Bacterial ghosts – biological particles as delivery ... · PDF fileBacterial ghosts – biological particles as delivery systems for antigens, nucleic acids and drugs Chakameh Azimpour

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,

A-1090, Vienna, Austria2 BIRD-C GmbH&CoKEG, Schonborngasse 12/12, A-1080 Vienna,

Austria3 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 a

Current

ntigen-presenting cell

DOX d
oxorubicin
DDS d
rug delivery system
MHC m
ajor histocompatibility complex
OmpA o
uter membrane protein A
IntroductionNew 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

Opinion in Biotechnology 2004, 15:530–537

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 systemsVarious 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 disadvantageofviruses,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

www.sciencedirect.com

Bacterial ghosts Tabrizi et al. 531

Box 2 Particles used for immunization and drug delivery.

Biological particles

Live attenuated or inactivated bacteria [14,15,17,31��,33�,51–53]

Bacterial ghosts [28–30,54]

Live attenuated or inactivated viruses [55–58]

Virus-like particles [5�,36,37,59,60�]

Table 1

Advantages and limitations of biological particles as delivery vehicles.

Biological particle Advantages Limitations

Live attenuated or

inactivated bacteria

Activation of innate immune system via pattern recognition receptors Reversion to virulence

Generation of humoral and cell-mediated immune response Horizontal gene transfer

Used as carrier for antigens Stability of recombinant phenotype

Pre-existing immunity against carrier strain

Antibiotic markers

Live GRAS bacteria Non-pathogenic Poorly immunogenic

Virosomes Biodegradable, contain no preservatives or detergents

and present fewer localized adverse events

Pathogenic for immunocompromised

recipients

Limited capacity to encapsulate foreign

antigens or DNA

Live attenuated or

inactivated viruses

Improved long-term expression of target genes

using viral integration system into host genome

Incomplete inactivation

Revertants to virulence

Edible vaccines from

transgenic plants

Low-cost production, ease of use Requirement for strong adjuvants

Erythrocytes Biocompatibility, complete biodegradability Different serotypes

Hazard for blood transfusion

Bacterial ghosts Non-living carriers Presence of lipopolysaccharides

Carriage of different antigens, DNA and drugs simultaneously

Strong adjuvant properties

Good recognition and uptake by antigen-presenting cells

High loading capacity for DNA

Targeting properties for different tissues

Ideal vaccine characteristics are summarized in Box 2. GRAS, generally recognized as safe.

(immune-stimulating complexes) are nanometer-sized

micellar assemblies composed of saponin, cholesterol

and phospholipids that contain amphiphilic membrane

protein antigens [11��]. Nanoparticles and microparticles

based on liposomal or vesicular (niosomes) systems might

find a broad range of application in the future [12,13].

Bacterial delivery systemsIt is well accepted that live attenuated and inactivated

whole-cell bacteria constitute an effective delivery sys-

tem for recombinant antigens and nucleic acids [14].

The advantage of live or inactivated bacteria is that innate

immune cells recognize highly conserved structures on

microorganisms — termed pathogen-associated molecu-

lar patterns (e.g. lipopolysaccharides, CpG and outer

membrane protein A [OmpA]) — via pattern-recognition

receptors, such as the mannose receptor and Toll-like

Box 1 Ideal vaccine characteristics.

Safe in all ages and in immunocompromised patients

Efficacious in all ages (from young infants to the elderly)

Efficacious single-dose vaccine to be administered soon after birth

Early onset of protection (day 8)

Long-lived protection (years)

Cold-chain independent (i.e. can be stored and processed without

refrigeration)

Needle-free delivery (e.g. mucosal route of immunisation)

Practical, simple formulation (e.g. stable dry powder for non-invasive

administration)

Multiple vaccine combinations


receptors [15]. Thus, heterologous antigens aside, carrier

bacteria can generate both humoral and cell-mediated

immunity. As a constituent of the bacteria, the hetero-

logous antigens are not only targeted to appropriate path-

ways of major histocompatibility complex (MHC) class I

and class II antigen processing and presentation, but also

generate an adequate cytokine milieu for promoting

antigen-specific responses [16].

Despite the strong and well-documented immunological

advantages of bacterial delivery vectors, there are safety

Edible vaccines from transgenic plants [5�]

Erythrocytes [43�]

Phage display of antigens [61]

Synthetic particles

Virosomes [3,4��,62]

Liposomes [6,7,8�,9,10,11��,32��]

Peptides and proteins [44�,63]

Proteosomes [11��,64]

Niosomes [12,13]

ISCOM (immune-stimulating complexes) [11��,65,66]

Chitosan microspheres [11��,67,68]

Polylactide/polyglycolide microspheres [69–71]

Cochleates, phospholipids-calcein precipitates [11��]


532 Pharmaceutical biotechnology

Figure 1

Scanning electron micrograph of an E. coli bacterial ghost produced using modified protein E lysis procedure [28]. A large opening enables the

bacterial ghost envelope complex to be revealed.

limitations with the bacterial adjuvant/delivery system.

Traditionally, pathogenic bacteria like Listeria spp.,

Salmonella spp., Yersinia spp., Shigella spp. and Mycobac-teria spp. are the most frequently used bacterial carriers.

The most significant problems in their usage are the

possibility of reversion to virulence, horizontal gene

transfer, the stability of the recombinant phenotype,

pre-existing immunity against the carrier strain and the

presence of antibiotic markers [17]. An alternative

approach is the use of live GRAS (generally recognized

as safe) bacteria, which are non-pathogenic and are inves-

tigated solely as delivery vectors (see Table 1; Box 1) [18].

Bacterial ghosts as delivery systemsBacterial ghosts represent empty non-denaturated envel-

opes derived from Gram-negative bacteria by protein-

E-mediated lysis, which retain all morphological and

structural features of the natural cell (for a review see

[19]). They can be used as vaccine candidates per se with

intrinsic adjuvant properties based on well-known

immune-stimulating compounds such as lipopolysacchar-

ides, lipid A and peptidoglycan (Figures 1 and 2). Alter-

natively, they can be employed as a delivery system for

proteins/antigens, nucleic acids, drugs and soluble com-

pounds for various medical and technical applications

[20–27]. Not only can bacterial ghosts act as delivery

vehicles for inner and/or outer membrane tethered anti-

gens, but they can also deliver water-soluble drugs or

antigens (see Figure 2; Table 2) [28–30]. The results of

several in vivo and in vitro studies confirm the potential of

bacterial ghosts as dual carrier/adjuvant technology for

modern vaccine development (for a review see [28]). The

cellular and tissue tropism of bacterial ghosts in combina-

tion with excellent carrier capacity in several cellular

compartments (Table 2) offers much potential for anti-


gen, nucleic acid and drug delivery. Bacterial ghosts are

taken up very effectively by antigen-presenting cells

(APCs) such as macrophages and dendritic cells and

are particularly suited as vaccines for mucosal adminis-

tration by oral, intranasal or aerogenic routes, resulting in

the induction of humoral and cellular immune responses

[28,30].

Delivery systems for antigensBecause new-generation vaccines based on recombinant

proteins are often less immunogenic than traditional

vaccines, they require specific choices of delivery parti-

cles and adjuvants to improve their presentation and

targeting and to thus induce an appropriate protective

immune response. This is particularly important at the

mucosa, the most effective site for immune stimulation.

The choice of carriers and/or adjuvants and of the antigen

itself have the potential to modulate the immune

response (i.e. predominantly B-cell or T-cell based)

appropriate for a particular pathogen.

The development of efficacious vaccines against intra-

cellular bacteria, parasites and viruses requires the induc-

tion of T-cell mediated responses. One ingenious

delivery system makes use of antigen fusions with the

listeriolysin enzyme of Listeria monocytogenes to help

induce a CD8+ T-cell response against phagosomal and

cytosolic antigens [31��]. Listeriolysin expression disrupts

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��].



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


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



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-


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-

vants (being inactivated whole-cell bacteria), thereby

promoting efficient maturation and activation of dendritic

cells. Thus, bacterial ghosts constitute a promising tech-

nology platform for the development of more efficient

DNA vaccines.

More recently, a new delivery system based on bacterial

ghosts has been developed in which, following E-

mediated lysis, DNA is tethered via a DNA-binding

membrane-anchored protein to the ghost inner mem-

brane. This system is the subject of continuing studies

(P Mayrhofer et al., unpublished).



Delivery systems for drugsMany diseases and cancers require the systemic admin-

istration of highly aggressive drugs to already immuno-

compromised patients. Deleterious and often severe side

effects result from a lack of cellular and tissue selectivity.

Another major issue is the poor solubility of some drugs

used in cancer treatment. Considering this, the develop-

ment of a safer and more efficient drug delivery system

(DDS) is the priority for future prophylactic treatments.

The DDS has three major goals: enhancement of drug

permeability for crossing physiological barriers; targeting

of drugs to the point of action; and the controlled release

of drugs [42]. Several biological DDSs are currently

employed, although none is ideal for all applications.

The use of erythrocytes in drug targeting has many

advantages, including biocompatibility, complete biode-

gradability and lack of toxic products, longer life-span

compared with synthetic carriers and a relatively inert

intracellular environment. Applications for this system

include intravenous slow drug release (e.g. antineo-

plasms, vitamins and antibiotics), enzyme therapy, target-

ing the reticuloendothelial system (e.g. adriamycin and

bleomycin against hepatic tumors and antileishmanial or

antiamoebial drugs against parasitic disease), and the

improvement of oxygen delivery to tissues [43�]. Another

DDS that exploits host constituents is the use of fusions

with the plasma protein, albumin. Albumin meets several

requirements of a drug carrier and shows accumulation in

tumors and in inflamed joints in patients with rheumatoid

arthritis [44�].

In a similar drug-packaging mechanism to erythrocytes,

liposomes have been utilized as drug delivery vehicles

for cancer treatment [45,46]. An ideal liposome formula-

tion, optimized for stability, will improve drug delivery

by decreasing the required dose and increasing the

efficacy of the entrapped drug at the target organ or

tissue [47].

More recently, several novel DDSs have been developed.

One of these involves nanoscale hepatitis B virus surface

antigen (HBsAg) L particles, which have many properties

that make them useful as in vivo gene transfer vectors and

as a DDS. The display of various cell-binding molecules

on the surface makes L particles particularly useful for

cell- and tissue-specific gene/drug delivery [48].

Bacteria might also offer a solution for drug delivery,

particularly through their tropic capacity. As a naturally

tissue tropic delivery system, bacterial ghosts have shown

early promise as a DDS. Recently, bacterial ghosts made

from the colonic commensal M. haemolytica were used for

the in vitro delivery of doxorubicin (DOX) to human

colorectal adenocarcinoma (Caco-2) cells. Adherence

studies showed that the M. haemolytica ghosts targeted

the Caco-2 cells and released the loaded DOX within the


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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30. Jechlinger W, Haidinger W, Paukner S, Mayrhofer P, Riedmann E,Marchart J, Mayr U, Haller C, Kohl G, Walcher P et al.: Bacterialghosts as carrier and targeting systems for antigen delivery. InVaccine Delivery Strategies. Edited by Goebel W. HorizonScientific Press; 2002:163-184.

31.��

Samuelson P, Gunneriusson E, Nygren PA, Stahl S: Display ofproteins on bacteria. J Biotechnol 2002, 96:129-154.

This review gives an overview of the basic principles of various bacterialdisplay systems and highlights current uses and possible future trends intheir technological application.

32.��

Dietrich G, Viret JF, Gentschev I: Haemolysin A and listeriolysin –two vaccine delivery tools for the induction of cell-mediatedimmunity. Int J Parasitol 2003, 33:495-505.

This review demonstrates the use of haemolysin A and listeriolysin asvaccine delivery tools and highlights their use for vaccination againstprotozoan parasites by induction of cell-mediated immunity.

33.�

Mauriello EM, Duc le H, Isticato R, Cangiano G, Hong HA,De Felice M, Ricca E, Cutting SM: Display of heterologousantigens on the Bacillus subtilis spore coat using CotCas a fusion partner. Vaccine 2004, 22:1177-1187.

In this paper the authors describe the bacterial spore as an efficientvehicle for mucosal immunization and demonstrate the advantages of thisnovel antigen delivery strategy.

34. Eko FO, Lubitz W, McMillan L, Ramey K, Moore TT, Ananaba GA,Lyn D, Black CM, Igietseme JU: Recombinant Vibrio choleraeghosts as a delivery vehicle for vaccinating against Chlamydiatrachomatis. Vaccine 2003, 21:1694-1703.

35. Riedmann EM, Kyd JM, Smith AM, Gomez-Gallego S, Jalava K,Cripps AW, Lubitz W: Construction of recombinant S-layerproteins (rSbsA) and their expression in bacterial ghosts – adelivery system for the nontypeable Haemophilus influenzaeantigen Omp26. FEMS Immunol Med Microbiol 2003, 37:185-192.

36. Martinez X, Regner M, Kovarik J, Zarei S, Hauser C, Lambert PH,Leclerc C, Siegrist CA: CD4-independent protective cytotoxic Tcells induced in early life by a non-replicative delivery systembased on virus-like particles. Virology 2003, 305:428-435.

37. Koutsky LA, Ault KA, Wheeler CM, Brown DR, Barr E, Alvarez FB,Chiacchierini LM, Jansen KU: A controlled trial of a humanpapillomavirus type 16 vaccine. N Engl J Med 2002,347:1645-1651.



38. Gentschev I, Dietrich G, Spreng S, Kolb-Maurer A, Brinkmann V,Grode L, Hess J, Kaufmann SH, Goebel W: Recombinantattenuated bacteria for the delivery of subunit vaccines.Vaccine 2001, 19:2621-2628.

39.��

Weiss J: Transfer of eukaryotic expression plasmids tomammalian host cells by salmonella spp. Int J Med Microbiol2003, 293:95-106.

This comprehensive review addresses the principles and application ofbacteria-mediated gene transfer. The paper highlights the ability ofattenuated intracellular bacterial, such as S. typhimurium, S. typhi andS. flexneri, L. monocytogenes, to transfer plasmid DNA into the host cell.Recombinant E. coli and Agrobacterium tumeophaciens baterial ghostsare described in this context. In addition, the author thoroughly evaluatesthe effectiveness and potential of Salmonella-mediated and mucosalDNA vaccination.

40. Xu F, Hong M, Ulmer JB: Immunogenicity of an HIV-gag DNAvaccine carried by attenuated Shigella. Vaccine 2003,21:644-648.

41. Ebensen T, Paukner S, Link C, Kudela P, de Domenico C, Lubitz W,Guzman CA: Bacterial ghosts are an efficient delivery systemfor DNA vaccines. J Immunol 2004, 172:6858-6865.

42. Yasukawa T, Ogura Y, Tabata Y, Kimura H, Wiedemann P,Honda Y: Drug delivery systems for vitreoretinal diseases.Prog Retin Eye Res 2004, 23:253-281.

43.�

Hamidi M, Tajerzadeh H: Carrier erythrocytes: an overview.Drug Deliv 2003, 10:9-20.

An excellent review covering most aspects of erythrocytes as carriervehicles. Besides the descriptions of various carrier applications it givesan overview of methods for encapsulation of drugs and bioactive agents,advantages of erythrocytes for drug delivery, in vitro characteristics andstorage of carrier erythrocytes and information about drug release.

44.�

Wunder A, Muller-Ladner U, Stelzer EH, Funk J, Neumann E,Stehle G, Pap T, Sinn H, Gay S, Fiehn C: Albumin-based drugdelivery as novel therapeutic approach for rheumatoidarthritis. J Immunol 2003, 170:4793-4801.

An interesting publication concerning a new approach in the therapy ofrheumatoid arthritis using methothrexat coupled to human serum albu-min. Results of both in vitro and in vivo experiments are shown.

45. Pignatello R, Puleo A, Puglisi G, Vicari L, Messina A: Effect ofliposomal delivery on in vitro antitumor activity of lipophilicconjugates of methotrexate with lipoamino acids.Drug Deliv 2003, 10:95-100.

46. Mandal AK, Sinha J, Mandal S, Mukhopadhyay S, Das N:Targeting of liposomal flavonoid to liver in combatinghepatocellular oxidative damage. Drug Deliv 2002, 9:181-185.

47. Anderson M, Omri A: The effect of different lipid components onthe in vitro stability and release kinetics of liposomeformulations. Drug Deliv 2004, 11:33-39.

48. Yamada T, Ueda M, Seno M, Kondo A, Tanizawa K, Kuroda S:Novel tissue and cell type-specific gene/drug delivery systemusing surface engineered hepatitis B virus nano-particles.Curr Drug Targets Infect Disord 2004, 4:163-167.

49. Paukner S, Kohl G, Lubitz W: Bacterial ghosts as noveladvanced drug delivery systems: antiproliferative activity ofloaded doxorubicin in human Caco-2 cells. J Control Release2004, 94:63-74.

50. Huter V, Szostak MP, Gampfer J, Prethaler S, Wanner G, Gabor F,Lubitz W: Bacterial ghosts as drug carrier and targetingvehicles. J Control Release 1999, 61:51-63.

51. Dietrich G, Kolb-Maurer A, Spreng S, Schartl M, Goebel W,Gentschev I: Gram-positive and Gram-negative bacteria ascarrier systems for DNA vaccines. Vaccine 2001, 19:2506-2512.

52. Weiss S: Transfer of eukaryotic expression plasmids tomammalian hosts by attenuated Salmonella spp. Int J MedMicrobiol 2003, 293:95-106.

53. Spreng S, Dietrich G, Goebel W, Gentschev I: Protection againstmurine listeriosis by oral vaccination with recombinantSalmonella expressing protective listerial epitopes within asurface-exposed loop of the TolC-protein. Vaccine 2003,21:746-752.


54. Riedmann EM, Kyd JM, Cripps AW, Lubitz W: Adjuvantproperties of bacterial ghosts. 2004, in press.

55. Rayner JO, Dryga SA, Kamrud KI: Alphavirus vectors andvaccination. Rev Med Virol 2002, 12:279-296.

56. Lundstrom K: Alphavirus vectors for vaccine production andgene therapy. Expert Rev Vaccines 2003, 2:447-459.

57. Balasuriya UB, Heidner HW, Davis NL, Wagner HM, Hullinger PJ,Hedges JF, Williams JC, Johnston RE, David Wilson W, Liu IKet al.: Alphavirus replicon particles expressing the two majorenvelope proteins of equine arthritis virus induce high levelprotection against challenge with virulent virus in vaccinatedhorses. Vaccine 2002, 20:1609-1617.

58. Bramson J, Dayball K, Evelegh C, Wan YH, Page D, Smith A:Enabling topical immunization via microporation: anovel method for pain-free and needle-free deliveryof adenovirus-based vaccines. Gene Ther 2003,10:251-260.

59. Yao Q, Bu Z, Vzorov A, Yang C, Compans RW: Virus-like particleand DNA-based candidate AIDS vaccines. Vaccine 2003,21:638-643.

60.�

Reed SG, Campos-Neto A: Vaccines for parasitic and bacterialdiseases. Curr Opin Immunol 2003, 15:456-460.

This paper outlines recent developments in vaccine strategies againstcommon parasitic diseases such as malaria and leishmaniasis, as well asbacterial diseases like tuberculosis and meningitis.

61. McGrath S, Fitzgerald GF, van Sinderen D: The impact ofbacteriophage genomics. Curr Opin Biotechnol 2004,15:94-99.

62. Cusi MG, Zurbriggen R, Valassina M, Bianchi S, Durrer P,Valensin PE, Donati M, Gluck R: Intranasal immunization withmumps virus DNA vaccine delivered by influenza virosomeselicits mucosal and systemic immunity. Virology 2000,277:111-118.

63. Tung CH, Weissleder R: Arginine containing peptides asdelivery vectors. Adv Drug Deliv Rev 2003, 55:281-294.

64. Jones T, Allard F, Cyr SL, Tran SP, Plante M, Gauthier J,Bellerose N, Lowell GH, Burt DS: A nasal proteosome influenzavaccine containing baculovirus-derived hemagglutinininduces protective mucosal and systemic immunity.Vaccine 2003, 21:3706-3712.

65. Agrawal L, Haq W, Hanson CV, Rao DN: Generating neutralizingantibodies, Th1 response and MHC non-restrictedimmunogenicity of HIV-I env and gag peptides in liposomesand ISCOMs with in-built adjuvanticity. J Immune Based TherVaccines 2003, 1:5.

66. Demana PH, Davies NM, Berger B, Rades T: Incorporation ofovalbumin into ISCOMs and related colloidal particlesprepared by the lipid film hydration method. Int J Pharm 2004,278:263-274.

67. Sinha VR, Singla AK, Wadhawan S, Kaushik R, Kumria R, Bansal K,Dhawan S: Chitosan microspheres as a potential carrier fordrugs. Int J Pharm 2004, 274:1-33.

68. Jiang HL, Park IK, Shin NR, Yoo HS, Akaike T, Cho CS: Controlledrelease of Bordetella Bronchiseptica Dermonecrotoxin (BBD)vaccine from BBD-loaded chitosan microspheres in vitro.Arch Pharm Res 2004, 27:346-350.

69. Peyre M, Fleck R, Hockley D, Gander B, Sesardic D: In vivouptake of an experimental microencapsulated diphtheriavaccine following sub-cutaneous immunisation. Vaccine 2004,22:2430-2437.

70. Stanley AC, Buxton D, Innes EA, Huntley JF: Intranasalimmunisation with Toxoplasma gondii tachyzoite antigenencapsulated into PLG microspheres induces humoraland cell-mediated immunity in sheep. Vaccine 2004,22:3929-3941.

71. Keegan ME, Whittum-Hudson JA, Mark Saltzman W: Biomimeticdesign in microparticulate vaccines. Biomaterials 2003,24:4435-4443.


www.elsevier.com/locate/jconrel NE

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Journal of Controlled Releas

GE

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

Medicine, Veterin7rplatz 1, A-1210, Vienna, Austria. Tel.: +43 1

25077 2104; fax: +43 1 25077 2190.

E-mail address: [email protected]

(W. Jechlinger).

1. Introduction

The ability of DNA vaccination to induce immune

response has been shown in a range of infectious

disease models [1]. However, using naked DNA, it

has become apparent that high doses and/or multiple

immunizations are required to induce immune

responses in larger animals and humans [2,3]. There-

fore much effort is now focused on increasing the

e 102 (2005) 725–735

P. Mayrhofer et al. / Journal of Controlled Release 102 (2005) 725–735726

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efficiency of DNA vaccines. The various approaches

being taken to improve the potential of this emerging

technology are: (i) modification of vector DNA to

increase expression levels or to affect the type of

immune response [4], (ii) use of adjuvants to enhance

the immune response elicited by DNA vaccines [5,6],

and (iii) development of new delivery systems for

specific targeting and/or better DNA uptake [7–9]. As

the major role of antigen presenting cells (APCs) in

vaccination strategies became clear, the latter

approach especially deals with the recruitment of

APC’s to the application site or with the increase of

the transfection efficiency [10,11].

Delivery of vaccine antigens using bacterial

carriers has resulted in the elicitation of effective

humoral and cellular immune response due to the

adjuvant properties of these vector systems [7]. In this

report, we describe the development of a new, non-

living bacterial DNA delivery system based on the

bacterial ghost technology. Bacterial ghosts are empty

envelopes of Gram-negative bacteria, which are

produced by controlled expression of the cloned lysis

gene E of bacteriophage fX174 [12]. Expression of

gene E results in the formation of a protein E specific

transmembrane tunnel structure [13]. During this

process the cytoplasmic content is expelled through

this lysis tunnel due to the osmotic pressure difference

between the cytoplasm and the culture medium [13].

The bacterial ghosts retain the structural integrity of

native cell envelopes therefore representing excellent

vaccine candidates endowed with the intrinsic adju-

vant properties. In the extended bacterial ghost

system, heterologous proteins are either mixed with

ghosts, exported into the periplasmic space, or they

are attached to the cytoplasmic membrane via specific

hydrophobic membrane anchor-peptides [14–16]. In

addition to proteins, this system can be used for the

packaging of drugs, nucleic acids or other compounds

for numerous applications [9,16,17].

In this study we developed a novel technique to

immobilize plasmid DNA to the inner membrane of

bacterial ghosts. This plasmid DNA (the Self-Immo-

bilizing Plasmid, pSIP) carries a tandem repeat of a

modified lactose operator sequence (the lacOs sites),

which is recognized by a fusion protein composed of

the repressor of the lactose operon (LacI) and a

hydrophobic membrane anchoring sequence (LV)[14,15] derived from the lysis protein of phage

MS2. The LacI-LVfusion protein produced from pSIP

is immobilized in the cytoplasmic membrane of

Escherichia coli via the hydrophobic sequences of

the truncated lysis protein (LV), whereby the LacI

repressor domain simultaneously binds to the lacOs

elements on the pSIP. During the lysis process most of

the cytoplasmic proteins and nucleic acids are

expelled through the E-specific lysis tunnel but the

anchored plasmid DNA is retained in the bacterial cell

envelopes.

This novel platform technology is designed to

combine three essential steps in production of non-

living bacterial vaccine vectors for gene delivery: (i)

the preparation of a non-living bacterial delivery

vehicle with adjuvant properties, (ii) the amplification

of plasmid DNA, and (iii) the loading of this non-

living bacterial vector with plasmid DNA.

Using the SIP technology these three steps can be

performed in vivo in a single cost-efficient process

using the appropriate bacterial carrier co-transformed

with an E-specific lysis vector and the self-immobi-

lizing plasmid. The proof of principle of the SIP

technology has been demonstrated in E. coli by

comparing the supernatant and pellet fractions of

lysed bacterial cultures, which were transformed

either with the pSIP or two control plasmids, each

lacking an essential feature of the immobilization

system. Quantitative analysis using real-time PCR

reveals that only a negligible percentage of the SIP-

DNA is expelled during the lysis process, whereas

about 50% of control plasmids can be found in the

supernatants of the bacterial lysates.

2. Materials and methods

2.1. Bacterial strains, plasmids and growth conditions

Plasmids pAWJ [18], pBAD24 [19], pPHB-LV[20]and pBBR122 [21] as well as E. coli strains MC4100

[22] and MG1655 [23] have been described. Plasmid

pREP-4 was purchased from QIAgen (Hilden,

Germany). Bacteria were grown in Luria broth

(LB) supplemented with ampicillin (200 Ag/ml),

and kanamycin (50 Ag/ml) as required. For gene

expression from vectors derived from pBAD24 the

medium was supplemented with 0.5% l-arabinose

final concentration. Expression of the lysis gene E

P. Mayrhofer et al. / Journal of Controlled Release 102 (2005) 725–735 727

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from vector pKLys36 was achieved by a temperature

shift from 35 8C to 42 8C. Growth and lysis of

bacterial cells was monitored by measuring the

optical density at 600 nm (OD600 nm). To determine

the E-mediated inactivation of bacterial cultures,

colony forming unit counts were performed as

previously described [24].

2.2. DNA manipulations

Preparation of plasmid DNA and isolation of DNA

fragments was carried out using kits from QIAgen

(PCR Purification Kit and Gel Extraction Kit) and

Peqlab (E.Z.N.A. Plasmid Miniprep Kit I; PEQ-

LAB—Biotechnologie, Erlangen, Germany). Trans-

formation of bacterial strains and electrophoresis of

DNA were performed as described previously [25]. If

not otherwise stated, restriction enzymes, DNA-

modifying enzymes and nucleotides were obtained

from New England Biolabs (Frankfurt am Main,

Germany) or Roche Diganostics (Vienna, Austria).

The Pfu ploymerase used for PCR reactions was

purchased from Promega (Mannheim, Germany) and

used as specified by the manufacturer. For real time

PCR analysis, Dynazyme polymerase (Finnzymes Oy,

Espoo, Finland) and SYBR-Green I (Molecular

probes; Invitrogen, Lofer, Austria) were used accord-

ing to the instructions of the manufacturers.

2.3. Construction of plasmid pLacOs

A 306 bp PCR fragment containing an unrelated

spacer sequence derived from the archea phage

fCH1 [26] flanked by lacOs sites (underlined

sequence in primers lacos5 and lacos3) was

generated by PCR amplification using plasmid

pQE32a/h (a kind gift of Dr. Angela Witte;

unpublished data) as template and primers lacos5

(5V-CAGCAGATCGATAATTGTGAGCGCTCA-

CAATTGGAACTCAATACGACGGC-3V) and lacos3

(5V-CTGCTGATCGATAATTGTGAGCGCTCA-

CAATTAGCCGTGCCGGAGTA-3V) to introduce

ClaI restriction sites at the termini. The PCR reaction

containing 0.01 Ag/Al primer DNA, 0.2 mM dNTPs,

template DNA, 0.05 U/Al Pfu polymerase in Pfu

polymerase buffer was subjected to the following

conditions: 3 min 94 8C pre-denaturation, 30 cycles:

30 s 94 8C, 30 s 55 8C, 1 min 72 8C. The PCR

fragment was digested with ClaI and subsequently

cloned in the corresponding single site of the plasmid

pBAD24 resulting in the vector pLacOs.

2.4. Construction of plasmid pLacOsI

Using pREP-4 as template a 1118 bp PCR fragment

containing the lacI gene was obtained by PCR

amplification. Oligonucleotides laci5 (5V-CAGCAGC-CATGGGTAAACCAGTAAC GTATACGATGTC-3V)and laci3 (5V-TGCTGCCTGCAGCTGCTGTCATC-TAGACTGCC CGCTTTCCA-3V) containing NcoI

(laci5) and XbaI/PstI (laci3) as terminal restriction

sites were used as primers. As the NcoI site was used,

the lacI sequence starts with ATG as the first codon

thereby removing the original lacI start codon GGT.

The laci3 primer contains a stop codon in between the

XbaI and PstI sites. Amplifying the lacI sequence with

these primers resulted in a sequence with an additional

serine and arginine codon (derived from the XbaI site)

at the 3V-end. The PCR reaction was performed using

the Expand Long Template PCR System from Roche

according to the instruction of the manufacturer. The

reaction mixture was subjected to the following

conditions: 2 min 94 8C pre-denaturation, 30 cycles:

30 s 94 8C, 30 s 53 8C, 2 min 68 8C and finally 5 min

elongation at 68 8C. The PCR fragment was digested

with NcoI and PstI and subsequently cloned in the

corresponding single sites of plasmid pLacOs resulting

in vector pLacOsI. The vector pLacOsI contains the

lacI gene under the control of the araB promoter.

2.5. Construction of plasmid pSIP

A 196 kb fragment containing the membrane

anchoring sequence, encoding the 56 C-terminal amino

acids of the lysis protein L derived from the phage

MS2, was generated by PCR amplification. Plasmid

pPHB-LVwas used as template and primers ms2l5 and

ms2l3 to introduce terminal XbaI and PstI restriction

sites, respectively: ms215 (5V-CAG CAGTCTA-

GAGGGCCATTCAAACATGA-3V); ms2l3 (5V-TGCTGCCTGCAGTTAAGTA AGCAATTGCTG-

TAAAGTC-3V). PCR was performed as described

above (see Construction of plasmid pLacOs) except

for the annealing temperature, which was 53 8C. Theresulting PCR fragment was digested with XbaI and

PstI and subsequently cloned in the corresponding


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single sites of plasmid pLacOsI resulting in the vector

pSIP. The vector pSIP contains the lactose repressor

fused to 56 C-terminal amino acids of the lysis protein

L under control of the araB promoter.

2.6. Construction of plasmid pDSIP

The vector pDSIP was constructed by digesting

pSIP with ClaI. The vector was eluted from an agarose

gel to remove the fragment containing the lacOs sites

and the spacer sequence. The eluted plasmid fragment

was religated resulting in the vector pDSIP.

2.7. Construction of plasmid pKLys36

A 1.98 kb fragment containing an E-specific lysis

cassette (cI857 repressor-EpRmut-gene E) of plasmid

pAWJwasgeneratedbyPCRamplification(948C,1min;

55 8C, 1 min; 72 8C 2 min; 30 cycles). The following

primers were used to introduce NcoI restriction sites:

sense (cI-Pvu) 5V-TTCCCCCCATGGCGATCGGCT-CAATTGTTATCAGC–3V; anti-sense (E-Pvu) 5V-TTAAAAACCATGGCGATCGCTCGGTACGGT-

CAGGC–3V. The PCR fragment was cloned in the

corresponding restriction site of the broad-host range

low copy number plasmid pBBR122. The resulting

plasmid pKLys36 contains the E-specific lysis cassette,

a broad-host rangeoriginof replication andakanamycin

resistancegene.

2.8. DNA precipitation from culture broth and pellet

of lysed E. coli cultures

Twenty-milliliter samples were taken at each time

point (onset and end of lysis). These samples were

centrifuged at 10,000�g for 15 min. The supernatants

of the lysed culture were collected in a SS-34

centrifuge tube, whereas the pellets were washed three

times with 500 Al of 10 mM Tris–Cl; pH 8.00. After

washing the pellet, the supernatant of the washing step

was added to the previously collected culture super-

natant samples. Plasmid DNA from the washed pellets

was isolated using the Peqlab E.Z.N.A Plasmid

Miniprep Kit I and plasmid DNA of the supernatant

sample were extracted using the CTAB method as

previously described [27] with some modifications.

Briefly, the supernatant was filtered using 0.22-Amfilter to avoid contamination with cellular debris

before 2.5 ml of 5% CTAB (w/v in 0.5 N NaCl) was

added to 20 ml supernatant of the lysates. After

centrifugation at 10,000�g for 15 min at 4 8C, thepellet was resuspended in 400 Al 1.2 N NaCl and

transferred to a 1.5-ml tube. The DNAwas precipitated

with 2.5 volumes of absolute Ethanol, washed with 1

ml 70% EtOH and air-dried. To remove the bulk of

chromosomal DNA, the preparation was further

purified with QIAgen Extraction kit according to the

instructions of the manufacturer.

The amount of DNA recovered from the super-

natant using the CTAB method was evaluated.

Duplicates of six DNA standards in 20 ml LB were

extracted using the CTAB method and quantified by

real-time PCR in duplicates. This revealed that

93F3.2% of the DNA could be recovered using this

CTAB protocol. The evaluation of the Peqlab mini

preparation kit was performed as well by parallel

extractions of six DNA standards in duplicates

followed by quantification in duplicates. This revealed

that 84.6F12.7 of the loaded DNA was extracted.

2.9. Quantification of plasmid DNA using real time

PCR

A real time PCR approach was optimized to

quantify the amount of pKLys36, pSIP, pDSIP and

pLacOsI in the pellet as well as in the supernatant of

lysed cultures. The template DNA was prepared from

supernatants and pellets of lysed bacterial cultures as

described above. Real time PCR was performed in

200 Al reaction tubes containing 200 AM dNTP, 1

AM of each primer, 2.5 Al of polymerase buffer

(10�), 5 Al of 1:100 diluted extracted DNA as

template, 0.25 Al SYBR-Green I and 0.25 AlDynazyme polymerase (2 U/Al) in a final volume

of 25 Al. Primers binding to the ampicillin resistance

gene (fwd: 5V-ATGAGTATTCAACATTTCCGTGTC-3V; rev: 5V-TTACCAATGCTTAATCAGTGAGG-3V)of plasmids pSIP, pDSIP and pLacOsI, respectively,

were used to amplify a 860 bp PCR fragment. To

quantify the plasmid pKLys36, a pair of primers was

designed to amplify gene E as follows: Fwd: 5VGCTGGACTTGGGATAC-3V, rev: 5V-GACATTACAT-CACTCCTTCTGC-3V. Tenfold serial dilutions

(10�2–10�6) of the plasmid pLacOsI, which was

prepared using a DNA minipreparation kit (Peqlab)

were used as the standard for reactions amplifying


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the ampicillin resistance gene. As with the pLacOsI

standard, tenfold serial dilutions (10�2–10�6) of the

plasmid pKLys36 were used as standard for reactions

amplifying gene E. For all PCR reactions the

following conditions were applied: 1 min 94 8C, 1min 60 8C and 1 min 72 8C (30 cycles). The data

shown in Fig. 4 represents an average of 3 measure-

ments for each construct. Samples for real time PCR

analysis were taken at the first time-point when the

OD600 of the cultures was decreasing (onset of lysis)

or at the last time-point measured (end of lysis). The

Rotor Gene 2000, real time Cycler (Corbett

Research, Mortlake, Australia) was used as specified

by the manufacturer. Data had been analysed with

special software, supplied by the manufacturer.

2.10. SDS-PAGE and Western blotting

Protein samples were heated to 95 8C for 5 min in

sample buffer and separated on a 12% SDS-PAGE

gel according to Laemmli [28]. Proteins were trans-

ferred to nitrocellulose membranes by semidry

electro blotting. Membranes were blocked for 1 h

in 5% low fat milk powder in TBS. Proteins were

detected either with polyclonal rabbit antiserum to

the Lac Repressor or to the MS2 lysis protein L. The

a-LacI antiserum was purchased from Stratagene

(Strategene Europe, Amsterdam, The Netherlands),

the a-MS2 serum was a kind gift from Joachim-

Volker Hfltje (Max-Planck-Institute for Evolutionary

Biology in Tqbingen, Germany). Anti Lac Repressor

serum was diluted 1:3000, anti L serum was diluted

1:350 in TBS, 0.5% BSA, 0.05% NaN3. Alkaline

phosphatase coupled to goat anti-rabbit antibodies

from Sigma was used in a 1:5000 dilution. The

membranes were incubated for 1 h with the

respective antiserum dilution. To stain the antigen–

antibody complex, BCIP and NTB from Roche in

alkaline phosphatase buffer were used as recom-

mended by the supplier. Antisera were pre-treated

with acetone powder from the appropriate E. coli

strain as described previously to avoid unspecific

binding [29].

2.11. Preparation of membrane fractions from E. coli

Five-hundred-milliliter cultures of E. coli MC4100

transformed either with pLacOsI or pSIP were grown

at 28 8C. Expression of the repressor protein LacI

from plasmid pLacOsI as well as the LacI-LV fusionprotein from plasmid pSIP was induced with l-

arabinose at an OD600 of 0.3. Cells were harvested

after 2 h of induction. Membrane fractions were

prepared according to Schnaitman [30,31] with a

slight modification. Briefly, prior to cell disruption,

the buffer was supplemented with the complete mini

protease inhibitor cocktail from Roche, according to

the manufacturer’s instructions. The cells were

opened by two times passing through the French

Press. The membranes were subsequently collected

by high-speed centrifugation (1 h, 4 8C, 105,000�g).

The proteins in the supernatant fraction of the

centrifugation step were considered as the soluble

cytoplasmic protein fraction. The pellet after high-

speed centrifugation, the membrane fraction, was

further treated according to Schnaitman to separate

the inner (cytoplasmic) and outer membrane fraction.

After separation a 1-ml aliquot of the inner mem-

brane band in the sucrose gradient was collected for

further analysis.

3. Results

3.1. Expression and membrane anchoring of protein

LacI-LV

Vectors pLacOsI and pSIP (Fig. 1) were analyzed

for their ability to express the cloned LacI or the LacI-

LVfusion proteins in E. coli MC4100, respectively. To

determine whether the fusion protein is anchored in

the cytoplasmic membrane, membrane fractions were

prepared after protein expression and subjected to

SDS-PAGE and Western Blot analysis. Aliquots of the

inner membrane fraction, the cytoplasmic protein

fraction as well as whole bacterial cells were

analyzed. The expressed proteins were detected with

both, antibodies to the LacI protein as well as

antibodies to the LV-peptide part of the fusion protein.

There is no detectable signal when proteins expressed

from E. coli MC4100 (pLacOsI) were analyzed using

anti-LV antibodies (Fig. 2A, lanes 1–3). Analysis of

samples prepared from E. coli MC4100 (pSIP)

revealed a signal at the molecular weight expected

for the LacI-LVhybrid protein. This 45 kDa protein

can be detected in whole bacterial cells, in the inner

Fig. 1. The Self-Immobilizing Plasmid pSIP (A) and the control plasmids each lacking essential features for immobilization pDSIP (B) and

pLacOsI (C), respectively. Ap, ampicillin resistance gene; ColE1, origin of replication; lacI, lac repressor gene; LV, amino acids 21–75 of MS2 L

protein; PBAD, arabinose-inducible promoter; araC, repressor/inducer of the PBAD promoter; Os, lac operator sites with a tenfold higher binding

affinity for the lac repressor than the wild-type operator sites; spacer, unrelated spacer sequence derived from the archea phage fCH1.


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membrane fraction as well as in the cytoplasmic

fraction (see Fig. 2A, lanes 4–6).

Aliquots of the same samples were applied for

protein detection with anti-LacI antiserum (Fig. 2B).

The LacI protein expressed from MC4100 (pLacOsI)

can be detected at the expected molecular weight of 38

kd in whole bacterial cells (Fig 2B, lane 1), in the

cytoplasmic fraction (Fig. 2B, lane 3) but not in the

inner membrane fraction (Fig. 2B, lane 2). As expected

Fig. 2. Western blot analysis for the detection of LacI-LVwith anti-L antiser

MC4100 (pLacOsI) whole cells; lane 2, E. coli MC4100 (pLacOsI) inner

fraction; lane 4, E. coli MC4100 (pSIP) whole cells; lane 5, E. coli MC41

cytoplasmic fraction.

analysis of the LacI-LVsamples derived from E. coli

MC4100 (pSIP) using anti-LacI antiserum revealed the

same signals as when detection was performed with

anti-LV-antiserum (Fig. 2A, lanes 4–6).

The Western blot analyses show that the LacI-LVfusion protein, but not the lactose repressor alone, can

be found in the membrane fraction, indicating that the

fusion protein is associated with the cytoplasmic

membrane via the LVsequence.

um (A) and with anti-LacI antiserum (B), respectively: lane 1, E. coli

membrane fraction; lane 3, E. coli MC4100 (pLacOsI) cytoplasmic

00 (pSIP) inner membrane fraction; lane 6, E. coli MC4100 (pSIP)


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3.2. Self-immobilization of plasmid pSIP and

quantification of DNA by real time PCR

A schematic system description is provided in

Fig. 3A. Co-transformed E. coli MC4100 harboring

the lysis plasmid pKLys36 and the vectors pLacOsI,

pDSIP or pSIP, respectively, were monitored for the

lysis and immobilization behavior. Control plasmids

pDSIP and pLacOsI should not be immobilized in the

cytoplasmic membrane as they lack the lacI recog-

nition sites or the LV anchor sequence, respectively.

Protein expression from the araBAD promoter was

induced with l-arabinose at an optical density of

about OD600 0.1. Induced cells were grown to an

OD600 of about 0.3 and subsequently shifted to 42

8C to induce expression of the lysis protein (Fig.

3B). Expression of the LacI-LVhybrid protein for 75

min in MC4100 (pSIP) and MC4100 (pDSIP) does

not significantly influence the lysis behavior com-

pared to MC4100 (pLacOsI) (Fig. 3B). Analyzing

Fig. 3. Schematic system description (A) and growth/lysis experiments with

pKLys36 (B). (A) Cells transformed with pSIP and the lysis plasmid pK

promoter. The hybrid protein integrates in the bacterial membrane and bind

inactivate the cI857 repressor for lysis induction. The cytoplasmic content i

the bacterial ghost. (B) Growth and lysis of E. coli MC4100 (pKLys36) co-

by arrows cells were grown at 35 8C to an OD600 of approximately 0.1 fo

shifted to 42 8C for induction of lysis. Open symbols indicate the ghost f

CFU counts revealed that at least 99.9% of bacteria

were killed by protein E mediated lysis.

DNA was precipitated from the culture super-

natant as described in Materials and methods. The

control vectors pDSIP and pLacOsI could be precipi-

tated from the culture supernatant after lysis, whereas

no visible band was found when a corresponding

volume of the supernatant preparation derived from a

lysed MC4100 (pSIP) culture was applied on the

agarose gel (data not shown). For the initial experi-

ments E. coli MC4100, which is a D(arg-lac)U169

strain, was chosen to avoid interference with the

endogenous lac operon. Performing the same experi-

ments with an E. coli wild type strain (MG1655) led

to the same results as with MC4100 suggesting that

the principle of self-immobilization is independent

from the genetic background of the host. These

findings indicate that the plasmid pSIP is immobilized

in the cytoplasmic membrane of the bacterial ghosts,

whereas the control plasmids are expelled in the

E. coli MC4100 harbouring pSIP or control plasmids together with

Lys36 are grown at 35 8C and LacI-LVis expressed from the PBADs to lacOs sites on the pSIP vector. The culture is shifted to 42 8C to

ncluding the lysis plasmid is expelled. The pSIP vector is retained in

transformed with pLacOsI (E), pSIP (n) or pDSIP (x). As indicatedr induction of protein expression with l-arabinose and subsequently

ormation.


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culture broth during the lysis process. However, to

give clear evidence of the self-immobilization these

DNA preparations were further analyzed. The amount

of pSIP DNA and control plasmids in the pellet and in

the supernatant of bacterial lysates were quantified by

real time PCR. To analyze the general ratio of pDNA

expelled to the culture broth and plasmids unspecifi-

cally retained after protein E mediated lysis, real-time

PCR was performed with samples derived from E.

coli MC4100 (pKLys36) at first. As it is shown in Fig.

4, row 4, by onset of the lysis process, only low

amounts of plasmid pKLys36 are detectable, whereas

at the end of lysis process around 50% of the lysis

plasmid were expelled. The same experiments were

performed with E. coli MC4100, which harbored the

lysis plasmid together with either pSIP (Fig. 4, row 1)

or pLacOsI (Fig. 4, row 2) or pDSIP (Fig. 4, row 3).

Comparable to pKLys36, about 45% of the control

plasmid pDSIP and pLacOsI were lost during the lysis

process, whereas the loss of immobilized plasmid

Fig. 4. Quantification of plasmid DNA in the pellet and in the

supernatant of lysed E. coli MC4100 cultures by real time PCR.

100% represents the total amount of plasmid DNA measured in the

pellet and supernatant of the corresponding lysates. Row 1, E. coli

MC4100 (pSIP, pKLys36); row 2, E. coli MC4100 (pLacOsI,

pKLys36); row 3, E. coli MC4100 (pDSIP, pKLys36); row 4, E. coli

MC4100 (pKLys36). Dark-gray bars represent the percentage of

plasmid DNA measured in the pellets of lysed cells, whereas white

bars indicate the percentage of plasmid DNA detected in the

supernatants of lysates. Samples for real time PCR analysis were

taken at the first time-point when the OD600 of the cultures was

decreasing (onset of lysis) or at the last time-point measured (end of

lysis) (Fig. 3). The error bars indicate the standard deviation of three

individual experiments.

pSIP was on average 7%. We calculated that roughly

10 Ag pSIP DNAwas immobilized per mg of bacterial

ghosts.

4. Discussion

Immobilization of the target sequence in the

cytoplasmic membrane of a bacterial delivery vector

is the key element of this novel platform technology.

The combination of the SIP technology and protein E

mediated lysis results in a bself-loadingQ, non-livingbacterial DNA carrier system. Using the SIP, the target

DNA can be amplified and retained in the bacterial

ghosts, while the bulk of the cytoplasmic content is

expelled into the culture broth during the lysis

process.

The central element of the SIP technology is the

LacI-LV hybrid protein. Results indicate that the

fusion of the highly hydrophobic membrane span-

ning sequence (56 amino acids) used in this study

does not impair the DNA binding functionality of the

lactose repressor protein. The results of the Western

Blot analyses show that LacI-LV is associated with

the inner membrane solely due to its hydrophobic

sequence. Since the LacI-LVfusion protein proves to

be functional, we conclude that the membrane

anchoring fragment does not interfere with the

DNA binding properties of the repressor protein

and vice versa (e.g. by inducing conformational

changes in the DNA binding domain due to

improper folding or by inhibiting the essential

oligomerization).

The LacI-LVhybrid has also been detected in the

cytoplasmic fraction after high-speed centrifugation,

indicating that the hybrid is still soluble although

the repressor protein was fused to a highly hydro-

phobic sequence. This might result from the fact

that a 38.5 kDa protein was fused to a 6.4 kDa

peptide and that the larger protein simply plays the

dominant role in terms of physico-chemical charac-

teristics including solubility. The harsh mechanical

disruption of bacterial cells by two times passing

through the French Press for membrane fraction

preparation (described in Sections 2.11. and 3.1)

completely destroys the integrity of the membrane

system, thereby releasing associated proteins in the

cytoplasmic fraction. Apart from the formation of a


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trans-membrane tunnel, the protein E mediated lysis

process does not alter the bacterial envelope and will

therefore not release that much, if any immobilized

hybrid proteins.

The repressor of the lactose operon was chosen

because the structural and functional stability of this

molecule has been demonstrated in various studies,

e.g. (i) it has been shown that fusions to the C-

terminus of LacI do not alter the DNA binding

properties of the protein even if the fusion protein is

as large as h-galactosidase [32], (ii) several mod-

ifications of this repressor had been performed

without significant loss of the DNA binding

capability [33–35] and (iii) it has been demonstrated

that plasmid expressed LacI fusion proteins bind to

the vector encoding them, when the lactose operator

sequence is integrated in this construct [36,37].

Apart from its functional stability, the high stability

of the protein/DNA complex is another advanta-

geous feature of the lactose operator/repressor

system. The repressor tetramer has two DNA

binding sites and exhibits strong cooperative bind-

ing to DNA molecules containing two suitably

spaced operator sequences, thereby forcing the

intervening DNA into a loop structure [38,39]. This

complex is further stabilized using a symmetric

variant of the wild type operator with a tenfold

higher affinity, the lacOs site [40,41].

Pellet as well as supernatant DNA preparations

corresponding to an identical volume of culture broth

was subjected to real-time PCR analysis. The DNA

content of both fractions calculated on basis of the

real-time PCR assays was summarized and defined as

100%. Therefore the calculations made are based on

isolated DNA only, i.e. losses during the preparation

are not considered. Furthermore, different methods

have been applied to isolate DNA from the culture

broth (CTAB method) and the lysed bacterial pellet

(Peqlab kit). Therefore, the calculated ratio of plasmid

DNA might not properly reflect the exact ratio in the

culture broth and the pellets, even though the

comparison of the methods revealed very similar

yields if they are used to isolate DNA from the

supernatant or the bacterial ghost pellet, respectively.

Although the bacterial ghost pellets have been

washed several times, it cannot be excluded that

there is still plasmid DNA unspecifically associated

with these cell envelopes. Evaluation of the lysis

efficiency revealed only 0.1% unlysed cells indicating

only a negligible contribution of these survivors to

the overall result. Based on these findings it is

probably not possible to exactly determine the

percentage of control plasmid DNA (pLacOSI,

pDSIP) retained in bacterial ghosts compared to a

DNA content of 100%, which is the number of

plasmids in intact (unlysed) bacterial cells. However,

the experimental setup for this study and the

calculations based on it were designed to answer

the question if there is more pSIP retained in bacterial

ghosts than control plasmids. As the conditions

mentioned above are similar for each investigated

plasmid, the results for the different control plasmids

and the pSIP can naturally be compared to each other.

The significant higher level of retained pSIP clearly

demonstrates the efficiency of the self-immobilization

system. It has been shown that there is still residual

genomic DNA as well as unspecifically associated

plasmid DNA in ghost preparations, if not treated

with a nuclease [24]. However, many immunization

studies have been performed with bacterial ghosts

[9,42–44]. In none of these studies there has been

evidence for a negative effect of the residual DNA on

the immune response.

Recently it has been demonstrated that bacterial

ghosts are excellent vehicles for the delivery of in

vitro loaded plasmid DNA [9]. They showed high

efficiency in the transfection of macrophages and

primary dendritic cells (52% to 60%). Ghost-mediated

DNA delivery resulted in the elicitation of more

efficient humoral and cellular immune responses than

using equal amounts of naked DNA [9]. At present

there is no evidence whether the strong interaction

between the membrane-anchored LacI protein and the

plasmid DNA hampers or even improves immune

responses. Immunization studies are planned which

will compare ghosts mediated immune responses

against target antigens encoded on pSIP or on

conventional in vitro loaded plasmids. To formulate

a non-living DNA delivery system, normally the

production of the carrier, the pDNA as well as the

loading of the carrier with the pDNA are separate,

laborious and therefore cost intensive processes. In

this report we demonstrated that the SIP system is able

to combine these essential steps in a single in vivo

process, resulting in a non-living bacterial DNA

delivery vehicle.


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Appendix A. Supplementary data

Supplementary data associated with this article can

be found, in the online version, at doi:10.1016/

j.jconrel.2004.10.026.

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www.elsevier.com/locate/addr

DTD 5

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Advanced Drug Delivery Rev

OFBacterial ghosts as antigen delivery vehicles

Ulrike Beate Mayra, Petra Walchera, Chakameh Azimpoura, Eva Riedmanna,b,

Christoph Hallera, Werner Lubitza,b,TaInstitute of Microbiology and Genetics, University of Vienna, A-1090 Vienna, Austria

bBIRD-C GmbH and CoKEG, Schoenborngasse 12, A-1080 Vienna, Austria

Received 31 January 2004; accepted 25 January 2005
O
ORRECTED PR

Abstract

The bacterial ghost system is a novel vaccine delivery system unusual in that it combines excellent natural intrinsic adjuvant

properties with versatile carrier functions for foreign antigens. The efficient tropism of bacterial ghosts (BG) for antigen

presenting cells promotes the generation of both cellular and humoral responses to heterologous antigens and carrier envelope

structures. The simplicity of both BG production and packaging of (multiple) target antigens makes them particularly suitable

for use as combination vaccines. Further advantages of BG vaccines include a long shelf-life without the need of cold-chain

storage due to their freeze-dried status, they are safe as they do not involve host DNA or live organisms, they exhibit improved

potency with regard to target antigens compared to conventional approaches, they are versatile with regards to DNA or protein

antigen choice and size, and as a delivery system they offer high bioavailability.

D 2005 Elsevier B.V. All rights reserved.

Keywords: Target antigen; Gram-negative bacterial envelope; Particle presentation technology; DNA vaccine; Adjuvant; Delivery system;

Bacterial ghosts

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

2. Production of bacterial ghosts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

3. Bacterial ghosts as candidate vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

3.1. Parenteral immunization with bacterial ghosts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

U0169-409X/$ - s

doi:10.1016/j.ad

Abbreviation

dendritic cells;

membrane; OMP

target antigen; T

T Correspondi

+43 1 406 50 9

E-mail addr

iews xx (2005) xxx–xxx

NC

ee front matter D 2005 Elsevier B.V. All rights reserved.

dr.2005.01.027

s: APC, antigen presenting cells; APP, Actinobacillus pleuropneumoniae; BG, bacterial ghosts; CPS, cytoplasmic space; DC,

GFP, green fluorescent protein; IM, inner membrane; LPS, lipopolysaccharide; MBP, maltose binding protein; OM, outer

, outer membrane protein; PBMC, peripheral blood derived monocytic cells; PPS, periplasmic space; StrpA, streptavidin; TA,

CP, toxin-co-regulated pili; VCG, Vibrio cholerae ghosts.

ng author. Institute of Microbiology and Genetics, University of Vienna, A-1090 Vienna, Austria. Tel.: +43 1 4277 54670; fax:

3.

esses: [email protected], [email protected] (W. Lubitz).

ADR-11331; No of Pages 11

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3.2. Induction of cytokines by bacterial ghosts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

3.3. Mucosal immunizations with bacterial ghosts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

4. Bacterial ghost system as carrier of foreign target antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

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UNCORREC

1. Introduction

Subunit vaccines composed of purified compo-

nents can be produced from many microorganisms;

however, they are often poorly immunogenic neces-

sitating an appropriate adjuvant in the vaccine

formulation. Similarly, for DNA vaccines to reach

their full potential, new vaccine delivery systems need

to be developed which better activate mucosal

immune responses.

The bacterial ghost system is one such vaccine

delivery system, combining targeting of antigen

components to antigen presenting cells (APC) and

providing the required adjuvant activity without the

need for further additions. BG are produced by protein

E-mediated lysis of Gram-negative bacteria. They are

non-living bacterial envelopes, which maintain the

cellular morphology and native surface antigenic

structures including bioadhesive properties of the

natural cell. Lipopolysaccharide (LPS) present in the

outer membrane (OM) does not limit the use of BG as

vaccine candidates because of the minimal toxicity of

cell-associated LPS compared to free LPS. The

intrinsic adjuvant properties of BG enhances T-cell

activation and systemic, mucosal and cellular immun-

ity to target antigens.

E-mediated lysis has been achieved in various

Gram-negative bacteria, including Escherichia coli

K12, enterohaemorrhagic (EHEC) and entertoxigenic

(ETEC) strains, Actinobacillus pleuropneumoniae,

Bordetella bronchiseptica, Erwinia cypripedii, Heli-

cobacter pylori, Klebsiella pneumoniae, Mannheimia

haemolytica, Pasteurella multocida, Pseudomonas

putida, Ralstonia eutropha, Salmonella typhimurium

and enteritidis strains, and Vibrio cholerae. This broad

spectrum of bacteria shows that E-mediated lysis most

probably works in every Gram-negative bacterium,

provided that the E specific lysis cassette can be

introduced into the new recipient by an appropriate

vector allowing tight repression and induction control

PROOFof lethal gene E. Although BG have been used as

vaccine candidates against their own envelope struc-

tures, a more practical use remains versatile carrier and

adjuvant vehicles for foreign target antigens of

bacterial or viral origin. As described in more detail

in the following sections, BG have the capacity for

pre-lysis localization of target antigens in or a

combination of the OM, the inner membrane (IM),

the periplasmic space (PPS) and the internal lumen of

the cytoplasmic space (CPS). The choice of antigen

compartmentalization gives the bacterial ghost system

significant potential for the challenges of constructing

subunit or DNA human and veterinary vaccines.

ED2. Production of bacterial ghosts

BG are produced by expression of cloned gene E

from bacteriophage PhiX174 resulting cell lysis in

Gram-negative bacteria. Expression of gene E can be

placed under transcriptional control of either the

thermosensitive EpL/pR-cI857 promoter, or under

chemical inducible promoter repressor systems, like

lacPO or the tol expression system [1–3]. Mutations to

the EpR promoter/operator regions have resulted in

new expression systems, which stably repress gene E

expression at temperatures of up to 37 8C, but stillallowed induction of cell lysis at a temperature range

of 39–42 8C [4]. Alternatively, by combining the EpRpromoter/cI repressor system with the lacI/lacPO for

control of gene E expression, a cold-sensitive system

for ghost formation by lowering the growth temper-

ature of the bacteria from 37 8C or higher to 28 8C or

lower has been obtained [5].

Gene E codes for a membrane protein of 91 amino

acids, which is able to fuse inner and outer mem-

branes of Gram-negative bacteria [6,7], forming an E-

specific lysis tunnel through which the cytoplasmic

content is expelled [8]. The remaining empty CPS

(internal lumen) of the bacteria is largely devoid of

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nucleic acids, ribosomes or other constituents,

whereas the IM and OM structures of the BG are

well preserved [6,9]. The IM remains intact during

expulsion of cytoplasmic material and electron micro-

graphs clearly show a sealed PPS [6,8]. The protein E-

induced occurrence of lysophosphatidylethanol-amine

in the host cell membrane most probably facilitates

the IM and OM fusion [10]. Although E-mediated

lysis is dependent on activities of the autolytic system

of the bacteria peptidoglycan, sacculi prepared from

E-lysed cells remain intact emphasizing that the

overall composition of this rigid layer is not changed

by the E-mediated process. The diameter of the E-

specific transmembrane tunnel structure ranges

between 40 and 200 nm and is determined by the

mesh size of the surrounding murein [11]. Electron

microscopic studies emphasize that the E-specific

transmembrane tunnel structure is not randomly

distributed over the cell envelope but is restricted to

areas of potential division sites, predominantly in the

middle of the cell or at polar sites [6,8]. Analysis of E-

mediated lysis in bacterial mutant strains with defects

in cell division suggest that initiation of cell division

rather than specific functions of the septosome plays

an essential role in protein E-mediated lysis [9,11].

The E-specific membrane fusion process can be

divided into three phases including the integration of

protein E into the IM, followed by a conformational

change of protein E and assembly into multimers at

potential cell division sites [12]. The mechanism for

the conformational change is most probably a cis–

trans isomerization of the proline 21 residue within

the first membrane-embedded a-helix of protein E

[13]. The local fusion of the IM and OM is achieved

by a transfer of the C-terminal domain of protein E

towards the surface of the OM of the bacterium.

BG have been developed for envelope and/or

heterologous antigen presentation from a range of

important Gram-negative bacterial pathogens includ-

ing Francisella tularensis, Brucella melitensis, enter-

otoxigenic and enterohemaorrhagic E. coli (EHEC,

ETEC), and V. cholerae. To date, immune responses

against P. multocida, Mannheimia (former Pasteur-

ella) haemolytica, A. pleuropneumoniae and V.

cholerae have been assessed in several animal models

for parenteral, oral and aerogenic modes of delivery,

in view of human and veterinary applications. The BG

particle presentation technology for target antigens to

induce an immune response against the target antigens

has also been studied extensively by our group

(reviews in [14–17]) and promising results of recent

studies will be presented below.

ED PROOF

3. Bacterial ghosts as candidate vaccines

3.1. Parenteral immunization with bacterial ghosts

Bovine pneumonic pasteurellosis caused by M.

haemolytica is a serious disease leading to death in

cattle if it remains untreated. Pilot subcutaneous BG

immunization studies of mice and rabbits with either

P. multocida- or M. haemolytica-ghosts induced

antibodies cross-reactive to heterologous Pasteurella

strains. The number of proteins in Pasteurella whole-

cell protein extracts recognized by the sera constantly

increased during the observation period of 50 days.

More importantly, dose-dependent protection against

homologous nasal challenge was observed in mice

immunized with P. multocida ghosts [18].

Following on from this work, M. haemolytica

ghosts cattle immunization studies were performed

using a cattle lung challenge model. Protective

immunity of cattle against homologous challenge

was induced by alum-adjuvanted M. haemolytica

ghosts. It is important to note that BG do not need

additional adjuvants to induce protective immunity.

However, alum was added to the ghost vaccine

preparation in this study to compare its antigenicity

with a commercially available vaccine [19].

Bacterial ghosts have been tested as a vaccine

against swine pleuropneumonia, a disease with a high

mortality rate in pigs. Intramuscular immunization of

pigs with A. pleuropneumoniae (APP) ghosts or

formalin-inactivated APP whole-cell bacteria pro-

tected for clinical disease in both vaccination groups.

The protective efficacy was evaluated by clinical,

bacteriological, serological and post-mortem exami-

nations. Immunization with BG did not cause clinical

side-effects. After aerosol challenge, the control group

of pigs developed fever and pleuropneumonia. In both

vaccination groups, animals were fully protected

against clinical disease and lung lesions, whereas

colonization of the respiratory tract with APP was

prevented by BG immunization alone. The induction

of specific mucosal antibodies as detected in the

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bronchoalveolar lavage suggests that immunization

with BG induces antibody populations specific to

non-denaturated surface antigens. In this study at

least, APP-BG are more efficacious in protecting pigs

against colonization and infection than the inactivated

whole-cell vaccine [20]. Indications for a cross-

protective potential of the ghost vaccine were sup-

ported by studies on rabbit hyperimmune sera [21].

Cholera is a significant cause of morbidity and

mortality in humans and a vaccine is very much

needed. To this end, V. cholerae ghosts were produced

and assessed in a rabbit model. Rabbits were immu-

nized s.c./i.m. with ghosts prepared from V. cholerae

strains of O1 or O139 serogroup following growth

under culture conditions which favour or repress the

production of toxin-co-regulated pili (TCP). Immuno-

blotting confirmed the TCP status of V. cholerae

ghosts (VCG), which retained the cellular morphol-

ogy and surface component profile of viable bacteria.

Sera from immunized rabbits was assayed for anti-

bodies to lipopolysaccharide (LPS) and to TCP.

Regardless of the TCP status of the VCG preparations

used for immunization, all animals produced anti-

bodies to LPS as demonstrated in bactericidal assays.

Anti-LPS antibodies were likely responsible for

conferring passive immunity in the infant mouse

cholera model to challenge with the homologous

O139 strain. Cross-protective anti-TCP antibody was

generated only in rabbits immunized with TCP-

positive VCG. This sera induced protection against

heterologous challenge [22].

3.2. Induction of cytokines by bacterial ghosts

To investigate the activation of APC by BG we

studied the in vitro uptake of VCG and E. coli BG in

dendritic cells (DC) and RAW macrophages and the

induction of inflammatory mediators in the THP-1

human macrophage cell line. The synthesis of inflam-

matory mediators such as TNF-a in the THP-1 cell line

was stimulated by a hundred-fold higher dose of VCG

than the corresponding amount of free LPS [23,24].

These results support in vivo experiments in rabbits

with intravenous administration of E. coli BG. Below

a threshold dose, no toxic effects of BG administration

could be detected whilst the doses used stimulated

significant humoral immune responses [25]. Signifi-

cant production of IL-12 in DC was induced by E. coli

ED PROOF

BG. Secretion in DC of cytokines TNFa and IL-12

was increased 37 and 18-fold, respectively, whereas in

peripheral blood monocytes the secretion of TNFa and

IL-12 increased only twofold. These results suggest

that BG stimulate the activation of cellular Th1

immune responses. In addition, maturation of DC is

a prerequisite for efficient stimulation of T cells and

exposure of DC to BG resulted in a marked increase in

their ability to activate T cells. Thus, BG are promising

carrier and adjuvants for target antigens.

3.3. Mucosal immunizations with bacterial ghosts

Different routes of mucosal immunizations with

BG (aerogen, oral, intranasal, intravaginal, intraocular

or rectal) have been assessed in various animal models.

Binding and uptake of BG into APC is dependent on

surface structures of the envelope being recognized by

toll like receptors on human or animal cells.

Inhalation and deposition of BG within the airways

are the initial steps preceding adherence of the vaccine

candidates to the respiratory tract. Once BG are

deposited in the lung lining fluids (mucosa), they

are rapidly cleared by alveolar macrophages and

translocation of deposited particles in the mucus also

lead to clearance via the gastrointestinal tract [26].

APP-BG, after evaluation in the pig lung infection

model was then further assessed in an aerosol

immunization model [27]. The model utilized com-

puter-controlled standardized inhalation conditions for

the recipient pigs. APP-BG aerosol immunization has

been shown to induce complete protection against

pleuropneumonia in pigs [28].

The capability of BG to induce a T-cell-mediated

immune response was studied following uptake of

APP ghosts by primary APC of pigs. Specific T-cell

responses were detected after in vitro re-stimulation of

primed blood T cells with APP ghosts. In addition, we

investigated uptake of APP BG by DC and subse-

quent DC activation. DC are known to be phagocytic

in specific immature stages of development. Follow-

ing the internalization and processing of the antigens,

increased expression of MHC class II molecules in

APC was shown 12 h after their exposure to BG.

Together with the specific T-cell response to the

antigen processed by the APC, it could be demon-

strated that porcine APC have the capacity to

stimulate antigen-specific T cells after internalization

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and processing of the antigen. The data suggest that

BG effectively stimulate monocytes and macrophages

to induce TH1-type cytokine directed immune

responses. DC stimulated by BG can be used for

active immunization and immunotherapy in situ [24].

The immunological and protective efficacy of V.

cholerae ghosts (VCG) expressing TCP (VCG-TCP)

from V. cholerae serogroups O1 and O139 has been

investigated in the reversible intestinal tie adult rabbit

diarrhea (RITARD) model. Rabbits were immunized 3

times intragastrically with a mixture of lyophilized

VCG-TCP from serogroup O1 and serogroup O139

and were challenged 30 days after the first immuniza-

tion with virulent V. cholerae O1 and V. cholerae

O139 strains.

Serum vibriocidal antibodies were observed in all

immunized animals and it could be shown that adult

rabbits were protected against diarrhea and death

following intralumen challenge with fully virulent V.

cholerae serogroups O1 and O139 [29]. Animal

models indicate that VCG induce humoral and cellular

immune responses against cell envelope constituents

including protective immunity against challenge

infections. All oral ghost vaccination experiments

were carried out with freeze-dried ghosts resuspended

in saline without the addition of adjuvants, stabilizers

or other substances [29]. VCG have the advantage of

ease of production by simple fermentation under

conditions, which favour the expression of TCP.

367368369370371372373374375376377378379380381382383384385

UNCORRE4. Bacterial ghost system as carrier of foreign

target antigens

Foreign target antigens can be tethered to the OM or

IM, exported into the PPS or can be expressed as S-

layer fusion proteins, which form shell-like self

assembly structures filling either the PPS or CPS

(Fig. 1). The OM (Fig. 1) is an asymmetric lipid bilayer

with LPS in the outer leaflet and phospholipids in the

inner leaflet. The polysaccharide moieties of LPS,

filaments and pili extend from the OM to the environ-

ment. The role of TCP to confer cross-protective

immunity in VCG has been mentioned earlier.

Outer membrane target antigen expression exploits

outer membrane proteins (OMP), which can be

modified to incorporate unrelated sequences [30]. In

a recent study, hepatitis B virus core 149 antigen was

ED PROOF

incorporated into OMP-A as fusion protein and

displayed on the surface of E. coli BG. These ghosts

induced a significant immune response against the

HBC 149 core antigen in mice [31].

Localization of target antigens in the PPS offers

several advantages. Target antigens exported to this

compartment are not only protected from external

degradation processes but are also immersed in a sugar-

rich environment of membrane-derived oligosacchar-

ides, which protect TA during lyophilization. Further-

more, soluble target antigens can be expressed in the

PPS of BG as the E-lysis tunnel seals the IM and OM.

MalE fusion proteins have been constructed which

secrete the target antigens into the PPS either as a

soluble protein or as part of a S-layer self-assembly

structure (Fig. 1). Site-directed mutagenesis of the S-

layer genes sbsA and sbsB and structural/functional

analysis of S-layer domains essential for intra- and/or

inter-molecular interactions [32–34] revealed flexible

surface loops in both proteins that accept foreign

target antigens sequences coding for up to 600 aa [35].

Such recombinant S-layer fusion proteins within a BG

consist of several hundred thousand monomers per

cell and because of their ability to assemble into a

superstructure, they do not form inclusion bodies.

Depending on the specific aim, multiple presenta-

tion of target antigens within the S-layer structure

could have beneficial effects compared to the soluble

form of the corresponding antigen (Fig. 1).

Electron microscopic pictures show sheet like self-

assembly structures of recombinant SbsA–Omp26

subunits in the PPS of E. coli ghosts [36]. The

Omp26 of non-typeable Haemophilus influenzae

(NTHi) was carried within the superstructure and E.

coli BG harboring this construct were highly immu-

nogenic for Omp26 when administered intraperito-

neally to mice [33].

The potential of E. coli ghosts carrying MalE–

Omp26 or MalE–SbsA–Omp26 fusion proteins in the

PPS was assessed as a delivery system for mucosal

immunization in a rat model and different routes of

immunization were evaluated. Animals were muco-

sally immunized targeting either gut only or gut and

lung mucosal sites. In the gut/lung regime, two initial

gut targeted inoculations with BG were followed by an

intratracheal (IT) boost with purified Omp26. The gut

only immunization regime showed a moderate

enhancement of bacterial clearance following pulmo-

UNCORRECTED PROOF

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OMP A

Pori

n

LPS

L

L

OM IM

PPS

CPS

OM

PPS

IM

CPS CPS

IM

IM

OM

Fig. 1. Bacterial ghost as carriers of autologous or foreign target antigens. Target antigens are compartmentalized into the outer membrane (OM),

the periplasmic space (PPS), the inner membrane (IM) and the cytoplasmic space (CPS). Target antigens (TA) can be BG components themselves

(e.g. pili, LPS, OMP, inner membrane proteins (IMP) and flagella). Foreign target antigens are displayed on the BG surface as a fusion protein

with OMP-A (upper left corner), or are exported to the sealed PPS as maltose binding protein (malE) or malE-sbsA/sbsB S-layer fusion proteins

(upper right corner). Target antigens can be anchored to the inner membrane via EV, LV or EV and LV anchor sequences. Membrane anchored StrpA

(EV-StrpA) can bind any biotinylated target antigen to the inner membrane. DNA carrying a lac operator sequence can bind to a membrane

anchored lacI (LacI-LV) repressor molecule (lower left and right corners). Recombinant S-layer proteins carrying foreign target antigens can fill

up the CPS of the BG. By loading the bacterial lumen with cccDNA plasmids, BG can act as carrier for DNA vaccines (lower left corner) .

Pilus Maltose binding protein S-layer

Outer membrane protein Outer membrane protein A carrying a target antigen Inner membrane protein

LPS Target antigen Porin

Phospholipid E’-Anchor L’-Anchor

Penicillin binding protein Flagellum E’anchored StrpA

L’anchored LacI repressor cccDNA ccc-DNA carrying thelac operator site

Biotin Biotinylated target antigen Bacterial ghost

Peptidoglycan


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nary challenge whereas the gut/lung immunization

regime resulted in significantly enhanced pulmonary

clearance of NTHi. Both immunization regimes

induced high levels of Omp26 specific antibodies in

the serum of immunized rats, with higher levels in the

groups that received the IT boost with purified Omp26.

Analysis of IgG isotypes present in serum suggest that

the immune response was predominantly of a T-helper1

type. Additionally, immunization induced a significant

cellular immune response with lymphocytes from

animals vaccinated using the gut/lung regime respond-

ing significantly to Omp26 when compared to control

groups. In summary, mucosal immunization with

recombinant Omp26 in E. coli ghosts followed by a

boost with purified Omp26 induced a specific and

protective immune response [37].

Bacterial ghosts have also been produced to

express target antigens in the CPS. Expression of

SbsA or SbsB fusion proteins in the CPS followed by

E-mediated lysis of the bacteria results in crystalline

planar arrays of S-layer proteins not released to the

surrounding medium (Fig. 1).

The CPS of BG can be filled either with water

soluble subunit antigens or emulsions such that the

target antigen itself or a matrix can be coupled to

appropriate anchors on the inside of the IM of BG

(Fig. 2). For example, BG with streptavidin anchored

on the inside of the IM can be filled by resuspending

lyophilized BG in solutions carrying biotinylated TA

[38].

For membrane anchoring of target antigens or of

acceptor proteins like streptavidin to the cytoplasmic

side of the IM, a membrane targeting system was

developed [39]. By cloning foreign DNA sequences

into the membrane targeting vector pMTV5, any gene

of interest can be expressed as a hybrid protein with

N-, C- or N-/C-terminal (EV-, LV-, EV-LV; Figs. 1 and 2)

membrane anchors directing and attaching the fusion

proteins to the cytoplasmic side of the IM of the

bacteria prior to E-mediated lysis. The current list of

membrane anchored target proteins comprises various

viral core or envelope proteins and bacterial target

antigens or enzymes. For the latter, it could be shown

that the enzymatic activities of h-galactosidase, PHB-synthase or alkaline phosphatase were not impaired

indicating that the membrane anchors do not interfere

with the proper folding of the target proteins and that

clustering and self-assembly (for example for h-

ED PROOF

galactosidase) is possible. The IM anchored HIV1-

RT and HIV1-gp41 target antigens carried by BG

induced humoral as well as a cellular immune

responses in animal models [40,41].

Any BG can be used as carrier for foreign antigens.

In a recent study [42], VCG have been successful used

to immunize against Chlamydia trachomatis. In

accordance with the new paradigm for vaccine design,

an efficacious anti-chlamydial vaccine should elicit a

genital mucosal Th1 response. To design a candidate

vaccine against Chlamydia based on the BGS, the

gene encoding the major OMP, omp1, of C. tracho-

matis was expressed in V. cholerae, as an IM-anchored

protein. Intranasal and intramuscular immunization of

naive mice with V. cholerae ghosts expressing OMP1

induced a strong Th1 immune response in the genital

mucosa. The ability of this vaccine delivery system to

protect susceptible animals from chlamydial infection

offers potential for the future development of effica-

cious vaccines capable of protecting human against

pathogens causing intracellular infections. In addition,

immune T cells from immunized mice could transfer

partial protection against a C. trachomatis genital

challenge to naRve mice. These results suggest that

VCG expressing chlamydial proteins may constitute a

suitable subunit vaccine for inducing an efficient

mucosal T-cell response that protects against C.

trachomatis infection [42]. In this example, VCG

offer the opportunity for designing TA vaccines within

the context of a cell envelope which is also able to

induce protective immunity against cholera.

Bacterial ghosts have been more recently developed

for delivery of antigens such as DNA. The internal

space of BG can be filled with a substituted matrix, e.g.

biotinylated dextran or polylysine which then binds the

target antigens of interest (Fig. 2a). For DNAvaccines,

it has been shown that plasmid DNA complexed with

polylysine can be efficiently packaged into BG [38]. If

the lac repressor proteins (LacI) is membrane anchored

(Fig. 1), it is still able to bind lac operator sequences

carried on plasmid DNA. Plasmids bound to the

membrane by this specific interaction are retained in

BG and are not expelled to the culture medium

following induction of E-mediated lysis.

It has also been observed that plasmid DNA

associates unspecifically with the inside of the IM.

Purified covalent closed circular DNA (cccDNA) can

be loaded to BG by resuspension of freeze dried BG

UNCORRECTED PROOF

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StrpA

BiotinylatedTA

StrpA

BiotinylatedPolymer

a b

c d

e f

ETTS

Fig. 2. Targeting membrane vesicles on top of the E-specific transmembrane tunnel (ETTS) structure of bacterial ghosts. (a) BG with

streptavidin-biotin coupled target antigens (TA) or biotinylated polymer in the CPS with open E-specific transmembrane tunnel structure. (b)

Sealing of inside-out vesicles of Gram-negative bacteria to the E-specific transmembrane tunnel structure of BG. Protein E fusion proteins (c) in

vivo biotinylated ( ) or (d) extended with streptavidin ( ) using the specific biotin–streptavidin interactions ( ) to position the membrane

vesicle of the E-specific transmembrane tunnel structure. (e) Multimers of streptavidin–biotin molecules (for simplicity only one construct is

shown) can form chimney like structures between the BG and the targeted vesicle. (f) Soluble target antigens of other substances ( ) can be

carried in the CPS of BG.


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in DNA solutions. Depending on the DNA concen-

tration roughly 2000–4000 plasmids per single BG

can be attached to the IM.

When BG carrying plasmids encoding the green

fluorescent protein (GFP) were exposed under tissue

culture conditions to Caco-2 cells, DC or macro-

phages the cells expressed high levels of GFP [43].

As the latter two cell types are well known for their

high capacity to degrade phagocytosed material, it is

even more astonishing that GFP plasmids delivered by

BG transferred the DNA from the endosome/lyso-

some to the nucleus without any help of endo-

somolytic agents or of nuclear localization signals.

In an initial immunization study, plasmids encoding

the TA LacZ loaded in BG revealed strong humoral as

well as a Th1 help-mediated immune responses

against h-galactosidase in mice [44].

The feasibility of plugging the E-lysis tunnel of BG

to entrap target antigens in the CPS following loss of

cellular cytoplasmic constituents has been assessed.

Using a vesicle-to-ghost membrane fusion system,

BG can be plugged in order to use BG as carrier and

adjuvant systems for soluble, non-attached, hydro-

philic TA. The sealing process of ghosts requires

inside-out vesicles of gram-negative bacteria and

fuses the vesicles to the inner membrane at the edges

of the lysis tunnel of the ghost carrier. Ortho-

nitrophenyl-galactoside (ONPG), calcein and fluores-

cein-labeled DNAwere used as reporter substances to

test that BG can be sealed by restoring membrane

integrity (Fig. 2b) [45].

The technique of loosely closing BG is under

optimization and, as can be seen in Fig. 2c–f, antigen

carriers can be obtained by targeting a vesicle on top

of the E-specific transmembrane tunnel. In the most

simple model, vesicles can be targeted to the E-

specific transmembrane tunnel by specific interaction

of biotinylated protein E with membrane anchored

streptavidin on the surface of inside out vesicles (Fig.

2c) or vice versa by using E-streptavidin fusion

proteins for creation of the E-specific transmembrane

tunnel and inside out vesicles with membrane

anchored biotinylated receptor sequences (Fig. 2d).

In an alternative model, both receptor sequences on

the BG as well as on the inside out vesicle display

streptavidin on the surface and free biotin is used as

coupling agent (Fig. 2e). This traps the vesicle on top

of the E-specific transmembrane tunnel and can be

ED PROOF

used to construct BG carrying back packed envelope

fragments from other bacteria or viruses being either

biotinylated or modified with streptavidin.

With such constructs soluble drugs or antigens filled

into BG may be able to leak out through the tiny cleft

between the vesicle and the BG carrier (Fig. 2f). The

release rate of the enclosed substances can be regulated

by the distance between the BG carrier and vesicle

attached by adding various amounts of free biotin and

streptavidin, forming chimney-like structures which

can be constructed with different release properties.

Target antigens embedded in BG can be regarded

as subunit vaccine candidates fully equipped with a

whole bacterial cell adjuvant for better uptake by APC

by pattern recognition. The bacterial ghost antigen

presentation technology combines target antigen(s) on

a carrier, which elicits an efficient immune response.

The formulation of the target antigens packaged into

the ghost envelope structures is dependant on their

own physiochemical properties and it may be of

benefit to either loosely package them into the inner

space of the envelopes or to fix them to a matrix.

Clearly, different combinations of substances may

need to be located simultaneously in various compart-

ments of the BGS for optimal formulation. The

bacterial ghost technology warrants further investiga-

tion as it has great strategic potential in areas of

vaccine development against viral and bacterial

threats for which conventional vaccines do not exist

or are not sufficiently efficient.

Acknowledgements

This work was supported by grant GZ 309.049/1-

VI/6/2003 from the Austrian Ministry of Science. The

technical assistance of Beate Bauer, Alisa Lajta,

Roland N. Leitner and John McGrath for preparing

the manuscript is highly appreciated.

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