Plague is a deadly infectious disease that has killed

over 200 million people worldwide. Formalin-inactivated

Yersina pestis vaccine USP that is given by the
intramuscular route has been used for plague prevention.
However, the production of licensed Plague vaccine USP
has been discontinued due to lack of efficacy against
pneumonic plague, and its reactogenicity. We now report
that intranasal administration of paraformaldehyde-
inactivated Y. pestis CO92 is immunogenic and efficacious
in protection against pneumonic plague. Mice were
immunized intranasally with 10
8
CFU of inactivated Y.pestis
with or without 1g IL-12 on days 0 and 21. Immunized mice
were challenged intranasally on day 60 with lethal Y.pestis
CO92. The immune mice generated robust antibody
responses and showed 100% survival from lethal challenge.
This inactivated vaccine also induced BALT-like structures in
the lung that corresponded with increased levels of B-
lymphocytes, T-lymphocytes, and MHC-II+ cells in to the
lung parenchyma. Further studies are underway to
characterize the protective mechanisms.
Inactivated Whole-Cell Vaccine Delivered Intranasally Protects Mice against Pneumonic Plague
Devender Kumar and Dennis W. Metzger
Center for Immunology and Microbial Disease, Albany Medical College, Albany, NY 12208

CONCLUSIONS
We thank Michelle Wyland-O'Brien, Sharon
Salmon, and Sherie OConnell for ABSL-3
assistance.
We thank Yili Lin & Bibiana Iglesias of the AMC
Immunology Core for their help with flow cytometry
and histology.
The authors received financial support from the
DOD-ONR (Award no. N00014-06-1-1176).
ABSTRACT
INTRODUCTION
RESULTS
Fig.1. Intranasal vaccination with iYp vaccine along with
IL-12 enhances protection from pneumonic plague
Fig. 2. 10
7
CFU of intranasal iYp vaccine have limited toxicity
A. Percent body weight of mice after vaccination
B. Lung TNF- levels after vaccination
Fig. 4. IL-12 enhances the antibody titers of intranasal iYp whole-cell
vaccine
A. Serum IgG
B. Day 35 Lung antibodies
Fig. 3. Intranasal iYp vaccine induces cellular infiltration in lungs

Fig. 5. IL-12 enhances the appearance of inducible BALT-like
structures in the lungs of vaccinated mice
B. B220+ B-cells are present in Inducible BALT-like structures in
lung sections. Immunofluorescence, 40x
C. IL-12 enhances levels of B-cells, T-cells, and MHC-II + cells in
lung parenchyma after intranasal iYp vaccination. Analysis was
done by flow cytometry.
A. Lung sections, H&E staining, 40x
Plague is an exceptionally virulent and zoonotic disease.
Y. pestis is classified as a Category A select agent by the
CDC, that could to be used as a biological weapon.
Pneumonic plague has a high fatality rate even if antibiotic
treatment is started within 20 hours of infection.
Plague vaccine USP does not prevent primary pneumonic
plague in humans or mice when administered
intramuscularly.
No licensed vaccine is presently available.
Interleukin-12 (IL-12) is an extremely potent mucosal
adjuvant that enhances humoral and cell-mediated immunity.
HYPOTHESIS
Intranasal vaccination of mice with inactivated Y. pestis
vaccine adjuvanted with IL-12 will generate local and
systemic immune responses that will protect against
primary pneumonic plague.

METHODS
Vaccine: 0.25% paraformaldehyde inactivated Y. pestis
CO92 (iYp)
Animal model: BALB/c mice (n=8/gp)
Vaccine dose: 10
5
-10
8
CFU
Vaccine route: Intranasal
Vaccine volume: 30 l/mouse
Vaccination protocol:
Intranasally-administered inactivated whole-cell
Y. pestis can be used as a safe and potent
vaccine against primary pneumonic plague in
mice.
Vaccination generates robust serum and lung
antibodies.
IL-12 is an effective mucosal adjuvant for
intranasal vaccination against pneumonic plague.
Acknowledgements
Day 0 Day 21 Day 42/60
Prime Boost
Intranasal 10
4
Y.pestis CO92
Challenge
Survival
analysis
0 2 4 6 8 10 12 14
0
20
40
60
80
100
10
8
+ IL-12 10
8
PBS
0 2 4 6 8 10 12 14
0
20
40
60
80
100
10
7
+IL-12 10
7
PBS
0 2 4 6 8 10 12 14
0
20
40
60
80
100
10
6
+IL-12 10
6 PBS
0 2 4 6 8 10 12 14
0
20
40
60
80
100
10
5
+IL-12 10
5 PBS
Days post-infection
P
e
r
c
e
n
t

s
u
r
v
i
v
a
l
10
8
iYp 10
7
iYp
10
6
iYp 10
5
iYp
0 1 2 3 4 5 6 7 8 9 10 11
80
90
100
110
10
8
+ IL-12 10
8
PBS
0 1 2 3 4 5 6 7 8 9 10 11
80
90
100
110
10
7
+ IL-12 10
7
PBS
Days post-vaccination
P
e
r
c
e
n
t

W
e
i
g
h
t
10
8
iYp
10
7
iYp
1 3 5
0
500
1000
1500
2000
2500
3000
3500
4000
10
7
iYp + IL-12
10
7
iYp
PBS
Days post-immunization
T
N
F
-

(
p
g
/
m
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10
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7
5
3
D
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p
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0 7 14 21 28 35 42
10
100
1000
10000
100000
10
7
iYp + IL-12 10
7
iYp PBS
Days post-vaccination
S
e
r
u
m

I
g
G

T
i
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s
Ig
M
Ig
G
Ig
G
1
Ig
G
2
a
Ig
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2
b
Ig
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3
Ig
A
1
10
100
1000
10000
10
7
iYp + IL-12 10
7
iYp PBS
L
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g

A
n
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b
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y

T
i
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D
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p
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10
7
iYp + IL-12 10
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iYp PBS
B-cells T-cells MHC-II+ cells
0
1.010
6
2.010
6
3.010
6
4.010
6
5.010
6
10
7
iYp + IL-12
10
7
iYp
PBS
A
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