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.     

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.     

Immune response mediated by liposome-associated protein antigens. II. Comparison of the effectiveness of vesicle-entrapped and surface-associated antigen in immunopotentiation.

The immunogenicity of different preparations of liposome-associated bovine serum albumin (LSM-BSA) was compared in order to examine the significance of the site of association between a protein antigen and the liposomal carrier. Animals immunized with liposomes containing both entrapped and surface-adsorbed BSA. (LSM-BSA) e + a, were found to give a BSA-specific plaque-forming cell (PFC) response comparable with that elicited by animals immunized with trypsinized (LSM-BSA) e + a which contained mainly entrapped BSA. In contrast, the PFC response elicited by animals immunized with liposomes containing BSA adsorbed on the membrane surface, (LSM-BSA)a, was significantly less than that of the above two groups. Finally, animals injected with trypsinized (LSM-BSA)a only elicited a marginally detectable PFC response. Results of this study demonstrate that liposomes can enhance the antibody response of a protein antigen regardless of whether the antigen is entrapped within the vesicle or absorbed on the outer surface. Entrapped antigen, however, proved to be more immunogenic than that which was surface-adsorbed.     

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.     

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.     

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.     

Archaeosome vaccine adjuvants induce strong humoral, cell-mediated, and memory responses: comparison to conventional liposomes and alum

Ether glycerolipids extracted from various archaeobacteria were formulated into liposomes (archaeosomes) possessing strong adjuvant properties. Mice of varying genetic backgrounds, immunized by different parenteral routes with bovine serum albumin (BSA) entrapped in archaeosomes ( approximately 200-nm vesicles), demonstrated markedly enhanced serum anti-BSA antibody titers. These titers were often comparable to those achieved with Freund's adjuvant and considerably more than those with alum or conventional liposomes (phosphatidylcholine-phosphatidylglycerol-cholesterol, 1. 8:0.2:1.5 molar ratio). Furthermore, antigen-specific immunoglobulin G1 (IgG1), IgG2a, and IgG2b isotype antibodies were all induced. Association of BSA with the lipid vesicles was required for induction of a strong response, and >80% of the protein was internalized within most archaeosome types, suggesting efficient release of antigen in vivo. Encapsulation of ovalbumin and hen egg lysozyme within archaeosomes showed similar immune responses. Antigen-archaeosome immunizations also induced a strong cell-mediated immune response: antigen-dependent proliferation and substantial production of cytokines gamma interferon (Th1) and interleukin-4 (IL-4) (Th2) by spleen cells in vitro. In contrast, conventional liposomes induced little cell-mediated immunity, whereas alum stimulated only an IL-4 response. In contrast to alum and Freund's adjuvant, archaeosomes composed of Thermoplasma acidophilum lipids evoked a dramatic memory antibody response to the encapsulated protein (at approximately 300 days) after only two initial immunizations (days 0 and 14). This correlated with increased antigen-specific cell cycling of CD4(+) T cells: increase in synthetic (S) and mitotic (G(2)/M) and decrease in resting (G(1)) phases. Thus, archaeosomes may be potent vaccine carriers capable of facilitating strong primary and memory humoral, and cell-mediated immune responses to the entrapped antigen.     

Primary immune response to liposomal tetanus toxoid in mice: the effect of mediators.

Primary immune response (IgG1) to tetanus toxoid entrapped in liposomes composed of equimolar egg phosphatidylcholine and cholesterol, and the effect of a variety of physiological and non-physiological mediators (co-entrapped with the toxoid or entrapped in separate liposomes) on such responses, were studied in BALB/c mice. Results show that (i) primary responses, not detectable with the free antigen, were elicited with the same amount of antigen given in liposomes within a range (37.4:1-2857:1) of liposomal phospholipid to toxoid mass ratios. At a higher ratio (17,804:1) response was reduced to very low levels. Immune responses obtained with liposomal toxoid were maintained at measurable levels 24 weeks after immunization. (ii) Interleukin-2 (IL-2), interferon-gamma (IFN-gamma) and N-acetyl muramyl-L-threonyl-D-isoglutamine ([Thr1]MDP), but not its liposoluble 6-O-stearoyl derivative (6-O-S-[Thr1]MDP), co-entrapped with the toxoid at appropriate phospholipid to toxoid ratios, generally reduced primary response to levels below those achieved with liposomes containing the antigen alone. Further, responses obtained with 6-O-S[Thr1]MDP coentrapped with the toxoid or separately entrapped were higher than those seen with [Thr1]MDP in similar formulations. The significance of these findings is discussed in conjunction with the structural characteristics of liposomes.     

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.     

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 pH-sensitive polymer-modified liposomes with cationic lipid inclusion as antigen delivery carriers for cancer immunotherapy

Cationic lipid-incorporated liposomes modified with pH-sensitive polymers were prepared by introducing 3, 5-didodecyloxybenzamidine as a cationic lipid to egg yolk phosphatidylcholine liposomes modified with 3-methylglutarylated hyperbranched poly(glycidol) (MGlu-HPG) as a pH-sensitive polymer. These liposomes were stable at neutral pH, but were destabilized below pH 6.0 because MGlu-HPG changed its characteristics from hydrophilic to hydrophobic in response to the pH decrease. Cationic lipid inclusion improved their pH sensitivity at weakly acidic pH and association of liposomes with murine dendritic cell (DC) lines. Cationic lipid-incorporated liposomes delivered entrapped ovalbumin (OVA) molecules not only to cytosol but also to endosome/lysosome. Treatment with cationic lipid-incorporated liposomes induced up-regulation of antigen presentation-involved molecules on DCs, the promotion of cytokine production, and antigen presentation via both major histocompatibility complex (MHC) class I and II molecules. Especially, antigen presentation via MHC class II was promoted by cationic lipid inclusion, which might correspond to efficient endosome/lysosome delivery of OVA. Subcutaneous administration of OVA-loaded cationic lipid-incorporated liposomes induced antigen-specific antibody production in serum and Th1-dominant immune responses in the spleen. Furthermore, administration of the cationic lipid-incorporated liposomes to mice bearing E.G7-OVA tumor more significantly reduced the tumor volume than liposomes without cationic lipids. Therefore, cationic lipid inclusion into pH-sensitive polymer-modified liposomes, which can achieve both efficient antigen intracellular delivery and activation of antigen presenting cell, is an effective approach to develop antigen carriers for efficient cancer immunotherapy.     

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.     

Memory antibody response from antigen loaded polymer particles and the effect of antigen release kinetics

Memory antibody response is the hallmark of long lasting immunity. In this study, we report the generation of memory antibody response while immunizing with single dose of polymer particle entrapped antigens. Immunization with admixture of alum and polylactide (PLA) polymer particles (2–8 μm) entrapping antigens not only elicited long lasting primary antibody response but also very high levels of memory antibody titer upon re-exposure to very small amount of soluble antigen. In the case of tetanus toxoid (TT), the memory antibody titers from PLA particle based immunization were almost four times higher than that achieved from two doses of alum adsorbed antigen and sustained at a higher level for a longer period of time. Memory antibody response was detected even after challenging the animals after 18 months of primary immunization. Similar enhanced memory antibody response was also observed in the case of immunization with PLA particle entrapping diphtheria toxoid (DT). Memory antibody response generated from polymeric formulations was highly antigen specific. Polymer particles with different release profile of antigen were used as a model system to evaluate the role of antigen on immunological memory. The results suggest that slow and continuous release of antigen from polymer particles plays a critical role in eliciting improved memory antibody response from single point immunization.     

The complement lysozyme sequence in immune bacteriolysis

Suspensions of Escherichia coli were not affected by lysozyme alone, but in solutions of appropriate pH and ionic strength some lysozyme was bound to the bacterial surface and remained available for action if the bacteria were subsequently treated with antibody and complement. In the presence of antibody, complement had a relatively prolonged action on E. coli ending in lysis. However, from an early stage in the reaction this lysis could be accelerated by adding lysozyme. The results suggest that human complement acted on the outer, lipoprotein—lipopolysaccharide layers of the bacterial cell wall and so gave lysozyme access to the deeper mucopeptide.

When the number of bound lysozyme molecules per bacterium was less than 10,000–20,000 the lysozyme effect decreased rapidly. However, many of these molecules may have been inactivated by K antigen.

     

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 intrasplenic circulation of three formulations of the same protein antigen

This paper presents a kinetic study of the intrasplenic circulation of three formulations of the protein antigen conalbumin including the soluble form and two liposomal formulations, one encapsulated in the internal aqueous milieu and one surface-linked to the liposomal vehicle. These formulations differ not only in their physical status but also in their immunostimulating properties and were chosen in an attempt to correlate the movements of antigen in lymphoid tissues with the immune response elicited. The presence of conalbumin was followed over a period of 21 days using, as a detection system, an antibody that we developed and which allows for the visualization of antigenic peptides such as those presented at the surface of antigen-presenting cells (APC). The results demonstrate that the amount of antigen accessing to the spleen, its time of residency and the pathway it follows are all profoundly influenced by the form under which it penetrates the immune system. The results also indicate that the marked initial preferences of an antigen for either B cells, marginal zone macrophages (MZM) or metallophilic macrophages (MM) are of fundamental importance in determining the fate of this antigen in the spleen. It is concluded that the exact formulation of an antigen is as crucial to the regulation of the immune response as is the nature of this antigen. It is further concluded that liposomes can be used efficiently to modify the formulation of an antigen and can contribute as such to the induction of specific immune functions by driving the antigen towards some privileged immune cell populations.     

Role of sustained antigen release from nanoparticle vaccines in shaping the T cell memory phenotype

Particulate vaccines are emerging promising technologies for the creation of tunable prophylactics against a wide variety of conditions. Vesicular and solid biodegradable polymer platforms, exemplified by liposomes and polyesters, respectively, are two of the most ubiquitous platforms in vaccine delivery studies. Here we directly compared the efficacy of each in a long-term immunization study and in protection against a model bacterial antigen. Immunization with poly(lactide-co-glycolide) (PLGA) nanoparticles elicited prolonged antibody titers compared to liposomes and alum. The magnitude of the cellular immune response was also highest in mice vaccinated with PLGA, which also showed a higher frequency of effector-like memory T cell phenotype, leading to an effective clearance of intracellular bacteria. The difference in performance of these two common particulate platforms is shown not to be due to material differences but appears to be connected to the kinetics of antigen delivery. Thus, this study highlights the importance of sustained antigen release mediated by particulate platforms and its role in the long-term appearance of effector memory cellular response.     

Large liposome agglutination technique for the serological detection of syphilis

In response to tissue invasion by Treponema pallidum, an antibody complex, called reagin, appears in the serum of the syphilitic patients. Reagin has the ability to combine with cardiolipin, therefore, cardiolipin has traditionally been used as the antigen in different test configurations for the serological detection of syphilis: the VDRL (Venereal Disease Research Laboratory) flocculation test and the RPR (rapid plasma reagin) test. Here we introduce a liposome preparation composed of cardiolipin, lecithin and cholesterol and containing a colored dye which upon mixing with syphilitic serum on a slide or a plastic card results in clearly visible liposome agglutination. We show that the liposomes in the size range of 1–10 μm are essential for proper sensitivity. The liposomes are colored with either a water-soluble dye entrapped in the aqueous space of liposomes or a lipophilic dye embedded in the membrane. By virtue of the liposome size and coloration the agglutination pattern is easily visible within 3–5 min without the use of a mechanical rotator. The approach described here can be extended to the detection of other antibodies and polyvalent antigens.     

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.