Synthetic chimeric vaccinia virus

Abstract

To provide synthetic chimeric vaccinia virus.SOLUTION: The invention relates in various aspects to a synthetic chimeric vaccinia virus or compositions comprising such viruses, and the development and use of systems and methods for producing such synthetic chimeric vaccinia viruses. The synthetic chimeric vaccinia viruses are well suited, among others, as virus vaccines or to generate an oncolytic response and pharmaceutical formulations. The disclosure in one aspect is based on the finding that a synthetic chimeric vaccinia virus such as scVACV can be produced from chemically synthesized overlapping fragments of the vaccinia virus genome.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The Sequence Listing accompanying this application has been submitted electronically via EFS-Web in text format and is hereby incorporated by reference in its entirety. The text file containing the Sequence Listing is named 104545-0031-WO-SequenceListing.txt. The text file, created on May 2, 2019, is 288,652 bytes in size. [Background technology]

[0002] Poxviruses (members of the family Poxviridae) are double-stranded DNA viruses that can infect both humans and animals. Poxviruses are classified into two subfamilies based on their host range. The subfamily Chordopoxviridae, which infects vertebrate hosts, consists of eight genera, four of which (Orthopoxvirus, Parapoxvirus, Molluscipoxvirus, and Yatapoxvirus) are known to infect humans. Smallpox is caused by infection with variola virus (VARV), a member of the Orthopoxvirus (OPV) genus. The OPV genus includes several genetically related but morphologically identical viruses, including camelpox virus (CMLV), cowpox virus (CPXV), ectromelia virus (ECTV, "mousepox agent"), horsepox virus (HPXV), monkeypox virus (MPXV), rabbitpox virus (RPXV), raccoonpox virus, skunkpox virus, gerbilpox virus, Uasin Gishū disease virus, vaccinia virus (VACV), variola virus (VARV), and volepox virus (VPV). Besides VARV, at least three other OPVs are known to infect humans, including VACV, MPXV, and CPXV. To date, vaccination with "live" VACV is the only proven protection against smallpox. Aggressive vaccination programs led to the eradication of smallpox in 1980, and routine public vaccination was discontinued. However, there remains a need to find new, safe and effective means of vaccinating individuals against VARV and other OPVs.

[0003] Various preparations of VACV have been used as smallpox vaccines. Most of these contain several closely related viruses (e.g., Dryvax), and one contains a single molecular clone, ACAM2000. However, like Dryvax and other VACV vaccines, even ACAM2000 is associated with significant side effects, including cardiomyopathy and pericarditis. To reduce risk, the ACAM2000 vaccine, like other live vaccines, has numerous contraindications that exclude individuals with cancer, immunodeficiencies, organ transplant recipients, atopic dermatitis, eczema, psoriasis, cardiac conditions, and those taking immunosuppressive medications. It is estimated that 15–50% of the U.S. population will fall into one of these categories, confirming the need for the development of safer vaccines or vaccination protocols (Kennedy et al., 2007). Kennedy R, Poland GA. 2007. T-Cell epitope discovery. for variola and vaccinia viruses. Rev Med Viroll 7: 93-113). Therefore, there is a need for the development of a vaccine that is similar in effectiveness to Dryvax or ACAM2000™, but is safer.

[0004] The production of safe, pure, potent, and effective vaccines requires quality assurance procedures to ensure uniformity and consistency in the vaccine manufacturing process. In the past, embryonated chicken eggs or primary chicken embryo fibroblast cell cultures have been used to grow viruses for the production of vaccines against yellow fever, influenza, measles, and mumps. These media were considered acceptable because it was believed that adventitious agents that might infect chickens would not be infectious or pathogenic to humans (FDA Briefing Document Vaccines and Related Biological Products Advisory Committee Meeting. September 19, 2012). However, if the virus tropism changes, safety could be compromised.

[0005] Other culture media, such as calf lymph for smallpox vaccines, have been used to grow viruses for vaccine production. After inoculating calves with smallpox, lymph containing white blood cells is extracted and stored in capillary tubes. This is then used to vaccinate people against smallpox. However, there is a risk of contamination with bovine spongiform encephalopathy or scrapie prions. Modern vaccine regulations and guidelines state that all materials used must be from BSE-free sources, but nothing about scrapie-free sources. Of particular concern is the fact that the Dryvax vaccine, produced between 1980 and 1982, has not been scrutinized using modern methods. Specifically, these stocks have never been tested for adventitious agents (Murphy and Osburn, Emerging Infectious Diseases, www.cdc.gov / eid). Vol. 11, No. 7, July 2005).

[0006] Therefore, there is a need for the development of a vaccine similar in effectiveness to the existing Dryvax or ACAM2000™ vaccines, but which is safer, reproducible, and free of residual cells, residual DNA, prions, and adventitious agents. The present application provides a safe, reproducible, and contaminant-free chimeric vaccinia virus assembled and replicated from chemically synthesized DNA. Because chemical genome synthesis does not rely on a natural template, extensive structural and functional modifications of the viral genome are possible. Chemical genome synthesis is particularly advantageous when natural templates are unavailable for gene replication or modification by conventional molecular biology methods. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Kennedy R et al. Rev Med Virol (2007)l7:93~113 Summary of the Invention [Means for solving the problem]

[0008] Aspects of the present invention provide synthetic chimeric vaccinia viruses, methods for producing such viruses, and the use of such viruses, for example, as immunogens in immunogenic formulations in in vitro assays, as vehicles for heterologous gene expression, or as oncolytic agents for the treatment of cancer. The synthetic chimeric vaccinia viruses of the present application are characterized by one or more modifications compared to wild-type vaccinia viruses.

[0009] The present disclosure is based, in one aspect, on the discovery that synthetic chimeric vaccinia viruses (eg, scVACVs) can be produced from chemically synthesized overlapping fragments of the vaccinia virus genome.

[0010] Thus, in one aspect, the present invention relates to a synthetic chimeric vaccinia virus (e.g., scVACV) that is replicated and reactivated from DNA derived from synthetic DNA, wherein the viral genome of said virus differs from the wild-type genome of said virus in that it is characterized by one or more modifications, which modifications are derived from the group including chemically synthesized DNA, cDNA or genomic DNA.

[0011] In another aspect, the present invention relates to a method for producing a synthetic chimeric vaccinia virus (scVACV), comprising the steps of: (i) chemically synthesizing overlapping DNA fragments corresponding to substantially all of the viral genome of a vaccinia virus; (ii) transfecting the overlapping DNA fragments into helper virus-infected cells; (iii) culturing the cells to produce a mixture of helper virus and synthetic chimeric vaccinia particles in the cells; and (iv) plating the mixture on host cells specific for the scVACV to recover the scVACV.

[0012] In another aspect, the invention relates to synthetic chimeric vaccinia viruses (scVACVs) produced by the methods of the present disclosure.

[0013] In another aspect, the present invention relates to a pharmaceutical composition comprising a synthetic chimeric vaccinia virus (scVACV) of the present disclosure and a pharmaceutically acceptable carrier.

[0014] In another aspect, the present invention relates to a method for inducing an oncolytic response in a subject, the method comprising administering to the subject a composition comprising a scVACV of the present disclosure.

[0015] In another aspect, the present invention relates to a method for expressing a heterologous protein in a host cell, the method comprising the steps of introducing a heterologous nucleic acid sequence into a scVACV of the present disclosure, infecting a host cell with the scVACV, and culturing the host cell under conditions for expression of the heterologous protein.

[0016] In another aspect, the present invention relates to a method of inducing or boosting an immune response to vaccinia virus, comprising administering to a subject in need thereof a composition comprising a scVACV of the present disclosure.

[0017] In another aspect, the present invention relates to a method of inducing or boosting an immune response against smallpox virus infection, comprising administering to said subject a composition comprising a scVACV of the present disclosure.

[0018] In another aspect, the present invention relates to a method of inducing or boosting an immune response to monkeypox virus infection, comprising administering to said subject a composition comprising a scVACV of the present disclosure.

[0019] In another aspect, the present invention relates to a method of immunizing a human subject to protect said subject from smallpox virus infection, comprising administering to said subject a composition comprising a scVACV of the present disclosure.

[0020] In another aspect, the present invention relates to a method of treating a smallpox virus infection, comprising administering to said subject a composition comprising a scVACV of the present disclosure.

[0021] In another aspect, the present invention relates to a method of treating cancer in a subject, comprising administering to a subject in need thereof a composition comprising a scVACV of the present disclosure.

[0022] This patent application contains at least one drawing executed in color. Copies of this patent application with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0023] BRIEF DESCRIPTION OF THE DRAWINGS The foregoing summary, as well as the following detailed description of the present disclosure, will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the present disclosure, there are shown in the drawings, presently preferred embodiment(s). It should be understood, however, that the disclosure is not limited to the precise arrangements and instrumentalities shown. [Brief explanation of the drawings]

[0024] [Figure 1-1] Figures 1A and 1B show schematic diagrams of the linear dsDNA VACV genome strain ACAM2000; Genbank accession AY313847. Figure 1A illustrates the unmodified genomic sequence of the VACV ACAM2000 genome, with the naturally occurring AarI and BsaI restriction sites indicated. Figure 1B depicts the modified VACV ACAM2000 genome used to chemically synthesize large dsDNA fragments. Overlapping scVACV ACAM2000 genomic fragments are depicted in blue. The engineered BsaI restriction sites, which were not silently mutated in the left inverted terminal repeat (LITR) and right inverted terminal repeat (RITR), are also shown. [Figure 1-2] Same as above. [Figure 1-3] Same as above. [Figure 1-4] Same as above.

[0025] [Figure 2-1] Figures 2A-2C show detailed schematics of the first approximately 1500-3000 bp of the published genomes of (A) the VACV WR strain and (B) the VACV ACAM2000. The tandem repeat regions are indicated by red (70 bp repeat), blue (125 bp repeat), and green (54 bp repeat) boxes. The ORF corresponding to the gene C23L is also indicated in each genome. (C) Schematic of the direct repeat region containing the 70 bp repeat of VACV WR. This sequence was synthesized to contain a SapI restriction site at the 5' end and an NheI restriction site at the 3' end for ligation of the hairpin / duplex portion and the VACV ACAM2000 ITR fragment, respectively. [Figure 2-2] Same as above. [Figure 2-3] Same as above. [Figure 2-4] Same as above. [Figure 2-5] Same as above. [Figure 2-6] Same as above.

[0026] [Figure 3-1] Figures 3A and 3B show the assembly of vaccinia virus terminal hairpin loops with double-stranded DNA into the initial 70-bp repeat sequence. (A) Phosphorylated oligonucleotide sequences ordered to create WR double-stranded DNA are shown. (B) Gel electrophoresis of WR strain double-stranded DNA (lane 2) and hairpin DNA alone (lane 3) followed by ligation (lane 4). The ligation product (arrow) was then excised from the gel and purified so that it could be ligated into the 70-bp repeat sequence to mimic the sequence of the wtVACV ACAM2000 sequence. [Figure 3-2] Same as above. [Figure 3-3] Same as above. [Figure 3-4] Same as above.

[0027] [Figure 4-1]Figure 4 shows the ligation of a SapI / NheI-digested 70-bp repeat fragment to the WR strain hairpin / duplex DNA fragment. The 70-bp repeat fragment was digested with SapI and NheI and then gel-purified prior to ligation with the hairpin / duplex DNA fragment at a 5:1 molar ratio of hairpin / duplex DNA to the 70-bp fragment. The upward shift in the approximately 2300-bp band in lanes 4 and 5 indicates successful addition of the hairpin / duplex fragment. These bands were then gel-extracted from the gel prior to ligation to the digested VACV ACAM2000 ITR fragment. [Figure 4-2] Same as above.

[0028] [Figure 5-1] Figure 5 shows the digestion of scVACV ACAM2000 fragments. The ITR fragment was digested with both NheI and I-SceI at 37°C for 2 hours, followed by dephosphorylation with alkaline phosphatase to remove the phosphate groups and facilitate more efficient ligation of this fragment to the terminal hairpin loop / duplex / 70 bp tandem repeat fragment. Other scVACV ACAM2000 DNA plasmids were linearized with I-SceI at 37°C for 2 hours, followed by heat inactivation of the restriction enzymes at 65°C for 10 minutes. [Figure 5-2] Same as above.

[0029] [Figure 6-1] Figure 6 shows the in vitro growth characteristics of scVACV ACAM2000-WR DUP / HP. Multistep growth kinetics measured in monkey kidney epithelial cells (BSC-40). Cells were infected at a multiplicity of infection of 0.03, virus was harvested at the indicated times, and virus was titrated on BSC-40 cells. Data are representative of three independent experiments. Error bars indicate the standard error of the mean (SEM). [Figure 6-2] Same as above.

[0030] [Figure 7-1]Figure 7 shows the in vitro growth characteristics of scVACV ACAM2000-WR DUP / HP and scVACV ACAM2000-ACAM2000 DUP / HP compared to scVACV ACAM2000-WR DUP / HP and scVACV ACAM2000-ACAM2000 DUP / HP, in which the YFP-gpt marker has been replaced with the J2R gene sequence (VAC_WRΔJ2R), and to wtVACV ACAM2000. Multistep growth kinetics measured in monkey kidney epithelial cells (BSC-40). Cells were infected at a multiplicity of infection of 0.03, and virus was harvested at the indicated times. Virus was titrated on BSC-40 cells. Error bars indicate the standard error of the mean (SEM). [Figure 7-2] Same as above.

[0031] [Figure 8-1]Figure 8 shows restriction endonuclease mapping of reactivated scVACV ACAM2000-WR DUP / HP clones. Pulsed-field gel electrophoresis analysis. Two independent scVACV ACAM2000-WR DUP / HP clones, as well as a VACV WR control in which the YFP-gpt marker was replaced with the J2R gene sequence (VAC_WRΔJ2R) and a wtVACV ACAM2000 control (VAC_ACAM2000), were purified and then either left undigested or digested with BsaI, HindIII, or NotI and PvuI. The expected absence of nearly all BsaI sites in the scVACV ACAM2000 clones was evident. Minor differences in HindIII-digested scVACV ACAM2000 genomic DNA were observed compared to VAC_WRΔJ2R and VACV_ACAM2000. Genomic DNA digested with NotI and PvuI excised the 70-bp tandem repeat fragment found in the left and right ITR sequences. In VAC_WRΔJ2R, the approximate size of the 70-bp repeat sequence was approximately 3.6 kbp. Interestingly, in two independent scVACV ACAM2000 clones, two bands of different sizes corresponding to the 70-bp tandem repeat were observed (marked with *), although the full-length 70-bp tandem repeat element was ligated to the ITR fragment. When ACAM2000 genomic DNA was digested with NotI and PvuI, a band of approximately 4.7 kbp was observed, which may indicate the size of the 70-bp repeat sequence in ACAM2000. [Figure 8-2] Same as above.

[0032] [Figure 9-1] Figure 9 shows nucleotide sequence variations between sequences of VACV strains. Figure 9A depicts VACV nucleotide sequence variations within the duplex regions in the ITRs (SEQ ID NOS: 15-18). Figure 9B depicts the VACV ACAM2000 secondary hairpin loops covalently linked to the ends of the ACAM2000 linear dsDNA genome (S-form SEQ ID NOS: 19 and F-form SEQ ID NOS: 20). The terminal loop sequences are highlighted in green. [Figure 9-2] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0033] General technology Unless otherwise specified herein, scientific and technical terms used in this application shall have the meanings commonly understood by those skilled in the art. Generally, the nomenclature used in connection with and techniques of pharmacology, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, genetics, and protein and nucleic acid chemistry described herein are those well known and commonly used in the art. In case of conflict, the present specification, including definitions, will control.

[0034] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques can be found in, for example, Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JE Cellis, ed., 1998) Academic Press; Animal Cell Culture (RI Freshney, ed., 1987); Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell, eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, NY (2002); Harlow and Lane Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1998); Coligan et al., Short Protocols in Protein Science, John Wiley & Sons, NY (2003); Short Protocols in Molecular Biology (Wiley and Sons, 1999) are fully described in.

[0035] It should be noted that the original text "に完全に説明されている" in line 8 is not grammatically correct in Japanese. The corrected translation should be "are fully described in". If this is a special term or jargon in a specific field, please double-check according to the actual situation.Enzymatic reactions and purification techniques are carried out according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with analytical chemistry, biochemistry, immunology, molecular biology, organic synthetic chemistry, and medicinal and pharmaceutical chemistry described herein, as well as the laboratory methods and techniques thereof, are well known and commonly used in the art. Standard techniques are used for chemical synthesis and chemical analysis.

[0036] Throughout this specification and the embodiments, the word "comprise" or variations such as "comprises" or "comprising" refer to the specified integer or integers. It is understood that any integer or group of integers is intended to imply the inclusion of any other integer or group of integers, but not the exclusion of any other integer or group of integers.

[0037] Whenever an embodiment is described herein with the word "comprising," it is understood that other similar embodiments described with the terms "consisting of" and / or "consisting essentially of" are also provided.

[0038] The term "including" is used to mean "including but not limited to." "Including" and "including but not limited to" are used interchangeably.

[0039] Any example(s) following the term "eg" or "for example" are not intended to be exhaustive or limiting.

[0040] Unless the context otherwise requires, singular terms shall include pluralities and plural terms shall include the singular.

[0041] The articles "a," "an," and "the" are used herein to refer to one or to more than one (i.e., at least one) of the grammatical object of the article. By way of example, "an" means one element or more than one element. Reference herein to a value or parameter with "about" includes (and describes) embodiments that are directed to that value or parameter itself. For example, a reference to "about X" includes a reference to "X." Numerical ranges include the numbers defining the range.

[0042] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the present disclosure are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. However, any numerical value inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Moreover, all ranges disclosed herein should be understood to encompass all subranges subsumed therein. For example, a specified range of "1 to 10" should be considered to include (inclusively) every subrange between the minimum value of 1 and the maximum value of 10; that is, all subranges beginning with a minimum value of 1 or greater, e.g., 1 to 6.1, and ending with a maximum value of 10 or less, e.g., 5.5 to 10.

[0043] Although exemplary methods and materials are described herein, methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this application. The materials, methods, and examples are illustrative only and not limiting.

[0044] definition The following terms, unless otherwise indicated, shall be understood to have the following meanings: As used herein, the terms "wild-type virus," "wild-type genome," "wild-type protein," or "wild-type nucleic acid" refer to an amino acid or nucleic acid sequence that occurs naturally in a particular population (e.g., a particular virus species, etc.).

[0045] The terms "chimeric" or "engineered" or "modified" (e.g., chimeric vaccinia, engineered polypeptide, modified polypeptide, engineered nucleic acid, modified nucleic acid) or grammatical variations thereof are used interchangeably herein and refer to a non-naturally occurring sequence that has been engineered to have one or more changes compared to the naturally occurring sequence.

[0046] As used herein, "synthetic virus" refers to a virus that is initially derived from synthetic DNA (e.g., chemically synthesized DNA, PCR-amplified DNA, engineered DNA, polynucleotides containing nucleoside analogs, etc., or combinations thereof), including its progeny, which may not necessarily be completely identical (in morphology or total genomic DNA) to the original parent synthetic virus due to natural, accidental, or deliberate mutations. In some embodiments, a synthetic virus refers to a virus in which substantially all of the viral genome is initially derived from synthetic DNA (e.g., chemically synthesized DNA, PCR-amplified DNA, engineered DNA, polynucleotides containing nucleoside analogs, etc., or combinations thereof). In a preferred embodiment, the synthetic virus is derived from chemically synthesized DNA.

[0047] As outlined elsewhere herein, certain positions in the viral genome can be altered. As used herein, "position" refers to a position in the genome sequence. Corresponding positions are generally determined through alignment with other parent sequences.

[0048] As used herein, the term "residue" in the context of a polypeptide refers to an amino acid unit in a linear polypeptide chain, which is the remainder of each amino acid, i.e., -NH-CHR-C-, after water is removed in forming a polypeptide from the α-amino acid, i.e., NH-CHR-COOH.

[0049] As known in the art, "polynucleotide" or "nucleic acid," as used interchangeably herein, refers to a chain of nucleotides of any length, including DNA and RNA. The nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a chain by DNA or RNA polymerase. A polynucleotide can contain modified nucleotides, such as methylated nucleotides and their analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the chain. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, for example, by conjugation with a labeling component. Other types of modifications include, for example, "caps," substitutions of one or more analogs of naturally occurring nucleotides; internucleotide modifications, such as those with uncharged bonds (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and those with charged bonds (e.g., phosphorothioates, phosphorodithioates, etc.); those containing pendant moieties, such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.); those with intercalators (e.g., acridine, psoralen, etc.); those containing chelators (e.g., metals, radioactive metals, boron, oxidizing metals, etc.); those containing alkylating agents; those with modified linkages (e.g., alpha-anomeric nucleic acids, etc.); as well as unmodified forms of polynucleotide(s). Additionally, any of the hydroxyl groups normally present on the sugar can be replaced with, for example, phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or conjugated to a solid support. The 5' and 3' terminal OH can be phosphorylated or substituted with amine or organic capping group moieties of 1 to 20 carbon atoms. Other hydroxyls can also be derivatized to standard protecting groups.Polynucleotides may also contain analogous forms of ribose or deoxyribose sugars commonly known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro-, or 2'-azido-ribose, carbocyclic sugar analogs, alpha- or beta-anomeric sugars, epimeric sugars such as arabinose, xylose, or lyxose, pyranose sugars, furanose sugars, sedoheptulose, acyclic analogs, and abasic nucleoside analogs, such as methyl riboside. One or more phosphodiester linkages may be replaced with alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which phosphate is replaced with P(O)S ("thioate"), P(S)S ("dithioate"), (O)NR2 ("amidate"), P(O)R, P(O)OR', CO, or CH2 ("formacetal"), where each R or R' is independently H or a substituted or unsubstituted alkyl (1-20C), optionally containing an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl. Not all linkages in a polynucleotide need be identical. The above description applies to all polynucleotides, including RNA and DNA, referred to herein.

[0050] The terms "polypeptide," "oligopeptide," "peptide," and "protein" are used interchangeably herein to refer to a chain of amino acids of any length. The chain can be linear or branched, can contain modified amino acids, and / or can be interrupted by non-amino acids. The term also encompasses amino acid chains that are modified naturally or by intervention; for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. For example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art, are also included within this definition. It is understood that a polypeptide can exist as a single chain or associated chains.

[0051] "Homologous," in all its grammatical forms and spelling variations, refers to the relationship between two proteins that share a "common evolutionary origin," including proteins from a superfamily of organisms of the same species as well as homologous proteins from organisms of different species. Such proteins (and their encoding nucleic acids) have sequence homology, as reflected by their sequence similarity, whether in terms of percent identity or by the presence of specific residues or motifs and conserved positions. "Homologous" can also refer to nucleic acids that are native to viruses.

[0052] However, in common usage and in this application, the term "homologous" when modified by an adverb such as "highly" can refer to sequence similarity, with or without regard to a common evolutionary origin.

[0053] "Heterologous," in all its grammatical forms and spelling variations, can refer to nucleic acid that is non-native to the virus. It means that the nucleic acid is derived from a different species or strain than the nucleic acid of the organism to which it is described as heterologous. In a non-limiting example, the viral genome of a scVACV contains a heterologous terminal hairpin loop. The heterologous terminal hairpin loop can be derived from a different viral species or a different VACV strain.

[0054] The term "sequence similarity," in all its grammatical forms, refers to the degree of identity or correspondence between nucleic acid or amino acid sequences that may or may not share a common evolutionary origin.

[0055] "Percent (%) sequence identity" or "sequence % identical to" with respect to a reference polypeptide (or nucleotide) sequence is defined as the percentage of amino acid residues (or nucleic acids) in a candidate sequence that are identical to those in the reference polypeptide (nucleotide) sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and after not considering any conservative substitutions as part of the sequence identity. Alignment to determine percent amino acid sequence identity can be accomplished in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximum alignment over the entire length of the sequences being compared.

[0056] As used herein, a "host cell" includes an individual cell or cell culture that may be or has been a recipient of a virus of the present disclosure. A host cell includes progeny of a single host cell, which progeny may not necessarily be completely identical (in morphology or total genomic DNA) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells that have been transfected and / or transformed in vivo by a poxvirus of this disclosure.

[0057] As used herein, " vector " refers to a construct that can deliver and preferably express one or more genes or sequences of interest in host cells.Examples of vectors include, but are not limited to, virus vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells such as producer cells.

[0058] As used herein, an "isolated molecule" (e.g., a polypeptide, polynucleotide, or fragment thereof) is a molecule that, by virtue of its origin or source, (1) is not associated with one or more naturally associated components that naturally accompany it; (2) is substantially free of one or more other molecules from the same species; (3) is expressed by cells from a different species; or (4) is not naturally occurring. Thus, a molecule that is chemically synthesized or expressed in a cellular system different from the cell from which it naturally originates is "isolated" from its naturally associated components. Molecules can also be rendered substantially free of naturally associated components by isolation using purification techniques well known in the art. Molecular purity or homogeneity can be tested by several means well known in the art. For example, the purity of a polypeptide sample can be tested using polyacrylamide gel electrophoresis and gel staining to visualize the polypeptide using techniques well known in the art. For certain purposes, HPLC or other means well known in the art for purification can be used to provide higher resolution.

[0059] As used herein, the term "isolated," in the context of viruses, refers to viruses that are derived from a single parent virus. Viruses can be isolated using routine methods known to those skilled in the art, including, but not limited to, those based on plaque purification and limiting dilution.

[0060] As used herein, the phrase "multiplicity of infection" or "MOI" is the average number of virus per infected cell. The MOI is determined by dividing the number of virus added (ml added x plaque forming units (PFU)) by the number of cells added (ml added x cells / ml).

[0061] As used herein, "purify" and grammatical variations thereof refer to the complete or partial removal of at least one impurity from a mixture containing a polypeptide and one or more impurities, thereby improving the level of purity of the polypeptide in the composition (i.e., by lowering the amount (ppm) of the impurity(s) in the composition). As used herein, "purified" in the context of a virus refers to a virus that is substantially free of cellular material and culture medium from the cell or tissue source from which the virus is derived. The term "substantially free of cellular material" includes preparations of a virus in which the virus is separated from cellular components of the cells from which it was isolated or recombinantly produced. Thus, a virus that is substantially free of cellular material includes a protein preparation having less than about 30%, 20%, 10%, or 5% (by dry weight) of cellular proteins (also referred to herein as "contaminating proteins"). The virus is also substantially free of culture medium, i.e., the culture medium occupies less than about 20%, 10%, or 5% of the volume of the virus preparation. The virus can be purified using routine methods known to those skilled in the art, including but not limited to chromatography and centrifugation.

[0062] As used herein, "substantially pure" refers to a material that is at least 50% pure (i.e., free from contaminants), more preferably at least 90% pure, more preferably at least 95% pure, even more preferably at least 98% pure, and most preferably at least 99% pure.

[0063] The terms "patient," "subject," or "individual" are used interchangeably herein and refer to a human or non-human animal. These terms include mammals, such as humans, primates, farm animals (including cows, pigs, camels, etc.), companion animals (e.g., dogs, cats, etc.), and rodents (e.g., mice and rats).

[0064] As used herein, the terms "prevent," "preventing," and "prevention" refer to delaying or reducing the recurrence or onset of one or more symptoms of a disease (e.g., a poxvirus infection) in a subject as a result of administration of a therapy (e.g., a prophylactic or therapeutic agent). For example, in the context of administering a therapy to a subject for an infection, "prevent," "preventing," and "prevention" refer to inhibiting or reducing the development or onset of an infection (e.g., a poxvirus infection or a condition associated therewith) in a subject, or preventing the recurrence, onset, or development of one or more symptoms of an infection (e.g., a poxvirus infection or a condition associated therewith), resulting from administration of a therapy (e.g., a prophylactic or therapeutic agent) or combination of therapies (e.g., a combination of prophylactic or therapeutic agents).

[0065] As used herein, the terms "treat," "treating," or "treatment" refer to treating a condition or patient and to taking steps to obtain beneficial or desired results, including clinical results. With respect to infectious diseases (e.g., poxvirus or variola virus infections), treatment refers to eradication or control of replication of an infectious agent (e.g., poxvirus or variola virus), reduction in the number of infectious agents (e.g., reduction in viral titer), reduction or amelioration of the progression, severity, and / or duration of an infection (e.g., poxvirus / smallpox infection or a condition or symptom associated therewith), or amelioration of one or more symptoms, resulting from administration of one or more therapies (including, without limitation, administration of one or more prophylactic or therapeutic agents). With respect to cancer, treatment refers to eradication, removal, alteration, or control of primary, localized, or metastatic cancer tissue, resulting from administration of one or more therapeutic agents of the present disclosure. In certain embodiments, such terms refer to minimizing or slowing the spread of cancer resulting from the administration of one or more therapeutic agents of the present disclosure to a subject with such a disease. In other embodiments, such terms refer to the elimination of pathogenic cells.

[0066] "Administering" or "administration" of a substance, compound, or agent to a subject can be performed using one of a variety of methods known to those skilled in the art. For example, the compound or agent can be administered sublingually or intranasally, by inhalation into the lungs, or rectally. Administering can also be performed, for example, once, multiple times, and / or over one or more extended periods of time. In some embodiments, administering includes both direct administration, including self-administration, and indirect administration, including the act of prescribing a drug. For example, as used herein, a physician who instructs a patient to self-administer a drug or have another administer the drug, and / or who provides a patient with a prescription for the drug, is administering the drug to the patient.

[0067] Each embodiment described herein can be used individually or in combination with any other embodiment described herein.

[0068] overview Poxviruses are large (approximately 200 kbp) DNA viruses that replicate in the cytoplasm of infected cells. The Orthopoxvirus (OPV) genus contains several poxviruses that vary greatly in their ability to infect different hosts. Vaccinia virus (VACV), for example, can infect a wide range of hosts, whereas variola virus (VARV), the causative agent of smallpox, only infects humans. A feature common to many, if not all, poxviruses is their ability to nongenetically "reactivate" within the host. Nongenetic reactivation refers to the process by which cells infected with one poxvirus can promote the recovery of a second, "dead" virus (e.g., one inactivated by heat) that would otherwise be noninfectious.

[0069] Purified poxvirus DNA is not infectious because the viral life cycle requires transcription of early genes via a virally encoded RNA polymerase that is packaged into virions. However, this defect can be overcome if viral DNA is transfected into cells that have previously been infected with a helper poxvirus, providing the necessary factors required to transcribe, replicate, and package the transfected genome in trans (Sam CK, Dumbell KR. Expression of poxvirus DNA in coinfected cells and marker rescue of thermosensitive mutants by subgenomic fragments of DNA. Ann Virol (Inst Past). 1981;132:135-50). this generates mixed viral progeny, but this problem can be overcome by performing the reactivation reaction in a cell line that supports the growth of both viruses and then eliminating the helper virus by plating the virus mixture on cells that do not support the growth of the helper virus (Scheiflinger F, Dorner F, Falkner FG. Construction of chimeric vaccinia viruses by molecular cloning and packaging. Proceedings of the National Academy of Sciences of the United States of America. 1992;89(21):9977-81).

[0070] Previously, Yao and Evans described a method in which high-frequency recombination and replication reactions catalyzed by the leporipoxvirus, Shope fibroma virus (SFV), can be coupled with an SFV-catalyzed reactivation reaction to rapidly assemble recombinant vaccinia strains using multiple overlapping fragments of viral DNA (Yao XD, Evans DH. High-frequency genetic recombination and reactivation of orthopoxviruses from DNA fragments transfected into leporipoxvirus-infected cells. Journal of Virology. 2003;77(13):7281-90). For the first time, the reactivation and characterization of a functional synthetic chimeric vaccinia virus [scVACV] using chemically synthesized overlapping double-stranded DNA fragments is described.

[0071] Synthetic chimeric vaccinia viruses of the present disclosure In one aspect, the present invention provides functional synthetic chimeric vaccinia viruses (scVACVs) that are initially replicated and assembled from chemically synthesized DNA. The viruses that can be produced by the methods of the present disclosure can be any vaccinia virus whose genome has been sequenced or can be largely sequenced, or whose natural isolates are available. Various embodiments of the scVACVs can be based on the genomic sequence of a naturally occurring strain, a variant or mutant, a mutagenized virus, or a genetically engineered virus. In some embodiments, the viral genome of the scVACV contains one or more modifications compared to the wild-type genome or the base genomic sequence of the virus. The modifications can include one or more deletions, insertions, substitutions, or a combination thereof. In one embodiment, the modifications can include the insertion of one or more multiple cloning sites to allow for the insertion of exogenous DNA. It is understood that modifications can be introduced by any number of methods commonly known in the art. The modified portions of the genome can be derived from chemically synthesized DNA, cDNA, or genomic DNA. In another embodiment, the viral genome of the scVACV of the present disclosure contains one or more modifications to add or restore one or more unique restriction sites. Modifications that add or restore one or more restriction sites can be performed at restriction sites that have been eliminated to facilitate clonal selection.

[0072] Chemical genome synthesis is particularly beneficial when a natural template is unavailable for genetic modification, amplification, or replication using conventional molecular biology methods. The genome sequence for wtVACV (NYCBH strain, clone ACAM2000) has been described and published, but it is incomplete. The sequence of the terminal hairpin loop has not been determined, and only four 54-bp repeat sequences have been identified. The presence of 70-bp, 125-bp, and 54-bp tandem repeat sequences was confirmed in a wild-type isolate of VACV ACAM2000 after sequencing, indicating that the currently published sequence of ACAM2000 was incomplete. We have constructed a functional synthetic chimeric VACV (scVACV). Specifically, we successfully constructed a functional scVACV strain, NYCBH, clone ACAM2000, by substituting a terminal hairpin loop based on the wtVACV telomere of a different strain for the VACV's own terminal hairpin loop sequence.In some embodiments, the viral genome of the VACV virus is selected from the group consisting of Western Reserve, clone 3, Tian Tian, ​​Tian Tian clone TP5, Tian Tian clone TP3, NYCBH, NYCBH clone Acambis 2000, Wyeth, Copenhagen, Lister, Lister 107, Lister-LO, Lister GL-ONC1, Lister GL-ONC2, Lister GL-ONC3, Lister GL-ONC4, Lister CTC1, Lister IMG2 (Turbo FP635), IHD-W, LC16m18, Lederle, Tashkent clone TKT3, Tashkent clone TKT4, USSR, Evans, Praha, L-IVP, V-VET1 or LIVP6.1.1, Ikeda, EM-63, Malbran, Duke, 3737, CV-1, Connaught Laboratories, Serro The virus genome is a strain selected from the group consisting of 2, CM-01, NYCBH Dryvax clone DPP13, NYCBH Dryvax clone DPP15, NYCBH Dryvax clone DPP20, NYCBH Dryvax clone DPP17, NYCBH Dryvax clone DPP21, VACV-IOC, chorioallantoic vaccinia virus Ankara (CVA), modified vaccinia Ankara (MVA), and MVA-BN. In a preferred embodiment, the viral genome is based on the NYCBH strain. More preferably, the viral genome is derived from the NYCBH strain, clone Acambis 2000, or ACAM2000. New VACV strains are still being discovered continuously. It is understood that the scVACV of the present disclosure can be based on such newly discovered VACV strains.

[0073] Dryvax® is derived from the New York City Board of Health strain of vaccinia virus (Wyeth Laboratories, Marietta, PA), grown on the skin of calves, and then virtually freeze-dried for storage.

[0074] VACV strain ACAM2000, a live smallpox (vaccinia) vaccine, is a live vaccinia virus derived from plaque-purified cloning from Dryvax®, propagated in African green monkey kidney (Vero) cells, and tested free of adventitious agents (Osborne JD et al. Vaccine. 2007; 25(52):8807-32).

[0075] V-VET1 or LIVP6.1.1 was developed by Genelux. It was isolated from a wild-type stock of the Lister strain of vaccinia virus (Lister strain, Institute of Viral Preparations (LIVP), Moscow, Russia) and represents a "natural" virus (no genetic manipulation has been performed). The thymidine kinase (tk) gene of the LIVP6.1.1 virus is inactive (Shvalov AN et al. Genome Announc. 2016 May-Jun;4(3):e00372-16).

[0076] GLV-1h68 (called GL-ONC1 when produced for clinical investigation) was developed by Genelux from the Lister strain by inserting three expression cassettes encoding Renilla luciferase-Aequorea green fluorescent protein fusion (Ruc-GFP), LacZ, and β-glucuronidase into the F14.5L, J2R (thymidine kinase), and A56R (hemagglutinin) loci of the viral genome, respectively (Zhang Q et al. Cancer Res. 2007;67(20):10038-46.).

[0077] Chemical viral genome synthesis also opens the possibility of introducing numerous beneficial modifications into the resulting genome or into specific portions thereof. Modifications can improve the ease of cloning to generate viruses, provide sites for the introduction of recombinant gene products, improve the ease of identifying reactivated viral clones, and / or confer a plethora of other beneficial characteristics (e.g., introducing desired antigens, generating oncolytic viruses, etc.). In some embodiments, modifications can include attenuation or deletion of one or more virulence factors. In some embodiments, modifications can include the addition or insertion of one or more virulence-regulating genes or gene-encoded regulatory factors.

[0078] Traditionally, terminal hairpins of poxviruses have been difficult to clone and sequence, so it is not surprising that some published genome sequences (e.g., VACV, ACAM2000, and HPXV MNR-76) are incomplete. Specifically, the genome sequences of wtVACV, strain NYCBH, and clone ACAM2000 have been described and published, but they are incomplete. The sequence of the terminal hairpin loop has not been determined, and only four 54-bp repeat sequences have been identified. Because the published sequences of the wtVACV strain NYCBH and clone ACAM2000 genomes are incomplete, the hairpins cannot be replicated accurately, and prior to the present application, it was unknown whether VACV could be replicated and assembled from polynucleotides based solely on the known portions of the wtVACV genome. It was also unknown whether hairpins from one strain of virus would be functional in another strain. The present inventors created a functional synthetic chimeric VACV (scVACV) ACAM2000 by substituting terminal hairpin loops based on the wtVACV telomere of a different strain for VACV's own terminal hairpin loop sequence. In an exemplary embodiment, a ssDNA fragment was chemically synthesized using the published sequence of the VACV WR strain telomere as a guide and ligated onto a dsDNA fragment containing the left and right ends of the VACV strain NYCBH. In some embodiments, the terminal hairpins are based on the terminal hairpins of any VACV strain whose genome has been completely sequenced or whose natural isolate is available for genome sequencing.In some embodiments, the terminal hairpin loop is selected from the group consisting of Western Reserve, clone 3, Tian Tian, ​​Tian Tian clone TP5, Tian Tian clone TP3, NYCBH, NYCBH clone Acambis 2000, Wyeth, Copenhagen, Lister, Lister 107, Lister-LO, Lister GL-ONC1, Lister GL-ONC2, Lister GL-ONC3, Lister GL-ONC4, Lister CTC1, Lister IMG2 (Turbo FP635), IHD-W, LC16m18, Lederle, Tashkent clone TKT3, Tashkent clone TKT4, USSR, Evans, Praha, L-IVP, V-VET1 or LIVP6.1.1, Ikeda, EM-63, Malbran, Duke, 3737, CV-1, Connaught Laboratories, Serro 2, CM-01, NYCBH Dryvax clone DPP13, NYCBH Dryvax clone DPP15, NYCBH Dryvax clone DPP20, NYCBH Dryvax clone DPP17, NYCBH. The scVACV of the present disclosure is based on a strain selected from the group consisting of Dryvax clone DPP21, VACV-IOC, chorioallantoic vaccinia virus Ankara (CVA), modified vaccinia Ankara (MVA), and MVA-BN. In a preferred embodiment, the terminal hairpin loop is based on the Western Reserve strain of VACV (WR strain). New VACV strains are still being discovered continuously. It is understood that the scVACV of the present disclosure can be based on such newly discovered VACV strains.

[0079] In another embodiment, the viral genome of a scVACV of the present disclosure comprises homologous or heterologous terminal hairpin loops and tandem repeat regions (70 bp, 125 bp, and 54 bp tandem repeats) located downstream of the hairpin loops, wherein the tandem repeat region contains a different number of repeat sequences from that of wtVACV (i.e., a naturally occurring virus). The numbers of repeat sequences of the 70 bp, 125 bp, and 54 bp tandem repeats found in the VACV virus strain WR were 22, 2, and 8, respectively. In another embodiment, the number of tandem repeat regions varies among different poxviruses, different vaccinia viruses, and different vaccinia virus strains. The term homologous terminal hairpin loops means that the terminal hairpin loops are derived from the same virus species / strain, and the term heterologous terminal hairpin loops means that the terminal hairpin loops are derived from different virus species / strains.

[0080] In some embodiments, the modification can include the deletion of one or more restriction sites. In some embodiments, the modification can include the introduction of one or more restriction sites. In some embodiments, the restriction sites deleted from or added to the genome include, but are not limited to, AanI, AarI, AasI, AatI, AatII, AbaSI, AbsI, Acc65I, AccI, AccII, AccIII, AciI, AclI, AcuI, AfeI, AflII, AflIII, AgeI, AhdI, AleI, AluI, AlwI, AlwNI, ApaI, ApaLI, ApeKI, ApoI, AscI, AseI, AsiSI, AvaI, AvaII, AvrII, Bae GI, BaeI, BamHI, BanI, BanII, BbsI, BbvCI, BbvI, BccI, BceAI, BcgI, BciVI, BclI, BcoDI, BfaI, BfuAI, BfuCI, BglI, BglII, BlpI, BmgBI, Bm rI, BmtI, BpmI, Bpu10I, BpuEI, BsaAI, BsaBI, BsaHI, BsaI, BsaJI, BsaWI, BsaXI, BseRI, BseYI, BsgI, BsiEI, BsiHKAI, BsiWI, BslI, BsmAI, B smBI, BsmFI, BsmI, BsoBI, Bsp1286I, BspCNI, BspDI, BspEI, BspHI, BspMI, BspQI, BsrBI, BsrDI, BsrFαI, BsrGI, BsrI, BssHII, BssSαI, Bst API, BstBI, BstEII, BstNI, BstUI, BstXI, BstYI, BstZ17I, Bsu36I, BtgI, BtgZI, BtsαI, BtsCI, BtsIMutI, Cac8I, ClaI, CspCI, CviAII, CviK I-1, CviQI, DdeI, DpnI, DpnII, DraI, DrdI, EaeI, EagI, EarI, EciI, Eco53kI, EcoNI, EcoO109I, EcoP15I, EcoRI, EcoRV, FatI, FauI, Fnu4HI , FokI, FseI, FspEI, FspI, HaeII, HaeIII, HgaI, HhaI, HincII, HindIII, HinfI, HinP1I, HpaI, HpaII, HphI, Hpy166II, Hpy188I, Hpy188III,Hpy99I, HpyAV, HpyCH4III, HpyCH4IV, HpyCH4V, I-CeuI, I-SceI, KasI, KpnI, LpnPI, MboI, MboII, MfeI, MluCI, MluI, MlyI, MmeI, MnlI, MscI, MseI, MslI, MspA1I, MspI, MspJI, MwoI, NaeI, NarI, NciI, NcoI, NdeI, NgoMIV, NheI, NlaIII, NlaIV, NmeAIII, NotI, NruI, NsiI, NspI, PacI, PaeR7I, PciI, PflFI, PflMI, PleI, PluTI, PmeI, PmlI, PpuMI, PshAI, Restriction sites may be selected from one or more of: PsiI, PspGI, PspOMI, PspXI, PstI, PvuI, PvuII, RsaI, RsrII, SacI, SacII, SalI, SapI, Sau3AI, Sau96I, SbfI, ScrFI, SexAI, SfaNI, SfcI, SfiI, SfoI, SgrAI, SmaI, SmlI, SnaBI, SpeI, SphI, SrfI, SspI, StuI, StyD4I, StyI, Swal, TaqαI, TfiI, TseI, Tsp45I, TspMI, TspRI, Tth111I, XbaI, XcmI, XhoI, XmaI, XmnI or ZraI. It is understood that any desired restriction site(s) or combination of restriction sites can be inserted or mutated into and / or eliminated from the genome. In some embodiments, one or more AarI sites are deleted from the viral genome. In some embodiments, one or more BsaI sites are deleted from the viral genome. In some embodiments, one or more restriction sites are completely eliminated from the genome (e.g., all AarI sites in the viral genome can be eliminated). In some embodiments, one or more AvaI restriction sites are introduced into the viral genome. In some embodiments, one or more StuI sites are introduced into the viral genome. In some embodiments, the one or more modifications can include the integration of a recombination target, including, but not limited to, a loxP or FRT site.

[0081] In some embodiments, the modification may include a fluorescent marker, such as, but not limited to, a green fluorescent protein (GFP), enhanced GFP, yellow fluorescent protein (YFP), cyan / blue fluorescent protein (BFP), red fluorescent protein (RFP) or variants thereof; a selection marker, such as, but not limited to, a drug resistance marker (e.g., E. coli xanthine-guanine phosphoribosyltransferase gene (gpt), Streptomyces alboniger puromycin acetyltransferase gene (pac), neomycin phosphotransferase I gene (nptI), neomycin phosphotransferase gene II (nptII), hygromycin phosphotransferase (hpt), sh ble gene, etc.; protein or peptide tags, for example, but not limited to, MBP (maltose binding protein), CBD (cellulose binding domain), GST (glutathione-S-transferase), poly(His), FLAG, V5, c-Myc, HA (hemagglutinin), NE-tag, CAT (chloramphenicol acetyltransferase), DHFR (dihydrofolate reductase), HSV (herpes simplex virus), VSV-G (vesicular stomatitis virus glycoprotein), luciferase, protein A, protein G, streptavidin, T7, thioredoxin, yeast two-hybrid tag, e.g., B The modifications may include the introduction of a selection tag, such as a marker such as 42, GAL4, LexA, or VP16; a localization tag, such as an NLS-tag, a SNAP-tag, a Myr-tag, etc. It is understood that other selection markers and / or tags known in the art can be used. In some embodiments, the modifications include one or more selection markers (e.g., a fluorescent marker such as YFP, a drug selection marker such as gpt, etc.) to aid in the selection of reactivated viral clones. In some embodiments, the one or more selection markers are deleted from the reactivated clones after the selection step.

[0082] In one aspect, the scVACV of the present invention can be used as a vaccine to protect against pathogenic poxvirus infections (e.g., VARV, MPXV, MCV, ORFV, Ausdyk virus, BPSV, sealpox virus, etc.), as a therapeutic agent to treat or prevent pathogenic poxvirus infections (e.g., VARV, MPXV, MCV, ORFV, Ausdyk virus, BPSV, sealpox virus, etc.), as a vehicle for heterologous gene expression, or as an oncolytic agent. In some embodiments, the scVACV can be used as a vaccine to protect against VARV infection. In some embodiments, the scVACV can be used to treat or prevent VARV infection.

[0083] Method for producing synthetic chimeric VACV In one aspect, the present invention provides systems and methods for synthesizing, reactivating, and isolating functional synthetic chimeric VACV (scVACV) from chemically synthesized overlapping double-stranded DNA fragments of the viral genome. Recombination of the overlapping DNA fragments of the viral genome and reactivation of the functional scVACV are carried out in cells previously infected with a helper virus. Briefly, overlapping DNA fragments encompassing all or substantially all of the viral genome of the scVACV are chemically synthesized and transfected into helper virus-infected cells. The transfected cells are cultured to generate mixed viral progeny containing the helper virus and the reactivated scVACV. The mixed viral progeny are then plated onto host cells that do not support helper virus growth but allow the growth of the synthetic chimeric vaccinia virus to eliminate the helper virus and recover the synthetic chimeric vaccinia virus. In some embodiments, the helper virus does not infect the host cells. In some embodiments, the helper virus can infect host cells but does not grow well in them. In some embodiments, the helper virus grows more slowly in the host cell compared to the scVACV.

[0084] In some embodiments, substantially all of the synthetic chimeric vaccinia virus genome is derived from chemically synthesized DNA. In some embodiments, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, more than 99%, or 100% of the synthetic chimeric vaccinia virus genome is derived from chemically synthesized DNA. In some embodiments, the vaccinia virus genome is derived from a combination of chemically synthesized DNA and naturally occurring DNA. In some embodiments, all of the fragments comprising the vaccinia virus genome are chemically synthesized. In some embodiments, one or more of the fragments are chemically synthesized, and one or more of the fragments are derived from naturally occurring DNA (e.g., by PCR amplification or by established recombinant DNA techniques).

[0085] The number of overlapping DNA fragments used in the disclosed methods depends on the size of the vaccinia virus genome. Practical considerations, such as, on the one hand, decreased recombination efficiency with increasing numbers of fragments, and, on the other hand, the difficulty in synthesizing very large DNA fragments with decreasing numbers of fragments, also inform the number of overlapping fragments used in the disclosed methods. In some embodiments, a synthetic chimeric vaccinia virus genome can be synthesized as a single fragment. In some embodiments, a synthetic chimeric vaccinia virus genome is assembled from 2 to 14 overlapping DNA fragments. In some embodiments, a synthetic chimeric vaccinia virus genome is assembled from 4 to 12 overlapping DNA fragments. In some embodiments, a synthetic chimeric vaccinia virus genome is assembled from 6 to 12 overlapping DNA fragments. In some embodiments, a synthetic chimeric vaccinia virus genome is assembled from 8 to 11 overlapping DNA fragments. In some embodiments, a synthetic chimeric vaccinia virus genome is assembled from 8 to 10, 10 to 12, or 10 to 14 overlapping DNA fragments. In some embodiments, the synthetic chimeric vaccinia virus genome is assembled from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 overlapping DNA fragments. In a preferred embodiment, the synthetic chimeric vaccinia virus genome is assembled from 9 overlapping DNA fragments. In an exemplary embodiment of the present disclosure, a synthetic vaccinia virus (scVACV) is reactivated from 9 chemically synthesized overlapping double-stranded DNA fragments. In some embodiments, terminal hairpin loops are synthesized separately and ligated onto fragments comprising the left and right ends of the vaccinia virus genome. In some embodiments, the terminal hairpin loops can be derived from naturally occurring templates. In some embodiments, the terminal hairpin of the scVACV is derived from wtVACV. In some embodiments, the terminal hairpin is derived from a wtVACV terminal hairpin of a different strain instead of the VACV's own terminal hairpin loop sequence.In some embodiments, the terminal hairpin is based on the terminal hairpin of any wtVACV whose genome has been completely sequenced or whose natural isolate is available for genome sequencing.

[0086] The size of the overlapping fragments used in various aspects of the methods of the present invention depends on the size of the vaccinia virus genome. It is understood that there can be wide variation in fragment size, and various practical considerations, such as the ability to chemically synthesize very large DNA fragments, will inform the selection of fragment size. In some embodiments, the fragments are within the size range of about 2,000 bp to about 50,000 bp. In some embodiments, the fragments are within the size range of about 3,000 bp to about 45,000 bp. In some embodiments, the fragments are within the size range of about 4,000 bp to 40,000 bp. In some embodiments, the fragments are within the size range of about 5,000 bp to 35,000 bp. In some embodiments, the largest fragments are about 18,000bp, 20,000bp, 21,000bp, 22,000bp, 23,000bp, 24,000bp, 25,000bp, 26,000bp, 27,000bp, 28,000bp, 29,000bp, 30,000bp, 31,000bp, 32,000bp, 33,000bp, 34 In an exemplary embodiment of the present disclosure, scVACV is reactivated from nine chemically synthesized overlapping double-stranded DNA fragments ranging in size from about 10,000 bp to about 32,000 bp (Table 1).

[0087] The helper virus can be any poxvirus capable of providing the trans-acting enzymatic machinery necessary to reactivate the poxvirus from the transfected DNA. The helper virus can have a different or narrower host cell range than the scVACV to be generated (e.g., Shope fibroma virus (SFV) has a very narrow host range compared to orthopoxviruses such as vaccinia virus (VACV) or HPXV). The helper virus can have a different plaque phenotype compared to the scVACV to be generated. In some embodiments, the helper virus is a leporipoxvirus. In some embodiments, the leporipoxvirus is SFV, tularensis fibroma virus, rabbit fibroma virus, squirrel fibroma virus, or myxoma virus. In a preferred embodiment, the helper virus is SFV. In some embodiments, the helper virus is an orthopoxvirus. In some embodiments, the Orthopoxvirus is Camelpox virus (CMLV), Cowpox virus (CPXV), Ectromelia virus (ECTV, "Mousepox agent"), HPXV, Monkeypox virus (MPXV), Rabbitpox virus (RPXV), Raccoonpox virus, Skunkpox virus, Gerbilpox virus, Uasin Gishū disease virus, VACV, and Volepox virus (VPV). In some embodiments, the helper virus is Avipoxvirus, Capripoxvirus, Cervidpoxvirus, Crocodylipoxvirus, Molluscipoxvirus, Parapoxvirus, Suipoxvirus, or Yatapoxvirus. In some embodiments, the helper virus is Fowlpoxvirus. In some embodiments, the helper virus is Alphaentomopoxvirus, Betaentomopoxvirus, or Gammaentomopoxvirus. In some embodiments, the helper virus is a psoralen-inactivated helper virus. In an exemplary embodiment of the present disclosure, scVACV is reactivated from overlapping DNA fragments that are transfected into SFV-infected BGMK cells.SFV is then eliminated by plating the mixed virus progeny onto BSC-40 cells.

[0088] The skilled artisan will understand that the appropriate host cells used for reactivation of scVACV, and the selection and / or isolation of scVACV, will depend on the particular combination of helper virus and chimeric poxvirus generated by various aspects of the disclosed methods. Any host cells that support the growth of both the helper virus and scVACV can be used for the reactivation step, and any host cells that do not support the growth of the helper virus can be used to eliminate the helper virus and select and / or isolate the scVACV. In some embodiments, the helper virus is a leporipoxvirus, and the host cells used for the reactivation step can be selected from rabbit kidney cells (e.g., LLC-RK1, RK13, etc.), rabbit lung cells (e.g., R9ab), rabbit skin cells (e.g., SF1Ep, DRS, RAB-9), rabbit corneal cells (e.g., SIRC), rabbit carcinoma cells (e.g., Oc4T / cc), rabbit skin / carcinoma cells (e.g., CTPS), monkey cells (e.g., Vero, BGMK, etc.), or hamster cells (e.g., BHK-21, etc.). In a preferred embodiment, the host cell is a BGMK cell.

[0089] In some embodiments, scVACV can be grown in any culture medium that allows the virus to grow to a titer that allows for the use of scVACV as described herein. In one embodiment, the culture medium allows the scVACV to grow to a titer equivalent to that determined for the corresponding wild-type virus. In some embodiments, scVACV can be grown in cells that are susceptible to infection by VACV (e.g., avian cells, bat cells, bovine cells, camel cells, canary cells, feline cells, deer cells, equine cells, chicken cells, gerbil cells, goat cells, human cells, monkey cells, porcine cells, rabbit cells, raccoon cells, seal cells, sheep cells, skunk cells, vole cells, etc.). Such methods are well known to those of skill in the art. Representative mammalian cells include, but are not limited to, BHK, BGMK, BRL3A, BSC-40, CEF, CEK, CHO, COS, CVI, HaCaT, HEL, HeLa cells, HEK293, human osteosarcoma cell line 143B, MDCK, NIH / 3T3, and Vero cells. For virus isolation, scVACV is generally removed from the cell culture and separated from cellular components by well-known purification procedures, such as density gradient centrifugation and column chromatography, and can be further purified, if desired, using procedures well known to those skilled in the art, such as plaque assays.

[0090] In another aspect of the invention, a method for producing a synthetic chimeric vaccinia virus (scVACV) comprises the steps of (i) chemically synthesizing overlapping DNA fragments corresponding to substantially all of the viral genome of a vaccinia virus and chemically synthesizing a terminal hairpin loop from another strain of vaccinia virus; (ii) transfecting the overlapping DNA fragments into helper virus-infected cells; (iii) culturing the cells to produce a mixture of helper virus and synthetic chimeric vaccinia virus particles in the cells; and (iv) plating the mixture on host cells specific for the scVACV to recover the scVACV. In some embodiments, the scVACV of the method is derived from the NYCBH strain, clone Acambis2000, and the terminal hairpin loop is derived from the Western Reserve strain of vaccinia virus.

[0091] Polynucleotides of the present disclosure In one aspect, the present invention provides polynucleotides (e.g., double-stranded DNA fragments) for producing functional synthetic chimeric poxviruses (scVACVs). In some embodiments, the present invention provides methods for producing functional scVACVs from synthetic DNA (e.g., chemically synthesized DNA, PCR-amplified DNA, engineered DNA, polynucleotides containing nucleoside analogs, etc.). In some embodiments, the present invention provides methods for producing functional scVACVs from chemically synthesized overlapping double-stranded DNA fragments of the viral genome. Polynucleotides of various aspects of the present invention can be designed based on published genome sequences. If natural isolates of vaccinia virus are readily available, the viral genome can be sequenced before selecting and designing polynucleotides of the present disclosure. Alternatively, if a partial DNA sequence of vaccinia virus is available, for example, from a clinical isolate from material associated with an infected individual, from a forensic sample, or from PCR-amplified DNA, the partial viral genome can be sequenced before selecting and designing polynucleotides of the present disclosure. In one aspect, the scVACV of the present invention, and therefore the polynucleotides of the present disclosure, can be based on the genomic sequence of a naturally occurring strain, a variant or mutant, a mutagenized virus, or a genetically engineered virus.

[0092] In one aspect, the present invention provides an isolated polynucleotide comprising a nucleotide sequence that is at least 90% identical (e.g., at least 91%, 92%, 93%, or 94% identical), at least 95% identical (e.g., at least 96%, 97%, 98%, or 99% identical), or 100% identical to all or a portion of a reference VACV genomic sequence or its complement. The isolated polynucleotides of the present disclosure can comprise at least 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, 20000, 25000, 30000, 35000, 40000, 45000 bp or more of contiguous or non-contiguous nucleotides of a reference polynucleotide molecule (e.g., a reference VACV genome or fragment thereof). One of skill in the art will understand that nucleic acid sequences complementary to the nucleic acids and variants of the nucleic acids are also within the scope of the present application. In further embodiments, the nucleic acid sequences of the present disclosure can be isolated, recombinant, and / or fused to heterologous nucleotide sequences, or can be in a DNA library.

[0093] In some embodiments, the invention provides polynucleotides for producing a scVACV, wherein the VACV is selected from the following strains: Western Reserve, clone 3, Tian Tian, ​​Tian Tian clone TP5, Tian Tian clone TP3, NYCBH, NYCBH clone Acambis 2000, Wyeth, Copenhagen, Lister, Lister 107, Lister-LO, Lister GL-ONC1, Lister GL-ONC2, Lister GL-ONC3, Lister GL-ONC4, Lister CTC1, Lister IMG2 (Turbo FP635), IHD-W, LC16m18, Lederle, Tashkent clone TKT3, Tashkent clone TKT4, USSR, Evans, Praha, L-IVP, V-VET1 or LIVP6.1.1, Ikeda, EM-63, Malbran, Duke, 3737, CV-1, Connaught Laboratories, Serro 2, CM-01, NYCBH Dryvax Clone DPP13, NYCBH Dryvax Clone DPP15, NYCBH Dryvax Clone DPP20, NYCBH Dryvax Clone DPP17, NYCBH Dryvax clone DPP21, VACV-IOC, chorioallantoic vaccinia virus Ankara (CVA), modified vaccinia Ankara (MVA) and MVA-BN. In a preferred embodiment, the scVACV is derived from the NYCBH strain clone Acambis 2000 or ACAM2000.

[0094] In one aspect, the present invention provides polynucleotides for producing synthetic chimeric vaccinia viruses (scVACVs). In a specific embodiment, the scVACV genome can be based on the published genome sequence described for VACV strain NYCBH clone ACAM2000 (GenBank accession AY313847; Osborne JD et al. Vaccine. 2007; 25(52):8807-32). In various aspects of the invention, the methods of the present disclosure can be used to produce scVACVs. It has been shown that terminal hairpin loops from vaccinia virus (VACV) strain WR can be ligated onto the ends of the VACV genome strain NYCBH clone ACAM2000 to produce functional scVACV particles. In some embodiments, terminal hairpin loops from vaccinia virus (VACV) strain ACAM2000 can be ligated onto the ends of the VACV genome strain NYCBH clone ACAM2000 to produce functional scVACV particles using the methods of the present disclosure. The scVACV genome can be divided into nine overlapping fragments, as described in the Examples of the present disclosure and as shown in Table 1. In some embodiments, the VACV genome can be divided into 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 overlapping fragments. In some embodiments, the entire genome can be provided as a single fragment. Fragment sizes are shown in Table 1. In some embodiments, the VACV genome can be divided into 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 overlapping fragments. In some embodiments, the entire genome can be provided as a single fragment. Fragment sizes are shown in Table 1. Polynucleotides of various aspects of the present invention comprise nucleic acid sequences that are at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 1-9. In some embodiments, isolated polynucleotides of the present invention include variants of these sequences, where such variants can include missense mutations, nonsense mutations, duplications, deletions, and / or additions. SEQ ID NOs: 13 and 14 represent the nucleotide sequences of the VACV (WR strain) terminal hairpin loop. SEQ ID NO:19 and SEQ ID NO:20 represent the nucleotide sequences of the VACV (ACAM2000 strain) terminal hairpin loop. In some embodiments, the terminal hairpin loop comprises a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:13 or SEQ ID NO:14.In some embodiments, the terminal hairpin loop comprises a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:19 or SEQ ID NO:20.

[0095] In other embodiments, the scVACV genome is from Western Reserve (Genbank accession NC006998; Genbank accession AY243312), CL3 (Genbank accession AY313848), Tian Tian (Genbank accession AF095689.1), Tian Tian clone TP5 (JX489136), TP3 (Genbank accession KC207810) and TP5 (Genbank accession KC207811), NYCBH, Wyeth, Copenhagen (Genbank accession M35027), NYCBH clone Acambis 2000 (Genbank accession AY313847), Lister 107 (Genbank accession DQ121394), Lister-LO (Genbank accession AY678276), modified vaccinia virus Ankara (MVA) (Genbank accession U94848; Genbank accession AY603355), MVA-BN (Genbank accession DQ983238), Lederle, Tashkent clone TKT3 (Genbank accession KM044309), and TKT4 (Genbank accession KM044309). 4310), USSR, Evans, Praha, LIVP, Ikeda, IHD-W (Genbank accession KJ125439), LC16m8 (AY678275), EM-63, IC, Malbran, Duke (Genbank accession DQ439815), 3737 (Genbank accession DQ377945), VACV-IOC (Genbank accessions KT184690 and KT184691), CV-1, Connaught Laboratories, based on VACV strains selected from CVA (Genbank accession AM501482), Serro 2 virus (Genbank accession KF179385), Cantaglo virus isolate CM-01 (Genbank accession KT013210), and Dryvax clones DPP15 (Genbank accession JN654981), DPP20 (Genbank accession JN654985), DPP13 (Genbank accession JN654980), DPP17 (Genbank accession JN654983), and DPP21 (Genbank accession JN654986).

[0096] In one aspect, the present invention provides an isolated polynucleotide comprising a nucleotide sequence that is at least 90% identical (e.g., at least 91%, 92%, 93%, or 94% identical), at least 95% identical (e.g., at least 96%, 97%, 98%, or 99% identical), or 100% identical to all or a portion of a reference wtVACV genomic sequence. In some embodiments, the isolated polynucleotides of the present disclosure comprise variants of the reference sequence, where such variants include missense mutations, nonsense mutations, duplications, deletions, and / or additions. In some embodiments, an isolated polynucleotide of the present invention can comprise at least 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, 20000, 25000, 30000, 35000, 40000, 45000 bp or more of contiguous or non-contiguous nucleotides of a reference polynucleotide molecule (e.g., a reference wtVACV genome).

[0097] Polynucleotides complementary to any of the polynucleotide sequences disclosed herein are also encompassed by the present application. Polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be DNA (genomic or synthetic) or RNA molecules. RNA molecules include mRNA molecules. Additional coding or non-coding sequences can, but need not, be present in the polynucleotides of the present disclosure, and polynucleotides can, but need not, be linked to other molecules and / or supporting materials.

[0098] Two polynucleotide or polypeptide sequences are said to be "identical" if the sequence of nucleotides or amino acids in the two sequences is the same when aligned for maximum correspondence as described below. Comparison between two sequences is typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. As used herein, a "comparison window" refers to a segment of at least about 20, usually 30 to about 75, or 40 to about 50, contiguous positions, in which a sequence can be compared to a reference sequence over the same number of contiguous positions after optimally aligning the two sequences. Additionally, or alternatively, a polynucleotide or variant may be substantially homologous to the polynucleotides provided herein. Such polynucleotide variants are capable of hybridizing to the polynucleotides (or their complements) of the present disclosure under moderately stringent conditions.

[0099] Suitable "moderately stringent conditions" include a prewash in a solution of 5x SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); overnight hybridization at 50-65°C in 5x SSC; followed by two washes for 20 minutes each in 2x, 0.5x, and 0.2x SSC containing 0.1% SDS at 65°C.

[0100] As used herein, "highly stringent conditions," or "high stringency conditions," include (1) those using low ionic strength and high temperature for washing, e.g., 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate at 50°C; (2) those using a denaturing agent, e.g., formamide, e.g., 50 (v / v)% formamide with 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 750 mM sodium chloride, 75 mM sodium citrate, 50 mM sodium phosphate buffer, pH 6.5, at 42°C during hybridization; or (3) those using 50% formamide, 5x SSC (0.75M NaCl, 0.075 M sodium citrate, 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5x Denhardt's solution, sonicated salmon sperm DNA (50 µg / ml), 0.1% SDS, and 10% dextran sulfate at 42°C, followed by washes with 0.2x SSC (sodium chloride / sodium citrate) at 42°C and 50% formamide at 55°C, followed by a high stringency wash consisting of 0.1x SSC containing EDTA at 55°C. Those skilled in the art will recognize how to adjust temperature, ionic strength, etc. as necessary to accommodate factors such as probe length.

[0101] The polynucleotide of this disclosure can be obtained by chemical synthesis, recombinant method or PCR.The method of chemical synthesis of polynucleotide is well known in the art and does not need to be described in detail herein.Those skilled in the art can use the sequences provided herein and commercially available DNA synthesizers to prepare desired DNA sequences.

[0102] To prepare a polynucleotide using recombinant methods, a polynucleotide containing the desired sequence can be inserted into a suitable vector, which can then be introduced into a suitable host cell for replication and amplification, as further discussed herein. Polynucleotides can be inserted into host cells by any means known in the art. Cells are transformed by introducing an exogenous polynucleotide by direct uptake, endocytosis, transfection, F-mating, or electroporation. Once introduced, the exogenous polynucleotide can be maintained in the cell as a non-integrated vector (such as a plasmid) or can be integrated into the host cell genome. Such amplified polynucleotide can be isolated from the host cell by methods well known in the art. See, for example, Sambrook et al., 1989.

[0103] Instead, PCR allows for the reproduction of DNA sequences. PCR technology is well known in the art and is described in U.S. Patent Nos. 4,683,195, 4,800,159, 4,754,065 and 4,683,202, and in PCR: The Polymerase Chain Reaction, Mullis et al. eds., Birkauswer Press, Boston, 1994.

[0104] RNA can be obtained by using the isolated DNA in an appropriate vector and inserting it into a suitable host cell. When the cell replicates and the DNA is transcribed into RNA, the RNA can then be isolated using methods well known to those skilled in the art, for example, as set forth in Sambrook et al., 1989, supra.

[0105] In other embodiments, the nucleic acids of the present invention also include nucleotide sequences that hybridize to the nucleotide sequences set forth in SEQ ID NOS: 1-9 under highly stringent conditions or sequences complementary thereto. Those skilled in the art will readily appreciate that appropriate stringency conditions promoting DNA hybridization can vary. For example, hybridization can be performed in 6.0x sodium chloride / sodium citrate (SSC) at approximately 45°C, followed by a wash in 2.0x SSC at 50°C. For example, the salt concentration in the wash step can be selected from low stringency conditions of approximately 2.0x SSC at 50°C to high stringency conditions of approximately 0.2x SSC at 50°C. Furthermore, the temperature in the wash step can be increased from low stringency conditions at room temperature of approximately 22°C to high stringency conditions at approximately 65°C. Both temperature and salt can be varied, or temperature or salt concentration can be held constant while the other variable is varied. In one embodiment, the invention provides nucleic acids that hybridize under low stringency conditions of 6×SSC at room temperature, followed by a wash at 2×SSC at room temperature.

[0106] Isolated nucleic acids that differ due to the degeneracy of the genetic code are also within the scope of some embodiments of the present invention. For example, some amino acids are specified by more than one triplet. Codons or synonyms that specify the same amino acid (e.g., CAU and CAC are synonyms for histidine) can result in "silent" mutations that do not affect the amino acid sequence of the protein. Those skilled in the art will understand that these variations in one or more nucleotides (up to about 3-5% of the nucleotides) of a nucleic acid encoding a particular protein can exist between members of a given species due to natural allelic variation. All such nucleotide variations and resulting amino acid polymorphisms are within the scope of this application.

[0107] An aspect of the present invention further provides recombinant cloning and expression vectors useful for cloning the polynucleotides of the present disclosure. An aspect of the present invention further provides transformed host cells containing the polynucleotide molecules or recombinant vectors, and novel strains or cell lines derived therefrom.

[0108] The host cell may be a bacterial cell, a yeast cell, a filamentous fungal cell, an algae cell, an insect cell, or a mammalian cell. In some embodiments, the host cell is E. coli. A variety of different vectors have been developed for specific use in each of these host cells, including phages, high copy number plasmids, low copy number plasmids, and shuttle vectors, among others, any of which can be used to practice the present disclosure.

[0109] Suitable cloning vectors can be constructed by standard techniques or selected from a large number of cloning vectors available in the art. While the cloning vector of choice may vary depending on the host cell intended for use, useful cloning vectors will generally be capable of autonomous replication, may have a single target for a specific restriction endonuclease, and / or may have a marker gene that can be used in the selection of clones containing the vector. Suitable examples include plasmids and bacterial viruses, e.g., pBAD18, pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA, and shuttle vectors such as pSA3 and pAT28. These and many other cloning vectors are available from commercial vendors, e.g., BioRad, Stratagene, and Invitrogen.

[0110] To facilitate selection of host cells transformed or transfected with the cloning vectors of the present disclosure, the vectors can be engineered to further include coding sequences for reporter gene products or other selectable markers. Such coding sequences are preferably operably linked to regulatory element coding sequences, as described above. Reporter genes useful in some embodiments of the present invention are well known in the art and include those encoding green fluorescent protein, luciferase, xylE, and tyrosinase, among others. Nucleotide sequences encoding selectable markers are well known in the art and include those encoding gene products that confer antibiotic or antimetabolite resistance or that supply auxotrophic requirements. Examples of such sequences include those encoding resistance to ampicillin, erythromycin, thiostrepton, or kanamycin, among others.

[0111] A vector containing a polynucleotide of interest and / or the polynucleotide itself can be introduced into a host cell by any of several suitable means, including electroporation, transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; particle bombardment; lipofection; and infection (e.g., when the vector is an infectious agent such as vaccinia virus). The choice of introduction vector or polynucleotide often depends on the characteristics of the host cell.

[0112] One aspect of the present invention further provides transformed host cells containing the polynucleotide molecules or recombinant vectors, and novel strains or cell lines derived therefrom. In some embodiments, host cells useful in practicing the present invention are E. coli cells. E. coli strains such as E. coli TOP10, E. coli BL21(DE3), DH5α, etc., available from the American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, Va. 20110, USA, and commercial sources, can be commonly used. In some embodiments, other prokaryotic or eukaryotic cells can be used. In some embodiments, the host cell is a member of a genus selected from Clostridium, Zymomonas, Escherichia, Salmonella, Serratia, Erwinia, Klebsiella, Shigella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Schizosaccharomyces, Kluyveromyces, Yarrowia, Pichia, Candida, Pichia, or Saccharomyces. Such transformed host cells generally include, but are not limited to, microorganisms, such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA vectors, or yeast transformed with recombinant vectors, among others. Preferred eukaryotic host cells include yeast cells, although mammalian or insect cells can also be effectively utilized. Suitable host cells include prokaryotes (for example, E. coli, B. subtillis, S. lividans or C. glutamicum) and yeasts (for example, S. cerevisae, S. pombe, P. pastoris or K. lactis).

[0113] In one embodiment, the present invention also includes the genome of scVACV, recombinants thereof, or functional portions thereof. A functional portion of a viral genome can be a portion of the genome encoding a protein or portion thereof (e.g., a domain, epitope, etc.), a portion comprising a regulatory element or a component of a regulatory element, such as a promoter, enhancer, cis- or trans-acting element, etc. Such viral sequences can be used to identify or isolate the virus or recombinants thereof, for example, using PCR, hybridization techniques, or by establishing an ELISA assay.

[0114] Pharmaceutical Compositions of the Present Disclosure In one aspect, the present invention relates to a pharmaceutical composition comprising a scVACV of the present disclosure and a pharmaceutically acceptable carrier.

[0115] The term "pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a pharmaceutical composition (e.g., an immunogenic or vaccine formulation) is administered. Saline and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. The formulation should suit the mode of administration.

[0116] In some embodiments, the pharmaceutical compositions of the present invention can be administered by standard routes of administration. Many methods can be used to introduce the formulation into a subject, including, but not limited to, intranasal, intratracheal, oral, intradermal, intramuscular, intraperitoneal, intravenous, conjunctival, and subcutaneous routes.

[0117] Exemplary Uses Prevention or treatment of pathogenic poxvirus infections In some embodiments, the synthetic chimeric vaccinia viruses (scVACVs) of the present invention can be used in immunization or to induce or boost a subject's immune response against a pathogenic poxvirus infection. In another embodiment, the scVACVs can be used to induce or boost an immune response against a vaccinia virus. In another embodiment, the scVACVs can be used to induce or boost an immune response against a variola virus. In another embodiment, the scVACVs can be used to induce or boost an immune response against a monkeypox virus. In another embodiment, the scVACVs can be used to prevent, manage, or treat one or more pathogenic poxvirus infections in a subject, for example, to treat a variola virus infection. In some embodiments, the scVACV is selected from the following strains of vaccinia virus: Western Reserve, Clone 3, Tian Tian, ​​Tian Tian Clone TP5, Tian Tian Clone TP3, NYCBH, NYCBH Clone Acambis 2000, Wyeth, Copenhagen, Lister, Lister 107, Lister-LO, Lister GL-ONC1, Lister GL-ONC2, Lister GL-ONC3, Lister GL-ONC4, Lister CTC1, Lister IMG2 (Turbo FP635), IHD-W, LC16m18, Lederle, Tashkent clone TKT3, Tashkent clone TKT4, USSR, Evans, Praha, L-IVP, V-VET1 or LIVP6.1.1, Ikeda, EM-63, Malbran, Duke, 3737, CV-1, Connaught Laboratories, Serro 2, CM-01, NYCBH Dryvax clone DPP13, NYCBH Dryvax clone DPP15, NYCBH Dryvax clone DPP20, NYCBH Dryvax clone DPP17, NYCBH Dryvax clone DPP21, VACV-IOC, chorioallantoic vaccinia virus Ankara (CVA), modified vaccinia Ankara (MVA), and MVA-BN. In a preferred embodiment, the scVACV is derived from the NYCBH strain clone Acambis 2000 or ACAM2000.

[0118] In one aspect, the scVACV of the present invention can be used in immunogenic formulations, e.g., vaccine formulations. The formulations can be used to prevent, manage, neutralize, treat, and / or ameliorate pathogenic poxvirus infections. The immunogenic formulations can include live or inactivated scVACV. The scVACV can be inactivated by methods well known to those skilled in the art. Common methods use formalin and heat for inactivation. In some embodiments, the immunogenic formulation comprises a live vaccine. Production of such live immunogenic formulations can be achieved using conventional methods, including propagation of scVACV in cell culture followed by purification. For example, as can be determined by the skilled artisan, scVACV can be cultured in BHK, BGMK, BRL3A, BSC-40, CEF, CEK, CHO, COS, CVI, HaCaT, HEL, HeLa cells, HEK293, human osteosarcoma cell line 143B, MDCK, NIH / 3T3, Vero cells, etc.

[0119] In one aspect, the scVACVs of the invention can be used to prevent, manage, or treat smallpox. In another aspect, the scVACVs of the invention can be used as vaccines for the prevention of smallpox in individuals or populations that have been exposed to, are potentially exposed to, or are at risk for exposure to smallpox. The scVACVs of various aspects of the invention can be used to form a new national stockpile of smallpox vaccines. In some embodiments, the scVACVs of the invention can be administered prophylactically to defense personnel, first responders, and the like.

[0120] In one embodiment, a composition comprising a scVACV of the present invention is used as a smallpox vaccine. In one aspect, the scVACV of the present invention produced by the methods of the present disclosure has a small-plaque phenotype. A small-plaque phenotype is generally considered to reflect attenuation. Thus, the scVACV produced by various methods of the present invention provide a safe alternative to existing smallpox vaccines. In some embodiments, the vaccine may be safe for administration to immunosuppressed subjects (e.g., HIV patients, patients undergoing chemotherapy, patients being treated for cancer, rheumatic disorders, or autoimmune disorders, patients undergoing or who have undergone organ or tissue transplants, immunodeficient patients, children, pregnant women, patients with atopic dermatitis, eczema, psoriasis, cardiac conditions, and patients taking immunosuppressive medications) who may suffer severe complications from existing smallpox vaccines and therefore for whom existing smallpox vaccines are contraindicated. In some embodiments, the vaccine can be used in combination with one or more antiviral treatments to suppress viral replication. In some embodiments, the vaccine can be used in combination with brincidofovir treatment to suppress viral replication. In some embodiments, the vaccine can be used in combination with tecovirimat / SIGA-246 treatment to suppress viral replication. In some embodiments, the vaccine can be used in combination with an acyclic nucleoside phosphonate (cidofovir), an oral alkoxyalkyl prodrug of an acyclic nucleoside or phosphonate (brincidofovir or CMX001). In some embodiments, the vaccine can be used in combination with vaccinia immune globulin (VIG). In some embodiments, the vaccine can be used in subjects previously immunized with a peptide or protein antigen derived from VACV, VARV, or HPXV. In some embodiments, the vaccine can be used in subjects previously immunized with killed or inactivated VACV.In some embodiments, the vaccine can be used in subjects who have previously been immunized with a replication-deficient / defective VACV virus strain, MVA (Modified Virus Ankara). In some embodiments, vaccine formulations comprising the scVACV of the present invention can include live or inactivated scVACV.

[0121] In one embodiment, a composition comprising the scVACV of the present disclosure is used as a smallpox vaccine. The scVACVs are ACAM2000 (Genbank accession AY313847), Western Reserve (Genbank accession NC006998; Genbank accession AY243312), CL3 (Genbank accession AY313848), Tian Tian (Genbank accession AF095689.1), Tian Tian clone TP5 (JX489136), TP3 (Genbank accession KC207810) and TP5 (Genbank accession KC207811), NYCBH, Wyeth, Copenhagen (Genbank accession M35027), NYCBH clone Acambis 2000 (Genbank accession AY313847), Lister 107 (Genbank accession DQ121394) Lister-LO (Genbank accession AY678276), modified vaccinia virus Ankara (MVA) (Genbank accession U94848; Genbank accession AY603355), MVA-BN (Genbank accession DQ983238), Lederle, Tashkent clones TKT3 (Genbank accession KM044309) and TKT4 (KM044310), USSR, Evans, Praha, LIVP, Ikeda, IHD-W (Genbank accession KJ125439), LC16m8 (AY678275), EM-63, IC, Malbran, Duke (Genbank accession DQ439815), 3737 (Genbank accession DQ377945), CV-1, Connaught The strains can be based on VACV strains selected from: Laboratories, CVA (Genbank accession AM501482), Serro 2 virus (Genbank accession KF179385), Cantaglo virus isolate CM-01 (Genbank accession KT013210), Dryvax clones DPP15 (Genbank accession JN654981), DPP20 (Genbank accession JN654985), DPP13 (Genbank accession JN654980), DPP17 (Genbank accession JN654983), DPP21 (Genbank accession JN654986), and IOC (Genbank accessions KT184690 and KT184691).In one embodiment, the scVACV used as a smallpox vaccine is based on the ACAM2000 strain (Genbank accession AY313847). In one embodiment, the scVACV used as a smallpox vaccine is based on the VACV-IOC strain (Genbank accessions KT184690 and KT184691). In one embodiment, the scVACV used as a smallpox vaccine is based on the MVA strain (Genbank accession U94848; Genbank accession AY603355). In one embodiment, the scVACV used as a smallpox vaccine is based on the MVA-BN strain (Genbank accession DQ983238). In some embodiments, vaccine formulations comprising the scVACV of the present disclosure can include live or inactivated scVACV.

[0122] In some embodiments, compositions comprising scVACV of the invention are used as vaccines against VACV, MPXV or CPXV infection.

[0123] In some embodiments, scVACVs of the invention can be engineered to express heterologous antigens or epitopes and can be used as vaccines against the source organism of such antigens and / or epitopes.

[0124] Immunogenic formulations (e.g., vaccines) of the present disclosure comprise an effective amount of scVACV and a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other recognized pharmacopeia for use in animals, and more particularly, in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a pharmaceutical composition (e.g., an immunogenic or vaccine formulation) is administered. Saline and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. The formulation should suit the mode of administration. This may depend on whether the scVACV is live or inactivated. In some embodiments, the purified scVACV of the present invention can be lyophilized for later use or immediately prepared into a pharmaceutical solution. The scVACV can also be diluted in a physiologically acceptable solution, such as sterile saline, with or without an adjuvant or carrier.

[0125] In one aspect, the immunogenic formulations (e.g., vaccines) of the present invention can be administered to patients by scarification. Vaccines can also be administered by any other standard route of administration. Many methods can be used to introduce the immunogenic formulations (e.g., vaccines), including, but not limited to, intranasal, intratracheal, oral, intradermal, intramuscular, intraperitoneal, intravenous, conjunctival, and subcutaneous routes. In birds, the method can further include choanal inoculation. As an alternative to parenteral administration, aspects of the present invention also encompass routes of mass administration for agricultural purposes, such as through drinking water or in sprays. Instead, it may be preferable to introduce the scVACV of the present disclosure through its natural route of infection. In some embodiments, the immunogenic formulations of the present invention are administered as an injectable solution, a consumable transgenic plant expressing the vaccine, a sustained-release gel or implantable encapsulated composition, a solid implant, or a nucleic acid. Immunogenic formulations can also be administered in creams, lotions, ointments, skin patches, lozenges, or oral liquids, such as suspensions, solutions, and emulsions (oil-in-water or water-in-oil). The accepted route of administration for live replicating smallpox vaccines is skin scarification, which creates a virus-shedding lesion at the vaccination site that persists for several days. The lesion is a potential source of contact transmission of the vaccine to individuals for whom administration of a live vaccine may be contraindicated. Therefore, intramuscular administration of immunogenic formulations can offer advantages. In a preferred embodiment, administration of scVACV ACAM2000 is intramuscular. In another preferred embodiment, administration is by skin scarification. Intramuscular administration can also be used for other synthetic chimeric orthopoxviruses, such as synthetic chimeric horsepox virus (scHPXV). For intramuscular administration, it is important to use a needle of the correct length to reach the muscle mass and avoid penetration into the subcutaneous tissue. When administering intramuscular injections, the needle should be inserted at a 90° angle.

[0126] In certain embodiments, the immunogenic formulations (e.g., vaccines) of the present disclosure do not provide complete protection from infection, but do result in lower titers or reduced numbers of pathogens (e.g., pathogenic poxviruses) compared to untreated subjects. In certain embodiments, administration of the immunogenic formulations of the present disclosure results in a 0.5-fold, 1-fold, 2-fold, 4-fold, 6-fold, 8-fold, 10-fold, 15-fold, 20-fold, 25-fold, 50-fold, 75-fold, 100-fold, 125-fold, 150-fold, 175-fold, 200-fold, 300-fold, 400-fold, 500-fold, 750-fold, or 1,000-fold or greater reduction in pathogen titer compared to untreated subjects. The benefits of a reduction in pathogen titer, number, or overall burden include, but are not limited to, less severity of symptoms of infection and a reduction in the length of disease or condition associated with the infection.

[0127] In certain embodiments, the immunogenic formulations (e.g., vaccines) of the present disclosure do not provide complete protection from infection, but do result in a fewer number of symptoms or a decrease in the intensity of symptoms, or reduced morbidity or mortality compared to untreated subjects.

[0128] In various embodiments, the immunogenic formulations (e.g., vaccines) of the invention or antibodies produced by the scVACVs of the present disclosure are administered to a subject in combination with one or more other therapies (e.g., antiviral or immunomodulatory therapies) for the prevention of an infectious disease (e.g., a pathogenic poxvirus infection). In other embodiments, the immunogenic formulations or antibodies produced by the scVACVs of the invention are administered to a subject in combination with one or more other therapies (e.g., antiviral or immunomodulatory therapies) for the treatment of an infectious disease (e.g., a pathogenic poxvirus infection). In yet other embodiments, the immunogenic formulations or antibodies produced by the scVACVs of the invention are administered to a subject in combination with one or more other therapies (e.g., antiviral or immunomodulatory therapies) for the management and / or amelioration of an infectious disease (e.g., a pathogenic poxvirus infection). In a specific embodiment, the immunogenic formulations or antibodies produced by the scVACVs of the invention are administered to a subject in combination with one or more other therapies (e.g., antiviral or immunomodulatory therapies) for the prevention of smallpox. In another specific embodiment, the immunogenic preparations or antibodies produced by the scVACV of the present invention are administered to a subject in combination with one or more other therapies (e.g., antiviral or immunomodulatory therapies) for the treatment of smallpox. In some embodiments, the vaccine can be used in combination with one or more antiviral treatments to suppress viral replication. In some embodiments, the vaccine can be used in combination with brincidofovir treatment to suppress viral replication. In some embodiments, the vaccine can be used in combination with tecovirimat / SIGA-246 treatment to suppress viral replication. In some embodiments, the vaccine can be used in combination with an acyclic nucleoside phosphonate (cidofovir), an oral alkoxyalkyl prodrug of an acyclic nucleoside or phosphonate (brincidofovir or CMX001). In some embodiments, the vaccine can be used in combination with vaccinia immune globulin (VIG).In some embodiments, the vaccine can be used in subjects who have previously been immunized with peptide or protein antigens derived from VACV, VARV, or HPXV. In some embodiments, the vaccine can be used in subjects who have previously been immunized with killed or inactivated VACV. In some embodiments, the vaccine can be used in subjects who have previously been immunized with a replication-deficient / defective VACV virus strain, MVA (Modified Virus Ankara).

[0129] Any antiviral agent known to those skilled in the art can be used in the formulations (e.g., vaccine formulations) and methods of various aspects of the present invention. Non-limiting examples of antiviral agents include proteins, polypeptides, peptides, fusion proteins, antibodies, nucleic acid molecules, organic molecules, inorganic molecules, and small molecules that inhibit and / or reduce viral attachment to its receptor, viral internalization into cells, viral replication, or viral release from cells. Specifically, antiviral agents include, but are not limited to, antiviral drugs that block extracellular viral maturation (tecovirimat / SIGA-246), acyclic nucleoside phosphonates (cidofovir), oral alkoxyalkyl prodrugs of acyclic nucleoside phosphonates (brincidofovir or CMX001), or vaccinia immune globulin (VIG). In some embodiments, antiviral agents include, but are not limited to, nucleoside analogs (e.g., zidovudine, acyclovir, gangcyclovir, vidarabine, idoxuridine, trifluridine, and ribavirin), foscarnet, amantadine, rimantadine, saquinavir, indinavir, ritonavir, alpha-interferon and other interferons, and AZT.

[0130] Dosage and administration regimens can be determined by those skilled in the art based on the needs of the subject being treated. A skilled practitioner can consider factors such as the subject's age or weight, the severity of the disease or condition being treated, and the subject's response to treatment. In some embodiments, the compositions of the present invention can be administered, for example, as needed or daily. Administration can be carried out over a variety of periods. For example, the administration regimen can last for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, or longer. In some embodiments, the administration regimen lasts for 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or longer.

[0131] In some embodiments, the scVACV of the present invention can also be used to produce antibodies useful for passive immunotherapy, diagnostic or prognostic immunoassays, etc. Methods for producing antibodies are well known in the art. Antibodies can be further modified (e.g., chimerized, humanized, etc.) before use in immunotherapy.

[0132] Oncolytic agents As used in this disclosure, an "oncolytic virus" or "oncolytic agent" is considered to be any virus that is generally capable of killing tumor cells (non-resistant) by infecting said tumor cells.

[0133] In one aspect, the synthetic chimeric poxviruses (scVACVs) of the present invention can be used as oncolytic agents that selectively replicate in and kill cancer cells. In another aspect, the present invention relates to a method for inducing an oncolytic response in a subject, comprising administering to the subject a composition comprising a scVACV of the present disclosure. Rapidly dividing cells, such as cancer cells, are generally more permissive to poxvirus infection than non-dividing cells. Many characteristics of poxviruses, such as safety in humans, ease of producing high-titer strains, stability of viral preparations, and the ability to induce anti-tumor immunity after replication in tumor cells, make them desirable oncolytic agents. The scVACVs produced by various methods of the present invention can contain one or more modifications that make them suitable for the treatment of cancer. Accordingly, in one aspect, the present disclosure provides a method for inducing death in cancer cells, comprising contacting the cells with an isolated scVACV or a pharmaceutical composition comprising a scVACV of the present disclosure. In one aspect, the present disclosure provides a method of treating cancer, comprising administering a therapeutically effective amount of a scVACV of the present disclosure to a patient in need thereof. Another aspect includes a scVACV or composition described herein for use in treating cancer or inducing death in neoplastic disorders. Another aspect includes the use of a scVACV or composition described herein for inducing death in neoplastic disorder cells, such as cancer cells, or for treating neoplastic disorders, such as cancer. In some embodiments, poxvirus oncolytic therapy is administered in combination with one or more conventional cancer therapies (e.g., surgery, chemotherapy, radiation therapy, hyperthermia, and biological / immunological therapy). In a specific embodiment, the oncolytic virus is scVACV NYCBH strain, clone Acambis 2000, or ACAM2000.

[0134] Using the methods of the present application, one or more desirable genes can be easily introduced, and one or more undesirable genes can be easily deleted from the scVACV genome. In some embodiments, scVACVs of the present invention for use as oncolytic agents are designed to express transgenes to enhance their immune reactivity, anti-tumor targeting and / or efficacy, cell-to-cell spread, and / or cancer specificity. In some embodiments, scVACVs of the present invention are designed or engineered to express immunomodulatory genes (e.g., viral genes that block GM-CSF or TNF function). In some embodiments, scVACVs of the present invention are designed to contain genes that express factors that attenuate pathogenicity. In some embodiments, scVACVs of the present invention are designed or engineered to express therapeutic agents (e.g., hEPO, BMP-4, antibodies against specific tumor antigens or portions thereof, etc.). In some embodiments, scVACVs of the present invention are designed or engineered to contain the gmCSF gene. In some embodiments, scVACVs of the present invention are modified for attenuation. In some embodiments, the scVACV of the present invention is designed or engineered to lack the viral thymidine kinase (TK) gene. In some embodiments, the scVACV of the present invention is designed or engineered to lack the ribonucleotide reductase gene. In some embodiments, the scVACV of the present invention is designed or engineered to lack the vaccinia growth factor gene. In some embodiments, the scVACV of the present invention is designed or engineered to lack the hemagglutinin gene.

[0135] In one aspect, the scVACV of the present invention is useful for treating various neoplastic disorders and / or cancers. In some embodiments, the types of cancer include, but are not limited to, bone cancer, breast cancer, bladder cancer, cervical cancer, colorectal cancer, esophageal cancer, glioma, gastric cancer, gastrointestinal cancer, head and neck cancer, liver cancer, e.g., hepatocellular carcinoma, leukemia, lung cancer, lymphoma, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, skin cancer, e.g., melanoma, testicular cancer, etc., or any other tumor or pre-neoplastic lesion that can be treated.

[0136] In another embodiment, the method further comprises detecting the presence of the administered scVACV in neoplastic or cancerous cells and / or in a sample from a subject administered an isolated or recombinant virus or composition described herein. For example, the subject can be tested before and / or after administration of a scVACV or composition described herein, e.g., to monitor the progression of infection. In some embodiments, the scVACV of the present disclosure comprises a detection cassette, and detecting the presence of the administered chimeric VACV comprises detecting a protein encoded by the detection cassette. For example, if the detection cassette encodes a fluorescent protein, the subject or sample is imaged using a method that visualizes fluorescence.

[0137] In one embodiment, the oncolytic formulation of the present invention comprises an effective amount of the scVACV of the present disclosure and a pharmaceutically acceptable carrier, the term "pharmaceutically acceptable" having been already explained above in the previous section.

[0138] In some embodiments, the compositions of the present invention are administered at a poxvirus treatment facility. In certain aspects, a poxvirus treatment facility is a facility where subjects in need of immunization or treatment with a composition or method of the present disclosure can be immunized or treated in an environment where they are isolated from other subjects (e.g., caregivers and family members) who are not scheduled for immunization or treatment or who could potentially be infected by the treated subject. In some embodiments, subjects who are not scheduled for immunization or who could be infected by the treated subject include HIV patients, patients undergoing chemotherapy, patients being treated for cancer, rheumatic disorders, or autoimmune disorders, patients undergoing or who have undergone organ or tissue transplants, immunodeficient patients, children, pregnant women, patients with atopic dermatitis, eczema, psoriasis, cardiac conditions, and patients taking immunosuppressive medications, etc. In some embodiments, the poxvirus treatment facility is an orthopoxvirus treatment facility. In some embodiments, the poxvirus treatment facility is a smallpox treatment facility.

[0139] In some embodiments, compositions of the invention comprising scVACV are administered by a specialist in smallpox adverse events, including, but not limited to, eczema vaccinatum, progressive vaccinia, post-vaccinal encephalitis, myocarditis, and dilated cardiomyopathy.

[0140] Viral vectors for recombinant gene expression In one aspect, the synthetic chimeric poxvirus (scVACV) of the present invention can be engineered to contain heterologous sequences. The heterologous sequences can be from different poxvirus species or from any non-poxvirus source. In one aspect, the heterologous sequences are antigenic epitopes selected from any non-poxvirus source. As used herein, non-poxvirus sources refer to organisms other than poxviruses. In some embodiments, the recombinant virus can express one or more antigenic epitopes from non-poxvirus sources, including, but not limited to, Plasmodium falciparum, mycobacteria, Bacillus anthracis, Vibrio cholerae, MRSA, rhabdoviruses, influenza viruses, flaviviruses, paramyxoviruses, hepatitis viruses, and viruses from the human immunodeficiency virus family, or viruses that cause hemorrhagic fever, such as hantaviruses or filoviruses, i.e., Ebola or Marburg viruses. In another aspect, the heterologous sequences are antigenic epitopes from different poxvirus species. These viral sequences can be used to modify the host spectrum or immunogenicity of scVACV.

[0141] In some embodiments, the scVACV of the present invention can encode a heterologous gene / nucleic acid that expresses a therapeutic nucleic acid (e.g., an antisense nucleic acid) or a therapeutic peptide (e.g., a peptide or protein with a desired biological activity).

[0142] In some embodiments, expression of the heterologous nucleic acid sequence is preferably, but not exclusively, under the transcriptional control of a poxvirus promoter. In some embodiments, the heterologous nucleic acid sequence is preferably inserted into a non-essential region of the viral genome. Methods for inserting heterologous sequences into the genome of poxviruses are known to those skilled in the art. In some embodiments, the heterologous nucleic acid is introduced by chemical synthesis. In an exemplary embodiment, the heterologous nucleic acid can be cloned into the VACV105 / J2R locus of the scVACV of the present disclosure.

[0143] In one aspect of the present invention, the scVACV can be used for the introduction of heterologous nucleic acid sequences into target cells, where the sequences are homologous or heterologous to the target cell. Introduction of the heterologous nucleic acid sequence into target cells can be used to produce heterologous peptides or polypeptides and / or complete viruses encoded by the sequences in vitro. In one embodiment, the method comprises infecting host cells with the scVACV of the present invention; culturing the infected host cells under suitable conditions; and isolating and / or enriching the peptides, proteins, and / or viruses produced by the host cells. Suitable conditions for culturing scVACV-infected host cells to express heterologous peptides or polypeptides are well known in the art and will vary depending on the host cell used (see, for example, Molecular Cloning: A Laboratory Manual). Manual, second edition (Sambrook et al., 1989).

[0144] It is to be understood that the embodiments of the present application described are merely illustrative of some of the applications of the principles of the present application. Numerous modifications can be made by those skilled in the art based on the teachings presented herein without departing from the true spirit and scope of the present application.

[0145] The following examples are presented as representative of the present application and should not be construed as limiting the scope of the invention, since these and other equivalent embodiments will become apparent in light of the present disclosure, drawings, and accompanying examples. [Example]

[0146] Example 1: Selection and design of overlapping segments of viral genomes Synthetic chimeric VACV ACAM2000 containing hairpin and duplex sequences of the VACV WR strain (scVACV ACAM2000-WR DUP / HP) The design of the scVACV genome was based on the previously described genome sequence of VACV ACAM2000 [GenBank accession AY313847] (Osborne JD et al. Vaccine. 2007;25(52):8807-32). The genome was divided into nine overlapping fragments (Figure 1). These fragments were designed to share at least 1.0 kbp of overlapping sequence (i.e., homology) with each of their adjacent fragments, providing sites for homologous recombination to facilitate assembly of the full-length genome (Table 1). These overlapping sequences provided sufficient homology to accurately perform recombination between the co-transfected fragments (Yao XD, Evans DH. Journal of Virology. 2003;77(13):7281-90). Table 1: VACV ACAM2000 genome fragments used in this study. Sizes and sequences within the VACV ACAM2000 genome [GenBank accession AY313847] are listed. [Table 1]

[0147] To aid in the subcloning of these fragments, the AarI and BsaI restriction sites were silently mutated in all fragments except for the two ITR-encoding fragments, which were not mutated because these regions contain nucleotide sequence-specific recognition sites that are important for efficient DNA replication and concatemer resolution.

[0148] To facilitate visualization of reactivated VACV ACAM2000 (VACV ACAM2000 YFP-gpt::105) under a fluorescent microscope, a YFP / gpt cassette under the control of a poxvirus early-late promoter was introduced into the thymidine kinase locus. The gpt locus also provided a potential tool for selection of reactivated virus using drug selection.

[0149] Traditionally, terminal hairpins have been difficult to clone and sequence, so it is not surprising that the published sequence of the VACV ACAM2000 genome is incomplete. Examination of the published extreme terminal regions of the VACV ACAM2000 strain revealed some differences between ACAM2000 and the well-characterized VACV WR strain (GenBank accession number AY243312) (Figure 2). In the WR strain, immediately downstream of the covalently closed hairpin loops located at the 5' and 3' ends of the VACV genome, are 70-bp tandem repeat sequences. These are followed by two 125-bp repeat sequences and eight 54-bp repeat sequences (Figure 2A). However, only four 54-bp repeat sequences were identified in the published VACV ACAM2000 sequence (Figure 2B). The presence of 70-bp, 125-bp, and 54-bp repeat sequences was confirmed in wild-type isolates of VACV ACAM2000 after sequencing (using Illumina), indicating that the currently published sequence of ACAM2000 is incomplete. Due to the short read length (<300 nucleotides) of the Illumina read data, we were unable to precisely determine what the actual ACAM2000 genomic sequence within this approximately 3 kbp region was. Instead, we decided to recreate a VACV ACAM2000 virus that had a sequence similar to the VACV WR from the terminal hairpin of the C23L gene to just before the stop codon (Figure 2). This contained both 125-bp and 54-bp tandem repeat sequences that are not included in the published ACAM2000 sequence but were detected when we performed next-generation Illumina sequencing of wtVACV ACAM2000. The 5' ends of the modified VACV ACAM2000 left and right ITR fragments also contained NheI restriction sites, which would allow for the direct attachment of 70 bp tandem repeat sequences to the ITR ends (discussed in Example 2). The F- and S-terminal hairpin loop sequences of wtVACV ACAM2000 are shown in Figure 9 and in SEQ ID NOs: 20 and 19, respectively.

[0150] Synthetic chimeric VACV ACAM2000 (scVACV ACAM2000-ACAM2000 DUP / HP) containing hairpin and duplex sequences of the VACV ACAM2000 strain The design of the scVACV genome was based on the previously described genome sequence of VACV ACAM2000 [GenBank accession AY313847] (Osborne JD et al. Vaccine. 2007; 25(52):8807-32). The genome was divided into nine overlapping fragments (Fig. 1). These fragments were designed to share at least 1.0 kbp of overlapping sequence (i.e., homology) with each of their adjacent fragments, providing sites for homologous recombination to facilitate assembly of the full-length genome (Table 1). These overlapping sequences provided sufficient homology to accurately perform recombination between the co-transfected fragments (Yao XD, Evans DH. Journal of Virology. 2003;77(13):7281-90).

[0151] To aid in the subcloning of these fragments, the AarI and BsaI restriction sites were silently mutated in all fragments except for the two ITR-encoding fragments, which were not mutated because these regions contain nucleotide sequence-specific recognition sites that are important for efficient DNA replication and concatemer resolution.

[0152] To facilitate visualization of reactivated VACV ACAM2000 (VACV ACAM2000 YFP-gpt::105) under a fluorescent microscope, a YFP / gpt cassette under the control of a poxvirus early-late promoter was introduced into the thymidine kinase locus. The gpt locus also provided a potential tool for selection of reactivated virus using drug selection.

[0153] The F- and S-terminal hairpin loop sequences of wtVACV ACAM2000 are shown in Figure 9 and in SEQ ID NOs: 20 and 19, respectively.

[0154] Example 2 Ligation of the F- and S-terminal hairpin loops of VACV WR to the right and left ITR fragments of VACV ACAM2000 A 70-bp repeat fragment identical to that of the VACV WR strain was synthesized (Fig. 2C; SEQ ID NO: 10). SapI and NheI restriction sites were included at the 5' and 3' ends of the 70-bp tandem repeat fragment to facilitate ligation to the VACV WR hairpin sequence and the right and left ITR fragments of VACV ACAM2000, respectively. Before the VACV WR terminal hairpin loop could be ligated to the 70-bp tandem repeat fragment, the loop had to be extended an additional 58 bp using a double-stranded sequence synthesized by IDT technology (Fig. 3A). This was due to the extra sequence immediately downstream of the concatemer resolution site before the first 70-bp repeat found in VACV strain WR. The double-stranded sequence was generated by synthesizing two single-stranded DNA molecules that, when annealed together, produced a double-stranded DNA molecule with a 5'-TGT overhang at the 5' end and a 5'-GGT overhang at the 3' end (Fig. 3A; SEQ ID NO: 11 and SEQ ID NO: 12). Because the F- and S-terminal hairpin loops of VACV WR generate 3'-ACA overhangs at their terminal loops, a 58-bp duplex was ligated to the hairpin to generate a terminal hairpin loop of approximately 130 bp that appeared identical to the sequence found in VACV WR strains up to the beginning of the 70-bp repeat (Fig. 3B). This hairpin / duplex fragment was gel-purified and then subsequently ligated to the SapI-digested ends of the 70-bp repeat fragment. Digestion of the 70-bp tandem repeat fragment with SapI created a three-base overhang (5'-CCA) that is complementary to the 5' GGT overhang of the terminal hairpin / duplex structure. The 70-bp tandem repeat was mixed with either the F-terminal hairpin / duplex structure (Fig. 4, lane 4) or the S-terminal hairpin / duplex structure (Fig. 4, lane 5) in approximately a 5-fold molar excess over the 70-bp tandem repeat fragment in the presence of DNA ligase. This resulted in an upward shift in the DNA electrophoresis gel compared to the 70 bp only reaction (Fig. 4, lane 3), indicating that the terminal hairpin / duplex had been successfully ligated to the 70 bp tandem repeat fragment (Fig. 4).

[0155] This terminal hairpin / duplex / 70 bp tandem repeat fragment was then ligated onto a 70 bp ACAM2000 left or right ITR fragment, the ends of which had previously been modified to contain an NheI restriction site. When this fragment was digested, a 5'-CTAG overhang was left at its 5' end. At the 3' end of the 70 bp tandem repeat fragment, an NheI site was used to directly ligate this fragment to the LITR and RITR regions of a VACV ACAM2000 DNA fragment. After digestion of the VACV ACAM2000 left and right ITR fragments, the S-terminal hairpin / duplex / 70 bp tandem repeat fragment or the F-terminal hairpin / duplex / 70 bp tandem repeat fragment was separately ligated to the left or right ITR fragment using DNA ligase in a 1:1 molar ratio at 16°C overnight. The DNA ligase was then heat-inactivated at 65°C before transfection into Shope fibroma virus (SFV)-infected BGMK cells.

[0156] Ligation of the F- and S-terminal hairpin loops of VACV ACAM2000 to the right and left ITR fragments of VACV ACAM2000 A 70-bp repeat fragment identical to that of the VACV ACAM2000 strain was synthesized. SapI and NheI restriction sites were included at the 5' and 3' ends of the 70-bp tandem repeat fragment to facilitate ligation to the VACV ACAM2000 hairpin sequence and the VACV ACAM2000 right and left ITR fragments, respectively. Before the VACV ACAM2000 terminal hairpin loop could be ligated to the 70-bp tandem repeat fragment, the loop had to be extended an additional 58 bp using a double-stranded sequence synthesized by IDT technology. This was due to the extra sequence immediately downstream of the concatemer resolution site before the first 70-bp repeat found in VACV strain ACAM2000. The double-stranded sequence was generated by synthesizing two single-stranded DNA molecules (SEQ ID NO:21 and SEQ ID NO:22) that, when annealed together, produced a double-stranded DNA molecule with a 5'-TGT overhang at the 5' end and a 5'-GGT overhang at the 3' end. Because the F- and S-terminal hairpin loops of VACV ACAM2000 generate 3'-ACA overhangs at their terminal loops, a 58-bp duplex was ligated to the hairpin to generate a terminal hairpin loop of approximately 130 bp. This hairpin / duplex fragment was gel-purified and then ligated to the SapI-digested ends of the 70-bp repeat fragment. Digestion of the 70-bp tandem repeat fragment with SapI created a three-base overhang (5'-CCA) that is complementary to the 5' GGT overhang of the terminal hairpin / duplex structure. The 70-bp tandem repeat was mixed with either the F-terminal hairpin / duplex structure or the S-terminal hairpin / duplex structure in approximately a 5-fold molar excess relative to the 70-bp tandem repeat fragment in the presence of DNA ligase. This resulted in an upward shift in the DNA electrophoresis gel compared to the 70 bp only reaction, indicating that the terminal hairpin / duplex had been successfully ligated to the 70 bp tandem repeat fragment.

[0157] This terminal hairpin / duplex / 70 bp tandem repeat fragment was then ligated onto the left or right ITR fragment of ACAM2000, whose ends had previously been modified to contain an NheI restriction site. When the left or right ITR fragment was digested, a 5'-CTAG overhang was left at its 5' end. At the 3' end of the 70 bp tandem repeat fragment, an NheI site was used to directly ligate this fragment to the LITR and RITR regions of the VACV ACAM2000 DNA fragment. After digestion of the VACV ACAM2000 left and right ITR fragments, the S-terminal hairpin / duplex / 70 bp tandem repeat fragment or the F-terminal hairpin / duplex / 70 bp tandem repeat fragment was separately ligated to the left or right ITR fragment using DNA ligase at a 1:1 molar ratio overnight at 16°C. The DNA ligase was then heat-inactivated at 65°C before transfection into Shope fibroma virus (SFV)-infected BGMK cells.

[0158] Example 3: Preparation of overlapping DNA fragments of VACV ACAM2000 Using the restriction enzyme I-SceI, each of the overlapping DNA fragments of VACV ACAM2000 in Table 1 was cloned into a plasmid provided by GeneArt. Prior to transfection of these synthetic DNA fragments into BGMK cells, the plasmid was digested with I-SceI and the products were run on a gel to confirm successful linearization of the DNA fragments (Figure 5). After 2 hours of digestion at 37°C, the reaction was subsequently heat-inactivated at 65°C. Samples were stored on ice or at 4°C until the terminal hairpin / duplex / 70 bp tandem repeat / ITR fragments were formed (described above).

[0159] Example 4: Reactivation from chemically synthesized dsDNA fragments SFV strains Kasza and BSC-40 were originally obtained from the American Type Culture Collection. Buffalo green monkey kidney (BGMK) cells were obtained from G. McFadden (University of Florida). BSC-40 and BGMK cells are grown at 37°C in 5% CO2 in minimal essential medium (MEM) supplemented with L-glutamine, non-essential amino acids, sodium pyruvate, antibiotics and antimycotics, and 5% fetal calf serum (FCS; Thermo Fisher Scientific).

[0160] Reactivation of scVACV ACAM2000-WR DUP / HP or scVACV ACAM2000-ACAM2000 DUP / HP in Shope Fibroma Virus-infected cells Buffalo green monkey kidney (BGMK) cells were grown in 60 mm tissue culture dishes containing MEM until they reached approximately 80% confluency. Cells were infected with Shope Fibroma virus (SFV) at an MOI of 0.5 in serum-free MEM for 1 hour at 37°C. The inoculum was replaced with 3 ml of warm MEM containing 5% FCS and returned to the incubator for an additional hour. Meanwhile, transfection reactions were set up as follows: After approximately 2 hours at 37°C, linearized VACV ACAM2000 fragments were transfected into SFV-infected BGMK cells (using Lipofectamine 2000) in molar equivalent amounts based on the length of each fragment containing the VACV ACAM2000 genome. Different amounts of total DNA were tested; 5, 6, and 7.5 μg of DNA were successfully able to reactivate ACAM2000 from these overlapping DNA fragments. The complex was incubated at room temperature for 10 minutes and then added dropwise to BGMK cells previously infected with SFV. Approximately 24 hours after infection, the medium was replaced with fresh MEM containing 5% FCS. The cells were cultured at 37°C for an additional 3–4 days (4–5 days total). Viral particles were harvested by scraping infected cells into cell culture medium and performing three cycles of freezing and thawing. The crude extract was diluted to 10% with serum-free MEM. -2The reactivated scVACV ACAM2000 YFP-gpt::105 was harvested by diluting the inoculum to 100% and plating 4 ml of the inoculum onto 9–16 150 mm tissue culture plates of BSC-40 cells. One hour after infection, the inoculum was replaced with MEM containing 5% FCS and 0.9% Noble agar. Yellow fluorescent plaques were visualized under an inverted microscope, and individual plaques were selected for further analysis. The scVACV ACAM2000 YFP-gpt::105 plaques were plaque-purified three times with yellow fluorescent selection. After four days, infected plates containing both SFV and VACV ACAM2000 clones were harvested and subsequently subjected to three freeze-thaw cycles to release the virus, which was then serially diluted and plated onto BSC-40 cells, which preferentially promote the growth of VACV ACAM2000 virus compared to SFV virus. Three rounds of plaque purification were performed, followed by expansion of the virus stock in 10-150 mm tissue culture plates. Virus was then lysed from these cells, separated on a 36% sucrose cushion, and further purified on a 24%-40% sucrose density gradient. Genomic DNA was isolated from these purified genomes, and next-generation Illumina sequencing was performed to confirm the sequence of the synthetic viral genome.

[0161] Example 4 Growth characteristics compared to wild-type ACAM2000 virus In vitro multistep growth curves of the isolated synthetic chimeric VACV ACAM2000-WR DUP / HP, scVACV ACAM2000-ACAM2000 DUP / HP, and wild-type VACV ACAM2000 viruses were performed in monkey kidney epithelial (BSC-40) cells. Cells were infected at a multiplicity of infection (MOI) of 0.03, and virus was harvested at the indicated times (3, 6, 12, 21, 48, and 72 h). Virus titration was performed on BSC-40 cells. Data shown in Figure 6 represent three independent experiments. As shown in Figure 6, scVACV ACAM2000-WR DUP / HP and wtVACV ACAM2000 viruses grew with indistinguishable growth kinetics over a 72-h period.

[0162] Comparison of the growth curves of scVACV ACAM2000-WR DUP / HP (YFP-gpt marker), scVACV ACAM2000-ACAM2000 DUP / HP (YFP-gpt marker), scVACV ACAM2000-WR DUP / HP (no marker) (YFP-gpt marker replaced by the J2R gene sequence), scVACV ACAM2000-ACAM2000 DUP / HP (no marker) (YFP-gpt marker replaced by the J2R gene sequence), and wtVACV ACAM2000 shows that there are no statistically any differences in the growth characteristics of these viruses compared to wtACAM2000 VACV (Fig. 7).

[0163] Example 5: Verification of the scVACV ACAM2000-WR DUP / HP YFP-gpt::105 Genomic Sequence by PCR and Restriction Fragment Analysis Further analysis of the scVACV ACAM2000 YFP-gpt::105 genome by restriction digestion followed by pulsed-field gel electrophoresis (PFGE) was performed on genomic DNA isolated using sucrose gradient purification (Yao XD, Evans DH. Methods Mol Biol. 2004;269:51-64). Two independent scVACV ACAM2000-WR The DUP / HP clones, as well as the VACV_WRΔJ2R control, in which the J2R gene sequence was replaced with a YFP-gpt marker, and the wtVACV ACAM2000 control (VAC_ACAM2000), were purified and then either left undigested or digested with BsaI, HindIII, or NotI and PvuI. Isolated genomic DNA from both the scVACV ACAM2000-WR DUP / HP and wtVACV ACAM2000 were digested with BsaI and HindIII. Because most of the BsaI sites in the scVACV ACAM2000 genome were silently mutated, a nearly intact approximately 200-kbp fragment was observed after BsaI digestion (Figure 8, lanes 8 and 9). This differs from the wtVACV ACAM2000 and wtVACV WR control (VAC_WRΔJ2R) genomes, which were extensively digested when treated with BsaI (Figure 8, lanes 6 and 7). To confirm that the scVACV ACAM2000-WR DUP / HP genomes could still be digested with other enzymes, these genomes were digested with HindIII, which generated multiple bands from the scVACVACAM2000-WR DUP / HP clones (Fig. 8, lanes 12 and 13). To confirm the presence of the 70-bp tandem repeat element within the ITR region, genomic DNA was digested with NotI and PvuI (Fig. 8, lanes 14–17). In the wtVACV WR control (VAC_WRΔJ2R) sample, a band of approximately 3.6 kbp (marked with an asterisk) was detected, encompassing all of the 70-bp tandem repeats of the VACV WR strain. Given that the VACV WR strain was used as a template to design the synthesis of the ITR repeat elements, we expected to detect several bands in the NotI / PvuI-digested scVACVACAM2000 clones at approximately the same size as those observed in the WR strain. When comparing the two scVACV ACAM2000-WR DUP / HP clones, differences in the size of this region were observed, suggesting that not all of the 70-bp repeats were integrated into each reconstructed genome (Figure 7, lanes 16 and 17). This is not unexpected, given that others have shown that these repeat elements can expand and contract under selective pressure in cell culture (Paez and Esteban (1988). Virology;163(1):145-54).

[0164] Overall, in vitro analysis of the scVACV ACAM2000-WR DUP / HP YFP-gpt::105 genome demonstrated that VACV expression from chemically synthesized DNA fragments is significantly different from that from the scVACV ACAM2000-WR DUP / HP YFP-gpt::105 genome. These results suggest that ACAM2000-WR DUP / HP reactivation was successful and that scVACV ACAM2000-WR DUP / HP virus behaves like wtVACV ACAM2000 virus in vitro.

[0165] Example 6: Confirmation of the scVACV ACAM2000 YFP-gpt::105 genome sequence by whole genome sequence analysis Two clones of scVACV ACAM2000-WR DUP / HP and two clones of scVACV ACAM2000-ACAM2000 DUP / HP were sequenced. Illumina reads were assembled de novo using CLC Genomics Workstation (version 11) with a word size of 35 or 61. The assembled contigs were then imported into Snapgene software and aligned to a reference sequence of the predicted scACAM2000 sequence based on the synthetic fragment provided by GeneArt.

[0166] For scVACV ACAM2000-WR DUP / HP clone 1, contig 1 was 16,317 bp and corresponded to most of the ITR region (excluding the tandem repeat sequences). Contig 2 was 167,020 bp and aligned with the central conserved region of the genome (nucleotide positions 19,467 to 186,486). For scVACV ACAM2000-WR DUP / HP clone 2, contig 3 was 16,322 bp and corresponded to most of the ITR region (excluding the tandem repeat sequences). Contig 1 was 167,020 bp and aligned with the central conserved region of the genome (nucleotide positions 19,467 to 186,486). The clone 2 contig contained a single nucleotide substitution (C to A) at nucleotide position 136791. This corresponds to nucleotide position 156,256 of the scACAM2000 genomic sequence and resulted in an amino acid change from Asp to Tyr (A41L) in VAC_ACAM2000_177.

[0167] For scVACV ACAM2000-ACAM2000 DUP / HP clone 1, contig 1 was 167,020 bp and aligned with the central conserved region of the genome (nucleotide positions 19,469 to 186,488). Contig 2 was 16,150 bp and corresponded to most of the ITR region (excluding the tandem repeats). When this contig was mapped to the Snapgene reference genome, gaps in the sequence were observed at positions 2633 to 3417 and 15,175 to 15,220. The first gap region corresponded to a 54-bp repeat region, likely due to the inability to accurately assemble these regions using novel assembly tools. Direct mapping of the raw Illumina reads to the reference genome did not result in any gaps within either region. For scVACV ACAM2000-ACAM2000 DUP / HP clone 2, contig 1 was 16,075 bp and corresponded to most of the ITR region (excluding the tandem repeat sequences). Contig 2 was 167,078 bp and aligned with the central conserved region of the genome (nucleotide positions 19,469 to 186,546). There was a gap observed in contig 2 between nucleotide positions 15,176 and 15,220. However, direct mapping of the raw Illumina read data to the reference genome did not yield any gaps within this region. None of the sequenced clones of scVACV ACAM2000-ACAM2000 DUP / HP showed any other nucleotide variations at any position within the genome.

[0168] Illumina reads were also mapped to the CLC Genomics reference map. The Illumina reads covered the full length of the reference sequence, with average coverage of 1925 and 2533 for clones 1 and 2 of scVACV ACAM2000-WR DUP / HP, and 2195 and 1602 for clones 1 and 2 of scVACV ACAM2000-ACAM2000 DUP / HP, respectively.

[0169] Overall, the sequencing data corroborate the in vitro genomic analysis data and confirm that scVACV ACAM20000-WR DUP / HP and scVACV ACAM2000-ACAM2000 DUP / HP were successfully reactivated in SFV-infected cells.

[0170] Example 7 Removal of the YFP / gpt selection marker After reactivation of scVACV ACAM2000 YFP-gpt::105, the yfp / gpt selection marker at the thymidine kinase locus can be removed.

[0171] Example 8 Nucleotide sequence variation within the terminal hairpin and duplex regions of the ITRs among various VACV strains Nucleotide sequence variations in the "duplex" region immediately downstream of the concatemer resolution site in the VACV WR, ACAM 2000, Dryvax, and Copenhagen strains are shown in Figure 9. The sequence variations are seen as four nucleotide substitutions and three nucleotide deletions between the wtACAM2000, Dryvax DPP15, TianTan, and Copenhagen strains compared to the WR strain.

[0172] Example 9 Determination of Pathogenicity in a Mouse Intranasal Model or Through Tail Scarification The toxic effects of scVACV ACAM2000-WR DUP / HP and scVACV ACAM2000-ACAM2000 DUP / HP were determined in this study. For this experiment, six groups of Balb / c mice were administered three different doses of scVACV ACAM20000-WR DUP / HP and scVACV ACAM2000-ACAM2000 DUP / HP, as described in Examples 1-7, and compared to a PBS control group, as well as wtVACV (WR) and wtVACV ACAM2000 control groups (a total of 12 treatment groups). Three additional mice were included in this experiment, receiving no treatment during the study period. All mice were sampled for blood at predetermined time points throughout the experiment, with additional mice serving as baseline for serum analysis.

[0173] Prior to inoculation of Balb / c mice, all virus strains were propagated in BSC-40 cells (African green monkey kidney), harvested by trypsinization, washed with PBS, extracted from the cells by Dounce homogenization, purified by ultracentrifugation through a 36% sucrose cushion, and resuspended in PBS to a final concentration of 10 7 PFU / ml to 10 9 Titrate to be between PFU / ml.

[0174] The dose selected for this study (10 5 PFU / dose, 10 6 PFU / dose and 10 7The PFU / dose is based on previous studies using known vaccine strains of VACV, including Dryvax and IOC (Medaglia ML, Moussatche N, Nitsche A, Dabrowski PW, Li Y, Damon IK, et al. Genomic Analysis, Phenotype, and Virulence of the Historical Brazilian Smallpox Vaccine Strain IOC: Implications for the Origins and Evolutionary Relationships of Vaccinia Virus. Journal of Virology. 2015;89(23):11909-25; Qin L, Favis N, Famulski J, Evans DH. Evolution of and evolutionary relationships between extant vaccinia virus strains. Journal of Virology. 2015;89(3):1809-24).

[0175] Virus is administered intranasally or via tail scarification. See details in Examples 10 and 11 below.

[0176] Example 10: Determine whether scVACV administered via intranasal inoculation confers immune protection against a lethal VACV-WR challenge Since weight loss is used as a measure of virulence in mice, wtVACV (WR strain) leads to approximately 20-30% weight loss, 5 × 10 3 VACV Dryvax clone DPP15 was also administered intranasally at a dose of 10 PFU. 7Because it was administered intranasally at PFU / dose, the pathogenicity of this well-known smallpox vaccine could be directly compared to the synthetic versions scVACV ACAM20000-WR DUP / HP and scVACV ACAM2000-ACAM2000 DUP / HP. Mice were purchased from Charles River Laboratories and allowed to acclimate to their environment for at least 1 week after receipt and prior to virus challenge.

[0177] Each mouse receives a single dose (approximately 10 μl) of virus administered via intranasal injection under anesthesia. Mice are monitored daily for a period of 30 days for signs of infection, such as swelling, excretion, or other abnormalities. Each mouse is specifically monitored for weight loss daily following virus administration. Mice that lose more than 25% of their body weight in addition to other morbidity factors are euthanized according to the University of Alberta's animal health care facility protocol.

[0178] Even the highest doses of scVACV ACAM20000-WR DUP / HP and scVACV ACAM2000-ACAM2000 DUP / HP tested may not produce any overt signs of disease in Balb / c mice. One of the VACV strains (Brazilian smallpox vaccine strain IOC) may produce 10 7 PFU did not cause any disease (Medaglia ML, Moussatche N, Nitsche A, Dabrowski PW, Li Y, Damon IK, et al. Genomic Analysis, Phenotype, and Virulence of the Historical Brazilian Smallpox Vaccine Strain IOC: Implications for the Origins and Evolutionary Relationships of Vaccinia Virus. Journal of virology. 2015;89(23):11909-25). 10 9Have a titer greater than PFU / mL Due to the difficulty of producing purified strains, it is impractical to test doses much higher than this.

[0179] Thirty days after virus inoculation, a lethal dose (10 6 Mice are then challenged with 1000 PFU / dose (1000 PFU / dose). Mice are closely monitored for signs of infection as described above. Mice are weighed daily, and any mice that lose more than 25% of their body weight in addition to other morbidity factors are euthanized. Mice inoculated with PBS prior to administration of a lethal dose of VACV-WR are expected to show significant weight loss and signs of other morbidity factors within 7-10 days of inoculation. Approximately 14 days after lethal challenge with VACV-WR, all mice are euthanized and blood is collected to confirm the presence of VACV-specific neutralizing antibodies in the serum by standard plaque reduction assay.

[0180] Example 11 Determine whether scVACV administered via tail scarification confers immune protection against lethal VACV-WR challenge Prior to the start of the tail scarification procedure, immunocompetent Balb / C animals are anesthetized. A series of 15-20 scratch / poke wounds are made at the base of the tail over a length of 1-2 cm using the tip of a 25-gauge needle. Varying volumes of 3-5 μL of virus are applied to the scarification site.

[0181] Mice remain anesthetized until the virus is expected to be absorbed into the scarification site. Over a period of 28 days, mice are monitored daily for signs of weight loss. A pustule forms at the site of the tail scarification approximately 8-10 days after scarification (known as "acquisition").

[0182] Twenty-eight days after virus inoculation, a lethal dose (10 6Mice are then challenged with 1000 PFU / dose). Mice are closely monitored for signs of infection as described above. Mice are weighed daily, and any mice that lose more than 25% of their body weight in addition to other morbidity factors are euthanized. Mice inoculated with PBS prior to administration of a lethal dose of VACV-WR are expected to show significant weight loss and signs of other morbidity factors within 7-10 days of inoculation. Approximately 14 days after lethal challenge with VACV-WR, all mice are euthanized and blood is collected to confirm the presence of VACV-specific neutralizing antibodies in the serum by standard plaque reduction assay.

[0183] All of the non-vaccinated animals die from this lethal dose of VACV WR within 7 days of virus challenge. The present invention provides, for example, the following items. (Item 1) A synthetic chimeric vaccinia virus (scVACV) replicated and reactivated from DNA derived from synthetic DNA, wherein the viral genome of said virus differs from the wild-type genome of said virus in that it is characterized by one or more modifications. (Item 2) 2. The scVACV of item 1, wherein the synthetic DNA is selected from one or more of chemically synthesized DNA, PCR-amplified DNA, engineered DNA, and polynucleotides comprising nucleoside analogs. (Item 3) The scVACV according to item 1, wherein the synthetic DNA is a chemically synthesized DNA. (Item 4) 4. The scVACV according to any one of items 1 to 3, wherein the one or more modifications comprise one or more deletions, insertions, substitutions or a combination thereof. (Item 5) 5. The scVACV according to any one of items 1 to 4, wherein the one or more modifications comprise one or more modifications to eliminate one or more unique restriction sites. (Item 6) 5. The scVACV according to any one of items 1 to 4, wherein the one or more modifications comprise one or more modifications to add or restore one or more unique restriction sites. (Item 7) 6. The scVACV according to any one of items 1 to 5, wherein the one or more modifications include one or more modifications to eliminate one or more AarI restriction sites. (Item 8) 6. The scVACV according to any one of items 1 to 5, wherein the one or more modifications include one or more modifications to eliminate all AarI restriction sites. (Item 9) 6. The scVACV according to any one of items 1 to 5, wherein the one or more modifications include one or more modifications to eliminate one or more BsaI restriction sites. (Item 10) 10. The scVACV of any one of items 1 to 9, wherein the viral genome comprises a heterologous terminal hairpin loop. (Item 11) 11. The scVACV according to any one of items 1 to 10, wherein the viral genome comprises terminal hairpin loops derived from different vaccinia virus strains. (Item 12) 12. The scVACV according to any one of items 1 to 11, wherein the viral genome comprises a terminal hairpin loop derived from the VACV WR strain. (Item 13) 10. The scVACV of any one of items 1 to 9, wherein the viral genome comprises homologous or heterologous terminal hairpin loops and the tandem repeat region comprises a different number of repeat sequences than in wtVACV. (Item 14) The viral genome may be selected from the group consisting of Western Reserve, Clone 3, Tian Tian, ​​Tian Tian Clone TP5, Tian Tian Clone TP3, NYCBH, NYCBH Clone Acambis 2000, Wyeth, Copenhagen, Lister, Lister 107, Lister-LO, Lister GL-ONC1, Lister GL-ONC2, Lister GL-ONC3, Lister GL-ONC4, Lister CTC1, Lister IMG2 (Turbo FP635), IHD-W, LC16m18, Lederle, Tashkent clone TKT3, Tashkent clone TKT4, USSR, Evans, Praha, L-IVP, V-VET1 or LIVP6.1.1, Ikeda, EM-63, Malbran, Duke, 3737, CV-1, Connaught Laboratories, Serro 2, CM-01, NYCBH Dryvax clone DPP13, NYCBH Dryvax clone DPP15, NYCBH Dryvax clone DPP20, NYCBH Dryvax clone DPP17, NYCBH Dryvax clone DPP21, VACV-IOC, chorioallantoic vaccinia virus Ankara (CVA), modified vaccinia Ankara (MVA), and MVA-BN. (Item 15) 15. The scVACV according to item 14, wherein the viral genome of the scVACV is based on the genome of the NYCBH strain, clone Acambis 2000. (Item 16) 15. The scVACV according to item 14, wherein the viral genome of the scVACV is based on the genome of the NYCBH strain, clone Dryvax. (Item 17) 15. The scVACV according to item 14, wherein the viral genome of the scVACV is based on the genome of the Lister strain, V-VET1. (Item 18) 15. The scVACV according to item 14, wherein the viral genome of the scVACV is based on the genome of the modified virus Ankara (MVA) strain. (Item 19) 15. The scVACV according to item 14, wherein the viral genome of the scVACV is based on the genome of the MVA-BN strain. (Item 20) 15. The scVACV according to item 14, wherein the viral genome of the scVACV is based on the genome of the IOC strain. (Item 21) 21. The scVACV according to any one of items 1 to 20, wherein the left and right terminal hairpin loops a) comprise a slow and a fast form of the vaccinia virus terminal hairpin loop, respectively, b) comprise a fast and a slow form of the vaccinia virus terminal hairpin loop, respectively, c) both comprise slow form of the vaccinia virus terminal hairpin loop, or d) both comprise fast form of the vaccinia virus terminal hairpin loop. (Item 22) 22. The scVACV of item 21, wherein the slow form comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO: 13 or SEQ ID NO: 19, and the fast form comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO: 14 or SEQ ID NO: 20. (Item 23) 23. The scVACV of item 22, wherein the slow form comprises a nucleotide sequence that is at least 90% identical to the sequence of SEQ ID NO: 13 or SEQ ID NO: 19, and the fast form comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 14 or SEQ ID NO: 20. (Item 24) 24. The scVACV of item 23, wherein the slow form comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: 13 or SEQ ID NO: 19, and the fast form comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: 14 or SEQ ID NO: 20. (Item 25) 25. The scVACV according to item 24, wherein the slow type consists of the nucleotide sequence of SEQ ID NO: 13 or SEQ ID NO: 19, and the fast type consists of the nucleotide sequence of SEQ ID NO: 14 or SEQ ID NO: 20. (Item 26) 26. The scVACV of any one of items 1 to 25, wherein the virus is replicated and reactivated from overlapping chemically synthesized DNA fragments corresponding to substantially all of the viral genome of the scVACV. (Item 27) 27. The scVACV according to item 26, wherein the virus is replicated and reactivated from 2 to 14 overlapping fragments. (Item 28) 28. The scVACV according to item 27, wherein the virus is replicated and reactivated from 6 to 12 overlapping fragments. (Item 29) 29. The scVACV of item 28, wherein the virus is replicated and reactivated from nine overlapping segments. (Item 30) 30. The scVACV of any one of items 1 to 29, wherein the virus is reactivated using leporipoxvirus-catalyzed recombination and reactivation. (Item 31) 31. The scVACV according to item 30, wherein the leporipoxvirus is selected from the group consisting of Shope fibroma virus (SFV), Tularensis fibroma virus, Rabbit fibroma virus, Squirrel fibroma virus and Myxoma virus. (Item 32) 1. A method for producing a synthetic chimeric vaccinia virus (scVACV), comprising: (i) chemically synthesizing overlapping DNA fragments corresponding to substantially all of the viral genome of said vaccinia virus; (ii) transfecting the overlapping DNA fragments into helper virus-infected cells; (iii) culturing the cells to produce a mixture of helper virus and synthetic chimeric vaccinia virus particles in the cells; (iv) plating the mixture on host cells specific for the scVACV to recover the scVACV; A method comprising: (Item 33) 33. The method of claim 32, wherein the helper virus is selected from the group consisting of leporipoxvirus, fowlpox virus, and psoralen-inactivated helper virus. (Item 34) 34. The method of claim 33, wherein the leporipoxvirus is selected from the group consisting of Shope fibroma virus (SFV), tularensis fibroma virus, rabbit fibroma virus, squirrel fibroma virus, and myxoma virus. (Item 35) 35. The method of claim 34, wherein the leporipoxvirus is SFV. (Item 36) 36. The method according to any one of items 32 to 35, wherein the helper virus-infected cells are BGMK cells. (Item 37) 37. The method of any one of items 32 to 36, wherein step (i) further comprises chemically synthesizing terminal hairpin loops from another strain of VACV and ligating them onto a fragment comprising the left and right ends of the viral genome. (Item 38) the overlapping DNA fragments are (i) a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NOs: 1 to 9; (ii) a nucleotide sequence that is at least 90% identical to the sequence of SEQ ID NOs: 1 to 9; (iii) a nucleotide sequence that is at least 95% identical to the sequence of SEQ ID NOs: 1 to 9; or (iv) Nucleotide sequences consisting of SEQ ID NOs: 1 to 9 38. The method according to any one of Items 32 to 37, comprising: (Item 39) 39. A synthetic chimeric vaccinia virus (scVACV) produced by the method of any one of items 32 to 38. (Item 40) 32. A pharmaceutical composition comprising the scVACV according to any one of items 1 to 31 and a pharmaceutically acceptable carrier. (Item 41) 41. The pharmaceutical composition of claim 40, wherein the scVACV is inactivated. (Item 42) 42. The pharmaceutical composition according to item 41, wherein the inactivation is carried out by heat, UV or formalin. (Item 43) A method for inducing an oncolytic response in a subject, the method comprising administering to the subject a composition comprising the scVACV described in any one of items 1 to 31 or a pharmaceutical composition described in any one of items 40 to 42. (Item 44) 32. A method for expressing a heterologous protein in a host cell, the method comprising the steps of introducing a heterologous nucleic acid sequence into a scVACV according to any one of items 1 to 31, infecting the host cell with the scVACV, and culturing the host cell under conditions for expression of the heterologous protein. (Item 45) 45. The method of claim 44, wherein the heterologous nucleic acid sequence is derived from a different poxvirus species or any non-poxvirus source. (Item 46) Item 47. A method for inducing or boosting an immune response against vaccinia virus, comprising administering to a subject in need thereof a composition comprising the scVACV of any one of Items 1 to 31 or the pharmaceutical composition of any one of Items 40 to 42. 42. A method for inducing or boosting an immune response against smallpox virus, comprising administering to a subject in need thereof a composition comprising a scVACV according to any one of items 1 to 31 or a pharmaceutical composition according to any one of items 40 to 42. (Item 48) 42. A method for inducing or boosting an immune response against monkeypox virus, comprising administering to a subject in need thereof a composition comprising a scVACV according to any one of items 1 to 31 or a pharmaceutical composition according to any one of items 40 to 42. (Item 49) 43. A method for immunizing a human subject to protect said subject from smallpox virus infection, comprising administering to said subject a composition comprising scVACV according to any one of items 1 to 31 or a pharmaceutical composition according to any one of items 40 to 42. (Item 50) 42. A method for treating smallpox virus infection, comprising administering to a subject in need thereof a composition comprising scVACV according to any one of items 1 to 31 or a pharmaceutical composition according to any one of items 40 to 42. (Item 51) A method for treating cancer in a subject, comprising administering to the subject in need thereof a composition comprising a scVACV according to any one of items 1 to 31 or a pharmaceutical composition according to any one of items 40 to 42. (Item 52) 52. The method of any one of items 43 or 46 to 51, wherein the administration can be selected from skin scarification, intramuscular or intravenous administration. (Item 53) 53. The method of any one of items 43 or 46-52, wherein the composition is administered in a poxvirus treatment facility. (Item 54) 54. The method of any one of items 43 or 46-53, wherein the composition is administered by a smallpox adverse event specialist. (Item 55) 55. The method of claim 54, wherein the smallpox adverse event is selected from vaccinia eczema, progressive vaccinia, post-vaccinal encephalitis, myocarditis, and dilated cardiomyopathy.

Claims

[Claim 1] The invention as set forth in the drawings.

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