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We have investigated the possibility of a T-cell epitope peptide providing help for a B-cell epitope peptide when both peptides are co-entrapped in the same liposomes. Epitope models used were a 28 amino acid peptide from the S region of the hepatitis B surface antigen (HBsAg) (subtype adw) containing an H-2′. Th-cell epitope, and a 33 amino acid peptide from the pre-S1 region of the HBsAg (subtype adw) designed to exclude an adjacent H-2′ T-cell epitope, the latter (pre-S1) peptide being recognized by SJL (H-2′) mice as a B-cell epitope. SJL(H-2′) mice were immunized twice intramuscularly with S or pre-S1 peptide alone, co-entrapped in the same liposomes or entrapped in separate liposomes which were mixed before injection. Analysis of sera for anti-peptide IgG1 antibodies revealed that the Th-cell peptide provided help for the pre-S1 peptide only when the two peptides were co-entrapped in the same vesicles. This helper effect was found to correlate with the ability of S peptide (co-entrapped with the pre-S1) to stimulate T-cell proliferation in vitro. There was no IgGl response against pre-S1 peptide in mice immunized with a mixture of the free peptides or a mixture of separately entrapped peptides. A helper effect, albeit much weaker, was also observed in mice immunized with the two peptides emulsified in incomplete Freund's adjuvant. Antisera from mice immunized with both peptides co-entrapped in liposomes were found to bind to full length (pre-S1 containing) recombinant HBsAg. Moreover, binding values were much higher than those seen with antisera from animals immunized with the liposomal S peptide above, presumably because of full access of anti-pre-S1 antibodies to the pre-S1 region of the rHBsAg. It is concluded that liposomes could serve not only as an immunological adjuvant for peptides but also as a carrier for Th- and B-cell epitopes thus eliminating the need for covalent linkage to a carrier protein. [ABSTRACT FROM AUTHOR]

Primary immune response (IgG1) to tetanus toxoid entrapped in liposomes composed of equimolar egg phosphatidylcholine and cholesterol, and the effect of a variety of physiological and non- physiological mediators (co-entrapped with the toxoid or entrapped in separate liposomes) on such responses, were studied in BALB/c mice. Results show that (i) primary responses, not detectable with the free antigen, were elicited with the same amount of antigen given in liposomes within a range (37·4:1–2857:1) of liposomal phospholipid to toxoid mass ratios. At a higher ratio (17,804:1) response was reduced to very low levels. Immune responses obtained with liposomal toxoid were maintained at measurable levels 24 weeks after immunization. (ii) Interleukin-2 (IL-2), interferon- gamma (IFN-γ) and N-acetyl muramyl-L-threonyl-D-isoglutamine ([Thr1]MDP), but not its liposoluble 6-O-stearoyl derivative (6-O-S-[Thr1]MDP), co-entrapped with the toxoid at appropriate phospholipid to toxoid ratios, generally reduced primary response to levels below those achieved with liposomes containing the antigen alone. Further, responses obtained with 6-O-S[Thr1]MDP co- entrapped with the toxoid or separately entrapped were higher than those seen with [Thr1]MDP in similar formulations. The significance of these findings is discussed in conjunction with the structural characteristics of liposomes. [ABSTRACT FROM AUTHOR]

The effect of various manipulations of liposomes composed of equimolar phospholipid and cholesterol on immune responses to the incorporated immunopurified tetanus toxoid was investigated in BALB/c mice. In studies designed to establish proper dosage for immunization and to reveal the roles of the liposomal phospholipid to toxoid mass ratio, gel-liquid crystalline transition temperatures (Tc) of the liposomal phospholipids and mode of antigen incorporation into liposomes on immune responses, animals were injected intramuscularly with various amounts of the toxoid, free, entrapped in multilamellar dehydration-rehydration vesicles (DRY) or covalently linked to the surface of multilamellar vesicles (MLV) prepared by the classical procedure. Two identical injections separated by 4 weeks were given and IgG1 and IgG2b antibodies specific for the toxoid assayed in sera by an enzyme-linked immunosorbent assay. Results suggest that (i) the adjuvant effect of liposomes is improved considerably when liposomal phospholipid to toxoid mass ratios are as high as 2049:1; however, adjuvanticity is reduced to reach very low levels for much higher ratios (e.g. 90,361 : 1); (ii) antibody responses are similar for liposomes (phospholipid to toxoid mass ratios: 143- 331) made of a variety of phospholipids with Tcs ranging from -32° to 41.5° but are low or non- existent when liposomes are made of distearoyl phosphatidylcholine (Tc, 54°) (however, see Discussion); (ii) there are no differences in antibody responses between liposomes with entrapped and surface-linked toxoid. [ABSTRACT FROM AUTHOR]

The active component of the exoantigens of malarial parasites which stimulates macrophages to secrete tumour necrosis factor (TNF) has been shown to depend upon a phospholipid, the activity of which was blocked by phosphatidylinositol (PI) and inositol monophosphate (IMP) in competitive inhibition studies. Antisera made against the exoantigens of Plasmodium yoelii, which inhibited their induction of TNF, were found by an ELISA assay to contain antibody against several other phospholipids. However, the inhibitory antibody was removed specifically by adsorption with liposomes containing PI, but not other phospholipids. Furthermore, PI was the only phospholipid in non-liposomal form which induced thc production of inhibitory antisera. Mice immunized with IMP, but not inositol, also produced inhibitory antisera. When incorporated into liposomes several other phospholipids did give rise to inhibitory antibodies but, in contrast to the antisera against parasite exoantigcns, PI and IMP, the inhibitory activity was removed by adsorption with heteroiogous phospholipid liposomes, suggesting that it was directed against a common determinant, presumably the phosphate ester head group. Inhibitory antibodies in the antisera tested were predominantly IgM and titres were not increased after repeated injections. Antisera raised against PI, IMP or the crossreacting phospholipid liposomes also inhibited TNF secretion by macrophages stimulated by exoantigens of the human parasites P. falciparum and P. vivax, but not by bacterial lipopolysaccharide. These findings confirm our conclusion that exoantigens from these different species contain phosphate bound to inositol in their TNF-inducing moiety. [ABSTRACT FROM AUTHOR]

The effect of the ligand mannosylated albumin, covalently coupled to the surface of tetanus toxoid-containing dehydration-rehydration vesicles (DRV), on their adjuvanticity was investigated. Toxoid-containing DRV coated with mannosylated albumin bound in vitro to mouse (BALB/c) peritoneal macrophages selectively and to a greater extent than non-mannosylated DRV. ELISA assays of anti-toxoid IgG1 and IgG2b in the sera of BALB/c mice immunized with various toxoid-containing DRV preparations suggest that mannosylated liposomes are superior in immunoadjuvant action to conventional ones. Such targeted adjuvanticity appears to be influenced by the number of ligand molecules available on the surface of liposomes rather than the number of mannose residues on albumin. [ABSTRACT FROM AUTHOR]

Previously, we showed that CD206-targeted liposomal delivery of co-encapsulated immunodominant myelin basic protein (MBP) sequences MBP 46-62 , MBP 124-139 and MBP 147-170 (Xemys) suppressed experimental autoimmune encephalomyelitis in dark Agouti rats. The objective of this study was to assess the safety of Xemys in the treatment of patients with relapsing-remitting multiple sclerosis (MS) and secondary progressive MS, who failed to achieve a sustained response to first-line disease-modifying therapies. In this phase I, open-label, dose-escalating, proof-of-concept study, 20 patients with relapsing-remitting or secondary progressive MS received weekly subcutaneously injections with ascending doses of Xemys up to a total dose of 2.675 mg. Clinical examinations, including Expanded Disability Status Scale score, magnetic resonance imaging results, and serum cytokine concentrations, were assessed before the first injection and for up to 17 weeks after the final injection. Xemys was safe and well tolerated when administered for 6 weeks to a maximum single dose of 900 μg. Expanded Disability Status Scale scores and numbers of T2-weighted and new gadolinium-enhancing lesions on magnetic resonance imaging were statistically unchanged at study exit compared with baseline; nonetheless, the increase of number of active gadolinium-enhancing lesions on weeks 7 and 10 in comparison with baseline was statistically significant. During treatment, the serum concentrations of the cytokines monocyte chemoattractant protein-1, macrophage inflammatory protein-1β, and interleukin-7 decreased, whereas the level of tumor necrosis factor-α increased. These results provide evidence for the further development of Xemys as an antigen-specific, disease-modifying therapy for patients with MS.

Background: The need for lifelong, daily insulin injections can have a dramatic effect on patient compliance, can be painful, and runs the risk of local infections. Furthermore, needle-stick injuries are common, and the issue of needle disposal is troublesome. Injecting a long-acting insulin analog with needle-free administration would be a significant improvement for diabetic subjects, but is not currently feasible. To achieve a constant, reliable delivery of a novel, long-acting insulin analog, Lipoxen's SuliXen (polysialylated insulin) in a solid dosage form capable of being delivered without a needle has been developed. The aim of this study was to evaluate the feasibility of Lipoxen's SuliXen delivery with the Glide solid dose injector, Glide SDI., Materials and Methods: A formulation containing 14 kDa polysialic acid (PSA)-recombinant human insulin conjugate was manufactured at Lipoxen PLC and transferred to Glide Pharma. The PSA-insulin conjugate solution was incorporated into different excipients at Glide Pharma (excipients 1 and 2), and formulations were manufactured containing implants with doses of 0.3 and 1.0 IU of insulin, respectively. Two different polymeric excipients were investigated for their suitable release profiles. The physicomechanical properties of the formulations were characterized in terms of solid dosage form strength (via three-point bend and compression) and disintegration time at 37 degrees C. A preclinical efficacy study was performed in a nondiabetic rat model (Sprague-Dawley)., Results: The study demonstrated successful incorporation of PSA-insulin conjugate into formulations compatible for use with the solid dose injector. Physicochemical characterization indicated that each formulation produced was physically robust. For excipient 1, the compressive stress and three-point-bend-test values recorded for the 0.3 IU formulation were 106.99 +/- 14.3 MPa and 30.6 +/- 1.4 N (force in newtons), respectively. Corresponding values for the 1.0 IU dose were 53.10 +/- 10.2 MPa and 16.66 +/- 1.0 N. For excipient 2, the compressive stress and three-point-bend-test values recorded for the 0.3 IU dose were 53.10 +/- 10.2 MPa and 7.64 +/- 0.9 N, respectively, whereas the corresponding values recorded for the 1.0 IU dose were 41.61 +/- 7.4 MPa and 13.18 +/- 1.3 N. Each formulation successfully penetrated a laboratory substrate, achieving 100% penetration in each case. In vivo analysis demonstrated that PSA-insulin conjugate shows prolongation of activity (at least two-fold more compared to insulin) for more than 5 hours in the rat model., Conclusion: Even though additional work may be required, for example, to develop several fixed dose formulations, the preliminary results show that solid dosage forms incorporating PSA-insulin conjugate maintained the prolongation of PSA-insulin conjugate activity in the rat model. Convenient and easy to use, the solid dose injector will not only ensure diabetic patient compliance and trust but also provide cost-effective solutions for safe, reliable, and controlled needle-free injection of PSA-insulin conjugate., ((c) 2010 Diabetes Technology Society.)

Numerous reports have indicated that intramuscular injection of antigen-coding naked plasmid DNA can trigger humoral and cell-mediated protective immunity against infection. This follows DNA uptake by muscle fibres, leading to the expression and extracellular release of the antigen. Here it is shown for the first time that intramuscular immunization of mice with pRc/CMV HBS (encoding the S region of hepatitis B antigen; HBsAg) entrapped into positively charged (cationic) liposomes leads to greatly improved humoral and cell-mediated immunity. These cationic liposome-entrapped DNA vaccines generate titres of anti-HBsAg IgG1 antibody isotype in excess of 100-fold higher and increased levels of both IFN-gamma and IL-4 when compared with naked DNA or DNA complexed with preformed similar (cationic) liposomes. It is likely that immunization with liposome-entrapped plasmid DNA involves antigen-presenting cells locally or in the regional draining lymph nodes.

The adjuvanticity of liposomes on two different modes of presentation of polio virus subunit peptides was demonstrated by incorporating the poorly immunogenic synthetic polio peptides, W1 and W2, into the internal space of and covalently-linked to the surface of dehydration-rehydration vesicles (DRV). It was found that for both peptides, liposome association in either mode boosted the primary and secondary IgG1 responses against 5 micrograms peptide as compared to controls in which free peptide was administered. Surface-linkage of peptides (both W1 and W2) exhibited an initially more rapid rise in antibody levels, as compared to internal entrapment of the peptides, but elicited no observable secondary response. However, although encapsulated W1 showed a milder primary response when compared to the surface-linked formulation, it later elicited a strong secondary response. These results suggested that it may be advantageous to administer liposomal virus subunit vaccines in both surface-linked and internally entrapped formulations to achieve adequate initial antibody levels followed by an anamnestic response.

A novel positively charged lipid, 1,2-bis(hexadecylcycloxy)-3-trimethylaminopropane-HCl (BisHOP), when incorporated into the bilayers of phosphatidylcholine (PC) and distearoyl phosphatidylcholine (DSPC) dehydration-rehydration vesicles (DRV), was shown to have a powerful effect in enhancing the IgG1 response to tetanus toxoid encapsulated within the liposomes. The adjuvant effect was significantly greater when 20% BisHOP was incorporated as compared to 10% incorporation and to control PC and DSPC DRV. Plain, uncharged DSPC DRV were found to have a greater adjuvant effect than plain PC DRV on the entrapped tetanus toxoid after a single intramuscular injection. Even though antibody levels at 8 weeks post-injection were similar for 20% BisHOP PC and DSPC DRV, the rate of rise of antibody titres was more rapid for 20% BisHOP DSPC than for 20% BisHOP PC DRV. These results suggest that faster and higher titers of antibodies may be obtained by optimal manipulation of the charged and non-charged lipid components of liposomes.

Small unilamellar neutral, negatively and positively charged liposomes composed of egg phosphatidylcholine, various amounts of cholesterol and, when appropriate, phosphatidic acid or stearylamine and containing 6-carboxyfluorescein were injected into mice, incubated with mouse whole blood, plasma or serum or stored at 4 degrees C. Liposomal stability, i.e. the extent to which 6-carboxyfluorescein is retained by liposomes, was dependent on their cholesterol content. (1) Cholesterol-rich (egg phosphatidylcholine/cholesterol, 7:7 molar ratio) liposomes, regardless of surface charge, remained stable in the blood of intravenously injected animals for up to at least 400min. In addition, stability of cholesterol-rich liposomes was largely maintained in vitro in the presence of whole blood, plasma or serum for at least 90min. (2) Cholesterol-poor (egg phosphatidylcholine/cholesterol, 7:2 molar ratio) or cholesterol-free (egg phosphatidylcholine) liposomes lost very rapidly (at most within 2min) much of their stability after intravenous injection or upon contact with whole blood, plasma or serum. Whole blood and to some extent plasma were less detrimental to stability than was serum. (3) After intraperitoneal injection, neutral cholesterol-rich liposomes survived in the peritoneal cavity to enter the blood circulation in their intact form. Liposomes injected intramuscularly also entered the circulation, although with somewhat diminished stability. (4) Stability of neutral and negatively charged cholesterol-rich liposomes stored at 4 degrees C was maintained for several days, and by 53 days it had declined only moderately. Stored liposomes retained their unilamellar structure and their ability to remain stable in the blood after intravenous injection. (5) Control of liposomal stability by adjusting their cholesterol content may help in the design of liposomes for effective use in biological systems in vivo and in vitro.

Chronic treatment (10 i.p. injections over 20 days) of Balb/c mice with SM liposomes led to 50 and 300% enlargement of the liver and spleen respectively. No such effect was observed after similar treatment with PC liposomes. Biochemical analysis of the enlarged tissues showed no significant changes in the concentrations of glycolipid, phospholipid and certain hydrolytic enzymes. However, the increase in tissue size was paralleled by an increase in protein content. Light and electron microscopy studies of the enlarged tissues revealed an increase in the number of Kupffer cells and collections of inflammatory cells in the liver and widespread granulomatous inflammation in the spleen. We conclude that SM liposomes, although probably toxic for use as a drug carrier, may serve as a model agent in the study of tissue granulomatous inflammation.

Non-entrapped and liposome-entrapped Clostridium perfringens neuraminidase (0.5-0.6 unit) was injected into rats and its fate as well as its effect on plasma and erythrocyte N-acetylneuraminic acid was investigated. The following observations were made. (1) Although removal of both non-entrapped and liposome-entrapped neuraminidase from the circulation was completed within 5h after injection, their recovery in tissues was distinctly different; 7-10% of the injected non-entrapped enzyme was found in the liver and none in the liver lysosomal fraction or the spleen. In contrast, 20-26% of the liposome-entrapped enzyme was found in the liver of which 60-69% was in the lysosomal fraction. Spleen contained 3.6-5.0% of the enzyme. (2) The presence of the non-entrapped neuraminidase in blood led to the extensive desialylation of plasma and to a decrease in the concentration or total removal from the circulation of some of the plasma glycoproteins. (3) Injection of non-entrapped neuraminidase also led to the partial desialylation of erythrocytes the life span of which was diminished and their uptake by the liver and spleen augmented. (4) Entrapment of neuraminidase in liposomes before its injection prevented the enzyme from acting on its substrate in plasma or on the erythrocyte surface, and values obtained for plasma glycoproteins and erythrocyte survival were similar to those observed in control rats. (5) Entrapment in liposomes of therapeutic hydrolases intended for the degradation of substances stored within the tissue lysosomes of patients with storage diseases could prevent the potentially hazardous enzymic action of hydrolases in blood and at the same time direct the enzymes to the intracellular sites where they are needed.

Human blood lymphocytes were coated with increasing amounts of human kappa chain (2-85mug/10(7) cells) through the linking reagent CrCl(3). These cells were then exposed to small unilamellar liposomes composed of egg phosphatidylcholine, cholesterol and phosphatidic acid (molar proportions 7:7:1) containing carboxyfluorescein and/or (111)In-labelled bleomycin and bearing (131)I-labelled affinity chromatography-purified or non-purified anti-(kappa-chain) immunoglobulin G (IgG) [see the preceding paper, Gregoriadis, Meehan & Mah (1981) Biochem. J.200, 203-210]. In some experiments liposomes contained [(14)C]phosphatidylcholine. (1) Lymphocytes (10(7)) coated with 2-85mug of kappa chain and exposed to liposomes devoid of IgG or bearing non-purified anti-(kappa chain) IgG bound only a small proportion of the liposomal markers. Even with liposomes bearing the purified anti-(kappa chain) IgG, uptake of the labels improved only slightly for cells coated with up to 10mug of kappa chain. However, with higher concentrations of the antigen on the cell surface, binding was improved considerably to reach values of 31% ((111)In-labelled bleomycin) and 43% ((131)I-labelled IgG) of added liposomes for cells coated with 85mug of kappa chain. (2) Lymphocytes coated with kappa chain were exposed to liposomes bearing increasing amounts (0-180mug/0.9mg of egg phosphatidylcholine) of purified anti-(kappa chain) IgG. It was found that under the present conditions, binding of all three markers ((111)In-labelled bleomycin, (131)I-labelled IgG and [(14)C]phosphatidylcholine) was directly proportional to the concentration of IgG on the liposomal surface. However, uptake values remained unchanged above 90mug of IgG. (3) Antibody-mediated uptake of liposomes by cells coated with the corresponding antigen without loss of their metabolic activities may provide a method of efficient targeting.

1. A glucocerebroside beta-glucosidase-rich detergent-free preparation was obtained from human placentas by a rapid method combining affinity chromatography on concanavalin A-Sepharose and organic-solvent precipitation. In a typical preparation about 11000 units of the enzyme purified 1500-fold were obtained from five placentas in 2 days. 2. The enzyme preparation also contained other hydrolases, but the extent of their purification was much smaller. 3. Studies on entrapment in liposomes showed that all glucocerebroside beta-glucosidase activity used could be incorporated in neutral egg phosphatidylcholine-cholesterol liposomes. Association with liposomes appeared to discriminate against other proteins, including some of the hydrolases, thus contributing to further purification of the enzyme. More than 95% of the liposome-associated enzyme activity was latent.