Replication-inducible poxvirus and uses therefor

Abstract

Provided herein is a nucleic acid including a gene for expressing a transactivator, wherein the activity of the transactivator is controlled by the presence or absence of tetracycline or a derivative of tetracycline; and a regulated promoter controlling transcription of a gene required for replication of the recombinant, live poxvirus; and a transcription insulator in cis with the promotor; and a gene required for replication of the recombinant, live poxvirus in cis with the transcription insulator..

Description

Attorney Docket No. 06527-2505243REPLICATION-INDUCIBLE POXVIRUS AND USES THEREFORCROSS-REFERENCE TO RELATED APPLICATION

[0001] The present application claims priority to U. S. Provisional Patent Application No. 63 / 752,016, filed January 31, 2025, the disclosure of which is hereby incorporated by reference in its entirety.REFERENCE TO A SEQUENCE LISTING

[0002] The Sequence Listing associated with this application is filed in electronic format via Patent Center and is hereby incorporated by reference into the specification in its entirety. The name of the file containing the Sequence Listing is 2505243. xml. The size of the file is 30,261 bytes, and the file was created on January 26, 2026.BACKGROUNDField of the Invention

[0003] Provided herein are compositions, in particular replication-inducible viruses, and methods of using the same.Description of Related Art

[0004] Monkeypox virus (MPXV) was declared a global health emergency of international concern with over 96,000 cases globally in 117 countries. Various nations, including the USA, UK, and Canada, have implemented a "ring vaccination" strategy to curb MPXV spread, a selective vaccination approach successful in containing smallpox and Ebola outbreaks. The Centers for Disease Control and Prevention (CDC) recommends vaccination for individuals at high risk of MPXV infection. However, there is currently no vaccine specifically developed against MPXV. Smallpox vaccines, assumed to be 85% effective and providing cross-protective immunity, are recommended by the WHO and CDC. Despite minimal testing against MPXV, these vaccines, including JYNNEOS (Imvanex or Imvamune) and ACAM2000, have been applied to prevent MPXV infection. JYNNEOS, a live non-replicating virus vaccine, is administered in two injections over four weeks, while ACAM2000, a live replication-competent virus preparation, is given by pricking the skin surface. Both vaccines have side effects, with JYNNEOS causing mild reactions like headache, fatigue, nausea, and injection site reactions. ACAM2000, on the other hand, presents more adverse complications, including fever, rash, lymph node swelling, and serious concerns such as high rates of myocarditis and pericarditis. It is considered unsafe for specific populations, such as pregnant women, infants, immunocompromised 16AG1309. DOCXAttorney Docket No. 06527-2505243individuals, or those living with HIV. JYNNEOS stands out for its lower reactogenicity compared to traditional smallpox vaccines.

[0005] Stable poxvirus vaccination compositions and devices, as well as vaccination protocols, are needed that require only a single dose of a poxvirus vaccine that result in long-lasting immunity and minimal side-effects.SUMMARY

[0006] Provided herein is a nucleic acid including a gene for expressing a transactivator, wherein the activity of the transactivator is controlled by the presence or absence of tetracycline or a derivative of tetracycline; and a regulated promoter controlling transcription of a gene required for replication of the recombinant, live poxvirus; and a transcription insulator in cis with the promotor; and a gene required for replication of the recombinant, live poxvirus in cis with the transcription insulator.

[0007] Also provided herein is a method of immunizing a patient to an antigen comprising administering to the patient a composition comprising recombinant poxvirus as described, thereby eliciting an immune response against a poxvirus.

[0008] Also provided herein is a kit comprising the poxvirus as described, wherein the poxvirus is lyophilized; and a solution for reconstituting lyophilized poxviruses; and a bifurcated needle.

[0009] Also provided herein is a device comprising a microneedle array wherein one or more microneedles comprise the poxvirus of as described.

[0010] Also provided herein is a kit comprising the device as described and tetracycline or a derivative of tetracycline.

[0011] Further non-limiting embodiments are set forth in the following numbered clauses:

[0012] Clause 1: A nucleic acid comprising; a gene for expressing a transactivator, wherein the activity of the transactivator is controlled by the presence or absence of tetracycline or a derivative of tetracycline; a regulated promoter controlling transcription of a gene required for replication of a recombinant, live poxvirus; a transcription insulator in cis with the regulated promotor; and the gene required for replication of the recombinant, live poxvirus, in cis with the transcription insulator.

[0013] Clause 2: The nucleic acid of clause 1, wherein the nucleic acid is a poxvirus genome.

[0014] Clause 3: The nucleic acid of clause 1 or clause 2, wherein the gene required for replication of the poxvirus is A6L.26AG1309. DOCXAttorney Docket No. 06527-2505243

[0015] Clause 4: The nucleic acid of any of clauses 1-3, wherein the gene required for replication of the poxvirus is A3L.

[0016] Clause 5: The nucleic acid of any of clauses 1-4, wherein the poxvirus is of the genus Orthopoxvirus.

[0017] Clause 6: The nucleic acid of any of clauses 1-5, wherein the poxvirus is of the species Orthopoxvirus vaccinia.

[0018] Clause 7: The nucleic acid of any of clauses 1-6, wherein the poxvirus is of the NYBOH strain of Orthopoxvirus vaccinia, or a strain derived therefrom.

[0019] Clause 8: The nucleic acid of any of clauses 1-7, wherein the poxvirus is of the ACAM2000 strain of Orthopoxvirus vaccinia.

[0020] Clause 9: The nucleic acid of any of clauses 1-8, wherein the transactivator is a reverse tetracycline-controlled transactivator.

[0021] Clause 10: The nucleic acid of any of clauses 1-9, wherein the promotor is an inducible promotor.

[0022] Clause 11: The nucleic acid of any of clauses 1-10, wherein the inducible promotor comprises a tetracycline-on transcription response element (TRE).

[0023] Clause 12: The nucleic acid of any of clauses 1-11, wherein the transcription insulator is a barrier insulator.

[0024] Clause 13: The nucleic acid of any of clauses 1 -12, wherein the transcription insulator is sea urchin arylsulfatase insulator.

[0025] Clause 14: The nucleic acid of any of clauses 1 -13, wherein the transcription insulator is a fluorescent protein gene.

[0026] Clause 15: The nucleic acid of any of clauses 1 -14, wherein the transcription insulator is YFP-gpt.

[0027] Clause 16: The nucleic acid of any of clauses 1-15, further comprising a gene for expressing a non-poxvirus antigen.

[0028] Clause 17: The nucleic acid of any of clauses 1-16, wherein the antigen is from a pathogenic species.

[0029] Clause 18: The nucleic acid of any of clauses 1-17, wherein the antigen is from a non-poxvirus of the genus Ebolavirus.

[0030] Clause 19: The nucleic acid of any of clauses 1-18, wherein the antigen is from the species Zaire ebolavirus.

[0031] Clause 20: The nucleic acid of any of clauses 1-19, wherein the antigen is from the species Sudan ebolavirus.36AG1309. DOCXAttorney Docket No. 06527-2505243

[0032] Clause 21: A nucleic acid comprising a poxvirus genome comprising a cassette, wherein the cassette comprises a first promoter, a tet operator, a transcription insulator, a second promoter, a third promoter, and a tetR.

[0033] Clause 22: The nucleic acid of clause 21, wherein the first promoter is a P11 promoter, the second promoter is a pE / L promoter, and the third promoter is a p7.5 promoter.

[0034] Clause 23: The nucleic acid of clause 21 or clause 22, wherein the transcription insulator is flanked by loxP sites.

[0035] Clause 24: The nucleic acid of any of clauses 21-23, wherein the transcription insulator comprises a fluorescent protein.

[0036] Clause 25: The nucleic acid of any of clauses 21 -24, wherein the fluorescent protein is YFP-gpt.

[0037] Clause 26: The nucleic acid of any of clauses 21-25, wherein the transcription insulator is sea urchin arylsulfatase insulator.

[0038] Clause 27: The nucleic acid of any of clauses 21-26, having the sequence SEQ ID NO: 1.

[0039] Clause 28: The nucleic acid of any of clauses 21-27, further comprising a gene for expressing a non-poxvirus antigen.

[0040] Clause 29: The nucleic acid of any of clauses 21-28, wherein the nonpoxvirus antigen is from a pathogenic species.

[0041] Clause 30: The nucleic acid of any of clauses 21-29, wherein the nonpoxvirus antigen is from the genus Ebolavirus.

[0042] Clause 31: The nucleic acid of any of clauses 21-30, wherein the nonpoxvirus antigen is from the species Zaire ebolavirus.

[0043] Clause 32: The nucleic acid of any of clauses 21-31, wherein the nonpoxvirus antigen is from the species Sudan ebolavirus.

[0044] Clause 33: The nucleic acid of any of clauses 21-32, having the sequence SEQ ID NO: 2.

[0045] Clause 34: A method of immunizing a patient to an antigen comprising administering to the patient a composition comprising a poxvirus virion comprising the nucleic acid of any of clauses 1-33, thereby eliciting an immune response against a poxvirus.

[0046] Clause 35: The method of clause 34, wherein the composition is administered intradermally.46AG1309. DOCXAttorney Docket No. 06527-2505243

[0047] Clause 36: The method of clause 34 or clause 35, wherein the composition has previously been lyophilized, and the lyophilized composition is reconstituted in a liquid solution prior to administration.

[0048] Clause 37: The method of any of clauses 34-36, wherein the reconstituted composition is administered via scarification.

[0049] Clause 38: The method of any of clauses 34-37, wherein the composition comprising the recombinant poxvirus is delivered with a microneedle of a microneedle array.

[0050] Clause 39: The method of any of clauses 34-38, wherein the composition comprising the recombinant poxvirus is received in or on a microneedle comprising tetracycline or an analog thereof.

[0051] Clause 40: A kit comprising: a lyophilized composition comprising a poxvirus virion comprising the nucleic acid of any of clauses 1-33; a solution for reconstituting lyophilized composition; and a bifurcated needle.

[0052] Clause 41: The kit of clause 40, further comprising tetracycline or a derivative of tetracycline.

[0053] Clause 42: A device comprising a microneedle array wherein one or more microneedles comprise a poxvirus virion comprising the nucleic acid of any of clauses 1-33.

[0054] Clause 43: The device of clause 42, wherein the tetracycline is incorporated into the one or more microneedles comprising the nucleic acid.

[0055] Clause 44: A kit comprising the device of clause 42 and tetracycline or a derivative of tetracycline.

[0056] Clause 45: A recombinant poxvirus virion, comprising a nucleic acid comprising; a tetracycline repressor (TetR); a tetracycline operator (TetO); a gene required for replication of a recombinant, live poxvirus, wherein the gene is A3L and / or A6L; and a transcription insulator in cis with the TetO and the gene required for replication of the recombinant, live poxvirus, wherein the insulator prevents expression of the gene required for replication of the recombinant, live poxvirus in the absence of tetracycline or a derivative thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0057] FIG. 1 Tet-On system. The Tet-On system can be used to control the expression of a gene-of-interest. Only in the presence of tetracycline, TetR can remove56AG1309. DOCXAttorney Docket No. 06527-2505243from the tetO promoter and activate gene expression. Production of the encoded protein can thus be switched ON by the administration of tetracycline.

[0058] FIG. 2 Replication-inducible vaccinia virus vectors with enhanced safety. (A) Genome of the ACAM2000 of VACV showing Hindlll restriction fragments A through P and the location of the A6L genes. ITR, inverted terminal repeat. (B) Cassettes containing the P11 promoter, followed by the tet operator (02) were inserted upstream of the A6L genes to generate the recombinant ACAM2000 with P11tetO2. The cassettes also contain the tetR gene and the YFP-gpt fusion linked lox gene under VACV promoters (p7.5 and pE / L). PCR analysis of viral DNAfrom TetYFP. ACAM2000 conforming homologous recombination (C) PCR analysis of viral DNA from Tet. ACAM2000 (see, e.g., Guo ZS, et al. Rapid Generation of Multiple Loci-Engineered Marker-free Poxvirus and Characterization of a Clinical-Grade Oncolytic Vaccinia Virus. Mol Ther Methods Clin Dev. 2017 Sep 30;7:112-122) confirming deletion of YFP-gpt following recombination with Cre8.

[0059] FIG. 3 Replication-inducible vaccinia virus forms plaques only in the presence of DOX with enhanced safety. CV-1 cells monolayers were infected with serial diluted ACAM2000(parental VACV), TetYFP. ACAM2000 and Tet. ACAM2000 in the absence or presence of 1ug / ml DOX and cells were stained with crystal violet 3 DPI (A, B, and C) or imaged by brightfield (phase) and fluorescence microscopy (D, E, and F). In the absence of DOX only single YFP+ cells were observed 3 DPI for TetYFP. ACAM2000 (E), and under higher magnification, YFP expression was contained to single cells and was the only indication of infection (red inset), suggesting abortive infections.

[0060] FIG. 4 Experiment 1 Schedule. Immunization and monitor viral replication using In vivo image system for first few weeks until gone.

[0061] FIG. 5 Experiment 2 Schedule. Immunization on Day 0 and conduct continued bleeding (red drop) for ELISA and neutralization assay.

[0062] FIG. 6 In-Vivo Experiment 3 Schedule. Immunization on Day 0 and Day 28 of CAST / EiJ followed by challenge with MPXV on day 56.

[0063] FIG. 7 In-Vivo Experiment 4-5 Schedule. Immunization on Day 0 and Day 28 of C57BL / 6 (Exp.4) or A / Ncr (Exp.5) followed by challenge with ECTV on day 56.

[0064] FIG. 8 Replication-inducible vaccinia virus vectors with enhanced safety. (A) Genome of the ACAM2000 of VACV showing Hindlll restriction fragments A through P and the location of the A6L genes. ITR, inverted terminal repeat. (B) Cassettes 66AG1309. DOCXAttorney Docket No. 06527-2505243containing the P 11 promoter, followed by the tet operator (02) were inserted upstream of the A6L genes to generate the recombinant ACAM2000 with P11tetO2. The cassettes also contain the tetR gene and the YFP-gpt fusion linked lox gene under VACV promoters (p7.5 and pE / L). PCR analysis of viral DNA from YFP. AGAM2024 (TetYFP. ACAM2000) conforming homologous recombination (C) PCR analysis of viral DNA from AGAM2024 (TetARS. ACAM2000) conforming replacement of YFP-gpt to ARS (arylsulfatase), a gene insulator from sea urchin.

[0065] FIGS. 9A and 9B Replication-inducible vaccinia virus forms plaques only in the presence of DOX with enhanced safety. CV-1 cells monolayers were infected with serial diluted ACAM2000(parental VACV), YFP. AGAM2024 (TetYFP. ACAM2000), Tet. ACAM2000, and AGAM2024 (TetARS. ACAM2000) in the absence or presence of 1ug / ml DOX and cells were stained with crystal violet 3 DPI. In the absence of DOX, no plaques were observed 3 DPI for YFP. AGAM2024 and AGAM2024, while size-reduced plaques were seen for Tet. ACAM2000. The typical plaques were detected for ACAM2000 in both the absence and presence of DOX.

[0066] FIG. 10 (A) Cassettes containing the Pn promoter, followed by the tet operator (O2) were inserted upstream of the A6L genes to generate the recombinant ACAM2000 with PntetO2. The cassettes also contain the tetR gene and the EBOVzaireGP gene under VACV promoters (p7.5 and pE / L). (B) PCR analysis of viral DNA from AGAM2024. EBOV-GP conforming replacement of YFP-gpt to EBOVzaireGP gene.

[0067] FIG. 11 Replication-inducible vaccinia virus forms plaques only in the presence of DOX with enhanced safety. CV-1 cells monolayers were infected with serial diluted AGAM2024. EBOV-GP in the absence or presence of 1ug / ml DOX and cells were stained with crystal violet 3 DPI. In the absence of DOX, no plaques were observed 3 DPI for AGAM2024. EBOV-GP, while the typical plaques were detected in the presence of DOX.

[0068] FIGS.12A-12C Flow cytometry assay of Expi293 cells expressing EBOV-GP at the cell surface. Expi293 cells were infected with ACAM2000(parental VACV), AGAM2024 (TetARS. ACAM2000), and AGAM2024. EBOV-GP. At 30 hrs postinfection, binding to EBOV-GP at the cell surface was analyzed by incubation with mice sera obtained at week 6 after immunization with Ad5. EBOV-GPf followed by staining with FITC-conjugated anti-mouse IgG. AGAM2024. EBOV-GP induced EBOV-GP expression on cell surface.76AG1309. DOCXAttorney Docket No. 06527-2505243

[0069] FIG. 13 provides a schematic and a continuous exemplary nucleic acid sequence of SEQ ID NO: 1.

[0070] FIG. 14 provides a schematic and a contiguous exemplary nucleic acid sequence of SEQ ID NO: 2.DESCRIPTION OF THE INVENTION

[0071] The use of numerical values in the various ranges specified in this application, unless expressly indicated otherwise, are stated as approximations as though the minimum and maximum values within the stated ranges are both preceded by the word “about”. In this manner, slight variations above and below the stated ranges can be used to achieve substantially the same results as values within the ranges. Also, unless indicated otherwise, the disclosure of ranges is intended as a continuous range including every value between the minimum and maximum values. As used herein, “a” and “an” refer to one or more.

[0072] As used herein, the term “comprising” is open-ended and may be synonymous with ‘including’, ‘containing’, or ‘characterized by’. The term "consisting essentially of" limits the scope of a claim to the specified materials or steps, and those that do not materially affect basic and novel characteristic(s). The term “consisting of" excludes any element, step, or ingredient not specified in the claim. As used herein, embodiments "comprising" one or more stated elements or steps also include but are not limited to embodiments "consisting essentially of" and "consisting of" these stated elements or steps.

[0073] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Stated numbers of bases and amino acids, and molecular weight or molecular mass values for nucleic acids and polypeptides are exemplary and may be approximate, and are provided for illustration of various examples, embodiments, or aspects of the present disclosure. Unless otherwise indicated, polymer molecular weight is expressed as number-average molecular weight (Mn). Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below.86AG1309. DOCXAttorney Docket No. 06527-2505243

[0074] Unless stated otherwise, nucleotide sequences are recited herein in a 5’ to 3’ direction, and amino acid sequences are recited herein in an N-terminal to C-terminal direction according to convention.

[0075] As used herein, the term “patient” or “subject” refers to members of the animal kingdom including but not limited to human beings, and “mammal” refers to all mammals, including, but not limited to human beings.

[0076] A nucleic acid molecule (a nucleic acid) refers to a polymeric forms of nucleotides, which may include, for example and without limitation, RNAor DNAin any form, including c synthetic forms and mixed polymers of the above. A nucleotide may be a ribonucleotide, deoxynucleotide or a modified form of either type of nucleotide. The term “nucleic acid molecule” as used herein is synonymous with “nucleic acid” and “polynucleotide.” The term includes single- and double-stranded forms of DNA. A polynucleotide may include either or both naturally occurring and modified nucleotides linked together by naturally occurring and / or non-naturally occurring nucleotide linkages.

[0077] A recombinant nucleic acid refers to a nucleic acid molecule (or protein or virus) that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by broadly-known genetic engineering techniques (see, e.g., Lanigan TM, et al. Principles of Genetic Engineering. Genes (Basel). 2020 Mar 10; 11 (3):291., van der Oost J, Patinios C. The genome editing revolution. Trends Biotechnol. 2023 Mar;41(3):396-409., and Khalil AM. The genome editing revolution: review. J Genet Eng Biotechnol. 2020 Oct 29;18(1):68.). The term recombinant includes nucleic acids and proteins that have been altered solely by addition, substitution, or deletion of a portion of a natural nucleic acid molecule or protein. In non-limiting embodiments, a recombinant nucleic acid may include sequences from species other than those from the family Poxviridae, referred to as non-poxvirus sequences. In one non-limiting example, a recombinant poxvirus virion nucleic acid may contain one or more non-poxvirus sequences, such as from an Ebolavirus. In non-limiting examples, non-poxvirus sequences may encode nonpoxvirus antigens. In non-limiting examples, non-poxvirus antigen is from a pathogenic species, such as Ebolavirus.96AG1309. DOCXAttorney Docket No. 06527-2505243

[0078] By "expression" or “gene expression,” it is meant the overall flow of information from a gene or functional / structural RNA, to produce a gene product (typically a protein, optionally post-translationally modified, or a functional / structural RNA). A “gene” refers to a functional genetic unit for producing a gene product, such as RNA or a protein in a cell, or other expression system encoded on a nucleic acid and comprising: a transcriptional control sequence, such as a promoter and other c / s-acting elements, such as transcriptional response elements (TREs) and / or enhancers; an expressed sequence that may encode a protein (referred to as an open-reading frame or ORF), and a polyadenylation sequence. By "expression of genes under transcriptional control of," or alternately "subject to control by," a designated sequence such as TRE or transcription control element, it is meant gene expression from a gene containing the designated sequence operably linked (functionally attached, typically in c / s) to the gene. A "gene for expression of" a stated gene product is a gene capable of expressing that stated gene product when placed in a suitable environment--that is, for example, when transformed, transfected, transduced, etc. into a cell, and subjected to suitable conditions for expression.

[0079] A promoter may be an array of c / s-acting nucleic acid control sequences which direct transcription of a nucleic acid. A promoter includes necessary nucleic acid sequences near the start site of transcription and optionally includes distal enhancer or repressor elements. A “constitutive promoter” is a promoter that is continuously active and is not subject to regulation by external signals or molecules. In the case of a constitutive promoter "suitable conditions" for expression of a gene product means that the gene typically need only be introduced into an appropriate host cell. In contrast, the activity of an “regulated promoter” is regulated by an external signal or molecule (for example, a transcription factor). Regulated promoters may be either inducible promoters or repressible promoters. In the case of a regulated promoter, "suitable conditions" for expression of a gene product means when factors that regulate transcription, such as DNA-binding proteins, are present or absent - for example an amount of the respective inducer is available to the expression system (e.g., cell), or factors causing suppression of a gene are unavailable or displaced -effective to cause expression of the gene. With an inducible promoter, expression of a gene product occurs when factors that regulate transcription are present. With a repressible promoter, expression of a gene product occurs when factors that regulate transcription are absent.106AG1309. DOCXAttorney Docket No. 06527-2505243

[0080] To immunize a subject, e.g., to vaccinate the subject, refers to a process by which an active or adaptive immune response is elicited in a patient, such as humoral or cell-mediated immune response against an antigen, for example, of a microbe or other pathogen, or a specific cell type, such as a cancer cell. In one example, a goal of immunization may be to render a subject partly or fully protected from an infectious disease, or to reduce a number of cancer cells in a patient.

[0081] A vaccine may refer to a preparation of immunogenic material capable of stimulating an immune response, administered for the prevention, inhibition, amelioration, or treatment of infectious, such as Mpox, infections, or other types of disease. Immunogenic material may include live, modified, or attenuated microorganisms (such as bacteria or viruses) or inactivated (killed) microorganisms, or antigenic proteins, peptides or DNA derived from them. A modified virus refers to a virus that is genetically-modified, e.g. by recombinant or mutagenesis. Vaccines may elicit prophylactic (preventative or protective) or therapeutic immune responses. Routes of administration of vaccines may vary according to the nature of the vaccine, but may include inoculation, ingestion, inhalation, scarification, or other forms of administration. Vaccines may be administered with an adjuvant to boost the immune response. Vaccines may also be administered with another biologically active agent, such as an antibiotic (e.g., tetracycline or an analog thereof). “Treating” and like terms refer to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition in a patient. “Ameliorating” and like terms refer to the reduction in the number or severity of one or more signs or symptoms of a disease or infection in a patient.

[0082] An immunogen refers to a compound, composition, or substance which is capable, under appropriate conditions, of stimulating an immune response, such as the production of antibodies, such as neutralizing antibodies, or a T-cell response in an animal, including compositions that are injected or absorbed into an animal. As used herein, an “immunogenic composition” is a composition comprising an immunogen. An immunogen may be, in non-limiting embodiments, an antigen.

[0083] An immune response is a response of a cell of the immune system of a patient, such as a B-cell, T-cell, macrophage or polymorphonucleocyte, to a stimulus, such as an antigen of a live or modified virus. An immune response in a patient may include any cell of the body involved in a host defense response, including, for example and without limitation, an epithelial cell that secretes an interferon or a cytokine.116AG1309. DOCXAttorney Docket No. 06527-2505243

[0084] " Therapeutically effective amount," as used herein, is intended to include the amount of a therapeutic agent, such as an immunogen, as described herein that, when administered to a subject having a disease, is sufficient to effect treatment of the disease (e.g., by diminishing, ameliorating, or maintaining the existing disease or one or more symptoms of disease). The "therapeutically effective amount" may vary depending on compound or composition, how it is administered, the disease and its severity and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the subject to be treated.

[0085] A "therapeutically-effective amount" also includes an amount of an agent that produces some desired local or systemic effect at a reasonable benefit / risk ratio applicable to any treatment. Compounds and compositions described herein may be administered in a sufficient amount to produce a reasonable benefit / risk ratio applicable to such treatment. For example, a therapeutically-effective amount of a virus vaccine useful for eliciting an immune response in a subject against an immunogen, such as a protein of the virus or expressed by the virus. In the context of the present disclosure, a therapeutically effective amount of a poxvirus, e.g., a vaccinia virus vaccine, for example, is an amount sufficient to increase resistance to, ameliorate, and / or otherwise treat infection caused by a poxvirus, e.g., a vaccinia virus in a subject without causing a substantial cytotoxic effect in the subject. The effective amount of a poxvirus vaccine, e.g., as described herein, useful for increasing resistance to, ameliorating, and / or treating infection in a subject will be dependent on, for example, the subject being treated, the manner of administration of the therapeutic composition, and other factors.

[0086] Dosage can be varied by an attending clinician to maintain a desired concentration at a target site (for example, systemic circulation). Higher or lower concentrations can be selected based on the mode of delivery, for example, epicutaneous, intradermal, trans-epidermal, percutaneous, intramuscular, rectal, oral, pulmonary, or intranasal delivery. The actual dosage of disclosed immunogen will vary according to factors such as the disease indication and particular status of the subject (for example, the subject's age, size, fitness, extent of symptoms, susceptibility factors, and the like), time and route of administration, other drugs or treatments being administered concurrently, as well as the specific pharmacology of the composition for eliciting the desired activity or biological response in the subject. Dosage regimens 126AG1309. DOCXAttorney Docket No. 06527-2505243can be adjusted to provide an optimum prophylactic or therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental side effects of the disclosed immunogen and / or other biologically active agent is outweighed in clinical terms by therapeutically beneficial effects.

[0087] As used herein, administering a composition (e.g. an immunogenic composition, such as a vaccine) to a subject means to give, apply or bring the composition into contact with the subject. Administration can be accomplished by any of a number of routes, such as, for example, topical, oral, subcutaneous, intradermal, intramuscular, transdermal, mucosal, intraperitoneal, intravenous, intrathecal, and intramuscular. Scarification, for example with a bifurcated needle, may be another mode of delivery, as is well known for vaccinia-based vaccines.

[0088] Microneedle arrays may take any of a number of forms, however, they generally comprise a plurality of microneedles or microinjectors, attached to a backing that permits a user to press the array onto the skin of a patient so that the plurality of microneedles enter the skin and facilitate dermal or subdermal delivery of an active agent (e.g., deliverable), such as a compound, composition, or organism such as a recombinant virus particle. In one example, the array comprises a plurality of dissolvable microneedles that contain the active agent, and on piercing the skin, the microneedles dissolve - thereby releasing the active agent.

[0089] In one non-limiting example, the microneedles are prepared and used as described in International Patent Publication No. WO2012081933, Kim, J. D et al. Droplet-Born Air Blowing: Novel Dissolving Microneedle Fabrication. J Control Release 2013, 170, 430-436; Erdos G, et al. Improved Cutaneous Genetic Immunization by Microneedle Array Delivery of an Adjuvanted Adenovirus Vaccine. J Invest Dermatol. 2020 Dec; 140(12):2528-2531.e2; Lee MH, et al. Protection against tuberculosis achieved by dissolving microneedle patches loaded with live Mycobacterium paragordonae in a BCG prime-boost strategy. Front Immunol. 2023 Jun 16; 14:1178688; Kim, E., et al. Fourth Dose of Microneedle Array Patch of SARS-CoV-2 S1 Protein Subunit Vaccine Elicits Robust Long-Lasting Humoral Responses in Mice. Int Immunopharmacol 2024, 129, 111569; Kim E, et al. The Long-Term Immunity of a Microneedle Array Patch of a SARS-CoV-2 S1 Protein Subunit Vaccine Irradiated by Gamma Rays in Mice. Vaccines. 2025; 13(1 ):86, in which a recombinant poxvirus virion or a nucleic acid, according to any aspect, embodiment, or example provided136AG1309. DOCXAttorney Docket No. 06527-2505243herein, is incorporated into one or more dissolvable microneedles of a microneedle delivery device as described.

[0090] Therapeutic compositions, including those containing the binding reagents or any pharmaceutically-acceptable salt thereof disclosed herein, may comprise a pharmaceutically acceptable carrier, or excipient. An excipient is an inactive substance used as a carrier for the active ingredients of a medication. Although "inactive," excipients may facilitate and aid in increasing the delivery or bioavailability of an active ingredient in a drug product. Non-limiting examples of useful excipients include: adjuvants, anti-adherents, binders, rheology modifiers, carriers, coatings, disintegrants, emulsifiers, oils, buffers, salts, acids, bases, fillers, diluents, solvents, flavors, colorants, glidants, lubricants, preservatives, antioxidants, sorbents, vitamins, sweeteners, etc., as are available in the pharmaceutical / compounding arts.

[0091] The compositions, methods, and dosage forms disclosed herein may include one or more adjuvants or molecules with immune stimulant or adjuvant effect. In other examples, an adjuvant is not included in the composition but is separately administered to a subject (for example, in combination with a composition disclosed herein) before, after, or substantially simultaneously with administration of one or more of the immunogen-containing compositions disclosed herein. Adjuvants are agents that increase or enhance an immune response in a subject administered an antigen, compared to administration of the antigen in the absence of an adjuvant. One example of an adjuvant is an aluminum salt, such as aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate, or aluminum hydroxyphosphate. Other adjuvants include biological adjuvants, such as cytokines (for example, IL-2, IL-6, IL-12, RANTES, GM-CSF, TNF-a, or IFN-y), growth factors (for example, GM-CSF or G-CSF), one or more molecules such as OX-40L or 4-1 BBL, immunostimulatory oligonucleotides (for example, CpG oligonucleotides, for example, see U. S. Pat. Nos.6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116; 6,339,068; 6,406,705; and 6,429,199), Toll-like receptor agonists (for example, TLR2, TLR4, TLR7 / 8, or TLR9 agonists), and bacterial lipopolysaccharides or their derivatives (such as 3D-MPL). Additional adjuvants include oil and water emulsions, squalene, or other agents. An adjuvant may be a water-in-oil emulsion in which antigen solution is emulsified in mineral oil (for example, Freund's incomplete adjuvant), sometimes with the inclusion of killed mycobacteria (Freund's complete adjuvant) to further enhance antigenicity. In one example, the adjuvant is a mixture of stabilizing detergents, micelle-forming agent,146AG1309. DOCXAttorney Docket No. 06527-2505243and oil available under the name PROVAX® (IDEC Pharmaceuticals, San Diego, Calif.). One of skill in the art can select a suitable adjuvant or combination of adjuvants to be included in the compositions disclosed herein or administered to a subject in combination with the compositions disclosed herein. Molecules with immune stimulant or adjuvant effects, include, without limitation: TLR3 agonists such as Poly(l: C) or Poly-ICLC; TLR 4 agonists such as LPS or monophosphoryl lipid derivatives; TLR 5 agonists such as flagellin derivatives; TLR 7 / 8 agonists such as imiquimod or R848; TLR 9 agonists such as CpG sequences; Stimulator of Interferon Genes (STING) pathway agonists such as ADU-S100; stimulatory neuroimmune mediators such as calcitonin gene-related peptide (CGRP); neurokinin 1 (NK1) receptor agonists such as Hemokinin 1 and Substance P; saponin related adjuvants such as QS-21 (Quillaja saponaria); purinoergic receptor agonists such as ATP; or oil-in-water emulsion adjuvants such as MF59.

[0092] Useful dosage forms for the binding reagents or any pharmaceutically-acceptable salt thereof disclosed herein include, for example and without limitation: parenteral, intravenous, intramuscular, intraocular, or intraperitoneal solutions, oral tablets or liquids, topical drops, ointments, or creams, and transdermal devices (e.g., patches). The compound may be a sterile solution comprising the active ingredient (drug or compound), and a solvent, such as water, saline, lactated Ringer's solution, or phosphate-buffered saline (PBS). Additional excipients, such as polyethylene glycol, emulsifiers, salts and buffers may be included in the solution.

[0093] Suitable dosage forms may include single-dose, or multiple-dose vials or other containers, such as medical syringes or droppers, e.g., eye droppers.

[0094] Pharmaceutical formulations adapted for administration include aqueous and non-aqueous sterile solutions which may contain, in addition to the active pharmaceutical ingredient or drug, for example and without limitation, adjuvants, antioxidants, buffers, bacteriostats, lipids, liposomes, lipid nanoparticles, emulsifiers, suspending agents, and rheology modifiers. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use. Extemporaneous solutions and suspensions may be prepared from sterile powders, granules and tablets.156AG1309. DOCXAttorney Docket No. 06527-2505243

[0095] Therapeutic / pharmaceutical compositions as described herein may be prepared in accordance with acceptable pharmaceutical procedures, such as described in Remington: The Science and Practice of Pharmacy, 21st edition, ed. Paul Beringer et al., Lippincott, Williams & Wilkins, Baltimore, MD Easton, Pa. (2005) (see, e.g., Chapters 37, 39, 41, 42 and 45 for examples of powder, liquid, parenteral, intravenous and oral solid formulations and methods of making such formulations).

[0096] Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. For example, sterile injectable solutions can be prepared by incorporating the active agent in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, typical methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

[0097] Sterile powders, such as, without limitation, lyophilized viruses, may be reconstituted in liquid solutions, such as a sterile solution of glycerine, phenol, and water (e.g., 50% glycerin, 0.25% phenol, and water).

[0098] The phrase "pharmaceutically-acceptable carrier" as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject being treated. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such 166AG1309. DOCXAttorney Docket No. 06527-2505243as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium state, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; and (22) other non-toxic compatible substances employed in pharmaceutical formulations. Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, Pa., 21st Edition (2005) (see above), describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic compositions, such as a chimeric virus, and additional pharmaceutical agents.

[0099] In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (for example, powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

[0100] Transdermal drug delivery methods use various methods to permeate the stratum corneum, and include microneedle or microneedle array, thermal ablation, microdermabrasion, electroporation, and cavitational ultrasound methods and related dosage forms for delivery of therapeutic agents through the stratum corneum. Certain transdermal delivery methods may be preferred for delivery of larger virus particles, such as dermabrasion and microneedle / microneedle array delivery methods (See,176AG1309. DOCXAttorney Docket No. 06527-2505243e.g., Prausnitz, M. R., et al. Transdermal Drug Delivery Nat Biotechnol. 2008 November; 26(11): 1261-1268. doi:10.1038 / nbt.1504 and Alkilani, A. Z., et al. Transdermal Drug Delivery: Innovative Pharmaceutical Developments Based on Disruption of the Barrier Properties of the stratum corneum. Pharmaceutics 2015, 7, 438-470; doi:10.3390 / pharmaceutics7040438). Also useful in the methods described herein is dermal (e.g., intradermal and / or epicutaneous) delivery, such as by scarification of the skin and application of virus particles to the scarified skin during scarification, or after scarification, as with traditional poxvirus vaccination methods.

[0101] Poxviruses are a large family of DNA viruses that can infect a wide range of hosts. The most studied poxvirus, vaccinia virus (VACV) is the prototypic member of the Orthopoxvirus genus, which also includes Cowpox virus, Monkeypox virus (MPXV), and Variola virus, the causative agent of smallpox. The poxvirus genome, about 190 kb in size, can be modified genetically and can accommodate inserts of DNA fragments exceeding 25 kb in size without losing infectivity and other functions (Guo ZS, et al. Rapid Generation of Multiple Loci-Engineered Marker-free Poxvirus and Characterization of a Clinical-Grade Oncolytic Vaccinia Virus. Mol Ther Methods Clin Dev. 2017 Sep 30;7:112-122.). Various strains of VACV have been used as vaccines for the eradication of smallpox in the world. These include the New York City Board of Health (NYCBOH) strain, from which Dryvax was derived (Belongia EA, Naleway AL. Smallpox vaccine: the good, the bad, and the ugly. Clin Med Res. 2003 Apr;1(2):87-92.). Further fears about the use of smallpox as an agent of biological terrorism resulted in the development of ACAM2000 from Dryvax (Monath TP, et al. ACAM2000 clonal Vero cell culture vaccinia virus (New York City Board of Health strain)-a second-generation smallpox vaccine for biological defense. Int J Infect Dis.2004 Oct;8 Suppl 2: S31-44.). ACAM2000 is US FDA-approved (Nalca A, Zumbrun EE. ACAM2000: the new smallpox vaccine for United States Strategic National Stockpile. Drug Des Devel Ther. 2010 May 25;4:71-79.). The full genomic sequence of ACAM2000 is known and is broadly-available. For example and without limitation, an exemplary genomic sequence of ACAM2000 is provided in GenBank Reference No. AY313847.1 (Vaccinia virus strain Acambis clone 2000, complete genome) (Osborne JD, et al. Genomic differences of Vaccinia virus clones from Dryvax smallpox vaccine: The Dryvax-like ACAM2000 and the mouse neurovirulent Clone-3. Vaccine. 2007 Oct 8;25:8807-8832).186AG1309. DOCXAttorney Docket No. 06527-2505243

[0102] A number of poxvirus gene products are required for replication of the virus, examples of which include essential genes A3, A11, H7, L2, A6, and A30.5 (see, e.g., O'Connell CM, et al. Replication-inducible vaccinia virus vectors with enhanced safety in vivo. PLoS One. 2020 Apr 2;15(4):e0230711. and Meng X, et al. Vaccinia Virus A6 Is a Two-Domain Protein Requiring a Cognate N-Terminal Domain for Full Viral Membrane Assembly Activity. J Virol. 2017 Apr 28;91(10):e02405-16, indicating that genetic studies with VACV revealed a number of viral proteins that are essential for crescent formation, including A11, H7, L2, A6, and A30.5). In one example, A6 is a virion component expressed late in infection and appears to be essential in virion morphogenesis. In another example, A3 is present both in intracellular mature virions (MVs) and extracellular enveloped virions (EVs) and is conserved among all Chordopoxviruses. The designations A6, A6L, and OPG132 may be considered synonymous.

[0103] Immunization with VACV led to the successful eradication of smallpox worldwide and since then, VACV has been used as a viral vector for the development of recombinant vaccines for humans and animals. Due to the cross-protective immunity provided by VACV based vaccines against other Orthopoxviruses, multiple VACV based vaccines have been recommended for use to prevent MPXV infection. Cross-protection against MPXV is inferred based on high genetic conservation within immunogenic protein regions and epitopes between MPXV and VACV. Additionally, 85% protection against MPXV has been observed during historic MPXV outbreaks. The recommended VACV vaccines for protection against MPXV include JYNNEOS, a live non-replicating virus vaccine derived from the Ankara strain of VACV, and ACAM2000, a live replication-competent virus preparation derived from the NYBOH (New York Board of Health) strain of VACV via plaque purification of VACV in the Dryvax vaccine. (O'Connell CM, etal. Replication-inducible vaccinia virus vectors with enhanced safety in vivo. PLoS One. 2020 Apr2;15(4):e0230711. and Ahmed SF, et al. Vaccinia-Virus-Based Vaccines Are Expected to Elicit Highly Cross-Reactive Immunity to the 2022 Monkeypox Virus. Viruses. 2022 Sep 3; 14(9): 1960.)

[0104] The Tet-On system described here may be based on the transposon Tn 10 operon that confers tetracycline resistance in bacteria (Hillen W, et al. Mechanisms underlying expression of Tn10 encoded tetracycline resistance. Annu Rev Microbiol.1994; 48:345-69.). In the tet operon, the Tet repressor (TetR) is unable to bind to tet operators in the presence of tetracycline, allowing transcription of the tetracycline 196AG1309. DOCXAttorney Docket No. 06527-2505243resistance gene. The tet operon has been adapted to develop the replication-inducible VACV. Control of gene expression in VACV has been achieved by expressing the TetR gene (tetR) constitutively and inserting a tet operator element (O2) after the transcriptional start site of VACV genes, allowing their expression to be regulated by tetracyclines (see, e.g., Traktman P, et al. Elucidating the essential role of the A14 phosphoprotein in vaccinia virus morphogenesis: construction and characterization of a tetracycline-inducible recombinant. J Virol. 2000 Apr;74(8):3682-95, Unger B, Traktman P. Vaccinia virus morphogenesis: a13 phosphoprotein is required for assembly of mature virions. J Virol. 2004 Aug;78(16):8885-901, Hagen CJ, et al. Antibiotic-dependent expression of early transcription factor subunits leads to stringent control of vaccinia virus replication. Virus Res. 2014 Mar 6; 181:43-52, O’Connell CM, et al. (2020) Replication-inducible vaccinia virus vectors with enhanced safety in vivo. PLoS ONE 15(4): e0230711 ) Therefore, we propose to improve the safety of VACV in vivo by using tetR and tetO2 to control the transcription of genes that are required for viral replication. Ideally, these replication-inducible VACVs should replicate to wild-type levels in the presence of tetracyclines such as doxycycline (DOX) and would be unable to replicate in the absence of these antibiotics. Basal expression from the tetracycline can occur, resulting in undesirable expression of the target gene without its inducer (e.g., Dox) present. In the context of replication-inducible poxvirus, leakiness would result in undesirable replication.

[0105] Insulator sequences are c / s-regulatory elements that protect gene expression from accidental activation (e,g., enhancer-blocking insulators), and / or accidental heterochromatin-mediated gene silencing (e.g., barrier insulators), which act as protective boundaries for the gene environment from silencing or enhancing occurring in adjacent native gene environments. Barrier insulators can be utilized in systems for controlling genes by preventing the accidental override of the molecular switches that control the genes of interest. This is especially important in systems with a high potential of risk if disordered gene expression occurs (as in poxvirus replication control) and those systems where gene leakiness is already a known issue (as with Tet-On systems). Barrier insulators may be coding or non-coding sequences. Potential barrier insulators include the sea urchin derived arylsulfatase (ARS) insulator, used herein. Other suitable insulators include, without limitation: CTCF (CCCTC-binding factor) insulators (e.g., Yang J, Corces VG. Insulators, long-range interactions, and genome function. CurrOpin Genet Dev. 2012 Apr;22(2):86-92., Liu, M. et al. Genomic 206AG1309. DOCXAttorney Docket No. 06527-2505243discovery of potent chromatin insulators for human gene therapy. Nat. Biotechnol. 2015 Feb;33(2): 198-203), B1 and B2 SINEs (Short Interspersed Nuclear Elements) (e.g., Roman AC, et al. Dioxin receptor and SLUG transcription factors regulate the insulator activity of B1 SINE retrotransposons via an RNA polymerase switch. Genome Res. 2011 Mar;21(3):422-32, Lunyak VV, et al. Developmentally regulated activation of a SINE B2 repeat as a domain boundary in organogenesis. Science. 2007 Jul 13;317(5835):248-51), cHS4 (chicken hypersensitive site 4) insulator (e.g., Chung JH, et al. A 5' element of the chicken beta-globin domain serves as an insulator in human erythroid cells and protects against position effect in Drosophila. Cell. 1993 Aug 13;74(3):505-14.), ALOXE3 (arachidonate lipoxygenase 3) tDNA insulator (e.g., Raab JR, et al. Human tRNA genes function as chromatin insulators. EMBO J. 2012 Jan 18;31(2):330-50.), chiMARs (matrix attachment regions localized upstream the chicken lysozyme gene) (e.g., Loc PV, et al. The matrix attachment regions of the chicken lysozyme gene co-map with the boundaries of the chromatin domain. EMBO J. 1988 Mar;7(3):655-64.). Barrier insulators that are coding sequences may, in non-limiting embodiments, be transcribed and translated into proteins, including proteins that further undergo post-translational modifications, for example glycoproteins and / or nucleoproteins. These proteins and related molecules may be immunogens, antigens, and / or components of an immunogenic composition. In non-limiting embodiments, the antigen may be from an Ebolavirus. In non-limiting embodiments, the antigen may be an Ebolavirus nucleoprotein, an Ebolavirus glycoprotein, VP24, VP30, VP35, VP40, and / or like antigens known to those of skill in the art. Additional random sequence (e.g., DNA) inserts may be utilized, and which will not be expected to affect expression of other genes.

[0106] Provided herein are compositions including nucleic acids, for example nucleic acids of and / or encoding replication-controllable viruses, such as poxviruses, as well as kits including the same and methods of using the same. Such compositions allow for improved vaccination, treatment, and testing of treatments for viruses, such as poxviruses and / or Ebolaviruses. To this end, a composition as described herein may include a recombinant poxvirus virion, for example a recombinant, live poxvirus virion, where expression of a gene required for replication may be controlled.

[0107] In non-limiting embodiments, the poxvirus is of the genus Orthopoxvirus. In non-limiting embodiments, the poxvirus is of the species Orthopoxvirus vaccinia. In non-limiting embodiments, the poxvirus is of the NYBOH strain of Orthopoxvirus 216AG1309. DOCXAttorney Docket No. 06527-2505243vaccinia, or a strain derived therefrom. In non-limiting embodiments, the poxvirus is of the ACAM2000 strain of Orthopoxvirus vaccinia.

[0108] In non-limiting embodiments, the nucleic acid further includes a gene encoding a non-poxvirus antigen. In non-limiting embodiments, the gene encoding a non-poxvirus antigen is from a pathogenic species. In non-limiting embodiments, the antigen is from a non-poxvirus of the genus Ebolavirus. In non-limiting embodiments, the antigen is from the species Zaire ebolavirus. In non-limiting embodiments, the antigen is from the species Sudan ebolavirus. In non-limiting embodiments, the antigen may be an Ebolavirus nucleoprotein, an Ebolavirus glycoprotein, VP24, VP30, VP35, VP40, and / or like antigens known to those of skill in the art

[0109] In non-limiting embodiments the composition includes a nucleic acid including one or more segments including and / or encoding a transactivator, a promoter, a transcription insulator, and a gene / protein required for replication of the poxvirus.

[0110] As used herein, a “transactivator” is a protein that stimulates gene transcription by binding to a nucleic acid, such as DNA, or by interacting with transcription proteins. In non-limiting embodiments, the activity of the transactivator is controlled by the presence or absence of a compound. In non-limiting embodiments, the compound is an antibiotic. In non-limiting embodiments, the compound is tetracycline or a derivative of tetracycline. In non-limiting embodiments, the transactivator is a reverse tetracycline-controlled transactivator.

[0111] In non-limiting embodiments, the nucleic acid may further include a promoter, such as a regulated promoter. In non-limiting embodiments, the regulated promoter may control transcription of a gene required for replication of the recombinant, live poxvirus. In non-limiting embodiments, the promotor is an inducible promotor and / or a repressible promotor. In non-limiting embodiments, the inducible promotor comprises a tetracycline-on transcription response element (TRE).

[0112] In non-limiting embodiments, the nucleic acid may include and / or encode a transcription insulator (e.g., an insulator sequence), which may be in cis with the promotor. In non-limiting embodiments, the transcription insulator is a barrier insulator. In non-limiting embodiments, the transcription insulator is sea urchin arylsulfatase insulator. In non-limiting embodiments, the transcription insulator is a fluorescent protein gene. In non-limiting embodiments transcription insulator is YFP-gpt.226AG1309. DOCXAttorney Docket No. 06527-2505243

[0113] In non-limiting embodiments, the nucleic acid may include and / or encode a protein required for replication of the recombinant, live poxvirus, which may be in cis with the transcription insulator. Such proteins are described herein and may include, without limitation, A6L and / or A3L. In non-limiting embodiments, the gene required for replication of the recombinant, live poxvirus is A6L. In non-limiting embodiments, the gene / protein required for replication of the recombinant, live poxvirus is A3L.

[0114] Also provided herein is a nucleic acid including a poxvirus genome and a cassette. In non-limiting embodiments, the cassette may include a first promoter, an operator sequence, a transcription insulator, a second promoter, a third promoter, and a repressor. In non-limiting embodiments, the operator and repressor may be an antibiotic-based sequence, such as a tetracycline-on transcription response element (TRE). In non-limiting embodiments, the cassette may include one or more tetO (e.g., tetO2) regions and one or more tetR regions.

[0115] In non-limiting embodiments, the various promoters may be one or more of a P11 promoter, an early-late promoter (e.g., a pE / L promoter), and / or a p7.5 promoter. In non-limiting embodiments, the first promoter is a P11 promoter, the second promoter is a pE / L promoter, and the third promoter is a p7.5 promoter. The transcription insulator may be any of those described herein, including, for example, sea urchin arylsulfatase or a fluorescent protein gene (e.g., YFP-gpt). In non-limiting embodiments, the transcription insulator is under the control of a site-specific genome modification system, such as Cre / loxP. Accordingly, in non-limiting embodiments, the transcription insulator may be flanked by loxP sites. In non-limiting embodiments, the cassette has a sequence as set forth in SEQ ID NO: 1, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or greater sequence identity to SEQ ID NO: 1, all values and subranges therebetween inclusive.

[0116] In non-limiting embodiments, the cassette may further include a gene for expressing a non-poxvirus antigen. In non-limiting embodiments the non-poxvirus antigen is from a pathogenic species. In non-limiting embodiments, the non-poxvirus antigen is from the genus Ebolavirus. In non-limiting embodiments, the non-poxvirus antigen is from the species Zaire ebolavirus and / or Sudan ebolavirus. In non-likmiting embodiments, the antigen is an Ebolavirus nucleoprotein, an Ebolavirus glycoprotein, VP24, VP30, VP35, VP40, and / or like antigens known to those of skill in the art. In non-limiting embodiments, the cassette has a sequence as set forth in SEQ ID NO: 2,236AG1309. DOCXAttorney Docket No. 06527-2505243or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or greater sequence identity to SEQ ID NO: 1, all values and subranges therebetween inclusive.

[0117] Also provided herein is a method of immunizing a patient to an antigen, using a composition as described herein. In non-limiting embodiments, the method includes administering to the patient a composition including a recombinant poxvirus as described herein, thereby eliciting an immune response against a poxvirus. In nonlimiting embodiments, the composition is administered intradermally and / or transdermally. In non-limiting embodiments, the composition (e.g., the recombinant poxvirus) has previously been lyophilized, and the lyophilized composition is reconstituted in a liquid solution prior to administration. In non-limiting embodiments, the composition is administered via scarification. In non-limiting embodiments, the composition including the recombinant poxvirus is delivered with a microneedle of a microneedle array. In non-limiting embodiments, the microneedle includes a composition including the recombinant poxvirus as described herein and tetracycline or an analog thereof.

[0118] Also provided herein is a kit including a composition as described herein. In non-limiting embodiments, the composition (e.g., the poxvirus) is lyophilized. In non-limiting embodiments, the kit includes a solution for reconstituting the lyophilized composition. In non-limiting embodiments, the kit includes a bifurcated needle. In nonlimiting embodiments, the kit further includes tetracycline ora derivative of tetracycline.

[0119] Also provided herein is a device, for example a microneedle array, where one or more microneedles include a composition as described herein. In non-limiting embodiments, tetracycline is incorporated into one or more of the one or more microneedles, for example a microneedle including the composition.

[0120] The following examples are provided to illustrate certain particular features and / or embodiments. These examples should not be construed to limit the disclosure to the particular features or embodiments described.EXAMPLES

[0121] The following examples are intended to be illustrative and exemplary. Example 1Preliminary Data: Generation of tetracyclines replication-inducible AGAM2024 Mpox vaccine.

[0122] Tetracycline-controlled Tet-On gene expression systems are an established system for the regulation of expression of genes in various organisms (FIG. 1). A6L is 246AG1309. DOCXAttorney Docket No. 06527-2505243a VACV viral membrane assembly protein and minor virion component expressed late in infection and essential in virion morphogenesis. Importantly, VACV with inducible expression of A6L in the presence of tetracyclines replicated at same level as wildtype VACV in the presence of tetracycline but was not detected in the absence of tetracycline.

[0123] Building upon this finding we have recently generated a tetracyclinedependent replication-inducible live New York city board of health live VACV vaccine strain ACAM2000 MPXV vaccine through the creation of a cassette containing the P11 promoter, followed by the tet operator, upstream of the A6L genes (FIGS. 2A and 8A).Included in the cassette are the tetR gene and YFPgpt fusion gene for combined selection of recombinant virus (FIGS. 2B and 8B). The recombinant VACV was generated by standard homologous recombination after transfection of the shuttle vectors into HEK293 cell monolayers infected 2 hours previously with ACAM2000. Recombinant TetYFP. ACAM2000 (YFP. AGAM2024) was plaque purified from transfection of lysates in CV-1 cells in the presence of 1 ug / mL doxycycline (DOX) and visualized by fluorescent microscopy for YFP. The PCR products (2961 bp) using primers A6L-F and A7L-R designed to amplify the spanning the site of recombination confirmed the insertion of TetR-YFPgpt, while 308 bp fragments was amplified from the genome unrecombined ACAM2000 virus (FIGS. 2B and 8B). After the four plaque purifications cycles, the YFPgpt gene in the recombinant YFP. AGAM2024 was replaced with ARS (arylsulfatase), a gene insulator from sea urchin in HEK293 cells transfected with DNA fragment of TetR-ARS. The replacement of the YFP-gpt gene was confirmed by PCR using genomic DNA from six plaques, resulting in 2320bp (FIG.8C). We also confirmed the recombination by genome sequencing. Thus, we have generated a tetracycline-inducible Tet. ACAM2000 (AGAM2024) MPXV vaccine candidate.

[0124] The ability of the putative inducible viruses to replicate in the absence or presence of inducer was first investigated by performing standard plaque assays in CV-1 cells, either in the absence or presence of DOX (1 pg / ml), followed by crystal violet staining 3 days postinfection (DPI). Isolated plaques that formed 3 DPI in the presence of DOX by YFP. AGAM2024 and AGAM2024 were typical (FIGS. 3B / 3E, 3C / 3F, and 9) and identical to parental ACAM2000 plaques in size (FIGS. 3A, 3D, and 9). However, in the absence of DOX, no plaques could be detected 3 DPI for YFP. AGAM2024 and AGAM2024 (FIGS. 3B / 3E, 3C / 3F, and 9). Moreover, plaque 256AG1309. DOCXAttorney Docket No. 06527-2505243formation in CV-1 cells was also investigated in unfixed cells by brightfield and fluorescence microscopy. Under fluorescence microscopy, YFP. AGAM2024 formed typical plaques in the presence of DOX. However, only single YFP+ cells could be detected in the absence of DOX (FIG. 3E). Under high magnification, the single YFP+ cells appeared normal and there was no evidence of YFP expression in the neighboring cells (FIG. 3E). Taken together, these observations are indicative of abortive infections. In addition, detection of high levels of YFP expression in these abortively infected cells suggests that late gene expression from the pE / L promoter was not compromised in the absence of DOX.Experimental Design

[0125] Six groups of ten 4-6-week-old (five) and $ (five) C57BL / 6 mice will be vaccinated by scarification of tetracycline-dependent replication-inducible live pox marker vaccine YFP. AGAM2024 and DOX, along with controls (FIG. 4). Specifically, three experimental groups will be vaccinated with either 1 x 106plaque-forming units (pfu) of tetracycline-dependent replication-inducible live Mpox vaccine, YFP. AGAM2024 by scarification with or without 1 ug or 10 ug of tetracycline. Three control groups will be vaccinated with 1 x 106pfu of replication competent live Mpox vaccine ACAM2000 by scarification with or without 1 ug or 10 ug of tetracycline. The goal of this initial experiment is to evaluate the effect in vivo replication of different doses of DOX on the replication of the tetracycline inducible AGAM2024 Mpox vaccine and its immunogenicity when compared with the parent replication competent FDA approved Mpox vaccine ACAM2000. In this experiment we will evaluate YFP. AGAM2024vaccine delivery and DOX induced replication through fluorescent live animal imaging and epifluorescence microscopy. This will be essential to identifying the kinetics of YFP. AGAM2024 viral replication in the presence of tetracycline in vivo and ensuring efficient localized replication. Once determined the efficacy of AGAM2024 in vivo replication and the optimal dosage of DOX, we will perform a second experiment using the experimental doses range derived from the human vaccination dose of ACAM2000, which consists of administering 0.0025 mL of live vaccinia virus with 2.5 - 12.5 x 105pfu. Additionally, we included a 5X higher dose, enabling assessment of both in vivo induction of replication or its use as replication incompetent vaccine. ACAM2000™ will be included as a positive vaccination control while AGAM2024 without DOX will be used to evaluate the efficacy as replication incompetent vaccine. Eight groups of ten 4-6-week-old (five) and $ (five) C57BL / 6266AG1309. DOCXAttorney Docket No. 06527-2505243mice will be vaccinated by scarification of 2.5, 12.5, and 62.5 x 105pfu of AGAM2024 ± DOX, along with controls. We will collect sera from mice at weeks 0, 2, 4, 6, 8, 10, and 12 and each month there on to assess the breadth and longevity of MPXV antibody response through ELISA endpoint titer measurement (FIG. 5). Briefly, we will coat plates with inactivated MPXV strain Zaire 79, NR-2324 (BEI Resources, Manassas, VA) and determine endpoint IgG titers. We will also measure the neutralizing antibodies by plaque reduction neutralization (PRNTso) assay. The heat-inactivated sera (56°C for 30 min) will be serially diluted and incubated with ~50 pfu of wild-type MPXV Zaire 79 for 1 h at 37°C in 5% CO2. The virus-serum mixtures will be added onto pre-formed Vero E6 cell monolayers and incubated, followed by removing supernatant and cell monolayers covered with methylcellulose overlay. After 72 hours the plate will be fixed and then cell monolayers stained with 1 % crystal violet, before plaques are counted and photographed. The neutralizing antibody titers will be defined as the serum dilutions resulting in a 50% reduction relative to the total number of plaques counted without antibody, according to the Behrens-Karber formula (4408718). Titers will be standardized to the standard preparation of human Vaccinia Immune Globulin CNJ-016 (BEI Research Repository Resource). When these mice reach their life endpoint, approximately 1 year, they will be sacrificed, and their bone marrow used to assess memory antibody-producing response through Enzyme-linked immunosorbent spot (ELISpot) assay for insights into the memory antibody response to vaccination. We will than compare safety and efficacy of the AGAM2024 vaccine to the approved pox vaccines ACAM2000 and JYNNEOS in challenge studies in C57BL / 6, A / Ncr and CAST / EiJ mice. After selection of the optimal dose in Experiment 2, in Experiment 3 (FIG. 6) we will conduct a challenge study in the Mpox susceptible murine strains CAST / EiJ to evaluate the protective efficacy of AGAM2024 in comparison with ACAM2000 both by scarification and intramuscular JYNNEOS. Briefly, five groups of sixteen 4-6-week-old (eight) and $ (eight) CAST / EiJ mice will be vaccinated at Day 0 with the dose determined in Experiment 2 of AGAM2024 (+DOX), ACAM2000™ control, 108pfu of JYNNEOS control, and AGAM2024 (--DOX) without tetracycline as non-replicative vaccine control and saline negative control. JYNNEOS vaccinated animals will receive a boost vaccine on day 28. On day 56, mice will be inoculated with 104pfu of MPXV-Z79-CB2 by the intranasal route. Weight loss and death will be monitored to plot survival curves for each vaccine candidate. In Experiment 4 (FIG. 7), five groups of sixteen 4-6 week-old (eight) and $ (eight)276AG1309. DOCXAttorney Docket No. 06527-2505243C57BL / 6mice, and in Experiment 5 (FIG. 7), five groups of sixteen 4-6- week-old (eight) and $ (eight) A / Ncr mice will be vaccinated at Day 0 with the dose determined in Experiment 2 the AGAM2024 (+DOX), ACAM2000™ control, JYNNEOS control, and AGAM2024 (-DOX) as non-replicative vaccine control and PBS as a negative control. JYNNEOS vaccinated animals will receive a boost vaccine on day 28. On day 56, mice will be inoculated with 103pfu of ectromelia mousepox virus (ECTV). In Experiments 3, 4, and 5 (FIGS. 6 and 7), after euthanasia of mice (timepoint dependent on weight loss and clinical symptoms of distress) we will collect blood samples, spleens, and lungs from mice to measure viral titer using RT-PCR and compare histopathological changes in each organ with hematoxylin and eosin staining. This study represents the initial efficacy evaluation of the AGAM2024 vaccine on pox viral challenge.Expected outcomes, potential problems, and alternative approaches

[0126] We do not anticipate major technical difficulties in vaccinating mice and elucidating the immune responses to Mpox vaccines. Due to the superior immunogenicity conferred by traditional live ACAM2000™ vaccines, we anticipate immunogenicity to be high after vaccination with the AGAM2024 vaccine. If the immunogenicity of AGAM2024 is suboptimal, we will evaluate the integration of adjuvants such as Poly(l: C), PCEP, or squalene (MF59). If the immunogenicity of AGAM2024 is substantial, we will further evaluate in future studies our vaccine strategy in alternative murine models. If AGAM2024 (+DOX) and (-DOX) vaccine arms have similar response in Experiment 3, we will perform a dose escalation study to determine the (-DOX) dose that does not confer protection.Data Analysis, Statistics, and Power Assessment

[0127] For the characterization of AGAM2024 virus, the resulting plaque assay titer, and plaque radius will be assessed using two-way ANOVA followed by Tukey's multiple comparisons. To assess the immunogenicity of AGAM2024 in-vivo; Mpox IgG titer, neutralization titer, will be analyzed using one-way AN OVA fol lowed by Dunnett’s test. For assessment of the necessary sample size per group for in-vivo experiments outlined we performed a power analysis using G*Power 3.1 software, with a set at 0.05, based on comparable IgG-endpoint data. Power analysis showed that a sample size of 4 was necessary to confidently detect an effect between groups if one is present. Therefore, all in-vivo experiments outlined have a minimum total n of 4.286AG1309. DOCXAttorney Docket No. 06527-2505243

[0128] Having described this invention, it will be understood to those of ordinary skill in the art that the same can be performed within a wide and equivalent range of conditions, formulations and other parameters without affecting the scope of the invention or any embodiment thereof. References incorporated herein by reference are incorporated for their technical disclosure and only to the extent that they are consistent with the present disclosure.Example 2

[0129] A replication-inducible vaccinia virus expressing Ebola virus glycoprotein, AGAM2024. EBOV-GP, was generated by replacing the YFP-gpt with the EBOVzaireGP gene, creating a dual vaccine targeting MPOX and EBOV. CV-1 cells monolayers were infected with serial dilutions of AGAM2024. EBOV-GP in the absence or presence of 1ug / ml DOX and stained with crystal violet at 3 DPI. In the absence of DOX, no plaques were observed at 3 DPI, whereas typical vaccinia virus plaques were detected in the presence of DOX (FIG. 11). We next examined whether AGAM2024. EBOV-GP expressed membrane-bound EBOV-Gp by flow cytometry. Expi293 cells were infected with AGAM2024. EBOV-GP, ACAM2000(parental VACV), or AGAM2024 (TetARS. ACAM2000) and cultured for 30 hrs post-infection in the presence of DOX. Cell-surface expression of EBOV-GP was analyzed by incubation with 1:20 diluted mice sera, collected at week 6 after immunization with Ad5. EBOV-GPf, along with pre-immune sera collected at week 0 used as a negative control. FITC-conjugated antimouse IgG was used as a secondary antibody. Ad5. EBOV-GPf is a recombinant adenovirus type 5 expressing the extracellular domain of Ebola virus glycoprotein fused to the T4 fibritin (foldon) trimerization domain (Kim E, et al. The Long-Term Immunity of a Microneedle Array Patch of a SARS-CoV-2 S1 Protein Subunit Vaccine Irradiated by Gamma Rays in Mice. Vaccines. 2025; 13(1):86). EBOV-GP surface expression was detected only in cells infected with AGAM2024. EBOV-GP, but not in those infected with ACAM2000 or AGAM2024. No specific antibody binding was observed using week 0 sera (FIG. 12).Sequences

[0130] SEQ ID NO: 1 is a cassette that includes nucleic acids encoding membrane assembly protein A6L (SEQ ID NO: 3), promoter-tet operator PntetOz (SEQ ID NO: 4), loxP sites (SEQ ID NO: 5), sea urchin arylsulfatase (ARS) (SEQ ID NO: 6), promoter p7.5 (SEQ ID NO: 7), promoter pE / L (SEQ ID NO: 8), tetR (SEQ ID NO: 9), andA7L (SEQ ID NO: 10).296AG1309. DOCXAttorney Docket No. 06527-2505243

[0131] SEQ ID NO: 2 is a cassette that includes nucleic acids encoding membrane assembly protein A6L (SEQ ID NO: 11), promoter Pn and tet operator tetO2 (PntetO2) (SEQ ID NO: 12), Ebola virus glycoprotein (EBOVzaireGP) (SEQ ID NO: 13), promoter p7.5 (SEQ ID NO: 14), promoter pE / L (SEQ ID NO: 15), tetR (SEQ ID NO: 16), and A7L (SEQ ID NO: 17).

[0132] The present invention has been described with reference to certain exemplary embodiments, dispersible compositions and uses thereof. However, it will be recognized by those of ordinary skill in the art that various substitutions, modifications or combinations of any of the exemplary embodiments may be made without departing from the spirit and scope of the invention. Thus, the invention is not limited by the description of the exemplary embodiments, but rather by the appended claims as originally filed.306AG1309. DOCX

Claims

Attorney Docket No. 06527-2505243Claims:

1. A nucleic acid comprising;a gene for expressing a transactivator, wherein the activity of the transactivator is controlled by the presence or absence of tetracycline or a derivative of tetracycline;a regulated promoter controlling transcription of a gene required for replication of a recombinant, live poxvirus;a transcription insulator in cis with the regulated promotor; and the gene required for replication of the recombinant, live poxvirus, in cis with the transcription insulator.

2. The nucleic acid of claim 1, wherein the nucleic acid comprises a poxvirus genome.

3. The nucleic acid of any of claim 1, wherein the gene required for replication of the poxvirus isA6L.

4. The nucleic acid of claim 1, wherein the gene required for replication of the poxvirus isA3L.

5. The nucleic acid of claim 1, wherein the recombinant, live poxvirus is of the genus Orthopoxvirus.

6. The nucleic acid of claim 1, wherein the recombinant, live poxvirus is of the species Orthopoxvirus vaccinia.

7. The nucleic acid of claim 1, wherein the recombinant, live poxvirus is of the NYBOH strain of Orthopoxvirus vaccinia, or a strain derived therefrom.

8. The nucleic acid of claim 1, wherein the recombinant, live poxvirus is of the ACAM2000 strain of Orthopoxvirus vaccinia.316AG1309. DOCXAttorney Docket No. 06527-25052439. The nucleic acid of claim 1, wherein the transactivator is a reverse tetracycline-controlled transactivator.

10. The nucleic acid of claim 1, wherein the promotor is an inducible promotor.

11. The nucleic acid of claim 1, wherein the inducible promotor comprises a tetracycline-on transcription response element (TRE).

12. The nucleic acid of claim 1, wherein the transcription insulator is a barrier insulator.

13. The nucleic acid of claim 1, wherein the transcription insulator is sea urchin arylsulfatase insulator.

14. The nucleic acid of claim 1, wherein the transcription insulator is a fluorescent protein gene.

15. The nucleic acid of claim 1, wherein the transcription insulator is YFP-gpt.

16. The nucleic acid of claim 1, further comprising a gene for expressing a non-poxvirus antigen.

17. The nucleic acid of claim 16, wherein the antigen is from a pathogenic species.

18. The nucleic acid of claim 16, wherein the antigen is from a nonpoxvirus of the genus Ebolavirus.

19. The nucleic acid of claim 18, wherein the antigen is from the species Zaire ebolavirus.326AG1309. DOCXAttorney Docket No. 06527-250524320. The nucleic acid of claim 18, wherein the antigen is from the species Sudan ebolavirus.

21. A nucleic acid comprising a poxvirus genome comprising a cassette, wherein the cassette comprises a first promoter, a tet operator, a transcription insulator, a second promoter, a third promoter, and a tetR.

22. The nucleic acid of claim 21, wherein the first promoter is a P11 promoter, the second promoter is a pE / L promoter, and the third promoter is a p7.5 promoter.

23. The nucleic acid of claim 21, wherein the transcription insulator is flanked by loxP sites.

24. The nucleic acid of claim 21, wherein the transcription insulator comprises a fluorescent protein.

25. The nucleic acid of claim 24, wherein the fluorescent protein is YFP-gpt.

26. The nucleic acid of claim 21, wherein the transcription insulator is sea urchin arylsulfatase insulator.

27. The nucleic acid of claim 21, having the sequence SEQ ID NO: 1.

28. The nucleic acid of claim 21, further comprising a gene for expressing a non-poxvirus antigen.

29. The nucleic acid of claim 28, wherein the non-poxvirus antigen is from a pathogenic species.

30. The nucleic acid of claim 28, wherein the non-poxvirus antigen is from the genus Ebolavirus.336AG1309. DOCXAttorney Docket No. 06527-250524331. The nucleic acid of claim 28, wherein the non-poxvirus antigen is from the species Zaire ebolavirus.

32. The nucleic acid of claim 28, wherein the non-poxvirus antigen is from the species Sudan ebolavirus.

33. The nucleic acid of claim 28, having the sequence SEQ ID NO: 2.

34. A method of immunizing a patient to an antigen comprising administering to the patient a composition comprising a poxvirus virion comprising the nucleic acid of claim 1, thereby eliciting an immune response against a poxvirus.

35. The method of claim 34, wherein the composition is administered intradermally.

36. The method of claim 34, wherein the composition has previously been lyophilized, and the lyophilized composition is reconstituted in a liquid solution prior to administration.

37. The method of claim 36, wherein the reconstituted composition is administered via scarification.

38. The method of claim 37, wherein the composition comprising the recombinant poxvirus is delivered with a microneedle of a microneedle array.

39. The method of claim 37, wherein the composition comprising the recombinant poxvirus is received in or on a microneedle comprising tetracycline or an analog thereof.

40. A kit comprising:a. a lyophilized composition comprising a poxvirus virion comprising the nucleic acid of claim 1;b. a solution for reconstituting lyophilized composition; and c. a bifurcated needle.346AG1309. DOCXAttorney Docket No. 06527-250524341. The kit of claim 40, further comprising tetracycline or a derivative of tetracycline.

42. A device comprising a microneedle array wherein one or more microneedles comprise a poxvirus virion comprising the nucleic acid of claim 1.

43. The device of claim 42, wherein the tetracycline is incorporated into the one or more microneedles comprising the nucleic acid.

44. A kit comprising the device of claim 42 and tetracycline or a derivative of tetracycline.

45. A recombinant poxvirus virion, comprising a nucleic acid comprising;a tetracycline repressor (TetR);a tetracycline operator (TetO);a gene required for replication of a recombinant, live poxvirus, wherein the gene is A3L and / or A6L; anda transcription insulator in cis with the TetO and the gene required for replication of the recombinant, live poxvirus, wherein the insulator prevents expression of the gene required for replication of the recombinant, live poxvirus in the absence of tetracycline or a derivative thereof.356AG1309. DOCX

Images