Synthetic chimeric vaccinia virus

Abstract

The present invention relates in various aspects to the development and use of synthetic chimeric vaccinia viruses or compositions comprising such viruses, as well as systems and methods for producing such synthetic chimeric vaccinia viruses. The synthetic chimeric vaccinia viruses are particularly well suited as viral vaccines or to produce oncolytic reactions and pharmaceutical formulations.

Description

[0001] This application is a divisional application of patent application no. 201980029677.9. TECHNICAL FIELD

[0002] The present invention relates, in various aspects, to synthetic chimeric vaccinia viruses or compositions comprising the same, and the development and use of systems and methods for producing the same. The synthetic chimeric vaccinia viruses are particularly well suited as viral vaccines or to generate oncolytic responses and pharmaceutical formulations. BACKGROUND

[0003] The sequence listing associated with this application is being submitted electronically via EFS-Web and is hereby incorporated by reference in its entirety. The name of the text file containing the sequence listing is 104545-0031-WO-SequenceListing.txt. The text file was created on May 2, 2019 and is 288,652 bytes in size.

[0004] Poxviruses (members of the family Poxviridae) are double-stranded DNA viruses that can infect both humans and animals. Poxviruses are divided into two subfamilies based on host range. The Chordopoxviridae subfamily, which infects vertebrate hosts, is composed 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 a number of genetically related and morphologically identical viruses, including camelpox virus (CMLV), cowpox virus (CPXV), ectromelia virus (ECTV, "mousepox reagent"), horsepox virus (HPXV), monkeypox virus (MPXV), rabbitpox virus (RPXV), raccoonpox virus, skunkpox virus, Taterapox virus, Uasin Gishu disease virus, vaccinia virus (VACV), variola virus (VARV), and volepox virus (VPV). At least three other OPVs, including VACV, MPXV, and CPXV, are known to infect humans in addition to VARV. To date, vaccination with "live" VACV is the only proven protection against smallpox. An aggressive vaccination program led to the eradication of smallpox in 1980, and routine smallpox vaccination of the public was discontinued. However, there remains a need to find new safe and effective individual vaccination methods against VARV and other OPVs.

[0005] Multiple formulations of VACV have been used as smallpox vaccines. Most of these vaccines consist of multiple related viruses (e.g., Dryvax) and one, ACAM2000, contains a single molecular clone. However, similar to Dryvax and other VACV vaccines, even ACAM2000 is associated with serious side effects including myocarditis and pericarditis. To reduce the risk, the ACAM2000 vaccine, like other live vaccines, has many contraindications, excluding individuals with cancer, immunodeficiency, organ transplant recipients, patients with atopic dermatitis, eczema, psoriasis, heart disease, and patients taking immunosuppressive agents. It is estimated that 15-50% of the US population falls into one of these categories, demonstrating the need to develop a safer vaccine or vaccination regimen (Kennedy et al., 2007 Kennedy R, Poland GA. 2007. T-Cell epitope discovery for variola and vaccinia viruses. Rev Med Virol 7:93-113). Thus, there is a need to develop a vaccine that is as efficacious as Dryvax or ACAM2000 TM but safer.

[0006] Production of safe, pure, potent, and efficacious vaccines requires quality assurance programs to ensure consistency and coherence of the vaccine production process. In the past, embryonated chicken eggs or primary chicken embryo fibroblast cultures have been used to grow viruses to make vaccines against yellow fever, influenza, measles, and mumps. These substrates were considered acceptable because it was thought that exogenous agents that could infect chickens would not infect humans and would not be pathogenic to humans (FDA Briefing Document Vaccines and Related Biological Products Advisory Committee Meeting, September 19, 2012). However, if the viral tropism changes, safety can be compromised.

[0007] Other substrates have been used to culture viruses for vaccine production, such as calf lymph for smallpox vaccine. After a calf is inoculated with smallpox, lymph containing white blood cells is extracted and saved in capillary tubes. This is later used for human vaccination to prevent smallpox. However, there is a risk of contamination with the BSE or scrapie prions. Although regulations and guidelines for modern vaccines indicate that all materials used must come from BSE-free areas, there are no regulations and guidelines regarding the status of scrapie-free areas. Of particular concern is the fact that Dryvax vaccines produced in 1980-1982 were not scrutinized by modern methods. Specifically, these stocks were not tested for adventitious agents (Murphy and Osburn. Emerging Infectious Diseases. www.cdc.gov / eid. Vol. 11, No. 7, July 2005).

[0008] Thus, there is a need to develop a vaccine that is similar in efficacy to the existing Dryvax or ACAM2000 TM vaccines, but is safer, reproducible and free of residual cells, residual DNA, prions and adventitious agents.

[0009] The present application provides a chimeric vaccinia virus assembled and replicated from chemically synthesized DNA that is safe, reproducible and free of contaminants. Because chemical genome synthesis does not rely on a natural template, a variety of structural and functional modifications of the viral genome are possible. Chemical genome synthesis is particularly useful when no natural template is available for genetic replication or modification by conventional molecular biology methods. SUMMARY

[0010] One aspect of the present application provides synthetic chimeric vaccinia viruses, methods for producing such viruses and uses of the viruses, e.g., as immunogens, in immunogenic formulations, in in vitro assays, as vectors 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 relative to the wild-type vaccinia virus.

[0011] In one aspect, the present application is based on the discovery that synthetic chimeric vaccinia viruses (e.g., scVACV) can be produced from chemically synthesized overlapping fragments of vaccinia virus genomes.

[0012] Thus, in one aspect, the present application relates to a synthetic chimeric vaccinia virus (e.g., scVACV) that is replicated and reactivated from DNA derived from chemically synthesized DNA, the viral genome of which differs from the wild-type genome of the virus by one or more modifications derived from a group comprising chemically synthesized DNA, cDNA or genomic DNA.

[0013] In another aspect, the present application relates to a method of making 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 a cell infected with a helper virus; (iii) culturing the cell to produce a mixture of helper virus and synthetic chimeric vaccinia particles in the cell; and (iv) plating the mixture on a host cell specific for scVACV to recover the scVACV.

[0014] In another aspect, the present application relates to a synthetic chimeric vaccinia virus (scVACV) produced by the method of the present application.

[0015] In another aspect, the present application relates to a pharmaceutical composition comprising a synthetic chimeric vaccinia virus (scVACV) of the present application and a pharmaceutically acceptable carrier.

[0016] In another aspect, the present application relates to a method of inducing an oncolytic response in a subject, comprising administering to the subject a composition comprising a scVACV of the present application.

[0017] In another aspect, the present application relates to a method for expressing a heterologous protein in a host cell, comprising introducing a heterologous nucleic acid sequence into a scVACV of the present application, infecting a host cell with the scVACV, and culturing the host cell under conditions for expression of the heterologous protein.

[0018] In another aspect, the present application relates to a method of triggering or enhancing an immune response against a vaccinia virus, comprising administering to a subject in need thereof a composition comprising a scVACV of the present application.

[0019] In another aspect, the present application relates to a method of triggering or enhancing an immune response against a smallpox virus infection, comprising administering to the subject a composition comprising a scVACV of the present application.

[0020] In another aspect, the present application relates to a method of triggering or enhancing an immune response against a monkeypox virus infection, comprising administering to the subject a composition comprising a scVACV of the present application.

[0021] In another aspect, the present application relates to a method of immunizing a human subject to protect the subject from a smallpox virus infection, comprising administering to the subject a composition comprising a scVACV of the present application.

[0022] In another aspect, the present application relates to a method of treating a smallpox virus infection, comprising administering to the subject a composition comprising a scVACV of the present application.

[0023] In another aspect, the present application 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 application. BRIEF DESCRIPTION OF DRAWINGS

[0024] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0025] The above summary of the present application and the following detailed description of the application will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the present application, there is shown in the drawings a presently preferred embodiment. It should be understood, however, that the present application is not limited to the precise arrangements and instrumentalities shown.

[0026] Figure 1A and 1B Diagram of the linear dsDNA VACV genome of the ACAM2000 strain (Genbank accession number AY313847). Figure 1A Unmodified genomic sequence of the VACV ACAM2000 genome is illustrated, with the naturally occurring Aarl and Bsal restriction sites shown. Figure 1B Modified VACV ACAM2000 genome is described, which was used for chemical synthesis of large dsDNA fragments. Overlapping scVACV ACAM2000 genome fragments are depicted in blue. Also shown are engineered Bsal restriction sites in the left inverted terminal repeat (LITR) and right inverted terminal repeat (RITR) that are not mutated.

[0027] Figures 2A-2C (A) Detailed schematic of the first ~1500-3000 bp of the published genomes of (A) VACV WR strain and (B) VACV ACAM2000. The tandem repeat regions are indicated with red (70 bp repeat), blue (125 bp repeat) and green (54 bp repeat) boxes. The ORF corresponding to gene C23L is also indicated in each genome. (C) Schematic of the direct repeat region containing the 70 bp repeat in VACV WR. This sequence was synthesized to contain a Sail restriction site at the 5' end and a Nhel restriction site at the 3' end, to allow ligation of the hairpin / duplex fragment and the VACV ACAM2000 ITR fragment, respectively.

[0028] Figure 3A and 3B. Assembly of vaccinia virus terminal hairpin loop with duplex DNA to the first 70 bp repeat sequence. (A) Depiction of the ordered phosphorylated oligonucleotide sequence that was used to generate the WR duplex DNA. (B) Depiction of gel electrophoresis of the WR strain duplex DNA (lane 2) and the hairpin DNA alone (lane 3) and after ligation (lane 4). The ligation product (arrow) was subsequently excised from the gel and purified, enabling it to be ligated to the 70 bp repeat sequence to mimic the sequence of the wt VACV ACAM2000 sequence.

[0029] Figure 4 . Ligation of the 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 gel purified before being ligated to the hairpin / duplex DNA fragment at a 5: 1 molar ratio of hairpin / duplex DNA: 70 bp fragment. Successful addition of the hairpin / duplex fragment is indicated by the upward shift in the band at ~ 2300 bp in lanes 4 and 5. These bands were subsequently gel extracted from the gel before being ligated to the digested VACV ACAM2000 ITR fragment.

[0030] Figure 5 . Digestion of the scVACV ACAM2000 fragments. The ITR fragment was digested with both NheI / I-SceI for 2 hours at 37°C before being dephosphorylated with alkaline phosphatase to remove the phosphate groups and facilitate more efficient ligation of the fragment to the terminal hairpin loop / duplex / 70 bp tandem repeat fragment. The other scVACV ACAM2000 DNA plasmids were linearized with I-SceI for 2 hours at 37°C followed by heat inactivation of the restriction enzyme for 10 minutes at 65°C.

[0031] Figure 6 . In vitro growth properties of scVACV ACAM2000-WR DUP / HP. Multi-step growth kinetics were 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 and titrated on BSC-40 cells. Data represent three independent experiments. Error bars represent the standard error of the mean (SEM).

[0032] Figure 7In vitro growth properties of scVACVACAM2000-WR DUP / HP and scVACVACAM2000-ACAM2000 DUP / HP compared to scVACVACAM2000-WR DUP / HP and scVACVACAM2000-ACAM2000 DUP / HP in which the YFP-gpt marker has been replaced with the J2R gene sequence (VAC_WRΔJ2R) and wtVACVACAM2000. Multi-step growth kinetics were 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 and titrated on BSC-40 cells. Error bars represent the standard error of the mean (SEM).

[0033] Figure 8 Restriction enzyme mapping of reactivated scVACVACAM2000-WR DUP / HP clones. Pulse field gel electrophoresis analysis. Two independent scVACVACAM2000-WR DUP / HP clones plus VACV WR control (in which the YFP-gpt marker has been replaced with the J2R gene sequence (VAC_WRΔJ2R)) and wtVACVACAM2000 control (VAC_ACAM2000) were purified and then left undigested, digested with BsaI, HindIII, or NotI and PvuI. The expected loss of almost all BsaI sites in the scVACVACAM2000 clones is evident. Smaller differences in the HindIII digested scVACVACAM2000 genomic DNA compared to VAC_WRΔJ2R and VACV_ACAM2000 were observed. The 70 bp tandem repeat fragment found at the left ITR and right ITR sequences was cleaved with NotI and PvuI digestion of genomic DNA. The approximate size of the 70 bp repeat sequence in VAC_WRΔJ2R is close to 3.6 kbp. Interestingly, in both independent scVACVACAM2000 clones, two different size bands corresponding to the 70 bp tandem repeat were observed (labeled with *), even though the full length 70 bp tandem repeat element is linked to the ITR fragment. When ACAM2000 genomic DNA was digested with NotI and PvuI, a band at ~4.7 kbp was observed, which can indicate the size of the 70 bp repeat sequence in ACAM2000.

[0034] Figure 9 Nucleotide sequence changes between VACV strain sequences. Figure 9 A depicts VACV nucleotide sequence changes within duplex regions in ITRs (SEQ ID NOs: 15-18). Figure 9B depicts the VACV ACAM2000 secondary hairpin loop, which is covalently attached to the end of the linear dsDNA genome of ACAM2000 (SEQ ID NO: 19 for the S-form and SEQ ID NO: 20 for the F-form). The terminal loop sequence is highlighted in green. DETAILED DESCRIPTION

[0035] General Techniques

[0036] Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings that are commonly understood by one of ordinary skill in the art. Generally, nomenclature used in connection with, and techniques related to, 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 the event of a conflict between the definitions in the specification (including the definitions) and those in the incorporated documents, the definitions in the specification shall control.

[0037] The practice of the present application will employ, unless otherwise indicated, conventional molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology techniques, which are within the skill of the art. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) Cold Spring Harbor Laboratory; Oligonucleotide Synthesis (M. J. Gait ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis ed., 1998) Academic Press; Animal Cell Culture (R. I. Freshney ed., 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell eds., 1993-1998) J. Wiley & 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); and Current Protocols in Immunology (J. E. Coligan et al. eds., 1991).Ausubel et al. (eds.), 1987); PCR: The Polymerase Chain Reaction (Mullis et al. (eds.), 1994); Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rded., 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 & Sons, 1999).

[0038] Enzymatic reactions and purification techniques were performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclature used in connection with, and the laboratory procedures and techniques of, analytical chemistry, biochemistry, immunology, molecular biology, synthetic organic chemistry, and medicinal and pharmacological chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses and chemical analyses.

[0039] Throughout this specification and the embodiments, the word "comprise," or variations such as "comprises" or "comprising," will be understood to imply the inclusion of the stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0040] It should be understood that any place where the language "comprise" is used herein to describe an embodiment, other similar embodiments described in terms of "consist of and / or "consist essentially of are also provided.

[0041] The term "comprising" is used to mean "including, but not limited to." "Comprising" and "including" are used interchangeably.

[0042] Any examples following the terms "such as" or "for example" are not meant to be exhaustive or limiting.

[0043] The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise.

[0044] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to one or at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0045] Notwithstanding that the numerical ranges and parameters setting forth the broadest scope of the application are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Moreover, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein. For example, a stated range of "1 to 10" should be considered to include any and all subranges between (and including) the minimum value of 1 and the maximum value of 10; that is, all subranges beginning with a minimum value of 1 or more, e.g. 1 to 6.1, and ending with a maximum value of 10 or less, e.g., 5.5 to 10.

[0046] Exemplary methods and materials are described herein, although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. The materials, methods, and examples are illustrative only and not intended to be limiting.

[0047] Definitions

[0048] Unless otherwise indicated, the following terms are understood to have the following meanings:

[0049] As used herein, the term "wild-type virus," "wild-type genome," "wild-type protein," or "wild-type nucleic acid" refers to a sequence of amino acids or nucleic acids that naturally occurs within a population, e.g., a particular viral species, etc.

[0050] The terms "chimeric" or "engineered" or "modified" (e.g., chimeric vaccinia virus, engineered polypeptide, modified polypeptide, engineered nucleic acid, modified nucleic acid) or grammatical variations thereof are used interchangeably herein to refer to a non-native sequence that has been manipulated to have one or more changes relative to the native sequence.

[0051] As used herein, "synthetic virus" refers to a virus that is originally derived from synthetic DNA (e.g., chemically synthesized DNA, PCR-amplified DNA, engineered DNA, polynucleotides comprising nucleotide analogs, etc., or combinations thereof) and includes progeny thereof, and progeny can not necessarily be identical (in morphology or genomic DNA complement) to the original parent synthetic virus due to natural, accidental, or intentional mutation. In some embodiments, synthetic virus refers to a virus in which substantially all of the viral genome is originally derived from synthetic DNA (e.g., chemically synthesized DNA, PCR-amplified DNA, engineered DNA, polynucleotides comprising nucleotide analogs, etc., or combinations thereof). In a preferred embodiment, the synthetic virus is derived from chemically synthesized DNA.

[0052] As outlined elsewhere herein, certain positions of the viral genome can be altered. As used herein, "position" refers to a position in the genomic sequence. Corresponding positions are generally determined by alignment with other parental sequences.

[0053] As used herein, the term "residue" in the context of a polypeptide refers to an amino acid unit in a straight polypeptide chain. This is the remainder of each amino acid after removal of water in forming the polypeptide from an alpha-amino acid (i.e., NH2-CHR-COOH) (i.e., -NH-CHR-C-).

[0054] As known in the art, "polynucleotide" or "nucleic acid," used interchangeably herein, refers to a chain of nucleotides of any length and includes DNA and RNA. The nucleotides can 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 comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modifications to the nucleotide structure can be imparted before or after the assembly of the chain. The nucleotide sequence can be interrupted by non-nucleotide components. The polynucleotide can be further modified, such as by conjugation with a labeling component. Other types of modifications include, for example, "caps," substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, amino phosphonates, phosphoramidates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing chiral linkages (e.g., alkenyl phosphonates), those containing pendant debranched or branched chains having other

[0055] 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 straight or branched, it can contain modified amino acids and / or can be interrupted by non-amino acids. The term also encompasses an amino acid chain that has been modified, either naturally or by intervention; for example, disulfide formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides that contain one or more analogs of an amino acid (including, for example, unnatural amino acids and the like) and other modifications known in the art. It is understood that the polypeptide can exist as a single chain or as associated chains.

[0056] "Homologous," in all its grammatical forms and spelling variations, refers to the relationship between two proteins that have a "common evolutionary origin" (including proteins from the same organism species' superfamily, as well as homologous proteins from different organism species). These proteins (and their encoding nucleic acids) have sequence homology, as reflected by their sequence similarity, whether by percent identity or by the presence of particular residues or motifs and conserved positions. "Homologous" can also refer to a nucleic acid that is natural to the virus.

[0057] However, in common usage and in this application, the term "homologous," when modified by an adverb such as "highly," can refer to sequence similarity and can or can not involve a common evolutionary origin.

[0058] "Heterologous," in all its grammatical forms and spelling variations, can refer to a nucleic acid that is non-native to the virus. It means derived from a different species or a different strain than the nucleic acid relative to which it is described as heterologous. In a non-limiting example, the viral genome of a scVACV comprises a heterologous terminal hairpin loop. The heterologous terminal hairpin loop can be derived from a different virus species or from a different VACV strain.

[0059] The term "sequence similarity," in all its grammatical forms, refers to the degree of identity or correspondence between nucleic acid or amino acid sequences, which can or can not share a common evolutionary origin.

[0060] "Percent (%) sequence identity" or "% sequence identity" 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 with the amino acid residues (or nucleic acids) in the reference polypeptide (nucleotide) sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0061] As used herein, "host cell" includes an individual cell or cell culture that can be or has been a recipient for a virus of the application. Host cells include the progeny of the original host cell, and the progeny can not necessarily be identical to the original parent cell either phenotypically or at the genomic DNA level. Host cells include cells in vivo transduced and / or transformed with a poxvirus of the application.

[0062] As used herein, "vector" refers to a construct capable of delivering and preferably expressing one or more genes or sequences of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors bound to cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.

[0063] As used herein, an "isolated molecule" (where the molecule is, for example, a polypeptide, polynucleotide, or fragment thereof) is one which, with respect to its origin or derivation, (1) is not associated with one or more naturally associated components with which it is associated in its native state, (2) is substantially free of one or more other molecules normally found in association with it in nature, (3) is expressed by a cell from a different species, or (4) does not occur in nature. Thus, a molecule that is chemically synthesized or expressed in a cell system different from the cell from which it naturally originates will be "isolated" from its naturally associated components. A molecule can also be rendered substantially free of naturally associated components by isolation using purification techniques known in the art. Molecule purity or homogeneity can be determined by a number of means well known in the art. For example, the purity of a polypeptide sample can be analyzed using polyacrylamide gel electrophoresis and gel staining to visualize the polypeptide using techniques well known in the art. Greater resolution can be provided by using HPLC or other means for purification well known in the art for certain purposes.

[0064] As used herein, in the context of viruses, the term "isolated" refers to a virus that is derived from a single parental virus. Viruses can be isolated using routine methods known to those of skill in the art, including but not limited to those based on plaque purification and limiting dilution methods.

[0065] As used herein, the phrase "multiplicity of infection" or "MOI" is the average number of viruses per infected cell. MOI is determined by dividing the number of viruses added (ml added x plaque forming units (PFU)) by the number of cells added (ml added x cells / ml).

[0066] As used herein, "purified" and grammatical variations thereof refer to the removal (whether completely or partially) of at least one impurity from a mixture containing a polypeptide and one or more impurities, thereby increasing the level of purity of the polypeptide in the composition (i.e., by decreasing the amount (ppm) of impurities in the composition). As used herein, in the context of viruses, "purified" refers to a virus that is substantially free of cellular material and media from the cellular or tissue source from which the virus is derived. The language "substantially free of cellular material" includes virus preparations that contain less than about 30%, 20%, 10%, or 5% (by dry weight) of cells proteins (also referred to herein as "contaminant proteins") of the cells from which the virus is isolated or recombinantly produced. The virus is also substantially free of media, i.e., the media comprises less than about 20%, 10%, or 5% of the volume of the virus preparation. Viruses can be purified using routine methods known to those of skill in the art, including but not limited to chromatography and centrifugation.

[0067] As used herein, "substantially pure" refers to a material that is at least 50% pure (i.e., free of contaminants), more preferably at least 90% pure, more preferably at least 95% pure, still more preferably at least 98% pure, and most preferably at least 99% pure.

[0068] 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, livestock animals (including bovines, porcines, camels, etc.), companion animals (e.g., canids, felines, etc.), and rodents (e.g., mice and rats).

[0069] As used herein, the term “prevent” refers to the delay in the recurrence or onset of one or more symptoms of a disease (e.g., a poxvirus infection) or the alleviation of one or more symptoms in a subject as a result of the administration of a therapy (e.g., a prophylactic or therapeutic agent). For example, in the context of the administration of a therapy to a subject against an infection, “prevent” refers to the inhibition or alleviation of the development or onset of an infection (e.g., a poxvirus infection or a condition associated therewith) or the prevention of the recurrence, onset, or development of one or more symptoms of an infection (e.g., a poxvirus infection or a condition associated therewith) in a subject as a result of the administration of a therapy (e.g., a prophylactic or therapeutic agent) or the administration of a combination of therapies (e.g., a combination of prophylactic or therapeutic agents).

[0070] As used herein, the term “treat” refers to treating a condition or a patient, and refers to taking steps to obtain a beneficial or desired result, including clinical results. With respect to an infection (e.g., a poxvirus infection or a smallpox virus infection), treatment refers to the eradication or control of the replication of an infectious agent (e.g., a poxvirus or a smallpox virus), the reduction in the number of infectious agents (e.g., reduction in viral titer), the alleviation or amelioration of the progression, severity, and / or duration of an infection (e.g., a poxvirus / smallpox infection or a condition or symptom associated therewith), or the amelioration of one or more symptoms as a result of the administration of one or more therapies, including but not limited to the administration of one or more prophylactic or therapeutic agents. With respect to cancer, treatment refers to the eradication, removal, alleviation, or control of primary, local, or metastatic cancerous tissue as a result of the administration of one or more therapeutic agents of the application. In certain embodiments, the term refers to the minimization or delay of the spread of cancer as a result of the administration of one or more therapeutic agents of the application to a subject having such a disease. In other embodiments, the term refers to the elimination of the cells that cause the disease.

[0071] “Administering” a substance, compound, or agent to a subject can be performed using one of a variety of methods known to those of skill in the art. For example, a compound or agent can be administered sublingually or intranasally, by inhalation into the lungs, or rectally. Administration can also be performed, for example, once, multiple times, and / or over one or more extended periods. In some aspects, administration includes direct administration (including self-administration) and indirect administration (including the act of prescribing). For example, as used herein, a physician who instructs a patient to self-administer a drug or administers a drug to a patient through another person administers the drug to the patient.

[0072] Each embodiment described herein can be used individually or in combination with any other embodiment described herein.

[0073] SUMMARY

[0074] Poxviruses are large (~200 kbp) DNA viruses that replicate in the cytoplasm of infected cells. The Orthopoxvirus (OPV) genus includes many poxviruses that vary greatly in their ability to infect different hosts. For example, vaccinia virus (VACV) can infect a broad host group, while the causative agent of smallpox, variola virus (VARV), only infects humans. A common feature of many, if not all, poxviruses is their ability to be "reactivated" non-genetically within a host. Non-genetic reactivation refers to a process in which a cell infected with one poxvirus can facilitate the recovery of a second, "dead" virus (e.g., heat-inactivated virus) that is not infectious by itself.

[0075] Purified poxvirus DNA is not infectious because the viral life cycle requires a virus-encoded RNA polymerase to transcribe early genes packaged in the virion. However, this defect can be overcome if the viral DNA is transfected into a cell previously infected with a helper poxvirus, which provides the essential factors required for transcribing, replicating, and packaging the infectious 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). Although this generates mixed viral progeny, the problem can be overcome by performing the reactivation reaction in a cell line that supports the propagation of both viruses, and subsequently eliminating the helper virus by plating the viral 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).

[0076] Previously, Yao and Evans described a method for rapidly assembling recombinant vaccinia strains using multiple overlapping fragments of viral DNA by catalyzing high- frequency recombination reactions and replication reactions by rabbitpox virus, Shope fibroma virus (SFV), which can be coupled with SFV-catalyzed reactivation reactions (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). The first description of reactivation and characterization of functional synthetic chimeric vaccinia viruses [scVACVs] using chemically synthesized overlapping double-stranded DNA fragments.

[0077] Synthetic chimeric vaccinia viruses of the invention

[0078] In one aspect, the present application provides a functional synthetic chimeric vaccinia virus (scVACV) that is initially replicated and assembled from chemically synthesized DNA. The virus that can be produced according to the methods of the present application can be any vaccinia virus for which the genome has been sequenced or can be mostly sequenced or for which a natural isolate is available. The scVACVs of various embodiments can be based on the genomic sequence of a naturally occurring strain, variant or mutant, mutagenized virus or genetically engineered virus. In some embodiments, the viral genome of the scVACV includes one or more modifications relative to the wild-type genome or base genomic sequence of the virus. The modifications can include one or more deletions, insertions, substitutions or combinations thereof. In one embodiment, the modifications can include the insertion of one or more multiple cloning sites such that foreign DNA can be inserted. It will be appreciated that the modifications can be introduced in a variety of ways that are well 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 application includes one or more modifications to add or repair one or more unique restriction sites. The modifications to add or repair one or more restriction sites can be made on restriction sites that have been eliminated to facilitate clonal selection.

[0079] Chemical genome synthesis is particularly useful when natural templates are not available for genetic modification, amplification, or replication by conventional molecular biology methods. The genome sequence of wtVACV (strain NYCBH, clone ACAM2000) has been described and published, although it is incomplete. The sequence of the terminal hairpin loop was not determined, only four 54 bp repeat sequences were identified. Following sequencing, the presence of 70 bp, 125 bp and 54 bp tandem repeats was confirmed in the wild-type isolate of VACV ACAM2000, indicating that the currently published ACAM2000 sequence is incomplete. The present inventors generated a functional synthetic chimeric VACV (scVACV). Specifically, the present inventors successfully generated a functional scVACV strain NYCBH, clone ACAM2000 by using terminal hairpin loops based on the telomeres of different strains of wtVACV instead of the terminal hairpin loop sequence of VACV itself. In some embodiments, the viral genome of the VACV virus is a strain 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 LIVP 6.1.1, 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, Vaccinia Calara (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. It will be appreciated that the scVACV of the present application can be based on this newly discovered VACV strain.

[0080] It was derived from the New York City Health Commission vaccinia virus strain (Wyeth Laboratories, Marietta, PA) and cultured on calf skin, then essentially freeze-dried for storage.

[0081] Live VAV ACAM2000 strain and smallpox (vaccination) vaccine are derived from The plaque-purified clone of live vaccinia virus was cultured in African green monkey kidney (Vero) cells and tested to be free of exogenous factors (Osborne JD et al., Vaccine. 2007; 25(52):8807-32).

[0082] V-VET1 or LIVP 6.1.1 was developed by Genelux. It was isolated from a wild-type reservoir of the Lister strain of vaccinia virus (Lister strain, Institute of Viral Preparations (LIVP), Moscow, Russia) and represents a “natural” virus (without genetic manipulation). The thymidine kinase (tk) gene of the LIVP 6.1.1 virus is inactive (Shvalov AN et al., Genome Announc. 2016 May-June; 4(3):e00372-16).

[0083] Genelux developed GLV-1h68 (named GL-ONC1, produced for clinical research) from the Lister strain by inserting three expression cassettes encoding Renilla luciferase-jellyfish 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.).

[0084] Chemical viral genome synthesis also opens up the possibility of introducing a wide range of useful modifications into the resulting genome or specific portions thereof. Modifications can increase the ease of cloning to produce viruses, provide sites for introducing recombinant gene products, improve the ease of identifying reactivated viral clones, and / or confer multiple other useful characteristics (e.g., introduction of desired antigens, production of oncolytic viruses, etc.). In some embodiments, modifications may include weakening or deleting one or more virulence factors. In some embodiments, modifications may include adding or inserting one or more virulence-regulating genes or genes encoding regulatory factors.

[0085] Traditionally, the terminal hairpins of poxviruses are 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 sequence of wt VACV strain NYCBH clone ACAM2000 has been described and published, but it is not complete. The sequence of the terminal hairpin loop was not determined, and only four 54 bp repeat sequences were identified. Because the published genome sequence of wt VACV strain NYCBH clone ACAM2000 is incomplete, the hairpin cannot be precisely replicated, and prior to the present application, it was not known whether VACV could replicate and assemble from polynucleotides based solely on the known portion of the wt VACV genome. It was also not known that a hairpin from one strain would be effective in another strain. The present inventors generated a functional synthetic chimeric VACV (scVACV) ACAM2000 by using terminal hairpin loops based on the telomeres of different strains of wt VACV in place of the terminal hairpin loop sequence of VACV itself. In exemplary embodiments, ssDNA fragments were chemically synthesized using the published telomere sequence of VACV WR strain as a guide and ligated to dsDNA fragments containing the left and right ends of VACV strain NYCBH. In some embodiments, the terminal hairpin is based on the terminal hairpin of any VACV strain for which the genome has been completely sequenced or for which a naturally isolated strain is available for genome sequencing.In some embodiments, the terminal hairpin loop is based on a strain selected from the group 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 LIVP 6.1.1, 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, Vaccinia Calara (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. It will be appreciated that the scVACV of the application can be based on such newly discovered VACV strains.

[0086] In another embodiment, the viral genome of the scVACV of the application comprises a homologous or heterologous terminal hairpin loop and a tandem repeat region (70bp, 125bp and 54bp tandem repeats) located downstream of the hairpin loop, wherein the tandem repeat region comprises a different number of repeats than the wtVACV (i.e. naturally occurring virus). The number of repeats of the 70bp, 125bp and 54bp tandem repeats found in the VACV virus (strain WR) are 22, 2 and 8, respectively. In another embodiment, the number of tandem repeats region is variable among different poxviruses, different vaccinia viruses and different vaccinia virus strains. The term homologous terminal hairpin loop means that the terminal hairpin loop is from the same virus species / same strain, whereas the term heterologous terminal hairpin loop means that the terminal hairpin loop is from a different virus species / different strain.

[0087] In some embodiments, the modification can comprise deletion of one or more restriction sites. In some embodiments, the modification can comprise introduction of one or more restriction sites. In some embodiments, the restriction site to be deleted from the genome or added to the genome can be selected from one or more restriction sites such as, but 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, BaeGI, BaeI, BamHI BanI, BanII, BbsI, BbvCI, BbvI, BccI, BceAI, BcgI, BciVI, BclI, BcoDI, BfaI, BfuAI, BfuCI, BglI, BglII, BlpI, BmgBI, BmrI, BmtI, BpmI, Bpu10I, BpuEI, BsaAI, BsaBI, BsaHI, BsaI, BsaJI, BsaWI, BsaXI, BseRI, BseYI, BsgI, BsiEI, BsiHKAI, BsiWI, BslI, BsmAI, BsmBI, BsmFI, BsmI, BsoBI, Bsp1286I, BspCNI, BspDI, BspEI, BspHI, BspMI, BspQI, BsrBI, BsrDI, BsrFαI, BsrGI, BsrI, BssHII, BssSαI, BstAPI, BstBI, BstEII, BstNI, BstUI, BstXI, BstYI, BstZ17I, Bsu36I, BtgI, BtgZI, BtsαI, BtsCI, BtsIMutI, Cac8I, ClaI, CspCI, CviAII, CviKI-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-Ceul, I-SceI, KasI, Kpnl, LpnPI, MboI, MboII, Mfel, MluCI, Mlul, Mlyl, Mmel, MnlI, MscI, Msel, MslI, MspAl I, MspI, MspJI, Mwol, Naell, NarI, Ncill, Ncol, Ndel, NgoMIV, Nhel, NlaIII, NlaIV, NmeAIII, NotI, NruI, Nsil, NspI, PacI, PaeR7I, Pcil, PflFI, PflMI, PleI, PluTI, Pmel, PmlI, PpuMI, PshAI, Psil, PspGI, PspOMI, PspXI, PstI, PvuI, PvuII, Rsal, RsrII, SacI, SacII, Sail, SapI, Sau3AI, Sau96I, SbfI, ScrFI, SexAI, SfaNI, SfcI, Sfil, SfoI, SgrAI, Smal, SmlI, SnaBI, Spel, SphI, SrfI, SspI, Stul, StyD4I, StyI, Swal, Taqal, Tfil, TseI, Tsp45I, TspMI, TspRI, Tthlll, Xbal, Xcml, Xhol, Xmal, Xmnl, or Zral. It will be appreciated that any desired restriction site or combination of restriction sites can be inserted into or mutated 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 Bsal sites are deleted from the viral genome. In some embodiments, one or more restriction sites are eliminated entirely from the genome (e.g., all AarI sites can be eliminated from the viral genome). In some embodiments, one or more Aval restriction sites are introduced into the viral genome. In some embodiments, one or more Stul sites are introduced into the viral genome. In some embodiments, one or more modifications can include incorporation of recombination engineering targets, including but not limited to loxP or FRT sites.

[0088] In some embodiments, the modification can include the introduction of a fluorescent marker, such as but not limited to green fluorescent protein (GFP), enhanced GFP, yellow fluorescent protein (YFP), cyan / blue fluorescent protein (BFP), red fluorescent protein (RFP), or variants thereof, etc.; a selectable marker, such as but not limited to a drug resistance marker (e.g., E. coli xanthine-guanine phosphoribosyltransferase gene (gpt), Streptomyces alboniger purine acetyltransferase gene (pac), neomycin phosphotransferase I gene (nptl), neomycin phosphotransferase gene II (nptll), hygromycin phosphotransferase (hpt), sh ble gene, etc.); a protein or peptide tag, such as 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 tags such as B42, GAL4, LexA, or VP16; a localization tag, such as NLS-tag, SNAP-tag, Myr-tag, etc. It will be appreciated that other selectable markers and / or tags known in the art can be used. In some embodiments, the modification includes one or more selectable markers to aid in selection of reactivated clones (e.g., a fluorescent marker such as YFP, a drug selection marker such as gpt, etc.) to aid in selection of reactivated viral clones. In some embodiments, the one or more selectable markers are removed from the reactivated clones after the selection step.

[0089] In one aspect, the scVACV of the application can be used as a vaccine to protect against a pathogenic poxvirus infection (e.g., VARV, MPXV, MCV, ORFV, Ausdyk virus, BPSV, Sealpox virus, etc.), as a therapeutic to treat or prevent a pathogenic poxvirus infection (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.

[0090] Methods of producing synthetic chimeric VACVs

[0091] In one aspect, the present application 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. The recombination of overlapping DNA fragments of the viral genome and reactivation of functional scVACV is performed 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 produce a mixed viral progeny comprising the helper virus and reactivated scVACV. Next, the mixed viral progeny is plated onto host cells that do not support the growth of the helper virus but allow the growth of the synthetic chimeric vaccinia virus so as 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 the host cells but grows poorly in the host cells. In some embodiments, the helper cells grow more slowly in the host cells compared to the scVACV.

[0092] 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 fragments are derived from naturally occurring DNA (e.g., by PCR amplification or by accepted recombinant DNA techniques).

[0093] The number of overlapping DNA fragments used in the methods of the application will depend on the size of the vaccinia virus genome. Practical considerations, such as the decrease in recombination efficiency with increasing number of fragments on one hand, and difficulties in synthesizing large DNA fragments with decreasing number of fragments on the other hand, will also provide information on the number of overlapping fragments used in the methods of the application. In some embodiments, the synthetic chimeric vaccinia virus genome can be synthesized as a single fragment. In some embodiments, the synthetic chimeric vaccinia virus genome is assembled from 2-14 overlapping DNA fragments. In some embodiments, the synthetic chimeric vaccinia virus genome is assembled from 4-12 overlapping DNA fragments. In some embodiments, the synthetic chimeric vaccinia virus genome is assembled from 6-12 overlapping DNA fragments. In some embodiments, the synthetic chimeric vaccinia virus genome is assembled from 8-11 overlapping DNA fragments. In some embodiments, the synthetic chimeric vaccinia virus genome is assembled from 8-10, 10-12, or 10-14 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 exemplary embodiments of the application, a synthetic vaccinia virus (scVACV) is reactivated from 9 chemically synthesized overlapping double-stranded DNA fragments. In some embodiments, the terminal hairpin loops are synthesized separately and ligated to the fragments comprising the left and right terminal 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 a wtVACV. In some embodiments, the terminal hairpin is derived from the terminal hairpin of a different strain of wtVACV, instead of the VACV own terminal hairpin loop sequence. In some embodiments, the terminal hairpin is that of any wtVACV whose genome has been completely sequenced or for which a naturally isolated strain is available for genome sequencing.

[0094] The size of the overlapping fragments used in various aspects of the methods of the application will depend on the size of the vaccinia virus genome. It will be appreciated that the fragment size can vary widely, and various practical considerations, such as the ability to chemically synthesize large DNA fragments, will inform the choice of fragment size. In some embodiments, the fragment size ranges from about 2,000 bp to about 50,000 bp. In some embodiments, the fragment size ranges from about 3,000 bp to about 45,000 bp. In some embodiments, the fragment size ranges from about 4,000 bp to 40,000 bp. In some embodiments, the fragment size ranges from about 5,000 bp to 35,000 bp. In some embodiments, the largest fragment is about 18,000 bp, 20,000 bp, 21,000 bp, 22,000 bp, 23,000 bp, 24,000 bp, 25,000 bp, 26,000 bp, 27,000 bp, 28,000 bp, 29,000 bp, 30,000 bp, 31,000 bp, 32,000 bp, 33,000 bp, 34,000 bp, 35,000 bp, 36,000 bp, 37,000 bp, 38,000 bp, 39,000 bp, 40,000 bp, 41,000 bp, 42,000 bp, 43,000 bp, 44,000 bp, 45,000 bp, 46,000 bp, 47,000 bp, 48,000 bp, 49,000 bp, or 50,000 bp. In an exemplary embodiment of the application, scVACV is reactivated from 9 chemically synthesized overlapping double-stranded DNA fragments ranging in size from about 10,000 bp to about 32,000 bp (Table 1).

[0095] The helper virus can be any poxvirus capable of providing the trans-acting enzymatic machinery required to reactivate poxvirus from transfected DNA. The helper virus can have a different or narrower host cell range compared to the scVACV to be produced (e.g., the 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 produced. In some embodiments, the helper virus is a lagomorpha poxvirus. In some embodiments, the lagomorpha poxvirus is SFV, Shope fibroma virus, cottontail fibroma virus, rabbit 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 camel poxvirus (CMLV), cowpox virus (CPXV), ectromelia virus (ECTV, "mousepox agent"), HPXV, monkeypox virus (MPXV), rabbitpox virus (RPXV), raccoonpox virus, skunkpox virus, gerbilinepox virus, Uasin Gishu disease virus, VACV, and woodchuckpox virus (VPV). In some embodiments, the helper virus is an aviopoxvirus, capripoxvirus, cervidpoxvirus, crocodylipoxvirus, molluscipoxvirus, parapoxvirus, suipoxvirus, or yatapoxvirus. In some embodiments, the helper virus is an aviopoxvirus. In some embodiments, the helper virus is an alpha, beta, or gamma insect poxvirus. In some embodiments, the helper virus is a psoralen-inactivated helper virus. In an exemplary embodiment of the application, the scVACV is reactivated from overlapping DNA fragments transfected into SFV-infected BGMK cells. The SFV is subsequently eliminated by plating the mixed virus progeny on BSC-40 cells.

[0096] Those skilled in the art will appreciate that suitable host cells are used for reactivation of the scVACV, and the selection and / or isolation of the scVACV will depend on the particular combination of helper virus and chimeric poxvirus produced by aspects of the methods of the application. Any host cell that supports the growth of both the helper virus and the scVACV can be used for the reactivation step, and any host cell that does not support the growth of the helper virus will be used to eliminate the helper virus and select and / or isolate the scVACV. In some embodiments, the helper cell is a rabbit poxvirus, 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.

[0097] In some embodiments, the scVACV can be propagated in any substrate that allows the virus to grow to a titer that permits use of the scVACV described herein. In one embodiment, the substrate allows the scVACV to grow to a titer comparable to those determined for the corresponding wild-type virus. In some embodiments, the scVACV can be grown in cells susceptible to infection by VACV (e.g., avian cells, bat cells, bovine cells, camel cells, canary cells, cat cells, deer cells, equine cells, poultry cells, gerbil cells, goat cells, human cells, monkey cells, porcine cells, rabbit cells, raccoon cells, seal cells, sheep cells, skunk cells, vole cells, etc.). These methods are well known to those skilled 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 osteogenic sarcoma cell line 143B, MDCK, NIH / 3T3, and Vero cells. For virus isolation, the scVACV is typically removed from the cell culture and separated from the cellular components by well-known clarification procedures (e.g., such as gradient centrifugation and column chromatography), and further purified as needed using procedures well known to those skilled in the art, such as plaque assays.

[0098] In another aspect of the application, a method of 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 terminal hairpin loops from another vaccinia virus strain; (ii) transfecting the overlapping DNA fragments into a cell infected with a helper virus; (iii) culturing the cell to produce a mixture of helper virus and synthetic chimeric vaccinia virus particles in the cell; and (iv) plating the mixture on host cells specific for scVACV to recover scVACV. In some embodiments, the scVACV of the method is derived from the NYCBH strain clone Acambis 2000 and the terminal hairpin loops are derived from the vaccinia virus Western Reserve strain.

[0099] Polynucleotides of the invention

[0100] In one aspect, the application provides polynucleotides (e.g., double-stranded DNA fragments) for producing functional synthetic chimeric poxviruses (scVACV). In some embodiments, the application provides methods for producing functional scVACV from synthetic DNA (e.g., chemically synthesized DNA, PCR-amplified DNA, engineered DNA, polynucleotides comprising nucleotide analogs, etc.). In some embodiments, the application provides methods for producing functional scVACV from chemically synthesized overlapping double-stranded DNA fragments of a viral genome. The polynucleotides of aspects of the application can be designed based on publicly available genomic sequences. When a natural isolate of vaccinia virus is readily available, the viral genome can be sequenced prior to selecting and designing the polynucleotides of the application. Alternatively, when partial DNA sequences of a vaccinia virus (e.g., PCR-amplified DNA from a clinical isolate, from a forensic sample, or from material associated with an infected human) are available, the partial viral genome can be sequenced prior to selecting and designing the polynucleotides of the application. In one aspect, the scVACV of the application, and thus the polynucleotides of the application, can be based on the genomic sequence of a naturally occurring strain, variant, or mutant, mutagenized virus, or genetically engineered virus.

[0101] In one aspect, the present application 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 the complement thereof. The isolated polynucleotide of the present application 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 ordinary skill in the art will appreciate that nucleic acid sequences complementary to nucleic acids and variants of nucleic acids also fall within the scope of the present application. In further embodiments, the nucleic acid sequences of the present application can be isolated, recombinant, and / or fused to a heterologous nucleotide sequence, or in a DNA library.

[0102] In some aspects, the present application provides a polynucleotide 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 (TurboFP635), IHD-W, LC16m18, Lederle, Tashkent Clone TKT3, Tashkent Clone TKT4, USSR, Evans, Praha, L-IVP, V-VET1 or LIVP 6.1.1, 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, Vaccinia Calara Chorioallantoic Membrane (CVA), Modified Vaccinia Ankara (MVA), and MVA-BN. In a preferred embodiment, the scVACV is derived from strain NYCBH Clone Acambis 2000 or ACAM2000.

[0103] In one aspect, the present application provides polynucleotides for producing synthetic chimeric vaccinia virus (scVACV). In one particular embodiment, the scVACV genome can be based on the published genome sequence described for VACV strain NYCBH clone ACAM2000 (GenBank Accession No. AY313847; Osborne JD et al. Vaccine. 2007; 25(52):8807-32). Various aspects of the present application show that using the methods of the present application, the terminal hairpin loop from vaccinia virus (VACV) strain WR can be ligated to the ends of VACV genome strain NYCBH clone ACAM2000 to produce a functional scVACV particle. In some embodiments, using the methods of the present application, the terminal hairpin loop from vaccinia virus (VACV) strain ACAM2000 can be ligated to the ends of VACV genome strain NYCBH clone ACAM2000 to produce a functional scVACV particle. The scVACV genome can be divided into 9 overlapping fragments, as described in the working examples of the present application and 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 one 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 one fragment. Fragment sizes are shown in Table 1. The polynucleotides of various aspects of the present application comprise a nucleic acid sequence that is 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, the isolated polynucleotides of the present application comprise variants of these sequences, where such variants can include missense mutations, nonsense mutations, duplications, deletions, and / or additions. SEQ ID NO: 13 and SEQ ID NO: 14 describe the nucleotide sequence of the terminal hairpin loop of VACV (strain WR). SEQ ID NO: 19 and SEQ ID NO: 20 describe the nucleotide sequence of the terminal hairpin loop of VACV (strain ACAM2000). 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 to 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 to SEQ ID NO: 20.

[0104] In other embodiments, the scVACV genome is based on a viral strain selected from the group consisting of Western Reserve (Genbank accession number NC 006998; Genbank accession number AY243312), CL3 (Genbank accession number AY313848), Tian Tian (Genbank accession number AF095689.1), Tian Tian clone TP5 (JX489136), TP3 (Genbank accession number KC207810) and TP5 (Genbank accession number KC207811), NYCBH, Wyeth, Copenhagen (Genbank accession number M35027), NYCBH clone Acambis 2000 (Genbank accession number AY313847), Lister 107 (Genbank accession number DQ121394) Lister-LO (Genbank accession number AY678276), Modified Vaccinia Virus Ankara (MVA) (Genbank accession number U94848; Genbank accession number AY603355), MVA-BN (Genbank accession number DQ983238), Lederle, Tashkent clone TKT3 (Genbank accession number KM044309) and TKT4 (KM044310), USSR, Evans, Praha, LIVP, Ikeda, IHD-W (Genbank accession number KJ125439), LC16m8 (AY678275), EM-63, IC, Malbran, Duke (Genbank accession number DQ439815), 3737 (Genbank accession number DQ377945), VACV-IOC (Genbank accession numbers KT184690 and KT184691), CV-1, Connaught Laboratories, CVA (Genbank accession number AM501482), Serro 2 virus (Genbank accession number KF179385), Cantaglo virus isolate CM-01 (Genbank accession number KT013210), Dryvax clone DPP15 (Genbank accession number JN654981), DPP20 (Genbank accession number JN654985), DPP13 (Genbank accession number JN654980), DPP17 (Genbank accession number JN654983), DPP21 (Genbank accession number JN654986).

[0105] In one aspect, the present application 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 polynucleotide of the present application comprises a variant of a reference sequence, where such variants can include missense mutations, nonsense mutations, duplications, deletions, and / or additions. In some embodiments, the isolated polynucleotide of the present application 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 bp of a reference polynucleotide molecule (e.g., a reference wtVACV genome) of contiguous or non-contiguous nucleotides.

[0106] The present application also encompasses polynucleotides complementary to any of the polynucleotide sequences disclosed herein. The polynucleotides can be single-stranded (coding or antisense) or double-stranded, and can be DNA (genomic or synthetic) or RNA molecules. RNA molecules include mRNA molecules. Additional coding or non-coding sequences can be, but need not be, present in the polynucleotides of the present application, and the polynucleotides can be, but need not be, attached to other molecules and / or supporting materials.

[0107] Two polynucleotide or polypeptide sequences are considered "identical" if the nucleotide or amino acid sequences in the two sequences are identical when aligned for maximum correspondence as described below. Comparison of two sequences is typically performed by comparing sequences over a comparison window to identify and compare sequence similarity local regions. A "comparison window" as used herein, is a segment of at least about 20 contiguous positions, usually 30 to about 75 or 40 to about 50 contiguous positions, wherein the sequence can be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. A polynucleotide or variant can also or alternatively be substantially homologous to a polynucleotide provided by the present application. Such polynucleotide variants are capable of hybridizing to a polynucleotide of the present application (or its complement) under moderately stringent conditions.

[0108] Appropriate "moderate stringency conditions" include prewashing in a solution of 5X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50°C-65°C in 5X SSC; followed by washing in 65°C in 2X, 0.5X, and 0.2X SSC containing 0.1% SDS, each for 20 minutes.

[0109] As used herein, "high stringency conditions" or "high stringency" are as follows: (1) using low ionic strength and high temperature for washing, for example, 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium lauryl sulfate at 50°C; (2) employing a denaturing agent, such as formamide, during hybridization, for example, 50% (v / v) formamide with 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer at pH 6.5, and 750 mM sodium chloride, 75 mM sodium citrate at 42°C; or (3) using 50% formamide, 5x SSC (0.75 M 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, with washes at 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 of skill in the art will know how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length.

[0110] Polynucleotides of the present application can be obtained using chemical synthesis, recombinant methods, or PCR. Methods of chemical polynucleotide synthesis are well known in the art and need not be described in detail here. Those of skill in the art can use the sequences provided herein and commercial DNA synthesizer suppliers to generate the desired DNA sequences.

[0111] To prepare polynucleotides using recombinant methods, a polynucleotide comprising the desired sequence can be inserted into an appropriate vector, and the vector can then be introduced into an appropriate host cell for replication and amplification, as further described herein. The polynucleotide can be inserted into the host cell by any means known in the art. Cells are transformed by the introduction of exogenous polynucleotides by direct uptake, endocytosis, transfection, F- conjugation, or electroporation. Once introduced, the exogenous polynucleotide can be maintained within the cell either as a non-integrated vector, such as a plasmid, or integrated into the host cell genome. Polynucleotides so amplified can be isolated from the host cell by methods well known in the art. See, e.g., Sambrook et al., 1989.

[0112] Alternatively, PCR allows for the replication of DNA sequences. PCR techniques are well known in the art and 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.

[0113] RNA can be obtained by using the isolated DNA in an appropriate vector and inserting it into an appropriate host cell. When the cell replicates and the DNA is transcribed into RNA, the RNA can then be isolated using methods well known in the art, e.g., as set forth in Sambrook et al., 1989, supra.

[0114] In other embodiments, the nucleic acids of the application also include nucleotide sequences that hybridize to the nucleotide sequences set forth in SEQ ID NOs: 1-9, or the complement thereof, under highly stringent conditions. Those of ordinary skill in the art will readily appreciate that the appropriate stringency conditions that promote DNA hybridization can be varied. For example, hybridization can be performed at about 45°C in 6.0x sodium chloride / sodium citrate (SSC) followed by a wash of 2.0x SSC at 50°C. For example, the salt concentration in the wash steps can be selected from a low stringency of about 2.0x SSC at 50°C to a high stringency of about 0.2x SSC at 50°C. In addition, the temperature of the wash steps can range from a low stringency of room temperature, about 22°C, to a high stringency of about 65°C. Both the temperature and the salt concentration can be varied, or one can remain constant while the other is varied. In one embodiment, the application provides nucleic acids that hybridize under low stringency conditions of 6x SSC at room temperature followed by a wash of 2x SSC at room temperature.

[0115] Isolated nucleic acids that differ due to degeneracy in the genetic code also fall within the scope of some aspects of the application. For example, a number of amino acids are designated by more than one triplet. Codons that specify the same amino acid, or synonymous codons (e.g., CAU and CAC are synonymous codons for histidine), can give rise to "silent" mutations that do not affect the amino acid sequence of a protein. Those of skill in the art will appreciate that these variations in one or more nucleotides of a nucleic acid encoding a particular protein can exist due to natural allelic variation in members of a given species. Any and all such nucleotide variations and resulting amino acid polymorphisms are within the scope of the present application.

[0116] The present application in one aspect additionally provides recombinant cloning and expression vectors containing one or more of the polynucleotide molecules of the present application. The present application in one aspect further provides host cells into which a polynucleotide molecule or recombinant vector has been introduced and new strains or cell lines derived therefrom.

[0117] The host cell can be a bacterial cell, a yeast cell, a filamentous fungal cell, an algal cell, an insect cell, or a mammalian cell. In some embodiments, the host cell is E. coli. A wide variety of different vectors have been developed for each of these host cells specifically, including bacteriophages, high copy number plasmids, low copy number plasmids, and shuttle vectors, among others, and any of these can be used to practice the present application.

[0118] Appropriate cloning vectors can be constructed according to standard techniques, or can be selected from the large number of cloning vectors available in the art. Although the cloning vector selected can vary depending on the host cell intended to be used, useful cloning vectors typically have the ability to self-replicate, can have a single target for a particular restriction endonuclease, and / or can carry a gene for a marker that can be used in the selection of clones containing the vector. Appropriate examples include plasmids and bacterial viruses such as pBAD18, pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mp 18, mp 19, pBR322, pMB9, Col El, pCRl, RP4, phage DNAs, and shuttle vectors such as pSA3 and pAT28. These and many other cloning vectors are available from commercial suppliers such as BioRad, Stratagene, and Invitrogen.

[0119] To aid in the selection of host cells transformed or transfected with the cloning vectors of the present application, the vector can be engineered to include a coding sequence for a reporter gene product or other selectable marker. As described above, such coding sequences are preferably operably associated with a regulatory element coding sequence. Reporter genes for use in some aspects of the present application are well known in the art and include those genes 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 a gene product that confers resistance to an antibiotic or an antimetabolite or that provides a nutritional auxotrophy. Examples of these sequences include those encoding resistance to ampicillin, erythromycin, thiostrepton, or kanamycin, among others.

[0120] The vector and / or polynucleotide containing the polynucleotide of interest can be introduced into a host cell by any of more than one appropriate means, including electroporation, transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; microprojectile bombardment; lipofection; and infection (e.g., when the vector is an infectious agent such as vaccinia virus). The choice of introducing the vector or polynucleotide will often depend on the characteristics of the host cell.

[0121] One aspect of the application further provides transformed host cells comprising the polynucleotide molecule or recombinant vector, and novel strains or cell lines derived therefrom. In some embodiments, the host cells used to practice the application are E. coli cells. E. coli strains such as, for example, E. coli TOP10 or E. coli BL21(DE3), DH5a, and the like, which are available from the American Type Culture Collection (ATCC) (10801 University Blvd., Manassas, Va. 20110, USA) and commercial sources, can be typically 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 and the like. Preferred eukaryotic host cells include yeast cells, but mammalian cells or insect cells can also be effectively utilized. Suitable host cells include prokaryotes (such as E. coli, B. subtillis, S. lividans, or C. glutamicum) and yeasts (such as S. cerevisae, S. pombe, P. pastoris, or K. lactis).

[0122] In one aspect, the present application also includes a genome of a scVACV, a recombinant thereof, or a functional portion thereof. A functional portion of a viral genome can be a portion of the genome that encodes a protein or a portion thereof (e.g., a domain, an epitope, etc.), a portion that comprises a regulatory element or a component of a regulatory element (such as a promoter, an enhancer, a cis- or trans-acting element, etc.). The viral sequence can be used to identify or isolate the virus or a recombinant thereof, for example, by using PCR, hybridization techniques, or by establishing an ELISA assay.

[0123] Pharmaceutical compositions of the invention

[0124] In one aspect, the present application relates to a pharmaceutical composition comprising a scVACV of the present application and a pharmaceutically acceptable carrier.

[0125] The term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition (e.g., immunogenic or vaccine formulation) is administered. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectables. 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 formulations should suit the mode of administration.

[0126] In some embodiments, the pharmaceutical compositions of the present application can be administered by standard routes of administration. A number of methods can be used to introduce the formulation into the subject, these include, but are not limited to, intranasal, intratracheal, oral, intradermal, intramuscular, intraperitoneal, intravenous, conjunctival, and subcutaneous routes.

[0127] Illustrative uses

[0128] Preventing or treating pathogenic poxvirus infections

[0129] In some embodiments, the synthetic chimeric vaccinia virus (scVACV) of the present invention can be used for immunization or to trigger or enhance an immune response in a subject to a pathogenic poxvirus infection. In another embodiment, scVACV can be used to trigger or enhance an immune response against a vaccinia virus. In another embodiment, scVACV can be used to trigger or enhance an immune response against smallpox virus. In another embodiment, scVACV can be used to trigger or enhance an immune response against monkeypox virus. In yet another embodiment, scVACV can be used to prevent, control, or treat one or more pathogenic poxvirus infections in a subject, such as for treating smallpox virus infection. In some implementations, scVACV is selected from the following vaccinia virus 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 LIVP 6.1.1, 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, SCVACV-IOC, Ankara chorioallantoic vaccinia virus (CVA), modified Ankara vaccinia virus (MVA), and MVA-BN. In a preferred embodiment, scVACV is derived from NYCBH clone Acambis 2000 or ACAM2000.

[0130] On one hand, the scVACV of the present invention can be used in immunogenic preparations, such as vaccine preparations. The preparations can be used to prevent, control, neutralize, treat, and / or improve pathogenic poxvirus infections. Immunogenic preparations can include live or inactivated scVACV. scVACV can be inactivated by methods known to those skilled in the art. Common methods use formalin and heat for inactivation. In some embodiments, the immunogenic preparation includes a live vaccine. Such a live immunogenic preparation can be produced using conventional methods, including propagating scVACV in cell cultures and subsequently purifying it. For example, 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., as determined by those skilled in the art.

[0131] On the one hand, the scVACV of the present invention can be used for the prevention, control, or treatment of smallpox. On the other hand, the scVACV of the present invention can be used as a vaccine to prevent smallpox in individuals or groups who have been exposed, are potentially exposed, or are at risk of exposure to smallpox. The scVACV of various aspects of the present invention can be used to generate domestic stockpiles of new smallpox vaccines. In some embodiments, the scVACV of the present invention can be administered prophylactically to defense personnel, first responders, etc.

[0132] In one embodiment, a composition comprising the scVACV of the present invention is used as a smallpox vaccine. On one hand, the scVACV of the present invention, produced according to the method according to the invention, will have a small plaque phenotype. Generally, the small plaque phenotype is considered to reflect attenuation. Therefore, the scVACV produced according to various methods of the present invention provides a safe alternative to existing smallpox vaccines. In some embodiments, the vaccine can be safely administered to immunosuppressed subjects (e.g., HIV patients, patients undergoing chemotherapy, patients undergoing cancer treatment, patients with rheumatic diseases or autoimmune diseases, patients undergoing or having received organ or tissue transplants, patients with immunodeficiency, children, pregnant women, patients with atopic dermatitis, eczema, psoriasis, heart disease, and patients using immunosuppressants, etc.), who may suffer from serious complications from existing smallpox vaccines and are therefore contraindicated to using existing smallpox vaccines. In some embodiments, the vaccine can be used in combination with one or more antiviral treatments to inhibit viral replication. In some embodiments, the vaccine can be used in combination with brincidofovir treatment to inhibit viral replication. In some embodiments, the vaccine can be used in combination with tevoirimat / SIGA-246 treatment to inhibit viral replication. In some embodiments, the vaccine may be used in combination with acyclic nucleoside phosphate (cidofovir), an oral alkoxyalkyl prodrug of acyclic nucleoside or phosphonate (brincidofovir or CMX001). In some embodiments, the vaccine may be used in combination with vaccinia immunoglobulin (VIG). In some embodiments, the vaccine may 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 may be used in subjects who have previously been immunized with killed or inactivated VACV. In some embodiments, the vaccine may be used in subjects who have previously been immunized with a replication-deficient / defective VACV virus strain MVA (modified Ankara vaccinia virus). In some embodiments, vaccine formulations comprising the scVACV of the present invention may comprise live or inactivated scVACV.

[0133] In one embodiment, the composition comprising the scVACV of the present invention is used as a smallpox vaccine. The scVACV may be based on a VCV strain selected from the following: ACAM2000 (Genbank accession number AY313847), Western Reserve (Genbank accession number NC 006998; Genbank accession number AY243312), CL3 (Genbank accession number AY313848), Tian Tian (Genbank accession number AF095689.1), Tian Tian clone TP5 (JX489136), TP3 (Genbank accession number KC207810) and TP5 (Genbank accession number KC207811), NYCBH, Wyeth, Copenhagen (Genbank accession number M35027), NYCBH clone Acambis 2000 (Genbank accession number AY313847), and Lister. 107 (Genbank accession number DQ121394), Listener-LO (Genbank accession number AY678276), Modified Ankara Vaccine Virus (MVA) (Genbank accession number U94848; Genbank accession number AY603355), MVA-BN (Genbank accession number DQ983238), Lederle, Tashkent clones TKT3 (Genbank accession number KM044309) and TKT4 (KM044310), U SSR, Evans, Praha, LIVP, Ikeda, IHD-W (Genbank registration number KJ125439), LC16m8 (AY678275), EM-63, IC, Malbran, Duke (Genbank registration number DQ439815), 3737 (Genbank registration number DQ377945), CV-1, Connaught Laboratories, CVA (Genbank registration number AM501482), Serro 2 viruses (Genbank accession number KF179385), Cantaglo virus isolate CM-01 (Genbank accession number KT013210), Dryvax clones DPP15 (Genbank accession number JN654981), DPP20 (Genbank accession number JN654985), DPP13 (Genbank accession number JN654980), DPP17 (Genbank accession number JN654983), DPP21 (Genbank accession number JN654986), and IOCs (Genbank accession numbers KT184690 and KT184691).In one embodiment, the scVACV used as a smallpox vaccine is based on strain ACAM2000 (Genbank accession number AY313847). In one embodiment, the scVACV used as a smallpox vaccine is based on strain VAV-IOC (Genbank accession numbers KT184690 and KT184691). In one embodiment, the scVACV used as a smallpox vaccine is based on strain MVA (Genbank accession number U94848; Genbank accession number AY603355). In one embodiment, the scVACV used as a smallpox vaccine is based on strain MVA-BN (Genbank accession number DQ983238). In some embodiments, the vaccine formulation comprising the scVACV of the present invention may comprise live or inactivated scVACV.

[0134] In some embodiments, compositions containing scVACV of the present invention are used as vaccines against VACV infection, MPXV infection, or CPXV infection.

[0135] In some embodiments, the scVACV of the present invention can be designed to express heterologous antigens or epitopes and can be used as a vaccine against the source organism of such antigens and / or epitopes.

[0136] The immunogenic formulations (e.g., vaccines) of this invention 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 pharmacopoeia for use in animals, and more specifically, in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or medium administered with the pharmaceutical composition (e.g., an immunogenic or vaccine formulation). Saline solutions and aqueous solutions of dextran and glycerol may 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, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, glycol, water, ethanol, etc. Examples of suitable pharmaceutical carriers are described in EWMartin's "Remington's Pharmaceutical Sciences". The formulation should be suitable for the route of administration. Specific formulations may also 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 can be prepared immediately in a pharmaceutical solution. scVACV can also be diluted in physiologically acceptable solutions such as sterile saline, with or without excipients or carriers.

[0137] On one hand, the immunogenic preparations (e.g., vaccines) of the present invention can be administered to patients via scratch administration. Vaccines can also be administered via any other standard route of administration. Numerous methods can be used to introduce the immunogenic preparations (e.g., vaccines), including but not limited to intranasal, intratracheal, oral, intradermal, intramuscular, intraperitoneal, intravenous, conjunctival, and subcutaneous routes. In birds, the method may further include posterior nasal inoculation. As an alternative to parenteral administration, the present invention also includes large-scale administration routes for agricultural purposes, such as via drinking water or spray. Alternatively, scVACV of the present invention can preferably be introduced via its natural route of infection. In some embodiments, the immunogenic preparations of the present invention are administered as injections, consumable transgenic plants expressing vaccines, sustained-release gels or implantable encapsulation compositions, solid implants, or nucleic acids. The immunogenic preparations can also be administered in creams, lotions, ointments, skin patches, lozenges, or oral solutions such as suspensions, solutions, and emulsions (oil-in-water or water-in-oil). An acceptable route of administration for live, replicating smallpox vaccines is percutaneous pricking, which creates viral shedding lesions at the vaccination site that last for several days. These lesions are a potential source of vaccine exposure to individuals who may be contraindicated to receiving live vaccines. Therefore, intramuscular administration of the immunogenic formulation can offer advantages. In one preferred embodiment, scVACV ACAM2000 is administered intramuscularly. In another preferred embodiment, administration is via percutaneous pricking. Intramuscular administration can also be used for other synthetic chimeric orthopox viruses, such as synthetic chimeric horsepox virus (scHPXV). In the case of intramuscular administration, it is important to use a needle of the correct length to reach the muscle mass without penetrating into the subcutaneous tissue. When administering an intramuscular injection, the needle should be inserted at a 90° angle.

[0138] In some embodiments, the immunogenic formulation of the present invention (e.g., a vaccine) does not result in complete protection against infection, but causes a lower titer or a reduced number of pathogens (e.g., pathogenic poxviruses) compared to untreated subjects. In some embodiments, administration of the immunogenic formulation of the present invention results in a reduction of pathogen titer by 0.5 times, 1 time, 2 times, 4 times, 6 times, 8 times, 10 times, 15 times, 20 times, 25 times, 50 times, 75 times, 100 times, 125 times, 150 times, 175 times, 200 times, 300 times, 400 times, 500 times, 750 times, or 1,000 times or greater relative to untreated subjects. The benefits of reducing pathogen titer, number, or total burden include, but are not limited to, a reduction in the severity of infection symptoms and a shorter duration of infection-related illness or condition.

[0139] In some embodiments, the immunogenic preparations (e.g., vaccines) of the present invention do not result in complete protection against infection, but result in a reduction in the number or intensity of symptoms, or a reduction in morbidity or mortality compared to untreated subjects.

[0140] In various embodiments, the immunogenic agent of the present invention (e.g., a vaccine) or an antibody generated by the scVACV of the present invention is administered to a subject in combination with one or more other therapies (e.g., antiviral or immunomodulatory therapies) for the prevention of infection (e.g., pathogenic poxvirus infection). In other embodiments, the immunogenic agent or an antibody generated by the scVACV of the present invention is administered to a subject in combination with one or more other therapies (e.g., antiviral or immunomodulatory therapies) for the treatment of infection (e.g., pathogenic poxvirus infection). In still other embodiments, the immunogenic agent or an antibody generated by the scVACV of the present invention is administered to a subject in combination with one or more other therapies (e.g., antiviral or immunomodulatory therapies) for the control and / or improvement of infection (e.g., pathogenic poxvirus infection). In a specific embodiment, the immunogenic agent or an antibody generated by the scVACV of the present invention is administered to a subject in combination with one or more other therapies (e.g., antiviral or immunomodulatory therapies) for the prevention of smallpox. In yet another specific embodiment, the immunogenic agent or an antibody generated by the scVACV of the present invention is 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 may be used in combination with one or more antiviral treatments to inhibit viral replication. In some embodiments, the vaccine may be used in combination with brincidofovir treatment to inhibit viral replication. In some embodiments, the vaccine may be used in combination with tevirima / SIGA-246 treatment to inhibit viral replication. In some embodiments, the vaccine may be used in combination with acyclic nucleoside phosphates (cidofovir), or oral alkoxyalkyl prodrugs of acyclic nucleosides or phosphonates (brincidofovir or CMX001). In some embodiments, the vaccine may be used in combination with vaccinia immunoglobulin (VIG). In some embodiments, the vaccine may be used in subjects who have previously been immunized with peptide or protein antigens derived from VAV, VARV, or HPXV. In some embodiments, the vaccine may be used in subjects who have previously been immunized with killed or inactivated VAV. In some embodiments, the vaccine may be used in subjects who have previously been immunized with replication-deficient / defective VAV virus strains, or MVA (modified Ankara vaccinia virus).

[0141] Any antiviral agent known to those skilled in the art can be used in formulations (e.g., vaccine formulations) and methods of various aspects of this invention. Non-limiting examples of antiviral agents include proteins, polypeptides, peptides, fusion protein antibodies, nucleic acid molecules, organic molecules, inorganic molecules, and small molecules that inhibit and / or reduce viral binding to its receptors, viral internalization into cells, viral replication, or viral release from cells. Specifically, antiviral agents include, but are not limited to, antiviral agents that block extracellular viral maturation (tevirima / SIGA-246), acyclic nucleoside phosphates (cidofovir), oral alkoxyalkyl prodrugs of acyclic nucleosides or phosphonates (brincidofovir or CMX001), or vaccinia immunoglobulin (VIG). In some implementations, antiviral agents include, but are not limited to, nucleoside analogues (e.g., zidovudine, acyclovir, gangcyclovir, vidarabine, idoxuridine, trifluridine, and ribavirin), foscarnet, amantadine, rimantadine, saquinavir, indinavir, ritonavir, alpha-interferon, and other interferons and AZT.

[0142] Dosage and administration regimen can be determined by those skilled in the art based on the needs of the subject to be treated. Those skilled in the art 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 invention are administered, for example, as needed or daily. Dosage administration can be performed at different time intervals. 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 will last 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.

[0143] In some aspects, the scVACV of the present invention can also be used to generate antibodies suitable for passive immunotherapy, diagnostic or prognostic immunoassays, etc. Methods for generating antibodies are well known in the art. Antibodies can be further modified (e.g., chimeric, humanized, etc.) before use in immunotherapy.

[0144] Oncolytic agents

[0145] The “oncolytic virus” or “oncolytic agent” used in this invention is typically any virus that can kill said tumor cells (non-resistant) by infecting them.

[0146] On the one hand, the synthetic chimeric poxvirus (scVACV) of the present invention can be used as an oncolytic agent for selectively replicating and killing cancer cells in cancer cells. On the other hand, the present invention relates to a method for inducing an oncolytic response in a subject, comprising administering to the subject a composition containing the scVACV of the present invention. Rapidly dividing cells (such as cancer cells) are generally more susceptible to poxvirus infection than undividing cells. Many characteristics of poxviruses, such as safety in the human body, ease of production of high-titer stock solutions, stability of viral preparations, and ability to induce antitumor immunity after replication in tumor cells, make poxviruses ideal oncolytic agents. scVACVs produced according to various methods of the present invention may contain one or more modifications that make them suitable for treating cancer. Thus, on one hand, the present invention provides a method for inducing death in cancer cells, comprising contacting cells with isolated scVACV or a pharmaceutical composition containing the scVACV of the present invention. On one hand, the present invention provides a method for treating cancer, comprising administering to a patient in need a therapeutically effective amount of the scVACV of the present invention. On the other hand, it includes the scVACV or compositions described herein for treating cancer or inducing death in neoplastic conditions. On the other hand, this includes the use of scVACV or compositions described herein to induce cell death in neoplastic disease cells (such as cancer cells) or to treat neoplastic diseases (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 / immunotherapy). In specific embodiments, the oncolytic virus is scVACV NYCBH strain, clone Acambis 2000, or ACAM2000.

[0147] Using the methods of this application, one or more desired genes can be readily introduced and one or more unwanted genes can be deleted from the scVACV genome. In some embodiments, the scVACVs of the present invention, used as oncolytic agents, are designed to express transgenes to enhance their immunoreactivity, antitumor targeting and / or potency, cell-to-cell diffusion, and / or cancer specificity. In some embodiments, the scVACVs of the present invention are designed or engineered to express immunomodulatory genes (e.g., GM-CSF or viral genes that block TNF function). In some embodiments, the scVACVs of the present invention are designed to include genes expressing attenuated virulence factors. In some embodiments, the 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, the scVACVs of the present invention have been designed or engineered to include the gmCSF gene. In some embodiments, the scVACVs of the present invention have been modified to attenuate virulence. In some embodiments, the scVACVs of the present invention are designed or engineered to lack the viral thymidine kinase (TK) gene. In some embodiments, the scVACVs of the present invention are 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.

[0148] On one hand, the scVACV of the present invention is applicable to the treatment of a wide variety of neoplastic conditions and / or cancers. In some embodiments, the cancer types 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 such as hepatocellular carcinoma, leukemia, lung cancer, lymphoma, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, skin cancer such as melanoma, testicular cancer, etc., or any other treatable tumors or precancerous lesions.

[0149] In another embodiment, the method further includes detecting the presence of the administered scVACV in a neoplastic condition or cancer cells and / or in a sample of a subject who has received the isolated or recombinant virus or composition described herein. For example, the subject may be tested before and / or after administration of the scVACV or composition described herein to assess, for example, the progression of an infection. In some embodiments, the scVACV of the present invention includes a detection kit, and detecting the presence of the administered chimeric VACV includes detecting a protein encoded by the detection kit. For example, where the detection kit encodes a fluorescent protein, the subject or sample is imaged using a method that visualizes fluorescence.

[0150] In one aspect, the oncolytic formulation of the present invention comprises an effective amount of the scVACV of the present invention and a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" has already been explained in the foregoing sections.

[0151] In some embodiments, the compositions of the present invention are administered in a poxvirus treatment facility. In some aspects, a poxvirus treatment facility is a facility in which subjects requiring immunization or treatment with the compositions or methods of the present invention can be immunized or treated in an environment that isolates them from other subjects who are not intended to be immunized or treated, or other subjects potentially infected by the treated subject (e.g., caregivers and family members). In some embodiments, subjects not intended to be immunized or potentially infected by the treated subject include HIV patients, patients undergoing chemotherapy, patients undergoing treatment for cancer, rheumatic diseases, or autoimmune diseases, patients receiving or having received organ or tissue transplants, patients with immunodeficiency, children, pregnant women, patients with atopic dermatitis, eczema, psoriasis, heart disease, and patients using immunosuppressants, 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.

[0152] In some embodiments, the composition of the present invention containing scVACV is administered by an expert in smallpox adverse events. In some embodiments, smallpox adverse events include, but are not limited to, vaccine-induced eczema, progressive pox, post-vaccination encephalitis, myocarditis, and dilated cardiomyopathy.

[0153] Viral vectors for recombinant gene expression

[0154] On the one hand, the synthetic chimeric poxvirus (scVACV) of the present invention can be engineered to carry a heterologous sequence. The heterologous sequence can originate from a different poxvirus species or from any non-poxvirus source. On the other hand, the heterologous sequence is an antigenic epitope selected from any non-poxvirus source. As used in this application, a non-poxvirus source refers to an organism different from a poxvirus. 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, rhabdovirus, influenza virus, viruses of the Flaviviridae family, paramyxovirus, hepatitis virus, human immunodeficiency virus, or viruses that cause hemorrhagic fever (such as hantaviruses or filoviruses, i.e., Ebola or Marburg virus). On the other hand, the heterologous sequence is an antigenic epitope from a different poxvirus species. These viral sequences can be used to modify the host spectrum or immunogenicity of scVACV.

[0155] In some embodiments, the scVACV of the present invention can encode a heterologous gene / nucleic acid expressing a therapeutic nucleic acid (e.g., an antisense nucleic acid) or a therapeutic peptide (e.g., a peptide or protein having the desired biological activity).

[0156] In some embodiments, the expression of the heterologous nucleic acid sequence is preferably, but not exclusively, under the transcriptional control of the vaccinia virus promoter. In some embodiments, the heterologous nucleic acid sequence is preferably inserted into a non-essential region of the viral genome. Methods for inserting the heterologous sequence into the vaccinia virus genome are known to those skilled in the art. In some embodiments, the heterologous nucleic acid is introduced by chemical synthesis. In exemplary embodiments, the heterologous nucleic acid can be cloned into the VACV105 / J2R locus of the scVACV of the present invention.

[0157] The scVACV of one aspect of this invention can be used to introduce a heterologous nucleic acid sequence into target cells, the sequence being homologous or heterologous to the target cell. Introducing the heterologous nucleic acid sequence into target cells can be used to generate, in vitro, heterologous peptides or polypeptides encoded by said sequence and / or intact viruses. In one embodiment, the method includes infecting host cells with the scVACV of this invention; culturing the infected host cells under appropriate conditions; and isolating and / or enriching the peptides, proteins, and / or viruses produced by the host cells. Those skilled in the art are familiar with appropriate conditions for culturing scVACV-infected host cells to express heterologous peptides or polypeptides, and these conditions vary depending on the host cells used (see, for example, Molecular Cloning: A Laboratory Manual, 2nd edition (Sambrook et al., 1989)).

[0158] It should be understood that the embodiments of this application described herein merely illustrate some applications of the principles of this application. Based on the teachings provided herein, those skilled in the art can make many modifications without departing from the true spirit and scope of this application.

[0159] The following embodiments are illustrated to represent this application. These embodiments should not be construed as limiting the scope of the invention, as these and other equivalent embodiments will be apparent from the present disclosure, the accompanying drawings, and the appended embodiments.

[0160] Examples

[0161] Example 1. Selection and design of overlapping segments of viral genome

[0162] Synthetic chimeric VACV ACAM2000 (scVACV ACAM2000-WR DUP / HP) containing VACV WR strain hairpin and double-stranded sequences.

[0163] The scVACV genome was designed based on the previously described genome sequence of VCV ACAM2000 [GenBank accession number AY313847] (Osborne JD et al., Vaccine. 2007; 25(52):8807-32). The genome was divided into nine overlapping segments (Figure 1). These segments were designed to share at least 1.0 kbp of overlapping sequence (i.e., homology) with each adjacent segment to provide sites for homologous recombination to drive the assembly of the full-length genome (Table 1). These overlapping sequences provided sufficient homology for accurate recombination between co-transfected segments (Yao XD, Evans DH. Journal of Virology. 2003; 77(13):7281-90).

[0164] Table 1: VAV ACAM2000 genome fragments used in this study. The size and sequence within the VAV ACAM2000 genome [GenBank accession number AY313847] are described.

[0165] Fragment name Size (bp) Sequence GA_LITR ACAM2000 18,525 SEQ ID NO: 1 GA_FRAG_1 ACAM2000 24,931 SEQ ID NO: 2 GA_FRAG_2 ACAM2000 23,333 SEQ ID NO: 3 GA_FRAG_3 ACAM2000 26,445 SEQ ID NO: 4 GA_FRAG_4 ACAM2000 26,077 SEQ ID NO: 5 GA_FRAG_5 ACAM2000 24,671 SEQ ID NO: 6 GA_FRAG_6 ACAM2000 25,970 SEQ ID NO: 7 GA_FRAG_7 ACAM2000 28,837 SEQ ID NO: 8 GA_RITR ACAM2000 17,641 SEQ ID NO: 9

[0166] To facilitate subcloning of these fragments, the AarI and BsaI restriction sites were silenced in all fragments (except for the two ITR-coding fragments). In cases where these regions contain nucleotide sequence-specific recognition sites important for efficient DNA replication and multiply resolution, the BsaI restriction sites in these two ITR-coding fragments were not mutated.

[0167] By introducing the YFP / gpt box, controlled by the early and late promoters of poxviruses, into the thymidine kinase locus, the reactivation of VACV ACAM2000 (VACV ACAM2000 YFP-gpt::105) can be easily visualized under a fluorescence microscope. The Gpt locus also provides a potential tool for selecting reactivated viruses using drug selection.

[0168] Traditionally, terminal hairpins are difficult to clone and sequence, so it is not surprising that the published sequence of the VCV ACAM2000 genome is incomplete. Upon examining the extremely terminal regions of the published VCV ACAM2000 strain, some differences appear between ACAM2000 and the highly characterized VCV WR strain (Genbank accession number #AY243312) (Figure 2). In the WR strain, there is a 70 bp tandem repeat immediately downstream of a covalently closed hairpin loop located at the 5' and 3' ends of the VCV genome. These are followed by two 125 bp repeats and eight 54 bp repeats (…). Figure 2A However, among the published VACV ACAM2000 sequences, only four 54bp repeat sequences were identified. Figure 2BSequencing (using Illumina) confirmed the presence of 70bp, 125bp, and 54bp repetitive sequences in the wild-type isolate of VCV ACAM2000, indicating that the currently published ACAM2000 sequence is incomplete. Due to the short read length of Illumina reads (<300 nucleotides), the inventors could not accurately determine which actual ACAM2000 genome sequence was in this ~3kbp form. Instead, the inventors decided to reconstruct the VACVACAM2000 virus (Figure 2) with a sequence similar to VACV WR, extending from the terminal hairpin to just before the stop codon of the C23L gene. This included both the 125bp and 54bp tandem repeat sequences, which, although not included in the published ACAM2000 sequence, were detected during next-generation Illumina sequencing of wtVACV ACAM2000. At the 5' ends of the modified VCV ACAM2000 left and right ITR fragments, NheI restriction sites are also included, allowing direct ligation of a 70 bp tandem repeat sequence to the ITR end (discussed in Example 2). The F and S-terminal hairpin loop sequences of wtVACV ACAM2000 are shown respectively... Figure 9 And in SEQ ID NO:20 and 19.

[0169] Synthetic chimeric VACV ACAM2000 (scVACVACAM2000-ACAM2000 DUP / HP) containing hairpin and double-stranded sequences of VACV ACAM2000 strain.

[0170] The scVACV genome was designed based on the previously described VACV ACAM2000 genome sequence [GenBank accession number AY313847] (Osborne JD et al., Vaccine. 2007; 25(52):8807-32). The genome was divided into nine overlapping segments (Figure 1). These segments were designed to share at least 1.0 kbp of overlapping sequence (i.e., homology) with each adjacent segment to provide sites for homologous recombination to drive the assembly of the full-length genome (Table 1). These overlapping sequences provided sufficient homology to allow for accurate recombination between co-transfected segments (Yao XD, Evans DH. Journal of Virology. 2003; 77(13):7281-90).

[0171] To facilitate subcloning of these fragments, the AarI and BsaI restriction sites were silenced in all fragments (except for the two ITR-coding fragments). The BsaI restriction sites in these two ITR-coding fragments were not mutated, even though these regions contain nucleotide sequence-specific recognition sites important for efficient DNA replication and concatenator resolution.

[0172] By introducing the YFP / gpt box, controlled by the early and late promoters of poxviruses, into the thymidine kinase locus, the reactivation of VACV ACAM2000 (VACV ACAM2000 YFP-gpt::105) can be easily visualized under a fluorescence microscope. The Gpt locus also provides a potential tool for selecting reactivated viruses using drug selection.

[0173] The F and S terminal hairpin loop sequences of wtVACV ACAM2000 were respectively in Figure 9 As shown in SEQ ID NO:20 and 19.

[0174] Example 2

[0175] Attach the VCV WR F and S end hairpins to the right and left ITR segments of the VCV ACAM2000.

[0176] Synthesize the same 70bp repeat fragment as VACV WR strain ( Figure 2C (SEQ ID NO: 10). SapI and NheI restriction sites are included at the 5' and 3' ends of the 70bp tandem repeat to facilitate ligation to the VACV WR hairpin sequence and the right and left ITR fragments of VACVACAM2000, respectively. Before the VACV WR terminal hairpin loop can be ligated to the 70bp tandem repeat, the loop must be extended by an additional 58bp using a double-stranded sequence synthesized via IDT. Figure 3A This is because the extra sequence immediately follows the polysynthetic resolution site, preceding the first 70 bp repeat sequence found in the VCV strain WR. The double-stranded sequence is generated by synthesizing two single-stranded DNA molecules, which, when annealed together, produce a double-stranded DNA with a 5'-TGT overhang at the 5' end and a 5'-GGT overhang at the 3' end. Figure 3A (SEQ ID NO:11 and SEQ ID NO:12). Because the F and S terminal hairpin loops of VACV WR produce 3'-ACA protrusions at their terminal loops, a 58 bp double strand is attached to the hairpin to generate a ~130 bp terminal hairpin loop that appears identical to the sequence found in the VACV WR strain up to 70 bp at the beginning of the repeating sequence. Figure 3BThe hairpin / double-stranded fragment was gel-purified and then subsequently ligated to the SapI-digested ends of a 70 bp repeat fragment. Digestion of the 70 bp tandem repeat fragment with SapI yielded a tribase overhang (5'-CCA), complementary to the 5'-GGT overhang in the terminal hairpin / double-stranded structure. The 70 bp tandem repeat was then ligated to the F-terminal hairpin / double-stranded structure (…). Figure 4 Lane 4) or S-terminal hairpin / double chain structure ( Figure 4 Lane 5) was mixed with a ~5-fold molar excess of the 70bp tandem repeat fragment in the presence of DNA ligase. This was compared with a reaction containing only 70bp ( Figure 4 Compared to lane 3, this produces an upward shift in the DNA electrophoresis gel, indicating that the terminal hairpin / double strand has been successfully ligated to the 70bp tandem repeat fragment. Figure 4 ).

[0177] The terminal hairpin / double-stranded / 70bp tandem repeat fragment was then ligated to the 70bp ACAM2000 left or right ITR fragment (previously modified at their ends to include NheI restriction sites). During digestion, the 5'-CTAG overhang remained at their 5' ends. At the 3' end of the 70bp tandem repeat fragment, the fragment was directly ligated to the LITR and RITR regions of the VACV ACAM2000 DNA fragment using the NheI site. After digestion of the VACV ACAM2000 left and right ITR fragments, the S-terminal hairpin / double-stranded / 70bp tandem repeat fragment or the F-terminal hairpin / double-stranded / 70bp tandem repeat fragment was 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, followed by transfection into Scyphoblastic fibroma virus (SFV)-infected BGMK cells.

[0178] Attach the F and S end hairpins of the VCV ACAM2000 to the right and left ITR segments of the VCV ACAM2000.

[0179] A 70 bp repeat fragment identical to that of the VCV 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 VCV ACAM2000 hairpin sequence and the right and left ITR fragments of VCV ACAM2000, respectively. Before the VCV ACAM2000 terminal hairpin loop could be ligated to the 70 bp tandem repeat fragment, the loop had to be extended by an additional 58 bp using a double-stranded sequence synthesized by IDT Technologies. This is because the extra sequence is immediately downstream of the multiplex resolution site, preceding the first 70 bp repeat sequence found in the VCV strain ACAM2000. The double-stranded sequence was synthesized by synthesizing two single-stranded DNA molecules, which, when annealed together, would produce a double-stranded DNA molecule (SEQ ID NO:21 and SEQ ID NO:22) with a 5'-TGT overhang at the 5' end and a 5'-GGT overhang at the 3' end. Because VACV ACAM2000F and S-terminal hairpin loops produce 3'-ACA overhangs on their terminal loops, a 58 bp doublet was ligated to the hairpin to produce a ~130 bp terminal hairpin loop. This hairpin / doublet fragment was gel-purified and then subsequently ligated to the SapI-digested end of a 70 bp repeat fragment. Digestion of the 70 bp tandem repeat fragment with SapI produced a tribase overhang (5'-CCA), which is complementary to the 5'-GGT overhang in the terminal hairpin / doublet structure. The 70 bp tandem repeat was mixed with either the F-terminal or S-terminal hairpin / doublet structure in a ~5-fold molar excess relative to the 70 bp tandem repeat fragment in the presence of DNA ligase. This produced an upshift in the DNA electrophoresis gel compared to a reaction with only 70 bp, indicating that the terminal hairpin / doublet was successfully ligated to the 70 bp tandem repeat fragment.

[0180] The terminal hairpin / double-stranded / 70bp tandem repeat fragment was then ligated to the left or right ITR fragment of ACAM2000 (which had previously been modified at their ends to include NheI restriction sites). When the left or right ITR fragment was digested, the 5'-CTAG overhang remained at their 5' ends. At the 3' end of the 70bp tandem repeat fragment, the fragment was directly ligated to the LITR and RITR regions of the VACV ACAM2000 DNA fragment using the NheI site. After digestion of the left and right ITR fragments of VACV ACAM2000, the S-terminal hairpin / double-stranded / 70bp tandem repeat fragment or the F-terminal hairpin / double-stranded / 70bp tandem repeat fragment was ligated to the left or right ITR fragment, respectively, using DNA ligase at a 1:1 molar ratio and overnight at 16°C. The DNA ligase was then heat-inactivated at 65°C, followed by transfection into Scyphoblastic fibromatosis virus (SFV)-infected BGMK cells.

[0181] Example 3. Preparation of VACV ACAM2000 overlapping DNA fragments

[0182] Each VACV ACAM2000 overlapping DNA fragment from Table 1 was cloned into a plasmid provided by GeneArt using the restriction enzyme I-SceI. Before transfecting these synthesized DNA fragments into BGMK cells, the plasmids were digested with I-SceI, and the products were run on a gel to confirm successful linearization of the DNA fragments. Figure 5 After digestion at 37°C for 2 hours, the reactants were subsequently heat-inactivated at 65°C. The samples were stored on ice or at 4°C until terminal hairpins / double strands / 70bp tandem repeats / ITR fragments formed (as described above).

[0183] Example 4. Reactivation from chemically synthesized dsDNA fragments

[0184] SFV strains Kasza and BSC-40 were originally obtained from the American Center for Type Culture Collection. Buffalo green monkey kidney (BGMK) cells were obtained from G. McFadden (University of Florida). BSC-40 and BGMK cells were propagated at 37°C in 5% CO2 in minimal basal medium (MEM) supplemented with L-glutamine, non-essential amino acids, sodium pyruvate, antibiotics and antifungal agents (antimycotics), and 5% fetal bovine serum (FCS; Thermo Fisher Scientific).

[0185] Reactivation of scVACV ACAM2000-WR DUP / HP or scVACV ACAM2000-ACAM2000 DUP / HP in Hepburn fibromatosis virus-infected cells

[0186] Buffalo green monkey kidney (BGMK) cells were cultured in 60 mm tissue culture dishes containing MEM until they reached approximately 80% confluence. Cells were infected with Schipper's fibromatosis virus (SFV) at 0.5 MOI in serum-free MEM at 37°C for 1 hour. The inoculum was replaced with 3 ml of warm MEM containing 5% FCS and returned to the incubator for another hour. Meanwhile, the transfection reaction was set up as follows. After approximately 2 hours at 37°C, linearized VAV ACAM2000 fragments were transfected into SFV-infected BGMK cells at molar equivalents based on the length of each fragment containing the VAV ACAM2000 genome (using Lipofectamine 2000). Different amounts of total DNA were experimented with, and 5, 6, and 7.5 μg of DNA successfully reactivated ACAM2000 from these overlapping DNA fragments. The complexes were incubated at room temperature for 10 minutes and then added dropwise to the previously SFV-infected BGMK cells. Approximately 24 hours after infection, replace the culture medium with fresh MEM containing 5% FCS. Incubate the cells at 37°C for another 3-4 days (4-5 days in total).

[0187] Viral particles were recovered by scraping infected cells into cell culture medium and performing three freeze-thaw cycles. The crude extract was diluted 10⁻⁶ in serum-free MEM. -2 4 ml of inoculum was plated onto 9-16 150 mm tissue culture plates containing BSC-40 cells to recover reactivated scVACV ACAM2000 YFP-gpt::105. One hour post-infection, the inoculum was replaced with MEM containing 5% FCS and 0.9% Noble agar. Yellow fluorescent spots were observed under an inverted microscope, and individual spots were picked for further analysis. The scVACV ACAM2000YFP-gpt::105 spots were purified three times using yellow fluorescent selection.

[0188] Four days later, infected plates containing both SFV and VACV ACAM2000 clones were harvested, followed by three freeze-thaw cycles to release the virus. The resulting products were then serially diluted and plated onto BSC-40 cells, which preferentially promoted the growth of VACV ACAM2000 virus compared to SFV. Three rounds of plaque purification were performed, followed by amplification of the viral stockpile in 10–150 mm tissue culture plates. The virus was subsequently lysed from these cells and isolated on a 36% sucrose pad, followed by further purification on a 24%–40% sucrose density gradient. Genomic DNA was isolated from these purified genomes and subjected to next-generation Illumina sequencing to confirm the sequence of the synthesized viral genome.

[0189] Example 4. Growth properties compared to wild-type ACAM2000 virus

[0190] In vitro multi-step growth curves of synthetic chimeric VCV ACAM2000-WR DUP / HP, scVACV ACAM2000-ACAM2000 DUP / HP, and wild-type VCV ACAM2000 virus isolated from monkey kidney epithelial (BSC-40) cells. Cells were infected with a multiplicity of infection of 0.03, and the virus was harvested at specified time points (3h, 6h, 12h, 21h, 48h, and 72h), and the virus was titrated on BSC-40 cells. Figure 6 The data shown represents three independent experiments. For example... Figure 6 As shown, during the 72-hour period, scVACV ACAM2000-WR DUP / HP and wtVACV ACAM2000 viruses grew with indistinguishable growth kinetics.

[0191] Comparison of growth curves between scVACV ACAM2000-WR DUP / HP (YFP-gpt marker), scVACV ACAM2000-ACAM2000DUP / HP (YFP-gpt marker), scVACV ACAM2000-WR DUP / HP (no marker) (YFP-gpt marker replaced with J2R gene sequence), scVACV ACAM2000-ACAM2000 DUP / HP (no marker) (YFP-gpt marker replaced with J2R gene sequence), and wtVACV ACAM2000 showed no statistically significant difference in growth properties compared to wtACAM2000 VACV. Figure 7 ).

[0192] Example 5. The genomic sequence of scVACV ACAM2000-WR DUP / HP YFP-gpt::105 was confirmed by PCR and restriction fragment analysis.

[0193] Genomic DNA isolated using sucrose gradient centrifugation was further analyzed by restriction digestion followed by pulsed-field gel electrophoresis (PFGE) on the scVACV ACAM2000YFP-gpt::105 genome (Yao XD, Evans DH. Methods Mol Biol. 2004; 269:51-64). Two independent scVACV ACAM2000-WR DUP / HP clones were purified, along with a VACV_WRΔJ2R control (where the J2R gene sequence had been replaced with the YFP-gpt marker) and a wtVACV ACAM2000 control (VAC_ACAM2000), and then kept undigested and digested with BsaI, HindIII, or NotI and PvuI. Genomic DNA isolated from scVACV ACAM2000-WR DUP / HP and wtVACV ACAM2000 was digested with BsaI and HindIII. Since most BsaI sites in the scVACV ACAM2000 genome have been silenced, most of the intact ~200kbp fragments were 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 fully digested when treated with BsaI. Figure 8 Lanes 6 and 7). To confirm that the scVACV ACAM2000-WR DUP / HP genome could still be digested with another enzyme, these genomes were digested with HindIII, which produced numerous bands from the scVACV ACAM2000-WR DUP / HP clone ( Figure 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 ( Figure 8 (Lane 14 to 17).

[0194] In the wtVACV WR control (VAC_WRΔJ2R) sample, a band at approximately 3.6 kbp (marked with an asterisk) was detected, which contains all 70 bp tandem repeats in the VACV WR strain. Given that the VACV WR strain is used as a template for the synthesis of ITR repeat elements, some bands of approximately the same size as those observed in the WR strain were expected to be detected in NotI / PvuI-treated scVACVACAM2000 clones. A difference in the size of this region was observed when comparing two scVACV ACAM2000-WR DUP / HP clones, suggesting that not all 70 bp repeats were incorporated into each reconstructed genome. Figure 7Lanes 16 and 17). Given that others have shown that these repetitive elements can expand and contract under selection pressure in cell cultures, this is not surprising (Paez and Esteban (1988). Virology; 163(1):145-54).

[0195] In summary, in vitro analysis of the scVACV ACAM2000-WR DUP / HP YFP-gpt::105 genome suggests that reactivation of VACV ACAM2000-WR DUP / HP from chemically synthesized DNA fragments is successful, and the scVACV ACAM2000-WR DUP / HP virus behaves similarly to the wtVACV ACAM2000 virus in vitro.

[0196] Example 6. The genome sequence of scVACV ACAM2000YFP-gpt::105 was confirmed by whole-genome sequence analysis.

[0197] Two scVACV ACAM2000-WR DUP / HP clones and two scVACV ACAM2000-ACAM2000DUP / HP clones were sequenced. Illumina reads were assembled de novo using a CLC Genome Workstation (version 11) with a word length of 35 or 61. The assembled contigs were then imported into Snapgene software and aligned with a reference sequence based on the expected scACAM2000 sequence of the synthesized fragment provided by GeneArt.

[0198] For clone 1 of scVACV ACAM2000-WR DUP / HP, contiguous group 1 is 16,317 bp and corresponds to most of the ITR region (except for tandem repeats). Contiguous group 2 is 167,020 bp and aligns with the central conserved region of the genome (nucleotide positions 19,467 to 186,486). For clone 2 of scVACV ACAM2000-WR DUP / HP, contiguous group 3 is 16,322 bp and corresponds to most of the ITR region (except for tandem repeats). Contiguous group 1 is 167,020 bp and aligns with the central conserved region of the genome (nucleotide positions 19,467 to 186,486). A single nucleotide substitution (C to A) is present at nucleotide position 136791 in the contiguous group of clone 2. This corresponds to nucleotide positions 156,256 in the scACAM2000 genome sequence and results in an amino acid change from Asp to Tyr in VAC_ACAM2000_177(A41L).

[0199] For clone 1 of scVACV ACAM2000-ACAM2000 DUP / HP, contiguous group 1 is 167,020 bp and aligns with the central conserved region of the genome (nucleotide positions 19,469 to 186,488). Contiguous group 2 is 16,150 bp and corresponds to most of the ITR region (except for tandem repeat sequences). When this contiguous group is mapped to the reference genome in Snapgene, gaps in the sequence are observed at positions 2633 to 3417 and nucleotide positions 15,175 to 15220. The first gap region corresponds to a 54 bp repeat region and is most likely due to the inaccurate assembly of these regions using de novo assembly tools. Directly mapping the raw Illumina reads to the reference genome does not result in any gaps in either region. For clone 2 of scVACVACAM2000-ACAM2000DUP / HP, contiguous group 1 is 16,075 bp and corresponds to most of the ITR region (except for tandem repeat sequences). Contiguous group 2 is 167,078 bp and aligned with a central conserved region of the genome (nucleotide positions 19,469 to 186,546). A gap was observed in contiguous group 2 from nucleotide positions 15,176 to 15,220. However, directly mapping the raw Illumina reads to the reference genome did not result in any gap in this region. Clones sequenced by scVACVACAM2000-ACAM2000 DUP / HP showed no additional nucleotide mutations at any location in the genome.

[0200] Illumina readings were also mapped to a reference map in the CLC genome. The Illumina readings 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, respectively, and average coverage of 2195 and 1602 for clones 1 and 2 of scVACVACAM2000-ACAM2000DUP / HP, respectively.

[0201] Overall, the sequencing data confirmed the in vitro genomic analysis data and demonstrated the successful reactivation of scVACV ACAM20000-WR DUP / HP and scVACV ACAM2000-ACAM2000 DUP / HP in SFV-infected cells.

[0202] Example 7. Removal of YFP / gpt selection markers

[0203] After reactivating scVACV ACAM2000 YFP-gpt::105, the yfp / gpt selection marker in the thymidine kinase locus can be removed.

[0204] Example 8. Nucleotide sequence variations among various VCV strains within the double-stranded regions of the terminal hairpins and ITRs.

[0205] Figure 9 This study shows nucleotide sequence changes in the "double-stranded" region directly downstream of the multiply resolving site in VCV WR, ACAM 2000, Dryvax, and Copenhagen strains. Compared to the WR strain, the sequence changes observed in wtACAM2000, DryvaxDPP15, TianTan, and Copenhagen strains are 4 nucleotide substitutions and 3 nucleotide deletions.

[0206] Example 9. Determination of toxicity in a mouse intranasal model or by tail scratch method

[0207] This study determined the toxic effects of scVACV ACAM20000-WR DUP / HP and scVACV ACAM2000-ACAM2000DUP / HP. For this experiment, six groups of Balb / c mice were administered three different doses of scVACV ACAM20000-WR DUP / HP and scVACV ACAM2000-ACAM2000DUP / HP as described in Examples 1-7, and compared with a PBS control group, a wtVACV(WR) control group, and a wtVACV ACAM2000 control group (a total of 12 treatment groups). Three additional mice that did not receive any treatment during the study period were included in this experiment. Blood samples were taken from all mice at predetermined points throughout the experiment, and these additional mice were used as the baseline for serum analysis.

[0208] Prior to inoculation of Balb / c mice, all viral strains were cultured in BSC-40 cells (African green monkey kidneys), harvested by trypsin digestion, washed in PBS, extracted from cells by Dounce homogenization, purified by ultracentrifugation via a 36% sucrose pad, resuspended in PBS, and titrated to a final concentration of 10. 7 PFU / ml up to 10 9 PFU / ml.

[0209] Based on previous studies using known VACC vaccine virus strains (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), the dose (10) used in this study was selected. 5 PFU / dosage, 10 6 PFU / dosage and 10 7 PFU / dosage).

[0210] The virus is administered intranasally or via tail scratching. See Examples 10 and 11 below for details.

[0211] Example 10. Determining whether intranasal administration of scVACV confers immune protection against lethal VACV-WR challenge.

[0212] Since weight loss was used as a measure of toxicity in mice, the result was calculated at 5 x 10⁻⁶. 3 Intranasal administration of PFU at doses of wtVACV (WR strain) resulted in approximately 20-30% weight loss. Also, at 10... 7 PFU / dose intranasal administration of the VCV Dryvax clone DPP15 allows the virulence of this well-known smallpox vaccine to be directly comparable to the synthetic versions scVACV ACAM20000-WRDUP / HP and scVACV ACAM2000-ACAM2000 DUP / HP. Mice were purchased from Charles River Laboratories and acclimatized to their environment for at least one week upon receipt before virus administration.

[0213] Each mouse was simultaneously administered a single dose of virus (~10 μl) via intranasal injection under anesthesia. Mice were monitored daily for signs of infection (such as swelling, excretion, or other abnormalities) for 30 days. Weight loss in each mouse was specifically monitored daily following virus administration. Mice that experienced a weight loss exceeding 25% (excluding other pathogenic factors) were euthanized according to the University of Alberta's animal health care facility protocol.

[0214] Even at the highest tested doses of scVACV ACAM20000-WR DUP / HP and scVACV ACAM2000-ACAM2000 DUP / HP, Balb / c mice showed no obvious signs of disease. One of the VACV strains (Brazilian smallpox vaccine strain IOC), in some cases, at 10... 7 PFU does not cause 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). Due to the difficulty in preparing titers exceeding 10 9 The purified stock solution contains PFU / mL, so testing doses higher than this is impractical.

[0215] Thirty days after viral vaccination, a lethal dose of VACV-WR (10) was administered intranasally. 6 Mice were challenged with PFU (dose). Mice were closely monitored for signs of infection as described above. Mice were weighed daily, and those showing a weight loss exceeding 25% (excluding other pathogenic factors) were euthanized. Mice expected to show significant weight loss and other signs of pathogenic factors within 7–10 days prior to administration of a lethal dose of VAV-WR were anticipated. Approximately 14 days after lethal challenge with VAV-WR, all mice were euthanized, and blood was collected to confirm the presence of VAV-specific neutralizing antibodies in serum by a standard plaque reduction assay.

[0216] Example 11. Determining whether scVACV administered via the tail scratch method confers immune protection against lethal VCV-WR challenge.

[0217] Anesthetize immune-active Balb / C mice before beginning the tail scratching procedure. At the base of the tail, use the tip of a 25-gauge needle to make a series of 15-20 scratches / punctures over a length of 1-2 cm. Apply 3-5 μL of a different virus to the scratch sites.

[0218] Mice were anesthetized until the virus had a chance to be absorbed into the scratch site. Signs of weight loss in mice were monitored daily over a 28-day period. Pustules (known as "acquired") formed at the scratch site on the tail approximately 8–10 days post-scratching.

[0219] Twenty-eight days after viral vaccination, a lethal dose of VACV-WR (10) was subsequently administered intranasally. 6 Mice were challenged with PFU (dose). Mice were closely monitored for signs of infection as described above. Mice were weighed daily, and those showing a weight loss exceeding 25% (excluding other pathogenic factors) were euthanized. Mice expected to show significant weight loss and other signs of pathogenic factors within 7–10 days prior to administration of a lethal dose of VAV-WR were anticipated. Approximately 14 days after lethal challenge with VAV-WR, all mice were euthanized, and blood was collected to confirm the presence of VAV-specific neutralizing antibodies in serum by a standard plaque reduction assay.

[0220] All unvaccinated animals died within 7 days of viral challenge due to the lethal dose of VACV WR.

[0221] This application provides the following implementation scheme:

[0222] 1. A synthetic chimeric vaccinia virus (scVACV), said virus being replicated and reactivated from synthetic DNA, said virus having a viral genome that differs from the wild-type genome of said virus in that it is characterized by one or more modifications.

[0223] 2. The scVACV according to embodiment 1, wherein the synthetic DNA is selected from one or more of the following: chemically synthesized DNA, PCR-amplified DNA, engineered DNA, and polynucleotides containing nucleoside analogs.

[0224] 3. The scVACV according to embodiment 1, wherein the synthetic DNA is chemically synthesized DNA.

[0225] 4. The scVACV according to any one of embodiments 1-3, wherein the one or more modifications include one or more deletions, insertions, substitutions or combinations thereof.

[0226] 5. The scVACV according to any one of embodiments 1-4, wherein the one or more modifications include one or more modifications that eliminate one or more unique restriction sites.

[0227] 6. The scVACV according to any one of embodiments 1-4, wherein the one or more modifications include one or more modifications that add or repair one or more unique restriction sites.

[0228] 7. The scVACV according to any one of embodiments 1-5, wherein the one or more modifications include one or more modifications that eliminate one or more AarI restriction sites.

[0229] 8. The scVACV according to any one of embodiments 1-5, wherein the one or more modifications include one or more modifications that eliminate all AarI restriction sites.

[0230] 9. The scVACV according to any one of embodiments 1-5, wherein the one or more modifications include one or more modifications that eliminate one or more BsaI restriction sites.

[0231] 10. The scVACV according to any one of embodiments 1-9, wherein the viral genome comprises a heterologous terminal hairpin loop.

[0232] 11. The scVACV according to any one of embodiments 1-10, wherein the viral genome contains a terminal hairpin loop derived from different vaccinia virus strains.

[0233] 12. The scVACV according to any one of embodiments 1-11, wherein the viral genome contains a terminal hairpin loop derived from the VACV WR strain.

[0234] 13. The scVACV according to any one of embodiments 1-9, wherein the viral genome contains homologous or heterologous terminal hairpin loops, and wherein the tandem repeat region contains a different number of repeats than wtVACV.

[0235] 14. The scVACV according to any one of embodiments 1-13, wherein the viral genome is the genome of a VCV strain 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, Lister107, 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 LIVP. 6.1.1, 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, VCV-IOC, Ankara chorioallantoic vaccinia virus (CVA), Modified Ankara vaccinia virus (MVA), and MVA-BN.

[0236] 15. The scVACV according to embodiment 14, wherein the viral genome of the scVACV is based on the genome of the NYCBH strain clone Acambis 2000.

[0237] 16. The scVACV according to embodiment 14, wherein the viral genome of the scVACV is based on the genome of the NYCBH strain clone Dryvax.

[0238] 17. The scVACV according to embodiment 14, wherein the viral genome of the scVACV is based on the genome of the Lister strain V-VET1.

[0239] 18. The scVACV according to embodiment 14, wherein the viral genome of the scVACV is based on the genome of a modified Ankara vaccinia virus (MVA) strain.

[0240] 19. The scVACV according to embodiment 14, wherein the viral genome of the scVACV is based on the genome of the MVA-BN strain.

[0241] 20. The scVACV according to embodiment 14, wherein the viral genome of the scVACV is based on the genome of the IOC strain.

[0242] 21. The scVACV according to any one of embodiments 1-20, 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 a slow form of the vaccinia virus terminal hairpin loop; or d) both comprise a fast form of the vaccinia virus terminal loop.

[0243] 22. The scVACV according to embodiment 21, wherein the slow form comprises at least 85% identical nucleotide sequences to the nucleotide sequences of SEQ ID NO:13 or SEQ ID NO:19, and the fast form comprises at least 85% identical nucleotide sequences to the nucleotide sequences of SEQ ID NO:14 or SEQ ID NO:20.

[0244] 23. The scVACV according to embodiment 22, wherein the slow form comprises at least 90% identical nucleotide sequences to the sequence of SEQ ID NO:13 or SEQ ID NO:19, and the fast form comprises at least 90% identical nucleotide sequences to the nucleotide sequences of SEQ ID NO:14 or SEQ ID NO:20.

[0245] 24. The scVACV according to embodiment 23, wherein the slow form comprises at least 95% identical nucleotide sequences to the nucleotide sequences of SEQ ID NO:13 or SEQ ID NO:19, and the fast form comprises at least 95% identical nucleotide sequences to the nucleotide sequences of SEQ ID NO:14 or SEQ ID NO:20.

[0246] 25. The scVACV according to embodiment 24, wherein the slow form consists of the nucleotide sequence of SEQ ID NO:13 or SEQ ID NO:19, and the fast form consists of the nucleotide sequence of SEQ ID NO:14 or SEQ ID NO:20.

[0247] 26. The scVACV according to any one of embodiments 1 to 25, wherein the virus is replicated and reactivated from overlapping chemically synthesized DNA fragments corresponding to substantially the entire viral genome of scVACV.

[0248] 27. The scVACV according to embodiment 26, wherein the virus is replicated and reactivated from 2-14 overlapping fragments.

[0249] 28. The scVACV according to embodiment 27, wherein the virus is replicated and reactivated from 6-12 overlapping fragments.

[0250] 29. The scVACV according to embodiment 28, wherein the virus is copied and reactivated from nine overlapping fragments.

[0251] 30. The scVACV according to any one of embodiments 1 to 29, wherein the virus is reactivated using recombination and reactivation catalyzed by rabbitpox virus.

[0252] 31. The scVACV according to embodiment 30, wherein the rabbitpox virus is selected from the group consisting of: Schipper's fibroma virus (SFV), wild rabbit fibroma virus, domestic rabbit fibroma virus, squirrel fibroma virus, and myxoma virus.

[0253] 32. A method for preparing synthetic chimeric vaccinia virus (scVACV), comprising the following steps:

[0254] (i) Chemical synthesis of overlapping DNA fragments corresponding to essentially the entire viral genome of vaccinia virus;

[0255] (ii) Transfect the overlapping DNA fragment into cells that help the virus infect;

[0256] (iii) Culturing the cells to produce a mixture of helper virus and synthetic chimeric vaccinia virus particles in the cells; and

[0257] (iv) The mixture is plated on host cells that are specific to the scVACV to recover the scVACV.

[0258] 33. The method according to embodiment 32, wherein the helper virus is selected from the group consisting of: rabbitpox virus, fowlpox virus and psoralen-inactivated helper virus.

[0259] 34. The method according to embodiment 33, wherein the rabbitpox virus is selected from the group consisting of: Shoemaker-Schönlein fibroma virus (SFV), wild rabbit fibroma virus, domestic rabbit fibroma virus, squirrel fibroma virus, and myxoma virus.

[0260] 35. The method according to embodiment 34, wherein the rabbitpox virus is SFV.

[0261] 36. The method according to any one of embodiments 32 to 35, wherein the cells assisting in viral infection are BGMK cells.

[0262] 37. The method according to any one of embodiments 32 to 36, wherein step (i) further comprises chemically synthesizing a terminal hairpin loop from another VAV strain and attaching it to a fragment containing the left and right ends of the viral genome.

[0263] 38. The method according to any one of embodiments 32 to 37, wherein the overlapping DNA fragment comprises:

[0264] (i) A nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO:1-9;

[0265] (ii) A nucleotide sequence that is at least 90% identical to the sequence of SEQ ID NO:1-9;

[0266] (iii) A nucleotide sequence that is at least 95% identical to the sequence of SEQ ID NO:1-9; or

[0267] (iv) A nucleotide sequence consisting of the sequences of SEQ ID NO:1-9.

[0268] 39. A synthetic chimeric vaccinia virus (scVACV) produced by any one of embodiments 32 to 38.

[0269] 40. A pharmaceutical composition comprising scVACV as described in any one of embodiments 1 to 31 and a pharmaceutically acceptable carrier.

[0270] 41. The pharmaceutical composition according to embodiment 40, wherein the scVACV is inactivated.

[0271] 42. The pharmaceutical composition according to embodiment 41, wherein the inactivation is carried out by heating, UV or formalin.

[0272] 43. A method for inducing an oncolytic response in a subject, comprising administering to the subject a composition comprising scVACV as described in any one of embodiments 1 to 31 or a pharmaceutical composition as described in any one of embodiments 40 to 42.

[0273] 44. A method for expressing a heterologous protein in a host cell, comprising introducing a heterologous nucleic acid sequence into a scVACV according to any one of embodiments 1 to 31, infecting a host cell with the scVACV, and culturing the host cell under conditions expressing the heterologous protein.

[0274] 45. The method according to embodiment 44, wherein the heterologous nucleic acid is derived from a different poxvirus species or from any non-poxvirus source.

[0275] 46. ​​A method of triggering or enhancing an immune response against vaccinia virus, comprising administering to a subject in need a composition comprising scVACV as described in any one of embodiments 1 to 31 or a pharmaceutical composition as described in any one of embodiments 40 to 42.

[0276] 47. A method of triggering or enhancing an immune response against smallpox virus, comprising administering to a subject in need a composition comprising scVACV as described in any one of embodiments 1 to 31 or a pharmaceutical composition as described in any one of embodiments 40 to 42.

[0277] 48. A method of triggering or enhancing an immune response against monkeypox virus, comprising administering to a subject in need a composition comprising scVACV as described in any one of embodiments 1 to 31 or a pharmaceutical composition as described in any one of embodiments 40 to 42.

[0278] 49. A method of immunizing a human subject to protect the subject from smallpox virus infection, comprising administering to the subject a composition comprising scVACV of any one of embodiments 1 to 31 or a pharmaceutical composition of any one of embodiments 40 to 42.

[0279] 50. A method of treating smallpox virus infection, comprising administering to a subject in need a composition comprising scVACV as described in any one of embodiments 1 to 31 or a pharmaceutical composition as described in any one of embodiments 40 to 42.

[0280] 51. A method of treating cancer in a subject, comprising administering to a subject in need a composition comprising scVACV as described in any one of embodiments 1 to 31 or a pharmaceutical composition as described in any one of embodiments 40 to 42.

[0281] 52. The method according to any one of embodiments 43 or 46 to 51, wherein the application may be selected from skin incision, intramuscular or intravenous application.

[0282] 53. The method according to any one of embodiments 43 or 46 to 52, wherein the composition is applied in a poxvirus treatment facility.

[0283] 54. The method according to any one of embodiments 43 or 46 to 53, wherein the composition is applied by an expert in smallpox adverse events.

[0284] 55. The method according to embodiment 54, wherein the smallpox adverse event is selected from: cowpox eczema, progressive pox, post-vaccination encephalitis, myocarditis, and dilated cardiomyopathy.

Claims

1. A synthetic chimeric vaccinia virus (scVACV) that is replicated and reactivated from DNA derived from synthetic DNA, the viral genome of the virus differing from the wild-type genome of the virus by one or more modifications.

2. A method of making 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 cells infected with a helper virus; (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 scVACV.

3. A synthetic chimeric vaccinia virus (scVACV) produced by the method of claim 2.

4. A pharmaceutical composition comprising the scVACV of claim 1 and a pharmaceutically acceptable carrier.

5. Use of a composition comprising the scVACV of claim 1 or the pharmaceutical composition of claim 4 in the manufacture of a medicament for inducing an oncolytic response in a subject.

6. A method of expressing a heterologous protein in a host cell comprising introducing a heterologous nucleic acid sequence into the scVACV of claim 1, infecting a host cell with the scVACV and culturing the host cell under conditions to express the heterologous protein.

7. Use of a composition comprising the scVACV of claim 1 or the pharmaceutical composition of claim 4 in the manufacture of a medicament for triggering or enhancing an immune response against one or more of vaccinia virus, variola virus, and monkeypox virus.

8. Use of a composition comprising the scVACV of claim 1 or the pharmaceutical composition of claim 4 in the manufacture of a medicament for immunizing a human subject to protect the subject from one or more of variola virus infection, vaccinia virus infection, and monkeypox virus infection.

9. Use of a composition comprising the scVACV of claim 1 or the pharmaceutical composition of claim 4 in the manufacture of a medicament for treating one or more of variola virus infection, vaccinia virus infection, and monkeypox virus infection.

10. Use of a composition comprising the scVACV of claim 1 or the pharmaceutical composition of claim 4 in the manufacture of a medicament for treating cancer in a subject.

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