Synthetic lipopeptides incorporated in liposomes: In vitro stimulation of the proliferation of murine splenocytes and in vivo induction of an immune response against a peptide antigen (original) (raw)
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Synthetic lipopeptides formulated in liposomes: effect on their immune stimulatory capacity in vitro
2006 International Conference on Nanoscience and Nanotechnology, 2006
Conjugation of antigenic peptides to lipoamino acids (LAA) has been shown to increase the membrane permeability of peptides and protects them from enzymatic degradation, but the resulting LAA-conjugated peptides were poorly soluble in both aqueous and organic solvents. To overcome the formulation issue and to further enhance the bioactivity, lipopeptide constructs (LAA-LCMV 33-41 ) have been encapsulated within liposomes with high entrapment efficiency. The purpose of this study was to assess the immune stimulatory capacity of these lipopeptides and their liposomal formulations. Because dendritic cells (DCs) play a key role as antigen-presenting cells in immune responses, the in vitro cellular activities of the lipopeptides and their liposomal formulations were tested by measuring the up-regulation of the surface markers CD80, CD86 and MHCII on DCs. We found that conjugation of lipoamino acid with peptide antigen enhanced its immune stimulatory capacity compared with the unmodified peptide. However, encapsulation of lipopeptides within liposomes appears to compromise their immuno-stimulatory capacity at least at a loading of 10%.
Bioconjugate Chemistry, 2005
Synthetic analogues of triacylated and diacylated lipopeptides derived from the N-terminal domain of respectively bacterial and mycoplasmal lipoproteins are highly potent immunoadjuvants when administered either in combination with protein antigens or covalently linked to small peptide epitopes. Because of their amphipathic properties, lipopeptides, such as S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-N-palmitoyl-(R)-cysteinyl-alanyl-glycine (Pam(3)CAG), can be conveniently incorporated into liposomes and serve as anchors for antigens that are linked to them. To design vaccination constructs based on synthetic peptides and liposomes as vectors. we have accordingly synthesized a series of lipopeptides that differ by the number (Pam(3)C vs Pam(2)C) and nature of the acyl chains (palmitoyl vs oleoyl) and by the presence at their C-terminus of thiol-reactive functions, such as maleimide or bromoacetyl. When incorporated into liposomes, these latter functionalized lipopeptides allow, in aqueous media, a well controlled chemoselective conjugation of HS-peptides to the surface of the vesicles. Using a BALB/c mice splenocyte proliferation assay ([(3)H]thymidine incorporation), we have measured the lymphocyte activation potency of the different lipopeptides. We found that, compared to their free (emulsified) forms, the liposomal lipopeptides were endowed with enhanced mitogenic activities; i.e., up to 2 orders of magnitude for Pam(3)CAG which was more potent than Pam(2)CAG. The impact of functionalization on the cellular activity of Pam(3)CAG was dependent on the thiol-reactive group introduced: whereas the bromoacetyl derivative retained its full activity, the presence of a maleimide group virtually abolished the lymphocyte activation of the lipopeptide. Finally, the substitution of saturated palmitoyl chains by unsaturated oleoyl chains was inhibitory. Thus, thiol-reactive Ol(3)CAG derivatives were the least active mitogens in our assay. Taken together, our findings are of importance for the further optimization of antigen-specific liposomal-based synthetic vaccines; the bromoacetyl derivative of Pam(3)CAG should be a promising lipopeptide derivative serving as an anchor for peptide epitopes while retaining its lymphocyte activation activity.
Effect of synthetic lipopeptides formulated in liposomes on the maturation of human dendritic cells
Molecular Immunology, 2005
Diacylated (e.g. MALP-2) and triacylated (Pam(3)Cys derivatives) lipopeptides, deriving from the N-terminal moiety of respectively mycoplasmal and E. coli lipoproteins, are powerful adjuvants recognized by Toll-like receptors (TLR) which have been used successfully to trigger cell activation and immune responses. To design liposome-based vaccination constructs in which Th and CTL epitopes are conjugated to synthetic lipopeptide analogues anchored into the bilayers of the vesicles, the peptide moieties of the lipopeptides were functionalized with thiol-reactive groups, such as maleimide (Mal) or bromoacetyl, incorporated into liposomes and reacted with thiol carrying peptide epitopes. Because dendritic cells (DCs) play a key role as antigen-presenting cells in immune responses, in the present study we have evaluated the impact of the functionalization of lipopeptide analogues Pam(2)CAG, Pam(3)CAG and Ol(3)GAG on the phenotypic maturation of human monocyte-derived DCs. The intrinsic cellular activities of the lipopeptide analogues incorporated into liposomes were monitored, in vitro, by measuring the up-regulation of the cell-surface markers CD80, CD83, CD86 and HLA-DR. We found that in some cases their functionalization with thiol-reactive groups led to a loss of activity. The stimulatory potency can be ranked in the following order: Pam(3)CAG>/=Pam(2)CAG-Mal-Th approximately Pam(2)CAG-Mal>Pam(3)CAG-Mal-Th (where Th is a HS-peptide) and no appreciable activity was detected for Pam(3)CAG-Mal, Ol(3)CAG-Mal and Ol(3)CAG-Mal-Th. Our findings indicate that subtle modifications in the peptide moiety of lipopeptides have a great impact on the immunomodulatory properties of these molecules. For the engineering of liposome/lipopeptide-based vaccines, the maleimide derivative of Pam(2)CAG appears to be the best candidate.
Use of a liposome antigen delivery system to alter immune responses in vivo
Journal of Pharmaceutical Sciences, 1998
0 It has been reported that a certain peptide encompassing residues 129−140 of the hepatitis B virus core antigen (HBcAg) leads to a Th2-type response in C57BL/10 mice. We postulated that by formulating the peptide in liposomes along with an immune modulator known as MPLA the immune response could be directed toward a Th1-type response. If these liposomes could deliver the peptide along with MPLA to antigen presenting cells, then the immune response generated could be polarized to a Th1 response. The type of immune response initiated after immunization with the peptide HBcAg (126− 140) in different formulations was determined by an ex vivo T cell proliferation assay and by analysis of the cytokine profile of the proliferating T cells. A group of C57BL/6 mice immunized with peptide plus MPLA in a liposome formulation displayed a strong T cell proliferative response. The T cell subset was identified as Th1 based on the cytokine profile. The cytokine profiles showed significant production of interferon-γ (IFN-γ, a Th1-type cytokine) and extremely low levels of interleukin-4 (IL-4, a Th2-type cytokine). The control group of C57BL/6 mice immunized with peptide plus alum showed a very low level of T cell proliferation, and no increase was seen in IFN-γ or IL-4 production. These data signify that a Th1-type response occurred in mice treated with peptide in a liposome formulation but not in mice treated with the control formulation.
Liposomal Vaccine Adjuvant Formulations
2016
Liposomes known as Army Liposome Formulation (ALF), which are used as a potent vaccine adjuvant formulation, have a lipid bilayer bulk composed of phospholipids, usually dimyristoyl phosphatidylcholine and dimyristoyl phosphatidylglycerol, in which the hydrocarbon chains have a melting temperature in water of ≥23°C. Cholesterol is present in the bilayer as a stabilizer, and monophosphoryl lipid A (MPLA) as an immunostimulator. Using synthetic congeners of MPLA we demonstrate that with ALF liposomes containing a cholesterol-binding saponin as an additional adjuvant, differences in the number and placement of 14-carbon hydrocarbon chains attached to the diglucosamine phosphate headgroup of the MPLA do not significantly impact the magnitude of the immune response to an HIV-1 envelope protein.
Immunological adjuvants: a role for liposomes
Immunology today, 1990
Recent technological advances have resulted in the production of safe subunit and synthetic small peptide vaccines. These vaccines are weakly or non-immunogenic and cannot, therefore, be used effectively in the absence of immunological adjuvants (agents that can induce strong immunity to antigens). Owing to the toxicity of adjuvants, only one (aluminium salts) has hitherto been licensed for use in humans, and it is far from ideal. In this article, Gregory Gregoriadis discusses the use of liposomes as an alternative safe, versatile, universal adjuvant that can induce humoral- and cell-mediated immunity to antigens when administered parenterally or enterally.
Journal of Pharmacy and Pharmacology, 2006
Vesicular adjuvant systems composing dimethyldioctadecylammonium (DDA) can promote both cell-mediated and humoral immune responses to the tuberculosis vaccine fusion protein in mice. However, these DDA preparations were found to be physically unstable, forming aggregates under ambient storage conditions. Therefore there is a need to improve the stability of such systems without undermining their potent adjuvanticity. To this end, the effect of incorporating non-ionic surfactants, such as 1-monopalmitoyl glycerol (MP), in addition to cholesterol (Chol) and trehalose 6,6 0 -dibehenate (TDB), on the stability and efficacy of these vaccine delivery systems was investigated. Differential scanning calorimetry revealed a reduction in the phase transition temperature (T c ) of DDA-based vesicles by $12 C when MP and cholesterol (1:1 molar ratio) were incorporated into the DDA system. Transmission electron microscopy (TEM) revealed the addition of MP to DDA vesicles resulted in the formation of multi-lamellar vesicles. Environmental scanning electron microscopy (ESEM) of MP-Chol-DDA-TDB (16:16:4:0.5 mol) indicated that incorporation of antigen led to increased stability of the vesicles, perhaps as a result of the antigen embedding within the vesicle bilayers. At 4 C DDA liposomes showed significant vesicle aggregation after 28 days, although addition of MP-Chol or TDB was shown to inhibit this instability. Alternatively, at 25 C only the MP-based systems retained their original size. The presence of MP within the vesicle formulation was also shown to promote a sustained release of antigen in-vitro. The adjuvant activity of various systems was tested in mice against three subunit antigens, including mycobacterial fusion protein Ag85B-ESAT-6, and two malarial antigens (Merozoite surface protein 1, MSP1, and the glutamate rich protein, GLURP). The MPand DDA-based systems induced antibody responses at comparable levels whereas the DDAbased systems induced more powerful cell-mediated immune responses.