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Infection and Immunity, June 2006, p. 3607-3617, Vol. 74, No. 6
0019-9567/06/$08.00+0     doi:10.1128/IAI.01836-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Topical Application of Escherichia coli-Vectored Vaccine as a Simple Method for Eliciting Protective Immunity

Jianfeng Zhang, Zhongkai Shi, Fan-kun Kong, Edward Jex, Zhigang Huang, James M. Watt, Kent R. Van Kampen, and De-chu C. Tang*

Vaxin Inc., 2800 Milan Court, Birmingham, Alabama 35211

Received 9 November 2005/ Returned for modification 9 December 2005/ Accepted 2 March 2006


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ABSTRACT
 
We report here that animals can be protected against lethal infection by Clostridium tetani cells and Bacillus anthracis spores following topical application of intact particles of live or {gamma}-irradiated Escherichia coli vectors overproducing tetanus and anthrax antigens, respectively. Cutaneous {gamma}{delta}T cells were rapidly recruited to the administration site. Live E. coli cells were not found in nonskin tissues after topical application, although fragments of E. coli DNA were disseminated transiently. Evidence suggested that intact E. coli particles in the outer layer of skin may be disrupted by a {gamma}{delta}T-cell-mediated innate defense mechanism, followed by the presentation of E. coli ligand-adjuvanted intravector antigens to the immune system and rapid degradation of E. coli components. The nonreplicating E. coli vector overproducing an exogenous immunogen may foster the development of a new generation of vaccines that can be manufactured rapidly and administered noninvasively in a wide variety of disease settings.


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INTRODUCTION
 
Noninvasive epicutaneous vaccination without pain, fear, and tissue damage (35, 38) offers distinct advantages over conventional vaccination regimens in that it can be administered by nonmedical personnel and potentially has a higher compliance rate. Administration of vaccines to the surface of skin may also trigger efficient antigen presentation, as the outer layer of skin is more immunocompetent than deep tissue (9, 29). To date, both animals and humans have been immunized against a wide variety of antigens and pathogens by topical application of adenovirus-vectored vaccines (4, 17, 22, 29, 35, 38) and bacterial toxin-adjuvanted proteins (11-13).

To counteract unpredicted disease outbreaks and bioterrorist attacks, vaccines have to be not only safe and efficacious but also amenable to rapid, large-scale production. The Escherichia coli bacterium is fully defined at the molecular level (3) and has proven to be a simple and efficient vector system for the production of exogenous proteins since its first use, which marked the advent of the recombinant DNA era (1, 19). Recombinant plasmid DNA isolated from transformed E. coli vectors is also effective in eliciting an immune response when used as a genetic vaccine (33, 37). We report here that there is no need to biochemically purify recombinant protein or DNA as a vaccine from E. coli vectors. Topical application of intact E. coli particles overproducing pathogen-derived antigens can effectively mobilize the immune repertoire toward beneficial immune protection against relevant pathogens through the controlled activation of an E. coli-targeted defense mechanism in the outer layer of skin. Production and administration of this new class of vaccines are less dependent upon medical resources than any other vaccination regimens. Moreover, this demonstration provides compelling evidence that a cutaneous defense mechanism that rapidly disrupts invading bacterial cells indeed exists along the precarious skin barrier to ward off infections.


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MATERIALS AND METHODS
 
Recombinant E. coli vectors. Plasmid pTET-nir (provided by J. VanCott and J. McGhee), encoding a codon-optimized tetanus toxin C fragment (TetC) (24) driven by the E. coli nirB promoter (7), was transformed into E. coli DH10B cells (Stratagene, La Jolla, CA) to generate the EnirB-tetC vector. Plasmid pnirBVaxin, with the E. coli nirB promoter inserted upstream from a multiple cloning site (MCS), was constructed as follows. The nirB promoter, including its ATG initiation codon and ribosome binding site, was amplified by PCR from plasmid pTET-nir using primers 5'-CTCGACATGTCTATTCAGGTAAATTTGATG-3' and 5'-TATCCTCGAGCATCAGAAAGTCTCCTGTGG-3', followed by an insertion of the amplified nirB promoter into the AflIII-XhoI site of plasmid pZErO-2 (Invitrogen Corp., Carlsbad, CA), to generate plasmid pZErO-nirB. The MCS was amplified from the plasmid pBluescript II KS(+) (Stratagene) using primers 5'-CTCGTATCCTCGAGGTCGACGGTATCGA-3', and 5'-ATATAGGCCTGAGCTCCACCGCGGTGGC-3', followed by the insertion of the amplified MCS into the XhoI-StuI site of pZErO-nirB, to generate plasmid pZErO-nirB-MCS. A T7 terminator was generated by annealing synthetic oligonucleotides 5'-CCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGG-3', and 3'-TCGAGGTATTGGGGAACCCCGGAGATTTGCCCAGAACTCCCCAAAAAACGACTTTCCTCC-5'. The synthetic T7 terminator was inserted into the SacI-StuI site of pZErO-nirB-MCS to generate plasmid pnirBVaxin.

Plasmid p{lambda}PRVaxin was constructed by replacing the nirB promoter in pnirBVaxin with a fragment containing the bacteriophage lambda PR promoter-cro ribosome binding site-ATG codon and the {lambda}cI857 variant of the {lambda}cI gene from plasmid pCQV2 (28) (provided by C. Queen). The cI857 product represses PR at 32°C but allows overexpression from the PR promoter at 42°C (28). The lambda PR promoter-cI857 repressor unit was amplified from plasmid pCQV2 using primers 5'-GAATTCACATGTTTGACAGCTTATCATCGA-3' and 5'-AGATCTCTCGAGCATACAACCTCCTTAGTA-3', followed by insertion into the AflIII-XhoI site of pnirBVaxin to replace the nirB promoter.

The Bacillus anthracis protective antigen (PA) gene corresponding to the protease-cleaved PA63 fragment was excised from pCPA (a plasmid encoding the PA63 gene driven by the human cytomegalovirus [CMV] early promoter) (27) (provided by D. Galloway) with XhoI-XbaI, followed by insertion into the XhoI-XbaI site of pnirBVaxin and p{lambda}PRVaxin to generate plasmids pnirB-PA63 (PA63 driven by the nirB promoter) and p{lambda}PR-PA63 (PA63 driven by the lambda PR promoter), respectively.

The full-length PA83 gene (41) was amplified from B. anthracis DNA using primers 5'-GAATTCGGATCCGAAGTTAAACAGGAGAACCGG-3' and 5'-GGTACCCTCGAGTAATTTAAAAATCACCTAGAA-3', with built-in BamHI and XhoI restriction sites, followed by the insertion of the PA83 gene into the BamHI-XhoI site of the plasmid pCAL-n-FLAG (Stratagene), to generate plasmid pCAL-PA83. A BamHI-SacI fragment containing the full-length PA83 gene was subsequently excised from pCAL-PA83 and inserted into the BamHI-SacI site of p{lambda}PRVaxin to generate plasmid p{lambda}PR-PA83, with PA83 driven by the lambda PR promoter.

The immunogenic but atoxic fragment of the B. anthracis lethal factor (LF) (LF7 fragment) was amplified from plasmid pAdApt-LF7 (provided by M. Bell and D. Galloway) using primers 5'-ACAGTAGGATCCGCGGGCGGTCATGGTGAT-3' and 5'-GTCGACCTCGAGTTATGAGTTAATAATGAA-3'. The amplified LF7 gene was inserted into the BamHI-XhoI site of pCAL-n-FLAG to generate plasmid pCAL-LF7. The LF7 fragment was subsequently excised from pCAL-LF7 with BamHI and SacI, followed by insertion into the BamHI-SacI site of pnirBVaxin and p{lambda}PRVaxin, to generate plasmids pnirB-LF7 (LF7 driven by the nirB promoter) and p{lambda}PR-LF7 (LF7 driven by the lambda PR promoter), respectively.

The LF4 fragment in pCLF4 (27) (provided by D. Galloway) was replaced by the LF7 fragment to generate plasmid pCMV-LF7 (LF7 driven by the CMV promoter) by eliminating the XbaI fragment encompassing the LF4 fragment, followed by insertion of a KpnI-XbaI fragment containing the full-length LF7 fragment from pAdApt-LF7 into the KpnI-XbaI site downstream from the CMV promoter.

All plasmids encoding anthrax antigens were transformed into E. coli BL21-CodonPlus-RIL cells (Stratagene) to generate EnirB-PA63, E{lambda}PR-PA63, ECMV-PA63, E{lambda}PR-PA83, EnirB-LF7, E{lambda}PR-LF7, and ECMV-LF7 vectors, respectively. Unlike TetC, whose codon usage was adapted to the codon bias of E. coli genes by changing DNA sequences to make the codon usage match the available tRNA pool within E. coli cells without changing the amino acid sequences (24), the B. anthracis codons in PA and LF genes were not optimized in this study.

Vaccination. Young (approximately 3-month-old) female ICR (Harlan, Indianapolis, IN), BALB/c, and A/J (Jackson Laboratory, Bar Harbor, ME) mice were immunized by topical application of 0.1 ml of replicating or nonreplicating E. coli-vectored vaccines as a thin film onto the surface of abdominal skin as described previously (29). Briefly, the skin was prepared by depilation with an electric trimmer in conjunction with gentle brushing using a soft-bristle toothbrush without inducing erythema (Draize scores of ≤1), and unabsorbed E. coli particles were washed away 1 h postadministration, except for specific experiments (Fig. 1). Vectors were allowed to incubate with the naked skin under a piece of a Tegaderm patch (3M). E. coli vectors were harvested during the mid-log phase of growth. Some E. coli particles were {gamma}-irradiated at a lethal dose of 2,000 Gy, as described previously (34), prior to administration. No colonies were formed when 2 x 109 irradiated EnirB-tetC particles were incubated on Luria-Bertani (LB) agar plates. Some E. coli vectors were lyophilized by resuspending cells at a density of 1 x 1010 CFU/ml in phosphate-buffered saline (PBS) containing 24% trehalose, followed by rapid freezing in liquid nitrogen and drying on a Labconco Freeze Dry system (FREEZEONE 6) at –50°C to a final pressure of 10 to 20 mtorr for 1 day. Lyophilized E. coli powder was kept at room temperature without exposure to visible light. Prior to immunization, dried E. coli particles were resuspended in water, followed by {gamma}-irradiation at a dose of 2,000 Gy. E. coli vectors harboring plasmids containing the lambda PR promoter were cultivated at 32°C until entering mid-log phase and shifted to 42°C for an additional 2 h when activation of the lambda PR promoter was required. The nirB promoter was not induced in this study. All E. coli particles were washed with PBS twice prior to administration to eliminate any residual antibiotics. All experiments using mice were performed according to institutional guidelines.


Figure 1
Figure 1
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  		FIG.1. Immunization of mice against the TetC protein by topical application of live EnirB-tetC vectors. (A) Overexpression of a codon-optimized TetC fragment in E. coli. Soluble proteins extracted from E. coli DH10B cells with or without pTET-nir were fractionated on a 8 to 16% SDS-polyacrylamide gradient gel. Lane 1, proteins extracted from control E. coli DH10B cells without plasmids (5 µg); lane 2, proteins extracted from EnirB-tetC cells expressing TetC (5 µg). The arrow indicates the position of TetC. The gel was stained with Coomassie blue. Mr (in thousands) is indicated on the left. (B) Dose-response of anti-TetC antibody titers following topical application of live EnirB-tetC vectors. (C) Elicitation of anti-TetC antibody titers following incubation of naked skin with live EnirB-tetC vectors at a dose of 1 x 1010 CFU for various amounts of time. (D) Effects of stratum corneum ablation on the potency of E. coli-vectored epicutaneous vaccines. Live EnirB-tetC vectors were administered onto skin at a dose of 5 x 109 CFU. None, hair was not removed; Depilation, skin was depilated by an electric trimmer prior to vaccination; Brush, depilated skin was further brushed prior to vaccination as described previously (29). (E) Persistence of antibody titers induced by EnirB-tetC-vectored epicutaneous vaccines. Live EnirB-tetC vectors were administered onto skin at a dose of 1 x 109 CFU. Sera were collected from vaccinated animals 1 and 8 months postimmunization for analysis of anti-TetC titers. ICR mice were immunized in a single-dose regimen. Pre- and postimmunization paired sera were analyzed for anti-TetC ELISA titers as described previously (29). Animals immunized by topical application of E. coli DH10B cells without plasmids served as negative controls. Naïve animals and mice immunized with control E. coli particles all had anti-TetC titers of ≤100 as previously shown (29). The results are plotted as the log ELISA titers. Triangles, anti-TetC titers in individual mice 3 weeks postimmunization; diamonds, anti-TetC titers in individual mice 1 month postimmuniza- tion; circles, anti-TetC titers in individual mice 3 months postimmunization; squares, anti-TetC titers in individual mice 8 months postimmunization; bars, anti-TetC geometric mean log ELISA titer.

Recovery of live E. coli reporter vectors from tissues. Live E. coli XL1-Blue MRF' cells transformed with the plasmid pBluescript II KS(+) expressing ß-galactosidase (Stratagene) were administered to ICR mice by topical application at a dose of 1 x 109 CFU and intranasal instillation at a dose of 1 x 108 CFU. At indicated time points, individual organs (approximately 200 mg of tissue per organ) were homogenized in 1 ml of sterile PBS, followed by inoculation of 0.1 ml of the tissue supernatant or whole blood onto LB agar plates containing ampicillin, chloro-3-indolyl-ß-isopropyl-ß-D-thiogalactopyranoside (IPTG), and 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-gal). A dark-blue ampicillin-resistant colony represented an E. coli reporter vector that had infiltrated into the host tissue. To avoid cross-contamination, surgical instruments were sterilized by flame following soaking in 70% ethanol.

Amplification of disseminated E. coli DNA fragments. ICR mice were immunized by topical application of EnirB-tetC cells at a dose of 1 x 109 CFU. At the indicated time points, total DNA from individual organs was extracted and subjected to nested PCR, as described previously (29), to amplify a subfragment of the E. coli adenine phosphoribosyltransferase (aptE. coli) gene using the primers 5'-GCGACTGCACAGCAGCTTGAGTATCTC-3' and 5'-CAGTTTAACGGTCGCTTCGATAGTGCC-3' for the first cycle of amplification, followed by a second amplification cycle using the primers 5'-AAGCTTACGCTCTCAGCATCGACTTGC-3' and 5'-AACGTGGATCTCCAGCTGATCGGTGCC-3'. Amplified DNA fragments were fractionated in agarose gel and visualized, as described previously (29), for detection of the diagnostic 224-bp subfragment of the aptE. coli gene. Plasmid pUC8apt, containing the aptE. coli gene (14) (provided by M. Taylor), was included in the PCR analysis as a positive control. Samples were scored as negative if no signals were visualized.


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RESULTS
 
Elicitation of a systemic immune response by topical application of live E. coli-vectored vaccines. ICR mice were vaccinated by topical application of intact particles of a live E. coli vector (EnirB-tetC) overexpressing the 50-kDa TetC protein driven by the E. coli nirB promoter (Fig. 1A). As shown in Fig. 1B, a serum antibody response to TetC was elicited by the administration of a single dose in a dose-dependent manner with the anti-TetC antibody titers peaking in the dose range of 109 to 1010 CFU. There was a trend that the longer an E. coli-vectored vaccine patch was incubated with skin, the higher an antibody titer was induced (Fig. 1C), although the difference between data at 1 h and data at 24 h was statistically insignificantly (P > 0.05 by Mann-Whitney rank-sum test). As a result, the incubation time with E. coli particles on the skin was standardized to 1 h for other experiments in this study. The effectiveness of the approach in hosts with a different genetic background was tested by epicutaneous vaccination. Anti-TetC antibodies were also elicited in BALB/c mice by topical application of EnirB-tetC particles (data not shown).

Ablation of the stratum corneum appeared to be an essential step in ensuring effective epicutaneous vaccination using intact, recombinant E. coli particles. As shown in Fig. 1D, only a small number of animals (3/10 mice) exhibited seroconversion (≥10-fold elevated enzyme-linked immunosorbent assay [ELISA] titers) when E. coli vectors were administered onto skin that was depilated, but not brushed, prior to vaccine administration. All of the animals (10/10 mice) were effectively immunized when depilated skin was further brushed prior to vaccination. Moreover, the anti-TetC titers in animals in which the stratum corneum had been ablated by brushing were significantly higher than those in their nonablated counterparts (P ≤ 0.001 by Mann-Whitney rank-sum test).

The anti-TetC immune response persisted for at least 8 months after a single-dose administration of EnirB-tetC-vectored epicutaneous vaccine without significantly losing antibody titers over time (P > 0.05 by Mann-Whitney rank-sum test) (Fig. 1E).

Elicitation of a systemic immune response by topical application of {gamma}-irradiated, sonicated, fixed, and lyophilized E. coli-vectored vaccines. Vaccination by topical application of {gamma}-irradiated nonreplicating EnirB-tetC particles also elicited an immune response with a magnitude comparable to that induced by their live counterparts (Fig. 2A).


Figure 2
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  		FIG. 2. Immunization of mice against the TetC protein by topical application of irradiated, sonicated, fixed, and lyophilized EnirB-tetC vectors. (A) Effects of {gamma}-irradiation on the potency of E. coli-vectored epicutaneous vaccines. Both live EnirB-tetC vectors and their {gamma}-irradiated counterparts were administered to ICR mice by topical application at a dose of 1 x 109 particles. Anti-TetC antibody titers were determined by ELISA 3 weeks and 3 months postimmunization. (B) Inactivation of E. coli-vectored epicutaneous vaccines by sonication and formalin fixation. ICR mice were immunized by topical application of 1 x 1010 live EnirB-tetC particles, lysates made from 1 x 1010 sonicated EnirB-tetC particles, and 1 x 1010 formalin-fixed EnirB-tetC particles, respectively. Anti-TetC antibody titers were determined by ELISA 3 weeks postimmunization. EnirB-tetC cells were harvested from log-phase cultures and resuspended in PBS at a concentration of 1 x 1011 CFU/ml after two washes in PBS. An aliquot was sonicated at maximum intensity for 240 cycles, with each cycle comprising 15 s of sonication followed by 15 s of cooling on ice using the Sonicator 3000 apparatus (Misonix). Another aliquot was fixed in 10% buffered formalin for 1 h, followed by three washes in PBS. Live, anti-TetC log ELISA titer induced by live EnirB-tetC vectors; Sonication, anti-TetC log ELISA titer induced by sonicated EnirB-tetC lysates; Formalin, anti-TetC log ELISA titer induced by formalin-fixed EnirB-tetC particles. (C) Immunization by topical application of lyophilized-reconstituted-irradiated E. coli-vectored vaccines. EnirB-tetC cells were resuspended at a density of 1 x 1010 CFU/ml in PBS containing 24% trehalose, followed by lyophilization and storage of dry E. coli powder in the dark at room temperature for 1 month. Only 10% of cells could form colonies after reconstitution. EnirB-tetC cells reconstituted from dry powder and their nonlyophilized counterparts were both {gamma}-irradiated at a dose of 2,000 Gy prior to immunization. +, anti-TetC log ELISA titer in mice 3 weeks after topical application of 1 x 108 lyophilized-reconstituted-irradiated EnirB-tetC particles (density of E. coli particles was based on colony counts after reconstitution); –, anti-TetC log ELISA titer in mice 3 weeks after topical application of 1 x 108 irradiated but nonlyophilized EnirB-tetC particles. (D) Elicitation of anti-TetC antibody titers by intramuscular injection of a licensed tetanus-diphtheria vaccine (Td). ICR mice were immunized by injection of 50 µl of diluted Td (Aventis Pasteur Inc., Swiftwater, PA) into the hind-leg quadriceps, and anti-TetC antibody titers were determined by ELISA. Td was diluted in PBS. Intramuscular injection of 0.5 ml undiluted Td containing 5 Lf of tetanus toxoid is recommended for immunizing a human adult. Negative controls and symbols are described in the Fig. 1 legend.

Although {gamma}-irradiation did not decrease the potency of E. coli-vectored epicutaneous vaccines, disruption of E. coli cells by sonication (P < 0.05 by Mann-Whitney rank-sum test) or fixation of these particles by using formalin (P ≤ 0.001 by Mann-Whitney rank-sum test) significantly abolished the immunogenicity of this new class of vaccines (Fig. 2B).

The E. coli-vectored vaccine can potentially be stored and shipped without the requirement for a cold chain. After lyophilization of EnirB-tetC cells, the dry E. coli powder was stored in the dark at room temperature. Only approximately 10% of lyophilized EnirB-tetC cells were capable of forming colonies after reconstitution 1 month after lyophilization. Reconstituted EnirB-tetC particles were subsequently {gamma}-irradiated prior to immunization of mice. As shown in Fig. 2C, the potency of the lyophilized-reconstituted-irradiated EnirB-tetC-vectored epicutaneous vaccine was comparable to that of its irradiated but nonlyophilized counterpart, with a mean titer falling within a range between those induced by intramuscular injection of 0.015 and 0.15 flocculation units (Lf) of a licensed tetanus toxoid (Fig. 2D).

The effectiveness of the approach in terms of vaccination using different antigens was tested by topical application of E. coli vectors expressing anthrax antigens. The response to two different anthrax antigens was compared: B. anthracis protective antigen (PA63 or PA83) and an immunogenic but atoxic fragment of lethal factor (LF7) whose toxicity was abolished by mutating the catalytic site of B. anthracis LF (21). As shown in Fig. 3A, the potency of the E. coli-vectored epicutaneous anthrax vaccine was determined, at least in part, by the strength of the promoter driving antigen expression in E. coli cells. An immune response was not elicited when PA63 and LF7 genes were under the transcriptional control of the eukaryotic CMV promoter; anti-PA and anti-LF antibody titers were augmented significantly when a temperature-sensitive lambda PR promoter (28) driving the expression of these antigens was induced at 42°C prior to {gamma}-irradiation and vaccine administration, with respect to its uninduced counterpart and the E. coli nirB promoter (P < 0.05 by Mann-Whitney rank-sum test).


Figure 3
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  		FIG. 3. Immunization of mice against anthrax antigens by topical application of E. coli vectors expressing PA and LF7. (A) Correlation between promoter strength and the potency of E. coli-vectored epicutaneous vaccines. A/J mice were immunized by topical application of an E. coli vector expressing PA63 mixed with another E. coli vector expressing LF7 driven by the same promoter in a single-dose regimen. Both vectors were {gamma}-irradiated and administered at a dose of 5 x 109 particles per vector. Pre- and postimmunization paired sera were analyzed for anti-PA and anti-LF ELISA titers, as described previously (29), using PA and LF proteins (List Biological Laboratories, Inc., Campbell, CA) as the capture antigen, respectively. CMV, ECMV-PA63 plus ECMV-LF7 with antigens driven by the CMV promoter; NirB, EnirB-PA63 plus EnirB-LF7 with antigens driven by the nirB promoter; {lambda}32, E{lambda}PR-PA63 plus E{lambda}PR-LF7 with antigens driven by the lambda PR promoter at 32°C; {lambda}42, E{lambda}PR-PA63 plus E{lambda}PR-LF7 with antigens driven by the lambda PR promoter induced at 42°C as described in Materials and Methods. Symbols are described in the Fig. 1 legend. (B) Effect of booster application on the potency of E. coli-vectored epicutaneous anthrax vaccines. A/J mice were immunized by topical application of heat-induced {gamma}-irradiated E{lambda}PR-PA83 and E{lambda}PR-LF7 vectors at a dose of 1 x 109 particles per vector. Anti-PA and anti-LF antibody titers were determined by ELISA 1 month and 2 months postimmunization. –, mice were immunized in a single-dose regimen without booster application; +, mice were boosted once 1 month after primary immunization; inverted triangle, antibody titer in individual mice 2 months postimmunization. Other symbols are described in the Fig. 1 legend. (C) Toxin-neutralizing antibody titers induced by topical application of E. coli-vectored anthrax vaccines. A/J mice were immunized by topical application of {gamma}-irradiated E. coli-vectored anthrax vaccines at a dose of 5 x 109 particles per vector. Animals were boosted twice at intervals of 1 month. Postimmunization sera were collected for analyzing toxin-neutralizing antibody titers 1 month after the last booster application. *E, control mice immunized by irradiated E. coli-BL21-CodonPlus-RIL cells; *E-PA63, E{lambda}PR-PA63; *E-PA83, E{lambda}PR-PA83; *E-LF7, E{lambda}PR-LF7; *E-PA83+*E-LF7, E{lambda}PR-PA83 plus E{lambda}PR-LF7 (asterisk, {gamma}-irradiation). The lambda PR promoter was induced at 42°C as described in Materials and Methods. Serum samples were analyzed for toxin-neutralizing antibody titers as described previously (27), with modifications. Briefly, sera from all immunized animals in a group (n = 10 mice) were pooled in equal amounts, serially diluted, and incubated with 0.06 µg PA and 0.12 µg LF (List Biological Laboratories). Neutralized toxins and nonneutralized controls were added to 1 x 105 mouse monocyte macrophage RAW 264.7 cells (American Type Culture Collection, Manassas, VA) in a well of a 96-well plate, followed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay 1 day later. Shown is the percent cell viability. Survival of control cells without exposure to PA and LF was arbitrarily defined as 100%.

As shown in Fig. 3B, the potency of E{lambda}PR-PA83 (E. coli cells expressing PA83 driven by the heat-induced lambda PR promoter)-vectored and E{lambda}PR-LF7 (E. coli cells expressing LF7 driven by the heat-induced lambda PR promoter)-vectored anthrax vaccines could be further enhanced by a booster application (P < 0.05 by Mann-Whitney rank-sum test when antibody titers in animals without a booster application were compared to those in their boosted counterparts 2 months after primary immunization).

Toxin-neutralizing antibodies were elicited in mice following topical application of {gamma}-irradiated E. coli vectors expressing PA63 (E{lambda}PR-PA63), PA83 (E{lambda}PR-PA83), and LF7 (E{lambda}PR-LF7) under the transcriptional control of the heat-induced lambda PR promoter (Fig. 3C). PA83 appeared to be more immunogenic than PA63.

Protection of animals against tetanus and anthrax by topical application of E. coli-vectored vaccines. To determine whether the immune response elicited by epicutaneous administration of the E. coli-vectored vaccines can protect animals against live pathogens in a disease setting, ICR mice were vaccinated by topical application of live EnirB-tetC particles and then challenged by footpad injection of a lethal dose of Clostridium tetani cells as described previously (29). As shown in Fig. 4A, all of the animals (10/10 mice) vaccinated by a single topical application of 1 x 1010 CFU of live EnirB-tetC cells survived the challenge, with 90% (9/10) and 40% (4/10) of mice surviving when the dose was reduced to 1 x 109 and 1 x 108 CFU, respectively. Naïve control animals all died within 5 days. The protection afforded by topical application of a dose range between 109 and 1010 CFU reached statistical significance when it was compared to that of the naïve control group (P ≤ 0.001 by Fisher's exact test).


Figure 4
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  		FIG. 4. Protection of mice against pathogenic bacteria in disease settings. (A) ICR mice were immunized by topical application of live EnirB-tetC particles in a single-dose regimen and challenged by footpad injection of 6 x 103 C. tetani cells 3 months postimmunization as described previously (29). Naïve, no immunization; 10E8, mice immunized at a dose of 1 x 108 CFU; 10E9, mice immunized at a dose of 1 x 109 CFU; 10E10, mice immunized at a dose of 1 x 1010 CFU. Antibody titers prior to challenge are shown in Fig. 1B. (B) ICR mice were immunized by topical application of {gamma}-irradiated EnirB-tetC particles in a single-dose regimen and challenged as described in A. *10E5, mice immunized at a dose of 1 x 105 irradiated EnirB-tetC particles; *10E9, mice immunized at a dose of 1 x 109 irradiated EnirB-tetC particles; 10E9, mice immunized at a dose of 1 x 109 live EnirB-tetC particles. Antibody titers for mice in groups *10E9 and 10E9 prior to challenge are shown in Fig. 2A; those in group *10E5 were not detectable (≤100). (C) A/J mice were immunized by topical application of nonreplicating E. coli vectors producing anthrax antigens at a dose of 5 x 109 {gamma}-irradiated particles per vector; two booster applications were administered at intervals of 1 month, and animals were challenged by intranasal instillation of B. anthracis Sterne spores at a dose of 1 x 105 CFU 1 month after the last booster application. This B. anthracis Sterne 34F2 vaccine strain is nonencapsulated (pXO2–) but toxigenic (pXO1+) with PA and LF expressed from the pXO1 plasmid. Spores were produced and purified as described previously (16). *E, irradiated E. coli BL21-CodonPlus-RIL-vaccinated control mice; *E-PA83, E{lambda}PR-PA83; *E-LF7, E{lambda}PR-LF7; *E-PA83+*E-LF7, E{lambda}PR-PA83 plus E{lambda}PR-LF7 (asterisk, {gamma}-irradiation). Antibody titers prior to challenge are shown in Fig. 3C. The lambda PR promoter was induced at 42°C as described in Materials and Methods. All data are plotted as percent survival versus number of days after challenge. Numbers in parentheses represent the number of animals in each group. Results represent two independent experiments in A and C, respectively.

The level of protection afforded by a single dose of the {gamma}-irradiated, nonreplicating E. coli vectors was comparable to that induced by their live counterparts, with 78% (7/9) of mice surviving lethal challenge after topical application of 1 x 109 irradiated EnirB-tetC particles (Fig. 4B), which was significantly higher than that of the group immunized with 1 x 105 particles (P ≤ 0.001 by Fisher's exact test).

To test whether topical application of E. coli-vectored anthrax vaccines is protective, A/J mice that are susceptible to B. anthracis Sterne spores (39) were vaccinated. In this case, the mice were administered the vaccine three times at intervals of 1 month and then challenged by intranasal instillation of a lethal dose of B. anthracis Sterne spores. As shown in Fig. 4C, significant protection was not afforded by topical vaccination with irradiated E{lambda}PR-PA83 particles alone (44% survival; 4/9 mice) or irradiated E{lambda}PR-LF7 particles (13% survival; 1/8 mice). However, a vaccination protocol in which irradiated E{lambda}PR-PA83 and E{lambda}PR-LF7 particles were coadministered three times at monthly intervals provided statistically significant protection (55% survival; 11/20 mice) compared to the vector control group (P < 0.01 by Fisher's exact test).

Recruitment of {gamma}{delta}T cells to the administration site following topical application of E. coli-vectored vaccines. The recruitment of {gamma}{delta}T cells to the site of administration was evaluated by immunofluorescent staining of skin sections with a monoclonal antibody against the {delta} chain of the {gamma}{delta}T-cell receptor ({gamma}{delta}TCR) after topical application of live EnirB-tetC cells. Within 1 day of application, a large number of {gamma}{delta}T cells were recruited to the administration site (Fig. 5A). Mass recruitment of {gamma}{delta}T cells to the outer layer of skin was not observed upon topical application of an adenovirus vector encoding TetC (Fig. 5B). The density of {gamma}{delta}T cells in the skin of naïve control mice was also low (Fig. 5C).


Figure 5
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  		FIG. 5. Recruitment of {gamma}{delta}T cells to the administration site after topical application of E. coli-vectored vaccines. (A) Skin resected from the administration site of a representative animal 1 day after topical application of live EnirB-tetC vectors at a dose of 1 x 109 CFU. {gamma}{delta}T cells appeared as infiltrating fluorescent red cells. (B) Skin resected from the administration site of a representative animal 1 day after topical application of AdCMV-tetC (an E1/E3-defective adenovirus vector encoding TetC driven by the human CMV promoter) (29) at a dose of 1 x 1010 viral particles. Note that the number of {gamma}{delta}T cells in the skin was significantly lower than that found in A. (C) Abdominal skin resected from a representative naïve animal. The number of {gamma}{delta}T cells in the skin was as low as that in B. BALB/c mice were immunized by topical application of EnirB-tetC and AdCMV-tetC vectors encoding the same TetC fragment, respectively. Skin samples were sectioned and stained, as described previously (15), with a biotinylated hamster anti-mouse {gamma}{delta}TCR monoclonal antibody (clone GL3; BD Biosciences Pharmingen, San Diego, CA), followed by staining with streptavidin-tetramethyl rhodamine isothiocyanate. Sections were examined on a fluorescent Zeiss Axioskop2 Plus microscope using a x40 objective lens in conjunction with an Axiocam digital camera. Each section is representative of three mice.

Fate of E. coli particles in animals following topical application. To determine the fate of E. coli particles after epicutaneous vaccination, the presence of live reporter E. coli vectors expressing ß-galactosidase, as well as E. coli vector DNA, was determined at the administration site, in the bloodstream, and in internal organs at various intervals postadministration. As shown in Table 1, viable E. coli cells expressing ß-galactosidase could be recovered from the skin at the administration site 1 h and 1 day following topical application but could not be recovered from the skin 1 month postadministration. Reporter E. coli cells could not be recovered from the blood or any internal organs at any time point. In contrast, E. coli reporter cells instilled intranasally could be recovered from a wide variety of organs 1 h after administration, followed by rapid eradication of invading bacteria.


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  		TABLE 1. Fate of live E. coli reporter vectors following topical application and intranasal instillationa

Although the aforementioned results suggested that intact E. coli particles were unable to penetrate the skin, wide dissemination of E. coli DNA fragments was detected following epicutaneous vaccination. Subfragments of the aptE. coli gene (14) could be amplified by PCR from a variety of tissues 1 day after topical application (Table 2 and Fig. 6) and was amplifiable in blood as early as 1 h postadministration. One month postadministration, however, the E. coli DNA was undetectable in any of the tissues examined. The presence of E. coli DNA in blood and internal organs in the absence of live E. coli cells suggested that the E. coli particles may be rapidly disrupted upon application to the surface of the skin and that this disruption could be associated with systemic dissemination of intravector DNA fragments for a brief period of time.


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  		TABLE 2. Fate of aptE. coli DNA in mice following topical application of E. coli particlesa


Figure 6
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  		FIG. 6. Amplification of a subfragment of the aptE. coli gene by nested PCR from tissue extracts following topical application of E. coli-vectored vaccines. (A) One hour postadministration. (B) One day postadministration. ICR mice were immunized by topical application of live EnirB-tetC particles at a dose of 1 x 109 CFU. Total DNA was extracted from individual tissues at the indicated time points. Nested PCR was performed to amplify a diagnostic 224-bp subfragment of the aptE. coli gene as described in Materials and Methods. Lane 1, skin at the administration site; lane 2, blood; lane 3, brain; lane 4, heart; lane 5, lung; lane 6, kidney; lane 7, liver; lane 8, spleen; lane 9, inguinal, cervical, and brachial pooled lymph nodes; lane 10, ovary; lane 11, pUC8apt plasmid as a positive control; lane 12, template-free negative control. Shown are PCRs from representative animals. DNA extracted from the abdominal skin of a naïve mouse was also used as a negative control. No signals were detected in the negative controls or any tissues 1 month postadministration.


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DISCUSSION
 
We show here that animals can be effectively vaccinated against live pathogens following noninvasive administration of replicating or nonreplicating E. coli-vectored epicutaneous vaccines (Fig. 1 to 4). Unlike genetic immunization (33, 37), elicitation of an immune response by an E. coli-vectored epicutaneous vaccine was not mediated by the expression of antigens in host cells, because {gamma}-irradiation, which fragments DNA (34), did not appreciably affect the potency of the vaccine (Fig. 2 and 4). Moreover, the titers of the antigen-specific antibodies in the sera of the vaccinated animals showed a correlation with the potency of promoters used to drive antigen production in E. coli instead of mammalian cells (Fig. 3A).

Although animals have preexisting immunity to E. coli due to constant interaction with this bacterium in the gastrointestinal tract (31), any preexisting E. coli-targeted immunosurveillance may conceivably enhance the potency of E. coli-vectored epicutaneous vaccines, because the destruction of E. coli vectors by an anti-E. coli immune reaction could lead to the release of more vector constituents for antigen presentation. The potential synergy between E. coli-vectored epicutaneous vaccines and preexisting anti-E. coli immunity should thus allow this new class of vaccines to be administered repeatedly without diminishing efficacy.

The finding that E. coli DNA was disseminated systemically after topical application of intact E. coli particles suggested that E. coli vectors may be disrupted in the outer layer of skin soon after administration, with perhaps a fraction of intravector components being captured by cutaneous antigen-presenting cells that trafficked systemically. The transient nature of this noninvasive vaccination modality could potentially strengthen an adaptive immune response by minimizing antigen-induced apoptosis of T lymphocytes (32). It is possible that the disruption of bacterial particles in the outer layer of skin followed by the degradation of bacterial components and the elicitation of an immune response represent a cascade of defense reactions in counteracting transdermal invasion of microbes. Since E. coli has been coevolving with animals as a commensal, the immune system must have a constant memory for E. coli epitopes. It has been shown that an array of E. coli ligands are capable of inducing a specific gene expression profile in dendritic cells (18) and may be associated with the activation of the Toll-like receptor signaling pathway (2, 36). Conceivably, the high immunogenicity of endogenous E. coli ligands may thus promote the effectiveness of E. coli-vectored epicutaneous vaccines by activating an E. coli-targeted defense mechanism as natural adjuvants.

The rapid accumulation of {gamma}{delta}T cells underneath the vaccine patch after topical application of E. coli particles (Fig. 5A) provides compelling evidence that this novel vaccination modality is able to activate cutaneous immunity in a noninvasive and timely manner. The {gamma}{delta}T-cell response is considered to be an innate bactericidal reaction in the skin, since a higher number of viable Staphylococcus aureus cells can be recovered from infected skin of TCR{delta}–/– mice than from the skin of TCR{delta}+/– congenic control mice (26). It has previously been shown that human {gamma}{delta}T cells can recognize a nonprotein E. coli antigen in a major histocompatibility complex-independent manner (10) and kill mycobacteria by the production of granulysin (8). In addition to the capacity for {gamma}{delta}T cells to recognize and disrupt bacterial cells, this class of T cells may also function as antigen-presenting cells by processing and displaying antigens and providing costimulatory signals for the induction of naïve {alpha}ßT-cell proliferation and differentiation (5). The finding that topical application of an adenovirus vector encoding the same TetC fragment did not result in the recruitment of {gamma}{delta}T cells (Fig. 5B) suggested that adenovirus-vectored epicutaneous vaccines (29, 35, 38) and their E. coli-vectored counterparts may activate the cutaneous immune system through different pathways.

As discussed above, some in situ disruption of E. coli particles after administration may be essential for presenting intravector antigens to the immune system; however, we found that lysis of E. coli particles prior to epicutaneous immunization may be counterproductive. Topically applied lysates made from sonicated EnirB-tetC particles were less effective than live E. coli cells in eliciting an anti-TetC antibody response (Fig. 2B). Neither topical application of purified TetC protein without toxin adjuvants (12) nor administration of formalin-fixed EnirB-tetC particles to the surface of skin (Fig. 2B) elicited a robust immune response to TetC. These findings collectively underscore the necessity for a native configuration of E. coli cells in triggering an immune response against antigens embedded within the vector following topical application. Changing the shape or texture of E. coli particles may interfere with E. coli-targeted immune reactivity. Vaccination by topical application of intact E. coli particles has the advantage not only that the antigen conformation and the antigen-E. coli ligand complexes essential for activation of the cutaneous immune system are not perturbed prior to epicutaneous vaccination but also that this modality can respond rapidly to a large escalation in vaccine demand during disease outbreaks by avoiding the time-consuming purification steps.

The anti-TetC antibody titer induced by the topical application of replicating or nonreplicating EnirB-tetC particles (Fig. 1 and 2) was higher than that induced by intramuscular injection of 0.015 Lf of a licensed tetanus vaccine (Fig. 2D) or 100 µg of a plasmid encoding TetC in mice (29). Although it has previously been reported that mice can be protected against an injection of 10 ng of tetanus toxin and inhalation of 1.9 x 104 anthrax spores following multiple applications of bacterial toxin-adjuvanted TetC (12) and PA (20) proteins, respectively, to the surface of skin, it is difficult to draw conclusions at this time regarding the comparative efficacy of these different approaches due to the differences in the reagents and methods used for analysis of protective immunity.

The efficacy of the EnirB-tetC-vectored epicutaneous vaccine was demonstrated by 100% protection of immunized animals against a lethal dose of C. tetani after a single administration of E. coli particles to the surface of the skin (Fig. 4A). The high potency was correlated with an overproduction of the TetC protein in E. coli cells, as shown by sodium dodecyl sulfate (SDS) gel analysis (Fig. 1A). The TetC codon usage in the EnirB-tetC vector was optimized to match the E. coli tRNA pool in order to overcome the low yield caused by a difference in codon bias between C. tetani and E. coli (24). In contrast to the EnirB-tetC vector, only partial protection against intranasal instillation of 1 x 105 anthrax spores was achieved following three applications of E{lambda}PR-PA83- and E{lambda}PR-LF7-vectored epicutaneous vaccines. The level of protection (Fig. 4C) was well correlated with that of neutralizing antibody titers in sera (Fig. 3C). Although the relatively low level of protection against anthrax spores may be attributed to an overdose of spores and/or a defective immune system in the A/J mouse strain (40), the difference in codon bias between B. anthracis and E. coli, as suggested by sequence information (41), may also be accountable, since neither PA63 nor LF7 proteins could be produced in E. coli cells to a detectable level in an SDS gel (data not shown), indicating that these B. anthracis proteins were not produced as abundantly in E. coli cells as the codon-optimized TetC fragment (Fig. 1A). The immunogenicity of the E. coli-vectored anthrax vaccine is expected to rise when the yield of an anthrax antigen is augmented in E. coli cells by removing B. anthracis-associated rare codons. In addition to codon optimization, the potency of E. coli-vectored epicutaneous vaccines may also be enhanced by the development of transstratum corneum enhancers, new epicutaneous adjuvants, bioengineered E. coli particles with a less-rigid cell wall, or a combination thereof.

The failure of the live E. coli cells to disperse beyond the skin (Table 1) suggested that the skin is a tight physical barrier and that epicutaneous vaccination should not pose any risk greater than normal exposure to bacteria in the environment. The laboratory E. coli bacteria are attenuated strains with excellent biosafety records (30). Unlike pathogenic E. coli strains, internalized E. coli cells of a laboratory strain do not induce apoptosis of professional phagocytes (6). The safety profile can be further improved by depleting the replicative potential of E. coli vectors as well as inactivating any antibiotic resistance genes through the use of {gamma}-irradiation.

Viable E. coli cells can be stored at ambient temperatures as lyophilized powder (23, 25), and therefore, E. coli-vectored vaccines can be stockpiled and shipped without reliance on an unbroken cold chain. It is promising that the lyophilized-irradiated E. coli-vectored epicutaneous vaccine (Fig. 2C) may emerge as the most economical class of vaccines for mass immunization. Its potency can potentially be further improved by increasing the survival rate of E. coli cells during lyophilization as described previously (25).

Overall, the use of intact E. coli particles eliminates the time-consuming and deleterious requirement for the biochemical purification of antigens, the hazard of contemporary adjuvants, and the intrinsic problems associated with needle injections. It is amenable to large-scale, rapid, low-cost production, distribution, and administration. Thus, epicutaneous administration of E. coli-vectored vaccines holds promise as the preferred modality for vaccine administration in general and for mass immunizations specifically.

To our knowledge, this is the first proof-of-principle demonstration that controlled exposure of the skin to pathogen-derived antigens expressed in the context of E. coli particles following topical application of replicating or nonreplicating E. coli vectors can elicit a protective immune response against live pathogens in animal models without any appreciable side effects. The finding that the outer layer of skin is an immunocompetent organ inherently capable of extracting antigens from intact E. coli particles provides the foundation for mitigating disease outbreaks and bioterrorist attacks in a simple, rapid, effective, economical, painless, and safe manner.


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ACKNOWLEDGMENTS
 
We thank the University of Alabama at Birmingham for providing research facilities; D. Galloway, M. Bell, C. Queen, J. VanCott, J. McGhee, and M. Taylor for providing plasmids; and F. Hunter and D. Grove for critical reading of the manuscript.

This work was supported by Office of Naval Research contract N00014-02-2-0003 and grant N00014-01-1-0945 and National Institutes of Health grants 1-R43-AI-47558 and 2-R42-AI/HD-44520. D.C.T. was also supported by the Year 2000 Wallace H. Coulter Award for Innovation and Entrepreneurship.


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FOOTNOTES
 
* Corresponding author. Mailing address: Vaxin Inc., 2800 Milan Court, Birmingham, AL 35211. Phone: (205) 909-3738. Fax: (205) 943-6656. E-mail: tang{at}vaxin.com. Back

Editor: D. L. Burns


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Infection and Immunity, June 2006, p. 3607-3617, Vol. 74, No. 6
0019-9567/06/$08.00+0     doi:10.1128/IAI.01836-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.




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