Bacterial ghosts – biological particles as delivery systems for antigens, nucleic acids and drugs Chakameh Azimpour Tabrizi 1 , Petra Walcher 1 , Ulrike Beate Mayr 1 , Thomas Stiedl 1,2 , Matthias Binder 1,2 , John McGrath 1,3 and Werner Lubitz 1,2,Despite the exponential rate of discovery of new antigens and DNA vaccines resulting from modern molecular biology and proteomics, the lack of effective delivery technology is a major limiting factor in their application. The bacterial ghost system represents a platform technology for antigen, nucleic acid and drug delivery. Bacterial ghosts have significant advantages over other engineered biological delivery particles, owing to their intrinsic cellular and tissue tropic abilities, ease of production and the fact that they can be stored and processed without the need for refrigeration. These particles have found both veterinary and medical applications for the vaccination and treatment of tumors and various infectious diseases. Addresses 1 Institute of Microbiology and Genetics, Section Microbiology and Biotechnology, University of Vienna, Althanstrasse 14, UZAII, 2B 522, A-1090, Vienna, Austria 2 BIRD-C GmbH&CoKEG, Schonborngasse 12/12, A-1080 Vienna, Austria 3 Gadi Research Centre, University of Canberra, ACT 2601, Australia e-mail: [email protected]Current Opinion in Biotechnology 2004, 15:530–537 This review comes from a themed issue on Pharmaceutical biotechnology Edited by Carlos A Guzman and Giora Z Feuerstein Available online 28th October 2004 0958-1669/$ – see front matter # 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2004.10.004 Abbreviations APC antigen-presenting cell DOX doxorubicin DDS drug delivery system MHC major histocompatibility complex OmpA outer membrane protein A Introduction New generation recombinant protein and DNA vaccines are generally poorly immunogenic, thus there is an urgent need to develop improved delivery systems and adjuvant formulations. This requirement has fueled intense world- wide research into biological particles as novel delivery systems for antigens, nucleic acids and drugs. The ambi- tious aim of future vaccines is to provide maximum efficacy with minimum number of doses, delivered safely and easily [1 ] (see Table 1; Box 1). Several biological and synthetic systems ranging from fusion proteins, lipid spheres and sugar particles to virus-like particles and whole-cell bacteria are in use or being investigated as dual-carrier molecules and adjuvants for antigens, nucleic acids and drugs (Box 2). This review will focus on current strategies for antigen, nucleic acid and drug delivery by biological particles with an emphasis on the use of bacterial ghosts as delivery systems. Non-bacterial delivery systems Various non-bacterial biological delivery systems are listed in Box 2. Of these, live attenuated or inactivated viruses and virus-derived particles are the best documented deliv- ery systems, as they are obligate parasites of eukaryotic cells. So far, live attenuated viruses, virus-like particles and virosomes have been developed [2]. Viral vectors can improve the long-term expression of target genes through the natural integration of the viral genome into that of the host. Virus-like particles have been engineered using viral structural proteins and nucleic acids as an alternative deliv- ery system. One disadvantage of viruses, virus-like particles and virosomes alike, is that their capacity to encapsulate foreign antigens or DNA is restricted. Virosomes are immu- nopotentiating reconstituted influenza virus envelopes of approximately 150 nm in diameter, which comprise the influenza surface glycoproteins haemagglutinin and neur- aminidase (NA) and a mixture of natural and synthetic phospholipids [3]. Safety concerns owing to the possibility of incomplete inactivation, immunocompromised vaccine recipients or revertants further limit the use of virosomes and live attenuated viruses [4 ]. Edible vaccines from transgenic plants offer a safer deliv- ery system, but unfortunately this system requires strong adjuvants to be immunogenic [5 ] and much work is still needed to improve the practical aspects of this approach. Non-organism alternatives are being developed as deliv- ery vehicles with greater success, especially for drug delivery. Liposomes are spherical phospholipid bilayers with an internal space that allows the incorporation of hydrophilic antigens. To enhance the delivery capacity of liposomes, different receptor molecules have been included in the bilayer [6,7,8 ,9,10,11 ]. ISCOMs Current Opinion in Biotechnology 2004, 15:530–537 www.sciencedirect.com
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Bacterial ghosts – biological particles as delivery systemsfor antigens, nucleic acids and drugsChakameh Azimpour Tabrizi1, Petra Walcher1, Ulrike Beate Mayr1,Thomas Stiedl1,2, Matthias Binder1,2, John McGrath1,3 and Werner Lubitz1,2,�
Despite the exponential rate of discovery of new antigens and
DNA vaccines resulting from modern molecular biology and
proteomics, the lack of effective delivery technology is a
major limiting factor in their application. The bacterial ghost
system represents a platform technology for antigen, nucleic
acid and drug delivery. Bacterial ghosts have significant
advantages over other engineered biological delivery particles,
owing to their intrinsic cellular and tissue tropic abilities,
ease of production and the fact that they can be stored and
processed without the need for refrigeration. These
particles have found both veterinary and medical applications
for the vaccination and treatment of tumors and various
infectious diseases.
Addresses1 Institute of Microbiology and Genetics, Section Microbiology and
Biotechnology, University of Vienna, Althanstrasse 14, UZAII, 2B 522,
the phagosomal membrane, releasing the target antigen
into the cytoplasm for MHC I presentation and T-cell
activation. Similarly, other groups have employed the
Escherichia coli a-haemolysin (HlyA) secretion system
for delivery of heterologous antigens and a large number
of hybrid proteins have been generated by gene fusion
with the C-terminal end of HlyA [32��].
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Bacterial ghosts Tabrizi et al. 533
Figure 2
Outer membrane
Periplasm
Inner membrane and cytoplasm
Phospholipid
Lipopolysaccharide
Peptidoglycan
Mannose
Maltose-binding proteinTarget antigen
S-layerprotein
Omp A
Streptavidin Biotin
DNA Drug
E′
LacI LacOs
Membrane proteinsPorin
Lipoprotein
Pilus
Current Opinion in Biotechnology
L′ Membrane anchor
Schematic of the localisation of different molecules in bacterial ghosts. The outer membrane, the periplasmic space and the inner membrane
facing the cytoplasmic lumen with integrated target antigens and different structural elements are drawn in a cartoon with specific emphasis
on the potential locations of target antigens and their carrier proteins.
Another intracellular antigen delivery system has been
developed with virus-like particles as well as virosomes
[4��]. Peptide vaccination with virosome carriers has been
investigated in several disease models including malaria,
melanoma and hepatitis C [3].
Compared with simple virus-like particle carriers, the
bacterial cell offers several compartments for the delivery
of immunogenic antigens and has a greater capacity.
Expression of an antigen in the cytosol, periplasm or
outer membrane of the carrier bacteria can have a pro-
found impact on the elicited immune response. For
Table 2
Applications of bacterial ghosts as delivery systems.
Display compartment Display of antigens
Outer membrane Surface presentation by OmpA fusion or
through fusion with pili structures
Periplasmic space Presentation of foreign antigens by MalE fusion
Inner membrane Anchoring of foreign proteins specific with
N0-, C0- or N0- and C0- membrane
anchors to the inner membrane
Cytoplasmic space Paracristalline fusion protein sheets,
which remain in the cytoplasmic lumen
after E-mediated lysis of the carrier bacteria
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example, surface-exposed expression or secretion of anti-
gens leads to a better induction of specific antibodies [14].
Antigenic epitopes have also been inserted into flagellin,
fimbriae or in the outer membrane or periplasmic proteins
MalE, LamB, OmpA and PhoE in different Salmonellastrains [31��]. An alternative to conventional bacterial
delivery system approaches has been reported in the
development of a surface-display system based on the
use of the spore coat of Bacillus subtilis [33�]. This has an
interesting advantage in that bacterial spores can survive
extremes of temperature, desiccation and exposure to
solvents and other noxious chemicals.
Therapeutic proteins
or peptides
Nucleic acids Drugs
Binding of hydrophobic
drugs by affinity to
membranes
Membrane-bound
enzymes
Filling with DNA
plasmids from 4 to
5000 copies/ghost
Sealing bacterial
ghosts for water
soluble drugs
Current Opinion in Biotechnology 2004, 15:530–537
534 Pharmaceutical biotechnology
Bacterial ghosts offer a safe, easy to manipulate and
straightforward to produce alternative to traditional anti-
gen bacterial carrier systems, with all of the advantages of
the latter. Foreign protein localisation within bacterial
ghosts is performed by fusion with specific anchor
sequences for attachment on the inside of the inner
membrane, export into the periplasmic space by fusion
to the MalE signal sequence or attachment to the outer
membrane as fusion proteins with OmpA or pili (Table 2;
Figure 2) [34]. Together with heterologously expressed
S-layer proteins SbsA and SbsB, which form shell-like
self-assembly structures filling the periplasmic and/or the
internal lumen cytoplasmic space, the capacity of ghost
vectors to function as carriers of polypeptides is vastly
extended [35].
The suitability of bacterial ghost technology for designing
an antichlamydial vaccine was evaluated by constructing
a candidate vaccine based on a Vibrio cholerae vector
expressing major outer membrane proteins. The efficacy
of the vaccine was assessed in a murine model of Chla-midia trachomatis genital infection [34]. Intramuscular
delivery of the vaccine candidate induced elevated local
genital mucosal as well as systemic T helper 1 (Th1)
responses. In addition, immune T cells from immunized
mice could transfer partial protection against a C. tracho-matis genital challenge to naıve mice. These results
suggest that V. cholerae ghosts expressing chlamydial
proteins might constitute a suitable subunit vaccine for
inducing an efficient mucosal T-cell response that pro-
tects against C. trachomatis infection.
Importantly for conformation-dependent B-cell epitope
presentation, it has been shown that the enzymatic activ-
ities of membrane-attached b-galactosidase and polyhy-
droxybutyrate synthase in bacterial ghosts is not impaired
by the attachment. This indicates that the membrane
anchors do not interfere with the proper folding of the
target proteins and that self-assembly of subunits (e.g. for
b-galactosidase) is possible [30].
Delivery systems for nucleic acidsAlthough no DNA vaccine has yet been approved for
routine human or veterinary use, the potential of this
vaccination strategy has been repeatedly demonstrated in
experimental animal models. Because of the simplicity
and versatility of these vaccines, various routes and modes
of delivery are used to elicit the desired immune
response; however, the need for large amounts of DNA
and numerous doses for optimal vaccination has led to the
search for delivery systems better able to target cells and
improve currently poor immunogenicity.
The intracellular nature of viruses has been exploited as a
tool for DNA delivery with the development of virus-like
particles. The abilities of a non-replicative DNA delivery
system based on parvovirus-like particles to induce cyto-
Current Opinion in Biotechnology 2004, 15:530–537
toxic T lymphocyte responses in the neonatal period has
been shown recently [36]. Results from phase I and phase
II human clinical trials indicated that virus-like particles
are safe, well tolerated and immunogenic when adminis-
tered parenterally [5�,37]. In a more complex approach,
the efficacy of an intranasally administered mumps DNA-
vaccine delivered using cationic virosomes as carrier and
E. coli heat-labile toxin as adjuvant has been demon-
strated in a mouse model [3].
The development of bacterial carriers somewhat extends
the application of DNA vaccines for mucosal immuniza-
tion [28]. Significant humoral and cellular immune
responses against bacterial, viral and tumor antigens have
been induced by in vivo delivery of DNA vaccines in
small-animal models. Encouragingly, results have been
demonstrated with a broad spectrum of Gram-positive
and Gram-negative bacterial vectors, including L. mono-cytogenes [38], Salmonella typhimurium [39��], Salmonellatyphi, Shigella flexneri [39��,40] and invasive E. coli [38].
A delivery system based on bacterial ghosts has also
proven effective for DNA vaccines. In vitro studies
showed that Mannheimia haemolytica ghosts loaded with
a plasmid carrying the gene encoding green fluorescent
protein are efficiently taken up by APCs with high (52–
60%) transfection rates [41]. Subsequent in vivo vaccina-
tion studies in Balb/c mice demonstrated that M. haemo-lytica ghost-mediated DNA delivery by intradermal or
intramuscular route of a eukaryotic expression plasmid
encoding for b-galactosidase under the control of a cyto-
megalovirus promoter (pCMVbeta), stimulated more effi-
cient antigen-specific humoral and cellular (CD4+ and
CD8+) immune responses than naked DNA. It was shown
that the use of bacterial ghosts as DNA carriers allowed
for modulation of the major T-helper cell response (from
a mixed Th1/Th2 to a more dominant Th2 pattern) to the
b-galactosidase gene product compared with the naked
DNA. Moreover, intravenous immunization with dendri-
tic cells loaded ex vivo with pCMVbeta-containing ghosts
elicited b-galactosidase-specific responses [41]. The
results are certainly encouraging considering the primary
role of dendritic cells as APCs. Bacterial ghosts not only
target the DNA vaccine construct to APCs, but also
provide a strong danger signal by acting as natural adju-
studies showed that the M. haemolytica ghosts targeted
the Caco-2 cells and released the loaded DOX within the
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cells. Cytotoxicity assays showed a two-log enhancement
in cytotoxic and antiproliferative activity in cells incu-
bated with DOX-loaded ghosts compared with DOX
directly added to the culture media [49].
Current work with bacterial ghosts lies in the investiga-
tion of the carrier capacity of the cytoplasmic lumen. This
intracellular space of bacterial ghosts can be filled either
with water-soluble substances or emulsions such that the
drug(s) of interest can be coupled to streptavidin
anchored on the inside of the cytoplasmic membrane.
For some purposes, it is advantageous to fill the internal
space of the ghost with a substituted matrix, which then
binds the drug(s) of interest. In model experiments,
biotinylated fluorescence-labelled dextran has been used
to completely fill the internal space of streptavidin-ghosts
[50]. As substituted dextran has a high capacity for bind-
ing peptides, drugs or other substances, therapy and
prevention might yet prove feasible with bacterial ghosts
as tropic carriers. Also, bacterial ghosts can be filled and
sealed for the delivery of fluid, non-anchored substances.
In a recent study, E. coli ghosts were filled with the
reporter substance calcein and were sealed by fusion with
membrane vesicles to maintain inner membrane integr-
ity. Adherence and uptake studies showed that murine
macrophages and human Caco-2 cells took up the bacter-
ial ghosts and calcein was released within the cell [29].
ConclusionsBacterial ghosts are very useful non-living carriers, as they
can carry foreign antigens, nucleic acids and drugs in one
or more cellular locations simultaneously. Their ease of
manufacture, the fact that they can be stored and pro-
cessed without the need for refrigeration and their excel-
lent safety profile — even when administered at high
doses — are important considerations for a broad spec-
trum of applications. The identical surface receptors of
bacterial ghosts and their living counterparts are being
exploited for specific cellular and tissue targeting. Few
other biological delivery systems offer such excellent
carrier qualities in combination with application-based
tropism in humans, animals or plant tissues.
AcknowledgementsThe technical assistance of Beate Bauer for preparing the manuscriptis greatly appreciated. This work was supported by grantGZ 309.049/1-VI/6/2003.
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Current Opinion in Biotechnology 2004, 15:530–537
www.elsevier.com/locate/jconrel NE
DELIV
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Journal of Controlled Releas
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Immobilization of plasmid DNA in bacterial ghosts
Peter Mayrhofera,b,c, Chakameh Azimpour Tabrizia, Petra Walchera,b,
Wolfgang Haidingera,b, Wolfgang Jechlingera,b,c,*, Werner Lubitza,b
aInstitute of Microbiology and Genetics, Section Microbiology and Biotechnology, University of Vienna, UZA II, 2B522,
Althanstrasse 14, A-1090 Wien, AustriabBIRD-C GmbH and CoKEG, Schonborngasse 12, A-1080 Vienna, Austria
cMayrhofer and Jechlinger OEG, Strozzigasse 38/12, A-1080 Vienna, Austria
Received 8 July 2004; accepted 21 October 2004
Available online 14 November 2004
Abstract
The development of novel delivery vehicles is crucial for the improvement of DNA vaccine efficiency. In this report, we
describe a new platform technology, which is based on the immobilization of plasmid DNA in the cytoplasmic membrane of a
bacterial carrier. This technology retains plasmid DNA (Self–Immobilizing Plasmid, pSIP) in the host envelope complex due to
a specific protein/DNA interaction during and after protein E-mediated lysis. The resulting bacterial ghosts (empty bacterial
envelopes) loaded with pDNA were analyzed in detail by real time PCR assays. We could verify that pSIP plasmids were
retained in the pellets of lysed Escherichia coli cultures indicating that they are efficiently anchored in the inner membrane of
bacterial ghosts. In contrast, a high percentage of control plasmids that lack essential features of the self-immobilization system
were expelled in the culture broth during the lysis process. We believe that the combination of this plasmid immobilization
procedure and the protein E-mediated lysis technology represents an efficient in vivo technique for the production of non-living
DNA carrier vehicles. In conclusion, we present a bself-loadingQ, non-living bacterial DNA delivery vector for vaccination
endowed with intrinsic adjuvant properties of the Gram-negative bacterial cell envelope.
D 2004 Elsevier B.V. All rights reserved.
Keywords: Bacterial ghosts; Self-immobilization; DNA carrier vehicle; DNA delivery system; Gene transfer
0168-3659/$ - see front matter D 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.jconrel.2004.10.026
* Corresponding author. Institute of Bacteriology, Mycology
and Hygiene, Department of Pathobiology, University of Veterinary