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Immune Responses and Vaccine Development
29Oct2012
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www.globalsecurity.org www.manbir-online.com
History of Vaccination
Smallpox – Variola Only disease to be eradicated
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History of Vaccination
China late 1600s The Golden Mirror of Medicine Listed 4 methods of smallpox inoculation Plug the nose with powdered scabs on
cotton wool Powdered scabs blown into the nose Undergarments of an infected child put on
a healthy child for several days Cotton smeared with the contents of a
vesicle and stuffed into the nose.
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History of Vaccination
Variolation Introduction of dried pus from smallpox
pustules into the skin of the patient. India, Turkey, India, America in the
1700’s George Washington ordered recruits to
the continental army to undergo variolation to prevent smallpox.
2-3% of people variolated died of smallpox
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History of Vaccination
Vaccination: Live attenuated organisms late 1700s – early 1800s
Benjamin Jesty, Edward Jenner Inoculation of humans with cowpox
to prevent smallpox infection. Louis Pasteur Inoculation of animals/humans with
attenuated anthrax, cholera, and rabies.
Louis Pasteur
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History of Vaccination
Vaccination – Killed whole organisms Late 1800s
Protein vaccines Early 1900’s Diphtheria and Tetanus toxoids
Recombinant proteins/conjugates Modern Era Hepatitis B, HPV – virus like particles Hib, pneumo, meninge – conjugates Flumist, Varivax – live attenuated virus
Flumist-vaccine.com
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Success of Vaccination
Eradication of smallpox
Near eradication of polio
Reduced rates of vaccine preventable diseases
poliosurvivorsnetwork.org.uk
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Basic Immunology
What happens after vaccination?
Activation of Innate and Adaptive immunity Motherspace.in
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The Course of an Infection Le
vel o
f Inf
ecti
on
Duration of Infection
Innate Response
Infection Cleared
Initiation of adaptive response
adaptive response
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Innate Immunity
First line of defense. Response is not microorganism specific. Granulocytes are the primary innate immune
cells Macrophages Neutrophils Eosinophils Basophils Mast Cells Macrophage ingesting lysteria
Pathology.wustl.edu
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Pathogen Recognition Cells of innate immunity have receptors that
recognize pathogens. Toll-like receptors Mannose receptor Glycan receptor
Pathogen recognition results in engulfment and cytokine/chemokine release. Cause inflammation Recruit additional leukocytes to the site of
infection Direct the form of the adaptive immune
response.
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Toll-like Receptors (TLRs) Recognize Conserved Molecular Patterns on Pathogens
TLR2 TLR1 TLR6
TLR4 TLR5
TLR3
TLR9
MyD88 TRIF
MyD88
NF-κB, ISREs, AP1
Cytokines, Chemokines, IFNs
• 1/2/6 – peptidoglycan • 3 – double stranded RNA • 4 – LPS • 5 – Flagellin •7/8 – single stranded RNA • 9 – CpG DNA
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Antigen Presenting Cells
Are a link between innate and adaptive immunity.
Professional APCs Dendritic cells
Macrophages
B-cells
Antigens are degraded and presented to T-cells on MHC I & II molecules.
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What Happens Next?
Migration to lymph nodes for presentation to T-cells.
Afferent Lymphatics
Efferent Lymphatics
B-cells
T-cells
Plasma cells
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Immunological Synapse formed in the lymph node
Pathmicro.med.sc.edu
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CD4 Co-Receptor Stabilizes interactions with MHC II.
T-cell
Antigen Presenting Cell
Activation of transcription factors
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CD8 Co-Receptor Stabilizes interactions with MHC I.
T-cell
Antigen PresentingCell
Activation of transcription factors
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T-cell activation is not Complete Without Co-Stimulation B7 on the APC binds with CD28 on the T-cell to provide the co-
stimulation signal.
IL-2 induces T-cell proliferation.
T-cell
Antigen PresentingCell
Activation of transcription factors
IL-2
B7 CD28
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Antigen Recognition Without Co-Stimulation Results in Anergy Cells are unable to produce IL-2 for proliferation
Ensures tolerance to self tissues
T-cell
Antigen PresentingCell
Activation of transcription factors
IL-2
B7 (CD80/86) CD28
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Activated CD4+ T-cells Differentiate Th1 – Cell mediated immunity
Th2 – Humoral mediated immunity
Th17 – Implicated in auto-immunity, protection from bacterial & fungal infection
Differentiation dependent on cytokines and peptide binding strength
Th1 Th2 Th17
IL-12, INF-γ
IL-4 IL-10 IL-21, IL-23
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Intracellular microorganism destroyed
T-Helper Type 1
Activation
Th1
Th1
Macrophage
Macrophage
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T-Helper Type 2
Activation
Neutralizing antibody secretion
Th2
Th2
B-cell
B-cell Y Y Y Y Y
Y Y
Y Y
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Activation of CD8+ T-cells
IL-2 induces T-cell proliferation.
Differentiates into a cytotoxic T-cell
T-cell
Antigen PresentingCell
Activation of transcription factors
IL-2
B7 CD28
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T-cell Mediated Cytotoxicity If CTL matches its target antigen it induces apoptosis in the
target cell.
Apoptosis is induced by release of lytic granules and expression of Fas ligand
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T-cell Mediated Cytotoxicity
Lytic granules contain: Perforin: Forms membrane pores in target cell
Serine proteases: degrades cellular proteins
Lytic Granule Release
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T-cell Mediated Cytotoxicity
CTL migrates to new target.
Target cell dies.
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Components of Vaccine Formulations
Antigen – entity in which it is desired to direct the immune response against.
Adjuvant – Enhances the desired immune response.
Buffer – Maintains appropriate pH.
Excipients – Isotonicity and stabilization
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A Rational, Systematic Approach for the Development of Vaccine Formulations
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Biophysical Characterization
Utilizing diverse analytical techniques to characterize the physical stability of an antigen.
Empirical phase diagram approach Define suitable pH, Buffer species, ionic strength
pH
Tem
pera
ture
(oC
)
6.5 7 7.5 8 30
40
50
60
70
80
90
6.0
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Stabilizer Screening
High Throughput Screening of GRAS excipients.
Excipient 2 would be
selected as a stabilizer for further development
Excipient Class Examples
Amino Acids Arginine, Aspatate, Glycine, Glutamate, Lysine, Proline,
Antioxidants Ascorbic Acid, EDTA, Malic Acid
Cyclodextrins α−cyclodextrin, β or γ 2-hydroxypropyl-cyclodextrin
Proteins Albumin, Gelatin
Sugars/Sugar Alcohols Sucrose, Trehalose, Lactose, Dextrose, Glycerol, Sorbitol, Mannitol
Surfactants Brij, Pluronic, Tween
0
0.2
0.4
0.6
0.8
1
0 20 40 60 80 100
Temperature (oC)
Opt
ical
Den
sity
.
ControlExcipient 1Excipient 21+2
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Adjuvants
Enhance the potency of vaccines.
Aluminum salts are the most common.
Emulsions have been approved in the EU.
Bayerite
Gibbsite
Boehmite
MF59 -Novartis Aluminum hydroxide adjuvant
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Aluminum Adjuvant Interactions Adsorption profile with
aluminum adjuvants Adsorption stability Importance of adsorption for
immunogenicity Differentiation of the immune
response with alternative adjuvants
10
100
1000
10000
Aluminum TLR4 agonist Combo
GM
T
IgG1IgG2Total IgG
1
10
100
1000
10000
Solution Adsorbed Non-Adsorbed
GM
T
0
20
40
60
80
100
-40 -20 0 20 40
Adjuvant Zeta Potential (mV)
% A
dsor
bed
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Product Contact Material Interactions
Interaction with product contact materials can impact vaccine formulations Filters, glass, stainless
steel, plastic, etc..
Leachables and Extractables
0
5
10
15
20
25
30
35
0 2 4 6 8 10 12
Time (min)
Pres
sure
(psi
)
CAPESPVDF
0
20
40
60
80
100
120
0 10 20 30 40
Time (months)
% L
oss
VialSyringe
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Real Time/Accelerated Stability
Correlation of real time and accelerated conditions.
Early decisions on whether formulation is suitable to obtain shelf life goals
Support for temperature excursions during shipment and storage of clinical material
-4.5
-4
-3.5-3
-2.5
-2
-1.5-1
-0.5
00.003 0.003 0.003 0.003 0.003 0.003 0.003 0.003 0.003
1/T
Ln(k
)
85
90
95
100
105
0 10 20 30 40
Time (months)
Dos
e
ActualPredicted
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Product Support
Vaccines are not always stored properly.
Issues with cold chain Human error
Power outage
Lack of space
WH
O
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Thank You
Questions?
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