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Bacterial ghosts as delivery systems

4/24/2012. 2. Introduction . Current delivery systems include liposomes, micelles, polymers Recently microorganisms have been exploited as diverse delivery systems. 4/24/2012. 3. Introduction. Many studies have been done to provide different delivery systems for different classes of drugs in ord

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Bacterial ghosts as delivery systems

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    1. 4/24/2012 1 Bacterial ghosts as delivery systems

    2. 4/24/2012 2 Introduction Current delivery systems include liposomes, micelles, polymers Recently microorganisms have been exploited as diverse delivery systems New generation of recombinant protein and DNA vaccines are generally poorly immunogenic thus there is an urgent need to develop improved DS The aim of future vaccines is to provide maximum efficacy with minimum number of doses, delivered safely and easilyNew generation of recombinant protein and DNA vaccines are generally poorly immunogenic thus there is an urgent need to develop improved DS The aim of future vaccines is to provide maximum efficacy with minimum number of doses, delivered safely and easily

    3. 4/24/2012 3 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.

    4. 4/24/2012 4 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.

    5. 4/24/2012 5 What is microorganism delivery system A system incorporating a micro-organism (bacteria or viruses) as a means of delivering a product to the recipient.

    6. 4/24/2012 6 Viruses as delivery systems Live attenuated viruses Inactivated viruses Virus like particles Vaccinia virus……..

    7. 4/24/2012 7 Bacterial delivery systems Live attenuated bacteria Inactivated bacteria Bacterial ghost

    8. 4/24/2012 8 Bacterial ghosts Definition: non-living bacterial envelopes, which maintain the cellular morphology and native surface antigenic structures including bioadhesive properties of the natural cell.

    9. 4/24/2012 9 Advantages and limitations of biological particles as delivery vehicles.

    10. 4/24/2012 10 Advantages and limitations of biological particles as delivery vehicles.

    11. 4/24/2012 11 Different species of microorganisms have been used to produce ghosts like: Bacterial ghosts

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    13. 4/24/2012 13 BG are produced by the controlled expression of the plasmid-encoded lysis gene E of bacteriophage PhiX174 in gram-negative bacteria. How to prepare BG

    14. 4/24/2012 14 How to prepare BG: Gene E product is a hydrophobic protein of 91 amino acids. It exerts its lytic function by its integration into the inner membrane, followed by fusion of inner and outer membrane and formation of a transmembrane tunnel of 40–80 nm in diameter, through which all cytoplasmic contents are expelled. Protein E-specific lysis does not cause any physical or chemical denaturation to bacterial surface structers. How to prepare BG: Gene E product is a hydrophobic protein of 91 amino acids. It exerts its lytic function by its integration into the inner membrane, followed by fusion of inner and outer membrane and formation of a transmembrane tunnel of 40–80 nm in diameter, through which all cytoplasmic contents are expelled. Protein E-specific lysis does not cause any physical or chemical denaturation to bacterial surface structers.

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    16. 4/24/2012 16 Microscopical appearance of BG Ghost can be distinguished from their living unlysed cells using light microscopic examination by their:

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    18. 4/24/2012 18 Differential interference contrast micrographs of unlysed E. coli NM522 (A) and ghosts created by alternative lysis (B). Ghosts can be clearly distinguished from unlysed cells by their ‘flat’ appearance due to their greater transparency. Ghost cells broken into two halves or with blast off polar caps, caused by the explosion-like lysis induced by the alternative lysis protocol, are indicated by arrows. Differential interference contrast micrographs of unlysed E. coli NM522 (A) and ghosts created by alternative lysis (B). Ghosts can be clearly distinguished from unlysed cells by their ‘flat’ appearance due to their greater transparency. Ghost cells broken into two halves or with blast off polar caps, caused by the explosion-like lysis induced by the alternative lysis protocol, are indicated by arrows.

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    21. 4/24/2012 21 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. Ref: ghost paper, bgdc paper and other in brief Different studies have been done including DNA vaccine DS against many diseases BG as DDS like study of doxorubicinRef: ghost paper, bgdc paper and other in brief Different studies have been done including DNA vaccine DS against many diseases BG as DDS like study of doxorubicin

    22. 4/24/2012 22 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)

    23. 4/24/2012 23 Different approaches to utilizing the ghosts OM is an asymmetric lipid bilayer with lipopolysaccharide (LPS) in the outer leaflet and phospholipids in theinner leaflet.

    24. 4/24/2012 24 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.

    25. 4/24/2012 25 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.

    26. 4/24/2012 26 Applications of bacterial ghosts as delivery systems

    27. 4/24/2012 27 Bacterial ghost as carrier of autologous or foreign target antigens. The BG can be divided into four different compartments carrying TA, namely, the OM, the PPS, the IM and the CPS which are presented in detail on the corners of the schematic BG in the centre. TA can be BG components themselves (e.g. pili, LPS, OMP, IMP and flagella) or represent foreign TA displayed on the BG surface as fusion protein with the 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). TA can be anchored into the IM via EV, LV or EV and LV anchor sequences (lower right corner). Membrane-anchored StrpA (EVStrpA) can bind any biotinylated TA to the inner membrane (lower left corner). DNA carrying a lac operator sequence can bind to a membrane anchored lacI (LacI-LV) repressor molecule (lower left corner). Recombinant S-layer proteins carrying foreign TA can fill up the CPS of the BG (lower left corner). Loading the bacterial lumen with cccDNA plasmids, BG can act as carrier for DNA vaccines (lower left corner). Bacterial ghost as carrier of autologous or foreign target antigens. The BG can be divided into four different compartments carrying TA, namely, the OM, the PPS, the IM and the CPS which are presented in detail on the corners of the schematic BG in the centre. TA can be BG components themselves (e.g. pili, LPS, OMP, IMP and flagella) or represent foreign TA displayed on the BG surface as fusion protein with the 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). TA can be anchored into the IM via EV, LV or EV and LV anchor sequences (lower right corner). Membrane-anchored StrpA (EVStrpA) can bind any biotinylated TA to the inner membrane (lower left corner). DNA carrying a lac operator sequence can bind to a membrane anchored lacI (LacI-LV) repressor molecule (lower left corner). Recombinant S-layer proteins carrying foreign TA can fill up the CPS of the BG (lower left corner). Loading the bacterial lumen with cccDNA plasmids, BG can act as carrier for DNA vaccines (lower left corner).

    28. 4/24/2012 28 Bacterial ghosts as adjuvant and:or carriers for foreign target antigens. (a) Ghosts are mixed with different antigens (**) and act as carrier and adjuvant for the induction of the immune response. (b) Antigens are attached to the inside of the cytoplasmic membrane via (É) N-terminal-, (Č) C-terminal or (ÉČ) N-and C-terminal anchor sequences. (c) Membrane anchored streptavidin (Y) can bind any biotinylated target antigen to the inner membrane. (d) Target antigens can be exported to the periplasmic space (pp) either by their own signal sequence () or as fusion partner with polypeptides carrying a signal sequence ( ). (e) Ghost with a combination of membrane anchored target antigens and antigens exported to the pp. (f) Recombinant S-layer proteins rSbsA (--) or rSbsB (--) as carriers of foreign antigens filling the cytoplasmic space of ghosts. (g) rSbsA, rSbsB proteins with foreign target antigens exported to the pp. (h) Combination of membrane anchored antigens and rSbsA-, rSbsB- target antigens in the inner lumen of the ghosts. (i) Combination of membrane anchored target antigens and rSbsA- and rSbsB antigens in the pp of ghosts. (j) Combination of target antigens on rSbsA-, rSbsB- carriers in the inner lumen, the pp and on the outer surface of ghosts. (k) Combination of target antigens immobilizied by membrane anchors or on rSbsA-, rSbsB-carriers in the inner lumen, pp and outer surface of ghosts. (l) Ghost with membrane anchored streptavidin and biotinylated polymer filling the inner lumen of the ghosts together with soluble target antigens. Bacterial ghosts as adjuvant and:or carriers for foreign target antigens. (a) Ghosts are mixed with different antigens (**) and act as carrier and adjuvant for the induction of the immune response. (b) Antigens are attached to the inside of the cytoplasmic membrane via (É) N-terminal-, (Č) C-terminal or (ÉČ) N-and C-terminal anchor sequences. (c) Membrane anchored streptavidin (Y) can bind any biotinylated target antigen to the inner membrane. (d) Target antigens can be exported to the periplasmic space (pp) either by their own signal sequence () or as fusion partner with polypeptides carrying a signal sequence ( ). (e) Ghost with a combination of membrane anchored target antigens and antigens exported to the pp. (f) Recombinant S-layer proteins rSbsA (--) or rSbsB (--) as carriers of foreign antigens filling the cytoplasmic space of ghosts. (g) rSbsA, rSbsB proteins with foreign target antigens exported to the pp. (h) Combination of membrane anchored antigens and rSbsA-, rSbsB- target antigens in the inner lumen of the ghosts. (i) Combination of membrane anchored target antigens and rSbsA- and rSbsB antigens in the pp of ghosts. (j) Combination of target antigens on rSbsA-, rSbsB- carriers in the inner lumen, the pp and on the outer surface of ghosts. (k) Combination of target antigens immobilizied by membrane anchors or on rSbsA-, rSbsB-carriers in the inner lumen, pp and outer surface of ghosts. (l) Ghost with membrane anchored streptavidin and biotinylated polymer filling the inner lumen of the ghosts together with soluble target antigens.

    29. 4/24/2012 29 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.

    30. 4/24/2012 30 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.

    31. 4/24/2012 31 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.

    32. 4/24/2012 32 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 (green ) or (d) extended with streptavidin (orange ) using the specific biotin–streptavidin interactions (both ) 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 ( blue) can be carried in the CPS of BG. 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 (green ) or (d) extended with streptavidin (orange ) using the specific biotin–streptavidin interactions (both ) 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 ( blue) can be carried in the CPS of BG.

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    36. 4/24/2012 36 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.

    37. 4/24/2012 37 Does the endotoxicity limit the use of BG All the studies performed suggest that endotoxicity of BG is not a limiting factor if : Safety is an important criterion for the assessment of vaccines, endotoxicity causes spesific problems when components or whole cell of Gram-ve bacteria are used. Lipopolysaccharide LPS is a major component of BG as the envelope structure is not altered by the production process. Lipid A, the major toxic component of the bacterial LPS is known to induce a variety of endotoxic effect. LPS in the membrane elicit different immune & inflammatory reactions in human. LPS induce TNFasecretion by macrophages is known to be mediated by binding to the LPS binding proteins and CD14 receptorSafety is an important criterion for the assessment of vaccines, endotoxicity causes spesific problems when components or whole cell of Gram-ve bacteria are used. Lipopolysaccharide LPS is a major component of BG as the envelope structure is not altered by the production process. Lipid A, the major toxic component of the bacterial LPS is known to induce a variety of endotoxic effect. LPS in the membrane elicit different immune & inflammatory reactions in human. LPS induce TNFasecretion by macrophages is known to be mediated by binding to the LPS binding proteins and CD14 receptor

    38. 4/24/2012 38

    39. 4/24/2012 39 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.

    40. 4/24/2012 40 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. Bg as a delivery system for drugs

    41. 4/24/2012 41 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.

    42. 4/24/2012 42 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 Enzyme immobilization has been massively used in industry to get enzymatic product without B interferance Bacterial ghosts as carriers of enzymes For specific applications membrane-anchored enzymes like polyhydroxybutyrate synthase are able to produce a polymer matrix within the ghosts when fed with the precursor molecule butyryl-CoA. It is envisaged that formation of polymers within the ghost can be coupled with packaging of chemicals, drugs or nucleic acids to the matrix material. Any biotinylated material can be bound within the lumen of ghosts carrying membrane-anchored streptavidin. When biotinylated alkaline phosphatase was bound to such ghosts the enzymatic activity of the enzyme was not impaired.  Enzyme immobilization has been massively used in industry to get enzymatic product without B interferance Bacterial ghosts as carriers of enzymesFor specific applications membrane-anchored enzymes like polyhydroxybutyrate synthase are able to produce a polymer matrix within the ghosts when fed with the precursor molecule butyryl-CoA. It is envisaged that formation of polymers within the ghost can be coupled with packaging of chemicals, drugs or nucleic acids to the matrix material.Any biotinylated material can be bound within the lumen of ghosts carrying membrane-anchored streptavidin. When biotinylated alkaline phosphatase was bound to such ghosts the enzymatic activity of the enzyme was not impaired. 

    43. 4/24/2012 43 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.

    44. 4/24/2012 44 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 don’t involve live organisms. Also, as a delivery system they offer high bioavailability.

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