N-fragment of edema factor as a candidate antigen for immunization against anthrax

Vaccine 24 (2006) 662–670

N-fragment of edema factor as a candidate antigen for immunization against anthrax Mingtao Zeng ∗ , Qingfu Xu, Eric D. Hesek, Michael E. Pichichero Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Box 672, Rochester, NY 14642, USA Received 11 February 2005; received in revised form 27 June 2005; accepted 5 August 2005 Available online 26 August 2005

Abstract The nontoxic N-terminal fragment of Bacillus anthracis edema factor (EF) was evaluated as a candidate antigen in an anthrax vaccine using a replication-incompetent adenoviral vector. An E1/E3 deleted adenovirus (Ad/EFn) encoding the N-terminal region 1–254 amino acids of the edema factor (EFn) was constructed using the native DNA sequence of EFn. Intramuscular immunization three times with 108 plaque forming units (pfu)/dose of Ad/EFn in A/J mice resulted in 37% and 57% protection against a subcutaneous challenge with B. anthracis Sterne strain spores at a dosage of 200 × LD50 and 100 × LD50 , respectively. EF-specific serum IgG responses (including total IgG, IgG1, and IgG2a isotype titers) were robust in the Ad/EFn immunized animals. Interestingly, anti-EF antibodies cross-reacted with anthrax lethal factor (LF), and had a neutralizing capability against both anthrax lethal toxin (Letx) and edema toxin (Edtx), as demonstrated by in vitro toxin neutralization assays using J774A.1 mouse macrophage and Chinese hamster ovary cell (CHO), respectively. Our data suggest that EF plays a role in eliciting protective immunity against anthrax, and that it should be included in a new generation multi-component subunit vaccine. © 2005 Elsevier Ltd. All rights reserved. Keywords: Anthrax vaccine; Edema factor; Protective immunity; Replication-incompetent adenovirus

1. Introduction The etiological agent of anthrax, Bacillus anthracis, is a gram-positive, nonmotile, aerobic or facultatively anaerobic, spore-forming, rod-shaped bacterium [1–3]. The recent bioterrorist attacks using B. anthracis spores caused five deaths in the United States. This tragedy has resulted in a great urgency for the development of more effective anthrax vaccines. Virulent strains of B. anthracis are characterized by a polyglutamic acid capsule and the production of a threecomponent toxin [e.g., protective antigen (PA), lethal factor (LF) and edema factor (EF)]. All strains of unattenuated B. anthracis carry two plasmids, pX01 and pX02, which encode the three toxin components and enzymes for capsule biosynthesis, respectively. The toxin components 83 kDa PA, 85 kDa LF, and 89 kDa EF are encoded by genes pag, ∗

Corresponding author. Tel.: +1 585 275 1003; fax: +1 585 273 1289. E-mail address: Mingtao [email protected] (M. Zeng).

0264-410X/$ – see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2005.08.056

lef and cya, respectively, in pX01 [4–6]. PA binds to host cell anthrax toxin receptors (ATRs), such as tumour endothelial marker 8 (TEM8) and human capillary morphogenesis protein 2 (CMG2) and is cleaved by cell surface furin to produce a 63-kDa fragment PA63 [7–10]. ATRs-bound PA63 oligomerizes to a heptamer and acts to translocate the catalytic moieties of the toxin, LF and EF, from endosomes to the cytosol [9,11]. LF appears to be a metalloprotease [12]. The combination of PA and LF, named anthrax lethal toxin (Letx), kills certain cultured cells and animals due to the action of LF. Recent evidence suggests that LF is able to enter cells without PA but needs PA binding to function as a toxin [13]. EF is an adenylate cyclase that generates cAMP. The combination of PA and EF, named edema toxin (Edtx), disables phagocytes and other cells due to the intracellular adenylate–cyclase activity of EF [14,15]. PA has been shown to be an essential component of a vaccine [16]. Interestingly, anti-PA antibody specific immunity has anti-spore activity and might have a role in impeding the

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early stages of infection with B. anthracis spores [17]. Human vaccination with the anthrax vaccine BioThraxTM (BioPort Corp., Lansing, MI) requires six immunizations followed by annual boosters [18,19]; 1% systemic and 3.6% local adverse events in humans have been reported [2]. This underscores the need for development of new, improved anthrax vaccines. To date, there have been many attempts to improve the safety profile and immunogenicity of the anthrax vaccine using PA as an antigen, including the formulation of PA in adjuvants [20–22], conjugating capsular poly-gamma-dglutamic acid (PGA) to PA [23,24], the use of purified PA [12] and its C-terminal domain 4 (PA-D4) [25], the development of PA-based DNA vaccines [26,27], and expression of PA in adenovirus, Salmonella typhimurium, Bacillus subtilis, vaccinia viral vector, and venezuelan equine encephalitis virus [28–33]. The PA-D4 (residues 596–735 in PA) is responsible for binding cellular receptors, the anthrax toxin receptors (ATRs), and was recently shown to contain the dominant protective epitopes of PA [25,34]. Previous research indicated that immunization with plasmid expression vectors in a combination of PA and N-terminal region truncated LF (LFn, residues 10–254 of the mature LF protein) provides better protection than PA alone [35,36]. Immunization with somatic components of B. anthracis, such as capsule, surface polysaccharides, and cell-associated antigens EA1 and EA2 has not been shown to provide protective immunity [37]. However, PA vaccines provide less protection than live spore vaccines against lethal challenge with several strains of B. anthracis. Moreover, the direct correlation between anti-PA titers and protection is not fully established [38–41]. It has been suggested that, in addition to PA, LF and EF also play an important role in providing immunity [42]. Also, studies of spore vaccines suggested that some other B. anthracis antigens may contribute in a significant manner to protective immunity [6,43]. The N-terminal regions of EF (EFn) and LF (LFn) share similar structural similarities. Both are responsible for the binding of the N-domain of PA to facilitate their entry into host cells, but both domains lack enzymatic activity [6,15,44–48]. Therefore, host immune responses against these domains may prevent PA, LF, and EF from gaining access into target cells. Replication-incompetent adenoviruses are currently available and represent efficient vehicles for gene transfer in vitro and in vivo [49]. Adenovirus-vectored recombinant vaccines expressing a wide array of antigens have been constructed, and protective immunity against different pathogens has been demonstrated in animal models [28,50–52]. In this research, we have evaluated the role of EFn in eliciting immunity to anthrax using an adenovirus-vectored vaccine. Our rationale for choosing EFn are: (1) host immune response against EF may be able to neutralize EF and further inhibit its function to generate cAMP; (2) EFn is not toxic to the host cell if it is expressed intracellularly after genetic immunization with the adenoviral vector; (3) based on the structural similarities between LFn and EFn, we also hypothesize that antibodies

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against EF would cross-react with LF and further protect host cells from both toxins.

2. Materials and methods 2.1. Construction of adenoviral vector encoding EFn The AdMax System (Microbix, Canada) was used to construct the replication-incompetent adenoviral vector in this study. This adenoviral vector was based on human adenovirus serum type 5 (Ad5) with E1 and E3 deletion. The nucleotides 1–762 of cya gene encoding the mature EF were amplified from the plasmid pSE42 [53] by PCR using Platinum® Pfx DNA polymerase (Invitrogen, CA). The primers 5EF (5 GCCGTCGACACCATGGGGATGAATGAACATTACACTGAG-3 ) and 3EF (5 -GCCGTCGACTTACACCTTCTTTCTTCAAACTT-3 ) for the PCR included a SalI restriction site at each 5 end (there is no SalI site in the region of the nucleotides 1–762 of the cya gene) [53]. The PCR products were digested with SalI and inserted into a shuttle plasmid pDC316 at its SalI site. An extra C (underlined) was accidentally placed behind the TTA stop codon in the 3EF primer. Therefore, the gene transcription continued until a new stop codon at position 1223–1225 in the pDC316 vector, resulting in a final protein product of the amino acid 1–254 of EF plus the extra 68 amino acids at the C-terminus of EFn (VSRLRATCLLQLIMVTNKAIASQISQIKHFFHCILVVVCPNSSMYLIMSGSSSIEDPITSYSIHYTKL). After Cre-mediated recombination between pDC316/EFn and an adenoviral plasmid pBHGlox/delta E1, E3Cre in the permissive 293 cells, the recombinant adenovirus Ad/EFn was rescued and propagated. A single virus plaque was isolated and used for virus purification/production. Adenovirus was produced by CsCl gradient centrifugation method. Purified adenovirus was dialyzed and stored in aliquots in an adenovirus storage buffer containing 10 mM Tris pH 7.5, 135 mM NaCl, 5 mM KCl, 1 mM MgCl2 , and 1 M sucrose in a −80 ◦ C freezer. To verify the EFn insert in the adenoviral genome, the viral genomic DNA from purified adenovirus was isolated with DNAZol reagent (Invitrogen, CA) and used as a template in a PCR to amplify the EFn insert with the primer pair 5mCMV(5 -CATTCTATTGGCTGAGCTCC-3 ) and 3EF. The 5mCMV primer is located in the murine cytomegalovirus (mCMV) promoter region upstream from the EFn coding sequence and the 3EF is located at the 3 end of the EFn sequence. Thus, the positive PCR results indicated that the EFn was cloned into the adenoviral vector with the correct orientation downstream of the mCMV promoter. In addition, the unique 1.7 kb Ad5 fiber gene fragment in the adenoviral vector backbone was co-amplified by PCR with the primers 5Fb (5 -CCGTCTGAAGATACCTTCAA-3 ) and 3Fb (5 -ACCAGTCCCATGAAAATGAC-3 ), indicating that the recombinant adenovirus Ad/EFn was constructed. PCR products were fractionated in a 1% agarose gel, stained with ethidium bromide, and visualized with the Kodak

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for the challenged animals were the occurrence of severe anthrax symptoms or 2 weeks after spore injection. Any animals that developed severe symptoms of anthrax, such as ruffled coat, hunching, shivering, uncoordinated movements, dehydration, respiratory difficulties, and skin lesions, were euthanized promptly during the 2 weeks. 2.4. Measurement of serum antibody titers

Fig. 1. Validation of adenoviral vector encoding EFn by PCR. Lane 1, DNA marker, ␭-DNA digested with HindIII; lane 2, plasmid pBHG10X/delta E1, E3Cre as DNA template in PCR (positive control for Ad5 Fiber); lane 3, pDC316/EFn (positive control for EFn); lane 4 is a negative control for PCR, no template DNA were added into the PCR reaction mixture; lanes 5–7, genomic DNA of adenovirus Ad/EFn. The primers used in the PCR are indicated in the figure.

Imaging System 440CF (Fig. 1). The final adenovirus Ad/EFn encodes the 1–254 amino acids of EFn plus the 68 amino acid tag under the transcriptional control of mCMV early promoter. 2.2. Animal immunization and serum collection Four to six-week-old female A/J mice were purchased from Jackson Laboratory (Bar Harbor, ME), allotted into treatment and control groups, and housed under BSL2 pathogen-free conditions in the animal facility at the University of Rochester (four mice/cage). The mice were immunized by intramuscular injection into the hind-leg quadriceps with 1 × 108 pfu/dose adenovirus Ad/EFn in treatment groups or an equal volume of adenovirus storage buffer in control groups. Animals were immunized with Ad/EFn one time (week 0), two times (week 0, 4), or three times (week 0, 4, 8). Animal sera were collected every 2 weeks by retro-orbital bleeding and stored at −20 ◦ C until assayed. The animal research herein reported was conducted in facilities with programs accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care. 2.3. Challenge with B. anthracis Sterne spores The animals were challenged by subcutaneous injection with 1.85 × 105 to 6.78 × 105 spores of B. anthracis Sterne 12 weeks after the first immunization. The B. anthracis Sterne strain spores were from the anthrax spore vaccine (nonencapsulated live culture, US vet. License No. 188, Colorado Serum Company, Denver, CO). This represents a dose of approximately 100–600 times the 50% lethal dose (LD50 ) for A/J mice [54]. The animals were monitored twice a day for 1 week after spore challenge, and daily thereafter. The endpoints

Serum antibody titers against EF or LF were measured by the enzyme-linked immunosorbent assay (ELISA). 0.1 ml of coating solution containing 1 ␮g/ml recombinant EF or LF (List Biological Laboratories, CA and the ATCC BEI Resources, VA) in 0.1 M carbonate buffer (pH 9.6) was added into each well of 96-well Nunc-Immuno plates (Nalge Nunc International, Denmark), and incubated overnight at 4 ◦ C. The plates were then washed twice with washing buffer (0.05% Tween 20 in PBS) and blocked with 1% (w/v) bovine serum albumin in PBS for 1 h at room temperature. After three washes, 100 ␮l serial dilutions of mice sera in PBS containing 0.05% Tween 20 were added to each well (starting with 1:100 dilution), and incubated for 2 h at room temperature. The plates were washed with washing buffer five times and incubated with 100 ␮l/well of 1:10,000 dilution of goat anti-mouse IgG, IgG1 or IgG2a conjugated to horseradish peroxidase (Calbiochem, CA) for 1 h at room temperature. Unbound antibodies were removed by washing five times with washing buffer, and the bound antibody was detected following an incubation for 30 min with peroxidase substrate ABTS [2,2 -azinobis (3-ethylbenzhiazolinesulfonic acid)] (Sigma–Aldrich, MO). The absorbance values were obtained using a Dynatech MR 4000 model microplate reader with a 405 nm filter. The endpoint was calculated as the dilution of serum producing the same optical density at 405 nm as a 1:100 dilution of preimmune serum [50]. 2.5. Anthrax edema toxin neutralization assay Edtx (EF + PA) is able to increase the intracellular levels of cyclic AMP (cAMP) in Chinese hamster ovary (CHO) cells [14]. Neutralizing antibodies to EF were measured by the ability of sera to neutralize this cytotoxicity of Edtx in CHO cells. The CHO-K1 cells (ATTC #CCL-61) were seeded in a 96-well tissue culture plate at a density of 40,000 cells/well in 100 ␮l Khaigan’s modified F-12 medium containing 10% fetal bovine serum, and incubated at 37 ◦ C, 5% CO2 for 24 h as previously described [55]. In a new 96-well culture plate, serum sample pools from eight mice immunized with Ad/EFn were serially diluted with cell culture medium. PA or/and EF (ATCC/BEI Resources, VA) were diluted with cell culture medium and added to corresponding wells at a final concentration of 1 and 0.25 ␮g/ml and then incubated with supplement of 5% CO2 at 37 ◦ C for 1 h. Cell culture medium was then removed from the CHO cells and 100 ␮l of the neutralization reaction mixtures were transferred to the corresponding wells in the plate. One hour after incu-

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bation with neutralization reaction mixtures at 37 ◦ C, cells were lysed by addition of 100 ␮l 0.1M HCl and incubated at room temperature for 20 min. cAMP concentration was detected by using Cyclic AMP (low pH) Immunoassay Kit (R&D System Inc., MN) according to manufacturer’s instructions. Briefly, 50 ␮l of neutralizing reagent, 50 ␮l alkaline phosphate-labeled cAMP, 50 ␮l anti-cAMP antibody solution, and 100 ␮l samples or the cAMP standards were added to a 96-well microplate coated with a goat anti-rabbit polyclonal antibody. The plate was incubated for 2 h at room temperature with constant shaking. The plate was then rinsed five times with washing buffer and then incubated with 200 ␮l pNPP (p-nitrophenyl phosphate) substrate for 1 h. After the addition of 50 ␮l stop solution, the optical density (OD) was determined immediately by a Dynatech MR 4000 microplate reader at 405 nm with 580 nm as a reference wavelength. Cellular protein concentration was determined by BCA protein assay kit (Pierce, IL). cAMP concentrations (pmol/mg protein) were calculated by the standard curve obtained in the parallel assay. 2.6. Anthrax lethal toxin neutralization assay Neutralizing antibodies to LF were measured by the ability of sera to neutralize the cytotoxicity of Letx (LF + PA) for J774A.1 mouse macrophage cells. 5 × 104 J774A.1 cells in 100 ␮l of Dulbecco’s Modified Eagles’s medium (DMEM) containing 10% FBS, 4.5 g/l glucose, and 2 mM l-glutamine were added into each well in 96-well culture plates and incubated with 5% CO2 at 37 ◦ C for 24 h. Pooled sera from animal groups were diluted in two-fold series with the medium and incubated with recombinant LF protein (List Biological Laboratories, CA) at a final concentration of 40 ng/ml at 37 ◦ C for 1 h to allow for neutralization to occur. Recombinant PA protein (List Biological Laboratories, CA; at 60 ng/ml final concentration) was added to the LF and sera mixture and incubated at room temperature for 1 h, and then the 100 ␮l LF-sera-PA mixture was added to each well on the plates. Equal volumes of medium and media containing PA only, LF only, and PA plus LF were added onto the cells in control wells. After an incubation for 7 h at 37 ◦ C, 20 ␮l of 3-[-4,5-dimethl-thiazol-2-y]-2-5-diphenyltrazo-lium bromide (MTT; Sigma–Aldrich, MO) at 5 mg/ml in PBS was added to each well and then incubated for an additional 2 h. Then 50 ␮l of 20% SDS in 50% dimethylformamide were added into each well. Optical densities were measured at 570 nm with a reference wavelength at 690 nm using a microplate reader (Dynatech MR 4000 model). The ratio of LF plus sera and PA versus the medium alone, expressed as percentage of cell viability, was calculated for each dilution. 2.7. Statistical analysis Antibody titers among different groups at different time points were compared and analyzed using the LSD test, ANOVA/MANOVA, STATISTICA 6.0 (StatSoft, Tulsa,

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OK); in comparing groups, those with P-values <0.05 and <0.01 were considered to be significant and very significant, respectively. Survival data from spore challenge experiments were analyzed by the LIFETEST procedure in SAS 8.02 (SAS Institute Inc., Cary, NC). To test the difference between challenge groups, the log-rank test was used.

3. Results 3.1. Antibody response to anthrax toxin components after immunization with Ad/EFn Groups (eight mice/group) of female A/J mice were immunized with Ad/EFn by intramuscular injection. After one (week 0), two (week 0, 4), and three (week 0, 4, 8) immunizations with a dosage of 1 × 108 pfu Ad/EFn vector, antibody responses in animal sera were measured by ELISA. Table 1 summarizes antibody responses against EF and LF after single and multiple immunizations with Ad/EFn. As we expected, the anti-EF antibodies cross-reacted with LF in a dose dependent manner. IgG, IgG1, and IgG2a antibody titers against EF and LF had significant increases in week 6 after a booster immunization in week 4 (P < 0.05). The rise in antigen specific IgG1 and IgG2a after Ad/EFn immunization suggested that both Th2 and Th1 immune responses against EF and LF were elicited. However, the third immunization in week 8 did not significantly enhance anti-EF and anti-LF IgG, IgG1 and IgG2a immune responses in comparison to the two dose immunization (P > 0.05). Therefore, the first booster immunization is necessary and the second booster immunization may not be required to achieve significantly higher antibody responses. 3.2. In vitro neutralization against anthrax edema toxin To determine the functionality of anti-EF antibodies elicited by the immunization, an in vitro assay for neutralization of anthrax edema toxin was performed. Neither EF nor PA alone is capable of elevating cAMP concentrations in CHO cells. Only EF combined with PA (Edtx) can increase intracellular cAMP concentrations (positive control). In comparison to the positive control, we observed that four-fold diluted anti-EF anti-sera could completely neutralize EF and inhibit Edtx for generation of cAMP in CHO cells (Fig. 2). The results demonstrate that anti-EF antibodies elicited by immunization with Ad/EFn are functional neutralizing antibodies against EF and are able to inhibit the activity of Edtx. 3.3. In vitro protection against anthrax lethal toxin In order to determine whether anti-EF/anti-LF antibodies in the sera were capable of neutralizing anthrax Letx, an in vitro protection assay was performed using J774A.1 mouse macrophage cells, which are sensitive to Letx. As shown in Fig. 3A, a 1:2 dilution of pooled sera from immu-

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Table 1 Summary of antibody titers in sera from A/J mice immunized with Ad/EFn Antibody

Time (weeks)

Three immunizations Range

Anti-EF IgG1

Two immunizations Average

Range

One immunization Average

Range

Average

2 4 6 8 12

100–200 100–400 400–1600 800–3200 1600–6400

113aX 275aX 1200bX 1800bX** 3200cX**

100–100 100–400 800–1600 800–3200 800–1600

100aX 188aX 1200bcX** 1800cX** 1000bY*

100–100 100–200 100–800 100–800 100–400

100aX 150aX 388aY 313aZ 188aZ

IgG2a

2 4 6 8 12

100–100 100–200 200–1600 400–3200 800–3200

100aX 125aX 775bX* 1250bcX** 1700cX**

100–200 100–200 400–1600 400–3200 800–3200

113aX 125aX 700bX 1250cY** 1600cY**

100–200 100–200 200–800 100–400 100–400

125aX 125aX 350aY 213aZ 188aZ

IgG

2 4 6 8 12

100–400 400–3200 1600–6400 3200–6400 1600–6400

250aX 850aX 3000bX** 3600bcX** 4200cX**

100–400 400–1600 1600–6400 1600–3200 800–3200

238aX 900aX 3200bX** 2600bX** 2300bX**

100–400 400–1600 400–1600 400–800 200–1600

225aX 850aX 800aX 550aY 725aY

2 4 6 8 12

100–200 100–400 400–1600 1600–3200 1600–6400

113aX 188aX 750bXY** 2200cX** 2400cX**

100–100 100–200 400–1600 800–3200 800–1600

100aX 163aX 800bY** 2100cX** 1200bY**

100–100 100–200 200–800 200–800 100–400

100aX 163aX 375aX 350aY 188aZ

IgG2a

2 4 6 8 12

100–100 100–200 200–800 400–1600 400–3200

100aX 125aX 425aX* 950bX** 1350cX**

100–100 100–200 200–800 400–3200 400–1600

100aX 125aX 425aX** 950bX** 1150bX**

100–100 100–200 200–400 100–400 100–400

100aX 125aX 275aX 250aY 238aY

IgG

2 4 6 8 12

200–200 200–1600 800–6400 3200–6400 1600–6400

200aX 600aX 2900bX 4000cX** 3600bcX**

100–200 200–800 800–6400 800–3200 400–6400

138aX 425aX 2900bX 2700bX** 2200bY**

100–200 200–800 100–1600 400–800 100–1600

162aX 550aX 563aY 750aZ 963aZ

Anti-LF IgG1

Values in columns without the same letters (a, b, c) differ significantly between different time points in the same immunization schedule (P < 0.05, n = 8). Values in rows without the same capital letters (X, Y, Z) differ significantly between different immunization schedules at the same time points (P < 0.05, n = 8); * P < 0.05, ** P < 0.01 in comparison with the negative control group at the same time point. Data from the control group (100–200) are not shown in this table. Data were analyzed using LSD test, ANOVA/MANOVA, STATISTICA 6.0 (StatSoft Inc., Tulsa, OK).

nized mice (eight mice/group) after a single immunization with 1 × 108 pfu Ad/EFn conferred no protection against the cytotoxic effects of Letx in comparison with the control group (about 6% protection). However, after a second and third booster immunization, the in vitro protection rate of 1:2 diluted sera increased to 30% and 66%, respectively. As indicated in Fig. 3B, the in vitro protection rate at two times dilution increased steadily from 24% (week 4) to 48% (8 weeks) and 66% (week 12) in the three dose immunization group (week 0, 4, 8). This suggested that booster immunization elicited more potent neutralizing antibody responses in the animals. 3.4. Protective immunity elicited by adenovirus expressing EFn To explore whether the EFn-based adenovirus-vectored vaccine can provide protection against B. anthracis infec-

tion, the immunized A/J mice were challenged with 100–600 × LD50 of B. anthracis Sterne spores subcutaneously. The results from the challenge experiments show that immunization with Ad/EFn had partial protection against the spore challenge (Fig. 4). Fig. 4A shows that although one or two vaccine doses could not protect animals against the challenge with 200 × LD50 of B. anthracis Sterne spores, immunization significantly changed the survival distribution in the four groups (P-value 0.0062). The mean survival time was 6.4 days in the control group and 6.9, 7.3 and 8.1 days for one, two and three immunization groups, respectively. Pairwise comparison showed that three immunizations with Ad/EFn significantly changed the survival when compared to the control group (P-value 0.0071). This indicated that booster immunizations enhanced the protective immunity in animals. Fig. 4B shows that the survival rate of the immunized animals is highly dependent on the dosage of spore used in the challenge experiments. After animals were immunized

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three times with Ad/EFn, 57% could survive for 2 weeks from a 100 × LD50 spore challenge; however, only 37% survived from 200 × LD50 spore challenge. Although none survived from 600 × LD50 spore challenge, the survival rates were different between the dosage groups (P-value 0.004). In particular, the survival in the 200 × LD50 spore challenge group was significantly different from the control group (P-value 0.005).

4. Discussion Fig. 2. Neutralization of anthrax edema toxin by sera from immunized mice. The CHO-K1 cells were used for the assay in a 96-well tissue culture plate at a density of 40,000 cells/well. The anti-EF serum pools were from eight mice in week 10 after immunization with Ad/EFn in 0, 4, and 8 weeks. Various dilutions of pooled sera were pre-incubated with recombinant EF and PA, and then added into the CHO cells. Cellular protein concentration was determined by BCA protein assay kit (Pierce, IL). The intracellular cAMP concentrations were detected by Cyclic AMP Immunoassay Kit (R&D System Inc., MN).

In this research, using an adenoviral vector as an antigen delivery vehicle, we provide evidence that the nontoxic Nfragment (amino acid 1–254) of EF is capable of eliciting significant Th2 and Th1 immune responses against both EF and LF (Table 1) as well as protective immunity against B. anthracis spore challenges (Fig. 3). The data also demonstrate that anti-EF/anti-LF antibodies inhibit the toxicity of Letx and Edtx (Figs. 2 and 3). Furthermore, the protective immune

Fig. 3. Neutralization of anthrax lethal toxin by sera from immunized mice. Various dilutions of pooled sera (n = 8) were pre-incubated with recombinant LF and PA, and then added to J774A.1 mouse macrophage cells. The cell viability was measured. Panel A: sera from different groups immunized with Ad/EFn 12 weeks after the primary immunization. Mice were immunized one (week 0), two (week 0, 4), three (week 0, 4, 8) times with Ad/EFn as indicated. Mice in control group were injected with adenovirus storage buffer three times (week 0, 4, 8). Panel B: sera pool from the same group at different time points after immunized one to three times in week 0, 4, 8 with Ad/EFn.

Fig. 4. Protection against B. anthracis spore challenge in mice immunized with Ad/EFn. Panel A: mice were immunized one (week 0), two (weeks 0, 4), or three (week 0, 4, 8) times as indicated, and then challenged with 200 × LD50 B. anthracis Sterne spore in week 12. Panel B: mice were immunized three times (week 0, 4, 8) and challenged with 100–600 × LD50 B. anthracis Sterne spore as indicated in week 12. Mice in control groups were injected with adenovirus storage buffer three times (week 0, 4, 8).

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responses can be enhanced by additional immunizations. The animal survival time and rate are dependent on the number of immunizations and the dosage of the B. anthracis spores administered in the challenge (Fig. 4). Therefore, protective immunity in animals appears to be dose dependent. Cattle and other livestock are commonly vaccinated with viable unencapsulated but toxigenic spores of B. anthracis Sterne strain, which carries plasmid pXO1 but lacks pXO2. The efficacy and duration of protective immunity conferred by the B. anthracis Sterne strain is much greater than that elicited by cell-free PA vaccines, although antibody titers against PA induced by the live vaccine are often lower than those induced by PA-based cell-free vaccines [40]. Immunization of mice with a mutant Sterne strain expressing EF but lacking LF and PA conferred up to 25% protection against 1–100 × LD50 spore challenge, while mutant Sterne strain without EF, LF, and PA provided no immunoprotection [42]. This indicates that host immune responses against EF may contribute to immunity to anthrax. However, due to safety concerns, live spore anthrax vaccines are unlikely to be licensed for human use. Although PA is the major component in the currently licensed anthrax vaccine (BioThraxTM ), the requirement of multiple injections highlights the inefficiency of the vaccine. Other vaccine candidates are needed, and perhaps more effective anthrax vaccines would contain multiple antigens. Immunization with a plasmid vector encoding the N-terminal fragment (amino acid 10–254) of LF confers protection against Letx challenge, and the combination of LFn and PA provides better protection against anthrax than using LFn or PA alone [35,36]. The N-terminal fragments of EF and LF are responsible for binding of PA and further translocating the toxic parts of these toxin factors into the cytosol. Since EF and LF bind to the same sequence of PA, and both EFn and LFn regions display high similarity (44% similarity) in amino acid sequences, this explains why anti-EF antibodies elicited by Ad/EFn react with EF and cross-react with LF. In this research, we show that anti-EF antibody has neutralizing capability against Edtx and Letx. In addition, since anti-EF immune responses specifically block the entry of EF into target cells and inhibit its function, this vaccine displays the potential to decrease the onset of symptoms of anthrax that result from edema factor in B. anthracis infection. On the other hand, previous research by Little et al. showed that anti-LF monoclonal antibodies also reacted with EF [56]. Our results strongly support the role of EF in eliciting host immunity against EF and LF, although the adenovirus Ad/EFn used in this study expresses the 254 amino acids of EFn plus a 68 amino acid tag at its C-terminus. We have constructed the adenovirus Ad/rEFn encoding only the 1–254 amino acids of EF. Our preliminary results show that immunization with Ad/rEFn elicits serum antibody responses against EF and LF at comparable levels as immunization with Ad/EFn (data not shown). It is likely that the 68 amino acid tag co-expressed with EF1–254 does not affect the immune

response to EF. After a sequence comparison, we were able to determine that the 68 amino acid tag does not contain amino acid sequences found in the LF. We recognize that the anti-EF and anti-LF antibody responses are rather weak compared to those generally seen after immunization with adenoviral vectors. We believe that the EFn expression level is low because the native EFn DNA sequence is used in this adenovirus. The codon usage of the native EF gene reflects a high A + T (71%) base composition for its DNA [53]. The EFn DNA sequence is probably not optimal for expression in mammalian cells, and the EFn is not secreted from the cells. Therefore, we are now constructing a high-level EFn expression vector which will express a signal peptide of human tissue plasminogen activator (tPA) with human codonoptimized EFn [57,58]. We envision that the secretory EFn will be expressed at a higher level in host cells and may be presented to antigen presenting cells (APCs) more efficiently after immunization. Other strategies to enhance the immune response to EFn could be the application of adjuvant, such as CpG motifs in the vector DNA sequence or inclusion of a cytokine (IL4, GMCSF, or IL12) expression vector in the vaccine [57]. Since we used the human Ad5 derived replicationincompetent adenovirus for vaccine delivery, the issue of host pre-existing immunity against adenoviruses needs to be addressed in further experiments. Application of alternative serotype adenoviral vectors, such as human Ad35 or Ad11, to which the majority of human populations have very low pre-existing immunity, could be a possible solution to this problem [59,60]; adenoviral vectors derived from animals, such as ovine and chimpanzee adenoviruses may also be used as alternative vaccine delivery vectors [61,62]. In summary, our data demonstrate that an EFn-based genetic vaccine is capable of eliciting host immunity against anthrax. The inclusion of the nontoxic portion of EF in addition to PA in a third generation anthrax vaccine has merit for further study.

Acknowledgements This work was supported by the US Public Service research grant AI053598 (M.Z.) from the National Institute of Allergy and Infectious Diseases. Additional support was also provided by the Porter Anderson Immunobiology Fellowship from the University of Rochester (M.Z.). We are grateful to Maria Alevia, Peter Sidor, and Natasha Girgis for their assistance in animal sera preparation and adenovirus construction, and to Peter Salzman and Xin Tu for performing statistic analysis. We thank Stephen Leppla for providing EF plasmid, EF, LF, PA proteins, and antisera for this research. We greatly appreciate the support from Stephen Dewhurst, Barbara H. Iglewski, and Robert C. Rose. The ATCC BEI Resources (funded by NIAID/NIH) has provided recombinant EF, LF, and PA, and the US Department of Defense provided AAV (BioThraxTM ) for our research.

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