VERO CELL LINES STABLY EXPRESSING HSV ICP0 PROTEIN

Abstract

Provided herein are Vero cell lines that stably express Herpes Simplex Virus (HSV) ICP0 protein. These cells have the same morphology of Vero cells, exhibit stable expression of HSV ICP0 protein, and also efficiently complement replication of HSV ICP0 deficient virus for greater than 20, 30, or even 40 cell passages.

Claims

1. A Vero cell line comprising a nucleotide sequence that encodes for a Herpes Simplex virus (HSV) ICP0 protein operably linked to a promoter, wherein the promoter comprises SEQ ID NO:1.

2. The Vero cell line of claim 1, wherein the promoter comprises SEQ ID NO: 2.

3. The Vero cell line of claim 1, wherein the nucleotide sequence encodes for HSV-1 ICP0 that comprises the amino acid sequence of SEQ ID NO: 5.

4. The Vero cell line of claim 1, wherein, the cell line exhibits Vero cell morphology.

5. The Vero cell line of claim 1, wherein the cell line is capable of maintaining ICP0 complementation efficiency within 2 standard deviations as measured by a viral replication assay for greater than 20 cell passages, 30 cell passages, or 40 cell passages.

6. (canceled)

7. (canceled)

8. The Vero cell line of claim 1, wherein the nucleotide sequence that encodes for a Herpes Simplex virus (HSV) ICP0 protein operably linked to a promoter comprises or consists essentially of SEQ ID NO: 4.

9. (canceled)

10. The Vero cell line of claim 1, wherein the cell line further comprises an additional nucleic acid that encodes a tetracycline repressor protein (tetR) operably linked to a promoter.

11. The Vero cell line of claim 10, wherein the promoter operably linked to the tetracycline repressor protein comprises SEQ ID NO: 9.

12. The Vero cell line of claim 10, wherein the nucleic acid that encodes a tetracycline repressor protein encodes the amino acid sequence of SEQ ID NO: 11.

13. The Vero cell line of claim 10, wherein the nucleic acid that encodes tetracycline repressor protein (tetR) operably linked to a promoter comprises or consists essentially of SEQ ID NO: 7.

14. (canceled)

15. The Vero cell line of claim 9, which is V0-584 or V0R-124.

16. (canceled)

17. The Vero cell line of claim 1, further comprising a gene encoding antibiotic resistance.

18. The Vero cell line of claim 1, further comprising an ICP0 deficient HSV virus.

19. The Vero cell line of claim 1, further comprising a nucleic acid encoding a recombinant protein of interest that is operably linked to a promoter.

20. The Vero cell line of claim 19, wherein the recombinant protein of interest is a therapeutic protein.

21. The Vero cell line of claim 20, wherein the therapeutic protein is a vaccine protein.

22. A method of producing a viral vaccine of interest comprising propagating a virus to be used for vaccination in a Vero cell line of claim 1.

23. The method of claim 22, wherein the virus to be used for vaccination is an ICP0 deficient HSV virus.

24. The method of claim 22, wherein the virus is an adenovirus.

25. (canceled)

26. (canceled)

27. A method of producing ICP0 deficient HSV virus comprising i) infecting a Vero cell line of any of claim 1, with an ICP0 deficient HSV virus, ii) incubating the cell line in a tissue culture medium; and iii) collecting the ICP0 deficient virus produced by the cell line.

Description

BRIEF DESCRIPTION OF FIGURES

[0026] FIG. 1 is a graph that shows plaque-forming efficiency of N2-lacZ, an HSV-2 ICP0 null mutant, in Vero cells, V0-584 cells and U2OS cells. Vero cells and V0-584 cells were seeded at 610e5 cells per 60 mm dish and U2OS cells were seeded at 1.2510.sup.6 cells per 60 mm dish. At 46-48 h post-seeding, triplicate dishes of U2OS cells, V0-584 cells, and Vero cells were infected with N2-lacZ at various PFU/dish. After 1.5 h incubation at 37 C. in an inoculation volume of 0.5 ml/dish, methylcellulose was added. Plaques were stained with neutral-red at 72 h post-infection and counted a day later. The number of input PFU was based on the titer on U2OS cell monolayers.

[0027] FIG. 2 is a graph that shows the plaque-forming efficiency of CJ2-gD2 in V0-584 cells, V0R-124 cells and U2CEP4R-11 cells. V0-584 cells and V0R-124 were seeded at 610.sup.5 cells per 60 mm dish and U2CEP4R-11 cells were seeded at 1.2510.sup.6 cells per 60 mm dish. At 46-48 h post-seeding, triplicate dishes of U2CEP4R-11 cells, V0-584 cells and V0R-124 cells were infected with CJ2-gD2 at various PFU/dish. After 1.5 h incubation at 37 C in an inoculation volume of 0.5 ml/dish, methylcellulose was added. Plaques were stained with neutral-red at 72 h post-infection and counted a day later. The number of input PFU was based on the titer on U2CEP4R-11 cell monolayers.

[0028] FIG. 3 is a light microscope photograph panel that indicates V0R-124 cells are morphologically similar to Vero cells. Vero cells and V0R-124 cells were seeded at 610.sup.5 cells per 60 mm dish. Cells were photographed under the light microscope at 48 h post-seeding.

[0029] FIG. 4 is a graph of plaque-forming efficiency of CJ2-gD2 in V0R-124 cells in either the absence or presence of tetracycline. V0R-124 were seeded at 610.sup.5 cells per 60 mm dish. At 45 h post-seeding, triplicate dishes of V0R-124 cells were infected with CJ2-gD2 at 150 PFU/dish for plaque assay in the absence of doxycycline and at 1.510.sup.4 PFU/dish for plaque-assay in the presence of doxycycline. Plaques were stained with neutral-red at 72 h post-infection and counted a day later. The number of input PFU was based on the titer on V0R-124 cell monolayers.

[0030] FIG. 5 is a fluorescent microscope photo panel that indicate regulation of eGFP expression from the tetO-containing HCMV major immediate-early promoter in V0R-124 cells in the absence or the presence of doxycycline. V0R-124 cells and Vero cells were seeded at 510.sup.5 cells per 60 mm dish. At 21 h post-seeding, duplicate dishes of V0R-124 cells and Vero cells were transfected with 0.1 ug/dish of pCDNA4TO-eGFP and 1.5 ug/dish of pCDNA3 by Lipofectinamine 2000. Transfection medium was removed at 4 h post-transfection followed by addition of growth medium with or without doxycycline. Expression of EGFP was photographed under the fluorescence microscopy at 24 h and 48 h post-transfection.

DESCRIPTION

[0031] Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0032] The practice of embodiments of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, Current Protocols in Immunology (J. E. Coligan et al., eds., 1999, including supplements through 2016); Current Protocols in Molecular Biology (F. M. Ausubel et al., eds., 1987, including supplements through 2016); Short Protocols in Molecular Biology, F. M. Ausubel et al., eds., fifth edition 2002, including supplements through 2016; Molecular Cloning: A Laboratory Manual, third edition (Sambrook and Russel, 2001); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); The Immunoassay Handbook (D. Wild, ed., Stockton Press NY, 1994); Bioconjugate Techniques (Greg T. Hermanson, ed., Academic Press, 1996); Methods of Immunological Analysis (R. Masseyeff, W. H. Albert, and N. A. Staines, eds., Weinheim: VCH Verlags gesellschaft mbH, 1993), Harlow and Lane Using Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1999; and Beaucage et al. eds., Current Protocols in Nucleic Acid Chemistry John Wiley & Sons, Inc., New York, 2000, including supplements through 2016).

[0033] As used herein, the term cell line refers to a tissue cultured cell that has been cloned and can be represented by a single cell or a cell population propagated from the single cell clone.

[0034] As used herein the term Vero cell line refers to a cell line derived from the kidney of a normal adult African green monkey. For example, on Mar. 27, 1962, a cell line derived from the kidney of a normal adult African green monkey named CCL-81 was isolated by Y. Yasumura and Y. Kawakita at the Chiba University in Chiba, Japan; ATCC CCL-81 (Manassas, Va. 20110 USA). An optional growth medium for a Vero cell line is Sigma's Delbecco's modified Eagle's Medium (Sigma, ST. LOUIS, Mo., 63103, USA), Catalog No. D5796. Complete growth medium, is made by adding the following components to the base medium: fetal bovine serum to a final concentration of 10%. Cells are passaged normally, which is by cell dilution at a time when there is about 100% confluence in a tissue culture dish.

[0035] As used herein Vero cell line morphology refers to the phenotypic shape and membrane structure of a Vero cell which can be determined by microscope (see for example FIG. 3, that shows the morphology of Vero Cells by light microscope).

[0036] As used herein the term herpes simplex virus (HSV) refers to both HSV type 1 and HSV type 2. See e.g. Fatahzadeh M1, Schwartz R A. Human herpes simplex virus infections: epidemiology, pathogenesis, symptomatology, diagnosis, and management, J Am Acad Dermatol. 2007 November; 57(5):737-63, ATCC holdings (Manassas, Va. 20110 USA) include a number of HSV-1 and HSV-2 strains, including for example: HSV-1 HF; HSV-1 MacIntyre; HSV-1 KOS; HSV-1 GHSV-UL46; HSV-1 ATCC-2011-9; HSV-2 MS; HSV-2 G; HSV-2 ATCC-2011-2.

[0037] As used herein, the term ICP0 protein refers to the HSV protein that is an immediate-early protein which possesses E3 ubiquitin ligase activity. ICP0 activates HSV-1 gene expression, disrupts nuclear domain (ND) 10 structures, mediates the degradation of cellular proteins, and enables evasion of the host's antiviral defenses. As used herein the term ICP0 deficient HSV refers to a recombinant HSV vector whose genome does not encode active ICP0 or fully functional ICP0, i.e. ICP0 with normal wild type function. Activity of ICP0 can be monitored using any of the means known to those in the art, See e.g. Miles C Smith et al, HSV-1 ICP0: paving the way for viral replication Future Virol. 2011 April; 6(4): 421-429; Mirna P Lanfranca et al., HSV-1 ICP0: An E3 Ubiquitin Ligase that counteracts host intrinsic and immunity, Cells 2014 3:438-454.

[0038] There are many variants of HSV ICP0 protein, e.g. some of HSV-1 ICP0, strain KOS variants are: Genebank Accession: P08393.1 GI: 124134; Accession: AFI23590.1 GI: 384597746; Accession: AFI23649.1 GI: 384597805; Accession: AFE62827.1 GI: 380776964; Accession: AFE62886.1 GI: 380777023; Accession: ADM22381.1 GI: 304318198; Accession: AL018731.1 GI: 952947655; Accession: ALO18672.1 GI: 952947596; Accession: ALO18655.1 GI: 952947578; Accession: ALO18596.1 GI: 952947519; Accession: AKH80472.1 GI: 822581062; Accession: AKH80399.1 GI: 822580988; Accession: AKG61929.1 GI: 820021112; Accession: AKG61857.1 GI: 820021035; etc. and the like. Each strain of HSV1 or of HSV2 have multiple variants, all with functional ICP0. These variants are well known in the art and can be found in protein databases. Such variants may be used in methods of the invention. Examples of HSV-2 ICP0 variants, include but are not limited to: Accession: YP_009137210: YP_009137210.1 GI:820945210; Accession: YP_009137151.1 GI: 820945151; Accession: AEV91397.2 GI: 556197555; Accession: AEV91338.2 GI: 556197550; Accession: ADG01890.1 GI: 295322885; Accession: ADG01889.1 GI: 295322883; Accession: ADG01888.1 GI: 295322881; Accession: ADG01887.1 GI: 295322879; Accession: ADG01885.1 GI: 295322875; Accession: ADG01886.1 GI: 295322877; etc, and the like.

[0039] As used herein, the term variant or in the context of polypeptides or proteins refers to a polypeptide or protein that comprises an amino acid sequence which has been altered by the introduction of amino acid residue substitutions, deletions and/or additions. Typically substitutions are conservative amino acid substitutions, however non-conservative substitutions can be made that do not destroy the functionality of the protein, e.g. HSV ICP0. Conservative amino acid substitutions refers to replacing one amino acid with another having similar structural and/or chemical properties, e.g. such as the replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine, or glycine with another small amino acid residue. Conservative substitution tables providing functionally similar amino acids are well known in the art. As used herein, the term non-conservative refers to substituting an amino acid residue for a different amino acid residue that has different chemical properties. The non-conservative substitutions include, but are not limited to aspartic acid (D) being replaced with glycine (G); asparagine (N) being replaced with lysine (K); or alanine (A) being replaced with arginine (R). For purposes of embodiments of the invention non-conservative substitutions may reduce but does not destroy the proteins normal function.

[0040] As used herein the term comprising or comprises is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the invention, yet open to the inclusion of unspecified elements, whether essential or not.

[0041] As used herein the terms, consisting essentially of, or variations such as consists essentially of, or consist essentially of refer to the inclusion of any recited elements, or group of elements, and the optional inclusion of other elements, of similar or different nature than the recited elements, that do not materially change the basic properties of the claimed elements. For example, a nucleotide sequence that consists essentially of a recited sequence may also include additional one or more nucleic acid additions, deletions, or substitutions that do not materially change by a statistically significant amount the expression level of ICP0 and the ability of the ICP0 protein to complement HSV replication at high efficiency. For example, substitutions may correlate to the degenerative amino acid code.

[0042] The term consisting of refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment. For example, the nucleotide sequence has no additions, deletions or substitutions.

[0043] As used herein, the terms protein are used interchangeably and refer to a polymer or oligomer of consecutive amino acid residues.

[0044] As used herein, the terms nucleotide sequence refers to DNA molecule sequences (e.g., cDNA or genomic DNA.

[0045] As used herein, the term promoter refers to regulatory control nucleic acid sequences involved in transcription of nucleotide coding sequences, which may or may not include enhancer elements. Such a promoter may be inducible or constitutive. The term operably linked refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A promoter operably linked to a coding sequence is ligated in such a way that expression of the coding sequence is achieved.

[0046] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term about. The term about when used in connection with percentages may mean 10%, or even e.g. 20%, preferably 10%, more preferably 5%, still more preferably 1%. In addition, the singular terms a, an, and the include plural referents unless context clearly indicates otherwise. Similarly, the word or is intended to include and unless the context clearly indicates otherwise. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. The abbreviation, e.g. is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation e.g. is synonymous with the term for example.

[0047] The term statistically significant or significantly refers to statistical significance and generally means a two standard deviation (2SD) above or below a normal or reference level. The term refers to statistical evidence that there is a difference. The decision is often made using the p-value. If within two standard deviations than there is not a statistically significant difference.

[0048] Embodiments of the invention are based, in part, upon the surprising successful generation of a Vero cell line that encodes ICP0 protein at levels able to complement ICP0 deficient HSV replication at high efficiency, yet at levels that the ICP0 protein remains stably expressed for greater than 20, greater than 30, and even greater than 40 passages. The stability and low toxicity of expression of the ICP0 protein while still being able to complement ICP0 deficient HSV replication at high efficiency (e.g. as evidenced by plaque formation assay) is quite surprising. In particular, we have identified a promoter system that enables the optimal balance of ICP0 expression in Vero cells. The promoter system uses a promoter that comprises SEQ ID NO: 1.

[0049] In certain embodiments, the promoter comprises the addition of VP16 responsive elements, e.g. one, two or three VP16 responsive elements, e.g. see SEQ ID NO: 2.

[0050] In particular, cell lines can now be produced that complement replication of ICP0 deficient HSV at high efficiency for greater than 20, greater than 30 and even greater than 40 passages.

[0051] In embodiments of the invention efficiency of complementation can be determined using any HSV viral replication assay known to those of skill in the art. One such assay is a plaque forming assay also referred to as a plaque forming efficiency assay. HSV plaque assays determine the number of plaque forming units (pfu) in a virus sample, which is one measure of virus quantity and replication, e.g. if measuring the amount of virus produced by a particular Vero cell line. For example, a confluent monolayer of host cells can be infected with HSV ICP0 deficient virus, e.g. produced from a Vero Cell line described herein, at varying dilutions and covered with a semi-solid medium, such as methylcellulose or agar, to prevent the virus infection from spreading indiscriminately. A viral plaque is formed when a virus infects a cell within the fixed cell monolayer. The virus infected cell will lyse and spread the infection to adjacent cells where the infection-to-lysis cycle is repeated. The infected cell area will create a plaque (an area of infection surrounded by uninfected cells) which can be seen by neutral-red staining or with an optical microscope. Plaque formation can take 3-5 days, depending on the virus being analyzed. Plaques are generally counted manually and the results, in combination with the dilution factor used to prepare the plate, are used to calculate the number of plaque forming units per sample unit volume (pfu/mL). The pfu/mL result represents the number of infective particles within the sample and is based on the assumption that each plaque formed is representative of one infective virus particle. Other assays to monitor replication efficiency include but are not limited to, e.g. a focus forming assay (FFA), protein assays, enzyme-linked immunosorbent assay (ELISA), quantitative polymerase chain reaction (qPCR), and flow cytometry, such assays are well known in the art (e.g. Kaufmann, S. H.; Kabelitz, D. (2002). Methods in Microbiology Vol. 32: Immunology of Infection; Martin, S. J. (1978). The Biochemistry of Viruses. Cambridge University Press; Flint, S. J.; Enquist, W.; Racaniello, V. R.; Skalka, A. M. (2009). Virological Methods. Principles of Virology. ASM Press). The HSV replication assay (e.g. an assay to determine how much virus is produced by the Vero cell) that is used to determine ICP0 complementation efficiency of an ICP0 expressing Vero cell line described herein, can include a control reference for High efficiency of ICP0 complementation, such as a control cell line, e.g. U2OS or U2CEP4R-11 cells are known to produce infectious virus at concentration (titer) of 7-8 log.sub.10 PFU/ml. For comparison the control U2OS cell line value should be seeded to normalize cell number taking inconsideration the growth rates of the cells as to represent the same number of cells producing virus as the tested Vero cell line. As used herein, high efficiency refers to having a plaque-forming efficiency for ICP0 null mutants that is equal to, or less than, that observed with U2OS cells, but no more than 10 fold less than that observed with U2OS cells. In one embodiment, the plaque forming efficiency for ICP0 null mutants in the Vero cell line is no more than 5 fold less, or 4 fold less, or 2 fold less, than what is observed with U2OS cells. U2OS cells are available from the American Type Culture Collection (ATCC); (U-2 OS ATCC HTB96).

[0052] In certain embodiments, the Vero cell line that stably expresses ICP0 has a parental Vero cell morphology. Cell lines may be visualized by light microscopy to assess morphology, see for example FIG. 3. Cell growth kinetics can also be monitored. In one embodiment, the cell growth kinetics of the Vero cell line expressing ICP0 has the same or close to growth kinetics as the parental Vero cells.

[0053] In certain embodiments, the HSV ICP0 is selected from HSV-1 ICP0, or variant thereof, and HSV-2 ICP0, or variant thereof. In one embodiment, the nucleotide sequence encodes HSV-1 ICP0, or variant thereof. In one embodiment the nucleotide sequence encodes HSV-1 ICP0 that comprises the amino acid sequence of SEQ ID NO: 5. In one embodiment, the nucleotide sequence encodes HSV-2 ICP0 that comprises the amino acid sequence of SEQ ID NO: 6.

[0054] In one aspect, a Vero cell line is provided that comprises a nucleotide sequence that encodes for Herpes Simplex Virus (HSV) ICP0 protein, wherein the nucleotide sequence comprises SEQ ID NO: 4. In one embodiment, the Vero cell line exhibits the same Vero cell morphology as the cell line from which it was derived (the parental Vero Cell line morphology). In certain embodiments, the Vero cell line is capable of maintaining ICP0 complementation efficiency, e.g. within 1 or within 2 standard deviations as measured by plaque forming efficiency assay, for greater than 20 cell passages, or for greater than 30 cell passages, or for even for greater than 40 cell passages.

[0055] In one aspect, a Vero cell line is provided that comprises a nucleotide sequence that encodes for a Herpes Simplex Virus (HSV) ICP0 protein e.g. a HSV-1 ICP0 protein or a HSV-2 ICP0 protein, operably linked to a promoter wherein the promoter comprises SEQ ID NO: 2.

[0056] In one aspect, a Vero cell line is provided that comprises a nucleotide sequence that encodes for a Herpes Simplex Virus (HSV) ICP0 protein e.g. a HSV-1 ICP0 protein or a HSV-2 ICP0 protein, operably linked to a promoter wherein the promoter consists essentially of SEQ ID NO: 2.

[0057] In one aspect, a Vero cell line is provided that comprises the nucleotide sequence of SEQ ID NO: 4, which is a sequence that encodes for a HSV-1 ICP0 protein operably linked to the promoter of SEQ ID NO: 2.

[0058] In certain embodiments of each of these aspects, the Vero cell line further comprises an additional nucleic acid that encodes a tetracycline repressor protein (tetR) operably linked to a promoter.

[0059] As used herein, a tetracycline repressor protein (tetR) refers to a transcriptional repressor protein that regulates transcription (See e.g Ramos, Juan L et al. (2005-06-01). The TetR Family of Transcriptional Repressors. Microbiology and Molecular Biology Reviews 69 (2): 326-356, 2005). Many tetR proteins are known in the art and are suitable for use in embodiments of the invention. In one embodiment, the nucleic acid that encodes a tetracycline repressor protein that encodes the amino acid sequence of SEQ ID NO: 11, or variant thereof.

[0060] Any promoter known to those of skill in the art may be operably linked to the TetR nucleic acid in embodiments of the invention. In one embodiment, the promoter operably linked to the nucleic acid encoding tetR, comprises SEQ ID NO: 9. In certain embodiments, a beta-globin intron is inserted between the promoter and the tetR nucleotide sequence that encodes the tetR protein (of SEQ ID NO: 7).

[0061] In another aspect of the invention, a method of using these Vero cell lines to produce recombinant ICP0 deficient Herpes Simplex Virus (HSV) is provided. The method comprises propagating an ICP0 deficient HSV virus in a Vero cell line of any of each of these aspects of the invention. In one embodiment, the method comprises i) infecting the Vero cell line with an ICP0 deficient HSV virus, ii) incubating the cell line in a tissue culture medium; and iii) collecting the ICP0 deficient virus produced by the cell line. Methods for producing and isolation HSV virus are well known to those of skill in the art, See e.g. Goins WF1, et al. Construction and production of recombinant herpes simplex virus vectors, Methods Mol Biol. 2008;433:97-113; and Herpes Simplex Virus: Propagation, Quantification, and Storage, Current protocols in microbiology by John Wiley & Sons, Inc. Chapter 14, 14. E.1-14E.23, contributed by John A. Blaho, Elise R. Morton, and Jamie C. Yedowitz, October 2005). The Vero cell lines described herein use the same medium as suggested for Vero cells.

[0062] Methods for generating Vero cell lines that stably expresses HSV ICP0 protein and that efficiently complements ICP0 deficient HSV are also provided. The methods comprise contacting Vero cells with a nucleic acid that encodes HSV ICP0 (e.g. HSV1 or HSV2 ICP0 ) operably linked to a promoter, e.g. wherein the promoter comprises SEQ ID NO: 1 or SEQ ID NO: 2, and screening for complementation of HSV ICP0 deficient virus. Standard transfection protocols known in the art can be used for contacting the Vero cells with the nucleic acids described herein. See e.g. Current Protocols in Molecular Biology (F. M. Ausubel et al., eds., 1987, including supplements through 2016). Complementation efficiently can be monitored using any HSV replication efficiency assay known to those in the art, e.g. a plaque forming assay or ELISA assay.

[0063] In one embodiment, the method comprises contacting Vero cells with a nucleotide sequence that encodes for a Herpes Simplex virus (HSV) ICP0 protein operably linked to a promoter, wherein the promoter comprises SEQ ID NO:1. In one embodiment, the method comprises contacting Vero cells with a nucleotide sequence that encodes for a Herpes Simplex virus (HSV) ICP0 protein operably linked to a promoter, wherein the promoter comprises SEQ ID NO:2. In one embodiment, the method comprises contacting Vero cells with a nucleotide sequence that encodes for a Herpes Simplex virus (HSV) ICP0 protein that is operably linked to a promoter, wherein the promoter consists essentially of SEQ ID NO: 2. In one embodiment, the HSV ICP0 is HSV-1 ICP0, e.g. SEQ ID NO 5, or variant thereof. In one embodiment, the HSV ICP0 is HSV-2 ICP0, e.g. SEQ ID NO 6, or variant thereof.

[0064] It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0065] Embodiments of the invention are further described in the following numbered paragraphs. [0066] 1. A Vero cell line comprising a nucleotide sequence that encodes for a Herpes Simplex virus (HSV) ICP0 protein operably linked to a promoter, wherein the promoter comprises SEQ ID NO:1. [0067] 2. The Vero cell line of paragraph 1, wherein the promoter comprises SEQ ID NO: 2. [0068] 3. The Vero cell line of any one of paragraphs 1-2, wherein the nucleotide sequence encodes for HSV-1 ICP0 that comprises the amino acid sequence of SEQ ID NO: 5. [0069] 4. The Vero cell line of any one of paragraphs 1-3, wherein, the cell line exhibits Vero cell morphology. [0070] 5. The Vero cell line of any one of paragraphs 1-4, wherein the cell line is capable of maintaining ICP0 complementation efficiency within 2 standard deviations as measured by a viral replication assay for greater than 20 cell passages. [0071] 6. The Vero cell line of any one of paragraphs 1-5, wherein the cell line is capable of maintaining ICP0 complementation efficiency within 2 standard deviations as measured by a viral replication assay for greater than 30 cell passages. [0072] 7. The Vero cell line of any one of paragraphs 1-6, wherein the cell line is capable of maintaining ICP0 complementation efficiency within 2 standard deviations as measured by a viral replication assay for greater than 40 cell passages. [0073] 8. The Vero cell line of any one of paragraphs 1-7, wherein the nucleotide sequence that encodes for a Herpes Simplex virus (HSV) ICP0 protein operably linked to a promoter comprises SEQ ID NO: 4. [0074] 9. The Vero cell line of any one of paragraphs 1-7, wherein the nucleotide sequence that encodes for a Herpes Simplex virus (HSV) ICP0 protein operably linked to a promoter consists essentially of SEQ ID NO: 4. [0075] 10. The Vero cell line of any one of paragraphs 1-9, wherein the cell line further comprises an additional nucleic acid that encodes a tetracycline repressor protein (tetR) operably linked to a promoter. [0076] 11. The Vero cell line of paragraph 10, wherein the promoter operably linked to the tetracycline repressor protein comprises SEQ ID NO: 9. [0077] 12. The Vero cell line of any one of paragraphs 10-11, wherein the nucleic acid that encodes a tetracycline repressor protein encodes the amino acid sequence of SEQ ID NO: 11. [0078] 13. The Vero cell line of any one of paragraphs 10-12, wherein the nucleic acid that encodes tetracycline repressor protein (tetR) operably linked to a promoter comprises SEQ ID NO: 7. [0079] 14. The Vero cell line of any one of paragraphs 10-12, wherein the nucleic acid that encodes a tetracycline repressor protein (tetR) operably linked to a promoter consists essentially of SEQ ID NO: 7. [0080] 15. The Vero cell line of paragraph 9, which is V0-584. [0081] 16. The Vero cell line of paragraph 14, which is V0R-124. [0082] 17. The Vero cell line of any one of paragraphs 1-16, further comprising a gene encoding antibiotic resistance. [0083] 18. The Vero cell line of any one of paragraphs 1-17, further comprising an ICP0 deficient HSV virus. [0084] 19. The Vero cell line of any one of paragraphs 1-17, further comprising a nucleic acid encoding a recombinant protein of interest that is operably linked to a promoter. [0085] 20. The Vero cell line of paragraph 19, wherein the recombinant protein of interest is a therapeutic protein. [0086] 21. The Vero cell line of paragraph 20, wherein the therapeutic protein is a vaccine protein. [0087] 22. A method of producing a viral vaccine of interest comprising propagating a virus to be used for vaccination in a Vero cell line of any one of paragraphs 1-21. [0088] 23. The method of paragraph 22, wherein the virus to be used for vaccination is an ICP0 deficient HSV virus. [0089] 24. The method of paragraph 22, wherein the virus is an adenovirus. [0090] 25. A method of producing a recombinant protein of interest comprising propagating the Vero cell line of any one of paragraphs 19-21, for sufficient time to allow for expression of the protein. [0091] 26. A method of producing ICP0 deficient HSV virus comprising propagating an HSV ICP0 deficient HSV virus in a Vero cell line of paragraph 1. [0092] 27. A method of producing ICP0 deficient HSV virus comprising i) infecting a Vero cell line of any of paragraph 1, with an ICP0 deficient HSV virus, ii) incubating the cell line in a tissue culture medium; and iii) collecting the ICP0 deficient virus produced by the cell line.

[0093] All references, publications and patents described herein, in the Examples and throughout the Specification, are incorporated herein by reference in their entirety. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

EXAMPLES

Materials and Methods

[0094] Cells: African Green Monkey Kidney CCL-81 (Vero) cells (ATCC) and the human osteosarcoma line U2OS cells were grown and maintained in Dulbecco's Modified Eagle's Medium (DMEM; Sigma Aldrich) supplemented with 10% fetal bovine serum (FBS) in the presence of 100 U/ml penicillin G and 100 g/ml streptomycin sulfate (GIBCO, Carlsbad, Calif.) (Yao, et al., J. Virol. 69:6249-58 (1995)). U2OS cells are able to complement functionally for the HSV-1 ICP0 deletion (Yao, et al., J. Virol. 69:6249-58 (1995)). U2CEP4R11 cells are tetR-expressing U2OS cells that were maintained in DMEM plus 10% FBS and hygromycin B at 50 g/ml (Yao, et al., Hum. Gene Ther. 9:1939-50 (1998)).

[0095] Plasmids: pMF3-ICP0 is an HSV-1 ICP0-expressing plasmid that contain no HSV-1 sequence flanking the HSV-1 ICP0 ORF (SEQ ID NO: 3). pMF3-ICP3 encodes ICP0 ORF under the control of modified HCMV minimal promoter consisting of 3 VP16 responsive elements plus 2CAAAT/SP1 elements 5 to the HCMV TATA element (SEQ ID NO: 4). pcDNA3 and pCDNA4/TO were obtained from Invitrogen (Carlsbad, Calif.). pcDNA3 encodes neomycin-resistant gene under the control of SV40 promoter. pCDNA4/TO encodes Zeocin-resistant gene. pCDNA4TO-eGFP is an eGFP (enhanced green fluorescent protein)-expressing plasmid.

[0096] Plasmid pMF-tetR contains a synthesized DNA fragment consisting of an optimized tetR transcription unit that includes: 1) a modified HCMV major immediate-early promoter that is lack of HCMV promoter sequence from 174 to 370 bp and contains two HSV-1 VP16 responsive elements with the first VP16 responsive element at 149 bp upstream of the HCMV TATA element and the second VP16 responsive element at 274 bp upstream of the HCMV TATA element, 2) beta-globin intron, and 3) a codon optimized tetR coding sequence followed by SV40 poly A signal sequence (SEQ ID NO: 7).

[0097] Viruses: N2-lacZ is an HSV-2 ICP0 null mutant, in which the Xho I-ICP0 coding sequence in both copies of the ICP0 gene in the HSV-2 genome are replaced by the Lac Z gene. CJ2-gD2 is an HSV-2 ICP0-deletion mutant-based non-replicating dominant-negative HSV-2 recombinant virus in which both copies of the lacZ gene in N2-lacZ are replaced by DNA sequences encoding the gD2 gene driven by the tetO-bearing HSV-1 major immediate-early ICP4 promoter, while the gene encoding UL9-C535C is under the control of the tetO-containing hCMV major immediate-early promoter in an opposite orientation of the inserted gD2 gene (Akhrameyeva, J. Virol. 85:5036-47 (2011)). N2-lacZ was propagated in U2OS cells, while CJ2-gD2 was propagated and plaque assayed in U2CEP4R11 cells.

Example 1

Establishment of an HSV-1 ICP0 Expressing Stable Cell Line that can Complement the Plaque Forming Efficiency of HSV-2 ICP0 Null Mutant Comparable to that of a Human Osteosacarma Cell Line, U2OS Cells

[0098] We were able to successfully minimize the cytotoxic effect of ICP0 by using a minimal promoter pMF-3 (SEQ ID NO: 01). To establish the ICP0-expressing stable cell lines, we constructed an ICP0-expressing plasmid, pMF3-ICP0. Linearized pMF3-ICP0 plasmid was then transfected into CCL-81 Vero cells along with linearized pcDNA3 by Lipofectamine 2000 (Invitrogen Inc.). At 30 hours post-transfection, cells were seeded into 100 mm dishes at various cell density and were grown in DMEM growth medium containing G418 at 400 ug/ml. G418 resistant colony cells were assayed by its ability to complement the growth of HSV-2 ICP0 null mutant, N2-lacZ, benchmarked against U2OS cells. Among more than 1000 G418-resistant colonies we selected, V0-584 cells represent the only G418-resistant and ICP0-expressing stable cell line that can complement the plaque-formation of N2-lacZ at level comparable to U2OS cells. The results in FIG. 1 show that the plaque-forming efficacy of N2-lacZ in passage 7 V0-584 cells is 2-fold lower than that of U2OS cells and 120-fold higher than that of normal CCL-81 cells. The complementing efficiency of V0-584 cells for N2-lacZ remains 2-3 fold lower than that of U2OS cells at passage 33.

Example 2

Establishment of a tetR- and ICP0-Expressing Vero Cells that can Complement the Plaque Forming Efficiency of CJ2-gD2 Efficiently

[0099] We have synthesized a DNA sequence at GeneArt (Invitrogen Inc.), which contains an optimized tetR transcription unit that includes: 1) a modified HCMV major immediate-early promoter, which contain the first VP16 responsive element at position 149 bp and the second VP16 responsive element at position 274 bp upstream of the HCMV TATA element, respectively (SEQ ID NO: 9), 2) beta-globin intron (SEQ ID NO: 10), and 3) a codon optimized tetR coding sequence (SEQ ID NO: 8) followed by SV40 poly A signal sequence. The plasmid that contains above mentioned DNA sequence was named pMF-tetR.

[0100] To establish tetR- and ICP0-expressing stable CCL-81 Vero cells, V0-584 cells were transfected with linearized pMF-tetR along with linearized pCDNA4/TO (Invitrogen) by Lipofectamine 2000. Transfected cells were then seeded into 100 mm dishes at various cell density and were grown in DMEM growth medium containing G418 at 400 ug/ml and Zeocin at 200 ug/ml. G418- and Zeocin-resistant colony cells were assayed by its ability to complement the growth of CJ2-gD2, benchmarked against U2CEP4R-11 cells. V0R-124 cells represent a G418/Zeocin-resistant stable cell line that can complement the plaque-formation of CJ2-gD2 efficiently even at passage 41. The results in FIG. 2 show that the plaque-forming efficacy of CJ2-gD2 in V0R-124 cells is 4.5-fold lower than that of U2CEP4R-11 cells and is 3,378-fold higher than that of V0-584 cells. The plaque-forming efficiency of N2-lacZ on V0R-124 cells is similar to that of V0-584 cells (data not shown). Notably, V0R-124 cells are morphologically similar to CCL-81 cells and exhibit similar growth kinetics as CCL-81 cells (FIG. 3).

[0101] FIG. 4 represents the plaque-forming efficiency of CJ2-gD2 in V0R-124 cells in either the absence or presence of doxycycline. The result shows that the plaque-forming efficiency of CJ2-gD2 in V0R-124 cells in the presence of tetracycline is 2,418-fold lower than in the absence of tetracycline, demonstrating further that like in tetR-expressing Vero cell line, VCEP4R-28 cells (Akhrameyeva, J. Virol. 85:5036-47 (2011)), tetR expressed in V0R-124 cells can effectively suppresses the expression of UL9-C535C in CJ2-gD2-infected V0R-124 cells in the absence of doxycycline.

Example 3

Regulation of Gene Expression from the tetO-Containing HCMV Major Immediate-Early Promoter in V0R-124 Cells

[0102] To further examine the effectiveness of tetR expressed in V0R-124 cells in regulating gene expression from the tetO-containing promoters, V0R-124 cells were seeded into 60 mm dishes at 610.sup.5 cells/dish. At 21 h post-seeding, cells were transfected with pCDNA4TO-eGFP with Lipofectamine 2000. Transfection medium was removed at 4 h post-transfection followed by addition of normal growth medium with no tetracycline or with doxycycline at a concentration of 0.2 ug/ml. Expression of EGFP was visualized directly under the fluorescence microscopy at 24 h and 48 h post-transfection. As shown in FIG. 5 while a significant number of green cells were seen in V0R-124 cells grown in the presence of doxycycline, a few faint green cells were detected under un-induced conditions, showing that gene expression of the tetO-containing promoter can be tightly regulated by doxycycline in V0R-124 cells.

[0103] Moreover, given the intensity of eGFP detected in transfected Vero cells and V0R-124 cells in the presence of tetracycline, the results suggest further that expression of ICP0 in V0R-124 cells can effectively elevate gene expression following transient transfection, consistent with the previously demonstrated potent trans-activating function of ICP0 on gene expression.

TABLE-US-00001 SEQUENCELISTING SEQIDNO:1MinimalPromoterSequenceinpMF3-ICP0 CCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAG(SEQIDNO:1) SEQIDNO:2PromotersequenceinpMF3-ICP0(with3xVP16responsiveelements (underlinedandhighlight)plus2xCAAATGGGCGGcis-actingelements(SEQIDNO:12)) usedfortheexpressionofHSV-1ICP0codingsequence. ATGCTAATGATATACATGCCACGTACTTATGGTGTCTATGCTAATGATATTCGCAAATGGGC GGTAGACCGGTGAATTCATGCTAATGATATTCTTTGGTACCATTGACGCAAATGGGCGGTAG GCGTGTACGGTGGGAGGTCTATATAAG(SEQIDNO:2) SEQIDNO:3HSV-1ICP0codingsequencefollowedbySV40polyAsignalsequencein plasmidspMF3-ICP0. ATGGAGCCCCGCCCCGGAGCGAGTACCCGCCGGCCTGAGGGCCGCCCCCAGCGCGAGGTGA GGGGCCGGGCGCCATGTCTGGGGCGCCATATTGGGGGGCGCCATATTGGGGGGCGCCATGT TGGGGGACCCCCGACCCTTACACTGGAACCGGCCGCCATGTTGGGGGACCCCCACTCATAC ACGGGAGCCGGGCGCCATGTTGGGGCGCCATGTTAGGGGGCGTGGAACCCCGTGACACTAT ATATACAGGGACCGGGGGCGCCATGTTAGGGGGTGCGGAACCCCCTGACCCTATATATACA GGGACCGGGGTCGCCCTGTTGGGGGTCGCCATGTGACCCCCTGACTTTATATATACAGACCC CCAACACATACACATGGCCCCTTTGACTCAGACGCAGGGCCCGGGGTCGCCGTGGGACCCC CTGACTCATACACAGAGACACGCCCCCACAACAAACACACAAGGACCGGGGTCGCCGTGTT GGGGGCGTGGTCCCCACTGACTCATACGCAGGCCCCCCTTACTCACACGCATCTAGGGGGGT GGGGAGGAGCCGCCCGCCATATTTGGGGGACGCCGTGGGACCCCCGACTCCGGTGCGTCTG GAGGGCGGGAGAAGAGGGAAGAAGAGGGGTCGGGATCCAAAGGACGGACCCAGACCACCT TTGGTTGCAGACCCCTTTCTCCCCCCTCTTCCGAGGCCAGCAGGGGGGCAGGACTTTGTGAG GCGGGGGGGGGAGAGGGGGAACTCGTGGGTGCTGATTGACGCGGGAAATCCCCCCCCATTC TTACCCGCCCCCCTTTTTTCCCCTTAGCCCGCCCCGGATGTCTGGGTGTTTCCCTGCGACCGA GACCTGCCGGACAGCAGCGACTCTGAGGCGGAGACCGAAGTGGGGGGGCGGGGGGACGCC GACCACCATGACGACGACTCCGCCTCCGAGGCGGACAGCACGGACACGGAACTGTTCGAGA CGGGGCTGCTGGGGCCGCAGGGCGTGGATGGGGGGGCGGTCTCGGGGGGGAGCCCCCCCC GCGAGGAAGACCCCGGCAGTTGCGGGGGCGCCCCCCCTCGAGAGGACGGGGGGAGCGACG AGGGCGACGTGTGCGCCGTGTGCACGGATGAGATCGCGCCCCACCTGCGCTGCGACACCTT CCCGTGCATGCACCGCTTCTGCATCCCGTGCATGAAAACCTGGATGCAATTGCGCAACACCT GCCCGCTGTGCAACGCCAAGCTGGTGTACCTGATAGTGGGCGTGACGCCCAGCGGGTCGTT CAGCACCATCCCGATCGTGAACGACCCCCAGACCCGCATGGAGGCCGAGGAGGCCGTCAGG GCGGGCACGGCCGTGGACTTTATCTGGACGGGCAATCAGCGGTTCGCCCCGCGGTACCTGA CCCTGGGGGGGCACACGGTGAGGGCCCTGTCGCCCACCCACCCGGAGCCCACCACGGACGA GGATGACGACGACCTGGACGACGGTGAGGCGGGGGGCGGCAAGGACCCTGGGGGAGGAGG AGGAGGAGGGGGGGGGAGGGAGGAATAGGCGGGCGGGCGAGGAAAGGGCGGGCCGGGGA GGGGGCGTAACCTGATCGCGCCCCCCGTTGTCTCTTGCAGCAGACTACGTACCGCCCGCCCC CCGCCGGACGCCCCGCGCCCCCCCACGCAGAGGCGCCGCCGCGCCCCCCGTGACGGGCGGG GCGTCTCACGCAGCCCCCCAGCCGGCCGCGGCTCGGACAGCGCCCCCCTCGGCGCCCATCG GGCCACACGGCAGCAGTAACACCAACACCACCACCAACAGCAGCGGCGGCGGCGGCTCCC GCCAGTCGCGAGCCGCGGCGCCGCGGGGGGCGTCTGGCCCCTCCGGGGGGGTTGGGGTTGG GGTTGGGGTTGTTGAAGCGGAGGCGGGGCGGCCGAGGGGCCGGACGGGCCCCCTTGTCAAC AGACCCGCCCCCCTTGCAAACAACAGAGACCCCATAGTGATCAGCGACTCCCCCCCGGCCT CTCCCCACAGGCCCCCCGCGGCGCCCATGCCAGGCTCCGCCCCCCGCCCCGGGCCCCCCGCG TCCGCGGCCGCGTCGGGACCCGCGCGCCCCCGCGCGGCCGTGGCCCCGTGCGTGCGAGCGC CGCCTCCGGGGCCCGGCCCCCGCGCCCCGGCCCCCGGGGCGGAGCCGGCCGCCCGCCCCGC GGACGCGCGCCGTGTGCCCCAGTCGCACTCGTCCCTGGCTCAGGCCGCGAACCAAGAACAG AGTCTGTGCCGGGCGCGTGCGACGGTGGCGCGCGGCTCGGGGGGGCCGGGCGTGGAGGGTG GGCACGGGCCCTCCCGCGGCGCCGCCCCCTCCGGCGCCGCCCCGCTCCCCTCCGCCGCCTCT GTCGAGCAGGAGGCGGCGGTGCGTCCGAGGAAGAGGCGCGGGTCGGGCCAGGAAAACCCC TCCCCCCAGTCCACGCGTCCCCCCCTCGCGCCGGCAGGGGCCAAGAGGGCGGCGACGCACC CCCCCTCCGACTCAGGGCCGGGGGGGCGCGGCCAGGGTGGGCCCGGGACCCCCCTGACGTC CTCGGCGGCCTCCGCCTCTTCCTCCTCTGCCTCTTCCTCCTCGGCCCCGACCCCCGCGGGGGC CGCCTCTTCCGCCGCCGGGGCCGCGTCCTCCTCCGCTTCCGCCTCCTCGGGCGGGGCCGTCG GTGCCCTGGGAGGGAGACAAGAGGAAACCTCCCTCGGCCCCCGCGCTGCTTCTGGGCCGCG GGGGCCGAGGAAGTGTGCCCGGAAGACGCGCCACGCGGAGACTTCCGGGGCCGTCCCCGC GGGCGGCCTCACGCGCTACCTGCCCATCTCGGGGGTCTCTAGCGTGGTCGCCCTGTCGCCTT ACGTGAACAAGACTATCACGGGGGACTGCCTGCCCATCCTGGACATGGAGACGGGGAACAT CGGGGCGTACGTGGTCCTGGTGGACCAGACGGGAAACATGGCGACCCGGCTGCGGGCCGCG GTCCCCGGCTGGAGCCGCCGCACCCTGCTCCCCGAGACCGCGGGTAACCACGTGATGCCCC CCGAGTACCCGACGGCCCCCGCGTCGGAGTGGAACAGCCTCTGGATGACCCCCGTGGGGAA CATGCTGTTCGACCAGGGCACCCTAGTGGGCGCCCTGGACTTCCGCAGCCTGCGGTCTCGGC ACCCGTGGTCCGGGGAGCAGGGGGCGTCGACCCGGGACGAGGGAAAACAATAACAGAACT TGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAA GCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCT GAAGCTTGGC(SEQIDNO:3) SEQIDNO:42PromotersequenceinpMF3-ICP0(with3xVP16responsiveelements (underlined)plus2xCAAATGGGCGGcis-actingelements(SEQIDNO:12))usedforthe expressionofHSV-1ICP0codingsequenceplustheHSV-1ICP0codingsequence. ATGCTAATGATATACATGCCACGTACTTATGGTGTCTATGCTAATGATATTCGCAAATGGGC GGTAGACCGGTGAATTCATGCTAATGATATTCTTTGGTACCATTGACGCAAATGGGCGGTAG GCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGG AGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCG GCCGGGAACGGTGCATTGGAACGGACTCTAGAGGATCCATGGAGCCCCGCCCCGGAGCGAG TACCCGCCGGCCTGAGGGCCGCCCCCAGCGCGAGGTGAGGGGCCGGGCGCCATGTCTGGGG CGCCATATTGGGGGGCGCCATATTGGGGGGCGCCATGTTGGGGGACCCCCGACCCTTACAC TGGAACCGGCCGCCATGTTGGGGGACCCCCACTCATACACGGGAGCCGGGCGCCATGTTGG GGCGCCATGTTAGGGGGCGTGGAACCCCGTGACACTATATATACAGGGACCGGGGGCGCCA TGTTAGGGGGTGCGGAACCCCCTGACCCTATATATACAGGGACCGGGGTCGCCCTGTTGGG GGTCGCCATGTGACCCCCTGACTTTATATATACAGACCCCCAACACATACACATGGCCCCTT TGACTCAGACGCAGGGCCCGGGGTCGCCGTGGGACCCCCTGACTCATACACAGAGACACGC CCCCACAACAAACACACAAGGACCGGGGTCGCCGTGTTGGGGGCGTGGTCCCCACTGACTC ATACGCAGGCCCCCCTTACTCACACGCATCTAGGGGGGTGGGGAGGAGCCGCCCGCCATAT TTGGGGGACGCCGTGGGACCCCCGACTCCGGTGCGTCTGGAGGGCGGGAGAAGAGGGAAG AAGAGGGGTCGGGATCCAAAGGACGGACCCAGACCACCTTTGGTTGCAGACCCCTTTCTCC CCCCTCTTCCGAGGCCAGCAGGGGGGCAGGACTTTGTGAGGCGGGGGGGGGAGAGGGGGA ACTCGTGGGTGCTGATTGACGCGGGAAATCCCCCCCCATTCTTACCCGCCCCCCTTTTTTCCC CTTAGCCCGCCCCGGATGTCTGGGTGTTTCCCTGCGACCGAGACCTGCCGGACAGCAGCGAC TCTGAGGCGGAGACCGAAGTGGGGGGGCGGGGGGACGCCGACCACCATGACGACGACTCC GCCTCCGAGGCGGACAGCACGGACACGGAACTGTTCGAGACGGGGCTGCTGGGGCCGCAG GGCGTGGATGGGGGGGCGGTCTCGGGGGGGAGCCCCCCCCGCGAGGAAGACCCCGGCAGT TGCGGGGGCGCCCCCCCTCGAGAGGACGGGGGGAGCGACGAGGGCGACGTGTGCGCCGTG TGCACGGATGAGATCGCGCCCCACCTGCGCTGCGACACCTTCCCGTGCATGCACCGCTTCTG CATCCCGTGCATGAAAACCTGGATGCAATTGCGCAACACCTGCCCGCTGTGCAACGCCAAG CTGGTGTACCTGATAGTGGGCGTGACGCCCAGCGGGTCGTTCAGCACCATCCCGATCGTGAA CGACCCCCAGACCCGCATGGAGGCCGAGGAGGCCGTCAGGGCGGGCACGGCCGTGGACTTT ATCTGGACGGGCAATCAGCGGTTCGCCCCGCGGTACCTGACCCTGGGGGGGCACACGGTGA GGGCCCTGTCGCCCACCCACCCGGAGCCCACCACGGACGAGGATGACGACGACCTGGACGA CGGTGAGGCGGGGGGCGGCAAGGACCCTGGGGGAGGAGGAGGAGGAGGGGGGGGGAGGG AGGAATAGGCGGGCGGGCGAGGAAAGGGCGGGCCGGGGAGGGGGCGTAACCTGATCGCGC CCCCCGTTGTCTCTTGCAGCAGACTACGTACCGCCCGCCCCCCGCCGGACGCCCCGCGCCCC CCCACGCAGAGGCGCCGCCGCGCCCCCCGTGACGGGCGGGGCGTCTCACGCAGCCCCCCAG CCGGCCGCGGCTCGGACAGCGCCCCCCTCGGCGCCCATCGGGCCACACGGCAGCAGTAACA CCAACACCACCACCAACAGCAGCGGCGGCGGCGGCTCCCGCCAGTCGCGAGCCGCGGCGCC GCGGGGGGCGTCTGGCCCCTCCGGGGGGGTTGGGGTTGGGGTTGGGGTTGTTGAAGCGGAG GCGGGGCGGCCGAGGGGCCGGACGGGCCCCCTTGTCAACAGACCCGCCCCCCTTGCAAACA ACAGAGACCCCATAGTGATCAGCGACTCCCCCCCGGCCTCTCCCCACAGGCCCCCCGCGGC GCCCATGCCAGGCTCCGCCCCCCGCCCCGGGCCCCCCGCGTCCGCGGCCGCGTCGGGACCC GCGCGCCCCCGCGCGGCCGTGGCCCCGTGCGTGCGAGCGCCGCCTCCGGGGCCCGGCCCCC GCGCCCCGGCCCCCGGGGCGGAGCCGGCCGCCCGCCCCGCGGACGCGCGCCGTGTGCCCCA GTCGCACTCGTCCCTGGCTCAGGCCGCGAACCAAGAACAGAGTCTGTGCCGGGCGCGTGCG ACGGTGGCGCGCGGCTCGGGGGGGCCGGGCGTGGAGGGTGGGCACGGGCCCTCCCGCGGC GCCGCCCCCTCCGGCGCCGCCCCGCTCCCCTCCGCCGCCTCTGTCGAGCAGGAGGCGGCGGT GCGTCCGAGGAAGAGGCGCGGGTCGGGCCAGGAAAACCCCTCCCCCCAGTCCACGCGTCCC CCCCTCGCGCCGGCAGGGGCCAAGAGGGCGGCGACGCACCCCCCCTCCGACTCAGGGCCGG GGGGGCGCGGCCAGGGTGGGCCCGGGACCCCCCTGACGTCCTCGGCGGCCTCCGCCTCTTC CTCCTCTGCCTCTTCCTCCTCGGCCCCGACCCCCGCGGGGGCCGCCTCTTCCGCCGCCGGGG CCGCGTCCTCCTCCGCTTCCGCCTCCTCGGGCGGGGCCGTCGGTGCCCTGGGAGGGAGACAA GAGGAAACCTCCCTCGGCCCCCGCGCTGCTTCTGGGCCGCGGGGGCCGAGGAAGTGTGCCC GGAAGACGCGCCACGCGGAGACTTCCGGGGCCGTCCCCGCGGGCGGCCTCACGCGCTACCT GCCCATCTCGGGGGTCTCTAGCGTGGTCGCCCTGTCGCCTTACGTGAACAAGACTATCACGG GGGACTGCCTGCCCATCCTGGACATGGAGACGGGGAACATCGGGGCGTACGTGGTCCTGGT GGACCAGACGGGAAACATGGCGACCCGGCTGCGGGCCGCGGTCCCCGGCTGGAGCCGCCG CACCCTGCTCCCCGAGACCGCGGGTAACCACGTGATGCCCCCCGAGTACCCGACGGCCCCC GCGTCGGAGTGGAACAGCCTCTGGATGACCCCCGTGGGGAACATGCTGTTCGACCAGGGCA CCCTAGTGGGCGCCCTGGACTTCCGCAGCCTGCGGTCTCGGCACCCGTGGTCCGGGGAGCA GGGGGCGTCGACCCGGGACGAGGGAAAACAATAA(SEQIDNO:4) SEQIDNO:5HSV-1ICP0aminoacidsequence AminoAcidSequenceforHSV-1ICP0-strainKOSfore.g.variants.Seee.g.also:Accession: P08393.1GI:124134;Accession:AFI23590.1GI:384597746;Accession:AFI23649.1GI:384597805; Accession:AFE62827.1GI:380776964;Accession:AFE62886.1GI:380777023;Accession: ADM22381.1GI:304318198;Accession:ALO18731.1GI:952947655;Accession:ALO18672.1GI: 952947596;Accession:ALO18655.1GI:952947578;Accession:ALO18596.1GI:952947519; Accession:AKH80472.1GI:822581062;Accession:AKH80399.1GI:822580988;Accession: AKG61929.1GI:820021112;Accession:AKG61857.1GI:820021035;etc.andthelike. MEPRPGASTRRPEGRPQREPAPDVWVFPCDRDLPDSSDSEAETE VGGRGDADHHDDDSASEADSTDTELFETGLLGPQGVDGGAVSGGSPPREEDPGSCGGA PPREDGGSDEGDVCAVCTDEIAPHLRCDTFPCMHRFCIPCMKTWMQLRNTCPLCNAKL VYLIVGVTPSGSFSTIPIVNDPQTRMEAEEAVRAGTAVDFIWTGNQRFAPRYLTLGGH TVRALSPTHPEPTTDEDDDDLDDADYVPPAPRRTPRAPPRRGAAAPPVTGGASHAAPQ PAAARTAPPSAPIGPHGSSNTNTTTNSSGGGGSRQSRAAVPRGASGPSGGVGVVEAEA GRPRGRTGPLVNRPAPLANNRDPIVISDSPPASPHRPPAAPMPGSAPRPGPPASAAAS GPARPRAAVAPCVRAPPPGPGPRAPAPGAEPAARPADARRVPQSHSSLAQAANQEQSL CRARATVARGSGGPGVEGGHGPSRGAAPSGAAPSGAPPLPSAASVEQEAAVRPRKRRG SGQENPSPQSTRPPLAPAGAKRAATHPPSDSGPGGRGQGGPGTPLTSSAASASSSSAS SSSAPTPAGATSSATGAASSSASASSGGAVGALGGRQEETSLGPRAASGPRGPRKCAR KTRHAETSGAVPAGGLTRYLPISGVSSVVALSPYVNKTITGDCLPILDMETGNIGAYV VLVDQTGNMATRLRAAVPGWSRRTLLPETAGNHVTPPEYPTAPASEWNSLWMTPVGNM LFDQGTLVGALDFRSLRSRHPWSGEQGASTRDEGKQ(SEQIDNO:5) SEQIDNO:6HSV-2ICP0aminoacidsequenceACCESSIONYP_009137210 VERSIONYP_009137210.1GI:820945210[note:fore.g.Variants,seealsoe.g.Accession: YP_009137151.1GI:820945151;Accession:AEV91397.2GI:556197555;Accession:AEV91338.2GI: 556197550;;Accession:ADG01890.1GI:295322885;;Accession:ADG01889.1GI:295322883; Accession:ADG01888.1GI:295322881;Accession:ADG01887.1GI:295322879;Accession: ADG01885.1GI:295322875;Accession:ADG01886.1GI:295322877;etc,andthelike]] 1 meprpgtssradpgperpprqtpgtpaaphawgmlndmqwlassdseeetevgisdddlh 61 rdstseagstdtemfeaglmdaatpparppaerqgsptpadaqgscgggpvgeeeaeagg 121 ggdvcavctdeiapplrcqsfpclhpfcipcmktwiplrntcplcntpvaylivgvtasg 181 sfstipivndprtrveaeaavragtavdfiwtgnqrtaprslslgghtvralsptppwpg 241 tddedddladvdyvppaprraprrggggagatrgtsqpaatrpappgaprssssggaplr 301 agvgsgsgggpavaavvprvaslppaagggraqarrvgedaaaaegrtppagqpraaqep 361 pivisdspppsprrpagpgplsffssssaqvssgpgggglpqssgraarpraavaprvrs 421 ppraaaapvvsasadaagpappavpvdahraprsrmtqaqtdtqaqslgragatdargsg 481 gpgaeggpgvprgtntpgaaphaaegaaarprkrrgsdsgpaasssasssaaprsplapq 541 gvgakraaprrapdsdsgdrghgplapasagaappsaspssqaavaaaasssssaassss 601 saasssssaasssssaassssaassssssssasssaggaggsvasasgagerretslgpr 661 aaaprgprkcarktrhaeggpepgardpapgltrylpiagvssvvalapyvnktvtgdcl 721 pvldmetghigayvvlvdqtgnvadllraaapawsrrtllpeharncyrppdyptppase 781 wnslwmtpvgnmlfdqgtlvgaldfhglrsrhpwsreqgapapagdapaghge SEQIDNO:7OptimizedtetRtranscriptionunit:modifiedHCMVmajorimmediate-early promoterwithVP16responsiveelements,beta-globinIntron,codonoptimizedtetRcoding sequence,andSV40polyAsignalsequence. 5- GAATTCACGCGTCCGTGCATGCTAATGATATTCCGCCCAACGACCCCCGCCCATTGACGTCA ATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGA CTATTTACGGTAAACTGCATGCTAATGATATTCTTTGACTCACGGGGATTTCCAAGTCTCCAC CCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCG TAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATA AGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCT CCATAGAAGACACCGGGACCGATCCAGCCTCCGTCGCGAGGTGAGTTTGGGGACCCTTGAT TGTTCTTTCTTTTTCGCTATTGTAAAATTCATGTTATATGGAGGGGGCAAAGTTTTCAGGGTG TTGTTTAGAATGGGAAGATGTCCCTTGTATCACCATGGACCCTCATGATAATTTTGTTTCTTT CACTTTCTACTCTGTTGACAACCATTGTCTCCTCTTATTTTCTTTTCATTTTCTGTAACTTTTTC GTTAAACTTTAGCTTGCATTTGTAACGAATTTTTAAATTCACTTTTGTTTATTTGTCAGATTGT AAGTACTTTCTCTAATCACTTTTTTTTCAAGGCAATCAGGGTATATTATATTGTACTTCAGCA CAGTTTTAGAGAACAATTGTTATAATTAAATGATAAGGTAGAATATTTCTGCATATAAATTC TGGCTGGCGTGGAAATATTCTTATTGGTAGAAACAACTACATCCTGGTCATCATCCTGCCTT TCTCTTTATGGTTACAATGATATACACTGTTTGAGATGAGGATAAAATACTCTGAGTCCAAA CCGGGCCCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTGGATCCGTGTTCC AACCACGGTCACGCTTCGGTGGCCACCATGAGCAGACTGGACAAGAGCAAGGTGATCAACA GCGCCCTGGAGCTGCTGAACGAGGTGGGCATCGAGGGCCTGACCACCAGAAAGCTGGCCCA GAAGCTGGGCGTGGAGCAGCCCACCCTGTACTGGCACGTGAAGAACAAGAGAGCCCTGCTG GACGCCCTGGCCATCGAGATGCTGGACAGACACCACACCCACTTCTGCCCCCTGGAGGGCG AGAGCTGGCAGGACTTCCTGAGAAACAACGCCAAGAGCTTCAGATGCGCCCTGCTGAGCCA CAGAGACGGCGCCAAGGTGCACCTGGGCACCAGACCCACCGAGAAGCAGTACGAGACCCT GGAGAACCAGCTGGCCTTCCTGTGCCAGCAGGGCTTCAGCCTGGAGAACGCCCTGTACGCC CTGAGCGCCGTGGGCCACTTCACCCTGGGCTGCGTGCTGGAGGACCAGGAGCACCAGGTGG CCAAGGAGGAGAGAGAGACCCCCACCACCGACAGCATGCCCCCCCTGCTGAGACAGGCCAT CGAGCTGTTCGACCACCAGGGCGCCGAGCCCGCCTTCCTGTTCGGCCTGGAGCTGATCATCT GCGGCCTGGAGAAGCAGCTGAAGTGCGAGAGCGGCAGCTAAATAGGTAGGTAGTCGACCC GGGACGAGGGAAAACAATAACAGAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAG CAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTC CAAACTCATCAATGTATCTTATCATGTCTGAAGCTTCTGCAG-3(SEQIDNO:7) SEQIDNO:8CodonoptimizedtetRDNAcodingsequenceplusKozakconsensussequence 5- GCCACCATGAGCAGACTGGACAAGAGCAAGGTGATCAACAGCGCCCTGGAGCTGCTGAACG AGGTGGGCATCGAGGGCCTGACCACCAGAAAGCTGGCCCAGAAGCTGGGCGTGGAGCAGC CCACCCTGTACTGGCACGTGAAGAACAAGAGAGCCCTGCTGGACGCCCTGGCCATCGAGAT GCTGGACAGACACCACACCCACTTCTGCCCCCTGGAGGGCGAGAGCTGGCAGGACTTCCTG AGAAACAACGCCAAGAGCTTCAGATGCGCCCTGCTGAGCCACAGAGACGGCGCCAAGGTGC ACCTGGGCACCAGACCCACCGAGAAGCAGTACGAGACCCTGGAGAACCAGCTGGCCTTCCT GTGCCAGCAGGGCTTCAGCCTGGAGAACGCCCTGTACGCCCTGAGCGCCGTGGGCCACTTC ACCCTGGGCTGCGTGCTGGAGGACCAGGAGCACCAGGTGGCCAAGGAGGAGAGAGAGACC CCCACCACCGACAGCATGCCCCCCCTGCTGAGACAGGCCATCGAGCTGTTCGACCACCAGG GCGCCGAGCCCGCCTTCCTGTTCGGCCTGGAGCTGATCATCTGCGGCCTGGAGAAGCAGCTG AAGTGCGAGAGCGGCAGCTAA(SEQIDNO:8) SEQIDNO:9ModifiedHCMVmajorimmediate-earlypromoter,whichhasHCMVpromoter sequencefrom-174to-370bpdeletedandcontainstwoHSV-1VP16responsiveelements thefirstVP16responsiveelementatposition149bpandthesecondVP16elementat position274bpupstreamoftheHCMVTATAelement: 5- ATGCTAATGATATTCCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCC ATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTGC ATGCTAATGATATTCTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGG GAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCAT TGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGT GAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACAC CGGGACCGATCCAGCCTCCG(SEQIDNO:9) Underlined-_VP16responsive SEQIDNO:10Beta-globinintron 5- AGGTGAGTTTGGGGACCCTTGATTGTTCTTTCTTTTTCGCTATTGTAAAATTCATGTTATATG GAGGGGGCAAAGTTTTCAGGGTGTTGTTTAGAATGGGAAGATGTCCCTTGTATCACCATGGA CCCTCATGATAATTTTGTTTCTTTCACTTTCTACTCTGTTGACAACCATTGTCTCCTCTTATTT TCTTTTCATTTTCTGTAACTTTTTCGTTAAACTTTAGCTTGCATTTGTAACGAATTTTTAAATT CACTTTTGTTTATTTGTCAGATTGTAAGTACTTTCTCTAATCACTTTTTTTTCAAGGCAATCAG GGTATATTATATTGTACTTCAGCACAGTTTTAGAGAACAATTGTTATAATTAAATGATAAGG TAGAATATTTCTGCATATAAATTCTGGCTGGCGTGGAAATATTCTTATTGGTAGAAACAACT ACATCCTGGTCATCATCCTGCCTTTCTCTTTATGGTTACAATGATATACACTGTTTGAGATGA GGATAAAATACTCTGAGTCCAAACCGGGCCCCTCTGCTAACCATGTTCATGCCTTCTTCTTTT TCCTACAGCT-3(SEQIDNO:10) SEQIDNO:11CodonoptimizedtetRDNAcodingsequenceplusKozakconsensussequenceand 28bpofICP275UTR(underlined) GTGTTCCAACCACGGTCACGCTTCGGTGGCCACC ATGAGCAGACTGGACAAGAGCAAGGTGATCAACAGCGCCCTGGAGCTGCTGAACGAGGTG GGCATCGAGGGCCTGACCACCAGAAAGCTGGCCCAGAAGCTGGGCGTGGAGCAGCCCACC CTGTACTGGCACGTGAAGAACAAGAGAGCCCTGCTGGACGCCCTGGCCATCGAGATGCTGG ACAGACACCACACCCACTTCTGCCCCCTGGAGGGCGAGAGCTGGCAGGACTTCCTGAGAAA CAACGCCAAGAGCTTCAGATGCGCCCTGCTGAGCCACAGAGACGGCGCCAAGGTGCACCTG GGCACCAGACCCACCGAGAAGCAGTACGAGACCCTGGAGAACCAGCTGGCCTTCCTGTGCC AGCAGGGCTTCAGCCTGGAGAACGCCCTGTACGCCCTGAGCGCCGTGGGCCACTTCACCCT GGGCTGCGTGCTGGAGGACCAGGAGCACCAGGTGGCCAAGGAGGAGAGAGAGACCCCCAC CACCGACAGCATGCCCCCCCTGCTGAGACAGGCCATCGAGCTGTTCGACCACCAGGGCGCC GAGCCCGCCTTCCTGTTCGGCCTGGAGCTGATCATCTGCGGCCTGGAGAAGCAGCTGAAGTG CGAGAGCGGCAGCTAAATAGGTAGGTA(SEQIDNO:11)