Antibodies to Lipids and Liposomes: Immunology and Safety

Naturally occurring antibodies to phospholipids and cholesterol are widespread; they occur commonly during the course of acute infections; they are not causally related to the anti-phospholipid syndrome; they have been associated with other clinical entities only as an epiphenomenon; and they have not been implicated as causing any clinical syndrome or disease. There are theoretical and experimental reasons to believe that normal cells and tissues are protected from binding of antibodies to bilayer lipids by steric hindrance due to adjacent larger molecules, such as large or charged adjacent glycolipids or proteins on the cell surface. There are also reasons to believe that certain natural antibodies to lipids can even serve useful normal functions. Antibodies to liposomal lipids induced by liposomes containing lipid A appear to have characteristics that are similar or identical to naturally occurring antibodies to lipids, and it is therefore believed that such antibodies would not cause adverse clinical effects. Numerous Phase I and II human clinical trials of experimental vaccines containing liposomes and lipid A have shown a high level of safety.     

Antibodies to lipids and liposomes: immunology and safety

Naturally occurring antibodies to phospholipids and cholesterol are widespread; they occur commonly during the course of acute infections; they are not causally related to the anti-phospholipid syndrome; they have been associated with other clinical entities only as an epiphenomenon; and they have not been implicated as causing any clinical syndrome or disease. There are theoretical and experimental reasons to believe that normal cells and tissues are protected from binding of antibodies to bilayer lipids by steric hindrance due to adjacent larger molecules, such as large or charged adjacent glycolipids or proteins on the cell surface. There are also reasons to believe that certain natural antibodies to lipids can even serve useful normal functions. Antibodies to liposomal lipids induced by liposomes containing lipid A appear to have characteristics that are similar or identical to naturally occurring antibodies to lipids, and it is therefore believed that such antibodies would not cause adverse clinical effects. Numerous Phase I and II human clinical trials of experimental vaccines containing liposomes and lipid A have shown a high level of safety.     

Immunological cross reactions between polysaccharides of Streptococci groups A and L]

Antibodies on sepharose immunosorbents containing A-polysaccharide-sepharose or synthetic beta-N-acetylglucosamine, were isolated from the sera of rabbits immunized with streptococci, group A, by means of affinity chromatography. Antibodies obtained from some sera with both immunosorbents reacted with streptococcus, group A and L polysaccharides. Partial identity of these polysaccharides was revealed by the immunodiffusion test. Absorption of antibodies with polysaccharides, group A and L, showed their different specificities. These antibodies could apparently be directed against the end parts of molecules of streptococcus, group A polysaccharide.     

Interaction of spermine with polyphosphoinositides containing liposomes and myo-inositol 1,4,5 triphosphate

The interaction of spermine with liposomes containing 2% phosphatidylinositol, phosphatidylinositol 4 phosphate and phosphatidylinositol 4,5 biphosphate was inferred from the ability of these liposomes to interfere with spermine binding to the resin heparin-Sepharose. The inositol phospholipids tested showed different affinities for spermine: the order of binding strength appear to be phosphatidylinositol phosphatidylinositol 4 phosphate phosphatidylinositol 4,5 biphosphate. The ability of vesicles containing 2% polyphosphoinositides to interact with spermine is comparable to that of either single stranded RNAs or highly negatively charged liposomes. Myo-inositol 1,4,5 triphosphate has a much lower ability to bind spermine.     

Antibodies to liposomal phosphatidylserine and phosphatidic acid

Polyclonal antisera to phosphatidylserine or phosphatidic acid were induced in rabbits by injecting liposomes containing phosphatidylserine or phosphatidic acid and lipid A. Adsorption of antisera with liposomes containing different phospholipids revealed that some degree of reactivity with one or more phospholipids other than the immunizing phospholipid was often observed. However, cross-reactivity with other phospholipids was not a universal phenomenon, and one antiserum to phosphatidylserine failed to cross-react (i.e., was not adsorbed) with liposomes containing other phospholipids. All of the antisera were inhibited by soluble phosphorylated haptens (e.g., phosphocholine but not choline), but one of the antisera to phosphatidylserine was inhibited both by phosphoserine and by serine alone. Liposomal membrane composition influenced the activity of antiserum to phosphatidylserine. Regardless of whether unsaturated (beef brain) or saturated (dimyristoyl) phosphatidylserine was used in the immunizing liposomes, the antisera reacted more vigorously with liposomes containing unsaturated than saturated phosphatidylserine. We conclude that liposomes containing lipid A can serve as vehicles for stimulating polyclonal antisera to phosphatidylserine and phosphatidic acid. Although cross-reactivity with certain other phospholipids can be observed, sera from selected animals apparently can exhibit a high degree of specific activity to the immunizing phospholipid antigen.     

Immunological comparison of the b and c1 cytochromes from bovine heart mitochondria and the photosynthetic bacterium Rhodopseudomonas sphaeroides R-26

Antibodies against cytochromes b and c1 of bovine heart mitochondria and the photosynthetic bacterium, Rhodopseudomonas sphaeroides R-26, were raised in rabbits. The purified antibodies showed high titers against their respective antigens in enzyme-linked immunosorbent assays. Less than 15% cross-reactivity between the mitochondrial and bacterial cytochromes was detected. Although antibodies against mitochondrial cytochrome b did not inhibit the mitochondrial cytochrome b-c1 complex, a 70% inhibition was obtained when these antibodies were incubated with delipidated mitochondrial cytochrome b-c1 complex prior to reconstitution with phospholipids indicating that the catalytic site(s) of mitochondrial cytochrome b are masked by phospholipids. On the other hand, antibodies against bacterial cytochrome b showed significant inhibition of the intact bacterial cytochrome b-c1 complex, indicating that some of the catalytic site epitopes of bacterial cytochrome b are exposed to the hydrophilic environment. Similar to antibodies against mitochondrial cytochrome b, antibodies against bacterial cytochrome b inhibited 50% activity of the mitochondrial cytochrome b-c1 complex only when they were incubated with the delipidated mitochondrial cytochrome b-c1 complex prior to reconstitution with phospholipids, indicating that the common epitopes between the cytochromes b are masked by phospholipids. Antibodies against mitochondrial and bacterial cytochromes c1 completely inhibited their respective cytochrome b-c1 complexes but no cross-immunoinhibition was observed. However, when antibodies against bacterial cytochrome c1 were incubated with the delipidated mitochondrial cytochrome b-c1 complex before reconstitution with phospholipids, a 65% inhibition was observed, indicating that the common epitopes between the cytochromes c1 were also somewhat masked by phospholipids. Antibodies against mitochondrial cytochrome c1 inhibited 70% of the succinate oxidase activity in the intact mitochondria preparation, but no inhibition was observed in submitochondrial particles, indicating that some mitochondrial cytochrome c1 epitopes are exposed to the cytoplasmic side.     

Oral and parenteral immunization with synthetic retro-inverso peptides induce antibodies that cross-react with native peptides and parent antigens

The objective of this study was to determine whether certain retro-inverso peptides have the potential to act as synthetic vaccines in mice, when immunized by injection or orally. Immunization of mice parenterally with conjugates of three such retro-inverso peptides and orally with the unconjugated peptides elicited generally high titres of anti-peptide antibodies. Antibodies against the same three peptides cross-reacted by binding strongly in ELISA to the native peptides and vice versa, regardless of the mode of immunization. Antibodies against a retro-inverso diphtheria peptide also reacted strongly with diphtheria toxin. Seven of 8 mice, immunized by injection of the conjugate of a retro-inverso derivative of robustoxin [a lethal spider (Atrax robustus) venom toxin] were protected from challenge involving injection with twice the minimum lethal dose of A. robustus venom containing the toxin.     

Preparation and properties of antisera to glycolipid of guinea pig erythrocyte membrane

Antibodies against a glycolipid of guinea pig erythrocyte membranes were prepared in rabbits by immunization with guinea pig erythrocyte stroma or the purified glycolipid, gangliotriaosylceramide. The antibodies agglutinated guinea pig erythrocytes. The specificity of antibodies could be revealed by several immunochemical methods, including inhibition of hemagglutination, immunodiffusion, agglutination of liposomes, and complement fixation. The antibodies were specific for gangliotriaosylceramide.     

Immunological studies on the localization of phosphatidylglycerol in the membranes of Mycoplasma hominis.

Phosphatidylglycerol is the main component (87%) of the membrane phospholipids of Mycoplasma hominis. It is immunologically active. Antibodies directed against phosphatidylglycerol were detected in rabbits intravenously immunised with native M. hominis or isolated M. hominis membranes. The intravenous method of immunisation was chosen in order to select for a response to surface antigenic determinants. Anti-phosphatidylglycerol antibodies were induced in rabbits by intravenously injecting the flocculated complexes of methylated bovine serum albumin and a phosphatidylglycerol/phosphatidylcholine/cholesterol mixture. These antibodies were specifically bound to intact M. hominis, as shown by complement fixation and Coombs tests. Native M. hominis were not agglutinated by anti-phosphatidylglycerol antibodies; but after partial digestion of the membrane proteins with Pronase, the mycoplasmas were heavily agglutinated by the anti-phosphatidylglycerol antibodies. The same amount of anti-phosphatidylglycerol antibodies was bound to intact M. hominis, containing 600 mug of phosphatidylglycerol as to 6 mug of phosphatidylglycerol in the optimal configurational arrangement of a mixed phosphatidylglycerol/phosphatidylcholine/cholesterol micelle. It is concluded that the major part of the phosphatidylglycerol in native M. hominis membranes is masked, probably by membrane proteins, and is not accessible to the anti-phosphatidylglycerol antibodies.     

Rabbit acrosin: immunological dissimilarity to rabbit trypsin (1).

Antibodies obtained from guinea pigs injected with rabbit pancreatic trypsin together with antibodies raised in rabbits against bovine acrosin or bovine pancreatic trypsin were reacted against various mammalian trypsins and acrosins in double diffusion tests. The results of immunodiffusion analyses reveal antigenic dissimilarity between rabbit acrosin and rabbit trypsin.     

Letters

Abstract

Long-term effects of life-long (>2 year) repeated intravenous injections (up to 17) of high doses of liposomes, lipid A, or liposomes containing lipid A were assessed in BALB/c mice. the liposomes contained dipalmitoylphosphatidylcholine, and cholesterol (1/0.75). When compared with mice injected only with normal saline, there were no statistical differences in life spans observed between the different groups. Animals injected with liposomes or liposomes containing lipid A gradually developed “ruffled” fur, but the animals did not appear sick otherwise, and no differences were observed in the mean weights of the animals in the different groups. All of the animals that were tested in each group, including those injected with normal saline, developed IgG antibodies against one or more of nine lipid antigens. the antibodies were detected by a solid-phase enzyme-linked immunosorbent assay (ELisA) and the antigens consisted of either lipid A or one of eight different phospholipids. After 765 days, when approximately half of the animals had died spontaneously, the surviving animals were sacrificed and subjected to extensive histopathological analysis. In each group (including the normal saline group) certain animals exhibited pathological changes in liver, spleen, or lung that might be expected to occur in highly aged animals. Approximately half of the animals in each group had lymphoproliferative disorders (hyperplasia and/or lymphoma). However, there were no bone marrow abnormalities, and no hepatic or splenic granulomatous reactions were found in any of the animals. From a pathological standpoint the groups were indistinguishable from each other. We conclude that life-long repeated injection of liposomes, lipid A, or liposomes containing lipid A does not alter longevity or cause any overt pathological changes in mice. We also conclude that antibodies to lipid A and a variety of phospholipids occur spontaneously in aged mice that have been injected only with normal saline.     

Specific binding of concanavalin A to free inositol and liposomes containing phosphatidylinositol

We previously reported that concanavalin A could bind specifically to liposomes containing phospholipids and lacking glycoconjugates (Biochem. Biophys. Res. Comm. 74, 208, 1977). In the present study we show that the binding of concanavalin A to the liposomes was greatly increased (up to 5 fold) by the presence of phosphatidylinositol in the liposomes. Furthermore, the binding of concanavalin A to phosphatidylinositol-liposomes was specific and could be inhibited by either alpha-methyl mannoside or by myo-inositol. We also found that concanavalin A-induced lymphocyte mitogenesis could be inhibited either by alpha-methyl mannoside or by myo-inositol. Simultaneous addition of both inhibitors to concanavalin A and liposomes showed that inhibition was non-competitive: alpha-methyl mannoside was more inhibitory to liposomes lacking phosphatidylinositol, and myo-inositol was more inhibitory to liposomes containing phosphatidylinositol. This suggests that the binding site for inositol might be different than that for mannose. Equilibrium dialysis and Scatchard plots revealed 4 binding sites each for inositol and mannose at neutral pH. The binding constants of concanavalin A were 0.13 X 10(4) and 0.25 X 10(4) liters/mole respectively for inositol and mannose. We conclude that concanavalin A binds specifically to the inositol portion of phosphatidylinositol.     

Prostaglandin and thromboxane in liposomes: suppression of the primary immune response to liposomal antigens

Liposomes containing lipid A as adjuvant and also containing prostaglandin E2 or thromboxane B2 were examined for the ability to influence induction of humoral immunity against liposomal protein or lipid antigens in rabbits. The protein antigen consisted of cholera toxin that was bound to ganglioside GM1 on the surface of the liposomes. High titers of anti-cholera toxin antibodies were produced and IgM and IgG responses were detected. When the immunizing liposomes contained either prostaglandin E2 or thromboxane B2 as part of the lipid bilayer, the primary immune response, involving both IgM and IgG antibodies, was greatly reduced. The secondary immune response observed after a boosting immunization was not suppressed by liposomal eicosanoids. A similar inhibitory effect on the primary response was observed when liposomal lipid antigens were examined instead of cholera toxin. An inhibitory effect of liposomal prostaglandin E2 on the phagocytic uptake of opsonized liposomes by cultured macrophages was also observed, suggesting that liposomal eicosanoids can have direct local effects on macrophages that might influence the immune response to liposomal antigens.     

Membrane-damaging action of staphylococcal alpha-toxin on phospholipid-cholesterol liposomes

The mechanism of membrane damage by staphylococcal alpha-toxin was studied using carboxyfluorescein (internal marker)-loaded multilamellar liposomes prepared from various phospholipids and cholesterol. Liposomes composed of phosphatidylcholine or sphingomyelin and cholesterol bound alpha-toxin and released carboxyfluorescein in a dose dependent manner, when they were exposed to alpha-toxin of concentrations higher than 1 or 8 micrograms/ml, respectively. In contrast, the other liposomes composed of phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol or phosphatidylinositol plus cholesterol were not susceptible to the toxin even at high concentrations up to 870 micrograms/ml. The insensitive liposomes containing either phosphatidylserine or phosphatidylglycerol were made sensitive to alpha-toxin by inserting phosphatidylcholine into the liposomal membranes. In addition, phosphorylcholine inhibited the toxin-induced marker release from liposomes. These results indicated that the choline-containing phospholipids are required for the interaction between alpha-toxin and liposomal membranes. Susceptibility of liposomes containing phosphatidylcholine or sphingomyelin increased with the increase in cholesterol contents of the liposomes. Based on these results, we propose that the choline-containing phospholipids are possible membrane components or structures responsible for the toxin-membrane interaction, which leads to damage of membranes. Furthermore, cholesterol may facilitate the interaction between alpha-toxin and membrane as a structural component of the membrane.     

Human hybridoma lupus anticoagulants distinguish between lamellar and hexagonal phase lipid systems

Antibodies to phospholipids may have important physiological and biological functions. Lupus anticoagulants represent a subclass of anti-phospholipid antibodies which are characterized by their ability to prolong the clotting time in in vitro coagulation assays measuring partial thromboplastin time (PTT) (Thiagarajan, P., Shapiro, S. S., and DeMarco, L. (1980) J. Clin. Invest. 66, 397-405). In the present study, we produced hybridomas by fusing lymphocytes from 13 systemic lupus erythematosus patients with the GM 4672 lymphoblastoid line. Of the resulting 67 hybridoma autoantibodies, 14 (21%) were found to prolong a modified PTT assay, and 11 of these antibodies were analyzed further. Competition experiments, using a modified PTT assay, demonstrated that hexagonal phase phospholipids, including natural and synthetic forms of phosphatidylethanolamine, were able to neutralize the lupus anticoagulant activity of all 11 hybridoma antibodies. In contrast, lamellar phospholipids, such as phosphatidylcholine and synthetic lamellar forms of phosphatidylethanolamine, had no effect on the anticoagulant activity. Thus, these antibodies are capable of recognizing phospholipids on purely structural criteria. The demonstration that anti-phospholipid antibodies are able to distinguish between different structural arrangements of phospholipid may have important implications regarding the immunoregulation of autoimmunity.     

A fluorescent sterol probe study of cholesterol/phospholipid membranes

The behavior of dehydroergosterol in -α-dimyristoylphosphatidylcholine (DMPC) unsonicated multilamellar liposomes was characterized by absorption spectroscopy and fluorescence measurements. Dehydroergosterol exhibited a lowered absorption coefficient in multilamellar liposomes whiel the steady-state fluorescence anisotropy of dehydroergosterol in these membranes decreased significantly with increasing dehydroergosterol concentration, suggesting membrane sterol-sterol interactions. The comparative steady-state anisotropy of 0.9 mole percent dehydroergosterol in multilamellar liposomes was lower than in small unilamellar vesicles suggesting different sterol environments for dehydroergosterol. Dehydroergosterol fluorescence lifetime was relatively independent of membrane sterol content and yielded similar values in sonicated and unsonicated model membranes. In multilamellar liposomes containing 5 mole percent cholesterol, the gel-to-liqui crystalline phase transition of DMPC detected by 0.9 mole percent dehydroergosterol was significantly broadened when compared to the phase transition detected by dehydroergosterol in the absence of membrane cholesterol (Smutzer, G. et al. (1986) Biochim. Biophys. Acta 862, 361–371). In multilamellar liposomes containing 10 mole percent cholesterol, the major fluorescence lifetime of dehydroergosterol did not detect the gel-to-liquid crystalline phase transition of DMPC. Time-correlated fluorescence anisotropy decays of dehydroergosterol in DMPC multilamellar liposomes in the absence and presence of 5 mole percent cholesterol exhibited a single rotational correlation time near one nanosecond that was relatively independent of temperature and low concentrations of membrane cholesterol. The limiting anisotropy of 0.9 mole percent dehydroergosterol decreased above the gel-to-liquid crystalline phase transition in membranes without cholesterol and was not significantly affected by the phase transition in membranes containing 5 mole percent cholesterol. These results suggested hindered rotational diffusion of dehydroergosterol in multilamellar liposomes. Lifetime and time-correlated fluorescence measurements of 0.9 mole percent dehydroergosterol in multilamellar liposomes further suggested this fluorophore was detecting physical properties of the bulk membrane phospholipids in membranes devoid of cholesterol and was detecting sterol-rich regions in membranes of low sterol concentration.     

Antibodies to the scrapie protein decorate prion rods

Scrapie is a degenerative, transmissible neurologic disease of sheep and goats which occurs in the absence of any detectable host immune response. Antibodies to the scrapie agent have been produced after immunization of rabbits with either scrapie prions or the prion protein, PrP 27-30, purified from infected hamster brain. Immunoreactivity of the antisera was assessed by dot and Western immunoblots with purified prions and PrP 27-30. Antibodies raised against infectious prions were more immunoreactive with native than denatured preparations, whereas those raised against PrP 27-30 were more reactive with denatured prion preparations. As determined by second antibody-colloidal gold, both antisera were found to decorate scrapie prion rods in purified preparations. Antibodies to cellular filamentous proteins failed to react with PrP 27-30 or the scrapie prion rods; conversely, antibodies to PrP 27-30 did not exhibit immunoreactivity with cellular filamentous proteins. The monospecificity of the rabbit antiserum raised against PrP 27-30 was established by its reactivity after affinity purification. The purified antibodies reacted with PrP 27-30 on Western blots and with the prion rods. Considerable evidence indicates that the scrapie rods are aggregates of infectious prions; the findings presented here provide an immunologic demonstration that PrP 27-30 is a structural component of the prion rods.     

Immunochemical analysis of cartilage proteoglycans. Antigenic determinants of substructures.

Antibodies were raised in rabbits by injection of cartilage proteoglycan monomers, isolated hyaluronic acid-binding region, polysaccharide-peptides prepared by trypsin digestion of proteoglycans and link-protein. The rabbits injected with the proteoglycan monomers made antibodies reacting with the intact proteoglycan. The antiserum contained antibodies specific for, and also reacting with, the isolated hyaluronic acid-binding region and the keratan sulphate-rich region. In addition there were probably antibodies reacting with other structures of the proteoglycan monomer. When isolated hyaluronic acid-binding region was used for immunization the antibodies obtained reacted specifically with the hyaluronic acid-binding region. The antibodies obtained from rabbits immunized with the polysaccharide-peptides reacted with the proteoglycan monomers and showed a reaction identical with that of the chondroitin sulphate-peptides isolated after trypsin digestion of proteoglycans. The antibodies prepared with the link-protein as the antigen reacted only with the link-protein and not with any preparation from the proteoglycan monomer. Neither did any of the antisera raised against the proteoglycan monomer or its substructures react with the link-protein. Separately it was shown that the peptide 'maps' prepared from trypsin digests of the link-protein and the hyaluronic acid-binding region were different. Therefore it appears that the link-protein is not structurally related to the proteoglycan or the hyaluronic acid-binding region. Digestion of proteoglycan monomers or isolated hyaluronic acid-binding region with trypsin did not destroy the antigenic sites of the hyaluronic acid-binding region. In contrast trypsin digests of previously reduced and alkylated preparations did not react with the anti-(hyaluronic acid-binding region). The trypsin digests, however, reacted with both the antibodies directed against the chondroitin sulphate-peptides and those against the keratan sulphate-peptides. Trypsin digestion of the link-proteins destroyed the antigenic site and the reactivity with the antibodies. By combining immunoassay of proteoglycan preparations before and after trypsin digestion it is feasible to quantitatively determine its substructures by using the antisera described above.     

Monoclonal antibodies reacting with serogroup and serovar specific epitopes on different lipopolysaccharide subunits of Leptospira interrogans serovar pomona

Monoclonal antibodies with two kinds of specificities, produced against Leptospira interrogans serovar pomona, were studied by agglutination and immunoblotting. Antibodies reacted either exclusively with serovar pomona or with all members of the Pomona serogroup, but none of the antibodies reacted with representative serovars of other serogroups. Both antibodies recognized epitopes on purified lipopolysaccharide (LPS) from serovar pomona. In immunoblotting experiments the serogroup specific antibody recognized both the major LPS bands of 21 kDa and 26 kDa whereas the serovar specific antibodies reacted only with the 26 kDa band, thus localizing serovar specificity in the 26 kDa band and serogroup specific epitopes on at least two different LPS subunits.     

Production of polyclonal antibodies to the trichothecene mycotoxin 4,15-diacetylnivalenol with the carrier-adjuvant cholera toxin.

The trichothecene mycotoxin 4,15-diacetylnivalenol (DNIV) was conjugated to cholera toxin (DNIV-CT) for use as an immunogen and as an adjuvant for specific antibody production. Repeated intravenous injection of 7.5 micrograms of the conjugate was effective at generating specific antibodies to DNIV in rabbits as determined by enzyme-linked immunosorbent assay (ELISA). When small amounts (1 to 10 micrograms per animal) of DNIV-CT were used to immunize mice, polyclonal antibodies were observed as early as 4 weeks of immunization. The relative affinity of the antibodies to DNIV increased with the immunogen dose in mice. Antibodies were not detectable in either rabbits or mice that were injected with DNIV conjugated to the carrier protein bovine serum albumin or when DNIV-CT was blocked with glutaraldehyde. Competitive ELISA of mouse and rabbit serum revealed that the antibodies were most specific for DNIV but reacted to a small extent with fusarenone-X, deoxynivalenol, and nivalenol. No reactivity was observed with 3- or 15-acetyldeoxynivalenol. The results suggest that specific polyclonal antibodies can be prepared against a trichothecene when CT is used as an adjuvant and carrier protein. DNIV antibodies will be useful for monitoring the compound in food in conjunction with other trichothecene antibodies, detection of DNIV-producing cultures, and investigation of 8-ketotrichothecene biosynthesis.