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Generation of Recombinant Vaccinia Viruses

Patricia L. Earl1,  Bernard Moss1,  Linda S. Wyatt1,  Miles W. Carroll2

1National Institute of Allergy and Infectious Diseases, Bethesda, Maryland
2Oxford BioMedica, Oxford, United Kingdom


Publication Name: 
Current Protocols in Molecular Biology
Unit Number: 
Unit 16.17
DOI: 
10.1002/0471142727.mb1617s43
Online Posting Date: 
May, 2001
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Abstract

This unit first describes how to infect cells with vaccinia virus and then transfect them with a plasmid-transfer vector to generate a recombinant virus. Methods are also presented for purifying vaccinia virus and for isolating viral DNA, which can be used during transfection. Also presented are selection and screening methods used to isolate recombinant viruses and a method for the amplification of recombinant viruses. Finally, a method for live immunostaining that has been used primarily for detection of recombinant modified vaccinia virus Ankara (MVA) is presented.

     
 
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Table of Contents

  • Unit Introduction
  • Basic Protocol 1: Transfection of Infected Cells with a Vaccinia Vector
  • Support Protocol 1: Purification of Vaccinia Virus
  • Support Protocol 2: Isolation of Vaccinia Virus DNA
  • Basic Protocol 2: Selection and Screening of Recombinant Virus Plaques
  • Basic Protocol 3: Amplification of a Plaque
  • Basic Protocol 4: Live Immunostaining of MVA Recombinants
  • Support Protocol 3: Coating Plates with Concanavalin A
  • Reagents and Solutions
  • Commentary
  • Bibliography
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Transfection of Infected Cells with a Vaccinia Vector

 Materials
  • pSC11, pRB21, pSC65, pLW9, or other suitable vector (Table 16.17.1)
     
    Table 16.17.1 Vaccinia Virus Transfer Vectors
    VectoraPromoterbCloning sitescInsertion sitesdSelection/screeningReference
    pGS20p7.5 (E/L)BamHI; SmaITKTK–Mackett et al., 1984
    pSC11p7.5 (E/L)SmaI; MCSTKTK–, -galChakrabarti et al., 1985; Earl et al., 1990; Bacik et al., 1994
    pMJ601, pMJ602psyn (L)MCSTKTK–, -galDavison and Moss, 1990
    pRB21psyn (E/L)MCSF12L/F13LPlaqueBlasco and Moss, 1995
    pMC02psyn (E/L)MCSTKTK–, GUSCarroll and Moss, 1995
    pSC59psyn (E/L)MCSTKTK–Chakrabarti et al., 1997
    pSC65psyn (E/L)MCSTKTK–, -galChakrabarti et al., 1997
    pJS4psyn (E/L) ×2MCSTKTK–Chakrabarti et al., 1997
    pJS5psyn (E/L) ×2MCSTKTK–, gptChakrabarti et al., 1997
    pG06psyn (E/L) ×2MCSDel IIITransient gpt eSutter et al., 1994
    pLW-7psyn (E/L)MCSDel IIITransient gpt eWyatt et al., 1996
    pMC03psyn (E/L)MCSDel IIIGUSCarroll and Moss, 1995
    pLW-9pH5 (E/L)MCSDel IIITransient gpt eWyatt et al., 1996
    pLW-17pH5 (E/L)MCSDel IINoneL. Wyatt and B. Moss, unpub. observ.
    pLW-21psyn (E/L)MCSDel IINoneL. Wyatt and B. Moss, unpub. observ.
    pLW-22psyn (E/L)MCSDel II-galL. Wyatt and B. Moss, unpub. observ.
    pLW-24p7.5 (E/L)MCSDel IINoneL. Wyatt and B. Moss, unpub. observ.
     a pRB21 was specifically designed for use with vaccinia virus vRB12, which has a deletion in the F13L gene. The plasmids pG06, pLW-7, pMC03, pLW-9, pLW-17, pLW-21, pLW-22, and pLW-24 were designed for MVA.
     b Abbreviations: E, early; L, late; E/L, early and late. The designation “×2” refers to two oppositely oriented promoters that can be used for expression of two genes.
     c SmaI digestion gives a blunt end for cloning any fragment that has been blunt-ended. MCS signifies multiple cloning sites.
     d Abbreviations: TK, thymidine kinase locus; F12L/F13L, between F12L and F13L open reading frames; Del III, site of natural deletion in MVA.
     e Transient selection in which XGPRT gene is deleted from recombinant vaccinia virus during recombination; see Background Information.
  • CV-1, BS-C-1, BHK-21, or CEF cells (UNIT 16.16)
  • Complete MEM-10 and -2.5 media (UNIT 16.16)
  • Vaccinia virus stock (UNIT 16.16)
  • 0.25 mg/ml trypsin (2× crystallized and salt-free; Worthington; filter sterilize and store at –20°C)
  • Transfection buffer (see recipe)
  • 2.5 M CaCl2
  • 20 mM HEPES, pH 7.4
  • DOTAP liposomal transfection reagent (Boehringer Mannheim)
  • OptiMEM medium (Life Technologies)
  • Dry ice/ethanol bath
  • 25-cm2 tissue culture flask
  • 12 × 75–mm polystyrene tubes
  • Disposable scraper or rubber policeman, sterile
  • 15-ml conical centrifuge tubes
  • Sorvall centrifuge with H-6000A rotor (or equivalent)
  • Additional reagents and equipment for subcloning (UNIT 3.16), isolation of plasmid (UNIT 1.7), and tissue culture (APPENDIX 3F)

NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO2 incubator unless otherwise specified.

NOTE: All reagents and equipment coming into contact with live cells must be sterile, and aseptic technique should be used accordingly.


Support Protocol 1: Purification of Vaccinia Virus

 Materials
  • Vaccinia virus stock (UNIT 16.16)
  • 0.25 mg/ml trypsin (2× crystallized and salt-free; Worthington; filter sterilize and store at –20°C)
  • HeLa S3 cells growing in suspension culture (UNIT 16.16)
  • Complete spinner medium–5 (UNIT 16.16)
  • 10 mM and 1 mM Tris·Cl, pH 9.0 (APPENDIX 2)
  • 36% (w/v) sucrose solution in 10 mM Tris·Cl, pH 9.0
  • 40%, 36%, 32%, 28%, and 24% (w/v) sucrose solutions in 1 mM Tris·Cl, pH 9.0
  • Sorvall centrifuge with H-6000A rotor (or equivalent)
  • 2-liter vented spinner flasks (microcarrier type; Bellco)
  • Dounce homogenizer, glass, with tight pestle
  • Cup sonicator (e.g., Ultrasonic Processor VC-600 from Sonics and Materials)
  • Ultracentrifuge with Beckman SW 27 or SW 28 rotor (or equivalent) and sterile centrifuge tubes
  • Spectrophotometer
  • Additional reagents and equipment for tissue culture and counting cells (APPENDIX 3F), and titering virus (UNIT 16.16)

NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO2 incubator unless otherwise specified.

NOTE: All reagents and equipment coming into contact with live cells must be sterile, and aseptic technique should be used accordingly.


Support Protocol 2: Isolation of Vaccinia Virus DNA

 Materials
  • Purified vaccinia virus (see Support Protocol 1)
  • 50 mM and 1 M Tris·Cl, pH 7.8 (APPENDIX 2)
  • 10% (w/v) sodium dodecyl sulfate (SDS)
  • 60% (w/v) sucrose
  • 10 mg/ml proteinase K
  • Buffered phenol: phenol equilibrated with 50 mM Tris·Cl, pH 7.8 (UNIT 2.1)
  • 1:1 (v/v) phenol/chloroform
  • 1 M sodium acetate, pH 7.0
  • 100% and 95% ethanol
  • Spectrophotometer
  • Sorvall centrifuge with H-6000A rotor (or equivalent)
  • Additional reagents and equipment for phenol extraction of DNA (UNIT 2.1) and measuring DNA concentration (APPENDIX 3D)

NOTE: Avoid vortexing the DNA throughout this protocol.

Basic Protocol 2: Selection and Screening of Recombinant Virus Plaques

 Materials
  • BS-C-1, HuTK– 143B, BHK-21, or CEF confluent monolayer cells (UNIT 16.16) and appropriate complete medium
  • Complete MEM-2.5 medium (UNIT 16.16)
  • Selective agents (for XGPRT selection; filter sterilize, and store at –20°C):     10 mg/ml (400×) mycophenolic acid (MPA; Calbiochem) in 0.1 N NaOH     10 mg/ml (40×) xanthine in 0.1 N NaOH     10 mg/ml (670×) hypoxanthine in 0.1 N NaOH
  • Transfected cell lysate (see Basic Protocol 1)
  • 2% LMP agarose (Life Technologies) in H2O, sterilized by autoclaving
  • Complete 2× plaque medium-5 (see recipe)
  • 5 mg/ml 5-bromodeoxyuridine (BrdU) in H2O (for TK selection; filter sterilize and store at –20°C)
  • 10 mg/ml neutral red in H2O
  • 4% Xgal in dimethylformamide (optional, for -galactosidase screening; Table 1.4.2)
  • 2% Xgluc in dimethylformamide (optional, for GUS screening)
  • Dry ice/ethanol bath
  • 6-well, 35-mm tissue culture plates
  • Cup sonicator (e.g., Ultrasonic Processor VC-600 from Sonics and Materials)
  • 45°C water bath
  • Cotton-plugged Pasteur pipets, sterile
  • Additional reagents and equipment for tissue culture and counting cells (APPENDIX 3F) and serial dilution of virus (UNIT 1.11)

NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO2 incubator unless otherwise specified.

NOTE: All reagents and equipment coming into contact with live cells must be sterile, and aseptic technique should be used accordingly.


Basic Protocol 3: Amplification of a Plaque

 Materials
  • Resuspended recombinant plaque (see Basic Protocol 2)
  • Confluent monolayer cultures of appropriate cells in both a 12-well, 22-mm tissue culture plate and a 25-cm2 tissue culture flask (UNIT 16.16)
  • Complete MEM-2.5 and -10 media (UNIT 16.16)
  • Selective agents (for XGPRT selection; filter sterilize, and store at –20°C):
        10 mg/ml (400×) mycophenolic acid (MPA; Calbiochem) in 0.1 N NaOH
        10 mg/ml (40×) xanthine in 0.1 N NaOH
        10 mg/ml (670×) hypoxanthine in 0.1 N NaOH
  • 5 mg/ml 5-bromodeoxyuridine (BrdU) in H2O (for TK selection; filter sterilize and store at –20°C)
  • Dry ice/ethanol bath
  • Spinner culture of HeLa S3 cells (UNIT 16.16)
  • Cup sonicator (e.g., Ultrasonic Processor VC-600 from Sonics and Materials)
  • 15-ml conical centrifuge tubes
  • Sorvall centrifuge with H-6000A rotor (or equivalent)
  • 150-cm2 tissue culture flask
  • Additional reagents and equipment for tissue culture and counting cells (APPENDIX 3F)

NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO2 incubator unless otherwise specified.

NOTE: All reagents and equipment coming into contact with live cells must be sterile, and aseptic technique should be used accordingly.


Basic Protocol 4: Live Immunostaining of MVA Recombinants

 Materials
  • 150-cm2 flask of confluent CEF (UNIT 16.16)
  • Complete MEM-2, -2.5, and -10 media (UNIT 16.16)
  • Transfected cell lysate (see Basic Protocol 1)
  • Dry ice/ethanol bath
  • Primary antibody to protein product of foreign gene
  • Horseradish peroxidase–conjugated secondary antibody (to species of primary antibody)
  • Concanavalin A–coated 6-well tissue culture plates (see Basic Protocol 3)
  • Cup sonicator (e.g., Ultrasonic Processor VC-600 from Sonics and Materials)
  • Inverted microscope
  • Sterile toothpicks
  • Cell scraper or plunger of 1-ml syringe
  • 75- and 150-cm2 tissue culture flasks
  • Additional reagents and equipment for culture, trypsinization, and immunostaining of CEF cells, titering of MVA, and preparation of MVA stocks (UNIT 16.16)

NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO2 incubator unless otherwise specified.

NOTE: All reagents and equipment coming into contact with live cells must be sterile, and aseptic technique should be used accordingly.


Support Protocol 3: Coating Plates with Concanavalin A

 Materials
  • Concanavalin A (Sigma)
  • Phosphate-buffered saline (PBS; APPENDIX 2)
  • 6-well, 35-mm tissue culture dishes
  • Plastic bags for storage

NOTE: All reagents and equipment coming into contact with live cells must be sterile, and aseptic technique should be used accordingly.

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Figures

  • Figure 16.17.1
    Homologous recombination between a transfected plasmid and the vaccinia virus genome. TKL and TKR are vaccinia virus DNA sequences flanking the foreign gene. p11 and p7.5 are promoters.

    View Image
  • Figure 16.17.2
    Plasmid transfer vector pSC11.

    View Image

Literature Cited

 Literature Cited
    Bacik, I., Cox, J.H., Anderson, R., Yewdell, J.W., and Bennink, J.R. 1994. TAP (transporter associated with antigen processing)–independent presentation of endogenously synthesized peptides is enhanced by endoplasmic reticulum insertion sequences located at the amino- but not carboxyl-terminus of the peptide. J. Immunol. 152:381-387.
    Bertholet, C., Drillien, R., and Wittek, R. 1985. One hundred base pairs of 5¢ flanking sequence of a vaccinia virus late gene are sufficient to temporally regulate late transcription. Proc. Natl. Acad. Sci. U.S.A. 82:2096-2100.
    Blasco, R. and Moss, B. 1995. Selection of recombinant vaccinia viruses on the basis of plaque formation. Gene 158:157-162.
    Carroll, M.W. and Moss, B. 1995. E. coli -glucuronidase (GUS) as a marker for recombinant vaccinia viruses. BioTechniques 19:352-355.
    Carroll, M.W. and Moss, B. 1997. Host range and cytopathogenicity of the highly attenuated MVA strain of vaccinia virus: Propagation and generation of recombinant viruses in a nonhuman mammalian cell line. Virology 238:198-211.
    Chakrabarti, S., Brechling, K., and Moss, B. 1985. Vaccinia virus expression vector: Coexpression of beta-galatosidase provides visual screening of recombinant virus plaques. Mol. Cell. Biol. 5:3403-3409.
    Chakrabarti, S., Sisler, J.R., and Moss, B. 1997. Compact, synthetic, vaccinia virus early/late promoter for protein expression. BioTechniques 23:1094-1097.
    Cochran, M.A., Puckett, C., and Moss, B. 1985. In vitro mutagenesis of the promoter region for a vaccinia virus gene: Evidence for tandem early and late regulatory signals. J. Virol. 54:30-37.
    Davison, A.J. and Moss, B. 1990. New vaccinia virus recombination plasmids incorporating a synthetic late promoter for high level expression of foreign proteins. Nucl. Acids Res. 18:4285-4286.
    Earl, P., Koenig, S., and Moss, B. 1990. Biological and immunological properties of human immunodeficiency virus type 1 envelope glycoprotein: Analysis of proteins with truncations and deletions expressed by recombinant vaccinia viruses. J. Virol. 65:31-41.
    Falkner, F.G. and Moss, B. 1988. Escherichia coli gpt gene provides dominant selection for vaccinia virus open reading frame expression vectors. J. Virol. 62:1849-1854.
    Falkner, F.G. and Moss, B. 1990. Transient dominant selection of recombinant vaccinia viruses. J. Virol. 64:3108-3111.
    Isaacs, S.N., Kotwal, G.J., and Moss, B. 1990. Reverse guanine phosphoribosyltransferase selection of recombinant vaccinia viruses. Virology 178:626-630.
    Mackett, M., Smith, G.L., and Moss, B. 1984. General method for production and selection of infectious vaccinia virus recombinants expressing foreign genes. J. Virol. 49:857-864.
    Meyer, H., Sutter, G., and Mayr, A. 1991. Mapping of deletions in the genome of the highly attenuated vaccinia virus MVA and their influence on virulence. J. Gen. Virol. 72:1031-1038.
    Merchlinsky, M., Eckert, D., Smith, E., and Zauderer, M. 1997. Construction and characterization of vaccinia direct ligation vectors. Virology 238:444-451.
    Patel, D.D., Ray, C.A., Drucker, R.P., and Pickup, D.J. 1988. A poxvirus-derived vector that directs high levels of expression of cloned genes in mammalian cells. Proc. Natl. Acad. Sci. U.S.A. 85:9431-9435.
    Pfleiderer, M., Falkner, F.G., and Dorner, F. 1995. A novel vaccinia virus expression system allowing construction of recombinants without the need for selection markers, plasmids and bacterial hosts. J. Gen. Virol. 76:2957-2962.
    Scheiflinger, F., Falkner, F.G., and Dorner, F. 1996. Evaluation of the thymidine kinase (tk) locus as an insertion site in the highly attenuated vaccinia MVA strain. Arch. Virol. 141:663-669.
    Sutter, G., Wyatt, L.S., Foley, P.L., Bennink, J.R., and Moss, B. 1994. A recombinant vector derived from the host range-restricted and highly attenuated MVA strain of vaccinia virus stimulates protective immunity in mice to influenza virus. Vaccine 12:1032-1040.
    Wyatt, L.S., Shors, S.T., Murphy, B.R., and Moss, B. 1996. Development of a replication-deficient recombinant vaccinia virus vaccine effective against parainfluenza virus 3 infection in an animal model. Vaccine 14:1451-1458.
 Key References
    Mackett et al., 1984. See above.
    Piccini, A., Perkus, M.E., and Paoletti, E. 1987. Vaccinia virus as an expression vector. Methods Enzymol. 153:545-563.
     
 
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