T cell-directed anticancer vaccine against oncovirus

Abstract

An immune-based approach for treating and preventing skin cancer by boosting T cell immunity against commensal HPV present in the skin. Accordingly, provided herein are compositions comprising: (i) a plurality of antigenic peptides, each comprising a sequence of 9 to 30 amino acids derived from a protein from a commensal human papillomavirus; (ii) a plurality of live or live-attenuated commensal human papillomaviruses; (iii) a plurality of antigenic proteins from a commensal human papillomavirus, preferably in a virus-like particle; and / or (iv) a plurality of nucleic acids encoding (a) a plurality of antigenic peptides, each comprising a sequence of 9 to 30 amino acids derived from a protein from a commensal human papillomavirus, or (b) a plurality of antigenic proteins from a commensal human papillomavirus; and optionally a T cell adjuvant that increases T cell responses to the antigenic peptides.

Description

Technical Field

[0001] Priority Claim This application claims the benefit of U.S. Patent Application No. 62 / 772,443, filed Nov. 28, 2018; U.S. Patent Application No. 62 / 831,691, filed Apr. 9, 2019; and U.S. Patent Application No. 62 / 909,698, filed Oct. 2, 2019. The entire contents of the foregoing are incorporated herein by reference.

[0002] Government Support of Research or Development This invention was made with government support under grant number OD021353 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] An immune-based approach for treating and preventing skin cancer by boosting T cell immunity against commensal HPV present in the skin.

Background Art

[0004] Non-melanoma skin cancers, including squamous cell carcinoma (SCC) and basal cell carcinoma (BCC), are the most common types of cancer 8 . Ultraviolet (UV) light is a preventable cause of skin cancer, yet the incidence of skin cancer in the United States has doubled from 1992 to 2012 9 . Skin cancer causes significant morbidities, including ulcers and cosmetic disfigurement. Importantly, the mortality rate of SCC is similar to that of melanoma in immunosuppressed patients, including solid organ transplant recipients (OTRs). 10~12 . In addition to those side effects, current skin cancer treatments represent an increasing public health burden, exceeding $1 billion annually in the United States 13 .

Summary of the Invention

Problems to be Solved by the Invention

[0005] Immunosuppression increases the risk of virus-induced cancers 1Among these, non-melanoma skin cancers are associated with beta-human papillomavirus (β-HPV), and in particular, in immunosuppressed patients, the risk of skin cancer increases >100-fold. 2~5 However, previous studies have not established a causal role for low-risk HPVs in skin cancer. Here, we provide an alternative explanation for this association by demonstrating that anti-papillomavirus immunity suppresses skin cancer in immunocompetent hosts and that loss of this immunity, rather than the carcinogenic effects of commensal HPVs, is the reason for the markedly increased risk of skin cancer in immunosuppressed patients. In clinical studies, we found that the anatomical distribution of skin cancers in immunosuppressed patients was significantly different from the warts caused by their HPVs but matched the distribution of skin cancers in immunocompetent patients. This pattern of skin cancer distribution suggested that ultraviolet (UV) light was the major cause of cancer in both populations. To experimentally investigate the effect of papillomavirus on carcinogen-induced skin cancer, we colonized immunocompetent wild-type (Wt) C57BL / 6, FVB, and SKH-1 mice with murine papillomavirus type 1 (MmuPV1). 6、7 Colonizing mice with innate or acquired immunity from immune mice against MmuPV1 acquired significant protection against chemical- and UV-induced skin carcinogenesis compared to their non-infected counterparts. RNA and DNA in situ hybridization for 25 commensal β-HPVs revealed a marked loss of viral activity and load in human skin cancer cells compared to adjacent normal skin. Finally, the β-HPV E7 peptide activated CD8 + T cells isolated from normal human skin. Our findings reveal the beneficial effects of commensal viruses and establish the basis for an immune-based approach to treating and preventing skin cancer by boosting T cell immunity against commensal HPVs present in all of our skin.

Means for Solving the Problems

[0006] Accordingly, provided herein is a composition comprising: (i) a plurality of antigenic peptides each comprising a sequence of 9 to 30 amino acids derived from a protein from a commensal human papillomavirus; (ii) a plurality of live or live-attenuated commensal human papillomaviruses; (iii) a plurality of antigenic proteins from a commensal human papillomavirus, preferably in virus-like particles; and / or (iv) (a) a plurality of antigenic peptides each comprising a sequence of 9 to 30 amino acids derived from a protein from a commensal human papillomavirus, or (b) a plurality of nucleic acids encoding a plurality of antigenic proteins from a commensal human papillomavirus; and optionally a T cell adjuvant that increases a T cell response to the antigenic peptides. In some embodiments, the commensal human papillomavirus is a low-risk α-HPV, β-HPV, γ-HPV, and / or μ-HPV strain, e.g., the commensal human papillomavirus is a low-risk α-HPV, β-HPV, γ-HPV, and / or μ-HPV strain listed in Table A.

[0007] In some embodiments, the plurality of antigenic peptides comprises peptides derived from one or more of the E1, E2, E4, E5, E6, or E7 proteins.

[0008] In some embodiments, the plurality of antigenic peptides comprises peptides derived from proteins from a plurality of commensal human papillomaviruses.

[0009] In some embodiments, the composition comprises at least 200 peptides each having a unique sequence, e.g., the composition comprises a plurality of peptides for each unique sequence.

[0010] In some embodiments, the composition comprises one or more virus vectors engineered to express a plurality of proteins or antigenic peptides, e.g., a virus vector selected from the group consisting of recombinant retroviruses, adenoviruses, adeno-associated viruses, alphaviruses, and lentiviruses.

[0011] In some embodiments, the T cell adjuvant comprises one or more of nanoparticles that enhance T cell responses, poly-ICLC (carboxymethylcellulose, polyinosinic acid-polycytidylic acid, and poly-L-lysine double-stranded RNA), imiquimod, CpG oligodeoxynucleotides and formulations (IC31, QB10), AS04 (aluminum salt formulated with 3-O-desacyl-4'-monophosphoryl lipid A (MPL)), AS01 (MPL and saponin QS-21), MPLA, STING agonists, other TLR agonists, Candida albicans skin test antigen (Candin), GM-CSF, Fms-like tyrosine kinase-3 ligand (Flt3L), and / or IFA (incomplete Freund's adjuvant). In some embodiments, the T cell adjuvant comprises topical resiquimod, and / or imiquimod, and / or topical 5-fluorouracil, and / or topical calcipotriene (calcipotriol), for example in combination with 5-fluorouracil.

[0012] Also provided herein is a method of treating or reducing the risk of developing skin cancer in a subject, the method comprising administering to the subject an effective amount of the composition described herein. In addition, provided herein is a composition described herein for use in a method of treating or reducing the risk of developing skin cancer in a subject.

[0013] In some embodiments, the subject has an increased risk of developing skin cancer or is immunocompromised, for example as a result of aging, or acquired immunodeficiency, primary immunodeficiency, or organ transplantation.

[0014] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials for use in the present invention are described herein, other suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database registrations, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0015] Other features and advantages of the present invention will be apparent from the following detailed description, the drawings, and the claims.

Brief Description of the Drawings

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[0017] In particular, human papillomavirus (HPV) belonging to the low-risk beta (β) genus is found in more than 80% of SCCs among OTRs. 2~5 Therefore, a viral cause of possible skin cancer has been proposed. 14 β-HPV is the cause of benign persistent warts and, together with other skin-tropic low-risk HPV genera, is ubiquitously present in the immunoresponsive adult skin as a normal flora. 3、4、15、16 In contrast to high-risk α-HPV, there is no major β-HPV subtype identified in skin cancer. 14 The β-HPV genome is rarely integrated into the DNA of cancer cells. 5 In addition, transcriptome analysis has not identified papillomavirus gene expression in SCCs of immunoresponsive or immunosuppressed patients. 4 In skin cancers positive for β-HPV, the viral load in tumor cells is less than 1 copy per cell. 14 Furthermore, the spread of β-HPV DNA in actinic keratosis (SCC precursor lesions) is higher than in SCCs in immunoresponsive patients, and most HPV is present in the surface layer rather than in the basal proliferative region of skin cancer. 17、18 These findings contribute to the "hit and run" theory to explain the role of HPV in skin carcinogenesis, where the virus promotes the initiation of skin cancer but is then lost during tumor maintenance. 5、19 .

[0018] The findings reported herein clarify a novel role for commensal HPV in the development of non-melanoma skin cancer. Clinical studies have demonstrated that immunosuppression has no impact on the anatomical distribution of skin cancer, which is closely related to areas of greatest sun damage. The different localization of skin cancers away from intermittently sun-damaged and sun-protected skin, including most of the warts caused by HPV, suggests that commensal HPV does not initiate skin cancer in immunosuppressed patients, either alone or in combination with UV. The MmuPV1 colony formation model enables mechanistic studies of the relationship between papillomavirus and skin cancer in the context of an intact immune system. Using this model, we show that MmuPV1 colony-forming immune-responsive mice are protected from chemically and UV-induced skin cancer compared to their uninfected counterparts. Furthermore, we demonstrate that T cell immunity against MmuPV1 confers protection to MmuPV1 colony-forming mice from carcinogen-induced skin tumors. Finally, our innovative approach for the detection of viral RNA and DNA in tissue sections at subcellular resolution reveals negative selection against β-HPV viral activity and load in malignant keratinocytes that form cancer, bypassing antiviral T cell immunity in the skin.

[0019] These findings support a new explanation for the role of low-risk commensal HPV in the development of skin cancer. The extremely low prevalence of warts in immunocompetent adults 24 highlights the ability of the functional immune system to target and eliminate HPV-infected proliferating cells. Similarly, anti-HPV immunity halts the development of skin cancer through the recognition of commensal HPV in pre-cancerous cells that share the antigenic / immunogenic properties of warts and are effectively eliminated. This protective immunity is impaired in immunosuppressed patients, leading to a marked increase in skin cancer, warts, and HPV viral load in this population. Thus, the increased risk of skin cancer in immunosuppression represents a loss of the protective effect of antiviral immunity rather than an acquired susceptibility to HPV-induced skin cancer.

[0020] The different anatomical distributions of skin cancer and warts indicate UV exposure as a major determinant of skin cancer risk in immunosuppressed patients. This finding is supported by SCC transcriptome analysis, which demonstrates indistinguishable patterns of mature genes in SCCs of immunosuppressed and immunocompetent patients. 25 If commensal HPV contributes significantly mutagenic effects to the development of SCC, immunosuppressed patients would be expected to have fewer UV-induced mutations and / or a distinct pattern of mutated genes. This is in stark contrast to high-risk oncogenic viruses such as Merkel cell polyomavirus (MCPyV) in the skin, which causes Merkel cell carcinomas with fewer mutations compared to highly mutated UV-induced MCPyV-negative Merkel cell carcinomas. 26、27 These observations, together with the low HPV load and lack of its transcriptional activity in skin cancer 4、14 provide ample evidence that the contribution of commensal HPV to the development of skin cancer is negligible.

[0021] Experimental studies on MmuPV1 in wild-type mice demonstrate a protective role of commensal papillomavirus against skin cancer in immunocompetent hosts. The suppression of MmuPV1-induced warts has been shown to be T cell-mediated. 22、28 Here, we show that virus-specific T cells are sufficient to render MmuPV1 colony-forming mice protected from carcinogen-induced skin cancer. Interestingly, MmuPV1 colony-forming SKH-1 mice are also protected from UV-induced epidermal dysplasia, which may suggest a role for commensal HPV in maintaining the homeostasis of highly mutated sun-damaged human skin. 29 Previous studies in animal models of HPV skin infections have implicated HPV as a driver of skin cancer. The cell-autonomous growth effect of commensal HPV in keratinocytes as a causative agent of warts is clear. 30 However, the use of animals with isolated transgenic expression of the viral E6 / 7 proteins 31 immunodeficient mice 30、or immunosuppressive doses of UV 23、32、33 has hampered published studies that fully investigate the role of low-risk commensal papillomaviruses in skin carcinogenesis due to the lack of a physiological immune response. These studies emphasize the importance of the use of fully infectious virus and antiviral immune responses to reach translational conclusions about the role of the virome in human disease.

[0022] Evidence of CD8 + T cell responsiveness to β-HPV peptides in normal adult skin supports a role for an HPV-specific adaptive immune response to the normal cutaneous bacterial flora. Although not wishing to be bound by theory, together with the loss of β-HPV activity and viral load in skin cancer compared to normal skin, commensal β-HPVs are thought to function as immunogenic tags in abnormally dividing keratinocytes. They are involved in the cytotoxic T cell response to any proliferative lesions caused by cells containing the active virus. Thus, T cell-based vaccines against β-HPV could provide an innovative approach to boosting antiviral immunity in the skin and could help prevent warts and skin cancer, particularly in high-risk populations, especially at the pre-transcriptional OTR. Recent B cell-based HPV vaccines block infection of epithelial cells by high-risk HPVs of the alpha genus 34 。In contrast, the goal of β-HPV vaccines is to take advantage of the beneficial effects of β-HPV colonization by enhancing cell-mediated antiviral immunity in colonized skin to prevent the development of warts and skin cancer. Different from peptides from β-HPV, which is the normal cutaneous bacterial flora 3、4、15、16 、high-risk HPV16 peptides did not induce any response from T cells present in the skin. This emphasizes the importance of cutaneous commensal HPV in the programming of the antitumor immune response and the need to boost this immunity for the prevention and treatment of skin cancer. Based on this data, we now understand that commensal viruses can be agents that help protect our immune system against cancer development, even in immunocompetent populations.

[0023] In summary, we demonstrate a novel and beneficial effect of commensal HPVs in skin cancer protection by alerting cytotoxic immunity against any proliferative lesions in the skin. Considering the emergence of diverse viromes in the skin 35 it is important to identify the composition of the viral communities present in the skin of immune-responsive and immunosuppressed individuals, and to determine how these viruses contribute to human health and disease.

[0024] T cell-based vaccines against β-HPV Current vaccines against cancer-causing viruses such as high-risk alpha human papillomavirus (α-HPV) are designed to activate B cells that, in the first stage, result in the production of antiviral antibodies that prevent viral infection in the target tissue (e.g., the cervix). However, we recently discovered that commensal viruses that colonize the target tissue immediately after birth in all individuals (e.g., low-risk beta-type HPV colonization of the skin) can play a protective role against carcinogen-induced cancers (e.g., skin cancer) by inducing antiviral T cell immunity that can eliminate any proliferative lesions, including HPV viruses. In other words, in adult individuals immune to warts (lesions caused by HPV in the skin), HPV infection functions as a tag in skin cells and alerts T cells as soon as cells begin to abnormally proliferate, either in the context of warts caused by HPV or skin cancer caused by carcinogens. Therefore, boosting T cell immunity with a T cell-directed vaccine enhances the protective effects of commensal viruses without eliminating their dormant colonization of the target tissue. Described herein are compositions that can be used to induce a T cell-based immune response against β-HPV, thereby reducing the risk that a subject will develop skin cancer. Instead of preventing or eliminating infection, the vaccine induces T cell immunity against commensal viruses already infecting the tissue, for the purpose of boosting the detection and elimination of early cancerous clones by T cells using the viruses present in all cells. Current high-risk HPV vaccines for the prevention of cervical and head and neck cancers are meant to prevent infection in the first stage and have minimal efficacy in individuals already infected with the virus.

[0025] Antigenic peptide In some embodiments, the composition comprises a plurality of antigenic peptides derived from proteins from commensal human papillomaviruses, such as low-risk cutaneous tropic α-HPV, β-HPV, γ-HPV, and / or μ-HPV strains, such as those listed in Table A, for example, peptides derived from the E1, E2, E6, or E7 proteins (i.e., continuous amino acids therefrom, i.e., including fragments). In some embodiments, the composition does not contain peptides derived from cancer-associated HPV types, such as high-risk HPVs, such as HPV16 or 18. See, for example, Ma et al., J Virol. 2014 May; 88(9): 4786-4797; Doorbar et al., Rev Med Virol. 2015 Mar; 25(Suppl Suppl 1): 2-23; Doorbar et al., The biology and life‐cycle of human papillomaviruses. Vaccine 2012; 30(Suppl 5): F55-F70; de Villiers, Virology 2013; 445(1-2): 2-10; and U.S. Patent No. 8,652,482, which are incorporated herein by reference. Other commensal HPV types that are not "high-risk" α-HPVs can be used, and Table A is an exemplary but not exhaustive list.

[0026]

Table 1-1

[0027]

Table 1-2

[0028] The peptide can be derived from any antigenic protein in the virus, and in some embodiments, the peptide is derived from the E1, E2, E4, E5, E6 or E7 protein. Sequences for these proteins in many coisolates are provided. In some embodiments, the composition includes at least 50 or more, 100, 150, 200, 250, 300, 350, 400, 450, 500 or more different peptides (i.e., peptides having different sequences). In some embodiments, the composition includes at least 50 or more, 100, 150, 200, 250, 300, 350, 400, 450, 500 or more different peptides from each virus strain, and peptides from two or more virus strains are included.

[0029] In some embodiments, the peptides are of a length optimized for MHC I / MHC II presentation, e.g., 9 - 30 amino acids, e.g., 12 - 25, 12 - 18, 12 - 16, 13 - 16, 14 - 16, or 15 amino acids. The sequence of the peptides can be, for example, bioinformatically identified to predict antigenicity and / or generated using a moving window of overlapping peptides that span the entire protein, such as synthetic long overlapping peptides, e.g., 15 - amino acid peptides with a 10 - amino acid overlap (similar to the "gene walk" method used to identify optimal antisense oligonucleotides). In some embodiments, overlapping synthetic long peptides (SLPs) are used (Zom et al., Cancer Immunol Res. 2014 Aug;2(8):756 - 64). The composition can include multiple peptides from one or more (e.g., multiple) different viral strains. The peptides are preferably synthetic peptides, and methods for synthesizing peptides are known in the art and include liquid - phase techniques and solid - phase peptide synthesis (SPPS). See, for example, Petrou and Sarigiannis, Ch. 1 - Peptide synthesis: Methods, trends, and challenges, In: Editor(s): Sotirios Koutsopoulos, Peptide Applications in Biomedicine, Biotechnology and Bioengineering, Woodhead Publishing, 2018, pages 1 - 21; and Chandrudu et al., Molecules 2013, 18, 4373 - 4388.

[0030] Antigen protein In some embodiments, the composition can include virus-like particles containing a plurality of proteins, such as those listed in Table A, of the commensal human papillomavirus, such as E1, E2, E6, or E7 proteins from low-risk α-HPV, β-HPV, γ-HPV, and / or μ-HPV strains (see, e.g., Yang et al., Virus Res 231, 148-165 (2017); Hancock et al., Therapeutic HPV vaccines. Best Pract Res Clin Obstet Gynaecol 47, 59-72 (Feb. 2018); Joh et al., Exp Mol Pathol.;93(3):416-21 (2012)).

[0031] Nucleic acid-based vaccine In some embodiments, the composition can include a plurality of DNA plasmids and / or RNA replicons containing nucleotide sequences that express a protein or antigenic peptide (i.e., containing a continuous amino acid, i.e., a fragment, derived from) a protein derived from a commensal human papillomavirus, such as those listed in Table A, such as an E1, E2, E6, or E7 protein from low-risk α-HPV, β-HPV, γ-HPV, and / or μ-HPV strains (see, e.g., Yang et al., Virus Res 231, 148-165 (2017); Hancock et al., Therapeutic HPV vaccines. Best Pract Res Clin Obstet Gynaecol 47, 59-72 (2018)).

[0032] Vector-based live vaccine In some embodiments, the composition can comprise a plurality of viral vectors engineered to express a protein or antigenic peptide that is derived from (i.e., comprises a contiguous amino acid sequence, i.e., a fragment of) a protein from a commensal human papillomavirus, such as a low-risk α-HPV, β-HPV, γ-HPV, and / or μ-HPV strain, such as those listed in Table A (see, e.g., Yang et al., Virus Res 231, 148-165 (2017); Hancock et al., Therapeutic HPV vaccines. Best Pract Res Clin Obstet Gynaecol 47, 59-72 (2018)).

[0033] Viral vectors for use in the methods and compositions include recombinant retroviruses, adenoviruses, adeno-associated viruses, alphaviruses, and lentiviruses.

[0034] A preferred viral vector system useful for the delivery of nucleic acids in the present method is adeno-associated virus (AAV). AAV is a small non-enveloped virus having a 25 nm capsid. There are no diseases known or shown to be associated with the wild-type virus. AAV has a single-stranded DNA (ssDNA) genome. AAV has been shown to exhibit long-term episomal transgene expression, and AAV has demonstrated excellent transgene expression in a number of tissues, including the brain, particularly neurons. Vectors containing AAV of about 300 base pairs can be packaged and integrated. The space for exogenous DNA is limited to about 4.7 kb. DNA can be introduced into cells using AAV vectors such as those described in Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985). Various nucleic acids have been introduced into different cell types using AAV vectors (see, for example, Hermonat et al., Proc. Natl. Acad. Sci. USA 81:6466-6470 (1984); Tratschin et al., Mol. Cell. Biol. 4:2072-2081 (1985); Wondisford et al., Mol. Endocrinol. 2:32-39 (1988); Tratschin et al., J. Virol. 51:611-619 (1984); and Flotte et al., J. Biol. Chem. 268:3781-3790 (1993)). A large number of alternative AAV variants (over 100 have been cloned) exist, and AAV variants have been identified based on desired properties. For example, AAV9 has been shown to efficiently cross the blood-brain barrier. Also, the AAV capsid can be genetically engineered to increase the efficiency and selectivity of transduction, such as biotinylated AAV vectors, directed molecular evolution, self-complementary AAV genomes. In some embodiments, AAV1 is used.

[0035] Alternatively, retroviral vectors and adeno-associated virus vectors can be used as recombinant gene delivery systems for the transfer of exogenous genes in vivo, particularly into humans. These vectors provide efficient delivery of genes into cells, and the transferred nucleic acid is stably integrated into the host chromosomal DNA. The development of special cell lines that produce only replication-defective retroviruses (referred to as "packaging cells") has increased the utility of retroviruses for gene therapy, and the defective retroviruses have been characterized for use in gene transfer for gene therapy purposes (see Miller, Blood 76:271 (1990) for a review). Replication-defective retroviruses can be packaged into virions, which can be used to infect target cells by using helper viruses by standard techniques. Protocols for producing recombinant retroviruses and for infecting cells with such viruses in vitro or in vivo can be found in Ausubel, et al., eds., Current Protocols in Molecular Biology, Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals. Examples of suitable retroviruses include pLJ, pZIP, pWE, and pEM, which are known to those of skill in the art. Examples of suitable packaging virus strains for preparing both amphotropic and xenotropic retrovirus systems include ΨCrip, ΨCre, Ψ2, and ΨAm.Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, in vitro and / or in vivo (see, e.g., Eglitis, et al. (1985) Science 230:1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA 87:6141-6145; Huber et al. (1991) Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; van Beusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay et al. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J. Immunol. 150:4104-4115; U.S. Patent No. 4,868,116; U.S. Patent No. 4,980,286; International Publication No. WO 89 / 07136; International Publication No. WO 89 / 02468; International Publication No. WO 89 / 05345; and International Publication No. WO 92 / 07573).

[0036] Another viral gene delivery system useful in the present method utilizes an adenovirus-derived vector. The adenovirus genome can be manipulated so that it encodes and expresses the gene product of interest, but is inactivated in terms of its ability to replicate in the normal lytic viral life cycle. See, for example, Berkner et al., BioTechniques 6:616 (1988); Rosenfeld et al., Science 252:431-434 (1991); and Rosenfeld et al., Cell 68:143-155 (1992). Suitable adenovirus vectors derived from the adenovirus strain Ad5 type dl324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7, etc.) are known to those skilled in the art. In certain situations, recombinant adenoviruses can be advantageous in that they cannot infect non-dividing cells and can be used to infect a wide range of cell types, including epithelial cells (Rosenfeld et al., (1992), supra). Furthermore, the viral particles are relatively stable, suitable for purification and concentration, and can be modified as described above to affect the spectrum of infection. In addition, the introduced adenovirus DNA (and the foreign DNA contained therein) does not integrate into the host cell genome but remains episomal, thereby avoiding potential problems that can occur as a result of in situ insertional mutagenesis when the introduced DNA integrates into the host genome (e.g., retroviral DNA). Also, the carrying capacity of the adenovirus genome for foreign DNA is large compared to other gene delivery vectors (up to 8 kilobases) (Berkner et al., supra; Haj-Ahmand and Graham, J. Virol. 57:267 (1986)).

[0037] Alphaviruses can also be used. Alphaviruses are enveloped single-stranded RNA viruses with a broad host range and, when used in gene therapy protocols, can provide high levels of transient gene expression. Exemplary alphaviruses include Semliki Forest virus (SFV), Sindbis virus (SIN), and Venezuelan equine encephalitis (VEE) virus, all of which have been genetically engineered to provide highly efficient replication-deficient and responsive expression vectors. Alphaviruses exhibit significant neurotropism and are thus useful for CNS-related diseases. See, for example, Lundstrom, Viruses. 2009 Jun; 1(1): 13-25; Lundstrom, Viruses. 2014 Jun;6(6): 2392-2415;Lundstrom, Curr Gene Ther. 2001 May;1(1):19-29;Rayner et al., Rev Med Virol. 2002 Sep-Oct;12(5):279-96.

[0038] Symbiotic HPV Vaccine Strategy The symbiotic HPV vaccine strategy can be used to optimally boost antiviral T cell immunity in the skin to prevent cancer development and to treat early SCC with live virus, including actinic keratosis, in situ SCC, and early invasive SCC. Described herein is a platform for generating and propagating live low-risk HPV in culture to produce live and live-attenuated HPV vaccines for use in patients.

[0039] This prophylactic cancer vaccine has the advantage of colonizing the skin extensively with a "good" virus for the prevention and treatment of skin cancer. The only T cell-based vaccine strategy with proven efficacy is the live attenuated varicella-zoster virus vaccine: Zostavax for the prevention of herpes zoster (Sullivan et al., Current opinion in immunology. 2019;59:25-30. Epub 2019 / 04 / 11). In the case of Zostavax, the target virus varicella-zoster is the cause of chickenpox and herpes zoster, and thus the attenuated virus has to be developed for vaccine safety. T cell-based vaccine strategies against commensal HPV target low-risk papillomaviruses, which are normal human bacterial flora. Therefore, there is no evidence of these viruses causing any serious disease in adult individuals other than benign skin warts and only in highly immunosuppressed patients. Thus, live commensal papillomavirus vaccine is an ideal platform for skin cancer prevention as it can efficiently infect cells and viral antigen peptides can be effectively presented to T cells in the major histocompatibility complex (HMC) while avoiding neutralizing antibodies.

[0040] Live HPV vaccine: Using an in vitro culture system, cutaneous tropic HPVs can be propagated. Commensal HPVs can be obtained using known methods, for example, they can be isolated from warts of adult immunosuppressed patients. Next, the purified virus (Kreider et al., Virology. 1990;177(1):415-7) is transferred into an organotypic raft culture model using human primary keratinocytes (low passage human foreskin keratinocytes (HKF) rather than immortalized cell lines (Bienkowska-Haba et al., PLoS Pathog. 2018;14(3):e1006846. Epub 2018 / 03 / 02; Ozbun et al., Curr Protoc Microbiol. 2014;34:14B 3 1-8. Epub 2014 / 08 / 02; Anacker et al., Journal of visualized experiments : JoVE. 2012(60). Epub 2012 / 03 / 08)). The difficulty in introducing the HPV genome into keratinocytes is solved by using an infection method from the extracellular matrix (ECM) to the cells since HPV preferentially binds to the basement membrane and the ECM secreted by keratinocytes in vivo and in vitro (Richards et al., Viruses. 2014;6(12):4856-79). This involves seeding the cells onto the surface of a collagen gel and then moving this gel together with the culture medium onto a stainless-steel grid to create an air-medium interface. The possibility of propagating commensal HPVs in culture enables the use of live commensal HPVs in our prophylactic cancer vaccines.

[0041] Live attenuated HPV vaccine: Live attenuated HPV vaccines can also be used. Low-risk co-existing HPV E6 protein has been shown to interfere with Notch signaling that drives keratinocyte differentiation and cell cycle arrest (Tan et al., Proceedings of the National Academy of Sciences of the United States of America. 2012;109(23):E1473-80. Epub 2012 / 05 / 024). Specifically, E6 binds to the C-terminal domain of the Mastermind-like (MAML1) protein, which is a member of the Notch transcription complex (ibid.). This enables suppression of keratinocyte differentiation, maintains a favorable cellular environment for low-risk HPV replication, and leads to the development of warts. As a result, mutations in the gene encoding the E6 protein of co-existing HPV at the binding site with the LXXLL domain of MAML-1 enable the development of a safe live attenuated virus for use in vaccines. Protein E6 contains four zinc-binding domains, each possessing two C-x-x-C motifs (Nomine et al., Mol Cell. 2006;21(5):665-78. Epub 2006 / 03 / 02). Specifically, the N-terminal domain has been suggested to be the binding site of the E6 protein (ibid.). In some embodiments, the virus contains one or more mutations from a C-x-x-C motif to an S-x-x-S motif in the amino-terminal domain of the E6 protein to prevent binding to MAML-1 and inhibit the development of warts upon infection with the mutant virus in human tissues. Indeed, these specific cysteine-to-serine mutations inhibit the binding of zinc ions to the zinc-binding domain of the protein, thereby preventing the binding ability of the protein.

[0042] HPV has also been shown to bind to the LXXLL consensus sequence of target proteins such as MAML-1 (Tungteakkhun et al., Arch Virol. 2008;153(3):397-408. Epub 2008 / 01 / 04). In some embodiments, the virus binds to the LXXLL binding motif (see, for example, Brimer et al., PLoS Pathog. 2017 Dec; 13(12): e1006781), for example, to the amino-terminal E6 zinc-binding domain and the carboxy-terminal zinc-binding domain (Vande Pol and Klingelhutz, Virology, 2013, 445(1-2):115-137), or contains mutations in one or more of 8S9A10T, I128T or Δ146~151 (White et al., J Virol. 2012 Dec; 86(24): 13174-13186).

[0043] Symbiotic HPV clinical isolates are attenuated as described above so that they can complete their entire life cycle without maintaining their pathogenic ability to cause warts. Before introducing the HPV clinical isolate into an in vitro culture system, the attenuated mutant is generated using oligonucleotide-directed site-specific mutagenesis. The oligonucleotide carrying the desired mutation is introduced into the HPV genome cloned into a plasmid or bacterial artificial chromosome (BAC), and the method has been described previously to yield infectious virions using an organotypic raft culture model (Meyers et al., Journal of virology. 2002;76(10):4723-33. Epub 2002 / 04 / 23). The recombinant viral genome is introduced into human primary keratinocytes. After transfection, the cells differentiate and grow using an organotypic culture model that supports the entire HPV life cycle.

[0044] T cell adjuvant The composition can also include an adjuvant that increases the T cell response. For example, as described in Stano et al., Vaccine (2012) 30:7541-6 and Swaminathan et al., Vaccine (2016) 34:110-9, nanoparticles that enhance the T cell response can be included, for example. See also Panagioti et al., Front. Immunol., 16 February 2018; doi.org / 10.3389 / fimmu.2018.00276. Alternatively, or in addition, poly-ICLC (carboxymethylcellulose, polyinosinic acid-polycytidylic acid, and poly-L-lysine double-stranded RNA), imiquimod, resiquimod (R-848), CpG oligodeoxynucleotides and formulations (IC31, QB10), AS04 (aluminum salt formulated with 3-O-desacyl-4'-monophosphoryl lipid A (MPL)), AS01 (MPL and saponin QS-21), MPLA, STING agonists, other TLR agonists, Candida albicans skin test antigen (Candin), GM-CSF, Fms-like tyrosine kinase-3 ligand (Flt3L), and / or an adjuvant containing IFA (incomplete Freund's adjuvant) can also be used. In some embodiments, topical imiquimod and / or topical 5-fluorouracil and / or topical calcipotriene (calcipotriol) (e.g., as described in Cunningham et al., J Clin Invest. 2017;127(1):106-116) in combination with 5-fluorofuracil function as an adjuvant for the vaccine (this is particularly applicable to subjects with pre-cancerous skin lesions commonly treated with these topical agents).See, for example, Khong and Willem, Journal for ImmunoTherapy of Cancer 4:56 (2016); Coffman et al., Immunity. 2010 Oct 29; 33(4): 492-503; Martins et al., EBioMedicine 3:67-78, 2016; and Del Giudice, Seminars in Immunology, 2018, doi.org / 10.1016 / j.smim.2018.05.001.

[0045] Composition The pharmaceutical composition typically includes a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes, among other things, saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., which are compatible with the administration of pharmaceuticals.

[0046] The pharmaceutical composition is typically formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, such as intravenous, intradermal, subcutaneous, intratumoral, intramuscular, or subcutaneous administration.

[0047] Methods of formulating suitable pharmaceutical compositions are known in the art; see, for example, Remington: The Science and Practice of Pharmacy, 21st ed., 2005; and the books in the Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY) series. For example, solutions or suspensions used for parenteral, intradermal, intramuscular or subcutaneous application can contain the following components: sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycol, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffering agents such as acetate, citrate or phosphate, and agents for adjusting isotonicity such as sodium chloride or dextrose. The pH can be adjusted using an acid or base such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be enclosed in ampoules, disposable syringes or multiple-dose vials made of glass or plastic.

[0048] A pharmaceutical composition suitable for use in injection can comprise a sterile aqueous solution (if water-soluble), or a dispersing agent for immediate preparation of a sterile injection or dispersion, and a sterile powder. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (trademark) (BASF, Parsippany, NJ), or phosphate buffered saline (PBS). In all cases, the composition must be sterile and be liquid to the extent that easy injectability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of a dispersing agent, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferable to include in the composition isotonic agents, such as sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride. Sustained absorption of an injectable composition can be caused by including in the composition agents that delay absorption, such as aluminum monostearate and gelatin.

[0049] Sterile solutions that can be injected can be prepared by incorporating the required amount of the active compound in a suitable solvent, optionally together with one or a combination of the ingredients listed above, and then subjecting them to filtration sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other ingredients from the ingredients listed above that are required. In the case of sterile powders for preparing injectable sterile solutions, a preferred method of preparation is vacuum drying and lyophilization, which yield a powder of the active ingredient and any additional desired ingredients from its sterile filtered solution in advance.

[0050] In one embodiment, the therapeutic compound is prepared with a carrier that protects the therapeutic compound against rapid elimination from the body, for example, a sustained release formulation including implant and microcapsule delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid can be used. Such formulations can be prepared using standard techniques or can be obtained commercially, for example, from Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions, including liposomes targeted to selected cells by monoclonal antibodies to cellular antigens, can also be used as pharmaceutically acceptable carriers. These can be prepared by methods known to those skilled in the art, for example, according to the method described in U.S. Patent No. 4,522,811.

[0051] The pharmaceutical composition can be contained in a container, pack, dispenser, together with instructions for use for administration.

[0052] subject The vaccine compositions described herein can be used to boost immunity against skin cancer in immunoresponsive subjects, as well as immunosuppressed or immunocompromised patients in whom T cell immunity against β-HPV is reduced and who are prone to developing multiple cutaneous warts and cancers (virus-loaded) with poor prognosis. In some embodiments, the subject does not have cancer (e.g., does not have skin cancer). In some embodiments, the subject is at high risk of developing skin cancer, such as non-melanoma, such as cutaneous squamous cell carcinoma (i.e., has a risk exceeding that of the general population). For example, the subject may have a family history of skin cancer, a personal history of excessive sun exposure / sunburn, fair skin, residence in sunny or high-altitude climates, exposure to carcinogens such as radiation or arsenic, moles, pre-cancerous skin lesions, or a family or personal history of skin cancer.

[0053] In some embodiments, the subject may be immunosuppressed, for example, due to organ transplantation, acquired immunodeficiency, such as HIV / AIDS, or primary human immunodeficiency. In some embodiments, the subject is immunosuppressed due to aging. Since aging is associated with immunosenescence, the methods and compositions are useful in aging individuals, and thus, even usually elderly individuals can benefit from vaccines that boost their antiviral immunity. Thus, in some embodiments, the subject is aged, for example, at least 50, 55, 60, 65, 70, 75, 80, 85 or 90 years old.

[0054] Subjects that can be treated using the methods include mammalian subjects, such as human and non-human animal subjects.

[0055] Method for inducing anti-cancer immunity The compositions can be used to reduce the risk of developing skin cancer, such as non-melanoma, such as cutaneous squamous cell carcinoma, in order to induce anti-cancer immunity. The method includes administering one or more doses of the vaccine compositions described herein to a subject, such as a subject in need thereof.

[0056] The composition is administered in an effective amount. An "effective amount" is an amount sufficient to produce a beneficial effect or a desired result. For example, an effective amount is an amount that achieves a desired therapeutic effect, e.g., an amount necessary to treat a disease or to reduce the risk of occurrence of a disease or symptoms of a disease (each also referred to as a therapeutically effective amount or a prophylactically effective amount). The effective amount can be administered in one or more administrations, applications, or dosages. The therapeutically effective amount (i.e., the effective dosage) of a therapeutic compound depends on the therapeutic compound selected. The composition can be administered once or multiple times per day, including once every other day, up to once or multiple times per week. One of ordinary skill in the art will understand that certain factors, including but not limited to the severity of the disease or disorder, previous treatment, overall health and / or age of the subject, and other diseases present, can affect the dosage and timing required to effectively treat the subject. Also, treatment of a subject with a therapeutically effective amount of a therapeutic compound described herein can include a single treatment or a series of treatments. For example, the method can include administering a first dose, followed by a second dose (e.g., a "booster" dose) at a later time point, e.g., 1, 2, 4, 6, 8, 12, 18, 24, or 52 weeks later.

[0057] The dosage, toxicity, and therapeutic efficacy of a therapeutic composition can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dosage ratio between toxicity and therapeutic effect is the therapeutic index and can be expressed as the LD50 / ED50 ratio. Compositions showing a high therapeutic index are preferred. Compositions showing toxic side effects can be used, but care must be taken to minimize and reduce the side effects.

[0058] Data obtained from cell culture assays and animal studies can be used in the formulation of dosage ranges for use in humans. The dosage of such compounds is preferably within a range of circulating concentrations that includes an ED50 with little or no toxicity. The dosage may be varied within this range depending on the dosage form utilized and the route of administration utilized. For any composition used in the methods described herein, a therapeutically effective dosage can first be estimated from cell culture assays. The dosage may be formulated in animal models. Such information can be used to more accurately determine useful dosages in humans.

[0059] The method can also include, for example, in a subject having skin cancer, administration of one or more treatments known in the art for skin cancer, or treatments for reducing the risk of developing skin cancer. For example, combination treatments with the compositions described herein and field treatments for actinic keratosis (for reducing the risk of developing skin cancer), such as topical 5-fluorouracil, topical imiquimod, topical calcipotriene plus 5-fluorouracil, ingenol mebutate, and photodynamic therapy. In some embodiments, these agents boost antigen presentation (innate signals) while the composition boosts antigen recognition by T cells. In a subject having skin cancer, surgical procedures (e.g., Mohs surgery, excision, curettage and electrocautery (electrosurgery), cryosurgery or laser surgery); radiation therapy; photodynamic therapy; topical drug therapy (e.g., topical 5-fluorouracil, topical imiquimod, topical calcipotriene plus 5-fluorouracil, or ingenol mebutate); or systemic drug therapy (e.g., semiprimab-rwlc, e.g., for a subject having metastatic squamous cell carcinoma of the skin) can be used in combination with the method.

Examples

[0060] The present invention will be further illustrated in the following examples, which do not limit the scope of the present invention described in the claims.

[0061] Method The following materials and methods were used in the examples below.

[0062] Human studies Participants in the clinical study consented to the review of their medical records, access to their archived skin cancers, and donation of excisional biopsy samples for research in the high-risk skin cancer clinic at Massachusetts General Hospital. Discarded, anonymized normal human skin samples were obtained through the Morse Surgery Clinic at Massachusetts General Hospital. Samples of skin lesions and normal skin were processed (a) for immune cell or RNA isolation and (b) fixed in formalin and embedded in paraffin for histological assays.

[0063] Animal studies All mice were housed under sterile conditions in accordance with animal care regulations at the animal facilities of Massachusetts General Hospital and the University of Louisville. Female C57BL / 6J (The Jackson Laboratory, Bar Harbor, ME, strain code: 000664), FVB (Charles River, Wilmington, MA, strain code: 207), and SKH-1 Elite (Charles River, strain code: 477) mice, 6 - 8 weeks of age, were used in the immunoresponsive arm of this study. CD4 - / - ;CD8 - / - mice were used as T cell-deficient hosts (provided by Dr. David G. DeNardo; CD8 - / - :The Jackson Laboratory, strain code: 032563). MmuPV1-infected mice were housed in a biological containment unit in accordance with animal care regulations at the animal facility of the University of Louisville.

[0064] Statistical analysis The exact probability test of Fisher's on both sides was used as the significance test for the results of the anatomical distribution of skin cancer and warts. Pearson's χ2 test was used for other categorical variables. The Mann-Whitney U test on both sides was used for tumor counts and T cell activation assays. The paired t test on both sides was used to compare the RNAish and DNAish signal counts between skin cancer and their adjacent normal skin. The unpaired T test on both sides was used for the epidermal thickness, immunostained T cell count, RNAish signal count comparing skin lesions with normal human skin, and other continuous variables. The log-rank test was used as the significance test for the results of the time to tumor onset. A P value of less than 0.05 was considered significant. All bar graphs show the mean + standard deviation.

[0065] MmuPV1 vaccination The MmuPV1 virus stock was prepared from MmuPV1-induced nasal papillomas of B6.Cg-Foxn1 nu / Foxn1 nu mice according to the previously described protocol. 36 Wt and CD4 - / - ;CD8 - / -The skin on the back of the mice was clipped with an electric clipper and waxed. Next, the skin was wounded by passing a nail file over the entire skin 10 - 20 times, causing minute abnormalities in the skin barrier and simultaneous erythema of the skin. 20 μl of virus inoculum was dispensed onto the wounded skin and spread evenly. The same virus inoculum was used for all infected mice, causing the development of confluent warts on the skin of the back of T - cell - deficient FVB mice. Sham - infected mice received 20 μl of sterile saline. Vaseline gauze (McKesson, San Francisco, CA, catalog number 61 - 20056) was cut to fit the site of the wound and applied under a standard band - aid. 0.5 mg / kg of meloxicam (Boehringer Ingelheim Vetmedica, St. Joseph, MO) was injected subcutaneously for analgesia and repeated the next day. The band - aid was removed at 48 hours, and 200 μl of sterile saline was injected subcutaneously into any lethargic mice.

[0066] PCR Detection of MmuPV1 in Mouse Skin To confirm skin colony formation after dorsal skin infection with MmuPV1, at the end of the carcinogenesis protocol, DNA was isolated from skin biopsies using the DNeasy Blood & Tissue kit (Qiagen, Hilden, Germany, catalog number 69506). PCR amplification of the MmuPV1 L1 protein was performed according to the previously described method (primers are listed in the following table). 1

[0067]

Table 2

[0068] Development of Warts Ten weeks after virus infection or sham - infection, the mice were monitored for the development of warts. As previously described 37, mice with warts lasting >2 months were considered to have "persistent" warts. We classified these mice as "non-immune" and excluded them from chemical and UV carcinogenesis studies. Mice that showed either no wart development or rejection of warts were classified as "immune" and included in the carcinogenesis study.

[0069] T cell isolation and transplantation MumPV1 colony-forming FVB mice (immune mice) that did not develop warts or showed spontaneous regression of warts by 10 weeks post-infection were used as T cell donors. CD4 + and CD8 + single cell suspensions of T cells were prepared using the EasySep™ Mouse T Cell Isolation Kit (Stemcell Technologies, Vancouver, Canada, catalog number 19851). One million T cells in 200 μl of sterile saline were intravenously injected into the tail vein of wart-bearing (non-immune) Wt FVB mice. Recipient mice were monitored for the resolution of warts on their skin. To assess the MmuPV1 specificity of T cells from MmuPV1 colony-forming immune mice, we transplanted their sorted CD4 + and CD8 + T cells into CD4 - / - ;CD8 - / - mice as recipient mice. Donor mice were intravenously injected with 2 μg of CD45-APC (BioLegend, San Diego, CA, catalog number 103112) 3 minutes prior to harvest to deplete any circulating naive T cells. At harvest, single cell suspensions of skin draining lymph nodes were stained with CD3e-PE-Cy7 (Biolegend, catalog number 100320), CD4-APC-Cy7 (Biolegend, catalog number 100414), CD8α-FITC (Biolegend, catalog number 100706, Table 5), and CD62L-PerCP / Cy5.5 (Biolegend, catalog number 104432, Table 5). The sorted CD45 - CD3 + CD4+ CD62 Low and CD45 - CD3 + CD8 + CD62 Low Donor memory T cells 38 129,600 cells (6:1 CD4 + :CD8 + (comparison) and CD4 - / - ;CD8 - / - Mice were intravenously injected with MmuPV1-specific T cells. As a control for MumPV1-specific T cells, a group of Wt FVB mice was vaccinated against an unrelated virus (mouse parvovirus type 1) to increase the population of T cells that would not respond to MmuPV1. This group of T cell donors was vaccinated 30 days and 3 days before harvest at 4 sites in the skin of the back with a cocktail of 50ug polyinosinic-polycytidylic acid (Poly(I:C), Sigma Aldrich, St. Louis, MO, Catalog No. P1530) combined with mouse parvovirus virus-like particles (VLPs) in 200μl of sterile saline (50μl per vaccination site) delivered by subcutaneous injection. 200 μl of 5% imiquimod (Sigma-Aldrich, Cat. No. 1338313) dissolved in dimethyl sulfoxide (DMSO) and diluted in 100% EtOH (Sigma-Aldrich, Cat. No. 276855) was applied topically after each vaccination. - / - ;CD8 - / - Mice and Wt FVB mice were infected with MmuPV1 2 days after T cell transfer, including mice that received T cells from donors treated with parvovirus vaccine plus topical imiquimod. MmuPV1-T cell recipients, T cell-deficient CD4 - / - ;CD8 - / -Another subgroup of Wt mice received an injection of the SCC cell line into their right flanks and were monitored for tumor growth (Figure 7A). Mice were closely monitored for 2 months for the development of warts in the MmuPV1-infected cohort and SCC growth in the tumor cohort, including photographs and measurements of tumor size. To examine the presence / absence of T cells in recipient mice, peripheral blood was collected from the mice 3 weeks after T cell transfer. Two to three drops of blood per mouse were collected via the submandibular vein into 10 ml of RBC lysis buffer (Biolegend, catalog number 420301), stained with CD3e-PE-Cy7, CD4-APC-Cy7, and CD8α-FITC, and examined by flow cytometry.

[0070]

Table 3

[0071] Chemical carcinogenesis protocol According to the evidence of infection and MmuPV1 immunity, C57BL / 6J and FVB mice received a skin chemical carcinogenesis protocol. All animals were shaved, and 7 days later, received a single dose of 100 μg of 7,12-dimethylbenz[a]anthracene (DMBA) (Sigma Aldrich, catalog number D3254) in 200 μl of acetone on the dorsal skin. One week later, treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) (Sigma Aldrich, catalog number P1585) dissolved in 200 μl of acetone was initiated (3 times a week for 30 weeks in the C57BL / 6J cohort and 2 times a week for 20 weeks in the FVB cohort). Throughout the carcinogenesis protocol, tumors were counted weekly and photographs were collected every other week. The final tumor burden was determined based on the total number of visible skin lesions that developed on the dorsal skin of the animals.

[0072] UV carcinogenesis protocol Following evidence of infection and MmuPV1 immunity, SKH-1 mice received a skin carcinogenesis protocol (Figure 11G). Mice received a single dose of 50 μg of DMBA in 200 μl of acetone on the dorsal skin. One week later, SKH-1 mice received narrowband ultraviolet B (UVB) (302–312 nm) three times per week for 25 weeks by a UVP Black-Ray® lamp (VWR, Radnor, PA, catalog number 36575-052) that was regularly calibrated using an International Light IL1400A digital illuminometer. Mice received 100 mJ / cm 2 of UVB at each time point of UV treatment. This is considered to be below the erythema dose of fair-skinned individuals of average sunburn capacity (Fitzpatrick skin types 2–3) and is close to 40–60 minutes of sunlight exposure at midday in Florida in the summer. 39、40 Over the entire carcinogenesis protocol, tumors were counted weekly and photographs were collected every other week. The final tumor burden was determined based on the total number of visible skin lesions that developed on the dorsal skin of the animals.

[0073] Histology and immunofluorescence staining The back skin samples were collected and fixed overnight at 4°C in 4% paraformaldehyde (PFA, Sigma Aldrich, catalog number P6148). Next, the tissues were dehydrated in ethanol, processed, and paraffin-embedded. Paraffin-embedded tissues were sectioned at 5 μm, deparaffinized, and stained with hematoxylin and eosin (H&E). For immunofluorescence staining, rehydrated tissue sections were permeabilized for 5 minutes with 1× PBS supplemented with 0.2% v / v Triton X-100 (Thermo Fisher Scientific, Waltham, MA, catalog number BP151). Antigen retrieval was performed at high pressure for 20 minutes in an antigen unmasking solution (Vector Laboratories, Burlingame, CA, catalog number H-3300) using a Cuisinart pressure cooker. The slides were washed three times for 3 minutes each in 1× PBS supplemented with 0.1% v / v Tween 20 (Sigma-Aldrich, catalog number P1379). The sections were blocked with 5% m / v bovine serum albumin (Fisher Scientific, Hampton, NH, catalog number BP1600) and 5% v / v goat serum (Sigma-Aldrich, catalog number G9023). The slides were stained overnight at 4°C with either 1:500 rat anti-CD3 and 1:500 rabbit anti-CD4 or 1:400 rabbit anti-CD8a (Table 5). The next day, the slides were washed as described above and incubated for 2 hours at room temperature with 1:200 goat anti-rat PE and 1:500 goat anti-rabbit FITC (Table 5). After washing as described above, the slides were incubated for 5 minutes at room temperature with 1:4000 DAPI (Invitrogen, Carlsbad, CA, catalog number D3571) and then washed as described above. The slides were mounted with Prolong Gold Antifade reagent (Invitrogen, catalog number P36930). After staining, ten randomly selected images of morphologically normal skin from each section were acquired at a total magnification of 200×. CD3 + , CD4 + and CD8 +Manual counting of cells blindly was performed using the ZEN Blue “event” tool (Zeiss, Oberkochen, Germany). Positive cells were determined by comparing the minimized background and fluorescence intensity using ZEN. Analyses were performed on the number of double-positive cells (e.g., CD3 + CD4 + ) in the epithelial compartment (i.e., epidermis and hair follicles) or dermis in each image, and based on the total number of CD3 + cells.

[0074] Serology Using previously described methods 41 , anti-MmuPV1-specific antibodies in mouse sera were detected by enzyme-linked immunosorbent assay (ELISA).

[0075] RNA and DNA in situ hybridization RNAish and DNAish were performed on formalin-fixed paraffin-embedded (FFPE) human and mouse tissue sections using RNAscope® probes and protocols (Supplementary Table 2; DNA probes were generated using the sense strand of viral DNA at the same RNA probe binding site; Advanced Cell Diagnostics, California, USA). 42We performed the RNAscope® assay hybridization and incubation steps using the HybEZ™ hybridization system. Briefly, 5-μm thick sections were baked in a dry oven at 60 °C for 1 hour, immediately deparaffinized in xylene, and subsequently rehydrated through an ethanol series. Epitope retrieval was performed by placing the slides in RNAscope® 1X Target Retrieval Reagent (Advanced Cell Diagnostics, catalog number 322000) at 102 °C for 15 minutes and then washing. Protease treatment was then performed by adding RNAscope® Protease Plus (Advanced Cell Diagnostics, catalog number 322331) to the sections and incubating at 40 °C for 30 minutes in a HybEZ™ Oven II (Advanced Cell Diagnostics, catalog number 321720). After probe hybridization with the target probe, preamplifier, and amplifier, the sections were stained with Fast RED reagent (RNAscope® 2.5 HD Detection Reagent-RED, Advanced Cell Diagnostics, catalog number 322360). 50% Hematoxylin plus 0.02% aqueous ammonia was used as a counterstain. Positive and negative probes were used in each assay to ensure appropriate controls. We used probes against peptidylprolyl isomerase B (PPIB, Advanced Cell Diagnostics, catalog number 313901), an endogenous housekeeping gene, and dapB (Advanced Cell Diagnostics, catalog number 310043), a bacterial gene, as positive and negative controls, respectively. We evaluated the red signals of RNAish and DNAish at 400× magnification under a standard brightfield microscope. Ten representative areas of skin cancer and normal skin from each slide were imaged at 400× magnification, and the positive RNAish / DNAish signals and keratinocyte nuclei were counted blindly in each image.

[0076] qRT-PCR RNA samples were extracted from human tissues stored at 4°C in Allprotect (Qiagen, catalog number 76405), and the rapidly frozen samples were stored at -80°C. Tissue pieces (about 50 - 100 mg) were washed using 1-fold sterile PBS and placed into tubes containing 5 mm TissueLyser beads. Subsequently, 600 μl of RLT and βME were added to the samples and beads. The tissue was homogenized mechanically for 5 minutes. The liquid was transferred to a new tube to which 1 ml of TRIzol was added. Using the standard Thermo Fisher protocol for TRIzol, the solution was mixed and centrifuged at 4°C for 10 minutes. The supernatant was collected, and 0.2 ml of chloroform / 1 ml of TRIzol was added. The mixture was centrifuged and the supernatant was recovered. For RNA extraction, we used the Allprep DNA / RNA Mini Kit (Qiagen, catalog number 80284). The supernatant was then added to an Allprep DNA spin column, and the flow-through was mixed with 1 volume of 70% ethanol. This solution was mixed and applied to an RNeasy spin column following the standard purification method including DNase digestion. RNA was quantified using a nanodrop, and 1 μg of RNA was used for the reverse transcriptase reaction using the SuperScript III RT Kit (ThermoFisher, catalog number 18080044). 1 μg of RNA was mixed with 0.25 mg / ml random primers, 10 mM dNTP mix and nuclease-free H2O to a total of 13 μl. This sample was incubated at 65°C for 5 minutes. A mixture of diluted 1× First Strand Buffer, 0.1 M DTT, 400 U / μl RNaseOUT and 200 U of superscript III was added to the nucleotide mixture. The sample was then incubated in a thermocycler. The program consisted of 5 minutes at 25°C, 1 hour at 50°C, and 15 minutes at 70°C. After PCR, the cDNA samples were diluted 1:9 using UltraPure™ DNase / RNase-Free distilled water. 3 μl of the 1:9 dilution was used in a total 10 μl qPCR reaction. For forward and reverse primers, 0.5 μl at a 10 μM concentration was used.The primers were purchased from IDT. 43 5 μl of SYBR® Green master mix was used together with 1 μl of UltraPure™ DNase / RNase-Free distilled water per reaction. qPCR was performed on a LightCycler 480 II (Roche, Basel, Switzerland, product number 05015278001). The qRT-PCR products were verified on a 1% agarose gel by electrophoresis at 120 V for 60 minutes.

[0077] Human T cell isolation and peptide stimulation T cells were isolated from human skin as previously described 44Briefly, discarded normal facial skin samples generated as part of the Mohs surgical repair were obtained. Subcutaneous adipose tissue was removed from the human facial skin tissue, and the remaining tissue was minced. Small fragments of the tissue were digested in RPMI 1640 containing 1% DNase-I (Sigma-Aldrich) and 0.2% collagenase-I (Fisher Scientific) at 37 °C for 2 h. The cells were then collected through a 40-μm cell strainer and incubated in RPMI 1640 containing 20% FBS, 1% penicillin / streptomycin, 1% glutamine, 0.00035% 2-mercaptoethanol, and 50 U / ml recombinant human IL-2 (BioLegend). Human skin T cells were seeded in 96-well plates and treated with a pool of five β-HPV E7 peptides (HPV5 / 8 / 9 / 20 / 38, 5 μg / mL of each peptide, custom peptide, JPT, Berlin, Germany), a pool of HPV16 E7 peptides (5 μg / mL of each peptide, PepMix™ HPV 16 (protein E7), JPT, product code PM-HPV16-E7), or 50 ng / ml phorbol 12-myristate 13-acetate (PMA) plus 500 ng / ml ionomycin (Ion). The peptide pools were generated as 15mers with 11 amino acids overlapping across the length of the E7 protein. Twenty-four hours after peptide exposure, the cells were collected, stained with antibodies against surface markers for T cell activation (Table 5), and examined by flow cytometry (BD LSRFortessa X-20). Flow data were analyzed using FlowJo software, Ashland, OR.

[0078] [Example 1] Immunity against commensal papillomaviruses protects from skin cancer To investigate whether cutaneous tropic HPVs contribute to the development of skin cancer, we conducted a clinical study mapping the anatomical localization of skin cancers and warts from 83 immunosuppressed participants. Eighty-six percent of warts caused by HPV occurred in sun-protected (SP) skin and intermittently sun-damaged (ISD) skin, whereas 41 of 74 (55%) skin cancers occurred in chronically sun-damaged skin of the head and neck (Tables 1A–B and Figures 4A–B). In contrast, the anatomical distribution of skin cancers in immunosuppressed patients matched that of immunocompetent patients (Tables 1A–B and Figures 4A–B). The absence of an increased skin cancer propensity at anatomical sites with high viral activity and wart occurrence suggested that UV was the dominant skin cancer initiator in immunosuppressed patients.

[0079]

Table 4

[0080]

Table 5

[0081] To determine the impact of papillomaviruses on carcinogen-induced skin cancers, we utilized the murine papillomavirus (MmuPV1), which has recently emerged as a robust tool in the study of HPV-related skin diseases. 6、7 We developed a method to infect the skin on the backs of animals with MmuPV1, which was T cell-deficient CD4 - / - ;CD8 - / -It caused confluent warts in the skin of the mouse back, but did not cause skin lesions in immunocompetent Wt animals (Figures 5A - B). Infection of Wt C57BL / 6 mice with MmuPV1 resulted in extensive colony formation on the skin of their backs without the development of warts in 100% of the animals (Figure 6A). Two months after infection, MmuPV1-infected and sham-infected mice were subjected to a standard skin chemical carcinogenesis protocol on the back skin using dimethylbenzanthracene (DMBA) once and 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment three times a week for 30 weeks. 20 Surprisingly, animals with MmuPV1-induced colony formation showed a significant delay in the onset of carcinogen-induced skin tumors compared to non-infected mice (p = 0.0020; Figure 1A). In addition, MmuPV1 colony-forming mice developed significantly fewer tumors over time (starting 22 weeks after DMBA; p < 0.05; Figure 1B) and ended the study with a significantly lower tumor burden compared to non-infected mice (p < 0.0001; Figures 1C, 6C). MmuPV1-infected Wt C57BL / 6J mice that did not receive DMBA-TPA remained free of skin tumors / warts during the 32-week follow-up period (Figures 1A - C).

[0082] More susceptible to chemical skin carcinogenesis 20To investigate the effect of papillomavirus on skin cancer in FVB strain mice, we infected the skin on the backs of Wt FVB mice as described above and achieved complete skin colony formation (Figure 6B). 23% of the infected mice showed complete immunity immediately after infection. However, 77% of the mice developed warts on the skin of their backs 5 weeks after infection (Figure 1D). 10 weeks after infection, the warts completely disappeared in 58% of the wart-bearing FVB mice, showing antiviral adaptive immunity. 42% of the animals continued to have persistent warts without a change in the number of their warts (Figure 1D). T cells transplanted from the skin draining lymph nodes of MmuPV1-immune mice (i.e., without warts) conferred immunity to mice with persistent warts and resulted in the rejection of warts 2 weeks after adoptive T cell transfer (Figure 1E). To examine whether memory T cells isolated from immune mice were MmuPV1-specific, we transplanted these T cells into CD4 - / - ;CD8 - / - mice and subsequently infected the skin on their backs with MmuPV1 (Figure 7A). CD4 - / - ;CD8 - / - mice that received T cells from MmuPV1-immune mice developed fewer warts after infection compared to CD4 - / - ;CD8 - / - mice depleted of T cells and CD4 - / - ;CD8 - / - mice that received T cells from parvovirus-vaccinated Wt mice (Figure 7B). In contrast to their protective effect against warts induced by MmuPV1, T cells from MmuPV1-immune mice had no effect on SCC tumor growth in CD4 - / - ;CD8 - / - mice (Figure 7C).

[0083] MmuPV1-colonized Wt FVB mice, which have innate and adaptive immunity to MmuPV1, were treated once with DMBA, followed 1 week later by TPA twice weekly for 20 weeks. Similar to C57BL / 6J animals, MmuPV1-colonized Wt FVB mice were protected from chemical carcinogenesis and showed a significant delay in the development of skin tumors (p<0.0001; Fig. 1f). MmuPV1-colonized mice developed fewer tumors over time (p<0.05 starting 7 weeks after DMBA; Fig. 1g), and they had a significantly lower tumor burden at the end of the study (p<0.01; Fig. 1h and i). Thus, MmuPV1-colonized immune FVB mice receiving 7,12-dimethylbenzanthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA) for 20 weeks were protected from chemical carcinogenesis compared to mock-infected mice. Furthermore, mice with acquired immunity after T cell transfer were also protected from chemical carcinogenesis (Fig. 1J). We detected MmuPV1 viral DNA in normal skin (Fig. 8A and B) and in blood with anti-MmuPV1 L1, E6, and E7 antibodies (Fig. 8C and D) of MmuPV1-colonized Wt C57BL / 6J and FVB mice at the end of the DMBA-TPA protocol. Importantly, MmuPV1-colonized mice were CD8 + Memory T (T RM ) vs. total CD3 + As shown by the ratio of CD8 T cells to the epithelial compartment of the skin compared to uninfected mice + We demonstrated increased T cell homing (p = 0.0004, Figure 5E-H). DMBA-TPA-induced skin tumors in MmuPV1-colonized mice exhibited similar growth and mutational properties to those in mock-infected mice and were devoid of MmuPV1 viral transcripts (Figures 8I-J, 9). We found that CD4 +No changes were detected in the T cell population (Figures 8E and F). Interestingly, we were able to detect MmuPV1 RNA in the normal skin of MmuPV1 colony-forming mice at the time of recovery (Figure 9). These findings demonstrate the protective effect of anti-MmuPV1 T cell immunity in the suppression of carcinogen-induced tumors in MmuPV1 colony-forming skin.

[0084] To determine the effect of papillomavirus colony formation on UV carcinogenesis, we studied hairless SKH-1 mice that are immunocompetent and develop skin tumors in response to UV irradiation. 21 After MmuPV1 infection, abundant MmuPV1 L2 RNA was detected 3 weeks after infection in the skin of immunized mice (i.e., without the development of warts), as well as in the skin and warts of animals lacking immediate anti-MmuPV1 immunity (Figure 7A). Eight MmuPV1-infected mice that received a single immunosuppressive dose of ultraviolet B (UVB; 300 mJ / cm2) 3 months after MmuPV1 infection developed warts9 and showed long-term persistence of MmuPV1 colony formation in the skin (Figures 10B–C). To avoid immunosuppressive UV exposure, MmuPV1- and sham-infected mice were treated with DMBA for 1 week before being treated with UVB (100 mJ / cm2) three times a week for 25 weeks. A small subset of SKH-1 mice with persistent warts 2 months after dorsal skin infection with MmuPV1 (Wang et al., PLoS Pathog. 11, e1005243 (2015)) were vaccinated intraperitoneally three times over a 2-week period with live MmuPV1 virus particles. Four weeks later, 5 of 9 mice developed immunity to MmuPV1 as demonstrated by rejection of their persistent warts (Figure 10D). Mice with acquired immunity to MmuPV1 developed significantly fewer skin tumors compared to non-immunized mice (P = 0.0159; Figures 10D–E). Furthermore, the total numbers of T cells and CD8+ T cells were significantly increased in the skin tumors of MmuPV1 colony-forming mice (Figures 10F–M, 11A–C). The levels of skin- and tumor-infiltrating CD3−CD45+ leukocytes and CD4+ T cells did not differ between the two groups (Figures 10F–M, 11D–F).

[0085] To determine the role of CD8+ T cells in mediating antitumor immunity induced by cutaneous papillomavirus colonization, SKH-1 mice were infected with MmuPV1 or mock-infected with MmuPV1 virus-like particles (mock (VLP)). MmuPV1- and mock (VLP)-infected mice underwent CD8+ T cell depletion mediated by anti-CD8 antibody in conjunction with the UV carcinogenesis protocol (Figures 11G–H). Notably, MmuPV1-colonized SKH-1 mice treated with IgG control developed significantly fewer tumors compared to MmuPV1-colonized mice that underwent T cell depletion, as well as compared to both IgG- and anti-CD8 antibody-treated control groups mock-infected with (VLP) (Figures 11I–J). Consistent with our findings in other immunocompetent strains of mice, MmuPV1-colonized Xpc− / − mice (Sands et al., Nature 377, 162–165 (1995)), which lack the ability to repair UV-induced DNA mutations, were protected from skin cancer compared to their mock-infected controls (Figures 11K–N).

[0086] To avoid highly immunosuppressive UV dosing 23 the dorsal skin was irradiated with 100 mJ / cm 2One week before receiving UVB treatment three times a week for 25 weeks, the mice were treated with a single dose of 50 μg of DMBA. The onset of skin tumors was not significantly delayed (Figure 2A), but MmuPV1 - colonized SKH-1 mice developed significantly fewer tumors over time (starting 17 weeks after DMBA; p < 0.05; Figure 2B) and had significantly fewer tumor burdens at the end of the study compared to non-infected mice (p < 0.005; Figures 2C and D). Interestingly, extensive epidermal dysplasia induced by DMBA - UV treatment in the dorsal skin of non-infected mice was not present in MmuPV1 - colonized animals (Figure 12A). This block in DMBA - UV - induced dysplasia was reflected by a reduction in epidermal thickness in MmuPV1 - colonized mice compared to their non-infected counterparts (Figure 12B). Analysis of immune cells revealed a significant increase in the ratio of the total number of CD8 + T cells and CD8 + T RM to total T cells at the end of the DMBA - UV protocol (p < 0.05, Figures 2e - g). No significant difference was observed in the total number of CD4 + T cells between the groups (Figures 12C - D). MmuPV1 DNA was detectable in the normal skin of MmuPV1 - colonized mice at the end of the DMBA - UV protocol (Figure 12E).

[0087] The protective effect of anti - MmuPV1 immunity against carcinogen - induced skin cancer in mice suggested that β - HPVs in the skin of immunocompetent individuals may play a similar protective role. To investigate this, we utilized a pool of β - HPV RNAish probes that detect E6 / 7 transcripts of 25 β - HPV types in tissue sections to provide new insights into the intracellular localization of the virus in the skin (Figure 13 and Table 2) 16RNAISH of β-HPV detected β-HPV transcripts in warts (positive control) from immunosuppressed patients and keratinocytes of skin cancers (Figures 14A - B). Normal skin from 18-year-old immunocompetent patients did not show detectable RNAISH signals of β-HPV (Figure 14B). qRT-PCR of HPV5, 9, and 15 E6 in RNA isolated from the same skin cancers and normal skin verified the results of RNAISH of β-HPV (Figure 14B).

[0088]

Table 6

[0089] Next, we performed RNAISH of β-HPV in skin cancers from our immunosuppressed and immunocompetent patients (Table 3).

[0090]

Table 7

[0091] β-HPV RNA was detectable in warts, verruca-associated hypertrophic actinic keratosis, and SCC from immunosuppressed patients (Figure 3a). β-HPV RNA expression was reduced in cancer cells compared to adjacent normal skin cells from immunosuppressed patients (Figure 3a). β-HPV RNA expression was mostly absent in cancer cells of SCC from immunocompetent patients, although low levels of β-HPV RNA expression were present in adjacent normal skin (Figure 3a). β-HPV RNAish signal counts across skin cancers revealed a significant reduction in β-HPV RNA expression in cancer cells compared to keratinocytes of adjacent normal skin in both immunocompetent and immunosuppressed patients (p < 0.001, Figure 3b). Skin cancer cells in immunosuppressed patients had significantly more viral transcripts compared to skin cancer cells in immunocompetent patients (Figure 15A). Additionally, skin lesions from immunosuppressed patients had significantly higher β-HPV RNA expression throughout the tissue compared to skin lesions from immunocompetent patients and normal facial skin samples (Figure 15B–C). β-HPV RNA expression was detectable in a significantly greater number of basal keratinocytes in normal skin from immunosuppressed patients compared to immunocompetent patients (Figure 16A–B). To examine the intracellular levels of β-HPV viral load in tissue sections, we utilized a pool of β-HPV DNA in situ hybridization (DNAish) probes that detect the sense strand of the E6 / 7 gene from 25 β-HPV types. β-HPV DNAish revealed the intracellular localization of β-HPV viral DNA in human skin (Figure 17). β-HPV DNAish showed high viral loads in warts, verruca-associated hypertrophic actinic keratosis, and SCC from immunosuppressed patients (Figure 18A). The viral load of β-HPV was reduced in cancer cells compared to adjacent normal skin in immunosuppressed patients (p < 0.05, Figure 18B). The reduction in the viral load of β-HPV in cancer cells was more evident in lesions from immunocompetent patients when compared to adjacent normal keratinocytes (p < 0.01, Figure 18C).Higher viral activity and loads in skin cancers of immunosuppressed patients correlated with significantly fewer tumors and skin-infiltrating CD8+ T and CD103+ CD8+ TRM cells in those skin cancers compared to samples from immunocompetent patients (Figures 19A - C).

[0092] Finally, we examined whether normal skin from sun-damaged sites contains T cells specific for β-HPV. T cells isolated from normal facial skin of immunocompetent adults were exposed to peptides derived from the E7 proteins of β-HPV types 5, 8, 9, 20, and 38 (Table 4). Skin-derived CD8 + cytotoxic T lymphocytes (CTLs) became activated, as indicated by a significant increase in CD69 + and CD137 + in CD69 + CTLs compared to negative controls (Figures 3C - D, 19D). Consistent with their activated state, we detected more CTL degranulation (CD107a + in CD8 + T cells) after exposure to β-HPV peptides compared to negative controls (Figure 19D). In contrast to β-HPV peptides, the high-risk HPV16 E7 peptide did not activate skin-derived CTLs (Figures 3C - D and Figure 19D).

[0093]

Table 8 - 1

[0094]

Table 8 - 2

[0095]

Table 8 - 3

[0096] To further develop the vaccine strategy, we use long overlapping peptides derived from the MmuPV1 E1, E2, E6, and E7 proteins, together with the poly-ICLC adjuvant, to vaccinate wild-type (Wt) mice after colony formation by MmuPV1. We then determine whether this vaccination reduces the risk of skin cancer upon exposure to carcinogens. In addition, we study the efficacy of a β-HPV multipeptide vaccine (peptides derived from the E1, E2, E4, E6, and E7 proteins of HPV 5, 8, 9, 17, 20, 38, 50, 75, 80, and 151) in inducing an immune response against skin cancer by subcutaneous injection into sites of early squamous cell carcinoma in high-risk patients under an IRB-approved protocol. This study enables a clinical trial to investigate vaccine application in high-risk patients for cancer prevention, for example, for use of the vaccine in solid organ transplant recipients before they receive a transplant.

[0097] To identify signals that result in antigen presentation of papillomavirus to T cells after abnormal keratinocyte proliferation, we performed RNA sequencing (RNA-seq) in skin warts, MmuPV1-infected DMBA-UV-treated skin and tumors, and sham-infected DMBA-UV-treated skin and tumors of SKH-1 mice (Figure 20a–c). Among 20 genes upregulated in both MmuPV1-induced warts and DMBA-UV-induced tumors (both from the MmuPV1-infected and sham-infected groups) compared to skin (also from both groups), there were several immune-related genes, including the damage-associated molecular pattern (DAMP) genes S100a8 and S100a9 (Figure 20c). In human SCC and warts, we confirmed the induction of S100 genes, where the S100A8 and S100A9 genes were downregulated compared to normal skin, compared to normal skin and benign skin growths of seborrheic keratosis (Figure 20d–f).

[0098] [Example 2] Live HPV Vaccine and Live Attenuated HPV Vaccine Using an in vitro culture system, skin-tropic HPVs are propagated. Commensal HPVs are isolated, for example, from warts of immunocompromised adult patients. Next, the purified virus is transferred into an organotypic raft culture model using human primary keratinocytes (low passage rather than immortalized cell lines (Bienkowska-Haba et al., PLoS Pathog. 2018;14(3):e1006846)). The difficulty in the transfer of the HPV genome into keratinocytes is resolved by using an infection method from the extracellular matrix (ECM) to cells because HPV preferentially binds to the basement membrane and the ECM secreted by keratinocytes in vivo and in vitro (Richards et al., Viruses. 2014;6(12):4856-79. Epub 2014 / 12 / 1). This involves seeding cells onto the surface of a collagen gel and then moving this gel together with the culture medium onto a stainless-steel grid to create an air-medium interface.

[0099] Mutations in the gene encoding the E6 protein of oncogenic HPV at the binding site to the LXXLL domain of MAML-1 enable the development of safe live attenuated viruses for use in vaccines. The complete HPV genome is inserted into a bacterial artificial chromosome (BAC) for stable maintenance of the HPV genome in Escherichia coli and for introduction of mutations into E6 at its binding site to MAML-1. The BAC sequence is flanked by loxP sites to allow removal of the bacterial sequences derived from the viral genome by Cre recombination prior to transplantation into human cells. Stepwise mutations of the E6 protein (as performed for MAML1 in Tan et al., Proceedings of the National Academy of Sciences of the United States of America. 2012;109(23):E1473-80. Epub 2012 / 05 / 024). The recombinant viral genome is then introduced into human primary keratinocytes. After transfection, the cells are differentiated and grown using an organotypic culture model that supports the entire HPV life cycle to determine the effect of each mutation on the binding of the E6 protein to MAML1.

[0100] References

[0101]

Table 9-1

[0102]

Table 9-2

[0103]

Table 9-3

[0104]

Table 9-4

[0105]

Table 9-5

[0106] Other embodiments The present invention has been described in conjunction with its detailed description, but it should be understood that the foregoing description is intended to illustrate and not limit the scope of the present invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. The inventions described in the original claims of this application are listed below. [Invention 1] A plurality of antigenic peptides, each containing a 9-30 amino acid sequence derived from (i) a protein from a commensal human papillomavirus, or (ii) a live or live-attenuated commensal human papillomavirus; and A T cell adjuvant that increases the T cell response to the antigenic peptide A composition comprising. [Invention 2] The composition according to Invention 1, wherein the commensal human papillomavirus is a low-risk α-HPV, β-HPV, γ-HPV and / or μ-HPV strain. [Invention 3] The composition according to Invention 1, wherein the commensal human papillomavirus is a low-risk α-HPV, β-HPV, γ-HPV and / or μ-HPV strain listed in Table A. [Invention 4] The composition according to Invention 1, wherein the plurality of antigenic peptides comprises peptides derived from one or more of the E1, E2, E4, E5, E6 or E7 proteins. [Invention 5] The composition according to Invention 1, wherein the plurality of antigenic peptides comprises peptides derived from proteins from a plurality of commensal human papillomaviruses. [Invention 6] The composition according to Invention 5, comprising at least 200 peptides each having a unique sequence. [Invention 7] The composition according to Invention 6, comprising a plurality of peptides for each unique sequence. [Invention 8] A plurality of antigenic proteins from a commensal human papillomavirus, preferably in virus-like particles; and A T cell adjuvant that increases the T cell response to the antigenic peptide A composition comprising. [Invention 9] The composition according to Invention 8, wherein the plurality of antigenic proteins comprises one or more of the E1, E2, E4, E5, E6 or E7 proteins. [Invention 10] The composition according to invention 8, wherein the co-existing human papillomavirus is a low-risk α-HPV, β-HPV, γ-HPV and / or μ-HPV strain. [Invention 11] The composition according to invention 8, wherein the co-existing human papillomavirus is a low-risk α-HPV, β-HPV, γ-HPV and / or μ-HPV strain listed in Table A. [Invention 12] (i) a plurality of antigenic peptides each containing a sequence of 9 to 30 amino acids derived from a protein derived from a co-existing human papillomavirus, or (ii) a plurality of nucleic acids encoding a plurality of antigenic proteins derived from a co-existing human papillomavirus; and a T cell adjuvant that increases the T cell response to the antigenic peptide A composition comprising. [Invention 13] The composition according to invention 12, wherein the plurality of antigenic proteins comprises one or more E1, E2, E4, E5, E6 or E7 proteins. [Invention 14] The composition according to invention 12, wherein the co-existing human papillomavirus is a low-risk α-HPV, β-HPV, γ-HPV and / or μ-HPV strain. [Invention 15] The composition according to invention 12, wherein the co-existing human papillomavirus is a low-risk α-HPV, β-HPV, γ-HPV and / or μ-HPV strain listed in Table A. [Invention 16] The composition according to invention 12, comprising one or more viral vectors engineered to express the plurality of proteins or antigenic peptides. [Invention 17] The composition according to invention 12, wherein the viral vector is selected from the group consisting of recombinant retroviruses, adenoviruses, adeno-associated viruses, alphaviruses and lentiviruses. [Invention 18] The composition according to any one of inventions 1 to 17, wherein the T cell adjuvant comprises one or more of nanoparticles that enhance T cell responses, poly-ICLC (carboxymethylcellulose, polyinosinic acid-polycytidylic acid, and poly-L-lysine double-stranded RNA), imiquimod, CpG oligodeoxynucleotides and formulations (IC31, QB10), AS04 (aluminum salt formulated with 3-O-desacyl-4'-monophosphoryllipid A (MPL)), AS01 (MPL and saponin QS-21), MPLA, STING agonists, other TLR agonists, Candida albicans skin test antigen (Candin), GM-CSF, Fms-like tyrosine kinase-3 ligand (Flt3L), and / or IFA (incomplete Freund's adjuvant). [Invention 19] The composition according to any one of inventions 1 to 17, wherein the T cell adjuvant comprises topical resiquimod, or topical imiquimod, or topical 5-fluorouracil, or topical calcipotriene (calcipotriol), or a combination thereof, for example, calcipotriene combined with 5-fluorouracil. [Invention 20] A method of treating cutaneous cancer in a subject or reducing the risk of its occurrence, the method comprising administering to the subject an effective amount of the composition according to any one of inventions 1 to 19. [Invention 21] The method according to invention 20, wherein the subject has an increased risk of developing cutaneous cancer or is immunocompromised. [Invention 22] The method according to invention 21, wherein the subject is immunocompromised as a result of aging, or acquired immunodeficiency, or organ transplantation. [Invention 23] The composition according to any one of inventions 1 to 19, for use in a method of treating cutaneous cancer in a subject or reducing the risk of its occurrence. [Invention 24] The composition for use according to invention 23, wherein the subject has an increased risk of developing cutaneous cancer or is immunocompromised. [Invention 25] The composition for use according to invention 24, wherein the subject is immunocompromised as a result of aging, or acquired immunodeficiency, or organ transplantation.

Claims

Claim 1 A plurality of (i) antigenic peptides, each peptide comprising a 9-30 amino acid fragment in one or more of the E1, E2, E4, E5, E6 or E7 proteins derived from the commensal HPV5, HPV8, HPV9, HPV12, HPV14, HPV15, HPV17, HPV19, HPV20, HPV21, HPV22, HPV23, HPV24, HPV25, HPV36, HPV37, HPV38, HPV47, HP V49, HPV75, HPV76, HPV80, HPV92, HPV93, HPV96, HPV107, HPV110, HPV111, HPV129, HPV151 and / or HPV164 human papillomavirus strains, or (ii) live or live-attenuated commensal low-risk PV5, HPV8, HPV9, HPV12, HPV14, HPV15, HPV17, HPV19, HPV20, HPV21, HPV22, HPV23, HPV24, HPV25, HPV36, HPV37, HPV38, HPV47, HP V49, HPV75, HPV76, HPV80, HPV92, HPV93, HPV96, HPV107, HPV110, HPV111, HPV129, HPV151 and / or HPV164 human papillomavirus strains-derived one or more of the E1, E2, E4, E5, E6 or E7 proteins; and A T cell adjuvant that increases the T cell response to said antigenic peptide A composition comprising. Claim 2 The composition according to claim 1, wherein said peptide comprises said protein derived from a plurality of said commensal human papillomavirus strains. Claim 3 The composition according to claim 2, comprising at least 200 peptides each having a different sequence. Claim 4 The composition according to claim 3, comprising a plurality of peptides for each different sequence. Claim 5 One or more E1, E2, E4, E5, E6 or E7 proteins derived from a plurality of human papillomavirus strains of co-infection low-risk PV5, HPV8, HPV9, HPV12, HPV14, HPV15, HPV17, HPV19, HPV20, HPV21, HPV22, HPV23, HPV24, HPV25, HPV36, HPV37, HPV38, HPV47, HP V49, HPV75, HPV76, HPV80, HPV92, HPV93, HPV96, HPV107, HPV110, HPV111, HPV129, HPV151 and / or HPV164; and A T cell adjuvant that increases the T cell response to said protein A composition comprising.

6. The composition according to claim 5, wherein the plurality of antigenic proteins are in virus-like particles.

7. (i) A plurality of antigenic peptides, each peptide comprising a 9-30 amino acid fragment of one or more E1, E2, E4, E5, E6 or E7 proteins derived from co-infection HPV5, HPV8, HPV9, HPV12, HPV14, HPV15, HPV17, HPV19, HPV20, HPV21, HPV22, HPV23, HPV24, HPV25, HPV36, HPV37, HPV38, HPV47, HP V49, HPV75, HPV76, HPV80, HPV92, HPV93, HPV96, HPV107, HPV110, HPV111, HPV129, HPV151 and / or HPV164 human papillomavirus strains, or (ii) A plurality of nucleic acids encoding one or more E1, E2, E4, E5, E6 or E7 proteins derived from co-infection low-risk PV5, HPV8, HPV9, HPV12, HPV14, HPV15, HPV17, HPV19, HPV20, HPV21, HPV22, HPV23, HPV24, HPV25, HPV36, HPV37, HPV38, HPV47, HP V49, HPV75, HPV76, HPV80, HPV92, HPV93, HPV96, HPV107, HPV110, HPV111, HPV129, HPV151 and / or HPV164 human papillomavirus strains And A T cell adjuvant that increases the T cell response to said antigenic peptide A composition comprising.

8. The composition according to claim 7, comprising one or more viral vectors engineered to express said plurality of said proteins or said antigenic peptides.

9. The composition according to claim 8, wherein the viral vector is selected from the group consisting of recombinant retrovirus, adenovirus, adeno-associated virus, alphavirus and lentivirus.

10. The composition according to any one of claims 1 to 9, wherein the T cell adjuvant comprises one or more of poly-ICLC, imiquimod, CpG oligodeoxynucleotide, IC31, QB10, AS04 (aluminum salt formulated with 3-O-desacyl-4'-monophosphoryllipid A (MPL)), AS01, MPLA, STING agonist, other TLR agonists, Candida albicans skin test antigen (Candin), GM-CSF, Fms-like tyrosine kinase-3 ligand (Flt3L), and IFA (incomplete Freund's adjuvant).

11. The composition according to any one of claims 1 to 9, wherein the T cell adjuvant comprises topical resiquimod, or topical imiquimod, or topical 5-fluorouracil, or topical calcipotriene, or a combination thereof.

12. The composition according to claim 11, wherein the T cell adjuvant comprises topical calcipotriene in combination with 5-fluorouracil.

13. A pharmaceutical composition for treating skin cancer in a subject or reducing the risk of its occurrence, comprising the composition according to any one of claims 1 to 12.

14. The pharmaceutical composition according to claim 13, wherein the subject has an increased risk of developing skin cancer or is immunocompromised.

15. The pharmaceutical composition according to claim 14, wherein the subject is immunocompromised as a result of aging, or acquired immunodeficiency, or organ transplantation.

16. The composition according to any one of claims 1 to 12, for use in a method of treating skin cancer in a subject or reducing the risk of its occurrence.

17. The composition for use according to claim 16, wherein the subject has an increased risk of developing skin cancer or is immunocompromised.

18. The composition for use according to claim 17, wherein the subject is immunocompromised as a result of aging, or acquired immunodeficiency, or organ transplantation.

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