Research challenges for the
delivery of vaccines
Prof Yvonne Perrie et al.
y.perrie@aston.ac.uk
1
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
The impact of
vaccination
Vaccine Failures
Malaria
225 million cases per years
Nearly 1 million deaths a year
HIV
25 million deaths
33 million people living with HIV
2.6 cases every year
1.8 million deaths per year
TB
9.6 million new cases a year
1.7 million deaths year
Pneumococcus
Papillomavirus
H influenza B
Polio (IPV)
successful
Meningococcus
unsuccessful
Measles
HAV
Ditheria
ANTBODIES
Challenge:
3 global killers
Tetanus
MMR
Polio
Malaria
TB
T CELL
HIV
ANTIGEN STABILITY
1 DAY
1 YEAR
10 YEARS
NO CHANGE
(Rappuoli R, Aderem A. (2011) Nature 437: 463-469)
Other considerations
Disease?
Species?
Route of administration?
Response?
Types of vaccines
1. Live attenuated
BCG, polio
polio
3. Sub-unit
Hep B
Safety
efficacy
2. Inactivated (dead)
What makes a
Vaccine?
VACCINE
Biological
Component
Delivery
Mechanism
Whole Organism
Protein
Carbohydrate
etc.
Adjuvant
Alum
AS009, AS008, MB666
Experimental Adjuvants
“raw”
recombinant protein
live viral vector
naked DNA
loaded onto APCs
liposomes
etc.
Challenge – improving subunit vaccine
efficacy
Most subunit vaccines require adjuvants in order to induce protective
immune responses to the targeted pathogens.
Adjuvant
Produced by
Disease
Aluminium salts
Various
Various
MF59® (squalene)
Novartis
Influenza
AS03 (squalene+
GSK biologicals
tocopherol)
AS04 (MPL+aluminium GSK biologicals
hydroxide)
Virosome
Crucell
Influenza
HPV, HBV
Influenza, HAV
Adjuvants
‘Immunologist’s dirty little secret’
‘
Challenge – designing adjuvants
Alarmins
Adjuvant:antigen
association
5. Depot-effect
1. Danger
MØ
4. Antigen delivery
MØ
Increase in co-stimulatory
molecules
Infiltrating phagocytes
2. Signal ‘0’
APC
MHC Cl
II
PRR:PAMP interactions
CD80
3. Recombinant signal 2
Research aim
To develop a sub-unit vaccine that offers protection against TB
1/3 of the world population has latent
TB
Second only to HIV as greatest
worldwide killer.
BCG is effective against severe
forms, but less effective in all forms.
In UK, most infections are in London
Replacing BCG
Need for a new safe, effective and affordable vaccine
Protect against all strains of TB (including MDR-TB)
Suitable for use in children, adolescents and adults
Safe for those living with HIV
SSI developed the Ag85B-ESAT6 fusion antigen
(fusion protein from mycobacterium tuberculosis).
Cationic liposomal adjuvants)
Cationic lipid
Dimethyldioctadecylammonium
(DDA)
moderate/strong TH2
Strong TH1
Carry antigen to APC
Immunostimulator
α,α’-trehalose 6,6´-dibehenate
(TDB)
Not effective without a
delivery system
Engages a TLRindependent Syk/Card9dependent pathway
Preparation of liposomes
4. Add Antigen
solution
Lipid
1.evaporation
2. Addition of Buffer
3. Warm + Vortexing
+-
+-
-+
+
-+
-
++
+
+-
+-
DDA:TDB liposomes
10μm
Size
(nm)
ZP
(mV)
Loading
(%)
DDA
488 ± 124
+46 ± 1.6
89 ± 10%
DDA:TDB
416 ± 40
+48 ± 5.1
87 ± 8%
DDA
46 oC
DDA:TDB
43 oC
Davidsen, Perrie et al., BBA, 1718 (2005) 22-31.
Cationic liposomes for vaccine delivery
DDA
Delivers antigen to DCs
TDB
Activates DCs through SykCard9 signalling pathway and
induces Th1 and Th17.
Log10 Reduction (CFU/lung)
1
0.8
Delivery
system
0.6
Adjuvant
0.4
0.2
0
DDA:TDB
BCG Danish
Mechanism of action?
??
2 immunisations, 28 days apart.
Log10 Reduction (CFU/lung)
Key measures for a TB vaccine:
Protection on challenge
IFNg production
1
0.8
0.6
0.4
0.2
0
DDA:TDB
BCG Danish
Liposomes for vaccine delivery:
Formulation & Function
Physical/chemical
Characteristics
-+
-
+
+-
-
Stability
++
-
+
-+
Size,
Surface properties
Antigen loading/ release
Thermal analysis
Chemical analysis
-
+
+
+-
Pharmacokinetics
and release
Biological function
Identify controlling
parameters
Considering delivery of liposomal adjuvants
Antigen:
Tyrosine
residues
Liposomes:
Antigen
Investigating the bio-distribution
Monocytes pre-stained
using pontamine blue
(2 – 3 days prior)
Gamma
counting
Antigen
levels
Dual labelled
antigen and
adjuvant
Tritium
counting
CAF
levels
Liposomes promote depot effect and TDB
promotes monocyte recruitment
Antigen only
DDA + Antigen
DDA:TDB + Antigen
% Dose at injection site
100
80
60
40
20
0
0
5
10
15
Time (days)
Henriksen., 2010a, Journal of Controlled Release.
Controlled release of the liposomes from the depot site?
DSPC - neutral
DDA - cationic
-+
+
+
-
+
-
+
+
-
+
-
-
-
+
DDA
-
+-
-
-
-
+
-
-
DSPC
Liposome & Antigen retention – site of injection
DDA:TDB
DSPC:TDB
% antigen dose
at SOI
- ++-+
+
++++
+ - +-
***
-
-
-
0
4
1
0
-
Neutral liposomes cleared quicker
***
-
***
Days post injection
20
-
0
DSPC/TDB
14
20
-
40
4
-
DDA/TDB
60
1
40
80
0
60
DDA:TDB
DSPC:TDB
Monocyte
influx
100
80
14
% liposome dose
at SOI
100
Days post injection
Better retention of bound antigen
Henriksen., 2010b, Journal of Controlled Release.
DDA/DSPC ratio:
Th1 responses are dependent,
Th2 responses are independent.
Formulation (µg/dose)
IFN-γ
DDA
DSPC
5000 4000 3000 2000 1000
pg/mL
0
DDA
DSPC
TDB
250
0
50
150
100
50
100
150
50
50
200
50
0
250
50
IL-5
0
500
pg/mL
Hussain et al., 2014. Journal of Pharmacy and Pharmacology
-+
+
-
Antigen needs to be adsorbed for co-delivery
+-
+-
+
-
++
+
+-
-
-
-
+
-
-
-
-
+-
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
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
Henriksen et al., 2011, Molecular Pharmaceutics.
Affordable?
Easy scale up
Heat stable
Scale-up - Microfluidics
Aqueous
Buffer (+
antigen)
Vaccine formulation
Controlling liposome characteristics:
Our DDA:TDB conc is locked
Kastner et al. 2015, in progress
Heat stable vaccines:
Long term stability of liposomes is a recognised problem.
Common format is a freezedried product.
3500
Vesicle size (nm)
3000
2500
H 20
>15%
antigen loss
2000
1500
γ-irradiate
1000
500
0
0
10
20
30
Sterile and stable
product?
Time (days)
Davidsen et al., BBA, 1718 (2005) 22-31.
Key Attributes of liposomal vaccine:
+ +
+
Ant
Excipients: Lipids & antigen
- Physico-chemical stability
The vaccine suspension
- Viscosity, pH
The freeze-dried sterile product
- water content
- long term stability
Antibody & Cellular
Liposome
Size
Surface
Loading
Release kinetics
Biological activity
Freeze-dried & Gamma Sterilisation:
charge
900
45
70
800
40
60
700
35
400
300
30
25
20
10
100
5
0
0
FD
Ster
3000
2500
CFS
50
40
30
2000
1500
1000
15
200
activity
IFN-g (pg/ml)
500
% cumulative release
50
600
release
80
1000
Zet Potential (mV)
Vesicle size (nm)
size
20
500
10
0
FD
Ster
0
0
25 50 75
time (hrs)
100
FD
Ster
Retained stability and biological efficacy.
Outcome:
DDA:TDB
Use Pattern recognition receptors,
including the Toll-like receptors (TLRs), to
enhance induction and activation of two
critical arms of the host defence
Summary – the challenges in the designing and delivery
of vaccines
Vaccine
type
Route
Adjuvant/
Delivery
Immune response
What is needed:
More effective
More affordable
Easier to use
Easier to access
Enhancing stability - Freeze-drying
Pressure (Pa)
The phase diagram of water
Critical
point
Liquid
A  B: freezing
B  C: reduced pressure
C  D: sublimation and drying
A
B
H20
Solid
Triple point
Vapour
610
C
D
0.0075
Temperature (°C)
Cryoprotection
Problem:
Membrane destabilisation
and aggregation
Solution:
Cryoprotect the liposomes
using carbohydrates
Stabilises by forming stable
boundaries
Vesicle Size (nm)
Vesicle Size (nm)
1500
1000
500
0
Fresh Liposomes
1500
1000
500
0
Freeze-dried
Mohammed et al., Methods, 40 (2006) 30-38.
Fresh Liposomes
Cyro-protected
Vaccine efficacy – protein activity not essential
There is no correlation
between immune response
and the retained activity
following lyophilization and
reconstitution.
Even though the activity
drops by about 50% in the
driest samples.
Antibody response in mice injected with AP vaccine as fraction of
retained AP activity in the reconstituted vaccines. Dashed line is a
linear regression of the data, with the R2 value shown.
Clausi, et al., (2008). J Pharm Sci
Protein activity after reconstitution
As the samples are dried
there is a drop in the protein
activity
however, the largest drop in
activity occurs when the
samples dry below 1 wt%
water
Fraction of enzymatic activity retained compared to activity in liquid
vaccine as a function of residual water following lyophilization.
Closed circles are FD, open circles are SFD samples.
Clausi, et al., (2008). J Pharm Sci.
Freeze-drying of the system
Probe 1 (0 mmol sucrose)
Probe 2 (200 mmol sucrose)
Shelf temperature
Condenser
Proposed end point of primary drying
Temperature (oC)
40
20
0
-20
-40
-60
Dried lipid product
-80
-100
-5
Frozen sample
5
15
25
35
45
55
Time (hours)
65
75
85
95
http://www.athenahealth.com/whitepapers/cost-of-vaccines/