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: 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: 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.     

Cellular immune response to the antigen administered as an immune complex in vivo.

Recently, it was shown that 10(2)- to 10(3)-fold lower doses of human serum albumin (HSA) are sufficient for the same T-cell response in vitro, if HSA is administered to the cultures bound in the immune complex rather than in the soluble form. In the present study, we analysed the capacity of HSA in the form of immune complexes to elicit specific cellular immune response in vivo. We found that antigen bound in the immune complex with murine, syngeneic polyclonal antibodies elicited the same T-cell response as fivefold higher doses of free antigen. On the other hand, HSA bound in the immune complexes with xenogeneic, rabbit polyclonal antibodies did not enhance anti-HSA cellular immune response. Our results indicate that binding of antigen in the immune complex could play an important role in enhancing an antigen-specific cellular immune response in vivo.     

Humoral Immune Response to the Antigen Administered as an Immune Complex

Antigen (HSA) bound in immune complexes at equivalence with syngeneic anti-HSA antibodies elicit much stronger humoral immune response then soluble HSA. On the other hand, administration of immune complexes formed with xenogeneic (rabbit) anti-HSA antibodies suppressed humoral immune response against HSA, but not against rabbit IgG in mice. We suggest that immunization with antigen bound in immune complex might represent a powerful tool in enhancing humoral immune responses.     

Cellular immune response to the antigen administered as an immune complex.

Immune complexes are specifically bound to the Fc receptors on the surface of various types of cells capable of presenting antigens. It was therefore determined if human serum albumin (HSA), bound in an immune complex, is presented more efficiently to the HSA-specific T cells than HSA alone. Primed, polyclonal murine T cells were stimulated in vitro with either HSA alone or HSA bound at equivalence to syngeneic, polyclonal anti-HSA antibodies of IgG class. The stoichiometric composition of the insoluble immune complex was AgAb2.91. The present study shows that 10(2)-10(3)-fold lower doses of HSA are sufficient for the same T-cell response in vitro, if HSA is administered to the cultures in the form of the immune complex rather than in the soluble form. The enhancement of T-cell proliferation in the presence of immune complex was blocked with monoclonal antibody (mAb) against Fc receptor. Our results indicate that binding of antigen in the immune complex could play an important role in enhancing an antigen-specific cellular immune response.     

pH-sensitive polymer-liposome-based antigen delivery systems potentiated with interferon-γ gene lipoplex for efficient cancer immunotherapy

Potentiation of pH-sensitive liposome-based antigen carriers with IFN-γ gene lipoplexes was attempted to achieve efficient induction of tumor-specific immunity. 3-Methylglutarylated poly(glycidol) (MGluPG)-modified liposomes and cationic liposomes were used, respectively, for the delivery of antigenic protein ovalbumin (OVA) and IFN-γ-encoding plasmid DNA (pDNA). The MGluPG-modified liposomes and the cationic liposome-pDNA complexes (lipoplexes) formed hybrid complexes via electrostatic interactions after their mixing in aqueous solutions. The hybrid complexes co-delivered OVA and IFN-γ-encoding pDNA into DC2.4 cells, a murine dendritic cell line, as was the case of MGluPG-modified liposomes for OVA or the lipoplexes for pDNA. Both the lipoplexes and the hybrid complexes transfected DC2.4 cells and induced IFN-γ protein production, but transfection activities of the hybrid complexes were lower than those of the parent lipoplexes. Subcutaneous administration of hybrid complexes to mice bearing E.G7-OVA tumor reduced tumor volumes, which might result from the induction of OVA-specific cytotoxic T lymphocytes (CTLs). However, the hybrid complex-induced antitumor effect was the same level of the MGluPG-modified liposome-mediated antitumor immunity. In contrast, an extremely strong antitumor immune response was derived when these liposomes and lipoplexes without complexation were injected subcutaneously at the same site of tumor-bearing mice. Immunohistochemical analysis of tumor sections revealed that immunization through the liposome–lipoplex combination promoted the infiltration of CTLs to tumors at an early stage of treatment compared with liposomes, resulting in strong therapeutic effects.     

Immunogenicity and safety of liposome-vaccine encapsulating hepatitis B surface antigen and phosphodiester CpG oligodeoxynucleotides

CpG oligodeoxynucleotides (CpG ODN) as adjuvant have been extensively studied in recent years. Phosphodiester CpG ODN (PO CpG ODN) can perfectly mimic bacterial DNA in enhancing immune response but are vulnerable to nucleases in vivo . This study aimed to evaluate the immunostimulatory potential and safety of phosphodiester CpG ODN encapsulated in nonphospholipid liposomes. BALB/c mice were immunized intramuscularly with different formulations of liposomes,CpG ODN and hepatitis B surface antigen (HBsAg). The results demonstrated that the encapsulated PO CpG ODN were protected against rapid degradation in vivo and retained their adjuvant activity. PO CpG ODN encapsulated with HBsAg in liposomes induced strong Th1-biased or Th1/Th2 mixed humoral immune response in mice with the magnitude similar to their phosphothioate equivalent in the same formulation. High IFN-gamma production induced by this formulation confirmed the generation of strong cellular immune response. Additionally, co-delivery of HBsAg and PO CpG ODN improved the immune response over that obtained with separate delivery. Safety experiment showed that liposome-encapsulaed PO CpG ODN and HBsAg caused mild systemic and moderate local adverse reaction. In conclusion, our data shows that PO CpG ODN encapsulated in liposomes fully exhibit their Th1-type adjuvant activity and act as a potential adjuvant for vaccines.     

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.     

The development of specific antibody-containing cells and the localization of extracellular antibody in the follicles of the spleen of rabbits after administration of free or liposome-associated albumin antigen

In order to study the localization pattern of specific antibody-containing cells and extracellular antibody in the spleen during a primary immune response, the antigen human serum albumin (HSA) was injected intravenously in rabbits, either free in solution, or associated with the surfaces of liposomes as a model of membrane-associated antigens. Demonstration of specific antibody-containing cells was performed by incubation of sections of spleen with HSA-HRP conjugates, followed by peroxidase cytochemistry. Specific anti-HSA antibody-containing cells were detected already at 4 days after injection of the antigen. The bulk of these cells localized initially in the outer parts of the periarteriolar lymphocyte sheaths (PALS) and around the terminal arteriolar branches. Both extracellular antibody and specific antibody-containing cells were also found in the follicles of the spleen. Arguments are given that extracellular antibody precedes the development of specific antibody-containing cells in the follicle. This extracellular antibody probably represents antigen-antibody complexes trapped in the follicles as soon as the antigen in the circulation is complexed by the first antibodies produced during the immune response. The localization pattern of specific antibody-containing cells and extracellular antibody did not differ markedly when rabbits injected with free or liposome-associated antigen were compared. Results are discussed particularly with respect to the role of follicles in the immune response.     

The production of polyclonal and monoclonal antibodies in mice using novel immunization methods

Two novel immunization methods (intrasplenic and intra-inguinal lymph node) have been developed for the production of polyclonal and monoclonal antibodies in mice. Freund's complete adjuvant and antigen were mixed in the ratio of 1 : 2 (v/v). Various concentrations of human serum albumin (HSA) were used as antigen. No primary immune response was induced with 0.1 μg of HSA in either of the methods studied. Intrasplenic immunization resulted in the strongest primary immune responses using all other doses of HSA. The primary immune response induced by intrasplenic immunization with 0.5 μg of HSA was higher than any response induced by subcutaneous immunization with various doses of HSA. Inguinal lymph node immunization was less effective than intrasplenic immunization but better than subcutaneous immunization with 1–50 μg of HSA. Comparisons were also made of the efficacy of different adjuvants when inducing primary immune responses with 1 μg of HSA. Freund's complete adjuvant resulted in a much stronger response than Freund's incomplete adjuvant and alum. Both intrasplenic and inguinal lymph node immunization using 1–5 μg of HSA were able to induce strong primary immune responses. Secondary immunization with either method or intravenous injection 3 days before fusion resulted in a higher frequency of specific monoclonal antibodies.     

Immunomodulating properties of substances to be used in combination with liposomes

Liposomes haptenated with tripeptide-enlarged dinitrophenyl (DNP) are known to act as thymus-independent antigens which induce a strong IgM response and only limited amounts of circulating IgG. When haptenated liposomes are used in vaccination studies, it is of practical importance to improve the immunogenicity of these complexes. Therefore, an evaluation was made of the potency of various substances to modulate the immune response in such a way that the total antibody production is increased, including a relative great increase of 2-mercaptoethanol (2-ME)-resistant antibodies and immunological memory is induced. The following substances were used: glycophorin A (GP-A), sialogangliosides (monosialo-, disialo- and trisialoganglioside), 6-0-stearoyl-MDP (MDP-SA) and lipid A (lip A). Lip A incorporated into liposomes was the only substance inducing considerable increases of both total and 2-ME-resistant haemagglutination (HA) titre after immunization. Depending on the dose tested, the sialic-acid-containing protein GP-A had a small and varying influence on the serum antibody response. Sialogangliosides transiently decreased in a dose-dependent manner the total antibody titre in serum. In contrast to lip A, the lipophilic bacterial adjuvant MDP-SA did not influence HA titres significantly. The number of plaque-forming cells (PFC) in the spleen was enhanced considerably after both primary and secondary immunization with liposomes containing lip A. The other substances tested induced only minor differences of the number of PFC. To some extent, lip A induced immunological memory. In conclusion, it can be stated that of the agents tested, only lip A is a potent and consistent stimulator of the humoral immune response to liposomes haptenated with DNP groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Surface charge distribution is a determinant of antigen deposition in the renal glomerulus: studies employing ''charge-hybrid'' molecules.

The deposition of antigens and immune complexes (IC) in the renal glomerulus is charge-dependent. The demonstration that molecules of net anionic charge, but with discrete positively charged regions, exhibit affinity for the glomerular basement membrane (GBM) extends this concept. Charge hybrid (polar) molecules were constructed by covalently coupling small polycations (lysozyme or linear poly-L-lysine chains with a mean of 17 and 20 residues) to larger polyanions (ovalbumin or human serum albumin (HSA]. Although the products were of overall net anionic charge they still bound to glomerular structures. Immunofluorescence studies performed after i.v. injection of the samples into rats revealed that HSA:poly-L-lysine had the highest affinity. Radioisotopic measurements showed uptake of HSA:poly-L-lysine to be a function of the number of lysine residues; binding of HSA:poly-L-lysine20 was 2.5 times higher than HSA:poly-L-lysine17 (P less than 0.01). Prior injection of a small competing polycation (polyethyleneimine 1200) reduced uptake of HSA:poly-L-lysine by 75%, indicating the charge-based nature of the interaction. HSA:poly-L-lysine20 alone was effectively eliminated from the glomeruli within 72 h. Administration of HSA:poly-L-lysine followed by anti-HSA antibody induced immune complex formation in the capillary wall, giving rise to a granular immunofluorescence pattern and discrete subendothelial and subepithelial deposits. Molecules with polar structure do occur naturally and may contribute to immune complex formation in glomerulonephritis.     

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.     

The development of specific antibody-containing cells in the spleen of rabbits during the secondary immune response against free or liposome-associated albumin antigen

In order to study the distribution pattern of specific antibody-containing cells in the spleen of rabbits during the secondary immune response, rabbits were given two intravenous injections of either free or liposome-associated human serum albumin (HSA) within an interval of 2 months. Demonstration of specific antibody-containing cells was performed by incubation of sections of spleen with HSA-horseradish peroxidase (HRP) conjugates, followed by peroxidase cytochemistry. Specific anti-HSA antibody-containing cells were detected already within 2 days after booster and peak numbers were found 4 days after booster. The bulk of these cells localized in the coaxial lymphocyte sheaths surrounding the terminal arterioles in the spleen. Specific antibody-containing cells were also found in the follicles. Using a double immunoenzyme technique we demonstrated that a majority of the specific antibody-containing cells produced immunoglobulin G(IgG) antibodies. From the results, it is also concluded that, after a priming injection with liposome-associated HSA, liposomes do not further enhance the secondary immune response, when they are also used for the booster injection.     

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.     

Antigen feeding modifies the course of antigen-induced immune complex disease.

Low affinity mice, prone to chronic immune complex disease (ICD) induced by daily injection of antigen, were fed 0.05% HSA in their drinking water for 7 days before the start of daily injections of HSA. Antigen feeding resulted in a marked decrease in the incidence of ICD despite the presence of high levels of circulating immune complexes. These complexes, which persisted in the circulation for long periods, were of low molecular weight and did not localize in the glomeruli. Antigen fed mice had lower levels of free antibody in their sera compared with control mice which may have favoured the formation of small latticed complexes in antigen excess. Antibody affinity was not apparently affected by prior feeding with antigen.     

Binding parameters of defined immune complexes to rat basophilic leukaemia (RBL) cells.

The binding parameters of chemically-defined immune complexes composed of rat IgGa-anti-human serum albumin (HSA) to rat basophilic leukaemia cells were analysed. It was demonstrated that the uptake of various sized immune complexes is time-, temperature- and pH-dependent. A higher binding rate was observed with more Fc portions available in the immune complex. A comparison of the binding rate for immune complexes with that of heat aggregates shows that immune complexes bind with greater affinity to the cells.