Oncolysate-loaded Escherichia coli bacterial ghosts enhance the stimulatory capacity of human dendritic cells.

The natural adjuvant properties of bacterial ghosts (BGs) lie within the presence of intact pathogen-associated molecular patterns on their surface. BGs can improve the direct delivery, natural processing and presentation of target antigens within dendritic cells (DCs). Moreover, sensitization of human DCs by cancer cell lysate (oncolysate)-loaded BGs in the presence of IFN-α and GM-CSF enhanced DC maturation as indicated by an increased expression of maturation markers and co-stimulatory molecules, higher production of IL-12p70 and stimulation of significantly increased proliferation of both autologous CD4+ and CD8+ T cells compared to DCs matured in the presence of purified lipopolysaccharide. The induced T cells efficiently recognized oncolysate-derived tumor-associated antigens expressed by cancer cells used for the production of oncolysate. Our optimized one-step simultaneous antigen delivery and DC maturation-inducing method emerges as a promising tool for the development and implementation of next-generation cellular cancer immunotherapies.

A novel method to recover inclusion body protein from recombinant E. coli fed-batch processes based on phage ΦX174-derived lysis protein E.

Production of recombinant proteins as inclusion bodies is an important strategy in the production of technical enzymes and biopharmaceutical products. So far, protein from inclusion bodies has been recovered from the cell factory through mechanical or chemical disruption methods, requiring additional cost-intensive unit operations. We describe a novel method that is using a bacteriophage-derived lysis protein to directly recover inclusion body protein from Escherichia coli from high cell density fermentation process: The recombinant inclusion body product is expressed by using a mixed feed fed-batch process which allows expression tuning via adjusting the specific uptake rate of the inducing substrate. Then, bacteriophage ΦX174-derived lysis protein E is expressed to induce cell lysis. Inclusion bodies in empty cell envelopes are harvested via centrifugation of the fermentation broth. A subsequent solubilization step reveals the recombinant protein. The process was investigated by analyzing the impact of fermentation conditions on protein E-mediated cell lysis as well as cell lysis kinetics. Optimal cell lysis efficiencies of 99% were obtained with inclusion body titers of >2.0 g/l at specific growth rates higher 0.12 h-1 and inducer uptake rates below 0.125 g/(g × h). Protein E-mediated cell disruption showed a first-order kinetics with a kinetic constant of -0.8 ± 0.3 h-1. This alternative inclusion body protein isolation technique was compared to the one via high-pressure homogenization. SDS gel analysis showed 10% less protein impurities when cells had been disrupted via high-pressure homogenization, than when empty cell envelopes including inclusion bodies were investigated. Within this contribution, an innovative technology, tuning recombinant protein production and substituting cost-intensive mechanical cell disruption, is presented. We anticipate that the presented method will simplify and reduce the production costs of inclusion body processes to produce technical enzymes and biopharmaceutical products.

Multi-parameter flow cytometry as a process analytical technology (PAT) approach for the assessment of bacterial ghost production.

Flow cytometry (FCM) is a tool for the analysis of single-cell properties in a cell suspension. In this contribution, we present an improved FCM method for the assessment of E-lysis in Enterobacteriaceae. The result of the E-lysis process is empty bacterial envelopes-called bacterial ghosts (BGs)-that constitute potential products in the pharmaceutical field. BGs have reduced light scattering properties when compared with intact cells. In combination with viability information obtained from staining samples with the membrane potential-sensitive fluorescent dye bis-(1,3-dibutylarbituric acid) trimethine oxonol (DiBAC4(3)), the presented method allows to differentiate between populations of viable cells, dead cells, and BGs. Using a second fluorescent dye RH414 as a membrane marker, non-cellular background was excluded from the data which greatly improved the quality of the results. Using true volumetric absolute counting, the FCM data correlated well with cell count data obtained from colony-forming units (CFU) for viable populations. Applicability of the method to several Enterobacteriaceae (different Escherichia coli strains, Salmonella typhimurium, Shigella flexneri 2a) could be shown. The method was validated as a resilient process analytical technology (PAT) tool for the assessment of E-lysis and for particle counting during 20-l batch processes for the production of Escherichia coli Nissle 1917 BGs.

Protection against Shiga-Toxigenic Escherichia coli by Non-Genetically Modified Organism Receptor Mimic Bacterial Ghosts.

Shiga-toxigenic Escherichia coli (STEC) causes severe gastrointestinal infections in humans that may lead to life-threatening systemic sequelae, such as the hemolytic uremic syndrome (HUS). Rapid diagnosis of STEC infection early in the course of disease opens a window of opportunity for therapeutic intervention, for example, by administration of agents that neutralize Shiga toxin (Stx) in the gut lumen. We previously developed a recombinant bacterium that expresses a mimic of the Stx receptor globotriaosyl ceramide (Gb3) on its surface through modification of the lipopolysaccharide (A. W. Paton, R. Morona, and J. C. Paton, Nat Med 6:265-270, 2000, http://dx.doi.org/10.1038/73111). This construct was highly efficacious in vivo, protecting mice from otherwise fatal STEC disease, but the fact that it is a genetically modified organism (GMO) has been a barrier to clinical development. In the present study, we have overcome this issue by development of Gb3 receptor mimic bacterial ghosts (BGs) that are not classified as GMOs. Gb3-BGs neutralized Stx1 and Stx2 in vitro with high efficiency, whereas alternative Gb3-expressing non-GMO subbacterial particles (minicells and outer membrane blebs) were ineffective. Gb3-BGs were highly efficacious in a murine model of STEC disease. All mice (10/10) treated with Gb3-BGs survived challenge with a highly virulent O113:H21 STEC strain and showed no pathological signs of renal injury. In contrast, 6/10 mice treated with control BGs succumbed to STEC challenge, and survivors exhibited significant weight loss, neutrophilia, and histopathological evidence of renal damage. Thus, Gb3-BGs offer a non-GMO approach to treatment of STEC infection in humans, particularly in an outbreak setting.

A novel one-step expression and immobilization method for the production of biocatalytic preparations.

BACKGROUND: Whole cell biocatalysts and isolated enzymes are considered as state of the art in biocatalytic preparations for industrial applications. Whole cells as biocatalysts are disadvantageous if substrate or products are toxic to the cells or undesired byproducts are formed due to the cellular metabolism. The use of isolated enzymes in comparison is more expensive due to the required downstream processing. Immobilization of enzymes after purification increases preparation costs for biocatalysts significantly, but allows for the efficient reuse of the enzymes in the biocatalytic process. For a more rapid processing one-step expression and immobilization is desirable. RESULTS: This study focused on the development of a new one-step expression and immobilization technique for enzymes on the example of the ß-galactosidase from Escherichia coli K12. The enzyme was expressed in E. coli with a C-terminal membrane anchor originating from cytochrome b5 from rabbit liver and was thus in situ immobilized to the inner surface of the cytosolic membrane. Then, the expression of a lytic phage protein (gene E from PhiX174) caused the formation of a pore in the cell wall of E. coli, which resulted in release of the cytosol. The cellular envelopes with immobilized enzymes were retained. Batch and fed-batch processes were developed for efficient production of these biocatalysts. It was possible to obtain cellular envelopes with up to 27,200 ± 10,460 immobilized enzyme molecules per cellular envelope (753 ± 190 U/gdry weight). A thorough characterization of the effects of membrane immobilization was performed. Comparison to whole cells showed that mass transfer limitation was reduced in cellular envelopes due to the pore formation. CONCLUSION: In this study the feasibility of a new one-step expression and immobilization technique for the generation of biocatalytic preparations was demonstrated. The technique could be a useful tool especially for enzyme systems, which are not suitable for whole-cell biocatalysts due to severe mass transfer limitations or undesired side reactions mediated by cytosolic enzymes.

Novel Salinomycin-Based Paramagnetic Complexes-First Evaluation of Their Potential Theranostic Properties.

Combining therapeutic with diagnostic agents (theranostics) can revolutionize the course of malignant diseases. Chemotherapy, hyperthermia, or radiation are used together with diagnostic methods such as magnetic resonance imaging (MRI). In contrast to conventional contrast agents (CAs), which only enable non-specific visualization of tissues and organs, the theranostic probe offers targeted diagnostic imaging and therapy simultaneously. METHODS: Novel salinomycin (Sal)-based theranostic probes comprising two different paramagnetic metal ions, gadolinium(III) (Gd(III)) or manganese(II) (Mn(II)), as signal emitting motifs for MRI were synthesized and characterized by elemental analysis, infrared spectral analysis (IR), electroparamagnetic resonance (EPR), thermogravimetry (TG) differential scanning calorimetry (DSC) and electrospray ionization mass spectrometry (ESI-MS). To overcome the water insolubility of the two Sal-complexes, they were loaded into empty bacterial ghosts (BGs) cells as transport devices. The potential of the free and BGs-loaded metal complexes as theranostics was evaluated by in vitro relaxivity measurements in a high-field MR scanner and in cell culture studies. RESULTS: Both the free Sal-complexes (Gd(III) salinomycinate (Sal-Gd(III) and Mn(II) salinomycinate (Sal-Mn(II)) and loaded into BGs demonstrated enhanced cytotoxic efficacy against three human tumor cell lines (A549, SW480, CH1/PA-1) relative to the free salinomycinic acid (Sal-H) and its sodium complex (Sal-Na) applied as controls with IC50 in a submicromolar concentration range. Moreover, Sal-H, Sal-Gd(III), and Sal-Mn(II) were able to induce perturbations in the cell cycle of treated colorectal and breast human cancer cell lines (SW480 and MCF-7, respectively). The relaxivity (r1) values of both complexes as well as of the loaded BGs, were higher or comparable to the relaxivity values of the clinically applied contrast agents gadopentetate dimeglumine and gadoteridol. CONCLUSION: This research is the first assessment that demonstrates the potential of Gd(III) and Mn(II) complexes of Sal as theranostic agents for MRI. Due to the remarkable selectivity and mode of action of Sal as part of the compounds, they could revolutionize cancer therapy and allow for early diagnosis and monitoring of therapeutic follow-up.

Iron-regulated lysis of recombinant Escherichia coli in host releases protective antigen and confers biological containment.

The use of a recombinant bacterial vector vaccine is an attractive vaccination strategy to induce an immune response to a carried protective antigen. The superiorities of live bacterial vectors include mimicry of a natural infection, intrinsic adjuvant properties, and the potential for administration by mucosal routes. Escherichia coli is a simple and efficient vector system for production of exogenous proteins. In addition, many strains are nonpathogenic and avirulent, making it a good candidate for use in recombinant vaccine design. In this study, we screened 23 different iron-regulated promoters in an E. coli BL21(DE3) vector and found one, P(viuB), with characteristics suitable for our use. We fused P(viuB) with lysis gene E, establishing an in vivo inducible lysis circuit. The resulting in vivo lysis circuit was introduced into a strain also carrying an IPTG (isopropyl-ß-d-thiogalactopyranoside)-inducible P(T7)-controlled protein synthesis circuit, forming a novel E. coli-based protein delivery system. The recombinant E. coli produced a large amount of antigen in vitro and could deliver the antigen into zebrafish after vaccination via injection. The strain subsequently lysed in response to the iron-limiting signal in vivo, implementing antigen release and biological containment. The gapA gene, encoding the protective antigen GAPDH (glyceraldehyde-3-phosphate dehydrogenase) from the fish pathogen Aeromonas hydrophila LSA34, was introduced into the E. coli-based protein delivery system, and the resultant recombinant vector vaccine was evaluated in turbot (Scophtalmus maximus). Over 80% of the vaccinated fish survived challenge with A. hydrophila LSA34, suggesting that the E. coli-based antigen delivery system has great potential in bacterial vector vaccine applications.

Effectiveness of engineering the nontypeable Haemophilus influenzae antigen Omp26 as an S-layer fusion in bacterial ghosts as a mucosal vaccine delivery.

The potential of empty bacterial cell envelopes (ghosts) as a delivery system for mucosal immunization was assessed in a rat model and different routes of immunization were evaluated. Animals were mucosally immunized targeting either gut only or gut and lung mucosal sites with Escherichia coli ghosts harbouring the nontypeable Haemophilus influenzae (NTHi) antigen Omp26. Omp26 was expressed as either a part of an S-layer fusion or as a soluble protein in the periplasm. In the gut/lung regime two initial gut targeted inoculations with the ghosts were followed by an intratracheal (IT) boost with purified Omp26. The gut only immunization regime showed a moderate enhancement of bacterial clearance following pulmonary challenge whereas the gut/lung immunization regime resulted in significantly enhanced pulmonary clearance of NTHi. Both immunization regimes induced high levels of Omp26 specific antibodies in the serum of immunized rats, with higher levels in the groups that received the IT boost with purified Omp26. Analysis of IgG isotypes present in serum suggest that the immune response was predominantly of a T-helper1 type. Additionally, immunization induced a significant cellular immune response with lymphocytes from animals vaccinated using the gut/lung regime responding significantly to Omp26 when compared to control groups. The results of this study show that mucosal immunization with recombinant Omp26 in E. coli ghosts followed by a boost with purified Omp26 can induce a specific and protective immune response in a rodent model of acute lung infection.

Escherichia coli ghosts promote innate immune responses in human keratinocytes.

Bacterial ghosts (BGs) as non-living bacterial envelopes devoid of cytoplasmic content with preserved and intact inner and outer membrane structures of their living counterparts have been used to study the ability of their surface components for the induction of antimicrobial peptides and pro-inflammatory cytokines in human primary keratinocytes (KCs). Quantitative real-time PCR analysis revealed that incubation of KCs with BGs generated from wild-type Escherichia coli induced the mRNA expression of antimicrobial psoriasin (S100A7c) in a BGs particle concentration-dependent manner. Using immunoblot analysis we showed that BGs generated from the flagellin-deficient (ΔFliC) E. coli strain NK9375 were as effective as its isogenic wild-type (wt) E. coli strain NK9373 to induce psoriasin expression when normalized to BG particles being taken up by KCs. However, results obtained from endocytic activity of KCs reflect that internalization of BGs is greatly dependent on the presence of flagellin on the surface of BGs. Moreover, BGs derived from wt E. coli NK9373 strongly induced the release of the pro-inflammatory cytokines IL-6 and IL-8, compared to ΔFliC E. coli NK9375 BGs. Taken together, obtained data demonstrate that non-living BGs possessing all bacterial bio-adhesive surface properties in their original state while not posing any infectious threat have the capacity to induce the expression of innate immune modulators and that these responses are partially dependent on the presence of flagellin.

Freezing from the inside: Ice nucleation in Escherichia coli and Escherichia coli ghosts by inner membrane bound ice nucleation protein InaZ.

Ice nucleation (IN) active bacteria such as Pseudomonas syringae promote the growth of ice crystals more effectively than any material known. Using the specialized ice nucleation protein (INP) InaZ, P. syringae-the well studied epiphytic plant pathogen-attacks plants by frost damage and, likewise fascinating, drives ice nucleation within clouds when airborne in the atmosphere by linkage to the Earth's water cycle. While ice nucleation proteins play a tremendous role for life on the planet, the molecular details of their activity on the bacterial membrane surface are largely unknown. Bacterial ghosts (BGs) derived from Escherichia coli can be used as simplified model systems to study the mode of action of InaZ. In this work, the authors used BGs to study the role of InaZ localization on the luminal side of the bacterial inner membrane. Naturally, P. syringae INPs are displayed on the surface of the outer membrane; so in contrast, the authors engineered an N-terminal truncated form of inaZ lacking the transport sequence for anchoring of InaZ on the outer membrane. This construct was fused to N- and C-terminal inner membrane anchors and expressed in Escherichia coli C41. The IN activity of the corresponding living recombinant E. coli catalyzing interfacial ice formation of supercooled water at high subzero temperatures was tested by a droplet-freezing assay and surface spectroscopy. The median freezing temperature (T50) of the parental living E. coli C41 cells without INP was detected at -20.1 °C and with inner membrane anchored INPs at a T50 value between -7 and -9 °C, demonstrating that the induction of IN from the inside of the bacterium by inner membrane anchored INPs facing the luminal inner membrane side is very similar to IN induced by bacterial INPs located at the outer membrane. Bacterial ghosts derived from these different constructs showed first droplet freezing values between -6 and -8 °C, whereas E. coli C41 BGs alone without carrying inner membrane anchored INPs exhibit a T50 of -18.9 °C. Sum frequency generation spectroscopy showed structural ordered water at the BG/water interface, which increased close to the water melting point. Together, this indicates that the more efficient IN of INP-BGs compared to their living parental strains can be explained by the free access of inner membrane anchored INP constructs to ultrapure water filling the inner space of the BGs.

Corrigendum: Subcutaneous Immunization of Dogs With Bordetella bronchiseptica Bacterial Ghost Vaccine.

[This corrects the article DOI: 10.3389/fimmu.2019.01377.].

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.

Subcutaneous Immunization of Dogs With Bordetella bronchiseptica Bacterial Ghost Vaccine.

The Bordetella species are Gram-negative bacterial pathogens that colonizes mammalian respiratory tract causing respiratory diseases in humans and animals. B. bronchiseptica causes clinical conditions in many mammals including immunocompromised humans. Using the dog model of respiratory infection, it has been shown in this study that a newly developed B. bronchiseptica Bacterial Ghost (BbBG) vaccine exhibited significant protection in the face of a severe pathogenic bacterial challenge in seronegative dogs. The protein E-specific lysis mechanism was used to produce BbBGs. Bacterial Ghosts (BGs) are the empty cell envelope of Gram-negative bacterium. They are genetically processed to form a microscopic hole in their membrane, through which all the cytoplasmic contents are expelled leaving behind intact empty bacterial shells. Due to the intact surface structures of BGs, they offer the safety of inactivated but efficacy of live attenuated vaccines. In this study, seronegative dogs were vaccinated subcutaneously (s/c) with two different doses of a newly developed BbBG vaccine [lower 10∧5 (BbBG - 5) and higher 10∧7 (BbBG - 7)] on day 0 and 21. The animals were challenged (by aerosol) with virulent live B. bronchiseptica strains 41 days after first vaccination. The dogs vaccinated s/c with BbBG - 7 vaccine had significantly lower spontaneous coughing scores (P = 0.0001) than dogs in negative control group. Furthermore, the tested BbBG - 7 vaccine was equivalent to the positive control vaccine Bronchicine CAe in terms of safety and efficacy. For the first time, we report the successful use of liquid formulated BGs vaccines in animal studies. Earlier reported studies using BGs vaccines were performed with resuspended freeze-dried BGs preparations.

Harnessing the Immunomodulatory Properties of Bacterial Ghosts to Boost the Anti-mycobacterial Protective Immunity.

Tuberculosis (TB) pathogenesis is characterized by inadequate immune cell activation and delayed T cell response in the host. Recent immunotherapeutic efforts have been directed at stimulating innate immunity and enhancing interactions between antigen presenting cells and T cells subsets to improve the protective immunity against TB. In this study, we investigated the immunostimulatory properties of bacterial ghosts (BG) as a novel approach to potentiate the host immunity against mycobacterial infection. BG are intact cytoplasm-free Escherichia coli envelopes and have been developed as bacterial vaccines and adjuvant/delivery system in cancer immunotherapy. However, BG have yet to be exploited as immunopotentiators in the context of infectious diseases. Here, we showed that BG are potent inducers of dendritic cells (DC), which led to enhanced T cell proliferation and differentiation into effector cells. BG also induced macrophage activation, which was associated with enhanced nitric oxide production, a key anti-mycobacterial weapon. We further demonstrated that the immunostimulatory capability of BG far exceeds that of LPS and involves both TLR4-dependent and independent pathways. Consistently, BG treatment, but not LPS treatment, reduced the bacterial burden in infected mice, which correlated with increased influx of innate and adaptive effector immune cells and increased production of key cytokines in the lungs. Finally and importantly, enhanced bacilli killing was seen in mice co-administered with BG and second-line TB drugs bedaquiline and delamanid. Overall, this work paves the way for BG as potent immunostimulators that may be harnessed to improve mycobacteria killing at the site of infection.

Fibroblast nemosis arrests growth and induces differentiation of human leukemia cells.

Interactive signaling between cancer cells and stroma plays an important role in determining tumor development. We recently found tumor cell-derived factors to induce fibroblast clustering, and that the high amounts of hepatocyte growth factor/scatter factor (HGF/SF) produced by these cell-cell contact-activated fibroblasts enhanced the invasiveness of c-Met-expressing cancer cells. We now observed that leukemia cells lacking c-Met respond to this novel type of fibroblast activation, nemosis, with growth arrest and differentiation to a dendritic cell-like phenotype. This effect was counteracted by introduction of c-Met expression into these cells. Moreover, those leukemia cell lines harboring properly processed c-Met showed no effect in response to nemosis. We propose this effect to be mediated by nemosis-derived cytokine signals, and present the potential candidates induced in the nemotic fibroblasts: interleukins-1, -6, -8, -11, leukemia inhibitory factor and granulocyte-macrophage-colony-stimulating factor. Our results emphasize the role of activated stromal fibroblasts in controlling progression of certain hematologic malignancies in a c-Met expression-dependent manner.

Development of a Chlamydia trachomatis bacterial ghost vaccine to fight human blindness.

Trachoma is the world's leading cause of preventable disease and the third most common cause of blindness after cataract and glaucoma, affecting an estimated 84 million people and leaving 590 million at risk. As a crippling disease, trachoma causes an enormous loss of productivity and constitutes a major socioeconomic burden. Although antibiotics are effective in treating active cases of the illness, they do not prevent re-infection, which occurs with high frequency in susceptible populations. Also, once infection and pathology are established, treatment may be less effective. Another major public health challenge posed by trachoma is that a large number of infected individuals are asymptomatic and readily infect those with whom they interact. Thus, an inexpensive and easy to deliver vaccine for trachoma would be highly effective in reducing the devastation caused by this disease. Development of an effective vaccine for controlling and preventing trachoma will require an understanding of the complex immunological mechanisms that occur during infection, identifying those antigens that elicit a protective immune response and designing effective vaccine delivery systems. Significant progress has been made in the delineation of the immune correlates of protection that will form the basis of vaccine evaluation. Recent advances in chlamydial genomics and proteomics has identified a number of protective antigens or epitopes that when appropriately delivered will produce an efficacious vaccine. The challenge at this time is the development of effective methods for vaccine delivery. We have developed an effective bacterial ghost (BG) delivery system possessing intrinsic adjuvant properties and capable of simultaneously delivering multiple antigens to the immune system. Such a flexible delivery system can produce an effective vaccine that will prevent the development of trachomatous conjunctivitis and blindness. The safety and relatively cheap production cost of BG-based vaccines offer a technological and manufacturing advantage for a vaccine needed on a global scale.

Bacterial ghosts as adjuvant to oxaliplatin chemotherapy in colorectal carcinomatosis.

Colorectal cancer (CRC) is one of the most commonly diagnosed cancers and a major cause of cancer mortality worldwide. At late stage of the disease CRC often shows (multiple) metastatic lesions in the peritoneal cavity which cannot be efficiently targeted by systemic chemotherapy. This is one major factor contributing to poor prognosis. Oxaliplatin is one of the most commonly used systemic treatment options for advanced CRC. However, drug resistance - often due to insufficient drug delivery - is still hampering successful treatment. The anticancer activity of oxaliplatin includes besides DNA damage also a strong immunogenic component. Consequently, the aim of this study was to investigate the effect of bacterial ghosts (BGs) as adjuvant immunostimulant on oxaliplatin efficacy. BGs are empty envelopes of gram-negative bacteria with a distinct immune-stimulatory potential. Indeed, we were able to show that the combination of BGs with oxaliplatin treatment had strong synergistic anticancer activity against the CT26 allograft, resulting in prolonged survival and even a complete remission in this murine model of CRC carcinomatosis. This synergistic effect was based on an enhanced induction of immunogenic cell death and activation of an efficient T-cell response leading to long-term anti-tumor memory effects. Taken together, co-application of BGs strengthens the immunogenic component of the oxaliplatin anticancer response and thus represents a promising natural immune-adjuvant to chemotherapy in advanced CRC.

Immunogenicity and protection against genital Chlamydia infection and its complications by a multisubunit candidate vaccine.

BACKGROUND AND PURPOSE: Genital infections due to Chlamydia trachomatis pose a considerable public health challenge worldwide and a vaccine is urgently needed to protect against these infections. We examined whether a vaccine composed of a combination of the major outer membrane protein (MOMP) and porin B protein (PorB) of C. trachomatis would have a protective advantage over a single subunit construct. METHODS: Single and multisubunit vaccines expressing MOMP and PorB were constructed and evaluated in the mouse model of genital infection. Thus, groups of female C57BL/6 mice were immunized intramuscularly with recombinant Vibrio cholerae ghosts (VCG) expressing the vaccine antigens or VCG alone and humoral and cell-mediated immune responses were evaluated. RESULTS: Significant levels of Chlamydia-specific secretory immunoglobulin A and immunoglobulin G2a were detected in vaginal washes and serum of immunized mice. The multisubunit construct induced a significantly higher level of T-helper Type 1 response than the single subunits as measured by the amount of interferon-gamma produced by immune T cells in response to re-stimulation with ultraviolet-irradiated elementary bodies in vitro. Three weeks after the last immunization, animals were challenged intravaginally with 10(7) inclusion-forming units of C. trachomatis serovar D. There was a significant difference in the intensity and duration of vaginal shedding between the vaccine-immunized mice and controls. All the animals immunized with the multisubunit vaccine had completely resolved the infection 2 weeks post-challenge. Higher numbers of embryos were observed in vaccinated animals than in controls, indicating protection against infertility. CONCLUSION: These results underscore the potential, albeit moderate, vaccine advantage of the multisubunit formulation.

Functional display of ice nucleation protein InaZ on the surface of bacterial ghosts.

In a concept study the ability to induce heterogeneous ice formation by Bacterial Ghosts (BGs) from Escherichia coli carrying ice nucleation protein InaZ from Pseudomonas syringae in their outer membrane was investigated by a droplet-freezing assay of ultra-pure water. As determined by the median freezing temperature and cumulative ice nucleation spectra it could be demonstrated that both the living recombinant E. coli and their corresponding BGs functionally display InaZ on their surface. Under the production conditions chosen both samples belong to type II ice-nucleation particles inducing ice formation at a temperature range of between -5.6 °C and -6.7 °C, respectively. One advantage for the application of such BGs over their living recombinant mother bacteria is that they are non-living native cell envelopes retaining the biophysical properties of ice nucleation and do no longer represent genetically modified organisms (GMOs).

Bacterial ghosts as adjuvants in syngeneic tumour cell lysate-based anticancer vaccination in a murine lung carcinoma model.

Instead of relying on external anticancer factors for treatment, immunotherapy utilizes the host's own immune system and directs it against given tumour antigens. This study demonstrated that it is possible to overcome the documented immunosuppressive properties of tumour cell lysate by supplementing it with appropriate adjuvant. Lewis lung carcinoma (LLC)­challenged C57BL/6 mice were treated with LLC cryo­lysate mixed with either bacterial ghosts (BGs) generated from E. coli Nissle 1917 or B. subtilis 70 kDa protein as adjuvants. Median and overall survival, the size of metastatic foci in lung tissue and levels of circulating CD8a+ T cells were evaluated and compared to the untreated control mice or mice treated with LLC lysate alone. After primary tumour removal, a course of three subcutaneous vaccinations with LLC lysate supplemented with BGs led to a significant increase in overall survival (80% after 84 days of follow­up vs. 40% in untreated control mice), a significant increase in circulating CD8a+ T cells (16.57 vs. 12.6% in untreated control mice) and a significant decrease in metastasis foci area and incidence. LLC lysate supplemented with B. subtilis protein also improved the inspected parameters in the treated mice, when compared against the untreated control mice, but not to a significant degree. Therefore, whole cell lysate supplemented with BGs emerges as an immunostimulatory construct with potential clinical applications in cancer treatment.