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Clinical and Vaccine Immunology, March 2009, p. 414-422, Vol. 16, No. 3
1071-412X/09/$08.00+0     doi:10.1128/CVI.00362-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Francisella tularensis Infection-Derived Monoclonal Antibodies Provide Detection, Protection, and Therapy {triangledown}

Anne G. Savitt, Patricio Mena-Taboada, Gloria Monsalve, and Jorge L. Benach*

Center for Infectious Disease, Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, New York 11794

Received 2 October 2008/ Returned for modification 24 November 2008/ Accepted 6 January 2009


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ABSTRACT
 
Francisella tularensis is the causative agent of tularemia and a potential agent of biowarfare. As an easily transmissible infectious agent, rapid detection and treatment are necessary to provide a positive clinical outcome. As an agent of biowarfare, there is an additional need to prevent infection. We made monoclonal antibodies to the F. tularensis subsp. holarctica live vaccine strain (F. tularensis LVS) by infecting mice with a sublethal dose of bacteria and, following recovery, by boosting the mice with sonicated organisms. The response to the initial and primary infection was restricted to immunoglobulin M antibody directed solely against lipopolysaccharide (LPS). After boosting with sonicated organisms, the specificity repertoire broadened against protein antigens, including DnaK, LpnA, FopA, bacterioferritin, the 50S ribosomal protein L7/L12, and metabolic enzymes. These monoclonal antibodies detect F. tularensis LVS by routine immunoassays, including enzyme-linked immunosorbent assay, Western blot analysis, and immunofluorescence. The ability of the antibodies to protect mice from intradermal infection, both prophylactically and therapeutically, was examined. An antibody to LPS which provides complete protection from infection with F. tularensis LVS and partial protection from infection with F. tularensis subsp. tularensis strain SchuS4 was identified. There was no bacteremia and reduced organ burden within the first 24 h when mice were protected from F. tularensis LVS infection with the anti-LPS antibody. No antibody that provided complete protection when administered therapeutically was identified; however, passive transfer of antibodies against LPS, FopA, and LpnA resulted in 40 to 50% survival of mice infected with F. tularensis LVS.


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INTRODUCTION
 
Francisella tularensis, a small gram-negative bacterium, has become the subject of intensive research in recent years due to its classification by the Centers for Disease Control as a category A potential agent of biowarfare (47). F. tularensis is a zoonotic agent, the causative agent of tularemia, which can be transmitted through inhalation of aerosolized bacteria, handling of infected animals, arthropod bites, and contaminated water across the northern hemisphere (8, 60).

Four subspecies of F. tularensis have been identified, namely F. tularensis subsp. tularensis, F. tularensis subsp. holarctica, "F. tularensis subsp. novicida," and F. tularensis subsp. mediasiatica (15). Of these subspecies, only two—the highly virulent type A F. tularensis subsp. tularensis, exemplified by the SchuS4 strain ("F. tularensis SchuS4"), and the less virulent type B F. tularensis subsp. holarctica, exemplified by the live vaccine strain ("F. tularensis LVS")—cause human disease, and both are endemic in the United States (15). F. tularensis subsp. holarctica has an intradermal (ID) 50% lethal dose of approximately 103 (12), and F. tularensis SchuS4 has a 50% lethal dose of <50 organisms (39). F. tularensis LVS, a derivative of F. tularensis subsp. holarctica, is attenuated for humans (reviewed in reference 46) but provides an effective murine model (25). The mechanism of attenuation for F. tularensis LVS remains to be defined (46), and F. tularensis LVS does not protect against exposure to large respiratory doses of the highly virulent type A strains (10).

Monoclonal antibodies (MAbs) are powerful tools for both diagnostics and therapeutics. The best source of antibody targeting an infectious agent is a natural infection (5, 41). Rapid antibody-based assays enable clinicians to quickly diagnose and treat infectious diseases, while humanized antibodies and antibody derivatives such as single-chain variable fragments may be useful in the treatment of infectious diseases by directly targeting the microorganism or targeting infected cells for delivery of toxic agents (7).

Passive antibody transfer provides immediate immunity (6), with the advantages of low toxicity and high specificity (7). Passive protection against tularemia has been demonstrated for a long time (18). Transfer of peritoneal leukocytes and serum from immune mice into naïve mice resulted in survival of 10% of the mice when challenged with fully virulent F. tularensis SchuS4; rechallenged 6 weeks later, all the surviving mice died (1). However, more than 40 years later, there have been only a few studies that investigated the ability of passively transferred antibodies to protect against infection, with results demonstrating that immune serum may be protective in the presence of a coordinated host response (21, 30, 52, 53).

In this study, we sought to identify bacterial antigens that induce a natural antibody response in mice. To accomplish this, mice were infected with a sublethal dose of F. tularensis LVS, followed by a boost with sonicated organisms. Spleen cells were fused to murine myelomas to produce antibody-secreting hybridomas. We obtained MAbs that are useful as diagnostic, therapeutic, or research tools, as well as identified antigens that may contribute to the efficacy of a multiantigen recombinant vaccine.


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MATERIALS AND METHODS
 
Reagents. Lipopolysaccharides (LPS) from Escherichia coli, Salmonella typhosa, and Salmonella enterica serovar Enteritidis were obtained from Sigma-Aldrich (St. Louis, MO). LPS from F. tularensis subsp. tularensis strain SchuS4 (type A) and F. tularensis subsp. holarctica strain 1547 (a type B clinical isolate) were provided by Martha Furie, Center for Infectious Disease, Stony Brook University. LPS from F. tularensis LVS, F. tularensis SchuS4, and Francisella tularensis subsp. novicida were purified by the hot phenol method and analyzed by silver staining of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, essentially as described for LPS from F. tularensis LVS (4).

Mice. All mice were purchased from Charles River Laboratories (Wilmington, MA) and were maintained in the facility of the Division of Laboratory Animal Resources at Stony Brook University. All animal procedures were approved by the institutional animal care and use committee.

Culture of F. tularensis LVS and F. tularensis SchuS4. F. tularensis LVS (ATCC 29684; Manassas, VA) was grown in broth culture as previously described (16). Briefly, frozen stocks were prepared from bacteria grown to mid-log phase in Mueller-Hinton broth (BD Biosciences, San Jose, CA) supplemented with 2% IsoVitaleX enrichment (BD Biosciences), 0.1% glucose, 63 mM CaCl2, 53 mM MgCl2, and 34 mM ferric pyrophosphate. An experimental stock plate was made by streaking a freshly thawed vial of bacteria on a chocolate agar plate (BD Biosciences). The bacteria were allowed to form colonies by growing for 2 to 3 days in an incubator at 37°C and 5% CO2. For infection experiments, a single colony was inoculated into supplemented Mueller-Hinton broth and grown to late log phase for 16 to 18 h at 37°C with shaking at 100 rpm in a 5% CO2 atmosphere. The number of viable bacteria in the suspensions was determined by plating 10 µl of serial dilutions onto Mueller-Hinton II agar plates and counting colonies 3 days later. F. tularensis SchuS4 was cultured following the same protocol as described above for F. tularensis LVS under BSL3 conditions at the Public Health Research Institute of the New Jersey Medical School, Newark, NJ.

Preparation of sonicated F. tularensis LVS. Approximately 1 x 1011 CFU of F. tularensis LVS were harvested from 500 ml of broth culture by centrifugation at 1,500 x g for 10 min at 4°C in a GSA rotor in a Sorvall RC5B refrigerated centrifuge. The pellet was washed three times with Dulbecco's phosphate-buffered saline (PBS; Gibco/Invitrogen, Carlsbad, CA), followed by centrifugation at 1,500 x g for 10 min. Following the final wash, the pellet was resuspended in 4 ml of 20 mM Tris-Cl (pH 8.0) with a protease inhibitor cocktail (Roche Applied Sciences, Indianapolis, IN). The bacteria were sonicated on ice for a 30-s pulse, followed by a 30-s rest, five times using a Microson XL ultrasonic cell disruptor (Misonix, Farmingdale, NY) at a power of 7 W. Protein concentration of the sonicate was determined by the Bradford protein assay (Bio-Rad Laboratories, Hercules, CA).

Generation of MAbs. Female BALB/c mice (6 to 8 weeks old; Charles River) were ID infected with a sublethal dose of F. tularensis LVS (105 CFU per mouse). Following recovery from the infection, sera from mice were tested by enzyme-linked immunosorbent assay (ELISA) for the presence of antibodies to F. tularensis LVS sonicate. Mice selected for splenectomy had a titer ratio of >1:1,000 to F. tularensis LVS sonicate. Prior to fusion, the mouse was boosted intraperitoneally with 0.28 mg of F. tularensis LVS sonicate. Four days following the booster, the mouse was sacrificed, and the spleen cells were isolated aseptically and fused with mouse myeloma cell line Sp2/0 (ATCC) at a ratio of five spleen cells to one myeloma cell by pelleting them together at 150 x g for 5 min in an IEC CR-6000 centrifuge in Dulbecco's minimal essential medium (DMEM; Invitrogen) containing penicillin and streptomycin (Invitrogen). Following aspiration of the medium, the cells were resuspended in 35% polyethylene glycol 1500 (Roche) in DMEM medium and centrifuged at 150 x g for 5 min. The polyethylene glycol was aspirated, and the fused cells were suspended in DMEM Glutamax (Invitrogen) supplemented with 15% fetal clone I serum (HyClone, Logan, UT), 10% NCTC109 (Invitrogen), nonessential amino acids (Invitrogen), penicillin and streptomycin, HAT buffer (10–4 M hypoxanthine, 4 x 10–7 M aminopterin, 1.6 x 10–5 M thymidine; Sigma), and 10% macrophage conditioned medium (44, 54) and distributed into 10 96-well plates. Half of the medium was replaced 3 days postfusion and again the day before screening.

Identification of MAbs. Hybridomas were screened by ELISA against F. tularensis LVS sonicate. Wells exhibiting both a positive response by ELISA and positive cell growth were grown up to 30 ml in culture, and three 10-ml aliquots were pelleted and resuspended in freezing medium (10% dimethyl sulfoxide, 90% fetal clone I) for cryostorage. ELISA-positive samples were screened by SDS-PAGE/Western blotting against F. tularensis LVS solubilized in Laemmli buffer, using a Miniblotter 45 (Immunetics) to screen 45 samples per blot. Hybridomas reacting with a specific protein antigen were selected for subcloning by limiting dilution. Subclones were screened by ELISA and Western blotting; positive subclones were expanded and cryopreserved as described above. Isotypes were determined using the MAb isotyping kit (Thermo Scientific, Waltham, MA).

Purification of MAbs. Hybridomas were grown in 70% animal-derived component-free medium (HyClone)-30% DMEM supplemented with nonessential amino acids and inoculated into CELLine CL1000 flasks (Integra Biosciences, Fernwald, Germany), and antibody-containing supernatants were harvested according to the manufacturer's instructions. Antibody was purified from the supernatants on Montage antibody spin columns with Prosep-G media (Millipore, Billerica, MA), following the manufacturer's instructions. The resulting samples were desalted and concentrated using an Amicon Ultra-15 centrifugal filter device with a 30,000-molecular-weight cutoff (Millipore) and resuspended in PBS. The protein yield was determined by optical density on a NanoDrop ND1000 spectrophotometer (NanoDrop, Wilmington, DE). The protein concentration was adjusted to 2 mg/ml in PBS, and the MAbs were stored at –20°C in 0.5-ml aliquots.

ELISA. F. tularensis LVS sonicate was applied to Maxisorp U-bottomed 96-well plates (Nunc) at 3.75 µg/ml in 0.1 ml coating buffer (10 mM Na2CO3, 10 mM NaHCO3 [pH 9.6]) overnight at 4°C. The wells were blocked for 1 h at room temperature (RT) with filter-sterilized PBS containing 0.05% Tween 20 (Sigma) and 1% bovine serum albumin (BSA; Fraction V, Sigma) (PBST-BSA); incubated for 2 h at 37°C with primary antisera diluted in PBST-BSA or undiluted hybridoma supernatants; washed with PBST; incubated for 1 h at 37°C with secondary antibody (goat anti-mouse IgG, Fc-specific, alkaline phosphatase conjugate; Jackson ImmunoResearch, West Grove, PA), diluted 1:2,000 in PBST-BSA; and washed with PBST followed by distilled H2O. Alkaline phosphatase substrate para-nitrophenyl phosphate (Sigma) in substrate buffer (10% diethanolamine, 1.5 mM MgCl2 [pH 9.6]) at 1 mg/ml was applied at 0.05 ml/well, and color development allowed to proceed for 1 h at 37°C. Results were evaluated by reading absorbance at 405 nm in an MRX microplate reader (Dynatech, Chantilly, VA).

SDS-PAGE/Western blotting. F. tularensis LVS harvested from broth culture was resuspended in Laemmli buffer with β-mercaptoethanol and boiled at 95°C for 5 min. Antigens were separated on a large format 10% SDS polyacrylamide gel and transferred to 0.22 µm PVDF-Plus (polyvinylidene difluoride) membranes (Osmonics, Inc.), essentially as described previously (56). The membrane was blocked overnight in 5% powdered milk in PBS and then inserted into a Miniblotter 45 (Immunetics, Boston, MA). Primary antibodies (undiluted hybridoma supernatants or parent mouse sera diluted 1:1,000 in PBST-1% BSA) were applied to the channels of the miniblotter, and the membrane was incubated for 2 h on a rocking platform at RT. After extensive washing with PBST, alkaline phosphatase-conjugated Fc-specific goat anti-mouse IgG secondary antibody (Jackson ImmunoResearch) was applied at a dilution of 1:2,000 in PBST-BSA, followed by incubation for 1 h on a rocking platform at RT. The membrane was then extensively washed first with PBST and then with distilled H2O and developed using nitroblue tetrazolium-BCIP (5-bromo-4-chloro-3-indolylphosphate) substrate (Roche).

Immunofluorescence assays. Murine macrophage-like cells J774A.1 (ATCC) were seeded onto 12-mm coverslips in a 24-well plate at 3.7 x 105 cells/ml of DMEM containing 10% heat-inactivated fetal bovine serum (HyClone) and plated at 1 ml per well the day before infection. The cells were infected with F. tularensis LVS at a multiplicity of infection of 10 CFU/cell, treated with 5 µg/ml gentamicin to kill extracellular bacteria, and incubated for a total of 20 h, essentially as described previously (16). For fluorescent microscopy, the cells were washed and fixed in 2.5% paraformaldehyde for 30 min at RT and then permeabilized with 0.5% Triton X-100 (Sigma) in PBS for 5 min at 37°C. The cells were blocked with 3% BSA in PBS for 5 min at 37°C, probed for 30 min at RT with MAbs in undiluted tissue culture supernatants and rabbit polyclonal antisera against F. tularensis LVS (43) diluted 1:100 in PBS-3% BSA or in MAb tissue culture supernatants for double-labeling experiments, and detected with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse secondary antibody or tetramethyl rhodamine isothiocyanate (TRITC)-conjugated goat anti-rabbit secondary antibody (Jackson ImmunoResearch) for 30 min at RT. Following washing with PBS-3% BSA, coverslips were mounted on slides by using Vectashield plus DAPI (4',6-diaminidino-2-phenylindole) mounting medium (Vector Labs, Burlingame, CA) and visualized on an inverted/DIC Zeiss Axiovert 200M microscope equipped with an AxioCam HRm camera (Zeiss, Thornwood, NY) and mercury arc lamp light source using a 63x Plan-Apochromat (numerical-aperture 1.40 oil objective) and operated using Axiovision version 4.5 (Zeiss) software. The following excitation and emission wavelengths were used for imaging: for DAPI, {lambda}ex of 360 ± 20 nm, {lambda}em of 460 ± 25 nm; for FITC, {lambda}ex of 480 ± 20 nm, {lambda}em of 535 ± 25 nm; and for Texas Red (for TRITC), {lambda}ex of 560 ± 25 nm, {lambda}em of 645 ± 35 nm.

Immunoprecipitation. To 500 µg of total sonicated F. tularensis LVS protein in 0.5 ml of NET-gel buffer (50 mM Tris-Cl [pH 7.5], 150 mM NaCl, 0.1% NP-40, 1 mM EDTA, 0.25% gelatin) (49), 1 µl of hybridoma supernatant or 1 µg of isotype control (BioLegend, San Diego, CA) was added, and the samples were rocked for 1 h at 4°C on ice. Protein G Sepharose beads (Zymed, San Francisco, CA) were prewashed in PBS, and 50 µl of beads at 50% in PBS was added to each sample. Following incubation for 1 h at 4°C on ice with rocking, the samples were spun for 20 s at RT and washed three times with 1 ml cold PBS, each wash incubating for 20 min at 4°C on a rocking platform. Following a 5-s centrifugation, the supernatant was aspirated, the beads were resuspended in 20 µl of 2x SDS-gel loading buffer containing ß-mercaptoethanol, and the samples were heated for 5 min at 95°C. The samples were centrifuged for 2 min to remove the beads, and the samples were loaded onto duplicate gels for Sypro Ruby staining (Invitrogen) and Western blotting. Sypro Ruby staining was performed following the manufacturer's instructions.

Protein identification. Antigen bands identified by Western blotting were excised from Sypro Ruby-stained SDS polyacrylamide gels. Proteolytic degradation, high-performance liquid chromatography separation (LCPackings, Bannockburn, IL), and liquid chromatography/tandem mass spectrometry analysis (API Qstar Pulsar I; Applied Biosystems, Foster City, CA) were performed by the Proteomics Center of the School of Medicine, Stony Brook University. Masses obtained from the mass spectroscopy analysis were subjected to database searches, while internal peptide sequences were aligned using a protein-to-protein Basic Local Alignment Search Tool, generating a probability-based Mowse score, where the score is –10 · log(P) (P is the probability that the observed match is a random event).

Protection assays. Six- to eight-week-old female C3H/HeN mice were used for all protection assays. Mice were inoculated with 50 µg of purified MAb in 200 µl PBS per dose for a series of three daily doses per mouse on the days described in the figure legends. Experiments with F. tularensis SchuS4 were conducted under animal P3 conditions at the Public Health Research Institute of the New Jersey Medical School, Newark, NJ. Mice were challenged ID with 7 x 107 CFU of F. tularensis LVS or 24 CFU of F. tularensis SchuS4 on day 0 and monitored daily for survival. Surviving mice were monitored for 3 months. Survival curves were analyzed by the Kaplan-Meier log-rank test, using GraphPad Prism software version 4.00 for Macintosh (GraphPad Software, San Diego, CA).

Organ burden assays. Mice were inoculated and infected with F. tularensis LVS as described in "Protection assays" above. On days 1, 4, 8, and 12, mice were sacrificed and their blood, livers, and spleens harvested. Livers and spleens were homogenized in PBS in sterile plastic stomacher bags (VWR, West Chester, PA). Blood and homogenates of livers and spleens were subjected to serial dilutions, and 10 µl of each dilution was plated onto chocolate agar plates and incubated at 37°C in a 5% CO2 atmosphere for 2 days, following which colonies were counted.


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RESULTS
 
Following recovery from sublethal infection, boosting with heat-killed organisms results in a monotypic IgM response to LPS. To derive hybridomas secreting MAbs representative of the natural murine response to infection with F. tularensis LVS, mice were infected ID with a sublethal dose of F. tularensis LVS and allowed to recover, and samples of their sera were tested against sonicated organisms ("F. tularensis LVS sonicate"). A mouse with a high titer ratio (>1:1,000) was selected for splenic fusion and boosted with heat-killed organisms. This resulted in the generation of hybridomas producing immunoglobulin M (IgM) antibodies only to LPS (data not shown). To broaden the antibody response to include different Ig classes and to include peptide antigens of F. tularensis in the response, mice recovering from sublethal infection were boosted with F. tularensis LVS sonicate. Of the 183 ELISA-positive hybridomas screened against total F. tularensis LVS proteins by immunoblotting, 177 yielded the LPS ladder typical of gram-negative bacteria, and many reacted with protein antigens. Selected hybridomas were subcloned by limiting dilution to obtain IgG class MAbs that recognized specific protein antigens as well as LPS.

The subcloned MAbs were used to immunoprecipitate their cognate antigens, which were then separated by SDS-PAGE, and the bands were excised and submitted for analysis by mass spectrometry. The results were screened against the F. tularensis LVS and F. tularensis SchuS4 genomic databases, and the protein antigens were unambiguously identified. Table 1 lists the MAbs, their isotypes and cognate antigens, as well as the probability-based Mowse scores and numbers of peptides identified following mass spectrometry analysis. Each protein was unambiguously identified based on having a Mowse score of >300, with the exception of LpnA. However, the identity of LpnA as the cognate antigen to MAb 164 was confirmed by Western blotting against an LpnA knockout strain of F. tularensis LVS (17). Consistent with previous reports, the predominant isotype of these MAbs is IgG2a (45).


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  		TABLE 1. List of MAbs and isotypes and antigen identification

Specificity of anti-LPS IgG MAb. The LPS of F. tularensis is different from other gram-negative bacteria both structurally (36) and immunologically (4, 11, 36, 42, 58). In fact, among Francisella spp., the LPS of F. tularensis LVS and the LPS of F. tularensis SchuS4 are identical, while that of F. tularensis subsp. novicida is different (36). In immunoblot assays, the anti-LPS MAb detects LPS from Francisella strains F. tularensis SchuS4 (type A) and F. tularensis 1547 (type B) but does not detect LPS from F. tularensis subsp. novicida (Fig. 1A). This antibody does not detect LPS from other gram-negative bacteria such as E. coli, S. enterica serovar Enteritidis, and S. typhosa (Fig. 1B).


Figure 1
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  		FIG. 1. Specificity of anti-F. tularensis LVS LPS MAb. Purified LPS from all strains were subjected to SDS-PAGE, transferred to nitrocellulose filters, and probed with MAb against the LPS of F. tularensis LVS. (A) F. tularensis subsp. holarctica strain LVS (lane 1), F. tularensis subsp. tularensis strain SchuS4 (lane 2), F. tularensis subsp. holarctica strain 1547 (lane 3), F. tularensis subsp. novicida (lane 4), and F. tularensis subsp. holarctica strain LVS (lane 5). (B) F. tularensis LVS (lane 1), E. coli (lane 2), S. enterica serovar Enteritidis (lane 3), and S. typhosa (lane 4).

MAbs are species specific to antigens of F. tularensis. Anti-F. tularensis LVS MAbs detect the orthologous antigens in lysate of F. tularensis SchuS4 (Fig. 2). With the exception of anti-Bfr, all of the antibodies react with the F. tularensis SchuS4 antigens. However, when tested against extracts of E. coli, Borrelia burgdorferi, and Yersinia pseudotuberculosis (Fig. 3), all of the MAbs are specific for the F. tularensis LVS proteins, except for anti-DnaK, which cross-reacts with the DnaK of E. coli but not with that of B. burgdorferi or Y. pseudotuberculosis.


Figure 2
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  		FIG. 2. MAbs to F. tularensis LVS recognize orthologous antigens from Francisella sp. strains. Lysates from F. tularensis LVS (lanes designated "L") and F. tularensis SchuS4 (lanes designated "S") were subjected to SDS-PAGE, transferred to nitrocellulose filters, and probed with anti-F. tularensis LVS MAbs against the proteins indicated at the top of each pair of lanes. (A) 10% SDS-PAGE. (B) 20% SDS-PAGE.


Figure 3
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  		FIG. 3. Specificity of anti-F. tularensis LVS MAbs. Lysates from F. tularensis LVS (lanes 1), E. coli (lanes 2), Y. pseudotuberculosis (lanes 3), and B. burgdorferi (lanes 4) were subjected to SDS-PAGE, transferred to nitrocellulose filters, and probed with anti-F. tularensis LVS MAbs against the proteins indicated at the top of each set. (A) 10% SDS-PAGE. (B) 20% SDS-PAGE.

MAbs can detect intracellular F. tularensis. MAbs were tested for their ability to identify intracellular bacteria in J774A.1 cells. The cells were probed with each MAb in combination with rabbit anti-F. tularensis LVS sera, followed by FITC-labeled goat anti-mouse IgG and TRITC-labeled goat anti-rabbit IgG secondary antibodies. Controls including uninfected cells, cells treated with each antibody alone, and cells treated with only secondary antibodies were negative (data not shown). The rabbit antibody (TRITC-labeled) identifies the infected cells, while the merged (yellow) images confirm the colocalization of the rabbit antibody with each of the MAbs (Fig. 4).


Figure 4
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  		FIG. 4. Immunofluorescence assay of F. tularensis LVS-infected, Triton X-100-permeabilized J774A.1 cells. Murine macrophage cells, J774A.1, were seeded onto coverslips and infected with F. tularensis LVS at a multiplicity of infection of 10 CFU/cell. After 20 h, the cells were fixed with paraformaldehyde, permeabilized with Triton X-100, and probed with MAbs and rabbit polyclonal antisera against F. tularensis LVS antigens. Secondary detection was by FITC-labeled antimouse and TRITC-labeled goat anti-rabbit antibodies. Nuclei of cells were visualized with DAPI.

Protection assays. Two procedures were used to determine whether these MAbs resulting from a natural infection can protect mice when administered prophylactically or therapeutically. When antibodies were administered prophylactically in three daily 50-µg doses per mouse on days –1, 0, and +1, with the mice challenged on day 0 with ID administration of 7 x 107 CFU of broth-grown F. tularensis LVS, anti-LPS conferred 100% protection from infection, while anti-FopA protected 90% of the mice (Fig. 5A). The other MAbs were not protective under these conditions, nor at an increased dosage of 200 µg/mouse (data not shown). Organ burden assays performed on mice treated under the same set of conditions, i.e., three daily 50-µg doses per mouse on days –1, 0, and +1 to each mouse with challenge on day 0 of ID administration of 7 x 107 CFU of broth-grown F. tularensis LVS, showed that bacteria were rapidly cleared from the liver and spleen of mice protected with anti-LPS, while bacteria from the liver and spleen of the mice treated with anti-FopA and the PBS-receiving control mice were not cleared. However, the anti-FopA-treated mice recovered, while the PBS-receiving control mice died (Fig. 5B). Prophylactic experiments mixing the anti-FopA MAb with anti-DnaK, anti-SucC, or anti-Bfr MAbs did not increase the efficacy of the anti-FopA antibody (data not shown).


Figure 5
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  		FIG. 5. MAb prophylaxis (F. tularensis LVS). Survival and organ burden of bacteria in mice inoculated with anti-F. tularensis LVS MAbs and infected with F. tularensis LVS. (A) C3H/HeN mice were inoculated with three daily doses of 50 µg of anti-LPS, anti-FopA, or PBS per mouse on days –1, 0, and +1, respectively, in groups of three or four mice, for a total of 13 mice per MAb or control; they were challenged on day 0 with an ID inoculation of F. tularensis LVS of 7 x 107 CFU per mouse. Survival was monitored daily, and survival curves were analyzed using the Kaplan-Meier log-rank test of GraphPad Prism 4. (B) Three C3H/HeN mice per time point, per MAb or control, were inoculated and infected as described for panel A. On days 1, 4, 8, and 12 postinfection, the surviving mice were sacrificed, their organs were harvested, and organ burden was enumerated by plating serial dilutions of homogenized organs onto chocolate agar plates.

Under the same conditions as those described for F. tularensis LVS, mice were administered the same antibodies prophylactically and then challenged on day 0 with 24 CFU of broth-grown F. tularensis SchuS4. In these experiments, the mice receiving PBS or anti-FopA rapidly succumbed to infection. The mice that received the anti-LPS, however, survived significantly longer before succumbing to the infection (Fig. 6).


Figure 6
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  		FIG. 6. MAb prophylaxis (F. tularensis SchuS4). Survival of mice inoculated with anti-F. tularensis LVS MAbs and infected with F. tularensis SchuS4. C3H/HeN mice were inoculated with anti-LPS, anti-FopA, or PBS, as described in the legend to Fig. 5; they were infected with F. tularensis SchuS4 at a multiplicity of infection of 24 CFU per mouse on day 0 in groups of 10 mice. Survival was monitored daily.

When MAbs were administered postchallenge, or therapeutically, in three daily 50-µg doses on days 1, 2, and 3 postinfection, none of the MAbs conferred complete protection. However anti-LpnA, anti-FopA, and anti-LPS significantly increased the survival of the mice (Fig. 7).


Figure 7
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  		FIG. 7. MAb therapeutics. Survival of mice infected with F. tularensis LVS and then treated with MAbs on days 1, 3, and 5 postinfection. C3H/HeN mice were infected ID with F. tularensis LVS at 7 x 107 CFU per mouse on day 0 and then treated with three daily doses of 50 µg of anti-LPS, anti-FopA, or anti-LpnA MAb or PBS per mouse on days 1, 3, and 5 postinfection. The mice were assessed daily for survival.


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DISCUSSION
 
We have identified a panel of MAbs to antigens—LPS, FopA, LpnA, DnaK, SucB, SucC, Bfr, and RplL—from F. tularensis LVS. All of these antibodies are useful research and diagnostic tools because they detect F. tularensis LVS in a variety of immunoassays, including ELISA, Western blotting, immunoprecipitation, and immunofluorescence. They also recognize the orthologous antigens from the virulent F. tularensis SchuS4, while distinguishing them from other bacteria. Two of the MAbs protect naïve mice from lethal challenge when administered prophylactically; three provide significant protection when administered therapeutically.

The ability of F. tularensis LVS LPS to induce protective immunity has been examined. Immunization with purified F. tularensis LVS LPS provided protection against lethal challenge with F. tularensis LVS (19, 20) but not F. tularensis SchuS4, although LPS-immunized mice boosted with F. tularensis LVS survived challenge with F. tularensis SchuS4 (21). The MAb to LPS completely protects mice against ID challenge with F. tularensis LVS, and not only do all of the mice survive, but there are virtually no bacteria detectable in the organs as early as 1 day postinfection.

Each of these MAbs recognizes a different protein antigen, and each of these antigens is a target for the murine or human antibody response to F. tularensis, suggesting that these antigens could contribute to the development of a subunit vaccine. Several groups have recently demonstrated that FopA (27, 28, 37), LpnA (27, 37), DnaK (27, 28, 37), SucB (28, 37, 40), Bfr (37, 40), and RplL (28) are components of the bacterial membrane. All six of these antigens were identified as reactive with serum from human tularemia patients (28) and five of them (all except Bfr) with murine serum (57). DnaK, SucC, and Bfr were among the proteins identified as being released into culture medium, and Bfr was shown to significantly stimulate T cells and production of gamma interferon (32). LpnA was also shown to stimulate T cells (24, 50), while FopA, LpnA, DnaK, and SucB were among the immunodominant antigens identified by protein microarray (14).

Two of the antigens identified by our MAbs, FopA and LpnA, are unique to Francisella. A comparison of the sequences of these antigens of the attenuated F. tularensis LVS and the virulent F. tularensis SchuS4 reveals that there is greater than 99% sequence identity for both proteins. Therefore, it is not surprising that the MAbs recognize these antigens from both strains. These two proteins were among the earliest antigens to be identified and have been studied as potential vaccine immunogens (19, 20, 50, 51, 62). Both proteins were expressed on the surface of S. enterica serovar Typhimurium (20, 51), and in both instances, partial protection resulted, much of which was attributed to the cross-reactivity between the surface antigens of S. enterica serovar Typhimurium and those of F. tularensis.

The five other antigens identified by our MAbs—DnaK, SucB, SucC, Bfr, and RplL—are not individually protective in passive transfer experiments. These proteins have all been identified as immunogens in other bacterial systems, and some of them have provided protection when delivered in a vaccine (2, 29, 33-35, 60a).

DnaK, also known as heat shock protein 70, has long been recognized as a chaperone protein, in both prokaryotic and eukaryotic systems, aiding in the proper folding of proteins during biosynthesis and unfolding misfolded proteins when necessary (22, 59). In F. tularensis, DnaK expression increases in response to heat and hydrogen peroxide (13); the DnaK molecular chaperone system of F. tularensis, first cloned in 1995, cross-reacts with polyclonal antisera raised against E. coli DnaK (61). Our anti-DnaK MAb 193 does not cross-react with DnaK from B. burgdorferi or Y. pseudotuberculosis but, like the polyclonal antisera, cross-reacts with E. coli DnaK.

Two components of the 2-oxoglutarate dehydrogenase complex have proven immunogenic—dihydrolipoamide succinyl transferase (SucB; MAb 195) and the beta subunit of succinyl coenzyme A synthetase (SucC; MAb 121). Both SucB and SucC have recently been determined to be immunogenic in organisms such as Bartonella spp. (23, 33), Brucella spp. (55, 62), and Coxiella burnetii (38), and SucC is considered a diagnostic target as well as a potential vaccine candidate in Leptospira spp. (48).

Bacterioferritin (Bfr) is the cognate antigen of MAb 163. An inorganic ion transport and metabolic protein, Bfr strongly stimulates T cells isolated from F. tularensis LVS-infected mice (32). This immunogenic protein has been shown to provide low but significant protection in a Brucella sp. vaccine, utilizing Yersinia enterocolitica as the delivery vehicle (2), and in a naked DNA vaccine (3).

The 50S ribosomal protein L7/L12 (RplL) is recognized by MAb 109. Although MAb 109 is not protective in our passive immunity assays, this antigen has been successfully used in vaccines for Brucella abortus (14, 34, 35). More importantly, however, mucosal immunization with the Streptococcus pneumoniae RplL protein provided 99% clearance of pulmonary infection within 5 hours following challenge (29).

Of critical interest for the defense against biological weapons, of course, is the issue of rapid response following exposure to a biological agent. While the laboratory and environmental strains of F. tularensis are sensitive to a broad range of antibiotics, it can be postulated that a strain engineered for biowarfare would be resistant to those antibiotics. Therefore, in the absence of an effective and widely available vaccine, it must be assumed that the only therapy that can provide immediate immunity against a biological agent is the passive administration of antibody (6), which may be combined with related therapies, such as adoptively transferred immune cells or growth factors and interferons (26). Interest in passive or adoptive transfer of immunity against F. tularensis has been building upon a foundation of studies of innate and humoral immunity in F. tularensis infection. Early work on F. tularensis demonstrated the efficacy of transferring immune peritoneal leukocytes or spleen cells to naïve mice to provide protection against the virulent SchuS5 strain (a streptomycin-resistant version of the now well-studied SchuS4 strain) (1). It has also been shown that both serum and spleen cells transferred from F. tularensis LVS-immunized mice, administered at the time of challenge, protect mice from lethal challenge (45). Likewise, IgG antibodies purified from immune serum provide protection which requires gamma interferon and T cells (45), and serum from mice immunized with LPS, administered 2 hours before challenge, is protective against intraperitoneal challenge with F. tularensis LVS (21). Immune serum is also protective against intranasal infection with F. tularensis LVS both when administered preinfection and as much as 48 h postinfection and every 3 days thereafter (30).

The therapeutic (postinfection) protective effect of anti-LPS, anti-FopA, and anti-LpnA is significant but incomplete. In summary, we have developed MAbs against a panel of F. tularensis LVS antigens consisting of LPS, LpnA, FopA, DnaK, SucB, SucC, RplL, and Bfr. These antibodies are specific for Francisella antigens from both F. tularensis LVS and F. tularensis SchuS4 (with the exception of DnaK, which cross-reacts with E. coli DnaK), and they detect F. tularensis LVS in numerous immunoassays. Two of the antibodies, anti-LPS and anti-FopA, protect naïve mice against ID infection with F. tularensis LVS when administered prophylactically, and anti-LPS protects naïve mice against ID infection with F. tularensis SchuS4. These two antibodies plus anti-LpnA provide significant, but incomplete, protection against ID infection when administered therapeutically.

From the perspective of defense against a potential agent of biowarfare, this work demonstrates that examining the murine antibody response to infection with F. tularensis LVS yielded information about the immunogenicity of F. tularensis LVS antigens and their potential efficacy in a recombinant multiantigen vaccine. Further, the ability of the anti-LPS MAb to extend time to death from challenge with F. tularensis SchuS4 suggests that LPS may be a valuable adjunct to a vaccine.


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ACKNOWLEDGMENTS
 
This work was supported by National Institutes of Health grant P01 AI055621 (to J.L.B.) and the Northeast Biodefense Center grant U54 AI 057158-Lipkin.

We thank the Proteomics Center of the Stony Brook University School of Medicine for assistance with mass spectroscopy, the Public Health Research Institute (PHRI) at the International Center for Public Health, New Jersey Medical School—UMDNJ for conducting the SchuS4 experiments, and Rebecca Rowehl of the Cell Culture/Hybridoma Facility at Stony Brook University School of Medicine for assistance with the hybridoma clones.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Molecular Genetics and Microbiology, State University of New York at Stony Brook, Stony Brook, NY 11794-5120. Phone: (631) 632-4225. Fax: (631) 632-4294. E-mail: jbenach{at}notes.cc.sunysb.edu Back

{triangledown} Published ahead of print on 28 January 2009. Back


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Clinical and Vaccine Immunology, March 2009, p. 414-422, Vol. 16, No. 3
1071-412X/09/$08.00+0     doi:10.1128/CVI.00362-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



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