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