Journal of Immune Based Therapies and Vaccines Evaluation of Recombinant Invasive, Non-pathogenic Eschericia Coli as a Vaccine Vector against the Intracellular Pathogen, Brucella

Background: 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.


Background
There is no safe, effective human vaccine against brucellosis [1]. 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][7][8][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][15][16][17]. Killed Brucella or subunit vaccines are not effective in eliciting long term protection [18]. 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 [19] however Th2 or humoral immunity also participates in protecting the host [20][21][22][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 [19]. In fact numerous studies have shown that a CTL response is key to effective Brucella immunity [26,[28][29][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 [31]. Introduction of inv confers E. coli invasion of host cells by binding the αβ1integrin 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 [32]. 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 [33]. 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 [34]. 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][35][36][37][38][39][40][41][42][43][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.

Mouse care and vaccination
BALB/c female mice (H-2 d ), 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 × 10 7 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-2 d ) expressing GFP (RAW/GFP; [45]) was subjected to gamma-irradation (2 KR) and 1 × 10 6 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.

E. coli vector vaccines
All Escherichia coli used in these studies were strain DH5α™ (Invitrogen) except for recombinants expressing pDEST17 vectors were we used BL21-AI™ (Invitrogen). Table 1 describes the recombinant E. coli vector vaccines.

Invasion and gene delivery assays
One day prior to cell infection, eukaryotic cell lines were seeded at 2 × 10 5 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 CO 2 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 × 10 6 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-2K d HYLSTQSAL were costained 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 (SouthernBi- otech), and R-PE and FITC anti-rat IgG2a and anti-rat IgGκ (BD Biosciences). Flow cytometry analysis was performed on 3.5 × 10 5 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.

Flow cytometry
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.

Statistical analysis
Student's t-test was performed and results expressed as the arithmetic mean with the variance of the mean (mean ± SE).

The recombinant E. coli vaccine vector efficiently infects cells
The objective of this study was to take a non-pathogenic organism such as Escherichia coli and genetically engineer it to mimic infectivity and intracellular antigen trafficking of a pathogen such as Brucella melitensis. ). An example with TB1 and RAW264.7 cells is shown in Figure 1. To further determine whether the invasive E. coli were intracellular, invasion assays were performed ( Table 2). Note non-invasive E. coli were not recovered unless a high MOI was used. In contrast, large numbers of invasive E. coli were recovered from all cell lines analyzed. Furthermore, electron microscopy showed invasive E. coli bound to the cell surface and engulfed by lamellipodia consistent with invasin-integrin interactions ( Figure 2). Non-invasive E. coli were also used in the TEM assay, but could not be detected within or surface-bound to any non-phagocytic cell line (data not shown).
Since our intent is to use the invasive E.coli as a live vaccine vector, we examined localization and persistence of the vector in vivo. We transformed lux operon expressing E. coli DH5α (constitutively bioluminescent) with the inv-hly encoding plasmid as our invasive E. coli (inv E. coli). Mice were intraperitoneal injected with non-invasive or invasive bioluminescent E. coli and analyzed by biophotonic imaging over time. Both bioluminescent species trafficked to the spleen. However, the invasive E. coli vector persisted longer at the site of injection suggesting an extended period of antigen delivery (Figure 3).

The recombinant E. coli vaccine vector efficiently delivers therapeutics
Unlike Escherichia, Brucella, after being engulfed by the cell, escape phagosome lysis and multiply at the endoplasmic reticulum. Most likely, this process leads to MHC class I presentation of Brucella antigens by the host cell [48]. Escherichia, in contrast, are phagocytosed and rapidly destroyed with antigens being presented by MHC class II [49,50]. Therefore, the inv expressing plasmid coexpresses hly (hemolysin) to enhance MHC class I presentation of antigens carried by the invasive E. coli vaccine vector. Hemolysin (hly) or LLO perforates phagosomal membranes at low pH and the contents of the vaccine are released into the cytosol of the cell [51]. To test the functionality of the hly gene product in the E. coli vector, we first examined delivery of a eukaryotic expression plasmid, pEYFP-N1 expressing yellow fluorescent protein (YFP) under control of the eukaryotic CMV promoter, using fluorescent microscopy.  Recombinant invasive E. coli infects phagocytic and non-phagocytic cells Data indicate that the early choice of a Th1 (cellular) or a Th2 (humoral) immune response is dependent mainly on the balance between interleukin-12 (IL12), favoring a Th1 response, and interleukin-4 (IL4), favoring a Th2 response [52,53]. Vaccine studies have demonstrated that co-deliverance of IL12 with the antigen increases Th1 response to the vaccine [54][55][56][57]. Thus, we included a murine IL12 eukaryotic expression plasmid in the invasive E. coli vaccine vector and tested for delivery and expression of IL12 in cell culture. Using human HeLa cells, microfluorimetry analysis demonstrated greater than 70% of E. coli vaccine infected cells were positive for murine IL12 (Figure 4). This compared favorably to endogenous murine IL12 production by mouse Raw264.7 macrophage cell positive control. Therefore, the E. coli vaccine vector was effective in delivering therapeutics to the host.

The recombinant E. coli vaccine vector induces a Th1 response
Since we were interested in preparing a vaccine that would stimulate cell mediated immunity, we analyzed for a Th1 cytokine profile and specific CD8 + T cells. Performing realtime PCR gene expression profiling analysis on splenocytes from mice 5 h following vaccination with invasive E. coli vaccine or non-invasive E. coli, we analyzed for differences in primary immune response profiles. This timepoint was chosen because typically, cytokines that promote T cell responses are measured 5 h post-immunization [58]. Table 4 lists fold gene expression from splenocytes of animals receiving recombinant E. coli vaccine compared to control E. coli. The data were difficult to interpret since both key Th1 and Th2 cytokines were upregulated in E. coli vaccine immunized animals compared to E. coli control immunized animals. Most likely, the complexity of the cytokine profile can be attributed to the highly stimulatory LPS of E. coli [58,59]. Comparison profiles of E.coli vaccinated animals to PBS control animals were also performed (data not shown), but the results were not relevant to our objective of determining whether the recombinant E. coli vaccine would elicit a different cytokine profile relative to control E. coli.
However, because of the mixed Th1/Th2 cytokine profile of the primary immune response, we decided to investigate whether the secondary immune response would give a more defining Th1 cytokine profile response to the antigen. RAW 264.7 macrophages were co-cultured with splenic T cells from groups of mice that had been immunized 4 weeks. Half of the cultures were supplemented with the H-2K d -binding peptide HYLSTQSAL of GFP and supernatants were measured for cytokines after three days. GFP nonamer treated cultures showed a large increase in Th1 cytokine levels in E. coli vaccine immunized T cell groups with negligible change or decrease in Th2 cytokine levels ( Table 5). Production of IFNγ significantly increased for the two specific invasive E. coli vaccines, GFPinv and GFPinvIL12 whereas production of IL4 increased for the negative control vaccines, GFP and ()invIL12 as well as significantly increasing in the PBS control samples. Although the primary response indicated a mixed Th1/Th2 profile, the secondary immune response indicates a shift to the Th1 profile. Identification of anti-gen specific CD8 + T cells would confirm a Th1 profile and generation of cell-mediated immunity.

Figure 2 Transmission Electron Microscopy shows recombinant invasive E. coli similarly engulfed by non-professional phagocytic cells (D17, HeLa) and phagocytic cells (Raw
To determine the proportion of CD8 + T cells specific for GFP antigen in the spleens of E. coli vaccine immunized BALB/c mice, we used H-2K d MHC class I pentamer complex combined with the GFP peptide HYLSTQSAL (designated MHC-GFP pentamer) co-stained with CD8 + antibody and analyzed by flow cytometry. As shown in Figure 5, the invasive E. coli vaccine induced GFP peptide specific CD8 + T cells at a significant level (p < 0.05) greater than the non-vaccinated (PBS) and empty vaccine (()inv IL12; invasive without GFP) controls and at similar levels to mice given syngeneic APC's constitutively expressing the antigen (RAW/GFP). However, the non-invasive E. coli vaccine control (GFP) also induced notable levels of CD8 + T cells not significantly different than the vaccines (GFP inv and GFP inv IL12). The high number of specific CD8 + T cells induced by the invasive E. coli vaccines correlated  with the Th1 cytokine up-regulation induced in the secondary immune response by these cells in vitro ( Table 5). As a confirmation of E. coli vaccine generated cell mediated immunity, we analyzed cytolytic T lymphocyte (CTL) response.

The recombinant E. coli vaccine vector induces specific CTL responses
Splenocytes of mice immunized with the invasive E. coli vaccine vector expressing the GFP antigen were used as effector cells in cytotoxicity assays against RAW/GFP target cell lines. As shown in Figure 6,  Cytotoxicity assays affirmed that CTLs generated by the invasive E. coli vaccine were specific to the expressed antigen of the vector (Figure 7).

Discussion
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][62][63][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][67][68][69][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 [66]. 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 longlived, 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 [41] 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 com- ponents of this micro domain [71]. The Brucella endocytic pathway is distinct from the classical endosome-lysosome pathway in that Brucella inhibit phagosome-lysosome fusion [10]. Further, smooth Brucella infection of macrophages is inefficient with only 40-60% of cells infected in vitro after 1 hour [72]. In contrast, E. coli are efficiently engulfed and processed through the classical endosomelysosome pathway. However, this leads to rapid destruction of the bacteria and MHC class II presentation of antigen [73]. 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 [74]. The result is more cells internalizing the vac-cine 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.

Microfluorimetry of supernatant of HeLa cells expressing murine IL12 indicate efficient plasmid delivery after infection by recombinant invasive E. coli vaccine
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 [51], 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 macro-  phages and dendritic cells [34,35,43,44]. In vivo, E. coli vaccines expressing LLO induced a very strong anti-tumor CTL response [43]. 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 [51]. Primary immune responses activate antigen induced Tregs limiting vaccine efficacy [75]. 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 longterm 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 [61]. 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 [78]. Both IL12 and IFNγ are considered essential for protection against brucellosis [10]. 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 bacte-ria 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.
Recombinant E. coli vaccine vector delivering GFP antigen induced higher CTL response Figure 6 Recombinant E. coli vaccine vector delivering GFP antigen induced higher CTL response. Effector splenocytes of mice immunized with E. coli-GFP (GFP), our recombinant vaccine vector E. coli-GFP expressing invasin and hly (GFPinv), the recombinant vaccine vector also carrying the eukaryotic muIL12 vector (GFPinvIL12), or diluent control (PBS) were incubated with Raw/GFP target cells and assayed for cytotoxicity. Error bars represent quadruplicate wells. *GFPinvIL12 generated T cytotoxicity was significantly greater than GFP or PBS controls (p < 0.05).
Recombinant invasive E. coli vaccine vector induces specific CTL response Figure 7 Recombinant invasive E. coli vaccine vector induces specific CTL response. Inv-hly E.coli vaccine vectors expressing B. mel ORF BmeII-1097 antigen (B7), GFP antigen (GFP), or no antigen (Empty), were used to immunize mice along with a negative (PBS) control. Splenocytes were isolated and used against target RAW macrophages expressing either GFP (Raw/GFP) or B7 (Raw/B7). Data demonstrate that CTLs generated by the E. coli vaccine were specific to antigen expressed by the vaccine. *GFP vs GFP and B7 vs B7 specific cytotoxicity were significantly greater (p < 0.05) than nonspecific controls.

Conclusion
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.