Evaluation of recombinant invasive, non-pathogenic Eschericia coli as a vaccine vector against the intracellular pathogen, Brucella
© Harms et al; licensee BioMed Central Ltd. 2009
Received: 17 September 2008
Accepted: 06 January 2009
Published: 06 January 2009
There is no safe, effective human vaccine against brucellosis. Live attenuated Brucella strains are widely used to vaccinate animals. However these live Brucella vaccines can cause disease and are unsafe for humans. Killed Brucella or subunit vaccines are not effective in eliciting long term protection. In this study, we evaluate an approach using a live, non-pathogenic bacteria (E. coli) genetically engineered to mimic the brucellae pathway of infection and present antigens for an appropriate cytolitic T cell response.
E. coli was modified to express invasin of Yersinia and listerialysin O (LLO) of Listeria to impart the necessary infectivity and antigen releasing traits of the intracellular pathogen, Brucella. This modified E. coli was considered our vaccine delivery system and was engineered to express Green Fluorescent Protein (GFP) or Brucella antigens for in vitro and in vivo immunological studies including cytokine profiling and cytotoxicity assays.
The E. coli vaccine vector was able to infect all cells tested and efficiently deliver therapeutics to the host cell. Using GFP as antigen, we demonstrate that the E. coli vaccine vector elicits a Th1 cytokine profile in both primary and secondary immune responses. Additionally, using this vector to deliver a Brucella antigen, we demonstrate the ability of the E. coli vaccine vector to induce specific Cytotoxic T Lymphocytes (CTLs).
Protection against most intracellular bacterial pathogens can be obtained mostly through cell mediated immunity. Data presented here suggest modified E. coli can be used as a vaccine vector for delivery of antigens and therapeutics mimicking the infection of the pathogen and inducing cell mediated immunity to that pathogen.
There is no safe, effective human vaccine against brucellosis . Brucellosis is a zoonotic disease causing chronic fatigue, arthritis, recurrent fever, endocarditis, and orchitis in humans [2, 3]. The etiologic agents for brucellosis are the closely related, facultative, gram-negative, intracellular coccobacilli, Brucella species [4, 5]. The ease with which Brucella can be transmitted by aerosolization, and the unpredictable timing of the onset of symptoms raise the specter of a potentially insidious bioterror attack [6–9]. During the course of infection, Brucella are actively phagocytosed by macrophages or other phagocytic cells where they prevent phagosome-lysosome fusion, persist and replicate in endocytic compartments that acquire endoplasmic reticulum membranes [10, 11]. Bacteremia occurs during an acute phase that is hard to define or detect [12, 13]. Live attenuated Brucella strains are widely used to vaccinate animals against brucellosis. However, these live Brucella vaccines can cause disease and are unsafe for humans [14–17]. Killed Brucella or subunit vaccines are not effective in eliciting long term protection . Therefore, a new vaccine approach is needed.
Eliciting a specific T cell response is necessary to fight Brucella infection. Numerous studies have shown that Th1 or cell mediated immunity is crucial for protection against brucellosis  however Th2 or humoral immunity also participates in protecting the host [20–23]. Adoptive transfer of Brucella immune T cells protects mice against virulent Brucella infection [24, 25] with both CD4+ and CD8+ T cells involved in immunity [26, 27]. Nevertheless, murine brucellosis is markedly exacerbated in MHC I knockout mice that lack CD8+ T cells compared to CD4+ T cell deficient mice or wild type mice . In fact numerous studies have shown that a CTL response is key to effective Brucella immunity [26, 28–30].
Our approach utilizes a non-pathogenic Escherichia coli to mimic the intracellular pathogen Brucella melitensis in delivery and presentation of antigens to stimulate a Th1 and CTL response. E. coli are normally extracellular while Brucella are intracellular bacteria. Therefore, we engineered E. coli (DH5α) to express a plasmid containing the inv gene from Yersinia pseudotuberculosis and the hly gene from Listeria monocytogenes . Introduction of inv confers E. coli invasion of host cells by binding the αβ1-integrin heterodimer. Upon clustering of integrins, invasin activates signaling cascades. One signaling pathway causes activation of components of focal adhesion complexes including Src, focal adhesion kinase, and cytoskeletal proteins, leading to the formation of pseudopods that engulf the bacteria into the host cell. Binding of invasin to β1-integrin is necessary and sufficient to induce phagocytosis of the bacteria even by non-professional phagocytic cells. A second pathway including activation of Rac1, NF-κB, and mitogen-activated protein kinase, leads to production of proinflammatory cytokines . After internalization, E. coli is taken into the phagosome/lysosome where lysis of the bacterium occurs. The hly gene product, along with other bacterial proteins, is release into the lysosomal vesicle. The sulfhydryl-activated hly, also known as listeriolysin O (LLO) is a pore-forming cytolysin capable of binding and perforating phagosomal membranes at low pH . The cytoplasmic contents of the bacteria can then escape into the cytosolic compartment of the mammalian cell through the pores generated by LLO. This critical step exports antigen from the E. coli into the cytosol where further processing by proteosomes and translocation by TAP into the endoplasmic reticulum lumen occurs for MHC class I presentation . LLO is sufficient for MHC class I presentation of Ag when co-expressed in E. coli that are phagocytosed by Antigen Presenting Cells (APC) such as macrophages and dendritic cells [34, 35]. Using similar recombinant E. coli, others have shown successful delivery of genes and molecules [31, 34–44]. In this study, we investigate the potential of inv-hly expressing recombinant E. coli as a vaccine vector for immunization against the intracellular pathogen, Brucella.
Cells were maintained in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal calf serum (FCS), 4.5% dextrose, 1 mM sodium pyruvate, and antibiotic-antimycotic solution (100 μ/ml penicillin G sodium, 100 μg/ml streptomycin sulfate, 0.25 μg/ml amphotericin B). In addition, drugs used for selection were: Blasticidin-S (Invivogen; 10 μg/ml) and G418-sulfate (Alexis Biochemical; 400 μg/ml). Cell lines included: D17 (ATCC CCL-183), TB1 (ATCC CCL-88), J774A.1 (ATCC TIB-67), HeLa S3 (ATCC CCL-2.2), RAW 264.7 (ATCC TIB-71), HEK 293 (ATCC CRL-1573), FLK , and the cytotoxicity target cell line RAW/YFP .
Mouse care and vaccination
BALB/c female mice (H-2d), 4–6 wks old were purchased from Jackson Laboratory and injected with 0.1 ml of PBS i.p. one day prior to E. coli vaccinations to prevent the mice from succumbing to LPS-induced endotoxic shock from live E.coli. Intraperatoneal (i.p.) route of vaccination was chosen to best deliver live E. coli vector vaccine to mice based on consistency of results and ease of method. Recombinant E. coli vaccines were injected i.p. with 2 × 107 E.coli in PBS. PBS was used for negative controls. For experiments examining primary immune response cytokine profiles, mice were injected with E. coli vector vaccine and after 5 h, euthanized and spleens removed. For experiments enumerating antigen-specific CD8+ T cells, RAW264.7 macrophages (H-2d) expressing GFP (RAW/GFP; ) was subjected to gamma-irradation (2 KR) and 1 × 106 cells in PBS were vaccinated in mice i.p. following the same protocol as the E. coli vaccines. Animals were boosted with the same dose two weeks later. Four weeks after the final boost, animals were euthanized and spleens harvested and processed for CTL assays. Live imaging was performed (IVIS; Caliper Biosciences, Inc.) with animals anesthetized using Isofluorane. IVIS image analysis was performed using Living Image 3.0 software (Caliper Biosciences). Each group of mice consisted of 4 animals. All animal experiments were conducted with approval from the Institutional Animal Care and Use Committee.
The prokaryotic expression vector pGB2Ωinv-hly (10.05 kb; spectinomycin resistance) was a gift from C. Grillot-Courvalin and expresses invasin from Yersinia pseudotuberculosis and listeriolysin O (LLO) from Listeria monocytogenes; pMC221 (4.9 kb; chloramphenicol resistance) expresses uvGFP; pXen-13 (pSK luxCDABE; 8.8 kb; ampicillin resistance) was obtained from Caliper Life Sciences and carries the luxCDABE operon for engineering bioluminescent Gram-negative bacteria. The eukaryotic expression vector pEYFP-N1 (4.7 kb; kanamycin resistance) was purchased from Clontech and expresses enhanced yellow fluorescent protein (EYFP); pORF-mIL12 (4.8 kb; ampicillin resistance) was purchased from Invivogen and expresses both chains of a functional murine IL-12 connected by a linker. The retroviral vector pLNCX2/EYFP (kanamycin/neomycin resistance) was engineered using pLNCX2 (BD Biosciences) and the EYFP from pEYFP-N1. The retroviral vector pLNCX2/BMEII1097 was engineered similarly using the Brucella BMEII1097 gene from pDONR201/BMEII1097 of the Brucella ORFeome purchased from OPEN Biosystems . BMEII1097 is a probable transcription regulator syrB. This retroviral vector was used to transduce Raw 264.7 cells to be used as targets for CTL assays. The prokaryotic expression vector pDEST17/BMEII1097 was engineered from pDEST17 (invtrogen) and pDONR201/BMEII1097.
E. colivector vaccines
E. coli vector vaccines
Invasive E. coli
E. coli gfp
E. coli gfp+inv
E. coli gfp+inv+IL12
pMC221, pGB2Ω-inv-hly, pORF-mIL12
E. coli inv+IL12
Bioluminescent non-invasive E. coli
Bioluminescent invasive E. coli
E. coli EYFP
E. coli inv EYFP
E. coli inv B7a
Invasion and gene delivery assays
One day prior to cell infection, eukaryotic cell lines were seeded at 2 × 105 cells/well in a six-well plate (or two well chambered coverslips for fluorescent microscopy) in 2 ml/well RPMI with 10% fetal calf serum (Invitrogen) and grown in a humidified CO2 incubator at 37°C. E. coli were grown overnight in a shaking incubator at 37°C in LB broth (Difco) supplemented with appropriate antibiotic for plasmid selection. The following day, bacteria were counted by 600 nm absorbance spectrometry and added to washed eukaryotic cells in fresh medium without antibiotic at the specified MOI. Bacteria were then centrifuged onto the monolayer at 2 krpm for 5 min at room temperature. Cells were incubated for 90 min, washed and fresh medium added supplemented with 100 μg/ml gentamicin to kill extracellular bacteria. For invasion assays, cells were incubated for an additional 90 min to kill extracellular bacteria, then washed and lysed in 200 μl of 1% triton X-100 for 5 min at room temperature. Finally, 800 μl of LB broth was added to each well and CFU were determined on LB agar plates supplemented with chloramphenicol, the selection drug for the GFP plasmid. For gene delivery assays, cells were incubated then analyzed by fluorescent microscopy. Random fields of cells were counted and scored for fluorescence at indicated times. For IL-12 assays, infected cells were fixed and permeabilized using Cytofix/Cytoperm™ (BD Biosciences) following the manufacturer's protocol. Samples were stained using IL-12 (p40/p70) PE conjugated monoclonal antibody (BD Biosciences) and analyzed by flow cytometry.
MHC class I pentamer staining and cytokine profiling
Pooled splenocytes from four mice per immunization group were isolated and density gradient purified (Fico/Lite-LM (Mouse); Atlanta Biologicals). Leukocytes were subjected to non-T cell depletion using a Pan T Cell Isolation Kit and MACS separation (Miltenyi Biotec) following the manufacturer's protocol. Aliquots of 2 × 106 T cells were then used for flow cytometry or cytokine profiling. R-PE labeled Pro5® MHC class I pentamers GFP antigen specific for T cell receptors of H-2Kd HYLSTQSAL were co-stained with FITC labeled rat anti-mouse CD8α and used for flow cytometry along with controls following the manufacturer's suggested protocol (Proimmune). Controls included R-PE labeled rat anti-mouse CD3ε (SouthernBiotech), and R-PE and FITC anti-rat IgG2a and anti-rat IgGκ (BD Biosciences). Flow cytometry analysis was performed on 3.5 × 105 cells for each immunization group. For cytokine profiling, T cells from immunized and control mice were incubated with gamma-irradiated (2 KR) RAW 264.7 macrophages on 6 well plates with or without the addition of 50 mM GFP peptide (HYLSTQSAL; A&A Labs LLC) for 3 days. Supernatant was harvested, centrifuged to remove cell debris and processed using a Th1/Th2 cytokine kit by cytometric bead array (BD Biosciences). Data acquisition and analysis was performed according to the manufacturer's instructions using flow cytometry.
Cell mediated cytotoxicity
Splenocytes from immunized mice were isolated and gradient purified (described above) for use as effector cells. Transduced RAW 264.7 cells expressing GFP or BMEII1097 were cloned by limiting dilution and used as target cells. Cytotoxic effector cells were expanded in vitro by growth on confluent 2 KR gamma-irradiated target cells in six-well plates supplemented with 10% T-stim without Con A (BD Biosciences) for three days. Effector cells were then washed and purified through a density gradient. Cells were counted and assayed using a CytoTox 96® Non-Radioactive Cytotoxicity kit (Promega) following the manufacturer's protocol with 4 h incubation.
Acquisition was performed on a FACSCalibur flow cytometer (BD Biosciences) and analyzed using FlowJo 8.7.1 software (Tree Star, Inc).
Cell transfection and transduction
Retrovirus-mediated gene transfer was accomplished using the BD Retro-X System (BD Biosciences) following the manufacturer's suggested protocol. Briefly, 100 × 20 mm tissue culture dishes (Falcon) were seeded with the packaging cell line GP2-293 at 70–90% confluency. GP2-293 cells were co-transfected with 5 μg each of retroviral vector and the envelope glycoprotein expression vector pVSV-G using 15 μl/transfection of Lipofectamine 2000 (Invitrogen) for 3 h in a total volume of 5 ml medium/dish. Subsequently, transfection medium was replaced with 10 ml growth medium, and the cells incubated for 72 h. Retrovirus-containing supernatant was harvested, filtered (0.45 μm), and concentrated by ultracentrifugation. Supernatant was removed and virus resuspended in the residue (~200 μl) and frozen (-80°C). Cells for transduction were plated on 6-well tissue culture plates (Falcon) at 50% confluency. Concentrated retrovirus (titer unknown) along with polybrene (8 μg/ml) were added to 1 ml/well cells and incubated overnight. Transduction medium was replaced with fresh growth medium, and the following day cells were split into appropriate selective medium.
Cell lines (2 × 105 cells/well) were incubated on glass coverslips in six-well plates overnight at 37°C in a CO2 humidified incubator. Using conditions as with invasion assays, invasive or non-invasive E. coli were incubated with the cells at MOI 100 for 90 min. The cells were thoroughly washed to remove extracellular bacteria followed by gentimycin incubation for an additional 90 min. Cells were washed in PBS and fixed in Karnovsky's Fixative (Electron Microscopy Sciences) following manufacturer's protocol. TEM was performed at the University of Wisconsin Medical School Electron Microscope Facility http://www.micro.wisc.edu/. Figures were imported using Adobe Photoshop CS3 10.0.1.
Student's t-test was performed and results expressed as the arithmetic mean with the variance of the mean (mean ± SE).
The recombinant E. colivaccine vector efficiently infects cells
Intracellular bacterial survival (× 104) per 2 × 105 eukaryotic cells
MOI 10 (1.5 h Infection)
MOI 100 (1.5 h Infection)
The recombinant E. colivaccine vector efficiently delivers therapeutics
YFP gene delivery for mammalian cell expression (Fluorescent cells/103 total cells)
Inv E.coli [pEYFP-N1]
The recombinant E. colivaccine vector induces a Th1 response
Immune response gene profile of splenocytes after 5 h immunization with E. coli vaccine.
Th2 Regulation Genes
Other Immune Response Genes
Three day cytokine production (pg/ml)a of vaccinated mouse splenic T cells cultured in macrophages with (+) or without (-) 50 μM GFP peptide HYLSTQSAL.
The recombinant E. colivaccine vector induces specific CTL responses
There is no safe, effective vaccine against human brucellosis. The ability of Brucella to chronically infect humans is related to its ability to avoid a protective Th1 response [61–64]. Chronic brucellosis patients display a Th2 immune response [64, 65]. Our objective was to analyze a novel vaccine approach engineering E.coli to mimic invasion, immunoregulation, and antigen expression of Brucella without the pathogenicity of Brucella.
Recombinant invasive E. coli have been used to deliver therapeutically relevant molecules to mouse and human professional and non-professional phagocytic cells [38, 66–70]. To date, use of recombinant E. coli as vectors has mainly been for delivering DNA for genetic vaccination. The ability to easily be engulfed by cells in addition to the absence of plasmid size restrictions make bacteria an interesting vector for gene therapy. In most cases, the recombinant invasive E. coli is used to efficiently enter eukaryotic cells where it is destroyed, releasing a eukaryotic vector to the host cell for expression of a therapeutic gene . Using this basic approach, we modified E. coli to be a live vaccine that would efficiently invade host cells, deliver a eukaryotic gene expression vector to help modulate the proper immune response, and release a large amount of antigen efficiently produced by the prokaryotic expression system. E. coli infection would not be long-lived, unlike live Brucella, being cleared by the host relatively rapidly. Nevertheless, we found our invasive E. coli could survive in host cells up to 72 h after infection compared to control E. coli surviving less than 3 h post-infection (data not shown). These data had been confirmed by others  and suggest an alternate pathway of infection for our recombinant vaccine E. coli.
Bacteria enter cells through a variety of receptors. Host cell receptor(s) for binding and internalization of Brucella have not been identified but involve lipid rafts and components of this micro domain . The Brucella endocytic pathway is distinct from the classical endosome-lysosome pathway in that Brucella inhibit phagosome-lysosome fusion . Further, smooth Brucella infection of macrophages is inefficient with only 40–60% of cells infected in vitro after 1 hour . In contrast, E. coli are efficiently engulfed and processed through the classical endosome-lysosome pathway. However, this leads to rapid destruction of the bacteria and MHC class II presentation of antigen . To avoid this destructive pathway, we modified our E. coli vector to express invasin from Yersinia. This effectively made the vector 80–100% invasive to not only professional phagocytic cells, but to all cells expressing β1-integrin (Table 2, Figure 1). Further, the endocytic pathway was changed as evidenced that live recombinant E. coli could be isolated from macrophages after 3 hours (Table 2) whereas wild-type E. coli were destroyed. The pathway seemed to mimic that of Yersinia as demonstrated by TEM (Figure 2) where the bacterium adheres to a filopodium then is internalized to individual endosomes . The result is more cells internalizing the vaccine with potential to express antigen in association with MHC class I. Of great interest was the fact that in vivo, the vaccine expressed the reporter gene (lux) for a prolonged period at the site of immunization (Figure 3) as only viable bacteria continue to express lux. This confirms broad cell-type internalization and probable increased antigen presentation.
In addition to invasin of Yersinia pseudotuberculosis our recombinant E. coli vaccine vector co-expressed the hly gene of Listeria monocytogenes on the same vector. Modification of the bacterial vaccine to express listeriolysin O (LLO) was to increase MHC I presentation of the expressed antigen delivered by the vaccine. As reported by others , the bacteria would be lysed in the phagosome/lysosome. Through the pore-forming action of LLO, the cytoplasmic contents of our bacterial vaccine vector (including the over expressed antigen) would then escape into the cytosol and thereby be processed by the proteasome. In vitro, this LLO-mediated process has been shown to improve MHC I presentation of antigens by macrophages and dendritic cells [34, 35, 43, 44]. In vivo, E. coli vaccines expressing LLO induced a very strong anti-tumor CTL response . We did not confirm improved MHC I presentation of GFP antigen by LLO in studies presented here. However, we did see less YFP gene delivery for mammalian cell expression using recombinant E. coli without LLO (Table 3; data not shown). Furthermore, a recent report demonstrated that the presence of LLO in a recombinant bacterial vaccine suppresses CD4+ regulatory T cell (Treg) inhibition of antigen-specific CD8+ T cell expansion . Primary immune responses activate antigen induced Tregs limiting vaccine efficacy . The cytokine profile of the primary immune response to our recombinant E.coli vaccine vector revealed a mixed Th1/Th2 profile suggesting a high population of CD4+ T cells and possibly Tregs (Table 4). However, the secondary immune response to the vaccine shifted to a Th1 dominant cytokine profile (Table 5) and subsequent generation of antigen specific CTLs (Figures 6 and 7). It would be interesting to determine whether LLO expression in our vaccine vector affected successful CTL generation and long-term CD8+ effector memory T cells.
Three major regulatory cytokines, TNFα, IL12, and IFNγ, were increased in expression relative to controls in both primary immune response (Table 4) and secondary immune response (Table 5) using our recombinant E. coli vaccine vector indicating DC maturation and cell mediated immunity. TNFα is a multipotent proinflammatory cytokine fundamental for defense against a variety of intracellular pathogens and is primarily involved in DC maturation [76, 77]. DCs infected with E. coli clearly show a high capacity to induce the response of naïve T cells, and TNFα secretion by DCs infected with Brucella as well as E. coli was directly implicated in the maturation of these cells, since TNFα blocking antibodies cause a strong maturation decrease . Invasive E. coli vaccine, similar to Brucella, initiates the first phase of a T cell dependent adaptive immune response inducing the secretion of IL12 from APCs. IL12 then potently stimulates IFNγ production by activated naïve T cells . Both IL12 and IFNγ are considered essential for protection against brucellosis . Our inclusion of a murine IL12 mammalian expression plasmid in the E. coli vaccine vector results in a high level of IL12 expression in the infected cell (Figure 4). This IL12 rich microenvironment surrounding the host antigen presenting cell (professional or non-professional; Table 2) may be involved in supporting the Th1 profile of the secondary immune response as indicated by the high levels of TNFα and IFNγ (Table 5). The resulting maturation of DCs and CD8+ T cells would lead to cell mediated immunity.
The initial host defense to infection is stimulated by pathogen associated molecular patterns (PAMPS) common to different groups of pathogens. The toll-like receptor (TLR) family has emerged as a major group of signaling receptors for PAMPs [79, 80]. Classical LPS activates macrophages and DCs through binding the TLR-4. Nevertheless, the respective effects of APC stimulation by isolated LPS or living bacteria are clearly distinct, even when the bacteria carry a highly active LPS like E. coli; the bacteria probably bind not only to TLR-4 but also to a set of various receptors. Our studies demonstrate a notable Th1, specific CTL response to antigen delivered by the invasive, recombinant E. coli vaccine vector. However, the highly active LPS and PAMPS of E. coli may over stimulate the immune response to the vector. Engineering the E. coli genome to make the organism less stimulatory to the host would greatly improve the usefulness of this novel vaccine approach.
We began our studies with the goal of developing a live vaccine vector using an organism (E. coli) that was not pathogenic to the host and engineering it to mimic the bacterial pathogen Brucella intracellular infection to stimulate a protective cellular immune response. Our data show that this vaccine vector could efficiently infect cells of multiple tissues. These vaccine infected cells acting as antigen presenting cells can stimulate a cellular immune response with Th1 cytokine profile and specific CTLs. Studies are now in progress to determine whether this recombinant invasive E. coli vaccine vector, expressing pools of immunodominant Brucella antigens, would be sufficient to induce a protective immune response in mice. Our studies show that this novel vaccine could be applied to any disease where cellular immunity is required.
This work was sponsored by the NIH/NIAID Regional Center of Excellence for Bio-defense and Emerging Infectious Diseases Research (RCE) Program. The authors wish to acknowledge membership within and support from the Region V 'Great Lakes' RCE (NIH award 1-U54-AI-057153 and NIH R01-AI-073558).
We thank Dr. Catherine Grillot-Courvalin (Institut Pasteur, Unite des Agent Anti-bacteriens, Paris, France) for the kind gift of pGB2Ωinv-hly.
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