liposomal dna vaccines - formulation...vaccines & public health ‘the two public health...
TRANSCRIPT
Vaccines & Public health
‘The two public health interventions that have had the greatest impact on the World’s health
are clean water and vaccines’
– The World Health Organisation
Types of vaccines
1. Live attenuated BCG, polio
2. Inactivated (dead) polio
3. Sub-unit Hep B
4. DNA vaccines?
Safety
efficacy
DNA vaccines
% D
NA
vacc
ine
Injection site Lymph node
Rapid degradation in vivo
Plasmid encoding HBsAg
Sub-unit antigen
Hep B
SUV MLV
Hydrophilic head
Lipophilic tail
Multilamellar vesicles (MLV)
Large unilamellar vesicles (LUV)
Small unilamellar vesicles (SUV)
Bilayer formation in aqueous environment
Size
Lipid molecule
Liposomes as delivery systems:
Entrapment of DNA into liposomes
Liposomes
DNA
SUV-DNA complexes
+ +
+
+ +
+
+ +
+ +
+
+ +
+
+ +
Dehydrate / rehydrate
+ +
+
+ +
+
+ +
+ +
+
+ +
+
+ +
DRV with entrapped DNA
DRV(DNA)
Cyro-electron microscopy of DRV(DNA)
Perrie & Gregoriadis, 2000
DNA vaccines – mimic a viral response
Perrie et al., 2001
Liposome formulations
+ + +
+
+ +
+ +
+
+
Disteroyl phosphaditylcholine (DSPC)
Phosphatidylcholine (PC)
Dioleoyl phosphatidylethanolamine (DOPE)
Dioleoyl trimethylammonium propane (DOTAP)
or
The effect of phospholipid Tc: Physicochemical characteristics
Liposomes DNA incorporation (% of used)
Zeta Potential
(mV)
Size (nm)
PC liposomes
94 ± 3
32.1 ± 0.3
679 ± 96
DSPC liposomes 91 ± 4 32.9 ± 0.4 1025 ± 153
Blood serum samples taken at time intervals and tested for anti-HBsAg (S region) IgG subclasses by ELISA.
0 14 28 days
IM injection 10 ug DNA
The effect of phospholipid Tc:
0
50
100
150
200
250
300
PC DSPC
Luci
fera
se a
ctiv
ity (%
Lip
ofec
tin)
Transfection efficiency (in vitro, cos7):
* p<0.05 (compared with PC:DOPE:DOTAP)
# p<0.001 (compared with DRV(DNA))
*
#
1
1.5
2
2.5
3
3.5
PC DSPC NakedDNA
Rec
ipro
cal s
era
end
poin
t dilu
tion
(log1
0)
Immune response:
0.000.050.100.150.200.250.300.350.400.450.50
Naked DNA DRV(DNA) control
IL-4
(ng/
sple
en)
Cell-mediated responses
02468
101214
Naked DNA DRV(DNA) control
IFN
-gam
ma
(ng/
sple
en)
IL-4 and IFN-gamma levels in the spleens of mice immunised with naked of liposome-entrapped pRc/CMV HBS
Gene Expression: Liposomal (pCMV.efgp) Liposomal DRV(DNA) – PC:DOPE:DOTAP
DRV(DNA) Naked DNA Control
Popl
iteal
In
guin
al
Mus
cle
Types of vaccines
1. Live attenuated BCG, polio
2. Inactivated (dead) polio
3. Sub-unit Hep B
Safety
efficacy
Adjuvants
Adjuvant Produced by Disease
Aluminium salts Various Various
MF59® (squalene) Novartis Influenza
AS03 (squalene+ tocopherol)
GSK biologicals Influenza
AS04 (MPL+aluminium hydroxide)
GSK biologicals HPV, HBV
Virosome Crucell Influenza, HAV
Adjuvants
Most subunit vaccines require adjuvants in order to induce protective immune responses to the targeted pathogens.
Adjuvants ‘Immunologist’s dirty little secret’
‘
Adjuvants
APC
CD80
MHC Cl II
MØ
MØ
1. Danger
2. Signal ‘0’
3. Recombinant signal 2
4. Antigen delivery
5. Depot-effect
Infiltrating phagocytes
Alarmins
PRR:PAMP interactions
Increase in co-stimulatory molecules
Adjuvant:antigenassociation
Dimethyldioctadecylammonium (DDA)
Cationic liposomal adjuvants)
α,α’-trehalose 6,6´-dibehenate (TDB)
Cationic lipid
Immunostimulator
moderate/strong TH2 Strong TH1 Carry antigen to APC
Not effective without a delivery system Engages a TLR-independent Syk/Card9-dependent pathway
Lipid
1.evaporation 2. Addition of Buffer 3. Warm + Vortexing
Preparation of liposomes
4. Add Antigen solution
+ + +
+
+ +
+ +
+
+
- -
- -
- -
- - -
-
DDA:TDB liposomes
Davidsen, Perrie et al., BBA, 1718 (2005) 22-31.
10μm
46 oC
43 oC
Size (nm)
ZP (mV)
Loading (%)
DDA 488 ± 124 +46 ± 1.6 89 ± 10%
DDA:TDB 416 ± 40 +48 ± 5.1 87 ± 8%
DDA
DDA:TDB
Cationic liposomes for vaccine delivery
DDA Delivers antigen to DCs
TDB Activates DCs through Syk-Card9 signalling pathway and induces Th1 and Th17.
Adjuvant Delivery system
Liposomes for vaccine delivery: Formulation & Function
Physical/chemical Characteristics
Biological function
Pharmacokinetics and release
Stability +
+ +
+
+ +
+ +
+
+
- -
- -
- -
- - -
-
Identify controlling parameters
Size, Surface properties Antigen loading/ release Thermal analysis Chemical analysis
Considering delivery of liposomal adjuvants
Antigen:
Antigen
Liposomes:
Tyrosine residues
Investigating the bio-distribution and cell recruitment
Dual labelled antigen and adjuvant
Monocytes pre-stained using pontamine blue (2 – 3 days prior)
Gamma counting
Tritium counting
Antigen levels
CAF levels
Liposomes promote depot effect and TDB promotes monocyte recruitment
0
20
40
60
80
100
0 5 10 15
% D
ose
at in
ject
ion
site
Time (days)
Antigen only DDA + Antigen DDA:TDB + Antigen
Henriksen., 2010a, Journal of Controlled Release.
Controlled release of the liposomes from the depot site?
+ +
+ +
+ +
+ +
+
+
- -
- -
- -
-
- -
- -
-
- -
-
-
-
-
-
-
DDA - cationic
DSPC - neutral
DSPC DDA
Characteristics: Role of DDA
Antigen loading (% of amount added)
84 ± 3 78 ± 4 80 ± 6 62 ± 4 15 ± 3
-25
0
25
50
75
100
0
500
1000
1500
2000
250/0/50 150/100/50 100/150/50 50/200/50 0/250/50
Zeta
pot
entia
l (m
V)
Size
(nm
)
DDA/DSPC/TDB (µg/dose)
500 nm
500 nm 1µm
DSPC DDA
Hussain et al., 2014. Journal of Pharmacy and Pharmacology
0 1 4 140
20
40
60
80
100 DDA:TDBDSPC:TDB
***
***
Days post injection
% li
poso
me
dose
at S
OI
0 1 4 140
20
40
60
80
100DSPC:TDBDDA:TDB
***
Days post injection
% a
ntig
en d
ose
at S
OI
+ + +
+
+ +
+ +
+
+
- -
- -
- -
-
- -
-
-
-
- -
-
-
-
-
-
-
Liposome & Antigen retention – site of injection
Neutral liposomes cleared quicker Better retention of bound antigen
DDA/TDB
DSPC/TDB
Monocyte influx
Henriksen., 2010b, Journal of Controlled Release.
Immunisation study time scale
Day 9, 24, 37, 49 measure Ab responses
Measure Cytokines and cell proliferation
28 days
0.5 1.5 2.5 3.5 4.5 5.50.51.52.53.54.55.5
Day 9Day 24Day 37Day 49
DDA DSPC TDB
250 0 50
150 100 50
100 150 50
50 200 50
0 250 50
Free antigen
Formulation (µg/dose) IgG2b antibody titre IgG1 antibody titre
***
***
***
***
***
***
***
Reciprocal end point serum dilution (log10)
DS
PC
D
DA
Hussain et al., 2014. Journal of Pharmacy and Pharmacology
DDA DSPC TDB
250 0 50
150 100 50
100 150 50
50 200 50
0 250 50
Formulation (µg/dose)
DDA/DSPC ratio: Th1 responses are dependent, Th2 responses are independent.
DS
PC
D
DA
Hussain et al., 2014. Journal of Pharmacy and Pharmacology
010002000300040005000
IFN-γ
0 500
IL-5
pg/mL pg/mL
Multivariate Data Analysis (MVDA) Data collection PCA PLS
Large amounts of data collected Multiple variables, observations and responses More than one variable
Principal Component Analysis Data Mining Clusters / Trends Outliers
Partial least square Analysis Multivariate regression analysis Selection of important
variables for a selected response
Importance of variables Correlation of variables to
responses
Chemometrics Projection Methods PCA, PLS
Variables K Underlying Assumptions - X-variables are not
independent - X-variables may have errors - Outlier detection - Residuals may be
structured
Data table
Variables: Immune responses Lipid composition Size of Liposomes (Zeta potential of liposomes)
Responses: Immunity IL-2 IL-5 IL-6 IL-10 INF-g Spleen Proliferation IgG IgG1 IgG2b
Link between Variables and responses Cluster in immune
responses Most influential variable
Obs
erva
tions
N
Data table
MVA analysis of vaccine efficacy
Clustering relationships – Th1 responses driven by DDA content
Kastner et al., 2014. Accepted.
+ + +
+
+ +
+ +
+
+
+
+
+
+
+
+
+ +
+ + +
+
+ +
+ +
+
+
- -
- -
- -
- - -
-
Antigen needs to be adsorbed for co-delivery
Henriksen et al., 2011, Molecular Pharmaceutics.
-
-
- -
-
-
-
-
-
-
Both retained at SOI Both drain from SOI Cationic liposomes retained, Ag drains
Monocytes recruitment x Monocytes recruitment
Good protection APC presentation, IFN-g and IL-17
Keep the DDA content and enhance the flow?
DDA:TDB
Aggregation = Depot formation = Strong Th1
response
Modify this depot effect?
Retain DDA but mask the charge?
DDA:TDB
Aggregation = Depot formation = Strong Th1
response
PEGylated DDA:TDB
Reduction of Aggregation = Depot
formation?
010203040506070
DDA:TDB 5% PEGDDA:TDB
10% PEGDDA:TDB
25% PEGDDA:TDB
Zeta
pot
entia
l (m
V)
0
20
40
60
80
100
DDA:TDB 5% PEGDDA:TDB
10% PEGDDA:TDB
25% PEGDDA:TDB
% A
ntig
en lo
aded
PEGylated DDA:TDB
Pegylation of the liposomes – masking the charge
10% PEG DDA:TDB
DDA:TDB 25% PEG DDA:TDB In
ject
ion
site
Day 1 Day 4 Day 140
25
50
75
10010% PEG SUV25% PEG SUV
DDA:TDB SUVp<0.05
p<0.001
p<0.001
Days post injection
% li
poso
me
dose
at t
he S
OI
~120 nm
PLN
1 4 140.0
0.1
0.2
DDA:TDB SUV10% PEG SUV25% PEG SUV
0.3
0.4
0.5
0.6
Days post injection
% a
ntig
en d
ose/
mg
tissu
e fr
om t
he P
LN
1 4 140.0
0.1
0.2
0.3
0.4
0.5
0.6 DDA:TDB SUV10% PEG SUV25% PEG SUV
p<0.001
Days post injection
% li
po
som
e d
ose
/mg
tis
sue
fro
m t
he
PL
N
Small pegylated liposomes:
Kaur et al., 2012a and b, Journal of Controlled Release.
Kaur et al., 2012a and b, Journal of Controlled Release.
~120 nm
-5 -4 -3 -2 -1 0 1 2 3 4 5
DDA:TDB MLV
DDA:TDB SUV
10% PEG SUV
25% PEG SUV
Antigen
Naïve
Anti-Ag85-ESAT-6 IgG1 titres Anti-Ag85-ESAT-6 IgG2b titres
00 00 0
** **
0
Ear
ly a
ntib
ody
resp
onse
Day 12
PE
G
Pegylation promotes early immune responses
0 1000 2000 3000
0
10
25
IFN-g (pg/mL)
% P
EG
IFN
-g re
spon
se
PE
G
Summary – need co-delivery and possible controlled movement to lymphatics
Lymph nodes
Acknowledgements
Aston Team Fraser Crofts Dr Malou Henriksen Sameer Joshi Elisabeth Kastner Dr Randip Kaur Swapnil Khadke Behfar Moghaddam John Pollard Peter Stone Dr Jit Wilkhu Dr Alex Wilkinson
Statens Serum Institute Prof Peter Andersen Dr Else Marie Agger Dr Dennis Christensen Dr Karen Korsholm
We gratefully acknowledge our Sponsors:
BBSRC, EPSRC, European Commission contract No. LSHP-CT-2003-503367