lipid-a and phosphatidylinositol-4-phosphate

Natural IgG antibodies (NA) to lipids are ubiquitously distributed in sera of healthy humans and are believed to serve beneficial functions. Although NA to lipids generally exhibit germ line or near germ line binding specificities, the antibodies commonly increase transiently in the acute phases of most, if not all, infectious diseases and may serve as a first line of defense. In order to determine whether similar anti-lipid antibodies can be induced by a vaccine in humans, we examined stored sera obtained from volunteers who had previously received a candidate vaccine to Plasmodium falciparum. The vaccine had consisted of liposomes that contained both the recombinant protein antigen and also contained monophosphoryl lipid A (MPLA) as an adjuvant. All of the pre-immune sera contained NA to one or more of the liposomal lipids in the vaccine: dimyristol phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), cholesterol, and MPLA. After initial immunization, followed by a boost, increased levels of IgG antibodies to all of the liposomal lipids, especially DMPG and MPLA, were observed by ELISA. Antibodies to phosphatidylinositol-4-phosphate (PIP) above the normal pre-immune NA to PIP were also observed. Although PIP was not present in the immunizing liposomes, based on the adsorption of anti-PIP antibodies by DMPG the anti-PIP antibodies were thought to represent cross-reacting anti-DMPG antibodies. The immune response was apparently antigen-specific in that NA to unrelated lipids, other than PIP, that were not present in the liposomes, galactosyl ceramide and ganglioside GM1, were not increased by the immunization. We conclude that antibodies to DMPC, DMPG, PIP, cholesterol, and MPLA can be induced in humans by immunization with liposomes containing MPLA.

Topics: Adult; Antigens; Cholesterol; Cross Reactions; Enzyme-Linked Immunosorbent Assay; Humans; Immunization; Immunoglobulin G; Lipid A; Liposomes; Phosphatidylcholines; Phosphatidylglycerols; Phosphatidylinositol Phosphates; Vaccination; Vaccines

Lipid A derived from Gram-negative bacterial lipopolysaccharide is a potent adjuvant and antigen. Incorporation of lipid A into liposomes renders the liposomes themselves immunogenic, resulting in generation of specific antibodies that recognize either the individual liposomal lipids, or the unique pattern presented by the combination of lipids. Using liposomes containing lipid A, numerous polyclonal antisera and monoclonal antibodies have been produced against phospholipids, cholesterol, glycosphingolipids, and lipid A. Many of these antibodies have binding characteristics that are apparently similar to natural antibodies that are normally present in all human sera, and also antibodies that arise in response to various infections. Such antibodies probably represent a bridge between innate and adaptive immunity. The possible utility of liposomes containing lipid A as a constituent of certain types of novel vaccines was suggested by the observation that murine monoclonal antibodies to liposomal phosphatidylinositol-4-phosphate neutralized primary isolates of two different clades of HIV-1 in a human peripheral blood mononuclear cell neutralization assay.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Antiphospholipid; Antibodies, Monoclonal; Antibody Specificity; Cells, Cultured; HIV Antibodies; HIV-1; Humans; Lipid A; Liposomes; Mice; Phosphatidylinositol Phosphates; Toll-Like Receptor 4; Vaccines

Membranes of Rhizobium leguminosarum contain a 3-deoxy-D-manno-octulosonic acid (Kdo)-activated lipid A 4'-phosphatase required for generating the unusual phosphate-deficient lipid A found in this organism. The enzyme has been solubilized with Triton X-100 and purified 80-fold. As shown by co-purification and thermal inactivation studies, the 4'-phosphatase catalyzes not only the hydrolysis of (Kdo)2-[4'-32P]lipid IVA but also the transfer the 4'-phosphate of Kdo2-[4'-32P]lipid IVA to the inositol headgroup of phosphatidylinositol (PtdIns) to generate PtdIns-4-P. Like the 4'-phosphatase, the phosphotransferase activity is not present in Escherichia coli, Rhizobium meliloti, or the nodulation-defective mutant 24AR of R. leguminosarum. The specific activity for the phosphotransferase reaction is about 2 times higher than that of the 4'-phosphatase. The phosphotransferase assay conditions are similar to those used for PtdIns kinases, except that ATP and Mg2+ are omitted. The apparent Km for PtdIns is approximately 500 microM versus 20-100 microM for most PtdIns kinases, but the phosphotransferase specific activity in crude cell extracts is higher than that of most PtdIns kinases. The phosphotransferase is absolutely specific for the 4-position of PtdIns and is highly selective for PtdIns as the acceptor. The 4'-phosphatase/phosphotransferase can be eluted from heparin- or Cibacron blue-agarose with PtdIns. A phosphoenzyme intermediate may account for the dual function of this enzyme, since a single 32P-labeled protein species (Mr approximately 68,000) can be trapped and visualized by SDS gel electrophoresis of enzyme preparations incubated with Kdo2-[4'-32P]lipid IVA. Although PtdIns is not detected in cultures of R. leguminosarum/etli (CE3), PtdIns may be synthesized during nodulation or supplied by plant membranes, given that soybean PtdIns is an excellent phosphate acceptor. A bacterial enzyme for generating PtdIns-4-P and a direct link between lipid A and PtdIns-4-P biosynthesis have not been reported previously.

Topics: Carbohydrate Sequence; Chromatography, Ion Exchange; Chromatography, Thin Layer; Hot Temperature; Kinetics; Lipid A; Molecular Sequence Data; Phosphatidylinositol Phosphates; Phosphatidylinositols; Phosphotransferases; Rhizobium leguminosarum; Substrate Specificity