Role of Anthrax Toxins in Dissemination, Disease Progression, and Induction of Protective Adaptive Immunity in the Mouse Aerosol Challenge Model

Anthrax toxins significantly contribute to anthrax disease pathogenesis,and mechanisms by which the toxins affect host cellular responseshave been identified with purified toxins. However, the contributionof anthrax toxin proteins to dissemination, disease progression,and subsequent immunity after aerosol infection with sporeshas not been clearly elucidated. To better understand the roleof anthrax toxins in pathogenesis in vivo and to investigatethe contribution of antibody to toxin proteins in protection,we completed a series of in vivo experiments using a murineaerosol challenge model and a collection of in-frame deletionmutants lacking toxin components. Our data show that after aerosolexposure to Bacillus anthracis spores, anthrax lethal toxinwas required for outgrowth of bacilli in the draining lymphnodes and subsequent progression of infection beyond the lymphnodes to establish disseminated disease. After pulmonary exposureto anthrax spores, toxin expression was required for the developmentof protective immunity to a subsequent lethal challenge. However,immunoglobulin (immunoglobulin G) titers to toxin proteins,prior to secondary challenge, did not correlate with the protectionobserved upon secondary challenge with wild-type spores. A correlationwas observed between survival after secondary challenge andrapid anamnestic responses directed against toxin proteins.Taken together, these studies indicate that anthrax toxins arerequired for dissemination of bacteria beyond the draining lymphoidtissue, leading to full virulence in the mouse aerosol challengemodel, and that primary and anamnestic immune responses to toxinproteins provide protection against subsequent lethal challenge.These results provide support for the utility of the mouse aerosolchallenge model for the study of inhalational anthrax.

INTRODUCTION

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INTRODUCTION

Bacillus anthracis is a gram-positive, spore-forming bacteriumand the etiologic agent of anthrax (38). Three forms of thedisease exist, dependent on the route of exposure. Cutaneousanthrax occurs when spores are introduced through a skin woundand gastrointestinal intestinal disease occurs after ingestionof spores. The third manifestation, inhalational anthrax, resultsafter spores are inhaled into the lungs. Inhalational anthraxis the most severe form of the disease and is often associatedwith rapid disease progression and death (4, 25, 65). Althoughinhalational anthrax is extremely rare in humans, the use ofanthrax as a biological weapon in the fall of 2001 highlightedthe need to understand inhalational anthrax disease pathogenesisfor the generation of improved therapeutics and vaccines (15).

Protective antigen (PA) is the primary component of the currentU.S. licensed vaccine (anthrax vaccine absorbed [AVA]) (6).Functional anthrax toxins require the combination of PA withlethal factor (LF) for lethal toxin (LT) and PA with edema factor(EF) for edema toxin (ET). Genes encoding the toxin proteinsare carried on the virulence plasmid pXO1 (44). Once producedby the bacterium, PA binds to the eukaryotic cell surface receptorstumor endothelium marker 8 (TEM8) and capillary morphogenesisprotein 2 (CMG2), which then interact with host expressed LDL-receptor-relatedprotein 6 (LRP6) (7, 55). After PA binds to the host cell receptor,it is cleaved by furin and forms heptamers capable of bindingthree EF and/or LF molecules. After uptake of the toxin complexinto the cell by phagocytosis and subsequent acidification ofthe phagosome, the PA heptamer inserts into the membrane andmediates the translocation of the bound EF and/or LF into thecytoplasm of the host cell (64). The host cell repertoire thatanthrax toxins can target is large, since TEM8 and CMG2 areexpressed on a variety of cell types, including immune cells(3, 5). LF is a zinc-dependent metalloprotease capable of cleavinghost cell mitogen-activated protein kinase kinases in the cellcytosol (19, 62). Cleavage of mitogen-activated protein kinasekinases by LT has been shown to interrupt host cell signal transduction,consequently inhibiting the expression of some cytokines (8).However, spore infection of primary cells with toxin-producingstrains does not always result in the inhibition of cytokineproduction (9, 48). EF is an adenylate cyclase that increasesintracellular concentrations of cyclic AMP, which also disruptshost cell responses (31, 40, 59). The majority of work describingthe effects of anthrax toxins has been done using purified toxinsin vitro and in vivo (1, 12, 39, 40, 45, 64). Limited informationis available on the effects of anthrax toxins in the contextof an infection (17, 42, 49, 52). Detailed reviews on the mechanismsof toxin entry and the effects of toxins on host cell activityare available (1, 3, 45, 58, 60).

Fully virulent B. anthracis also carries a second virulenceplasmid, pXO2. The pXO2 plasmid harbors the genes for biosynthesisof the poly-D-glutamic acid capsule, which encases vegetativebacilli (28, 61). The function of capsule in disease pathogenesishas not been completely elucidated but is hypothesized to preventbacterial phagocytosis and act as a nonimmunogenic surface (17,56). Previously published reports have described the susceptibilityof mice to B. anthracis strains that express pXO2, even in theabsence of pXO1 (18, 27, 30), although the mechanism by whichthis occurs is unknown. Available data suggest that other animalspecies do not exhibit the same sensitivity to pXO1^– pXO2⁺strains as mice do, although published reports on this subjectare limited (32, 33).

Rabbits and nonhuman primates, challenged with fully virulentspores, are often used as models to study human anthrax pathogenesis.However, these animals can be costly and difficult to obtain,and such studies require costly biocontainment facilities. Miceprovide a practical model for studying disease pathogenesis,evaluating immune responses, and testing new vaccines and therapeutics.We recently described an inhalational murine model of anthraxthat recapitulates the disease pathogenesis observed in rabbitsand nonhuman primates challenged with fully virulent B. anthracis(42). In an effort to more fully validate this murine modeland understand the contribution of anthrax toxins to diseasepathogenesis in vivo, we used the murine aerosol challenge (42)and a series of isogenic toxin-deficient mutants (in a Sternestrain genetic background) (36) to examine the role of anthraxtoxins in virulence, dissemination, disease progression, andthe development of protective immunity. Our results show thatfunctional LT is required for the establishment of disseminateddisease and subsequent lethality and that ET contributes tothat process but is not required. In addition, our data showthat primary exposure to toxin components is required for immuneresponses that provide protection to a subsequent lethal challenge.The mice did not all exhibit elevated immunoglobulin titersin response to toxin proteins after primary exposure, but rapidanamnestic responses were evident and were likely involved inprotection against secondary lethal challenge.

MATERIALS AND METHODS

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MATERIALS AND METHODS

Aerosol challenge of A/J mice. Male and female A/J mice were purchased from the National CancerInstitute (Bethesda, MD). Mice between the ages of 6 and 12weeks of age were challenged as previously described (42, 49)with minimal modifications. Briefly, mice were exposed to aerosolizedspores for 70 min by using a nose-only exposure system (CH Technologies,Westwood, NJ), with fresh air supplied for 8 min before andafter spore exposure. The spore inoculum for each challenge,prepared from a toxin-deficient strain (triple knockout [TKO][ ${Delta}$ pag ${Delta}$ lef ${Delta}$ cya], double knockout [DKO] [ ${Delta}$ lef ${Delta}$ cya], or ${Delta}$ pag strain),or the parent strain B. anthracis Sterne (strain 7702) contained12 ml of 5 x 10⁹ spores/ml in distilled water with 0.01% Tween80. Generation of toxin-deficient strains was previously described,and the loss of toxin genes was confirmed with PCR using publishedprimers (36; data not shown). Generation and purification ofspores for all strains were carried out as previously described(22, 49). The 70-min aerosol exposure results in a retaineddose in the lungs of 2 x 10⁶ to 4 x 10⁶ spores, which is approximately10 times the 50% lethal dose (LD₅₀) calculated for strain 7702(42). For strain 7702 challenges, in which 1 LD₅₀ was desired,groups of A/J mice were exposed to an aerosolized spore inoculumof 5 x 10⁸ spores/ml for 45 min, with fresh air supplied for8 min before and after the challenge. Immediately after anychallenge, four mice were euthanized, and the lungs were removedand homogenized, serially diluted, and plated to determine theaverage number of spores retained in the lungs for that challenge.Values are reported as CFU. Mice were retained for survivalor rechallenge studies or euthanized at various times postchallengefor tissue collection. For rechallenge studies, mice were exposedto aerosols of strain 7702 (inoculum at 5 x 10⁹ spores/ml) for70 min. All mice for these studies were housed and maintainedat the Center for Biologics Evaluation and Research animal facilityunder the approval of the Institutional Animal Care and UseCommittee.

Measurement of serum antibody titers. Total serum immunoglobulin G (IgG) antibody titers to PA, LF,or EF were determined by using a quantitative anti-recombinantPA, LF, or EF enzyme-linked immunosorbent assay. Ninety-six-wellmicrotiter plates (Immunolon 2HB; ThermoLabsystems, Franklin,MA) were coated with 100 µl of recombinant PA, LF, orEF (1 µg/ml)/well overnight at 4°C. Plates were thenwashed (phosphate-buffered saline plus 0.05% Tween) and blockedwith 3% bovine serum albumin in phosphate-buffered saline for1 h at 37°C. Plates were incubated with 100 µl ofserially diluted (1:100 to 1:300,000 in blocking buffer) serumsamples at 37°C for 1 h. Plates were then incubated for30 min at room temperature with purified horseradish peroxidase-conjugatedgoat anti-mouse IgG (KPL, Gaithersburg, MD) diluted 1:1,000in blocking buffer. Finally, the plates were incubated for 15to 20 min at room temperature with 100 µl of ABTS [2,2'azinobis(3-ethylbenzthiazolinesulfonicacid); KPL). The reaction was stopped by adding 100 µlof ABTS peroxidase stop solution (KPL). Absorbance values wereobtained by using a Molecular Devices (Sunnyvale, CA) VersaMaxmicroplate reader at 405 nm. Samples were assayed in triplicate,and the endpoint antibody titers were expressed as the maximumdilution giving an absorbance of >0.2 (405 nm). The resultsare presented as the reciprocal of the dilution multiplied bythe absorbance value.

Bioluminescent B. anthracis and in vivo imaging. Luminescent strains of B. anthracis (Sterne 7702 or mutantsthereof) were created as follows. The luxCDABE operon from Photorhabdusluminescens was reconstructed by joining PCR fragments of theindividual genes, or portions thereof, and in the process, providingeach open reading frame with a strong gram-positive ribosome-bindingsite. The PCR template was pUTmini-Tn5kmlux (24). Fragmentswere assembled, without the introduction of unwanted additionalrestriction sites, through the use of the type IIs restrictionenzyme BsaI, essentially as described by Stemmer and Morris(57). A luxBADCE operon fragment, thus created, and flankedby XhoI and KpnI was cloned between the XhoI and KpnI sitesof pSS4030, a derivative of the temperature-sensitive vectorpBKJ236 (36). In a second step, a XhoI-digested PCR fragmentconsisting of the entire open reading frame of BA1951 was clonedinto the upstream XhoI site in the same orientation as the luxoperon. BA1951 is the open reading frame driven by the L-19(P_ntr) promoter, which was identified by a promoter screen inB. anthracis as a strong, constitutive promoter (26). In addition,complementary oligonucleotides encoding the strong, consensuspromoter trc-99 were inserted at the 3' end of the BA1951 openreading frame. The resulting plasmid, pSS4530, was introducedinto the Sterne 7702 strain by conjugation, and isolates inwhich the plasmid had integrated into the chromosome were selectedby growth at 37°C with selection for erythromycin resistance,as described previously (36). This insertion is predicted tohave no effect on the integrity of the BA1951 gene but placesluxBADCE under the control of the L-19 and trc-99 promoters.It was noted that, upon initial plating, colonies that wereeither more or less luminescent arose at a low frequency. Serialpassage of the more luminescent colonies to derive a highlyluminescent strain led to the isolation of BA679. In a similarway, highly luminescent derivatives of the toxin deletion strainsBA695 ( ${Delta}$ cya), BA723 ( ${Delta}$ lef), and BA781 ( ${Delta}$ pag ${Delta}$ lef ${Delta}$ cya) (36) werederived to yield BA680, BA681, and BA682, respectively. Thegenetic mechanism by which more luminescent derivatives aroseupon passage is unknown. However, these alterations exhibitedadequate phenotypic stability to allow their use in animal challenges.Furthermore, bacteria recovered from infected tissues of challengedanimals have demonstrated no diminution of either the levelof luminescence or the percentage of bacteria expressing thehighly luminescent phenotype (data not shown).

Images of mice were acquired by using the IVIS 100 in vivo imagingsystem (Xenogen). Mice were anesthetized by using 2.5% isofluoranemixed with oxygen using the XGI-8 gas anesthesia system suppliedwith the IVIS 100 (Xenogen). Images were acquired accordingto manufacturer's recommendations and analyzed by using LivingImage 2.5 software (Xenogen).

Tissue collection for dissemination studies. For tissue collection, groups of mice were euthanized at varioustimes postchallenge and lungs, cervical lymph nodes (cLNs),mediastinal lymph nodes (mLNs), diffuse and organized nasallyassociated lymphoid tissues (NALT), spleens, and livers werecollected. A 0.3-g piece of liver was collected for determiningCFU. Tissues used for CFU determination were homogenized inphosphate-buffered saline. In order to distinguish between sporesand spores plus bacilli, a fraction of each tissue homogenatewas heat treated (HT) at 65°C for 30 min to kill any vegetativebacilli. HT and untreated (UnT) samples were serially dilutedand plated on brain heart infusion agar to determine CFU numbers.Each figure distinguishes between UnT and HT samples for discerningthe number of spores plus bacilli (UnT) versus spores alone(HT). The limits of detection were 250 CFU for lungs, spleen,NALT, and liver and 10 CFU for cLNs and mLNs.

Statistical analysis. GraphPad Prism software (version 4.00; GraphPad Software, SanDiego, CA) was used for all statistical analysis. A log-ranktest was used to analyze differences in survival after aerosolchallenge. One-way analysis of variance with Tukey's multiplecomparison post-test was used for analyzing cytokine data.

RESULTS

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RESULTS

Role of B. anthracis toxin components in lethality of Sterne strain 7702 in A/J mice. Rabbits and nonhuman primates are often used as a model of anthraxdisease of humans. Studies with these animals have shown thatanthrax toxins (LT/ET) significantly contribute to anthrax pathogenesisand that PA-based immunity is critical for protection to lethalanthrax challenge. We have found that an inhalational murinemodel of anthrax in which complement-deficient A/J mice arechallenged with Sterne strain spores recapitulates the diseasepathogenesis observed in rabbits and NHP challenged with fullyvirulent B. anthracis (42). In an effort to further validatethis murine aerosol challenge model of anthrax, we examinedthe requirement for toxin expression for lethality in this model.An isogenic collection of B. anthracis strains, derived fromstrain 7702 and containing in-frame, unmarked deletions in thegenes encoding PA ( ${Delta}$ pag), LF ( ${Delta}$ lef), EF ( ${Delta}$ cya), LF and EF (DKO, ${Delta}$ lef ${Delta}$ cya), or LF, EF, and PA (TKO, ${Delta}$ pag ${Delta}$ lef ${Delta}$ cya) were utilizedfor these studies. A/J mice were challenged by the inhalationalroute with spores prepared from mutant strains or the parentstrain 7702, and survival was monitored for 10 days (Fig. 1A).Mice were challenged with 1 x 10⁶ to 5 x 10⁶ spores of eachstrain, which represents approximately 10 to 20 LD₅₀s for theparental strain 7702 (Fig. 1B). Figure 1A shows that deletionof either the gene coding for PA ( ${Delta}$ pag) or LF ( ${Delta}$ lef) resultedin complete attenuation of disease (100% survival of challengedmice). Although the strain lacking EF ( ${Delta}$ cya) was reduced in virulencecompared to 7702, it was not completely attenuated. Taken together,these data indicate that ET contributes to pathogenesis duringinhalational infection but is not absolutely required for lethality.In contrast, LT is required for lethality during inhalationalanthrax. These data also show that nontoxigenic derivativesof the Sterne strain are not lethal to A/J mice. The requirementfor toxins in the mouse aerosol challenge model provides anopportunity to evaluate the functional role of toxins and toxincomponents in vivo after aerosol exposure to spores.

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FIG. 1. Role of B. anthracis toxin components in the lethality of Sterne strain 7702 in A/J mice. (A) Groups of mice were exposed to aerosols of B. anthracis spores of Sterne strain 7702 or isogenic strains deficient for different toxin genes (TKO [ ${Delta}$ pag ${Delta}$ lef ${Delta}$ cya], DKO [ ${Delta}$ lef ${Delta}$ cya], ${Delta}$ pag, ${Delta}$ lef, and ${Delta}$ cya strains). (B) Average spore load in the lungs of exposed mice immediately after challenge. Lungs were collected from a fraction of the mice immediately after aerosol exposure to determine the average retained spore dose in the lungs. No significant difference between challenge groups was observed. The retained dose was 2 x 10⁶ to 5 x 10⁶ spores (approximately 10 to 20 LD₅₀s for strain 7702). The data shown were pooled from two independent experiments, with a minimum of eight mice in each challenge group for each experiment.

Exposure to toxin proteins after aerosol immunization contributes to protection against secondary lethal challenge. Since mice challenged with the DKO ( ${Delta}$ lef ${Delta}$ cya), TKO ( ${Delta}$ pag ${Delta}$ lef ${Delta}$ cya),and ${Delta}$ pag strains survived primary aerosol exposure, we were providedwith the opportunity to evaluate the relative contribution ofthe immune response to toxin proteins (PA or LF/EF) to protectiveimmunity generated after inhalational exposure to spores. Groupsof A/J mice were immunized by the aerosol route with sporesprepared from each of the toxin mutant strains and the 7702parent strain. For aerosol immunization, mice challenged withthe toxin-deficient strains received 10 times the LD₅₀ calculatedfor 7702; however, mice challenged with strain 7702 were exposedto spores at one LD₅₀ in order to have survivors for rechallenge(Fig. 2A). All of the mice challenged with a toxin-deficientstrain survived the primary immunization challenge, and ca.50% of mice challenged with one LD₅₀ of strain 7702 survivedprimary immunization (data not shown). At 30 days after theprimary aerosol exposure, all surviving mice, as well as a groupof naive A/J mice, were challenged by the aerosol route with10 LD₅₀s of strain 7702 spores (Fig. 2B). Survival was monitoredfor 10 days, as shown in Fig. 2C. Mice initially challengedwith mutant strains that still encoded either PA or LF/EF (the ${Delta}$ lef ${Delta}$ cya [DKO] or ${Delta}$ pag strain, respectively) had a significantlyhigher survival rate than mice immunized with a strain lackingall toxin genes ( ${Delta}$ pag ${Delta}$ lef ${Delta}$ cya [TKO]). Mice initially challengedwith strain 7702, which produces all toxin proteins, exhibitedthe greatest protection since 94% of the mice survived secondarylethal challenge (Fig. 2C). Mice initially challenged with the ${Delta}$ lef ${Delta}$ cya (DKO) or ${Delta}$ pag strain survived the secondary lethal challenge81 and 68% of the time, respectively. However, only 39% of miceinitially challenged with the ${Delta}$ pag ${Delta}$ lef ${Delta}$ cya (TKO) strain survivedthe lethal rechallenge. This survival rate was not significantlydifferent from that observed for naive mice. Although mice immunizedwith the ${Delta}$ pag strain had fewer survivors after lethal rechallengethan mice immunized with strain 7702, this difference was notsignificant (P = 0.0605). Taken together, these data indicatethat even in the context of an infection with spores, in whichthe host is exposed to a wide variety of spore and vegetativecell antigens, the development of protective immunity is dependentupon exposure to toxin components, PA and/or LF/EF.

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FIG. 2. Role of B. anthracis toxin components in eliciting protective immune responses for survival after rechallenge with wild-type spores. Groups of A/J mice were exposed to aerosols of B. anthracis spores of Sterne strain 7702, isogenic strains deficient for toxin genes (TKO [ ${Delta}$ pag ${Delta}$ lef ${Delta}$ cya], DKO [ ${Delta}$ lef ${Delta}$ cya], and ${Delta}$ pag strains), or pXO1^– pXO2^– strain 9131. (A) Average spore load in the lungs of exposed mice immediately after primary challenge. All mice received a dose of 2 x 10⁶ to 4 x 10⁶ spores (equivalent to 10 LD₅₀s for strain 7702), except mice exposed to strain 7702, which received 1 x 10⁵ to 2 x 10⁵ spores (equivalent to 1 LD₅₀)_. At 30 days after primary challenge the mice were rechallenged with 10 LD₅₀s of wild-type Sterne strain 7702 spores by the aerosol route. (B) The spore load in the lungs of exposed mice immediately after rechallenge is shown. (C) Survival after the secondary challenge was monitored for 10 days. The data shown were pooled from two independent experiments, with a minimum of eight mice in each primary challenge group for each experiment.

Antibody titers to PA and LF after primary aerosol challenge. Antibody responses to PA elicited after vaccination have beenshown to contribute significantly to protection upon lethalexposure to B. anthracis (34, 50, 51). To determine whethertiters of antibody to PA, LF, or EF were elevated after aerosolimmunization, groups of mice were immunized by aerosol challenge.On days 14 and 28 after aerosol immunization with live organism,sera were collected and assayed for total IgG to toxin components(Fig. 3). Only titers to PA and LF were measured due to thesmall amount of sera collected from each animal. Figure 3A indicatesthat anti-PA IgG titers were elevated in mice exposed to strain7702 and the DKO ( ${Delta}$ lef ${Delta}$ cya ${Delta}$ lef ${Delta}$ cya) strain. The increase in anti-PAIgG titers was not significant between these two groups, likelydue to the wide range of titers within each challenge group.Mice exposed to strains lacking PA ( ${Delta}$ pag ${Delta}$ lef ${Delta}$ cya [TKO] and ${Delta}$ pagstrains) did not show significant titers to PA, as a resultof the lack of this antigen. Mice exposed to the ${Delta}$ pag strain,which produces EF and LF, did not show elevated titers to LF(Fig. 3B), although these mice showed some level of protectionagainst lethal aerosol rechallenge (Fig. 2A).

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FIG. 3. Serum anti-PA, and anti-LF IgG titers 14 and 28 days after primary aerosol challenge. Groups of A/J mice were exposed to aerosols of B. anthracis spores of Sterne strain 7702 or an isogenic strain deficient for toxin genes (TKO [ ${Delta}$ pag ${Delta}$ lef ${Delta}$ cya], DKO [ ${Delta}$ lef ${Delta}$ cya], or ${Delta}$ pag strains), and serum samples were collected via the tail vein and assayed for levels of IgG against PA (A) and LF (B). Retained lung doses are as noted in Fig. 2B. All mice received a dose approximately equal to 10 LD₅₀s, except for the mice exposed to strain 7702, which received 1 LD_50. Each dot is representative of a single animal from one representative experiment, with a minimum of seven serum samples for each challenge group assayed. Titers are expressed as the reciprocal of the lowest dilution in which antibody was detected multiplied by the absorbance value.

Anamnestic antibody responses to toxin proteins after secondary lethal challenge. Mice that received an aerosol immunization with the spores oftoxin-deficient ${Delta}$ lef ${Delta}$ cya (DKO) or ${Delta}$ pag strains did not exhibitelevated titers of PA- or LF-specific IgG antibody relativeto mice that received an aerosol immunization with spores ofthe toxin-deficient ${Delta}$ pag lef cya (TKO) strain (Fig. 3). However,these mice were protected from a secondary lethal challenge,whereas mice that received an aerosol immunization with theTKO strain were not protected (Fig. 2C). In order to furtherunderstand the contribution of antibodies to toxin proteinsin protection, we measured the anamnestic antibody responseafter a secondary lethal challenge. We collected sera on day28 after aerosol immunization and on days 3 and 5 after secondarylethal challenge and measured the titers of IgG antibody toPA, LF, and EF. Figure 4 shows that titers of IgG to PA, LF,and EF were elevated on day 28 in mice immunized with strain7702, which is indicative of their survival rate to secondarylethal challenge (Fig. 2C). Conversely, anti-PA IgG titers ofmice immunized with the DKO ( ${Delta}$ lef ${Delta}$ cya) strain were not elevatedon day 28 after immunization but increased significantly byday 5 after lethal challenge (Fig. 4A). This correlated withresistance to secondary challenge (Fig. 2C). As previously noted,the percent survival of ${Delta}$ pag strain-immunized mice, followingsecondary lethal challenge, was less than for mice immunizedwith strain 7702, although the difference was not significant(P = 0.0605). This may be explained by the anamnestic antibodyresponse to LF after secondary challenge (Fig. 4B), since theanti-IgG titers increased slightly by day 5 after secondarychallenge. As expected, titers to PA, LF, and EF were not elevatedin mice immunized with the TKO ( ${Delta}$ pag ${Delta}$ lef ${Delta}$ cya) strain, since theseantigens were not present during priming (day 28, Fig. 4). Aftersecondary challenge, these mice succumbed to disease (Fig. 2C),which is likely explained by the lack of a primary responseto toxin components. Taken together, these data indicate thatalthough titers of IgG to toxin proteins may not be significantlyelevated after primary spore exposure, this does not necessarilycorrelate to a lack of protection to secondary lethal exposure.Priming of the immune response by the initial exposure can leadto a rapid, anamnestic response that contributes to protection.Initial exposure to toxin proteins is critical, since immunizationwith live spores that are deficient for toxin genes does notinduce protective immunity.

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FIG. 4. Serum IgG titers to PA, LF, and EF after rechallenge with aerosolized B. anthracis spores. For primary challenges, groups of A/J mice were exposed to aerosols of B. anthracis spores of Sterne strain 7702 or an isogenic strain deficient for toxin genes (TKO [ ${Delta}$ pag ${Delta}$ lef ${Delta}$ cya], DKO [ ${Delta}$ lef ${Delta}$ cya], or ${Delta}$ pag strain). Serum was collected 28 days (d28) after primary challenge, and on day 30 the same mice were rechallenged with 10 LD₅₀s of wild-type Sterne strain 7702 spores by the aerosol route. Serum was collected 3 (d3) and 5 (d5) days after rechallenge. Serum was assayed for levels of IgG against PA (A), LF (B), and EF (C). Each dot is representative of a single animal from one representative experiment, with a minimum of five serum samples for each challenge group assayed, except with the TKO group, since the mice died after rechallenge. Titers are expressed as the reciprocal of the lowest dilution in which antibody was detected multiplied by the absorbance value.

Role of anthrax toxins in dissemination and persistence after primary aerosol challenge. To better understand the dissemination of B. anthracis afteraerosol exposure and to investigate the contribution of theanthrax toxins to dissemination and persistence, we utilizedan in vivo bioluminescence imaging system to monitor the locationof vegetative bacilli in infected animals. This system allowstracking of an infection using a bacterial strain in which bioluminescentgenes from P. luminescens are genetically engineered to be constitutivelyexpressed by metabolically active bacterial cells. Sterne strainderivatives were generated, in which the bioluminescent genesunder the control of the L-19 and trc-99 promoters were crossedonto the chromosomes of the 7702, ${Delta}$ lef, ${Delta}$ cya, and TKO ( ${Delta}$ pag lefcya) strains. Groups of A/J mice were challenged by the aerosolroute with 10 LD₅₀s of the 7702-lux strain or each of the luminescenttoxin KO strains and were imaged daily for bioluminescence (Fig.5). Insertion of the lux operon into the chromosome and theexpression of the lux operon did not affect the virulence ofthe strains as evidenced by the survival curves after challengewith these strains (see Fig. S1 in the supplemental material).A minimum of 16 A/J mice were challenged with each luciferase-expressingstrain and were imaged daily. Figure 5 illustrates representativeimages from challenges of A/J mice with each of the luminescenttoxin KO strains. Representative images of two mice challengedwith 7702-lux are provided to illustrate that anthrax diseasein mice challenged with B. anthracis spores by the aerosol routedoes not progress synchronously (Fig. 5A). Bacilli were firstnoted in the NALT of all mice by day 1 postchallenge regardlessof challenge strain. In strain 7702-challenged mice, progressionto the cLNs was observed, followed by rapid dissemination todistal organs. In individual strain 7702-challenged mice, theday that progression to the cLNs was observed varied from day1 to day 4 (data not shown). Once widespread bacteremia wasapparent, mice succumbed to infection. Previously, emphasishas been placed only on the role of the lung and mLNs duringinhalational anthrax disease progression, but use of the bioluminescentstrain (Sterne-lux) with the in vivo imaging system led us toidentify the NALT and cLNs as an additional route of disseminationfollowing aerosol challenge. After challenge with LT-deficientstrains (the ${Delta}$ lef-lux and TKO-lux strains), bacilli were observedin the NALT of challenged mice by day 1 postchallenge (Fig.5C and D). However, luminescence was not observed in any tissuebeyond the NALT, and luminescence in the NALT diminished andwas lost over time in these mice. This result indicated thatin the absence of LT expression, the infection was containedwithin the draining lymphoid tissue and eventually cleared.The observation that ca. 50% of mice challenged with the ET-deficientstrain succumb to infection was reflected in these imaging experiments(Fig. 1A and Fig. 5B). Representative images of two mice challengedwith the ${Delta}$ cya-lux strain are provided to illustrate the two outcomesof infection observed (Fig. 5B). In the mice that ultimatelysurvived infection (e.g., mouse 1, Fig. 5B), the progressionobserved was similar to that observed in mice challenged withthe LT-deficient strains (Fig. 5C and D). In mice that succumbedto infection (e.g., mouse 2, Fig. 5B), the progression observedwas similar to that observed in mice challenged with the parental7702 strain (Fig. 5A).

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FIG. 5. Groups of A/J mice were exposed to aerosols of spores of luciferase-expressing the B. anthracis 7702-lux, ${Delta}$ lef-lux, ${Delta}$ cya ${Delta}$ lux, or TKO-lux strain and imaged daily. Representative pictures, exhibiting the typical dissemination pattern observed following infection with each B. anthracis strain, are shown. Units are given in photons/s/cm². No deaths were observed after day 6 among the group of mice shown.

In order to correlate studies in which luminescence was usedto monitor disease progression with studies in which tissueCFU were enumerated, we exposed groups of A/J mice to aerosolsof spores prepared from luciferase-expressing toxin-deficientstrains (the TKO, ${Delta}$ lef, and ${Delta}$ cya strains) or the parent strain(strain 7702) and imaged the mice each day. Mice were sacrificedat one of three stages of infection and the numbers of sporesplus bacilli (UnT) and spores only (HT) were enumerated forthe lung, NALT, cLNs, mLNs, liver, and spleen (Fig. 6). StageI was defined as the point in infection at which the luminescencewas first observed in the NALT (Fig. 6A). For all mice, regardlessof challenge strain, stage I was reached by day 1 postinfection.Stage II was defined as the point at which luminescence is firstobserved in the cLNs (Fig. 6B). For both strain 7702-challengedmice and mice challenged with the ${Delta}$ cya-lux strain, stage II wasattained between day 1 and day 4 postchallenge. No animal challengedwith either of the LT-deficient strains (TKO or ${Delta}$ lef strain)was observed to progress to stage II. Stage III was definedas the point in which infection beyond the cLNs was observed(Fig. 6C). Due to spore deposition from the aerosol challenge,CFU were always present in the lungs of infected animals (Fig.6). However, vegetative bacilli were only observed in the lungsof animals sacrificed at stage III, as evidenced by the reductionin CFU upon HT (Fig. 6C). This is consistent with the observationthat bacilli appear in the lung vasculature at late stages ofdisease progression after challenge with wild-type spores (42).Vegetative bacilli were also found in the lungs of mice challengedwith the ${Delta}$ cya strain at the late stage of disease (Fig. 6C).

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FIG. 6. Bacilli and spore load after aerosol challenge of A/J mice. Groups of mice were exposed to aerosols of the luciferase-expressing B. anthracis 7702-lux, ${Delta}$ lef ${Delta}$ lux, ${Delta}$ cya ${Delta}$ lux, or TKO-lux strain and imaged daily. Three stages of disease progression were examined. (A) Stage I was defined as the point in the infection at which luminescence is observed only in the NALT. (B) Stage II was defined as the point in the infection at which luminescence is observed in the NALT and cLNs. (C) Stage III was defined as the point in the infection at which luminescence is observed in tissues in addition to the NALT and cLNs. Representative images of 7702-lux-infected animals at each stage of infection are shown. Mice were sacrificed at each stage of infection, and the CFU numbers in the lungs, NALT, cLNs, mLNs, livers (0.3 g), and spleens were determined. Spore germination gives rise to heat-sensitive bacilli. To determine CFU resulting from spores or spores plus bacilli, a fraction of each homogenate was heat treated (HT) at 65°C for 30 min prior to diluting and plating. Untreated (UnT) CFU values represent spores plus bacilli, whereas HT CFU values represent spores only. The limits of detection were 250 CFU for lung, NALT, liver, and spleen and 10 CFU for the mLNs and cLNs. The dose used for all strains was 2 x 10⁶ to 4 x 10⁶ (= 10 LD₅₀s for strain 7702). Each dot is representative of a single animal, and data from two independent experiments are shown.

As shown in Fig. 6, CFU were present in the NALT, cLNs, andto a lesser extent in the mLNs of all animals at stage I ofinfection regardless of toxin expression. The small reductionseen in CFU upon heat treatment suggests that mixtures of sporesand bacilli were present in the NALT, cLNs, and mLNs. Overall,the presence or lack of toxin genes did not affect the initialdissemination to these draining lymphoid tissues. However, replicationand persistence of LT-deficient strains was limited in thesedraining lymphoid tissues, unlike with the 7702 parent strain(Fig. 5 and 6). LT-deficient strains failed to persist in theNALT or replicate to high numbers in the cLNs or mLNs, as evidencedby the failure of these strains to progress to stage II.

Anthrax toxin expression appears to have a significant rolein bacterial dissemination beyond the draining lymphoid tissue,since only mice challenged with the parent strain (strain 7702)or, at a lower frequency, mice challenged with the ${Delta}$ cya strainattained stage III of infection. Mice at this late stage ofinfection had high numbers of bacilli in the liver and spleen(Fig. 6C). This observation is supported by experiments in whichlarge numbers of mice challenged with the nonluminescent 7702and nonluminescent LT-deficient strains were all sacrificedon day 5. In these experiments, liver CFU counts in mice exposedto aerosols of 7702 averaged 1.7 x 10⁴ CFU, but bacilli werenot observed in the livers of mice challenged with any toxin-deficientstrain (see Fig. S2 in the supplemental material).

DISCUSSION

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DISCUSSION

Although cases of humans contracting inhalational anthrax arerare, the 2001 mail attacks highlighted the potential threatposed by the use of this agent in a bioterrorist or biowarfareattack. Thoroughly investigating and defining the involvementof various anthrax virulence factors, including toxins, is criticalfor the development of new vaccines and therapeutics. Whileutilizing in vitro models to investigate the mechanism by whichtoxins hinder host cell signaling and responses is important,in vitro experiments utilizing purified components do not alwaysaccurately model the complex interactions between host and pathogen.Thus, the availability and use of animal models is essentialto further our understanding of anthrax and the developmentand evaluation of new vaccines and therapeutics.

Rabbits and nonhuman primates often serve as models for studyinganthrax pathogenesis following exposure to virulent pXO1⁺ pXO2⁺strains of B. anthracis, and these models are thought to accuratelyreflect disease in humans (21, 25, 41, 51, 65). Mice can alsoserve as a model that is useful and affordable for the studyof anthrax disease pathogenesis. We utilize a mouse model inwhich complement-deficient mice are challenged with an unencapsulated,toxigenic strain of B. anthracis (pXO1⁺ pXO2^–). Confidencein any animal model of pathogenesis requires the accumulationof results from many studies that address different aspectsof disease. In previous work we described disease progression,innate cytokine responses, and histological changes followingaerosol challenge of complement-deficient mice with Sterne strainB. anthracis spores. Our previous studies demonstrated thatthe course of anthrax disease in complement-deficient mice (A/J)challenged with aerosolized Sterne spores is similar to thatobserved in rabbits and nonhuman primates challenged with fullyvirulent B. anthracis. More specifically, the bacterial disseminationand pathological changes observed in mice were similar to thoseobserved in rabbits and nonhuman primates (42). We and othershave demonstrated that this model can be used to evaluate toxin-basedvaccines (23, 37), and in the present study we demonstrate thatfunctional LT is required for disease progression in this mouseaerosol challenge model.

Previous studies have demonstrated that toxins are involvedin virulence and that host exposure to toxin is required forthe development of a protective immune response (10, 35, 43,46, 47). Our results are consistent with these earlier findings.We have extended our study to include an examination of thecontribution of the anthrax toxins to dissemination and persistenceof B. anthracis following pulmonary infection in vivo and ofthe role of the toxin components in eliciting protective primaryand secondary antibody responses.

Our examination of the contribution of the anthrax toxin proteinsto the induction of protective adaptive immunity demonstratedthat exposure to all three toxin proteins, in the context ofa sublethal aerosol challenge, provided the highest survivalrate to a subsequent, lethal aerosol challenge (Fig. 2C). Primaryexposure of mice to strains expressing either PA or LF/EF alsoaffords the animals protection to secondary lethal challenge.Our results suggest that primary exposure to PA provided thegreatest protection to secondary lethal challenge, since survivalafter rechallenge was not significantly different between strain7702, which expresses all three toxin components, and the DKO( ${Delta}$ cya-lef) strain, which expresses only PA. Taken together, ourresults indicate that after primary aerosol exposure, responsesto PA provide the greatest level of protection following secondarylethal challenge, but exposure to LF and EF contributes to protectionas well. It is striking that although the host was exposed toa variety of spore and vegetative cell antigens after aerosolimmunization, as evidenced by the detection of bacilli in theNALT and draining lymph nodes of challenged animals, developmentof protective immunity depended on expression of PA and/or LF/EF.

To further our understanding of the contribution of anthraxtoxins to disease pathogenesis, we utilized an in vivo bioluminescenceimaging system to monitor disease progression after aerosolspore exposure. Our results show that after challenge with 7702spores, luminescence is first observed in the NALT, indicatingthat vegetative bacilli are present in sufficient numbers tobe detected in this system (Fig. 5). At this same point in infection,bacilli and spores can be detected in the cLNs and to a lesserextent in the mLNs (Fig. 6A). Taken together, these resultsindicate that following aerosol exposure to anthrax spores,spores not only are deposited in the lungs but also are takenup in the NALT. From the NALT, spores and/or bacilli presumablydrain to the cLNs, and from the lungs, spores presumably trafficto the mediastinal lymph nodes. In our model, luminescence isnext observed in the cLNs, indicating that sufficient proliferationhas occurred at this site for the threshold level of detectionto be reached. When luminescence was observed in the cLNs oflive animals, it was also detectable in the mLNs of the sameanimal; however, in order to observe the luminescence in themLNs, it was necessary to dissect the animal and expose thelymph nodes ex vivo for imaging (data not shown). Furthermore,when mice with luminescence in the cLNs were sacrificed andthe CFU enumerated, CFU were observed in both the cLNs and themLNs (Fig. 6B). These results support the conclusion that disseminationoccurs by both the nose and the lung following aerosol exposureto spores.

Although bacilli are observed by in vivo imaging in the NALTof all challenged animals as early as day 1 postinfection, theday at which luminescence is first observed in the cLNs variedfrom as early as day 2 to as late as day 4. We speculate thatthe delay in disease progression observed at this step is dueto the engagement of the host innate immune response and theeffort of the host to contain the infection. The appearanceof luminescence in the cLN correlates with the failure of thehost innate immune response to control the infection since 100%of the animals that exhibited luminescence in the cLNs progressedto broadly disseminated disease and death.

We investigated the contribution of each toxin to dissemination,replication, and bacterial persistence during inhalational anthraxand discovered that neither LT nor ET was required for the initialdissemination to the NALT or to the draining lymph nodes (Fig.5 and 6). Work in the field has shown that both macrophagesand dendritic cells can traffic spores to the draining lymphnodes (8, 11, 29, 54). Thus, it is not surprising that toxinswould not be required for this step, since toxins would notbe expressed prior to germination of spores and the outgrowthof bacilli. Our finding that toxins do not contribute to theinitial dissemination to the draining lymph nodes is consistentwith previous observations indicating that germination doesnot occur in the lungs and suggests that the location wheretoxins first have an opportunity to be expressed and have animpact on disease or antigen presentation is in the draininglymphoid tissues (14, 30, 42, 49). Although LT did not affectthe initial appearance of B. anthracis spores and bacilli inthe NALT and draining lymph nodes, it was required for persistenceand replication to high numbers in both sites (Fig. 5 and 6).In animals challenged with strains lacking LT, luminescencewas never observed in the cLNs, and the luminescence observedinitially in the NALT diminished and was ultimately eliminated.This indicates that in the absence of LT, the bacterium cannotevade clearance by the host. This observation is consistentwith the growing body of data generated utilizing in vitro approachesthat suggests the anthrax toxins impair host innate immune responses,allowing the establishment of a productive infection (2, 3,13, 16, 20, 53, 59, 60, 63). Our results provide the first invivo demonstration that toxin expression is required early andthat the bacterial infection does not progress beyond the draininglymphoid tissue in the absence of LT. LT may be the primarycontributor to persistence and subsequent mortality, since challengewith the ET-deficient ( ${Delta}$ cya) strain was still lethal in 60% ofthe challenged animals, but challenge with an LT-deficient ( ${Delta}$ lef)strain was not lethal (Fig. 1A). The results using in vivo imagingsuggest that ET also contributes to evasion of the innate immuneresponse in that all animals that survived challenge with theET-deficient strain cleared the infection before high numbersof bacilli could be observed in the draining lymph nodes (Fig.5 and 6). Taken together, our results demonstrate that LT isrequired for the progression of disease beyond the draininglymphoid tissues and that ET contributes to that process butis not essential. Ongoing and future experiments are directedtoward the identification of LT-inhibited, innate immune functionsessential for the control of B. anthracis infection.

Our results demonstrating the requirement for LT and the contributionof ET to disease progression provide further evidence of therelevance and usefulness of this mouse aerosol challenge model.We have demonstrated the role of the anthrax toxins in evasionof the host response and the establishment of disseminated diseasein the context of an aerosol infection in mice. Further studiesusing the mouse model should allow for the characterizationof the interaction between the bacillus, the secreted toxins,and the host innate immune response.

ACKNOWLEDGMENTS

We thank Gopa Raychaudhuri for critical reading of the manuscript.

This project has been funded in whole or in part with Federalfunds from the National Institute of Allergy and InfectiousDiseases, National Institutes of Health, Department of Healthand Human Services, under IAAY1-A1-6153-01.

FOOTNOTES

* Corresponding author. Mailing address: Laboratory of Respiratory and Special Pathogens, DBPAP/CBER/FDA, Bldg. 29, Rm. 418, 29 Lincoln Dr., Bethesda, MD 20892. Phone: (301) 496-5564. Fax: (301) 402-2776. E-mail: merkel{at}cber.fda.gov

Published ahead of print on 27 October 2008.

Supplemental material for this article may be found at http://iai.asm.org/.

Editor: V. J. DiRita

Present address: Respiratory Diseases of Livestock ResearchUnit, USDA/ARS/NADC, 2300 Dayton Avenue B13, Ames, IA 50010.

REFERENCES

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