Mucosal immunoadjuvant activity of liposomes: role of alveolar macrophages.

Previously, we have reported on a liposomal adjuvant system for stimulation of both systemic IgG and mucosal s-IgA responses against viral antigens (influenza virus subunit antigen or whole inactivated measles virus) administered intranasally to mice. Immune stimulation is observed with negatively charged, but not with zwitterionic, liposomes and is independent of a physical association of the antigen with the liposomes. Furthermore, liposome-mediated immune stimulation requires deposition of the liposomes and the antigen in the lower respiratory tract. In the present study, it is shown that alveolar macrophages (AM) are the main target cells for negatively charged liposomes administered to the lungs of mice. AM isolated from animals, to which negatively charged liposomes were administered beforehand, showed large intracellular vacuoles, suggestive of massive liposome uptake. Under ex vivo conditions, both AM and RAW 264 cells exhibited a high capacity to take up negatively charged liposomes. The deposition of negatively charged liposomes, but not zwitterionic, liposomes in the lung reduced the phagocytic and migratory behaviour of AM, as assessed on the basis of transport of carbon particles to the draining lymph nodes of the lungs. Depletion of AM in vivo with dichloromethylene diphosphonate, facilitated an enhanced systemic and local antibody response against influenza subunit antigen deposited subsequently to the lower respiratory tract. In conclusion, these data provide support for the hypothesis that uptake of negatively charged liposomes blocks the immunosuppressive activity of AM, thereby facilitating local and systemic antibody responses.     

Stimulation of liposome-induced humoral immune responses by non-ionic block polymer surfactants in Xid mice.

Non-ionic block polymers (NBPs) have proved to be potent adjuvants for the humoral immune response against liposomes haptenated with tripeptide-enlarged dinitrophenyl groups (hapten J). Since both reversed triblocks and normal octablocks displayed adjuvant activity, reversed octablocks, in which structural properties of both groups are combined, were also tested for their adjuvant activity. The latter compounds displayed very strong adjuvant activity for J-haptenated liposomes, not only in normal BALB/c but also in (CBA/N x BALB/c)F1 progeny. To test the applicability of NBPs as adjuvants in semi-synthetic vaccines, the capacity of NBPs to stimulate the immune response against liposomes haptenated with Streptococcus pneumoniae type 3 capsular polysaccharide-derived oligosaccharides was analysed. In these studies, again NBPs proved potent adjuvants, stimulating antibody production to a large extent. In male (CBA/N x BALB/c)F1 mice, which carry a X-chromosome-linked immunodeficiency (Xid), antibody levels were stimulated to the largest extent by a normal octablock. Stimulation of antibody titres, however, did not result in increased protection in these Xid mice.     

Antibody responses to liposome-associated antigen

The humoral antibody response of CAF1 mice to low doses (1-100 micrograms) of egg albumin (EA) encapsulated in or covalently bound to the surface of liposomes was studied for three routes of administration. The liposome immunoadjuvant effect observed was found to depend on the location of the antigen, either on the liposome surface or entrapped inside the liposome, and on the number of immunizations. Following a single immunization, the highest antibody titers were elicited with liposomes having EA conjugated to their surface, regardless of the route of administration. For multiple immunizations given i.v. or i.p. EA conjugated to the surface of liposomes was also superior to either free or liposome-encapsulated EA. However, the antibody response to EA bound to the surface of liposomes was not enhanced as compared to free EA following multiple subcutaneous immunizations.     

Potentiation of the humoral response of intravenous antigen by splenotropic liposomes.

We have recently described that large liposomes composed of egg phosphatidylcholine (PC), cholesterol (chol) and monosialoganglioside GM1 show elevated accumulation in the red pulp of the spleen when they are i.v. administered into mice. Up to 50% of the injected dose was found in spleen at 4 h post injection. In this report, we have investigated the potential application of such liposomes in the stimulation of anti-lysozyme response in mice. Lysozyme entrapped in the splenotropic liposomes composed of PC/chol/GM1 showed higher efficiency in potentiating the humoral response than that of either free lysozyme or lysozyme entrapped in hepatotropic liposomes composed of PC/chol. The results demonstrate that high levels of i.v. antigen delivery by liposomes to the splenic macrophage instead of the liver Kupffer cells is important in the liposomal adjuvanticity. The antibody elicited by the liposome entrapped antigen was mainly IgG1 subtype.     

Allergens entrapped in liposomes reduce allergenicity and induce immunogenicity on repeated injections in mice

Liposomes which are nontoxic, biodegradable and biocompatible lipid vesicles are known to act as adjuvants and can be used to formulate sustained release preparation by encapsulation. In the present study, allergen entrapped in liposomes were injected at different time intervals in Swiss mice (made responders to IgE by injecting cyclophosphamide) and Balb/C mice (high IgE responders). Tissue distribution studies after intraperitoneal injection of allergen (entrapped and untrapped) revealed that liposome-entrapped allergen was retained for a longer time in all the tissues except kidney as compared to the free allergen given similarly. It was observed that serum specific IgG antibody levels were higher and specific IgE levels were lower in animals given repeated injections of liposomes-entrapped allergen as compared to those animals injected free allergen in the same manner. The entrapment of allergen in liposomes somehow suppressed the specific IgE response on repeated injections. The immunomodulatory effect of liposomes may be useful in the immunotherapy of respiratory allergic disorders.     

Liposomes, lipid A, and aluminum hydroxide enhance the immune response to a synthetic malaria sporozoite antigen.

A liposome-encapsulated cloned protein (R32tet32) containing sequences from the tetrapeptide repeat region of the circumsporozoite protein of Plasmodium falciparum sporozoites was examined for immunogenicity with rabbits and monkeys. Effects of adjuvants were tested by encapsulation of the antigen in liposomes either lacking or containing lipid A and adsorption with aluminum hydroxide (ALUM). When rabbits were immunized with R32tet32 alone, a primary antibody response was not seen and a secondary response did not appear until 32 to 36 weeks after boosting. Immunization with ALUM-adsorbed R32tet32 resulted in a minimal primary antibody response. A moderate secondary antibody response was detected within 2 weeks after boosting, but antibody levels decreased to preimmunization levels 8 weeks after boosting. When R32tet32 was encapsulated in liposomes containing lipid A, strong primary and secondary antibody responses were observed. Strong primary and secondary responses also were obtained when R32tet32 was encapsulated in liposomes either containing or lacking lipid A and the liposomes were adsorbed with ALUM. The strongest antibody response was obtained by immunization with ALUM-adsorbed liposomes containing lipid A and R32tet32, suggesting that the adjuvant effects of liposomes, lipid A, and ALUM were additive or synergistic.     

Flt3 ligand enhances the immunogenicity of a gag-based HIV-1 vaccine

Liposomes and Flt3 ligand (Flt3L), a ligand for the fms-like tyrosine kinase receptor Flt3/ FLK2, can augment the immune response to an HIV peptide vaccine. The HGP-30 peptide used in these studies is a synthetic peptide that corresponds to a highly conserved region of HIV-1 p17 gag (amino acids 86-115). Mice were immunized with HGP-30 or HGP-30 conjugated to keyhole limpet hemocyanin (KLH) and delayed-type hypersensitivity (DTH) responses, antibody (IgG) amount and antigen-specific proliferative responses by spleen cells were used to monitor the immune response. Daily injections of Flt3L prior to HGP-30 administration enhanced significantly an antigen-specific lymphocyte proliferation response when compared with Flt3L, HGP-30 alone or HGP-30 containing liposomes. Intravenous administration of HGP-30 was superior to intramuscular (i.m.) immunization for the induction of DTH responses. The HGP-30/KLH containing liposomes enhanced both DTH and antibody responses, while liposomes containing HGP-30 peptide elicited only T cell responses. In these studies, either Flt3L or liposomes increased DTH responses compared with the i.m. injection of the HGP-30 vaccine alone.     

The effect of surface sugars on liposomes in immunopotentiation

The effect of surface sugars of liposomes on the immunological responses to entrapped antigen has been investigated. alpha-Mannose and beta-galactose were grafted on the surface of liposomes containing lysozyme by covalent coupling of p-aminophenyl-D-glycosides to phosphatidyl ethanolamine liposomes using glutaraldehyde. Subcutaneous administration of antigen entrapped in beta-galactose liposomes stimulated an antibody response comparable to that elicited by sugar-free neutral liposomes. However, alpha-mannose bearing liposomes with entrapped lysozyme elicited an immune response similar to that induced by lysozyme in saline. Based on these observations it is suggested that alpha-mannose liposomes, that are specifically recognized by macrophages, are taken up rapidly by receptor mediated endocytosis and that the entrapped antigen is then rapidly degraded, resulting in low antibody production.     

HIV-1-specific cell-mediated immune responses induced by DNA vaccination were enhanced by mannan-coated liposomes and inhibited by anti-interferon-gamma antibody.

The adjuvant effect of mannan-coated liposomes on human immunodeficiency virus type-1 (HIV-1) DNA vaccine and the mechanism of this enhancement were studied. Coating of cationic liposomes with mannan significantly enhanced the ability of this vaccine to induce an HIV-specific delayed-type hypersensitivity (DTH) response. HIV-specific cytotoxic T-cell (CTL) activity elicited by DNA vaccination was also significantly enhanced with the mannan-liposome cocktail. This mannan-liposome-mediated activity was greatly inhibited by in vivo injection of anti-interferon (IFN)-gamma antibody, which suggests that IFN-gamma plays an important role in this HIV-specific immune response. The results of both isotype-specific antibody and cytokine analysis revealed that mannan-liposome-mediated DNA vaccination enhances Th1-mediated immunity.     

Antibody response to polyhistidine-tagged peptide and protein antigens attached to liposomes via lipid-linked nitrilotriacetic acid in mice

Particulate delivery systems enhance antibody responses to subunit antigens. However, covalent attachment of protein antigens can disrupt protein structure and mask critical epitopes, altering the antibody response to the antigen. In this report, we evaluate noncovalent metal chelation via nitrilotriacetic acid (NTA) as a nondestructive method to attach peptide and protein antigens to liposomes. Two model antigens, ovalbumin (OVA) and a peptide derived from the membrane-proximal region of HIV-1 gp41 (N-MPR), were polyhistidinylated and attached to liposomes via monovalent NTA (mono-NTA; K(D) [equilibrium dissociation constant], ～10 μM), trivalent NTA (tris-NTA; K(D), ～1 nM), or a covalent linkage. Attachment of N-MPR, but not OVA, to liposomes via an NTA lipid elicited stronger antibody responses in BALB/c mice than a formulation in which unassociated antigen was simply admixed with control liposomes lacking NTA. However, the tris-NTA linkage did not increase antibody responses to either N-MPR or OVA compared to the level for the mono-NTA linkage, despite the greater liposomal association of the antigen. For both antigens, covalently attaching them to a lipid elicited significantly stronger antibody responses than NTA-anchored antigens (OVA titer, 3.4 × 10(6) versus 1.4 × 10(6) to 1.6 × 10(6) [P < 0.001]; N-MPR titer, 4.4 × 10(4) versus 5.5 × 10(2) to 7.6 × 10(2) [P < 0.003]). The data indicate that NTA linkages may increase antibody titers to weak antigens such as N-MPR, but NTA-mediated attachment remains inferior to covalent conjugation. Moreover, enhancements in antigen-liposome affinity do not result in increased antibody titers. Thus, additional improvements of NTA-mediated conjugation technology are necessary to achieve an effective, nondestructive method for increasing the humoral response to antigens in particulate vaccines.     

The effect of surface-coupled antigen of liposomes in immunopotentiation

The effect of surface coupled antigens of liposomes on the immunological response has been investigated. Lysozyme was covalently coupled to neutral and positively charged liposomes using glutaraldehyde. Subcutaneous administration of these preparations stimulated a significant antibody response higher than that elicited by the antigen entrapped in neutral liposomes. Immunization by liposomal antigens together with complete Freund's adjuvant resulted in strong immune responses, highest with the antigen coupled to neutral and positively charged liposomes followed by the antigen entrapped in neutral liposomes. Primary and secondary immunization with lysozyme, both entrapped and coupled to liposomes, evoked an IgG response.     

An immune response to ovalbumin covalently coupled to liposomes is prevented when the liposomes used contain doxorubicin

It is now well established that liposomes with surface associated proteins are immunogenic. Repeated administration of protein coated liposomes elicits the generation of antibodies and the elimination of proteoliposome increases markedly in animals ‘immunized' with such liposomes. This immune response compromises the therapeutic potential of liposomal formulations that rely on the use of protein- or peptide-based targeting ligands to enhance cell specificity. Strategies to suppress or inhibit such immune responses must be developed if this technology is going to prove therapeutically viable. This study evaluates whether an immune response to a protein, covalently attached to liposomes by a thioether bond between N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP)-modified-protein and N-(4-(P-maleimidophenyl)butyryl) (MPB)-activated lipids, can be suppressed when the liposomes used contain the anti-cancer drug doxorubicin. To assess this, the highly immunogenic protein ovalbumin was conjugated onto liposomes composed of distearoylphosphatidylcholine/cholesterol (DSPC/Chol) with sufficient poly(ethylene glycol)-modified distearoyl phosphatidylethanolamine (PEG-DSPE) (2 mol%) to prevent liposome aggregation during protein coupling and to engender increased circulation lifetimes. The immune response to these liposomes with and without encapsulated doxorubicin was measured by: (1) monitoring liposome elimination after 3 weekly i.v. injections in C3H/HeJ mice and (2) measuring the anti-ovalbumin antibody levels by an ELISA assay. One week after a single dose of ovalbumin-coated PEG liposomes (50 μg protein/mouse) the immune response resulted in rapid elimination of a second dose of ovalbumin-coated PEG liposomes. Rapid liposome elimination was correlated to generation of high levels (>9 μg/ml plasma) of circulating anti-ovalbumin IgG. In contrast, anti-ovalbumin antibodies were not detected when the liposomes used contained doxorubicin. Plasma elimination of these drug loaded protein coated liposomes decreased following repeated weekly i.v. doses, an effect that is consistent with liposomal doxorubicin mediated suppression of phagocytic cells in the liver.     

Production of Antibodies to Dextran Using Liposome as Adjuvant

The kinetics and nature of the immune response to dextran-BSA entrapped into liposomes was studied in rabbit. The antibody titer in the secondary immunization was found to be higher as compared to that in the primary response. In the primary response antibodies were of both IgG and IgM type but following secondary immunization the IgG level was increased. The conjugate entrapped into liposomes could establish an immunological memory for dextran. In order to eliminate the carrier effect of BSA, dextran was either entrapped or coupled to liposome and the immune response was studied in mice. The results revealed that dextran entrapped into liposomes or coupled to liposome surface induced IgM type immune response and antibody titer did not increase on secondary immunization.     

Bioactive fragment of human interleukin-1b (163–171) modulates the immune response to synthetic peptides of RESA of Plasmodium falciparum

In this study a bioactive fragment, residues 163–171 of human interleukin-1β (hu IL-1β), that mimics the functions of the whole IL-1 molecule was chemically linked to two peptide sequences of the ring erythrocyte surface antigen (RESA), an asexual blood stage antigen of Plasmodium falciparum. The immunogenicity of different formulations was studied in different haplotypes of inbred mice. The peptide-IL-1β conjugates delivered in liposomes showed the maximal antibody production. Antigen entrapped in liposomes at a dose of 5 μg showed antibody levels comparable to that of peptide conjugate in alum. The IgG subclass profile with different RESA peptides and its conjugates at various doses in liposomes induced predominantly IgG2a/2b isotypes while alum-delivered formulations showed both IgG1 and IgG2a/2b isotypes. In vitro studies of merozoite reinvasion showed varying degree (50–85%) of inhibition, maximum being with the peptide conjugates in liposomes (80–87%). Thus, this approach suggests that linking of hu IL-1β is instrumental in augmenting the immune response against otherwise poorly immunogenic synthetic peptides. Received: 13 October 1997     

The Adjuvant Effect of Liposomes in Eliciting Anti-Galactosyl Antibodies

Negatively-charged, multilamellar liposomes, covalently coupled with p-aminophenyl-β-D-galactopyranoside, elicited in rabbits a nearly equivalent anti-galactosyl immune response in both the presence and the absence of complete Freund's-adjuvant (CFA). The antibody response obtained using liposomes both as the carrier and adjuvant was found to be better than that obtained through the conventional way of using protein carrier in CFA. The results suggest an effective role of liposomes as adjuvant in the production of sugar-specific antibodies.     

The Adjuvant Effect of Liposomes in Eliciting Anti-Galactosyl Antibodies

Negatively-charged, multilamellar liposomes, covalently coupled with p-aminophenyl-β-D-galactopyranoside, elicited in rabbits a nearly equivalent anti-galactosyl immune response in both the presence and the absence of complete Freund's-adjuvant (CFA). The antibody response obtained using liposomes both as the carrier and adjuvant was found to be better than that obtained through the conventional way of using protein carrier in CFA. The results suggest an effective role of liposomes as adjuvant in the production of sugar-specific antibodies.     

The effect of liposomal charge on the neutralizing antibody response against inactivated encephalomyocarditis and Semliki Forest viruses

The primary neutralizing antibody response to encephalomyocarditis (EMC) virus and Semliki Forest virus (SFV) is enhanced by addition of either negatively or positively charged liposomes. The purified u.v. light inactivated viruses were merely mixed with liposomes and injected intraperitoneally (i.p.) into mice. In contrast, neutral liposomes were unable to enhance the primary response to these viruses. Furthermore primary immunization with inactivated SFV mixed with either neutral, positively or negatively charged liposomes was associated with an enhancement of the secondary humoral response after i.p. booster injection of mice with inactivated virus alone. But neutral liposomes seemed to be less effective in this respect than either positively or negatively charged liposomes.     

The role of macrophages in the immunoadjuvant action of liposomes: effects of elimination of splenic macrophages on the immune response against intravenously injected liposome-associated albumin antigen.

The primary antibody response to intravenously administered and liposome-associated human serum albumin (HSA) was studied in mice under conditions where no response could be detected against the non-liposome-associated form of the antigen. The positive response against the antigen, entrapped in and/or exposed on the surfaces of liposomes, thus resulted from the adjuvant action of the liposomes. In mice intravenously injected with dichloromethylene diphosphonate (C12MDP) also entrapped in liposomes, all red pulp macrophages, marginal metallophilic macrophages and marginal zone macrophages had disappeared from the spleen 2 days after administration. Twenty-two days after such a treatment red pulp macrophages and marginal metallophilic macrophages had reappeared, but marginal zone macrophages were still absent. In mice injected with liposome-associated HSA at 2 days after treatment with the C12MDP liposomes, anti-HSA responses were severely depressed, but administration of the liposome-associated antigen 22 days after C12MDP liposomes elicited a normal response. These results point to a role of splenic macrophages in the processing of liposome-associated antigens, but marginal zone macrophages, which are located close to the open ends of the white pulp capillaries and thus are the first macrophages to meet the antigens arriving in the marginal zone are not required.     

Characterization of immunogenic properties of haptenated liposomal model membranes in mice. I. Thymus independence of the antigen.

This paper describes a rather simple coupling method for tripeptide enlarged haptens to phosphatidylethanolamine (PE) and the incorporation of these conjugates into liposomal model membranes (haptenated liposomes). These haptenated liposomes evoke a hapten-specific humoral immune response in mice. The magnitude of the response as measured by the appearance of direct plaque forming cells in the spleen is dependent on the route of immunization and the dose and epitope density of the hapten-PE derivatives. It was not possible to evoke an IgG response after either primary or secondary immunization with haptenated liposomes (as measured by the production of indirect plaques or mercaptoethanol-resistant antibody). These data, in addition to the observations that mice depleted of, or deficient in thymus-derived (T) lymphocytes respond to haptenated liposomes, indicate that these haptenated liposomes are T-cell independent antigens.     

Impairment of immunogenicity by antigen presentation in liposomes made from dimyristoylphosphatidylethanolamine linked to the secretion of prostaglandins by macrophages

The induction of antibody response in syngeneic rats by the Gross virus cell surface antigen (GCSAa) was dependent on the presentation of GCSAa into liposomes made from distearoylphosphatidylcholine (DSPC). GCSAa liposomes made from dimyristoylphosphatidylethanolamine (DMPE) were nonimmunogenic, even when used as anamnestic immunogens. Spleen cells, from rats twice immunized with GCSAa-DSPC-liposomes and used to transfer the anti-GCSAa immune response into naive recipients after a tertiary immunostimulation in vitro in the presence of naive peritoneal exudate cells (PEC), responded to soluble GCSAa only after irradiation at 500 rds and to GCSAa-DMPE-liposomes only when indomethacin was added during the in vitro stimulation. The preincubation of these cells with empty DMPE liposomes or the addition of supernatant from PEC fed with DMPE liposomes abrogated the response to GCSAa-DSPC liposomes. Using a specific radioimmunoassay, prostaglandin E2 was demonstrated to be produced by PEC when fed with DMPE liposomes, and not when fed with DSPC liposomes. This prostaglandin E2 secretion by PEC induced by DMPE liposomes was inhibited by indomethacin.