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: 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).     

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: The Immune Response Against Antigen-Containing Liposomes

Three ways of antigen administration to rabbits were compared with respect to the resulting immune responses.

Antigen was administered free in solution, entrapped in liposomes or free in solution but together with empty liposomes.

Liposomal material showed adjuvant activity when injected together with free antigen, but a much stronger adjuvant effect was found when antigen was injected entrapped in 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.     

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.     

Preparation of anti-polysaccharide antiserum using liposomes as immunological adjuvant

Bovine serum albumin (BSA) was coupled to dextran by controlled periodate oxidation followed by sodium borohydride reduction. The conjugate was entrapped in negatively charged, multilamellar phosphatidylcholine (lecithin) liposomes in which the polysaccharide remained surface-exposed, at least partially. Liposome-entrapped conjugate elicited in rabbits an appreciable anti-dextran response when compared with the sera raised in saline or in Freund's adjuvant. The anti-dextran antibodies belonged to both the IgM and IgG classes. The precipitin reaction of dextran with the antiserum raised in liposomes was determined.     

A specific enzyme-linked immunosorbent assay for definition of the IgG antibody response to disulphide-conjugated D-penicillamine in the rabbit

An enzyme-linked immunosorbent assay (ELISA) has been developed for unambiguous detection of antibodies against the sulphydryl drug D-penicillamine (PA) and its disulphide-conjugated metabolites. Disulphide-linked PA human serum albumin (PA-HSA) conjugates for use as coating antigens were prepared by a range of procedures employing oxidation with either potassium ferricyanide (0.1 M) or cupric sulphate (5 ppm). A satisfactory degree of conjugation was achieved by both oxidative procedures. Hapten density and antigenicity were increased when urea-denatured rather than non-denatured HSA was used. In 2 out of 3 rabbits, a specific IgG anti-PA response was detected following monthly injection of PA keyhole limpet haemocyanin (PA-KLH) in Freund's complete adjuvant. In the third rabbit, any anti-PA activity was obscured by a high level of binding to HSA. The anti-PA response was slow to develop in the 2 responder rabbits (requiring four injections) and was of low intensity (antibody titres less than 6,000). In contrast, the IgG antibody response to the structurally related drug captopril (CP), administered under identical conditions, was rapid in onset and of greater intensity (titres greater than 6,000 after one injection of CP-KLH). The hapten specificity of the IgG anti-PA-HSA antisera was defined by ELISA inhibition assays. Binding of IgG to PA-HSA was inhibited by PA, PA disulphide, PA cysteine and disulphide-linked PA-HSA conjugates, but not by PA acetone (thiazolidine ring-linked PA), CP, or unconjugated HSA. The inhibitory preparations were inactive in unrelated ELISAs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Water-soluble adjuvant obtained from Bacterionema matruchotii.

The adjuvant effect of a butanol-extracted water-soluble adjuvant (bu-WSA) obtained from Bacterionemia matruchotii, a gram-positive oral bacteria, was studied on the antibody response at the plaque-forming cell (PFC) level in murine spleens. Intraperitoneal injection of Bu-WSA caused significant increase in direct PFC numbers in spleens 1 to 3 days after the antigenic stimulation with sheep erythrocytes (SRBC). Injection of 100 to 800 microgram of Bu-WSA was effective, and 400 microgram of Bu-WSA seemed to be the optimum for induction of the adjuvant effect. The adjuvant effect was strongest when Bu-WSA was injected at the same time as the SRBC, but some effect was still observed when Bu-WSA was injected 7 days before or 1 day after the immunization. The adjuvant effect of Bu-WSA was greatest at high dose of antigen. The mice injected with Bu-WSA at the time of priming SRBC and then immunized with trinitrophenylated SRBC showed greater anti-trinitrophenyl PFC response than controls without the injection of Bu-WSA. These findings suggest that a part of the adjuvant effect of Bu-WSA depends on thymic cell function and another part does not.     

A novel positively-charged lipid 1,2-bis(hexadecylcycloxy)-3-trimethyl aminopropane (BisHOP) enhances the adjuvant effect of liposomes on encapsulated tetanus toxoid.

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.     

Enhanced mucosal priming by cholera toxin and procholeragenoid with a lipoidal amine adjuvant (avridine) delivered in liposomes

The mucosal adjuvant activity of avridine, a synthetic lipoidal amine [N,N-dioctadecyl-N',N'-(2-hydroxymethyl) propanediamine, previously designated CP-20,961), was studied in rats immunized intraintestinally with cholera toxin or procholeragenoid. Avridine was most efficient as an adjuvant when incorporated into liposomes; liposomes that lacked avridine had no adjuvant effect. Coadministration of avridine-containing liposomes with enteric priming doses of cholera toxin or procholeragenoid enhanced the efficiency of priming for secondary mucosal anti-cholera toxin responses, i.e., the establishment of memory, five- to sevenfold. Avridine-containing liposomes had no significant effect, however, on either the primary mucosal anti-cholera toxin response, when given with the primary dose of antigen, or on the secondary response, when given with the booster dose to previously primed animals. Little or no adjuvant effect occurred when avridine-containing liposomes were given concurrently with antigen, but at a separate mucosal site or parenterally, or at the site of enteric immunization, but 1 day earlier or later. These results support the notion that adjuvants may be developed which enhance the mucosal immunogenicity of locally applied antigens and suggest that liposomes may be effective vehicles for delivery of such adjuvants.     

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.     

家兔佐剂性关节炎致疼痛前后听觉P300的变化

目的：探讨家兔佐剂性关节炎致疼痛前后听觉事件相关电位P300成分的变化。方法：分别记录日本大耳白兔注射弗氏佐剂前,注射弗氏佐剂后第7天、第21天用吗啡止痛前后的听觉P300和痛阈。结果：家兔注射佐剂后第7天和第21天痛阈下降,听觉P300潜伏期均较正常时明显延长;运用吗啡止痛治疗后,痛阈上升,潜伏期明显缩短。注射佐剂后第7天P300波幅较正常时明显降低;运用吗啡止痛治疗后,波幅明显增高。注射佐剂后第21天波幅较第7天明显增高。结论：佐剂性关节炎兔的听觉P300与疼痛程度密切相关。     

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.     

Synthetic low-toxicity muramyl dipeptide and monophosphoryl lipid A replace Freund complete adjuvant in inducing growth-inhibitory antibodies to the Plasmodium falciparum major merozoite surface protein, gp195.

The Plasmodium falciparum major merozoite surface protein (gp195) is a protective antigen against lethal malaria. However, increasing evidence indicates that the efficacy of a malaria vaccine will require a strong adjuvant that is safe for human use. We compared the efficacies of two low-toxicity synthetic immunomodulators, B30-MDP (a lipophilic muramyl dipeptide derivative) and LA-15-PH (a synthetic equivalent of monophosphoryl lipid A), with that of Freund complete adjuvant (FCA) in eliciting an antibody response to gp195. Rabbits were immunized with native gp195 and B30-MDP, LA-15-PH, or the two in combination, with liposomes as the vehicle. Aluminum hydroxide and FCA were used as reference adjuvants. Results showed that adjuvant formulations based on B30-MDP alone or in combination with LA-15-PH induced high antibody titers to gp195, as compared with FCA. LA-15-PH alone was less effective. Aluminum hydroxide induced significantly lower antibody titers. The functional activity of the rabbit anti-gp195 antibodies induced by different adjuvants was evaluated in an in vitro parasite growth inhibition assay previously shown to correlate with anti-gp195 immunity in the Aotus monkey model. All rabbits immunized with B30-MDP-LA-15-PH and two of three rabbits immunized with B30-MDP alone produced sera that strongly inhibited parasite growth. The degree of growth inhibition was similar to that with FCA. The antibody titers of the rabbits receiving B30-MDP-LA-15-PH strongly correlated with the degree of in vitro growth inhibition. Our findings provided strong evidence that adjuvant formulations based on synthetic B30-MDP and LA-15-PH can replace FCA as adjuvants in stimulating protective immunity specific for gp195.     

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.     

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.     

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.     

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.