Incorporation of the TLR4 agonist monophosphoryl lipid A into the bilayer of DDA/TDB liposomes: physico-chemical characterization and induction of CD8+ T-cell responses in vivo.
ABSTRACT The combination of delivery systems like cationic liposomes and immunopotentiators such as Toll-like receptor (TLR) ligands is a promising approach for rational vaccine adjuvant design. The purpose of this study was to investigate how the incorporation of the poorly soluble TLR4 agonist monophosphoryl lipid A (MPL) into cationic liposomes based on dimethyldioctadecylammonium (DDA) and trehalose 6,6'-dibehenate (TDB) influenced the physicochemical and immunological properties of the liposomes.
The DDA/TDB/MPL liposomes were characterized with regard to particle size, poly dispersity, surface charge, stability and thermodynamic properties. The adjuvant formulations were tested in vivo in mice using ovalbumin (OVA) as model antigen.
Integration of MPL into the bilayer structure of DDA/TDB liposomes was evident from a decreased phase transition temperature, an improved membrane packing, and a reduction in surface charge. The particle size and favorable liposome storage stability were not affected by MPL. In mice, DDA/TDB/MPL liposomes induced an antigen-specific CD8(+) T-cell response and a humoral response.
Enhancing the solubility of MPL by inclusion into the bilayer of DDA/TDB liposomes changes the membrane characteristics of the adjuvant system and provides the liposomes with CD8(+) T-cell inducing properties without compromising humoral responses.
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ABSTRACT: Potent liposomal PorA formulations containing various lipopolysaccharide (LPS) derivatives were developed. The following adjuvants were compared: the commonly used aluminum phosphate (AlPO(4)), and three LPS like adjuvants: monophosphoryl lipid A (MPL), lipopolysaccharide (galE LPS) and the less toxic LPS mutant lpxL1. The immunogenicity in mice was evaluated and compared with that against an outer membrane vesicle (OMV) vaccine. The IgG isotype distribution and bactericidal activity were determined. Furthermore, PorA specific proliferation of lymph node cells after immunization and restimulation in vitro was studied with selected formulations. Both AlPO(4) and MPL were unable to improve the functional immunogenicity (i.e. bactericidal response) of liposomal PorA. Besides, when these adjuvants were used, the percentage of responders in the groups did not reach 100%. This was also observed with non adjuvated PorA-liposomes or OMV. Of the adjuvants studied, only galE LPS and lpxL1 LPS were capable of increasing the immunogenicity and avoid non responsiveness against PorA-liposomes. Importantly, the adjuvant activity of lpxL1 LPS was accompanied by an improved PorA specific proliferation of lymph node cells and a concomitant increase in IL-2 production. In conclusion and considering its lower toxicity, lpxL1 LPS adjuvated liposomes are superior to other formulations tested.Vaccine 11/2005; 23(43):5091-8. · 3.49 Impact Factor
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ABSTRACT: A randomised, double-blind study assessing the potential of four adjuvants in combination with recombinant hepatitis B surface antigen has been conducted to evaluate humoral and cell-mediated immune responses in healthy adults after three vaccine doses at months 0, 1 and 10. Three Adjuvant Systems (AS) contained 3-O-desacyl-4'-monophosphoryl lipid A (MPL) and QS21, formulated either with an oil-in-water emulsion (AS02B and AS02V) or with liposomes (AS01B). The fourth adjuvant was CpG oligonucleotide. High levels of antibodies were induced by all adjuvants, whereas cell-mediated immune responses, including cytolytic T cells and strong and persistent CD4(+) T cell response were mainly observed with the three MPL/QS21-containing Adjuvant Systems. The CD4(+) T cell response was characterised in vitro by vigorous lymphoproliferation, high IFN-gamma and moderate IL-5 production. Antigen-specific T cell immune response was further confirmed ex vivo by detection of IL-2- and IFN-gamma-producing CD4(+) T cells, and in vivo by measuring increased levels of IFN-gamma in the serum and delayed-type hypersensitivity (DTH) responses. The CpG adjuvanted vaccine induced consistently lower immune responses for all parameters. All vaccine adjuvants were shown to be safe with acceptable reactogenicity profiles. The majority of subjects reported local reactions at the injection site after vaccination while general reactions were recorded less frequently. No vaccine-related serious adverse event was reported. Importantly, no increase in markers of auto-immunity and allergy was detected over the whole study course. In conclusion, the Adjuvant Systems containing MPL/QS21, in combination with hepatitis B surface antigen, induced very strong humoral and cellular immune responses in healthy adults. The AS01B-adjuvanted vaccine induced the strongest and most durable specific cellular immune responses after two doses. These Adjuvant Systems, when added to recombinant protein antigens, can be fundamental to develop effective prophylactic vaccines against complex pathogens, e.g. malaria, HIV infection and tuberculosis, and for special target populations such as subjects with an impaired immune response, due to age or medical conditions.Vaccine 04/2008; 26(10):1375-86. · 3.49 Impact Factor
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ABSTRACT: Disaccharides are well-known reagents to protect biostructures like proteins and phospholipid-based liposomes during freezing and drying. We have investigated the ability of the two disaccharides trehalose and sucrose to stabilize a novel, non-phospholipid-based liposomal adjuvant composed of the cationic dimethyldioctadecylammonium (DDA) and trehalose 6,6'-dibehenate (TDB) upon freeze-drying. The liposomes were freeze-dried using a human dose concentration containing 2.5 mg/ml DDA and 0.5 mg/ml TDB with varying concentrations of the two sugars. The influence on particle size upon rehydration was investigated using photon correlation spectroscopy (PCS) and the gel to fluid phase transition was examined by differential scanning calorimetry (DSC). Data revealed that concentrations above 211 mM trehalose protected and preserved DDA/TDB during freeze-drying, and the liposomes were readily rehydrated. Sucrose was less efficient as a stabilizer and had to be used in concentrations above 396 mM in order to obtain the same effect. Immunization of mice with the tuberculosis vaccine candidate Ag85B-ESAT-6 in combination with the trehalose stabilized adjuvant showed that freeze-dried DDA/TDB liposomes retained their ability to stimulate both a strong cell-mediated immune response and an antibody response. These findings show that trehalose at isotonic concentrations protects cationic DDA/TDB-liposomes during freeze-drying. Since this is not the case for liposomes based on DDA solely, we suggest that the protection is facilitated via direct interaction with the headgroup of TDB and a kosmotropic effect, whereas direct interaction with DDA plays a minor role.Biochimica et Biophysica Acta 10/2007; 1768(9):2120-9. · 4.66 Impact Factor