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INFECTION AND IMMUNITY, Jan. 2009, p. 255–265 Vol. 77, No. 10019-9567/09/$08.00�0 doi:10.1128/IAI.00633-08

Role of Anthrax Toxins in Dissemination, Disease Progression, andInduction of Protective Adaptive Immunity in the Mouse

Aerosol Challenge Model�†Crystal L. Loving,1‡ Taruna Khurana,1 Manuel Osorio,2 Gloria M. Lee,1

Vanessa K. Kelly,1 Scott Stibitz,2 and Tod J. Merkel1*Laboratory of Respiratory and Special Pathogens1 and Laboratory of Enteric and Sexually Transmitted Diseases,2 Center for

Biologics Evaluation and Research, Food and Drug Administration, 8800 Rockville Pike, Bethesda, Maryland 20892

Received 22 May 2008/Returned for modification 30 July 2008/Accepted 16 October 2008

Anthrax toxins significantly contribute to anthrax disease pathogenesis, and mechanisms by which the toxinsaffect host cellular responses have been identified with purified toxins. However, the contribution of anthraxtoxin proteins to dissemination, disease progression, and subsequent immunity after aerosol infection withspores has not been clearly elucidated. To better understand the role of anthrax toxins in pathogenesis in vivoand to investigate the contribution of antibody to toxin proteins in protection, we completed a series of in vivoexperiments using a murine aerosol challenge model and a collection of in-frame deletion mutants lackingtoxin components. Our data show that after aerosol exposure to Bacillus anthracis spores, anthrax lethal toxinwas required for outgrowth of bacilli in the draining lymph nodes and subsequent progression of infectionbeyond the lymph nodes to establish disseminated disease. After pulmonary exposure to anthrax spores, toxinexpression was required for the development of protective immunity to a subsequent lethal challenge. However,immunoglobulin (immunoglobulin G) titers to toxin proteins, prior to secondary challenge, did not correlatewith the protection observed upon secondary challenge with wild-type spores. A correlation was observedbetween survival after secondary challenge and rapid anamnestic responses directed against toxin proteins.Taken together, these studies indicate that anthrax toxins are required for dissemination of bacteria beyondthe draining lymphoid tissue, leading to full virulence in the mouse aerosol challenge model, and that primaryand anamnestic immune responses to toxin proteins provide protection against subsequent lethal challenge.These results provide support for the utility of the mouse aerosol challenge model for the study of inhalationalanthrax.

Bacillus anthracis is a gram-positive, spore-forming bacte-rium and the etiologic agent of anthrax (38). Three forms ofthe disease exist, dependent on the route of exposure. Cuta-neous anthrax occurs when spores are introduced through askin wound and gastrointestinal intestinal disease occurs afteringestion of spores. The third manifestation, inhalational an-thrax, results after spores are inhaled into the lungs. Inhala-tional anthrax is the most severe form of the disease and isoften associated with rapid disease progression and death (4,25, 65). Although inhalational anthrax is extremely rare inhumans, the use of anthrax as a biological weapon in the fall of2001 highlighted the need to understand inhalational anthraxdisease pathogenesis for the generation of improved therapeu-tics and vaccines (15).

Protective antigen (PA) is the primary component of thecurrent U.S. licensed vaccine (anthrax vaccine absorbed[AVA]) (6). Functional anthrax toxins require the combination

of PA with lethal factor (LF) for lethal toxin (LT) and PA withedema factor (EF) for edema toxin (ET). Genes encoding thetoxin proteins are carried on the virulence plasmid pXO1 (44).Once produced by the bacterium, PA binds to the eukaryoticcell surface receptors tumor endothelium marker 8 (TEM8)and capillary morphogenesis protein 2 (CMG2), which theninteract with host expressed LDL-receptor-related protein 6(LRP6) (7, 55). After PA binds to the host cell receptor, it iscleaved by furin and forms heptamers capable of binding threeEF 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 membraneand mediates the translocation of the bound EF and/or LF intothe cytoplasm 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 signaltransduction, consequently inhibiting the expression of somecytokines (8). However, spore infection of primary cells withtoxin-producing strains does not always result in the inhibitionof cytokine production (9, 48). EF is an adenylate cyclase thatincreases intracellular concentrations of cyclic AMP, whichalso disrupts host cell responses (31, 40, 59). The majority of

* Corresponding author. Mailing address: Laboratory of Respira-tory 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: [email protected].

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

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

� Published ahead of print on 27 October 2008.

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work describing the effects of anthrax toxins has been doneusing purified toxins in vitro and in vivo (1, 12, 39, 40, 45, 64).Limited information is available on the effects of anthrax toxinsin the context of an infection (17, 42, 49, 52). Detailed reviewson the mechanisms of toxin entry and the effects of toxins onhost cell activity are available (1, 3, 45, 58, 60).

Fully virulent B. anthracis also carries a second virulenceplasmid, pXO2. The pXO2 plasmid harbors the genes forbiosynthesis of the poly-D-glutamic acid capsule, which encasesvegetative bacilli (28, 61). The function of capsule in diseasepathogenesis has not been completely elucidated but is hypoth-esized to prevent bacterial phagocytosis and act as a nonim-munogenic surface (17, 56). Previously published reports havedescribed the susceptibility of mice to B. anthracis strains thatexpress pXO2, even in the absence of pXO1 (18, 27, 30),although the mechanism by which this occurs is unknown.Available data suggest that other animal species do not exhibitthe same sensitivity to pXO1� pXO2� strains as mice do,although published reports on this subject are limited (32, 33).

Rabbits and nonhuman primates, challenged with fully vir-ulent spores, are often used as models to study human anthraxpathogenesis. However, these animals can be costly and diffi-cult to obtain, and such studies require costly biocontainmentfacilities. Mice provide a practical model for studying diseasepathogenesis, evaluating immune responses, and testing newvaccines and therapeutics. We recently described an inhala-tional murine model of anthrax that recapitulates the diseasepathogenesis observed in rabbits and nonhuman primates chal-lenged with fully virulent B. anthracis (42). In an effort to morefully validate this murine model and understand the contribu-tion of anthrax toxins to disease pathogenesis in vivo, we usedthe murine aerosol challenge (42) and a series of isogenictoxin-deficient mutants (in a Sterne strain genetic background)(36) to examine the role of anthrax toxins in virulence, dissem-ination, disease progression, and the development of protec-tive immunity. Our results show that functional LT is requiredfor the establishment of disseminated disease and subsequentlethality and that ET contributes to that process but is notrequired. In addition, our data show that primary exposure totoxin components is required for immune responses that pro-vide protection to a subsequent lethal challenge. The mice didnot all exhibit elevated immunoglobulin titers in response totoxin proteins after primary exposure, but rapid anamnesticresponses were evident and were likely involved in protectionagainst secondary lethal challenge.

MATERIALS AND METHODS

Aerosol challenge of A/J mice. Male and female A/J mice were purchased fromthe National Cancer Institute (Bethesda, MD). Mice between the ages of 6 and12 weeks of age were challenged as previously described (42, 49) with minimalmodifications. Briefly, mice were exposed to aerosolized spores for 70 min byusing a nose-only exposure system (CH Technologies, Westwood, NJ), with freshair supplied for 8 min before and after spore exposure. The spore inoculum foreach challenge, prepared from a toxin-deficient strain (triple knockout [TKO][�pag �lef �cya], double knockout [DKO] [�lef �cya], or �pag strain), or theparent strain B. anthracis Sterne (strain 7702) contained 12 ml of 5 � 109

spores/ml in distilled water with 0.01% Tween 80. Generation of toxin-deficientstrains was previously described, and the loss of toxin genes was confirmed withPCR using published primers (36; data not shown). Generation and purificationof spores for all strains were carried out as previously described (22, 49). The70-min aerosol exposure results in a retained dose in the lungs of 2 � 106 to 4 �106 spores, which is approximately 10 times the 50% lethal dose (LD50) calcu-

lated for strain 7702 (42). For strain 7702 challenges, in which 1 LD50 wasdesired, groups of A/J mice were exposed to an aerosolized spore inoculum of 5� 108 spores/ml for 45 min, with fresh air supplied for 8 min before and after thechallenge. Immediately after any challenge, four mice were euthanized, and thelungs were removed and homogenized, serially diluted, and plated to determinethe average number of spores retained in the lungs for that challenge. Values arereported as CFU. Mice were retained for survival or rechallenge studies oreuthanized at various times postchallenge for tissue collection. For rechallengestudies, mice were exposed to aerosols of strain 7702 (inoculum at 5 � 109

spores/ml) for 70 min. All mice for these studies were housed and maintained atthe Center for Biologics Evaluation and Research animal facility under theapproval of the Institutional Animal Care and Use Committee.

Measurement of serum antibody titers. Total serum immunoglobulin G (IgG)antibody titers to PA, LF, or EF were determined by using a quantitativeanti-recombinant PA, LF, or EF enzyme-linked immunosorbent assay. Ninety-six-well microtiter plates (Immunolon 2HB; ThermoLabsystems, Franklin, MA)were coated with 100 �l of recombinant PA, LF, or EF (1 �g/ml)/well overnightat 4°C. Plates were then washed (phosphate-buffered saline plus 0.05% Tween)and blocked with 3% bovine serum albumin in phosphate-buffered saline for 1 hat 37°C. Plates were incubated with 100 �l of serially diluted (1:100 to 1:300,000in blocking buffer) serum samples 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 1:1,000 in blockingbuffer. Finally, the plates were incubated for 15 to 20 min at room temperaturewith 100 �l of ABTS [2,2�azinobis(3-ethylbenzthiazolinesulfonic acid); KPL).The reaction was stopped by adding 100 �l of ABTS peroxidase stop solution(KPL). Absorbance values were obtained by using a Molecular Devices (Sunny-vale, CA) VersaMax microplate reader at 405 nm. Samples were assayed intriplicate, and the endpoint antibody titers were expressed as the maximumdilution giving an absorbance of �0.2 (405 nm). The results are presented as thereciprocal of the dilution multiplied by the absorbance value.

Bioluminescent B. anthracis and in vivo imaging. Luminescent strains of B.anthracis (Sterne 7702 or mutants thereof) were created as follows. The luxCDABEoperon from Photorhabdus luminescens was reconstructed by joining PCRfragments of the individual genes, or portions thereof, and in the process, pro-viding each open reading frame with a strong gram-positive ribosome-bindingsite. The PCR template was pUTmini-Tn5kmlux (24). Fragments were assem-bled, without the introduction of unwanted additional restriction sites, throughthe use of the type IIs restriction enzyme BsaI, essentially as described byStemmer and Morris (57). A luxBADCE operon fragment, thus created, andflanked by XhoI and KpnI was cloned between the XhoI and KpnI sites ofpSS4030, a derivative of the temperature-sensitive vector pBKJ236 (36). In asecond step, a XhoI-digested PCR fragment consisting of the entire open readingframe of BA1951 was cloned into the upstream XhoI site in the same orientationas the lux operon. BA1951 is the open reading frame driven by the L-19 (Pntr)promoter, which was identified by a promoter screen in B. anthracis as a strong,constitutive promoter (26). In addition, complementary oligonucleotides encod-ing the strong, consensus promoter trc-99 were inserted at the 3� end of theBA1951 open reading frame. The resulting plasmid, pSS4530, was introducedinto the Sterne 7702 strain by conjugation, and isolates in which the plasmid hadintegrated into the chromosome were selected by growth at 37°C with selectionfor erythromycin resistance, as described previously (36). This insertion is pre-dicted to have 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 were either more or less luminescent arose ata low frequency. Serial passage of the more luminescent colonies to derive ahighly luminescent strain led to the isolation of BA679. In a similar way, highlyluminescent derivatives of the toxin deletion strains BA695 (�cya), BA723 (�lef),and BA781 (�pag �lef �cya) (36) were derived to yield BA680, BA681, andBA682, respectively. The genetic mechanism by which more luminescent deriv-atives arose upon passage is unknown. However, these alterations exhibitedadequate phenotypic stability to allow their use in animal challenges. Further-more, bacteria recovered from infected tissues of challenged animals have dem-onstrated no diminution of either the level of luminescence or the percentage ofbacteria expressing the highly luminescent phenotype (data not shown).

Images of mice were acquired by using the IVIS 100 in vivo imaging system(Xenogen). Mice were anesthetized by using 2.5% isofluorane mixed with oxygenusing the XGI-8 gas anesthesia system supplied with the IVIS 100 (Xenogen).Images were acquired according to manufacturer’s recommendations and ana-lyzed by using Living Image 2.5 software (Xenogen).

Tissue collection for dissemination studies. For tissue collection, groups ofmice were euthanized at various times postchallenge and lungs, cervical lymphnodes (cLNs), mediastinal lymph nodes (mLNs), diffuse and organized nasally

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associated lymphoid tissues (NALT), spleens, and livers were collected. A 0.3-gpiece of liver was collected for determining CFU. Tissues used for CFU deter-mination were homogenized in phosphate-buffered saline. In order to distinguishbetween spores and spores plus bacilli, a fraction of each tissue homogenate washeat treated (HT) at 65°C for 30 min to kill any vegetative bacilli. HT anduntreated (UnT) samples were serially diluted and plated on brain heart infusionagar to determine CFU numbers. Each figure distinguishes between UnT andHT samples for discerning the number of spores plus bacilli (UnT) versus sporesalone (HT). The limits of detection were 250 CFU for lungs, spleen, NALT, andliver and 10 CFU for cLNs and mLNs.

Statistical analysis. GraphPad Prism software (version 4.00; GraphPad Soft-ware, San Diego, CA) was used for all statistical analysis. A log-rank test wasused to analyze differences in survival after aerosol challenge. One-way analysisof variance with Tukey’s multiple comparison post-test was used for analyzingcytokine data.

RESULTS

Role of B. anthracis toxin components in lethality of Sternestrain 7702 in A/J mice. Rabbits and nonhuman primates areoften used as a model of anthrax disease of humans. Studieswith these animals have shown that anthrax toxins (LT/ET)significantly contribute to anthrax pathogenesis and that PA-based immunity is critical for protection to lethal anthrax chal-lenge. We have found that an inhalational murine model ofanthrax in which complement-deficient A/J mice are chal-lenged with Sterne strain spores recapitulates the diseasepathogenesis observed in rabbits and NHP challenged withfully virulent 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 (�pag), LF (�lef), EF (�cya), LF and EF(DKO, �lef �cya), or LF, EF, and PA (TKO, �pag �lef �cya)were utilized for these studies. A/J mice were challenged by theinhalational route with spores prepared from mutant strains orthe parent strain 7702, and survival was monitored for 10 days(Fig. 1A). Mice were challenged with 1 � 106 to 5 � 106 sporesof each strain, which represents approximately 10 to 20 LD50sfor the parental strain 7702 (Fig. 1B). Figure 1A shows thatdeletion of either the gene coding for PA (�pag) or LF (�lef)resulted in complete attenuation of disease (100% survival ofchallenged mice). Although the strain lacking EF (�cya) wasreduced in virulence compared to 7702, it was not completelyattenuated. Taken together, these data indicate that ET con-tributes to pathogenesis during inhalational infection but is notabsolutely required for lethality. In contrast, LT is required forlethality during inhalational anthrax. These data also show thatnontoxigenic derivatives of the Sterne strain are not lethal toA/J mice. The requirement for toxins in the mouse aerosolchallenge model provides an opportunity to evaluate the func-tional role of toxins and toxin components in vivo after aerosolexposure to spores.

Exposure to toxin proteins after aerosol immunization con-tributes to protection against secondary lethal challenge.Since mice challenged with the DKO (�lef �cya), TKO (�pag�lef �cya), and �pag strains survived primary aerosol exposure,we were provided with the opportunity to evaluate the relativecontribution of the immune response to toxin proteins (PA orLF/EF) to protective immunity generated after inhalationalexposure to spores. Groups of A/J mice were immunized by the

aerosol route with spores prepared from each of the toxinmutant strains and the 7702 parent strain. For aerosol immu-nization, mice challenged with the toxin-deficient strains re-ceived 10 times the LD50 calculated for 7702; however, micechallenged with strain 7702 were exposed to spores at oneLD50 in order to have survivors for rechallenge (Fig. 2A). Allof the mice challenged with a toxin-deficient strain survived theprimary immunization challenge, and ca. 50% of mice chal-lenged with one LD50 of strain 7702 survived primary immu-nization (data not shown). At 30 days after the primary aerosolexposure, all surviving mice, as well as a group of naive A/Jmice, were challenged by the aerosol route with 10 LD50s ofstrain 7702 spores (Fig. 2B). Survival was monitored for 10days, as shown in Fig. 2C. Mice initially challenged with mutantstrains that still encoded either PA or LF/EF (the �lef �cya[DKO] or �pag strain, respectively) had a significantly highersurvival rate than mice immunized with a strain lacking alltoxin genes (�pag �lef �cya [TKO]). Mice initially challengedwith strain 7702, which produces all toxin proteins, exhibitedthe greatest protection since 94% of the mice survived second-ary lethal challenge (Fig. 2C). Mice initially challenged withthe �lef �cya (DKO) or �pag strain survived the secondarylethal challenge 81 and 68% of the time, respectively. How-ever, only 39% of mice initially challenged with the �pag �lef

FIG. 1. Role of B. anthracis toxin components in the lethality ofSterne strain 7702 in A/J mice. (A) Groups of mice were exposed toaerosols of B. anthracis spores of Sterne strain 7702 or isogenic strainsdeficient for different toxin genes (TKO [�pag �lef �cya], DKO [�lef�cya], �pag, �lef, and �cya strains). (B) Average spore load in thelungs of exposed mice immediately after challenge. Lungs were col-lected from a fraction of the mice immediately after aerosol exposureto determine the average retained spore dose in the lungs. No signif-icant difference between challenge groups was observed. The retaineddose was 2 � 106 to 5 � 106 spores (approximately 10 to 20 LD50s forstrain 7702). The data shown were pooled from two independentexperiments, with a minimum of eight mice in each challenge group foreach experiment.

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�cya (TKO) strain survived the lethal rechallenge. This sur-vival rate was not significantly different from that observed fornaive mice. Although mice immunized with the �pag strain hadfewer survivors after lethal rechallenge than mice immunizedwith strain 7702, this difference was not significant (P �0.0605). Taken together, these data indicate that even in thecontext of an infection with spores, in which the host is exposedto a wide variety of spore and vegetative cell antigens, thedevelopment of protective immunity is dependent upon expo-sure to toxin components, PA and/or LF/EF.

Antibody titers to PA and LF after primary aerosol chal-lenge. Antibody responses to PA elicited after vaccination havebeen shown to contribute significantly to protection uponlethal exposure to B. anthracis (34, 50, 51). To determinewhether titers of antibody to PA, LF, or EF were elevated afteraerosol immunization, groups of mice were immunized byaerosol challenge. On days 14 and 28 after aerosol immuniza-tion with live organism, sera were collected and assayed fortotal IgG to toxin components (Fig. 3). Only titers to PA andLF were measured due to the small amount of sera collectedfrom each animal. Figure 3A indicates that anti-PA IgG titerswere elevated in mice exposed to strain 7702 and the DKO(�lef �cya �lef �cya) strain. The increase in anti-PA IgG titerswas not significant between these two groups, likely due to thewide range of titers within each challenge group. Mice exposedto strains lacking PA (�pag �lef �cya [TKO] and �pag strains)did not show significant titers to PA, as a result of the lack ofthis antigen. Mice exposed to the �pag strain, which producesEF and LF, did not show elevated titers to LF (Fig. 3B),although these mice showed some level of protection againstlethal aerosol rechallenge (Fig. 2A).

Anamnestic antibody responses to toxin proteins after sec-ondary lethal challenge. Mice that received an aerosol immu-nization with the spores of toxin-deficient �lef �cya (DKO) or�pag strains did not exhibit elevated titers of PA- or LF-specific IgG antibody relative to mice that received an aerosolimmunization with spores of the toxin-deficient �pag lef cya(TKO) strain (Fig. 3). However, these mice were protectedfrom a secondary lethal challenge, whereas mice that receivedan aerosol immunization with the TKO strain were not pro-tected (Fig. 2C). In order to further understand the contribu-

tion of antibodies to toxin proteins in protection, we measuredthe anamnestic antibody response after a secondary lethalchallenge. We collected sera on day 28 after aerosol immuni-zation and on days 3 and 5 after secondary lethal challenge andmeasured the titers of IgG antibody to PA, LF, and EF. Figure4 shows that titers of IgG to PA, LF, and EF were elevated onday 28 in mice immunized with strain 7702, which is indicativeof their survival rate to secondary lethal challenge (Fig. 2C).Conversely, anti-PA IgG titers of mice immunized with theDKO (�lef �cya) strain were not elevated on day 28 afterimmunization but increased significantly by day 5 after lethalchallenge (Fig. 4A). This correlated with resistance to second-ary challenge (Fig. 2C). As previously noted, the percent sur-vival of �pag strain-immunized mice, following secondary le-thal challenge, was less than for mice immunized with strain7702, although the difference was not significant (P � 0.0605).This may be explained by the anamnestic antibody response toLF after secondary challenge (Fig. 4B), since the anti-IgGtiters increased slightly by day 5 after secondary challenge. Asexpected, titers to PA, LF, and EF were not elevated in miceimmunized with the TKO (�pag �lef �cya) strain, since theseantigens were not present during priming (day 28, Fig. 4).After secondary challenge, these mice succumbed to disease(Fig. 2C), which is likely explained by the lack of a primaryresponse to toxin components. Taken together, these data in-dicate that although titers of IgG to toxin proteins may not besignificantly elevated after primary spore exposure, this doesnot necessarily correlate to a lack of protection to secondarylethal exposure. Priming of the immune response by the initialexposure can lead to a rapid, anamnestic response that con-tributes to protection. Initial exposure to toxin proteins is crit-ical, since immunization with live spores that are deficient fortoxin genes does not induce protective immunity.

Role of anthrax toxins in dissemination and persistenceafter primary aerosol challenge. To better understand the dis-semination of B. anthracis after aerosol exposure and to inves-tigate the contribution of the anthrax toxins to disseminationand persistence, we utilized an in vivo bioluminescence imag-ing system to monitor the location of vegetative bacilli in in-fected animals. This system allows tracking of an infectionusing a bacterial strain in which bioluminescent genes from P.

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 [�pag�lef �cya], DKO [�lef �cya], and �pag strains), or pXO1� pXO2� strain 9131. (A) Average spore load in the lungs of exposed mice immediatelyafter primary challenge. All mice received a dose of 2 � 106 to 4 � 106 spores (equivalent to 10 LD50s for strain 7702), except mice exposed tostrain 7702, which received 1 � 105 to 2 � 105 spores (equivalent to 1 LD50). At 30 days after primary challenge the mice were rechallenged with10 LD50s of wild-type Sterne strain 7702 spores by the aerosol route. (B) The spore load in the lungs of exposed mice immediately after rechallengeis 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.



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luminescens are genetically engineered to be constitutively ex-pressed by metabolically active bacterial cells. Sterne strainderivatives were generated, in which the bioluminescent genesunder the control of the L-19 and trc-99 promoters werecrossed onto the chromosomes of the 7702, �lef, �cya, andTKO (�pag lef cya) strains. Groups of A/J mice were chal-lenged by the aerosol route with 10 LD50s of the 7702-lux strain

or each of the luminescent toxin KO strains and were imageddaily for bioluminescence (Fig. 5). Insertion of the lux operoninto the chromosome and the expression of the lux operon didnot affect the virulence of the strains as evidenced by thesurvival curves after challenge with these strains (see Fig. S1 inthe supplemental material). A minimum of 16 A/J mice werechallenged with each luciferase-expressing strain and were im-

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 aerosolsof B. anthracis spores of Sterne strain 7702 or an isogenic strain deficient for toxin genes (TKO [�pag �lef �cya], DKO [�lef �cya], or �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 inFig. 2B. All mice received a dose approximately equal to 10 LD50s, except for the mice exposed to strain 7702, which received 1 LD50. Each dotis 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.

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



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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, �lef-lux, �cya �lux, or TKO-luxstrain and imaged daily. Representative pictures, exhibiting the typical dissemination pattern observed following infection with each B. anthracisstrain, are shown. Units are given in photons/s/cm2. No deaths were observed after day 6 among the group of mice shown.



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aged daily. Figure 5 illustrates representative images fromchallenges of A/J mice with each of the luminescent toxin KOstrains. Representative images of two mice challenged with7702-lux are provided to illustrate that anthrax disease in micechallenged with B. anthracis spores by the aerosol route doesnot progress synchronously (Fig. 5A). Bacilli were first noted inthe NALT of all mice by day 1 postchallenge regardless ofchallenge strain. In strain 7702-challenged mice, progression tothe 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 bio-luminescent strain (Sterne-lux) with the in vivo imaging systemled us to identify the NALT and cLNs as an additional route ofdissemination following aerosol challenge. After challengewith LT-deficient strains (the �lef-lux and TKO-lux strains),bacilli were observed in the NALT of challenged mice by day1 postchallenge (Fig. 5C and D). However, luminescence wasnot observed in any tissue beyond the NALT, and lumines-cence in the NALT diminished and was lost over time in thesemice. This result indicated that in the absence of LT expres-sion, the infection was contained within the draining lymphoidtissue and eventually cleared. The observation that ca. 50% ofmice challenged with the ET-deficient strain succumb to infec-tion was reflected in these imaging experiments (Fig. 1A andFig. 5B). Representative images of two mice challenged withthe �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 suc-cumbed to infection (e.g., mouse 2, Fig. 5B), the progressionobserved was similar to that observed in mice challenged withthe parental 7702 strain (Fig. 5A).

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 toaerosols of spores prepared from luciferase-expressing toxin-deficient strains (the TKO, �lef, and �cya strains) or the parentstrain (strain 7702) and imaged the mice each day. Mice weresacrificed at one of three stages of infection and the numbersof spores plus bacilli (UnT) and spores only (HT) were enu-merated for the lung, NALT, cLNs, mLNs, liver, and spleen(Fig. 6). Stage I was defined as the point in infection at whichthe luminescence was first observed in the NALT (Fig. 6A).For all mice, regardless of challenge strain, stage I was reachedby day 1 postinfection. Stage II was defined as the point atwhich luminescence is first observed in the cLNs (Fig. 6B). Forboth strain 7702-challenged mice and mice challenged with the�cya-lux strain, stage II was attained between day 1 and day 4postchallenge. No animal challenged with either of the LT-deficient strains (TKO or �lef strain) was observed to progressto stage II. Stage III was defined as the point in which infectionbeyond the cLNs was observed (Fig. 6C). Due to spore depo-sition from the aerosol challenge, CFU were always present inthe lungs of infected animals (Fig. 6). However, vegetativebacilli were only observed in the lungs of animals sacrificed at

stage III, as evidenced by the reduction in CFU upon HT (Fig.6C). This is consistent with the observation that bacilli appearin the lung vasculature at late stages of disease progressionafter challenge with wild-type spores (42). Vegetative bacilliwere also found in the lungs of mice challenged with the �cyastrain at the late stage of disease (Fig. 6C).

As shown in Fig. 6, CFU were present in the NALT, cLNs,and to 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 ofspores and bacilli were present in the NALT, cLNs, and mLNs.Overall, the presence or lack of toxin genes did not affect theinitial dissemination to these draining lymphoid tissues. How-ever, replication and persistence of LT-deficient strains waslimited in these draining lymphoid tissues, unlike with the 7702parent strain (Fig. 5 and 6). LT-deficient strains failed to per-sist in the NALT or replicate to high numbers in the cLNs ormLNs, as evidenced by the failure of these strains to progressto stage II.

Anthrax toxin expression appears to have a significant role inbacterial 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 �cya strainattained stage III of infection. Mice at this late stage of infec-tion 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 nonluminescent7702 and nonluminescent LT-deficient strains were all sacri-ficed on day 5. In these experiments, liver CFU counts in miceexposed to aerosols of 7702 averaged 1.7 � 104 CFU, butbacilli were not observed in the livers of mice challenged withany toxin-deficient strain (see Fig. S2 in the supplemental ma-terial).

DISCUSSION

Although cases of humans contracting inhalational anthraxare rare, 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, invitro experiments utilizing purified components do not alwaysaccurately model the complex interactions between host andpathogen. Thus, the availability and use of animal models isessential to further our understanding of anthrax and the de-velopment and evaluation of new vaccines and therapeutics.

Rabbits and nonhuman primates often serve as models forstudying anthrax pathogenesis following exposure to virulentpXO1� pXO2� strains of B. anthracis, and these models arethought to accurately reflect disease in humans (21, 25, 41, 51,65). Mice can also serve as a model that is useful and afford-able for the study of anthrax disease pathogenesis. We utilizea mouse model in which complement-deficient mice are chal-lenged with an unencapsulated, toxigenic strain of B. anthracis(pXO1� pXO2�). Confidence in any animal model of patho-genesis requires the accumulation of results from many studiesthat address different aspects of disease. In previous work we



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described disease progression, innate cytokine responses, andhistological changes following aerosol challenge of comple-ment-deficient mice with Sterne strain B. anthracis spores. Ourprevious studies demonstrated that the course of anthrax dis-ease in complement-deficient mice (A/J) challenged with aero-solized Sterne spores is similar to that observed in rabbits andnonhuman primates challenged with fully virulent B. anthracis.More specifically, the bacterial dissemination and pathologicalchanges observed in mice were similar to those observed inrabbits and nonhuman primates (42). We and others havedemonstrated that this model can be used to evaluate toxin-based vaccines (23, 37), and in the present study we demon-strate that functional LT is required for disease progression inthis mouse aerosol challenge model.

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

Our examination of the contribution of the anthrax toxinproteins to the induction of protective adaptive immunity dem-onstrated that exposure to all three toxin proteins, in the con-text of a sublethal aerosol challenge, provided the highestsurvival rate to a subsequent, lethal aerosol challenge (Fig.2C). Primary exposure of mice to strains expressing either PAor LF/EF also affords the animals protection to secondarylethal challenge. Our results suggest that primary exposure toPA provided the greatest protection to secondary lethal chal-lenge, since survival after rechallenge was not significantly dif-ferent between strain 7702, which expresses all three toxincomponents, and the DKO (�cya-lef) strain, which expressesonly PA. Taken together, our results indicate that after pri-mary aerosol exposure, responses to PA provide the greatestlevel of protection following secondary lethal challenge, butexposure to LF and EF contributes to protection as well. It isstriking that although the host was exposed to a variety ofspore and vegetative cell antigens after aerosol immunization,as evidenced by the detection of bacilli in the NALT anddraining lymph nodes of challenged animals, development ofprotective immunity depended on expression of PA and/orLF/EF.

To further our understanding of the contribution of anthraxtoxins to disease pathogenesis, we utilized an in vivo biolumi-

nescence imaging system to monitor disease progression afteraerosol spore exposure. Our results show that after challengewith 7702 spores, luminescence is first observed in the NALT,indicating that vegetative bacilli are present in sufficient num-bers to be detected in this system (Fig. 5). At this same pointin infection, bacilli and spores can be detected in the cLNs andto a lesser extent in the mLNs (Fig. 6A). Taken together, theseresults indicate that following aerosol exposure to anthraxspores, spores not only are deposited in the lungs but also aretaken up in the NALT. From the NALT, spores and/or bacillipresumably drain to the cLNs, and from the lungs, sporespresumably traffic to the mediastinal lymph nodes. In ourmodel, luminescence is next observed in the cLNs, indicatingthat sufficient proliferation has occurred at this site for thethreshold level of detection to be reached. When luminescencewas observed in the cLNs of live animals, it was also detectablein the mLNs of the same animal; however, in order to observethe luminescence in the mLNs, it was necessary to dissect theanimal and expose the lymph nodes ex vivo for imaging (datanot shown). Furthermore, when mice with luminescence in thecLNs were sacrificed and the CFU enumerated, CFU wereobserved in both the cLNs and the mLNs (Fig. 6B). Theseresults support the conclusion that dissemination occurs byboth the nose and the lung following aerosol exposure tospores.

Although bacilli are observed by in vivo imaging in theNALT of all challenged animals as early as day 1 postinfection,the day at which luminescence is first observed in the cLNsvaried from as early as day 2 to as late as day 4. We speculatethat the delay in disease progression observed at this step isdue to the engagement of the host innate immune responseand the effort of the host to contain the infection. The appear-ance of luminescence in the cLN correlates with the failure ofthe host innate immune response to control the infection since100% of the animals that exhibited luminescence in the cLNsprogressed to broadly disseminated disease and death.

We investigated the contribution of each toxin to dissemi-nation, replication, and bacterial persistence during inhala-tional anthrax and discovered that neither LT nor ET wasrequired for the initial dissemination to the NALT or to thedraining lymph nodes (Fig. 5 and 6). Work in the field hasshown that both macrophages and dendritic cells can trafficspores to the draining lymph nodes (8, 11, 29, 54). Thus, it isnot surprising that toxins would not be required for this step,since toxins would not be expressed prior to germination ofspores and the outgrowth of bacilli. Our finding that toxins donot contribute to the initial dissemination to the draining

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, �lef �lux, �cya �lux, or TKO-lux strain and imaged daily. Three stages of disease progression were examined. (A) Stage I wasdefined 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 infectionat which luminescence is observed in the NALT and cLNs. (C) Stage III was defined as the point in the infection at which luminescence is observedin tissues in addition to the NALT and cLNs. Representative images of 7702-lux-infected animals at each stage of infection are shown. Mice weresacrificed at each stage of infection, and the CFU numbers in the lungs, NALT, cLNs, mLNs, livers (0.3 g), and spleens were determined. Sporegermination gives rise to heat-sensitive bacilli. To determine CFU resulting from spores or spores plus bacilli, a fraction of each homogenate washeat treated (HT) at 65°C for 30 min prior to diluting and plating. Untreated (UnT) CFU values represent spores plus bacilli, whereas HT CFUvalues represent spores only. The limits of detection were 250 CFU for lung, NALT, liver, and spleen and 10 CFU for the mLNs and cLNs. Thedose used for all strains was 2 � 106 to 4 � 106 (� 10 LD50s for strain 7702). Each dot is representative of a single animal, and data from twoindependent experiments are shown.



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lymph nodes is consistent with previous observations indicatingthat germination does not occur in the lungs and suggests thatthe location where toxins first have an opportunity to be ex-pressed and have an impact on disease or antigen presentationis in the draining lymphoid tissues (14, 30, 42, 49). AlthoughLT did not affect the initial appearance of B. anthracis sporesand bacilli in the NALT and draining lymph nodes, it wasrequired for persistence and replication to high numbers inboth sites (Fig. 5 and 6). In animals challenged with strainslacking LT, luminescence was never observed in the cLNs, andthe luminescence observed initially in the NALT diminishedand was ultimately eliminated. This indicates that in the ab-sence of LT, the bacterium cannot evade clearance by the host.This observation is consistent with the growing body of datagenerated utilizing in vitro approaches that suggests the an-thrax toxins impair host innate immune responses, allowing theestablishment of a productive infection (2, 3, 13, 16, 20, 53, 59,60, 63). Our results provide the first in vivo demonstration thattoxin expression is required early and that the bacterial infec-tion does not progress beyond the draining lymphoid tissue inthe absence of LT. LT may be the primary contributor topersistence and subsequent mortality, since challenge with theET-deficient (�cya) strain was still lethal in 60% of the chal-lenged animals, but challenge with an LT-deficient (�lef) strainwas not lethal (Fig. 1A). The results using in vivo imagingsuggest that ET also contributes to evasion of the innate im-mune response in that all animals that survived challenge withthe ET-deficient strain cleared the infection before high num-bers of bacilli could be observed in the draining lymph nodes(Fig. 5 and 6). Taken together, our results demonstrate that LTis required for the progression of disease beyond the draininglymphoid tissues and that ET contributes to that process but isnot essential. Ongoing and future experiments are directedtoward the identification of LT-inhibited, innate immune func-tions essential for the control of B. anthracis infection.

Our results demonstrating the requirement for LT and thecontribution of ET to disease progression provide further ev-idence of the relevance and usefulness of this mouse aerosolchallenge model. We have demonstrated the role of the an-thrax toxins in evasion of the host response and the establish-ment of disseminated disease in the context of an aerosolinfection in mice. Further studies using the mouse modelshould allow for the characterization of the interaction be-tween the bacillus, the secreted toxins, and the host innateimmune response.

ACKNOWLEDGMENTS

We thank Gopa Raychaudhuri for critical reading of the manuscript.This project has been funded in whole or in part with Federal funds

from the National Institute of Allergy and Infectious Diseases, Na-tional Institutes of Health, Department of Health and Human Ser-vices, under IAAY1-A1-6153-01.

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