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.     

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).     

Immunomodulation with liposomes: the immune response elicited by liposomes with entrapped dichloromethylene-diphosphonate and surface-associated antigen or hapten

Macrophages in the murine spleen were eliminated by the intravenous administration of dichloromethylene-diphosphonate (DMDP) encapsulated in liposomes. The immune response against an antigen (bovine serum albumin, BSA) or hapten (dinitrophenyl, DNP) associated with the surface of the DMDP liposomes was studied. Significant effects of macrophage elimination on the primary (IgM), but not on the secondary (IgG), anti-BSA response were found. Dramatic effects on the response against liposome-associated DNP were observed in macrophage-depleted mice. The number of anti-DNP antibody-forming cells in the spleen decreased from 300 to 28 per section, and anti-DNP serum titres dropped to 12% of their normal values. Since a similar phenomenon was observed for TNP-Ficoll, a thymus-independent type-2 antigen (and not for thymus-dependent or thymus-independent type-1 antigens), we suggest that this response should be classified as thymus-independent type-2 on grounds of its in vivo behaviour. We conclude that adjuvant activity (and memory formation) of liposomes with antigen exposed on their surface occurs irrespective of the presence or absence of splenic macrophages, and that DMDP liposomes could be useful in drug targeting with antigenic liposomes.     

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 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 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.     

Studies on antigenic competition: Effect of antigen dose on the immune response of mice injected simultaneously with human serum albumin and ferritin

Human serum albumin (HSA) and ferritin in Freund's incomplete adjuvant (FIA) were injected simultaneously, intraperitoneally into mice in various dose/ratios. The degree of suppression of the primary and secondary immune responses to a small dose of HSA was dependent on the dose of ferritin injected simultaneously. A totally suppressed primary response was associated with a secondary response which only attained the level of a primary response to HSA in FIA.

Similar results were obtained in experiments in which a constant dose of ferritin and varying amounts of HSA were injected simultaneously in FIA into mice.

Suppression of the immune response to alum-precipitated HSA in the presence of soluble ferritin was not as striking as when the antigens were injected with FIA. Nevertheless, suppression of the primary response was again associated with a reduced secondary response.

The results are discussed in the context of the possible mechanism(s) of antigenic competition.

     

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.     

Use of Liposomes as an Immunopotentiating Delivery System: in Perspective of Vaccine Development

Liposomes have been widely used to deliver antigens to the antigen-presenting cells (APCs) and also to modify their immunological behaviour in model animals. We recently demonstrated the potential of yeast lipid liposomes to undergo membrane-membrane fusion with cytoplasmic membrane of the target cells. Interestingly, studies in the present report revealed that antigen encapsulated in yeast lipid liposomes could be successfully delivered simultaneously into the cytosolic as well as endosomal processing pathways of APCs, leading to the generation of both CD4+ T helper and CD8+ cytotoxic T cells. In contrast, encapsulation of same antigen in egg phosphatidyl-choline (PC) liposomes, just like its free form, has inefficient access to the cytosolic pathway of major histocompatibility complex (MHC) I dependent antigen presentation and failed to generate antigen specific CD8+ cytotoxic T-cell response. However, both egg PC as well as yeast lipid liposomes have elicited strong antigen specific antibody responses in immunized animals. These results imply usage of liposome encapsulated antigen as potential candidate vaccine capable of eliciting both cell mediated as well as humoral immune responses.     

Liposome immune lysis assay (LILA). Application of sandwich method to determine a serum protein component with antibody-bearing liposomes

A complement-dependent liposome immune lysis assay (LILA) using carboxyfluorescein (CF)-entrapped liposomes bearing antibody was developed to measure C-reactive protein (CRP) antigen as a model of protein antigens in human sera. Goat anti-CRP antibody was covalently coupled to liposomes, and a specific lysis of the liposomes could be observed when the liposomes were incubated with both rabbit anti-CRP antibody (secondary antibody) and CRP antigen in sera in the presence of guinea pig complement. In this assay system, so-called sandwich assay, CRP (a multivalent antigen) bound to the liposomes bearing anti-CRP antibody and subsequently secondary antibody, which activated complement efficiently. The amount of CF released by a complement-dependent liposome immune lysis was proportional to CRP concentrations. This sandwich assay was simple, fast, highly sensitive, and covered the ranges 10-300 ng of CRP/ml in a homogeneous mode, that is, one where no separation step was employed. The results correlated well with those obtained by single radial immunodiffusion and enzyme immunoassay. This assay system would be applicable to the measurement of other protein antigens.     

Induction of primary antigen-specific immune reponses in SCID-hu-PBL by coupled T-B epitopes.

Adoptive transfer of human lymphoid cells into immunodeficient (SCID) mice lacking the ability to functionally rearrange T- and B-cell receptor genes constitutes a unique model to study and manipulate human immunocytes. We have investigated this model for the purpose of generating an antigen-specific primary humoral immune response. Peripheral blood lymphocytes (PBL) derived from blood donors were used to repopulate SCID mice, which subsequently were immunized with different B-cell epitopes coupled to either tetanus toxoid (TT), or to a promiscuous helper epitope of TT, or by incorporating the antigens into a liposome construct. By recruiting the necessary T-cell help found in the T-cell memory compartment against TT, primary immune responses were obtained against the hapten dinitrophenyl (DNP), the V3 loop peptide derived from glycoprotein (gp120) (HIV-1), the melanoma-associated GD2 ganglioside and ovine submaxillary mucin. The primary immune response against the GD2 ganglioside was induced by incapsulating TT into GD2-containing liposomes. These liposome constructs also allowed us to induce a high human IgG serotitre (3000-4000) against this normally not very immunogenic ganglioside.     

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.     

Antigen presentation as a factor in the protective immune response to renal infection.

Host protection against renal infection may be augmented by active immunization against the causative organism. In these experiments we have investigated the effect of varying amounts and methods of presentation of bacterial antigen on the seconday immune response. Primary immunization with varying amounts of both killed and live antigen did not affect the nature of the secondary immune response although active renal infection did have a noticeable effect on the titre of serum antibody during the primary immune response. The experiments confirmed the presence of immunological memory to the somatic antigen of E. coli and showed that memory persisted for at least 6 months after primary immunization. Experiments have also been carried out which have demonstrated that memory to the somatic antigen of E. coli is carried by the B lymphocyte.     

Resetting the Adaptive Immune System After Autologous Stem Cell Transplantation: Lessons from Responses to Vaccines

Autologous stem cell transplantation (ASCT) to treat autoimmune diseases (AID) is thought to reset immunological memory directed against autoantigens. This hypothesis can only be studied indirectly because the exact nature of the pathogenetic autoantigens is unknown in most AID. Therefore, 19 children with juvenile idiopathic arthritis (JIA) or systemic lupus erythematodes (SLE) and 10 adults with multiple sclerosis (MS) were vaccinated with the T-cell-dependent neoantigen rabies and the recall antigen tetanus toxoid after, respectively before, bone marrow harvest. Both vaccinations were repeated after ASCT. All except two of the responders mounted a primary antibody response to rabies after revaccination, and 44% of the responders mounted a primary antibody response to tetanus boost after ASCT. These data show that immunological memory to a neoantigen is lost in most patients with AID after immunoablative pretreatment; however, memory to a recall antigen boosted before bone marrow harvest is only lost in part of the patients. Disease progression was arrested in all patients with JIA/SLE except one, but only in a minority of MS patients. Clinical outcome on a per case basis was not associated with the profile of the immune response toward the vaccination antigens after ASCT.     

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).     

Development of immunological memory in rainbow trout (Oncorhynchus mykiss). I. An immunochemical and cellular analysis of the B cell response.

In vivo and in vitro analyses of the antibody responses of rainbow trout (Oncorhynchus mykiss) confirmed the existence of immunological memory in this species. An enhanced in vitro secondary antibody response was found to be due strictly to an expansion of the antigen-sensitive precursor pool without a concomitant increase in clone size. In contrast to the development of immunological memory in mammalian species, there was no evidence for affinity maturation during the primary or secondary response. A distinct shift in the fine specificity profiles of the antibodies, however, did occur during the generation of the secondary response. Additionally, more than a single injection of the priming antigen, TNP-KLH was required to produce an enhanced in vitro response to this T-dependent antigen. However, a second priming injection was not required to produce an enhanced secondary response to the T-independent form of antigen, TNP-LPS. These results indicate that memory in trout may be due to a simple expansion of the antigen-specific precursor pool without many of the qualitative changes in antibody or B cell function associated with the expression of memory in mammals.     

Immune response mediated by liposome-associated protein antigens. I. Potentiation of the plaque-forming cell response.

Mice, immunized with liposome-associated bovine serum albumin (LSM-BSA), showed a significantly higher BSA-specific plaque-forming cell (PFC) response than did mice injected with fluid BSA (fBSA). Physical association between the liposome carrier and the protein antigen is imperative for potentiating the PFC response, since the injection of empty liposomes, together with fBSA, was found to be ineffective in inducing an immune response. Liposome-associated protein antigen was found to be a potent stimulator of immunological memory, as demonstrated by the ability of LSM-BSA primed animals to generate a vigorous PFC response upon challenge with the weakly immunogenic fBSA. The injection of congenitally athymic homozygous nude (Nu/Nu) mice with LSM-BSA failed to induce significant antibody formation, whereas the heterozygous (Nu/+) littermates gave a normal PFC response to the same LSM-BSA preparation. Thus, BSA remains a T-cell-dependent antigen, despite its entrapment within liposomes, and T lymphocytes appear to play an obligatory role in providing synergistic interactions for eliciting a BSA-specific PFC response to the LSM-BSA.     

The acquired immune deficiency syndrome.

Liposomes prepared with human LS174T colon tumour cell membranes induce specific primary xenogeneic immune responses in BALB/c splenocytes in vitro. Characterization of the adjuvant role of these liposomes was accomplished by determining the effect on immune induction of several modifications on the liposomal carrier. The results showed that the carrier effect of liposomes was mediated primarily by tumour antigens exposed on the outer surface. Trypsin treatment of the liposomes eliminated 95% of the surface protein and significantly (P less than 0.05) reduced the ability of liposomes to induce cytotoxic splenocytes. The generation of cytolytic activity with liposomes was dose-dependent, with a 10 micrograms protein threshold and a maximal response at 100 micrograms. 'Rigid' liposomes were shown to be significantly (P less than 0.05) more efficacious than fluid liposomes in inducing cytotoxicity. In addition, the data indicate that the xenogeneic cell-mediated immunity exhibits identical classes of effector cells as found in murine-murine reactions. Lymphocytes bearing the THY-1, Lyt-1 and Lyt-2 surface markers were necessary for immune induction. The role of Lyt-123 subpopulation was suggested by the inability to achieve normal cytolytic levels by reconstitution with Lyt-1 plus Lyt-2 cells. Adherent cells were, as expected, necessary for the generation of primary immunity. Indeed, the interaction of I-A+ adherent cells with liposomes for at least 8 h was required to generate subsequent maximal T cell cytotoxic activity. The phenotype of the cytotoxic effector cell was Thy-1+, Lyt-2+, and I-Ad-. If this were an allo-or syngeneic, and not a xenogeneic system, this study would be of less interest. However, when coupled with the known molecular homologies between murine and human lymphocyte antigens, these results suggest that the concept of cross species major histocompatibility complex (MHC) restriction is tenable. Thus the liposome is not only an effective antigen carrier, but also a functional adjuvant for in vitro induced cell-mediated immunity.     

Neonatal thymectomy and the decrease in antigen-sensitivity of the primary response and immunological `memory' systems

Neonatal thymectomy of Swiss mice induced a 100-fold increase in antigen-dose needed to elicit a primary haemolysin response to sheep erythrocytes and a ten-fold increase in antigen-dose needed to elicit immunological `priming', when the mice were tested for antigen-sensitivity at 6 weeks of age. Using different antigen systems, other laboratories have reported that thymectomized mice exhibited a normal primary response and a ten-fold reduction in the level of antibody reached in the secondary response. It is suggested that the varying effect of thymectomy on the primary response is attributable to variations in the time of development of the capacity to give a primary antibody response against the different antigens.