The role of electrostatics in the adsorption process of proteins to preformed negatively-charged (phosphatidylcholine/phosphatidylglycerol) and neutral (phosphatidylcholine) liposomes was studied. The interaction was monitored at low ionic strength for a set of model proteins as a function of pH. The adsorption behavior of trypsin inhibitor (pI = 4.6), myoglobin (pI = 7.4), ribonuclease (pI = 9.6), and lysozyme (pI = 10.7) with preformed liposomes was investigated, along with changes in the electrophoretic mobility of liposomes through the adsorption of charged proteins. Mean protein charge was determined by acid/base titration. Significant adsorption of the proteins to negatively-charged liposomes was only found at pH values where the number of positive charge moieties exceeds the number of negative charge moieties on the protein by at least three charge units. Negligible adsorption to liposomes composed of zwitterionic lipids was observed in the pH range tested (4-9). The absolute value of the electrophoretic mobilities of negatively-charged, empty liposomes decreased after adsorption of positively-charged proteins. With increasing protein to phospholipid ratio, the drop in the electrophoretic mobility leveled off and reached a plateau; protein adsorption profiles showed a similar shape. Analysis of the data demonstrated that neutralization of the liposome charge due to the adsorption of the positively-charged proteins is the controlling factor in their adsorption. The plateau level reached depended on the type of protein and the pH of the incubation medium. This pH dependency could be ascribed to the mean positive charge of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

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Antigens presented on cell membranes or on liposomes are usually more immunogenic than antigens in soluble form, this being one of the reasons for the weak immunogenicity of extracted tumour-associated transplantation antigens (TATA). The main objective of this study is to solubilize TATA from tumour cells and to present them on a membrane-like structure to the immune system. Crude tumour cell membranes of SL2 lymphosarcoma cells (a spontaneously arising, weakly immunogenic tumour) were solubilized with octylglucoside or sodium deoxycholate, and reconstituted membranes (proteoliposomes) were prepared by detergent removal. Mice immunized s.c. with reconstituted membranes were protected against an i.p. challenge with tumour cells. Although octylglucoside solubilized only 41% of the membrane proteins, the reconstituted membranes were as immunoprotective as crude membranes. (Glyco)proteins were probably the major membrane components in the reconstituted membranes that induce immunoprotection, as mice immunized with preparations constituted of (glyco)lipids from SL2 cells could not reject SL2 cells. If Freund's complete adjuvant was used with the first immunization injection, no potentiation of the elicited immune responses was observed. Besides the membrane TATA, SL2 cells contained an apparently non-membrane-bound TATA, which was found in the cytoplasm. It is concluded that detergent solubilization of membranes and subsequent preparation of reconstituted membranes can be used to obtain membrane tumour-associated antigens that retain activity for induction of protective tumour immunity. The major advantage of this method is that membrane proteins are solubilized and are subsequently presented on a membrane-like structure that resembles the tumour cell membrane. On theoretical and practical grounds it provides a promising alternative for whole-cell vaccines.

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Reconstituted membranes consist of liposomal structures formed by removal of detergent from solubilized membrane constituents. The membrane-like configuration of reconstituted membranes makes them attractive as vehicles for presentation of tumor-associated antigens and induction of immune responses. In this study the potential of immunomodulators was assessed to enhance the specific immune response induced by immunization with reconstituted membranes prepared from SL2 lymphosarcoma cells. Reconstituted membranes containing muramyl tripeptide phosphatidylethanolamine (MTP-PE) provided better protection against a challenge with SL2 cells than did reconstituted membranes containing alternative immunomodulators. Local administration of IL-2 at the immunization sites further augmented the protection induced by reconstituted membranes with MTP-PE, but was ineffective when administered with plain reconstituted membranes. Immunity elicited by the triple modality of reconstituted SL2 membranes with MTP-PE and IL-2 was specific for SL2 cells. Systemic immunity was obtained against a challenge with a 100-fold higher number of SL2 cells than was reached after immunization with reconstituted membranes alone (10(5) vs. 10(3) SL2 cells). Macrophages isolated from the peritoneal cavity of immunized mice 5 to 7 days after tumor challenge expressed high in vitro cytotoxicity. However, in contrast to the observed specificity of the systemic immunity, macrophages killed both SL2 cells and non-related P815 cells. Neither major cytotoxic lymphocyte activity nor substantial cytotoxic antibody titers were detectable. These results clearly indicate that the approach using reconstituted membranes combined with particular immunomodulators warrants further exploration for the development of safe, well-characterized cancer vaccines.