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Microspheres as Delivery Systems for
Vaccines
Rosa Azhari, Ditza Levin,Yaffa MizrachiNebenzahl2,Eti Taub, Amir Dagan
Department of Biotechnology Engineering, ORT
Braude College, Karmiel, Israel
and 2Pediatric Infectious Disease Unit, Soroka
Medical Center, Beer Sheva, Israel
Vaccine Development is required
for:
• Infectious Diseases (HIV,
Hepatitis, SARS etc.)
• Cancer Immunotherapy
• Aoutoimmune Diseases
Control
• Allergy Suppression
Routes of Immunization
Oral
Imtramascular, Subcutaneous,
Intraperitoneal
Adjuvants
Enhancement of a specific immune response to vaccines
and
generation of long-term immune memory
Alum – the only adjuvant currently approved for use in
humans, (other adjuvants undergoing clinical trials at
different phases)
Limitations – not potent enough with the new generation
of antigens, directs towards one type of response (Th2)
Requirements from an
Adjuvant
Adjuvant Functions:
Strategy:
• Delivery to Antigen Presenting
Cells (APC)
• Antigen Encapsulation
in microparticles
• Active targeting
• Addition of immunomodulating agents
• Immuno-stimulation
• Slow Release
New Adjuvant Candidates:
• Particulates – liposomes
microspheres
virosomes
• Complexes with stimulating agents
Microspheres Formation by Complex
Coacervation
--
- -- - -
Interaction
Stabilization
--
+
+
+
+
+
+ + +
+ +
--+ +
-- ++- +- + +
- - + -+- +
Precipitation Microspheres
Formation
Complex Coacervation of Gelatin and
Chondroitin Sulfate
Gelatin – Derivative of Collagen
Positively charged at lower pH’s
pI=6.5
Chondroitin Sulfate - Polysaccharide
O
COOO
OH
OH
D- Glucuronic acid
CH2OH
OSO-3
O
O
Negatively charged
O
n
NHCOCH3
N-Acetyl- galactosamine-4-sulfate
SEM Micrographs of Ge-CS
Microspheres
Effect of CS Concentration on the size of
the Microspheres
0.2%
0.3%
0.1%
0.15%
Gelatin-Chondroitin sulfate (Ge-CS)
Microspheres’ Advantages for Vaccination:



Complex coacervation is a mild process that does not
damage the bioactivity of cytokines, antigens or
peptides.
Ge/CS MS are biodegradable and can be digested by
proteases in the extracellular matrix and in the
lysosomes of professional APC.
Ge and CS contain functional groups to which
targeting moieties can be attached.
Why is targeting important?
Antigen Presenting
Cells (APC):
MHC class II positive APC
Th
MQ
Ag
CD4
“Non-Self”
DC
Ag
Produce cytokines
that provide help for
humoral (antibodies)
and
cellular immune
responses
“Self”
Tc
CD8
BC
Ag
Kill pathogen-infected
cells and tumor cells.
MHC class I positive APC
Antigen Presenting
Cells (APC):
Th Immune Responses:
Cell Mediated Immunity
Th1
MQ
Ag
DC
BC
Ag
To
Viruses
Intracellular pathogens
Tumor Cells
T1Th1
Humoral Immunity
(Antibodies)
Ag
Th2
Bacteria
Toxins
Extracellular pathogens
The Role of Th1 and Th2 in the Pathology or
Resolution of Diseases
Th1
Infectious Disease
• Leishmaniasis
• Tuberculosis
• Leprosy
• AIDS
Th2
Protection
Protection
Protection
Protection
Damaging
Damaging
Damaging
Damaging
Autoimmune Disease
• Diabetes (IDDM)
• Multiple sclerosis
• Crohn’s disease
• Autoimmune thyroid
disease
Damaging
Damaging
Damaging
Damaging
Protection
Protection
Protection
Protection
Atopic Allergy
Protection
Damaging
Pregnancy
• Fetal Tolerance
• Unexplained recurrent
abortions
Damaging
Damaging
Protection
Protection
Gelatin-Chondroitin sulfate (Ge-CS)
Microspheres’ Advantages for Vaccination:


Activated T-cells express Ge receptors microspheres may attract T cells to the vaccination
site.
CS induces maturation of DC.
Can Ge/CS microspheres target
antigens to professional APC?
In-Vitro Presentation of Encapsulated Ag
Experimental system
CD4/CD8
OVA-specific
Hybridomas
BO-97-11
B3Z
OVA presentation
IL-2/ IL-2, LacZ
by APC
In-vitro Presentation of Encapsulated Ag
to CD4 or CD8 T Cells
Total
Spleen
Cells
MQ
DC
BC
CD4
OVA specific
Hybridomas
BO-97-11
APC + MS-OVA
or
APC + Soluble OVA
IL-2
MQ and DC can present the
Encapsulated Ag to CD4 Th Cells invitro
Sol-OVA
MS-OVA
MS-Ag
1.6
MQ
1.6
DC
1.2
Soluble-Ag
BC
1.2
0.8
0.8
0.4
0.4
0
0
10^2
10^3
10^4
10^5
10^6
10^2
APC/well
APC/well
10^3
10^4
10^5
10^6
Il-2 (pg/ml)
The MS target the Ag to the MQ which can
present it to CD8 T cells in-vitro
a MS-Ag
b peptide
MQ+DC
B cells
.
.
.
.
Ag (ug/ml)
c seperated MQ and DC
d peritoneal MQ
DC+MS-Ag
MS-Ag
MQ+MS-Ag
peptide
DC+peptide
MS empty+S-Ag
MQ+peptide
.
Ag (ug/ml)
.
.
.
Processing of Ge/CS MS in-vivo
Experimental system
MS-Ag
IV injection
Soluble-Ag
CD4
OVA-specific
Hybridoma
B0-97-11
2 hrs
C57BL/6 mice
APC
MQ+DC
BC
IL-2 ?
The Ge/Cs MS target the encapsulated Ag
to MQ and DC in-vivo
Presentation of Ag to CD4 T cells by APC challenged
in-vivo (IP) with MS-Ag
In vivo challenge with MS-Ag
In vivo challenge with S-Ag
DC
B cells
IL
-
/
ml
)
B cells
MQ
/
ml
)
Il-2(pg
(pg/ml)
MQ
IL
-
Il-2(pg
(pg/ml)
DC
.
APC/w ell (x ^ )
.
.
APC/well (x ^ )
.
The MS target the Ag to DC and MQ in-vivo and
they present it to CD8 T cells
In vivo challenge with MS-Ag presentation to
CD T cells
DC
Fluore s ce nt units
MQ
B cells
-
APC/well (x ^ )
Active Targeting
Targeting mAb
• DC
anti CD11C
•B cells
anti CD19
•DC and B cells
anti CD40
Active Targeting by Immunospheres
MS-Avidin preparation
O
MS
NH2
H
O
C CH2-CH2-CH2-C-H
Glutaraldehyde
Avidin
O
MS
NH C CH2-CH2-CH2-C-H
O
MS
O
O
NH C CH2-CH2-CH2-C-NH-
Avidin
Immunosphere Formation
O
MS
O
NH C CH2-CH2-CH2-C-NH-
Avidin
Ab-Biotin
O
MS
O
NH C CH2-CH2-CH2-C-NH-
Avidin
Fluorescent
label
Targeting to B cells
MS-CD19
72%
MS-Isotype
control
12 %
Targeting to DC
MS-CD11c
83-92%
MS-Hamster IgG
control
20-36%
Targeting of encapsulated Ag to DC or B cell
enhance the presentation of the Ag to CD4 T cells
Targeting to B cells
Targeting to DC
MS-anti B
%
100
50
0
0 0.625 1.25 2.5
Ag (ug/ml)
5
10
MS-Ig control
120
300
%
100
250
80
200
60
150
40
100
20
50
0
0
1.125 2.25
4.5
9
APC/well (x10^3)
18
% Enhancement
60
50
40
30
20
10
0
IL-2 (pg/ml)
MS-Ig control
% Enhancement
IL-2 (pg/ml)
150
MS-anti DC
Conclusions:

The MS target the Ag to MQ and DC for presentation to CD4 Th
cells in-vitro and in-vivo.

MS can deliver the Ag to MQ and DC for presentation to CD8 Tc
cells

Small MS can deliver the Ag to DC for presentation to CD8 Tc
cells

B-cells are not effective in presenting the encapsulated Ag, invitro or in-vivo.

Immunospheres efficiently target the Ag to B cells and enhance
their presentation to CD4 Th cells

Immunospheres efficiently target the Ag to DC. Might improve the
presentation to CD8 Tc cells.
Other Results :


Vaccination with the Microspheres: Induces
activation of Th cells, cytokines production, long
term antibody production. (Adjuvant properties)
The vaccine can be administered either Orally,
Intraperitonealy, or Subcutaneously.
Streptococcus pneumoniae
(S. pneumoniae)
• Gram-positive bacterium
• Found in 70-80% of infants and children
• 500,000 cases of pneumococcal diseases annually reported
in the USA
• 5-7% are fatal
• Capsular polysaccharide vaccine is only 60% effective in
the elderly and non-effecive in young children
17 Surface proteins identified as
having antigenic properties

Ling E., Feldman G., Portnoy M., Dagan R.,
Overweg K., Mulholland F., Chalifa-Casp V.,
Wells J. and Mizrachi-Nebenzahl Y. (2004),
Clin. Exp. Immunol. 138, 290-298
A combined Th1 and Th2 response is required to provide protection
PsipB
Pneumococcal surface immunogenic protein B
PsipB was cloned in E.coli, and the Ag isolated
on NiNTA from cell lysate.
PsipB with Alum provided 30% protection
following 2 immunizations, 21 days after
challenge with a lethal dose of Pnc strain
WU2
Encapsulation of PsipB


PsipB was encapsulated within CS/Ge
microspheres in order to identify the
preferred immune response.
Efficiency of encapsulation was 60%.
Immunization with PsipB
Survival plot
120
100
PsipB+MS
PsipB+Alum
MS
Alum
No. of mice
PsipB/MS
80
60
40
Non-immunized
20
0
0


2
4
6
Two IP immunizations, 3 weeks apart
Intranasal challenge with pathogen – 2 weeks
after 2nd vaccination
8
Future research

In-vivo targeting of Ag

Change of Ag
Changing the Microspheres’
Composition
Hyaluronic Acid (HA)




Biological polysaccharide,
~400 Dalton per monomer
Abundant in mammalian
tissues as a lubricant, in
higher MW (~106 Dalton)
Low MW (~103 Dalton) HA
activates the immune
system through dendritic
cells – Activity depends on
size
Carries negative charges in
a physiological environment
MaterialStudio –
Molecular Imaging of Interaction
Hyaluronic Acid
Gelatin
MaterialStudio – Calculation of Chi
Parameter
• Chi-parameter is the interaction parameter of two
compounds
• A very low and negative value suggests that the
two materials favour the coacervate state
O.D (absorbance)
at 640 nm.
OD 640)
Ends (DNS
Reducing
Hyaluronic Acid – Kinetics of Enzymatic
Digestion
1.2
1
0.8
0.6
0.4
0.2
0
0
200
400
600
800
1000
1200
1400
1600
Reaction Time (min.)
2000U
5000U
7500U
10000U
20000U
500U
1000U
HA Digestion Products
500U/5hr
500U/10min
Blank
500U/60min
5,000U/60min
5,000U/10min
Original HA
5,000U/24hr
GA
Blank
HA Digestion Products
20,000U/
24hr
2,000U/
10min
2,000U/
2,000U/
30min
5hr
2,000U/24hr
CS
Original
HA
Glucuronic
Acid
Blank
Blank
Future Research



Characterization of the mechanism of
HA enzymatic digestion.
Feasibility study of microencapsulation
of antigens within HA/Gelatin
microspheres.
Study of the effect of HA MW on
immunization
Acknowledgments

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The Israeli Ministry of Health
Einav Danino
Lilia Kotlierevski
Alex Kushnir
Nina Rodman
Svetlana Varshitzki
Sagiv Berkowitz
Neta-Lee Shwartzman
Eti Evzada
Ran Armoza