Generation of Recombinant Vaccinia Viruses

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

1 National Institute of Allergy and Infectious Diseases, Bethesda, Maryland, 2 Oxford BioMedica, Oxford, null
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 5.13
DOI:  10.1002/0471140864.ps0513s13
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

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.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

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
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

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

 Materials
  • pSC11, pRB21, pSC65, pLW9, or other suitable vector (Table 5.13.1)
  • CV-1, BS-C-1, BHK-21, or CEF cells (unit 5.12)
  • Complete MEM-10 and -2.5 media (unit 5.12)
  • Vaccinia virus stock (unit 5.12)
  • 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 and isolation of plasmid (appendix 4C), and tissue culture (appendix 3C)

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.
 
Table 5.13.1 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 gpteSutter et al., 1994
pLW-7psyn(E/L)MCSDel IIITransient gpteWyatt et al., 1996
pMC03psyn (E/L)MCSDel IIIGUSCarroll and Moss, 1995
pLW-9pH5 (E/L)MCSDel IIITransient gpteWyatt 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.
 apRB21 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.
 bAbbreviations: 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.
 cSmaI digestion gives a blunt end for cloning any fragment that has been blunt-ended. MCS signifies multiple cloning sites.
 dAbbreviations: TK, thymidine kinase locus; F12L/F13L, between F12L and F13L open reading frames; Del III, site of natural deletion in MVA.
 eTransient selection in which XGPRT gene is deleted from recombinant vaccinia virus during recombination; see Background Information.

Support Protocol 1: Purification of Vaccinia Virus

 Materials
  • Vaccinia virus stock (unit 5.10)
  • 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 5.12)
  • Complete spinner medium–5 (unit 5.12)
  • 10 mM and 1 mM Tris·Cl, pH 9.0 (appendix 2E)
  • 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 3C), and titering virus (unit 5.12)

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 2E)
  • 10% (w/v) sodium dodecyl sulfate (SDS; appendix 2E)
  • 60% (w/v) sucrose
  • 10 mg/ml proteinase K
  • Buffered phenol: phenol equilibrated with 50 mM Tris×Cl, pH 7.8 (appendix 2E)
  • 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 (appendix 4E) and measuring DNA concentration (appendix 4K)

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 5.12) and appropriate complete medium
  • Complete MEM-2.5 medium (unit 5.12)
  • 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% low-melting point (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)
  • 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 3C)

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 5.12)
  • Complete MEM-2.5 and -10 media (unit 5.12)
  • 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 5.12)
  • 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 3C)

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 5.12)
  • Complete MEM-2, -2.5, and -10 media (unit 5.12)
  • 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 Support 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 5.12)

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 2E)
  • 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.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

  •  FigureFigure 5.13.1 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
  •  FigureFigure 5.13.2 Plasmid transfer vector pSC11.
    View Image

Videos

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.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library