Immunization of mice against the TetC protein by topical application of irradiated, sonicated, fixed, and lyophilized EnirB-tetC vectors. (A) Effects of γ-irradiation on the potency of E. coli-vectored epicutaneous vaccines. Both live EnirB-tetC vectors and their γ-irradiated counterparts were administered to ICR mice by topical application at a dose of 1 × 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 × 10¹⁰ live EnirB-tetC particles, lysates made from 1 × 10¹⁰ sonicated EnirB-tetC particles, and 1 × 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 × 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 × 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 γ-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 × 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 × 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.

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 × 10³ C. tetani cells 3 months postimmunization as described previously (29). Naïve, no immunization; 10E8, mice immunized at a dose of 1 × 10⁸ CFU; 10E9, mice immunized at a dose of 1 × 10⁹ CFU; 10E10, mice immunized at a dose of 1 × 10¹⁰ CFU. Antibody titers prior to challenge are shown in Fig. 1B. (B) ICR mice were immunized by topical application of γ-irradiated EnirB-tetC particles in a single-dose regimen and challenged as described in A. *10E5, mice immunized at a dose of 1 × 10⁵ irradiated EnirB-tetC particles; *10E9, mice immunized at a dose of 1 × 10⁹ irradiated EnirB-tetC particles; 10E9, mice immunized at a dose of 1 × 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 × 10⁹ γ-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 × 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, Eλ_PR-PA83; *E-LF7, Eλ_PR-LF7; *E-PA83+*E-LF7, Eλ_PR-PA83 plus Eλ_PR-LF7 (asterisk, γ-irradiation). Antibody titers prior to challenge are shown in Fig. 3C. The lambda P_R 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.

Amplification of a subfragment of the apt_{E. 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 × 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 apt_{E. 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.

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. M_r (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 × 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 × 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 × 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.

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 γ-irradiated and administered at a dose of 5 × 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; λ32, Eλ_PR-PA63 plus Eλ_PR-LF7 with antigens driven by the lambda P_R promoter at 32°C; λ42, Eλ_PR-PA63 plus Eλ_PR-LF7 with antigens driven by the lambda P_R 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 γ-irradiated Eλ_PR-PA83 and Eλ_PR-LF7 vectors at a dose of 1 × 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 γ-irradiated E. coli-vectored anthrax vaccines at a dose of 5 × 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, Eλ_PR-PA63; *E-PA83, Eλ_PR-PA83; *E-LF7, Eλ_PR-LF7; *E-PA83+*E-LF7, Eλ_PR-PA83 plus Eλ_PR-LF7 (asterisk, γ-irradiation). The lambda P_R 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 × 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%.

Recruitment of γδT 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 × 10⁹ CFU. γδT 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 × 10¹⁰ viral particles. Note that the number of γδT cells in the skin was significantly lower than that found in A. (C) Abdominal skin resected from a representative naïve animal. The number of γδT 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 γδTCR 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 ×40 objective lens in conjunction with an Axiocam digital camera. Each section is representative of three mice.