Skin-targeted ferritin nanoparticle vaccines, uses and manufacturing thereof

Abstract

The present disclosure relates to the field of vaccines, as well as preparations, articles of manufacture, and methods of their use in the treatment and / or prevention of diseases (e.g., infectious diseases, cancer, etc.). In certain embodiments, the disease is a coronavirus infection related disease. In certain embodiments, the coronavirus can be a P-coronaviruses (e.g., SARS- CoV-2).

Description

[0001] 072396.1082

[0002] PATENT

[0003] SKIN-TARGETED FERRITIN NANOPARTICLE VACCINES, USES AND MANUFACTURING THEREOF

[0004] CROSS-REFERENCES TO RELATED APPLICATIONS

[0005] The application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 641,805, filed May 2, 2024, the content of which is incorporated by reference in its entirety, and to which priority is claimed.

[0006] SEQUENCE LISTING

[0007] A Sequence Listing conforming to the rules of WIPO Standard ST.26 is hereby incorporated by reference. Said Sequence Listing has been filed as an electronic document via PatentCenter encoded as XML in UTF-8 text. The electronic document, created on May 2, 2025, is entitled “072396.1082_ST26.xml”, and is 83,792 bytes in size.

[0008] GRANT INFORMATION

[0009] This invention was made with government support under HT9425-23-1-0427, and W81XWH- 18-2-0040 awarded by the Defense Health Agency, Medical Research and Development Branch, and AR079233 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0010] TECHNICAL FIELD

[0011] The present disclosure relates to the field of vaccines, as well as preparations, articles of manufactures, and methods of their use in the treatment and / or prevention of viral or bacterial infection related diseases. In certain embodiments, the present disclosure is related to coronavirus infection diseases. In certain embodiments, the coronavirus can be a P-coronaviruses (e.g., SARS- CoV-2). In certain embodiments, the present disclosure is related to arenavirus infection diseases. In certain embodiments, the present disclosure is related to HIV-1 infection diseases.

[0012] BACKGROUND OF THE INVENTION

[0013] In response to the COVID-19 pandemic, record-breaking vaccine development and widespread vaccination allowed the prevention of death of an estimated population of approximately 14 million people in the first year (Watson et al., The Lancet Infectious Diseases 22.9 (2022): 1293-1302). Nonetheless, there remains an urgent public health need for vaccine interventions against existing and novel infectious pathogens. For example, affordability, low-temperature storage and transport, suboptimal vaccine accessibility and acceptability, limited protection against emerging new viral variants, and waning of previous immune response remain ACTIVE 508396272 1 072396.1082

[0014] PATENT current and unmet needs. The present disclosure provides vaccine platforms (e.g., skin-targeted vaccine platforms) that can address these challenges.

[0015] SUMMARY OF THE INVENTION

[0016] The present disclosure provides compositions and articles of manufacture that can be used to treat or prevent infections (e.g., infections caused by SARS-CoV-2).

[0017] In certain non-limiting embodiments, the present disclosure provides a vaccine platform comprising a base and a plurality of microneedles extending from the base, wherein the plurality of microneedles comprises a vaccine comprising a nanoparticle-forming polypeptide, an antigenic polypeptide, and an adjuvant.

[0018] In certain embodiments, the nanoparticle-forming polypeptide comprises a ferritin polypeptide, a lumazine synthase polypeptide, a SpyTag polypeptide, a SpyCatcher polypeptide, or a combination thereof. In certain embodiments, the nanoparticle-forming polypeptide comprises a ferritin polypeptide. In certain embodiments, the ferritin polypeptide is a Helicobacter pylori ferritin polypeptide. In certain embodiments, the ferritin polypeptide comprises an amino acid sequence that is at least about 80%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, the ferritin polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1.

[0019] In certain embodiments, the ferritin polypeptide comprises a substitution of the glutamic acid residue (E) at position 13 of SEQ ID NO: 1. In certain embodiments, the ferritin polypeptide comprises an amino acid sequence that is at least about 80%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 3. In certain embodiments, the ferritin polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 3.

[0020] In certain embodiments, the antigenic polypeptide is a polypeptide obtained or derived from a virus selected from adenovirus, herpesvirus, Epstein Barr virus, herpes simplex type 1, herpes simplex type 2, human herpes virus simplex type 8, human cytomegalovirus, varicellazoster virus, poxvirus, parvovirus, papillomavirus, picomavirus, coxsackie virus, hepatitis A virus, poliovirus, togavirus, rubella virus, flavivirus, hepatitis C virus, yellow fever virus, dengue virus, west Nile virus, coronavirus, orthomyxovirus, influenza virus, rhabdovirus, paramyxovirus, measles virus, mumps virus, parainfluenza virus, respiratory syncytial virus, metapneumovirus,

[0021] ACTIVE 508396272 2 072396.1082

[0022] PATENT arenaviruses, Lassa virus, LuJo virus, Junin virus, Machupo virus, Bear Canyon virus, Wenzhou virus, Guanarito virus, Latino virus, Sabia virus, Chapare virus, Whitewater Arroyo virus, Pichinde virus, LCMV virus, Mopeia virus, Mobala, virus, Ippy virus, Mobala virus, and Morogoro virus, rhabdovirus, rabies virus, ebola, human immunodeficiency virus (HIV), hepadnavirus, hepatitis B, or a combination thereof.

[0023] In certain embodiments, the antigenic polypeptide is a polypeptide obtained or derived from a bacterium selected from A. duodenale, Anisakis spp., Ascaris lumbricoides, Balantidium coli, Cestoda spp., Cimicidae spp., Clonorchis sinensis, Dicrocoelium dendriticum, Dicrocoelium hospes, Diphyllobothrium latum, Dracunculus spp., E. granulosus, E. multilocularis, Entamoeba histolytica, Enterobius vermicularis, F. hepatica, F. magna, F. gigantica, F. jacksoni, Fasciolopsis buski, Giardia lamblia, Gnathostoma spp., H. nana, H. diminuta, Leishmania spp., Loa loa, M. conjunctus, M. albidus, Necator americanus, Oestroidea spp., Onchocercidae spp., O. viverrini, O.felineus, O. guayaquilensis, and O. nover ca, P. falciparum, Protofasciola robusta, Parafasciolopsis fasciomorphae, Paragonimus westermani, S. mansoni, S. japonicum, S. mekongi, S. haematobium, Spirometra erinaceieuropaei, Strongyloides stercoralis, T. saginata, T. solium, T. canis, T. cati, T. gondii, Trichobilharzia regenti, Trichinella spiralis, Trichuris trichiura, Trombiculidae spp. , Trypanosoma spp. , Tunga penetrans, Wuchereria bancrofti, or a combination thereof.

[0024] In certain embodiments, the antigenic polypeptide is a polypeptide obtained or derived from a parasite selected from A. duodenale, Anisakis spp., Ascaris lumbricoides, Balantidium coli, Cestoda spp., Cimicidae spp., Clonorchis sinensis, Dicrocoelium dendriticum, Dicrocoelium hospes, Diphyllobothrium latum, Dracunculus spp., E. granulosus, E. multilocularis, Entamoeba histolytica, Enterobius vermicularis, F. hepatica, F. magna, F. gigantica, F. jacksoni, Fasciolopsis buski, Giardia lamblia, Gnathostoma spp., H. nana, H. diminuta, Leishmania spp., Loa loa, M. conjunctus, M. albidus, Necator americanus, Oestroidea spp., Onchocercidae spp., O. viverrini, O. felineus, O. guayaquilensis, and O. noverca, P. falciparum, Protofasciola robusta, Parafasciolopsis fasciomorphae, Paragonimus westermani, S. mansoni, S. japonicum, S. mekongi,

[0025] S. haematobium, Spirometra erinaceieuropaei, Strongyloides stercoralis, T. saginata, T. solium,

[0026] T. canis, T. cati, T. gondii, Trichobilharzia regenti, Trichinella spiralis, Trichuris trichiura, Trombiculidae spp., Trypanosoma spp., Tunga penetrans, Wuchereria bancrofti, or a combination thereof.

[0027] In certain embodiments, the antigenic polypeptide is a polypeptide obtained or derived from a tumor antigen selected from gplOO, MART-l / Melan A, gp75 (TRP-I), tyrosinase, NY- ESO-I, melanoma proteoglycan, MAGE family antigens, BAGE family antigens, GAGE family

[0028] ACTIVE 508396272 3 072396.1082

[0029] PATENT antigens, RAGE family antigens, N- acetylglucosaminyltransferase-V, pl 5, P-catenin, MUM- 1, cyclin dependent kinase-4 (CDK4), p21-ras, BCR- abl, p53, pl85 HER2 / neu, epidermal growth factor receptor (EGFR), carcinoembryonic antigens (CEA), carcinoma- associated mutated mucins, EBNA gene products of EBV (i.e., EBNA-I), E7, E6 proteins of human papillomavirus, prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), idiotypic epitopes or antigens, for example, immunoglobulin idiotypes or T cell receptor idiotypes, KSA, kinesin 2, HIP-55, TGFP-1 anti- apoptotic factor, tumor protein D52, HIFT, NY-BR-I, NY-BR-62, NY-BR- 75, NY-BR-85, NY-BR-87, NY-BR-96, or a combination thereof.

[0030] In certain embodiments, the antigenic polypeptide is an antigenic arenavirus polypeptide. In certain embodiments, the antigenic polypeptide is an antigenic arenavirus gpl polypeptide. In certain embodiments, the antigenic polypeptide is an antigenic arenavirus stabilized GPC polypeptide.

[0031] In certain embodiments, the antigenic polypeptide is an antigenic arenavirus polypeptide or a functional fragment thereof comprising an amino acid sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 46. In certain embodiments, the antigenic arenavirus polypeptide or a functional fragment thereof comprises the amino acid sequence set forth in SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 46.

[0032] In certain embodiments, the antigenic arenavirus polypeptide or a functional fragment thereof comprises from amino acid 59 to amino acid 262 of the amino acid sequence set forth in SEQ ID NO: 44, from amino acid 263 to amino acid 496 of the amino acid sequence set forth in SEQ ID NO: 44, or from amino acid 59 to amino acid 496 of the amino acid sequence set forth in SEQ ID NO: 44. In certain embodiments, the antigenic arenavirus polypeptide or a functional fragment thereof comprises from amino acid 59 to amino acid 21 of the amino acid sequence set forth in SEQ ID NO: 45, from amino acid 252 to amino acid 285 of the amino acid sequence set forth in SEQ ID NO: 45, or from amino acid 59 to amino acid 485 of the amino acid sequence set forth in SEQ ID NO: 45. In certain embodiments, the antigenic arenavirus polypeptide or a functional fragment thereof comprises from amino acid 59 to amino acid 245 of the amino acid sequence set forth in SEQ ID NO: 46, from amino acid 246 to amino acid 479 of the amino acid sequence set forth in SEQ ID NO: 46, or from amino acid 59 to amino acid 479 of the amino acid sequence set forth in SEQ ID NO: 46.

[0033] In certain embodiments, the antigenic polypeptide is an antigenic HIV-1 polypeptide. In certain embodiments, the antigenic polypeptide is an antigenic HIV-1 Env gpl20 polypeptide. In

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[0035] PATENT certain embodiments, the antigenic polypeptide is an antigenic HIV-1 stabilized Env trimer polypeptide. In certain embodiments, the antigenic polypeptide is an antigenic HIV-1 gpl40 polypeptide.

[0036] In certain embodiments, the antigenic polypeptide is an antigenic coronavirus polypeptide. In certain embodiments, the antigenic coronavirus polypeptide comprises a coronavirus spike protein or a functional fragment thereof, a receptor-binding domain (RBD) or a functional fragment thereof, an N-terminal domain (NTD) or a functional fragment thereof, a receptorbinding domain (RBD)-N-terminal domain chimera or a functional fragment thereof, an SI domain or a functional fragment thereof, a stabilized extracellular spike S-2P domain or a functional fragment thereof, a stabilized extracellular spike S polypeptide or a functional fragment thereof, a stabilized extracellular spike S-trimer or a functional fragment thereof, a receptorbinding domain (RBD)-S-trimer chimera, a RBD-RBD-S-trimer chimera, or a combination thereof.

[0037] In certain embodiments, the antigenic coronavirus polypeptide comprises a coronavirus spike protein or a functional fragment thereof. In certain embodiments, the coronavirus spike protein or a functional fragment thereof comprises an amino acid sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or at least 100% identical to the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, the coronavirus spike protein or a functional fragment thereof comprises the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, the coronavirus spike protein or a functional fragment thereof comprises from amino acid 1 to amino acid 685 of the amino acid sequence set forth in SEQ ID NO: 4, from amino acid 14 to amino acid 685 of the amino acid sequence set forth in SEQ ID NO: 4, or from amino acid 686 to amino acid 1273 of the amino acid sequence set forth in SEQ ID NO: 4.

[0038] In certain embodiments, the antigenic coronavirus polypeptide comprises a receptorbinding domain (RBD) or a functional fragment thereof. In certain embodiments, the RBD polypeptide comprises an amino acid sequence that is at least about 80%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 5. In certain embodiments, the RBD polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 5.

[0039] In certain embodiments, the antigenic coronavirus polypeptide comprises an N-terminal domain (NTD) or a functional fragment thereof. In certain embodiments, the NTD polypeptide comprises an amino acid sequence that is at least about 80%, at least about 80%, at least about

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[0041] PATENT

[0042] 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 6. In certain embodiments, the NTD polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 6.

[0043] In certain embodiments, the antigenic coronavirus polypeptide comprises an SI domain or a functional fragment thereof. In certain embodiments, the SI polypeptide comprises an amino acid sequence that is at least about 80%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. In certain embodiments, the SI polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9

[0044] In certain embodiments, the antigenic coronavirus polypeptide comprises a stabilized extracellular spike S-2P domain or a functional fragment thereof. In certain embodiments, the S- 2P polypeptide comprises an amino acid sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or at least 100% identical to the amino acid sequence set forth in SEQ ID NO: 10. In certain embodiments, the S-2P polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 10.

[0045] In certain embodiments, the vaccine further comprises a second antigenic polypeptide. In certain embodiments, the first antigenic polypeptide and the second antigenic polypeptide are different. In certain embodiments, the first antigenic polypeptide and the second coronavirus polypeptide are the same.

[0046] In certain embodiments, the nanoparticle-forming polypeptide and the antigenic polypeptide are coupled by a linker to thereby generate a fusion protein. In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23.

[0047] In certain embodiments, the adjuvant is selected from ADU-S100, saponin, poly(I:C), Army Liposomal Formulation ALF, Army Liposomal Formulation ALFA, Army Liposomal Formulation ALFQ, alhydrogel, monophosphoryl lipid A (MPLA), oil in water emulsions, ADJUPLEX™, ADDAVAX™, CARBOPOL®, Poly ICLC, Flagellin, Iscomatrix, CpG oligodeoxynucleotides, virosomes, MF59, AS04, or a combination thereof. In certain embodiments, the adjuvant is Army Liposomal Formulation ALFQ. In certain embodiments, the adjuvant is ADU-S100. In certain embodiments, the adjuvant is selected from QS-21, QS-18, QS-

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[0049] PATENT

[0050] 7, or QS-9. In certain embodiments, the adjuvant is QS-21. In certain embodiments, the adjuvant is poly(I:C).

[0051] In certain embodiments, the vaccine and the adjuvant are loaded in the same microneedles. In certain embodiments, the vaccine and the adjuvant are pre-mixed and loaded in the same microneedles. In certain embodiments, the vaccine and the adjuvant are loaded in different microneedles. In certain embodiments, the vaccine is loaded on a metal-organic framework. In certain embodiments, the vaccine and the adjuvant are loaded on a metal-organic framework. In certain embodiments, the vaccine and the adjuvant are loaded on different metal-organic frameworks.

[0052] In certain embodiments, the base comprises poly (lactic-co-glycolic acid) (PLGA) or carboxymethylcellulose (CMC). In certain embodiments, each microneedle is dissolvable or water-soluble. In certain embodiments, each microneedle comprises carboxymethyl cellulose, trehalose, sucrose, gelatin, glucose, lactose, collagen, chitosan, poly-y-glutamate, polyvinylpyrrolidone, maltodextrin, silk, hyaluronic acid, poly (lactic-co-glycolic acid), poly(lactic acid), poly(vinyl alcohol), polyethylene glycol, or a combination thereof. In certain embodiments, each microneedle comprises carboxymethyl cellulose and trehalose. In certain embodiments, each microneedle comprises a layered structure comprising a tip. In certain embodiments, the vaccine is located at the tip of the microneedle.

[0053] In certain non-limiting embodiments, the present disclosure provides a method of manufacturing an administration device disclosed herein. In certain embodiments, the method comprises forming or providing a production mold of a flexible material, wherein the production mold comprising a plurality of cavities that are shaped to define a plurality of respective microneedles having a stem, a head, a filleted base, and at least one undercut feature. In certain embodiments, the method comprises delivering a first dissolvable or water-soluble material into at least the microneedle head portion defined by the respective cavities of the production mold, and prior to or during delivery of the first dissolvable or water-soluble material into at least the microneedle head portion, incorporating a vaccine into the first dissolvable or water-soluble material to produce a dissolvable or water-soluble matrix. In certain embodiments, the method comprises delivering the first dissolvable or water-soluble material and / or one or more additional dissolvable or water-soluble materials into the cavity and forming a plurality of microneedles in the production mold that include the dissolvable or water-soluble matrix. In certain embodiments, the method comprises removing the microneedles from the production mold by pulling the microneedles out of the mold, wherein the flexible material of the production mold has sufficient elasticity to allow for the molded microneedle array to be removed from the production mold. In

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[0055] PATENT certain embodiments, the method comprises loading a vaccine comprising a nanoparticle-forming polypeptide, an antigenic polypeptide in each microneedle, and an adjuvant.

[0056] In certain embodiments, the production mold defines at least one undercut feature in the microneedles directly below a microneedle head. In certain embodiments, the stem is formed from the first dissolvable or water-soluble material. In certain embodiments, the first dissolvable or water-soluble material comprises carboxymethyl cellulose, trehalose, polyvinylpyrrolidone, maltodextrin, silk, hyaluronic acid, poly (lactic-co-glycolic acid), poly (lactic acid), poly (vinyl alcohol), polyethylene glycol, or a combination thereof. In certain embodiments, at least a portion of the stem is formed from a non-dissolvable material. In certain embodiments, the method further delivering a second dissolvable material into the production mold to form a dissolving layer at a portion of the stem. In certain embodiments, said portion of the stem is adjacent to the microneedle head. In certain embodiments, the second dissolvable material of the dissolving layer is a material that dissolves more quickly than first dissolvable or water-soluble material.

[0057] In certain non-limiting embodiments, the present disclosure provides an administration device prepared by the methods disclosed herein.

[0058] In certain non-limiting embodiments, the present disclosure provides an article of manufacture comprising a device of disclosed herein. In certain embodiments, the article of manufacture further comprises instructions for applying the device to a subject. In certain embodiments, the article has extended stability. In certain embodiments, the article has extended stability at room temperature.

[0059] In certain non-limiting embodiments, the present disclosure provides a method of preventing and / or treating an infectious disease in a subject in need thereof. In certain embodiments, the method comprises inserting the plurality of microneedles of the device disclosed herein, wherein the inserting comprises penetrating the stratum comeum to deliver an effective amount of the vaccine to the epidermis and / or dermis without systemic exposure.

[0060] In certain embodiments, the infectious disease is a viral disease, a bacterial disease, or a parasitic disease. In certain embodiments, the infectious disease is COVID-19.

[0061] In certain non-limiting embodiments, the present disclosure provides a method of inducing an antibody response to a pathogen in a subject in need thereof. In certain embodiments, the method comprises inserting the plurality of microneedles of the device disclosed herein, wherein the inserting comprises penetrating the stratum corneum to deliver an effective amount of the vaccine to the epidermis and / or dermis without systemic exposure.

[0062] In certain non-limiting embodiments, the present disclosure provides a method of inducing a neutralizing immune response to a pathogen in a subject in need thereof. In certain

[0063] ACTIVE 508396272 8 072396.1082

[0064] PATENT embodiments, the method comprises inserting the plurality of microneedles of the device disclosed herein, wherein the inserting comprises penetrating the stratum comeum to deliver an effective amount of the vaccine to the epidermis and / or dermis without systemic exposure.

[0065] In certain embodiments, the pathogen is a virus, a bacterium, or a parasite. In certain embodiments, the pathogen is an adenovirus, a herpesvirus, an Epstein Barr virus, a herpes simplex type 1, a herpes simplex type 2, a human herpes virus simplex type 8, a human cytomegalovirus, a varicella-zoster virus, a poxvirus, a parvovirus, a papillomavirus, a reovirus, a picomavirus, a coxsackie virus, a hepatitis A virus, a poliovirus, a togavirus, a rubella virus, a flavivirus, a hepatitis C virus, a yellow fever virus, a dengue virus, a west Nile virus, a coronavirus, a orthomyxovirus, an influenza virus, a rhabdovirus, a paramyxovirus, a measles virus, a mumps virus, a parainfluenza virus, a respiratory syncytial virus, a metapneumovirus, a Lassa virus, a LuJo virus, a Junin virus, a Machupo virus, a Bear Canyon virus, a Wenzhou virus, a Guanarito virus, a Latino virus, a Sabia virus, a Chapare virus, a Whitewater Arroyo virus, a Pichinde virus, an LCMV virus, a Mopeia virus, a Mobala virus, an Ippy virus, a Mobala virus, a Morogoro virus, a rhabdovirus, a rabies virus, ebola, a retrovirus, a human immunodeficiency virus (HIV), a hepadnavirus, or a hepatitis B virus. In certain embodiments, the pathogen is SARS-CoV-2.

[0066] In certain embodiments, the pathogen is selected from Bacillus spp., Bordetella spp., Borrelia spp., Brucella spp., Campylobacter spp., Chlamydia spp., Clostridium spp., Corynebacterium spp., Enterococcus spp., Escherichia spp., Francisella spp., Haemophilus spp., Helicobacter spp., Legionella spp., Leptospira spp., Listeria spp., Mycobacterium spp., Mycoplasma spp., Neisseria spp., Pseudomonas spp., Rickettsia spp., Salmonella spp., Shigella spp., Staphylococcus spp., Streptococcus spp., Treponema spp., Vibrio spp., and Yersinia spp.. In certain embodiments, the pathogen is selected from Ancylostoma spp., Anisakis spp., Ascaris lumbricoides, Balantidium coli. Cestoda spp., Cimicidae spp., Clonorchis sinensis, Dicrocoelium dendrilicum, Dicrocoelium hospes. Diphyllobothrium latum, Dracunculus spp., Echinococcus spp., Entamoeba histolytica, Enter obius vermicular is, Fasciola spp., Fasciolopsis buski, Giardia spp., Gnathostoma spp., Hymenolepis spp., Leishmania spp., Loa loa, Metorchis spp., Necator americanus, Oestroidea spp., Onchocercidae spp., Opisthorchis spp., Plasmodium spp., Protofasciola robusta, Parafasciolopsis fasciomorphae, Paragonimus westermani, Schistosoma spp., Spirometra erinaceieuropaei, Strongyloides stercoralis, Taenia spp., Toxocara spp., Toxoplasma spp., Trichobilharziaregenti, Trichinella spiralis, Trichuris trichiura, Trombiculidae spp., Trypanosoma spp., Tunga penetrans, and Wuchereria bancrofti.

[0067] In certain non-limiting embodiments, the present disclosure provides a method of primeboost vaccination against a pathogen in a subject in need thereof comprising inserting the plurality

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[0069] PATENT of microneedles of a prime device and a boost device. In certain embodiments, each one of the prime device and the boost device is the administration device disclosed herein, wherein the inserting comprises penetrating the stratum comeum to deliver an effective amount of the vaccine to the epidermis and / or dermis without systemic exposure.

[0070] In certain non-limiting embodiments, the present disclosure provides a method of primeboost vaccination against a pathogen in a subject in need thereof comprising inserting the plurality of microneedles of a prime device disclosed herein, wherein the inserting comprises penetrating the stratum comeum to deliver an effective amount of the vaccine to the epidermis and / or dermis without systemic exposure. In certain non-limiting embodiments, the present disclosure provides a method of prime-boost vaccination against a pathogen in a subject in need thereof comprising inserting the plurality of microneedles of a boost device disclosed herein, wherein the inserting comprises penetrating the stratum corneum to deliver an effective amount of the vaccine to the epidermis and / or dermis without systemic exposure.

[0071] In certain embodiments, the pathogen is an adenovirus, a herpesvirus, an Epstein Barr virus, a herpes simplex type 1, a herpes simplex type 2, a human herpes virus simplex type 8, a human cytomegalovirus, a varicella-zoster virus, a poxvirus, a parvovirus, a papillomavirus, a reovirus, a picomavirus, a coxsackie virus, a hepatitis A virus, a poliovirus, a togavirus, a rubella virus, a flavivirus, a hepatitis C virus, a yellow fever virus, a dengue virus, a west Nile virus, a coronavirus, a orthomyxovirus, an influenza virus, a rhabdovirus, a paramyxovirus, a measles virus, a mumps virus, a parainfluenza virus, a respiratory syncytial virus, a metapneumovirus, a Lassa virus, a LuJo virus, a Junin virus, a Machupo virus, a Bear Canyon virus, a Wenzhou virus, a Guanarito virus, a Latino virus, a Sabia virus, a Chapare virus, a Whitewater Arroyo virus, a Pichinde virus, an LCMV virus, a Mopeia virus, a Mobala virus, an Ippy virus, a Mobala virus, a Morogoro virus, a rhabdovirus, a rabies virus, ebola, a retrovirus, a human immunodeficiency virus (HIV), a hepadnavirus, or a hepatitis B virus. In certain embodiments, the pathogen is SARS-CoV-2. In certain embodiments, the method further comprises identifying the subject as having COVID-19.

[0072] In certain embodiments, the pathogen is selected from Bacillus spp., Bordetella spp., Borrelia spp., Brucella spp., Campylobacter spp., Chlamydia spp., Clostridium spp., Corynebacterium spp., Enterococcus spp., Escherichia spp., Francisella spp., Haemophilus spp., Helicobacter spp., Legionella spp., Leptospira spp., Listeria spp., Mycobacterium spp., Mycoplasma spp., Neisseria spp., Pseudomonas spp., Rickettsia spp., Salmonella spp., Shigella spp., Staphylococcus spp., Streptococcus spp., Treponema spp., Vibrio spp., and Yersinia spp.. In certain embodiments, the pathogen is selected from Ancylostoma spp., Anisakis spp., Ascaris

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[0074] PATENT lumbricoides. Balantidium coli, Cestoda spp., Cimicidae spp., Clonorchis sinensis, Dicrocoelium dendrilicum, Dicrocoelium hospes. Diphyllobothrium latum, Dracunculus spp., Echinococcus spp., Entamoeba histolytica, Enter obius vermicular is, Fasciola spp., Fasciolopsis buski, Giardia spp., Gnathostoma spp., Hymenolepis spp., Leishmania spp., Loa loa, Metorchis spp., Necator americanus, Oestroidea spp., Onchocercidae spp., Opisthorchis spp., Plasmodium spp., Protofasciola robusta, Parafasciolopsis fasciomorphae, Paragonimus westermani, Schistosoma spp., Spirometra erinaceieuropaei, Strongyloides stercoralis, Taenia spp., Toxocara spp., Toxoplasma spp., Trichobilharziaregenti, Trichinella spiralis, Trichuris trichiura, Trombiculidae spp., Trypanosoma spp., Tunga penetrans, and Wuchereria bancrofti.

[0075] In certain non-limiting embodiments, the present disclosure provides a method of preventing and / or treating a tumor or cancer in a subject in need thereof. In certain embodiments, the method comprises inserting the plurality of microneedles of the device disclosed herein, wherein the inserting comprises penetrating the stratum corneum to deliver an effective amount of the vaccine to the epidermis and / or dermis without systemic exposure.

[0076] In certain non-limiting embodiments, the present disclosure provides a method of inducing an immune response against a tumor or cancer in a subject in need thereof. In certain embodiments, the method comprises inserting the plurality of microneedles of the device disclosed herein, wherein the inserting comprises penetrating the stratum corneum to deliver an effective amount of the vaccine to the epidermis and / or dermis without systemic exposure.

[0077] In certain non-limiting embodiments, the present disclosure provides a method of primeboost vaccination against a tumor or cancer in a subject in need thereof comprising inserting the plurality of microneedles of a prime device and a boost device, wherein each one of the prime device and the boost device is the administration device disclosed herein, wherein the inserting comprises penetrating the stratum corneum to deliver an effective amount of the vaccine to the epidermis and / or dermis without systemic exposure.

[0078] In certain non-limiting embodiments, the present disclosure provides a method of primeboost vaccination against a tumor or cancer in a subject in need thereof comprising inserting the plurality of microneedles of a prime device disclosed herein, wherein the inserting comprises penetrating the stratum corneum to deliver an effective amount of the vaccine to the epidermis and / or dermis without systemic exposure.

[0079] In certain non-limiting embodiments, the present disclosure provides a method of primeboost vaccination against a tumor or cancer in a subject in need thereof comprising inserting the plurality of microneedles of a boost device disclosed herein, wherein the inserting comprises

[0080] ACTIVE 508396272 11 072396.1082 PATENT penetrating the stratum corneum to deliver an effective amount of the vaccine to the epidermis and / or dermis without systemic exposure.

[0081] In certain embodiments, the tumor or cancer is selected from melanoma, neuroblastoma, ovarian cancer, synovial sarcoma, myxoid / round cell liposarcoma, breast cancer, squamous cell carcinoma, mesothelioma, adult and pediatric sarcomas, hematological cancers, prostate cancer, lung adenocarcinoma, esophageal squamous cell carcinoma, gastric cancer, bladder cancer, hepatocellular carcinoma, brain cancer, multiple myeloma, Ewing's sarcoma, rhabdomyosarcoma, germ cell tumors, Burkitt lymphoma, osteosarcoma, renal cell cancer, colon cancer, acute myeloid leukemia, colorectal cancer, glioblastoma, pancreatic cancer, chronic myeloid leukemia, nasopharyngeal carcinoma, cervical cancer, lymphomas, or renal cell carcinoma. In certain embodiments, the tumor or cancer is associated with a tumor antigen selected from gp 100, MART- 1 / Melan A, gp75 (TRP-I), tyrosinase, NY-ESO-I, melanoma proteoglycan, MAGE family antigens (i.e., MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-6, and MAGE-12), BAGE family antigens, GAGE family antigens (i.e., GAGE-1, GAGE-2), RAGE family antigens, N- acetylglucosaminyltransf erase- V, pl 5, P-catenin, MUM-I, cyclin dependent kinase-4 (CDK4), p21-ras, BCR- abl, p53, pl85 HER2 / neu, epidermal growth factor receptor (EGFR), carcinoembryonic antigens (CEA), carcinoma- associated mutated mucins (i.e., MUC-1 gene products), EBNA gene products of EBV (i.e., EBNA-I), E7, E6 proteins of human papillomavirus, prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), idiotypic epitopes or antigens, for example, immunoglobulin idiotypes or T cell receptor idiotypes, KSA, kinesin 2, HIP-55, TGFP-1 anti- apoptotic factor, tumor protein D52, HIFT, NY-BR-I, NY-BR-62, NY-BR- 75, NY-BR-85, NY-BR-87 and NY-BR-96

[0082] In certain embodiments, the subject is a human subject.

[0083] BRIEF DESCRIPTION OF THE DRAWINGS

[0084] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0085] Figure 1 shows a schematic of the presently disclosed vaccines.

[0086] Figures 2A-2F illustrate dissolvable microarray patch ferritin nanoparticle vaccines. Figure 2A shows dissolvable microarray patch (MAP)-based skin-targeted vaccine delivery platform. Figure 2B shows dissolvable MAP Spike protein Ferritin Nanoparticle (SpFN) vaccine. Figure 2C shows dissolvable MAP ADU-SlOO-adjuvanted SpFN vaccine. Figure 2D shows

[0087] ACTIVE 508396272 12 072396.1082

[0088] PATENT dissolvable MAP QS-21-adjuvanted SpFN vaccine. Figure 2E shows dissolvable MAP Poly(I:C)- adjuvanted SpFN vaccine. Figure 2F shows dissolvable MAP ALFQ adjuvanted SpFN vaccine. Scale bars in Figures 2B-2F = 500 pm.

[0089] Figures 3 A and 3B illustrate antibody responses elicited by dissolvable MAP Spike protein Ferritin Nanoparticle (SpFN) vaccine. Figure 3 A shows dissolvable microarray patches (MAPs) loaded with Spike protein Ferritin Nanoparticle (SpFN) vaccine induced robust and potent SARS- CoV-2-specific binding antibody responses that are superior to those elicited by the intramuscular injection of SpFN. Mice were immunized using MAPs loaded with SpFN (3 pg) or by intramuscular (IM) inj ection of SpFN (3 pg) on days 0 and 21. SARS-CoV-2 S 1 -specific total IgG concentrations in serum of immunized mice at 2 weeks and 5 weeks after primary immunization were measured by ELISA. Figure 3B shows dissolvable microarray patches (MAPs) loaded with Spike protein Ferritin Nanoparticle (SpFN) vaccine induce potent SARS-CoV-2-specific neutralizing antibodies that are superior to those elicited by intramuscular injection of SpFN. Mice were immunized using MAPs loaded with SpFN (3 pg) or by IM inj ection of SpFN (3 pg) on days 0 and 21. SARS-CoV-2-specific (Wuhan strain) neutralizing antibody titers (ID50) from mice 5 weeks after primary immunization were determined using an in vitro pseudovirus neutralization assay.

[0090] Figures 4A and 4B illustrate dissolvable microarray patches (MAPs) loaded with Spike protein Ferritin Nanoparticle (SpFN) vaccine induced potent SARS-CoV-2-specific humoral immune responses, such as binding antibody titers and neutralizing antibody levels, which are superior to those elicited by intramuscular injection of SpFN. Mice were immunized using MAPs loaded with SpFN (3 pg) or by IM injection of SpFN (3 pg) through different immunization schedules. SARS-CoV-2 SI -specific total IgG concentrations in serum of immunized mice were measured by ELISA and SARS-CoV-2-specific (Wuhan strain) neutralizing antibody titers (ID50) were determined using an in vitro pseudovirus neutralization assay. Figure 4A shows neutralization data at day 19. Figure 4B shows neutralization data at week 5.

[0091] Figure 5 shows dissolvable microarray patches (MAPs) loaded with Spike protein Ferritin Nanoparticle (SpFN) vaccine retain their immunogenicity for at least 45 days without refrigeration. Mice were immunized (single dose) by fresh SpFN (3 pg) MAPs that were freshly prepared or stored for 45 days at room temperature (RT) after fabrication, and SARS-CoV-2 Sl- specific total IgG concentrations in serum of immunized mice at 2 weeks after primary immunization were measured by ELISA.

[0092] Figures 6A-6E illustrate the characterization of humoral immune response with different adjuvants. Figures 6A and 6B show dissolvable microarray patches (MAPs) loaded with the Army

[0093] ACTIVE 508396272 13 072396.1082

[0094] PATENT

[0095] Liposomal Formulation ALFQ-adjuvanted Spike protein Ferritin Nanoparticle (SpFN) vaccine induce potent SARS-CoV-2-specific humoral immune responses, such as binding antibody titers and neutralizing antibody levels, which are superior or comparable to those elicited by intramuscular injection of ALFQ-adjuvanted SpFN. Mice were immunized using MAPs loaded with SpFN+ALFQ or by IM injection of SpFN+ALFQ through different immunization schedules. SARS-CoV-2 SI -specific total IgG concentrations in serum of immunized mice were measured by ELISA, and SARS-CoV-2-specific (Wuhan strain) neutralizing antibody titers (ID50) were determined using an in vitro pseudovirus neutralization assay. Figures 6C-6E show dissolvable microarray patches (MAPs) loaded with the adjuvanted (with the STING pathway agonist ADU- S100, the saponin adjuvant QS-21, and the TLR3 agonist Poly(I:C)) Spike protein Ferritin Nanoparticle (SpFN) vaccine induced potent SARS-CoV-2-specific humoral immune responses. Mice were immunized through different immunization schedules. SARS-CoV-2 SI -specific total IgG concentrations in serum of immunized mice were measured by ELISA. Figure 6A shows neutralization data at day 19 with vaccines including ALFQ. Figure 6B shows neutralization data at week 5 with vaccines including ALFQ. Figure 6C shows neutralization data with vaccines including ADU-S100. Figure 6D shows neutralization data with vaccines including QS-21. Figure 6E shows neutralization data with vaccines including Poly(I:C).

[0096] Figures 7A-7D illustrate characterization of T cell immune response with different adjuvants. Mice were immunized on Week 0 and boosted on Week 2. 5 days after boost, splenocytes were obtained and stimulated with antigen-specific PepTivator, followed by intracellular cytokine staining (ICS) and flow cytometry, to determine systemic cellular immune responses in terms of frequencies of antigen-specific CD4+ and CD8+ T cells in spleens. Figure 7A shows dissolvable microarray patches (MAPs) loaded with Spike protein Ferritin Nanoparticle (SpFN) vaccine, with or without Army Liposomal Formulation ALFQ, induced potent SARS- CoV-2-specific systemic polyfunctional T-cell immune responses. Figure 7B shows dissolvable microarray patches (MAPs) loaded with Spike protein Ferritin Nanoparticle (SpFN) vaccine, with or without Army Liposomal Formulation ALFQ, induced potent SARS-CoV-2-specific pulmonary polyfunctional T-cell immune responses. Figures 7C shows dissolvable microarray patches (MAPs) loaded with the STING agonist ADU-SlOO-adjuvanted Spike protein Ferritin Nanoparticle (SpFN) vaccine induced potent SARS-CoV-2-specific polyfunctional T-cell immune responses in spleen. Figure 7D shows dissolvable microarray patches (MAPs) loaded with the STING agonist ADU-SlOO-adjuvanted Spike protein Ferritin Nanoparticle (SpFN) vaccine induced potent SARS-CoV-2-specific polyfunctional T-cell immune responses in lung.

[0097] ACTIVE 508396272 14 072396.1082

[0098] PATENT

[0099] Figures 8A-8C illustrate characterization of the elicited humoral pseudovirus neutralizing immune response by MAPs containing 0.1 pg SpFN alone or with different adjuvants, against diverse sarbecoviruses. Figure 8A shows dissolvable microarray patches (MAPs) loaded with the Army Liposomal Formulation ALFQ-adjuvanted Spike protein Ferritin Nanoparticle (SpFN) vaccine induced potent SARS-CoV-2-specific neutralizing antibody levels against WA-1, Alpha, Beta, Delta, and Omicron BA. l SARS-CoV-2 variants, and SARS-CoV-1 Urbani strain. Figure 8B shows that the use of SpFN alone at a low dose can elicit neutralizing responses. Addition of adjuvant increases the neutralization responses against SARS-CoV-2 variants, while neutralization responses are high and consistent with the ALFQ adjuvanted group. Figure 8C shows the neutralization response for the four 0.1 pg SpFN groups either non-adjuvanted or adjvanted tested against the SARS-CoV-1 pseudovirus strain. There were statistically significant differences in the neutralizing responses across the four groups with the ADU-S100 and ALFQ groups showing the highest responses^

[0100] Figures 9A-9F depict engineering and quality control of microneedle patch ferritin nanoparticle vaccines. Figure 9A shows a representative photo of a globally deployable skin- targeted immunization platform with an array (10x10) of CMC / Trehalose MNP loaded with SpFN and ALFQ (scale bar = 10 mm). Figure 9B shows an optical stereomicroscopy image of CMC / Trehalose microneedles integrating SpFN and ALFQ (scale bar = 500 pm). Figure 9C shows a scanning electron microscopy (SEM) image of a CMC / Trehalose obelisk-shaped microneedle incorporating SpFN and ALFQ (scale bar = 100 pm). Figure 9D shows an optical stereomicroscopy image of CMC / Trehalose microneedles loaded with SpFN+ALFQ after application to the abdominal skin of a mouse in vivo for 15 min, revealing the remaining needle materials upon murine skin penetration and nearly complete dissolution (scale bar = 500 pm). Figure 9E shows an SEM image of CMC / Trehalose microneedles integrating SpFN and ALFQ after application to the abdominal skin of a mouse in vivo for 15 min, revealing the remaining microneedle materials upon murine skin penetration and nearly complete dissolution (scale bar = 250 pm). Figure 9F shows a graph representative of total endotoxin content in CMC / Trehalose MNPs incorporating SpFN and SpFN+ALFQ, determined by chromogenic limulus amebocyte lysate (LAL) assay through dissolving these MNPs in endotoxin-free water (Data = Mean+Standard Deviation from n = 6 CMC / Trehalose MNPs loaded with SpFN and SpFN+ALFQ).

[0101] Figures 10A-10F depict the production and quality control of microneedle patch ferritin nanoparticle vaccines. Figure 10A shows an optical stereomicroscopy image of a CMC / Trehalose MNP loaded with SpFN-AF647 and ALFQ-CM-Dil (scale bar = 500 pm). Figure 10B shows a

[0102] ACTIVE 508396272 15 072396.1082

[0103] PATENT brightfield microscopy image of a high-quality CMC / Trehalose microneedle integrating fluorescent-labeled vaccine components, SpFN-AF647 and ALFQ-CM-Dil (scale bar = 100 pm). Figure 10C shows a fluorescent microscopy image of a high-integrity CMC / Trehalose microneedle loaded with SpFN-AF647 and ALFQ-CM-Dil, with a filter corresponding to AF647 (scale bar = 100 pm). Figure 10D shows a fluorescent microscopy image of a high-quality CMC / Trehalose microneedle loaded with SpFN-AF647 and ALFQ-CM-Dil, with a filter corresponding to CM-Dil (scale bar = 100 pm). Figure 10E shows a merged microscopy image of a high-quality CMC / Trehalose microneedle loaded with SpFN-AF647 and ALFQ-CM-Dil (scale bar = 100 pm). Figure 10F shows quantitative data of the reproducibility of FN vaccine loading in MNPs (Data = Mean±Standard Deviation (SD) from n =5 SpFN-AF647+ALFQ-CM- Dil MNPs).

[0104] Figures 11A-11C depict skin-targeted delivery of ferritin nanoparticle vaccines (e.g., ALFQ adjuvanted spike protein ferritin nanoparticle-SpFN) using dissolvable microneedle patches in vivo in mice. Figures 11A and 11B show an in vivo fluorescent image of the whole mouse using the filters corresponding to SpFN-AF647 and ALFQ-CM-Dil after in vivo skin application (15 min) and removal of a SpFN-AF647+ALFQ-CM-Dil MNP (scale bars = 10 mm). Figure 11C shows epi-fluorescence microscopy images of the cryo-sectioned SpFN- AF647+ALFQ-CM-Dil MNP -treated murine skin shortly after the removal of MNP (scale bars = 100 pm).

[0105] Figures 12A-12D depict time-dependent pro-inflammatory skin microenvironment responses to microneedle patch ferritin nanoparticle vaccines in vivo in mice. Figure 12A shows fold changes in the expression levels of CCL2. Figure 12B shows fold changes in the expression levels of IFN-p. Figure 12C shows fold changes in the expression levels of CCL5. Figure 12D shows fold changes in the expression levels of CXCL10.

[0106] Figures 13A and 13B depict skin-targeted ferritin-nanoparticle (SpFN±adjuvant MNPs) vaccine-induced total IgG antibody responses in both young and aged mice. Figure 13 A shows a schematic of the experimental procedures. Figure 13B shows serum levels of total IgG antibodies elicited by MNP-FN vaccines in young and aged mice.

[0107] Figure 14A and 14B depict skin-targeted ferritin-nanoparticle vaccine (SpFN±adjuvant MNPs)-induced neutralizing antibody responses. Figure 14A shows a schematic of the experimental procedures. Figure 14B shows serum levels of WA-1 strain-specific and Betastrain-specific neutralizing antibody titers elicited by MNP-FN vaccines in mice.

[0108] Figures 15A and 15B depict the kinetics of the serum levels of skin-targeted ferritin- nanoparticle vaccine (SpFN±ADU-S100 MNPs)-induced total IgG antibody responses. Figure

[0109] ACTIVE 508396272 16 072396.1082 PATENT

[0110] 15A shows a schematic of the experimental procedures. Figure 15B shows serum levels of antigen-specific total IgG antibodies induced by MNP-FN vaccines in mice.

[0111] Figures 16A and 16B depict skin-targeted ferritin-nanoparticle vaccine-induced total IgG (MgpFN±adjuvant MNPs) antibody responses. Figure 16A depicts a schematic of the experimental procedures. Figure 16B shows serum levels of total IgG antibody elicited by MNP- FN MACV vaccines in mice.

[0112] Figures 17A-17D show skin-targeted ferritin-nanoparticle vaccine (JgpFN+ALFQ MNPs and GgpFN+ALFQ MNPs)-induced total IgG antibody responses with respect to intramuscularly injected ferritin-nanoparticle vaccine (JgpFN+ALFQ IM and GgpFN+ALFQ IM)-induced total IgG antibody responses in mice. Figure 17A depicts a schematic of the experimental procedures using JgpFN+ALFQ MNPs. Figure 17B shows serum levels of total IgG antibodies elicited by JgpFN+ALFQ MNPs. Figure 17C depicts a schematic of the experimental procedures using GgpFN+ALFQ MNPs. Figure 17D shows serum levels of total IgG antibodies elicited by GgpFN+ALFQ MNPs.

[0113] Figure 18A and 18B show systemic (spleen) and pulmonary (lungs) cellular responses induced by skin-targeted ferritin nanoparticle vaccines (SpFN+adjuvant MNPs) in both young and aged mice. Figure 18A shows a schematic of the experimental procedures. Figure 18B shows systemic antigen-specific CD4+T-cell responses elicited by our skin-targeted FN vaccines in young and aged mice. Figure 18C shows systemic antigen-specific CD8+T-cell responses elicited by our skin-targeted FN vaccines in young and aged mice. Figure 18D shows pulmonary antigenspecific CD4+T-cell responses induced by our skin-targeted FN vaccines in young and aged mice. Figure 18E shows pulmonary antigen-specific CD8+T-cell responses induced by our skin-targeted FN vaccines in young and aged mice.

[0114] Figure 19A and 19B show time-dependent serum levels of the cytokine IL-6 after immunization with skin-targeted ferritin nanoparticle vaccines (SpFN+ALFQ MNPs) in both young and aged mice. Figure 19A shows a schematic of the experimental procedures. Figure 19B shows serum levels of IL-6.

[0115] Figures 20A and 20B show the thermostability of skin-targeted ferritin nanoparticle vaccines (SpFN+ALFQ MNPs). Figure 20A shows a schematic of the experimental procedures. Figure 20B shows a histogram of the anti-spike total IgG detected at different time points.

[0116] DETAILED DESCRIPTION OF THE INVENTION

[0117] The emergence of SARS-CoV-2, also named COVID-19, marks the seventh coronavirus to be isolated from humans, and the third to cause a severe disease after severe acute respiratory

[0118] ACTIVE 508396272 17 072396.1082 PATENT syndrome (SARS) and Middle East respiratory syndrome (MERS). The rapid spread of SARS- CoV-2 in 2020 prompted the World Health Organization to declare the COVID-19 outbreak to be a pandemic. The rapidly evolving and constantly changing epidemiology of this pandemic raises the need to elucidate the molecular biology of these coronaviruses. The present disclosure provides compositions and articles of manufacture that can be used to treat or prevent infectious diseases (e.g., coronavirus infection).

[0119] The present disclosure is based, in part, on the demonstration that application of ferritin nanoparticle vaccines using dissolvable microarrays (e.g., dissolvable microarray patch) induced immune responses characterized by robust clonal expansion and functional diversification. Surprisingly, the observed immune responses were significantly higher compared to the intramuscular administration of the same vaccine.

[0120] Importantly, as demonstrated in the examples below, the presently disclosed subject matter can elicit immune responses by using a desired antigenic polypeptide. Thus, the presently disclosed subject matter will be able to elicit an immune response independently of the type of antigen. Non-limiting embodiments of the present disclosure are described by the present specification and Examples. For purposes of clarity of disclosure and not by way of limitation, the detailed description is divided into the following subsections:

[0121] 1. Definitions;

[0122] 2. Vaccines;

[0123] 3. Adjuvants;

[0124] 4. Administration Devices;

[0125] 5. Methods of Treatment; and

[0126] 6. Articles of Manufacture.

[0127] / . Definitions

[0128] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this disclosed subject matter belongs. The following references provide one of skill with a general definition of many of the terms used in this disclosed subject matter: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

[0129] ACTIVE 508396272 18 072396.1082

[0130] PATENT

[0131] As used herein, the use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification can mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0132] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The present disclosure also contemplates other embodiments “comprising,” “consisting of,” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0133] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 3 or more than 3 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, c.g, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, c.g, within 5-fold, or within 2-fold, of a value.

[0134] An “individual” or “subject” herein is a vertebrate, such as a human or non-human animal, for example, a mammal. Mammals include, but are not limited to, humans, non-human primates, farm animals, sport animals, rodents, and pets. Non-limiting examples of non-human animal subjects include rodents such as mice, rats, hamsters, and guinea pigs; rabbits; dogs; cats; sheep; pigs; goats; cattle; horses; and non-human primates such as apes and monkeys.

[0135] As used herein, the term “disease” refers to any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ.

[0136] An “effective amount” or “therapeutically effective amount” is an amount effective, at dosages and for periods of time necessary, that produces a desired effect, c.g, the desired therapeutic or prophylactic result. In certain embodiments, an effective amount will not be effective in treating or preventing the infections described herein, even though such dosage is deemed to be a therapeutically effective amount by those of skill in the art. For convenience only, exemplary dosages, drug delivery amounts, therapeutically effective amounts, and therapeutic levels are provided herein. The therapeutically effective amount may vary based on the route of administration and dosage form, the age and weight of the subject, and / or the subject’s condition, including the type and severity of the coronavirus infection. In certain embodiments, an effective amount can be formulated and / or administered in a single dose. In certain embodiments, an effective amount can be formulated and / or administered in a plurality of doses, for example, as part of prime-boost protocol.

[0137] ACTIVE 508396272 19 072396.1082

[0138] PATENT

[0139] As used herein, the term “prime-boost” refers to a vaccination protocol that involves the administration of multiple doses and / or different types of vaccines (e.g., different antigenic polypeptides, different epitopes, and the like) in sequential doses to improve the immune response. The first dose, the "prime", initiates an immune response, while the second dose, the "boost," amplifies the immune memory and increases the magnitude and breadth of the response. This strategy is often used to achieve stronger and more durable immunity. In certain embodiments, prime and boost can have different administration routes.

[0140] As used herein, the term “treating,” or “treatment” refers to clinical intervention in an attempt to alter the disease course of the individual or cell being treated and can be performed either for prophylaxis or during the course of clinical pathology. Therapeutic effects of treatment include, without limitation, preventing occurrence or recurrence of disease (e.g., COVID-19), alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing cancer, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. By “preventing” is intended precluding or reducing the risk of an infection from developing in a subject exposed to a coronavirus, or to precluding or reducing the risk of developing a high viral load of coronavirus or reducing or eliminating histopathology or virus presence in the airways or lungs. Prevention can also refer to the prevention of a subsequent infection once an initial infection has been treated or cured. Prevention may also refer to the prevention of or reduction of risk of transmission of virus from one subject host to another subject host.

[0141] By “increase” is meant to alter positively by at least about 5%. A positive alteration can be an increase of about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, about 100% or more.

[0142] By “reduce” is meant to alter negatively by at least about 5%. A negative alteration can be a decrease of about 5%, about 10%, about 25%, about 30%, about 50%, about 75% or more, even by about 100%.

[0143] The terms “nucleic acid sequence” and “polynucleotide,” as used herein, refer to a single or double-stranded covalently-linked sequence of nucleotides in which the 3’ and 5’ ends on each nucleotide are joined by phosphodiester bonds. The polynucleotide can include deoxyribonucleotide bases or ribonucleotide bases, and can be manufactured synthetically in vitro or isolated from natural sources.

[0144] The terms “polypeptide,” “peptide,” “amino acid sequence” and “protein,” used interchangeably herein, refer to a molecule formed from the linking of at least two amino acids. The link between one amino acid residue and the next is an amide bond and is sometimes referred

[0145] ACTIVE 508396272 20 072396.1082

[0146] PATENT to as a peptide bond. A polypeptide can be obtained by a suitable method known in the art, including isolation from natural sources, expression in a recombinant expression system, chemical synthesis, or enzymatic synthesis. The terms can apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

[0147] As used herein, the term “mutation” refers to a mutation in an amino acid sequence or in a nucleic acid sequence. In certain embodiments, a mutation in an amino acid sequence can be a substitution (replacement), an insertion (addition), or a deletion (truncation) of at least one amino acid in the amino acid sequence. In certain embodiments, a mutation in a nucleic acid sequence can be a substitution (replacement), an insertion (addition), or a deletion (truncation) of at least nucleotide of the nucleic acid sequence.

[0148] As used herein, “a functional fragment” of a molecule or polypeptide includes a fragment of the molecule or polypeptide that retains at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% of the primary function of the molecule or polypeptide.

[0149] As used herein, the term “substantially identical” or “substantially homologous” refers to a polypeptide or a nucleic acid molecule exhibiting at least about 50% identical or homologous to a reference amino acid sequence (for example, any of the amino acid sequences described herein) or a reference nucleic acid sequence (for example, any of the nucleic acid sequences described herein). In certain embodiments, such a sequence is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% identical or homologous to the amino acid sequence or the nucleic acid sequence used for comparison.

[0150] As used herein, “conservative” amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid within the same group. For example, amino acids can be classified by charge: positively-charged amino acids include lysine, arginine, histidine, negatively-charged amino acids include aspartic acid, glutamic acid, neutral charge amino acids include alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Amino acids can also be classified by polarity: polar amino acids include arginine (basic polar), asparagine, aspartic acid (acidic polar), glutamic acid (acidic polar), glutamine, histidine (basic polar), lysine (basic polar), serine, threonine, and tyrosine; non-polar amino acids include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. In certain

[0151] ACTIVE 508396272 21 072396.1082 PATENT embodiments, no more than one, no more than two, no more than three, no more than four, no more than five residues within a specified sequence are altered. Exemplary conservative amino acid substitutions are shown in Table 1 below.

[0152] Table 1

[0153] As used herein, the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (z.e., % homology = # of identical positions / total # of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. The percent homology between two amino acid

[0154] ACTIVE 508396272 22 072396.1082

[0155] PATENT sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent homology between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0156] As used herein, the term “isolated” refers to any biological molecules (e.g., peptide, antibody) that have been substantially separated or purified away from other biological components in the environment (such as a cell) in which the component naturally occurs, i.e., other proteins or cellular components.

[0157] The term “dosage” is intended to encompass a formulation expressed in terms of total amounts for a given timeframe, for example, as pg / kg / hr, pg / kg / day, mg / kg / day, or mg / kg / hr. The dosage is the amount of an ingredient administered in accordance with a particular dosage regimen. A “dose” is an amount of an agent administered to a mammal in a unit volume or mass, e.g., an absolute unit dose expressed in mg of the agent. The dose depends on the concentration of the agent in the formulation, e.g., in moles per liter (M), mass per volume (m / v), or mass per mass (m / m). The two terms are closely related, as a particular dosage results from the regimen of administration of a dose or doses of the formulation. The particular meaning, in any case, will be apparent from the context.

[0158] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 as well as all intervening decimal values between the aforementioned integers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. Ranges disclosed herein, for example, “between about X and about Y” are, unless specified otherwise, inclusive of range limits about X and about Y as well as X and Y. With respect to sub-ranges, “nested sub-ranges” that extend from either endpoint of the range are specifically contemplated. For example, a nested sub-range of an exemplary range of 1 to 50 can include 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

[0159] ACTIVE 508396272 23 072396.1082 PATENT

[0160] As used herein, the term “pharmaceutical formulation” refers to a composition or preparation that allows an effective the biological activity of the active ingredient to be effective. Pharmaceutical formulations can include excipients (e.g., pharmacologically inactive substances used as a carrier for the active ingredient. In certain embodiments, the pharmaceutical formulations are sterile.

[0161] As used herein, the term “sterile” refers to a formulation free or essentially free from all living microorganisms or other life forms.

[0162] 2. Vaccines

[0163] The present disclosure provides vaccines useful for the treatment and / or prevention of infection-related diseases. Additionally or alternatively, the present disclosure provides vaccines useful for the treatment and / or prevention of a cancer. Furthermore, the present disclosure provides vaccines useful for eliciting an immune response against an antigen of interest. In certain embodiments, the present disclosure provides vaccines useful for the treatment and / or prevention of coronavirus infection related diseases.

[0164] In certain embodiments, the vaccines include a nanoparticle including certain immunogenic molecules. In certain embodiments, the vaccines include a fusion protein that is capable of self-assembly. In certain embodiments, the fusion protein includes a nanoparticleforming polypeptide and an antigenic polypeptide. In certain embodiments, the nanoparticleforming polypeptide is capable of self-assembly into a nanoparticle. In certain embodiments, the antigenic polypeptide includes at least one antigenic polypeptide. For example, but without any limitation, the antigenic polypeptide can include a first antigenic polypeptide and a second antigenic polypeptide. In certain embodiments, the nanoparticle-forming peptide and the antigenic peptide are connected by a linker.

[0165] 2.1. Nanoparticle-Forming Polypeptide

[0166] The nanoparticle-forming polypeptide of the presently disclosed vaccine can be any suitable nanoparticle-forming polypeptide. Use of nanoparticle-forming polypeptide allows the display of viral antigens is an effective approach that can enhance immunogenicity and the immune response. Non-limiting examples of nanoparticle-forming polypeptides include ferritin, lumazine synthase, and polypeptides of the SpyTag: SpyCatcher system. In certain embodiments, the nanoparticle-forming polypeptides include ferritin. In certain embodiments, the nanoparticleforming polypeptides include lumazine synthase. In certain embodiments, the nanoparticleforming polypeptides include polypeptides of the SpyTag: SpyCatcher system.

[0167] ACTIVE 508396272 24 072396.1082

[0168] PATENT

[0169] In certain embodiments, the nanoparticle-forming polypeptide is a ferritin polypeptide. In certain embodiments, the ferritin polypeptide is a Helicobacter pylori ferritin polypeptide or a functional fragment thereof.

[0170] In certain embodiments, the ferritin polypeptide includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, the ferritin polypeptide includes substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence set forth in SEQ ID NO: 1, that do not significantly alter the function or activity of the ferritin polypeptide.

[0171] In certain embodiments, the ferritin polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, the ferritin polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 1, which is provided below. ESQVRQQFSKDIEKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLI I FLNENNVPVQLTS I SAPEHKFEGLTQI FQKAYEHEQHI SES INNIVDHAIKSKDHATFNFLQW YVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKSGS [ SEQ ID NO : 1 ]

[0172] In certain embodiments, the ferritin polypeptide comprises a substitution of the glutamic acid residue (E) at position 13 of SEQ ID NO: 1. In certain embodiments, the ferritin polypeptide includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 3. In certain embodiments, the ferritin polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 3. In certain embodiments, the ferritin polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 3, which are provided below: DI IKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLI I FLNENNVPV QLTS I SAPEHKFEGLTQI FQKAYEHEQHI SES INNIVDHAIKSKDHATFNFLQWYVAEQHEEEV LFKDILDKIELIGNENHGLYLADQYVKGIAKSRKSGS [ SEQ ID NO : 2 ] SKDI IKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLI I FLNENNV PVQLTS I SAPEHKFEGLTQI FQKAYEHEQHI SES INNIVDHAIKSKDHATFNFLQWYVAEQHEE EVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKSGS [ SEQ ID NO : 3 ]

[0173] Additional examples of nanoparticle-forming polypeptides encompassed by the present disclosure can be found in International Patent Publication No. WO 2021 / 178987, the contents of which are incorporated by reference in their entireties.

[0174] 2.2. Antigenic Polypeptides

[0175] In certain embodiments, the present disclosure provides vaccines and articles of manufacture useful for the treatment and / or prevention of an infectious disease.

[0176] ACTIVE 508396272 25 072396.1082

[0177] PATENT

[0178] The antigenic polypeptide of the presently disclosed vaccine can be any suitable antigenic polypeptide that is able to trigger a humoral (innate) or cell-mediated immune response. In certain embodiments, the antigenic polypeptide is derived from a virus, a bacterium, a parasite, a plant, a protozoan, a fungus, a tissue, or a transformed cell (e.g., a cancer cell). In certain embodiments, the antigenic polypeptide is associated with one or more diseases or conditions including, for example and without any limitation, infectious diseases, autoimmune diseases, and cancer.

[0179] In certain embodiments, the antigenic polypeptide is isolated from any portion of a virus. For example, but without any limitation, the antigenic polypeptide can be a natural viral capsid structure or a portion thereof, or a composite of a structure of multiple strains of the virus.

[0180] Non-limiting examples of viruses from which an antigenic polypeptide is obtained or derived include dsDNA viruses (e.g., adenovirus, herpesvirus, Epstein Barr virus, herpes simplex type 1, herpes simplex type 2, human herpes virus simplex type 8, human cytomegalovirus, varicella-zoster virus, poxvirus), ssDNA viruses (e.g., parvovirus, papillomavirus (e.g., El, E2, E3, E4, E5, E6, E7, E8, BPV1, BPV2, BPV3, BPV4, BPV5 and BPV6), dsRNA viruses (e g., reovirus), (+)ssRNA viruses (e.g., picornavirus, coxsackie virus, hepatitis A virus, poliovirus, togavirus, rubella virus, flavivirus, hepatitis C virus, yellow fever virus, dengue virus, west Nile virus, coronavirus), (-)ssRNA viruses (e.g., orthomyxovirus, influenza virus, rhabdovirus, paramyxovirus, measles virus, mumps virus, parainfluenza virus, respiratory syncytial virus, metapneumovirus, arenaviruses (including Lassa virus, LuJo virus, Junin virus, Machupo virus, Bear Canyon virus, Wenzhou virus, Guanarito virus, Latino virus, Sabia virus, Chapare virus, Whitewater Arroyo virus, Pichinde virus, LCMV virus, Mopeia virus, Mobala virus, Ippy virus, Mobala virus, and Morogoro virus, rhabdovirus, rabies virus, ebola), ssRNA-RT viruses (e.g. retrovirus, human immunodeficiency virus (HIV)), and dsDNA-RT viruses (e.g. hepadnavirus, hepatitis B). As one of skill in the art would appreciate, antigenic polypeptides can be derived from other viruses not listed above.

[0181] In certain embodiments, the antigenic polypeptide is isolated from any portion of bacteria. For example, but without any limitation, the antigenic polypeptide can be a polypeptide of the cell membrane of the bacteria. Non-limiting examples of bacteria from which an antigenic polypeptide is obtained or derived include Bacillus spp. (e.g., Bacillus anthracis), Bordetella spp. (e.g., Bordetella pertussis), Borrelia spp. (e.g., Borrelia burgdorferi), Brucella spp. (e.g., Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis), Campylobacter spp. (e.g., Campylobacter jejuni), Chlamydia spp. (e.g., Chlamydia pneumoniae, Chlamydia psittaci, Chlamydia trachomatis), Clostridium spp. (e.g., Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani), Corynebacterium spp. (e.g., Corynebacterium

[0182] ACTIVE 508396272 26 072396.1082

[0183] PATENT diplheriae). Enterococcus spp. (e.g., Enterococcus fctecalis. Enterococcus faecum), Escherichia spp. (e.g., Escherichia coli), Francisella spp. (e.g., Francisella lularensis . Haemophilus spp. (e.g., Haemophilus influenza). Helicobacter spp. (e.g., Helicobacter pylori), Legionella spp. (e.g., Legionella pneumophila), Leptospira spp. (e.g., Leptospira interrogans), Listeria spp. (e.g., Listeria monocytogenes), Mycobacterium spp. (e.g., Mycobacterium leprae, Mycobacterium tuberculosis), Mycoplasma spp. (e.g., Mycoplasma pneumoniae), Neisseria spp. (e.g., Neisseria gonorrhea, Neisseria meningitidis), Pseudomonas spp. (e.g., Pseudomonas aeruginosa , Rickettsia spp. (e.g., Rickettsia rickettsii), Salmonella spp. (e.g., Salmonella typhi, Salmonella typhinurium), Shigella spp. (e.g., Shigella sonnei), Staphylococcus spp. (e.g., Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus), Streptococcus spp. (e.g., Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyrogenes), Treponema spp. (e.g., Treponema pallidum , Vibrio spp. (e.g., Vibrio cholerae), and Yersinia spp. (Yersinia pestis). As one of skill in the art would appreciate, antigenic polypeptides can be derived from other bacteria not listed above.

[0184] In certain embodiments, the antigenic polypeptide is isolated from any portion of a parasite. Non-limiting examples of parasites from which an antigenic polypeptide is obtained or derived include Ancylostoma spp. (e.g., A. duodenale), Anisakis spp., Ascaris lumbricoides, Balantidium coli, Cestoda spp., Cimicidae spp., Clonorchis sinensis, Dicrocoelium dendriticum, Dicrocoelium hospes, Diphyllobothrium latum, Dracunculus spp., Echinococcus spp. (e.g., E. granulosus, E. multilocularis), Entamoeba histolytica, Enter obius vermicularis, Fasciola spp. (e.g., F. hepatica, F. magna, F. gigantica, F. jacksoni), Fasciolopsis buski, Giardia spp. (Giardia lamblia), Gnathostoma spp., Hymenolepis spp. (e.g., H. nana, H. diminuta), Leishmania spp., Loa loa, Metorchis spp. (M. conjunctus, M. albidus), Necator americanus, Oestroidea spp. (e.g., botfly), Onchocercidae spp., Opisthorchis spp. (e.g., 0. viverrini, O. felineus, O. guayaquilensis, and 0. noverca), Plasmodium spp. (e.g., P. falciparum), Protofasciola robusta, Parafasciolopsis fasciomorphae, Paragonimus westermani, Schistosoma spp. (e.g., S. mansoni, S. japonicum, S. mekongi, S. haematobium , Spirometra erinaceieuropaei, Strongyloides stercoralis, Taenia spp. (e.g., T. saginata, T. solium , Toxocara spp. (e.g., T. canis, T. cati), Toxoplasma spp. (e.g., T. gondii , Trichobilharzia regenti, Trichinella spiralis, Trichuris trichiura, Trombiculidae spp., Trypanosoma spp., Tunga penetrans, and Wuchereria bancrofti. As one of skill in the art would appreciate, antigenic polypeptides can be derived from other parasites not listed above.

[0185] In certain embodiments, the present disclosure provides vaccines and articles of manufacture useful for the treatment and / or prevention of a tumor. In certain embodiments, the antigenic polypeptide is isolated from any portion of a tumor, also known as tumor antigen (TA).

[0186] ACTIVE 508396272 27 072396.1082

[0187] PATENT

[0188] Non-limiting examples of tumor antigens from which an antigenic polypeptide is obtained or derived include gplOO, MART-l / Melan A, gp75 (TRP-I), tyrosinase, NY-ESO-I, melanoma proteoglycan, MAGE family antigens (i.e., MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-6, and MAGE-12), BAGE family antigens, GAGE family antigens (i.e., GAGE-1, GAGE-2), RAGE family antigens, N- acetylglucosaminyltransferase-V, pl 5, P-catenin, MUM-I, cyclin dependent kinase-4 (CDK4), p21-ras, BCR- abl, p53, pl85 HER2 / neu, epidermal growth factor receptor (EGFR), carcinoembryonic antigens (CEA); carcinoma- associated mutated mucins (i.e., MUC-1 gene products), EBNA gene products of EBV (i.e., EBNA-I), E7, E6 proteins of human papillomavirus, prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), idiotypic epitopes or antigens, for example, immunoglobulin idiotypes or T cell receptor idiotypes, KSA, kinesin 2, HIP-55, TGFP-1 anti- apoptotic factor, tumor protein D52, HIFT, NY-BR-I, NY- BR-62, NY-BR-75, NY-BR-85, NY-BR-87 and NY-BR-96. As one of skill in the art would appreciate, antigenic polypeptides can be derived from other public antigens not listed above.

[0189] In certain embodiments, the antigenic polypeptide is a viral antigenic polypeptide isolated from a Coronaviridae (e.g., Coronavirus, such as severe acute respiratory syndrome (SARS) virus).

[0190] 2,2,1 Coronaviruses

[0191] Coronaviruses are a family of viruses (e.g., the Coronaviridae family) responsible for respiratory infections in mammals and characterized by a genome of about 30 kb encoding 28 proteins.

[0192] The Coronaviridae family includes a-coronaviruses and P-coronaviruses, which can infect several animals including bats, humans, camels, and rabbits. Certain P-coronaviruses are responsible for the development of severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and COVID- 19. Additional examples of P-coronaviruses related diseases include OC44 and HKE11. Non-limiting examples of a-coronaviruses related diseases include, but are not limited to, 229E and NL63.

[0193] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible and pathogenic coronavirus that emerged in late 2019 and shares genetic and morphologic features with others in the Coronaviridae family (e.g., P-coronaviruses). The genome of SARS-CoV-2 shares 82% nucleotide identity with human SARS-CoV (SARS-CoV-1) and 89% with bat SARS- like-CoVZXC21. The spike (S) glycoprotein is clinically important because it bears significant structural homology with SARS-CoV-1 compared to other coronaviruses such as MERS-CoV and other merb ecoviruses. Like SARS-CoV-1, the surface Spike (S) glycoprotein of SARS-CoV-2 binds the same host receptor, ACE-2, to mediate cell entry. Spike (S) glycoprotein, a class I fusion

[0194] ACTIVE 508396272 28 072396.1082

[0195] PATENT protein, is also a critical determinant of viral host range and tissue tropism and the primary target of the host immune response. As such, most coronavirus vaccine candidates are based on S or one of its sub-components. Coronavirus S glycoproteins contain three segments: a large ectodomain, a single-pass transmembrane anchor and a short intracellular tail. The ectodomain consists of a receptor-binding subunit, SI, which contains two sub-domains: one at the N-terminus and the other at the C-terminus. The latter comprises the receptor-binding domain (RBD), which serves the vital function of attaching the virus to the host receptor and triggering a conformational change in the protein that results in fusion with the host cell membrane through the S2 subunit. Multiple technology platforms are currently advancing SARS-CoV-2 vaccine development, including nucleic acid vaccines, whole virus vaccines, recombinant protein subunit vaccines and nanoparticle vaccines. Of these vaccine platform types, nanoparticle technologies have previously been shown to improve antigen structure and stability, as well as vaccine targeted delivery, immunogenicity, and safety.

[0196] 2.2.1.1 Antigenic Coronavirus Polypeptides

[0197] In certain embodiments, the antigenic polypeptide comprises an antigenic coronavirus polypeptide. In certain embodiments, the antigenic coronavirus polypeptide comprises a coronavirus spike protein (also known as “S protein” or “glycoprotein S”) polypeptide, which is responsible, among other functions, for viral entry into a host cell. In certain embodiments, the antigenic coronavirus polypeptide includes at least one antigenic coronavirus polypeptide. For example, but without any limitation, the antigenic coronavirus polypeptide can include a first antigenic coronavirus polypeptide and a second antigenic coronavirus polypeptide.

[0198] S protein is capable of inducing a humoral response characterized by neutralizing antibodies. The S protein includes two functional subunits responsible for binding to the host cell receptor (SI subunit) and fusion of the viral and cellular membranes (S2 subunit). In certain embodiments, the antigenic coronavirus polypeptide includes the entire spike protein or a functional fragment thereof. In certain embodiments, the antigenic coronavirus polypeptide includes the SI subunit or a functional fragment thereof. In certain embodiments, the antigenic coronavirus polypeptide includes the S2 subunit or a functional fragment thereof. In certain embodiments, the antigenic coronavirus polypeptide includes any SARS-CoV-2 antigenic polypeptide or a functional fragment thereof.

[0199] In certain embodiments, the antigenic coronavirus polypeptide includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the

[0200] ACTIVE 508396272 29 072396.1082

[0201] PATENT amino acid sequence set forth in NCBI Reference Sequence: YP 009724390.1. In certain embodiments, the antigenic coronavirus polypeptide includes substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence set forth in NCBI Reference Sequence: YP 009724390.1, that do not significantly alter the function or activity of the antigenic coronavirus polypeptide.

[0202] In certain embodiments, the antigenic coronavirus polypeptide comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 4 or a functional fragment thereof. In certain embodiments, the antigenic coronavirus polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 4 or a functional fragment thereof. In certain embodiments, the antigenic coronavirus polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 4 or a functional fragment thereof. SEQ ID NO: 4 provided below.

[0203] In certain embodiments, the antigenic coronavirus polypeptide comprises from amino acid 1 to amino acid 1273 of the amino acid sequence set forth in SEQ ID NO: 4 or a functional fragment thereof. In certain embodiments, the antigenic coronavirus polypeptide consists of from amino acid 1 to amino acid 1273 of the amino acid sequence set forth in SEQ ID NO: 4 or a functional fragment thereof. In certain embodiments, the antigenic coronavirus polypeptide comprises from amino acid 1 to amino acid 685 of the amino acid sequence set forth in SEQ ID NO: 4 or a functional fragment thereof. In certain embodiments, the antigenic coronavirus polypeptide consists of from amino acid 1 to amino acid 685 of the amino acid sequence set forth in SEQ ID NO: 4 or a functional fragment thereof. In certain embodiments, the antigenic coronavirus polypeptide comprises from amino acid 14 to amino acid 685 of the amino acid sequence set forth in SEQ ID NO: 4 or a functional fragment thereof. In certain embodiments, the antigenic coronavirus polypeptide consists of from amino acid 14 to amino acid 685 of the amino acid sequence set forth in SEQ ID NO: 4 or a functional fragment thereof. In certain embodiments, the antigenic coronavirus polypeptide comprises from amino acid 686 to amino acid 1273 of the amino acid sequence set forth in SEQ ID NO: 4 or a functional fragment thereof. In certain embodiments, the antigenic coronavirus polypeptide consists of from amino acid 686 to amino acid 1273 of the amino acid sequence set forth in SEQ ID NO: 4 or a functional fragment thereof. MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTW FHAf f 1VSGTNGTKRFDNPVLPFNDGVYFASTEKSNI IRGWI FGTTLDSKTQSLLIVNNATNW IKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLRE FVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSS SGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFR VQPTES IVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVS

[0204] ACTIVE 508396272 30 072396.1082

[0205] PATENT

[0206] PTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGG NYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRW VLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDA VRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTG SNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQS I IAYTMSLGAEN SVAYSNNS IAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRAL TGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADA GFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQ I P FAMQMAYRFNG I GVTQNVL YENQKL I ANQFNSAI GKI QDS LS S TASALGKLQDWNQNAQAL NTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASAN LAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGWFLHVTYVPAQEKNFTTAPAICHDGKA HFPREGVFVSNGTHWFVTQRNFYEPQI ITTDNTFVSGNCDWIGIVNNTVYDPLQPELDSFKEE LDKYFKNHTSPDVDLGDISGINASWNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWY IWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT [ SEQ ID NO : 4 ]

[0207] In certain embodiments, the antigenic coronavirus polypeptide includes an S polypeptide or a functional fragment thereof. In certain embodiments, the antigenic coronavirus polypeptide includes a receptor-binding domain (RBD) polypeptide or a functional fragment thereof. In certain embodiments, the antigenic coronavirus polypeptide includes an N-terminal domain (NTD) polypeptide or a functional fragment thereof. In certain embodiments, the antigenic coronavirus polypeptide includes a receptor-binding domain (RBD)-N-terminal chimera polypeptide or a functional fragment thereof. In certain embodiments, the antigenic coronavirus polypeptide includes an SI polypeptide or a functional fragment thereof. In certain embodiments, the antigenic coronavirus polypeptide includes a stabilized extracellular spike S-2P polypeptide or a functional fragment thereof. In certain embodiments, the antigenic coronavirus polypeptide includes a stabilized extracellular spike S polypeptide or a functional fragment thereof. In certain embodiments, the antigenic coronavirus polypeptide includes a stabilized extracellular spike S- trimer or a functional fragment thereof.

[0208] In certain embodiments, the antigenic coronavirus polypeptide includes a receptor-binding domain (RBD) polypeptide or a functional fragment thereof. In certain embodiments, the RBD polypeptide includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 5 or a functional fragment thereof. In certain embodiments, the RBD polypeptide includes substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence set forth in SEQ ID NO: 5, that do not significantly alter the function or activity of the RBD polypeptide.

[0209] In certain embodiments, the RBD polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 5 or a functional fragment thereof. In certain embodiments, the RBD polypeptide

[0210] ACTIVE 508396272 31 072396.1082

[0211] PATENT consists of the amino acid sequence set forth in SEQ ID NO: 5 or a functional fragment thereof. SEQ ID NO: 5 is provided below.

[0212] NITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTN VYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRK SNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVWLSFELLHAPA TVCGP [ SEQ ID NO : 5 ]

[0213] In certain embodiments, the RBD polypeptide includes substitutions or modifications made to reduce “sticky” hydrophobic regions in order to increase expression in recombinant systems and / or the ability to form nanoparticles. Non-limiting examples of modifications of the RBD polypeptide encompassed by the present disclosure are described in International Patent Publication No. WO 2021 / 178971, the contents of which are incorporated by reference in their entirety.

[0214] In certain embodiments, the antigenic coronavirus polypeptide includes an N-terminal domain (NTD) polypeptide or a functional fragment thereof. In certain embodiments, the NTD polypeptide includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 6 or a functional fragment thereof. In certain embodiments, the NTD polypeptide includes substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence set forth in SEQ ID NO: 6, that do not significantly alter the function or activity of the NTD polypeptide.

[0215] In certain embodiments, the NTD polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 6 or a functional fragment thereof. In certain embodiments, the NTD polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 6 or a functional fragment thereof. SEQ ID NO: 6 is provided below.

[0216] QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTK RFDNPVLPFNDGVYFASTEKSNI IRGWI FGTTLDSKTQSLLIVNNATNWIKVCEFQFCNDPFL GVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYS KHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGY LQPRTFLLKYNENGTITDAVDCALDPLSETKCTL [ SEQ ID NO : 6 ]

[0217] In certain embodiments, the antigenic coronavirus polypeptide includes an SI polypeptide or a functional fragment thereof. In certain embodiments, the SI polypeptide includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or a functional fragment thereof. In certain embodiments, the SI polypeptide includes substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence set forth

[0218] ACTIVE 508396272 32 072396.1082

[0219] PATENT in SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, that do not significantly alter the function or activity of the SI polypeptide.

[0220] In certain embodiments, the SI polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or a functional fragment thereof. In certain embodiments, the SI polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or a functional fragment thereof. SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9 are provided below.

[0221] VNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRF DNPVLPFNDGVYFASTEKSNI IRGWI FGTTLDSKTQSLLIVNNATNWIKVCEFQFCNDPFLGV YYFKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKI YSKH TPINLVRDLPQGFSALEPLVDLPIGESilTRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYL QPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTES IVRFPNITN LCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYAD SFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLK PFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVWLSFELLHAPATVCG PKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPC SFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAf flADQLTPTWRVYSTGSNVFQTRAGCLIGA EHVNNSYECDIPIGAGICASYQTQT [ SEQ ID NO : 7 ]

[0222] QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTK RFDNPVLPFNDGVYFASTEKSNI IRGWI FGTTLDSKTQSLLIVNNATNWIKVCEFQFCNDPFL GVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYS KHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGY LQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTES IVRFPNIT NLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYA DSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNL KPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVWLSFELLHAPATVC GPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITP CSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGA EHVNNSYECDIPIGAGICASYQTQTNSPRRAR [ SEQ ID NO : 8 ]

[0223] SSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNG TKRFDNPVLPFNDGVYFASTEKSNI IRGWI FGTTLDSKTQSLLIVNNATNWIKVCEFQFCNDP FLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKI YSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYV GYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTES IVRFPN ITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNV YADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKS NLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVWLSFELLHAPAT VCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDI TPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLI GAEHVNNSYECDIPIGAGICASYQTGGSQS I IAYT [ SEQ ID NO : 9 ]

[0224] In certain embodiments, the antigenic coronavirus polypeptide includes an S-2P polypeptide or a functional fragment thereof. An S-2P sequence is a stabilized version of the spike ectodomain that includes two proline substitutions and stabilizes the prefusion conformation. Specifically, S-2P comprises proline modifications K986P and V987P, as well as the removal of

[0225] ACTIVE 508396272 33 072396.1082

[0226] PATENT the Furin cleavage site (RRAS to GSAS; SEQ ID NO: 42 and SEQ ID NO: 43, respectively). In certain embodiments, the S-2P polypeptide includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 10 or a functional fragment thereof. In certain embodiments, the S-2P polypeptide includes substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence set forth in SEQ ID NO: 10, that do not significantly alter the function or activity of the S-2P polypeptide.

[0227] In certain embodiments, the S-2P polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 10 or a functional fragment thereof. In certain embodiments, the S-2P polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 10 or a functional fragment thereof. SEQ ID NO: 10 is provided below.

[0228] RSVASQS I IAYTMSLGAENSVAYSNNS IAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTE CSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKP SKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSA LLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSS TASALGKLQDWNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSL QTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGWFLHVTYV PAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQI ITTDNTFVSGNCDWIGI VNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASWNIQKEIDRLNEVAKNLNES LIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDD SEPVLKGVKLHYT [ SEQ ID NO : 10 ]

[0229] In certain embodiments, the antigenic coronavirus polypeptide includes a stabilized extracellular spike S-2P polypeptide or a functional fragment thereof, a stabilized extracellular spike S polypeptide or a functional fragment thereof, or a stabilized extracellular spike S-trimer or a functional fragment thereof. Additional information on antigenic coronavirus polypeptides encompassed by the present disclosure can be found in International Patent Publication No. WO 2021 / 178971 and in International Patent Publication No. WO 2021 / 243219, the contents of which of each are incorporated by reference in their entirety.

[0230] In certain embodiments, the antigenic coronavirus polypeptide includes a HexaPro polypeptide, a stabilized S-2P variant with six prolines. Additional information on HexaPro polypeptide can be found in International Patent Publication No. WO 2021 / 178971; Hsieh et al. Science 369.6510 (2020): 1501-1505; and Lu et al., Proceedings of the National Academy of Sciences 119.35 (2022): e2110105119, the contents of each of which are incorporated by reference in their entirety.

[0231] Additionally or alternatively, the antigenic coronavirus polypeptide includes a SARS- CoV-1 spike protein (S protein) or a functional fragment thereof. Additional information on

[0232] ACTIVE 508396272 34 072396.1082 PATENT antigenic coronavirus polypeptides encompassed by the present disclosure can be found in International Patent Publication No. WO 2021 / 178971, the contents of which are incorporated by reference in their entirety.

[0233] 2,2,2 Arenaviruses

[0234] In certain embodiments, the antigenic polypeptide is isolated from an arenavirus. Arenaviruses (e.g., Machupo virus, Junin virus, and Guanarito virus) exhibit segmented RNA genomes that are integral to their replication and pathogenic mechanisms. These viruses possess two single-stranded RNA segments: the small (S) segment and the large (L) segment. The S segment encodes the nucleoprotein (NP) and the glycoprotein precursor (GPC), which undergoes proteolytic cleavage to yield GP1 and GP2, facilitating viral attachment and entry into host cells. The L segment encodes the RNA-dependent RNA polymerase (RdRP) and the matrix protein (Z), both of which are essential for viral RNA synthesis and assembly. The segmented nature of these genomes enables genetic reassortment, thereby enhancing viral adaptability and potential virulence. In certain embodiments, the arenavirus is a Machupo virus, a Junin virus, or a Guanarito virus.

[0235] In certain embodiments, the arenavirus is a Machupo virus. The Machupo virus is an arenavirus endemic to Bolivia that causes the disease commonly called Bolivian hemorrhagic fever. It was first discovered in 1959 in an outbreak in San Joaquin, Bolivia. This virus, like the other New World hemorrhagic viruses, is also spread by a murine vector, specifically, the Calomys callosus, also commonly known as the large vesper mouse. Clinically, Bolivian hemorrhagic fever is very similar to Argentine hemorrhagic fever. It has a constellation of symptoms that can include signs of increasing capillary leak, proteinuria, mucosal hemorrhage, narrow pulse pressure, and resultant vasoconstriction causing shock. With both the Junin and Machupo viruses, petechiae, facial edema, and hyperesthesia of the skin are more commonly observed than they are with the other New World hemorrhagic viruses. Survivors often have no permanent sequelae, except for mild alopecia and orthostatic hypotension that can last for a few weeks.

[0236] In certain embodiments, the arenavirus is a Junin virus. Junin virus was discovered in 1958 in remote agricultural regions of Argentina. Its clinical syndrome was appropriately named Argentine hemorrhagic fever. The virus is spread to humans through contact with bodily fluids and aerosolized excrement particles from the mammalian reservoir, Calomys musculinus (drylands vesper mouse). Those most at risk are rural populations in endemic areas who come into contact with the mouse during agricultural work or in their homes. The syndrome caused by the Argentine hemorrhagic fever virus is very similar to the other illnesses described in

[0237] ACTIVE 508396272 35 072396.1082 PATENT this chapter. The incubation period is approximately 7 to 14 days, after which patients develop fever, headache, myalgias, and anorexia. During days 3 to 5 of the disease, patients can develop petechial rashes on the soft palate and trunk. Disease can progress to generalized edema, with development of pleural effusions and bleeding from gums, nose, GI system, and uterus, as well as neurologic symptoms. Argentine hemorrhagic fever is also associated with autonomic instability, and patients often exhibit signs of postural hypotension, as well as intermittent flushing and diaphoresis.

[0238] In certain embodiments, the arenavirus is a Guanarito virus. Guanarito virus is an arenavirus that was first identified in rural areas of Venezuela in 1989. It is associated with Venezuelan hemorrhagic fever, a disease that occurs due to the clearing of forests and subsequent land preparation for farming. The virus is transmitted by a rodent host called the short-tailed cane mouse, Zygodontomys brevicauda.

[0239] 2.2.2.1 Antigenic Arenavirus Polypeptides

[0240] In certain embodiments, the antigenic polypeptide comprises an antigenic arenavirus polypeptide. In certain embodiments, the antigenic arenavirus polypeptide includes at least one antigenic arenavirus polypeptides. For example, but without any limitation, the antigenic arenavirus polypeptide can include a first antigenic arenavirus polypeptide and a second antigenic arenavirus polypeptide.

[0241] Arenavirus glycoproteins, particularly GP1 and GP2, are crucial for viral entry and act as primary targets for neutralizing antibodies. Thus, vaccination against these glycopeoteins could induce effective immune responses. In certain embodiments, the antigenic arenavirus polypeptide includes a GP1 or GP2 polypeptide or a functional fragment thereof. In certain embodiments, the antigenic arenavirus polypeptide includes the GP1 polypeptide or a functional fragment thereof. In certain embodiments, the antigenic arenavirus polypeptide includes the GP2 polypeptide or a functional fragment thereof. In certain embodiments, the antigenic arenavirus polypeptide includes any antigenic polypeptide derived from an arenavirus or a functional fragment thereof.

[0242] In certain embodiments, the antigenic arenavirus polypeptide includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in UniProt Reference Sequence: Q6IUF7. In certain embodiments, the antigenic arenavirus polypeptide includes substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence set forth in NCBI Reference Sequence:

[0243] ACTIVE 508396272 36 072396.1082

[0244] PATENT

[0245] Q6IUF7, that do not significantly alter the function or activity of the antigenic coronavirus polypeptide.

[0246] In certain embodiments, the antigenic arenavirus polypeptide comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 44 or a functional fragment thereof. In certain embodiments, the antigenic arenavirus polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 44 or a functional fragment thereof. In certain embodiments, the antigenic arenavirus polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 44 or a functional fragment thereof. SEQ ID NO: 44 is provided below.

[0247] In certain embodiments, the antigenic arenavirus polypeptide comprises from amino acid 1 to amino acid 496 of the amino acid sequence set forth in SEQ ID NO: 44 or a functional fragment thereof. In certain embodiments, the antigenic arenavirus polypeptide comprises from amino acid 2 to amino acid 58 of the amino acid sequence set forth in SEQ ID NO: 44 or a functional fragment thereof. In certain embodiments, the antigenic arenavirus polypeptide comprises from amino acid 2 to amino acid 496 of the amino acid sequence set forth in SEQ ID NO: 44 or a functional fragment thereof. In certain embodiments, the antigenic arenavirus polypeptide comprises from amino acid 59 to amino acid 262 of the amino acid sequence set forth in SEQ ID NO: 44 or a functional fragment thereof. In certain embodiments, the antigenic arenavirus polypeptide comprises from amino acid 263 to amino acid 496 of the amino acid sequence set forth in SEQ ID NO: 44 or a functional fragment thereof. In certain embodiments, the antigenic arenavirus polypeptide comprises from amino acid 59 to amino acid 496 of the amino acid sequence set forth in SEQ ID NO: 44 or a functional fragment thereof.

[0248] In certain embodiments, the antigenic arenavirus polypeptide includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in UniProt Reference Sequence: P26313. In certain embodiments, the antigenic arenavirus polypeptide includes substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence set forth in NCBI Reference Sequence: P26313, that do not significantly alter the function or activity of the antigenic coronavirus polypeptide.

[0249] In certain embodiments, the antigenic arenavirus polypeptide comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the

[0250] ACTIVE 508396272 37 072396.1082

[0251] PATENT amino acid sequence set forth in SEQ ID NO: 45 or a functional fragment thereof. In certain embodiments, the antigenic arenavirus polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 45 or a functional fragment thereof. In certain embodiments, the antigenic arenavirus polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 45 or a functional fragment thereof. SEQ ID NO: 45 is provided below.

[0252] In certain embodiments, the antigenic arenavirus polypeptide comprises from amino acid 1 to amino acid 496 of the amino acid sequence set forth in SEQ ID NO: 45 or a functional fragment thereof. In certain embodiments, the antigenic arenavirus polypeptide comprises from amino acid 2 to amino acid 58 of the amino acid sequence set forth in SEQ ID NO: 45 or a functional fragment thereof. In certain embodiments, the antigenic arenavirus polypeptide comprises from amino acid 2 to amino acid 485 of the amino acid sequence set forth in SEQ ID NO: 45 or a functional fragment thereof. In certain embodiments, the antigenic arenavirus polypeptide comprises from amino acid 59 to amino acid 251 of the amino acid sequence set forth in SEQ ID NO: 45 or a functional fragment thereof. In certain embodiments, the antigenic arenavirus polypeptide comprises from amino acid 252 to amino acid 485 of the amino acid sequence set forth in SEQ ID NO: 45 or a functional fragment thereof. In certain embodiments, the antigenic arenavirus polypeptide comprises from amino acid 59 to amino acid 485 of the amino acid sequence set forth in SEQ ID NO: 45 or a functional fragment thereof.

[0253] In certain embodiments, the antigenic arenavirus polypeptide includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in UniProt Reference Sequence: Q8 AYW 1. In certain embodiments, the antigenic arenavirus polypeptide includes substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence set forth in NCBI Reference Sequence: Q8AYW1, that do not significantly alter the function or activity of the antigenic coronavirus polypeptide.

[0254] In certain embodiments, the antigenic arenavirus polypeptide comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 46 or a functional fragment thereof. In certain embodiments, the antigenic arenavirus polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 46 or a functional fragment thereof. In certain embodiments, the antigenic arenavirus polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 46 or a functional fragment thereof. SEQ ID NO: 46 is provided below.

[0255] ACTIVE 508396272 38 072396.1082 PATENT

[0256] In certain embodiments, the antigenic arenavirus polypeptide comprises from amino acid 1 to amino acid 496 of the amino acid sequence set forth in SEQ ID NO: 46 or a functional fragment thereof. In certain embodiments, the antigenic arenavirus polypeptide comprises from amino acid 2 to amino acid 58 of the amino acid sequence set forth in SEQ ID NO: 46 or a functional fragment thereof. In certain embodiments, the antigenic arenavirus polypeptide comprises from amino acid 2 to amino acid 479 of the amino acid sequence set forth in SEQ ID NO: 46 or a functional fragment thereof. In certain embodiments, the antigenic arenavirus polypeptide comprises from amino acid 59 to amino acid 245 of the amino acid sequence set forth in SEQ ID NO: 46 or a functional fragment thereof. In certain embodiments, the antigenic arenavirus polypeptide comprises from amino acid 246 to amino acid 479 of the amino acid sequence set forth in SEQ ID NO: 46 or a functional fragment thereof. In certain embodiments, the antigenic arenavirus polypeptide comprises from amino acid 59 to amino acid 279 of the amino acid sequence set forth in SEQ ID NO: 46 or a functional fragment thereof.

[0257] ACTIVE 508396272 39 072396.1082

[0258] PATENT

[0259] 2.3. Linker

[0260] In certain embodiments, the nanoparticle-forming polypeptide and the antigenic polypeptide (e.g., antigenic coronavirus polypeptide) are connected by a linker. The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility. In certain embodiments, the linker can link the C-end of the antigenic peptide (e.g., antigenic coronavirus polypeptide) and the N-end of the nanoparticle-forming polypeptide. In certain embodiments, the linker can link the C-end of the nanoparticle-forming polypeptide and the N-end of the antigenic peptide (e.g., antigenic coronavirus polypeptide). Non-limiting examples of linkers are disclosed in Shen et al., Anal. Chem. 80(6): 1910-1917 (2008) and International Patent Publication No. WO 2021 / 178971, the contents of which are incorporated by reference in their entirety. In certain embodiments, the linker is a G4S linker. In certain embodiments, the linker comprises the amino sequence set forth in SEQ ID NO: 11. In certain embodiments, the linker comprises the amino sequence set forth in SEQ ID NO: 12. In certain embodiments, the linker comprises the amino sequence set forth in SEQ ID NO: 13. In certain embodiments, the linker comprises the amino sequence set forth in SEQ ID NO: 14. In certain embodiments, the linker comprises the amino sequence set forth in SEQ ID NO: 15. In certain embodiments, the linker comprises the amino sequence set forth in SEQ ID NO: 16. In certain embodiments, the linker comprises the amino sequence set forth in SEQ ID NO: 17. In certain embodiments, the linker comprises the amino sequence set forth in SEQ ID NO: 18. In certain embodiments, the linker comprises the amino sequence set forth in SEQ ID NO: 19. In certain embodiments, the linker comprises the amino sequence set forth in SEQ ID NO: 20. In certain embodiments, the linker comprises the amino sequence set forth in SEQ ID NO: 21. In certain embodiments, the linker comprises the amino sequence set forth in SEQ ID NO: 22. In certain embodiments, the linker comprises the amino sequence set forth in SEQ ID NO: 23. SEQ ID Nos: 11-23 are provided below.

[0261] GSGG [ SEQ ID NO : 11 ]

[0262] GSGGSG [ SEQ ID NO : 12 ]

[0263] GSGGEMKQIEDKIEEILSKIYHIENEIARIKKLIGRGSGGSG [ SEQ ID NO : 13 ] LDS IKEELDKIHKNGSGGSG [ SEQ ID NO : 14 ] IDS IKEEIDKIHKNGSGGSG [ SEQ ID NO : 15 ] MKQIEDKIEEILSKIYHIENEIARIKKLIGRGSGGSG [ SEQ ID NO : 16 ] GSGGGG [ SEQ ID NO : 17 ]

[0264] ACTIVE 508396272 40 072396.1082

[0265] PATENT

[0266] GSG [ SEQ ID NO : 18 ]

[0267] GSGGSGGSGGSGGG [ SEQ ID NO : 19 ] GGGSGGGSGG [ SEQ ID NO : 20 ] GGGG [ SEQ ID NO : 21 ] GGG [ SEQ ID NO : 22 ] SGG [ SEQ ID NO : 23 ]

[0268] 2.4. Fusion Proteins and Vaccines

[0269] In certain embodiments, the presently disclosed vaccines include multiple fusion proteins that are capable of self-assembly into a nanoparticle. In certain embodiments, the fusion protein includes a nanoparticle-forming polypeptide and an antigenic polypeptide (e.g., an antigenic coronavirus polypeptide).

[0270] In certain embodiments, the nanoparticle-forming polypeptide is a ferritin polypeptide or a functional fragment thereof (e.g., one disclosed in Section 2.1). In certain embodiments, the nanoparticle-forming polypeptide is a H. pylori ferritin polypeptide or a functional fragment thereof. In certain embodiments, the nanoparticle-forming polypeptides has the ability of selfassembly. For example, but without any limitation, a H. pylori ferritin polypeptide or a functional fragment thereof can self-assemble into a 24-member spherical particle, made up of multiple three-fold, four-fold, and / or two-fold axes. Thus, the resulting nanoparticle can include a 3-fold axis, a 4-fold axis, or a 2-fold axis. For example, but without any limitation, using the 3-fold axes, 8 antigenic trimeric coronavirus peptides can be presented on the surface of the self-assembling protein nanoparticle surface. In another example including the use of monomeric antigens (e.g., RBD), 24 coronavirus peptides can be presented on the surface of the self-assembling protein nanoparticle surface.

[0271] In certain embodiments, the presently disclosed vaccines include multiple fusion proteins that are capable of self-assembly into a nanoparticle. In certain embodiments, the fusion protein includes a nanoparticle-forming polypeptide and an antigenic polypeptide. In certain embodiments, the antigenic polypeptide is isolated from any portion of a virus. For example, but without any limitation, the antigenic polypeptide can be a natural viral capsid structure or a portion thereof, or a composite of a structure of multiple strains of the virus. Non-limiting examples of viruses from which an antigenic polypeptide is obtained or derived include dsDNA viruses (e.g., adenovirus, herpesvirus, Epstein Barr virus, herpes simplex type 1, herpes simplex type 2, human herpes virus simplex type 8, human cytomegalovirus, varicella-zoster virus, poxvirus), ssDNA viruses (e.g., parvovirus, papillomavirus (e.g., El, E2, E3, E4, E5, E6, E7, E8, BPV1, BPV2, BPV3, BPV4, BPV5 and BPV6), dsRNA viruses (e.g., reovirus), (+)ssRNA viruses (e.g., picomavirus,

[0272] ACTIVE 508396272 41 072396.1082

[0273] PATENT coxsackie virus, hepatitis A virus, poliovirus, togavirus, rubella virus, flavivirus, hepatitis C virus, yellow fever virus, dengue virus, west Nile virus, coronavirus), (-)ssRNA viruses (e.g., orthomyxovirus, influenza virus, rhabdovirus, paramyxovirus, measles virus, mumps virus, parainfluenza virus, respiratory syncytial virus, metapneumovirus, arenaviruses (including Lassa virus, LuJo virus, Junin virus, Machupo virus, Bear Canyon virus, Wenzhou virus, Guanarito virus, Latino virus, Sabia virus, Chapare virus, Whitewater Arroyo virus, Pichinde virus, LCMV virus, Mopeia virus, Mobala virus, Ippy virus, Mobala virus, and Morogoro virus, rhabdovirus, rabies virus, ebola), ssRNA-RT viruses (e.g. retrovirus, human immunodeficiency virus (HIV)), and dsDNA-RT viruses (e.g. hepadnavirus, hepatitis B). As one of skill in the art would appreciate, antigenic polypeptides can be derived from other viruses not listed above.

[0274] In certain embodiments, the presently disclosed vaccines include multiple fusion proteins that are capable of self-assembly into a nanoparticle. In certain embodiments, the fusion protein includes a nanoparticle-forming polypeptide and an antigenic polypeptide. In certain embodiments, the antigenic polypeptide is an antigenic arenavirus polypeptide disclosed above.

[0275] In certain embodiments, the presently disclosed vaccines include multiple fusion proteins that are capable of self-assembly into a nanoparticle. In certain embodiments, the fusion protein includes a nanoparticle-forming polypeptide and an antigenic polypeptide. In certain embodiments, the antigenic polypeptide is isolated from any portion of bacteria. For example, but without any limitation, the antigenic polypeptide can be a polypeptide of the cell membrane of the bacteria. Non-limiting examples of bacteria from which an antigenic polypeptide is obtained or derived include Bacillus spp. (e.g., Bacillus anthracis), Bordetella spp. (e.g., Bordetella pertussis), Borrelia spp. (e.g., Borrelia burgdorferi), Brucella spp. (e.g., Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis), Campylobacter spp. (e.g., Campylobacter jejuni), Chlamydia spp. (e.g., Chlamydia pneumoniae, Chlamydia psittaci, Chlamydia trachomatis), Clostridium spp. (e.g., Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani), Corynebacterium spp. (e.g., Corynebacterium diptheriae), Enterococcus spp. (e.g., Enterococcus faecalis, Enterococcus faecum), Escherichia spp. (e.g., Escherichia coll), Francisella spp. (e.g., Francisella tularensis), Haemophilus spp. (e.g., Haemophilus influenza), Helicobacter spp. (e.g., Helicobacter pylori), Legionella spp. (e.g., Legionella pneumophila), Leptospira spp. (e.g., Leptospira interrogans), Listeria spp. (e.g., Listeria monocytogenes), Mycobacterium spp. (e.g., Mycobacterium leprae, Mycobacterium tuberculosis), Mycoplasma spp. (e.g., Mycoplasma pneumoniae), Neisseria spp. (e.g., Neisseria gonorrhea, Neisseria meningitidis), Pseudomonas spp. (e.g., Pseudomonas aeruginosa), Rickettsia spp. (e.g., Rickettsia rickettsii), Salmonella spp. (e.g., Salmonella typhi, Salmonella

[0276] ACTIVE 508396272 42 072396.1082

[0277] PATENT typhinurium), Shigella spp. (e.g., Shigella sonnei), Staphylococcus spp. (e.g., Staphylococcus aureus, Staphylococcus epidermidis. Staphylococcus saprophyticus), Streptococcus spp. (e.g., Streptococcus agalacliae. Streptococcus pneumoniae, Streptococcus pyrogenes'), Treponema spp. (e.g., Treponema pallidum), Vibrio spp. (e.g., Vibrio cholerae), and Yersinia spp. (Yersinia pestis). As one of skill in the art would appreciate, antigenic polypeptides can be derived from other bacteria not listed above.

[0278] In certain embodiments, the presently disclosed vaccines include multiple fusion proteins that are capable of self-assembly into a nanoparticle. In certain embodiments, the fusion protein includes a nanoparticle-forming polypeptide and an antigenic polypeptide. In certain embodiments, the antigenic polypeptide is isolated from any portion of a parasite. Non-limiting examples of parasites from which an antigenic polypeptide is obtained or derived include Ancylostoma spp. (e.g., A. duodenale), Anisakis spp., Ascaris lumbricoides, Balantidium coli, Cestoda spp., Cimicidae spp., Clonorchis sinensis, Dicrocoelium dendriticum, Dicrocoelium hospes, Diphyllobothrium latum, Dracunculus spp., Echinococcus spp. (e.g., E. granulosus, E. multilocularis), Entamoeba histolytica, Enterobius vermicularis, Fasciola spp. (e.g., F. hepatica, F. magna, F. gigantica, F. jacksoni), Fasciolopsis buski, Giardia spp. (Giardia lamblia), Gnathostoma spp., Hymenolepis spp. (e.g., H. nana, H. diminuta), Leishmania spp., Loa loa, Metorchis spp. (M. conjunctus, M. albidus), Necator americanus, Oestroidea spp. (e.g., botfly), Onchocercidae spp., Opisthorchis spp. (e.g., 0. viverrini, O. felineus, O. guayaquilensis, and 0. noverca), Plasmodium spp. (e.g., P. falciparum , Protofasciola robusta, Parafasciolopsis fasciomorphae, Paragonimus westermani, Schistosoma spp. (e.g., S. mansoni, S. japonicum, S. mekongi, S. haematobium , Spirometra erinaceieuropaei, Strongyloides stercoralis, Taenia spp. (e.g., T. saginata, T. solium , Toxocara spp. (e.g., T. canis, T. cati), Toxoplasma spp. (e.g., T. gondii , Trichobilharzia regenti, Trichinella spiralis, Trichuris trichiura, Trombiculidae spp., Trypanosoma spp., Tunga penetrans, and Wuchereria bancrofti. As one of skill in the art would appreciate, antigenic polypeptides can be derived from other parasites not listed above.

[0279] In certain embodiments, the presently disclosed vaccines include multiple fusion proteins that are capable of self-assembly into a nanoparticle. In certain embodiments, the fusion protein includes a nanoparticle-forming polypeptide and an antigenic polypeptide. In certain embodiments, the antigenic polypeptide is isolated from any portion of a tumor or can be a tumor antigen (TA). Non-limiting examples of tumor antigens from which an antigenic polypeptide is obtained or derived include gplOO, MART-l / Melan A, gp75 (TRP-I), tyrosinase, NY-ESO-I, melanoma proteoglycan, MAGE family antigens (i.e., MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-6, and MAGE-12), BAGE family antigens, GAGE family antigens (i.e., GAGE-1, GAGE-

[0280] ACTIVE 508396272 43 072396.1082

[0281] PATENT

[0282] 2), RAGE family antigens, N- acetylglucosaminyltransferase-V, pl 5, P-catenin, MUM-I, cyclin dependent kinase-4 (CDK4), p21-ras, BCR- abl, p53, pl85 HER2 / neu, epidermal growth factor receptor (EGFR), carcinoembryonic antigens (CEA), carcinoma- associated mutated mucins (i.e., MUC-1 gene products), EBNA gene products of EBV (i.e., EBNA-I), E7, E6 proteins of human papillomavirus, prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), idiotypic epitopes or antigens, for example, immunoglobulin idiotypes or T cell receptor idiotypes, KSA, kinesin 2, HIP-55, TGFP-1 anti- apoptotic factor, tumor protein D52, HIFT, NY-BR-I, NY- BR-62, NY-BR-75, NY-BR-85, NY-BR-87 and NY-BR-96. As one of skill in the art would appreciate, antigenic polypeptides can be derived from other public antigens not listed above.

[0283] In certain embodiments, the presently disclosed vaccines include multiple fusion proteins that are capable of self-assembly into a nanoparticle. In certain embodiments, the fusion protein includes a nanoparticle-forming polypeptide and an antigenic polypeptide. In certain embodiments, the antigenic polypeptide is an antigenic coronavirus polypeptide (e.g., one disclosed in Section 2.2) or a functional fragment thereof. In certain embodiments, the antigenic coronavirus polypeptide can be a coronavirus spike protein polypeptide or a functional fragment thereof, a receptor-binding domain (RBD) polypeptide or a functional fragment thereof, an N- terminal domain (NTD) polypeptide or a functional fragment thereof, a receptor-binding domain (RBD)-N-terminal chimera polypeptide or a functional fragment thereof, an SI polypeptide or a functional fragment thereof, a stabilized extracellular spike S-2P polypeptide or a functional fragment thereof, a stabilized extracellular spike S polypeptide or a functional fragment thereof, a stabilized extracellular spike S-trimer or a functional fragment thereof, or a combination thereof.

[0284] In certain embodiments, the fusion protein comprises a coronavirus spike protein polypeptide or a functional fragment thereof and a ferritin polypeptide. In certain embodiments, the fusion protein comprises a receptor-binding domain (RBD) polypeptide or a functional fragment thereof and a ferritin polypeptide. In certain embodiments, the fusion protein comprises an N-terminal domain (NTD) polypeptide or a functional fragment thereof and a ferritin polypeptide. In certain embodiments, the fusion protein comprises a receptor-binding domain (RBD)-N-terminal chimera polypeptide or a functional fragment thereof and a ferritin polypeptide. In certain embodiments, the fusion protein comprises an SI polypeptide or a functional fragment thereof and a ferritin polypeptide. In certain embodiments, the fusion protein comprises a stabilized extracellular spike S-2P polypeptide or a functional fragment thereof and a ferritin polypeptide. In certain embodiments, the fusion protein comprises a stabilized extracellular spike S polypeptide or a functional fragment thereof and a ferritin polypeptide. In

[0285] ACTIVE 508396272 44 072396.1082

[0286] PATENT certain embodiments, the fusion protein comprises a stabilized extracellular spike S-trimer or a functional fragment thereof and a ferritin polypeptide.

[0287] In certain embodiments, the fusion protein includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 24. In certain embodiments, the fusion protein includes substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence set forth in SEQ ID NO: 24, that do not significantly alter the function or activity of the fusion protein. In certain embodiments, the ferritin polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 24. In certain embodiments, the ferritin polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 24.

[0288] In certain embodiments, the fusion protein includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 25. In certain embodiments, the fusion protein includes substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence set forth in SEQ ID NO: 25, that do not significantly alter the function or activity of the fusion protein. In certain embodiments, the ferritin polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 25. In certain embodiments, the ferritin polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 25.

[0289] In certain embodiments, the fusion protein includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 26. In certain embodiments, the fusion protein includes substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence set forth in SEQ ID NO: 26, that do not significantly alter the function or activity of the fusion protein. In certain embodiments, the ferritin polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 26. In certain embodiments, the ferritin polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 26.

[0290] In certain embodiments, the fusion protein includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 27. In certain embodiments, the fusion protein includes substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence set forth

[0291] ACTIVE 508396272 45 072396.1082

[0292] PATENT in SEQ ID NO: 27, that do not significantly alter the function or activity of the fusion protein. In certain embodiments, the ferritin polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 27. In certain embodiments, the ferritin polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 27.

[0293] In certain embodiments, the fusion protein includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 28. In certain embodiments, the fusion protein includes substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence set forth in SEQ ID NO: 28, that do not significantly alter the function or activity of the fusion protein. In certain embodiments, the ferritin polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 28. In certain embodiments, the ferritin polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 28.

[0294] In certain embodiments, the fusion protein includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 29. In certain embodiments, the fusion protein includes substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence set forth in SEQ ID NO: 29, that do not significantly alter the function or activity of the fusion protein. In certain embodiments, the ferritin polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 29. In certain embodiments, the ferritin polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 29.

[0295] In certain embodiments, the fusion protein includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 30. In certain embodiments, the fusion protein includes substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence set forth in SEQ ID NO: 30, that do not significantly alter the function or activity of the fusion protein. In certain embodiments, the ferritin polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 30. In certain embodiments, the ferritin polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 30.

[0296] In certain embodiments, the fusion protein includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth

[0297] ACTIVE 508396272 46 072396.1082

[0298] PATENT in SEQ ID NO: 31. In certain embodiments, the fusion protein includes substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence set forth in SEQ ID NO: 31, that do not significantly alter the function or activity of the fusion protein. In certain embodiments, the ferritin polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 31. In certain embodiments, the ferritin polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 31.

[0299] In certain embodiments, the fusion protein includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 32. In certain embodiments, the fusion protein includes substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence set forth in SEQ ID NO: 32, that do not significantly alter the function or activity of the fusion protein. In certain embodiments, the ferritin polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 32. In certain embodiments, the ferritin polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 32.

[0300] In certain embodiments, the fusion protein includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 33. In certain embodiments, the fusion protein includes substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence set forth in SEQ ID NO: 33, that do not significantly alter the function or activity of the fusion protein. In certain embodiments, the ferritin polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 33. In certain embodiments, the ferritin polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 33.

[0301] In certain embodiments, the fusion protein includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 33. In certain embodiments, the fusion protein includes substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence set forth in SEQ ID NO: 33, that do not significantly alter the function or activity of the fusion protein. In certain embodiments, the ferritin polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 33. In certain embodiments, the ferritin polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 33.

[0302] ACTIVE 508396272 47 072396.1082

[0303] PATENT

[0304] In certain embodiments, the fusion protein includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 34. In certain embodiments, the fusion protein includes substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence set forth in SEQ ID NO: 34, that do not significantly alter the function or activity of the fusion protein. In certain embodiments, the ferritin polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 34. In certain embodiments, the ferritin polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 34.

[0305] In certain embodiments, the fusion protein includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 35. In certain embodiments, the fusion protein includes substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence set forth in SEQ ID NO: 35, that do not significantly alter the function or activity of the fusion protein. In certain embodiments, the ferritin polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 35. In certain embodiments, the ferritin polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 35.

[0306] In certain embodiments, the fusion protein includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 36. In certain embodiments, the fusion protein includes substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence set forth in SEQ ID NO: 36, that do not significantly alter the function or activity of the fusion protein. In certain embodiments, the ferritin polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 36. In certain embodiments, the ferritin polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 36.

[0307] In certain embodiments, the fusion protein includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 37. In certain embodiments, the fusion protein includes substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence set forth in SEQ ID NO: 37, that do not significantly alter the function or activity of the fusion protein. In certain embodiments, the ferritin polypeptide comprises the amino acid sequence set forth in SEQ

[0308] ACTIVE 508396272 48 072396.1082

[0309] PATENT

[0310] ID NO: 37. In certain embodiments, the ferritin polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 37.

[0311] In certain embodiments, the fusion protein includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 38. In certain embodiments, the fusion protein includes substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence set forth in SEQ ID NO: 38, that do not significantly alter the function or activity of the fusion protein. In certain embodiments, the ferritin polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 38. In certain embodiments, the ferritin polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 38.

[0312] In certain embodiments, the fusion protein includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 39. In certain embodiments, the fusion protein includes substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence set forth in SEQ ID NO: 39, that do not significantly alter the function or activity of the fusion protein. In certain embodiments, the ferritin polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 39. In certain embodiments, the ferritin polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 39.

[0313] In certain embodiments, the fusion protein includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 40. In certain embodiments, the fusion protein includes substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence set forth in SEQ ID NO: 40, that do not significantly alter the function or activity of the fusion protein. In certain embodiments, the ferritin polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 40. In certain embodiments, the ferritin polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 40.

[0314] In certain embodiments, the fusion protein includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 41. In certain embodiments, the fusion protein includes substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence set forth

[0315] ACTIVE 508396272 49 072396.1082 PATENT in SEQ ID NO: 41, that do not significantly alter the function or activity of the fusion protein. In certain embodiments, the ferritin polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 41. In certain embodiments, the ferritin polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 41. SEQ ID Nos: 24-41 are provided below:

[0316] ACTIVE 508396272 50 072396.1082

[0317] PATENT

[0318] ACTIVE 508396272 51 072396.1082

[0319] PATENT

[0320] ACTIVE 508396272 52 072396.1082

[0321] PATENT

[0322] ACTIVE 508396272 53 072396.1082

[0323] PATENT

[0324] ACTIVE 508396272 54 072396.1082

[0325] PATENT

[0326] ACTIVE 508396272 55 072396.1082

[0327] PATENT

[0328] ACTIVE 508396272 56 072396.1082

[0329] PATENT

[0330] ACTIVE 508396272 57 072396.1082

[0331] PATENT

[0332] Additional examples of fusion proteins and vaccines encompassed by the present disclosure can be found in International Patent Publication No. WO 2021 / 178971 and International Patent Publication No. WO 2024 / 044108, the contents of each of which are incorporated by reference in their entirety.

[0333] In certain embodiments, the vaccine comprises from about 1 pg to about 1000 pg of a fusion protein disclosed herein (e.g., a fusion protein including a nanoparticle-forming polypeptide and an antigenic polypeptide). In certain embodiments, the vaccine comprises from about 1 pg to about 1000 pg, from about 5 pg to about 1000 pg, from about 10 pg to about 1000 pg, from about 15 pg to about 1000 pg, from about 20 pg to about 1000 pg, from about 30 pg to about 1000 pg, from about 40 pg to about 1000 pg, from about 50 pg to about 1000 pg, from about 100 pg to about 1000 pg, from about 150 pg to about 1000 pg, from about 200 pg to about 1000 pg, from about 250 pg to about 1000 pg, from about 300 pg to about 1000 pg, from about 350 pg to about 1000 pg, from about 400 pg to about 1000 pg, from about 500 pg to about 1000

[0334] ACTIVE 508396272 58 072396.1082

[0335] PATENT pg, from about 550 pg to about 1000 pg, from about 600 pg to about 1000 pg, from about 650 pg to about 1000 pg, from about 700 pg to about 1000 pg, from about 750 pg to about 1000 pg, from about 800 pg to about 1000 pg, from about 900 pg to about 1000 pg, or from about 950 pg to about 1000 pg of a fusion protein disclosed herein (e.g., a fusion protein including a nanoparticleforming polypeptide and an antigenic polypeptide).

[0336] In certain embodiments, the vaccine comprises from about 10 pg to about 200 pg, from about 20 pg to about 200 pg, from about 30 pg to about 200 pg, from about 40 pg to about 200 pg, from about 50 pg to about 200 pg, from about 60 pg to about 200 pg, from about 70 pg to about 200 pg, from about 80 pg to about 200 pg, from about 90 pg to about 200 pg, from about 100 pg to about 200 pg, from about 110 pg to about 200 pg, from about 120 pg to about 200 pg, from about 130 pg to about 200 pg, from about 140 pg to about 200 pg, from about 150 pg to about 200 pg, from about 160 pg to about 200 pg, from about 170 pg to about 200 pg, from about 180 pg to about 200 pg, or from about 190 pg to about 200 pg of a fusion protein disclosed herein (e.g., a fusion protein including a nanoparticle-forming polypeptide and an antigenic polypeptide). In certain embodiments, the vaccine comprises from about 10 pg to about 150 pg, from about 20 pg to about 150 pg, from about 30 pg to about 150 pg, from about 40 pg to about 150 pg, from about 50 pg to about 150 pg, from about 60 pg to about 150 pg, from about 70 pg to about 150 pg, from about 80 pg to about 150 pg, from about 90 pg to about 150 pg, from about 100 pg to about 150 pg, from about 110 pg to about 150 pg, from about 120 pg to about 150 pg, from about 130 pg to about 150 pg, or from about 140 pg to about 150 pg of a fusion protein disclosed herein (e.g., a fusion protein including a nanoparticle-forming polypeptide and an antigenic polypeptide). In certain embodiments, the vaccine comprises from about 10 pg to about 150 pg, from about 10 pg to about 30 pg, from about 10 pg to about 40 pg, from about 10 pg to about 50 pg, from about 10 pg to about 60 pg, from about 10 pg to about 70 pg, from about 10 pg to about 80 pg, from about 10 pg to about 90 pg, from about 10 pg to about 100 pg, from about 10 pg to about 110 pg, from about 10 pg to about 120 pg, from about 10 pg to about 130 pg, or from about 10 pg to about 140 pg of a fusion protein disclosed herein (e.g., a fusion protein including a nanoparticleforming polypeptide and an antigenic polypeptide). In certain embodiments, the vaccine comprises from about 50 pg to about 60 pg, from about 50 pg to about 70 pg, from about 50 pg to about 80 pg, from about 50 pg to about 90 pg, from about 50 pg to about 100 pg, from about 50 pg to about 110 pg, from about 50 pg to about 120 pg, from about 50 pg to about 130 pg, or from about 50 pg to about 140 pg of a fusion protein disclosed herein (e.g., a fusion protein including a nanoparticle-forming polypeptide and an antigenic polypeptide).

[0337] ACTIVE 508396272 59 072396.1082

[0338] PATENT

[0339] In certain embodiments, the vaccine comprises from about 1 pg to about 10 pg, from about 2 pg to about 10 pg, from about 3 pg to about 10 pg, from about 4 pg to about 10 pg, from about 5 pg to about 10 pg, from about 6 pg to about 10 pg, from about 7 pg to about 10 pg, from about 8 pg to about 10 pg, from about 9 pg to about 10 pg, from about 1 pg to about 2 pg, from about 2 pg to about 3 pg, from about 3 pg to about 4 pg, from about 4 pg to about 5 pg, from about 5 pg to about 6 pg, from about 6 pg to about 7 pg, from about 7 pg to about 8 pg, from about 1 pg to about 3 pg, from about 1 pg to about 4 pg, from about 1 pg to about 5 pg, from about 1 pg to about 6 pg, from about 1 pg to about 7 pg, from about 1 pg to about 8 pg, or from about 1 pg to about 9 pg of a fusion protein disclosed herein (e.g., a fusion protein including a nanoparticleforming polypeptide and an antigenic polypeptide).

[0340] In certain embodiments, prior to loading onto the plurality of microneedles of the administration device (e.g., one disclosed in Section 4 below), the presently disclosed vaccine can be included in metal-organic frameworks (MOFs). As used herein, “metal-organic frameworks” (MOFs) are organic-inorganic hybrid crystalline porous materials characterized by a regular array of positively charged metal ions surrounded by organic linker molecules. In MOFs, the metal ions form nodes that bind the arms of the linkers together to form a repeating, cage-like structure. Due to this hollow structure, MOFs have an extraordinarily large internal surface area.

[0341] MOFs offer unique structural diversity in contrast to other porous materials. For example, MOFs are characterized by uniform pore structures, atomic-level structural uniformity, tunable porosity, extensive varieties, flexibility in network topology, flexibility in network geometry, flexibility in network dimension, and flexibility in network chemical functionality.

[0342] 3. Adjuvants

[0343] In certain embodiments, the presently disclosed vaccine comprises an adjuvant. As used herein, the term “adjuvant” refers to an ingredient used to elicit and induce a stronger immune response in subject receiving the vaccine.

[0344] In certain embodiments, the adjuvant comprises ADU-S100, saponins (e.g., QS-21, QS- 18, QS-7, and QS-9), poly(I:C), Army Liposomal Formulation ALF, Army Liposomal Formulation ALFA, Army Liposomal Formulation ALFQ, alhydrogel, synthetic monophosphoryl lipid A (MPLA), oil in water emulsions, ADJUPLEX™, ADDA VAX™, CARBOPOL®, Poly ICLC, CpG oligodeoxynucleotides, Flagellin, Iscomatrix, virosomes, MF59, AS04, or a combination thereof.

[0345] In certain embodiments, the adjuvant comprises Army Liposome Formulation (ALF). ALF is composed of a family of suitable anionic liposome-based adjuvants, in which the liposomes contain synthetic phospholipids having dimyristoyl fatty acyl groups, cholesterol, and

[0346] ACTIVE 508396272 60 072396.1082

[0347] PATENT synthetic monophosphoryl lipid A (MPLA). ALF can be used in combination with another vaccine adjuvant, such as an aluminum salt, such as aluminum hydroxide (AH), to form ALFA. Liposomes containing the saponin Q21 (ALFQ) have also been combined with AH to form ALFQA.

[0348] In certain embodiments, the adjuvant comprises Army Liposomal Formulation ALFA (ALFA). In certain embodiments, the adjuvant comprises from about 1 mg / ml to about 20 mg / ml of ALFA. In certain embodiments, the adjuvant comprises from about 1 mg / ml to about 20 mg / ml, from about 2 mg / ml to about 20 mg / ml, from about 3 mg / ml to about 20 mg / ml, from about 4 mg / ml to about 20 mg / ml, from about 5 mg / ml to about 20 mg / ml, from about 6 mg / ml to about 20 mg / ml, from about 7 mg / ml to about 20 mg / ml, from about 8 mg / ml to about 20 mg / ml, from about 9 mg / ml to about 20 mg / ml, from about 10 mg / ml to about 20 mg / ml, from about 11 mg / ml to about 20 mg / ml from, about 12 mg / ml to about 20 mg / ml, from about 13 mg / ml to about 20 mg / ml, from about 14 mg / ml to about 20 mg / ml, from about 15 mg / ml to about 20 mg / ml, from about 3 mg / ml to about 15 mg / ml, from about 5 mg / ml to about 15 mg / ml, from about 8 mg / ml to about 15 mg / ml, from about 10 mg / ml to about 15 mg / ml, from about 11 mg / ml to about 15 mg / ml, from about 12 mg / ml to about 15 mg / ml, from about 13 mg / ml to about 15 mg / ml, from about 13 mg / ml to about 14 mg / ml of ALFA. In certain embodiments, the adjuvant comprises about 13.9 mg / ml of ALFA. In certain embodiments, the adjuvant comprises about 13.98 mg / ml of ALFA.

[0349] In certain embodiments, the adjuvant comprises Army Liposomal Formulation ALFQ (ALFQ). ALFQ was developed by the U.S. Army. ALFQ is an Army-Liposome- Formulation (ALF) containing high amounts of cholesterol together with the QS21 saponin (ALFQ). ALFQ has been used in numerous animal studies and with a variety of immunogens and has shown effectiveness in eliciting robust immune responses. In certain embodiments, the ALFQ adjuvant is a liposomal formulation containing monophosphoryl lipid A (MPLA) and QS-21 saponin. Additional information on the ALFQ adjuvant encompassed by the present disclosure can be found in International Patent Publication No. WO 2021 / 178971; U.S. Patent No. 10,434,167; and in Alving et al. Expert review of vaccines 19.3 (2020): 279-292; the contents of each of which are incorporated by reference in their entirety.

[0350] In certain embodiments, the adjuvant comprises from about 1 mg / ml to about 20 mg / ml of ALFQ. In certain embodiments, the adjuvant comprises from about 1 mg / ml to about 20 mg / ml, from about 2 mg / ml to about 20 mg / ml, from about 3 mg / ml to about 20 mg / ml, from about 4 mg / ml to about 20 mg / ml, from about 5 mg / ml to about 20 mg / ml, from about 6 mg / ml to about 20 mg / ml, from about 7 mg / ml to about 20 mg / ml, from about 8 mg / ml to about 20 mg / ml, from about 9 mg / ml to about 20 mg / ml, from about 10 mg / ml to about 20 mg / ml, from about 11 mg / ml

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[0352] PATENT to about 20 mg / ml from, about 12 mg / ml to about 20 mg / ml, from about 13 mg / ml to about 20 mg / ml, from about 14 mg / ml to about 20 mg / ml, from about 15 mg / ml to about 20 mg / ml, from about 3 mg / ml to about 15 mg / ml, from about 5 mg / ml to about 15 mg / ml, from about 8 mg / ml to about 15 mg / ml, from about 10 mg / ml to about 15 mg / ml, from about 11 mg / ml to about 15 mg / ml, from about 12 mg / ml to about 15 mg / ml, from about 13 mg / ml to about 15 mg / ml, from about 13 mg / ml to about 14 mg / ml of ALFQ. In certain embodiments, the adjuvant comprises about 13.5 mg / ml of ALFQ. In certain embodiments, the adjuvant comprises about 13.48 mg / ml of ALFQ.

[0353] In certain embodiments, the adjuvant comprises from about 1 pg to about 1000 pg of ALFQ. In certain embodiments, the adjuvant comprises from about 1 pg to about 1000 pg, from about 5 pg to about 1000 pg, from about 10 pg to about 1000 pg, from about 15 pg to about 1000 pg, from about 20 pg to about 1000 pg, from about 30 pg to about 1000 pg, from about 40 pg to about 1000 pg, from about 50 pg to about 1000 pg, from about 100 pg to about 1000 pg, from about 150 pg to about 1000 pg, from about 200 pg to about 1000 pg, from about 250 pg to about 1000 pg, from about 300 pg to about 1000 pg, from about 350 pg to about 1000 pg, from about 400 pg to about 1000 pg, from about 500 pg to about 1000 pg, from about 550 pg to about 1000 pg, from about 600 pg to about 1000 pg, from about 650 pg to about 1000 pg, from about 700 pg to about 1000 pg, from about 750 pg to about 1000 pg, from about 800 pg to about 1000 pg, from about 900 pg to about 1000 pg, or from about 950 pg to about 1000 pg of ALFQ.

[0354] In certain embodiments, the adjuvant comprises from about 1 pg to about 200 pg, from about 20 pg to about 200 pg, from about 30 pg to about 200 pg, from about 40 pg to about 200 pg, from about 50 pg to about 200 pg, from about 60 pg to about 200 pg, from about 70 pg to about 200 pg, from about 80 pg to about 200 pg, from about 90 pg to about 200 pg, from about 100 pg to about 200 pg, from about 110 pg to about 200 pg, from about 120 pg to about 200 pg, from about 130 pg to about 200 pg, from about 140 pg to about 200 pg, from about 150 pg to about 200 pg, from about 160 pg to about 200 pg, from about 170 pg to about 200 pg, from about 180 pg to about 200 pg, or from about 190 pg to about 200 pg of ALFQ. In certain embodiments, the adjuvant comprises from about 10 pg to about 150 pg, from about 20 pg to about 150 pg, from about 30 pg to about 150 pg, from about 40 pg to about 150 pg, from about 50 pg to about 150 pg, from about 60 pg to about 150 pg, from about 70 pg to about 150 pg, from about 80 pg to about 150 pg, from about 90 pg to about 150 pg, from about 100 pg to about 150 pg, from about 110 pg to about 150 pg, from about 120 pg to about 150 pg, from about 130 pg to about 150 pg, or from about 140 pg to about 150 pg of ALFQ. In certain embodiments, the adjuvant comprises from about 10 pg to about 150 pg, from about 10 pg to about 30 pg, from about 10 pg to about 40 pg, from about 10 pg to about 50 pg, from about 10 pg to about 60 pg, from about 10 pg to

[0355] ACTIVE 508396272 62 072396.1082 PATENT about 70 gg, from about 10 gg to about 80 gg, from about 10 gg to about 90 gg, from about 10 gg to about 100 gg, from about 10 gg to about 110 gg, from about 10 gg to about 120 gg, from about 10 gg to about 130 gg, or from about 10 gg to about 140 gg of ALFQ. In certain embodiments, the adjuvant comprises from about 50 gg to about 60 gg, from about 50 gg to about 70 gg, from about 50 gg to about 80 gg, from about 50 gg to about 90 gg, from about 50 gg to about 100 gg, from about 50 gg to about 110 gg, from about 50 gg to about 120 gg, from about 50 gg to about 130 gg, or from about 50 gg to about 140 gg of ALFQ.

[0356] In certain embodiments, the adjuvant is ADU-S100. ADU-S100 is a synthetic cyclic dinucleotide (CDN) agonist (activator) of Stimulator of Interferon Genes (STING), a receptor crucial to activate the innate (endogenous) immune system. ADU-S100 can activate all known human and mouse STINGs, and induces the expression of cytokines and chemokines, leading to a robust and durable antigen-specific T-cell mediated immune response against the antigenic polypeptide.

[0357] In certain embodiments, the adjuvant comprises from about 5 gg to about 50 gg of ADU- S100. In certain embodiments, the adjuvant comprises from about 50 gg to about 3500 gg, from about 100 gg to about 3500 gg, from about 200 gg to about 3500 gg, from about 300 gg to about 3500 gg, from about 400 gg to about 3500 gg, from about 500 gg to about 3500 gg, from about 600 gg to about 3500 gg, from about 700 gg to about 3500 gg, from about 800 gg to about 3500 gg, from about 900 gg to about 3500 gg, from about 1000 gg to about 3500 gg, from about 1250 gg to about 3500 gg, from about 1500 gg to about 3500 gg, from about 1750 gg to about 3500 gg, from about 2000 gg to about 3500 gg, from about 2250 gg to about 3500 gg, from about 2500 gg to about 3500 gg, from about 2750 gg to about 3500 gg, from about 3000 gg to about 3500 gg, from about 3250 gg to about 3500 gg, from about 100 gg to about 500 gg, from about 500 gg to about 1000 gg, from about 1000 gg to about 1500 gg, from about 1500 gg to about 2000 gg, from about 2000 gg to about 2500 gg, from about 2500 gg to about 3000 gg, or from about 3000 gg to about 3500 gg of ADU-S100.

[0358] In certain embodiments, the adjuvant is a saponin. Saponins are steroid or triterpene glycosides widely distributed in the plant and marine animal kingdoms. Saponins are noted for forming colloidal solutions in water which foam on shaking, and for precipitating cholesterol. When saponins are near cell membranes they create pore-like structures in the membrane which cause the membrane to burst. In certain embodiments, the saponin is selected from QS-21, QS18, QS-7, or QS-9.

[0359] In certain embodiments, the adjuvant is QS21. QS-21 is an acylated saponin at the 4- hydroxyl position on fucose with two linked 3,5 dihydroxy-6-methyloctanoic acids. QS21 induces

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[0361] PATENT inflammatory cytokines and imparts a Thl bias in vaccine responses. The ability to lyse cell membranes, hemolysis and injection site pain are major limiting factors in its use.

[0362] In certain embodiments, the adjuvant comprises from about 1 pg to about 1000 pg of QS- 21. In certain embodiments, the adjuvant comprises from about 1 pg to about 1000 pg, from about 5 pg to about 1000 pg, from about 10 pg to about 1000 pg, from about 15 pg to about 1000 pg, from about 20 pg to about 1000 pg, from about 30 pg to about 1000 pg, from about 40 pg to about 1000 pg, from about 50 pg to about 1000 pg, from about 100 pg to about 1000 pg, from about 150 pg to about 1000 pg, from about 200 pg to about 1000 pg, from about 250 pg to about 1000 pg, from about 300 pg to about 1000 pg, from about 350 pg to about 1000 pg, from about 400 pg to about 1000 pg, from about 500 pg to about 1000 pg, from about 550 pg to about 1000 pg, from about 600 pg to about 1000 pg, from about 650 pg to about 1000 pg, from about 700 pg to about 1000 pg, from about 750 pg to about 1000 pg, from about 800 pg to about 1000 pg, from about 900 pg to about 1000 pg, or from about 950 pg to about 1000 pg of QS-21.

[0363] In certain embodiments, the adjuvant is poly(I:C). Polyinosinic-polycytidylic acid (Poly(I:C)), a synthetic double-stranded homopolymer, binds TLR3 and activates the transcription factor interferon regulator factor 3 (IRF3) following the initiation of TIR domain-containing adaptor protein (TRIF)-dependent TLR signaling. Activation of IRF3 by poly(I:C) leads to the production of type I interferons. Poly(I:C) also activates the RNA helicases MDA-5 and RIG-1.

[0364] In certain embodiments, the adjuvant comprises from about 1 pg to about 1000 pg of poly(I:C). In certain embodiments, the adjuvant comprises from about 1 pg to about 1000 pg, from about 5 pg to about 1000 pg, from about 10 pg to about 1000 pg, from about 15 pg to about 1000 pg, from about 20 pg to about 1000 pg, from about 30 pg to about 1000 pg, from about 40 pg to about 1000 pg, from about 50 pg to about 1000 pg, from about 100 pg to about 1000 pg, from about 150 pg to about 1000 pg, from about 200 pg to about 1000 pg, from about 250 pg to about 1000 pg, from about 300 pg to about 1000 pg, from about 350 pg to about 1000 pg, from about 400 pg to about 1000 pg, from about 500 pg to about 1000 pg, from about 550 pg to about 1000 pg, from about 600 pg to about 1000 pg, from about 650 pg to about 1000 pg, from about 700 pg to about 1000 pg, from about 750 pg to about 1000 pg, from about 800 pg to about 1000 pg, from about 900 pg to about 1000 pg, or from about 950 pg to about 1000 pg of poly(I:C).

[0365] In certain embodiments, similarly to the vaccine described above, the adjuvants can be included in metal-organic frameworks (MOFs) prior to loading onto microarray.

[0366] 4. Administration Devices

[0367] The present disclosure provides an administration device which enables efficient and safe drug and vaccine delivery to the skin and mucosal surfaces. As used herein, the term

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[0369] PATENT

[0370] “administration device” refers to a device which is adapted or designed to administer a drug, for example, a presently disclosed vaccine, or pharmaceutical formulation.

[0371] In certain embodiments, the administration device is a skin-targeted administration device. As used herein, the term “skin-targeted” refers to intracutaneous drug delivery that allows the delivery of an active ingredient (e.g., a vaccine of Section 2) to immunologically rich skin layers (e.g., viable epidermis and dermis), thereby obtaining an effective intracutaneous immunization. Non-limiting examples of skin-targeted delivery strategies include STAR particles, spherical nucleic acids (SNAs), microneedle arrays (MNAs), and MNA-delivered particles. Additional information on skin-targeted drug delivery strategy can be found in Korkmaz et al., Expert opinion on drug delivery 18.2 (2021): 151-167.

[0372] In certain embodiments, the administration device is a patch delivery system. In certain embodiments, the administration device administers a certain volume of the active ingredient (e.g., vaccine disclosed in Section 2). For example, but without any limitation, the administration device administers about 0.001 mL, about 0.01 mL, about 0.1 mL, about 0.2 mL, about 0.3 mL, about 0.4 mL, about 0.5 mL, about 0.7 mL, about 1.0 mL, about 1.25 mL, about 1.4 mL, about 1.5 mL, about 1.75 mL, about 2.0 mL, or about 5.0 mL.

[0373] In certain embodiments, the administration device comprises a microneedle array. In certain embodiments, the microneedle array is a dissolvable microneedle array. In certain embodiments, the dissolvable microneedle array is an aqueous-soluble microneedle array. In certain embodiments, the dissolvable microneedle array is for transdermal insertion, e.g., local cutaneous delivery. In certain embodiments, the array includes a base portion and a plurality of microneedles extending from the base portion and containing a presently disclosed vaccine (e.g., one disclosed in Section 2). In certain embodiments, the microneedles further comprise at least one adjuvant (e.g., one disclosed in Section 3).

[0374] In certain embodiments, the microneedle array is from about 0.5 cm2to about 10 cm2, from about 0.6 cm2to about 10 cm2, from about 0.7 cm2to about 10 cm2, from about 0.8 cm2to about 10 cm2, from about 0.9 cm2to about 10 cm2, from about 1.0 cm2to about 10 cm2, from about 1.1 cm2to about 10 cm2, from about 1.2 cm2to about 10 cm2, from about 1.3 cm2to about 10 cm2, from about 1.4 cm2to about 10 cm2, from about 1.5 cm2to about 10 cm2, from about 1.6 cm2to about 10 cm2, from about 1.7 cm2to about 10 cm2, from about 1.8 cm2to about 10 cm2, from about 1.9 cm2to about 10 cm2, from about 2.0 cm2to about 10 cm2, from about 2.2 cm2to about 10 cm2, from about 2.5 cm2to about 10 cm2, from about 2.7 cm2to about 10 cm2, from about 3.0 cm2to about 10 cm2, from about 3.5 cm2to about 10 cm2, from about 4.0 cm2to about 10 cm2, from about 4.5 cm2to about 10 cm2, from about 5.0 cm2to about 10 cm2, from about 6.0 cm2to

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[0376] PATENT about 10 cm2, from about 7.0 cm2to about 10 cm2, from about 8.0 cm2to about 10 cm2, or from about 9.0 cm2to about 10 cm2. In certain embodiments, the microneedle array is from about from about 1.0 cm2to about 2 cm2, from about 1.1 cm2to about 2 cm2, from about 1.2 cm2to about 2 cm2, from about 1.3 cm2to about 2 cm2, from about 1.4 cm2to about 2 cm2, from about 1.5 cm2to about 2 cm2, from about 1.6 cm2to about 2 cm2, from about 1.7 cm2to about 2 cm2, from about 1.8 cm2to about 2 cm2, from about 1.9 cm2to about 2 cm2, from about 2.0 cm2to about 3 cm2, from about 2.2 cm2to about 3 cm2, from about 2.5 cm2to about 3 cm2, from about 2.7 cm2to about 3 cm2, from about 3.0 cm2to about 4 cm2, from about 3.2 cm2to about 4 cm2, from about 3.5 cm2to about 4 cm2, or from about 3.7 cm2to about 4 cm2. In certain embodiments, the plurality of microneedles includes from about 10 microneedles to about 106microneedles, from about 102microneedles to about 106microneedles, from about 103microneedles to about 106microneedles, from about 104microneedles to about 106microneedles, from about 105microneedles to about 106microneedles, from about 10 microneedles to about 105microneedles, from about 102microneedles to about 105microneedles, from about 103microneedles to about 105microneedles, from about 104microneedles to about 105microneedles, from about 10 microneedles to about 104microneedles, from about 102microneedles to about 104microneedles, from about 103microneedles to about 104microneedles, from about 10 microneedles to about 103microneedles, from about 102microneedles to about 103microneedles, or from about 10 microneedles to about 102microneedles.

[0377] In certain embodiments, each microneedle has a length between about 5 mm and about 0.1 mm. In certain embodiments, each microneedle has a length of about 5 mm or less, about 4 mm or less, about 3 mm or less, about 2 mm or less, about 1 mm or less, about 0.95 mm or less, about 0.90 mm or less, about 0.85 mm or less, about 0.80 mm or less, about 0.75 mm or less, about 0.70 mm or less, about 0.65 mm or less, about 0.60 mm or less, about 0.55 mm or less, about 0.50 mm or less, about 0.45 mm or less, about 0.40 mm or less, about 0.35 mm or less, about 0.30 mm or less, about 0.25 mm or less, about 0.20 mm or less, about 0.15 mm or less, or about 0.10 mm or less.

[0378] In certain embodiments, each microneedle includes a vaccine disclosed herein. In certain embodiments, the vaccine is loaded on a metal-organic framework. In certain embodiments, the vaccine is not loaded on a metal-organic framework.

[0379] In certain embodiments, each microneedle includes a vaccine and an adjuvant disclosed herein. In certain embodiments, the vaccine is loaded on a metal-organic framework and the adjuvant is loaded on the same metal-organic framework. In certain embodiments, the vaccine is loaded on a first metal-organic framework and the adjuvant is loaded on a second metal-organic

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[0381] PATENT framework. In certain embodiments, the vaccine is loaded on a metal-organic framework and the adjuvant is not loaded on a metal-organic framework. In certain embodiments, the vaccine is not loaded on a metal-organic framework and the adjuvant is loaded on a metal-organic framework. In certain embodiments, the vaccine and the adjuvant are not loaded on a metal-organic framework.

[0382] In certain embodiments, each microneedle includes either a vaccine disclosed herein, or an adjuvant disclosed herein. For example, but without any limitation, the plurality of microneedles disclosed herein can include a first set of microneedles comprising the vaccine disclosed herein and a second set of microneedles including the adjuvant. In another example, but without any limitation, the plurality of microneedles disclosed herein can include a first set of microneedles comprising the vaccine disclosed herein, a second set of microneedles including the adjuvant, and a third set of microneedles including both vaccine and adjuvant.

[0383] Figures 2A-2F illustrate exemplary shapes and dimensions of microneedles and microneedle arrays encompassed by the present disclosure. Based on the illustrative sizes shown in Figures 2A-2F, a microneedle array that comprises an active ingredient homogenously distributed throughout the array exhibits active ingredient waste of greater than 40 percent. For example, if the entire area of the array is 61 mm2and the microneedle array area is 36 mm2, then the percent utilization of the active ingredient is less than 60 percent. Although the dimensions reflected in Figures 2A-2F illustrate a particular size array and shape of microneedles, it should be understood that similar waste is present in any other size microneedle array in which the active ingredient is homogenously distributed throughout the array, regardless of the size of the array or the shape of the microneedles involved.

[0384] In certain embodiments, the microneedle array is a fully dissolvable microneedle array. In certain embodiments, the microneedle array comprises substrates and geometries that enable effective delivery of a broad range of active ingredients, including, for example and without any limitation, a broad range of protein and / or small molecule medicines and vaccines.

[0385] In certain embodiments, the microneedle array includes needle tips loaded with an active ingredient (e.g., a vaccine disclosed in Section 2). By localizing the active ingredients in this manner, the remainder of the microneedle array volume can be prepared using less expensive matrix material that is non-active and generally regarded as safe. The net result is greatly improved efficiency of drug delivery based on (1) reduced waste of non-deliverable active ingredients incorporated into the non-needle portions of the microneedle array, and (2) higher drug concentration in the skin penetrating needle tips. This results in dramatically improved economic

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[0387] PATENT feasibility proportional to the cost of drug cargo and increased effective cargo delivery capacity per needle of these novel microneedle arrays.

[0388] In certain embodiments, the plurality of microneedles can be pre-formed to have a shape that comprises a first cross-sectional dimension at a top portion, a second cross-sectional dimension at a bottom portion, and a third cross-sectional dimension at an intermediate portion. In certain embodiments, the intermediate portion is located between the top portion and the bottom portion. In certain embodiments, the third cross-sectional dimension is greater than the first and second cross-sectional dimensions. In certain embodiments, each microneedle includes a plurality of layers of dissoluble biocompatible material. For example, but without any limitation, each microneedle includes a plurality of layers of carboxymethylcellulose.

[0389] In certain embodiments, the dissolvable microneedle array can be prepared by a fabrication technology that results in various active ingredients to be incorporated into the needle tips. By this way, it is possible to use less expensive matrix material that is non-active and generally regarded as safe in the remainder of the microneedle array volume. The net result is greatly improved efficiency of drug delivery based on reduced waste of non-deliverable active ingredients incorporated into the non-needle portions of the microneedle array, and higher drug concentration in the skin penetrating needle tips. Additional information can be found in U.S. Pat. No. 10,0441,768; U.S. Pat. No. 8,834423; U.S. Pat. No. 11,744,927; U.S. Pat. No. 9,944,019; U.S. Pat. No. 11,744,889; U.S. Pat. No. 11,684,763; International Patent Publication No. WO 2020 / 232394; International Patent Publication No. WO 2021 / 243219; International Patent Publication No. WO 2021 / 207483; International Patent Publication No. WO 2022 / 236107; International Patent Application No. PCT / US2023 / 069282; and U.S. Provisional Application No. 63 / 490,670; the contents of each of which are incorporated by reference in their entirety.

[0390] In certain embodiments, the active ingredient (e.g., a vaccine disclosed in Section 2) is concentrated in the microneedle tips of the respective arrays. In certain embodiments, the active ingredient (e.g., a vaccine disclosed in Section 2) is concentrated in the upper half of the individual microneedles in the array. In certain embodiments, the active ingredient (e.g., a vaccine disclosed in Section 2) is concentrated in the tip of the microneedle, with the tip being defined by an area of the microneedle that extends from a base portion in a narrowing and / or tapered manner. The base portion, in turn, extends from the supporting structure of the array.

[0391] In certain embodiments, individual microneedles can include active ingredients (e.g., a vaccine disclosed in Section 2) only in the upper half of the microneedle. In certain embodiments, individual microneedles can include active ingredients (e.g., a vaccine disclosed in Section 2) only in the tips or in a narrowing portion near the tip of the microneedle. In certain embodiments,

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[0393] PATENT individual needles can include active ingredients (e.g., a vaccine disclosed in Section 2) throughout the entire microneedle portion that extends from the supporting structure. Additional information on the ways individual microneedles include active ingredients can be found in U.S. Pat. No. 10,0441,768; U.S. Pat. No. 8,834423; U.S. Pat. No. 11,744,927; U.S. Pat. No. 9,944,019; U.S. Pat. No. 11,744,889; U.S. Pat. No. 11,684,763; International Patent Publication No. WO 2020 / 232394; International Patent Publication No. WO 2021 / 243219; International Patent Publication No. WO 2021 / 207483; International Patent Publication No. WO 2022 / 236107; International Patent Application No. PCT / US2023 / 069282; and U.S. Provisional Application No. 63 / 490,670; the contents of each of which are incorporated by reference in their entirety.

[0394] In certain embodiments, the microneedle arrays can be configured to penetrate the stratum comeum to deliver their cargo (e.g., biologies or active ingredients) to the epidermis and / or dermis, while minimizing pain and bleeding by preventing penetration to deeper layers that may contain nerve endings and vessels. In certain embodiments, the microneedle array can include pyramidal CMC-microneedles. In certain embodiments, pyramidal CMC -microneedles effectively can penetrate the stratum corneum, epidermis, and dermis of living human skin, and thus can be used for cutaneous delivery. Additional information can be found in U.S. Pat. No. 10,0441,768; U.S. Pat. No. 8,834423; U.S. Pat. No. 11,744,927; U.S. Pat. No. 9,944,019; U.S. Pat. No. 11,744,889; U.S. Pat. No. 11,684,763; International Patent Publication No. WO 2020 / 232394; International Patent Publication No. WO 2021 / 243219; International Patent Publication No. WO 2021 / 207483; International Patent Publication No. WO 2022 / 236107; International Patent Application No. PCT / US2023 / 069282; and U.S. Provisional Application No. 63 / 490,670; the contents of each of which are incorporated by reference in their entirety.

[0395] To construct the microneedle arrays, a base material can be used to form portions of each microneedle that have active ingredients and portions that do not. As outlined above, each microneedle can include active ingredients only in the microneedles, only in the upper half of the microneedles, or only in a portion of the microneedle that tapers near the tip. Therefore, in order to control the delivery of the active ingredient(s) (e.g., a vaccine disclosed in Section 2) and to control the cost of the microneedle arrays, each microneedle can include a portion with an active ingredient and a portion without the active ingredient. In certain embodiments, the portion without the active ingredient includes the supporting structure of the microneedle array and, in some embodiments, a base portion (e.g., a lower half) of each microneedle in the array.

[0396] Various materials can be used as the base material for the microneedle arrays. The structural substrates of biodegradable solid microneedles can include poly (lactic-co-glycolic acid) (PLGA) or carboxymethylcellulose (CMC) based formulations, and the like.

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[0398] PATENT

[0399] In certain embodiments, CMC can be used as the base material of the microneedle arrays. This is because PLGA based devices can limit drug delivery and vaccine applications due to the relatively high temperature (e.g., 135 degrees Celsius or higher) and vacuum required for fabrication. In contrast, a CMC-based matrix can be formed at room temperature in a simple spincasting and drying process, making CMC-microneedle arrays more desirable for incorporation of sensitive biologies, peptides, proteins, nucleic acids, and other various active ingredients. CMC- hydrogel may be prepared from low viscosity sodium salt of CMC with or without active ingredients (as described below) in sterile dH2O. In certain exemplary embodiments, CMC can be mixed with sterile distilled water (dH2O) and with the active ingredients (e.g., a vaccine disclosed in Section 2) to achieve about 25 wt % CMC concentration. The resulting mixture can be stirred to homogeneity and equilibrated at about 4° C. for 24 hours. During this period, the CMC and any other components can be hydrated, and a hydrogel can be formed. The hydrogel may be degassed in a vacuum for about an hour and centrifuged at about 20,000 g for an hour to remove residual micro-sized air bubbles that might interfere with a spin casting / drying process of the CMC-microneedle arrays. The dry matter content of the hydrogel can be tested by drying a fraction (10 g) of it at 85° C for about 72 hours. The ready-to-use CMC-hydrogel is desirably stored at about 4° C. until use.

[0400] In certain embodiments, the active ingredients, with or without an adjuvant, can be incorporated in a hydrogel of CMC at a relatively high (20-30%) CMC-dry biologies weight ratio before the spin-casting process. Arrays can be spin-cast at room temperature, making the process compatible with the functional stability of a structurally broad range of active ingredients. Since the master and production molds can be reusable for a large number of fabrication cycles, the fabrication costs can be greatly reduced. The resulting dehydrated CMC-microneedle arrays are generally stable at room temperature or slightly lower temperatures (such as about 4° C ), and preserve the activity of the incorporated biologies, facilitating easy, low-cost storage and distribution.

[0401] In certain embodiments, the surface of the production molds can be covered with about 50 pl (for molds with 11 mm diameter) of CMC-hydrogel and spin-casted by centrifugation at 2,500 g for about 5 minutes. After the initial CMC-hydrogel layer, another 50 pl CMC-hydrogel can be layered over the mold and centrifuged for about 4 hours at 2,500 g. At the end of a drying process, the CMC-microneedle arrays are separated from the molds, trimmed off from excess material at the edges, collected and stored at about 4° C. The production molds may be cleaned and reused for further casting of microneedle arrays.

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[0403] PATENT

[0404] CMC-solids can be formed with layers that do not contain active ingredients and layers that contain active ingredients. For example, but without any limitation, CMC-solids with different shapes and embedded active cargos on an upper layer which becomes, after micromilling, the portions of the microneedle with the active ingredients. Additionally or alternatively, CMC solids can be milled to dimensions for further processing. It should be understood that the geometries are not intended to be limiting. Any geometry can be used with the active ingredients disclosed herein. Additional information of the shapes and embedding of the microarray can be found in U.S. Pat. No. 10,0441,768; U.S. Pat. No. 8,834423; U.S. Pat. No. 11,744,927; U.S. Pat. No. 9,944,019; U.S. Pat. No. 11,744,889; U.S. Pat. No. 11,684,763; International Patent Publication No. WO 2020 / 232394; International Patent Publication No. WO 2021 / 243219; International Patent Publication No. WO 2021 / 207483; International Patent Publication No. WO 2022 / 236107; International Patent Application No. PCT / US2023 / 069282; and U.S. Provisional Application No. 63 / 490,670; the contents of each of which are incorporated by reference in their entirety.

[0405] Exemplary microneedle arrays and methods of making and using microneedle arrays can be found in U.S. Pat. No. 10,0441,768; U.S. Pat. No. 8,834423; U.S. Pat. No. 11,744,927; U.S. Pat. No. 9,944,019; U.S. Pat. No. 11,744,889; U.S. Pat. No. 11,684,763; International Patent Publication No. WO 2020 / 232394; International Patent Publication No. WO 2021 / 243219; International Patent Publication No. WO 2021 / 207483; International Patent Publication No. WO 2022 / 236107; International Patent Application No. PCT / US2023 / 069282; and U.S. Provisional Application No. 63 / 490,670; the contents of each of which are incorporated by reference in their entirety. For example, apparatuses and methods are described for fabricating dissolvable microneedle arrays using master molds formed by micromilling techniques. For example, microneedle arrays can be fabricated based on a mastermold (positive) to production mold (negative) to array (positive) methodology. Micromilling technology can be used to generate various micro-scale geometries on virtually any type of material, including metal, polymer, and ceramic parts. Micromilled mastermolds of various shapes and configurations can be effectively used to generate multiple identical female production molds. The female production molds can then be used to microcast various microneedle arrays. Direct micromilling of mastermolds can replace other exemplary microneedle array production methods that involve expensive, complex, and equipment-sensitive SU-8 based lithography or laser etching techniques, which are conventionally used to create mastermolds for dissolvable needle arrays. In addition, as discussed below, micromilling can provide for the construction of more complex mastermold features than can conventional lithography and laser etching processes. Precision-micromilling systems can be

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[0407] PATENT used for fabricating a microneedle mastermold, using micro-scale (for example, as small as 10 pm (micrometers or microns)) milling tools within precision computer controlled miniature machine- tool platforms. The system can include a microscope to view the surface of the workpiece that is being cut by the micro-tool. The micro-tool can be rotated at ultra-high speeds (200,000 rpm) to cut the workpiece to create the desired shapes. Micromilling process can be used to create complex geometric features with many kinds of material, which are not possible using conventional lithographic or laser etching processes. Various types of tooling can be used in the micromilling process, including, for example, carbide micro-tools or diamond tools.

[0408] Mastermolds can be micromilled from various materials, including, for example, Cirlex® (DuPont, Kapton® polyimide). Mastermolds can be used to fabricate flexible production molds from a suitable material, such as a silicone elastomer, e.g., SYLGARD® 184 (Dow Coming). The mastermold is desirably formed of a material that is capable of being reused so that a single mastermold can be repeatedly used to fabricate a large number of production molds. Similarly, each production mold is desirably able to fabricate multiple microneedle arrays.

[0409] In another example, production molds are made from SYLGARD® 184 (Dow Corning) and are mixed at a 10: 1 SYLGARD® to curing agent ratio. The mixture is degassed for about 10 minutes and poured over the mastermold to form an approximately 8 mm layer, subsequently degassed again for about 30 minutes and cured at 85° C. for 45 minutes. After cooling down to room temperature, the mastermold is separated from the cured silicone, and the silicone production mold is trimmed. From a single mastermold, a large number of production molds (e.g., 100 or more) can be produced with very little, if any, apparent deterioration of the Cirlex® or acrylic mastermolds.

[0410] In a further example, to construct the microneedle arrays, a base material is used to form portions of each microneedle that have active ingredients and portions that do not. Of course, if desired, each microneedle can comprise only portions that contain active ingredients; however, to control the delivery of the active ingredient(s) and to control the cost of the microneedle arrays, each microneedle optionally is constructed such that a portion of the structure has an active ingredient and a portion does not include an active ingredient. Variations in the size, shape and number of the microneedles, and location of the active ingredient(s) in the microneedles, may be readily varied by varying the mastermold, or by varying the deposition and patterning of the materials used to produce the microarray.

[0411] Alternatively, the MNA may include microneedles that are not dissolvable, but that include the immunogen-containing composition coated thereon, or contained within a lumen or via thereof, which also allows for access to skin cells.

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[0413] PATENT

[0414] Various materials can be used as the plurality of microneedles. In certain embodiments, each microneedle comprises carboxymethyl cellulose, trehalose, sucrose, gelatin, glucose, lactose, collagen, chitosan, poly-y-glutamate, polyvinylpyrrolidone, maltodextrin, silk, hyaluronic acid, poly (lactic-co-glycolic acid), poly (lactic acid), poly (vinyl alcohol), polyethylene glycol, or a combination thereof.

[0415] A large variety of materials useful for preparation of the microneedle array are available, along with variation in the location of such materials in the microarray. Precise positioning and layering of the materials during, e.g., spin casting, of the microneedle array will yield any desired structure.

[0416] In certain embodiments, the presently disclosed administration devices can be obtained using additive manufacturing. As used herein, the term “additive manufacturing” refers to an advanced technique used for the manufacturing of complex geometries and structures by adding material in layer form, using 3D model data. Additive manufacturing is also known as rapid prototyping, or on-demand manufacturing, or digital fabrication, or desktop manufacturing, or solid freeform manufacturing, or layer manufacturing, or direct manufacturing technique, or 3D printing. Non-limiting examples of additive manufacturing techniques include stereo lithography (SLA), digital light processing (DLP), selective laser sintering (SLS), electron beam melting (EBM), fusion deposition modeling (FDM), multijet / polyjet 3D printing, selective laser melting (SLM), laminated object manufacturing (LOM), and the like. Additive manufacturing can create any complex 3D component of any shape by utilizing 3D model data, which is impossible to fabricate by traditional manufacturing techniques. Additional information on additive manufacturing can be found in Kumar et al., Journal of Manufacturing Processes 64 (2021): 828-850, the content of which is incorporated by reference in its entirety.

[0417] In certain embodiments, the presently disclosed administration devices can be obtained using additive manufacturing of master molds with micron-scale needles. In certain embodiments, the micron-scale needs include sharp tips and smooth edges.

[0418] In certain embodiments, the manufacturing of the presently disclosed administration devices can further require obtaining a negative production mold with microneedle-shaped wells obtained through micromolding of an elastomer with the master mold. In certain embodiments, the elastomer is polydimethylsiloxane (PDMS).

[0419] In certain embodiments, the manufacturing of the presently disclosed administration devices can further require preparation of the presently disclosed vaccines by spin-casting of vaccine components (e.g., antigenic polypeptides, adjuvants, etc.) along with water-soluble

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[0421] PATENT structural biomaterials into molds (e.g., PDMS molds). For example, and without any limitation, a combination of carboxymethyl cellulose (CMC) and Trehalose can be used.

[0422] 5. Methods of Treatment

[0423] The present disclosure provides methods of preventing and / or treating an infectious disease (e.g., COVID-19) in a subject in need thereof. In certain embodiments, the methods include administering an effective amount of a vaccine (e.g., one disclosed in Section 2) using a microneedle array (e.g., one disclosed in Section 4).

[0424] In certain embodiments, the subject is asymptomatic. In certain embodiments, the subject exhibits one or more symptoms selected from the group consisting of fever, cough, fatigue, shortness of breath (dyspnea), muscle and / or joint pain, sore throat, headache, conjunctivitis, diarrhea, lack or loss of appetite, vomiting, abdominal pain, chills, and a combination thereof. In certain embodiments, the subject exhibits one or more symptoms selected from the group consisting of fever, cough, and shortness of breath. In certain embodiments, the subject exhibits one or more digestive symptoms (e.g., extra-pulmonary symptoms) selected from the group consisting of diarrhea, lack or loss of appetite, vomiting, and abdominal pain.

[0425] In certain embodiments, the one or more symptoms appear from about 2 days to about 14 days after the subject's exposure to the pathogen (e.g., one described in Section 2, e.g., coronavirus). In certain embodiments, the one or more symptoms appear from about 2 days to about 3 days, from about 4 days to about 5 days, from about 6 days to about 10 days, from about 11 days to about 14 days after the subject's exposure to the pathogen (e.g., one described in Section 2, e.g., coronavirus).

[0426] In certain embodiments, the subject is a subject at risk. In certain embodiments, the subject is 60 years of age or older. In certain embodiments, the subject is 18 years of age or younger. In certain embodiments, the subject suffers from one or more preexisting medical conditions selected from the group consisting of lung disease, cardiovascular disease, diabetes, and a combination thereof. In certain embodiments, the subject is unresponsive to treatment with antivirals (e.g., remdesivir).

[0427] In certain embodiments, the subject is a human subject. In certain embodiments, the subject is a non-human subject, such as, but not limited to, a non-primate, a dog, a cat, a horse, a rabbit, a mouse, a rat, a guinea pig, a fowl, a cow, a goat, or a sheep.

[0428] In certain embodiments, the vaccine disclosed herein is included in pharmaceutical compositions to be administered to the subject. In certain embodiments, the pharmaceutical compositions further include a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers that can be used with the presently disclosed subject matter have the

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[0430] PATENT characteristics of not interfering with the effectiveness of the biological activity of the active ingredients and that is not toxic to the subject to whom it is administered. Non-limiting examples of suitable pharmaceutical carriers include phosphate-buffered saline solutions, water, emulsions, such as oil / water emulsions, various types of wetting agents, and sterile solutions. Additional nonlimiting examples of pharmaceutically acceptable carriers include gels, bioabsorbable matrix materials, implantation elements containing the inhibitor, and / or any other suitable vehicle, delivery, or dispensing mechanism or material. Such pharmaceutically acceptable carriers can be formulated by conventional methods and can be administered to the subject. In certain embodiments, the pharmaceutical acceptable carriers can include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as, but not limited to, octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3 -pentanol and m- cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). In certain embodiments, the suitable pharmaceutically acceptable carriers can include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, or combinations thereof.

[0431] In certain embodiments, the pharmaceutical compositions suitable for use in the presently disclosed subject matter can include compositions where the active ingredients, e.g., vaccine, are contained in an effective amount. The effective amount of an active ingredient can vary depending on the active ingredient, compositions used, the disease (e.g., COVID-19, cancer, etc.) and its severity, and the age, weight, etc., of the subject to be treated. In certain embodiments, a subject can receive an effective amount of the active ingredient in single or multiple administrations of one or more compositions, which can depend on the dosage and frequency as required and tolerated by the subject.

[0432] In certain embodiments, the methods include administering an effective amount of a vaccine (e.g., one disclosed in Section 2) using a microneedle array (e.g., one disclosed in Section 4) in a coordinate (or prime boost) vaccination protocol. In certain embodiments, coordinate immunization protocols employ separate vaccines or formulations, each directed toward eliciting

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[0434] PATENT an anti-viral immune response. In certain embodiments, separate immunogenic compositions that elicit the anti-viral immune response can be combined in a polyvalent immunogenic composition administered to a subject in a single immunization step. In certain embodiments, separate immunogenic compositions can be administered separately (e.g., in monovalent immunogenic compositions) in a coordinate (or prime boost) immunization protocol.

[0435] In certain embodiments, the coordinate (or prime boost) immunization protocol can include several boosts. In certain embodiments, each boost can be a different disclosed antigenic polypeptide. In certain embodiments, the boost can include the same antigenic polypeptide as another boost and / or the prime. In certain embodiments, the prime and boost can be administered as a single dose or multiple doses. For example, but without any limitations, the prime and boost can be administered as two doses, three doses, four doses, five doses, six doses, or more. In certain embodiments, the prime and boost can be administered to a subject over days, weeks, or months. In certain embodiments, different dosages can be used in a series of sequential immunizations. For example, but without any limitation, a relatively large dose in a primary immunization can be followed by a boost with relatively smaller doses.

[0436] In certain embodiments, the boost can be administered about two weeks, about three weeks, about four weeks, about five weeks, about six weeks, about seven weeks, or about eight weeks following the prime. In certain embodiments, the boost can be administered about several months after the prime. In certain embodiments, the boost can be administered about 5 months, about 6 months, about 7 months, about 8 months, about 10 months, about 12 months, about 18 months, about 24 months, or more after the prime. In certain embodiments, periodic additional boosts can also be used at appropriate time points to enhance the subject's “immune memory.” The adequacy of the vaccination parameters chosen, e.g., formulation, dose, regimen, and the like, can be determined by taking aliquots of serum from the subject and assaying antibody titers during the course of the immunization program. In addition, the clinical condition of the subject can be monitored for the desired effect, e.g., prevention of infection or improvement in disease state (e.g., reduction in viral load). If such monitoring indicates that vaccination is sub-optimal, the subject can be boosted with an additional dose of vaccine, and the vaccination parameters can be modified in a fashion expected to potentiate the immune response.

[0437] The amount utilized in a vaccine is selected based on the subject population (e.g., infant or elderly). An optimal amount for a particular composition can be ascertained by standard studies involving observation of antibody titers and other responses in subjects. It is understood that a therapeutically effective amount of a disclosed vaccine can include an amount that is ineffective

[0438] ACTIVE 508396272 76 072396.1082 PATENT at eliciting an immune response by administration of a single dose, but that is effective upon administration of multiple dosages, for example, in a prime-boost administration protocol.

[0439] Upon administration of a presently disclosed vaccine, the immune system of the subject typically responds by producing antibodies specific for the antigenic polypeptide included in the vaccine. Such a response signifies that an immunologically effective dose was delivered to the subject.

[0440] In certain embodiments, the antibody response of a subject can be determined in the context of evaluating effective dosages / immunization protocols. In most instances, it will be sufficient to assess the antibody titer in serum or plasma obtained from the subject. Decisions as to whether to administer booster inoculations and / or to change the amount of the therapeutic agent administered to the individual can be at least partially based on the antibody titer level.

[0441] The pathogen infection (e.g., coronavirus infection) does not need to be completely eliminated or reduced or prevented for the methods to be effective. For example, elicitation of an immune response to a coronavirus with one or more of the disclosed vaccines can reduce or inhibit infection with the pathogen (e.g., coronavirus) by a desired amount, for example, by at least about 10%, at least about 20%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, or even at least about 100% (elimination or prevention of detectable infected cells), as compared to infection with the pathogen (e.g., coronavirus) in the absence of the vaccine. In certain embodiments, pathogen replication (e.g., coronavirus replication) can be reduced or inhibited by the disclosed methods. Pathogen replication (e.g., coronavirus replication) does not need to be completely eliminated for the method to be effective. For example, the immune response elicited using one or more of the disclosed vaccines can reduce replication of the corresponding coronavirus by a desired amount, for example, by at least about 10%, at least about 20%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, or even at least about 100% (elimination or prevention of detectable replication of the pathogen, e.g., coronavirus), as compared to replication of the pathogen (e.g., coronavirus) in the absence of the immune response.

[0442] In certain embodiments, the disclosed vaccine is administered to the subject simultaneously with the administration of an adjuvant (e.g., one disclosed in Section 3).

[0443] In certain embodiments, administration of a therapeutically effective amount of one or more of the disclosed vaccines to a subject induces a neutralizing immune response in the subject. To assess neutralization activity, following immunization of a subject, serum can be collected from the subject at appropriate time points, frozen, and stored for neutralization testing. Methods

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[0445] PATENT to assay for neutralization activity are known to the person of ordinary skill in the art and include, but are not limited to, plaque reduction neutralization (PRNT) assays, microneutralization assays, flow cytometry-based assays, single-cycle infection assays. In certain embodiments, the serum neutralization activity can be assayed using a panel of coronavirus pseudoviruses.

[0446] The present disclosure provides methods of preventing and / or treating COVID-19 in a subject in need thereof. In another aspect, provided herein is a method of reducing the risk of developing COVID-19 in a subject at risk thereof, the method includes administering an effective amount of a vaccine (e.g., one disclosed in Section 2) using a microneedle array (e.g., one disclosed in Section 4).

[0447] In certain embodiments, the subject at risk of developing COVID-19 is a healthcare worker (e.g., emergency room physician or nurse, first responder). In certain embodiments, the subject at risk of developing COVID-19 is 60 years of age or older. In certain embodiments, the subject at risk of developing COVID-19 is 18 years of age or younger. In certain embodiments, the subject at risk of developing COVID-19 suffers from one or more preexisting medical conditions selected from the group consisting of lung disease, cardiovascular disease, and diabetes.

[0448] In certain embodiments, the subject at risk of developing COVID-19 is a resident of an assisted living facility or nursing home, a patient in a hospital for an unrelated treatment (i.e., not related to treatment for COVID-19), or a person incarcerated or working in a prison or jail setting. In certain embodiments, the subject at risk of developing COVID-19 is unresponsive to treatment with remdesivir. In certain embodiments, the subject at risk of developing COVID- 19 has been exposed to the virus or presumed to have been exposed to the virus.

[0449] In certain embodiments, a neutralizing immune response induced by the disclosed vaccines herein generates a neutralizing antibody against a coronavirus such as SARS-CoV-2. In certain embodiments, the neutralizing antibody herein binds to a cellular receptor or coreceptor of a coronavirus such as SARS-CoV-2 or component thereof. In certain embodiments, the viral receptor or coreceptor is a coronavirus receptor or coreceptor, preferably a pneumonia virus receptor or coreceptor, more preferably a human coronavirus receptor such as SARS-CoV-2 receptor or coreceptor. In certain embodiments, the neutralizing antibody herein modulates, decreases, antagonizes, mitigates, blocks, inhibits, abrogates and / or interferes with at least one coronavirus such as SARS-CoV-2 activity or binding, or with a coronavirus such as SARS-CoV- 2 receptor activity or binding, in vitro, in situ and / or in vivo, such as SARS-CoV-2 release, SARS- CoV-2 receptor signaling, membrane SARS-CoV-2 cleavage, SARS-CoV-2 activity, SARS-CoV- 2 production and / or synthesis. In certain embodiments, the disclosed vaccines herein induce

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[0451] PATENT neutralizing antibodies against SARS-CoV-2 that modulate, decrease, antagonize, mitigate, block, inhibit, abrogate and / or interfere with SARS-CoV-2 binding to a SARS-CoV-2 receptor or coreceptor, such as angiotensin converting enzyme 2 (ACE2), dipeptidyl peptidase 4 (DPP4), dendritic cell-specific intercellular adhesion molecule-3 -grabbing non integrin (DC-SIGN), and / or liver / lymph node-SIGN (L-SIGN).

[0452] The methods disclosed herein can further comprise a step of identifying a subject having COVID-19. Identification of a subject as having COVID-19 can include the detection of RNA from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a biological sample from the subject. In certain embodiments, the biological sample is a respiratory sample. Non-limiting examples of respiratory samples that can be used to detect SARS-CoV-2 include a nasopharyngeal swab sample, an oropharyngeal swab sample, a sputum sample, a bronchoalveolar lavage sample, a nasopharyngeal aspirate, a nasopharyngeal wash, a nasal aspirate, a nasal wash, and a lower respiratory tract aspirate. In certain embodiments, the biological sample is a fecal sample and / or an anal / rectal swab sample. In certain embodiments, polymerase chain reaction (PCR) is used to detect RNA from SARS-CoV-2 in a sample from a subject. Non-limiting examples of types of PCR that can be used to identify a subject as having COVID-19 include reverse transcription PCR (RT-PCR), real-time PCR (e.g., quantitative PCR (qPCR)), and realtime RT-PCR (rRT-PCR). In certain embodiments, a specific gene from SARS-CoV-2 is detected. For example, the E gene, RNA-dependent RNA polymerase gene (RdRp) gene, ORF la gene, ORF lb gene, N gene, or a combination thereof can be detected using primers or probes specific to the gene or a portion thereof. In certain embodiments, detection of RNA from SARS-CoV-2 can include using a kit comprising, for example, PCR reagents and primers and / or probes for detecting RNA from SARS-CoV-2 (e.g., primers and / or probes specific to the E gene, RNA- dependent RNA polymerase gene (RdRp) gene, ORF la gene, ORF lb gene, N gene, or a combination thereof). Many commercial kits are available to detect RNA from SARS-CoV-2. Non-limiting examples of such kits include PowerChek™ 2019-nCov RT-PCR kit (Kogene Biotech), RT-PCR Allpl ex 2019-nCoV Assay (Seegene); STANDARD M n-CoV Real-Time Detection Kit (SD Biosensor); rRT-PCR XPERT® Xpress SARS-CoV-2 (Cepheid Innovation); and Primerdesign Ltd COVID-19 GENESIG® Real-Time PCR assay.

[0453] In certain embodiments, the E gene and RdRp gene specific to SARS-CoV-2 is detected (see, e.g., PowerChek™ 2019-nCov RT-PCR kit; RT-PCR Allplex 2019-nCoV Assay; and STANDARD M n-CoV Real-Time Detection Kit (SD Biosensor)). In certain embodiments, the ORF la gene and N gene are detected (see, e.g., DiaPlexQ™ Novel Coronavirus Detection Kit (2019-nCoV) (SolGent Co., Ltd.)) In certain embodiments, the RdRP gene, E gene, and N gene

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[0455] PATENT are detected (see, e.g., Corman et al. Eurosurveillance, 25, 2000045 (2020)). In certain embodiments, the ORF1 ab genome region is detected (see, e.g., Primerdesign Ltd COVID-19 GENESIG® Real-Time PCR assay). In certain embodiments, the N2 gene and E gene are detected (see, e.g., rRT-PCR XPERT® Xpress SARS-CoV-2 (Cepheid Innovation)). In certain embodiments, primers and probes to detect the RdRp gene spanning nucleotides 12621-12727 and 14010-14116 (positions according to SARS-CoV, NC_004718) can be used to detect SARS-CoV- 2. See, e.g., the World Health Organization Protocol from the National Reference Center for Respiratory Viruses, Institut Pasteur, Paris (www.who.int / docs / default-source / coronaviruse / real- time-rt-pcr-assays-for-the-detection-of-sars-cov-2-institut-pasteur- paris.pdf?sfvrsn=3662fcb6_2). In certain embodiments, a first gene is detected in a screening test and a second gene is detected for confirmation. For example, the N gene from SARS-CoV-2 can be detected in a screening assay, and ORF lb from SARS-CoV-2 can be detected as a confirmatory assay.

[0456] In certain embodiments, the present disclosure provides methods of preventing and / or treating an infectious disease in a subject in need thereof. In certain embodiments, the methods comprise administering an administration device or article of manufacture disclosed herein. In certain embodiments, the methods comprise delivering an effective amount of a vaccine disclosed herein.

[0457] In certain embodiments, the infectious disease is a viral disease. In certain non-limiting embodiments, the viral disease is associated with viruses selected from dsDNA viruses (e.g., adenovirus, herpesvirus, Epstein Barr virus, herpes simplex type 1, herpes simplex type 2, human herpes virus simplex type 8, human cytomegalovirus, varicella-zoster virus, poxvirus), ssDNA viruses (e.g., parvovirus, papillomavirus (e.g., El, E2, E3, E4, E5, E6, E7, E8, BPV1, BPV2, BPV3, BPV4, BPV5 and BPV6), dsRNA viruses (e.g., reovirus), (+)ssRNA viruses (e.g., picomavirus, coxsackie virus, hepatitis A virus, poliovirus, togavirus, rubella virus, flavivirus, hepatitis C virus, yellow fever virus, dengue virus, west Nile virus, coronavirus), (-)ssRNA viruses (e.g., orthomyxovirus, influenza virus, rhabdovirus, paramyxovirus, measles virus, mumps virus, parainfluenza virus, respiratory syncytial virus, metapneumovirus, arenaviruses (including Lassa virus, LuJo virus, Junin virus, Machupo virus, Bear Canyon virus, Wenzhou virus, Guanarito virus, Latino virus, Sabia virus, Chapare virus, Whitewater Arroyo virus, Pichinde virus, LCMV virus, Mopeia virus, Mobala virus, Ippy virus, Mobala virus, and Morogoro virus, rhabdovirus, rabies virus, ebola), ssRNA-RT viruses (e.g. retrovirus, human immunodeficiency virus (HIV)), and dsDNA-RT viruses (e.g. hepadnavirus, hepatitis B). As one of skill in the art would appreciate, any virus not listed above can be encompassed by the present disclosure.

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[0459] PATENT

[0460] In certain embodiments, the infectious disease is a bacterial disease. In certain nonlimiting embodiments, the bacterial disease is associated with bacteria selected from Bacillus spp. (e.g., Bacillus anthracis), Bordetella spp. (e.g., Bordetella pertussis), Borrelia spp. (e.g., Borrelia burgdorferi), Brucella spp. (e.g., Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis), Campylobacter spp. (e.g., Campylobacter jejuni), Chlamydia spp. (e.g., Chlamydia pneumoniae, Chlamydia psittaci, Chlamydia trachomatis), Clostridium spp. (e.g., Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani), Corynebacterium spp. (e.g., Corynebacterium diptheriae), Enterococcus spp. (e.g., Enterococcus faecalis, Enterococcus faecum), Escherichia spp. (e.g., Escherichia coll), Francisella spp. (e.g., Francisella tularensis), Haemophilus spp. (e.g., Haemophilus influenza), Helicobacter spp. (e.g., Helicobacter pylori), Legionella spp. (e.g., Legionella pneumophila), Leptospira spp. (e.g., Leptospira interrogans), Listeria spp. (e.g., Listeria monocytogenes), Mycobacterium spp. (e.g., Mycobacterium leprae, Mycobacterium tuberculosis), Mycoplasma spp. (e.g., Mycoplasma pneumoniae), Neisseria spp. (e.g., Neisseria gonorrhea, Neisseria meningitidis), Pseudomonas spp. (e.g., Pseudomonas aeruginosa), Rickettsia spp. (e.g., Rickettsia rickettsii), Salmonella spp. (e.g., Salmonella typhi, Salmonella typhinurium), Shigella spp. (e.g., Shigella sonnei), Staphylococcus spp. (e.g., Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus), Streptococcus spp. (e.g., Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyrogenes), Treponema spp. (e.g., Treponema pallidum), Vibrio spp. (e.g., Vibrio cholerae), and Yersinia spp. (Yersinia pestis). As one of skill in the art would appreciate, any bacteria not listed above can be encompassed by the present disclosure.

[0461] In certain embodiments, the infectious disease is a parasitic disease. In certain nonlimiting embodiments, the parasitic disease is associated with parasites selected from Ancylostoma spp. (e.g., A. duodenale), Anisakis spp., Ascaris lumbricoides, Balantidium coli, Cestoda spp., Cimicidae spp., Clonorchis sinensis, Dicrocoelium dendriticum, Dicrocoelium hospes, Diphyllobothrium latum, Dracunculus spp., Echinococcus spp. (e.g., E. granulosus, E. multilocularis), Entamoeba histolytica, Enterobius vermicularis, Fasciola spp. (e.g., F. hepatica, F. magna, F. gigantica, F. jacksoni), Fasciolopsis buski, Giardia spp. (Giardia lamblia), Gnathostoma spp., Hymenolepis spp. (e.g., H. nana, H. diminuta), Leishmania spp., Loa loa, Metorchis spp. (M. conjunctus, M. albidus), Necator americanus, Oestroidea spp. (e.g., botfly), Onchocercidae spp., Opisthorchis spp. (e.g., 0. viverrini, O. felineus, O. guayaquilensis, and 0. noverca), Plasmodium spp. (e.g., P. falciparum), Protofasciola robusta, Parafasciolopsis fasciomorphae, Paragonimus westermani, Schistosoma spp. (e.g., S. mansoni, S. japonicum, S. mekongi, S. haematobium), Spirometra erinaceieuropaei, Strongyloides stercoralis, Taenia spp.

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[0463] PATENT

[0464] (e.g., T. saginala, T. solium), Toxocara spp. (e.g., T. canis, T. call . Toxoplasma spp. (e.g., T. gondii), Trichobilharzia regenti, Trichinella spiralis, Trichuris trichiura, Trombiculidae spp., Trypanosoma spp., Tunga penetrans, and Wuchereria bancrofti. As one of skill in the art would appreciate, any parasite not listed above can be encompassed by the present disclosure.

[0465] In certain embodiments, the disease is a tumor or cancer. For example, but without any limitation, the tumor or cancer is selected from melanoma, neuroblastoma, ovarian cancer, synovial sarcoma, myxoid / round cell liposarcoma, breast cancer, squamous cell carcinoma, mesothelioma, adult and pediatric sarcomas, hematological cancers, prostate cancer, lung adenocarcinoma, esophageal squamous cell carcinoma, gastric cancer, bladder cancer, hepatocellular carcinoma, brain cancer, multiple myeloma, Ewing's sarcoma, rhabdomyosarcoma, germ cell tumors, Burkitt lymphoma, osteosarcoma, renal cell cancer, colon cancer, acute myeloid leukemia, colorectal cancer, glioblastoma, pancreatic cancer, chronic myeloid leukemia, nasopharyngeal carcinoma, cervical cancer, lymphomas, or renal cell carcinoma.

[0466] In certain non-limiting embodiments, the tumor or cancer is associated with a tumor antigen (TA) selected from gplOO, MART-l / Melan A, gp75 (TRP-I), tyrosinase, NY-ESO-I, melanoma proteoglycan, MAGE family antigens (i.e., MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-6, and MAGE-12), BAGE family antigens, GAGE family antigens (i.e., GAGE-1, GAGE- 2), RAGE family antigens, N- acetylglucosaminyltransferase-V, pl 5, P-catenin, MUM-I, cyclin dependent kinase-4 (CDK4), p21-ras, BCR- abl, p53, pl85 HER2 / neu, epidermal growth factor receptor (EGFR), carcinoembryonic antigens (CEA), carcinoma- associated mutated mucins (i.e., MUC-1 gene products), EBNA gene products of EBV (i.e., EBNA-I), E7, E6 proteins of human papillomavirus, prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), idiotypic epitopes or antigens, for example, immunoglobulin idiotypes or T cell receptor idiotypes, KSA, kinesin 2, HIP-55, TGFP-1 anti- apoptotic factor, tumor protein D52, HIFT, NY-BR-I, NY- BR-62, NY-BR-75, NY-BR-85, NY-BR-87 and NY-BR-96. As one of skill in the art would appreciate, any tumor antigen not listed above can be encompassed by the present disclosure.

[0467] 6. Articles of Manufacture

[0468] An article of manufacture is provided which comprises the administration device and the vaccine disclosed herein and provides instructions for its use. In certain embodiments, the article of manufacture comprises a container.

[0469] In certain embodiments, the article of manufacture comprises an administration device (e.g., one disclosed in Section 4) which delivers to a subject a dose (e.g., an effective amount) of

[0470] ACTIVE 508396272 82 072396.1082

[0471] PATENT the presently disclosed vaccine. In certain embodiments, the administration device also delivers a dose of a presently disclosed adjuvant.

[0472] In certain embodiments, the article of manufacture is stable. As used herein, the term “stable” refers to an article of manufacture which retains its physical, chemical, and biological properties upon storage. In certain embodiments, the article of manufacture is stable when stored at room temperature. The storage period is generally selected based on the intended shelf-life of the formulation.

[0473] In certain embodiments, the article of manufacture has extended stability. As used herein, an article of manufacture has “extended stability” when it retains its physical, chemical, and biological properties upon storage at about 4°C for one year or more. Additionally or alternatively, an article of manufacture has “extended stability” when it retains its physical, chemical, and biological properties upon storage at room temperature for one month or more. In certain embodiments, the storage is at room temperature for about one month or more. In certain embodiments, the storage is at room temperature for about two months or more. As used herein, the room temperature is between about 20°C and about 22°C. In certain embodiments, the room temperature is about 20°C.

[0474] An article of manufacture “retains its physical properties” if the active ingredient (e.g., vaccine) retains its physical properties if it shows no signs or very little of aggregation, precipitation, and / or denaturation upon visual examination of color and / or clarity, or as measured by UV light scattering or by size exclusion chromatography.

[0475] An article of manufacture “retains its chemical properties” if the active ingredient (e.g., vaccine) at a given time is such that the active ingredient has not been modified (e.g., by oxidation, deamidation, reduction, etc.). Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Chemical alteration may involve size modification (e.g., clipping) which can be evaluated using size exclusion chromatography, SDS-PAGE and / or matrix-assisted laser desorption ionization / time-of-flight mass spectrometry (MALDI / TOF MS), for example. Other types of chemical alteration include charge alteration (e.g., occurring as a result of deamidation) which can be evaluated by ion-exchange chromatography or imaged capillary isoelectric focusing (icIEF), for example.

[0476] In certain embodiments, the article of manufacture includes a sterile container that contains the administration device including the vaccine; such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. In certain embodiments, the article of manufacture can include a single dose (e.g., a single administration device including the vaccine) or multiple doses (e.g., multiple doses of the

[0477] ACTIVE 508396272 83 072396.1082 PATENT administration device including the vaccine). Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments. A container can be any receptacle and closure suitable for storing, shipping, dispensing, and / or handling a pharmaceutical product.

[0478] In certain embodiments, the article of manufacture includes instructions for applying the device to a subject in order to deliver an effective of the vaccine. The instructions can include information about the use of the device, vaccines, etc.. In certain embodiments, the instructions include at least one of the following: description of the components (e.g., the administration device and the vaccine disclosed herein); dosage schedule and administration for immunization; precautions; warnings; indications; counter-indications; over dosage information; adverse reactions; animal pharmacology; clinical studies; and / or references. The instructions can be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.

[0479] In certain embodiments, the article of manufacture further includes instructions or supporting material that describe the use of the kit to diagnose COVID-19 and / or reference to a website or publication describing the same. In certain embodiments, the article of manufacture further includes instructions or supporting material that describe the use of the kit to determine a prognosis of COVID-19 and / or reference to a website or publication describing the same.

[0480] EXAMPLES

[0481] The present disclosure will be better understood by reference to the following Example, which is provided as exemplary of the presently disclosed subject matter, and not by way of limitation.

[0482] Example 1

[0483] The present example illustrates effective, safe, temperature stable, and broadly deployable vaccines that can provide long-lasting protection against targeted infectious pathogens. In particular, engineering of the immunoresponsive cutaneous microenvironment using the presently disclosed dissolvable microarray patches (MAPs) incorporating ferritin nanoparticle-decorated subunit antigens (e.g., Spike protein Ferritin Nanoparticle - SpFN) with or without vaccine adjuvants (e.g., the STING pathway agonist ADU-S100, the saponin adjuvant QS-21, the TLR3 agonist Poly(I:C), the Army Liposomal Formulation ALFQ) combined the highly efficient immune circuitry of the skin with emerging antigen, adjuvant, and vaccine delivery platforms.

[0484] In order to test the efficiency of the presently disclosed microarray patches, several microarray patches were generated. In particular, the following vaccines were generated: a

[0485] ACTIVE 508396272 84 072396.1082

[0486] PATENT dissolvable MAP Spike protein Ferritin Nanoparticle (SpFN) vaccine (Figure 2B); a dissolvable MAP ADU-SlOO-adjuvanted SpFN vaccine (Figure 2C); a dissolvable MAP QS-21-adjuvanted SpFN vaccine (Figure 2D); a dissolvable MAP Poly(I:C)-adjuvanted SpFN vaccine (Figure 2E); and a dissolvable MAP ALFQ adjuvanted SpFN vaccine (Figure 2F).

[0487] Next, mice were immunized with a prime dose (day 0) and a boost dose (week 3) using the Spike protein Ferritin Nanoparticle (SpFN) vaccines administered by intramuscular injection or the presently disclosed microarray patches. Anti-SARS-CoV-2 IgG were determined at 2 weeks and 5 weeks after the prime dose. As observed in Figure 3 A, while all the vaccines were able to elicit antibody production, a statistically significant increase was observed at week 5 upon administration of the presently disclosed microarray patches. Notably, these antibodies showed significant neutralization properties (Figure 3B). Further immunization schedules confirmed the ability of the presently disclosed microarray patches to elicit immune response (Figures 4A and 4B).

[0488] Next, it was determined whether lack of refrigeration could affect the efficacy of the presently disclosed patches. Mice were immunized with the presently disclosed patches that were store at room temperature after fabrication. As observed in Figure 5, no difference was observed in the antibody title between mice immunized with the vaccine stored in a refrigerated system and at room temperature.

[0489] Next, it was determined whether inclusion of adjuvants could improve the humoral immune response observed in the animal models. Spike protein Ferritin Nanoparticle (SpFN) vaccines including the Army Liposomal Formulation (ALFQ) were administered by intramuscular injection or microarray patches with different immunization schedules. As illustrated in Figure 6A-6E, the use of the ALFQ adjuvant elicited superior or comparable humoral immune response. Further, mice immunized with the patches including the STING pathway ADU-S100, the saponin adjuvant QS-21, and the ILT3 agonist Poly(I:C) also showed superior or comparable humoral immune response.

[0490] The immune response induced by the presently disclosed microarray patches including the ALFQ was further analyzed. As seen in Figures 7A-7D, systemic and pulmonary polyfunctional T-cell immune responses were observed in mice immunized with the presently disclosed patches. Moreover, similar results were observed in T cells in spleens and lungs. Thus, the presently disclosed microarray patches can induce a significant humoral response as well as a polyfunctional effect on T cells.

[0491] Finally, experiments to characterize the elicited humoral pseudovirus neutralizing immune response were performed. Briefly, MAPs containing 0.1 pg SpFN alone or with different

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[0493] PATENT adjuvants against diverse sarbecoviruses were tested. As seen in Figure 8A, dissolvable microarray patches (MAPs) loaded with the Army Liposomal Formulation ALFQ-adjuvanted Spike protein Ferritin Nanoparticle (SpFN) vaccine induced potent SARS-CoV-2-specific neutralizing antibody levels against WA-1, Alpha, Beta, Delta, and Omicron BA.l SARS-CoV-2 variants, and SARS-CoV-1 Urbani strain. Mice were immunized using MAPs loaded with the WA-1 SpFN+ALFQ, or without adjuvant, or with adjuvant Poly(I:C), or adjuvant ADU-S100, respectively. Figure 8B shows that the use of SpFN alone at a low dose can elicit neutralizing responses. Addition of adjuvant increases the neutralization responses against SARS-CoV-2 variants, while neutralization responses are high and consistent with the ALFQ adjuvanted group. Figure 8C shows the neutralization response for the four 0.1 pg SpFN groups either non- adjuvanted or adjvanted tested against the SARS-CoV-1 pseudovirus strain. There were statistically significant differences in the neutralizing responses across the four groups with the ADU-S100 and ALFQ groups showing the highest responses^

[0494] Overall, the present example describes a thermostable, needle-free, and self-administered skin-targeted vaccines for more effective, sustainable, and equitable global immunization campaigns against (re)-emerging infectious pathogens

[0495] Example 2

[0496] Engineering microneedle patch ferritin nanoparticle vaccines

[0497] To demonstrate the reproducible production of microneedle patch (MNP) ferritin nanoparticle (FN) vaccines, dissolvable MNPs integrating the Spike protein Ferritin Nanoparticle (SpFN) antigen and the ALFQ adjuvant (3D-PHAD+QS-21) were manufactured using microfabrication techniques. The presently disclosed MNP production technique involves (1) additive micromanufacturing of high-precision master molds with micron-scale needles with sharp tips and smooth edges; (2) fabrication of negative production molds with microneedleshaped wells obtained through micromolding of the elastomer polydimethylsiloxane (PDMS) with the master molds; and (3) engineering of final dissolving MNP-FN vaccines by spin-casting of vaccine components (e.g., antigen(s) and adjuvant(s)), along with water-soluble structural biomaterials of MNPs, into PDMS molds. A combination of two water-soluble biomaterials, carboxymethyl cellulose (CMC) and Trehalose, was used as the structural material of MNP-FN vaccines. CMC and Trehalose are designated as the Generally Recognized As Safe (GRAS) materials by the Food and Drug Administration (FDA) (i.e., safe for human use). Further, CMC is mechanically strong in its dry form, and trehalose offers ideal protein stabilizing characteristics, constituting an ideal biomaterial combination for the presently disclosed MNP-FN vaccines.

[0498] ACTIVE 508396272 86 072396.1082

[0499] PATENT

[0500] A representative photo of the presently disclosed globally deployable skin-targeted immunization platform with an array (10x10) of a CMC / Trehalose MNP loaded with SpFN and ALFQ is shown in Figure 9A (scale bar = 10 mm). An optical stereomicroscopy image of CMC / Trehalose microneedles integrating SpFN and ALFQ is shown in Figure 9B (scale bar = 500 pm). A scanning electron microscopy (SEM) image of a CMC / Trehalose obelisk-shaped microneedle incorporating SpFN and ALFQ is shown in Figure 9C (scale bar = 100 pm). An optical stereomicroscopy image of a CMC / Trehalose microneedle loaded with SpFN+ALFQ after application to the abdominal skin of a mouse in vivo for 15 minutes, revealing the remaining needle materials upon murine skin penetration and nearly complete dissolution, is shown in Figure 9D (scale bar = 500 pm). An SEM image of CMC / Trehalose microneedles integrating SpFN and ALFQ after application to the abdominal skin of a mouse in vivo for 15 minutes, revealing the remaining microneedle materials upon murine skin penetration and nearly complete dissolution is shown in Figure 9E (scale bar = 250 pm). Total endotoxin content in CMC / Trehalose MNPs incorporating SpFN and SpFN+ALFQ, determined by chromogenic limulus amebocyte lysate (LAL) assay through dissolving these MNPs in endotoxin-free water, is plotted in Figure 9F (Data = Mean+Standard Deviation from n = 6 CMC / Trehalose MNPs loaded with SpFN and SpFN+ALFQ).

[0501] To further demonstrate the production of high-quality skin-targeted MNP-FN vaccines, dissolvable MNPs integrating fluorescent-labeled vaccine components (e.g., FN antigen and adjuvant) were created using microfabrication methods described above. Dissolving MNPs with obelisk-shaped microneedles loaded with the SpFN antigen labeled with Alexa Fluor 647 fluorescent dye (SpFN-AF647) and the ALFQ adjuvant labeled with CM-Dil fluorescent dye (ALFQ-CM-Dil) were produced from CMC / Trehalose for better demonstration of the localization of vaccine components in MNPs.

[0502] An optical stereomicroscopy image of a CMC / Trehalose MNP loaded with SpFN-AF647 and ALFQ-CM-Dil is shown in Figure 10A (scale bar = 500 pm). Pink colored regions in the apex region of these microneedles demonstrate the precise localization of vaccine components (SpFN and ALFQ) in the tips of these obelisk-shaped microneedles. A brightfield microscopy image of a high-quality CMC / Trehalose microneedle integrating fluorescent-labeled vaccine components, SpFN-AF647 and ALFQ-CM-Dil is shown in Figure 10B (scale bar = 100 pm). A fluorescent microscopy image of a high-integrity CMC / Trehalose microneedle loaded with SpFN- AF647 and ALFQ-CM-Dil, with a filter corresponding to AF647 is shown in Figure 10C (scale bar = 100 pm). A fluorescent microscopy image of a high-quality CMC / Trehalose microneedle loaded with SpFN-AF647 and ALFQ-CM-Dil, with a filter corresponding to CM-Dil is shown in

[0503] ACTIVE 508396272 87 072396.1082

[0504] PATENT

[0505] Figure 10D (scale bar = 100 gm). A merged microscopy image of a high-quality CMC / Trehalose microneedle loaded with SpFN-AF647 and ALFQ-CM-Dil is shown in Figure 10E (scale bar = 100 pm). This merged image was obtained by combining the brightfield microscopy image (from Figure 10B) of a high-quality CMC / Trehalose microneedle integrating SpFN-AF647 and ALFQ- CM-Dil and fluorescent microscopy images (from Figure 10C and Figure 10D) of a CMC / Trehalose microneedle integrating SpFN-AF647 and ALFQ-CM-Dil. Quantitative data of the reproducibility of FN vaccine loading in MNPs, determined based on the standard curve obtained with the initial solution of SpFN-AF647+ALFQ-CM-Dil, which was used to manufacture SpFN-AF647+ALFQ-CM-Dil MNPs, with the samples obtained via dissolving SpFN-AF647+ALFQ-CM-Dil MNPs in endotoxin-free water is shown in Figure 10F (Data = Mean±Standard Deviation (SD) from n =5 SpFN-AF647+ALFQ-CM-Dil MNPs) indicate less than 10% SD in the loading of both FN antigen and adjuvant in MNPs.

[0506] Together, these data illustrate the engineering of high-quality skin-targeted FN vaccines in the form of high-integrity, mechanically strong, and sterile MNPs.

[0507] Cutaneous delivery of ferritin nanoparticle vaccines with microneedle patches

[0508] To demonstrate the successful cutaneous delivery of FN vaccines with MNPs, CMC / Trehalose MNPs integrating SpFN-AF647+ALFQ-CM-Dil were manufactured. These SpFN-AF647+ALFQ-CM-Dil MNPs were applied to murine skin in vivo for 15 minutes and then removed. Subsequently, in vivo live animal fluorescent imaging analysis of the corresponding mouse and epi-fluorescent imaging analysis of the cryo-sectioned SpFN-AF647+ALFQ-CM-Dil MNP-treated corresponding skin were performed. An in vivo fluorescent image of the whole mouse using the filters corresponding to SpFN-AF647 and ALFQ-CM-Dil after in vivo skin application (15 minutes) and removal of a SpFN-AF647+ALFQ-CM-Dil MNP, obtained using the IVIS system, is shown in Figure 11 A and Figure 1 IB, respectively (scale bars = 10 mm). Epifluorescence microscopy images of the cryo-sectioned SpFN-AF647+ALFQ-CM-Dil MNP- treated murine skin shortly after the removal of MNP are shown in Figure 11C (sections were also counterstained with DAPI; scale bars = 100 pm).

[0509] Pro-inflammatory skin microenvironment responses to ferritin nanoparticle vaccines

[0510] To evaluate the pro-inflammatory cutaneous microenvironment responses to FN vaccines in both young (8-10 weeks old) and aged (> 18 months old) mice, blank MNPs (no vaccine components), non-adjuv anted MNP-FN vaccines with the prototype antigen SpFN, adjuvanted MNP-FN vaccines with SpFN+ALFQ, or with SpFN+ADU-SlOO (a STING pathway agonist) were manufactured. These MNPs were administered to mouse ears (n = 5 per group) in vivo. At the indicated timepoints (6hr and 24 hr) after skin applications of these MNPs, those mice were

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[0512] PATENT sacrificed, the ear tissues isolated, and the time-dependent (6h, 24h) changes in the mRNA expression levels of key T helper 1 (Thl)-predominant pro-inflammatory mediators (e.g., CCL5, CCL2, CXCL10, and IFN-P) in the murine skin microenvironments measured using the qRT-PCR technique. Fold changes in the mRNA expression levels of these Th 1 -predominant pro- inflammatory chemokines / cytokines with respect to naive (or untreated) mouse ears are plotted in Figures 12A-12D. Specifically, fold changes in the expression levels of CCL2, IFN-P, CCL5, and CXCL10 with MNP treatments are presented in Figure 12A, Figure 12B, Figure 1C, and Figure 12D, respectively (Data = Mean±Standard Error of the Mean (SEM) from n = 5 mouse ears per group). Collectively, these results support that co-delivery of adjuvants with FN antigens in the same MNPs more efficiently upregulates targeted pro-inflammatory mediators in the vaccine- targeted skin microenvironments with respect to non-adjuvanted FN vaccines, with implications for a preferred skin milieu for improved vaccine efficacy.

[0513] Humoral responses induced by skin-targeted ferritin nanoparticle vaccines

[0514] To evaluate humoral responses generated by skin-targeted FN vaccines, SpFN MNPs, SpFN+ADU-SlOO MNPs, and SpFN+ALFQ MNPs were produced. Next, both young (8-10 weeks old) and aged (> 18 months) mice (n =5 per group) were immunized with these MNPs (naive mice served as unimmunized controls) as described in Figure 13 A, and probed vaccine- induced antigen-specific binding antibody responses in the serum of mice using the methods noted in Figure 13 A. Serum levels of total IgG antibodies elicited by MNP-FN vaccines in young and aged mice are measured and presented in Figure 13B. These results demonstrate that adjuvanted MNP-FN vaccines (e.g., SpFN+ADU-SlOO MNPs and SpFN+ALFQ MNPs) induce significantly increased antigen-specific antibody responses compared to a non-adjuvanted MNP-FN vaccine, which is consistent with a more pro-inflammatory skin milieu imprinted with adjuvanted MNP- FN vaccines (see Figures 12A-12D).

[0515] To further evaluate humoral responses generated by skin-targeted FN vaccines, the functional quality of MNP-FN vaccine-induced antibody responses was tested. To this end, SpFN MNPs, SpFN+ADU-SlOO MNPs, and SpFN+ALFQ MNPs were manufactured and young (8-10 weeks old) mice (n =5 per group) were immunized with these MNPs (naive mice served as unimmunized controls) as described in Figure 14A, and probed vaccine-induced neutralizing antibodies against WA-1 and Beta strains of SARS-CoV-2 using the methods noted in Figure 14A. Serum levels of WA-1 strain-specific and Beta-strain-specific neutralizing antibody titers elicited by MNP-FN vaccines in mice are presented in Figure 14B. These results demonstrate that adjuvanted MNP-FN vaccines (e.g., SpFN+ADU-SlOO MNPs and SpFN+ALFQ MNPs) induce significantly increased virus-specific neutralization activity compared with a non-adjuvanted

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[0517] PATENT

[0518] MNP-FN vaccine, which is consistent with a more pro-inflammatory skin milieu imprinted with adjuvanted MNP-FN vaccines (see Figures 12A-12D).

[0519] To evaluate the durability of humoral immune responses generated by skin-targeted FN vaccines, the kinetics of MNP-FN vaccine-induced antibody responses were monitored. Toward this end, SpFN MNPs and SpFN+ADU-SlOO MNPs were produced and young (8-10 weeks old) mice (n =5 per group) were immunized with these MNPs (naive mice served as unimmunized controls) as described in Figure 15 A. Vaccine-induced antigen-specific binding antibody responses in the serum of mice across 48 weeks were probed using the methods noted in Figure 15 A. Serum levels of antigen-specific total IgG antibodies induced by MNP-FN vaccines in mice are measured across 48 weeks and presented in Figure 15B. These results demonstrate that skin- targeted FN vaccines (e.g., SpFN MNPs and SpFN+ADU-SlOO MNPs) evoke long-lasting humoral responses, with implications that antigen-specific antibody levels elicited by adjuvanted MNP-FN vaccines remain at considerably higher levels compared to those generated by non- adjuvanted MNP-FN vaccines across 48 weeks, which is consistent with vaccine-induced durable immunity.

[0520] To validate the presently disclosed skin-targeted FN vaccine platforms for other viral families, with the purpose of demonstrating their broad applicability, a skin-targeted FN arenavirus vaccine was engineered and humoral responses induced by this vaccine were evaluated. First, novel MNP FN arenavirus vaccines were created by integrating Machupo virus (MACV) glycoprotein FN antigen (MgpFN) in MNPs, with or without adjuvants. Specifically, MgpFN MNPs, MgpFN+ADU-SlOO MNPs, and MgpFN+ALFQ MNPs were prepared. Young mice (8- 10 weeks old) were immunized with these MNPs (naive mice served as unimmunized controls) as described in Figure 16A, and probed vaccine-induced antigen-specific total IgG antibody levels in the serum of mice using the methods noted in Figure 16A. Serum levels of total IgG antibody elicited by MNP-FN MACV vaccines in mice are presented in Figure 16B. These results demonstrate that adjuvanted MNP-FN vaccines (e.g., MgpFN+ADU-SlOO MNPs and MgpFN+ALFQ MNPs) induced significantly increased virus-specific neutralization activity compared to non-adjuvanted MNP-FN vaccine, consistent with data obtained with SpFN.

[0521] To further validate the presently disclosed skin-targeted FN vaccine platforms for other viral families, additional skin-targeted FN arenavirus vaccines were engineered, and humoral responses elicited by these vaccines were determined. Specifically, skin-targeted arenavirus vaccines were produced by integrating either Junin virus (JUNV) glycoprotein FN antigen (JgpFN) in MNPs with ALFQ or by integrating Guanarito virus (GTOV) glycoprotein (GgpFN) in MNPs with ALFQ. Young mice (8-10 weeks old) were immunized with these MNPs (naive

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[0523] PATENT mice served as unimmunized controls and intramuscular injection of the same vaccines saved as another control) as described in Figure 17A and Figure 17C, and measured vaccine-induced antigen-specific total IgG antibody levels in the serum of mice using ELISA. Serum levels of total IgG antibodies elicited by these FN arenavirus vaccines in mice are presented in Figure 17B and Figure 17D. These results suggest that adjuvanted MNP-FN vaccines (e.g., JgpFN+ALFQ MNP and GgpFN+ALFQ MNP) induce significantly increased antigen-specific total IgG antibodies compared to traditional intramuscular immunization with the same vaccines.

[0524] Cellular responses induced by skin-targeted ferritin nanoparticle vaccines

[0525] To evaluate cell-mediated responses elicited by skin-targeted FN vaccines, SpFN MNPs, SpFN+ADU-SlOO MNPs, and SpFN+ALFQ MNPs were produced. Young (8-10 weeks old) and aged (> 18 months) mice (n =5 per group) were immunized with these MNPs (naive mice served as unimmunized controls) as described in Figure 18A, and probed vaccine-induced antigenspecific CD4+ and CD8+ T-cell responses in systemic (spleens) and mucosal (lungs) compartments using the methods noted in Figure 18 A.

[0526] Systemic (spleens) antigen-specific CD4+ and CD8+ T-cell responses elicited by the presently disclosed skin-targeted FN vaccines in young and aged mice are presented in Figure 18B and Figure 18C, respectively. Pulmonary (lungs) antigen-specific CD4+ and CD8+ T-cell responses induced by the presently disclosed skin-targeted FN vaccines in young and aged mice are shown in Figure 18D and Figure 18E, respectively. In general, adjuvanted MNP-FN vaccines generated more potent antigen-specific T-cell responses in both systemic and mucosal compartments in both young and aged mice compared to non-adjuvanted MNP-FN vaccines that failed to elicit robust T-cell immunity. SpFN+ALFQ MNPs were superior to SpFN+ADU-SlOO MNPs in inducing antigen-specific CD8+ T-cell responses. Further, adjuvanted MNP-FN vaccines helped to address immunosenescence for the induction of robust and potent antigenspecific T-cell responses in aged animals, thereby supporting the robust cellular immunogenicity characteristics of the presently disclosed skin-targeted FN vaccines.

[0527] Safety and thermostability of adjuvanted microneedle patch ferritin nanoparticle vaccines The presently disclosed skin-targeted FN vaccines are safe, thermostable, and needle-free vaccines that simplify the delivery, storage, distribution, disposal, and acceptability of vaccines compared to traditional heat-labile needle and syringe vaccines that require trained healthcare personnel for successful administration. To demonstrate the safety of the presently disclosed MNP-FN vaccines, SpFN MNPs and SpFN+ALFQ MNPs were produced, and their safety was tested in young (8-10 weeks old) and aged (> 18 months) mice (n =5 per group). Unlike needle and syringe injection of vaccine components, the presently disclosed skin-targeted FN vaccines

[0528] ACTIVE 508396272 91 072396.1082 PATENT minimized pro-inflammatory activities of vaccine components in space and time, providing a viable strategy to decouple the benefits of adjuvants from their side effects. Murine studies supported and showed the good safety profile of the presently disclosed skin-targeted FN vaccines since there was no significant changes in body weight and temperature of vaccinated mice, as well as no significant changes in serum levels of the pro-inflammatory mediator IL-6, which is an established marker of systemic toxicity and fever (Figure 19).

[0529] To validate the heat-stability of the MNP-FN vaccines, SpFN+ALFQ MNPs were manufactured, and thermostability studies were performed in young (8-10 weeks old) mice. These experiments supported that the presently disclosed skin-targeted FN vaccines retain activity for an extended period of storage. Specifically, SpFN+ALFQ MNPs maintained their immunogenicity at room temperature (RT, 22 C) for at least a year, indicated by non-significant differences in antigen-specific total IgG antibody levels induced by fresh SpFN+ALFQ MNPs and SpFN+ALFQ MNPs that had been stored in a sealed vial at RT for a year.

[0530] Together, the presently disclosed adjuvanted MNP-FN vaccines offer favorable globally deployable vaccine product characteristics, including safety for well-tolerability and thermostability for cost-effective storage and widespread distribution.

[0531] Although the presently disclosed subject matter and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, composition of matter, means, methods and steps described in the specification. As one of ordinary skill in the art will readily appreciate from the disclosure of the presently disclosed subject matter, processes, machines, manufacture, compositions of matter, means, methods, or steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein can be utilized according to the presently disclosed subject matter. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods, or steps.

[0532] Patents, patent applications, publications, product descriptions and protocols are cited throughout this application the disclosures of which are incorporated herein by reference in their entireties for all purposes.

[0533] ACTIVE 508396272 92

Claims

072396.1082PATENTWHAT IS CLAIMED IS:

1. A vaccine administration device comprising a base and a plurality of microneedles extending from the base, wherein the plurality of microneedles comprises a vaccine comprising a nanoparticle-forming polypeptide, an antigenic polypeptide, and an adjuvant.

2. The device of claim 1, wherein the nanoparticle-forming polypeptide comprises a ferritin polypeptide, a lumazine synthase polypeptide, a SpyTag polypeptide, a SpyCatcher polypeptide, or a combination thereof.

3. The device of claim 1 or 2, wherein the nanoparticle-forming polypeptide comprises a ferritin polypeptide.

4. The device of claim 3, wherein the ferritin polypeptide is a Helicobacter pylori ferritin polypeptide.

5. The device of claim 3 or 4, wherein the ferritin polypeptide comprises an amino acid sequence that is at least about 80%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 1.

6. The device of claim 5, wherein the ferritin polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1.

7. The device of claim 6, wherein the ferritin polypeptide comprises a substitution of the glutamic acid residue (E) at position 13 of SEQ ID NO: 1.

8. The device of claim 7, wherein the ferritin polypeptide comprises an amino acid sequence that is at least about 80%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 3.

9. The device of claim 8, wherein the ferritin polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 3.

10. The device of any one of claims 1-9, wherein the antigenic polypeptide is a polypeptide obtained or derived from a virus selected from adenovirus, herpesvirus, Epstein Barr virus, herpes simplex type 1, herpes simplex type 2, human herpes virus simplex type 8, human cytomegalovirus, varicella-zoster virus, poxvirus, parvovirus, papillomavirus, picornavirus, coxsackie virus, hepatitis A virus, poliovirus, togavirus, rubella virus, flavivirus, hepatitis C virus, yellow fever virus, dengue virus, west Nile virus, coronavirus, orthomyxovirus, influenza virus, rhabdovirus, paramyxovirus, measles virus, mumps virus, parainfluenza virus, respiratory syncytial virus, metapneumovirus, arenaviruses, Lassa virus, LuJo virus, JuninACTIVE 508396272 93072396.1082PATENT virus, Machupo virus, Bear Canyon virus, Wenzhou virus, Guanarito virus, Latino virus, Sabia virus, Chapare virus, Whitewater Arroyo virus, Pichinde virus, LCMV virus, Mopeia virus, Mobala, virus, Ippy virus, Mobala virus, and Morogoro virus, rhabdovirus, rabies virus, ebola, human immunodeficiency virus (HIV), hepadnavirus, hepatitis B, or a combination thereof.

11. The device of any one of claims 1-9, wherein the antigenic polypeptide is a polypeptide obtained or derived from a bacterium selected from Acinetobacter baumannii. Bacillus anlhracis. Bordetella pertussis, Borrelia burgdorferi, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia psittaci, Chlamydia trachomatis, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diptheriae, Enterococcus faecalis, Enterococcus faecum, Escherichia coli, Francisella tularensis, Haemophilus influenza, Helicobacter pylori, Legionella pneumophila, Leptospira interrogans, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycoplasma pneumoniae, Neisseria gonorrhea, Neisseria meningitidis, Pseudomonas aeruginosa, Rickettsia rickettsii, Salmonella typhi, Salmonella typhinurium, Shigella spp. , Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyrogenes, Treponema pallidum, Vibrio cholerae, Yersinia pestis, or a combination thereof.

12. The device of any one of claims 1-9, wherein the antigenic polypeptide is a polypeptide obtained or derived from a parasite selected from A. duodenale, Anisakis spp., Ascaris lumbricoides, Balantidium coli, Cestoda spp., Cimicidae spp., Clonorchis sinensis, Dicrocoelium dendriticum, Dicrocoelium hospes, Diphyllobothrium latum, Dracunculus spp.,E. granulosus, E. multilocularis, Entamoeba histolytica, Enterobius vermicularis, F. hepatica,F. magna, F. gigantica, F. jacksoni, Fasciolopsis buski, Giardia lamblia, Gnathostoma spp., H. nana, H. diminuta, Leishmania spp., Loa loa, M. conjunctus, M. albidus, Necator americanus, Oestroidea spp., Onchocercidae spp., O. viverrini, O. felineus, O. guayaquilensis, and O. noverca, P. falciparum, Protofasciola robusta, Parafasciolopsis fasciomorphae, Paragonimus westermani, S. mansoni, S. japonicum, S. mekongi, S. haematobium, Spirometra erinaceieuropaei, Strongyloides stercoralis, T. saginata, T. solium, T. canis, T. cati, T. gondii, Trichobilharzia regenti, Trichinella spiralis, Trichuris trichiura, Trombiculidae spp., Trypanosoma spp., Tunga penetrans, Wuchereria bancrofti, or a combination thereof.ACTIVE 508396272 94072396.1082PATENT13. The device of any one of claims 1-9, wherein the antigenic polypeptide is a polypeptide obtained or derived from a tumor antigen selected from gplOO, MART-l / Melan A, gp75 (TRP-I), tyrosinase, NY-ESO-I, melanoma proteoglycan, MAGE family antigens, BAGE family antigens, GAGE family antigens, RAGE family antigens, N- acetylglucosaminyltransf erase- V, pl 5, P-catenin, MUM-I, cyclin dependent kinase-4 (CDK4), p21-ras, BCR- abl, p53, pl85 HER2 / neu, epidermal growth factor receptor (EGFR), carcinoembryonic antigens (CEA), carcinoma- associated mutated mucins, EBNA gene products of EBV (i.e., EBNA-I), E7, E6 proteins of human papillomavirus, prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), idiotypic epitopes or antigens, for example, immunoglobulin idiotypes or T cell receptor idiotypes, KSA, kinesin 2, HIP-55, TGFP-1 anti- apoptotic factor, tumor protein D52, HIFT, NY-BR-I, NY-BR-62, NY-BR-75, NY-BR-85, NY-BR-87, NY-BR-96, or a combination thereof.

14. The device of any one of claims 1-10, wherein the antigenic polypeptide is an antigenic arenavirus polypeptide.

15. The device of any one of claims 1-10, wherein the antigenic polypeptide is an antigenic arenavirus gpl polypeptide.

16. The device of any one of claims 1-10, wherein the antigenic polypeptide is an antigenic arenavirus stabilized GPC polypeptide.

17. The device of any one of claims 14-16, wherein the antigenic polypeptide is an antigenic arenavirus polypeptide or a functional fragment thereof comprising an amino acid sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 46.

18. The device of claim 17, wherein the antigenic arenavirus polypeptide or a functional fragment thereof comprises the amino acid sequence set forth in SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 46.

19. The device of claim 18, wherein the antigenic arenavirus polypeptide or a functional fragment thereof comprises from amino acid 59 to amino acid 262 of the amino acid sequence set forth in SEQ ID NO: 44, from amino acid 263 to amino acid 496 of the amino acid sequence set forth in SEQ ID NO: 44, or from amino acid 59 to amino acid 496 of the amino acid sequence set forth in SEQ ID NO: 44.

20. The device of claim 18, wherein the antigenic arenavirus polypeptide or a functional fragment thereof comprises from amino acid 59 to amino acid 21 of the amino acid sequence set forth in SEQ ID NO: 45, from amino acid 252 to amino acid 285 of the amino acid sequence setACTIVE 508396272 95072396.1082PATENT forth in SEQ ID NO: 45, or from amino acid 59 to amino acid 485 of the amino acid sequence set forth in SEQ ID NO: 45.

21. The device of claim 18, wherein the antigenic arenavirus polypeptide or a functional fragment thereof comprises from amino acid 59 to amino acid 245 of the amino acid sequence set forth in SEQ ID NO: 46, from amino acid 246 to amino acid 479 of the amino acid sequence set forth in SEQ ID NO: 46, or from amino acid 59 to amino acid 479 of the amino acid sequence set forth in SEQ ID NO: 46.

22. The device of any one of claims 1-10, wherein the antigenic polypeptide is an antigenic HIV- 1 polypeptide.

23. The device of any one of claims 1-10, wherein the antigenic polypeptide is an antigenic HIV- 1 Env gpl20 polypeptide.

24. The device of any one of claims 1-10, wherein the antigenic polypeptide is an antigenic HIV- 1 stabilized Env trimer polypeptide.

25. The device of any one of claims 1-10, wherein the antigenic polypeptide is an antigenic HIV- 1 gpl40 polypeptide.

26. The device of any one of claims 1-10, wherein the antigenic polypeptide is an antigenic coronavirus polypeptide.

27. The device of claim 26, wherein the antigenic coronavirus polypeptide comprises a coronavirus spike protein or a functional fragment thereof, a receptor-binding domain (RBD) or a functional fragment thereof, an N-terminal domain (NTD) or a functional fragment thereof, a receptor-binding domain (RBD)-N-terminal domain chimera or a functional fragment thereof, an SI domain or a functional fragment thereof, a stabilized extracellular spike S-2P domain or a functional fragment thereof, a stabilized extracellular spike S polypeptide or a functional fragment thereof, a stabilized extracellular spike S-trimer or a functional fragment thereof, a receptor-binding domain (RBD)-S-trimer chimera, a RBD- RBD-S-trimer chimera, or a combination thereof.

28. The device of claim 27, wherein the antigenic coronavirus polypeptide comprises a coronavirus spike protein or a functional fragment thereof.

29. The device of claim 28, wherein the coronavirus spike protein or a functional fragment thereof comprises an amino acid sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% or at least 100% identical to the amino acid sequence set forth in SEQ ID NO: 4.

30. The device of claim 29, wherein the coronavirus spike protein or a functional fragment thereof comprises the amino acid sequence set forth in SEQ ID NO: 4.ACTIVE 508396272 96072396.1082PATENT31. The device of claim 23, wherein the coronavirus spike protein or a functional fragment thereof comprises from amino acid 1 to amino acid 685 of the amino acid sequence set forth in SEQ ID NO: 4, from amino acid 14 to amino acid 685 of the amino acid sequence set forth in SEQ ID NO: 4, or from amino acid 686 to amino acid 1273 of the amino acid sequence set forth in SEQ ID NO: 4.

32. The device of claim 27, wherein the antigenic coronavirus polypeptide comprises a receptorbinding domain (RBD) or a functional fragment thereof.

33. The device of claim 32, wherein the RBD polypeptide comprises an amino acid sequence that is at least about 80%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 5.

34. The device of claim 33, wherein the RBD polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 5.

35. The device of claim 27, wherein the antigenic coronavirus polypeptide comprises an N- terminal domain (NTD) or a functional fragment thereof.

36. The device of claim 35, wherein the NTD polypeptide comprises an amino acid sequence that is at least about 80%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 6.

37. The device of claim 36, wherein the NTD polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 6.

38. The device of claim 27, wherein the antigenic coronavirus polypeptide comprises an SI domain or a functional fragment thereof.

39. The device of claim 38, wherein the SI polypeptide comprises an amino acid sequence that is at least about 80%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.

40. The device of claim 39, wherein the SI polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO:

941. The device of claim 27, wherein the antigenic coronavirus polypeptide comprises a stabilized extracellular spike S-2P domain or a functional fragment thereof.

42. The device of claim 41, wherein the S-2P polypeptide comprises an amino acid sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least aboutACTIVE 508396272 97072396.1082PATENT37%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 10.

43. The device of claim 42, wherein the S-2P polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 10.

44. The device of any one of claims 1-40, further comprising a second antigenic polypeptide.

45. The device of claim 44, wherein the first antigenic polypeptide and the second antigenic polypeptide are different.

46. The device of claim 44, wherein the first antigenic polypeptide and the second coronavirus polypeptide are the same.

47. The device of any one of claims 1-46, wherein the nanoparticle-forming polypeptide and the antigenic polypeptide are coupled by a linker to thereby generate a fusion protein.

48. The device of claim 47, wherein the linker comprises the amino acid sequence set forth in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23.

49. The device of any one of claims 1-48, wherein the adjuvant is selected from ADU-S100, saponin, poly(I:C), Army Liposomal Formulation ALF, Army Liposomal Formulation ALFA, Army Liposomal Formulation ALFQ, alhydrogel, monophosphoryl lipid A (MPLA), oil in water emulsions, ADJUPLEX™, ADDAVAX™, CARBOPOL®, Poly ICLC, Flagellin, Iscomatrix, CpG oligodeoxynucleotides, virosomes, MF59, AS04, or a combination thereof.

50. The device of claim 49 , wherein the adjuvant is Army Liposomal Formulation ALFQ.

51. The device of claim 49 , wherein the adjuvant is ADU-S100.

52. The device of claim 49, wherein the adjuvant is selected from QS-21, QS-18, QS-7, or QS-9.

53. The device of claim 49 , wherein the adjuvant is QS-21.

54. The device of claim 49 , wherein the adjuvant is poly(I:C).

55. The device of any one of claims 1-54, wherein the vaccine and the adjuvant are loaded in the same microneedles.

56. The device of claim 55, wherein the vaccine and the adjuvant are pre-mixed and loaded in the same microneedles.

57. The device of any one of claims 1-54, wherein the vaccine and the adjuvant are loaded in different microneedles.

58. The device of any one of claims 1-56, wherein the vaccine is loaded on a metal-organic framework.ACTIVE 508396272 98072396.1082PATENT59. The device of any one of claims 1-58, wherein the vaccine and the adjuvant are loaded on a metal-organic framework.

60. The device of any one of claims 1-59, wherein the vaccine and the adjuvant are loaded on different metal-organic frameworks.

61. The device of any one of claims 1-60, wherein the base comprises poly (lactic-co-glycolic acid) (PLGA) or carboxymethylcellulose (CMC).

62. The device of any one of claims 1-61, wherein each microneedle is dissolvable or water- soluble.

63. The device of any one of claims 1-62, wherein each microneedle comprises carboxymethyl cellulose, trehalose, sucrose, gelatin, glucose, lactose, collagen, chitosan, poly-y-glutamate, polyvinylpyrrolidone, maltodextrin, silk, hyaluronic acid, poly (lactic-co-glycolic acid), poly(lactic acid), poly(vinyl alcohol), polyethylene glycol, or a combination thereof.

64. The device of claim 63, wherein each microneedle comprises carboxymethyl cellulose and trehalose.

65. The device of any one of claims 1-64, wherein each microneedle comprises a layered structure comprising a tip.

66. The device of claim 65, wherein the vaccine is located at the tip of the microneedle.

67. A method of manufacturing an administration device comprising a base and a plurality of microneedles, the method comprising: a) forming or providing a production mold of a flexible material, wherein the production mold comprises a plurality of cavities that are shaped to define a plurality of respective microneedles having a stem, a head, a filleted base, and at least one undercut feature; b) delivering a first dissolvable or water-soluble material into at least the microneedle head portion defined by the respective cavities of the production mold, and prior to or during delivery of the first dissolvable or water-soluble material into at least the microneedle head portion, incorporating a vaccine into the first dissolvable or water-soluble material to produce a dissolvable or water-soluble matrix; c) delivering the first dissolvable or water-soluble material and / or one or more additional dissolvable or water-soluble materials into the cavity and forming a plurality of microneedles in the production mold that include the dissolvable or water-soluble matrix; d) removing the microneedles from the production mold by pulling the microneedles out of the mold, wherein the flexible material of the production mold has sufficient elasticity to allow for the molded microneedle array to be removed from the production mold; andACTIVE 508396272 99072396.1082PATENT e) loading a vaccine comprising a nanoparticle-forming polypeptide, an antigenic polypeptide, and an adjuvant in each microneedle.

68. The method of claim 67, wherein the production mold defines at least one undercut feature in the microneedles directly below a microneedle head.

69. The method of claim 67 or 68, wherein the stem is formed from the first dissolvable or water- soluble material.

70. The method of any one of claims 67-69, wherein the first dissolvable or water-soluble material comprises carboxymethyl cellulose, trehalose, polyvinylpyrrolidone, maltodextrin, silk, hyaluronic acid, poly (lactic-co-glycolic acid), poly (lactic acid), poly (vinyl alcohol), polyethylene glycol, or a combination thereof.

71. The method of any one of claims 69 or 70, wherein at least a portion of the stem is formed from a non-dissolvable material.

72. The method of any one of claims 67-70, further comprising delivering a second dissolvable material into the production mold to form a dissolving layer at a portion of the stem.

73. The method of claim 72, wherein said portion of the stem is adjacent to the microneedle head.

74. The method of claim 72 or 73, wherein the second dissolvable material of the dissolving layer is a material that dissolves more quickly than the first dissolvable or water-soluble material.

75. An administration device prepared by the method of any one of claims 67-74.

76. An article of manufacture comprising the device of any one of claims 1-66 or 75.

77. The article of manufacture of claim 76 further comprising instructions for applying the device to a subject.

78. The article of manufacture of claim 76 or 77, wherein the article has extended stability.

79. The article of manufacture of claim 79, wherein the article has extended stability at room temperature.

80. A method of preventing and / or treating an infectious disease in a subject in need thereof, the method comprising inserting the plurality of microneedles of the device of any one of claims 1-66 or 75, wherein the inserting comprises penetrating the stratum comeum to deliver an effective amount of the vaccine to the epidermis and / or dermis without systemic exposure.

81. The method of claim 80, wherein the infectious disease is a viral disease, a bacterial disease, or a parasitic disease.

82. The method of claim 80 or 81, wherein the infectious disease is COVID-19.

83. A method of inducing an antibody response to a pathogen in a subject in need thereof, the method comprising inserting the plurality of microneedles of the device of any one of claimsACTIVE 508396272 100072396.1082PATENT1-66 or 75, wherein the inserting comprises penetrating the stratum comeum to deliver an effective amount of the vaccine to the epidermis and / or dermis without systemic exposure.

84. A method of inducing a neutralizing immune response to a pathogen in a subject in need thereof, the method comprising inserting the plurality of microneedles of the device of any one of claims 1-66 or 75, wherein the inserting comprises penetrating the stratum corneum to deliver an effective amount of the vaccine to the epidermis and / or dermis without systemic exposure.

85. The method of claim 83 or 84, wherein the pathogen is a virus, a bacterium, or a parasite.

86. The method of any one of claims 83-85, wherein the pathogen is an adenovirus, a herpesvirus, an Epstein Barr virus, a herpes simplex type 1, a herpes simplex type 2, a human herpes virus simplex type 8, a human cytomegalovirus, a varicella-zoster virus, a poxvirus, a parvovirus, a papillomavirus, a reovirus, a picomavirus, a coxsackie virus, a hepatitis A virus, a poliovirus, a togavirus, a rubella virus, a flavivirus, a hepatitis C virus, a yellow fever virus, a dengue virus, a west Nile virus, a coronavirus, a orthomyxovirus, an influenza virus, a rhabdovirus, a paramyxovirus, a measles virus, a mumps virus, a parainfluenza virus, a respiratory syncytial virus, a metapneumovirus, a Lassa virus, a LuJo virus, a Junin virus, a Machupo virus, a Bear Canyon virus, a Wenzhou virus, a Guanarito virus, a Latino virus, a Sabia virus, a Chapare virus, a Whitewater Arroyo virus, a Pichinde virus, an LCMV virus, a Mopeia virus, a Mobala virus, an Ippy virus, a Mobala virus, a Morogoro virus, a rhabdovirus, a rabies virus, ebola, a retrovirus, a human immunodeficiency virus (HIV), a hepadnavirus, or a hepatitis B virus.

87. The method of any one of claims 83-86, wherein the pathogen is SARS-CoV-2.

88. The method of any one of claims 83-85, wherein the pathogen is selected from Bacillus spp., Bordetella spp., Borrelia spp., Brucella spp., Campylobacter spp., Chlamydia spp., Clostridium spp., Corynebacterium spp., Enterococcus spp., Escherichia spp., Francisella spp., Haemophilus spp., Helicobacter spp., Legionella spp., Leptospira spp., Listeria spp., Mycobacterium spp., Mycoplasma spp., Neisseria spp., Pseudomonas spp., Rickettsia spp., Salmonella spp., Shigella spp., Staphylococcus spp., Streptococcus spp., Treponema spp., Vibrio spp., and Yersinia spp..

89. The method of any one of claims 83-85, wherein the pathogen is selected from Ancylostoma spp., Anisakis spp., Ascaris lumbricoides, Balantidium coli. Cestoda spp., Cimicidae spp., Clonorchis sinensis, Dicrocoelium dendrilicum, Dicrocoelium hospes. Diphyllobothrium latum, Dracunculus spp., Echinococcus spp., Entamoeba histolytica, Enter obius vermicular is, Fasciola spp., Fasciolopsis buski, Giardia spp., Gnathostoma spp., Hymenolepis spp., Leishmania spp., Loa loa, Metorchis spp., Necator americanus, Oestroidea spp.,ACTIVE 508396272 101072396.1082PATENTOnchocercidae spp., Opisthorchis spp., Plasmodium spp., Protofasciola robusta, Parafasciolopsis fasciomorphae, Paragonimus westermani, Schistosoma spp., Spirometra erinaceieuropaei, Strongyloides stercoralis, Taenia spp., Toxocara spp., Toxoplasma spp., Trichobilharzia regenti, Trichinella spiralis, Trichuris trichiura, Trombiculidae spp., Trypanosoma spp., Tunga penetrans, and Wuchereria bancrofti.

90. A method of prime-boost vaccination against a pathogen in a subject in need thereof comprising inserting the plurality of microneedles of a prime device and a boost device, wherein each one of the prime device and the boost device is the administration device of any one of claims 1-66 or 75, and wherein the inserting comprises penetrating the stratum corneum to deliver an effective amount of the vaccine to the epidermis and / or dermis without systemic exposure.

91. A method of prime-boost vaccination against a pathogen in a subject in need thereof comprising applying inserting the plurality of microneedles of a prime administration device of any one of claims 1-66 or 75, wherein the inserting comprises penetrating the stratum corneum to deliver an effective amount of the vaccine to the epidermis and / or dermis without systemic exposure.

92. A method of prime-boost vaccination against a pathogen in a subject in need thereof comprising inserting the plurality of microneedles of a boost administration device of any one of claims 1-66 or 75, wherein the inserting comprises penetrating the stratum corneum to deliver an effective amount of the vaccine to the epidermis and / or dermis without systemic exposure.

93. The method of any one of claims 90-92, wherein the pathogen is an adenovirus, a herpesvirus, an Epstein Barr virus, a herpes simplex type 1, a herpes simplex type 2, a human herpes virus simplex type 8, a human cytomegalovirus, a varicella-zoster virus, a poxvirus, a parvovirus, a papillomavirus, a reovirus, a picomavirus, a coxsackie virus, a hepatitis A virus, a poliovirus, a togavirus, a rubella virus, a flavivirus, a hepatitis C virus, a yellow fever virus, a dengue virus, a west Nile virus, a coronavirus, a orthomyxovirus, an influenza virus, a rhabdovirus, a paramyxovirus, a measles virus, a mumps virus, a parainfluenza virus, a respiratory syncytial virus, a metapneumovirus, a Lassa virus, a LuJo virus, a Junin virus, a Machupo virus, a Bear Canyon virus, a Wenzhou virus, a Guanarito virus, a Latino virus, a Sabia virus, a Chapare virus, a Whitewater Arroyo virus, a Pichinde virus, an LCMV virus, a Mopeia virus, a Mobala virus, an Ippy virus, a Mobala virus, a Morogoro virus, a rhabdovirus, a rabies virus, ebola, a retrovirus, a human immunodeficiency virus (HIV), a hepadnavirus, or a hepatitis B virus94. The method of any one of claims 90-92, wherein the pathogen is SARS-CoV-2.ACTIVE 508396272 102072396.1082PATENT95. The methods of 90-94, further comprising identifying the subject as having COVID-19.

96. The method of any one of claims 90-92, wherein the pathogen is selected from Bacillus spp., Bordetella spp., Borrelia spp., Brucella spp., Campylobacter spp., Chlamydia spp., Clostridium spp., Corynebacterium spp., Enterococcus spp., Escherichia spp., Francisella spp., Haemophilus spp., Helicobacter spp., Legionella spp., Leptospira spp., Listeria spp., Mycobacterium spp., Mycoplasma spp., Neisseria spp., Pseudomonas spp., Rickettsia spp., Salmonella spp., Shigella spp., Staphylococcus spp., Streptococcus spp., Treponema spp., Vibrio spp., and Yersinia spp..

97. The method of any one of claims 90-92, wherein the pathogen is selected from Ancylostoma spp., Anisakis spp., Ascaris lumbricoides, Balantidium coli. Cestoda spp., Cimicidae spp., Clonorchis sinensis, Dicrocoelium dendrilicum, Dicrocoelium hospes. Diphyllobothrium latum, Dracunculus spp., Echinococcus spp., Entamoeba histolytica, Enter obius vermicular is, Fasciola spp., Fasciolopsis buski, Giardia spp., Gnathostoma spp., Hymenolepis spp., Leishmania spp., Loa loa, Metorchis spp., Necator americanus, Oestroidea spp., Onchocercidae spp., Opisthorchis spp., Plasmodium spp., Protofasciola robusta, Parafasciolopsis fasciomorphae, Paragonimus westermani, Schistosoma spp., Spirometra erinaceieuropaei, Strongyloides stercoralis, Taenia spp., Toxocara spp., Toxoplasma spp., Trichobilharzia regenti, Trichinella spiralis, Trichuris trichiura, Trombiculidae spp., Trypanosoma spp., Tunga penetrans, and Wuchereria bancrofti.

98. A method of preventing and / or treating a tumor or cancer in a subject in need thereof, the method comprising inserting the plurality of microneedles of the device of any one of claims 1-66 or 75, wherein the inserting comprises penetrating the stratum comeum to deliver an effective amount of the vaccine to the epidermis and / or dermis without systemic exposure.

99. A method of inducing an immune response against a tumor or cancer in a subject in need thereof, the method comprising inserting the plurality of microneedles of primethe device of any one of claims 1-66 or 75, wherein the inserting comprises penetrating the stratum comeum to deliver an effective amount of the vaccine to the epidermis and / or dermis without systemic exposure.

100. A method of prime-boost vaccination against a tumor or cancer in a subj ect in need thereof comprising inserting the plurality of microneedles of a prime device and a boost device, wherein each one of the prime device and the boost device is the administration device of any one of claims 1-66 or 75, and wherein the inserting comprises penetrating the stratum corneum to deliver an effective amount of the vaccine to the epidermis and / or dermis without systemic exposure.ACTIVE 508396272 103072396.1082PATENT101. A method of prime-boost vaccination against a tumor or cancer in a subj ect in need thereof comprising inserting the plurality of microneedles of a prime device of any one of claims 1- 66 or 75, wherein the inserting comprises penetrating the stratum comeum to deliver an effective amount of the vaccine to the epidermis and / or dermis without systemic exposure.

102. A method of prime-boost vaccination against a tumor or cancer in a subj ect in need thereof comprising inserting the plurality of microneedles of a boost device of any one of claims 1-66 or 75, wherein the inserting comprises penetrating the stratum corneum to deliver an effective amount of the vaccine to the epidermis and / or dermis without systemic exposure.

103. The method of any one of claims 98-102, wherein the tumor or cancer is selected from melanoma, neuroblastoma, ovarian cancer, synovial sarcoma, myxoid / round cell liposarcoma, breast cancer, squamous cell carcinoma, mesothelioma, adult and pediatric sarcomas, hematological cancers, prostate cancer, lung adenocarcinoma, esophageal squamous cell carcinoma, gastric cancer, bladder cancer, hepatocellular carcinoma, brain cancer, multiple myeloma, Ewing's sarcoma, rhabdomyosarcoma, germ cell tumors, Burkitt lymphoma, osteosarcoma, renal cell cancer, colon cancer, acute myeloid leukemia, colorectal cancer, glioblastoma, pancreatic cancer, chronic myeloid leukemia, nasopharyngeal carcinoma, cervical cancer, lymphomas, or renal cell carcinoma.

104. The method of any one of claims 98-103, wherein the tumor or cancer is associated with a tumor antigen (TA) selected from gplOO, MART-l / Melan A, gp75 (TRP-I), tyrosinase, NY- ESO-I, melanoma proteoglycan, MAGE family antigens (i.e., MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-6, and MAGE-12), BAGE family antigens, GAGE family antigens (i.e., GAGE-1, GAGE-2), RAGE family antigens, N- acetylglucosaminyltransferase-V, pl 5, P- catenin, MUM-I, cyclin dependent kinase-4 (CDK4), p21-ras, BCR- abl, p53, pl85 HER2 / neu, epidermal growth factor receptor (EGFR), carcinoembryonic antigens (CEA), carcinoma- associated mutated mucins (i.e., MUC-1 gene products), EBNA gene products of EBV (i.e., EBNA-I), E7, E6 proteins of human papillomavirus, prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), idiotypic epitopes or antigens, for example, immunoglobulin idiotypes or T cell receptor idiotypes, KSA, kinesin 2, HIP-55, TGFP-1 anti- apoptotic factor, tumor protein D52, HIFT, NY-BR-I, NY-BR-62, NY-BR-75, NY-BR-85, NY-BR-87 and NY-BR-96105. The methods of any one of claims 90-104, wherein the subject is a human subject.ACTIVE 508396272 104

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