Introduction

Bacillus anthracis is a gram-positive, aerobic bacterium, and the causative agent of the disease anthrax. The pulmonary form of anthrax is of particular concern owing to the ability of bacterial endospores to be weaponized such that they are easily dispersed and inhaled. This form of the disease progresses rapidly and is highly lethal as evidenced by the contamination of the US mail system with anthrax spores in 2001, which ultimately resulted in five deaths. The lethality of B. anthracis is due to the endotoxins produced by the bacterium during infection.¹ Three subunits make up the anthrax toxins, protective antigen (PA), edema factor, and lethal factor. The toxins are AB-type toxins with the common subunit, PA, binding to a receptor on the surface of eukaryotic cells, oligomerizing, and then associating with edema factor or lethal factor to form edema toxin and lethal toxin (LeTx), respectively.² It is well established that antibodies generated against PA are sufficient for providing protection against toxin and spore challenge in animal models of anthrax.^{3, 4} Anthrax Vaccine Adsorbed (AVA) is the Food and Drug Administration-licensed anthrax vaccine available in the US, and it consists of a B. anthracis culture supernatant that has been adsorbed onto an aluminum adjuvant.⁵ This vaccine is able to stimulate antibodies against PA and can provide protection in animal models of anthrax.^{3, 6} However, in humans AVA requires six administrations over 18 months in addition to yearly boosts, a dosing regimen that is not ideal should rapid vaccination before, or in response to, bioterrorist events be necessary. This prolonged vaccination protocol, in addition to the local and systemic side effects that have been associated with the administration of AVA,^{7, 8, 9} have prompted the Institute of Medicine to recommend the development of additional vaccines against anthrax.

Heterologous prime-boost vaccination has emerged as an effective strategy for eliciting a robust immune response to target antigens.¹⁰ In this approach the immune system is primed by administering an antigen by one method and then boosted by subsequently delivering the same antigen using a different method. Prime-boost vaccination has been shown to stimulate potent cellular immunity by inducing high levels of antigen-specific CD4⁺ and CD8⁺ cells, although the molecular mechanisms underlying this response are unclear.^{11, 12, 13, 14, 15, 16} It has been speculated that activation of multiple immune pathways by antigens delivered using different vectors synergistically enhances the immune response to target antigens.¹⁰ One approach that has proven to be particularly effective is priming with plasmid DNA and boosting with a replication-incompetent adenovirus vector encoding the same antigen.^{11, 12, 13, 15} This approach has been used extensively in the development of vaccines against a number of pathogens including HIV, hepatitis C virus, Ebola virus, and Venezuelan equine encephalitis virus.^{11, 15, 17, 18}

Owing to its ability to elicit a potent cellular immune response, DNA priming followed by adenovirus boosting has been used almost exclusively for vaccinating against viral pathogens. However, there are reports indicating that this approach also stimulates a strong humoral immune response,^{15, 16} suggesting that this strategy may also be effective for providing protection against bacterial disease. In the present study, we evaluate heterologous and homologous prime-boost vaccination strategies using plasmid DNA and a replication-deficient adenovirus vector expressing a structural domain of B. anthracis PA for rapid immunization against anthrax. To our knowledge this is the first study in which DNA priming followed by adenovirus boosting has been evaluated as an approach for immunizing against a bacterial disease.

Results

Humoral response to prime-boost vaccination

To evaluate different heterologous and homologous prime-boost immunization strategies for vaccination against anthrax, we used a replication-incompetent adenovirus expressing domain four (D4) of B. anthracis PA (Ad.D4; referred to as Ad) and a plasmid expressing D4 (pUMVC6-D4; referred to as DNA). It has previously been shown that antibodies generated against D4 are sufficient for providing protection against spore and toxin challenge in mice.^{19, 20} In both Ad.D4 and pUMVC6-D4, a codon-optimized reading frame for D4 has been fused to a modified interleukin (IL)-2 signal peptide and placed under the control of the cytomegalovirus immediate early promoter (Figure 1a). As we wanted to develop a method for rapidly vaccinating against anthrax, we chose a relatively short dosing schedule in which mice were primed with DNA or Ad at time zero, boosted with either DNA or Ad 4 weeks later, and challenged with spores 19 days after boosting (Figure 1b). One group of mice was primed and boosted with phosphate-buffered saline (PBS) as a negative control. As a positive control a group of mice was primed and boosted with 25 l of AVA, a dose that we have shown is sufficient for providing protection from spore challenge (McConnell and Imperiale, unpublished data).

Figure 1.

Constructs used for prime-boost immunization and dosing schedule. (a) Schematic showing the replication defective adenovirus vector described previously²⁰ and the expression plasmid (pUMVC6-D4) used for prime-boost vaccination. ITR: inverted terminal repeat, CMV IE: cytomegalovirus immediate early promoter, optD4: codon optimized domain 4 of B. anthracis PA, IL-2sp: human IL-2 signal peptide. (b) Mice were primed with either 5 10⁹ vp of Ad.D4 or 100 g of pUMVC6-D4 at time zero and then boosted with either Ad.D4 or pUMVC6-D4 4 weeks later. Sera were collected from mice 3 and 6 weeks after priming for analysis (gray arrows). Mice were challenged with 8.1 10⁴ Sterne 34F₂ spores subcutaneously 19 days after boosting.

Full figure and legend (43K)

Indirect enzyme-linked immunosorbent assays were used to quantify the anti-PA antibody response in vaccinated mice. As expected, no mice had antibodies against PA before immunization (Figure 2a). Mice that were primed with Ad had high titers of anti-PA immunoglobulin (Ig) G 3 weeks after injection (9.2 10⁴6.8 10⁴; meanSEM), approximately 25 times higher than in mice that were primed with DNA (3.9 10³1.0 10³). Interestingly, at 3 weeks mice that were primed with Ad had approximately 30-fold more anti-PA IgG than did mice receiving AVA (2.7 10³2.4 10²). Mice that were primed with Ad and boosted with a second injection of Ad did not show any further increase in anti-PA IgG after boosting. Mice primed with DNA and boosted with DNA showed a modest increase in anti-PA IgG of approximately 9-fold. The group of mice that was primed with DNA and boosted with Ad, however, had a dramatic increase in anti-PA antibodies of over 80-fold. Furthermore, 6-week-antibody titers in DNA-primed/Ad-boosted mice (4.2 10⁵8.0 10⁴) were significantly higher than in mice that were Ad-primed/Ad-boosted (7.8 10⁴1.1 10⁴), DNA-primed/DNA-boosted (2.4 10⁴5.1 10³), or AVA-primed/AVA-boosted (4.4 10⁴3.9 10³; P<0.001 versus DNA-primed/Ad-boosted for all groups). In general, DNA vaccination resulted in greater variability in anti-PA titers than did vaccination with Ad or AVA.

Figure 2.

Anti-PA antibody response in vaccinated mice. (a) Total anti-PA IgG levels in sera from vaccinated mice taken before immunization (pre-immune), and at 3 and 6 weeks after priming. (b) Anti-PA IgG1, IgG2a, IgG2b, and IgG3 levels in sera taken 6 weeks after priming. Bars represent the average titer of each group with error bars representing the SEM of each group (n=10/group).

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In order to further characterize the immune response stimulated by different immunization strategies, levels of the four IgG subtypes were assessed after boosting (Figure 2b). DNA-primed/Ad-boosted mice had a robust IgG1, IgG2a, and IgG2b antibody response with low levels of IgG3. Anti-PA IgG1, IgG2a, IgG2b, and IgG3 were also detectable in DNA-primed/DNA-boosted and Ad-primed/Ad-boosted mice, although at levels lower than DNA-primed/Ad-boosted mice. Mice that were immunized with AVA had high levels of IgG1, but lower levels of IgG2a and IgG2b than mice in the other groups. No IgG3 was detected in mice immunized with AVA.

Virus-neutralizing antibodies

One possible explanation for the lack of increase in antibody levels in Ad-primed/Ad-boosted mice after boosting is the presence of virus-neutralizing antibodies generated during priming. Indirect enzyme-linked immunosorbent assays were used to detect anti-adenovirus antibodies in post-prime (3 week) sera. Only the group of mice that were primed with Ad had detectable antibodies against adenovirus proteins (anti-adenovirus titer=5.5 10⁴9.0 10³). In order to determine if these antibodies were able to neutralize virus infection, a virus-neutralization assay was performed. Sera from mice that were primed with PBS, AVA, or DNA were not able to neutralize virus infection even at the lowest dilution tested, whereas sera from mice primed with Ad neutralized virus infection even at a dilution of 1:160. These results indicate that there are significant levels of virus-neutralizing antibodies in sera from mice 3 weeks after a single injection of adenovirus and may explain the lack of increase in anti-PA IgG in Ad-primed/Ad-boosted mice.

Cell-mediated immunity

We next characterized the cell-mediated immune response that is generated with each of the prime-boost immunization strategies. IL-4 and interferon-gamma (IFN-) enzyme-linked immunosorbent spot (ELISPOT) assays were performed on splenocytes isolated from mice 15 days after boosting (Figure 3). The presence of IFN--secreting cells suggests a Th1-type immune response whereas IL-4-secreting cells indicate a Th2-type immune response.²¹ DNA-primed/Ad-boosted mice and Ad-primed/Ad-boosted mice had significantly higher levels of IFN--secreting cells (68768 and 59671 cells/10⁶ splenocytes, respectively) as compared to mice that were primed and boosted with DNA (22960 cells/10⁶ splenocytes; P<0.01). Interestingly, all groups had significantly higher levels of IFN--secreting cells than positive control mice that were immunized with AVA (3912 IFN--secreting cells/10⁶ splenocytes; P<0.05 versus all groups). All groups had detectable levels of IL-4-secreting cells. On average, DNA-primed/Ad-boosted, DNA-primed/DNA-boosted, and Ad-primed/Ad-boosted mice had lower levels of IL-4-secreting cells (366, 4214, and 172 cells/10⁶ splenocytes, respectively) than did mice that were immunized with AVA (11033 cells/10⁶ splenocytes).

Figure 3.

ELISPOT analysis using splenocytes from vaccinated mice. Spleens were harvested from immunized mice 15 days after boosting. Splenocytes were stimulated with 5 g/ml of PA or left unstimulated and the number of (a) IFN-- or (b) IL-4- secreting cells was measured by ELISPOT analysis. The number of spot forming cells (SFC) per 1 10⁶ splenocytes was determined by subtracting the number of spots formed by unstimulated cells from the number of spots formed in stimulated samples. Bars represent the average value for each group with error bars representing the SEM (n=3/group).

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Toxin neutralization

We next wanted to determine if the antibodies generated against PA were able to neutralize toxin in vitro. To this end, an assay was performed in which the ability of post-boost sera to protect the mouse macrophage cell line RAW 264.7 from LeTx-mediated lysis was assessed.²² As expected, sera from mice that received PBS were not able to neutralize LeTx even at the lowest dilution tested (Figure 4a). Toxin-neutralizing titers of sera from mice that were primed with DNA and boosted with Ad (1.7 10⁴5.0 10³) were significantly higher than neutralizing titers of sera taken from DNA-primed/DNA-boosted mice (1.4 10³4.6 10²; P<0.005) and Ad-primed/Ad-boosted mice (3.0 10³9.2 10²; P<0.01). Neutralizing titers of sera from the DNA-primed/Ad-boosted group were also about three times higher than neutralizing titers from positive control mice that received AVA (5.7 10³5.2 10²; P<0.05 versus DNA-primed/Ad-boosted).

Figure 4.

In vitro toxin neutralization and protection from spore challenge. (a) LeTx-neutralizing titers of sera from mice taken 2 weeks after boosting. Bars represent the average value for each group with error bars representing the SEM (n=10/group). (b) Nineteen days after boosting mice were challenged with 8.1 10⁴ Sterne 34F₂ spores (73.5 LD₅₀) by subcutaneous injection and monitored for survival over 14 days (n=7/group).

Full figure and legend (19K)

Protection from lethal spore challenge

To determine if the immune response generated against PA was sufficient for providing protection against anthrax, the remaining seven mice from each group were subjected to spore challenge 19 days after boosting. Mice received 8.1 10⁴ Sterne 34F₂ spores subcutaneously, a dose equivalent to 73.5 LD₅₀ in A/J mice,²³ and were monitored for survival over 14 days. As expected, all mice that were immunized with PBS succumbed to challenge by day 4 and all positive control mice that were immunized with AVA survived through day 14 (Figure 4b). Of the seven mice that were primed with DNA and boosted with DNA, six survived spore challenge. The mouse from this group that succumbed to challenge had undetectable levels of LeTx-neutralizing antibodies as determined by the macrophage protection assay. All mice that were Ad-primed/Ad-boosted or DNA-primed/Ad-boosted were completely protected from spore challenge.

To determine if lower doses of Ad.D4 could provide similar protection, mice were primed and boosted with either 5 10⁷, 5 10⁸, or 5 10⁹ vp (virus particles) of Ad.D4 at 0 and 4 weeks. Mice were then challenged 18 days after boosting and monitored for survival over 14 days. All vaccinated mice survived challenge indicating that lower doses of Ad.D4 can elicit protective immunity. Immunization with less Ad.D4 may help to limit vaccine-associated side effects when doses are scaled for human use.

Single-injection vaccination

The ability of an anthrax vaccine to quickly elicit protective immunity is highly desirable should rapid vaccination be necessary. As high levels of antibodies against PA were rapidly generated 3 weeks after priming with Ad (Figure 2a; Ad/Ad), we wanted to determine if a single injection of Ad.D4 could provide protection against lethal spore challenge. Mice were given a single injection of Ad 1, 2, or 3 weeks before spore challenge and sera were collected the day before challenge for analysis. Sera from mice injected 1 week before challenge did not have any detectable antibodies against PA, and these sera were not able to neutralize LeTx at the lowest dilution tested (Figure 5a and 5b). Antibodies against PA were detectable in sera from mice injected 2 weeks before challenge (1.2 10⁴1.3 10³), and sera from these mice were able to neutralize LeTx at low levels (1.2 10²5.9 10¹). Sera from mice injected 3 weeks before challenge had approximately 5-fold more anti-PA antibodies (5.6 10⁴1.3 10⁴) and 13-fold higher LeTx-neutralizing titers (1.5 10³5.9 10²) than sera from mice injected 2 weeks before challenge. One, two, or three weeks after vaccination mice were challenged with 1.5 10⁵ spores (137 LD₅₀) by subcutaneous injection and monitored for survival over 14 days (Figure 5c). All unvaccinated mice and mice challenged 1 week after immunization succumbed to challenge by day 4. However, three of five mice injected 2 weeks before challenge survived and mice injected 3 weeks before spore challenge were fully protected. These results demonstrate that a single injection of Ad.D4 provides significant protection from spore challenge after 2 weeks and complete protection after 3 weeks.

Figure 5.

Single-injection immunization. Mice were immunized with a single injection of 5 10⁹ particles of Ad.D4 1, 2, or 3 weeks before challenge, or left unimmunized (Naïve). (a) Anti-PA total IgG in sera taken from mice 1 day before spore challenge. (b) LeTx-neutralizing titers of sera taken from mice 1 day before spore challenge. Bars represent the average value for each group with error bars representing the SEM (n=5/group). (c) One, two, or three weeks after immunization mice were challenged with 1.5 10⁵ Sterne 34F₂ spores (137 LD₅₀) by subcutaneous injection and monitored for survival over 14 days.

Full figure and legend (20K)

Discussion

An incident involving the intentional release of B. anthracis spores could expose large numbers of individuals to a highly lethal pathogen. Owing to the rapid clinical progression of anthrax, post-exposure antibiotic therapy alone may be insufficient for preventing significant mortality. Such an incident may thus require the mass vaccination of large segments of the population before or after exposure to B. anthracis. An ideal vaccine would induce rapid protective immunity and have limited adverse side effects. Our results show that DNA-priming before Ad-boosting produces higher levels of antibodies than priming and boosting with Ad. The lack of effective boosting by a second injection of Ad may be due to neutralization of the vector by antibodies generated during priming. It is unclear what role virus-neutralizing antibodies play in the context of intramuscular injection, although it has been shown that immune responses against the virus capsid can inhibit transgene delivery to muscle tissue.²⁴ The finding that post-boost anti-PA titers were approximately 5-fold higher in DNA-primed/Ad-boosted mice than in mice that were Ad-primed/Ad-boosted suggests that the antibody response had not simply reached a maximum after a single injection of Ad. These data indicate that heterologous prime boosting is a superior approach for generating humoral immunity to PA than either single modality alone.

Although the results presented here indicate that adenovirus-based heterologous prime boosting induces a more robust antibody response than homologous prime boosting, Ad.D4 itself was able to elicit high levels of anti-PA IgG after a single injection. Our data demonstrate that a single injection can provide rapid protective immunity, suggesting that this approach may be beneficial not only as a prophylactic vaccine, but also as a therapeutic vaccine delivered after exposure has occurred. Recent work has shown that only 44% of non-human primates receiving ciprofloxacin alone after spore infection survived whereas 100% of primates receiving ciprofloxacin and AVA after infection survived, indicating that therapeutic vaccination can dramatically reduce mortality.²⁵ A vaccine that rapidly elicits high levels of anti-PA IgG would be ideally suited for such therapy.

Throughout these studies we used the Food and Drug Administration-licensed anthrax vaccine, AVA, as a positive control. The dose of AVA we used, when scaled for use in mice by weight/volume ratio, would be equivalent to approximately 150 times the dose approved for use in humans. In fact, when 1 scaled doses of the AVA were used to immunize mice, no antibodies against PA were produced (data not shown). Here we have shown that DNA-primed/Ad-boosted mice had 10-fold more anti-PA IgG than AVA-primed/AVA-boosted mice 2 weeks after boosting. This is in spite of the fact that AVA contains full-length PA whereas the DNA and Ad vectors encode only D4 of PA and that the enzyme-linked immunosorbent assays used here detect antibodies against full-length PA. More importantly, LeTx-neutralizing titers in DNA-primed/Ad-boosted mice were approximately 3-fold higher than in mice that received AVA. LeTx-neutralizing titers correlate well with survival in animal models, and vaccines that produce high toxin-neutralizing titers in mice perform well in non-human primate models.^{3, 4, 6}

The role of cell-mediated immunity in providing protection against anthrax is unclear. Although it is well established that antibodies against PA are sufficient for providing protection from anthrax in animal models,^{3, 4, 6} correlates of protective immunity have not been established in humans. The life cycle of B. anthracis involves a short-lived intracellular phase during which spores are engulfed by macrophages before germination.¹ This stage presents a window during which cytotoxic T cells may be effective in combating infection. In addition, because antigen-specific T cells are required for the stimulation of B cells, generating a robust cell-mediated immune response may result in increased antibody production. It may therefore be beneficial for a vaccine to induce not only a robust humoral response, but a cell-mediated response as well. Here we show Ad-primed/Ad-boosted mice and DNA-primed/Ad-boosted mice have high frequencies of IFN--secreting cells 2 weeks after boosting. This is in contrast to the low frequency of IFN--secreting cells seen in AVA-immunized mice. Analysis of IL-4-secreting cells showed that, on average, AVA-immunized mice had slightly higher levels than did genetically vaccinated mice. These data correlate well with the antibody subtypes seen in each group as IFN--secreting cells and IgG2a are associated with a Th1-type immune response and IL-4 and IgG1 are associated with a Th2-type response.²¹

We and others have previously reported the development of adenovirus-based vaccines against anthrax. We have reported that two administrations of Ad.D4 can provide protection from challenge with purified LeTx.²⁰ In addition, two previous reports have described the development of adenovirus vectors expressing full-length PA.^{26, 27} Although the latter studies showed that an anti-PA antibody response is elicited after vaccination, concerns have been raised about administering a potentially active toxin subunit to individuals at risk of being exposed to B. anthracis.²⁸ Because it is unknown how toxins made during B. anthracis infection will interact with PA produced from genetic vaccination, we chose to use only D4 of PA as an antigen. D4 is not functional as a toxin subunit as it lacks the domains necessary for oligomerization and interaction with the other toxin components, lethal factor and edema factor.²⁹ These reports also only evaluated protection from challenge with purified LeTx. The work presented here is therefore the first to demonstrate that adenovirus-based vaccines, which elicit anti-PA antibodies can provide protection from anthrax spore challenge. This is an important advance given a recent report demonstrating that the other anthrax toxin, edema toxin, may contribute significantly to mortality.³⁰ In addition to PA-based adenovirus vaccines, an adenovirus vector expressing the N-terminus of edema factor was able to provide partial protection from spore challenge in mice, further demonstrating the effectiveness of this vaccine strategy.³¹

There are a number of reports in the literature citing local and systemic side effects associated with administration of AVA, although some of these findings remain controversial.^{8, 9, 32} Ideally, a vaccine that could be used to immunize large numbers of people would have minimal adverse side effects. Adenovirus is not associated with serious human disease in healthy individuals and has an excellent safety record in human gene therapy trials. This is demonstrated by the current use of adenovirus as a vector in approximately one quarter of all gene therapy clinical trials (www.wiley.co.uk/genmed/clinical). Adenovirus has also been used previously for the effective mass vaccination of military recruits.³³ Doses of greater than 1 10¹³ vp have been safely administered in clinical trials, making the doses we tested here directly scalable for use in humans.^{34, 35} In addition, administration of adenovirus does not require the use of reactogenic adjuvants that can cause localized inflammation at the site of injection.

In summary, we have demonstrated that adenovirus-based prime-boost vaccination can provide protection against anthrax spore challenge. In addition, a single injection of an adenovirus vector-expressing D4 of PA was able to fully protect mice from spore challenge 3 weeks after vaccination. We also demonstrate that heterologous prime boosting with plasmid DNA and an adenovirus vector elicits a more robust humoral response than homologous prime boost with either single modality alone or the currently licensed vaccine. This strategy may therefore be effective for immunizing against bacterial as well as viral pathogens.

Materials and Methods

Cells and culture conditions. 293 cells are human embryonic kidney cells transformed by the adenovirus E1 region. RAW 264.7 cells are a murine macrophage cell line (American Type Culture Collection TIB-71). All cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, Carlsbad, CA) with 10% fetal bovine serum (DMEM-10), 100 U penicillin/ml, and 100 g streptomycin/ml at 37°C with 5% CO₂.

Viruses and plasmids. Construction of an E1-, E3- deleted adenovirus containing a codon-optimized reading frame of domain 4 (D4) of B. anthracis PA (Ad.D4) was described previously.²⁰ Ad.D4 was purified from infected 293 cells by double CsCl centrifugation and dialyzed into storage buffer (40 mM Tris; pH 8.1, 500 mM NaCl, 2 mM MgCl₂, 10% glycerol) before being stored at -80°C until injection. The concentration of vp was determined by measuring the absorbance at 260 nm, with 1 OD₂₆₀=1.0 10¹² particles. The concentration of infectious particles was determined by fluorescent focus assay.³⁶ The vp:infectious particle ratio for Ad.D4 used in this study was 86.1:1. A plasmid encoding D4 was constructed by amplifying the codon-optimized reading frame for D4 using primers D4for (5'-ATCGGGATCCTTCCACTATGACCGGAATAA-3') and D4rev (5'-TCAAGCTTTTAGCCGATTTCGATAGCCTT-3') and inserting the resulting polymerase chain reaction product into the pGEM-T Easy vector (Promega, Madison, WI) to make pGEM-D4. pGEM-D4 was digested with BamH1 and Apa1 and the product was inserted into the corresponding sites in pUMVC6 (University of Michigan Vector Core) to make pUMVC6-D4 (Figure 1a). In order to prepare pUMVC6-D4 for injection, plasmids were grown in Escherichia coli and purified using the Endofree Maxiprep Kit (Qiagen, Valencia, CA).

Animals and immunizations. For prime-boost immunization groups of 8-week-old female A/J mice (Jackson Laboratories, Bar Harbor, ME; n=10/group) were vaccinated intramuscularly via bilateral injection of the quadriceps muscle. Mice received either 100 g of pUMVC6-D4 (DNA) or 5 10⁹ vp of Ad.D4 diluted into 100 l PBS (Ad) at 0 and 4 weeks. As a negative control one group was mock-immunized with PBS at 0 and 4 weeks. As a positive control one group received 25 l of AVA subcutaneously at 0 and 4 weeks. Blood was collected from the retro-orbital sinus before the first injection (pre-immune) and at 3 and 6 weeks after the first injection. Serum was separated from blood cells and stored at -80°C until analysis. In order to evaluate the ability of lower doses of Ad.D4 to provide protective immunity, 8-week-old female A/J mice (n=3/group) were vaccinated intramuscularly with 5 10⁷, 5 10⁸, or 5 10⁹ vp of Ad.D4 at 0 and 4 weeks. To evaluate the ability of Ad.D4 to protect mice after a single injection, groups of 7–8-week-old female A/J mice (n=5/group) were vaccinated intramuscularly with 5 10⁹ vp of Ad.D4 1, 2, or 3 weeks before spore challenge. Serum was collected the day before spore challenge for analysis. All animal procedures were approved by the University of Michigan Committee on the Use and Care of Animals.

Serum Analysis. Anti-PA and anti-adenovirus enzyme-linked immunosorbent assays were performed as described previously.³⁷ Lethal toxin-neutralizing titers were determined for serum from each mouse as described previously.²⁶

Virus-neutralization assay. The day before the assay, 48-well plates were seeded with 8 10⁴ 293 cells/well. The day of the assay pooled serum from groups of mice was heat inactivated at 60°C for 20 min. Two-fold serial dilutions of serum were made in DMEM containing 2% fetal bovine serum (DMEM-2), and Ad.GFP (an E1-, E3-deleted adenovirus expressing enhanced green fluorescent protein under the control of the cytomegalovirus IE promoter; University of Michigan Vector Core) in DMEM-2 was added to each dilution to a final concentration of 5 10⁸ vp/ml. The virus/serum mixture was incubated at 37°C for 1 h to allow for antibody binding. Each dilution of 100 l was added to each well containing 293 cells and incubated for 1 h at 37°C to allow for infection. The serum/virus mixture was removed from the wells and the cells were overlaid with DMEM-10. The number of green fluorescent protein-positive cells/field was counted after approximately 20 h. Virus-neutralizing titer was defined as the highest dilution giving at least a 50% reduction in green fluorescent protein-positive cells.

ELISPOT assay. IFN- and IL-4 ELISPOT assays were performed as instructed by the manufacturer (BD Biosciences). Three mice from each group were euthanized 15 days after boosting and spleens were removed aseptically. Splenocytes were collected in RPMI medium (supplemented with 2 mM L-glutamine, 10 mM HEPES, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, and 0.05 mM 2-mercaptoethanol) by gently agitating spleens between two microscope slides. The resulting suspension was passed through a 70 m cell strainer and the cells washed two times with RPMI medium. After the second wash, cells were resuspended in red blood cell lysing buffer (Sigma, St Louis, MO) and incubated at room temperature for 2 min. Cells were washed twice with RPMI medium and diluted to the desired concentration with Rosewell Park Memorial Institute. Two-fold dilutions of cells from 1.1 10⁶ to 4.3 10³ cells/well were cultured in wells that had been coated with capture antibodies to either IFN- or IL-4. Cells were incubated for 24 h and either stimulated with 5 g/ml of PA or left unstimulated. After 24 h, splenocytes were removed and IFN- or IL-4 secretion was detected as recommended by the manufacturer. Spots were counted using ImmunoSpot software version 3.2 (CTL Analysis, Cleveland, OH). The number of spot-forming cells for each stimulated sample was determined by subtracting the number of spots formed by unstimulated cells from the same mouse.

Spore challenge. After prime-boost immunization, mice were challenged with spores of the Sterne 34F₂ strain of B. anthracis 19 days after boosting. Spores were diluted to the desired concentration in sterile water and injected subcutaneously after lightly anesthetizing mice using isoflurane. Mice were challenged with 8.1 10⁴ spores, which is equivalent to approximately 73.5 LD₅₀.²³ For dose response studies, mice were challenged 18 days after boosting with 4.9 10⁴ spores (44.5 LD₅₀). To evaluate the ability of Ad.D4 to protect mice after a single injection, mice were challenged with 1.5 10⁵ spores (137 LD₅₀) 1, 2, or 3 weeks after immunization.

Statistical analysis. Comparisons of antibody titers, toxin neutralization titers, and ELISPOT data between groups of mice were made using Student's unpaired t-test. A P<0.05 was considered significant.

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Acknowledgements

We thank members of the Imperiale lab for help with this work, Keith Bishop and members of his lab for assistance with ELISPOT analysis, Karla Passalacqua for assistance with spore preparation, Kathy Spindler and Sonja Gerrard for critical review of this paper, and the University of Michigan Vector and DNA Sequencing Cores for helpful advice and reagents provided under Grant 5 P30 CA46592 awarded to the University of Michigan Cancer Center. This work was supported by R21 AI059231 awarded to MJI from the NIH, the Faculty Research Venture Fund from Frederick G Novy III, MD and family in honor of Frederick G Novy. MJM was supported by T32 GM07863 and T32 GM08353 from the NIH.