Bacterial ghosts as delivery systems

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Bacterial ghosts as delivery systems
Prepared by Fadelah Sfouq
Supervised by Dr. Manal Baddour
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Introduction

• Current delivery systems include liposomes,
  micelles, polymers
• Recently microorganisms have been exploited as
  diverse delivery systems

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Introduction

• Many studies have been done to provide different
  delivery systems for different classes of drugs
  in order to
• Overcome the undesired effect without reducing
  the drug potency
• Also to allow site specific targeting of the
  drugs.

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Liposomes, micelles and polymers

• A colloidal delivery system which transports and
  releases the drugs at a controlled rate and then
  is biodegraded to nontoxic products capable of
  being metabolized or eliminated.

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What is microorganism delivery system

• A system incorporating a micro-organism (bacteria
  or viruses) as a means of delivering a product to
  the recipient.

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Viruses as delivery systems

• Live attenuated viruses
• Inactivated viruses
• Virus like particles
• Vaccinia virus..

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Bacterial delivery systems

• Live attenuated bacteria
• Inactivated bacteria
• Bacterial ghost

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Bacterial ghosts

• Definition
• non-living bacterial envelopes, which maintain
  the cellular morphology and native surface
  antigenic structures including bioadhesive
  properties of the natural cell.

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Advantages and limitations of biological
particles as delivery vehicles.
Limitations Advantages Biological particle
Reversion to virulence Horizontal gene transfer Stability of recombinant phenotype Pre-existing immunity against carrier strain Antibiotic markers Activation of innate immune system via pattern recognition receptors Generation of humoral and cell-mediated immune response Used as carrier for antigens Live attenuated or inactivated bacteria
Poorly immunogenic Pathogenic for immunocompromised recipients Limited capacity to encapsulate foreign antigens or DNA Non-pathogenic Biodegradable, contain no preservatives or detergents and present fewer localized adverse events Live GRAS bacteria or Virosomes
Requirement for strong adjuvant Low-cost production, ease of use Edible vaccines from transgenic plants
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Advantages and limitations of biological
particles as delivery vehicles.
Incomplete inactivation Reversion to virulence Improved long-term expression of target genes using viral integration system into host genome Live attenuated or inactivated viruses
Different serotypes Hazard for blood transfusion Biocompatibility, complete biodegradability Erythrocytes
Presence of lipopolysaccharides Non-living carriers 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 Bacterial ghosts
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Bacterial ghosts

• Different species of microorganisms have been
  used to produce ghosts like

• Salmonella sp

Actinobacillus pleuropneumoniae
Mannheimia haemolytica
Pasteurella haemolytica

• E-coli

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How to prepare BG

• BG are produced by the controlled expression of
  the plasmid-encoded lysis gene E of bacteriophage
  PhiX174 in gram-negative bacteria.

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Microscopical appearance of BG

• Ghost can be distinguished from their living
  unlysed cells using light microscopic examination
  by their

BG appear slightly elongated flat. Clearly
more transparent than intact bacteria. Large
holes
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Differential interference contrast micrographs
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High-resolution field emission scanning electron
micrograph of E. coli ghosts
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Application of BG

• Vaccine delivery and targeting system.
• DNA vaccines.
• Bacterial ghost system as carrier of foreign
  target antigens.
• Bacterial ghosts themselves have been tested as
  vaccine.
• Delivery systems for drugs.

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Different approaches to utilizing the ghosts

• Foreign target antigens (TA) can be tethered to
  the BG outer membrane (OM), inner membrane (IM)
  or exported into the periplasmic space (PPS).
• Also, it can be expressed as S-layer fusion
  proteins which form shell-like self assembly
  structure filling either PPS or cytoplasmic space
  (CPS)

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Different approaches to utilizing the ghosts

• OM is an asymmetric lipid bilayer with
  lipopolysaccharide (LPS) in the outer leaflet and
  phospholipids in theinner leaflet.

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Different approaches to utilizing the ghosts

• Localization of TA in the PPS offers several
  advantages
• TA protected from external degradation
  process
• Immersed in a sugar-rich environment which
  protect TA during lyophilization.

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Different approaches to utilizing the ghosts

• BG with streptavidin anchored on the inside of
  the IM can be filled by resuspending the
  lyophilized BG in solution carrying biotinylated
  TA.
• The CPS of BG can be filled either with water
  soluble subunit antigens or emulsion anchored to
  the inside of BG.

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Applications of bacterial ghosts as delivery
systems
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Drugs Nucleic acids Therapeutic proteins or peptides Display of antigens Display compartment
Binding of hydrophobic drugs by affinity to membranes Surface presentation by OmpA fusion or through fusion with pili structures Outer membrane
Membrane-bound enzymes 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 Periplasmic space
Sealing bacterial ghosts for watersoluble drugs Filling with DNA plasmids from 4 to 5000 copies/ghost Paracrystalline fusion protein sheets, which remain in the cytoplasmic lumen after E-mediated lysis of the carrier bacteria Cytoplasmic space
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Bacterial ghosts as adjuvant and/or carriers for
foreign target antigens.
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Different approaches to utilizing the ghosts

• Plugging of the E-lysis tunnel of BG can be use
  to entrap soluble, non-attached TA in the CPS.
• Using a vesicles-to-ghost membrane fusion system
  to plug BG.

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Different approaches to utilizing the ghosts

• Vesicles can be attached to E-tunnel either by
• Interaction of biotinylated protein E anchored
  with streptavidin.
• Using E-streptavidin fusion protein anchored to
  biotinylated receptors.

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Different approaches to utilizing the ghosts

• In an alternative model, using both sequences as
  coupling agent, used to construct BG carrying
  fragments from other microorganism being either
  biotinylated or modified with streptavidin.
• The release rate of the enclosed substances can
  be regulated by the distance between the BG and
  vesicle by adding various amount of free biotin
  and streptavidin.

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Targeting membrane vesicles on top of the
E-specific transmembrane tunnel (ETTS) structure
of bacterial ghosts.
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Target antigen(s)/ Protein structures  
Bacterial ghost Carrier  
Animal model  
Route of immunization/ Challenge
Immune Response  
Cell envelope   Toxin-coregulated Pilus (TCP)
Vibrio cholerae ghost (VCG)
Rabbit   Mouse
SC, IM   Intra Duodenal
Vibriocidal antibodies infant mouse
model Passive protection
Cell envelope TCP
VCG-O1 VCG-O139
Rabbit
Oral/RITARD model  
Vibriocidal titers, protection against
colonization/diarrhea
 
Cell envelope
Actinobacillus pleuropneumoniae (App)
Pig Rabbits
IM
Serum IgG, IgA, IgM
 
Cell envelope  
App  
Pig  
IM
Serum IgM, IgA, IgG
 
Cell envelope  
App  
Pig  
Aerogenic/oral homologous aerosol challenge  
BALF IgA, IgM, IgG, IgH , IgG2 Total/relative
number of CD2, CD4, CD8 T-cells
 
Cell envelope
Helicobacter. pylori  
Mouse  
Oral/intragastric challenge
Colonization assay, sterile immunity
 
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Subunit vaccine/ Location in BG
Bacterial ghost Carrier
Animal model  
Route of immunization/ Challenge
Immune Response
 
HIV-1 gp41 inner membrane anchored    
Piglets Mouse Mouse/ Rabbits  
SC Oral/lethal challenge IP, SC  
Serum IgG Survival time Serum IgG Serum IgG,
T-cell stimulation index
   
K. pneumoniae S. typhimurium E. coli
HIV-1 RT inner membrane anchored
E. coli
Mouse Mouse
IP SC
Serum IgG, T-cell stimulation index

Clamydia trachomatis MOMP inner membrane
anchored
V. cholera
Mouse
IM/ Intravaginal
IFN-g IgA, IgG2a in Genital tract, spleen

C. trachomatis MOMP/OMP2 inner membrane anchored
V. cholera
Mouse
IM/ Intravaginal
IFN-g IgA, IgG2a in Genital tract, T cell
proliferation in spleen
C. trachomatis MOMP/PorB inner membrane anchored
V. cholera
Mouse
IM/ Intravaginal
IFN-g IgA, IgG2a in vaginal wash outs, T-cell
proliferation in spleen
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sbsA-ZP3 cytoplasmic lumen
E. coli
Possum  
ID, IM Eye drops
Serum IgG,
SbsA-ntHaemophilus influenzae omp26 cytoplasmic
lumen
E. coli
Mouse
IM
Serum IgG
mal E-ntHi omp26 sbsA-ntHi omp26 periplasmic
space
E. coli
Rat
Oral, ID Intratracheal
Omp26 specific IgG, IgG1, 3gG2a in serum, Omp26
specific IgA in BAL, Omp26 specific lymphocytes
proliferation, bacterial recovery from BAL and
lung homogenates
E. coli
Mouse
IM
HBcAg-149 specific serum IgG
 
ompA- HBcAg-149 outer membrane anchored
DNA pEGFP-N1 DNA pCMVß cytoplasmic membrane
attached
M. haemolytica
Mouse
ID, IM
Ag-specific serum IgG, IFN-gamma ELISPOT, mixed
Th1/Th2 response, more dominant Th2 pattern
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Induction of immune response by BG

• Different studies have been done to investigate
  the activation of antigen presenting cells (APC)
  by measuring the uptake of BG by dendritic cells
  (DC) and macrophages.
• Exposing DC to BG resulted in marked increase in
  the ability of DC to activates T-cell immune
  response.

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Does the endotoxicity limit the use of BG

• All the studies performed suggest that
  endotoxicity of BG is not a limiting factor if

An appropriate dose of BG used
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Bacterial ghosts as carrier and targeting system
for DNA vaccines
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Bg as a delivery system for drugs

• Systemic application of different drugs often
  cause sever side effects.
• To reduce the undesired effects, advanced drug
  delivery systems are needed for specific
  targeting of these drugs.
• BG offer a solution for this problem.

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Bg as a delivery system for drugs

• Since anticancer drugs cause sever toxic effects
  BG have been used to reduce its toxicity by
  targeting these agents.
• BG from Mannheimia haemolytica to deliver
  target doxorubicin (DOX) to colorectal
  adenocarcinoma cells.

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Bg as a delivery system for drugs

• Recently, too many studies using BG as a delivery
  targeting system have been done in different
  drugs includes anticancer, for arthritis,
  different antibiotic.

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Enzyme immobilization

• Different enzymes can be immobilized within
  ghosts forming small units for compounds which
  can not be formed without interfering enzymatic
  activities of a cell

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Conclusion

• BG system is a novel delivery system in that it
  combines excellent natural intrinsic properties
  as a carrier for foreign antigen.
• Efficient BG system for vaccine delivery promotes
  the generation of both cellular humoral
  responses.

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Conclusion

• Advantages of BG includes the simplicity of both
  BG production and packaging of multiple target
  antigens.
• Further advantages of BG include long shelf-life
  duo to freeze-dried status.
• They are safe as they dont involve live
  organisms.
• Also, as a delivery system they offer high
  bioavailability.

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