Liposomes in Immunology: Further Evidence for the Adjuvant Activity of Liposomes

The immune response to HSA-phosphatidylcholine complexes administered to rabbits was not markedly enhanced when compared with the response to unmodified ESA.

It was found in earlier work that HSA entrapped in liposomes (mainly composed of phosphatidylcholine) evoked a strong immune response under conditions where no response was detected to free HSA.

The present results exclude the possibility that HSA- phosphatidylcholine complexes which may arise from liposome- encapsulated HSA may be responsible for the adjuvant activity of the liposomes.

The adjuvant activity of liposomes could also be established after administration of a liposome-associated strong antigen (BGG).     

Liposomes in Immunology: Impairment of the Adjuvant Effect of Liposomes by Incorporation of the Adjuvant Lysolecithin and the Role of Macrophages

The immune response against HSA (human serum albumin) was studied in rabbits after intravenous injection of various HSA preparations. When HSA was injected one day after, together with or coupled to lysolecithin, a late response was found in twelve out of thirteen rabbits, whereas a minority of the rabbits responded when lysolecithin was omitted.

These results confirm the adjuvant activity of lysolecithin. A rapid response starting on day 6 was found in rabbits injected with HSA entrapped in liposomes which had been composed of lecithin, phosphatidic acid and cholesterol (PPC liposomes). The response against liposome entrapped HSA was delayed for about one day when the phospholipid adjuvant lysolecithin was incorporated in the liposomes (LPPC liposomes).

Results lend support to the hypothesis that the adjuvant activity of lysolecithin and its opposite inhibition of the adjuvant activity of liposomes are mediated by the same mechanism, i.e. inhibition of enzymatic digestion in lysosomes of macrophages.     

Liposomes in Immunology: Multilamellar Phosphatidylcholine Liposomes as a Simple, Biodegradable and Harmless Adjuvant Without any Immunogenic Activity of its own

Multilamellar phosphatidylcholine liposomes were coated with the antigens human serum albumin (HSA) or bovine gamma globulin (BGG) simply by suspending the liposomes in a solution of the antigens.

Antigen coated phosphatidylcholine liposomes showed nearly the same adjuvant activity after intravenous injection as liposomes of a more complex phospholipid composition.

Since phosphatidylcholine liposomes are biodegradable, harmless, easily obtainable, have no immunogenic activity of their own and may be administered intravenously, they seem to be a promising immunoadjuvant.     

Liposomes in Immunology: Impairment of the Adjuvant Effect of Liposomes by Incorporation of the Adjuvant Lysolecithin and the Role of Macrophages

The immune response against HSA (human serum albumin) was studied in rabbits after intravenous injection of various HSA preparations. When HSA was injected one day after, together with or coupled to lysolecithin, a late response was found in twelve out of thirteen rabbits, whereas a minority of the rabbits responded when lysolecithin was omitted.

These results confirm the adjuvant activity of lysolecithin. A rapid response starting on day 6 was found in rabbits injected with HSA entrapped in liposomes which had been composed of lecithin, phosphatidic acid and cholesterol (PPC liposomes). The response against liposome entrapped HSA was delayed for about one day when the phospholipid adjuvant lysolecithin was incorporated in the liposomes (LPPC liposomes).

Results lend support to the hypothesis that the adjuvant activity of lysolecithin and its opposite inhibition of the adjuvant activity of liposomes are mediated by the same mechanism, i.e. inhibition of enzymatic digestion in lysosomes of macrophages.     

Liposomes as adjuvants with immunopurified tetanus toxoid: the immune response

Immune responses of BALB/c mice to immunopurified tetanus toxoid entrapped in dehydration-rehydration vesicles composed of equimolar egg phosphatidylcholine and cholesterol were compared to those of free toxoid. Animals were injected intramuscularly with the free or liposomal toxoid and identical injections were repeated 4 weeks and, in some experiments, 24 weeks later. Analysis of IgG1, IgG2a, IgG2b, IgG3 and IgM in the sera by an enzyme-linked immunosorbent assay suggested that adjuvanticity of liposomes is reflected in most antibody subclasses and that there is no shift in subclasses compared to the response obtained with the free antigen, thus establishing liposomes as a type I adjuvant. In other, appropriately designed experiments, the relative importance of events following the first and second injections in determining the adjuvant effect of liposomes was investigated. It was found that liposome adjuvanticity is the outcome of events following primary immunization.     

Liposomes in immunology: impairment of the adjuvant effect of liposomes by incorporation of the adjuvant lysolecithin and the role of macrophages.

The immune response against HSA (human serum albumin) was studied in rabbits after intravenous injection of various HSA preparations. When HSA was injected one day after, together with or coupled to lysolecithin, a late response was found in twelve out of thirteen rabbits, whereas a minority of the rabbits responded when lysolecithin was omitted. These results confirm the adjuvant activity of lysolecithin. A rapid response starting on day 6 was found in rabbits injected with HSA entrapped in liposomes which had been composed of lecithin, phosphatidic acid and cholesterol (PPC liposomes). The response against liposome entrapped HSA was delayed for about one day when the phospholipid adjuvant lysolecithin was incorporated in the liposomes (LPPC liposomes). Results lend support to the hypothesis that the adjuvant activity of lysolecithin and its opposite inhibition of the adjuvant activity of liposomes are mediated by the same mechanism, i.e. inhibition of enzymatic digestion in lysosomes of macrophages.     

The Secondary Immune Response Against Liposome Associated Antigens

In the present experiments, the secondary immune response against antigens is studied after priming with liposome associated antigens and booster injections with the antigen alone, in order to study the effect of liposomes on the generation of immunological memory against the associated antigens.

Liposomes show adjuvant activity with respect to both the primary and secondary immune response against associated human serum albumin (HSA). When the injected dose of liposome associated HSA was too low to elicit a primary immune response, generation of immunological memory against the antigen could not be detected. Horse radish peroxidase (HRP) associated with liposomes did not elicit a primary immune response, but immunological memory against the antigen was established.     

Liposomes in immunology: further evidence for the adjuvant activity of liposomes.

The immune response to HSA-phosphatidylcholine complexes administered to rabbits was not markedly enhanced when compared with the response to unmodified HSA. It was found in earlier work that HSA entrapped in liposomes (mainly composed of phosphatidylcholine) evoked a strong immune response under conditions where no response was detected to free HSA. The present results exclude the possibility that HSA-phosphatidylcholine complexes which may arise from liposome-encapsulated HSA may be responsible for the adjuvant activity of the liposome. The adjuvant activity of liposomes could also be established after administration of a liposome-associated strong antigen (BGG).     

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.     

Attempts to study the localization of liposomes and liposome entrapped antigen in the spleen.

Attempts were made to study the localization of intravenously injected liposomes and enclosed labelled antigens in the spleen of mice, using autoradiography. A more than hundred fold increased uptake of antigen by the spleen was obtained when liposome entrapped antigen was compared with free antigen or antigen-antibody complexes which have been used in earlier studies. No marked accumulation of the labelled antigen was found in the follicles of the spleen, although specific antibodies to the injected antigen have been injected simultaneously with the liposome entrapped antigen. It is argued that the bulk of the labelled antigen in the spleen 0.5 to 2 h after injection was still enclosed in the liposomes, whereas part of the label, arranged in patches in the red pulp and marginal zone, corresponds with labelled antigen in macrophages, released after digestion of the liposomal membranes. The present techniques did not enable the detection of the liposomes themselves.     

Liposomes as vehicle for allergen presentation in the immunotherapy of allergic diseases

Liposomes are non-toxic, biodegradable and weakly immunogenic lipid vesicles which can be used as immunomodulating agents. In the present study, multilamellar vesicles (MLV) and small unilamellar vesicles (SUV) were used to incorporate an allergenic protein from Artemisia scoparia pollen. MLV incorporated more allergenic protein than SUV. To assess the immunomodulating effect of allergen entrapped in liposomes, Swiss strain mice (made IgE responders) were injected with either free allergen or liposome-entrapped allergen (LEA) and their immune response was measured in terms of specific IgG and specific IgE levels. Results indicated that specific IgE response was significantly lower in mice injected LEA (P less than 0.02) than in mice injected free allergenic protein. Although specific IgG response was higher in mice injected LEA, there was no statistically significant difference between the two groups. Potential use of liposomes as non-immunogenic biocompatible vehicle for antigen presentation in immunotherapy will be discussed.     

Endotoxin Enhanced Adjuvant Effect of Liposomes, Particularly when Antigen and Endotoxin are Incorporated within the Same Liposome

Endotoxin has been shown to enhance the adjuvant effect of liposomes to protein antigens. When a weak antigen and endotoxin were associated with the same liposomes, antibody production could be detected 3 days (72 hours) after injection of the antigen-liposome-endotoxin preparation. When antigen and endotoxin were associated with different liposomes and these antigen-liposome and endotoxin-liposome preparations were injected simultaneously, the adjuvant effect of the liposomes was enhanced also, but not enough to detect antibody production on day 3.     

Endotoxin Enhanced Adjuvant Effect of Liposomes,Particularly when Antigen and Endotoxin are Incorporated within the Same Liposome

Endotoxin has been shown to enhance the adjuvant effect of liposomes to protein antigens. When a weak antigen and endotoxin were associated with the same liposomes, antibody production could be detected 3 days (72 hours) after injection of the antigen-liposome-endotoxin preparation. When antigen and endotoxin were associated with different liposomes and these antigen-liposome and endotoxin-liposome preparations were injected simultaneously, the adjuvant effect of the liposomes was enhanced also, but not enough to detect antibody production on day 3.     

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.     

Liposomes as immunological adjuvants in eliciting antibodies specific to the synthetic polypeptide poly(LTyr,LGlu)-poly(DLAla)–poly(LLys) with high frequency of site-associated idiotypic determinants

The antibody response to the synthetic polypeptide, poly(LTyr, LGlu)-poly(DLAla)–poly(LLys), [(T, G)-A–L], injected entrapped in liposomes which served as adjuvant, has been analyzed. The liposomes used were composed of phosphatidylcholine, cholesterol, dicetylphosphate and DL α-tocopherol (molar ratios as 4:3:0.1:0.5) and therefore, were negatively charged. Since the (T, G)-A–L is also negatively charged, no free complexes were formed. The (T, G)-A–L was found to be entrapped inside the enclosed volume of the liposomes, and no (T, G)-A–L antigenic determinants could be detected on the liposomal membranes. Injection of high-responder C3H.SW (H-2^b) mice with (T, G)-A–L-bearing liposomes demonstrated that the i.p. and the i.v. routes of immunization were efficient in eliciting (T, G)-A–L-specific antibodies, whereas the i.d. injection led to poor antibody responses. The latter route of immunization is the most effective when (T, G)-A–L is injected in complete Freund's adjuvant (CFA). When low doses (0.1 and 1 μg) of (T, G)-A–L were used for immunization, the liposomes were better adjuvants than CFA. The effectiveness of the liposomes as immunological adjuvants was also shown in their ability to induce high-potential, primed memory cells. The pattern of low (H-2^{k, a}) and high (H-2^b) responsiveness to (T, G)-A–L was retained following immunization with (T, G)-A–L entrapped in liposomes, as tested in two pairs of congenic strains. (T, G)-A–L-specific antibodies induced by injection with 1μ antigen entrapped in liposomes bear the (T, G)-A–L site-related idiotypic markers of C3H.SW (Igh-1^a) mice in a significantly higher frequency than the homologous idiotypes, namely the antibodies elicited in this strain against (T, G)-A–L in CFA. Thus, liposomes may serve as adjuvants for the production of relatively restricted (T, G)-A–L-specific antibodies of high qualitiy.     

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.     

Immunogenicity of hapten-carrier conjugates associated with liposomes

Immunogenicity of liposome-entrapped hapten-carrier conjugates was studied in guinea pigs. Animals immunized intravenously with a subimmunogenic dose of a hapten-carrier complex entrapped in liposomes exhibited cutaneous anaphylactic reactions to the hapten, even when the liposomes were treated with trypsin, but not delayed hypersensitivity (DH) reactions to the carrier. Animals immunized with a mixture of empty liposomes and antigen showed anaphylactic reactions to the hapten that appeared to be elicited by antigen adsorbed on the outer membrane of liposomes. The induction of anaphylaxis to the hapten by liposome-associated antigen was not due to an adjuvant property of liposomes. DH reactions to the carrier after injection of a liposome-entrapped subimmunogenic dose of a hapten-carrier conjugate could be observed only after activation of macrophages by Bacillus Calmette-Guérin.     

Humoral and cellular immunity to hepatitis B virus-derived antigens: comparative activity of Freund complete adjuvant alum, and liposomes.

Complete Freud adjuvant, aluminum gel, and liposomes were compared for their ability to enhance the immunogenicity of an intact 22-nm HBsAg particle vaccine and an HBsAg-derived polypeptide vaccine in guinea pigs. Both humoral and cell-mediated immune responses were evaluated. The greatest immune response was obtained with complete Freund adjuvant, regardless of the antigen preparation. Aluminum gel appeared to be a better adjuvant for 22-nm HBsAg particles, but the liposomes rendered polypeptide preparations more immunogenic. The possibility that various proportions were entrapped in aqueous compartments instead of being inserted into the lipid bilayers of liposomes might account for this difference. The development of both humoral and cellular immunity was dependent upon the use of an adjuvant, because aqueous preparations had poor immunogenicity.     

Liposomes as carriers for allergy immunotherapy

With the aim of obtaining an efficient but safer vaccine for allergy immunotherapy, the possibility of using liposomes as adjuvants has been considered given their proven low toxicity, adjuvant properties and ability to maintain the encapsulated substance in their interior for some time in vivo. Different methods of encapsulating allergenic extracts into neutral, positively, and negatively charged DPPC: cholesterol liposomes have been investigated and the immune response provoked by these in mice was studied and compared to the immune response to free allergen or other adjuvants such as aluminium hydroxide. The results obtained show that allergen encapsulated in all three types of liposomes elicit an increase in specific IgG levels higher than that provoked by free allergen, however, both encapsulation efficiency and specific IgG titre were higher when positively charged (DPPC: cholesterol stearylamine) liposomes were used. Specific IgE levels to allergen in positive and neutral liposomes was lower than to allergen in negative liposomes or adsorbed to Al(OH)3. No difference were found in the behaviour of liposomes prepared by different methods (the results were obtained from pooled sera from different groups of mice so there is no statistical data). These results confirm the immunoadjuvant effect of liposomes for allergy immunotherapy. Further studies to determine their lack of toxicity, pharmacokinetic studies and human clinical studies are necessary to confirm their adequacy for human use and advantage over current immunotherapy methods.