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Nasal Vaccines for Defense

Against Pneumonic Plague


Devender Kumar
11-11-2011

Acknowledgements

Metzger Lab:
Dr. Dennis Metzger
Dr. Girish Kirimanjeswara
Dr. Keer Sun
Michelle Wyland-O'Brien
Sharon Salmon
Shannan Ziles
Roberts Sean
Immunology Core:
Dr. Edmund Gosselin
Yili Lin





Department of Chemical and Biological
Engineering:
Dr. Balaji Narasimhan
Dr. Bret Ulrey

Department of Veterinary Microbiology and
Preventive Medicine:
Dr. Michael J. Wannemuehler
Dr. Amanda E. Ramer-Tait







Department of Microbiology and
Immunology :
Dr. Nejat Egilmez







Worldwide deaths from mucosal infections

Why intranasal immunization?
Nasal passage is major portal of
entry for lung pathogens
Generate mucosal and systemic
immunity
Generates immunity at other
mucosal surfaces
Immune responses occur at
pathogen-entry site
Prevents infections
Ease of administration/Mass
vaccination

Bacteriology
Yersinia pestis
Yersinia pestis Antiqua
Yersinia pestis Medievalis
Yersinia pestis Orientalis
1-2m x 0.5m, single cell or short chain
Gram-negative, Bipolar staining
Facultative anaerobe
Nonmotile, nonsporulating
Catalase+, oxidase-, urease-
Able to grow 4-40C, optimum 28 C
Flocculant growth in broth
Grey-white translucent colonies on Blood
Agar. Fried egg appearance in older
colonies on BA



Images: NY State Department of Health-Wadsworth Center, PHIL-CDC
Lcr
70kb
V Ag
Resistance to phagocytosis
Survival in macrophage
Yops
Phagocytosis
Platelet aggregation
Inflmmatory response
pFra
110kb
F1 Ag
Antiphagocytic
Pst
9.5kb
Pla
Degrade fibrin
& other extracellular
proteins
LPS
DIC
Endotoxic shock
Virulence Factors
Adhesins
The Global Distribution of Plague
From: Plague: Past, Present, and Future Stenseth NC, Atshabar BB, Begon M, Belmain SR, Bertherat E, et al. PLoS Medicine Vol. 5, No. 1, e3 doi:10.1371/journal.pmed.0050003
Symptoms
http://en.wikipedia.org/wiki/User:Mikael_Hggstrm/Diagrams
Plague Pathogenesis
H
u
m
a
n

M
i
c
e

H
u
m
a
n

Normal Lung Infected lungs
M
i
c
e

Possible Airborne Transmission Pathways of Y. pestis
after Its Intentional Aerosol Release
Metzger Lab, 2010; http://www.openclipart.org/
100-500 CFU
How to Prevent Pneumonic Plague?
Nanoparticles Whole-killed Cells Soluble Ags Microparticles
Natural bio-particles
Biomimetic
Multiple arrays of Ag
Assurance of inactivity


rF1-Vp10
rLsrB
rAil
rMalB
rYapM


rF1-Vp10
Controlled release
Single-dose
Long-term protection
Biodegradable
Modifiable to suit Ag release



V
a
c
c
i
n
e
s

A
d
j
u
v
a
n
t
s

Interleukin-12 Monophosphoryl Lipid A
TLR-4 agonist
Safe
Non-toxic
Ag presentation

Enhances cellular
and humoral immunity
Safe by i.n. route
Established efficacy
Inactivated Yersinia pestis CO92
Vaccine (iYp)
The i.n. Prime-Boost and Challenge Strategy for
iYp Vaccine
Day 0
i.n. iYp + IL-12 / iYp/ PBS
Day 21
i.n. iYp + IL-12 / iYp/ PBS
Day 80
i.n. Y. pestis CO92
Day 94
End of survival study
Administration of i.n. iYp + IL-12 is Safe
and Well-Tolerated
0 2 4 6 8 10 12
90
95
100
*
P<0.05
Days post-vaccination
%

B
o
d
y

w
e
i
g
h
t
1 3 5
2.5
3.0
3.5
4.0
10
7
iYp + IL-12
10
7
iYp
PBS
**
*
P<0.01
P<0.05
Days post-vaccination
T
N
F
-


(
p
g
/
m
l
)

l
o
g
1
0
A. B.
C.
Administration of i.n. iYp + IL-12 Protected
against Primary Pneumonic Plague
0 5 10 15 20 25
0
25
50
75
100
10
7
iYp+IL-12
10
7
iYp
PBS
ns
**
P=0.001
8.5 x 10
2
CFU, i.n.
Days post-challenge
BALB/c mice.
P
e
r
c
e
n
t

s
u
r
v
i
v
a
l
A.
B.
Days 0 21 80
1. 10
7
iYp + 0.5g IL-12
2. 10
7
iYp
3. PBS
BALB/c mice,
I.n. vaccination
Priming Boosting
94
Y. pestis CO92
challange, i.n.,
8.510
2
CFU
End of survival
observation
IL-12 Enhanced the Production of Anti-Y. pestis
Antibodies
0 10 20 30 40 50
1
2
3
4
5
*** *** ***
P

<

0
.
0
0
1
Sera
10
7
iYp + IL-12
10
7
iYp
PBS
Days post-vaccination
I
g
G

t
i
t
e
r
s

(
l
o
g
1
0
)
Sera
10
7
iYp + IL-12 10
7
iYp
3
4
5
Day 21 post-challenge
I
g
G

T
i
t
e
r
s

(
l
o
g
1
0
)
Lung
IgG IgA
0
1
2
3
4
5
*
**
*
P < 0.05
P < 0.01
P = 0.04
Day 35, post-vaccination
A
n
t
i
b
o
d
y

T
i
t
e
r
s

(
l
o
g
1
0
)
A. B. C.
The iYp + IL-12 Prime-Boost by i.n. Route
Prevented Pulmonary Colonization of Y. pestis
The i.n. iYp + IL-12 Prime-Boost Induced Cellular
Aggregations in Pulmonary Parenchyma
iYp + IL-12 PBS
D
a
y

3
5


p
o
s
t
-
v
a
c
c
i
n
a
t
i
o
n

A.







B.
D
a
y

2
8

p
o
s
t
-
v
a
c
c
i
n
a
t
i
o
n

B220 B220
7
2

h

p
o
s
t
-
c
h
a
l
l
e
n
g
e

iYp + IL-12 PBS
The i.n. iYp + IL-12 Prime-Boost Prevented
Development of Pulmonary Pathology
The i.n. iYp + IL-12 Prime-Boost Prevented Development of
Pulmonary Pathology
The i.n. iYp + IL-12 Prime-Boost Induced Protection is
Associated with Lower Levels of Pulmonary Cytokines After
i.n. Challenge
IL-6
iYp + IL-12 PBS Uninfected
0
1
2
3
4
5
*** ***
P < 0.001 P < 0.001

p
g
/
m
l

(
l
o
g
1
0
)
IL-10
iYp + IL-12 PBS Uninfected
0.8
0.9
1.0
1.1
1.2
iYp + IL-12
PBS
Uninfected
ns

p
g
/
m
l

(
l
o
g
1
0
)
MCP-1
iYp + IL-12 PBS Uninfected
0
1
2
3
4 ** *
P < 0.01 P < 0.05

p
g
/
m
l

(
l
o
g
1
0
)
IFN-
iYp + IL-12 PBS Uninfected
0
1
2
3 * **
P < 0.01 P < 0.05

p
g
/
m
l

(
l
o
g
1
0
)
TNF-
iYp + IL-12 PBS Uninfected
0
1
2
3
4
* *
P < 0.05 P < 0.05

(
p
g
/
m
l
)

l
o
g
1
0
IL-12p70
iYp + IL-12 PBS Uninfected
0.5
1.0
1.5
2.0
* *
P < 0.05 P < 0.05

p
g
/
m
l

(
l
o
g
1
0
)
The FcR are Required for Antibody-mediated
Passive and Active Immunity
0 5 10 15
0
25
50
75
100
*
P < 0.04
Days post-challenge
C57BL/6 mice
P
e
r
c
e
n
t

s
u
r
v
i
v
a
l
0 5 10 15
0
25
50
75
100
*
P < 0.02
Days post-infection
BALB/c mice
P
e
r
c
e
n
t

s
u
r
v
i
v
a
l
0 5 10 15
0
25
50
75
100
10
7
iYp + IL-12 Fc R
-/-
PBS
10
7
iYp + IL-12 Fc R
+/+
ns
*
P < 0.04
Days post-infection
C57BL/6 mice
P
e
r
c
e
n
t

s
u
r
v
i
v
a
l

Symbol BALB/c Mice Group IS / NMS transfer (Time)
Wild-type IS pre- and post -challenge -24, 0, +24, and +48h of infection

Wild-type IS post-challenge +18h of infection

Wild-type NMS -24, 0, +24, and +48h of infection
IS: Immune Serum; NMS: Normal mouse serum

Symbol C57BL/6 mice Group IS/NMS transfer (Time)
FcR
+/+
IS pre- and post -challenge -24, 0, +24, and +48h of infection

FcR
+/+
NMS pre- and post -challenge -24, 0, +24, and +48h of infection

FcR
-/-
IS pre- and post -challenge -24, 0, +24, and +48h of infection

FcR
-/-
NMS pre- and post -challenge -24, 0, +24, and +48h of infection
A.
B.
C.
The Complement Does Not Play Role in
Protection During Passively Transferred
Immunity
0 5 10 15
0
25
50
75
100
ns
P = 0.13
Days post-infection
C57BL/6 mice
P
e
r
c
e
n
t

s
u
r
v
i
v
a
l

Symbol C57BL/6 mice Group IS / NMS transfer (Time)
C3
+/+
IS pre- and post-challenge -24, 0, +24, and +48h of infection

C3
-/-
IS pre- and post-challenge -24, 0, +24, and +48h of infection

C3
+/+
NMS pre- and post-challenge -24, 0, +24, and +48h of infection

C3
-/-
NMS pre- and post-challenge -24, 0, +24, and +48h of infection
The CD4 and CD8 T Cells are Not Required for Protective
Immunity At Time of i.n. Challenge With Y. pestis
Days
0 21
Vaccination
Priming Boosting
Anti-mCD4 GK1.4 / Anti-mCD8 53-6.72 treatment, i.p.
70 73 76 79 83
80 90
1. 10
7
iYp + IL-12
2. 10
7
iYp + IL-12
3. 10
7
iYp + IL-12
4. 10
7
iYp + IL-12
5. PBS
1. 10
7
iYp + IL-12 (Rat IgG)
2. 10
7
iYp + IL-12 (Anti-CD4)
3. 10
7
iYp + IL-12 (Anti-CD8)
4. 10
7
iYp + IL-12 (Anti-CD4 + Anti-CD8)
5. PBS
Y. pestis CO92
challange, 1000 CFU,
i.n.
End of survival
observation
A.
B.
0 5 10
0
25
50
75
100
10
7
iYp + IL-12 (Rat IgG)
10
7
iYp + IL-12 (Anti-CD4)
10
7
iYp + IL-12 (Anti-CD8)
10
7
iYp + IL-12 (Anti-CD4 + Anti CD8)
PBS (Unvaccinated)
**
P = 0.003
Days post-challenge
BALB/c mice
P
e
r
c
e
n
t

s
u
r
v
i
v
a
l
Summary
Protect against pneumonic plague
IL-12 is required for inducing protection
Antibodies alone can provide protection which
is mediated through FcR
T-cells are dispensable at the time of infection
in iYp + IL-12 vaccinated mice
Single-Dose Intranasal
Polyanhydride Nanoparticles for
Vaccine Delivery
Material Properties Of 50:50 CPTEG:CPH Nanoparticles
Vaccination Regimen
Single-Dose Nanoparticles Induced Long-Term
Protection Against Pneumonic Plague
6 wk
23 wk
The Nanoparticles Prevented Colonization of Y. pestis and
Pulmonary Histopathology After 23 weeks of Vaccination
I.n. Vaccination of Nanoparticles Prevented pathology of Organs Post-challenge
Single-dose Administration of Nanoparticles Induced Long-
term Antibody Titers with High Avidity
Single-Dose Nanoparticle Priming Protected
Mice after 10 Months
Summary
Protect against pneumonic plague
Nanoparticles itself act as an adjuvant
Nanoparticles can abrogate the need for
multiple vaccinations and provide long-
duration immunity


Single-dose Intranasal Poly (lactic Acid)
Microspheres for Vaccine Delivery
Single-dose rF1-Vp10 Loaded PLA Microspheres Provides Long-
term Protection Against Pneumonic Plague
0 5 10 15
0
25
50
75
100
5 g FIV MP + IL-12 MP
7.5 g F1V MP + IL-12 MP
7.5 g F1V MP
PBS
*
P < 0.03
*
P < 0.01
Days post-infection
P
e
r
c
e
n
t

s
u
r
v
i
v
a
l
16 wk
PLA Microspheres induced Long-term Protection Against
Pneumonic Plague Correlates with IgG2a antibodies
Quorum Sensing Components as
Vaccine
I.n. Delivery of Quorum Sensing Components
Failed to Protect Against Pneumonic Plague
Limitations
Inactivated Vaccine
Literature
Duration of Immunity?
NHP trials not conducted
F1/V Ag based Vaccines
Doubt over efficacy in
Humans as it differentially
protect Cynomolgus
macaques and African
Green Monkeys
Ab levels does not
correlates with protection in
NHP
Improvements are Possible
Inactivated Vaccines
Optimize growth conditions
for maximum expression of
protective antigens
Better inactivation procedures
Encapsulation:
Safety,
Single-dose,
May abrogate the requirement
of adjuvant by encapsulating
high-dose and slow release


F1/V Ag based Vaccines
Encapsulation
Inclusion of novel protective
Ags / low dose iYp
Thank you