Adjuvanticity Of Liposomes Research Articles

Predicting the influence of liposomal lipid composition on liposome size, zeta potential and liposome-induced dendritic cell maturation using a design of experiments approach

In this study, the effect of liposomal lipid composition on the physicochemical characteristics and adjuvanticity of liposomes was investigated. Using a design of experiments (DoE) approach, peptide-containing liposomes containing various lipids (EPC, DOPE, DOTAP and DC-Chol) and peptide concentrations were formulated. Liposome size and zeta potential were determined for each formulation. Moreover, the adjuvanticity of the liposomes was assessed in an in vitro dendritic cell (DC) model, by quantifying the expression of DC maturation markers CD40, CD80, CD83 and CD86. The acquired data of these liposome characteristics were successfully fitted with regression models, and response contour plots were generated for each response factor. These models were applied to predict a lipid composition that resulted in a liposome with a target zeta potential. Subsequently, the expression of the DC maturation factors for this lipid composition was predicted and tested in vitro; the acquired maturation responses corresponded well with the predicted ones. These results show that a DoE approach can be used to screen various lipids and lipid compositions, and to predict their impact on liposome size, charge and adjuvanticity. Using such an approach may accelerate the formulation development of liposomal vaccine adjuvants.

Role of B-1 cells in the immune response against an antigen encapsulated into phosphatidylcholine-containing liposomes

B-1 lymphocytes comprise a unique subset of B cells that differ phenotypically, ontogenetically and functionally from conventional B-2 cells. A frequent specificity of the antibody repertoire of peritoneal B-1 cells is phosphatidylcholine. Liposomes containing phosphatidylcholine have been studied as adjuvants and their interaction with dendritic cells and macrophages has been demonstrated. However, the role of B-1 cells in the adjuvanticity of liposomes composed of phosphatidylcholine has not been explored. In the present work, we studied the contribution of B-1 cells to the humoral response against ovalbumin (OVA) encapsulated into dipalmitoylphosphatidylcholine (DPPC) and cholesterol-containing liposomes. BALB/X-linked immunodeficient (xid) mice, which are deficient in B-1 cells, showed quantitative and qualitative differences in the anti-OVA antibody response compared with wild-type animals after immunization with these liposomes. The OVA-specific immune response was significantly increased in the BALB/xid mice when reconstituted with B-1 cells from naive BALB/c mice. Our results indicate the internalization of DPPC-containing liposomes by these cells and their migration from the peritoneal cavity to the spleen. Phosphatidylcholine significantly contributed to the immunogenicity of liposomes, as DPPC-containing liposomes more effectively stimulated the anti-OVA response compared with vesicles composed of dipalmitoylphosphatidylglycerol. In conclusion, we present evidence for a cognate interaction between B-1 cells and phosphatidylcholine liposomes, modulating the immune response to encapsulated antigens. This provides a novel targeting approach to assess the role of B-1 cells in humoral immunity.

Subunit Vaccines: Distearoylphosphatidylcholine-Based Liposomes Entrapping Antigen Offer a Neutral Alternative to Dimethyldioctadecylammonium-Based Cationic Liposomes as an Adjuvant Delivery System

The adjuvanticity of liposomes can be directed through formulation to develop a safe yet potent vaccine candidate. With the addition of the cationic lipid dimethyldioctadecylammonium bromide (DDA) to stable neutral distearoylphosphatidylcholine (DSPC):cholesterol (Chol) liposomes, vesicle size reduces while protein entrapment increases. The addition of the immunomodulator, trehalose 6,6-dibehenate (TDB) to either the neutral or cationic liposomes did not affect the physiochemical characteristics of these liposome vesicles. However, the protective immune response, as indicated by the amount of IFN-γ production, increases considerably when TDB is present. High levels of IFN-γ were observed for cationic liposomes; however, there was a marked reduction in IFN-γ release over time. Conversely, for neutral liposomes containing TDB, although the initial amount of IFN-γ was slightly lower than the cationic equivalent, the overall protective immune responses of these neutral liposomes were effectively maintained over time, generating good levels of protection. To that end, although the addition of DSPC and Chol reduced the protective immunity of DDA:TDB liposomes, relatively high protection was observed for the neutral counterpart, DSPC:Chol:TDB, which may offer an effective neutral alternative to the DDA:TDB cationic system, especially for the delivery of either zwitterionic (neutral) or cationic molecules or antigens.

An Increased Adjuvanticity of Liposomes by the Inclusion of Phosphatidylserine in Immunization with Surface-Coupled Liposomal Antigen

Background: Exposure of phosphatidylserine (PS) on apoptotic cells is known to result in the enhanced recognition of apoptotic cells by phagocytes. By the inclusion of PS in the lipid component of liposomes, increased liposome immune adjuvant activity was expected. Methods: In the present study, two different liposome preparations containing either PS, i.e. PS-liposome, or phosphatidylcholine (PC), i.e. PC-liposome, were made, and macrophage recognition, processing, and antigen presentation of surface-coupled liposomal antigen were compared. Results: When ovalbumin-liposome conjugates were added to a culture of macrophages, enhanced recognition and processing of ovalbumin by the macrophages were observed by the inclusion of PS in the liposomes. The results correlated well with those regarding macrophage antigen presentation of liposome-coupled ovalbumin. Furthermore, in vivo immunization in mice with ovalbumin-liposome conjugates made with PS-liposomes induced a significantly higher level of anti-ovalbumin IgG antibody production than was induced by ovalbumin-liposome conjugates made with PC-liposomes. IgE-selective unresponsiveness was induced by ovalbumin-liposome conjugates regardless of the lipid components of liposomes. Conclusions: These results suggest that the inclusion of PS in liposomes enhances recognition and processing of surface-coupled liposomal antigen by macrophages, and increases liposome immune adjuvant activity.

The domain III fragment of Japanese encephalitis virus envelope protein: mouse immunogenicity and liposome adjuvanticity

The E protein of Japanese encephalitis virus (JEV) is the major antigen used to elicit neutralizing antibody response and protective immunity in hosts. In this study, the domain III protein of the attenuated strain CH2195LA was cloned to the pET32a expression vector and expressed as a thioredoxin (Trx) fusion protein in Escherichia coli. The recombinant protein was unique in forming a large fraction of the soluble recombinant protein in E. coli. The purified domain III fusion protein (TrxD3) was emulsified in Freund’s adjuvant (FA) as well as in different charged liposomes for immunization in mice. Immunization of TrxD3 fusion protein emulsified in Freund’s adjuvant and only the cationic liposome resulted in eliciting neutralizing antibodies and protective immunity in ICR mice. The cationic liposome can serve not only as a safer but also an effective adjuvant for the TrxD3 protein immunization. These studies can provide useful information for further developing the domain III recombinant protein vaccine against JEV.

Application of Liposomes in Antigen Presentation and Cytokine Delivery

AbstractIntroductionEver since the liposome has been proposed as an antigen carrier or vaccine adjuvant to enhance immune responses of various vaccines (1), a great deal of effort has been made to understand the physical and chemical properties of the liposome membranes that modulate the potency of liposomal adjuvants [for review, see (2)]. While no generally consistent conclusion can be drawn for all vaccine antigens, the role of lipid fluidity in liposome adjuvanticity has been investigated extensively. Kinsky (3) showed that trinitrophenyl (TNP)-sensitized liposomes composed primarily of gelphased lipids [defined by a gel-to-liquid phase-transition temperature (Tc) higher than 37°C] were more potent in eliciting B cell response. In this study, TNP is a lipid membrane-bound antigen. However, membrane fluidity does not appear to play a role in adjuvanticity with a water-soluble antigen. Six et al. (4) showed, using the water-soluble adenovirus type 5 hexon, that liposomes made of gel-phased lipids – dist...

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.

Mechanism of liposome adjuvanticity: An in vivo approach

The reputation of liposomes as adjuvant of the immune response is now firmly established despite the lack of information on the mechanisms involved in their immunopotentiating properties. The rapid targeting of massive doses of antigenic material to antigen-presenting cells, especially macrophages has, however, often been invoked as the principal source of liposomal adjuvanticity. In order to test this hypothesis, we analyzed the humoral response to antigen encapsulated in liposomes containing increasing amounts of surface-exposed mannose residues, ligand specific of an exclusive macrophagic receptor. Using BSA as a model antigen, we demonstrated that the humoral response is profoundly affected by mannosylation, being of prolonged duration and either inhibited or activated depending on the immunizing doses. These results suggest that the rapidity of antigen targeting is not the sole reason to liposome adjuvanticity and that the role of liposomes as antigenic depot is probably important to sustain substantial activation through successive restimulations. In this context, the increased rapidity in antigen targeting which favors the concentration of activation signals in time, results in an under-optimization of the response at high immunizing doses and in an optimization of this response at doses that would otherwise give rise to signal of sub-threshold intensity albeit during a longer period of time.

Liposome adjuvanticity: Influence of dose and protein: Lipid ratio on the humoral response to encapsulated and surface-linked antigen

The humoral response to bovine serum albumin either encapsulated in or surface-linked to liposomes was studied as a function of dose and protein: lipid ratio. Total immunoglobulin, total IgG, IgM, and the G isotypes, IgG1, IgG2a, and IgG3 were measured during the plateau phase of production after a boosting injection. Although the adjuvant character of liposomes was confirmed regardless of the mode of antigen association, important differences in the response to the two types of liposomal formulations were observed. Our results suggest that surface-linked antigen stimulates the immune system at lower doses than its encapsulated counterpart, is more sensitive to the protein:lipid ratios, and can stimulate the production of particular immunoglobulin isotypes in controlled conditions. Our data support the idea that different pathways of processing are utilized by the two forms of liposomal antigen.

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 IgG 1, IgG 2a, IgG 2b, IgG 3 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.