Generation of Recombinant Vaccinia Viruses

Linda S. Wyatt1, Patricia L. Earl1, Bernard Moss1

1 Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 16.17
DOI:  10.1002/cpmb.32
Online Posting Date:  January, 2017
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Abstract

This unit describes how to infect cells with vaccinia virus and then transfect them with a plasmid‐transfer vector or PCR fragment to generate a recombinant virus. Selection and screening methods used to isolate recombinant viruses and a method for the amplification of recombinant viruses are described. Finally, a method for live immunostaining that has been used primarily for detection of recombinant modified vaccinia virus Ankara (MVA) is presented. © 2017 by John Wiley & Sons, Inc.

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Generation of a Vaccinia Virus Vector by Homologous Recombination
  • Basic Protocol 2: Selection and Screening of Recombinant Virus Plaques
  • Basic Protocol 3: Amplification of Virus from a Plaque
  • Basic Protocol 4: Live Immunostaining and Amplification of MVA Recombinants
  • Support Protocol 1: PCR Amplification and DNA Sequencing of Inserted Gene
  • Support Protocol 2: Coating Plates with Concanavalin A
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Generation of a Vaccinia Virus Vector by Homologous Recombination

  Materials
  • Construct with gene of interest
  • pRB21, pSC11, pSC65, pLW‐44, pLAS‐1, pLW‐73, or other suitable vector (Table 16.17.1) BS‐C‐1, BHK‐21, or CEF cells (unit 16.16; Cotter et al., )
  • Vaccinia virus stock (unit 16.16; Cotter et al., )
  • Gel purification kit (QIAquick gel extraction kit; Qiagen, cat. no. 28704)
  • Complete MEM‐8 and ‐2.5 media (unit 16.16; Cotter et al., )
  • OptiMEM medium (Life Technologies)
  • Liposomal transfection reagent (e.g., Lipofectamine 2000, Life Technologies)
  • Transfection buffer (see recipe)
  • 2.5 M CaCl 2
  • Phosphate‐buffered saline (PBS; appendix 22)
  • Dry ice/ethanol bath
  • 12‐well tissue culture plates
  • 12 × 75–mm polystyrene tubes
  • Cup sonicator (e.g., Ultrasonic Processor VCX‐750 from Sonics and Materials)
  • Disposable scraper, plunger from a 1‐ml syringe, or rubber policeman, sterile
  • 2‐ml sterile microcentrifuge tubes (Sarstedt 2 ml screw‐cap tubes are optional)
  • Additional reagents and equipment for subcloning (Struhl, ), isolation of plasmid (Heilig et al., ), PCR amplification (Kramer and Coen, ), overlap PCR (see, e.g., Burrill et al., ), agarose gel electrophoresis (Voytas, ), and tissue culture ( appendix 3F; Phelan, )
NOTE: All reagents and equipment coming into contact with live cells must be sterile, and aseptic technique should be used accordingly. Culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

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; Cotter et al., ) and appropriate complete medium
  • Complete MEM‐2.5 medium (unit 16.16; Cotter et al., )
  • Methylcellulose (Sigma, cat. no. 274429‐500G)
  • Minimal essential medium (MEM), serum‐free, as solvent for methylcellulose
  • 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 ( protocol 1)
  • 5 mg/ml 5‐bromodeoxyuridine (BrdU) in H 2O (for TK selection; filter sterilize and store at –20°C)
  • 4% Xgal in dimethylformamide (optional, for β‐galactosidase screening)
  • 2% Xgluc in dimethylformamide (optional, for GUS screening)
  • Dry ice/ethanol bath
  • 6‐well, 35‐mm2 tissue culture plates
  • Cup sonicator (e.g., Ultrasonic Processor VCX‐750 from Sonics and Materials)
  • Fluorescence microscope (for GFP or other fluorescent protein screening)
  • Pipet tips or toothpicks, sterile
  • Sterile microcentrifuge tubes (Sarstedt 2 ml screw‐cap tubes are optional)
  • Additional reagents and equipment for tissue culture, including counting cells ( appendix 3F; Phelan, )
NOTE: All reagents and equipment coming into contact with live cells must be sterile, and aseptic technique should be used accordingly. Culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Basic Protocol 3: Amplification of Virus from a Plaque

  Materials
  • Resuspended recombinant plaque (see protocol 2)
  • Complete MEM‐2.5 and ‐8 media (unit 16.16; Cotter et al., )
  • Confluent monolayer cultures of appropriate cells in both a 6‐well and a 25‐cm2 tissue culture flask (unit 16.16; Cotter et al., )
  • 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 H 2O (for TK selection; filter sterilize and store at –20°C)
  • Spinner culture of HeLa S3 cells (unit 16.16; Cotter et al., )
  • Cup sonicator (e.g., Ultrasonic Processor VCX‐750 from Sonics and Materials)
  • 6‐well, 35‐mm2 tissue culture plates
  • 15‐ml conical centrifuge tubes
  • Sorvall centrifuge with H‐6000A rotor (or equivalent)
  • 162‐cm2 tissue culture flask
  • Additional reagents and equipment for tissue culture and counting cells ( appendix 3F; Phelan, )
NOTE: All reagents and equipment coming into contact with live cells must be sterile, and aseptic technique should be used accordingly. Incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Basic Protocol 4: Live Immunostaining and Amplification of MVA Recombinants

  Materials
  • 162‐cm2 flask of confluent CEF (unit 16.16; Cotter et al., )
  • Complete MEM‐2.5 and ‐8 media (unit 16.16; Cotter et al., )
  • Transfected cell lysate (see protocol 1)
  • Methylcellulose (Sigma, cat. no. 274429‐500G)
  • Primary antibody to protein product of foreign gene
  • Horseradish peroxidase–conjugated secondary antibody (to species of primary antibody)
  • Dry ice/ethanol bath
  • BHK‐21 cells (unit 16.16; Cotter et al., ; optional)
  • Concanavalin A–coated 6‐well tissue culture plates (see Support Protocol 3)
  • Cup sonicator (e.g., Ultrasonic Processor VCX‐750 from Sonics and Materials)
  • Inverted microscope
  • Sterile toothpicks
  • Cell scraper or plunger of 1‐ml syringe
  • 75‐ and 162‐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; Cotter et al., )
NOTE: All reagents and equipment coming into contact with live cells must be sterile, and aseptic technique should be used accordingly. Incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Support Protocol 1: PCR Amplification and DNA Sequencing of Inserted Gene

  Materials
  • Titered lysate of recombinant virus (see protocol 3)
  • 6‐well, 35‐mm2 tissue culture plate with appropriate confluent monolayer cells (unit 16.16; Cotter et al., )
  • Cell scraper or plunger of 1‐ml syringe
  • 2‐ml screw‐cap tubes
  • QIAamp DNA Blood Mini Kit (QIAgen, cat. no. 51104)
  • Appropriate PCR and sequencing primers
  • Additional reagents and equipment for PCR (Kramer and Coen, ), agarose gel electrophoresis (Voytas, ), and DNA sequencing (Shendure et al., )

Support Protocol 2: Coating Plates with Concanavalin A

  Materials
  • Concanavalin A (Sigma)
  • Phosphate‐buffered saline (PBS; appendix 22)
  • 6‐well, 35‐mm2 tissue culture plates
  • 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

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. doi: 10.1073/pnas.82.7.2096.
  Bisht, H., Roberts, A., Vogel, L., Bukreyev, A., Collins, P.L., Murphy, B.R., Subbarao, K., and Moss, B. 2004. Severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice. Proc. Natl. Acad. Sci. U.S.A. 101:6641‐6646. doi: 10.1073/pnas.0401939101.
  Blasco, R. and Moss, B. 1995. Selection of recombinant vaccinia viruses on the basis of plaque formation. Gene 158:157‐162. doi: 10.1016/0378‐1119(95)00149‐Z.
  Burrill, C.P., Strings, V.R., Schulte, M.B., and Andino, R. 2013. Poliovirus: Generation and characterization of mutants. Curr. Protoc. Microbiol. 29:15H.2.1‐15H.2.32.
  Carroll, M.W. and Moss, B. 1995. E. coli β‐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. doi: 10.1006/viro.1997.8845.
  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.
  Cotter, C.A., Earl, P.L., Wyatt, L.S., and Moss, B. 2017. Preparation of cell cultures and vaccinia virus stocks. Curr. Protoc. Mol. Biol. 117:16.16.1‐16.16.18. doi: 10.1002/cpmb.33
  Davison, A.J. and Moss, B. 1990. New vaccinia virus recombination plasmids incorporating a synthetic late promoter for high level expression of foreign proteins. Nucleic Acids Res. 18:4285‐4286. doi: 10.1093/nar/18.14.4285.
  Domi, A. and Moss, B. 2005. Engineering of a vaccinia virus bacterial artificial chromosome in Escherichia coli by bacteriophage lambda‐based recombination. Nat. Methods 2:95‐97. doi: 10.1038/nmeth734.
  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.
  Elroy‐Stein, O. and Moss, B. 2001. Gene expression using the vaccinia virus/T7 RNA polymerase hybrid system. Curr. Protoc. Mol. Biol. 43:16.19.1‐16.19.11.
  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.
  Heilig, J., Elbing, K.L., and Brent, R. 1998. Large‐scale preparation of plasmid DNA. Curr. Protoc. Mol. Biol. 41:1.7.1‐1.7.16.
  Isaacs, S.N., Kotwal, G.J., and Moss, B. 1990. Reverse guanine phosphoribosyltransferase selection of recombinant vaccinia viruses. Virology 178:626‐630. doi: 10.1016/0042‐6822(90)90367‐Z.
  Kingston, R.E., Chen, C.A., and Rose, J.K. 2003. Calcium phosphate transfection. Curr. Protoc. Mol. Biol. 63:9.1.1‐9.1.11.
  Kramer, M. F. and Coen, D. M. 2000. Enzymatic amplification of DNA by PCR: Standard procedures and optimization. Curr. Protoc. Mol. Biol. 56:15.1.1‐15.1.14.
  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.
  Merchlinsky, M., Eckert, D., Smith, E., and Zauderer, M. 1997. Construction and characterization of vaccinia direct ligation vectors. Virology 238:444‐451. doi: 10.1006/viro.1997.8828.
  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. doi: 10.1099/0022‐1317‐72‐5‐1031.
  Mortensen, R.M. and Kingston, R.E. 2009. Selection of transfected mammalian cells. Curr. Protoc. Mol. Biol. 86: 9.5.1‐9.5.13.
  Moss, B. and Earl, P.L. 2002. Overview of the vaccinia virus expression system. Curr. Protoc. Mol. Biol. 60:16.15.1‐16.15.5.
  Ourmanov, I., Brown, C.R., Moss, B., Carroll, M., Wyatt, L., Pleteva, L., Goldstein, S., Venson, D., and Hirsch, V.M. 2000. Comparative efficacy of recombinant modified vaccinia virus ankara expressing simian immunodeficiency virus (SIV) gag‐pol and/or env in macaque challenged with pathogenic SIV. J. Virol. 74:2740‐2751. doi: 10.1128/JVI.74.6.2740‐2751.2000.
  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. doi: 10.1073/pnas.85.24.9431.
  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. doi: 10.1099/0022‐1317‐76‐12‐2957.
  Phelan, M.C. 2006. Techniques for mammalian cell tissue culture. Curr. Protoc. Mol. Biol. 4:F:A.3F.1‐A.3F.18.
  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. doi: 10.1007/BF01718324.
  Shendure, J.A., Porreca, G.J., Church, G.M., Gardner, A.F., Hendrickson, C.L., Kieleczawa, J., and Slatko, B.E. 2011. Overview of DNA sequencing strategies. Curr. Protoc. Mol. Biol. 96:7.1.1‐7.1.23.
  Smith, G.L. and Moss, B. 1983. Infectious poxvirus vectors have capacity for at least 25,000 base pairs of foreign DNA. Gene 25:21‐28. doi: 10.1016/0378‐1119(83)90163‐4.
  Struhl, K. 1987. Subcloning of DNA fragments. Curr. Protoc. Mol. Biol. 13:3.16.1‐3.16.2.
  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. doi: 10.1016/0264‐410X(94)90341‐7.
  Voytas, D. 2000. Agarose gel electrophoresis. Curr. Protoc. Mol. Biol. 51:2.5A.1‐2.5A.9.
  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. doi: 10.1016/S0264‐410X(96)00072‐2.
   Wyatt, L.S., Earl, P.L., Xiao, W., Americo, J.A., Cotter, C.A., Vogt, J., and Moss, B. 2009. Elucidating and minimizing the loss by recombinant vaccinia virus of human immunodeficiency virus gene expression resulting from spontaneous mutations and positive selection. J. Virol. 83:7176‐7184. doi: 10.1128/JVI.00687‐09.
  Wyatt, L.S., Earl, P.L., Vogt, J., Eller, L.A., Chandran, D., Liu, J., Robinson, H.L., and Moss, B. 2008. Correlation of immunogenicities and in vitro expression levels of recombinant modified vaccinia virus Ankara HIV vaccines. Vaccine 26:486‐493. doi: 10.1016/j.vaccine.2007.11.036.
Key References
  Mackett et al., 1984. See above.
  Early report of the method used to construct recombinant virus expression vectors.
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