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We have investigated the possibility of a T-cell epitope peptide providing help for a B-cell epitope peptide when both peptides are co-entrapped in the same liposomes. Epitope models used were a 28 amino acid peptide from the S region of the hepatitis B surface antigen (HBsAg) (subtype adw) containing an H-2′. Th-cell epitope, and a 33 amino acid peptide from the pre-S1 region of the HBsAg (subtype adw) designed to exclude an adjacent H-2′ T-cell epitope, the latter (pre-S1) peptide being recognized by SJL (H-2′) mice as a B-cell epitope. SJL(H-2′) mice were immunized twice intramuscularly with S or pre-S1 peptide alone, co-entrapped in the same liposomes or entrapped in separate liposomes which were mixed before injection. Analysis of sera for anti-peptide IgG1 antibodies revealed that the Th-cell peptide provided help for the pre-S1 peptide only when the two peptides were co-entrapped in the same vesicles. This helper effect was found to correlate with the ability of S peptide (co-entrapped with the pre-S1) to stimulate T-cell proliferation in vitro. There was no IgGl response against pre-S1 peptide in mice immunized with a mixture of the free peptides or a mixture of separately entrapped peptides. A helper effect, albeit much weaker, was also observed in mice immunized with the two peptides emulsified in incomplete Freund's adjuvant. Antisera from mice immunized with both peptides co-entrapped in liposomes were found to bind to full length (pre-S1 containing) recombinant HBsAg. Moreover, binding values were much higher than those seen with antisera from animals immunized with the liposomal S peptide above, presumably because of full access of anti-pre-S1 antibodies to the pre-S1 region of the rHBsAg. It is concluded that liposomes could serve not only as an immunological adjuvant for peptides but also as a carrier for Th- and B-cell epitopes thus eliminating the need for covalent linkage to a carrier protein. [ABSTRACT FROM AUTHOR]

Primary immune response (IgG1) to tetanus toxoid entrapped in liposomes composed of equimolar egg phosphatidylcholine and cholesterol, and the effect of a variety of physiological and non- physiological mediators (co-entrapped with the toxoid or entrapped in separate liposomes) on such responses, were studied in BALB/c mice. Results show that (i) primary responses, not detectable with the free antigen, were elicited with the same amount of antigen given in liposomes within a range (37·4:1–2857:1) of liposomal phospholipid to toxoid mass ratios. At a higher ratio (17,804:1) response was reduced to very low levels. Immune responses obtained with liposomal toxoid were maintained at measurable levels 24 weeks after immunization. (ii) Interleukin-2 (IL-2), interferon- gamma (IFN-γ) and N-acetyl muramyl-L-threonyl-D-isoglutamine ([Thr1]MDP), but not its liposoluble 6-O-stearoyl derivative (6-O-S-[Thr1]MDP), co-entrapped with the toxoid at appropriate phospholipid to toxoid ratios, generally reduced primary response to levels below those achieved with liposomes containing the antigen alone. Further, responses obtained with 6-O-S[Thr1]MDP co- entrapped with the toxoid or separately entrapped were higher than those seen with [Thr1]MDP in similar formulations. The significance of these findings is discussed in conjunction with the structural characteristics of liposomes. [ABSTRACT FROM AUTHOR]

The effect of various manipulations of liposomes composed of equimolar phospholipid and cholesterol on immune responses to the incorporated immunopurified tetanus toxoid was investigated in BALB/c mice. In studies designed to establish proper dosage for immunization and to reveal the roles of the liposomal phospholipid to toxoid mass ratio, gel-liquid crystalline transition temperatures (Tc) of the liposomal phospholipids and mode of antigen incorporation into liposomes on immune responses, animals were injected intramuscularly with various amounts of the toxoid, free, entrapped in multilamellar dehydration-rehydration vesicles (DRY) or covalently linked to the surface of multilamellar vesicles (MLV) prepared by the classical procedure. Two identical injections separated by 4 weeks were given and IgG1 and IgG2b antibodies specific for the toxoid assayed in sera by an enzyme-linked immunosorbent assay. Results suggest that (i) the adjuvant effect of liposomes is improved considerably when liposomal phospholipid to toxoid mass ratios are as high as 2049:1; however, adjuvanticity is reduced to reach very low levels for much higher ratios (e.g. 90,361 : 1); (ii) antibody responses are similar for liposomes (phospholipid to toxoid mass ratios: 143- 331) made of a variety of phospholipids with Tcs ranging from -32° to 41.5° but are low or non- existent when liposomes are made of distearoyl phosphatidylcholine (Tc, 54°) (however, see Discussion); (ii) there are no differences in antibody responses between liposomes with entrapped and surface-linked toxoid. [ABSTRACT FROM AUTHOR]

The active component of the exoantigens of malarial parasites which stimulates macrophages to secrete tumour necrosis factor (TNF) has been shown to depend upon a phospholipid, the activity of which was blocked by phosphatidylinositol (PI) and inositol monophosphate (IMP) in competitive inhibition studies. Antisera made against the exoantigens of Plasmodium yoelii, which inhibited their induction of TNF, were found by an ELISA assay to contain antibody against several other phospholipids. However, the inhibitory antibody was removed specifically by adsorption with liposomes containing PI, but not other phospholipids. Furthermore, PI was the only phospholipid in non-liposomal form which induced thc production of inhibitory antisera. Mice immunized with IMP, but not inositol, also produced inhibitory antisera. When incorporated into liposomes several other phospholipids did give rise to inhibitory antibodies but, in contrast to the antisera against parasite exoantigcns, PI and IMP, the inhibitory activity was removed by adsorption with heteroiogous phospholipid liposomes, suggesting that it was directed against a common determinant, presumably the phosphate ester head group. Inhibitory antibodies in the antisera tested were predominantly IgM and titres were not increased after repeated injections. Antisera raised against PI, IMP or the crossreacting phospholipid liposomes also inhibited TNF secretion by macrophages stimulated by exoantigens of the human parasites P. falciparum and P. vivax, but not by bacterial lipopolysaccharide. These findings confirm our conclusion that exoantigens from these different species contain phosphate bound to inositol in their TNF-inducing moiety. [ABSTRACT FROM AUTHOR]

The effect of the ligand mannosylated albumin, covalently coupled to the surface of tetanus toxoid-containing dehydration-rehydration vesicles (DRV), on their adjuvanticity was investigated. Toxoid-containing DRV coated with mannosylated albumin bound in vitro to mouse (BALB/c) peritoneal macrophages selectively and to a greater extent than non-mannosylated DRV. ELISA assays of anti-toxoid IgG1 and IgG2b in the sera of BALB/c mice immunized with various toxoid-containing DRV preparations suggest that mannosylated liposomes are superior in immunoadjuvant action to conventional ones. Such targeted adjuvanticity appears to be influenced by the number of ligand molecules available on the surface of liposomes rather than the number of mannose residues on albumin. [ABSTRACT FROM AUTHOR]