Bacterial strain, plasmid construction, cultivation, and induction
For F1-VMN the E. coli strain BLR130 and the F1-V expression vector, pPW731 (USAMRIID), controlled under a T7 promoter, were used for F1-V expression [6
]. A growth medium of soytone, yeast extract, and glucose (J.T. Baker, Phillipsburg, NJ) and the antibiotics kanamycin (30 mg/L) and tetracycline (15 mg/L; Sigma, St. Louis, MO) were used in phosphate buffer, pH ~7.3. Sterile medium in shaker flasks (300 ml) was inoculated with 1 ml of the strain from a previously made glycerol stock and incubated for ~13 h at 37 °C with shaking at 220 rpm. Batch cultivations were carried out in a Bioflo 4500 (New England Biolabs, Ipswich, MA) equipped with a 15-L vessel and 10-L working volume. Growth medium (9.7 L) was inoculated with 300 ml of seed culture. The dissolved oxygen concentration was maintained above 15% air saturation at 37 °C by controlling the aeration and agitation rates through BIOCOMMAND software (New England Biolabs). Solution pH was kept between 7.2 and 7.4 by adding 0.1 N HCl or 30% NH4OH. After 3.5 h, the culture was induced with IPTG (1 mM) and harvested 2 h later by centrifugation. Cell paste aliquots were stored below –70 °C.
S, TOP10, BL21 (DE3), and BL21 Star (DE3) E. coli strains were from Invitrogen (Carlsbad, CA). BL21 Star cells carried a mutated rne gene that encoded a truncated RNase E protein lacking the capacity to degrade mRNA and leading to increased mRNA stability and enhanced protein expression. The F1-V pET24a(+) Cys425
expression plasmid (F1-VC42
4S) was prepared by site-directed mutagenesis of the original cysteine-containing caf1-lcrV gene
fusion (expressing F1-VSTD
) on source plasmid F1-VSTD
pET-24a (pPW731) [6
]. Site-directed mutagenesis was performed with the Quick-change site-directed mutagenesis kit (Stratagene, La Jolla, CA). Complementary mutagenic primers F1-V-CS-F (5′ - CT CAC TTT GCC ACC ACC TCC TCG GAT AAG TCC AGG CCG C - 3′) and F1-V-CS-R (5′ – GCG GCC TGG ACT TAT CCG AGG AGG TGG TGG CAA AGT GAG – 3′) were constructed with consideration of primer length (39 bp), % GC (59%), and melting temperature (85.8 °C). Each primer (125 ng) was combined with 50 ng of F1-V pET-24a (pPW731), along with the additional reaction chemistry as recommended by the manufacturer. Cycling parameters for the mutagenesis reaction included one cycle of 95 °C for 30 sec, followed by 12 cycles of 95 °C melting for 30 sec, 53 °C annealing for 1 min, and 68 °C extension for 7 min, concluding with a 4 °C hold. The mutagenesis reaction was then digested with 1 µl of Dpn
I at 37 °C for 1 h. The Dpn
I-digested mutagenesis reaction (1 µL) was used to transform chemically competent TOP10 E. coli
, and the transformed cells were grown on LB plates containing 50 µg/ml kanamycin. Positive clones were verified by bidirectional DNA sequence analysis on an ABI 3100 genetic analyzer (Applied Biosystems, Foster City, CA). The F1-VC424S
vector was transformed into BL21 Star cells for protein expression under control of the isopropyl-β-D-thiogalacto-pyranoside (IPTG)-inducible T7 promoter. F1-VC424S
-BL21 Star E. coli starter cultures were grown overnight in four 4-L shaker-flasks filled with a total of 10-L LB medium at 37 °C, and 250-rpm shaking in the presence of 50 mg/L of kanamycin. Starter cultures were then diluted 1:10 in fresh kanamycin-supplemented LB medium and grown at 37 °C, 250 rpm to an OD600
of 0.5 – 0.8. Protein expression was induced by adding IPTG (0.5 mM). After 3 h at 37 °C with 250-rpm shaking, cells pellets were collected by centrifugation at 10,000 × g for 20 min and stored at –70 °C.
Recovery of F1-VMN
For F1-VMN, combined wet cell paste from two fermentations was re-suspended to 40% w/v with 1.2 L of lysis buffer (50 mM Tris, 50 mM EDTA, 20 mM DTE, pH 9.0), sheared for 10 min with a HAAKE A82 (Thermo-Electron, Waltham, MA), and homogenized by three passages at 12,000 psi through a NS1001-L2K mechanical homogenizer (Niro-Soavi, S.p.A., Parma, Italy). The homogenizer was fitted with a chilled reservoir and cooling coil that was kept below 11 °C. The homogenized paste was adjusted to pH 8.3 +/- 0.2, clarified by centrifugation for 1 h at 10,000 RPM in a JA-10 rotor at 4 °C (Beckman Coulter, Fullerton, CA), and the supernatant was collected. F1-V was precipitated by a slow, well-mixed adjustment of the supernatant to pH 4.8 with 1 M acetic acid (pH 2.25). An off-white, granular pellet, enriched in F1-V, was collected by centrifugation for 1 h. The pellet was washed in an equal volume of 5 mM citric acid, pH 4.8 and then centrifuged, washed again, and the resulting pellet stored below –70 °C. The washed pellet was re-suspended in 2.5 volumes (~1 L) of solubilization buffer (10 mM Tris, 10 mM ethanolamine, 5 mM L-cysteine, 50 mM EDTA, pH 9.0) and mixed to disperse the pellet at 20 °C for 20 min. The solution was adjusted to pH 11.0 by a slow, drop-wise addition of 10 N NaOH and held for 5 min at 20 °C, then adjusted to pH 8.3 with vigorous mixing and slow addition of 1 M acetic acid. The F1-V-enriched supernatant was separated from a lower density, colorless precipitate, enriched in contaminants (notably 40 kDa E. coli membrane protein I, identified by N-terminal sequencing) by centrifugation. The supernatant was re-precipitated by slow adjustment to pH 4.8 and the F1-V enriched pellet was stored below –70 °C.
For F1-VC424S-MN, cell paste was re-suspended to 20% w/v with 50 mM Tris, 50 mM EDTA, pH 9.0, (without reducing agents) and homogenized by three passages through an EmulsiFlex-C5 MicroFluidizer (Avestin, Canada) at a backpressure of 10,000 to 15,000 psi. The homogenized paste was clarified by centrifugation for 35 min at 15,000 rpm in an SS-34 rotor at 4 °C (Beckman Coulter, Fullerton, CA). Using methods similar to those used for F1-V, except without the addition of reducing agents, F1-VC424S was recovered from the supernatant. The recovered F1-VC424S-enriched pellet was stored below –70 °C.
Columns and chromatography systems were cleaned and depyrogenated by exposure to 0.05 N NaOH for greater than 12 h or 0.5 N NaOH for 1 h followed by rinsing to neutral pH. For F1-VMN, F1-V-enriched pellet (400-g) was thawed at 20 °C and re-suspended 1:10 into 4 L of IEX-A buffer (10 mM Tris, 10 mM ethanolamine, 4.5 M urea, pH 8.3; then nitrogen sparged; and 5 mM fresh L-cysteine added). The load (~2.9 mS/cm) was held at 20 °C for ~3 h for F1-V dispersal and applied onto Q-Sepharose FF resin (BPG100/500, 10 cm D x 20 cm H bed, 90-µm bead size; GE Healthcare, Piscataway, NJ) and developed with one CV rinse and six CV linear gradient elutions at 60 cm/h to 3.5 M urea, 500 mM Gdn HCl in similar buffer (IEX-B). Monomer-enriched fractions, identified by HPLC-SEC analysis, were examined by SDS-PAGE to facilitate selection of the target monomeric F1-V species. The first major F1-V elution peak was collected between the 80- to 130-mM chloride ion (6.0 to 9.7 mS/cm) range. The Q-Sepharose FF elution pool was stored below –70 °C.
For F1-VC424S-MN, the F1-VC424S enriched pellet was re-suspended to 20-ml final volume with 10 mM Tris, 10 mM ethanolamine, 10 mM Gdn HCL, pH 8.3, and then then adjusted to pH 10.3, held for 30 min, and re-adjusted to pH 8.3 with 1 M acetic acid. High-purity solid urea was added to obtain a concentration of 4.5 M urea and the solution was held at 20 °C for 1 to 2 h before being loaded onto Q-Sepharose FF resin (1.6 × 10 cm, 90-µm bead size; GE Healthcare) equilibrated with 10 mM Tris, 10 mM ethanolamine, 4.5 M urea, 10 mM Gdn HCl, pH 8.3, followed by washing and linear gradient elution to 3.5 M urea / 500 mM Gdn HCl at 120 cm/h. The leading shoulder of a complex multi-peak structure was excluded from pooling to eliminate contaminants, identified by SDS-PAGE fraction analysis. F1-VC424S monomer-enriched fractions were collected and pooled from the first major peak eluting between 40 and 80 mM chloride, and stored below –70 °C. The second half (85 mg), was pooled separately and not processed further. The Q-Sepharose FF pool was diluted with high-quality water to 3.4 mS/cm (~2.5-fold), loaded onto Source 15Q resin (1.6 × 10 cm, 15-µm bead size, GE Healthcare) equilibrated with IEX-A buffer, and eluted with a linear gradient to 40% B over 16 CV at 120 cm/h (4 ml/min). The leading half of the main peak was pooled and stored below –70 °C.
For F1-VMN, buffers IEX-A and IEX-B were made as above except for replacement of L-cysteine with 1 mM DTT. To ensure complete protein reduction, DTT (5 mM) was added to the monomer-enriched pool. After 2.3-fold dilution (from ~9.5 mS/cm to 4.2 mS/cm, 4.75 L final volume) with IEX-A buffer, the pool was loaded onto Source 15Q resin (BPG100/500, 10 cm D x 20 cm H, 15-µm bead size; GE Healthcare), and eluted with a linear gradient to 40% IEX-B over eight CV at 60 cm/h. The F1-V monomer, eluting below 100 mM chloride ion, was pooled based on HPLC-SEC and SDS-PAGE fraction analysis. Contaminants present in a leading shoulder of a complex multi-peak structure were excluded from pooling. Two trailing shoulders, while also containing F1-V, were pooled separately and not processed further. The Source 15Q Elution Pool was separated into 5 × 200 mL aliquots and stored below –70 °C.
CHT affinity chromatography
For F1-VMN, CHT Type 2 resin (BPG100/500, 10 cm x 12 cm, 20-µm beads, BioRad, Hercules, CA), was charged with high phosphate buffer and equilibrated just before use with CHT-A buffer (10 mM Tris, 150 mM NaCl, 1 mM NaH2PO4, 0.1 mM CaCl2, pH 7.8; argon sparged; 1 mM DTE added, used immediately). For each of five CHT-T2 cycles, a 200 mL Source 15Q Elution Pool aliquot was thawed at ~20 ºC, adjusted to 1 mM NaH2PO4, 0.1 mM CaCl2, from 100 mM stocks, diluted fivefold into CHT-A buffer, applied to the column at 50 cm/h and eluted with a linear gradient to 50% Buffer CHT-B (CHT-A + 200 mM NaH2PO4) over 16 CV. CHT T2 elution fractions were collected into containers pre-loaded with L-arginine stock (1.3 M L-arginine, pH 10.0) to obtain a final concentration of 200 mM L-arginine in each collected fraction. An early-eluting, sharp, F1-V-containing peak was excluded from pooling. Center fractions within a broader major peak were pooled and concentrated to 8 to 9 mg/ml of total protein by A280 over a 1-ft2 PrepScale-TFF 10-kDa MW cut off spiral tangential flow filtration membrane (regenerated cellulose, Cat# CDUF001LG; Millipore, Billerica, MA). The concentrated (7.4 mg/ml) CHT-T2 pool was divided into 3 × 95 mL aliquots and stored below –70 °C.
For F1-VC424S (MN), CHT-T1 resin (1.6 × 10 cm, 20-µm bead size; BioRad, Hercules, CA) was equilibrated and developed similarly to CHT Type 2 resin above. The Source 15Q pool was thawed and processed through two CHT-T1 column cycles. During the first cycle, performed without trace phosphate added to the load, a portion of F1-VC424S did not bind. For the second cycle, 1 mM phosphate was added to the load, leading to complete F1-VC424S binding. For both cycles a single, notably sharp, concentrated elution peak, was pooled with a minor, extended tail excluded. The fractions were stored chilled.
Size exclusion chromatography formulation of F1-VMN
Each CHT-T2 aliquot (1.2% CV) was adjusted to pH 10.0, held overnight at 4 °C, loaded onto Superdex 200 PG resin (10 cm x 90 cm in a BPG 100/950 column, 34-µm bead size) and eluted with formulation buffer (20 mM L-arginine, 10 mM NaCl, argon, 1 ml of L-cysteine, pH 10.0) at a flow rate of 22 cm/h. The early eluting dimer-enriched fractions were pooled separately (252 mg) and stored below –70 °C. Fractions in the first half of the monomer peak, essentially free of contaminants, were 0.2-µm filtered, aliquoted, and stored below –70 °C. Trailing monomer-peak fractions, enriched in contaminants, were concentrated as above, re-fractionated, and combined with initial monomer-enriched fractions. This final pool was 0.2-µm filtered distributed into sterile cryo-vials; and stored below –70 °C.
For F1-VC424S-MN, main peak fractions were pooled (40 ml) and adjusted to 500 mM L-arginine by adding 3.5 g of solid L-arginine pre-dissolved in 7 ml water. After 10 min at pH 11.0, the pool was adjusted to pH 10.1 by the slow addition of HCl and held overnight at 4 °C. This yielded ~47 ml at 5.0 mg/mL or 235 mg of total protein. The adjusted CHT-T1 pool was fractionated by size-exclusion chromatography through Superdex 200 PG resin (two tandem columns, 10 cm x 90 cm in BPG 100/950 columns, 34-µm bead size),equilibrated and developed with 20 mM L-arginine, 10 mM NaCl, pH 10.0 (with no L-cysteine) at a flow rate of 22 cm/h. A 43-ml sample (0.3% of CV) was applied. Fractions in the first half of the monomer peak were pooled and stored below –70 °C; thawed; concentrated using YM-10 Centripreps (Millipore, Billerica, MA) at 4 °C; 0.2-µm filtered; distributed into sterile cryo-vials; and stored below –70 °C.
Conversion of monomeric F1-VMN to multimeric F1-VAG
An aliquot of formulated F1-VMN, at pH 10.0, was converted to F1-VAG by slow titration with acetic acid to pH 5.1, incubated overnight at 4 °C, and then stored below – 70 °C.
Optional freeze-drying of F1-VMN
F1-V in formulation buffer was adjusted to 2% w/v low endotoxin D-mannitol (Ferro Phanstiehl Laboratories, Inc., Waukegan, IL) added from a 20% D-mannitol stock dissolved in formulation buffer. The product was distributed into 3-ml glass vials, frozen at a plate temperature of –48 °C, and lyophilized in an AdVantange-ES Benchtop freeze-dryer (VirTis, Gardiner, NY) for 30 h at –45 °C, 8 h at –37 °C, followed by 15 h at +37 °C. Condenser coils were maintained at –80 °C. Vial stoppers were mechanically seated within the chamber while under vacuum and crimped externally. The vials were stored below –70 °C.
Total protein, Endotoxin and SDS-PAGE
Protein concentrations were measured by A280
divided by an absorption co-efficient of E = 0.468 A280, 1cm
per (mg of F1-V/ml), calculated using methods [13
] automated on the ExPASy Proteomic Server, ProtParm (2005 version, http://www.expasy.org
). For solubilized pellets, total protein was estimated with E = 1.0. For endotoxin measurement, the commercially available Charles River (Charleston, SC) kinetic chromogenic limulus amoebocyte lysate reactivity endotoxin kit was used, which had a lower detection limit of 0.005 EU/ml, established versus the provided endotoxin standard. For SDS-PAGE, 4– 12% Bis-Tris NuPAGE gels and reagents, Mark 12 size standards, and Sypro Ruby fluorescent stain were obtained from Invitrogen. Samples were reduced with 5% v/v 2-mercaptoethanol. Destained gels were scanned with a Molecular Dynamics model 595 scanning laser fluorimeter (GE Healthcare) and integrated with ImageMaster 1D Elite software (Version 4.1, GE Healthcare).
Size-exclusion chromatography coupled to multiangle laser light scattering (SEC-MALLS) was applied as previously reported for F1-V [6
], but with modifications as published [14
]. The HPLC pumps were Rainin HPXL 10 ml/min pumps (Varian, Walnut, CA) run at 0.4 ml/min. Fractionation was performed through two tandem G3000SWxl analytical size-exclusion chromatography columns (7.8 × 250 mm, 5-µM bead size, 250-Å pore size; Tosho Biosciences, Montgomeryville, PA), equilibrated with 0.2-µm filtered, helium-sparged mobile phase (0.1 M KH2
, 0.1 M Na2
, 0.3 M NaCl, pH 7.0). The light-scattering detector series consisted of a Rainin Dynamax UV-1 A280
detector (Varian); a Dawn EOS multi-angle, static, light-scattering detector (Wyatt Technology Corporation, Santa Barbara, CA); and an Optilab DSP interferometric refractometer (Wyatt). Average molar mass measurements were determined from aligned elution profiles within ASTRA for Windows software (Revision 4.90.07/QELS version 1.00, Wyatt). Bovine serum albumin (2 mg/ml, 10 µL injections) containing a mixture of solution forms (66.3 kDa monomer, 132.6 dimer kDa, and 198.9- kDa trimer; Pierce-Endogen, Rockford, IL) was used to normalize detectors, establish detector train delay times, set software parameters, and confirm system suitability before test sample analysis. According to previously reported methods [15
], the standard optical constant was calculated as K
* = 1.85 × 10–7
; as derived from (dn/dc) = 0.185 ml g–1
= 1.33; and λ0
= 681 nm; with the form factor set to unity.
For each sample, 100-µg aliquots were dried, re-solubilized to 1 M Gdn HCl in 0.1 M Tris, pH 8.0, divided in half and digested (1:30 enzyme-to-substrate ratio) with modified trypsin or chymotrypsin overnight at 37 °C with mixing by vortex at 1,200 rpm. The digest was quenched by acidification and the samples stored at 4 °C until analysis. The samples were injected onto a reverse-phase column (Grace Vydac LC/MS C18, 2.1 × 250 mm, C/N 218MS52, 35 °C; Hesperia, CA) fitted to an HPLC (Thermo Electron, Surveyor LC System, Waltham, MA) followed by a hold at 5% for 5 min and elution over 55 min at 0.2 ml/min using a 1% per min linear gradient of acetonitrile containing 0.08% trifluoroacetic acid and 0.02% formic acid with elution monitored at 214 nm. The effluent was directed into an ion trap mass spectrometer (Thermo Electron, LCQ-Deca MS) for detection by electrospray mass spectrometry (ESI-MS) in positive mode ionization with 250 °C capillary temperature, ~95 psi sheath gas pressure, ~5 psi auxiliary gas pressure, source at 5.5 kV with capillary at 44 V, lens offset by 50 V, multipole offset by –5.5 and –10.5V, inter multipole lens at –28V, entrance lens at –88V and a trap DC offset of –10V. MS/MS was performed using 35% collision energy. Sequential scanning, consisting of full-scan ESI-MS from m/z 500 to 2000 and triplicate MS/MS scans of the three most abundant base peak (BP) ions, was employed. Equine skeletal muscle myoglobin (Sigma-Aldrich, M0630, St. Louis, MO) was analyzed as a sample preparation and instrument performance standard. The resulting MS and MS/MS data sets were processed using Bioworks© (Thermo Electron, Version 3.1) and Xcaliber© Software (Thermo Electron, Version 1.3). Except where noted, fragment ion identity assignments were based upon automated software MS/MS analysis of primary-ion peak fragments with software default Xcorr thresholds set for assignment acceptance. The sequence coverage for the mutant myoglobin standard was 100%.
For disulfide-linked dimer dispersal scouting, a sub-fraction of purified F1-VMN formulated at ~0.7 mg/ml in 20 mM L-arginine, 10 mM NaCl, pH 9.9, without added 1 mM L-cysteine, was air oxidized to form ~22% disulfide-linked dimer. Reagents were added from un-adjusted, acidic, 100-mM stocks of freshly prepared DTE, L-cysteine, and IAA. For the two-reagent conditions, the reductant was added first, followed by a 10-min hold at 25 °C before adding IAA. Adjusted samples were held at 25 °C within the HPLC-SEC auto injector before analysis. Samples were analyzed through HPLC-SEC with two tandem columns (G3000SWxl) on an Agilent 1100 system (Agilent Technologies, Palo Alto, CA) eluted at 0.8 ml/min with 0.1 M KH2PO4, 0.1 M Na2SO4, 0.3 M NaCl, pH 7.0. Column performance was confirmed by running high MW size standards (BioRad). The percentage of integrated A230 eluting in each peak relative to total protein-related integrated absorbance was calculated within Chemstation 2.0 software (Agilent).
For non-covalently-linked multimer dispersal scouting, reagents were prepared as 10-fold stocks in high-quality water and adjusted as needed to ~pH 6.5. An aliquot of F1-VMN, initially formulated at ~0.7 mg/ml in 20 mM L-arginine, 10 mM NaCl, 1 mM L-cysteine, pH 9.9, was titrated by micro-addition of HCl to pH 6.5. In less than 5 min, the aliquots were divided and transferred with mixing into containers pre-loaded with 1/10th volume of additive stocks. Samples were held at 4 °C before HPLC-SEC analysis by methods similar to those described for disulfide-linked dimer dispersal scouting above.
Research was conducted in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals, and experiments involving animals were conducted according to the principles set forth in the Guide for the Care and Use of Laboratory Animals
]. The facility where this research was conducted is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. Groups of 10 female, 8- to 10-week-old outbred (Hsd:ND4) Swiss Webster mice were inoculated subcutaneously (s.c.) with purified, recombinant F1-VSTD
preparations. To evaluate the effect of aggregation state on the protective efficacy of F1-V as well as the efficacy of the new F1-VC424S,
various F1-V aggregation state formulations were produced. Vaccine candidate formulations included monomeric F1-VC424S-MN
, the cysteine-capped, monomeric F1-VMN
, and the converted multimer, F1-VAG
was produced by incubating F1-VMN
overnight at pH 5.1 and 4 °C to enhance F1-V aggregation. One group of 10 mice was inoculated s.c. with the previously reported mixed solution state F1-VStd
as a positive control [6
]. In order to maximize immunogenicity, each protein antigen was adsorbed to aluminum hydroxide adjuvant (Alhydrogel. 1.3%; Superfos Biosector, Vedbaek, Denmark; 0.19 mg of aluminum per dose), critically before exposure of adjuvant to injection buffer (1x PBS). Each antigen-adjuvant mixture (200 µL) containing 20 µg of each antigen was administered at a single subcutaneous site on the backs of the animals. After 30 days, the animals were boosted with an identical dose at the same injection site.
Measurement of serum antibody titer using ELISA
Mice were anesthetized with a mixture of 5 mg of xylazine (XYLA-JECT; Phoenix Pharmaceutical, Inc., St. Joseph, MO) per kg, 0.83 mg of acetylpromazine (Fermenta Animal Health Co., Kansas City, MO) per kg, and 67 mg of ketamine hydrochloride (Ketamine; Phoenix Pharmaceutical, Inc.) per kg administered intramuscularly. Blood was collected by retro-orbital sinus puncture for the determination of antibody titers 56 days after the initial injection by standard enzyme-linked immunosorbent assay (ELISA). Briefly, 100 ng of each purified protein in carbonate buffer, pH 9.4, was applied to each well of a 96-well microtiter plate and allowed to incubate overnight at 4 °C. Plates were then washed with 1x PBS + 0.05% Tween 20. Plates were blocked with 100 µl of assay diluent (1 x PBS, 1% bovine serum albumin, 0.05% Tween 20) for 1 h at 37 °C. Plates were washed again and serial dilutions of antiserum in assay diluent ranging from 1:50 to 1:2,048,000 were applied in triplicate. Plates were allowed to incubate at 37 °C for 1 h, washed, and a 1:5000 dilution of horseradish peroxidase-conjugated goat anti-mouse IgG was applied for 1 h at 37 °C. Plates were washed and the chromogenic substrate 3,3′,5,5′ tetramethylbenzidine (TMB; BD Biosciences, Pharmingen, San Diego, CA) was added. After a 30-min incubation at 37 °C in the dark, the reaction was stopped with 25 µl of 2 N sulfuric acid. Plates were read at an optical density of 450 nm (OD450).
Y. pestis lethal challenge
Each of the vaccinated animals designated to receive s.c. challenges was administered 104, 107, 108, or 109 50% lethal doses (LD50) of wild-type Y. pestis CO92, 30 days after the booster dose. The s.c. LD50 for adult mice challenged with CO92 is 1.9 colony-forming units (CFU) as determined by serial dilution and plating. The mice were observed daily for 28 days, at which time the survivors were killed. Fisher’s two-tailed exact tests were used to evaluate animal survival data. Mean time to death after lethal plague challenge was evaluated using Student’s t-tests. Significance in pair-wise comparisons of delayed time to death between groups was computed using Student’s t-tests.