Antigen-expressing immunostimulatory liposomes as a genetically programmable synthetic vaccine

Abstract

Liposomes are versatile (sub)micron-sized membrane vesicles that can be used for a variety of applications, including drug delivery and in vivo imaging but they also represent excellent models for artificial membranes or cells. Several studies have demonstrated that in vitro transcription and translation can take place inside liposomes to obtain compartmentalized production of functional proteins within the liposomes (Kita et al. in Chembiochem 9(15):2403-2410, 2008; Moritani et al.in FEBS J, 2010; Kuruma et al. in Methods Mol Biol 607:161-171, 2010; Murtas et al. in Biochem Biophys Res Commun 363(1):12-17, 2007; Sunami et al. in Anal Biochem 357(1):128-136, 2006; Ishikawa et al. in FEBS Lett 576(3):387-390, 2004; Oberholzer et al. in Biochem Biophys Res Commun 261(2):238-241, 1999). Such a minimal artificial cell-based model is ideal for synthetic biology based applications. In this study, we propose the use of liposomes as artificial microbes for vaccination. These artificial microbes can be genetically programmed to produce specific antigens at will. To show proof-of-concept for this artificial cell-based platform, a bacterial in vitro transcription and translation system together with a gene construct encoding the model antigen β-galactosidase were entrapped inside multilamellar liposomes. Vaccination studies in mice showed that such antigen-expressing immunostimulatory liposomes (AnExILs) elicited higher specific humoral immune responses against the produced antigen (β-galactosidase) than control vaccines (i.e. AnExILs without genetic input, liposomal β-galactosidase or pDNA encoding β-galactosidase). In conclusion, AnExILs present a new platform for DNA-based vaccines which combines antigen production, adjuvanticity and delivery in one system and which offer several advantages over existing vaccine formulations.

Keywords: Cell-free protein synthesis; DNA vaccination; Synthetic biology; Vaccine.

Fig. 1

Schematic representation of AnExIL formulations used in this study

Fig. 2

Determination of β-galactosidase expression by Western blotting under reducing conditions. lane1: S30 extract without DNA; lane 2: expressed β-galactosidase in IVTT mix; lane 3: expressed β-galactosidase after IMAC purification; lane 4: β-galactosidase from commercial source

Fig. 3

Serum anti-β-galactosidase antigen-specific total serum IgG responses in mice immunized i.m. with AnExIL-IN, AnExIL-ON, liposomal DNA vaccine (Lipo’s DNA vac) (a), liposomal protein vaccine (Lipo’s protein vac), liposomal protein/DNA vaccine (Lipo’s protein/DNA vac) and liposomal S30 extract (Lipo’s S30 extract) (b). Sera were collected 14 days after each immunization. Antibody titers are expressed as mean of the responding mice; the bars represent the 95% confidence intervals. The numbers above the columns indicate the number of responders per group. Asterisks indicate titers that are significantly (* P < 0.05; ** P < 0.001) higher than those of the group immunized (booster vaccination) with liposomal protein/DNA vaccines. Circles indicate titers that are significantly (° P < 0.05) higher than those of the group immunized (booster vaccination) with AnExIL-IN

Fig. 4

Serum anti-β-galactosidase antigen-specific total serum IgG2a (a) and IgG1 (b) responses and corresponding IgG2a/IgG1 ratios (prime vaccination c) and (booster vaccination d) of mice immunized i.m. with AnExIL-IN, AnExIL-ON, liposomal DNA vaccine (Lipo’s DNA vac), liposomal protein vaccine (Lipo’s protein vac) and liposomal protein/DNA vaccine (Lipo’s protein/DNA vac). Sera were collected 14 days after each immunization. Antibody titers are expressed as mean of the responding mice; the bars represent the 95% confidence intervals. The numbers above the columns indicate the number of responders per group. Asterisks indicate titers that are significantly (* P < 0.05; ** P < 0.001) higher than those of the group immunized (booster vaccination) with liposomal protein/DNA vaccines. Circles indicate titers that are significantly (° P < 0.05) higher than those of the group immunized (booster vaccination) with AnExIL-IN