Preparation of Immune Stimulating Complexes (ISCOMs) as Adjuvants

Allan McI. Mowat1, George Reid1

1 University of Glasgow, Glasgow, Scotland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 2.11
DOI:  10.1002/0471142735.im0211s16
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Purified proteins are often poorly immunogenic and in such cases the induction of primary immune responses requires use of an adjuvant. The immune stimulating complex (ISCOM) has a unique ability to provoke a full range of immune response to protein antigens, after both parenteral and oral immunization. This unit describes techniques for incorporating proteins into the ISCOM structure, a process that requires the presence of exposed hydrophobic regions on the protein. The basic protocol outlines a method for preparation of ISCOMs containing inherently nonhydrophobic proteins, to which palmitic acid has been attached covalently. Two alternate protocols are given that do not require covalent modification of the protein. In the first, hydrophobic groups are revealed by acid treatment of the protein. The second describes preparation of ISCOMs containing integral membrane proteins that therefore possess a hydrophobic transmembrane domain.

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

Table of Contents

  • Basic Protocol 1: Construction of ISCOMs Containing Palmitified Proteins
  • Support Protocol 1: Covalent Attachment of Palmitic Acid to Ovalbumin
  • Support Protocol 2: Quantitation of Protein Content by the Bradford Method
  • Support Protocol 3: Quantitation of Spikoside Content by Gel Filtration
  • Alternate Protocol 1: Incorporation of Integral Membrane Proteins into ISCOMs
  • Alternate Protocol 2: Incorporation of Acid‐Treated Proteins into ISCOMs
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Construction of ISCOMs Containing Palmitified Proteins

  Materials
  • 1 mg/ml palmitified ovalbumin (OVA) in recipeTris/NaDOC buffer (first protocol 2support protocol)
  • Sodium deoxycholate (NaDOC; BDH Chemicals #43035)
  • Decanoyl‐N‐methylglucamide (Mega 10; Sigma #D6277)
  • 100 mg/ml phosphatidylcholine (100 mg/ml stock in chloroform; Sigma #P2772)
  • Cholesterol (Sigma #C3137)
  • Chloroform (BDH Chemicals #1077)
  • Spikoside (Advet, AB)
  • 0.001% thiomersal in 50 mM Tris⋅Cl, pH 8.5 (BDH Chemicals, #30416)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 10% and 40% sucrose prepared in 50 mM Tris⋅Cl, pH 8.0 (prepare fresh)
  • Plastic film (Whatman #160/0026)
  • Plastic sealant (e.g., Plasticene)
  • Dialysis tubing (12,000‐14,000 MWCO; appendix 3A)
  • Electron microscope
  • Beckman SW‐41 rotor (or equivalent)
  • 0.22‐µm sterile filters
NOTE: All containers coming into contact with chloroform should be made of glass.

Support Protocol 1: Covalent Attachment of Palmitic Acid to Ovalbumin

  Materials
  • Ovalbumin (OVA; Sigma #A55033)
  • recipeDilution buffer
  • N‐(palmitoyloxy)succinimide (NPS; Sigma #P1162)
  • Dimethyl sulfoxide (DMSO) (Sigma #D5879)
  • 1 M Tris⋅Cl, pH 8.5 ( appendix 2A)
  • recipeTris/NaDOC buffer
  • Polyethylene glycol 20 (PEG; BDH Chemicals #29864)
  • Shaking water bath, 37°C
  • 1.5 × 130–cm Sephadex G‐100 column (Pharmacia)

Support Protocol 2: Quantitation of Protein Content by the Bradford Method

  Materials
  • 1 mg/ml bovine serum albumin (BSA; Sigma #A6793) in recipeTris/NaDOC buffer
  • Coomassie brilliant blue G‐250 (Sigma #B8522)
  • Sodium dodecyl sulfate (SDS; Sigma #L4509)
  • 95% ethanol in dH 2O
  • Orthophosphoric acid, supplied and used at 85% (Sigma #P6560)
  • recipeISCOMs sample ( protocol 1basic protocol)
  • Filter paper (Whatman #1001‐125)
NOTE: All glassware should be thoroughly rinsed in distilled water before use, as many laboratory detergents react with Bradfords solution.

Support Protocol 3: Quantitation of Spikoside Content by Gel Filtration

  Materials
  • Spikoside (Advet, AB)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Galactose oxidase (Sigma #G3385)
  • [3H]sodium borohydride (5 to 10 Ci/ml, specific activity 50 to 20 Ci/mmol; Amersham)
  • PD‐10 gel‐filtration column (Pharmacia)

Alternate Protocol 1: Incorporation of Integral Membrane Proteins into ISCOMs

  Additional Materials
  • Membrane extract
  • 2% (w/v) Mega 10 solution
  • Lipid mix (see step of protocol 1basic protocol)

Alternate Protocol 2: Incorporation of Acid‐Treated Proteins into ISCOMs

  Additional Materials
  • recipe1 M and 0.1 M glycine buffer, pH 2.5
  • Sonicating water bath
NOTE: Glass containers should be used exclusively in the following steps.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Bloom, B.R. 1989. Vaccines for the third world. Nature (Lond.) 342:115‐120.
   Chavali, S.R. and Campbell, J.B. 1987. Adjuvant effects of orally administered saponins on humoral and cellular immune responses in mice. Immunobiology. 174:347‐359.
   Cresswell, P. 1990. Questions of presentation. Nature (Lond.) 343:593‐594.
   Elson, C.O., Ealding, W., and Lefkowitz, J. 1984. A lavage technique allowing repeated measurement of IgA antibody in mouse intestinal secretions. J. Immunol. Methods 67:101‐108.
   Harding, C.V., Collins, D.S., Kanagawa, O., and Unanue, E.R. 1991. Liposome‐encapsulated antigens engender lysosomal processing for class II MHC presentation and cytosolic processing for class I presentation. J. Immunol. 147:2860‐2863.
   Heeg, K., Kuon., W., and Wagner, H. 1991. Vaccination of class I major histocompatibility complex (MHC)‐restricted murine CD8+ cytotoxic T lymphocytes toward soluble antigens: Immunostimulating‐ovalbumin complexes enter the class I MHC–restricted antigen pathway and allow sensitisation against the immunodominant peptide. Eur. J. Immunol. 21:1523‐1527.
   Helenius, A. and Simons, K. 1975. Solubilization of Membranes by Detergents. Biochem. Biophys. Acta. 415:29‐79
   Kensil, C.R., Patel, U., Lennick, M., and Marciani, D. 1991. Separation and characterization of saponins with adjuvant activity from Quillaja saponaria Molina cortex. J. Immunol. 146:431‐437.
   Lövgren, K. Kaberg, H., and Morein, B. 1990. An experimental influenza subunit vaccine (iscom): Induction of protective immunity to challenge infection in mice after intranasal or subcutaneous administration. Clin. Exp. Immunol. 82:435‐439.
   Lycke, N. and Holmgren, J. 1986. Strong adjuvant properties of cholera toxin on gut mucosal immune responses to orally presented antigens. Immunology 59:301‐308.
   Morein, B., Sundquist, B., Höglund, S., Dalsgaard, K., and Osterhaus, A. 1984. ISCOM, a novel structure for antigenic presentation of membrane proteins from enveloped viruses. Nature (Lond.) 308:457‐460.
   Morein, B., Lövgren, K., Höglund, S., and Sundquist, B. 1987. The ISCOM: An immunostimulating complex. Immunol. Today 8:333‐338.
   Morein, B., Fossum, C., Lövgren, K., and Höglund, S. 1990a. The ISCOM—a modern approach to vaccines. Semin.Virol. 1:49‐55.
   Morein, B., Ekstrom, J., and Lövgren, K. 1990b. Increased immunogenicity of a nonamphipathic protein (BSA) after inclusion into ISCOM. J. Immunol Methods 128:177‐181.
   Mowat, A. McI., Donachie, A.M., Reid, G., and Jarrett, O. 1991. Immune stimulating complexes containing Quil A and protein antigen prime class I MHC–restricted T lymphocytes in vivo and are immunogenic by the oral route. Immunology 72:317‐322.
   Mowat, A. McI. and Donachie, A.M. 1991. ISCOMs—a novel strategy for mucosal immunization? Immunol. Today 12:383‐385.
   Reid, G. 1992. Soluble proteins incorporate into ISCOMS after covalent attachment of fatty acids. Vaccine 9:597‐602.
   Takahashi, H., Takeshita, T., Morein, B., Putney, S., Germain, R.N., and Berzofsky, J.A. 1990. Induction of CD8+ cytotoxic T cells by immunization with purified HIV‐1 envelope protein in ISCOM. Nature (Lond.) 344:873‐875.
Key References
   Morein et al., 1984. See above.
  The initial description of ISCOMs which includes basic information on their chemistry and synthesis.
   Morein et al., 1990a. See above.
  Reviews the usefulness of ISCOMs in vaccination against infectious agents.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library