Ulrike Beate Mayr

Bacterial ghosts as an oral vaccine: a single dose of Escherichia coli O157:H7 bacterial ghosts protects mice against lethal challenge.

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) is a bacterial pathogen that is associated with several life-threatening diseases for humans. The combination of protein E-mediated cell lysis to produce EHEC ghosts and staphylococcal nuclease A to degrade DNA was used for the development of an oral EHEC vaccine. The lack of genetic material in the oral EHEC bacterial-ghost vaccine abolished any hazard of horizontal gene transfer of resistance genes or pathogenic islands to resident gut flora. Intragastric immunization of mice with EHEC ghosts without the addition of any adjuvant induced cellular and humoral immunity. Immunized mice challenged at day 55 showed 86% protection against lethal challenge with a heterologous EHEC strain after single-dose oral immunization and 93.3% protection after one booster at day 28, whereas the controls showed 26.7% and 30% survival, respectively. These results indicate that it is possible to develop an efficacious single-dose oral EHEC bacterial-ghost vaccine.

The bacterial ghost platform system: Production and applications

The Bacterial Ghost (BG) platform technology is an innovative system for vaccine, drug or active substance delivery and for technical applications in white biotechnology. BGs are cell envelopes derived from Gram-negative bacteria. BGs are devoid of all cytoplasmic content but have a preserved cellular morphology including all cell surface structures. Using BGs as delivery vehicles for subunit or DNA-vaccines the particle structure and surface properties of BGs are targeting the carrier itself to primary antigen-presenting cells. Furthermore, BGs exhibit intrinsic adjuvant properties and trigger an enhanced humoral and cellular immune response to the target antigen. Multiple antigens of the native BG envelope and recombinant protein or DNA antigens can be combined in a single type of BG. Antigens can be presented on the inner or outer membrane of the BG as well as in the periplasm that is sealed during BG formation. Drugs or supplements can also be loaded to the internal lumen or periplasmic space of the carrier. BGs are produced by batch fermentation with subsequent product recovery and purification via tangential flow filtration. For safety reasons all residual bacterial DNA is inactivated during the BG production process by the use of staphylococcal nuclease A and/or the treatment with b-propiolactone. After purification BGs can be stored long-term at ambient room temperature as lyophilized product. The production cycle from the inoculation of the pre-culture to the purified BG concentrate ready for lyophilization does not take longer than a day and thus meets modern criteria of rapid vaccine production rather than keeping large stocks of vaccines. The broad spectrum of possible applications in combination with the comparably low production costs make the BG platform technology a safe and sophisticated product for the targeted delivery of vaccines and active agents as well as carrier of immobilized enzymes for applications in white biotechnology.

Activation, stimulation and uptake of bacterial ghosts in antigen presenting cells.

Bacterial ghosts have been shown to be an innovative system to prepare vaccines of various bacteria with all features of the intact bacterial cell envelopes, especially all antigenic epitopes, but also to target recombinant proteins inserted in the cell envelopes of the ghost preparations to specific antigen presenting cells. To investigate the activation of the antigen presenting cell by bacterial ghosts in more detail we studied the uptake of bacterial ghosts in dendritic porcine cells and RAW macrophages and the induction of inflammatory mediators or mediators directing the immune response in THP-1 human macrophage cell line. The synthesis of inflammatory macrophage mediators such as TNFalpha in the THP1 cell line was stimulated by a hundred-fold higher dose of ghosts from Vibrio cholerae than the corresponding LPS using ELISA-analysis. These results confirm in vivo experiments indicating no toxic effects of ghosts in rabbits even after intravenous administration in doses stimulating significant humoral responses. We were also able to see a significant activation of IL-12 indicated by the analysis of IL-12(p70) synthesis and IL-12(p40) mRNA accumulation. This interleukine is of special importance in the activation of cellular TH1 immune responses. A rapid uptake of bacterial ghosts in macrophages within 10-30 min could be confirmed by electron microscopy. As antigen presentation is especially effective in porcine dendritic cells (DC) and even a low capacity of antigen uptake is sufficient for an induction of immune responses we investigated uptake and activation of bacterial ghosts by DC. DC are known to be phagocytic in specific immature stages. We found a significant uptake of bacterial ghosts from Actinobacillus pleuropneumoniae (App) and V. cholerae conjugated with FITC (fluorescinisothiocyanate) within 2 h. These data suggest that bacterial ghosts effectively stimulate monocytes and macrophages for the induction of TH1 directed immune responses and dendritic cells treated with bacterial ghosts may serve as a promising vehicle for active immunization and immunotherapy in situ.

Characterization and immunogenicity of Vibrio cholerae ghosts expressing toxin-coregulated pili

Bacterial ghosts are attractive for use as non-living vaccines and as carriers of heterologous antigens of vaccine relevance. Ghosts were prepared from Vibrio cholerae strains of O1 or O139 serogroup after growth under culture conditions, which favor or repress the production of toxin-coregulated pili (TCP). Immunoblotting confirmed the TCP status of these V. cholerae ghosts (VCG), which retained the cellular morphology and envelope sub-component profile of viable bacteria. Rabbits were immunized with VCGs prepared from O139 bacteria with TCP-positive or TCP-negative phenotypes and the resulting sera assayed for antibodies to lipopolysaccharide (LPS) and to TCP. Regardless of the TCP status of the VCG preparations used for immunization, all animals produced antibodies to LPS as demonstrated in bactericidal assays. These antibodies were probably responsible for the capacity of the antisera to confer passive immunity to challenge with the homologous O139 strain in the infant mouse cholera model (IMCM). Only following immunization with TCP-positive VCG, however, were antibodies to TCP generated, as judged by the potential of antisera to mediate protection against a challenge strain of heterologous serogroup.

BACTERIAL GHOSTS AS NOVEL EFFICIENT TARGETING VEHICLES FOR DNA DELIVERY TO THE HUMAN MONOCYTE-DERIVED DENDRITIC CELLS

Summary: Recombinant bacterial ghosts loaded with plasmids were tested as an antigen delivery system and as a potential mediator of maturation for human monocyte-derived dendritic cells (DCs). Bacterial ghosts are cell envelopes derived from Gram-negative bacteria; the intracellular content is released by the controlled expression of plasmid-encoded lysis gene E of PhiX174. All the cell surface structures of the native bacteria, including the outer membrane proteins, adhesins, LPS, lipid A, and peptidoglycans, are preserved. Co-incubation of immature DCs with ghosts resulted in decreased expression of CD1a, CD80, and CD83 molecules, while addition of maturation mix (TNF-α, IL-1β, IL-6, and PGE2) to the cultures enhanced expression of these molecules. No marked changes were observed in the expression of the CD11c, CD40, and CD86 surface molecules. The exposure of DCs to ghosts in combination with maturation mix resulted in a nonsignificant increase in their ability to activate T cells. DCs co-incubated with bacterial ghosts carrying plasmids encoding GFP in combination with maturation mix exhibited high expression levels of GFP (up to 85%). These results indicate that in addition to their well-established use as vaccines, bacterial ghosts can also be used as carriers of nucleic acid-encoded antigens.

Antigen discovery and delivery of subunit vaccines by nonliving bacterial ghost vectors.

The bacterial ghost (BG) platform system is a novel vaccine delivery system endowed with intrinsic adjuvant properties. BGs are nonliving Gram-negative bacterial cell envelopes which are devoid of their cytoplasmic contents, yet maintain their cellular morphology and antigenic structures, including bioadhesive properties. The main advantages of BGs as carriers of subunit vaccines include their ability to stimulate a high immune response and to target the carrier itself to primary antigen-presenting cells. The intrinsic adjuvant properties of BGs enhance the immune response to target antigens, including T-cell activation and mucosal immunity. Since native and foreign antigens can be carried in the envelope complex of BGs, combination vaccines with multiple antigens of diverse origin can be presented to the immune system simultaneously. Beside the capacity of BGs to function as carriers of protein antigens, they also have a high loading capacity for DNA. Thus, loading BGs with recombinant DNA takes advantage of the excellent bioavailability for DNA-based vaccines and the high expression rates of the DNA-encoded antigens in target cell types such as macrophages and dendritic cells. There are many spaces within BGs including the inner and outer membranes, the periplasmic space and the internal lumen which can carry antigens, DNA or mediators of the immune response. All can be used for subunit antigen to design new vaccine candidates with particle presentation technology. In addition, the fact that BGs can also carry piggyback large-size foreign antigen particles, increases the technologic usefulness of BGs as combination vaccines against viral and bacterial pathogens. Furthermore, the BG antigen carriers can be stored as freeze-dried preparations at room temperature for extended periods without loss of efficacy. The potency, safety and relatively low production cost of BGs offer a significant technical advantage over currently utilized vaccine technologies.

Bacterial ghosts as carriers of protein subunit and DNA‑encoded antigens for vaccine applications

Bacterial ghosts (BGs) represent vaccine delivery systems gifted with outstanding natural adjuvant properties. BGs are empty cell envelopes of Gram-negative bacteria lacking cytoplasmic content yet retaining all unaltered morphological and structural features of their living counterparts. The intact surface make-up of BGs is easily recognized by professional APCs through pattern-recognition receptors, making them ideal for mucosal administration through oral, ocular, intranasal or aerogenic routes, which represent the most desirable methods of application in advanced vaccine use. BGs have been designed to be used as carriers of active substances and foreign antigens (protein and/or DNA) for vaccine development. This review highlights the salient features of the BGs’ versatile multipurpose vaccine platform for application in a wide range of human and veterinary medicines.

Applications of Bacterial Ghosts in Biomedicine

Bacterial Ghosts (BG) are empty cell envelopes of Gram-negative bacteria which have been produced by E-mediated lysis. BG are devoid of cytoplasmic content and in combination with the expression of the nuclease SNUC, BG are also devoid of chromosomal and plasmid DNA. Proof of concept and proof of principle studies showed that BG candidate vaccines are highly immunogenic and in many instances induce protective immunity against lethal challenge in animal models. Due to their nature of being bacterial envelope complexes, BG are endowed with intrinsic natural adjuvant activity. BG are able to stimulate the innate and adaptive immune system without any addition of exogenous adjuvants. Although the use of plasmid encoded genetic information is essential for the final make up of BG, BG are not to be considered as genetically manipulated organisms (GMO), as they are nonliving and devoid of genetic information. The latter aspect is of great importance for safety, as no pathogenic islands or antibiotic resistance cassettes can be transferred to other bacteria by horizontal gene transfer. This is an important difference to other chemical-, heat- and pressure- or radiation-inactivated vaccine candidates, which also very often need artificial adjuvants to be added to improve their immunogenicity. The final BG vaccine preparations are freeze dried and are stable for many years at ambient temperature. BG can also be used as carrier and delivery vehicles for drugs or active substances in tumor therapy and due to specific targeting of tumor cells allow a higher specificity of treatment and a reduction of the total amount of drug per application. As carrier of enzymatic activity BG can be used for a new concept of probiotics which can synthesise active compounds from substrates of the environment where they are applied with a certain preference for the gut system. Thus, BG represent a promising technology platform for novel vaccines including combination or DNA vaccines, as drug carriers for therapeutic approaches in tumor treatment and as novel probiotics.

Effective gene transfer to melanoma cells using bacterial ghosts

Bacterial ghosts (BG) are cell envelopes preparations of Gram-negative bacteria devoid of cytoplasmic content produced by controlled expression of PhiX174 plasmid-encoded lysis gene E. Eight melanoma cell lines were investigated for their capacity to bind and phagocyte BG derived from Escherichia coli NM522 and Mannheimia haemolytica A23. High capability to bind BG was observed in almost all of the analyzed cell lines, furthermore cells were able to take up BG independently of the used bacterial species. Further, transfection efficiency of BG loaded with DNA in vitro was measured. The Bowes cells exhibited a high expression level of GFP and the incubation of cells with plasmid loaded BG led up to 82% transfection efficiency.

Proteomics and Bioinformatics Strategies to Design Countermeasures against Infectious Threat Agents

The potential devastation resulting from an intentional outbreak caused by biological warfare agents such as Brucella abortus and Bacillus anthracis underscores the need for next generation vaccines. Proteomics, genomics, and systems biology approaches coupled with the bacterial ghost (BG) vaccine delivery strategy offer an ideal approach for developing safer, cost-effective, and efficacious vaccines for human use in a relatively rapid time frame. Critical to any subunit vaccine development strategy is the identification of a pathogens proteins with the greatest potential of eliciting a protective immune response. These proteins are collectively referred to as the pathogens immunome. Proteomics provides high-resolution identification of these immunogenic proteins using standard proteomic technologies, Western blots probed with antisera from infected patients, and the pathogens sequenced and annotated genome. Selected immunoreactive proteins can be then cloned and expressed in nonpathogenic Gram-negative bacteria. Subsequently, a temperature shift or chemical induction process is initiated to induce expression of the PhiX174 E-lysis gene, whose protein product forms an E tunnel between the inner and outer membrane of the bacteria, expelling all intracellular contents. The BG vaccine system is a proven strategy developed for many different pathogens and tested in a complete array of animal models. The BG vaccine system also has great potential for producing multiagent vaccines for protection to multiple species in a single formulation.