INFECTION AND IMMUNITY, Aug. 2006, p. 4910–4914
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 74, No. 8
Immunogenicity and Protective Immunity against Bubonic Plague
and Pneumonic Plague by Immunization of Mice with the
Recombinant V10 Antigen, a Variant of LcrV
Kristin L. DeBord,1Deborah M. Anderson,1Melanie M. Marketon,1Katie A. Overheim,1
R. William DePaolo,2Nancy A. Ciletti,1Bana Jabri,2and Olaf Schneewind1*
Department of Microbiology1and Department of Pathology,2University of Chicago, Chicago, Illinois 60637
Received 14 November 2005/Returned for modification 27 January 2006/Accepted 24 April 2006
In contrast to Yersinia pestis LcrV, the recombinant V10 (rV10) variant (lacking residues 271 to 300) does not
suppress the release of proinflammatory cytokines by immune cells. Immunization with rV10 generates robust
antibody responses that protect mice against bubonic plague and pneumonic plague, suggesting that rV10 may
serve as an improved plague vaccine.
Yersinia pestis, the causative agent of bubonic plague and
pneumonic plague, is a gram-negative pathogen that infects
many animal species, including humans, and is transmitted by
arthropod vectors or aerosol droplets (16). Immunization with
purified recombinant LcrV (rLcrV) is sufficient to generate
protective immunity to both bubonic plague and pneumonic
plague in mice, guinea pigs, and non-human primates (6, 7, 10,
11, 13, 30). LcrV is a multifunctional protein that is required
for Y. pestis type III injection of effector proteins into immune
cells (4, 22). Y. pestis lacking LcrV is avirulent in mouse models
of plague disease (8, 17, 19, 20). In addition to its role in type
III secretion, LcrV displays immunosuppressive properties (2,
12–14, 24, 25, 28). Brubaker and colleagues showed that LcrV
injection of animals triggered release of interleukin-10 (13), a
cytokine that suppresses innate immune functions (21). rLcrV
also prevents the release of proinflammatory cytokines (gamma
interferon and tumor necrosis factor ?) in murine and human
macrophages (2, 12, 13). Considering the immune modulatory
properties of rLcrV, there are concerns regarding the safety of
rLcrV vaccines in humans (15).
We searched for variants with reduced immune modulatory
properties (15). rV10, a variant lacking amino acids 271 to 300
of LcrV, displayed a significant decrease in its ability to induce
interleukin-10 and to suppress tumor necrosis factor ? or
gamma interferon release (15). Immunization of mice with
rV10 protects against lethal plague infections caused by 1,000
mean lethal doses (MLD) of Y. pestis KIM5 (KIM D27) (15),
a ?pgm (pigmentation defective), attenuated strain that causes
plague infections only when inoculated into the bloodstream
(3). We sought to determine whether rV10 vaccination can
prevent plague disease in animal challenge studies with the
fully virulent isolate Y. pestis CO92 (15). Previous work deter-
mined the 50% lethal dose of Y. pestis CO92 to be ?1 to 2 CFU
via the subcutaneous route of infection (23, 29). We measured
a similar 50% lethal dose for Y. pestis CO92 in a subcutaneous
infection model in BALB/c mice (data not shown). Escherichia
coli BL21(DE3) carrying prLcrV or prV10 (15) was grown
overnight at 37 °C in Luria-Bertani medium (Difco) with 100
?g/ml ampicillin. Bacteria were diluted in fresh medium and
grown to an optical density at 600 nm of 0.8 to 1.0. T7 poly-
merase expression was induced with 1 mM isopropyl-?-D-thio-
galactopyranoside, and bacterial growth was continued for 3
hours at 37°C. Cells were harvested by centrifugation at 10,000
? g for 10 min. Bacterial sediment was suspended in 20 ml of
Tris-HCl (pH 7.5)–150 mM NaCl (column buffer) containing
100 ?M phenylmethylsulfonyl fluoride, and cells were dis-
rupted by two passages through a French pressure cell at
14,000 lb/in2. The lysate was subjected to ultracentrifugation at
40,000 ? g for 30 min, and the soluble fraction was applied to
a nickel nitrilotriacetic acid column (1-ml bed volume) pre-
equilibrated with 10 ml of column buffer. The column was
washed with 10 ml of the same buffer, followed by a second (10
ml of column buffer with 10% glycerol) and a third (10 ml of
column buffer with 10% glycerol and 20 mM imidazole) wash-
ing. Bound protein was eluted in 50 mM Tris-HCl (pH 7.5),
150 mM NaCl, and 10% glycerol containing 250 mM imidaz-
ole. Purified proteins were subjected to three sequential Triton
X-114 (Sigma) phase separations to remove endotoxins. Puri-
fied proteins were applied to a G-25 (Amersham) gel filtration
column to remove residual Triton X-114 and then retrieved by
phosphate-buffered saline elution. Lipopolysaccharide con-
tamination of purified proteins was assayed with Limulus ame-
bocyte lysate (QCL-1000; Cambrex, New Jersey) and deter-
mined to be less than 1 ng/100 ?g of purified protein. Protein
concentrations were determined by the bicinchoninic acid as-
say (Pierce Technology, Rockford, IL). Proteins were ali-
quoted at 1 mg/ml and stored at ?80 °C for further use.
Purified recombinant rLcrV and rV10 vaccine antigens were
emulsified with Alhydrogel. Groups of 10 BALB/c mice were
immunized with adjuvant alone or with 50 ?g of rLcrV or rV10
on day 0, followed by a booster with an equal dose on day 21.
Blood from 5 mice in each immunization set was taken on days
0, 14, 28, and 42 after primary immunization to measure the
generation of specific antibodies. On day 43, mice were chal-
lenged with 100,000 MLD of Y. pestis CO92 via subcutaneous
injection. rLcrV- or rV10-immunized mice were protected
against lethal challenge, whereas mice receiving adjuvant alone
* Corresponding author. Mailing address: Department of Microbi-
ology, University of Chicago, 920 E. 58th Street, Chicago, IL 60637.
Phone: (773) 834-9060. Fax: (773) 834-8150. E-mail: oschnee@bsd
succumbed to disease within 4 days after infection with an
average time-to-death of 2.5 days (Fig. 1 and Table 1).
These data demonstrate that, similar to rLcrV, rV10 immu-
nization of mice provides robust protection against bubonic
Pneumonic plague infections in mice can be precipitated via
aerosol inhalation or intranasal infection. Aerosol infection of
mice is technically demanding and requires high doses of Y.
pestis (1). To develop an intranasal infection model of Y. pestis
CO92, groups of 10 BALB/c mice were infected with bacterial
suspensions delivered by the intranasal route. Actual deposi-
tion in the lungs was determined by postmortem removal of
lungs 60 min after inoculation, followed by plating of tissue
homogenate and colony formation. After infection with 1.9 ?
105CFU of Y. pestis CO92, greater than 80% of inoculated
bacteria were found deposited in lung tissues. Animals were
monitored for 14 days for signs of lethal disease or death and
time-to-death was recorded. An average dose of 389 CFU
(MLD) caused lethal disease in half of all experimental ani-
mals, consistent with previous observations using Y. pestis bio-
var Medievalis strain KIM (26). The average time to death
varied, depending on dose, with high-dose animals succumbing
to infection on day 2, while animals infected with lower doses
developed lethal infections 4 days after inoculation. Groups of
10 BALB/c mice were immunized with rLcrV or rV10 accord-
ing to the two-dose regimen described above. On day 43 after
primary immunization, mice were challenged with 2,570 MLD
of Y. pestis CO92 (1,000,000 CFU) by intranasal inoculation.
rLcrV vaccination provided 70% protection in this experiment,
whereas mice immunized with rV10 were completely protected
(Fig. 2). These data suggest that rV10 vaccination is at least as
efficacious against lethal pneumonic plague challenge as rLcrV
immunization. Sera collected from immunized mice on day 42
FIG. 1. Vaccination of mice with rV10 provides protection against
bubonic plague. BALB/c mice were immunized intramuscularly with
adjuvant alone (Alhydrogel), rLcrV, or rV10 in a two-dose regimen (50
?g of purified, endotoxin-free antigen injected on day 0 and 21). On
day 43 postimmunization, mice were challenge with 100,000 MLD of Y.
pestis CO92 by subcutaneous injection, and survival was monitored.
FIG. 2. Vaccination of mice with rV10 provides protection against
pneumonic plague. (A) BALB/c mice were immunized intramuscularly
by following the standard two-dose regimen with adjuvant alone, rL-
crV, or rV10. On day 43 postimmunization, mice were challenged with
1,000,000 CFU (equivalent to 2,570 MLD) of Y. pestis CO92 via intra-
nasal instillation, and survival was monitored. (B) rLcrV-specific IgG1,
IgG2A, and IgG2B antibodies were measured by ELISA in sera of five
animals immunized with either rLcrV or rV10. rV10 immunization
generated significantly higher titers for IgG1 (P ? 0.001), IgG2A (P ?
0.0181), and IgG2B (P ? 0.0064). Statistical significance of differences
in IgG titers was interrogated with a paired Student’s t test, and P
values were recorded.
TABLE 1. Vaccine protection elicited by rV10 and rLcrV
immunization against intranasal challenge
with Y. pestis CO92
No. of surviving animals/total no. of animals in
cohort (avg time to death ? SD ?days?)b
Alhydrogel rLcrV rV10
4.5 ? 105
6.7 ? 106
5.6 ? 107
4.0 ? 108
2/9 (2.4 ? 1.2)
3/9 (4.2 ? 2.5)
aAnesthetized BALB/c mice were infected by intranasal inoculation and ob-
served for 14 days for the development of plague.
bStatistical significance of differences in average time to death at 4.0 ? 108
CFU infectious doses was interrogated with a paired Student’s t test, and the P
value was recorded (P ? 0.0531). NT, not tested.
VOL. 74, 2006NOTES 4911
after primary immunization were analyzed by enzyme-linked
immunosorbent assay (ELISA) for total immunoglobulin G
(IgG) specific for rLcrV or rV10. The data revealed a signifi-
cant increase in anti-rLcrV IgG antibody titer in rV10-vacci-
nated mice compared with rLcrV-immunized animals (1.3 ?
105[? 8.2 ? 103] for rV10 and 2.5 ? 104[? 0] for rLcrV; P ?
0.001, determined with a Student’s t test).
To compare rV10 and rLcrV vaccine efficacy, we investi-
gated breakthrough challenges in mice immunized with adju-
vant, rLcrV, or rV10. On day 43 after immunization, animals
FIG. 3. Effect of rLcrV and rV10 immunization on plague pathogenesis. (A) Y. pestis strain KIM5 carrying plasmid pMM83 (for expression and
type III injection of YopM-Bla) or pMM91 (for expression—but not type III secretion—of GST-Bla) were used for intravenous infection of mice
that had been immunized with adjuvant alone (Alhydrogel), rLcrV, or rV10. Bacterial load in the spleen of infected animals was quantified by
colony formation of tissue homogenate. The dashed line indicates the limit of detection. (B) Yersinia type III injection of splenic phagocytes
measured with CCF2-AM staining (blue fluorescence) and flow cytometry of cells isolated from representative animals from panel A immunized
with Alhydrogel (control), rLcrV, or rV10.
4912 NOTESINFECT. IMMUN.
were infected intranasally with doses ranging from 4.5 ? 105to
4.0 ? 108CFU. Adjuvant control mice succumbed to disease.
Mice vaccinated with rLcrV or rV10 were fully protected at a
challenge dose of 4.5 ? 105CFU. Mice immunized with rV10
and challenged with 6.7 ? 106or 5.6 ? 107CFU were also fully
protected; however, mice immunized with rLcrV were only
partially protected as 1/9 and 2/9 animals, respectively, suc-
cumbed to infection. At the highest dose, 4.0 ? 108CFU, rV10
offered partial protection as 6/9 mice succumbed to infection,
whereas rLcrV vaccination protected only 2/9 mice. Thus, in
comparison with rLcrV, rV10 vaccination offered at least equal
levels of plague vaccine protection.
rLcrV vaccination generates antibodies that provide protec-
tion against plague by improving the efficiency of host poly-
morphonuclear cell phagocytosis of Y. pestis and by blocking
bacterial type III injection of effector proteins into host cells
(18, 27). Immune sera from BALB/c mice vaccinated with
either rLcrV or rV10 were characterized for the immunoglob-
ulin isotype profile by ELISA. ELISA plates were coated with
100 ?l of the purified recombinant antigens at a concentration
of 1 ı `g/ml and incubated overnight at 4 °C. Serially diluted
mouse serum (100 ?l) was added to each well and assayed in
triplicate after blocking. The plates were incubated with horse-
radish peroxidase-conjugated anti-mouse IgG diluted at 1:10,000
(100 ?l per well), followed by washing, and were finally developed
with 3,3?,5,5?-tetramethybenzidine solution (100 ?l per well). The
reactions were stopped by adding 25 ?l of 2 M H2SO4, and the
plates were read at the optical density at 450 nm. An IgG1 or
IgG2A or IgG2B isotype-specific ELISA was conducted as de-
scribed above, using horseradish peroxidase-conjugated goat-
anti-mouse IgG1 or IgG2A or IgG2B (Jackson Immunore-
search, Pennsylvania) at a dilution of 1:1,000, 1:500, or 1:500,
respectively. Specific IgG titers were estimated as the maxi-
mum dilution of serum generating absorbance at 450 nm of 0.1
units over background. Using this antibody titer, mean anti-
body titers ? standard error of the mean were derived per
immunization group. Four independent experiments were per-
formed to verify reproducibility.
Animals receiving either one of these two vaccines re-
sponded with the predominant production of IgG1-type anti-
bodies (Fig. 2). Mice immunized with rLcrV showed little to no
IgG2a or IgG2b isotype (5). rV10 immunization generated
significantly increased humoral immune responses for IgG1,
2a, and 2b isotypes (Fig. 2). To characterize cellular immune
responses to vaccination, the proliferation of T cells isolated
from immunized mice was measured in response to incubation
with homologous or heterologous antigens. The data suggest
that rV10 booster immunizations generated elevated T-cell
responses compared to rLcrV booster immunizations.
Y. pestis strain KIM5 (pMM83) was used as a reporter to
analyze the effect of rV10 and rLcrV immunization on the type
III injection of effector Yops into immune cells. pMM83 en-
codes YopM-Bla, a hybrid between the YopM effector with a
C-terminal fusion to ?-lactamase (9). CCF2-AM is a mem-
brane-permeant ester with two fluorophores attached to ceph-
alosporin that exhibit fluorescence resonance energy transfer.
Excitation of coumarin (409 nm) results in green fluorescence
emission from fluorescein (520 nm) in intact CCF2-AM. When
the YopM-Bla is injected into host cells, ?-lactamase cleaves
CCF2-AM, thereby disrupting fluorescence resonance energy
transfer and establishing blue fluorescence emission that can
be measured by flow cytometry. Groups of seven mice were
immunized with adjuvant alone, rLcrV, or rV10 by following
the two-dose regimen. On day 43, mice were infected intrave-
nously with 1,000 CFU of Y. pestis KIM5 expressing either
YopM-Bla or GST-Bla, a control hybrid that cannot travel the
type III pathway (9). On day 2 postinfection, mice were eutha-
nized, and splenocytes were treated with CCF2-AM and sub-
jected to flow cytometry to analyze the injection of YopM-Bla
into immune cells (Fig. 3). Mice that received adjuvant devel-
oped high bacterial titers in the spleen, ranging from 106to 107
CFU. Only one of the mice that had been immunized with
LcrV developed colonization of the spleen, though no disease
symptoms were apparent in this animal. Mice that had been
immunized with rV10 harbored no detectable bacteria in the
spleen. When analyzed by flow cytometry, mice immunized
with adjuvant alone and then infected with Y. pestis expressing
YopM-Bla harbored a significant proportion of blue cells
(1.77% of splenocytes). As a control, adjuvant mice infected
with glutathione transferase (GST)-Bla Yersinia harbored no
blue cells. One mouse that had been vaccinated with LcrV
harbored Y. pestis KIM5 (pMM83) in the spleen, and 1% of its
splenocytes stained blue. No blue cells could be found in the
other two rLcrV-immunized mice that did not harbor bacteria
in the spleen. None of the rV10-vaccinated mice harbored blue
cells or were infected with Y. pestis, suggesting that rV10 vac-
cination efficiently clears the infection (Fig. 3).
In sum, using bubonic plague and pneumonic plague mod-
els, we observed high levels of protection afforded by rV10
immunization in mice that correlated with the development of
specific humoral and cellular immune responses that are at
least equivalent to those raised with rLcrV. As rV10 displays
reduced immune modulatory properties, this antigen may
serve as a safe and effective subunit vaccine for humans. To test
this prediction, rV10 vaccine efficacy and safety will need to be
examined in non-human primates with aerosol challenge of Y.
pestis CO92 as a measure for protection against pneumonic
The authors acknowledge membership within and support from the
Region V “Great Lakes” Regional Center of Excellence in Biodefense
and Emerging Infectious Diseases Consortium (NIH Award 1-U54-
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Editor: V. J. DiRita