Adjuvant Effect Of Liposomes Research Articles

Liposomes with differential lipid components exert differential adjuvanticity in antigen-liposome conjugates via differential recognition by macrophages.

We previously reported that liposomes having differential lipid components displayed differential adjuvant effects when antigen was coupled with liposomes via glutaraldehyde. In the present study, antigen-liposome conjugates prepared using liposomes having differential lipid components were added to the macrophage culture, and phagocytosis and the antigen digest of liposome-coupled antigen by macrophages were then investigated. Antigen presentation by macrophages to an antigen-specific T-cell clone was further investigated using the same conjugates. Antigen-liposome conjugates which induced higher levels of antibody production in vivo were recognized more often, and the liposome-coupled antigen was digested to a greater degree by macrophages than antigen-liposome conjugates which induced lower levels of antibody production. These results correlated closely with those regarding antigen presentation by macrophages; when antigen was coupled to liposomes showing higher adjuvant effect, macrophages cocultured with antigen-liposome conjugates activated antigen-specific T-cells at a higher degree. The concentration of OVA in the macrophage culture added as antigen-liposome conjugates was approximately 32 microg/mL. However, the extent of T-cell activation was almost equal to that when 800 microg/mL of soluble OVA was added to the culture. The results of the present study demonstrated that the adjuvant activity of liposomes observed primary in vivo correlated closely with the recognition of antigen-liposome conjugates and antigen presentation of liposome-coupled antigen by macrophages, suggesting that the adjuvant effects of liposomes are exerted at the beginning of the immune response, i.e., recognition of antigen by antigen-presenting cells.

Conformational stability and antibody response to the 18kDa heat-shock protein formulated into different vehicles.

Protein stability is one of the most important obstacles for successful formulation in the development of new-generation vaccines. Here, the 18kDa heat-shock protein (18kDa-hsp) was chemically modified though conjugation with bovine serum albumin or by esterification with N-hydroxysuccinimide ester of palmitic acid. The biologically active conformation of the protein was preserved after chemical modification. The immune responses to the recombinant 18kDa-hsp from Mycobacterium leprae were studied in different presentations: free, copolymerized with bovine serum albumin in aggregates (18kDa-hsp-BSA), and either surface linked to liposomes or entrapped into liposomes. Measuring the antibody production of immunized genetically selected mice has compared the adjuvant effects of liposomes and proteic copolymer. Among the two liposome preparations, the strongest response was obtained with the surface-exposed antigen-liposomes. The copolymer 18kDa-hsp-BSA conferred a high titer of antibody in injected mice, and persisted 70 d after immunization. This approach should prove very useful for designing more effective vaccines by using 18kDa-hsp as carrier protein.

Stimulating Role of Toxoids-laden Liposomes in Oral Immunization against Diphtheria and Tetanus Infections

Liposomes have been produced by injecting an ether solution of a mixture of lecithin and cholesterol into a diluted solution of prewarmed diphtheria and tetanus toxoids followed by elimination of the stream of ether vapour by vacuum.In a preliminary study, adjuvant effects of liposomes on the systemic and mucosal immune response have been studied. When a mixture of diphtheria toxoid (DT) and tetanus toxoid (TT) entrapped in liposomes were administered parenterally or orally in rabbit, a significant rise of specific antibodies against both toxoids was noticed. In monkeys receiving a mixture of DT and TT entrapped in liposomes orally, the antibody response after two and three ingestions of this product was mild but when liposomes containing toxoids were adsorbed with aluminium hydroxide in a similar experiment, a significant rise in the specific antibody response in monkey against both toxoids was recorded. Adult volunteers, similarly receiving a mixture of DT and TT, entrapped in liposomes and adsorbed with aluminium hydroxide have shown a significant rise in specific circulating antitoxins. In order to compare the efficacy of this technique of human oral immunization with the previous method, whereby a plant medicinal seed (LRS) was used as adjuvant in oral immunization of man, a second group of volunteers were simultaneously and similarly treated as suggested previously. The comparative results are discussed in the present report.

Interferon-γ inductive effect of liposomes as an immunoadjuvant

Adjuvant effect of liposomes was compared to that of aluminium hydroxide (Alum) using ovalbumin (OVA) as a model antigen, and the difference in adjuvanticity of liposomes from Alum has been evaluated on the basis of cytokine production. Both adjuvants enhanced the IgG levels, but a remarkable difference was observed in the production of IgG subclass; Alum enhanced IgG1 levels, and liposomes enhanced IgG2a, IG2b, and IG3 levels. Further, Alum enhanced antigen-specific IgE levels, whereas liposomes did not. To clarify the difference in adjuvant effect of these adjuvants, secretion of cytokines, especially interferon-γ (IFN-γ) and interleukin-4 (IL-4) which regulate IgE production, was estimated ex vivo. Overnight culture of splenic cells obtained from mice immunized with liposomes encapsulating OVA elicits IFN-γ secretion, but not IL-4 secretion. On the other hand, the production of both cytokines was elevated by the immunization with OVA-Alum complex. The results indicate that the difference of adjuvant activity between liposomes and Alum may come from the difference in the secretion of IL-4 and that, consequently, a different class of antibody response is developed. More importantly, negatively charged liposomes containing phosphatidylserine remarkably promoted IFN-γ secretion compared to neutral liposomes.

Adjuvant effect of liposomes and adamantylamide dipeptide on antigenicity of entrapped synthetic peptide derived from HIV-1 transmembrane region glycoprotein gp41

The synthetic peptide antigen (Ag) (the primary structure Tyr-Leu-Lys-Asp-Gln-Gln-Leu-Leu-Gly-Ile-Trp-Gly-Cys-Ser-Gly-Lys-Leu-Ile-Cys-Thr derived from the envelope glycoprotein gp41 of the human immunodeficiency virus type 1 (HIV-1) and exerting specificity with all HIV-1-positive sera available in the Czech Republic (and also in a panel of 10,000 sera from WHO)) was conjugated with bovine serum albumin (BSA) and encapsulated into liposomes. Adjuvant activities of liposomes with various lipid compositions were compared with Freund's complete adjuvant (FCA) and with aluminium hydroxide (AL). The immune response to BSA-Ag liposomes with co-entrapped adamantylamide dipeptide (AdDP) was comparable with that of FCA in terms of longevity and levels of specific antibodies in mouse sera.

Activation of systemic and mucosal immune response following nasal administration of liposomes.

Adjuvant effects of liposomes on systemic and mucosal immune response were investigated following nasal administration to Balb/c mice. Bovine serum albumin (BSA)-specific serum IgG and salivary IgA levels were significantly elevated when BSA-associated liposomes were administered intranasally twice at 4-week intervals. Systemic immune response was activated only by negatively charged liposomes, while activation of mucosal immune response was independent of liposomal charge. Antigen localization in liposomes affected immune adjuvant effect; the mucosal immune response could be activated only by liposomes to whose surface BSA was attached, but the systemic immune response was activated by both liposomes to which antigens were attached and in which the encapsulating antigens occurred. The results suggest that the contribution of antigen-presenting cells in activation of systemic and mucosal immunity following intranasal administration is different.

Adjuvant effects of liposomes containing lipid A: enhancement of liposomal antigen presentation and recruitment of macrophages.

Liposomes containing lipid A induced potent humoral immune responses in mice against an encapsulated malaria antigen (R32NS1) containing NANP epitopes. The immune response was not enhanced by lipid A alone or by empty liposomes containing lipid A. Experiments to investigate the adjuvant mechanisms of liposomes and lipid A revealed that liposome-encapsulated R32NS1 was actively presented by bone marrow-derived macrophages to NANP-specific cloned T cells. The degree of presentation was related to the amount of liposomal antigen added per macrophage in the culture medium. At high cell densities, poor presentation occurred when liposomes lacked lipid A but excellent presentation occurred when the liposomes contained lipid A. Liposomes containing lipid A and encapsulated antigen also activated gamma interferon-treated macrophages to produce nitric oxide. Macrophage activation and antigen presentation occurred with liposomes that could not be detected by the Limulus amebocyte lysis assay. Intraperitoneal injection of liposomal lipid A caused a marked increase in the recruitment of immature (peroxidase-positive) macrophages to the peritoneum. On the basis of these experiments, we propose that the mechanism of the adjuvant action of liposomal lipid A is partly due to increased antigen presentation by macrophages and partly due to recruitment of an increased number of macrophages serving as antigen-presenting cells.

Adjuvant effect of liposomes in the autoimmune response to rat male accessory glands

Wistar rats submitted to 3 intravenous (i.v.) or intraperitoneal (i.p.) immunizations with saline extract of rat male accessory glands (RAG) associated to liposomes developed a delayed type hypersensitivity (DTH) response to RAG after the first immunization, a remission state after the second immunization and a specific DTH response after the third injection. In a further study we transferred spleen mononuclear (SpM) cells from i.p. immunized rats taken 10 days after the second immunization (DTH negative) to normal or immunized recipients 24 h before or 10 days after the first immunization with RAG liposomes, respectively. The DTH response was reduced only in recipients previously immunized. Besides, it was possible to show that the transfer of SpM cells present when the response increased after the third injection in i.p. immunized donors reduced the suppression observed after the second injection. Rat accessory gland biopsies taken 10 days after the last immunization showed in nearly all cases mast cells, plasma cells and eosinophils with scarce lymphoid elements and increased acinar desquamation. This kind of infiltration had characteristics of cutaneous basophil hypersensitivity.

Liposomes, lipid A, and aluminum hydroxide enhance the immune response to a synthetic malaria sporozoite antigen.

A liposome-encapsulated cloned protein (R32tet32) containing sequences from the tetrapeptide repeat region of the circumsporozoite protein of Plasmodium falciparum sporozoites was examined for immunogenicity with rabbits and monkeys. Effects of adjuvants were tested by encapsulation of the antigen in liposomes either lacking or containing lipid A and adsorption with aluminum hydroxide (ALUM). When rabbits were immunized with R32tet32 alone, a primary antibody response was not seen and a secondary response did not appear until 32 to 36 weeks after boosting. Immunization with ALUM-adsorbed R32tet32 resulted in a minimal primary antibody response. A moderate secondary antibody response was detected within 2 weeks after boosting, but antibody levels decreased to preimmunization levels 8 weeks after boosting. When R32tet32 was encapsulated in liposomes containing lipid A, strong primary and secondary antibody responses were observed. Strong primary and secondary responses also were obtained when R32tet32 was encapsulated in liposomes either containing or lacking lipid A and the liposomes were adsorbed with ALUM. The strongest antibody response was obtained by immunization with ALUM-adsorbed liposomes containing lipid A and R32tet32, suggesting that the adjuvant effects of liposomes, lipid A, and ALUM were additive or synergistic.

Endotoxin enhanced adjuvant effect of liposomes, particularly when antigen and endotoxin are incorporated within the same liposome.

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