VHIR Seminar led by Gerrit Borchard, Section of Pharmaceutical Sciences University of Geneva, University of Lausanne Biopharmaceutical Sciences Geneva Switzerland. Abstract: In order to enhance the efficacy of vaccines, antigen and adjuvants are combined in particulate carrier systems resembling pathogens in size, shape and surface properties. These novelnano- and microcarriervaccines strategies, using DNA or subunit vaccines as antigens and specific ligands of receptors of the innate immune system,offer several advantages, such as enhanced immune recognition, direction of immune response bias, and enhancement of vaccine stability. We are focusing on eliciting protective immune responses against M. tuberculosis, a pathogen transmitted through inhalation, bydeveloping vaccine delivery systems composed of different materialsand administered by the mucosal route.

1. TLR
ligand
func.onalized
nanocarriers
to
enhance
immunogenicity
of
vaccines
J.
Poecheim
&
G.
Borchard,
Ph.D.
Vall
d’Hebron,
Ins.ut
di
Recerca
VHIR
Barcelona,
Catalunya
5.11.2013

2. Adjuvants…
“…the
immunologist’s
dirty
liPle
secrets”
C.A.
Janeway,
Cold
Spring
Harb
Symp
Quant
Biol
1989
Adjuvant

3. What
makes
viruses
immunogenic?
If
drugs
are
similar
or
iden.cal
with
respect
to
structure
and
mechanism
of
ac.on
(MOA)
to
endogenous
substances…
…should
drug
delivery
systems
not
resemble
their
“natural”
counterparts,
as
well?
Adjuvant

4. What
makes
viruses
immunogenic?
Viruses
Nature’s
best
(and
worst)
delivery
systems

5. Viruses
are
par.cles
• Uptake
by
an.gen-­‐presen.ng
cells
(APC)
depends
on
shape,
size
(10nm-­‐3µm),
surface
charge,
receptor
interac.ons,…
• Uptake
triggers
matura.on
of
dendri.c
cells,
trafficking
to
lymph
nodes
and
T-­‐cell
ac.va.on
• Viruses
interact
directly
with
B-­‐cells,
triggering
an.body
response
• Uptake
of
par.culate
an.gen
leads
to
cross-­‐presenta.on,
which
is
absent
in
soluble
an.gens

6. Viruses
show
repe..ve
structures
• Viruses
have
limited
gene.c
informa.on
for
proteins
• Viral
surface
is
quasi-­‐crystalline,
of
repe..ve
subunits
• Direct
ac.va.on
of
B-­‐cells,
breaking
tolerance
• T-­‐cell
independent
IgM

7. Viruses
replicate
• Sustained
an.gen
exposure
• Induc.on
of
T-­‐cell
memory,
important
at
re-­‐infec.on
• Size
of
T-­‐cell
memory
pool
is
dependent
on
dura.on
of
exposure
to
an.gen

8. Viruses
ac.vate
the
innate
immune
system
• Interac.on
with
pathogenic
paPern-­‐recogni.on
receptors
(PRRs),
e.g.,
Toll-­‐like
receptors
(TLRs)
• PRRs
are
expressed
on
many
cell
types,
including
APCs,
epithelial
and
B-­‐cells
• First
line
of
defense
against
infec.on
• Ac.va.on
of
adap.ve
immune
system

9. Adjuvants…
TLR,
Toll-­‐like
receptor
NLR,
nucleo.de-­‐binding
oligomeriza.on
domain
(NOD)-­‐like
receptor
RIG,
re.noic
acid-­‐inducible
gene
(RIG)-­‐1-­‐like
receptor
Adjuvant
Higgins
&
Mills,
Curr
Infect
Dis
Rep
2010

10. Toll-­‐like
receptors
C.
Nüsslein-­‐Volhard

11. 11

12. Pathogenic
paPern
recogni.on
receptors
(PRR)
Adjuvant
NOD:
Nucleo2de
Oligomeriza2on
Domain
TLR:
Toll-­‐like
Receptors

13. Adjuvants:
Toll-­‐like
receptor
agonists
• Insoluble
aluminum
salts
(alum)
and
uric
acid
crystals
poten.ally
ac.vate
the
NALP3
inflammasome,
as
does
chitosan
in
vitro
• Muramyl
dipep.de
(MDP,
NOD2),
minimum
effec.ve
component
of
complete
Freund’s
adjuvant,
pyrogenic
• Poly
I:C
(TLR3
and
RIG-­‐1),
synthe.c
analog
of
dsRNA,
Ampligen®,
in
clinical
trials
• LPS
(TLR4),
1955,
too
toxic
for
use
in
human
vaccines
• MPL
(TLR4),
modified
lipid
A
moiety
of
LPS,
included
in
Cervarix®
(HPV
vaccine)
as
AS04
(MPL
+
AlOH3)
• E6020,
synthe.c
and
selec.ve
TLR4
ligand
based
on
lipid,
in
combina.on
with
MF59
(squalene,
Tween
80,
Span
85
in
citrate
buffer)
o/w
emulsion

14. Par.culate
carriers
for
mucosal
immuniza.on
• TLRs
are
PaPern
Recogni.on
Receptors
present
on
diverse
cell
types
(epithelial,
immune
cells)
• Recognize
specific
molecular
paPerns
present
in
pathogens
like
bacteria,
viruses
or
fungi
• TLR
agonists
induce
matura.on
of
DC
and
ac.vate
the
immune
system
• Pam3Cys
(TLR-­‐2),
bacterial
recogni.on,
favor
T H 2,
produc.on
of
Ab
• IMQ
(TLR-­‐7),
viral
recogni.on,
favor
TH1,
cellular
IR
• Synergy?

15. Mucosal
immuniza.on:
a
real
challenge
• Protec.ve
mucosal
immune
responses
are
most
effec.vely
induced
by
mucosal
immuniza.on
• Protec.ve
immunity
against
mucosal
pathogens
requires
novel
vaccine
strategies
ac.va.ng
mul.ple
arms
of
the
innate
and
adap.ve
immune
systems
Successes
Poliovirus
Influenza
virus
S.ll
pending…
HIV
Herpes
virus
Mycobacterium
Lehner,
J
infect
Dis,
1999
-­‐
De
Magistris,
Adv
Drug
Deliv
Rev,
2006
Belyakov
IM,
J.
Immunol
(2009)

16. Use
of
nanopar.cles
for
mucosal
vaccina.on
-­‐ Protec.on
of
the
an.gen
against
degrada.on
-­‐ Avoid
an.gen
dilu.on
on
mucosa
-­‐ Targe.ng
of
an.gen-­‐presen.ng
cells
(APC)
-­‐ Increase
an.gen
uptake
by
immune
cells
-­‐ Failed
aPempts
using
synthe.c
biodegradable
NPs
(PLGA/
PLA):
No
induc.on
of
dendri.c
cell
matura.on
in
vitro
-­‐ Strategy:
Addi.on
of
immunos.mulatory
molecules
-­‐ Combina.on
of
different
PRR
ligands:
synergis.c
effect?

17. 17
VACCINE ADJUVANTS
“a
substance
used
to
stimulate
the
immune
system
to
provide
immunity
and
is
treated
to
act
as
an
antigen
without
inducing
the
disease”
Oxford dictionaries
Latin vaccinus, from vacca 'cow‘ (Edward Jenner, 1796)
"germ theory of disease“ (Louis Pasteur, 1880)
Latin adjuvare, meaning "to help“ (G.Ramon, 1925)
↑
specific immune responses to the antigen
special type of excipients

18. 18
MODERN VACCINE STRATEGIES
v Traditional vaccines: live-attenuated or whole-inactivated organisms.
→
Generally do not require adjuvants.
v “Modern
vaccines”:
subunit vaccines
Highly purified/ recombinant antigenic
proteins/ epitopes
DNA vaccines
Plasmid encoding antigenic protein
Safer, long-term protection, more specific
BUT: far less immunogenic than traditional vaccines
→ Need for improved, safe, and more powerful adjuvants!
www.niaid.nih.gov

19. 19
New generation vaccine formulation
Danger signals:
Pathogen products (e.g.
TLR ligands, NLR ligands)
Delivery system:
- Mineral salts (Alum)
- Micro- and nanoparticles
- Emulsions
- Liposomes
- Virosomes
- VLP
Vaccine antigens:
- Recombinant proteins
- Gene delivered antigens
Immune potentiators:
MPL, MDP, CpG ODNs,
Flagellin, Lipopeptides,
Saponins, dsRNA, small
molecule immune potentiators
(Imiquimod)

20. 3D
model
of
the
human
airway
barrier
PhD Defence 2010 / Heuking
Blank,
et
al.,
Am.
J.
Respir.
Cell
Mol.
Biol
(2007)
36,
669-­‐677.

21. Study design
Empty CTC NP
pGFP NP
CTC pGFP NP
+
+
nm
scale
CTPPC
+
+
nm
scale
Blank,
et
al.,
Am.
J.
Respir.
Cell
Mol.
Biol
(2007)
36,
669-­‐677.
+
nm
scale
+
21

22. Uptake
into
MDM:
CLSM
MDM
MDM
MDM
20
μm
20
μm
20
μm
Empty CTC NP
pGFP NP
CTC pGFP NP
+
+
nm
scale
CTPPC
+
+
nm
scale
+
nm
scale
+
22

23. 20
μm
CTPPC pDNA NP (N/P 3:1)

24. Uptake
into
MDDC:
CLSM
20
μm
20
μm
Empty CTC NP
CTC pDNA NP
+
+
nm
scale
20
μm
CTPPC pDNA NP
+
+
nm
scale
+
nm
scale
+

25. 20
μm
PhD Defence 2010 / Heuking
CTC pGFP NP (N/P 3:1)
25

26. Uptake pattern
100,0
Uptake
[%]
80,0
60,0
40,0
20,0
0,0
1
MDM
MDDC
EC
Uptake of pDNA NP into MDM, MDDC or epithelial cells (EC): unloaded CTC NP (white bar),
CTC pGFP NP (sheded bar) and CTPPC pGFP NP (dotted bar). Presented data are the mean ±
standard error of the mean of three independent experiments. Differences were considered
significant for * p<0.05.

27. Immune response: IL-8
*
IL-­‐8
[ng/ml]
20.0
*
15.0
NS
10.0
*
+
+
+
5.0
+
0.0
Medium
control
CTC
NP
CTC
pGFP
NP
CTPPC
pGFP
NP
ELISA: IL-8 release in the basolateral compartment from co-culture model due to pDNA NP
exposure. Differences were considered significant for * (p<0.05); NS, not significant.
Heuking,
et
al.
Nanobiotech.
11
(2013)
29

28. Immune
response:
TNF-­‐α
*
3.0
TNF-­‐alpha
[ng/ml]
*
NS
2.0
NS
+
+
1.0
+
+
0.0
Medium
control
CTC
NP
CTC
pGFP
NP
CTPPC
pGFP
NP
ELISA: TNF-α release in the basolateral compartment from co-culture model due to pDNA NP
exposure. Differences were considered significant for * (p<0.05); NS, not significant.
Heuking,
et
al.
Nanobiotech.
11
(2013)
29
28

29. Summary
(I)
+
+
+
+
+
+
nm
scale
***
IL-8 (ng/mL)
20
***
10
***
**
0
Medium
pDNA
NP CM25-TMC35
NP Conjugate
q Chemistry:
Successful
synthesis
of
TLR-­‐1/2
(Pam3Cys)
agonist
functionalized
chitosan
derivatives.
q Formulation:
Ability
of
Pam3Cys
decorated
pDNA
nanoparticles:
i) to
complex
DNA
(~400
nm,
~15-­‐20
mV),
by
forming
stable
particles
(release
study,
heparin
challenge),
ii) to
protect
the
plasmid
against
DNase
degradation
and
to
transfect
A549
and
HBE
cells.
q Immunogenicity
in
THP-­‐1
Φ:
Due
to
Pam3Cys
decoration
pDNA
nanoparticles
induced
higher
IL-­‐8
secretions
from
by
mTHP-­‐1
macrophages
and
3DCC.

30. Summary
(II)
IL-­‐8
[ng/ml]
20.0
15.0
10.0
5.0
0.0
Medium
control
CTC
NP
CTC
pGFP
NP
CTPPC
pGFP
NP
q For
pulmonary/bronchial
pDNA
vaccination,
the
use
of
CTTPC
versus
pDNA
alone
contributes
to
an
overall
higher
adjuvanticity:
q protection
against
enzymatic
degradation
q transfection
in
vitro
q transport
of
DNA
into
the
most
immune
competent
APC
type,
namely
dendritic
cells;
q increasing
the
overall
immune
response
(IL-­‐8,
TNF-­‐α).

31. 31
Enhancing cellular immune responses
Vaccine formulation
TLR and NLR signaling pathways
Nanocarrier
TLR 9 ligand
pDNA with CpG sequence
encoding antigen 85A
NOD 2 ligand
MDP
Muramyl dipeptide
Proinflammatory cytokines
Nucleus

32. 32
Presentation of the project
The aim is the preparation, characterization and in vitro testing of particulate carrier systems that
are able to target and stimulate immune cells by combinations of PRR ligands incorporated and/or
decorated on the particle surface.
Vector 1:
Trimethyl chitosan nanoparticles
Vector 2:
Squalene in water emulsion nanodroplets
Vector 3:
Cationorm ®
Antigen: Ag85A (Mycobacterium tuberculosis)
Immunostimulator #1: unmethyl. CpG sequence (TLR 9 ligand)
Immunostimulator #2: MDP (NOD 2 ligand)

33. 33
Nanoparticle preparation techniques
1) Trimethyl chitosan (TMC)/Chondroitin sulfate (CS) nanoparticles
Complex coacervation method:
Positively charged TMC 0.5%
+ negatively charged CS 0.1%
CS
Mean size:
283.3 nm ±
4.3
TMC
+
500 nm

34. 34
Nanoparticle preparation techniques
2) O/W emulsion preparation
1) homogenized for 1 min,
rpm
2) High shear processing
(Microfluidizer M110S)
DOTAP
Mean size:
129.5 nm ±
3.3
Distilled water
0.5% Span 85
0.5% Tween 80
5 % squalene
500 nm
10 000

35. 35
3) Cationorm ® cationic O/W emulsion
Cationorm ®
Oil
Mineral oil
Cationic agent
Cetalkonium chloride
Surfactants
Poloxamer, tyloxapol
Water
Water for injection
500 nm

36. 36
Size, zeta potential of nanocarriers
Carrier
Size [nm]
Zeta potential
[mV]
Polydispersity
TMC/CS
283.3 ±
4.3
33.0 ± 0.7
0.27
TMC/CS-pDNA
356.8 ± 33.4
16.9 ± 3.8
0.41
DOTAP-SWE
129.5 ±
3.3
22.8 ±
0.1
0.09
DOTSP-SWE-pDNA
165.8 ±
4.3
-17.5 ±
1.0
0.14
Cationorm ®
158.17 ± 2.28
14.53 ± 0.38
0.24
Cationorm ®-pDNA
216.62 ±
1.48
-35.65 ±
4.17
0.34

37. 37
Toxicity profile of nanocarriers
•
•
•
•
Cell
line:
RAW264.7
murine
macrophages
Dilu.on:
1:10
Incuba.on
.me:
24
h
Evalua.on:
XTT
prolifera.on
assay

38. 38
Immunogenicity of functionalized pDNA nanocarriers in vitro
•
•
•
•
Cell
line:
RAW264.7
murine
macrophages
Dilu.on:
1:10
Incuba.on
.me:
24
h
Evalua.on:
ELISA
mTNF-­‐α
***
***
***
Synergistic expression of TNF- α!
Values are means of 3 experiments; *** p <0.001

39. 39
Uptake of functionalized pDNA nanocarriers in vitro
•
•
•
•
Cell
line:
A549
human
alveolar
basal
epithelial
cells
Dilu.on:
1:10
Incuba.on
.me:
over
night
Evalua.on:
Confocal
microscopy
-­‐
Vectashield
moun.ng
media
containing
DAPI
-­‐
GFP-­‐pDNA
-­‐
MDP-­‐Rhodamine

40. 40
Summary
• The DNA vaccine formulations have been shown to be safe
• Both resulted in an increased pro-inflammatory cytokine release
by targeting TLR-9 and NLR-2.
• They elicited a synergistic enhancement as a result of delivering
two innate immune receptor ligands at the same time.
• Uptake and protein expression has been confirmed.

41. 41
Perspectives
a) In vitro: 2 questions to answer:
Repetition of synergistic studies: additive or synergistic effect
How do the ligands get into the cell/ into the nucleus?
→ Investigation of uptake mechanisms
b) In vivo:
- in Balb/c mice as immunological model for Th1 response
- Nod2 knock out mice: Synergistic effect NOD2-receptor dependent?

42. Immune response studied
§ Increase of anti-Ag85A antibodies by ELISA on serum:
Total IgG
§ Cellular responses : isolation of spleen
b1) ex-vivo protein stimulation : IFN-γ, IL-2, TNF-α, IL-4 (ELISA)
b2) Lymphocyte proliferation (XTT reagent)
b3) FACS – IFN-gamma, CD4+/CD8+
b4) ELISPOT
Restimulation of splenocytes with recombinant protein Ag85A in vitro, ev. with CD8+
specific peptide

43. 43
Acknowledgements
• UNIGE
• VFL
• IBCP
Dr. Nicolas Colin
Dr. Simon Heuking
Dr. Livia Brunner
Dr. Charlotte Primard
Prof. Gerrit Borchard
Dr. Christoph Bauer
Emmanuelle Sublet
Dr. Annasara Hansson
Dr. Leonardo Lauciello
Christian Reichert
Shqipe Kelmendi
Najoua Bennani