Introduction of the haemagglutinin transmembrane region in the influenza virus matrix protein facilitates its incorporation into ISCOM and activation of specific CD8(+) cytotoxic T lymphocytes.

The gene encoding the influenza virus A matrix (MA) protein was cloned into the bacterial expression vector pMalC with and without the sequence encoding the transmembrane region of the haemagglutinin (HA). With the resulting recombinant proteins, immune stimulating complexes (ISCOM) were prepared. The MA protein with the hydrophobic anchor region (rMAHA) associated more efficiently with ISCOM than the unmodified MA protein (rMA). A B-lymphoblastoid cell line (B-LCL) was lysed by an autologous CD8(+) cytotoxic T lymphocyte (CTL) clone specific for the MA protein after incubation with rMAHA-ISCOM but not after incubation with rMA, rMAHA, rMA-ISCOM or empty ISCOM. The B-LCL was also lysed by the CTL clone after incubation with empty ISCOM mixed with the respective MA proteins. Incubation of ISCOM with the rMAHA protein proved to be the most efficient in this respect. Addition of the proteasome inhibitors lactacystin or clasto-lactacystin beta-lactone to the B-LCL incubated with rMAHA-ISCOM or the MA proteins mixed with empty ISCOM dramatically decreased the lysis by the CD8(+) CTL clone. These results indicate that the addition of a hydrophobic anchor to hydrophilic proteins in combination with ISCOM facilitates their entry in the MHC class I processing and presentation pathway. This may be an attractive approach for the development of subunit vaccines aiming at the induction of CTL-mediated immunity.

Preparation and characterisation of quillaja saponin with less heterogeneity than Quil-A.

Immunisation against pathogens remains one of the most effective ways of preventing or reducing losses due to infectious diseases in animal husbandry. When inactivated vaccines are used, adjuvants are most often required to obtain satisfactory immune responses. One such type of adjuvant is saponin derived from the bark of Quillaja saponaria Molina, a tree of the rose family. A few different commercial sources exist, but due to the structural complexity and heterogeneity of these saponin preparations, it has been difficult to establish exactly which components are responsible for the adjuvant activity. By carefully selecting the bark source, we have succeeded in preparing a much less heterogeneous preparation of quillaja saponin. In this report we describe the preparation, in terms of structural complexity, hemolytic activity, adjuvant activity, and its ability to form ISCOM matrix. This new preparation could have implications for use per se, or as starting material for more effective preparation of pure substances.

Adjuvanticity and ISCOM formation by structurally diverse saponins.

Adjuvant activity and immunostimulating complex (ISCOM) formation by a series of saponins and glycoalkaloids differing in the structures of their aglycones and sugar chains were examined. The only two saponins apart from Quillaia that were adjuvant-active were Gypsophila and Saponaria, which resemble Quillaia in that they contain saponins with branched sugar chains attached to positions 3 and 28 of the aglycone. Glycoalkaloids with a branched sugar chain lacked adjuvant activity. Saponaria saponins formed irregular ISCOM-like structures, and Gypsophila produced a sheet of joined pore-like structures. The alfalfa hederagenin saponin and Quinoa also formed pore-sheets, despite lacking adjuvanticity.

Soluble proteins incorporate into ISCOMs after covalent attachment of fatty acid.

The immune stimulating complex (ISCOM) is a potent adjuvant which has the ability to induce both humoral and cellular immune reactions to protein antigens when they are physically associated with the ISCOM structure. However, in general only proteins with an exposed hydrophobic domain can associate with ISCOMs. As many soluble proteins are available as candidate subunit antigens there is a requirement for a method which promotes efficient incorporation of soluble protein into ISCOMs. Here it is demonstrated that following covalent attachment of palmitic acid, two soluble proteins, cytochrome C and ovalbumin, quantitatively incorporate into ISCOMs. ISCOMs containing ovalbumin prepared in this way have been shown to be highly immunogenic, generating humoral, delayed-type hypersensitivity and class I restricted T-cell immune responses following both parenteral and oral administration. The technique of incorporating soluble proteins into ISCOMs by covalent attachment of fatty acid should be generally applicable and extends the use of the ISCOM as an adjuvant.

Incorporation of soluble antigens into ISCOMs: HIV gp120 ISCOMs induce virus neutralizing antibodies.

Through a process of covalent attachment of palmitic acid, we have incorporated recombinant gp120 of HIV strain IIIB into ISCOMs. Rabbits immunized with ISCOMs incorporating 10 micrograms gp120 produced high levels of gp120-specific antibody, comparable to the response to ten times as much antigen in complete Freund's adjuvant. The ISCOM-induced antisera showed virus neutralizing activity against the homologous strain, but failed to neutralize two heterologous strains of HIV-1. The antisera recognized non-conformationally determined epitopes on gp120, and antibody binding to gp120 was affected by glycosylation of the viral glycoprotein.