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Update on influenza vaccination
using microneedle delivery
Ioanna Skountzou
Emory University School of Medicine
Emory Vaccine Center
Mark Prausnitz serves as a consultant and is an inventor on patents licensed to companies
developing products related to this presentation. This potential conflict of interest is being
managed by Georgia Tech and Emory University
Selected immunological advantages of skin
immunization
• Enhanced humoral immune responses
• Increased duration of immunity
• Increased breadth of immunity
Solid Metal
Avoid hypodermic needles
Painless
Enhanced stability
Rapid distribution
Dose sparing
Low cost
Potential self-administration
Advantages
Advantages of microneedle patch vaccine delivery
Polymer
“The dermal immune system”
Langerhans cells
Dermal dendritic cells
How does microneedle-based immunization initiate
immune responses?
MN immunization results in an increase of cytokines produced by the skin
resident populations important for neutrophils, monocytes and dendritic
cells recruitment, which are key players in the innate immune response
• Objective 1
• Determine the effectiveness of seasonal influenza subunit
vaccine strains encapsulated in dissolving microneedle
patches in murine model.
• Objective 2
• Determine contribution of Langerin+ cells in adaptive
responses elicited by skin immunization with dissolving
microneedle patches.
• Objective 3
• Confirm vaccine stability in dissolving microneedle patches (in
vitro and in vivo studies)
• Objective 4
• Define efficacy of subunit vaccine in non-human primates after
microneedle delivery
Objective 1: Efficacy of dissolving microneedles
before 1 min
10 min skin
• A/Brisbane/59/07 (H1N1)
• A/Victoria/210/09 (H3N2)
• B/Brisbane/60/08
• Cohorts of BALB/c mice were
immunized once with 3 µg HA
of monovalent vaccine
intramuscularly (unprocessed
vaccine, IM; vaccine mixed with
excipients, IM exc. ) or
cutaneously (mMN, pMN)
Vaccine and routes of delivery Gelatin microneedles
Immunization with H1N1 vaccine conferred complete survival
in all vaccinated cohorts
0 1 2 3 4 5 6 7 8 9 10 11 12 13 140
10
20
30
40
50
60
70
80
90
100
110Naive
IM exc
mMN
pMN
Days post-infection
Perc
ent surv
ival
0 1 2 3 4 5 6 7 8 9 10 11 12 1360
70
80
90
100
110Naive
mMN
IM exc
pMN
Days post-infection
Perc
ent B
W c
hanges o
f surv
ivors
Challenge with 5xLD50 mouse-
adapted A/Brisbane/59/07
H3N2 vaccine conferred complete survival in pMN cohorts, whereas
mMN and IM cohorts showed partial or no survival.
0 1 2 3 4 5 6 7 8 9 10 11 12 13 1460
70
80
90
100
110
IM exc
IM
pMN
mMN
Naive
Days post-infection
Perc
ent B
W c
hanges o
f surv
ivors
0 1 2 3 4 5 6 7 8 9 10 11 12 13 140
10
20
30
40
50
60
70
80
90
100
110
IM
IM exc
mMN
pMN
Naive
Days post-infection
Perc
ent surv
ival
Challenge with 5xLD50 mouse-
adapted A/Victoria/210/09
Influenza B vaccine induced robust, long lasting immune
responses by skin immunization
w k. 2 w k. 4 w k. 104
8
16
32
64
128
256
512
pMN
mMN
IM exc
IM
Naive
a
a
anti-
B/B
risbane/6
0/0
8
HA
I tite
rs G
mean 9
5%
CI
wk4 wk104
16
64
256
1024
4096
16384Naive
IM
IMexc
mMN
pMN
a,b
a,b
a,b
Anti-
B/B
risbane/6
0/0
8 N
T tite
rs
(Gm
ean95%
CI)
DT- DT- DT- DT+0
102030405060708090
100110120
0 hr
24 hr
30%
vaccine retention in skin
Pix
el i
nte
nsity
, %
V 1 2 3 4 5 6 7 8 9 1 0 11 12
-DT
+DT
0 1 2 3 4 5 6 7 8 9 10 11 12 13 140
10
20
30
40
50
60
70
80
90
100
Naive
DT-
DT+
Days post infection
Rate
s o
f surv
ival (%
)
Conclusion 2: Langerin+ cells contribute to adaptive responses
Langerin-DTR skin
V: A/Brisbane/59/07
1-3: skin alone
4-6: skin at 0h
7-9: skin at 24h w/ DT
10-12:skin at 24h w/o DT
Western blot of HA in skin lysate
Antigen removal ± DT
Protective immunity ± DT
Conclusion 3: Vaccine in pMN is stable for at least 90 days at 25oC
Stability of HA in gelatin patches
Protective immunity after 3 months at 25oC
0 1 2 3 4 5 6 7 8 9 10 11 12 13 140
10
20
30
40
50
60
70
80
90
100
110
Naive
pMN
pMN diss IM
5xLD50 A/Brisbane/59/07
Days post infection
Perc
ent surv
ival
0 10 20 30 40 50 60 70 80 900
50
100
150H1N1
B
H3N2* * *
Days in storage
pM
N p
atc
h c
onte
nt, %
fro
m in
itial
load (
Mean
+ S
D)
Macaca mullatta
(rhesus macaque)
2-year old
Day 0 Day 7 Day 14
Three groups of monkeys (4 animals each) received 45 μg of the
trivalent vaccine by:
Hypodermic needle injection (IM)
Metal microneedle arrays
Polymer microneedle patches
Week 8 Week 9 Day 21 Day 28 Week 7
Prime Boost
Blood and nasopharyngeal swabs
Week 52
Blood
Objective 4: Vaccine efficacy in hon-human primates
Samples collected
Whole blood for CBC and WBC phenotype
Serum for humoral responses (binding Abs, HAI, Neutralizing antibodies
and plaque reduction assay)
Plasma for chemokines/cytokines (Luminex)
Whole blood for T cell responses (FACS, ELISPOT assays) and B cell
responses (ASC and memory B cells)
Nasopharyngeal swabs for mucosal responses (IgA)
0 1 2 3 4 7 8 9 524
8
16
32
64
128IM
mMN
pMN
bo
ost
prim
e
weeks after immunization
Anti-B
/Brisbane/6
0/2
008 H
AI tite
rs
0 1 2 3 4 7 8 9 524
8
16
32
64
128IM
mMN
pMN
bo
ost
prim
e
weeks after immunization
an
ti-A
/Brisb
an
e/0
9/0
7 H
AI tite
rs
0 1 2 3 4 7 8 9 524
8
16
32
64
128
256
512IM
mMN
pMN
bo
ost
prim
e
weeks after immunization
an
ti-A
/Vic
toria
/21
0/2
009
HA
I tite
rs
H1N1 H3N2
B
All groups showed similar HAI titers against influenza A
viruses after boost
0 7 14 21 280.0
0.1
0.2
0.3
0.4IM
mMN
pMN
days post-prime
IgA
(naso
phary
ng
eal) O
.D.
0 7 14 21 280.0
0.1
0.2
0.3
0.4IM
mMN
pMN
days post-prime
IgA
(naso
phary
ng
eal) O
.D.
0 7 14 21 280.0
0.1
0.2
0.3
0.4
0.5IM
mMN
pMN
days post-prime
IgA
(naso
phary
ng
eal) O
.D.
H1N1 H3N2
B
Mucosal IgA responses were higher in the dissolving
microneedle group, with a peak between 14 and 21 days
H1N1 H3N2
B
d0
d7
d2
1
d0
d7
d2
1
d0
d7
d2
1
0
50
100
150IM
mMN
pMN
B/B
risb
ane
/60
/08
sp
ecifi
c
IgG
se
cre
ting
ce
lls
d0
d7
d2
1
d0
d7
d2
1
d0
d7
d2
1
0
50
100
150IM
mMN
pMN
A/B
risbane/5
9/0
7 s
pecifi
c
IgG
secre
ting c
ells
d0
d7
d2
1
d0
d7
d2
1
d0
d7
d2
1
0
50
100
150IM
mMN
pMN
A/V
icto
ria/2
10/0
9 s
pecifi
c
IgG
secre
ting c
ells
Animals immunized with pMN showed higher influenza-specific ASC numbers
three weeks after prime and flu specific memory B cells 2 weeks after boost
IM mMN pMN IM mMN pMN
0
50
100
150
200500
2500
4500
0
500
1000
1500
2000
2500
3000
3500
4000
4500d14 pb
TIV
specifi
c m
em
ory
B c
ells
/
10
6 P
BM
C
Tota
l IgG
mem
ory B
cells
/
10
6 PB
MC
wk0
wk1
wk2
wk3
wk4
wk7
wk8
wk9
wk5
2
4
8
16
32
64
128
256
512
1024
2048
IM
mMN
pMN
boost
prim
e
anti-B
/Brisbane/6
0/0
8 N
T titers
B: max titers 2048
wk0
wk1
wk2
wk3
wk4
wk7
wk8
wk9
wk5
2
4
8
16
32
64
128
256
512
IM
mMN
pMN
boost
prim
e
a-A
/Vic
toria/2
10/0
9 N
T titers
wk0
wk1
wk2
wk3
wk4
wk7
wk8
wk9
wk5
24
8
16
32
64
128
256
512
1024
2048
IM
mMN
pMN
boost
prim
e
a-A
/Brisb
an
e/5
9/0
7 N
T t
ite
rs
H1N1: max titers 512-1024 H3N2: max titers 128
Skin vaccination induced similar levels of neutralizing
antibody titers against all influenza viruses as the IM group
Cellular immune responses
• T cell immune responses were similar among all
vaccinated groups:
• Comparable frequencies of activated CD4+ and CD8+ TCM and
TEM cells among all vaccinated cohorts
• Comparable frequencies of proliferating CD8+ TCM and TEM cells
among all vaccinated cohorts
• The dissolving microneedle group demonstrated more consistent
proliferative capacity of CD4+ TEM cells
In the dissolving microneedle group we observed higher numbers of
IL-4 secreting T cells with a peak 3 weeks after prime
Conclusions
• Although the sample size of this study is too small to draw
conclusions the result gives us a hint of what we should expect
in clinical trials: diversity of responses and at least non-
inferiority between technologies
• More correlates of immunity should be included in vaccine
technology development:
Systemic humoral responses are not different between
intramuscular or skin vaccination but mucosal responses
may benefit from skin delivery
Skin vaccination may exert quantitative but not qualitative
advantages in T cell responses but in vaccines robustness of
immune response is critical.
Future plans
• Phase I clinical trials of dissolving microneedles in
2015
• Improving immune responses in high risk
populations
• Vaccine stability studies at various temperatures
and extended times
• Novel adjuvants for skin immunization
ACKNOWLEDGMENTS
I. Skountzou
R.W. Compans
E. V. Vassilieva
J. A. Pulit-Penaloza
C. Ibegbu
E. S. Esser
M. T. Taherbhai
S. Gill
D.G. Koutsonanos
M. del P. Martin Matos
W.C. Weldon
A. Stavropoulou
Z. Ashraf
M. McLausland
J. Jacob
L. Ye
M. P. Prausnitz
H.S. Gill
V. G. Zarnitsyn
S.P. Sullivan
J.W. Lee
S.O. Choi
N. Murthy
H. Kalluri
D. McAllister
W. Pewin
C. Edens
S. Henry
Derek O’ Hagan Ph.D.
Sushma Kommaredy
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