Vaccination of Mice with Baculovirus‐Infected Insect Cells Expressing Antigenic Proteins

Kimberly R. Jordan1, Fran Crawford2, John W. Kappler2, Jill E. Slansky1

1 University of Colorado, Denver, Colorado, 2 Howard Hughes Medical Institute, Denver, Colorado
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 2.15
DOI:  10.1002/0471142735.im0215s85
Online Posting Date:  April, 2009
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Abstract

Methods to induce antigen‐specific immune responses in mice using insect cells infected with recombinant baculoviruses are described in this unit. Although this vaccine strategy has been used to generate both antibody and T cell responses, it has been more thoroughly characterized for the peptide‐specific cytotoxic T cell responses. Nonspecific responses to the vaccine vehicle are controlled for by vaccinating with insect cells infected with baculoviruses encoding irrelevant antigens or no antigen. The baculovirus‐infected insect cells alone are an effective immune adjuvant to elicit antigen‐specific T cells. Overall, immune responses generated using this approach are similar to those generated by more conventional vaccine strategies. Curr. Protoc. Immunol. 85:2.15.1‐2.15.23. © 2009 by John Wiley & Sons, Inc.

Keywords: vaccine; immune responses; peptides; baculovirus; T cells; molecular cloning; insect cells

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Construction of Baculovirus Expression Vector Encoding Recombinant Peptide‐MHC Molecules or Antigen Protein
  • Basic Protocol 2: Transfection of the Peptide‐MHC (MHC‐TM‐Pepβ2M) Construct into Sf9 Insect Cells and Homologous Recombination of Plasmid DNA with Baculovirus DNA
  • Support Protocol 1: Confirmation of the Antigenic Peptide Sequence Encoded by the Baculovirus
  • Support Protocol 2: Growing Insect Cells
  • Support Protocol 3: Acid Washing Glassware
  • Basic Protocol 3: Cloning Baculoviruses that Express Peptide‐MHC by Limiting Dilution
  • Basic Protocol 4: Vaccination of Mice with Baculovirus‐Infected Insect Cells
  • Support Protocol 4: Quantification of Peptide‐MHC Expression in Baculovirus‐Infected Insect Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Construction of Baculovirus Expression Vector Encoding Recombinant Peptide‐MHC Molecules or Antigen Protein

  Materials
  • cDNA encoding MHC class I molecule of interest (investigator generated or commercially available)
  • Oligonucleotide primers for amplifying MHC (also see Fig. and unit 10.20): 5′ primer containing SalI site (primer no. 5; see Fig. ) and 3′ primer containing NheI site (primer no. 6; see Fig. ) in frame with the end of the α3 domain of the MHC molecule (Integrated DNA Technologies)
  • SalI, NheI, XbaI, BspEI restriction enzymes and buffers (New England Biolabs)
  • Modified pBacp10pH vector encoding LdTM‐AH1β2m (available from the authors upon request)
  • Gel extraction kit (optional): e.g., Qiagen Gel Extraction Kit
  • 5× T4 DNA ligase buffer (containing 5 mM ATP; Invitrogen)
  • 1 Weiss unit/µl T4 DNA ligase (Invitrogen)
  • Competent E. coli cells, DH5α (homemade or purchased)
  • LB plates (unit 10.19) containing 50 µg/ml ampicillin or carbenicillin
  • LB medium (unit 10.3) containing 50 µg/ml ampicillin or carbenicillin
  • PCR primers for subcloning antigenic peptide into vector (Integrated DNA Technologies; also see unit 10.20):
    • 5′ p10 primer (no. 7 in Fig. ) containing XbaI site
    • 3′ peptide primer 1 (no. 8 in Fig. )
    • 3′ peptide primer 2 (no. 9 in Fig. ) containing BspEI site
  • Qiagen Midiprep kit (or equivalent)
  • PCR purification columns (e.g., S300, GE Healthcare)
  • 14°C recirculating water bath
  • Culture tubes
  • Additional reagents and equipment for PCR amplification (unit 10.20), agarose gel electrophoresis (unit 10.4), restriction enzyme digestion (unit 10.8), DNA gel purification and quantitation with ethidium bromide (unit 10.5), isolation of plasmid DNA (miniprep; unit 10.3), and DNA sequencing (Chapter 7 in Ausubel et al., )

Basic Protocol 2: Transfection of the Peptide‐MHC (MHC‐TM‐Pepβ2M) Construct into Sf9 Insect Cells and Homologous Recombination of Plasmid DNA with Baculovirus DNA

  Materials
  • Sf9 insect cells (4 × 106 cells; protocol 4)
  • Supplemented Insect Cell Medium (see recipe and protocol 4
  • Transfection buffers A (see recipe) and B (see recipe)
  • Unsupplemented Grace's Insect Cell Medium (Invitrogen)
  • Linearized Sapphire Baculovirus DNA (Orbigen)
  • Plasmid encoding MHC‐TM‐pepβ2m ( protocol 1)
  • FACS buffer (see recipe)
  • Primary antibody: monoclonal antibody specific for MHC or β2m
  • Fluorescently‐labeled secondary antibody specific for the Ig class of the primary antibody
  • 6‐well tissue culture plates
  • Sealable plastic bag
  • 27°C incubator
  • 15‐ml conical centrifuge tubes
  • Centrifuge and microtiter plate carrier
  • 0.2 µm syringe filter and syringe
  • 1‐ml filtered pipet tips
  • 96‐well round‐bottom microtiter plate
  • Additional reagents and equipment for flow cytometry (Chapter 5)

Support Protocol 1: Confirmation of the Antigenic Peptide Sequence Encoded by the Baculovirus

  Materials
  • Sf9 insect cells ( protocol 4)
  • Supplemented Insect Cell Medium (see recipe and protocol 4)
  • Uncloned viral stock (see protocol 2, step 13)
  • Extraction buffer (see recipe)
  • 10 mg/ml proteinase K (Promega, prepared according to the manufacturer's instructions)
  • Taq DNA polymerase and reaction buffer (also see unit 10.20)
  • dNTP mix (unit 10.20)
  • Oligonucleotide primers for PCR (also see unit 10.20):
    • 5′ primer (no. 7 in Fig. )
    • 3′ primer (no. 10 in Fig. )
  • PCR purification columns (e.g., S300, GE Healthcare)
  • Sequencing primer (no. 3 in Fig. )
  • 24‐well plates
  • 27°C incubator
  • 56°C water bath
  • PCR tubes
  • Additional reagents and equipment for PCR (unit 10.20), agarose gel electrophoresis (unit 10.4), and DNA sequencing (Chapter 7 in Ausubel et al., )

Support Protocol 2: Growing Insect Cells

  Materials
  • Grace's Supplemented Insect Cell Medium (Invitrogen)
  • Fetal bovine serum (FBS)
  • 10% Pluronic F‐68 (Invitrogen)
  • 100× antibiotic/antimycotic (Invitrogen; store at –20°C in aliquots)
  • Frozen stock of Sf9 or Hi5 insect cells (Invitrogen)
  • Freezing medium (see recipe)
  • 0.2‐µm sterile filter devices
  • 15‐ml conical centrifuge tubes
  • 75‐cm2 tissue culture flask
  • 27°C incubator
  • 175‐cm2 tissue culture flask
  • Acid‐washed 1‐liter spinner flask (Bellco, see protocol 5 for acid washing procedure)
  • Magnetic stirrer (Bellco, 4‐position, 115v‐500 ml)
  • 1.5‐ml cryovials
  • Isopropanol freezing chamber (e.g., Mr. Frosty; Thermo Scientific)
  • Liquid nitrogen tank

Support Protocol 3: Acid Washing Glassware

  Materials
  • Bleach (6% sodium hypochlorite, standard stock solution) diluted 1:10 with deionized H 2O
  • 7X Cleaning Solution (Fisher Scientific)
  • Concentrated HCl
  • Glassware to be cleaned
  • Large aluminum tub or sink

Basic Protocol 3: Cloning Baculoviruses that Express Peptide‐MHC by Limiting Dilution

  Materials
  • Hi5 insect cells (Invitrogen)
  • Supplemented Insect Cell Medium (see recipe and protocol 4)
  • Filtered uncloned viral stock (from step 13 of protocol 2)
  • Sf9 insect cells (Invitrogen)
  • 15‐ and 50‐ml conical tubes
  • 96‐well flat‐bottom tissue culture plates
  • Sealable plastic bags
  • 27°C incubator
  • 6‐well tissue culture plates
  • Centrifuge
  • 0.2‐µm syringe filter and syringe
  • 175‐cm2 tissue culture flasks
  • Additional reagents and equipment for confirming that baculoviruses encode the protein of interest ( protocol 2), confirming the antigenic peptide sequence encoded by the baculovirus ( protocol 3), and determining cell viability by trypan blue exclusion ( appendix 3B)

Basic Protocol 4: Vaccination of Mice with Baculovirus‐Infected Insect Cells

  Materials
  • Sf9 insect cells (Invitrogen): 3 × 107 cells needed for one vaccination of five mice
  • Supplemented Insect Cell Medium (see recipe and protocol 4)
  • Cloned baculovirus stock (from step 23 of protocol 6)
  • 1× Hanks' Balanced Salt Solution (HBSS; Cellgro)
  • Inbred experimental mice (consider strain, genetic background, age, and sex that is appropriate for the experiment being performed)
  • 175‐cm2 tissue culture flasks
  • 27°C incubator
  • 50‐ml conical tubes
  • Centrifuge
  • 1‐ml syringes
  • 27‐G needles
  • Additional reagents and equipment for intraperitoneal injection of mice (unit 1.6)

Support Protocol 4: Quantification of Peptide‐MHC Expression in Baculovirus‐Infected Insect Cells

  Materials
  • Sf9 insect cells (Invitrogen)
  • Standard: peptide‐MHC, investigator‐generated or commercially purchased (e.g., from BD Biosciences)
  • 1× Hanks' Balanced Salt Solution (HBSS, Cellgro)
  • Insect cell lysis buffer (see recipe)
  • Anti‐MHC class I monoclonal antibody (30.7.5S for H‐2Ld protein) or anti‐β2m monoclonal antibody (BD Biosciences or ATCC)
  • Blocking buffer: 1% (v/v) fetal bovine serum (FBS) in PBS (see appendix 2A for PBS)
  • Wash buffer: PBS ( appendix 2A) containing 1% (v/v) FBS and 0.5% (v/v) Tween 20
  • Diluent buffer: PBS ( appendix 2A) containing 1% (v/v) FBS
  • Secondary anti–MHC class I monoclonal antibody conjugated to biotin (e.g., 28.18.4S for H‐2Ld protein; BD Biosciences or ATCC)
  • Streptavidin‐HRP conjugate (Pierce)
  • Final wash buffer: PBS ( appendix 2A) containing 0.5% (v/v) Tween 20
  • TMB One‐Step Substrate System (Promega)
  • 0.18 M sulfuric acid
  • 175‐cm2 tissue culture flask
  • 50‐ml conical centrifuge tubes
  • Centrifuge
  • End‐over‐end rotator
  • 96‐well ELISA plates: high‐protein‐binding, half‐area plates for enzyme immunoassay/radioimmunoassay (EIA/RIA; Costar)
  • 96‐well plate reader
  • Additional reagents and equipment for infecting insect cells with baculovirus ( protocol 7)
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Figures

Videos

Literature Cited

   Aichele, P., Unsoeld, H., Koschella, M., Schweier, O., Kalinke, U., and Vucikuja, S. 2006. CD8 T cells specific for lymphocytic choriomeningitis virus require type I IFN receptor for clonal expansion. J. Immunol. 176:4525‐4529.
   Alexopoulou, L., Holt, A.C., Medzhitov, R., and Flavell, R.A. 2001. Recognition of double‐stranded RNA and activation of NF‐kappaB by Toll‐like receptor 3. Nature 413:732‐738.
   Ausubel, F.A., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K. (eds.). 2009. Current Protocols in Molecular Biology. John Wiley & Sons, Hoboken, N.J.
   Crawford, F., Jordan, K.M., Stadinsky, B., Wang, Y., Huseby, E., Marrack, P., Slansky, J.E., and Kappler, J.W. 2006. Use of baculovirus MHC/peptide display libraries to characterize T‐cell receptor ligands. Immunol. Rev. 210:156‐170.
   Curtsinger, J.M., Valenzuela, J.O., Agarwal, P., Lins, D., and Mescher, M.F. 2005. Type I IFNs provide a third signal to CD8 T cells to stimulate clonal expansion and differentiation. J. Immunol. 174:4465‐4469.
   Huang, A.Y.C., Bruce, A.T., Pardoll, D.M., and Levitsky, H.I. 1996. In vivo cross‐priming of MHC class I‐restricted antigens requires the TAP transporter. Immunity 4:349‐355.
   Janssen, E.M., Lemmens, E.E., Wolfe, T., Christen, U., von Herrath, M.G., and Schoenberger, S.P. 2003. CD4+ T cells are required for secondary expansion and memory in CD8+ T lymphocytes. Nature 421:852‐856.
   Jordan, K.R., McMahan, R.H., Oh, J.Z., Pipeling, M.R., Pardoll, D.M., Kedl, R.M., Kappler, J.W., and Slansky, J.E. 2008. Baculovirus‐infected insect cells expressing peptide‐MHC complexes elicit protective antitumor immunity. J. Immunol. 180:188‐197.
   McMahan, R.H., McWilliams, J.A., Jordan, K.R., Dow, S.W., Wilson, D.B., and Slansky, J.E. 2006. Relating MHC‐peptide‐TCR affinity to immunogenicity for the rational design of tumor vaccines. J. Clin. Invest. 116:2543‐2551.
   Murphy, C.I., Piwnica‐Worms, H., Grunwald, S., Romanow, W.G., Francis, N., and Fan, H.Y. 2004. Overview of the baculovirus expression system. Curr. Protoc. Mol. Biol. 65:16.9.1‐16.9.10.
   Shedlock, D.J. and Shen, H. 2003. Requirement for CD4 T cell help in generating functional CD8 T cell memory. Science 300:337‐339.
   Slansky, J.E., Rattis, F.M., Boyd, L.F., Fahmy, T., Jaffee, E.M., Schneck, J.P., Margulies, D.H., and Pardoll, D.M. 2000. Enhanced antigen‐specific antitumor immunity with altered peptide ligands that stabilize the MHC‐peptide‐TCR complex. Immunity 13:529‐538.
   Sun, J.C. and Bevan, M.J. 2003. Defective CD8 T cell memory following acute infection without CD4 T cell help. Science 300:339‐342.
   Valmori, D., Souleimanian, N.E., Tosello, V., Bhardwaj, N., Adams, S., O'Neill, D., Pavlick, A., Escalon, J.B., Cruz, C.M., Angiulli, A., Angiulli, F., Mears, G., Vogel, S.M., Pan, L., Jungbluth, A.A., Hoffmann, E.W., Venhaus, R., Ritter, G., Old, L.J., and Ayyoub, M. 2007. Vaccination with NY‐ESO‐1 protein and CpG in Montanide induces integrated antibody/Th1 responses and CD8 T cells through cross‐priming. Proc. Natl. Acad. Sci. U.S.A. 104:8947‐8952.
   Wang, Y., Rubtsov, A., Heiser, R., White, J., Crawford, F., Marrack, P., and Kappler, J.W. 2005. Using a baculovirus display library to identify MHC class I mimotopes. Proc. Natl. Acad. Sci. U.S.A. 102:2476‐2481.
   Zaks, K., Jordan, M., Guth, A., Sellins, K., Kedl, R., Izzo, A., Bosio, C., and Dow, S. 2006. Efficient immunization and cross‐priming by vaccine adjuvants containing TLR3 or TLR9 agonists complexed to cationic liposomes. J. Immunol. 176:7335‐7345.
Key Reference
   Jordan et al., 2008. See above.
  This protocol was based on the experiments performed in this key paper.
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