Antigen-expressing immunostimulatory liposomes as a genetically programmable synthetic vaccine
Maryam Amidi
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Markus de Raad
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Daan J. A. Crommelin
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Wim E. Hennink
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Enrico Mastrobattista
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M. Amidi M. de Raad D. J. A. Crommelin W. E. Hennink E. Mastrobattista (&) Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University
, PO Box 80082, 3508 TB Utrecht,
The Netherlands
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 b-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 (b-galactosidase) than control vaccines (i.e. AnExILs without genetic input, liposomal b-galactosidase or pDNA encoding b-galactosidase).
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Synthetic biology is a rapidly emerging interdisciplinary
research field that aims to construct new biological parts and
systems with new functionalities through a process of
engineering and standardization. Vaccines may also benefit from
a synthetic biology-based design. With vaccination the aim
is to delude the immune system with an antigenic
formulation to make it believe it is dealing with a natural infection,
however, without causing illness. At present, the majority of
vaccines on the market consist of attenuated or inactivated
pathogens. Although effective, these systems are poorly
defined, suffer from batch-to-batch variation and can only be
used for pathogens that can be readily cultivated in the lab at
scales that permit vaccine production. Moreover, as the ratio
of antigenic compounds within the pathogen is more-or-less
fixed, there is poor control over the direction against which
antigenic compound an immune reaction will be evoked. The
bottom-up design of vaccines which consist of well-defined
antigenic compounds (e.g. proteins or nucleic acids) offers
better control over the evoked immune reaction, however,
the design of such vaccines is often empirical and in general
yields vaccines that are poorly immunogenic and rely on
adjuvants in order to be effective. Moreover, most vaccine
production schemes are rather time-consuming, and
therefore not suitable for rapid response intervention, for
example, to prevent the pandemic spread of a new influenza
strain in the human population.
Cell-free synthetic biology may offer new ways to design
potent and genetically programmable vaccines (Jewett et al.
2008; Tsuboi et al. 2008, 2010a, b; Zichel et al. 2010; Kanter
et al. 2007; Simpson 2006). It is based on the in vitro
transcription and translation of single or multiple gene constructs
in order to obtain a functional part or system. Applications of
cell-free biology include the production of membrane
proteins that are difficult to express in heterologous hosts
(Henderson et al. 2007; Junge et al. 2010; Beebe et al. 2010;
Nozawa et al. 2010; Reckel et al. 2010; Ishihara et al. 2005;
Berrier et al. 2004), high-throughput screening of protein
libraries by in vitro compartmentalization (Mastrobattista
et al. 2005), generation of artificial cells by encapsulation of
these complex biochemical reactions into cell-sized
compartments, like liposomes (Kita et al. 2008; Sunami et al.
2006, 2010; Yamaji et al. 2009; Murtas et al. 2007; Ishikawa
et al. 2004; Oberholzer et al. 1999). Recently, we have shown
that protein expression in liposomes can yield
microgramquantities of a model antigen (Amidi et al. 2010).
In this study, we propose the use of antigen-expressing
immunostimulatory liposomes (AnExILs) as artificial
microbes for vaccination (Fig. 1). The potential advantages
of such antigen-expressing immunostimulatory liposomes
(AnExILs) over conventional vaccines are numerous: the
specificity of the vaccine can be easily altered by simply
changing the DNA templates without having to ch (...truncated)