAGAAGCCAGACCCCCAAGGAGACCCTGAGCGAGAGGCTGAGCTGCGTGCAGGACAAGATCATCGACCACT ACGAGAATGACAGCAAGGACATCGACAGCCAGATCCAGTACTGGCAGCTGATCCGCTGGGAGAATGC CATCTTCTTCGCCGCCAGGGAGCACGGCATCCAGACCCTGAACCACCAGGTGGTGCCCGCCTACAACATT AGCAAGAGCAAGGCCCACAAGGCTATTGAGCTGCAGATGGCCCTGCAAGGCCTGGCCCAGAGCAGGTACA AGACCGAGGATTGGACCCTGCAGGACACCTGCGAGGAGCTGTGGAATACCGAGCCCACTCACTGCTTCAA GAAGGGCGGCCAGACCGTGCAGGTGTACTTCGATGGCAACAAGGACAATTGCATGACCTACGTGGCCTGG GACAGCGTGTACTACATGACTGATGCCGGCACATGGGACAAGACCGCTACCTGCGTGAGCCACAGGGGCC T G T AC T AC GT G AAG G AG G G GT AC AAC AC C T T C T AC AT C GAG T T C AAG AG C G AG T G C G AG AAG T AC G G G AA CACCGGCACCTGGGAGGTGCACTTCGGGAATAATGTGATTGATTGCAATGACTCTATGTGCAGCACCAGC GACGACACCGTGTCCGCTACTCAGCTGGTTAAGCAGCTGCAGCACACCCCCAGCCCCTACTCCAGCACCG TGTCCGTGGGCACCGCCAAGACCTACGGCCAGACCAGCGCTGCTACCAGGCCCGGCCACTGCGGCCTCGC GGAGAAGCAGCACTGCGGCCCCGTCAACCCCCTGCTCGGCGCCGCTACCCCCACCGGCAACAACAAGAGA CGGAAGCTCTGCAGCGGCAACACTACCCCCATCATCCACCTGAAGGGCGACAGAAACAGCCTGAAGTGCC TCCGGTACAGACTGAGGAAGCACAGCGACCACTATAGAGATATCAGCTCCACCTGGCACTGGACCGGCGC C G G C AAT GAG AAG AC CGGCATCCTGACTGTGACCTACCACAGCGAGACC C AG AGAAC C AAGT T C C T G AAT ACTGTTGCCATTCCCGATAGCGTGCAGATCCTGGTGGGCTACATGACCATGTAATGA SEQ ID NO: 148> SYNTHETIC_HPV18-6_647aa MDWTWILFLVAAATRVHSFEDPTRSGYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEKDLFVVYRD SIPHAACHKCIDFYSRIRELRHYSDSVYGDTLEKLTNTGLYNLLIRCLRCQKPLLRHLNEKRRFHNIAGH YRGQCHSCCNRARQERLQRRRETQVRGRKRRSHGPKATLQDIVLHLEPQNEIPVDLLGHGQLSDSEEEND EIDGVNHQHLPARRAEPQRHTMLCMCCKCEARIELVVESSADDLRAFQQLFLNTLSFVCPWCASQQRGRK RRSQTPKETLSERLSCVQDKI IDHYENDSKDIDSQIQYWQLIRWENAIFFAAREHGIQTLNHQWPAYNI SKSKAHKAIELQMALQGLAQSRYKTEDWTLQDTCEELWNTEPTHCFKKGGQTVQVYFDGNKDNCMTYVAW DSVYYMTDAGTWDKTATCVSHRGLYYVKEGYNTFYIEFKSECEKYGNTGTWEVHFGNNVIDCNDSMCSTS DDTVSATQLVKQLQHTPSPYSSTVSVGTAKTYGQTSAATRPGHCGLAEKQHCGPVNPLLGAATPTGNNKR RKLCSGNTTPI IHLKGDRNSLKCLRYRLRKHSDHYRDISSTWHWTGAGNEKTGILTVTYHSETQRTKFLN TVAI P D S VQ I L VG YMTM SEQ ID NO: 149> HPV18-E691MF-367038161Docket No.: 16553-20011.40 FEDPTRSGYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEKDLFWYRDSIPHAACHKCIDFYSRIR ELRHYSDSVYGDTLEKLTNTGLYNLLIRCLRCQKPLLRHLNEKRRFHNIAGHYRGQCHSCCNRARQERLQ RRRETQV SEQ ID NO: 150> HPV18-E7 RGRKRRSHGPKATLQDIVLHLEPQNEIPVDLLGHGQLSDSEEENDEIDGVNHQHLPARRAEPQRHTMLCM CCKCEARIELVVESSADDLRAFQQLFLNTLSFVCPWCASQQ SEQ ID NO: 151> HPV18- El QTPKETLSERLSCVQDKIIDHYENDSKDIDSQIQYWQLIRWENAIFFAAREHGIQTLNHQWPAYNISKS KAHKAIELQMALQGLAQSRYKTEDWTLQDTCEELWNTEPTHCFKKGGQTVQVYFDGNKDNCMTYVAWDSV YYMTDAGTWDKTATCVSHRGLYYVKEGYNTFYIEFKSECEKYGNTGTWEVHFGNNVIDCNDSMCSTSDDT VSATQLVKQLQHTPSPYSSTVSVGTAKTYGQTSAATRPGHCGLAEKQHCGPVNPLLGAATPTGNNKRRKL CSGNTTPIIHLKGDRNSLKCLRYRLRKHSDHYRDISSTWHWTGAGNEKTGILTVTYHSETQRTKFLNTVA IPDSVQILVGYMTM92MF-367038161

Claims

Docket No.: 16553-20011.40CLAIMSWe claim:

1. A composition comprising a first mRNA and a second mRNA encapsulated in a nanoparticle, wherein the first mRNA comprises a coding region of a cyclic GMP-AMP synthase (cGAS), the second mRNA comprises a coding region of a human papillomavirus (HPV) antigen.

2. A composition comprising a first mRNA encapsulated in a first nanoparticle and a second mRNA encapsulated in a second nanoparticle, wherein the first mRNA comprises a coding region of a cyclic GMP-AMP synthase (cGAS), and the second mRNA comprises a coding region of a human papillomavirus (HPV) antigen.

3. A composition comprising an mRNA encapsulated in a nanoparticle, wherein the mRNA comprises a first coding region and a second coding region separated by an intervening sequence, wherein the first coding region is a coding region of a cyclic GMP-AMP synthase (cGAS) and the second coding region is a coding region of a human papillomavirus (HPV) antigen or the first coding region is a coding region of a human papillomavirus (HPV) antigen and the second coding region is a coding region of a cyclic GMP-AMP synthase (cGAS), wherein the intervening sequence encodes a 2A-like peptide sequence, encodes a protease cleavage site, or is an internal ribosome entry site (IRES).

4. The composition of any one of claims 1-3, wherein the nanoparticle is a lipid nanoparticle (LNP) comprising an ionizable lipid, a pegylated lipid, a structural lipid, a phospholipid, or a combination thereof, optionally wherein the LNP comprises an ionizable lipid.

5. The composition of claim 4, wherein the LNP comprises the ionizable lipid, the pegylated lipid, the structural lipid and the phospholipid.

6. The composition of any one of claims 1-5, wherein the ionizable lipid comprises:i) 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1 -octylnonyl ester (SM-102) or analogs or derivatives thereof; and / orii) 6-((2-hexyldecanoyl)oxy)-N-(6-((2-hexyldecanoyl)oxy)hexyl)-N-(4-hydroxybutyl)hexan-l-aminium (ALC-0315) or analogs or derivatives thereof; and / oriii) (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA) or analogs or derivatives thereof.93MF-367038161Docket No.: 16553-20011.40 7. The composition of any one of claims 1-6, wherein the pegylated lipid is selected from the group consisting of a PEG-modified phosphatidyiethanolamine, a PEG-modified phosphatide acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglyerol, and combinations thereof.

8. The composition of any one of claims 1-6, wherein the pegylated lipid comprises polyethylene glycol [PEG] 2000 dimyristoyl glycerol [DMG].

9. The composition of any one of claims 1-8, wherein the structural lipid is selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha- tocopherol, and combinations thereof.

10. The composition of claim 9, wherein the structural lipid comprises cholesterol.

11. The composition of any one of claims 1-10, wherein the phospholipid comprises:i) a hydrophilic head moiety selected from the group consisting of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and sphingomyelin; andii) one or more fatty acid tail moieties selected from the group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, arachidic acid, arachidonic acid, phytanoic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.

12. The composition of any one of claims 1-10, wherein the phospholipid is selected from the group consisting of1.2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC),1.2-dimyristoyl-sn-glycero-phosphocholine (DMPC),1.2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1.2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),1.2-distearoyl-sn-glycero-3-phosphocholine (DSPC),1.2-diundecanoyl-sn-glycero-phosphocholine (DUPC),1 -palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),1.2-di-O-octadecenyl-sn-glycero-3-phosphocholine,l-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine,1.2-dilinolenoyl-sn-glycero-3 -phosphocholine,1.2-diarachidonoyl-sn-glycero-3 -phosphocholine,94MF-367038161Docket No.: 16553-20011.40 1.2-didocosahexaenoyl-sn-glycero-3-phosphocholine,1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),1.2-diphytanoyl-sn-glycero-3-phosphoethanolamine,1.2-distearoyl-sn-glycero-3-phosphoethanolamine,1.2-dilinoleoyl-sn-glycero-3-phosphoethanolamine,1.2-dilinolenoyl-sn-glycero-3 -phosphoethanolamine,1.2-diarachidonoyl-sn-glycero-3 -phosphoethanolamine,1.2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine,1.2-dioleoyl-sn-glycero-3-phospho-rac-(l -glycerol) sodium salt (DOPG),sphingomyelin, andcombinations thereof, optionally wherein the phospholipid comprises l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

13. The composition of any one of claims 1-3, wherein the nanoparticle is a polymeric nanoparticle comprising a non-lipid polymer.

14. The composition of claim 13, wherein the non-lipid polymer comprises a poly(β-amino ester) (PBAE), a cyclodextrin (CD), a polyethyleneimine (PEI), a poly(lactic-co-glycolic acid) (PLGA), a polyamidoamine (PAMAM) dendrimer or an ionizable amphiphilic Janus dendrimer.

15. The composition of any one of claims 1-3, wherein the nanoparticle is a protein nanoparticle, a viral particle, a virus-like particle (VLP) or a cationic nanoemulsion.

16. A composition comprising a first mRNA and a second mRNA complexed with one or more lipids (RNA-Lipoplex), wherein the first mRNA comprises a coding region of a constitutively active cyclic GMP-AMP synthase (cGAS), and the second mRNA comprises a coding region of a human papillomavirus (HPV) antigen, and the one or more lipids comprise a first lipid and a second lipid.

17. A composition comprising an mRNA complexed with one or more lipids (RNA-Lipoplex), wherein the mRNA comprises a first coding region and a second coding region separated by an intervening sequence, the first coding region is a coding region of a constitutively active cyclic GMP-AMP synthase (cGAS) and the second coding region is a coding region of a human papillomavirus (HPV) antigen or the first coding region is a coding region of a human papillomavirus (HPV) antigen and the second coding region is a coding region of a constitutively active cyclic GMP-AMP synthase (cGAS), and the one or more lipids comprise a first lipid and a second lipid, wherein the intervening sequence95MF-367038161Docket No.: 16553-20011.40 encodes a 2A-like peptide sequence, encodes a protease cleavage site, or is an internal ribosome entry site (IRES).

18. The composition of claim 16 or claim 17, wherein the first lipid is a cationic lipid, and the second lipid is a neutral or anionic lipid.

19. The composition of claim 18, wherein the cationic lipid comprises one or both of: i) 1,2-di-O-octadecenyl-3 -trimethylammonium propane (DOTMA) or analogs or derivatives thereof; andii) l,2-dioleoyl-3-trimethylammonium propane (DOTAP) or analogs or derivatives thereof.

20. The composition of claim 18 or claim 19, wherein the neutral or anionic lipid comprises: i) l,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE) or analogs or derivatives thereof; and / orii) cholesterol or analogs or derivatives thereof; and / oriii) l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or analogs or derivatives thereof.

21. The composition of any one of claims 1-20, wherein the composition does not comprise a TLR7 / 8 agonist.

22. The composition of any one of claims 1-20, further comprising a TLR7 / 8 agonist, wherein the TLR7 / 8 agonist is a small molecule with a molecule weight of 900 daltons or less.

23. The composition of claim 22, wherein the TLR7 / 8 agonist comprises an imidazoquinoline compound.

24. The composition of claim 23, wherein the TLR7 / 8 agonist comprises resiquimod (R848).

25. The composition of any one of claims 1-24, wherein the mRNA or the first mRNA and the second mRNA comprises a 5’ untranslated region (5’UTR) and a 3’ untranslated region (3’UTR).

26. The composition of any one of claims 1-25, wherein the mRNA comprises one or both of a 5' cap structure and a polyA tail.

27. The composition of any one of claims 1-26, wherein the mRNA comprises a modified nucleoside, optionally wherein the modified nucleoside comprises one or more of N1 -methyl -96MF-367038161Docket No.: 16553-20011.40 pseudouridine, 5-methyluridine, 2-thiouridine, pseudouridine and 5 -methylcytidine, optionally wherein the modified nucleoside comprises N1 -methyl -pseudouridine.

28. The composition of any one of claims 1-27, wherein the composition further comprises at least one excipient.

29. The composition of any one of claims 1-28, wherein the constitutively -active cGAS has a greater propensity to self DNA reactivity than its wild-type counterpart.

30. The composition of any one of claims 1-29, wherein the cGAS is a truncated cGAS devoid of an amino-terminal phosphoinositide-binding domain (cGAS AN).

31. The composition of claim 30, wherein the cGASΔN comprises the amino acid sequence of SEQ ID NO:1 or the amino acid sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:1, optionally wherein the cGASΔN has one, two, three or four conservative amino acid substitutions.

32. The composition of claim 31, wherein the cGASΔN comprises the consensus amino acid sequence of SEQ ID NO:9.

33. The composition of claim 31, wherein the cGASΔN is encoded by the nucleotide sequence of SEQ ID NO:41.

34. The composition of any one of claims 30-33, wherein the coding region of the cGASΔN is in operable combination with a start codon, optionally wherein the start codon is ATG, CUG, GUG or ACG, optionally wherein the start codon is ATG.

35. The composition of any one of claims 1-34, wherein the HPV antigen comprises: a) an E6 antigen, an E7 antigen and an E2 antigen of an HPV16 type, optionally wherein:(i) the E6 antigen comprises the amino acid sequence of SEQ ID NO: 18 or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:18; and(ii) the E7 antigen comprises the amino acid sequence of SEQ ID NO:21, or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:21; and(iii) the E2 antigen comprises the amino acid sequence of SEQ ID NO:25 or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25; orb) an E6 antigen, an E7 antigen and an E2 antigen of an HPV18 type, optionally wherein:(i) the E6 antigen comprises the amino acid sequence of SEQ ID NO: 149 or the amino acid sequence97MF-367038161Docket No.: 16553-20011.40 at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:43 or SEQ ID NO:149; and(ii) the E7 antigen comprises the amino acid sequence of SEQ ID NO:150, or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:44 or SEQ ID NO:150; and (iii) the E2 antigen comprises the amino acid sequence of SEQ ID NO: 151 or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:42 or SEQ ID NO:151; or c) both a) and b).

36. The composition of any one of claims 1-34, wherein the HPV antigen comprises one or both of an E6 antigen and an E7 antigen.

37. The composition of claim 36, wherein the HPV antigen comprises an E6 antigen and an E7 antigen, and wherein the E6 antigen and the E7 antigen are expressed as a fusion protein.

38. The composition of claim 36, wherein the HPV antigen comprises an E6 antigen and an E7 antigen, and wherein coding regions of the E6 antigen and the E7 antigen are separated by an intervening sequence, wherein the intervening sequence encodes a 2A-like peptide sequence, encodes a protease cleavage site, or is an internal ribosome entry site (IRES), optionally wherein the E6 antigen and the E7 antigen are expressed as separate antigens, optionally wherein the intervening sequence encodes a protease cleavage site, optionally wherein the protease cleavage site is a furin cleavable linker, optionally wherein the furin cleavable linker comprises the amino acid sequence of SEQ ID NO:28.

39. The composition of any one of claims 36-38, wherein the E6 antigen comprises:(i) the amino acid sequence of SEQ ID NO:18 or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:18;(ii) the amino acid sequence of SEQ ID NO:19, or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:19;(iii) the consensus amino acid sequence of SEQ ID NO: 20;(iv) the amino acid sequence of SEQ ID NO:43, or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:43; or(v) the amino acid sequence of SEQ ID NO:149, or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 149.

40. The composition of any one of claims 36-39, wherein the E7 antigen comprises:(i) the amino acid sequence of SEQ ID NO:21, or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:21;(ii) the amino acid sequence of SEQ ID NO:22, or the amino acid sequence at least 90%, 95%, 96%,98MF-367038161Docket No.: 16553-20011.40 97%, 98% or 99% identical to SEQ ID NO:22;(iii) the consensus amino acid sequence of SEQ ID NO: 23;(iv) the amino acid sequence of SEQ ID NO:44, or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:44; or(v) the amino acid sequence of SEQ ID NO: 150, or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 150.

41. The composition of any one of claims 1-40, wherein the HPV antigen further comprises one or both of an El antigen and an E2 antigen.

42. The composition of claim 41, wherein the HPV antigen comprises an El antigen and an E2 antigen, and wherein the El antigen and the E2 antigen are expressed as a fusion protein.

43. The composition of claim 41, wherein the HPV antigen comprises an El antigen and an E2 antigen, and wherein the coding regions of the El antigen and the E2 antigen are separated by an intervening sequence, wherein the intervening sequence encodes a 2A-like peptide sequence, encodes a protease cleavage site, or is an internal ribosome entry site (IRES), optionally wherein the El antigen and the E2 antigen are expressed as separate antigens, optionally wherein the intervening sequence encodes a protease cleavage site, optionally wherein the protease cleavage site is a furin cleavable linker, optionally wherein the furin cleavable linker comprises the amino acid sequence of SEQ ID NO:28.

44. The composition of any one of claims 41-43, wherein the El antigen comprises:(i) the amino acid sequence of SEQ ID NO:24 or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24; or(ii) the amino acid sequence of SEQ ID NO:41 or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:41.

45. The composition of any one of claims 41-44, wherein the E2 antigen comprises:(i) the amino acid sequence of SEQ ID NO:25 or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25; or(ii) the amino acid sequence of SEQ ID NO:42 or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:42; or(iii) the amino acid sequence of SEQ ID NO:151 or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:151.99MF-367038161Docket No.: 16553-20011.40 46. The composition of any one of claims 36-45, wherein the amino acid sequence of the E6 antigen or the E7 antigen is preceded by a methionine or the amino acid sequence of a signal peptide.

47. The composition of claim 46, wherein the signal peptide is an IgE leader comprising the amino acid sequence of SEQ ID NO:26.

48. The composition of any one of claims 1-47, wherein the HPV antigen and if present the IgE leader comprises:(i) the amino acid sequence of SEQ ID NO:35, or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:35; or(ii) the amino acid sequence of one of the group consisting of SEQ ID NOs:30-39, or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to one of the group consisting of SEQ ID NOs:30-39; and / or(iii) the amino acid sequence of SEQ ID NO:148, or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 148.

49. A pharmaceutical formulation comprising the composition of any one of claims 1-48, and a pharmaceutically acceptable excipient.

50. A method for production of hyperactivated dendritic cells, the method comprising contacting the dendritic cells with an effective amount of the composition of any one of claims 1-48, or the formulation of claim 49 to produce hyperactivated dendritic cells, wherein the hyperactivated dendritic cells contain apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC) speck(s).

51. The method of claim 50, wherein the dendritic cells are:(i) contacted in vivo with the composition; or(ii) contacted ex vivo with the composition.

52. The method of claim 50 or claim 51, wherein the hyperactivated dendritic cells:(i) secrete higher levels of one or more of IFNβ, RANTES, IP-10 and IFNα than unstimulated dendritic cells or dendritic cells contacted with empty LNPs; and / or(ii) express higher levels of at least one cell surface marker selected from the group consisting of CD40, CD86, CD69, MHC class II, MHC class I, CCR7, and combinations thereof than unstimulated dendritic cells or dendritic cells contacted with empty LNPs.100MF-367038161Docket No.: 16553-20011.40 53. A pharmaceutical formulation comprising at least 103, 104, 105or 106of the hyperactivated dendritic cells produced by the method of any one of claims 50-52, and a pharmaceutically acceptable excipient.

54. A method of stimulating an immune response against an HPV antigen, comprising administering an effective amount of the pharmaceutical formulation of claim 49 to an individual in need thereof to stimulate the immune response against the HPV antigen.

55. The method of claim 54, wherein the individual is a human subject infected with HPV.

56. The method of claim 54 or claim 55, wherein the human subject has precancerous genital dysplasia.

57. The method of claim 54 or claim 55, wherein the human subject has a cervical cancer, oropharyngeal cancer, vulvar cancer or anal cancer.

58. A method of treating precancerous genital dysplasia, comprising administering an effective amount of the pharmaceutical formulation of claim 49 to a human subject in need thereof to treat the genital dysplasia, wherein the human subject is infected with HPV.

59. The method of claim 58, wherein the precancerous genital dysplasia is one or both of cervical dysplasia and anal dysplasia.

60. A method of treating cancer, comprising administering an effective amount of the pharmaceutical formulation of claim 49 to a human subject in need thereof to treat the cancer, wherein the human subject is infected with HPV.

61. The method of claim 60, wherein the cancer is selected from the group consisting of cervical cancer, oropharyngeal cancer, vulvar cancer or anal cancer.

62. A method of treating or preventing an HPV -associated disease, comprising administering an effective amount of the pharmaceutical formulation of claim 49 to a human subject in need thereof to treat or prevent the HPV-associated disease.

63. The method of any one of claims 54-62, wherein the HPV is a high-risk type, optionally wherein the HPV is HPV-16 and / or HPV-18, optionally wherein the HPV is HPV-16.101MF-367038161Docket No.: 16553-20011.40 64. A method of preparing the composition of any one of claims 1-16, comprising encapsulating the mRNA or the first mRNA and the second mRNA in the particle.

65. A method of preparing the composition of any one of claims 17-21, comprising forming a complex between the mRNA or the first mRNA and the second mRNA and the one or more lipids.

66. An isolated mRNA encoding a human papillomavirus (HPV) antigen, wherein the HPV antigen comprises an E6 antigen and an E7 antigen, and one or both of an El antigen and an E2 antigen.

67. The mRNA of claim 66, wherein the mRNA encodes the E6 antigen and the E7 antigen as a fusion protein.

68. The mRNA of claim 66, wherein the coding regions of the E6 antigen and the E7 antigen are separated by an intervening sequence, wherein the intervening sequence encodes a 2A-like peptide sequence, encodes a protease cleavage site, or is an internal ribosome entry site (IRES), optionally wherein the E6 antigen and the E7 antigen are expressed as separate antigens, optionally wherein the intervening sequence encodes a protease cleavage site, optionally wherein the protease cleavage site is a furin cleavable linker, optionally wherein the furin cleavable linker comprises the amino acid sequence of SEQ ID NO:28.

69. The mRNA of any one of claims 66-68, wherein the E6 antigen comprises:(i) the amino acid sequence of SEQ ID NO:18 or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:18;(ii) the amino acid sequence of SEQ ID NO:19, or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:19;(iii) the consensus amino acid sequence of SEQ ID NO: 20;(iv) the amino acid sequence of SEQ ID NO:43, or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:43; or(v) the amino acid sequence of SEQ ID NO:149, or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 149.

70. The mRNA of any one of claims 66-69, wherein the E7 antigen comprises:(i) the amino acid sequence of SEQ ID NO:21, or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:21;(ii) the amino acid sequence of SEQ ID NO:22, or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22;102MF-367038161Docket No.: 16553-20011.40 (iii) the consensus amino acid sequence of SEQ ID NO: 23;(iv) the amino acid sequence of SEQ ID NO:44, or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:44; or(v) the amino acid sequence of SEQ ID NO:150, or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 150.

71. The mRNA of any one of claims 66-70, wherein the El antigen comprises:(i) the amino acid sequence of SEQ ID NO:24 or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24; or(ii) the amino acid sequence of SEQ ID NO:41 or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:41.

72. The mRNA of any one of claims 66-71, wherein the E2 antigen comprises:(i) the amino acid sequence of SEQ ID NO:25 or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25;(ii) the amino acid sequence of SEQ ID NO:42 or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:42; or(iii) the amino acid sequence of SEQ ID NO:151 or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:151.

73. The mRNA of any one of claims 66-72, wherein the HPV antigen comprises the E6 antigen, the E7 antigen, and the El antigen.

74. The mRNA of any one of claims 66-72, wherein the HPV antigen comprises the E6 antigen, the E7 antigen, and the E2 antigen, optionally wherein:a) the E6 antigen, the E7 antigen and the E2 antigen are of an HPV16 type, optionally wherein:(i) the E6 antigen comprises the amino acid sequence of SEQ ID NO: 18 or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:18; and(ii) the E7 antigen comprises the amino acid sequence of SEQ ID NO:21, or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:21; and(iii) the E2 antigen comprises the amino acid sequence of SEQ ID NO:25 or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25; orb) the E6 antigen, the E7 antigen and the E2 antigen are of an HPV 18 type, optionally wherein:(i) the E6 antigen comprises the amino acid sequence of SEQ ID NO: 149 or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:43 or SEQ ID NO:149; and103MF-367038161Docket No.: 16553-20011.40 (ii) the E7 antigen comprises the amino acid sequence of SEQ ID NO:150, or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:44 or SEQ ID NO:150; and (iii) the E2 antigen comprises the amino acid sequence of SEQ ID NO: 151 or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:42 or SEQ ID NO:151; or c) both a) and b).

75. The mRNA of any one of claims 66-74, wherein the HPV antigen comprises the E6 antigen, the E7 antigen, the El antigen and the E2 antigen.

76. The mRNA of claim 75, wherein the El antigen and the E2 antigen are expressed as a fusion protein.

77. The mRNA of claim 75, wherein the coding regions of the El antigen and the E2 antigen are separated by an intervening sequence, wherein the intervening sequence encodes a 2A-like peptide sequence, encodes a protease cleavage site, or is an internal ribosome entry site (IRES), optionally wherein the El antigen and the E2 antigen are expressed as separate antigens, optionally wherein the intervening sequence encodes a protease cleavage site, optionally wherein the protease cleavage site is a furin cleavable linker, optionally wherein the furin cleavable linker comprises the amino acid sequence of SEQ ID NO:28.

78. The mRNA of any one of claims 66-77, wherein the amino acid sequence of the E6 antigen, the E7 antigen, the El antigen or the E2 antigen is preceded by a methionine or the amino acid sequence of a signal peptide.

79. The mRNA of claim 78, wherein the signal peptide is an IgE leader comprising the amino acid sequence of SEQ ID NO:26.

80. The mRNA of any one of claims 66-79, wherein the HPV antigen, and if present the IgE leader, comprises:(i) the amino acid sequence of SEQ ID NO:35, or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:35; or(ii) the amino acid sequence of one of the group consisting of SEQ ID NOs:30-39, or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to one of the group consisting of SEQ ID NOs:30-39; and / or(iii) the amino acid sequence of SEQ ID NO:148, or the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 148.104MF-367038161Docket No.: 16553-20011.40 81. The mRNA of any one of claims 66-80, wherein the mRNA comprises a 5’ untranslated region (5’UTR) and a 3’ untranslated region (3’UTR).

82. The mRNA of any one of claims 66-81, wherein the mRNA further comprises one or both of a 5' cap structure and a poly A tail.

83. The mRNA of any one of claims 66-82, wherein the mRNA comprises a modified nucleoside, optionally wherein the modified nucleoside comprises one or more of N1 -methylpseudouridine, 5-methyluridine, 2-thiouridine, pseudouridine and 5 -methylcytidine, optionally wherein the modified nucleoside comprises N1 -methyl -pseudouridine.

84. The mRNA of any one of claims 66-83, wherein the HPV antigen does not comprise an LI antigen and / or an L2 antigen, or wherein the mRNA does not comprise a complete HPV genome.

85. The method of any one of claims 54-57, wherein stimulating the immune response against the HPV antigen comprises increasing HPV E2-specific secretion of IFNγ+ by immune cells of the subject.

86. The method of claim 85, wherein stimulating the immune response against the HPV antigen comprises increasing frequency of HPV E6-specific IFNγ+, CD4+ T-cells of the subject, and / or increasing frequency of HPV E7-specific IFNγ+, CD8+ T-cells of the subject.

87. The method of claim 85 or claim 86, wherein stimulating the immune response against the HPV antigen comprises increasing frequency of HPV E7-specific IFNγ+, TNFα+ CD8+ T-cells and / or IFNγ+, TNFa+, IL-2+ CD8+ T-cells of the subject.

88. The method of any one of claims 85-87, wherein stimulating the immune response against the HPV antigen comprises inducing presentation of MHC class I epitope(s) of the HPV antigen on antigen presenting cells of the subject.

89. The method of claim 88, wherein the subject expresses at least one HLA molecule of the group consisting of A*02:01, A*24:02, B*15:ll, B*35:01, and C*03:03 alleles, and the MHC class I epitope(s) comprises one or more of the amino acid sequences of SEQ ID NOs:47-53.

90. The method of claim 89, wherein the subject expresses at least one HLA molecule of the group consisting of A*02:01, A*24:02, B*15:ll, B*35:01, and C*03:03 alleles, and the MHC class I105MF-367038161Docket No.: 16553-20011.40 epitope(s) comprise one or more of SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:51 and SEQ ID NO:53.

91. The method of claim 88, wherein the subject expresses at least one HLA molecule of the group consisting of A*03:01, A*30:02, B*15:01, B*27:05, C*03:03 and C*05:01 alleles, and the MHC class I epitope(s) comprise one or more of the amino acid sequences of SEQ ID NOs:54-93.

92. The method of claim 88, wherein the subject expresses at least one HLA molecule of the group consisting of A*02:01, A*24:02, B*15:01, B*35:12, C*03:03 and C*04:01 alleles, and the MHC class I epitope(s) comprise one or more of the amino acid sequences of SEQ ID NOs:94-136.

93. The method of any one of claims 85-92, wherein stimulating the immune response against the HPV antigen comprises increasing one or more of the group consisting of HPV antigen expression, HPV antigen presentation, CD80 / CD86 / CD40 co-stimulation, CCR7 expression, secretion of Type I interferons, and secretion of T-cell chemokines.

94. The method of any one of claims 85-93, wherein the increase is relative to preadministration or control administration values, optionally wherein the control administration is administration of LNPs devoid of mRNA or administration of vehicle in which LNPs of the pharmaceutical formulation are suspended.

95. The method of any one of claims 85-93, wherein the increase is relative to administration of the mRNA comprising a coding region of a human papillomavirus (HPV) antigen in the absence of the mRNA comprising a coding region of a cyclic GMP-AMP synthase (cGAS).

96. The composition of any one of claims 1-48, wherein the composition does not comprise a lysophosphatidylcholine (LPC), optionally wherein the LPC has a single C13-C24 acyl chain.

97. The composition of claim 48, wherein the HPV antigen and if present the IgE leader is encoded by:(i) the nucleic acid sequence of SEQ ID NO: 142; or(ii) the nucleic acid sequence at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:142; or (iii) the nucleic acid sequence of one of the group consisting of SEQ ID NOs:137-147; or(iv) the nucleic acid sequence at least 95%, 96%, 97%, 98% or 99% identical to one of the group consisting of SEQ ID NOs:137-147.106MF-367038161Docket No.: 16553-20011.40 98. The mRNA of claim 80, wherein the HPV antigen and if present the IgE leader is encoded by:(i) the nucleic acid sequence of SEQ ID NO: 142; or(ii) the nucleic acid sequence at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:142; or (iii) the nucleic acid sequence of one of the group consisting of SEQ ID NOs:137-147; or(iv) the nucleic acid sequence at least 95%, 96%, 97%, 98% or 99% identical to one of the group consisting of SEQ ID NOs:137-147.

99. An isolated DNA comprising:(i) the nucleic acid sequence of SEQ ID NO: 142; or(ii) the nucleic acid sequence at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:142; or (iii) the nucleic acid sequence of one of the group consisting of SEQ ID NOs:137-147; or(iv) the nucleic acid sequence at least 95%, 96%, 97%, 98% or 99% identical to one of the group consisting of SEQ ID NOs:137-147.

100. An expression vector comprising the isolated DNA of claim 99 in operable combination with a promoter.

101. The expression vector of claim 100, wherein the vector is a plasmid.

102. Use of the composition of any one of claims 1-48, the formulation of claim 49 or the mRNA of any one of claims 66-84 in the manufacture of a medicament for inducing an immune response against the HPV antigen in an individual in need thereof.

103. The use of claim 102, wherein the individual is a human subject infected with HPV.

104. The use of claim 102 or claim 103, wherein the human subject has precancerous genital dysplasia.

105. The use of claim 102 or claim 103, wherein the human subject has cervical cancer, oropharyngeal cancer, vulvar cancer or anal cancer.

106. Use of the composition of any one of claims 1-48, the formulation of claim 49 or the mRNA of any one of claims 66-84 in the manufacture of a medicament for treating precancerous genital dysplasia in human subject infected with HPV.107MF-367038161Docket No.: 16553-20011.40 107. The use of claim 106, wherein the precancerous genital dysplasia is one or both of cervical dysplasia and anal dysplasia.

108. Use of the composition of any one of claims 1-48, the formulation of claim 49 or the mRNA of any one of claims 66-84 in the manufacture of a medicament for treating cancer in a human subject in need thereof, wherein the human subject is infected with HPV.

109. The use of claim 108, wherein the cancer is selected from the group consisting of cervical cancer, oropharyngeal cancer, vulvar cancer or anal cancer.

110. Use of the composition of any one of claims 1-48, the formulation of claim 49 or the mRNA of any one of claims 66-84 in the manufacture of a medicament for treating or preventing an HPV-associated disease in a human subject in need thereof, wherein the human subject is infected with HPV.

111. The use of any one of claims 102-110, wherein the HPV is a high-risk type, optionally wherein the HPV is HPV-16 and / or HPV-18, optionally wherein the HPV is HPV-16.

112. The composition of any one of claims 1-48 or the formulation of claim 49 or the method of use comprising the composition or the formulation, wherein the first mRNA comprising the coding region of a cyclic GMP-AMP synthase (cGAS) and the second mRNA comprising a coding region of a human papillomavirus (HPV) antigen are present ati) a weight / weight ratio of from about 0.4: 1 to about 25: 1, optionally from about 1: 1 to about 10:1, further optionally from about 1:1 to about 2:1; orii) a molar ratio of from 1: 1 to 2: 1 or about 1: 1.108MF-367038161

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