VACCINES

1. Introduction

2. The Immune System

3. Subunit & Peptide Vaccines

4. Attenuated Vaccines

5. Vector Vaccines

6. Bacterial Antigen delivery
systems


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Immunization: a procedure designed to increase
concentrations of antibodies and/or effector T-
cells which are reactive against infection (or
cancer).

Immunization procedure called vaccination and the
immunizing agent called vaccine (or serum in
historical references)

VACCINES
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Discovery of Vaccination
Discovered in 1796 by
Dr. Edward Jenner

Tested empirical knowledge:
mild cattle disease cowpox
protects against deadly human
disease smallpox

scratching liquid from cowpox
sores into the boy's skin -> full
protection against smallpox

VACCINES
Introduction
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VACCINES
The Immune system
Immune System
Innate
(Nonspecific)

Adaptive
(Specific)
Protects/re-exposure
Cellular Components Humoral Components Cellular Components Humoral Components
Interactions between the two systems
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Innate Immunity
Adaptive Immunity
Innate versus Adaptive Immunity
No memory
No time lag
Not antigen specific
A lag period
Antigen specific
Development of memory
VACCINES
The Immune system
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VACCINES
The Immune system
First-line defense: physical and chemical barrier
The first-line defense includes barriers to infection, such as skin and mucus coating of the gut and airways.

Second-line defense:
Phagocytic cells
phagocytic cells (Myeloid cells) (macrophages and neutrophil granulocytes) can destroy (phagocytose) foreign
substances. Phagocytosis involves digestion of the bacterium by using enzymes.

Anti-microbial proteins
Anti-microbial proteins are activated if a pathogen passes through the barrier offered by skin. There are several
classes of antimicrobial proteins, such as acute phase proteins (for example, proteins that enhance phagocytosis),
lysozyme, and the complement system.

The complement system is a very complex group of serum proteins, which is activated in a cascade fashion. Three
different pathways are involved in complement activation:


A cascade of protein activity follows complement activation -> destruction of the pathogen and inflammation

Interferons are also anti-microbial proteins.
-> secreted by virus-infected cells -> diffuse rapidly to neighboring cells -> inhibit the spread of the viral infection.
Innate Immune System
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The adaptive immune system, also called
the "acquired immune system",
ensures that most mammals that
survive an initial infection by a
pathogen are generally immune to
further illness caused by that same
pathogen.

The adaptive immune system is based on
dedicated immune cells termed
leukocytes (white blood cells) that are
produced by stem cells in the bone
marrow, and mature in the thymus
and/or lymph nodes. It is in the lymph
nodes where antigen is usually
presented to the immune system.

Adaptive Immune System
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VACCINES
The Immune system
Cells of the Immune System
Immune System
Myeloid Cells Lymphoid Cells
Granulocytic Monocytic T cells B cells
Neutrophils
Basophils
Eosinophils
Macrophages
Kupffer cells
Dendritic cells
Helper cells
Suppressor cells
Cytotoxic cells
Plasma cells
Natural Killer
cells
VACCINES
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Development of the Immune System
VACCINES
myeloid
Granulocyte
lymphoid
nk
thymus
CD8
+
CD4
+
CTL
TH2
TH1
Monocyte
B-Cells
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The Immune System
VACCINES
What Happens during an infection?
Innate Immunity activated

macrophages slip between cells [extravasation] to arrive

cytokine chemicals attract other troops [chemotaxis]

histamine chemicals dilate blood vessels for easier access to injury
[vasodilatation]
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The Immune System
VACCINES
Macrophages (The big eaters)
Phagocytic cells -
able to ingest small foreign invaders
neutrophils
monocyte

they release cytokines that enhance the immune
response


Mast cells /basophils
release histamine that dilates blood vessels

causes redness [erythrema], swelling
[edema], and heat [fever]

Call for help from the ADAPTIVE IMMUNE System
-> results in a coordinated successful defense !

Major players . . . the B lymphocytes

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The Immune System
VACCINES
The Adaptive Immune system

1. Humoral immune system: -> acts against bacteria and viruses using immunoglobulins
(also known as antibodies) -> produced by B cells.

2. Cellular immune system: -> destroys intracellular pathogens (such as virus-infected
cells and mycobacteria causing tuberculosis) using T cells (also called "T
lymphocytes"; "T" means they develop in the thymus).


There are two major types of T cells:

Cytotoxic T cells (TC cells): -> recognize infected cells by using T cell receptors to
probe cell surfaces (-> Major Histocompatability Complex [MHC]) . If they
recognize an infected cell, they release granzymes (proteases) to trigger that
cell to become apoptotic ("commit suicide")

Helper T cells (TH cells): -> activate macrophages and also produce cytokines
(interleukins) that induce the proliferation of B and T cells.
-> recognize infected cells of the immune system by using HelperT cell
receptors to probe cell surfaces (-> Major Histocompatability Complex [MHC])

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The Immune System
VACCINES
The Adaptive Immune system
Activated B cells differentiate into . . .
Antibody producing cells [attack mode]
Memory cells [remembers & future protection]
Antigen & T-helper cell
memory
antibodies
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Principle of Vaccination
VACCINES
A vaccine renders the recipient resistant to infection.

During vaccination a vaccine is injected or given orally.

The host produces antibodies for a particular pathogen.

Upon further exposure the pathogen is inactivated by the
antibodies and disease state prevented.

Generally to produce a vaccine the pathogen is grown in culture
and inactivated or nonvirulent forms are used for vaccination.
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Immunization:
When performed before exposure to an infectious agent (or
soon after exposure in certain cases), it is called
immunoprophylaxis,
intended to prevent the infection.

When performed during an active infection (or existing cancer),
it is called immunotherapy, intending to cure the infection (or
cancer)

VACCINES
Principle of Vaccination
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Types of Immunity:
VACCINES
Principle of Vaccination
-> Two mechanisms by which immunization can be achieved

Passive immunization:
Protective Abs --> non immune recipient
No immunological memory

Active immunization:
Induction of adaptive immune response, with protection and
memory.

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Passive versus active immunization:
VACCINES
Principle of Vaccination
-> TYPE ACQUIRED THROUGH Passive Immunization
-> Natural maternal serum/milk
-> Artificial immune serum


-> Type ACQURIED THROUGH Active Immunization
-> Natural infection
-> Artificial infection*:
Attenuated organisms (live)
inactivated organisms (dead)
Cloned genes of microbiological antigens
Purified microbial macromolecules
Synthetic peptides
DNA
*Artificial refers to steps involving human intervention

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Mechanism of Vaccination:
VACCINES
Principle of Vaccination
Establish resistance to virus/pathological organism by evoking an
immune response

1. Give host a foreign organism/protein in non-infectious form -> active
immunization

2. Antibodies are generated Ab binds to surface proteins of organism
-> passive immunization
Other vaccination components:
Adjuvant: chemicals in the vaccine solution that enhance the immune response
Alum Ag in the vaccine clumps with the alum such that the Ag is released
slowly, like a time-release capsule
gives more time for memory cells to form


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Principle of Vaccination
VACCINES
Traditional Vaccines:

Grow in animals (vaccinia in
calves for smallpox; rabbit
brains for rabies)

Simple bacterial culture
(Cholera vibrio) then
inactivation

Grow in eggs (influenza,
vaccinia) then inactivate
>100 million eggs used for
influenza in the USA every year
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Principle of Vaccination
VACCINES
Limitations To Traditional Vaccines:
-> cant grow all organisms in culture

-> safety to lab personnel

-> Expense

-> insufficient attentuation (living agent altered to become
harmless or less virulent)

-> reversion to infectious state

-> need refrigeration

-> do not work for all infectious agents

-> infants/children receive them immature immunity
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Principle of Vaccination
VACCINES
New Generation of Vaccines:
Recombinant DNA technology is being used to produce a new
generation of vaccines.

Virulence genes are deleted and organism is still able to stimulate
an immune response.
Live nonpathogenic strains can carry antigenic determinants from
pathogenic strains.
If the agent cannot be maintained in culture, genes of proteins for
antigenic determinants can be cloned and expressed in an
alternative host e.g. E. coli.
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Principle of Vaccination
VACCINES
Recombinant Vaccines:
1. Subunit Vaccines
peptide vaccines
Genetic immunization

2. Attenuated Vaccines

3. Vector Vaccines

4. Bacterial Antigen Delivery Systems
-> ghosts -> delivery systems (next semester)

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Principle of Vaccination
VACCINES
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Subunit/Peptide Vaccines
VACCINES
Do NOT use entire virus or bacteria (pathogenic agent)

Use components of pathogenic organism instead of whole organism

Advantage: no extraneous pathogenic particles i.e. DNA

Disadvantage: Is protein the same as in situ? -> Cost?

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Subunit/Peptide Vaccines
VACCINES
It has been showed that the capsid or envelope proteins are enough to
cause an immune response:
Herpes simplex virus envelop glycoprotein O.
Foot and mouth disease virus capsid protein (VP1)
Extracellular proteins produced by Mycobacterium tuberculosis.

Subunit Vaccines
Antibodies usually bind to surface proteins of the pathogen or proteins
generated after the disruption of the pathogen.
Binding of antibodies to these proteins will stimulate an immune response.
Therefore proteins can be use to stimulate an immune response.

Development of Subunit vaccines based on the following observation:
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Subunit/Peptide Vaccines
VACCINES
Example for a subunit vaccine -> Tuberculosis
Tuberculosis is caused by Mycobacterium tuberculosis.
The bacterium forms lessions in the tissues and organs -> causing cell death.
Often the lung is affected.
About 2 billion people are infected and there are 3 million deaths/year.
Currently tuberculosis is controlled by a vaccine called BCG (Bacillus Calmette-
Guerin) which is a strain of M. bovis.
M. bovis often responds to diagnostic test for M. tuberculosis.
Six extracellular proteins of M. tuberculosis were purified.
Separately and in combinations these proteins were used to immunized guinea
pigs.
These animals were then challenged with M. tuberculosis.
After 9-10 weeks examination showed that some combinations of the purified
proteins provided the same level of protection as the BCG vaccine.
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Subunit/Peptide Vaccines
VACCINES
Selection & delivery of vaccine peptides:

Use discrete portion (domain) of a surface
protein as Vaccine

These domains are epitopes (antigenic
determinants) -> are recognized by antibodies
CARRIER PROTEINS

Problem -> Small Peptides are often Digested
-> no strong immune response

-> Carrier Proteins Make more Stable + stronger
immune response

Make fusion protein of carrier + vaccine peptide
-> inert carrier or highly immunogenic carrier
(hepatitis B core protein)

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Attenuated Vaccines
VACCINES
Attenuated vaccines often consists of a pathogenic strains in
which the virulent genes are deleted or modified.

Live vaccines are more effective than a killed or subunit (protein)
vaccines.
Example -> Vaccine against Cholera
cholera is caused by infection withVibrio cholerae and is transmitted through
contaminated water.

V. cholerae produces a enterotoxin with an A1 subunit and 5 B subunits -> causes
disease

Presently the cholera vaccine consist of a phenol-killed V. cholerae and it only last
3-6 months.

A live vaccine would be more effective.
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Attenuated Vaccines
VACCINES
Example -> Vaccine against Cholera
550 bases deleted
of A1 peptide
The final result is V. cholerae with a 550 bp of the A peptide
deleted.

-> Currently being tested. 29
Vector Vaccines
VACCINES
Virus as Antigen Gene Delivery System !!!
Vaccinia good candidate for a live recombinant viral vaccine
benign virus
replicate in cytoplasm (viral replication genes)
easy to store


-> The vaccinia virus is generally nonpathogenic.
The procedure involves:
The DNA sequence for the specific antigen is inserted into a plasmid
beside the vaccinia virus promoter in the middle of a non-essential gene
e.g. thymidine kinase.
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Vector Vaccines
VACCINES
The procedure involves:
The plasmid is used to transform thymdine
kinase negative cells which were previously
infected with the vaccinia virus.

Recombination between the plasmid and vaccinia
virus chromosomal DNA results in transfer of
antigen gene from the recombinant plasmid to
the vaccinia virus.

Thus virus can now be used as a vaccine for the
specific antigen.


-> Recombinant Virus
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Vector Vaccines
VACCINES
A number of antigen genes have been inserted into the vaccinia
virus genome e.g.

Rabies virus G protein
Hepatitis B surface antigen
Influenza virus NP and HA proteins.

A recombinant vaccinia virus vaccine for rabies is able to elicit
neutralizing antibodies in foxes which is a major carrier of the
disease.
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Vector Vaccines
VACCINES
Control of Viral Vaccines Post Innoculation
Vaccinia virus is resistant to
interferon -> due to presence of
K3L protein

Use an interferon-sensitive
strain of vaccinia virus -> delete
K3L gene to create mutant

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Bacterial Antigen Delivery Systems
VACCINES
Antigen Gene
Bacterium
Antigen Proteins made on Bacterial cell
Vaccinate Patient
-> Use live nonpathogenic bacterium
which contains antigen (Salmonella or
epitope from cholera)

Insert antigen gene into flagellin gene

Epitope is expressed on the flagellum
surface

-> Flagellin-engineered bacteria is
VACCINE

Advantage - Oral Administration

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Vaccine Approval
VACCINES
Done by CBER (Center for Biologics Evaluation and Research),
an arm of the FDA

Generally same clinical trial evaluation as other biologics and
drugs

Site to learn more about vaccines:

http://www.fda.gov/cber/vaccine/vacappr.htm
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