lipid-a and 1-2-distearoyllecithin

Liposomes containing cholesterol (Chol) have long been used as an important membrane system for modeling the complex interactions of Chol with adjacent phospholipids or other lipids in a membrane environment. In this study we utilize a probe composed of QS-21, a saponin molecule that recognizes liposomal Chol and causes hemolysis of erythrocytes. The interaction of QS-21 with liposomal Chol results in a stable formulation which, after injection into the tissues of an animal, lacks toxic effects of QS-21 on neighboring cells that contain Chol, such as erythrocytes. Here we have used liposomes containing different saturated phospholipid fatty acyl groups and Chol, with or without monophosphoryl lipid A (MPLA), as model membranes. QS-21 is then employed as a probe to study the interactions of liposomal lipids on the visibility of membrane Chol. We demonstrate that changes either in the mole fraction of Chol in liposomes, or with different chain lengths of phospholipid fatty acyl groups, can have a substantial impact on the detection of Chol by the QS-21. We further show that liposomal MPLA can partially inhibit detection of the liposomal Chol by QS-21. The Limulus amebocyte lysate assay is used for binding to and detection of MPLA. Previous work has demonstrated that sequestration of MPLA into the liposomal lipid bilayer can block detection by the Limulus assay, but the binding site on the MPLA to which the Limulus protein binds is unknown. Changes in liposomal Chol concentration and phospholipid fatty acyl chain length influenced the detection of the liposome-embedded MPLA.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Adjuvants, Immunologic; Animals; Cell Extracts; Cholesterol; Dimyristoylphosphatidylcholine; Dose-Response Relationship, Drug; Hemolysis; Horseshoe Crabs; Humans; Lipid A; Liposomes; Phosphatidylcholines; Phosphatidylglycerols; Saponins

Vaccines that activate strong specific Th1-predominant immune responses are critically needed for many intracellular pathogens, including Leishmania. The requirement for sustained and efficient vaccination against leishmaniasis is to formulate the best combination of immunopotentiating adjuvant with the stable antigen (Ag) delivery system. The aim of the present study is to evaluate the effectiveness of an immunomodulator on liposomal Ag through subcutaneous (s.c.) route of immunization, and its usefulness during prime/boost against visceral leishmaniasis (VL) in BALB/c mice.. Towards this goal, we formulated recombinant GP63 (rGP63)-based vaccines either with monophosphoryl lipid A-trehalose dicorynomycolate (MPL-TDM) or entrapped within cationic liposomes or both. Combinatorial administration of liposomes with MPL-TDM during prime confers activation of dendritic cells, and induces an early robust T cell response. To investigate whether the combined formulation is required for optimum immune response during boost as well, we chose to evaluate the vaccine efficacy in mice primed with combined adjuvant system followed by boosting with either rGP63 alone, in association with MPL-TDM, liposomes or both. We provide evidences that the presence of either liposomal rGP63 or combined formulations during boost is necessary for effective Th1 immune responses (IFN-γ, IL-12, NO) before challenge infection. However, boosting with MPL-TDM in conjugation with liposomal rGP63 resulted in a greater number of IFN-γ producing effector T cells, significantly higher levels of splenocyte proliferation, and Th1 responses compared to mice boosted with liposomal rGP63, after virulent Leishmania donovani (L. donovani) challenge. Moreover, combined formulations offered superior protection against intracellular amastigote replication in macrophages in vitro, and hepatic and splenic parasite load in vivo.. Our results define the immunopotentiating effect of MPL-TDM on protein Ag encapsulated in a controlled release system against experimental VL.

Topics: Adjuvants, Immunologic; Animals; Cord Factors; Cytokines; Disease Models, Animal; Female; Humans; Injections, Subcutaneous; Leishmania donovani; Leishmaniasis Vaccines; Leishmaniasis, Visceral; Lipid A; Liposomes; Metalloendopeptidases; Mice; Mice, Inbred BALB C; Nitric Oxide; Phosphatidylcholines; Th1 Cells; Vaccination; Vaccines, Synthetic