INFECTION AND IMMUNITY, Apr. 2011, p. 1770–1778 Vol. 79, No. 40019-9567/11/$12.00 doi:10.1128/IAI.00605-10Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Antibodies Contribute to Effective Vaccination against RespiratoryInfection by Type A Francisella tularensis Strains�

Gopi Mara-Koosham,1 Julie A. Hutt,1,2 C. Rick Lyons,1 and Terry H. Wu1*Center for Infectious Disease & Immunity, Department of Internal Medicine, The University of New Mexico Health Science Center,

Albuquerque, New Mexico,1 and Lovelace Respiratory Research Institute, Albuquerque, New Mexico2

Received 4 June 2010/Returned for modification 28 June 2010/Accepted 25 January 2011

Pneumonic tularemia is a life-threatening disease caused by inhalation of the highly infectious intracellularbacterium Francisella tularensis. The most serious form of the disease associated with the type A strains can beprevented in experimental animals through vaccination with the attenuated live vaccine strain (LVS). Theprotection is largely cell mediated, but the contribution of antibodies remains controversial. We addressed thisissue in a series of passive immunization studies in Fischer 344 (F344) rats. Subcutaneous LVS vaccinationinduced a robust serum antibody response dominated by IgM, IgG2a, and IgG2b antibodies. Prophylacticadministration of LVS immune serum or purified immune IgG reduced the severity and duration of disease innaïve rats challenged intratracheally with a lethal dose of the virulent type A strain SCHU S4. The level ofresistance increased with the volume of immune serum given, but the maximum survivable SCHU S4 challengedose was at least 100-fold lower than that shown for LVS-vaccinated rats. Protection correlated with reducedsystemic bacterial growth, less severe histopathology in the liver and spleen during the early phase of infection,and bacterial clearance by a T cell-dependent mechanism. Our results suggest that treatment with immuneserum limited the sequelae associated with infection, thereby enabling a sterilizing T cell response to developand resolve the infection. Thus, antibodies induced by LVS vaccination may contribute to the defense of F344rats against respiratory infection by type A strains of F. tularensis.

Pneumonic tularemia is a highly debilitating disease causedby the Gram-negative coccobacillus Francisella tularensis.Strains classified under subspecies tularensis (type A) are themost virulent and pose the biggest challenge from a clinicalperspective (28), with a mortality rate estimated to exceed 30%in untreated patients (11). Prophylactic vaccination is the bestcountermeasure, and there is good historical evidence thatpneumonic tularemia can be prevented by vaccination with theattenuated F. tularensis live vaccine strain (LVS) (37). How-ever, LVS is unlikely to be licensed for mass vaccination be-cause the mechanism of attenuation has not been defined. Dueto the potential of a major public health threat, there is anurgent need to understand the protective mechanisms associ-ated with an effective immune response so that novel vaccinescan be developed.

Protective immunity against F. tularensis infection is usuallyattributed to an effective T cell response. However, F. tularen-sis has a significant extracellular phase, which makes it acces-sible to humoral immune responses (18). Indeed, there is am-ple evidence that B cells and antibodies are necessary for miceto develop their natural resistance to primary and secondaryLVS infections. Purified lipopolysaccharide (LPS) from LVSinduced a population of B1-a cells within 2 to 3 days of ad-ministration that protected mice against intraperitoneal (i.p.)LVS challenge (6, 7, 14). Consistent with these results, �MTmice lacking mature B cells exhibited increased susceptibilityto primary intradermal (i.d.) LVS infection and delayed bac-

terial clearance (15, 40). �MT mice were also more susceptibleto secondary i.p. LVS infection, and this defect was correctedby reconstitution with LVS-primed B cells (15). The contribu-tion of antibodies has been addressed repeatedly in passiveimmunization experiments, which showed that immune serumfrom humans and mice vaccinated with live or inactivated LVSprotected naïve mice against challenges with LVS or other lowvirulence strains given by a variety of routes (13, 19, 26, 29, 33,36, 40). The dominant antibody response was directed at LPS,but antibodies against protein antigens have also been found(17, 23, 31, 41, 43). Monoclonal antibodies specific for LPS orthe outer membrane protein FopA provided significant pro-tection against LVS challenge when given either prophylacti-cally (38) or therapeutically (30, 38). Together, these resultssuggest that antibodies contribute toward effective control ofattenuated or low-virulence F. tularensis strains.

It has been much more difficult to demonstrate antibody-mediated protection against type A strains in mice (1, 20, 21,38), even though they express many antigens recognized byLVS immune serum (13, 30). This is not surprising given thehistorical difficulties in generating protective immunity againsttype A strains in this animal model (5). However, Ray et al.recently showed that oral LVS vaccination protected miceagainst a pulmonary SCHU S4 challenge in an antibody-de-pendent manner (35). Klimpel et al. also reported a similarfinding using immune serum from mice cured of a lethal in-tranasal (i.n.) SCHU S4 infection with levofloxacin in a passiveimmunization model (27). Thus, the protective effects of anti-bodies appear not to be restricted only to low-virulence strainsbut may also contribute to the protection against highly viru-lent type A strains.

To further characterize the mechanism of antibody-medi-

* Corresponding author. Mailing address: Department of InternalMedicine, University of New Mexico, 1 University of New Mexico,MSC10 5550, Albuquerque, NM 87131. Phone: (505) 272-8593. Fax:(505) 272-9912. E-mail: twu@salud.unm.edu.

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ated protection, we utilized the recently characterized Fischer344 (F344) rat model (45). Since F344 rats developed muchstronger resistance to respiratory SCHU S4 challenge afterLVS vaccination than previously observed in mice, we specu-lated that antibodies may provide better protection in thismodel and allow us to define their protective mechanism morethoroughly. We now show in a passive immunization modelthat serum antibodies from LVS-vaccinated rats conferredprotection against a lethal intratracheal (i.t.) SCHU S4 chal-lenge. Protection correlated with reduced systemic bacterialgrowth and less severe histopathology during the early phase ofinfection and bacterial clearance by a T cell-dependent mech-anism. Thus, antibodies contribute to but are not sufficient forthe effective control of respiratory infections by fully virulenttype A strains. Our studies provide valuable insights into theprotective mechanisms of antibodies that will guide future de-velopment of tularemia vaccine candidates.

MATERIALS AND METHODS

Rats. Female F344 rats and athymic rnu/rnu rats were purchased from theNational Cancer Institute—Frederick (Frederick, MD). The animals werehoused in a specific-pathogen-free facility at the University of New MexicoAnimal Resource Facility. All animal procedures were reviewed and approved bythe Institutional Animal Care and Use Committee and the Biosafety Committeeat the University of New Mexico.

Bacteria. F. tularensis strains LVS and SCHU S4 were obtained from DynPortVaccine Company LLC (Frederick, MD). The original stock was expanded inChamberlain’s broth (Teknova, Hollister, CA) at 37°C for 48 h with gentleshaking, and aliquots of the culture were stored at �80°C without any preser-vative.

LVS vaccination and serum collection. Rats were lightly anesthetized withisoflurane (Abbott Laboratories, Chicago, IL) and vaccinated subcutaneously(s.c.) between the shoulder blades with 5 � 107 CFU LVS in 100 �l of phosphate-buffered saline (PBS). Four weeks after vaccination, rats were euthanized by CO2

overexposure and immune serum was collected and pooled. Normal serum wascollected in a similar manner or purchased from Charles River Laboratories(Wilmington, MA). Both normal and immune sera were heated to 55°C for 30min to inactivate complement, filter sterilized through a 0.22-�m syringe tip filter(Millipore, Billerica, MA), and stored at �80°C.

Purification of serum IgG and IgM. Serum IgG and IgM were purified usinga Melon gel purification kit (Thermo Scientific Pierce Protein Research, Rock-ford, IL) and Capture Select IgM affinity matrix (BAC B.V., Naarden, Nether-lands), respectively, following the manufacturer’s instructions with a few modi-fications. Briefly, normal and immune sera were diluted 10-fold in PBS and thenprecipitated slowly with 50% ammonium sulfate overnight at 4°C. The precipi-tate was separated from the supernatant by centrifugation at 3,000 � g for 20 minand was resuspended in Melon gel purification buffer for IgG purification or inPBS for IgM purification. The suspensions were subsequently dialyzed 3 timesagainst 300 volumes of Melon gel purification buffer for IgG or PBS for IgMusing a Slide-A-Lyzer dialysis cassette with a 10-kDa molecular weight cutoff(Thermo Scientific Pierce Protein Research, Rockford, IL). IgG was purifiedfrom the dialyzed samples following the manufacturer’s microcentrifuge spincolumn protocol using a Melon gel volume that was 1.25 times the undilutedserum volume. IgM was purified using IgM affinity matrix resin that was 0.5 timesthe undiluted serum volume. Before analyzing the purity and subsequent treat-ment of rats, purified IgG and IgM were dialyzed against PBS as describedabove. The concentration of total IgG and IgM was interpolated from a standardcurve generated with commercially available IgG and IgM of known concentra-tions (Sigma-Aldrich, St. Louis, MO), and the titers of anti-LVS IgG and IgM aswell as contaminating antibody isotypes were determined using an LVS-specificenzyme-linked immunosorbent assay (ELISA) as described below. The puritywas analyzed by 10% SDS-polyacrylamide gel electrophoresis (Bio-Rad, Hercu-les, CA).

ELISA for anti-LVS antibody titer. Maxisorp 96-well microtiter plates (Nunc,Rochester, NY) were coated with 2.5 � 106 to 5 � 106 CFU/ml of heat-killedLVS in PBS overnight at 4°C. After blocking with 5% nonfat dry milk–PBS for1 h at 37°C, 100 �l of rat serum was added in 2-fold serial dilutions and incubatedat 37°C for 1 h. Antibody classes and subclasses were detected by incubation with

horseradish peroxidase-conjugated goat antibodies against rat IgG (Calbiochem,San Diego, CA) and IgG1, IgG2a, IgG2b, IgG2c, IgM, and IgA (Thermo Sci-entific Pierce Protein Research Products, Rockford, IL) for 45 min at 37°C.Between each step, the plates were washed 5 times with 0.05% Tween 20–PBS.The plates were developed with a solution of 3,3�,5,5�-tetramethylbenzidine(TMB), the reaction was stopped with 1.8 N H2SO4, and the optical density (OD)was read at 450 nm. The antibody titer was defined as the reciprocal of thehighest dilution of immune serum that had a mean OD value that was at least 3standard deviations higher than the mean OD of normal serum at the samedilution.

Passive immunization and challenge. Rats were passively immunized by i.p.injection of 250 �l of serum, unless otherwise indicated in selected experiments.Purified IgG and IgM was used in some experiments; the volume given containedthe equivalent amount of LVS-specific IgG as in 250 �l of LVS-immune serum.At 24 h after passive immunization, rats were infected i.t. as described previously(45). Briefly, rats were immobilized on an inclined platform (Alpha Lab Supply,Albuquerque, NM) and intubated with a 20-gauge intravenous catheter (TerumoMedical Products, Somerset, NJ) with the help of a small animal laryngoscopewith a fiber optic light source (Penn-Century, Inc., Philadelphia, PA). In someexperiments 50 �l of inoculum premixed with self-illuminating quantum dots(Zymera, San Jose, CA) was delivered using a blunt-ended needle, followed by50 �l of Coelenterazine and a burst of �500 �l of air to ensure the delivery ofthe inoculum. The infected rats were imaged in vivo using the IVIS 100 opticalimaging system (Caliper Life Sciences, Hopkinton, MA). The health of theinfected animals was monitored daily along with weight loss and survival. Theclinical scores of infection were assigned as follows: 0, active, bright and alert,responsive to handling; 1, slight lethargy and weight loss; 2, decreased respon-siveness to handling, clear piloerection, more pronounced weight loss; 3, definitedecreased activity, ruffled coat, rapid and shallow breathing, hunched posture,eyes half closed and may have porphyrin secretion; 4, inactive and unresponsiveto handling, weak and/or ataxic, severe weight loss, eyes completely closed witha large amount of porphyrin secretion. Animals that succumbed to infection weregiven a maximum score of 4.

Bacterial burden analysis. To quantify the deposited bacteria, lungs wereaseptically removed 1 h after infection and homogenized in PBS using a hand-held or multisample homogenizer fitted with disposable plastic homogenizingprobes (Omni International, Marietta, GA). Lung homogenates were plated neator at appropriate dilutions onto selective cysteine heart agar plates with 5%rabbit blood, 100 U/ml penicillin G, and 100 U/ml polymyxin B (Remel, Lexena,KS), and bacterial colonies were quantified 4 to 5 days later using Qcount (SpiralBiotech, Bethesda, MD). A similar procedure was followed to determine thebacterial burden in lungs, spleen, and liver over the course of infection. When noorganism was found, a value equal to the limit of detection was used to calculatethe mean bacterial burden.

Histopathological evaluation. Following i.t. challenge, rats were euthanized byi.p. injection of 150 �l of Sleepaway (�100 mg/kg of body weight; Fort DodgeAnimal Health, Fort Dodge, IA) on days 1, 3, 5, 7, 10, 14, and 21 postchallenge.Lungs were removed from the thorax en bloc and inflated with 10% neutralbuffered formalin (NBF) via a tracheal cannula. Lungs, spleen, liver, and tra-cheo-bronchial lymph nodes were fixed in 10% NBF for 24 to 72 h and subse-quently trimmed for paraffin embedding. Paraffin-embedded tissues were sec-tioned at 5 �m and stained with hematoxylin and eosin for histological analysisby a board-certified veterinary pathologist. Lesions were graded in a blindedmanner on a semiquantitative scale based upon the severity and distribution oflesions (1, minimal; 2, mild; 3, moderate; 4, marked).

In vivo T cell depletion and flow cytometry. The hybridoma clones OX-8(mouse anti-rat CD8; IgG1), OX-38 (mouse anti-rat CD4; IgG2a), and 55-6(mouse anti-HIV-1 gp120; IgG2a) were obtained from the European Collectionof Cell Cultures (Salisbury, United Kingdom), and TS2/18.1.1 (mouse anti-human CD2; IgG1) was from the American Type Culture Collection (Manassas,VA). Ascites fluids were generated in female ICR SCID mice, and the IgGconcentrations were determined by high-performance liquid chromatography(Taconic, Albany, NY). For CD4 T cell depletion, rats were injected i.p. with 5mg/kg of CD4 T cell-depleting antibody OX-38 or isotype control antibody 55-6for five consecutive days and then with 1 mg/rat twice a week. For CD8 T celldepletion, 1 mg/rat of CD8 T cell-depleting antibody OX-8 or isotype controlantibody TS2/18.1.1 antibodies were administered once a week. One week afterthe start of antibody treatment, the depletion efficiency was confirmed by flowcytometric analyses of peripheral blood mononuclear cells collected by lateraltail vein bleed. In addition, the effects of CD4 T cell depletion on the populationsof NK cells, B cells, and CD8 T cells in the spleen, liver, and blood wereexamined. Rats were euthanized by CO2 overexposure and exsanguinated bycutting the inferior vena cava. Blood collected from the chest cavity was mixed

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with an equal volume of PBS supplemented with 50 U/ml of heparin and layeredover Lympholyte-M density separation medium (Cedarlane, Burlingtion, NC)following the manufacturer’s instructions. The lymphocytes were collected at theinterface of the density gradient medium. To isolate splenocytes, spleens werehomogenized between ground glass slides and passed through 70-�m nylonscreen (BD Biosciences, San Jose, CA). To isolate liver cells, the right lobes werehomogenized through a 200-gauge stainless steel mesh, resuspended in 40%Percoll, and layered over a 70% Percoll solution. The samples were centrifugedat 836 � g for 20 min at 4°C without break. Cells were harvested from the Percollinterface. All the cell preparations were treated with red blood cell lysis buffer(0.15 M NH4Cl, 1 mM KHCO3, 0.1 mM Na2-EDTA), washed with PBS, andresuspended in complete RPMI (RPMI 1640 medium supplemented with 10%heat-inactivated fetal bovine serum, 1 mM nonessential amino acids, 1 mML-glutamine, 1 mM sodium pyruvate) before staining. Cells were stained withbiotinylated anti-CD8b (mouse IgG1, �, clone 341) and anti-CD161a (mouseIgG1, �, clone 10/78), fluorescein isothiocyanate (FITC)-conjugated anti-CD45R(mouse IgG2b, �, clone HIS24), and allophycocyanin (APC)-conjugated anti-CD3 (mouse IgM, �, clone iF4) antibodies from BD Biosciences (San Jose, CA)and phycoerythrin (PE)-conjugated anti-CD8b (mouse IgG1, �, clone eBio341)and FITC-conjugated anti-CD4 (mouse IgG2a, clone OX-35) antibodies fromeBiosciences (San Diego, CA). Biotinylated cells were detected using peridininchlorophyll protein (PerCP)-conjugated streptavidin (Biolegend, San Diego,CA). Before staining the cells, nonspecific antibody binding was blocked byincubating with anti-rat CD32 (mouse IgG1, �, clone D34-485) according to themanufacturer’s instructions (BD Biosciences, San Jose, CA). Appropriate iso-type controls were used. The stained cells were fixed in 0.5% paraformaldehydeand analyzed by flow cytometry on a FACSCalibur (BD ImmunocytometrySystems, San Jose, CA). The flow cytometry data were analyzed using Winlist(Verity, Topsham, ME).

Statistics. Kaplan-Meier survival curves were analyzed by the log-rank test,and SCHU S4 burden following multiple serum treatments was analyzed bytwo-way analysis of variance (ANOVA). Total cell populations from the tissuesof isotype control-treated and CD4-depleting antibody-treated animals werecompared using a two-tailed unpaired t test. These analyses were performedusing GraphPad Prism version 5.01 software (GraphPad Software, San Diego,CA). SCHU S4 growth kinetics in experiments with single prophylactic serumtreatment were analyzed by fitting two-way ANOVA with interaction using thegeneral linear model in SAS (SAS Institute Inc., Cary, NC).

RESULTS

Development of serum antibody response after s.c. LVS vac-cination. We showed previously that Fischer 344 rats cleared avaccine inoculum of 5 � 107 LVS within 2 weeks of subcuta-

neous vaccination and were protected when challenged 2weeks later with SCHU S4 i.t. (45). To determine the antibodyresponse over this 4-week period, the serum concentrations ofLVS-specific IgM, IgG, and IgA were measured. SubcutaneousLVS vaccination induced robust serum IgM and IgG responsesin F344 rats (Fig. 1). The average IgM and IgG titers after 7days were 1:64,000 and 1:16,000, respectively. The IgM titerpeaked 1 week later at 1:32,000 and declined thereafter. TheIgG titers remained relatively stable at 1:16,000 to 1:32,000over the 4-week period. Serum IgA was detected in all vacci-nated rats, but the titer never exceeded 1:800 (data not shown).

Passive immunization with immune serum protects F344rats against pneumonic tularemia. To determine whetherLVS-specific serum antibodies can protect F344 rats against i.t.SCHU S4 challenge, naïve F344 rats were treated prophylac-tically with immune rat serum (IRS) and then challenged i.t.with SCHU S4. Immune sera were pooled from several F344rats 28 days after LVS vaccination, when no trace of LVS couldbe detected systemically. The predominant antibodies wereIgM, IgG2a, and IgG2b, and the titers of IgG1, IgG2c, and IgAwere at least 10-fold lower (Fig. 2). Naïve F344 rats weretreated i.p. with an arbitrary volume of 250 �l IRS, a volumewhich constituted �7% of the total serum volume in a recip-ient F344 rat weighing �150 g. At 24 h after serum transfer,the passively immunized rats were challenged i.t. with a smallbut lethal dose of �250 CFU, which was selected intentionallyto reduce the threshold required to detect any protective effectbrought about by the LVS-specific immune serum. Ratstreated with normal rat serum (NRS) and LVS-vaccinated ratswere used as negative and positive controls, respectively. Allrats were monitored for survival, weight loss, and clinical signsfor 5 weeks. The first indication of illness in the NRS-treatedrats was a slight weight loss and decreased alertness 3 to 4 dayspostinfection (p.i.). The disease progressed very quickly overthe next 48 to 72 h and was characterized by rapid loss of up to30% of body weight (Fig. 3A) and development of severelethargy, ruffled coat, eyelid ptosis, and hunched posture (Fig.3B). At the peak of disease severity, the rats were extremelyweak and unresponsive, and their eyes were completely closed

FIG. 1. Subcutaneous LVS vaccination induces a robust IgM andIgG serum antibody response. F344 rats (n � 3) were vaccinated s.c.with 5 � 107 CFU of LVS. Sera were collected on days 7, 15, 21, and28 postinoculation, and the total LVS-specific IgM, IgG, and IgAantibody titers were determined for each rat by ELISA using heat-killed LVS as the capture antigen. The antibody titer was defined asthe reciprocal of the highest dilution of immune serum that had amean OD value that was greater than 3 standard deviations above themean OD of normal serum at the same dilution. The data show thegeometric mean standard deviation.

FIG. 2. Antibody composition in the immune serum used for pas-sive immunization. F344 rats (n � 3 to 4) were vaccinated s.c. with 5 �107 CFU of LVS. Sera were collected 28 days postvaccination andanalyzed by ELISA for the presence of LVS-specific antibodies of theindicated isotypes and subclasses by using heat-killed LVS as the cap-ture antigen. Antibody titers were defined as described for Fig. 1. Thedata represent the geometric means standard deviations of samplescombined from two independent experiments.

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and surrounded by a large amount of ocular discharge. Mostinfected rats died within 2 weeks of infection (Fig. 3C). Al-though the IRS-treated rats exhibited some of the early signsof infection, weight loss occurred more gradually and rarelyexceeded 10% of the initial body weight. All signs of illnessresolved within 2 weeks of infection, and the rats remainedoutwardly healthy for the remaining 2 to 3 weeks of monitor-ing. LVS-vaccinated rats never lost weight or exhibited anyovert signs of disease. In five independent experiments, at aSCHU S4 challenge dose of 218 to 240 CFU, 27 of 29 ratstreated with IRS survived, while 29 of 30 NRS-treated rats died(Table 1). These results suggested that serum antibodies arecapable of mediating protection against a lethal respiratorySCHU S4 infection.

Purified IgG is sufficient for protection against SCHU S4infection. To verify that LVS-specific antibodies were responsiblefor the serum-mediated protection, IgG and IgM were purifiedfrom normal and immune sera. The purification process reducedthe titers of contaminating antibody isotypes to 1:100 and re-moved most contaminating proteins, except for a prominent 75-

kDa protein in the purified IgG fraction that has not been iden-tified (Fig. 4A and B). For passive immunization, F344 rats wereinjected with an amount of purified IgG and/or IgM that wasequivalent to that contained in 250 �l of serum. Similar to IRS,purified immune IgG provided significant protection against i.t.SCHU S4 challenge (Fig. 4C). In contrast, purified immune IgMoffered no protection and the treated rats succumbed to SCHUS4 infection at the same time as rats treated with normal serum.These results indicated that LVS-specific IgG is the principalprotective component in the immune serum. Since IRS and pu-rified immune IgG provided similar levels of protection, IRS wasused in all experiments described here.

Immune serum treatment of F344 rats limits SCHU S4growth. Since IRS treatment provided significant protectionagainst pulmonary SCHU S4 infection, we next investigatedthe effect of this treatment on SCHU S4 growth. IRS-treatedrats exhibited a pattern of SCHU S4 growth and disseminationthat was intermediate between the NRS-treated rats and theLVS-vaccinated rats. Bacterial expansion in the first 2 daysfollowing infection was identical between the IRS- and NRS-treated rats: in both groups, the number of lung bacteria in-creased to 107 CFU and systemic dissemination to the liver andspleen had occurred in the majority of animals (Fig. 5). Thetwo groups started to diverge on day 3, when fewer bacteriawere recovered from the IRS-treated rats. The bacterial bur-den in NRS-treated rats peaked on day 7 p.i., shortly beforethey died, with 8 � 108 CFU in the lungs, 4 � 108 CFU in theliver, and 2 � 107 CFU in the spleen. In contrast, the bacterialburden in the IRS-treated rats increased at a slower rate, to apeak on day 10 p.i. By day 14, the infection began to clear inthe IRS-treated rats, and the bacterial load in all three tissueshad dropped from their peak. These results suggest that IRSmay contribute to protection of naïve F344 rats by limitingbacterial growth and facilitating development of an immuneresponse that eventually clears the infection.

Protection by IRS is dependent on the SCHU S4 challengedose and the volume of IRS. To further characterize the po-

FIG. 3. Passive immunization with IRS protects naïve rats against a lethal i.t. SCHU S4 challenge. Groups (n � 6) of LVS-vaccinated rats andnaïve F344 rats were treated i.p. with 250 �l of heat-inactivated NRS or IRS and then challenged i.t. 24 h later with 240 CFU of SCHU S4. Thechallenge dose represents the actual lung deposition, determined within 1 h of infection. The infected rats were monitored daily for weight (A),clinical signs (B), and survival (C). In panel A, the results are presented as a percentage relative to the body weight, measured 24 h beforechallenge. A value of 100% indicates no weight change, and points above and below 100% represent weight gain and loss, respectively. In panelB, the disease severity was scored based on the criteria described in Materials and Methods. Data represent the averages of all survivors in eachgroup the standard deviations.

TABLE 1. Summary of survival results of F344 rats challengedintratracheally with SCHU S4 after prophylactic treatment

with 250 �l of serum

SCHU S4challenge

dose (CFU)a

Immune rat serum Normal rat serum

Survival ratio(no. alive/total)

MTDc

(days)Survival ratio

(no. alive/total)MTD(days)

130 6/6 1/5 7218–240b 27/29 11 1/30 9360 5/6 12 0/5 8727 5/6 14 0/6 71,496 1/6 113,525 0/6 13 0/6 810,083 0/6 10 0/6 4

a Based on bacteria recovered from the lungs 1 h after infection.b Data are from five separate experiments using a challenge dose within this

range.c MTD, mean time to death.

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tency of IRS, titrations of the SCHU S4 challenge dose and theIRS volume were performed. A single treatment with 250 �l ofIRS provided long-term protection to over 90% of naïve ratschallenged with up to �700 CFU SCHU S4 (Table 1). Themortality rate increased when the challenge dose was increasedto over 1.5 � 103 CFU, and all of the infected rats diedbetween 10 and 13 days after challenge. There was little cor-relation between the challenge dose and the time to death. Thecumulative results from seven independent experiments sug-gested that the i.t. 50% lethal dose (LD50) of SCHU S4 in F344rats treated with 250 �l of IRS was in the range of 700 to 1,500CFU; this was at least 100-fold less than the LD50 associatedwith s.c. LVS vaccination (45).

Reducing the IRS volume had a dose-dependent effect onthe level of protection against a fixed challenge dose. When theIRS volume was reduced to 25 �l, the treatment prolonged thesurvival of rats challenged i.t. with 360 CFU by 4 to 5 days, butthey eventually succumbed to infection (Fig. 6). All protectiveeffects were eliminated when the IRS volume was further re-duced to 2.5 or 0.25 �l. Increasing the IRS volume to 1 ml didnot substantially delay disease onset, improve clinical signs, oraccelerate resolution compared to the 250-�l treatment (datanot shown). Since the effectiveness of a single IRS treatmentmay be limited by the availability of target organisms at or

around the time of administration, rats were given repeatedIRS injections in an attempt to match the increasing bacterianumbers due to proliferation over the course of infection. F344rats were either treated with 250 �l of IRS once before SCHUS4 challenge or multiple times on days �1, 3, 6, 9, and 12relative to challenge. The total bacterial burden in the lungs,spleen, and liver of rats was determined on days 3, 5, 7, 10, and14 after challenge. As shown in Fig. 7, multiple antibody treat-ments did not alter the general pattern of SCHU S4 growthand dissemination compared to a single treatment (P � 0.05for all three organs).

Taken together, these results showed that a single IRS treat-ment was sufficient to protect F344 rats against i.t. challenge ofup to �700 SCHU S4 organisms. This protection required aminimum IRS volume of 250 �l, and any amount beyond thisvolume threshold given in a single or multiple treatments pro-vided little additional benefit.

Histopathology of serum-treated rats after i.t. challengewith SCHU S4. In order to determine how the IRS-treated ratssurvived a lethal pulmonary SCHU S4 challenge despite an ex-tremely high bacterial burden, we next evaluated whether IRStreatment limited the histopathology in the infected tissues. Lunglesions in both IRS- and NRS-treated rats were first detected onday 3 p.i. and consisted of neutrophilic and histiocytic inflamma-

FIG. 4. Passive transfer of purified LVS-immune IgG, but not IgM, protects naïve rats against a lethal i.t. SCHU S4 challenge. IgG and IgMwere purified from pooled NRS and IRS collected 28 days after s.c. LVS vaccination. The purities of enriched IgG (A) and IgM (B) preparationswere analyzed with SDS-PAGE gels stained with Coomassie blue dye. IRG, immune IgG; NRG, normal IgG; IRM, immune IgM; NRM, normalIgM. The titers of LVS-specific IgM, IgG, and IgA were determined by ELISA as described for Fig. 1, and the titer of contaminating antibodyisotypes in the enriched preparations was 1:100. (C) Groups of five F344 rats were treated with an amount of purified IgG or IgM that wasequivalent to 250 �l of serum and challenged i.t. 1 day later with 810 CFU of SCHU S4. Survival was monitored daily.

FIG. 5. IRS-treated rats exhibit a pattern of SCHU S4 growth intermediate between NRS-treated rats and LVS-vaccinated rats. LVS-vaccinated F344rats and naïve rats treated with 250 �l of heat-inactivated NRS or heat-inactivated IRS were challenged i.t. 1 day after serum treatment with 260 CFUof SCHU S4. Three to four rats were euthanized from each group on days 0, 1, 3, 5, 7, 10, 14, and 21 postchallenge to determine the total SCHU S4burden in the lungs, spleen, and liver. The numbers of lung bacteria on day 0 reflect the actual lung deposition determined within 1 h of infection, andthe dashed lines represent the limit of detection for each organ. Each data point represents the mean standard deviation.

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tion within alveoli, bronchioles, and bronchi and in the perivas-cular spaces of adjacent blood vessels. Over the next 4 days, thelung inflammation became progressively more necrotizing in bothexposure groups. Lung lesion severity was similar in the IRS- andNRS-treated rats during the first 7 days p.i., after which time theNRS-treated rats did not survive. For comparison, the lung le-sions in LVS-vaccinated rats were similar in nature and severity tothe IRS- and NRS-treated rats during the first 5 days p.i. butgradually decreased in severity starting at day 7 p.i.

Lesions in the liver and spleen were first detected in the IRS-and NRS- treated rats on day 3 p.i. Splenic and hepatic lesionsconsisted of multifocal, random neutrophilic and histiocyticinflammation on day 3 p.i. and frequently progressed to necro-tizing inflammation by day 5 p.i. In both the IRS- and NRS-treated rats, maximal hepatic and splenic lesion severity scoreswere achieved on day 5 p.i. However, the maximal severityscores for splenic and hepatic lesions in the IRS-treated ratswere lower than for the NRS- treated rats. Furthermore, thelesion severity decreased for the IRS-treated rats by day 7 p.i.,but not for the NRS-treated rats. For comparison, in the vac-cinated rats, lesions were sparse to nonexistent at all timepoints examined (Fig. 8A to F). These results demonstrate thatIRS treatment reduced inflammation in the tissues to which F.tularensis is known to disseminate.

T cells are critical for antibody-mediated protection. Theimportance of T cells in antibody-mediated protection wasdetermined in T cell-deficient athymic nude rats. Nude rats arederived from a heterogeneous genetic background and havenormal B cell function but an increased NK cell population.Nude rats were naturally more resistant to SCHU S4 infectionand lived 2 to 3 days longer than similarly infected F344 rats.Prophylactic treatment with 250 �l IRS significantly prolongedthe survival of infected nude rats by an additional 3 days (P 0.01), but all infected animals died by day 16 p.i. (Fig. 9A).These results suggested that a T cell-independent mechanismcan temporarily modify the disease process, but T cells arecritical for the long-term protection associated with immuneserum.

To determine the requirement for CD4 and CD8 T cells inpassively immunized F344 rats, we developed a very effective invivo depletion regimen using the anti-CD8 antibody OX-8 andthe anti-CD4 antibody OX-38. These antibody treatment reg-imens reduced and maintained the peripheral blood CD4 andCD8 T cell populations to 5 and 1% of normal levels,respectively, over the course of infection. As shown in Fig. 9B,depletion of CD8 T cells completely abolished the ability ofIRS to protect against SCHU S4 challenge. Depletion of CD4T cells had only a partial effect. This may be related to a slight(2-fold), but not statistically significant, increase in the totalnumber of CD8 T cells in the spleen, liver, and blood; nodifference in B cells (CD45R�) or NK cells (CD161�) wereobserved (data not shown). Indeed, the mortality rate of ratsdepleted of both CD4 and CD8 T cells was similar to theanimals depleted of CD8 T cells alone. Thus, these resultssuggest that CD8 T cells are essential for IRS to protect ratsagainst pulmonary infection with SCHU S4.

DISCUSSION

It is widely accepted that tularemia vaccines such as LVSmust induce a potent cell-mediated immunity to be effectiveagainst highly virulent type A strains of F. tularensis. LVSvaccination also induces a strong antibody response, but itsrole in protection has not been thoroughly addressed. We nowshow that the serum antibodies provided significant protection

FIG. 6. IRS-mediated protection is dose dependent. Groups of F344rats (n � 6) were treated i.p. with the indicated volumes of heat-inacti-vated IRS and 1 day later challenged i.t. with 360 CFU of SCHU S4. Fouradditional groups of rats were treated with equivalent volumes of NRS; allbut one rat treated with 250 �l died by day 10 of infection.

FIG. 7. Multiple IRS treatments have the same effect on SCHU S4 growth kinetics as a single treatment. Naïve F344 rats were either treatedwith a single dose of 250 �l of heat-inactivated IRS 1 day before challenge (day �1) or with multiple 250-�l doses on days �1 and �3, 6, 9, and12. One day after the first treatment, rats were challenged i.t. with 420 CFU of SCHU S4. On the indicated days, the bacterial burdens in the lungs,spleen, and liver were determined from three rats per group. The dashed lines indicate the detection limit for each tissue, and each data pointrepresents the mean standard deviation. There was no significant difference in the bacterial burden between the two groups (P � 0.05).

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against a lethal, respiratory SCHU S4 challenge in the F344 ratpassive immunization model. These results suggest that anti-bodies may be an important component in the overall defenseagainst pneumonic tularemia.

We previously showed that the F344 rat is a good animalmodel for studying human pneumonic tularemia (45). In thepresent study, F344 rats were vaccinated s.c. to reproducehuman vaccination by the scarification method currently used

under the Special Immunization Program at USAMRIID.Similar to humans vaccinated by scarification (16, 44), the s.c.vaccinated rats developed strong IgM and IgG responseswithin a week of vaccination. Further studies showed that onlypurified serum IgG but not IgM mediated protection againsti.t. SCHU S4 challenge in the rat. We have not ruled out thepotential contribution of IgA to protection, especially in thelungs or other mucosal surfaces. In fact, the majority of hu-

FIG. 8. IRS-treated rats develop less severe splenic inflammation than NRS-treated control rats at day 7 p.i. with SCHU S4. The spleens ofLVS-vaccinated (LVS vac), or IRS- and NRS-treated rats were examined histologically at day 7 after SCHU S4 challenge. The spleens ofvaccinated rats were without detectable lesions (A and B). The spleens of the NRS-treated rats exhibited nearly complete effacement of the redpulp with neutrophilic and histiocytic inflammation (C and D), while the spleens of the IRS-treated rats exhibited only multifocal neutrophilic andhistiocytic inflammation in the red pulp (E and F). Arrows point to paler-appearing areas of inflammation in the splenic red pulp.

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mans (16, 44) and mice (26) developed positive serum IgAtiters following scarification or i.n. vaccination, respectively. Theprotective capabilities of IgA have been demonstrated repeatedlyin vaccine studies with IgA-deficient mice, which did not developcomplete resistance against respiratory infections with LVS (2,34) and SCHU S4 (35). A significant amount of IgA was alsofound in the immune mouse serum that passively transferredimmunity against i.n. LVS infection (26). Thus, in addition to IgG,IgA may also play a role in protection against respiratory tulare-mia caused by type A strains.

It has been suggested that the morbidity and mortality as-sociated with pneumonic tularemia are caused by the damageinflicted on the extrapulmonary tissues (8). An extension ofthis idea is that control of bacterial dissemination and growthoutside the lungs would offer considerable survival advantage.Our results are consistent with this idea and suggest that pas-sive immunization modified the course of acute infection byenhancing the innate immune response to disseminated bac-teria. The presence of LVS-specific antibodies appeared to bemost critical during the early phase of infection, because IRSfailed to rescue infected rats when the treatment was delayedby 48 h (data not shown) and increasing the amount of serumor the treatment frequency did not improve the level of pro-tection. IRS treatment had little, if any, quantitative or quali-tative impact on the bacterial burden and the histopathology inthe lungs. Rather, the effects of IRS on bacterial growth weremost clearly observed in the infected liver and spleen. The liveris a major site of F. tularensis colonization and replication (9),and pneumonic tularemia is associated with hepatocellulardamage in multiple experimental animal models, including theF344 rat (8, 32, 42, 45). Although the level of damage fallsshort of liver failure and may be reversible, such damage maynevertheless compromise the liver’s ability to perform essentialmetabolic functions and to utilize its many innate immunemechanisms to control systemic bacterial growth (22, 25). Incontrast, IRS treatment reduced the bacterial burden and his-topathology in the liver and, in doing so, may have preservedmore liver functions in the infected rat. Similar passive immu-nization studies of mice with LVS point to a process thatinvolves Fc receptor and gamma interferon (IFN-�) (26, 36),and the liver is one of the richest sources of NK cells that arecapable of producing an IFN-� in response to F. tularensisinfection (4, 12). This may also explain the ability of immuneserum to delay the death of athymic nude rats and mice (36),

which express higher NK cell activity than wild-type animals(10, 24). By controlling the rapid growth rate and dissemina-tion of SCHU S4 and limiting the pathological damage asso-ciated with infection, IRS may have enabled the host to survivelong enough to develop an effective T cell response that elim-inated the infection. Indeed, LVS-vaccinated rats with both F.tularensis-specific antibodies and immune T cells had the low-est bacterial burden and showed the fewest histologicalchanges following SCHU S4 infection.

T cells are required for the long-term protective effects ofpassive immunization. In the absence of T cells, IRS onlydelayed the death of athymic nude rats by several days. CD8 Tcells appeared to play a more critical role than CD4 T cells,since depletion of CD8 T cells rendered rats completely sus-ceptible to SCHU S4 infection, while only a fraction of the ratsdepleted of CD4 T cells succumbed. A possible explanation forthe partial effect of CD4 T cell depletion is that the level ofdepletion may have been slightly different for each rat and thesurvivors had more residual cells. It is also possible that thechallenge dose varied slightly for each rat and the survivors hadbeen challenged with a lower dose.

With the growing awareness of the importance of antibodiesfor effective immunity against pneumonic tularemia, new vac-cine designs are beginning to incorporate antibodies or ele-ments that induce stronger antibody responses. For example,antibodies against LPS have been used to enhance antigenpresentation by targeting inactivated LVS to Fc receptors onmyeloid cells (34). Cholera toxin B (3) and a LVS O-polysac-charide–tetanus toxoid glycoconjugate (39) were used to in-duce a better antibody response to augment the cellular im-munity generated with inactivated or mutant LVS. Severalgroups have also used protein microarrays (17, 41) and immu-noproteomic approaches (23, 43) to identify immunoreactiveantibodies in serum from humans and mice with previous ex-posure to F. tularensis. In order to further improve these novelvaccine designs and to develop antibodies into potential ancil-lary therapeutic agents, it will be necessary to characterize theprotective response associated with any potential antibody candi-date. The fact that F344 rats were consistently protected by im-mune serum alone suggests that the F344 rat model will be avaluable tool not only to test the protective effect of these anti-bodies but also to characterize their mechanism of protection.

In conclusion, our studies showed that LVS vaccination in-duced serum antibodies that were protective against a lethal

FIG. 9. IRS-mediated protection is dependent on T cells. (A) T cell-deficient athymic (RNu) nude rats and immunocompetent Fischer 344 rats(n � 6 per group) were treated with 250 �l of heat-inactivated IRS or NRS and 1 day later challenged i.t. with 465 CFU SCHU S4. (B) Naïve F344rats (n � 5) were depleted of CD4, CD8, or both CD4 and CD8 T cells by i.p. injection of depleting antibodies OX-38 (CD4) and OX-8 (CD8).T cell depletion was maintained over the course of infection with additional treatments with depleting antibodies. The T cell-depleted rats weretreated with 250 �l of heat-inactivated IRS or NRS and 1 day later challenged i.t. with 810 CFU of SCHU S4. The rats were monitored daily forsurvival and clinical signs of illness.

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respiratory SCHU S4 infection. The protective responses de-fined in these studies provide valuable insights into the mech-anism of antibody-mediated protection and will help guide therational design of novel tularemia vaccines that induce not onlya robust cellular immunity but also a strong humoral immunity.

ACKNOWLEDGMENTS

We thank Jason Zsemyle and Gloria Statom for their excellenttechnical assistance, Ronald Schrader for assistance with statisticalanalysis of bacterial growth kinetics, and Mary F. Lipscomb for criticalreview of the manuscript and advice.

This project was funded with federal funds from the NationalInstitute of Allergy and Infectious Diseases, National Institutes ofHealth, Department of Health and Human Services, under contractno. HHSN266200500040C, and Public Health Service grant PO1AI056295.

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Editor: R. P. Morrison

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