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Preparation of Immune Stimulating Complexes (ISCOMs) as Adjuvants

Allan McI. Mowat¹, George Reid¹

¹University of Glasgow, Glasgow, Scotland, United Kingdom

Publication Name:

Current Protocols in Immunology

Unit Number:

Unit 2.11

DOI:

10.1002/0471142735.im0211s16

Online Posting Date:

May, 2001

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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.

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Unit Introduction
Basic Protocol: Construction of ISCOMs Containing Palmitified Proteins
Support Protocol: Covalent Attachment of Palmitic Acid to Ovalbumin
Support Protocol: Quantitation of Protein Content by the Bradford Method
Support Protocol: Quantitation of Spikoside Content by Gel Filtration
Alternate Protocol: Incorporation of Integral Membrane Proteins into ISCOMs
Alternate Protocol: Incorporation of Acid-Treated Proteins into ISCOMs
Reagents and Solutions
Commentary
Bibliography
Figures

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Materials

Basic Protocol: Construction of ISCOMs Containing Palmitified Proteins

Materials

1 mg/ml palmitified ovalbumin (OVA) in Tris/NaDOC buffer (first support 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: Covalent Attachment of Palmitic Acid to Ovalbumin

Materials

Ovalbumin (OVA; Sigma #A55033)
Dilution buffer
N-(palmitoyloxy)succinimide (NPS; Sigma #P1162)
Dimethyl sulfoxide (DMSO) (Sigma #D5879)
1 M Tris×Cl, pH 8.5 (appendix 2A)
Tris/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: Quantitation of Protein Content by the Bradford Method

Materials

1 mg/ml bovine serum albumin (BSA; Sigma #A6793) in Tris/NaDOC buffer
Coomassie brilliant blue G-250 (Sigma #B8522)
Sodium dodecyl sulfate (SDS; Sigma #L4509)
95% ethanol in dH₂O
Orthophosphoric acid, supplied and used at 85% (Sigma #P6560)
ISCOMs sample (basic 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: 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: Incorporation of Integral Membrane Proteins into ISCOMs

Additional Materials

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

Alternate Protocol: Incorporation of Acid-Treated Proteins into ISCOMs

Additional Materials

1 M and 0.1 M glycine buffer, pH 2.5
Sonicating water bath

NOTE: Glass containers should be used exclusively in the following steps.

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Figures

Figure 2.11.1

Ultrastructural appearance of SCOM containing ovalbumin, constructed according to the method described in the Basic Protocol. (73,000 ×).

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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.