Induction and detection of antibodies to squalene.

An enzyme-linked immunosorbent assay (ELISA) utilizing antigen coated on hydrophobic polyvinyldiene fluoride (PVDF) membranes is described for detecting antibodies that bind to squalene (SQE). Because of the prior lack of availability of validated antibodies to SQE, positive controls for the assay were made by immunization with formulations containing SQE to create monoclonal antibodies (mAbs) that reacted with SQE. Among eight immunogens tested, only two induced detectable murine antibodies to SQE: liposomes containing dimyristoyl phosphatidylcholine, dimyristoyl phosphatidylglycerol, 71% SQE, and lipid A [L(71% SQE+LA)], and, to a much lesser extent, an oil-in-water emulsion containing SQE, Tween 80, Span 85, and lipid A. In each case, lipid A served as an adjuvant, but neither SQE alone, SQE mixed with lipid A, liposomes containing 43% SQE and lipid A, nor several other emulsions containing both SQE and lipid A, induced antibodies that reacted with SQE. Monoclonal antibodies produced after immunizing mice with [L(71% SQE+LA)] served as positive controls for developing the ELISA. Monoclonal antibodies were produced that either recognized SQE alone but did not recognize squalane (SQA, the hydrogenated form of SQE), or that recognized both SQE and SQA. As found previously with other liposomal lipid antigens, liposomes containing lipid A also induced antibodies that reacted with the liposomal phospholipids. However, mAbs were also identified that reacted with SQE on PVDF membranes, but did not recognize either SQA or liposomal phospholipid. The polyclonal antiserum produced by immunizing mice with [L(71% SQE+LA)] therefore contained a mixed population of antibody specificities and, as expected, the ELISA of polyclonal antiserum with PVDF membranes detected antibodies both to SQE and SQA. We conclude that SQE is a weak antigen, but that antibodies that specifically bind to SQE can be readily induced by immunization with [L(71% SQE+LA)] and detected by ELISA with PVDF membranes coated with SQE.

Proteasome inhibitors block the entry of liposome-encapsulated antigens into the classical MHC class I pathway.

Liposome-encapsulated conalbumin (L(conalbumin)) is an antigen that is efficiently phagocytosed by bone marrow-derived macrophages and presented to effector cells as part of the major histocompatibility complex (MHC) class I complex. In this report, we show that the conalbumin component of L(conalbumin) is degraded to small peptide fragments and translocated to the area of the Golgi. Golgi localization is confirmed by co-localization of L(Texas red-conalbumin) (L(TR-conalbumin))with both NBD-ceramide, a lipid Golgi marker, and green fluorescent protein (GFP)-galactosyl transferase, a Golgi resident enzyme. Incubation of the cells with brefeldin A disrupts the Golgi and disperses the TR-conalbumin. Furthermore, when macrophages were incubated with another liposome-encapsulated antigen, L(ovalbumin), ovalbumin peptides were observed in the Golgi area and MHC class I-peptide complexes could be detected on the cell surface by both immunofluorescence microscopy and flow cytometry. The Golgi localization observed in vitro in cultured macrophages is mirrored by the in vivo uptake and Golgi localization of fluorescent L(conalbumin) in macrophages isolated from the spleen of a mouse injected with L(TR-conalbumin). The accumulation of peptide fragments in the Golgi is inhibited by the addition of the proteasome inhibitors, lactacystin and MG-132, demonstrating the role of the proteasome in this activity. In addition, when macrophages or a macrophage-derived cell line, are incubated with liposome-enccapsulated antigens and used as target cells in a cytotoxic T-cell (CTL) assay, the CTLs recognize the processed peptide-MHC complexes and kill the cells. In contrast, specific lysis of target cells by CTLs is inhibited when the target cells are first incubated with lactacystin. These results suggest that uptake and processing of L(antigen) follows the classical MHC class I pathway.

Naturally occurring antibodies to cholesterol: a new theory of LDL cholesterol metabolism.

Although low-density lipoprotein (LDL) receptors regulate the disposition of two-thirds of circulating serum LDL cholesterol, non-LDL receptor mechanisms account for removal of one-third. Here, Carl Alving and Nabila Wassef propose that naturally occurring antibodies to cholesterol in normal human plasma also contribute to LDL cholesterol turnover by opsonizing LDL and other lipoproteins containing 'bad' cholesterol for removal by complement receptors.

Hemodynamic changes induced by liposomes and liposome-encapsulated hemoglobin in pigs: a model for pseudoallergic cardiopulmonary reactions to liposomes. Role of complement and inhibition by soluble CR1 and anti-C5a antibody.

BACKGROUND: Intravenous administration of some liposomal drugs can trigger immediate hypersensitivity reactions that include symptoms of cardiopulmonary distress. The mechanism underlying the cardiovascular changes has not been clarified. METHODS AND RESULTS: Anesthetized pigs (n=18) were injected intravenously with 5-mg boluses of large multilamellar liposomes, and the ensuing hemodynamic, hematologic, and laboratory changes were recorded. The significant (P<0.01) alterations included 79+/-9% (mean+/-SEM) rise in pulmonary arterial pressure, 30+/-7% decline in cardiac output, 11+/-2% increase in heart rate, 236+/-54% increase in pulmonary vascular resistance, 71+/-27% increase in systemic vascular resistance, and up to a 100-fold increase in plasma thromboxane B2. These changes peaked between 1 and 5 minutes after injection, subsided within 10 to 20 minutes, were lipid dose-dependent (ED50=4. 5+/-1.4 mg), and were quantitatively reproducible in the same animal several times over 7 hours. The liposome-induced rises of pulmonary arterial pressure showed close quantitative and temporal correlation with elevations of plasma thromboxane B2 and were inhibited by an anti-C5a monoclonal antibody (GS1), by sCR1, or by indomethacin. Liposomes caused C5a production in pig serum in vitro through classic pathway activation and bound IgG and IgM natural antibodies. Zymosan- and hemoglobin-containing liposomes and empty liposomes caused essentially identical pulmonary changes. CONCLUSIONS: The intense, nontachyphylactic, highly reproducible, complement-mediated pulmonary hypertensive effect of minute amounts of intravenous liposomes in pigs represents a unique, unexplored phenomenon in circulation physiology. The model provides highly sensitive detection and study of cardiopulmonary side effects of liposomal drugs and many other pharmaceutical products due to "complement activation-related pseudoallergy" (CARPA).

Trafficking of liposomal antigen to the trans-Golgi of murine macrophages requires both liposomal lipid and liposomal protein.

Major histocompatibility complex (MHC) class I molecules found on antigen-presenting cells present peptides derived from cytoplasmic proteins to T cells. In contrast, peptides from exogenous proteins are mostly presented by class II molecules. It has been well established that liposomes can serve as an efficient delivery system for entry of exogenous protein antigens into the MHC class I pathway. Our previous studies utilizing fluorophore-labeled proteins encapsulated in liposomes demonstrated that after phagocytosis of the liposomes by bone marrow-derived macrophages (BMs), the processed peptides were subsequently visualized in the trans-Golgi, while free conalbumin was excluded from the trans-Golgi area. In the present study, we investigated whether liposomal lipids follow the same intracellular route as the liposomal proteins after phagocytosis by BMs. Multilamellar liposomes with different lipid compositions that also contained fluorescent phospholipids (empty liposomes) were incubated with murine BMs. Our results indicate that although empty liposomes were avidly phagocytosed by macrophages, the fluorescent liposomal lipids did not localize to any particular area of the cell but were distributed throughout the cell. In contrast, when a protein was encapsulated in the liposomes, the liposomal lipids were no longer dispersed throughout the cell, but were concentrated and localized in the trans-Golgi area. Furthermore, when the liposomes contained a fluorescent-labeled protein, the fluorescent peptides also localized to the trans-Golgi. These results demonstrate that the combination of both liposomal lipids and liposomal protein is required for Golgi-specific targeting of liposomal antigens. Transport of both liposomal lipids and liposomal proteins to the Golgi complex, a major subcellular organelle in the passage of MHC class I molecules, might explain why antigens encapsulated in liposomes readily induce cytotoxic T lymphocytes.

Liposomes containing lipid A serve as an adjuvant for induction of antibody and cytotoxic T-cell responses against RTS,S malaria antigen.

Encapsulation of soluble protein antigens in liposomes was previously shown to result in processing of antigen via the major histocompatibility complex class I pathway, as evidenced by costaining of the trans-Golgi region of murine bone marrow-derived macrophages (BMs) by fluorophore-labeled liposomal antigen and by a trans-Golgi-specific fluorescent lipid. Evidence is presented here that free or liposome-encapsulated RTS,S, a particulate malaria antigen consisting of hepatitis B particles coexpressed with epitopes from the Plasmodium falciparum circumsporozoite protein, also was localized in the trans-Golgi after incubation with BMs, suggesting processing by the class I pathway. An in vivo cytotoxic T-lymphocyte (CTL) response was detected, however, only after immunization with RTS,S encapsulated in liposomes containing lipid A and not after immunization with free RTS,S or with RTS,S encapsulated in liposomes lacking lipid A. Therefore, intracellular delivery of antigen containing CTL epitopes to the Golgi of BMs does not necessarily result in a CTL response in vivo unless an additional adjuvant, such as liposomes containing lipid A, is utilized. Encapsulation of RTS,S in liposomes containing monophosphoryl lipid A (MPL) resulted in a dose-dependent enhancement of the NANP-specific immunoglobulin G (IgG) antibody response compared to that of free RTS,S. The IgG1 and IgG2a subclasses predominated after immunization with RTS,S encapsulated in liposomes containing MPL. These results demonstrate that encapsulation of a lipid-containing particulate antigen, such as RTS, S, in liposomes containing lipid A can enhance both humoral and cellular immune responses.

HIV-1 neutralizing antibodies in the genital and respiratory tracts of mice intranasally immunized with oligomeric gp160.

Because mucosal surfaces are a primary route of HIV-1 infection, we evaluated the mucosal immunogenicity of a candidate HIV-1 vaccine, oligomeric gp160 (o-gp160). In prior studies, parenteral immunization of rabbits with o-gp160 elicited broad neutralizing serum Ab responses against both T cell line-adapted HIV-1 and some primary HIV-1 isolates. In this study, nasal immunization of mice with o-gp160, formulated with liposomes containing monophosphoryl lipid A (MPL), MPL-AF, proteosomes, emulsomes, or proteosomes with emulsomes elicited strong gp160-specific IgG and IgA responses in serum as well as vaginal, lung, and intestinal washes and fecal pellets. The genital, respiratory, and intestinal Abs were determined to be locally produced. No mucosal immune responses were measurable when the immunogen was given s.c. Abs from sera and from vaginal and lung washes preferentially recognized native forms of monomeric gp120, suggesting no substantial loss in protein tertiary conformation after vaccine formulation and mucosal administration. Inhibition of HIV-1MN infection of H9 cells was found in sera from mice immunized intranasally with o-gp160 formulated with liposomes plus MPL, proteosomes, and proteosomes plus emulsomes. Formulations of o-gp160 with MPL-AF, proteosomes, emulsomes, or proteosomes plus emulsomes elicited HIV-1MN-neutralizing Ab in lung wash, and formulations with proteosomes, emulsomes, or proteosomes plus emulsomes elicited HIV-1MN-neutralizing Ab in vaginal wash. These data demonstrate the feasibility of inducing both systemic and mucosal HIV-1-neutralizing Ab by intranasal immunization with an oligomeric gp160 protein.

Complement activation and thromboxane secretion by liposome-encapsulated hemoglobin in rats in vivo: inhibition by soluble complement receptor type 1.

Intravenous administration of liposome-encapsulated hemoglobin (LEH) in rats led to an early (within 15 min) decline of hemolytic complement (C) activity in the plasma along with a significant, parallel rise in thromboxane B2 (TXB2) levels. The TXB2 response was inhibited by co-administration of soluble C receptor type 1 (sCR1) with LEH, as well as by C depletion with cobra venom factor. These observations provide evidence for a causal relationship between LEH-induced C activation and TXB2 release, and suggest that sCR1 could be useful in attenuating the acute respiratory, hematological and hemodynamic side effects of LEH described earlier in the rat.

Complement activation in vitro by the red cell substitute, liposome-encapsulated hemoglobin: mechanism of activation and inhibition by soluble complement receptor type 1.

BACKGROUND: Liposome-encapsulated hemoglobin (LEH) has been developed as an emergency blood substitute, yet its effect on human complement has never been explored. Considering that complement activation is a major pathogenic factor in the respiratory distress syndrome that often develops in trauma and shock, LEH-induced complement activation may be a critical safety issue. STUDY DESIGN AND METHODS: Various LEH and corresponding empty liposomes were incubated with normal human sera, and various markers of complement activation (serum levels of C4d, Bb, SC5b-9, and CH50; C5a-induced granulocyte aggregation; membrane deposition of C3b) were measured. Incubations were also performed in the presence of (ethylene-bis[oxyethylenenitrilo]tetraacetic acid) (EGTA) and Mg++ (EGTA/Mg++) and soluble complement receptor type 1. RESULTS: LEH and liposomes activated human complement, as indicated by significant changes in one or more markers. The effect was primarily due to the presence of the phospholipid vehicle; small, unilamellar, highly homodispersed vesicles induced the greatest degree of complement activation. Complement activation was partially inhibited by EGTA/Mg++. The latter finding, together with the parallel increases in serum C4d and Bb, suggests activation of both the classical and alternative pathways. Soluble complement receptor type 1 (0.05-20 micrograms/mL) efficiently inhibited all vesicle-induced complement activation. CONCLUSION: Because of complement activation, the use of LEH for transfusion may require careful evaluation of safety. Soluble complement receptor type 1 may be useful as a prophylactic agent for complement activation-related complications of liposome infusions.

Visualization of peptides derived from liposome-encapsulated proteins in the trans-Golgi area of macrophages.

Exogenous proteins are generally not presented through the major histocompatibility complex (MHC) class I pathway, yet several recent studies show that particle-associated antigens induce a CD8+ T-cell response. Therefore, a pathway must exist in vivo for the presentation of exogenous antigens on class I molecules. In the present study, we investigated the intracellular fate of liposome-encapsulated Texas Red (TR)-conjugated protein in cultured bone marrow-derived macrophages (BMs). After phagocytosis of liposomes, the fluorescent liposomal protein, initially associated with the liposomal lipids in phagosomes, later entered the cytoplasm, and the processed protein was subsequently visualized in the trans-Golgi as a fluorescent peptide. Experiments performed with BMs from transporter associated with antigen processing (TAP1) knock-out mice demonstrated that the translocation of peptides into the trans-Golgi area was dependent upon TAP1 protein. We conclude that delivery of liposomal proteins or peptides to the cytoplasm of phagocytes and subsequent transport of peptides to the Golgi via the classical MHC class I pathway involving TAP proteins might explain the known propensity of liposomal antigens to induce cytotoxic T-lymphocytes (CTLs).

Complement activation in human serum by liposome-encapsulated hemoglobin: the role of natural anti-phospholipid antibodies.

In exploring the occurrence and mechanism of liposome-encapsulated hemoglobin (LEH)-induced complement (C) activation, we found that normal human serum contained low titers of IgG and IgM class natural antibodies with reactivity against LEH, and that the amount of vesicle-bound IgM significantly correlated with LEH-induced C consumption. IgM binding to LEH was inhibited by phosphocholine and ATP, but not by choline chloride. These data suggest that naturally occurring antibodies play a key role in LEH-induced C activation, and that a major portion of these antibodies are directed against the phosphate moiety on the phospholipid headgroups of liposome bilayers.

Liposomal subunit vaccines: effects of lipid A and aluminum hydroxide on immunogenicity.

Protein and peptide antigens frequently are only slightly immunogenic when utilized alone in vaccines. Formulation of these antigens in a carrier vehicle, particularly when an adjuvant is included, often results in markedly enhanced immune responses. Encapsulation of peptide and protein antigens in liposomes generally results in a relatively slight enhancement of antibody production compared with that observed with the antigen alone. However, when lipid A is included in the liposomes, immunogenicity is markedly increased compared both with antigen alone and with antigen encapsulated in liposomes lacking lipid A. The enhancement of the immune response caused by lipid A is dependent on the liposomal lipid A dose. Aluminum salts, such as aluminum hydroxide and aluminum phosphate, act as adjuvants for some antigens and are used in a variety of human vaccines. When liposomes containing encapsulated protein or peptide antigens were adsorbed with aluminum hydroxide, an enhancement of the antibody response was observed with some antigens, whereas with other antigens the presence of aluminum hydroxide either had no effect or resulted in a diminished antibody response. Immunogenicity of protein and peptide antigens can be enhanced by formulation in liposomes containing lipid A and, depending on the antigen, can be enhanced further by adsorption of the liposomal antigen formulation with aluminum salts.

Immunization with cholesterol-rich liposomes induces anti-cholesterol antibodies and reduces diet-induced hypercholesterolemia and plaque formation.

Immunization of rabbits with a protein-free formulation consisting of liposomes containing 71% cholesterol and lipid A as an adjuvant induced anticholesterol antibodies that caused complement-dependent lysis of liposomes lacking lipid A. The antibodies, immunoglobulin G (IgG) and immunoglobulin M (IgM), also recognized nonoxidized crystalline cholesterol as an antigen by enzyme-linked immunosorbent assay (ELISA). The effects of immunization against cholesterol on elevations in serum cholesterol and development of atherosclerosis were examined in rabbits fed a diet containing 0.5% to 1.0% cholesterol. Although the mean serum cholesterol level, mainly in the form of very-low-density lipoprotein cholesterol, rose as much as 60-fold in the nonimmunized rabbits, the elevation was significantly less--as much as 35% lower--in the immunized rabbits. Elevation of serum cholesterol was accompanied by an apparent drop in the level of antibodies on initiating the diet, followed by a rebound on stopping the diet, thus suggesting that the antibodies were adsorbed to cholesterol that was present in circulating lipoproteins. When lipoprotein fractions--composed of either very-low-density and intermediate-density lipoproteins derived from cholesterol-fed nonimmunized rabbits or human low-density lipoproteins--were tested as capture antigens by solid-phase ELISA, reactivity was observed with IgG and IgM antibodies present in the serum of immunized rabbits. Immunization also resulted in a marked decrease in the risk of developing atherosclerosis. Analysis of aortic atherosclerosis by quantitative histologic examination and fatty streaks by automated morphometric probability-of-occurrence mapping showed diminished atherosclerosis in most areas of the aorta in vaccine recipients. It is proposed that immunization with liposomes containing 71% cholesterol and lipid A can reduce diet-induced hypercholesterolemia and atherosclerosis.

Antibodies to cholesterol: biological implications of antibodies to lipids.

Injection of silicone gel or silicone oil intraperitoneally into BALB/c mice induced the formation of antibodies that reacted by ELISA with highly purified crystalline cholesterol and, to a much lesser extent, antibodies that reacted with a phospholipid (dimyristoyl phosphatidylglycerol). Although IgM and IgG antibodies to cholesterol were detected, the titers of IgG antibodies were low when compared with IgM. The titers of IgM antibodies to cholesterol in certain sera exhibited activities that reached baseline values at dilutions as high as 1:5000, thus making them equivalent to titers that have been previously published for ascites fluid containing murine monoclonal antibodies to cholesterol. The antibodies to cholesterol induced by silicone compounds are indistinguishable in their binding to crystalline cholesterol from naturally-occurring antibodies to cholesterol in normal human serum. They are also indistinguishable from antibodies induced by a proposed vaccine to cholesterol that is currently in late preclinical development for prevention of hypercholesterolemia in humans. The anti-cholesterol vaccine, which consists of liposomes heavily laden with cholesterol as an antigen and lipid A as an adjuvant, induces antibodies that react with low density lipoproteins (LDL) and opsonize them for removal by liver macrophages. It appears that silicone gel or silicone oil causes recruitment and adsorption of cholesterol at the injection site, and also serves as an adjuvant that may have immunostimulant properties similar to lipid A for inducing antibodies to lipids. Antibodies to lipids such as cholesterol or phospholipids are not harmful to intact cell membranes because of steric hindrance from surrounding lipids and larger macromolecules that block binding of the antibodies.

Antibody and cytotoxic T-lymphocyte responses to a single liposome-associated peptide antigen.

The development of peptide-based vaccines that elicit antibody (Ab) and cellular immune responses has been hampered by the lack of highly immunogenic formulations. In this study, we compared the induction of Ab and cytotoxic T-lymphocyte (CTL) responses to a peptide derived from the V3 loop of HIV-1 gp120 (P18 and its cysteine-glycine derivative (CG-P18)) when incorporated into liposomes with lipid A (LA) or mixed with aluminum hydroxide. P18-specific CTL were only observed with liposomes with LA. P18-specific Ab responses were found with liposomes containing CG-P18 but not P18. Increased surface expression of the former, resulted in enhancement of the Ab response without loss of CTL induction. Thus, the manner in which a peptide is localized can influence the outcome of the response induced by highly immunogenic liposome formulations.

Intracellular processing of liposome-encapsulated antigens by macrophages depends upon the antigen.

Two proteins, a recombinant malaria protein (R32NS1) and conalbumin, were encapsulated in separate liposomes. The mechanisms of presentation of unencapsulated and liposome-encapsulated R32NS1 and conalbumin to antigen-specific T-cell clones were investigated in in vitro antigen presentation assays using murine bone marrow-derived macrophages (BMs) as antigen-presenting cells. A much lower concentration of liposomal antigen than of unencapsulated antigen was required for T-cell proliferation. Liposome-encapsulated conalbumin required intracellular processing by BMs for antigen-specific T-cell proliferation, as determined by inhibition with chloroquine, NH4Cl, leupeptin, brefeldin A, monensin, antimycin A, NaF, and cycloheximide and by treatment of BMs with glutaraldehyde. Liposome-encapsulated conalbumin therefore follows the classical intracellular antigen processing pathway described for protein antigens. Similarly, unencapsulated conalbumin also required intracellular processing for presentation to antigen-specific T cells. In contrast, both unencapsulated R32NS1 and liposome-encapsulated R32NS1 were presented to T cells by BMs without undergoing internalization and intracellular processing. These results suggest that the antigen itself is the major element that determines whether a requirement exists for intracellular processing of liposomal antigens by macrophages.

A vaccine-elicited, single viral epitope-specific cytotoxic T lymphocyte response does not protect against intravenous, cell-free simian immunodeficiency virus challenge.

Protection against simian immunodeficiency virus (SIV) challenge was assessed in rhesus monkeys with a vaccine-elicited, single SIV epitope-specific cytotoxic T-lymphocyte (CTL) response in the absence of SIV-specific antibody. Strategies were first explored for eliciting an optimal SIV Gag epitope-specific CTL response. These studies were performed in rhesus monkeys expressing the major histocompatibility complex (MHC) class I gene Mamu-A*01, a haplotype associated with a predominant SIV CTL epitope mapped to residues 182 to 190 of the Gag protein (p11C). We demonstrated that a combined modality immunization strategy using a recombinant Mycobacterium bovis BCG-SIV Gag construct for priming, and peptide formulated in liposome for boosting, elicited a greater p11C-specific CTL response than did a single immunization with peptide-liposome alone. Vaccinated and control monkeys were then challenged with cell-free SIVmne by an intravenous route of inoculation. Despite a vigorous p11C-specific CTL response at the time of virus inoculation, all monkeys became infected with SIV. gag gene sequencing of the virus isolated from these monkeys demonstrated that the established viruses had no mutations in the p11C-coding region. Thus, the preexisting CTL response did not select for a viral variant that might escape T-cell immune recognition. These studies demonstrate that a potent SIV-specific CTL response can be elicited by combining live vector and peptide vaccine modalities. However, a single SIV Gag epitope-specific CTL response in the absence of SIV-specific antibody did not provide protection against a cell-free, intravenous SIV challenge.

Complement activation by liposome-encapsulated hemoglobin in vitro: the role of endotoxin contamination.

Incubation of liposome-encapsulated Hb (LEH) with rat serum at 37 degrees C led to accelerated decay of serum hemolytic complement (C) activity (CH50/ml). Empty liposomes (L) caused less decrease of CH50/ml, whereas free Hb had no effect on C activity. The LEH- and L-induced increases in C consumption were unlikely a consequence of endotoxin (LPS) contamination, as spiking of rat serum with LPS caused reduction in C only at levels significantly higher than those detectable in LEH or L. LPS-induced C consumption was not potentiated by free hemoglobin.

Complement activation in rats by liposomes and liposome-encapsulated hemoglobin: evidence for anti-lipid antibodies and alternative pathway activation.

Intravenous injection of hemoglobin-containing liposomes (LEH) caused a significant reduction in plasma hemolytic complement activity in rats on a time scale of minutes. Liposomes without hemoglobin also caused complement consumption, but less than LEH, while free hemoglobin was without effect. Consistent with complement activation, the LEH-induced drop in plasma hemolytic complement activity was closely paralleled by an increase in plasma thromboxane B2 level. Studies to determine the mechanism of complement activation demonstrated the presence of natural antibodies in rat serum against all lipid components of LEH, thus, the potential for classical pathway activation. Yet, in vitro incubation of LEH with rat serum showed that: 1) EGTA/Mg++, which inhibits complement activation through the classical pathway, did not inhibit complement consumption by LEH, and 2) the use of serum preheated at 50 degrees C, which inhibits C activation through the alternative pathway by selectively depleting factor B, effectively reversed the complement-consuming effect of LEH. Consequently, LEH-induced complement activation in rat serum seems to involve primarily the alternative pathway.

Liposomes as carriers for vaccines.

A liposome vaccine formulation that has been successfully used in both animal immunization studies and clinical trials is described. Issues concerning the choice of components for the liposomal vaccine formulation are discussed, especially with respect to the lipid components and the adjuvant. A procedure is described for manufacturing liposomal vaccines using Good Manufacturing Practices as promulgated by the U.S. Food and Drug Administration. Quality control testing for clinical use is described, with particular emphasis on aspects relevant to liposomes. Utilization issues are discussed, including injection volumes, antigen and adjuvant doses, and routes of administration.