Incorporation of hepatitis-B surface antigen (HBsAg) into liposomes.

Abstract

Previous work has shown that liposomes (Gregoriadis, 1976a) can act as immunological adjuvants to entrapped antigens (Allison & Gregoriadis, 1974). This, together with the apparent prevention of hypersensitivity reactions to their antigen contents on repeated challenge (Gregoriadis & Allison, 1974; Neerunjun & Gregoriadis, 1976), suggest that liposomes may satisfy the need for a safe and effective adjuvant for use in human and animal immunization programmes. Hepatitis-B surface antigen (HB,Ag) has been thought as a possible agent for active immunization against human viral hepatitis type B, and its immunogenicity has been investigated (Hilleman et al., 1975). The present paper deals with the possibility of enhancing such immunogenicity by the incorporation of the HB,Ag into liposomes. Pooled positive donor sera served as a source for the HB,Ag, which was purified by molecular-sieve chromatography (Sepharose 6B) followed by two CsCl,-gradient centrifugations in a Beckman SWSOL swinging rotor at 124000g for 72h. After dialysis against 0.01 M-sodium phosphate buffer, pH7.2, the final preparation consisted of the 22nm spherical particles. Partially purified HB,Ag was obtained after passing at room temperature a strongly positive serum through a Sepharose CLdB column, with Dulbecco’s phosphate-buffered saline. The antigen-rich preparation in the void volume contained 35pg of HB,Ag/ml. 1251-labelled antigen HB,Agwas from the AUSAB kit supplied by Abbott Ltd., Reading, U.K., andchromatographed as above before use. Incorporation of HB,Ag into liposomes, composed of 44pmol of egg phosphatidylcholine and 6.3ymol of cholesterol (neutral) or the same supplemented with 3.2pmol of either dicetyl phosphate (negative) or stearylamine (positive) liposomes (molar ratio 7 : 2 : l ) , was carried out as for other materials (Gregoriadis, 1976b), except that, after suspending the dry lipids with 2-3ml of phosphate-buffered saline containing antigen HB,Ag with or without labelled antigen HB,Ag, the suspension was transferred into a lOml flask and subjected to sonication in a bath-type sonicator for 1.5 h at 4°C. Shortly after, the free antigen was separated from the liposome-associated antigen by passing the suspension through a CL-2B Sepharose column ( 1 cm x 46cm) in phosphate-buffered saline. Antigen HB,Ag was measured by solid phase radioimmunoassay (AUSRIA 11, Abbott Ltd.). Quantification was made by reference to a standard curve based on a sample of known antigen HB,Ag concentration provided in the kit. Liposome-associated antigen HB,Ag was measured in the presence of sodium taurocholate ( 3 . 7 m ~ final concn.). At the concentration used, this detergent was judged superior to Triton X-100, sodium dodecyl sulphate and Nonidet LE in terms of liposome solubilization and non-interference (%