WO2003023040A2 - Vaccinia virus mva-e3l-knockout-mutants and use thereof - Google Patents
Vaccinia virus mva-e3l-knockout-mutants and use thereof Download PDFInfo
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- WO2003023040A2 WO2003023040A2 PCT/EP2002/010199 EP0210199W WO03023040A2 WO 2003023040 A2 WO2003023040 A2 WO 2003023040A2 EP 0210199 W EP0210199 W EP 0210199W WO 03023040 A2 WO03023040 A2 WO 03023040A2
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Classifications
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/24011—Poxviridae
- C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
- C12N2710/24141—Use of virus, viral particle or viral elements as a vector
- C12N2710/24143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/24011—Poxviridae
- C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
- C12N2710/24161—Methods of inactivation or attenuation
- C12N2710/24162—Methods of inactivation or attenuation by genetic engineering
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the present invention relates to mutant MVA vaccinia viruses, which are used for the generation of recombinant MVA vimses, as well as host cells, which have been infected with these mutant MVA vimses.
- the present invention further relates to DNA-vector constructs, and a method for the generation of recombinant MVA by using the mutant MVA viruses and the DNA-vector constructs.
- Vaccinia virus belongs to the genus Orthopoxvirus of the family of poxviruses. Certain strains of vaccinia virus have been used for many years as live vaccine to immunize against smallpox, for example the Elstree strain of the Lister Institute in the UK. Because of the complications which may derive from the vaccination (Schar, Zeitschr. fur Praventiv Kunststoff Kunststoff Kunststoff Kunststoff Kunststoff Kunststoff Kunststoff Kunststoff Kunststoff Kunststoff Kunststoff Kunststoff Kunststoff Kunststoff Kunststoff Kunststoff Kunststoff Kunststoff-39 [1973]), and since the declaration in 1980 by the WHO that smallpox had been eradicated nowadays only people at high risk are vaccinated against smallpox.
- Vaccinia viruses have also been used as vectors for production and delivery of foreign antigens (Smith et al., Biotechnology and Genetic Engineering Reviews 2, 383 -407 [1984]). This entails DNA sequences (genes) which code for foreign antigens being introduced, with the aid of DNA recombination techniques, into the genome of the vaccinia vimses. If the gene is integrated at a site in the viral DNA which is non-essential for the life cycle of the vims, it is possible for the newly produced recombinant vaccinia vims to be infectious, that is to say able to infect foreign cells and thus to express the integrated DNA sequence (EP Patent Applications No. 83, 286 and No. 110, 385).
- vaccinia vimses prepared in this way can be used, on the one hand, as live vaccines for the prophylaxis of infections, on the other hand, for the preparation of heterologous proteins in eukaryotic cells.
- Vaccinia virus is amongst the most extensively evaluated live vectors and has particular features in support of its use as recombinant vaccine: It is highly stable, cheap to manufacture, easy to administer, and it can accommodate large amounts of foreign DNA. It has the advantage of inducing both antibody and cytotoxic responses, and allows presentation of antigens to the immune system in a more natural way, and it was successfully used as vector vaccine protecting against infectious diseases in a broad variety of animal models. Additionally, vaccinia vectors are extremely valuable research tools to analyze structure-function relationships of recombinant proteins, determine targets of humoral and cell-mediated immune responses, and investigate the type of immune defense needed to protect against a specific disease.
- vaccinia vims is infectious for humans and its use as expression vector in the laboratory has been affected by safety concerns and regulations. Furthermore, possible future applications of recombinant vaccinia virus e.g. to generate recombinant proteins or recombinant viral particles for novel therapeutic or prophylactic approaches in humans, are hindered by the productive replication of the recombinant vaccinia vector. Most of the recombinant vaccinia viruses described in the literature are based on the Western Reserve (WR) strain of vaccinia vims. On the other hand, it is known that this strain is highly neuro virulent and is thus poorly suited for use in humans and animals (Morita et al., Vaccine 5, 65-70 [1987]).
- WR Western Reserve
- the MVA virus was deposited in compliance with the requirements of the Budapest Treaty at CNCM (Institut Pasteur, Collectione Nationale de Cultures de Microorganisms, 25, rue de Dondel Roux, 75724 Paris Cedex 15) on Dec. 15, 1987 under Depositary No. 1-721.
- the MVA virus has been analysed to determine alterations in the genome relative to the wild type CVA strain.
- Six major deletions ( deletion I, ⁇ , ⁇ i, IV, V, and NI ) have been identified (Meyer, H, Sutler, G. and Mayr A. (1991) J. Gen. Virol. 72, 1031 -1038).
- This modified vaccinia vims Ankara has only low virulence, that is to say it is followed by no side effects when used for vaccination. Hence it is particularly suitable for the initial vaccination of immunocompromised subjects.
- the excellent properties of the MVA strain have been demonstrated in a number of clinical trials (Mayr et al., Zbl. Bakt Hyg. I, Abt. Org. B 167, 375-390 [1987], Stickl et al., Dtsch. med. Wschr. 99, 2386 -2392 [1974]).
- MVA vaccinia vims strain of choice for vector development.
- MVA vaccines are already under clinical investigation in tumor immunotherapy and prophylaxis of human immunodeficiency virus infection.
- the MVA genome contains several open reading frames (ORFs) coding for viral regulatory factors (Antoine et al. 1998, Virology 244:365).
- ORFs open reading frames
- MVA ORF 050L Antoine et al. 1998, Virology 244:365
- VV gene E3L Goebel et al. 1990, Virology 179:247.
- the viral protein E3L is one of the key interferon (IFN) resistance factors encoded by VV (Smith et al. 1998, Sem. Virol. 8:409).
- heterologous markers into the MVA genome significantly improves the isolation of cloned recombinant viruses.
- selective or chromogenic agents such as mutagenic agent mycophenolic acid or X-Gal/DMFA, and/or the requirement for selective host cells.
- the maintenance of additional foreign gene sequences is not desireable for vector virases to be used in clinical applications, and further genetical engineering of the viral genome is necessary to remove unwanted markers
- a new MVA- Knock-out mutant is provided, which is characterized in that the MVA ORF 050L gene or functional parts thereof have been inactivated in the viral genome.
- Functional parts of the ORF 050L include e. g. the coding sequences for the carboxyterminal dsRNA binding domain of the E3L protein (Chang & Jacobs 1993, Virology 194:537) or the amino-terminal domain of the E3L protein which shares sequence similarities with cellular interferon response proteins, can bind Z-DNA, and is required for E3L function f n vzv0 (Brandt & Jacobs 2001, J. Virol. 75:850).
- a functional part of ORF 050L as used herein is defined as a fragment of the E3L protein, the inactivation of which in the MVA genome is leading to a lack of replication of the mutated MVA in CEF cells.
- the MVA ORF 050L gene or a functional part thereof has been inactivated by deletion from the viral genome.
- a recombinant MVA defective in E3L function may be generated by sequence mutagenesis, e.g. insertional mutagenesis, leading to the inactivation of functional E3L protein synthesis by frame-shift-introduction or through specific inhibition of E3L gene transcription.
- This recombinant MVA can advantageously be used in a method for the introduction of foreign genes and subsequent selection of transfected strains, i.e. in a method for the generation of recombinant MVA.
- Using recently established methodology of transient host range selection by consecutive cloning of engineered vimses in rabbit RK-13 and hamster BHK-21 cells Staib et al.
- mutant MVA were generated having E3L coding sequences deleted from the viral genome (MVA- ⁇ E3L).
- VVA- ⁇ E3L Western blot analysis of viral proteins made during infection of BHK-21 cells showed that the vimses were unable to produce E3L protein.
- MVA- ⁇ E3L replicated as efficiently as original nonmutated MVA F6 ( Figure 2D).
- E3L Precise reinsertion of the E3L coding sequence into the genome of MVA- ⁇ .
- E3L rescued the growth capacity of the vims on CEF resulting in revertant vimses MVA- ⁇ E3L-Rev ( Figure 2C, 2D).
- This data confirmed that E3L function was essential to allow for formation of MVA progeny in CEF.
- the cloning of E3L gene sequences into MVA plasmid vectors and transfection of the latter into MVA- ⁇ E3L- infected BHK-21 cells generated recombinant MVA which could be directly isolated by growth selection on CEF.
- this stringent growth selection of recombinant MVA (i) can be performed on CEF - a well established tissue culture suitable to produce MVA vaccines for clinical use, and (ii) is based on simple restoration of the original already well characterized MVA genotype.
- the principle underlying the invention is that as long as MVA is present on CEF without the appropriate DNA sequences having been introduced (E3L coding sequences and optionally further DNA sequences, which are, e.g. coding for heterologous proteins), a replication does not occur. Therefore, the present method/MVA knock-out mutant is suitable for the selection of recombinant MVA and therefore serves as a tool for the effective generation of recombinant MVA.
- the term “bulrecombinant MVA” means those MNA, which have been genetically altered, e.g. by D ⁇ A recombination techniques and which are provided for the use as a vaccine or as an expression vector.
- the recombinant MVA vaccinia viruses can be prepared as follows:
- a D ⁇ A-construct which contains a D ⁇ A-sequence which codes for E3L protein or an E3L-derived polypeptide and a D ⁇ A sequence encoding a foreign polypeptide both flanked by D ⁇ A sequences flanking a non-essential site, e. g. a naturally occuring deletion, e.g. deletion III, within the MVA genome, is introduced into cells, preferably eucaryotic cells.
- Preferred eucaryotic cells are BHK-21 (ATCC CCL-10), BSC-1 (ATCC CCL-26), CV-1 (ECACC 87032605) or MA104 (ECACC 85102918) cells) productively infected with MVA- ⁇ E3L, to allow homologous recombination.
- the D ⁇ A-construct to be inserted can be linear or circular.
- a circular D ⁇ A is preferably used. It is particularly preferable to use a plasmid.
- the DNA-construct may contain sequences flanking the left and the right side of a non- essential site, e.g. the site of deletion HI, within the MVA genome (Sutter, G. and Moss, B. (1992) Proc. Natl. Acad. Sci. USA 89, 10847-10851).
- the foreign DNA sequence may be inserted between the sequences flanking the non- essential site, e.g. the naturally occuring deletion.
- the foreign DNA sequence can be a gene coding for a therapeutic polypeptide, e.g. t-PA or interferon, or from a pathogenic agent.
- Pathogenic agents are to be understood to be virases, bacteria and parasites which may cause a disease, as well as tumor cells which multiply unrestrictedly in an organism and may thus lead to pathological growths. Examples of such pathogenic agents are described in Davis, B.D. et al, (Microbiology, 3rd ed. Harper International Edition).
- Preferred genes of pathogenic agents are those of influenza virases, of measles and respiratory syncytial viruses, of dengue vimses, of human immunodeficiency virases, for example HJV I and HIV II, of human hepatitis virases, e.g. HCV and HBV, of heroes virases, of papilloma virases, of the malaria parasite Plasmodium falciparum, and of the tuberculosis-causing Mycobacteria.
- tumor associated antigens are those of melanoma-associated differentiation antigens, e.g. tyrosinase, tyros inase-related proteins 1 and 2, of cancer testes antigens, e.g. MAGE-1,-2,-3, and BAGE, of non-mutated shared antigens overexpressed on tumors, e.g. Her-2/neu, MUC-1, and p53.
- melanoma-associated differentiation antigens e.g. tyrosinase, tyros inase-related proteins 1 and 2
- cancer testes antigens e.g. MAGE-1,-2,-3, and BAGE
- promoters are known to those skilled in the art, for example those of the vaccinia 11 kDa gene as are described in EP-A- 198, 328, and those of the 7.5 kDa gene (EP-A-110, 385).
- the DNA-construct can be introduced into the cells by transfection, for example by means of calcium phospate precipitation (Graham et al, Virol. 52, 456 -467 [1973]; Wigler et al. Cell 777-785 [1979] by means of electroporation (Neumann et al, EMBO J. 1, 841 -845 [1982]), by microinjection (Graessmann et al, Meth. Enzymology 101, 482 -492 (1983)), by means of liposomes (Straubinger et al. Methods in Enzymology 101, 512-527 (1983)), by means of spheroplasts (Schaffner, Proc. Natl. Acad. Sci. USA 77, 2163 -2167 (1980)) or by other methods known to those skilled in the art. Transfection by means of calcium phosphate precipitation is preferably used.
- the MVA vaccinia viruses generated according to the invention are converted into a physiologically acceptable form. This can be done based on the many years of experience in the preparation of vaccines used for vaccination against smallpox (Kaplan, Br. Med. Bull. 25, 131 -135 [1969]).
- about 10 O 7 particles of the recombinant MVA are freeze-dried in 100ml of phosphate-buffered saline (PBS) in the presence of 2% peptone and 1% human albumin in an ampoule, preferably a glass ampoule.
- PBS phosphate-buffered saline
- the lyophilisate can contain extenders (such as mannitol, dextran, sugar, glycine, lactose or polyvinylpyrrolidone) or other aids (such as antioxidants, stabilizers, etc.) suitable for parenteral administration.
- extenders such as mannitol, dextran, sugar, glycine, lactose or polyvinylpyrrolidone
- other aids such as antioxidants, stabilizers, etc.
- the lyophilisate can be dissolved in 0.1 to 0.2 ml of aqueous solution, preferably physiological saline, and administered parenterally, for example by intradermal inoculation.
- the vaccine according to the invention is preferably injected intracutaneously. Slight swelling and redness, sometimes also itching, may be found at the injection site (Stickl et al, supra).
- the mode of administration, the dose and the number of administrations can be optimized by those skilled in the art in a known manner. It is expedient where appropriate to administer the vaccine several times over a lengthy period in order to obtain a high level immune responses against the foreign antigen.
- the method of the present invention for the generation of recombinant MVA comprises the following steps: Infecting the host cells as described above with the recombinant MVA, trans fecting the host cells with a DNA-vector construct of the present invention and selecting restored MVA by growth on CEF cells or chicken embryo derived LSCC-H32 cells (Roth & Kaaden 1985 Appl Environ Microbiol 49:634-636) or avian cells (e.g. quail fibroblasts QT6 or QT35 cells).
- CEF cells or chicken embryo derived LSCC-H32 cells (Roth & Kaaden 1985 Appl Environ Microbiol 49:634-636) or avian cells (e.g. quail fibroblasts QT6 or QT35 cells).
- MVA- ⁇ E3L Construction and characterization of MVA- ⁇ E3L.
- A Schematic maps of the MVA genome and the plasmid p ⁇ KlL-E3L designed for the deletion of the E3L gene sequences. The n dIII restriction endonuclease sites of the MVA genome are indicated on the top.
- MVA-DNA sequences adjacent to the E3L gene flank-E3LI and flank- E3LII
- KIL gene expression allows selective growth of the instable intermediate virus MVA- ⁇ E3L+K1L in RK13-cells.
- the final mutant virus MVA- ⁇ E3L results after deletion of the KIL marker gene during a second homologous recombination involving additional repetitive sequences (LacZ).
- B PCR analysis of viral DNA.
- C Southern blot analysis of viral DNA.
- D Western blot analysis of lysates from CEF infected with MVA- ⁇ E3L or MVA in the presence or absence of AraC.
- the E3L protein band detected by the E3L-specific mouse monoclonal antibody TW9 is marked by an arrowhead.
- FIG. 2C shows the multiple-step growth of MVA- ⁇ E3L in comparison to MVA- E3rev and wildtype MVA in CEF cells.
- Figure 2D shows the multiple-step growth of MVA- ⁇ E3L in comparison to MVA-E3rev and wildtype MVA in BHK cells.
- Viral polypeptide synthesis BHK and (B) CEF cells were infected with MVA- ⁇ E3 , MVA or MVA-E3rev (lanes 9-12) and labeled with r 35 S]methionine for 30 min at the indicated hour post infection (hpi). Cell lysates were analyzed by gel electrophoresis on a 10% polyacrylamide gel and visualized by autoradiography. Protein standards (lane M) are indicated by their molecular masses (in kilo Daltons) on the left. Uninfected cells (U) served as control. Viral DNA synthesis (C, D).
- DNA isolated from BHK-21 (C) or CEF (D) at Oh, 2h, 4h, 6h and 8h after infection with MVA- ⁇ E3L or MVA was immobilized on a Hybond N + membrane and analyzed by hybridization of a 32 P-labeled MVA-DNA probe. Radioactivity was quantitated with a phophorimager analyzer.
- FIG. 4 Induction of apoptosis by MVA- ⁇ E3L in CEF cells.
- A Semiconfluent monolayers of CEF cells were either non-infected (lane 7) or infected with MVA- ⁇ E3L (lanes 1, 2), W ,MVA (lanes 3, 4) or MVA-E3rev (lanes 5, 6) with an MOI of 20 PFU/cell for DNA fragmentation analysis.
- Viral DNA extracts were obtained 16h post infection (lanes 1, 3, 5) and 24h post infection (lanes 2, 4, 6), separated by gel electrophoresis through 1% agarose, and visualized by ethidium bromide staining.
- C Apoptotic cells stained with Hoechst 3343 were counted in CEF cells infected with MVA- ⁇ E3L ( B
- FIG. 5 Dosis- and time-dependent extent of apoptosis.
- A The extent of apoptosis depending on the infectious dosis was measured by ELISA either in mock-infected CEF (light grey, 4 ⁇ position) or in cells infected with MVA- ⁇ E3L (B
- the cell death detection ELISA was performed according to the manufacturers instructions 16h post infection (Roche Diagnostics). Absorbance at 405 nm (Reference: 490 nm) was measured. Results are given as the mean/2 X the SEM.
- Genomic DNA isolated from eight different clones of recombinant MVA-P mII5 -gfp (recMVA), or fromnon- recombinant MVA (WT), and plasmid pIII-ESL-P ⁇ j -gfp DNA served as template DNAs for the amplification of characteristic DNA fragments.
- a 1-kb DNA ladder was used as standard for the molecular weights of the DNA fragments.
- the MVA virus is a greatly attenuated vaccinia virus produced by serial passages of the original CVA strain on chicken embryo fibroblast (CEF) cultures.
- CEF chicken embryo fibroblast
- baby hamster kidney cells (BHK-21), a well characterized, easily maintained cell line, supports MVA growth and as proficient ex- pression of recombinant genes as the highly efficient CEF and has been recommended for standardized MVA propagation during the development of expression vectors and live recombinant vaccines (Drexler et al. 1998, J. Gen. Virol, 79, 347-352).
- the MVA virus was normally grown on CEF cells, the host cell for which it had been adapted.
- CEF cells 11 -days old embryos were isolated from incubated chicken eggs, the extremities were removed, and the embryos were cut into small pieces and slowly dissociated in a solution composed of 25% trypsin at room temperature for 2 hours.
- the resulting cell suspension was diluted with one volume of medium I (MEM Eagle, for example obtainable from Gibco, Basle, Switzerland; Order No.
- the MVA- ⁇ E3L is routinely propagated in baby hamster kidney BHK-21 (American Type Culture Collection ATCC CCL-10) cells which were grown in minimal essential medium (MEM) supplemented with 10% fetal calf serum (FCS). BHK-21 cells were maintained in a humidified air-5% CO 2 atmosphere at 37 °C.
- MEM minimal essential medium
- FCS fetal calf serum
- the viruses were used for infection as follows. Cells were cultured in 175 cm 2 cell culture bottles. At 80-90% confluence, the medium was removed and the cells were incubated for one hour with an MVA virus suspension (0.01 infectious particles ( ⁇ pfu) per cell, 0.01 ml/cm 2 ) in phosphate-buffered saline (PBS/Dulbecco, for example Animed AG, Muttenz, Switzerland, Order No. 23.100.10). Then medium was added (0.2 ml/cm 2 ) and the bottles were incubated at 37°C for 2-3 days until about 80%> of the cells had rounded. The vims lysates were stored with the cells and medium, without treatment, in the cell culture bottles at -30°C before further processing (purification etc.)
- the sediment composed of viral and cell particles, was 5 suspended once in PBS (10-20 times the volume of the sediment), and the suspension was centrifuged as above.
- the new sediment was suspended in 10 times the volume of RSB buffer ( 1 OmM Tris-HCl pH 8.0, lOmM KC1, lmM MgC12), and the suspension was briefly treated with ultrasound (Labsonic 1510 equipped with a 4 mm diameter tip, obtainable from Bender and Hobein, Zurich, Switzerland; 2x10 seconds at 60 watts and l o room temperature) in order to disintegrate remai ning still intact cells and to liberate the virus particles from the cell membranes.
- RSB buffer 1 OmM Tris-HCl pH 8.0, lOmM KC1, lmM MgC12
- the cell nuclei and the larger cell debris were removed in the subsequent brief centrifugation of the suspension (Sorvall GSA rotor obtainable fromDuPont Co, D-6353 Bad Nauheim, FRG; 3 minutes at 3000 ⁇ m and 10° C).
- the sediment was once again suspended in RSB buffer, treated with ultrasound and
- 20 virus particles was taken up in 10ml of lOmM Tris-HCl, pH8.0, homogenized by brief treatment with ultrasound(2xl0 seconds at room temperature, apparatus as described above), and applied to a stepped gradient for further purification.
- the steps of the gradient were each composed of 5 ml of sucrose in lOmM Tris-HCl, pH 8.0 (sucrose concentration steps: 20%, 25%, 30%, 35% and 40%).
- the gradient was centrifuged in a Kontron TST
- MVA genomic DNA sequences flanking the E3L gene were amplified by polymerase chain reaction using DNA of MVA (cloned isolate F6, 582 nd passage on CEF) as a 0 template.
- the primers of the upstream flanking region of E3L were 5'-
- Mutant virus MVA ⁇ E3L was generated using previously described methodology (Staib et al. 2000, Biotechniques, 28: 1137-1148). Briefly, monolayers of lxl 0 6 confluent BHK- 21 cells grown in 6-well tissue-culture plates (Costar, Corning NY, USA) were infected with MVA at a multiplicity of infection (MOI) of 0.01 IU per cell. Ninety min after 5 infection cells were transfected with 10 ⁇ g of plasmid P ⁇ E3L-K1L DNA per well using calcium phosphate (CellPhect Transfection Kit, Amersham Pharmacia Biotech, Freiburg, Germany) as recommended by the manufacturer.
- CaI multiplicity of infection
- Niral D ⁇ A from cloned MVA isolates was routinely analyzed by PCR as described previously (Staib et al 2000).
- viras multiplication was monitored after infecting CEF or BHK-21 monolayers with 0.05 infectious units (IU) MVA, MVA- ⁇ E3L or MVA-E3rev per cell.
- IU infectious units
- MVA- ⁇ E3L MVA- ⁇ E3L
- MVA-E3rev MVA-E3rev
- MOI multiplicity of infection
- the infected cells were washed twice with RPMl 1640 and incubated with fresh RPMl 1640 medium containing 10% FCS at 37 °C in a 5%> CO2 atmosphere.
- RPMl 1640 medium containing 10% FCS at 37 °C in a 5%> CO2 atmosphere.
- viras was released by freeze-thawing and brief sonication.
- Serial dilutions of the resulting lysates were plated on confluent BHK-21 monolayers grown in 6-well plates as replicates of two.
- media were removed 48 hours p.i, cells were briefly fixed in acetone: methanol (1: 1, 1 ml/well).
- Cytoplasmic extracts of infected cell monolayers were prepared by adding 0.2 ml 0.5% Nonidet P-40 lysis buffer (20 mM Tris-HCL, 10 mM NaCl (pH 8.0)) for 10 min. Polypeptides from cell extracts were separated by 10% sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and analyzed by autoradiography.
- SDS sodium dodecyl sulfate
- PAGE polyacrylamide gel electrophoresis
- DNA fragmentation- CEF were mock-infected or infected with MVA, MVA- ⁇ E3L and MVA-E3rev at an MOI of 20.
- Cells were harvested at 16h and 24h p.i. and total DNA was extracted as described.
- Precipitated DNA was resuspended in 100 ⁇ l H 2 0, treated with RNase (final concentration: 1 mg/ml) for 15 min at 37°C and resolved in an 1% agarose gel. DNA fragments were visualized by staining with ethidium bromide.
- CEF cells grown to confluency in a 12-well plates containing 0 12 mm glass coverslips were either non-infected or infected with MVA, MVA- ⁇ E3 L or
- MVA-E3rev at MOIs of 5 or 20.
- Cells were stained at 16 or 24h p.i. with Hoechst 3343 for 30 min at room temperature and photographed under a fluorescence microscope.
- the ratio of apoptotic cells to non-apoptotic cells was determined by counting and presented as the mean and three times the standard e ⁇ or of the mean (mean/3 X the
- Nuclear events of apoptosis in vitro cell free mitotic extracts a model system for analysis of the active phase of apoptosis. J. Cell. Biol. 123: 7-22) containing 10 mM HEPES-KOH, 40 mM ⁇ -glycerophosphate, 50 mM NaCl, 2 mM MgCl ; 5 mM EGTA, and 1 mM dithiothreitol [DTT], (pH 7.0) supplemented with 0.1% CHAPS ⁇ 3'-[(3'-cholamidopiOpyl)-dimethylammonio]-l-propanesulfonate ⁇ ,100 ⁇ g of bovine serum albumin and acetyl-DEVD-7-amino-4-methylcoumarib (DEVD-AMC) (final concentration: 10 ⁇ M).
- novel vector plasmids were constructed on the basis of pHi plasmid vectors which contain MVA flanking sequences (flank HI-1 and flank ffl-2) to target insertion of recombinant DNA precisely to the site of the naturally occuring deletion HI within the MVA genome (Sutter & Moss 1992, Staib et al. 2000).
- a 627 bp DNA fragment containing the complete coding sequence of the MVA E3L gene under transcriptional control of its authentic promoter sequence was prepared by PCR, treated with Klenow and inserted between the flank HI-l and flank LTJ-2 DNA sequences in plasmid pLW9 via the restriction site BamHI (Wyatt, L. et al. 1996, Vaccine 14: 1451- 1458).
- a 723-bp DNA fragment containing the g p open reading frame was excised from pEGFP-Nl (CLONETECH Laboratories GmbH, Heidelberg, Germany) using the restriction endonucleases BamHI and Not! , treated with Klenow polymerase, and inserted into the restriction site Smal of p ⁇ i-E3-P sH5 to obtain pIII-E3-P sH5 -gfp.
- Recombinant MVA with deleted E3L gene sequences were isolated during several rounds of plaque purification on RK-13 and BHK-21 cell monolayers. During plaque cloning the expected genome alterations were monitored by PCR (Fig. IB). Primary viras stocks were amplified on BHK-21 cells, re-assessed by Southern blot analysis of viral DNA (Fig. IC), and designated MVA- ⁇ E3L. Western blotting of cell lysates which were prepared in the presence or absence of arabinoside C confirmed the synthesis of E3L protein in cells infected with wild-type MVA, whereas no E3L polypeptides could be detected after infection with MVA- ⁇ E3L (Fig. ID).
- plasmid vector pE3Lrev containing a DNA fragment comprising the complete E3L gene expression cassette together with its genomic flanking sequences for transfection of MVA- ⁇ E3L-infected BHK cells.
- Revertant virus MVA-E3rev could be readily isolated by plaque purification in CEF monolayers. Stable reinsertion of E3L was confirmed by PCR with genomic MNA-E3rev D ⁇ A, and Western blot analysis of cell lysates revealed synthesis of E3L protein at levels equal to wild-type MVA (data not shown).
- MVA- E3rev could be isolated without need for additional screening or selection suggested a successful reversion of the growth defect of MVA- ⁇ E3L in CEF.
- CEF and BHK cells were metabolically labeled with r 35 Simethionine at various times after infection with MVA, MVA- ⁇ E3L, or MVA- E3rev. After each labeling period, lysates were prepared and analyzed by SDS-PAGE and autoradiography. In BHK cells infected with MVA, late viral protein synthesis occurred at 5 h after infection and became prominent with profound shut-off of cell protein synthesis at 10 h after infection (Fig. 3A).
- FIG. 3A Similar patterns of viral proteins were found in BHK cells infected with MVA- ⁇ E3L or MVA-E3rev (Fig. 3A). In CEF infected with MVA or MVA-E3rev abundant late viral protein synthesis was found at times 5h and lOh after infection (Fig. 3B). By contrast, in CEF cells infected with MVA- ⁇ E3L we could hardly detect polypeptide bands specific for viral protein production (Fig. 3B). Some weak bands of polypeptides co-migrating with typical late viral proteins became visible with the prominent shut-off of host cell protein synthesis at times after 5h of infection.
- This infection phenotype being characterized by prominent shutdown of viral protein synthesis together with maintained capacity for viral DNA replication was pronounced of the non-permissive vaccinia viras infection of Chinese hamster ovary (CHO) cells (Spehner e f a j., 1988; Ramsey-Ewing and Moss, 1995).
- the abortive vaccinia virus- CHO infection is associated with induction of apoptosis that can be overcome by co- expression of the cowpox virus gene CHO h r (Ink e t a ⁇ 1995, Ramsey-Ewing and Moss, 1998).
- vaccinia E3L gene product was described to inhibit induction of apoptosis in vaccinia virus-infected HeLa cells (Lee and Esteban, 1994; Kibler ei a ⁇ ., 1997). Because we had observed an unusual cell shrinkage when monitoring CEF cultures by light microscopy within 24h of infection with MVA- ⁇ E3L, it appeared important to probe if the growth restriction of MVA- ⁇ E3L in CEF cells could be possibly linked to apoptosis. In a first standard assay for apoptosis we monitored for the characteristic cleavage of DNA into 180 bp-multimers co ⁇ esponding to a nucleosomal "DNA ladder" (Wyllie et al, 1980).
- Total cellular DNA was isolated from CEF cultures infected either with MVA- ⁇ E3L, MVA or MVA-E3rev, separated by agarose gel electrophoresis, and visualized upon ethidium bromide staining. We obseived the typical fragmentations of cellular DNA indicative for apoptosis in samples from CEF cells infected with MVA- ⁇ E3L for 16h and 24h. In co ntrast, no such DNA laddering was detectable in samples from CEF cells infected with MVA or revertant virus MVA-E3rev (Fig. 4A). Another hallmark for apoptosis is the appearance of extranuclear nucleosomes.
- vaccinia virus replication Upon infection of mammalian cells vaccinia virus replication has been found to be relatively resistant to IFN activity. Interestingly, this appears to be different in CEF cultures in which pretreatment with chicken interferon can inhibit vaccinia virus growth.
- cell monolayers were fixed and foci of virus infected cells were visualized using vaccinia virus-specific immunostaining (Fig. 7).
- MVA-infected cell foci upon IFN treatment was very comparable to plaque formation seen with vaccinia viras WR or CVA.
- MVA-infected cells in monolayers treated with up to 10 U IFN/ml medium. This latter amount clearly affected number and size of the foci, while higher IFN concentrations resulted in complete growth inhibition.
- sha ⁇ contrast there were no' detectable virus -infected cells in monolayers that were inoculated with MVA- ⁇ E3L irrespective of IFN treatment confirming the incapability of this mutant to productively replicate in CEF.
- Single gene dependent host range phenotypes in vaccinia viras infection can be elegantly used for efficient selection of recombinant virases through reinsertion of the host range gene into the mutant virus genome.
- MVA- ⁇ E3L in CEF To verify if the growth restriction of MVA- ⁇ E3L in CEF would allow for such host range selection we constructed an MVA insertion plasmid to target reintroduction of the E3L coding sequences under transcriptional control of its authentic promoter into the site of deletion III in the MVA genome. The addition of an expression cassette of the A.
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US6287570B1 (en) * | 1998-11-23 | 2001-09-11 | Patricia L. Foley | Vaccine against swine influenza virus |
KR101163816B1 (en) | 2005-09-22 | 2012-07-09 | 도쿄엘렉트론가부시키가이샤 | Plasma processing method and apparatus |
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WO1997002355A1 (en) * | 1995-07-04 | 1997-01-23 | GSF-Forschungszentrum für Umwelt und Gesundheit GmbH | Recombinant mva virus, and the use thereof |
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Also Published As
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CA2459754C (en) | 2011-05-10 |
AU2002337083A1 (en) | 2003-03-24 |
DK1425404T3 (en) | 2008-10-06 |
US7049145B2 (en) | 2006-05-23 |
EP1425404A2 (en) | 2004-06-09 |
DE60227268D1 (en) | 2008-08-07 |
JP4406561B2 (en) | 2010-01-27 |
DE10144664B4 (en) | 2005-06-09 |
WO2003023040A3 (en) | 2003-11-27 |
EP1425404B1 (en) | 2008-06-25 |
CA2459754A1 (en) | 2003-03-20 |
DE10144664A1 (en) | 2003-03-27 |
US20050028226A1 (en) | 2005-02-03 |
ATE399210T1 (en) | 2008-07-15 |
JP2005502360A (en) | 2005-01-27 |
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