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Recombinant bacterial ghosts: versatile targeting vehicles and promising vaccine candidates
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International Journal of Medical Microbiology 294 (2004) 303–311 www.elsevier.de/ijmm
REVIEW
Recombinant bacterial ghosts: versatile targeting vehicles and promising vaccine candidates Herbert Hoffelner, Rainer Haas Max von Pettenkofer Institut fu¨r Hygiene und Mikrobiologie, LMU Mu¨nchen, Pettenkoferstr. 9a, D-80336 Mu¨nchen, Germany Received 9 February 2004; received in revised form 14 April 2004; accepted 19 April 2004
Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303 Generation of bacterial ghosts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304 General principle of bacterial ghost formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304 Tightly controlled expression of PhiX174 lysis gene E . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304 Structural and functional aspects of bacterial ghosts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305 Application of the method to H. pylori . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305 Use of bacterial ghosts as vaccine delivery systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307 Specific targeting of bacterial ghosts to relevant cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307 Bacterial ghosts and their intrinsic adjuvant effect. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308 Induction of protective immunity by H. pylori ghosts in the mouse model. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309
Introduction Infectious diseases are still a leading cause of morbidity and death worldwide. Several diseases, thought to be under control for many years, have reemerged and a number of new diseases have emerged recently. Due to the often very rapid development of Corresponding author. Tel.: +49 89 5160 5255; fax: +49 89 5160 5223. E-mail address: [email protected] (R. Haas).
1438-4221/$ - see front matter r 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.ijmm.2004.04.003
microbial resistance to antimicrobial agents upon treatment of patients with drugs, the general management of a variety of diseases became much more difficult and expensive. The application of vaccines is, however, still a very efficient procedure in preventing infectious diseases. In principle, there are several different ways to design a vaccine. Modern approaches, applying recombinant DNA technologies, have so far exerted a limited impact on vaccine production, but the chances for a rational vaccine design using recombinant DNA technology are manifold and promising in the future.
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In general, vaccines might be grouped into different categories, the live vaccines, killed or inactivated vaccines, DNA- and subunit vaccines. Live bacterial vaccine carriers, such as attenuated Salmonella vaccine strains, can be equipped with defined recombinant antigens against pathogenic bacteria, as demonstrated by the production of Mycobacterium tuberculosis (Hess and Kaufmann, 1999), or Helicobacter pylori (GomezDuarte et al., 1999) antigens in attenuated Salmonella vaccine strains. The recombinant antigen(s) might reside within the Salmonella carrier strain (Gomez-Duarte et al., 1999), they might be secreted into the environment (Hess and Kaufmann, 1999), or they are injected into the host cells by a modified Salmonella type III secretion system (Ru¨ssmann et al., 1998). A rather novel approach is DNA vaccination. By this technology, naked plasmid DNA, that directs the synthesis and secretion of a vaccine antigen, is injected intramuscularly. The plasmid DNA is taken up by host cells and the encoded antigen is expressed and processed in the cell, comparable to a viral antigen. Subsequently, a humoral or cellular immune response will be triggered. Furthermore, killed pathogens, as substitutes for living infectious agents, have been widely used as a principle for vaccine development. Non-living vaccines are produced by chemical or physical inactivation of pathogenic bacteria. Here we discuss a group of rather novel vaccine candidates, grouped into the non-living, wholecell bacterial antigens, the so-called bacterial ghosts. Bacterial ghosts are Gram-negative bacterial cell envelopes, devoid of cytoplasmic contents, while maintaining their cellular morphology and native surface antigenic structures. Ghosts are suitable as a novel vaccine delivery system, endowed with intrinsic adjuvant properties, but also as general targeting vehicles for drugs. They are produced by PhiX174 protein E-mediated lysis of Gram-negative bacteria. The intrinsic adjuvant properties of bacterial ghost preparations enhance immune responses against envelope-bound antigens, including T-cell activation and mucosal immunity. Since native and foreign antigens might be produced in the envelope complex of ghosts before E-mediated lysis, multiple antigens of various origins can be presented to the immune system simultaneously, resulting in multivalent vaccine vehicles. The potency, safety and relatively low production cost of bacterial ghosts offer a significant technical advantage, especially when used as combination vaccines. The candidate ghost vaccines are supposed to be stable without the requirement of permanent cooling, easy to administer and effective. In this review we will discuss the new developments in the generation of ghosts from pathogenic bacteria and their application as vaccine candidates, with major emphasis on the human gastric pathogen H. pylori.
Generation of bacterial ghosts General principle of bacterial ghost formation Bacterial ghosts are inactivated, empty cells, that carry a natural outer surface, providing them with the original targeting functions of the pathogen they are derived from. They are thus able to induce a strong local or systemic immunity, depending on the mode of delivery. Ghosts are produced by controlled expression of the cloned bacteriophage PhiX174 lysis gene E (Witte et al., 1990a). The lysis gene E encodes a membrane protein of 91 amino acids, which is able to fuse inner and outer membranes of Gram-negative bacteria (Witte et al., 1990b) leading to the formation of a transmembrane tunnel structure through the cell envelope of pathogenic bacteria. For Escherichia coli, this tunnel varies in diameter between 40 and 200 nm (Witte et al., 1992). The cytoplasmic content of the bacteria is expelled upon opening, leaving an empty internal space devoid of nucleic acids, ribosomes or other constituents (Witte et al., 1992). The driving force for the release of cytoplasmic material is believed to depend on the osmotic pressure difference between the cytoplasm and the medium created by the opening of the tunnel structure.
Tightly controlled expression of PhiX174 lysis gene E The expression of the lysis gene E in the host bacterium has to be under tight genetic control, since a leaky expression status is lethal for the host bacterium, or induces suppressor mutations resulting in abrogation of gene E expression. Thus, the lysis gene E has been placed under transcriptional control of either the thermo-sensitive lpL =pR -cI857 promoter, or under the lacPO-lacIq promoter/repressor systems (Szostak et al., 1996). In an attempt to further extend the heat stability of the lpR promoter/cI857 repressor system, the repressor-binding operator region has been mutated. This resulted in a modified expression system, which stably repressed production of the lysis protein at temperatures of up to 37 1C, but still allowed induction of cell lysis at a temperature range between 39 1C and 42 1C (Jechlinger et al., 1999) (Fig. 1). The combination of the lpR promoter/cI repressor system with either the lacIq/lacPO resulted in a cold-sensitive system for ghost formation, which is induced by lowering the growth temperature of the bacteria from 37 to 28 1C or lower (Jechlinger et al., 1998). Since the structure and function of the Gram-negative cell envelope seems to be relatively conserved between bacteria, E-mediated lysis has been achieved in various Gram-negative bacteria, including E. coli K12 strains, Salmonella typhimurium, Salmonella
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Fig. 1. Production of bacterial ghosts by E-mediated lysis. The PhiX174 lysis gene E is under transcriptional control of the temperature-sensitive cI857 repressor and either the wild-type rightward lPR promoter (A) or the mutated lPRmut promoter (B) above the respective temperatures, gene E is expressed and a transmembrane tunnel structure is formed by fusion of the inner and outer membranes, through which the cytoplasmic content is expelled. cI857, lcI857 temperature-sensitive repressor gene; lPRmut , lPR , promoters of phage Lambda; E, PhiX174 lysis gene E.
enteritidis, Actinobacillus pleuropneumoniae, Klebsiella pneumoniae, Pasteurella haemolytica, Pasteurella multocida, Ralstonia eutropha, and Vibrio cholerae (Table 1) (Jalava et al., 2003). This broad spectrum of bacteria reveals that in principle E-mediated lysis works in many, if not all Gram-negative bacterial species. The major challenge is, that the E lysis cassette has to be introduced successfully into the recipient bacterium by an appropriate vector, or integrated into the chromosome, and that a tight repression and induction of gene E expression has to be achieved.
peptidoglycan layer remains intact after E-mediated lysis of bacteria (Witte et al., 1998). The protein Especific transmembrane tunnel structure appears to be not randomly distributed over the cell envelope, but restricted to areas of potential bacterial division sites, predominantly in the middle of the cell or at polar sites (Witte et al., 1990b, 1992). This non-destructive and specific mechanism of bacterial inactivation guarantees that ghosts share all functional properties of the outer surface with their living counterparts. Therefore, ghost carriers have superior qualities as tissue- or cell-specific delivery systems.
Structural and functional aspects of bacterial ghosts Application of the method to H. pylori A major advantage of bacterial ghosts, as compared with other procedures to inactivate bacteria, such as chemical or thermal inactivation, is the intact surface structure of bacterial ghosts. The 91-amino-acid lysis protein E forms a specific lysis tunnel by fusing the inner and outer membrane of Gram-negative bacteria. Interestingly, the part of the inner membrane not involved in tunnel formation remains intact during expulsion of cytoplasmic material. Electron microscopic studies clearly show a sealed periplasmic space at the border of the lysis tunnel (Witte et al., 1990b, 1992). The
Only recently, we have adapted the technology for generation of ghosts to the human bacterial pathogen H. pylori. H. pylori is a Gram-negative bacterium, which colonises the gastric mucosa of about 50% of the world population. The infection, usually acquired during childhood, persists chronically if not treated. Colonization of the gastric mucosa by H. pylori results in an acute inflammatory response and damage to the gastric epithelium. Inflammation can then progress to several disease states, ranging in severity from superficial
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Generation of bacterial ghosts in Gram-negative bacteria and their effectiveness as vaccine candidates Recombinant antigen
Type of delivery (animal)
Protection efficiency
Reference/reviewed in
Escherichia coli NM522 E. coli XL1-B
HIV-1RT HIV-1 gp41
n.d. (IgG response) n.d. (IgG response)
Szostak et al. (1996) Szostak et al. (1996)
E. coli serotype O78:K80 E. coli E. coli Salmonella typhimurium Salmonella enteritidis Actinobacillus pleuropneumoniae
— M. bovis vspA Bacillus sbsA/Haemophilus omp26 — — —
Klebsiella pneumoniae Pasteurella haemolytica (Mannheimia)
— —
Pasteurella multocida
—
Ralstonia eutropha Vibrio cholerae
— — C. trachomatis omp toxin co-regulated pili —
Subcutaneous (mice) Subcutaneous (rabbits) Intraperitoneal (mice) Intramuscular/peroral (chicken) Unknown (mice) Intraperitoneal (mice) Peroral (mice) — Intramuscular (pigs) Aerosol (pigs) Peroral (pigs) Subcutaneous (pigs) Subcutaneous (rabbits) Subcutaneous (cattle) Intraperitoneal (mice) Subcutaneous (rabbits) — Intragastric (rabbits) Intramuscular (mice) Intragastric (rabbits) Intragastric (mice)
Lower mortality n.d. n.d. Survival prolonged — Fully protected Partially protected Partially protected n.d. (IgG response) n.d. Fully protected up to 100% protection n.d. — Fully protected Fully protected n.d. Protected
Jalava et al. (2002) Jalava et al. (2002) Riedmann et al. (2003) Szostak et al. (1996) Szostak et al. (1997) Hensel et al. (1996, 2000) Huter et al. (2000) Katinger et al. (1999) Szostak et al. (1996) Marchart et al. (2003a) Marchart et al. (2003b) Marchart et al. (2003a) Marchart et al. (2003a) Schroll et al. (1998) Eko et al. (1994a, b) Eko et al. (2003a, b) Eko et al. (2000) Panthel et al. (2003)
Helicobacter pylori n.d., not determined.
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Carrier strain
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Table 1.
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gastritis, chronic atrophic gastritis, peptic ulceration, to mucosa-associated lymphoid tissue lymphoma (MALT) or adenocarcinoma (Blaser and Berg, 2001). The H. pylori infection is associated with 95% of duodenal and 70% of gastric ulcer cases (Lee et al., 1993), and the World Health Organization classified H. pylori as a Group 1 carcinogen (IARC, 1994). Gastric cancer is the second most common fatal malignancy in the world (Pisani et al., 1993), and it is estimated that by the year 2020 it will increase from fourteenth most common cause of death to the eighth (Murray and Lopez, 1997). The current therapy relies on the use of a proton-pump inhibitor and two antibiotics. The treatment is usually efficient, but problems may occur regarding compliance, antibiotic resistance, and possible recurrence of infection. Thus, vaccination is considered an important approach to control a bacterial infection widespread as the H. pylori infection. In the past, various approaches based on the use of selected bacterial antigens, mostly specific virulence factors, such as urease, the vacuolating cytotoxin (VacA), the cytotoxin-associated antigen (CagA), the neutrophil-activating protein (NAP), and others, conferred protection in animal models of infection (Del Giudice et al., 2001). In the H. pylori mouse model we and others demonstrated that these vaccine antigens were not only effective as prophylactic vaccines, but were also useful for the treatment of an already existing H. pylori infection as a therapeutic vaccine (Corthe´sy-Theulaz et al., 1995; Radcliff et al., 1997). Recently, our group managed to successfully generate ghosts of H. pylori (Panthel et al., 2003). This was achieved by cloning of the PhiX gene E lysis cassette into an E. coli–H. pylori shuttle vector, which was subsequently introduced under repressive conditions (o35 1C) into H. pylori strain P79. Temperatureinduction of the lysis gene cassette revealed a quantitative killing of the H. pylori bacteria without induction of lysis-resistant bacteria. Biochemical and transmission electron microscopic studies identified structurally intact H. pylori. This demonstrated that in principle the procedure works for H. pylori. For the future, however, the method has to be further optimised, to obtain a stronger repression of the lysis gene E and a better induction in H. pylori. Furthermore, for better handling of the system, the lysis gene cassette should be stably integrated into the bacterial chromosome.
Use of bacterial ghosts as vaccine delivery systems Specific targeting of bacterial ghosts to relevant cells The efficiency of a non-living vaccine is usually very much dependent on the method of delivery, the stability
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of the vaccine antigen in vivo and the adjuvant properties of the vaccine formulation. Concerning these criteria, bacterial ghosts are ideal vaccine candidates. Subunit vaccines, consisting of defined recombinant antigens, often have to be delivered by specific carrier systems, such as natural or synthetic polymers or liposomes, to reach their immune target cells. Bacterial ghosts combine both functions and act as vaccine antigens and carriers at the same time. The fact that bacterial ghosts preserve their native cell wall components, including bioadhesive structures such as pili, outer membrane proteins or polysaccharides makes them suitable for attachment to specific target tissues. For oral vaccination, the gastrointestinal (GI) tract has to be targeted. Bacterial fimbriae or pili, which are long, rod-like protein polymers found on the surface of many bacterial enteropathogens, are ideal structures for GI targeting of bacterial ghost vaccines. Bacterial ghosts prepared from Vibrio cholerae or EHEC appear to be excellent targeting vehicles for the GI tract of humans, since they contain fimbriae (toxin-co-regulated pilus, TCP and long polar fimbriae, lpf) interacting with epithelial cells of the GI tract (Taylor, 1991; Torres et al., 2002). Indeed, analysis of immune responses in an animal model indicated that V. cholerae ghosts induced humoral and cellular immune responses against cell envelope constituents including protective immunity against challenge infections (Eko et al., 2003b). Targeting of ghosts to the relevant immune cells, such as macrophages and dendritic cells (DCs), might also be achieved by recognition of the carbohydrates on the surface of ghosts. Bacteria-specific oligosaccharides, which are commonly found in the bacterial lipopolysaccharide (LPS) can be recognised by the 180-kDa mannose receptor (MR), a prototype of a family of multi-lectin receptors, functioning as pattern recognition receptor for foreign oligosaccharides. Macrophages and DCs are able to internalise MR-antigen complexes into acidic lysosomal compartments, where the antigens are degraded for antigen presentation (Astarie-Dequeker et al., 1999; Sallusto et al., 1995). Also the endothelium of human skin and dermal microvascular endothelial cells (DMEC) in vitro express MR. Interestingly, DMEC were able to take up E. coli ghosts into acidic phagosomes by ways of MR, but not ghosts of V. cholerae, which do not possess mannose in their LPS (Gro¨ger et al., 2000), demonstrating the high specificity of targeting bacterial ghosts to certain types of immune cells. In addition to the GI tract, also the respiratory tract has been successfully targeted by bacterial ghosts. For this purpose bacterial ghosts have been generated from Actinobacillus pleuropneumoniae (APP), a bacterial pathogen causing severe lung infections in pigs (Chiers et al., 2002). APP ghosts were used for aerosol immunization of pigs and proved to be safe and
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protective against a challenge infection with the lung pathogenic APP (Katinger et al., 1999). The uptake of APP ghosts by primary antigen-presenting cells (APC) was also studied in the pig model. For this purpose, APP ghosts were conjugated with fluorescein isothiocyanate (FITC) and incubated with isolated porcine DCs. A remarkable increase of FITC-labelling in the APC population and up-regulation of MHC class II molecules in APC was detectable, suggesting an efficient uptake of ghosts and the activation of DCs.
Bacterial ghosts and their intrinsic adjuvant effect In addition to the stability of the vaccine antigen in situ and the efficient delivery to the correct type of immune cells, an adjuvant effect is necessary to stimulate and potentiate the immune system. Such adjuvants are Alum, Freunds adjuvant, tapioca, cholera toxin (CT), E. coli heat labile toxin (LT), muramyl dipeptide, immune stimulating complexes (ISCOMS) and other bacterial cell wall components. In contrast to many subunit vaccines, bacterial ghosts contain some well-known immune-stimulating compounds, such as LPS and peptidoglycan, that are believed to enhance the activation of macrophages and DCs, which are major properties of adjuvants. Thus, bacterial ghosts combine both properties, to act as specific antigens and as necessary adjuvants to stimulate the immune response. Furthermore, a significant activation of cytokines, such as IL-12 and IL-18 was observed from DCs activated with bacterial ghosts, and DCs showed an increased ability to activate T-cells (Haslberger et al., 2000). These
data indicate that bacterial ghosts tend to stimulate a cellular TH1 immune response.
Induction of protective immunity by H. pylori ghosts in the mouse model Animal studies have shown that immunization with H. pylori whole-cell sonicates or purified components is efficient for the prevention of infection, and, even more importantly, for the treatment of pre-existing infections (Czinn and Nedrud, 1991; Corthe´sy-Theulaz et al., 1995; Ferrero et al., 1994; Michetti et al., 1994). All successful vaccination protocols included—in addition to the antigen—mucosal adjuvants, CT or LT. A major problem for development of an efficient vaccine against H. pylori in humans seems to be the choice of an appropriate mucosal adjuvant (Sutton and Lee, 2000). In a phase I–II clinical trial, where recombinant H. pylori urease was given orally to H. pylori-infected human volunteers, the dose of heat-labile toxin had to be reduced from 10 to 5 mg, due to intestinal toxicity (Michetti et al., 1999). Since CT and LT, and even the genetically detoxified forms of these adjuvants, induce diarrhea in humans (Kotloff et al., 2001; Michetti et al., 1999), it would be desirable to finally engineer a vaccine without the need of these adjuvants. We hypothesized that the use of H. pylori ghosts instead of purified antigens might avoid the use of adjuvant due to the rather acid- and protease-resistant nature of the bacterial ghost antigens and the presence of cell wall components with adjuvant properties. With one defined application dose of H. pylori ghosts we demonstrated in
Fig. 2. Prophylactic immunization with H. pylori ghosts. Two independent charges (A, B) of H. pylori ghosts were generated and used for vaccination experiments in the H. pylori mouse challenge model (Panthel et al., 2003). Oral immunizations were performed three times in weekly intervals and challenged with H. pylori three weeks later. Bacterial colonization was assessed by quantitative re-isolation of the challenge strain. Cfu/g, colony-forming units/gram of stomach tissue; naive, non-infected animals; PBS, shamimmunized, infection control; G, Ghosts; G+CT, Ghosts+cholera toxin; bars indicate the mean of the different groups (5 animals) and standard deviation. *indicates po0.05.
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a prophylactic vaccination experiment, indeed a protection against an H. pylori challenge strain in the mouse model without using an additional adjuvant (Fig. 2) (Panthel et al., 2003). By optimising gene expression of the E gene in H. pylori and by improving the treatment conditions after lysis, it might be feasible to generate in future H. pylori ghosts more reproducibly and completely free of cytosol. Thus, H. pylori ghosts have to be considered an interesting alternative to the vaccination techniques used so far to control the H. pylori infection.
Conclusions Vaccination with a killed microorganism enables the immune system to come into a risk-free contact with an otherwise dangerous pathogen. As the specific receptorrecognition properties of ghosts are the same as their living counterparts, they attach to tissue surfaces specific for the bacteria which they are derived from. This quality makes bacterial ghosts well adapted for targeting themselves and material entrapped within them to specific surfaces of host tissues. The efficient uptake of ghosts by APC facilitates the induction of an immune response against the original bacteria, or against foreign (recombinant) antigens, entrapped in the outer membrane, the periplasmic space or attached to the inner membrane of the bacterial ghost. In addition to a superior presentation of native antigens to the immune system, the ghost vaccination approach offers the unique possibility to design a multivalent vaccine by combining as many foreign antigens as necessary into a suitable bacterial carrier. Future vaccine trials will show whether candidate ghost vaccines are effective without the need of further adjuvants, are stable and are easy to administer, e.g. as an oral vaccine.
Acknowledgements The authors are grateful to W. Fischer for critical comments on the manuscript. The work on H. pylori vaccine development in the lab of the authors was funded by a grant of the Deutsche Forschungsgemeinschaft (DFG) to R. Haas (HA2697/4-2).
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REVIEW
Recombinant bacterial ghosts: versatile targeting vehicles and promising vaccine candidates Herbert Hoffelner, Rainer Haas Max von Pettenkofer Institut fu¨r Hygiene und Mikrobiologie, LMU Mu¨nchen, Pettenkoferstr. 9a, D-80336 Mu¨nchen, Germany Received 9 February 2004; received in revised form 14 April 2004; accepted 19 April 2004
Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303 Generation of bacterial ghosts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304 General principle of bacterial ghost formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304 Tightly controlled expression of PhiX174 lysis gene E . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304 Structural and functional aspects of bacterial ghosts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305 Application of the method to H. pylori . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305 Use of bacterial ghosts as vaccine delivery systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307 Specific targeting of bacterial ghosts to relevant cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307 Bacterial ghosts and their intrinsic adjuvant effect. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308 Induction of protective immunity by H. pylori ghosts in the mouse model. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309
Introduction Infectious diseases are still a leading cause of morbidity and death worldwide. Several diseases, thought to be under control for many years, have reemerged and a number of new diseases have emerged recently. Due to the often very rapid development of Corresponding author. Tel.: +49 89 5160 5255; fax: +49 89 5160 5223. E-mail address: [email protected] (R. Haas).
1438-4221/$ - see front matter r 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.ijmm.2004.04.003
microbial resistance to antimicrobial agents upon treatment of patients with drugs, the general management of a variety of diseases became much more difficult and expensive. The application of vaccines is, however, still a very efficient procedure in preventing infectious diseases. In principle, there are several different ways to design a vaccine. Modern approaches, applying recombinant DNA technologies, have so far exerted a limited impact on vaccine production, but the chances for a rational vaccine design using recombinant DNA technology are manifold and promising in the future.
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In general, vaccines might be grouped into different categories, the live vaccines, killed or inactivated vaccines, DNA- and subunit vaccines. Live bacterial vaccine carriers, such as attenuated Salmonella vaccine strains, can be equipped with defined recombinant antigens against pathogenic bacteria, as demonstrated by the production of Mycobacterium tuberculosis (Hess and Kaufmann, 1999), or Helicobacter pylori (GomezDuarte et al., 1999) antigens in attenuated Salmonella vaccine strains. The recombinant antigen(s) might reside within the Salmonella carrier strain (Gomez-Duarte et al., 1999), they might be secreted into the environment (Hess and Kaufmann, 1999), or they are injected into the host cells by a modified Salmonella type III secretion system (Ru¨ssmann et al., 1998). A rather novel approach is DNA vaccination. By this technology, naked plasmid DNA, that directs the synthesis and secretion of a vaccine antigen, is injected intramuscularly. The plasmid DNA is taken up by host cells and the encoded antigen is expressed and processed in the cell, comparable to a viral antigen. Subsequently, a humoral or cellular immune response will be triggered. Furthermore, killed pathogens, as substitutes for living infectious agents, have been widely used as a principle for vaccine development. Non-living vaccines are produced by chemical or physical inactivation of pathogenic bacteria. Here we discuss a group of rather novel vaccine candidates, grouped into the non-living, wholecell bacterial antigens, the so-called bacterial ghosts. Bacterial ghosts are Gram-negative bacterial cell envelopes, devoid of cytoplasmic contents, while maintaining their cellular morphology and native surface antigenic structures. Ghosts are suitable as a novel vaccine delivery system, endowed with intrinsic adjuvant properties, but also as general targeting vehicles for drugs. They are produced by PhiX174 protein E-mediated lysis of Gram-negative bacteria. The intrinsic adjuvant properties of bacterial ghost preparations enhance immune responses against envelope-bound antigens, including T-cell activation and mucosal immunity. Since native and foreign antigens might be produced in the envelope complex of ghosts before E-mediated lysis, multiple antigens of various origins can be presented to the immune system simultaneously, resulting in multivalent vaccine vehicles. The potency, safety and relatively low production cost of bacterial ghosts offer a significant technical advantage, especially when used as combination vaccines. The candidate ghost vaccines are supposed to be stable without the requirement of permanent cooling, easy to administer and effective. In this review we will discuss the new developments in the generation of ghosts from pathogenic bacteria and their application as vaccine candidates, with major emphasis on the human gastric pathogen H. pylori.
Generation of bacterial ghosts General principle of bacterial ghost formation Bacterial ghosts are inactivated, empty cells, that carry a natural outer surface, providing them with the original targeting functions of the pathogen they are derived from. They are thus able to induce a strong local or systemic immunity, depending on the mode of delivery. Ghosts are produced by controlled expression of the cloned bacteriophage PhiX174 lysis gene E (Witte et al., 1990a). The lysis gene E encodes a membrane protein of 91 amino acids, which is able to fuse inner and outer membranes of Gram-negative bacteria (Witte et al., 1990b) leading to the formation of a transmembrane tunnel structure through the cell envelope of pathogenic bacteria. For Escherichia coli, this tunnel varies in diameter between 40 and 200 nm (Witte et al., 1992). The cytoplasmic content of the bacteria is expelled upon opening, leaving an empty internal space devoid of nucleic acids, ribosomes or other constituents (Witte et al., 1992). The driving force for the release of cytoplasmic material is believed to depend on the osmotic pressure difference between the cytoplasm and the medium created by the opening of the tunnel structure.
Tightly controlled expression of PhiX174 lysis gene E The expression of the lysis gene E in the host bacterium has to be under tight genetic control, since a leaky expression status is lethal for the host bacterium, or induces suppressor mutations resulting in abrogation of gene E expression. Thus, the lysis gene E has been placed under transcriptional control of either the thermo-sensitive lpL =pR -cI857 promoter, or under the lacPO-lacIq promoter/repressor systems (Szostak et al., 1996). In an attempt to further extend the heat stability of the lpR promoter/cI857 repressor system, the repressor-binding operator region has been mutated. This resulted in a modified expression system, which stably repressed production of the lysis protein at temperatures of up to 37 1C, but still allowed induction of cell lysis at a temperature range between 39 1C and 42 1C (Jechlinger et al., 1999) (Fig. 1). The combination of the lpR promoter/cI repressor system with either the lacIq/lacPO resulted in a cold-sensitive system for ghost formation, which is induced by lowering the growth temperature of the bacteria from 37 to 28 1C or lower (Jechlinger et al., 1998). Since the structure and function of the Gram-negative cell envelope seems to be relatively conserved between bacteria, E-mediated lysis has been achieved in various Gram-negative bacteria, including E. coli K12 strains, Salmonella typhimurium, Salmonella
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Fig. 1. Production of bacterial ghosts by E-mediated lysis. The PhiX174 lysis gene E is under transcriptional control of the temperature-sensitive cI857 repressor and either the wild-type rightward lPR promoter (A) or the mutated lPRmut promoter (B) above the respective temperatures, gene E is expressed and a transmembrane tunnel structure is formed by fusion of the inner and outer membranes, through which the cytoplasmic content is expelled. cI857, lcI857 temperature-sensitive repressor gene; lPRmut , lPR , promoters of phage Lambda; E, PhiX174 lysis gene E.
enteritidis, Actinobacillus pleuropneumoniae, Klebsiella pneumoniae, Pasteurella haemolytica, Pasteurella multocida, Ralstonia eutropha, and Vibrio cholerae (Table 1) (Jalava et al., 2003). This broad spectrum of bacteria reveals that in principle E-mediated lysis works in many, if not all Gram-negative bacterial species. The major challenge is, that the E lysis cassette has to be introduced successfully into the recipient bacterium by an appropriate vector, or integrated into the chromosome, and that a tight repression and induction of gene E expression has to be achieved.
peptidoglycan layer remains intact after E-mediated lysis of bacteria (Witte et al., 1998). The protein Especific transmembrane tunnel structure appears to be not randomly distributed over the cell envelope, but restricted to areas of potential bacterial division sites, predominantly in the middle of the cell or at polar sites (Witte et al., 1990b, 1992). This non-destructive and specific mechanism of bacterial inactivation guarantees that ghosts share all functional properties of the outer surface with their living counterparts. Therefore, ghost carriers have superior qualities as tissue- or cell-specific delivery systems.
Structural and functional aspects of bacterial ghosts Application of the method to H. pylori A major advantage of bacterial ghosts, as compared with other procedures to inactivate bacteria, such as chemical or thermal inactivation, is the intact surface structure of bacterial ghosts. The 91-amino-acid lysis protein E forms a specific lysis tunnel by fusing the inner and outer membrane of Gram-negative bacteria. Interestingly, the part of the inner membrane not involved in tunnel formation remains intact during expulsion of cytoplasmic material. Electron microscopic studies clearly show a sealed periplasmic space at the border of the lysis tunnel (Witte et al., 1990b, 1992). The
Only recently, we have adapted the technology for generation of ghosts to the human bacterial pathogen H. pylori. H. pylori is a Gram-negative bacterium, which colonises the gastric mucosa of about 50% of the world population. The infection, usually acquired during childhood, persists chronically if not treated. Colonization of the gastric mucosa by H. pylori results in an acute inflammatory response and damage to the gastric epithelium. Inflammation can then progress to several disease states, ranging in severity from superficial
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Generation of bacterial ghosts in Gram-negative bacteria and their effectiveness as vaccine candidates Recombinant antigen
Type of delivery (animal)
Protection efficiency
Reference/reviewed in
Escherichia coli NM522 E. coli XL1-B
HIV-1RT HIV-1 gp41
n.d. (IgG response) n.d. (IgG response)
Szostak et al. (1996) Szostak et al. (1996)
E. coli serotype O78:K80 E. coli E. coli Salmonella typhimurium Salmonella enteritidis Actinobacillus pleuropneumoniae
— M. bovis vspA Bacillus sbsA/Haemophilus omp26 — — —
Klebsiella pneumoniae Pasteurella haemolytica (Mannheimia)
— —
Pasteurella multocida
—
Ralstonia eutropha Vibrio cholerae
— — C. trachomatis omp toxin co-regulated pili —
Subcutaneous (mice) Subcutaneous (rabbits) Intraperitoneal (mice) Intramuscular/peroral (chicken) Unknown (mice) Intraperitoneal (mice) Peroral (mice) — Intramuscular (pigs) Aerosol (pigs) Peroral (pigs) Subcutaneous (pigs) Subcutaneous (rabbits) Subcutaneous (cattle) Intraperitoneal (mice) Subcutaneous (rabbits) — Intragastric (rabbits) Intramuscular (mice) Intragastric (rabbits) Intragastric (mice)
Lower mortality n.d. n.d. Survival prolonged — Fully protected Partially protected Partially protected n.d. (IgG response) n.d. Fully protected up to 100% protection n.d. — Fully protected Fully protected n.d. Protected
Jalava et al. (2002) Jalava et al. (2002) Riedmann et al. (2003) Szostak et al. (1996) Szostak et al. (1997) Hensel et al. (1996, 2000) Huter et al. (2000) Katinger et al. (1999) Szostak et al. (1996) Marchart et al. (2003a) Marchart et al. (2003b) Marchart et al. (2003a) Marchart et al. (2003a) Schroll et al. (1998) Eko et al. (1994a, b) Eko et al. (2003a, b) Eko et al. (2000) Panthel et al. (2003)
Helicobacter pylori n.d., not determined.
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Carrier strain
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Table 1.
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gastritis, chronic atrophic gastritis, peptic ulceration, to mucosa-associated lymphoid tissue lymphoma (MALT) or adenocarcinoma (Blaser and Berg, 2001). The H. pylori infection is associated with 95% of duodenal and 70% of gastric ulcer cases (Lee et al., 1993), and the World Health Organization classified H. pylori as a Group 1 carcinogen (IARC, 1994). Gastric cancer is the second most common fatal malignancy in the world (Pisani et al., 1993), and it is estimated that by the year 2020 it will increase from fourteenth most common cause of death to the eighth (Murray and Lopez, 1997). The current therapy relies on the use of a proton-pump inhibitor and two antibiotics. The treatment is usually efficient, but problems may occur regarding compliance, antibiotic resistance, and possible recurrence of infection. Thus, vaccination is considered an important approach to control a bacterial infection widespread as the H. pylori infection. In the past, various approaches based on the use of selected bacterial antigens, mostly specific virulence factors, such as urease, the vacuolating cytotoxin (VacA), the cytotoxin-associated antigen (CagA), the neutrophil-activating protein (NAP), and others, conferred protection in animal models of infection (Del Giudice et al., 2001). In the H. pylori mouse model we and others demonstrated that these vaccine antigens were not only effective as prophylactic vaccines, but were also useful for the treatment of an already existing H. pylori infection as a therapeutic vaccine (Corthe´sy-Theulaz et al., 1995; Radcliff et al., 1997). Recently, our group managed to successfully generate ghosts of H. pylori (Panthel et al., 2003). This was achieved by cloning of the PhiX gene E lysis cassette into an E. coli–H. pylori shuttle vector, which was subsequently introduced under repressive conditions (o35 1C) into H. pylori strain P79. Temperatureinduction of the lysis gene cassette revealed a quantitative killing of the H. pylori bacteria without induction of lysis-resistant bacteria. Biochemical and transmission electron microscopic studies identified structurally intact H. pylori. This demonstrated that in principle the procedure works for H. pylori. For the future, however, the method has to be further optimised, to obtain a stronger repression of the lysis gene E and a better induction in H. pylori. Furthermore, for better handling of the system, the lysis gene cassette should be stably integrated into the bacterial chromosome.
Use of bacterial ghosts as vaccine delivery systems Specific targeting of bacterial ghosts to relevant cells The efficiency of a non-living vaccine is usually very much dependent on the method of delivery, the stability
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of the vaccine antigen in vivo and the adjuvant properties of the vaccine formulation. Concerning these criteria, bacterial ghosts are ideal vaccine candidates. Subunit vaccines, consisting of defined recombinant antigens, often have to be delivered by specific carrier systems, such as natural or synthetic polymers or liposomes, to reach their immune target cells. Bacterial ghosts combine both functions and act as vaccine antigens and carriers at the same time. The fact that bacterial ghosts preserve their native cell wall components, including bioadhesive structures such as pili, outer membrane proteins or polysaccharides makes them suitable for attachment to specific target tissues. For oral vaccination, the gastrointestinal (GI) tract has to be targeted. Bacterial fimbriae or pili, which are long, rod-like protein polymers found on the surface of many bacterial enteropathogens, are ideal structures for GI targeting of bacterial ghost vaccines. Bacterial ghosts prepared from Vibrio cholerae or EHEC appear to be excellent targeting vehicles for the GI tract of humans, since they contain fimbriae (toxin-co-regulated pilus, TCP and long polar fimbriae, lpf) interacting with epithelial cells of the GI tract (Taylor, 1991; Torres et al., 2002). Indeed, analysis of immune responses in an animal model indicated that V. cholerae ghosts induced humoral and cellular immune responses against cell envelope constituents including protective immunity against challenge infections (Eko et al., 2003b). Targeting of ghosts to the relevant immune cells, such as macrophages and dendritic cells (DCs), might also be achieved by recognition of the carbohydrates on the surface of ghosts. Bacteria-specific oligosaccharides, which are commonly found in the bacterial lipopolysaccharide (LPS) can be recognised by the 180-kDa mannose receptor (MR), a prototype of a family of multi-lectin receptors, functioning as pattern recognition receptor for foreign oligosaccharides. Macrophages and DCs are able to internalise MR-antigen complexes into acidic lysosomal compartments, where the antigens are degraded for antigen presentation (Astarie-Dequeker et al., 1999; Sallusto et al., 1995). Also the endothelium of human skin and dermal microvascular endothelial cells (DMEC) in vitro express MR. Interestingly, DMEC were able to take up E. coli ghosts into acidic phagosomes by ways of MR, but not ghosts of V. cholerae, which do not possess mannose in their LPS (Gro¨ger et al., 2000), demonstrating the high specificity of targeting bacterial ghosts to certain types of immune cells. In addition to the GI tract, also the respiratory tract has been successfully targeted by bacterial ghosts. For this purpose bacterial ghosts have been generated from Actinobacillus pleuropneumoniae (APP), a bacterial pathogen causing severe lung infections in pigs (Chiers et al., 2002). APP ghosts were used for aerosol immunization of pigs and proved to be safe and
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protective against a challenge infection with the lung pathogenic APP (Katinger et al., 1999). The uptake of APP ghosts by primary antigen-presenting cells (APC) was also studied in the pig model. For this purpose, APP ghosts were conjugated with fluorescein isothiocyanate (FITC) and incubated with isolated porcine DCs. A remarkable increase of FITC-labelling in the APC population and up-regulation of MHC class II molecules in APC was detectable, suggesting an efficient uptake of ghosts and the activation of DCs.
Bacterial ghosts and their intrinsic adjuvant effect In addition to the stability of the vaccine antigen in situ and the efficient delivery to the correct type of immune cells, an adjuvant effect is necessary to stimulate and potentiate the immune system. Such adjuvants are Alum, Freunds adjuvant, tapioca, cholera toxin (CT), E. coli heat labile toxin (LT), muramyl dipeptide, immune stimulating complexes (ISCOMS) and other bacterial cell wall components. In contrast to many subunit vaccines, bacterial ghosts contain some well-known immune-stimulating compounds, such as LPS and peptidoglycan, that are believed to enhance the activation of macrophages and DCs, which are major properties of adjuvants. Thus, bacterial ghosts combine both properties, to act as specific antigens and as necessary adjuvants to stimulate the immune response. Furthermore, a significant activation of cytokines, such as IL-12 and IL-18 was observed from DCs activated with bacterial ghosts, and DCs showed an increased ability to activate T-cells (Haslberger et al., 2000). These
data indicate that bacterial ghosts tend to stimulate a cellular TH1 immune response.
Induction of protective immunity by H. pylori ghosts in the mouse model Animal studies have shown that immunization with H. pylori whole-cell sonicates or purified components is efficient for the prevention of infection, and, even more importantly, for the treatment of pre-existing infections (Czinn and Nedrud, 1991; Corthe´sy-Theulaz et al., 1995; Ferrero et al., 1994; Michetti et al., 1994). All successful vaccination protocols included—in addition to the antigen—mucosal adjuvants, CT or LT. A major problem for development of an efficient vaccine against H. pylori in humans seems to be the choice of an appropriate mucosal adjuvant (Sutton and Lee, 2000). In a phase I–II clinical trial, where recombinant H. pylori urease was given orally to H. pylori-infected human volunteers, the dose of heat-labile toxin had to be reduced from 10 to 5 mg, due to intestinal toxicity (Michetti et al., 1999). Since CT and LT, and even the genetically detoxified forms of these adjuvants, induce diarrhea in humans (Kotloff et al., 2001; Michetti et al., 1999), it would be desirable to finally engineer a vaccine without the need of these adjuvants. We hypothesized that the use of H. pylori ghosts instead of purified antigens might avoid the use of adjuvant due to the rather acid- and protease-resistant nature of the bacterial ghost antigens and the presence of cell wall components with adjuvant properties. With one defined application dose of H. pylori ghosts we demonstrated in
Fig. 2. Prophylactic immunization with H. pylori ghosts. Two independent charges (A, B) of H. pylori ghosts were generated and used for vaccination experiments in the H. pylori mouse challenge model (Panthel et al., 2003). Oral immunizations were performed three times in weekly intervals and challenged with H. pylori three weeks later. Bacterial colonization was assessed by quantitative re-isolation of the challenge strain. Cfu/g, colony-forming units/gram of stomach tissue; naive, non-infected animals; PBS, shamimmunized, infection control; G, Ghosts; G+CT, Ghosts+cholera toxin; bars indicate the mean of the different groups (5 animals) and standard deviation. *indicates po0.05.
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a prophylactic vaccination experiment, indeed a protection against an H. pylori challenge strain in the mouse model without using an additional adjuvant (Fig. 2) (Panthel et al., 2003). By optimising gene expression of the E gene in H. pylori and by improving the treatment conditions after lysis, it might be feasible to generate in future H. pylori ghosts more reproducibly and completely free of cytosol. Thus, H. pylori ghosts have to be considered an interesting alternative to the vaccination techniques used so far to control the H. pylori infection.
Conclusions Vaccination with a killed microorganism enables the immune system to come into a risk-free contact with an otherwise dangerous pathogen. As the specific receptorrecognition properties of ghosts are the same as their living counterparts, they attach to tissue surfaces specific for the bacteria which they are derived from. This quality makes bacterial ghosts well adapted for targeting themselves and material entrapped within them to specific surfaces of host tissues. The efficient uptake of ghosts by APC facilitates the induction of an immune response against the original bacteria, or against foreign (recombinant) antigens, entrapped in the outer membrane, the periplasmic space or attached to the inner membrane of the bacterial ghost. In addition to a superior presentation of native antigens to the immune system, the ghost vaccination approach offers the unique possibility to design a multivalent vaccine by combining as many foreign antigens as necessary into a suitable bacterial carrier. Future vaccine trials will show whether candidate ghost vaccines are effective without the need of further adjuvants, are stable and are easy to administer, e.g. as an oral vaccine.
Acknowledgements The authors are grateful to W. Fischer for critical comments on the manuscript. The work on H. pylori vaccine development in the lab of the authors was funded by a grant of the Deutsche Forschungsgemeinschaft (DFG) to R. Haas (HA2697/4-2).
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