Immune Responses and Vaccine Development

29Oct2012

www.globalsecurity.org www.manbir-online.com

History of Vaccination

Smallpox – Variola Only disease to be eradicated

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.

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

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

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

Success of Vaccination

Eradication of smallpox

Near eradication of polio

Reduced rates of vaccine preventable diseases

poliosurvivorsnetwork.org.uk

Basic Immunology

What happens after vaccination?

Activation of Innate and Adaptive immunity Motherspace.in

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

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

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.

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

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.

What Happens Next?

Migration to lymph nodes for presentation to T-cells.

Afferent Lymphatics

Efferent Lymphatics

B-cells

T-cells

Plasma cells

Immunological Synapse formed in the lymph node

Pathmicro.med.sc.edu

CD4 Co-Receptor Stabilizes interactions with MHC II.

T-cell

Antigen Presenting Cell

Activation of transcription factors

CD8 Co-Receptor Stabilizes interactions with MHC I.

T-cell

Antigen PresentingCell

Activation of transcription factors

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

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

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

Intracellular microorganism destroyed

T-Helper Type 1

Activation

Th1

Th1

Macrophage

Macrophage

T-Helper Type 2

Activation

Neutralizing antibody secretion

Th2

Th2

B-cell

B-cell Y Y Y Y Y

Y Y

Y Y

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

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

T-cell Mediated Cytotoxicity

Lytic granules contain: Perforin: Forms membrane pores in target cell

Serine proteases: degrades cellular proteins

Lytic Granule Release

T-cell Mediated Cytotoxicity

CTL migrates to new target.

Target cell dies.

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

30

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

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

33

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

34

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

Product Support

Vaccines are not always stored properly.

Issues with cold chain Human error

Power outage

Lack of space

WH

O

Thank You

Questions?

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