VACCINES

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VACCINES
N7-2006 L. Duroux Slides assembled from diverse
sources
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Lecture Plan

1. Introduction
2. The Immune System
3. Subunit Peptide Vaccines
4. Attenuated Vaccines
5. Vector Vaccines

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1. INTRODUCTION
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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
• Inoculated 8-years-old boy with exudate from
  cowpox pustule full protection against smallpox

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Chicken !
BITCH !
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2. The Immune System
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Function of the Immune System(Self/Non-self
Discrimination)

• To protect from pathogens
• Intracellular (e.g. viruses and some bacteria and
  parasites)
• Extracellular (e.g. most bacteria, fungi and
  parasites)
• To eliminate modified or altered self

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The Invaders . . .

• Bacteria
• Viruses
• parasites
  such as fungi,
  
  protista, worms

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Our 1st Line of Defense...

• The Integumentary System
• Skin
• Mucous membranes
• Mucous
• provides a physical barrier preventing microbial
  access

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Other mechanisms of Defense...

• Physiological variables
• pH of our environment
• temperature of our environment
• chemical defenses
• nitric oxide, enzymes, proteins
• AND the IMMUNE SYSTEM

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Overview of the Immune System
Interactions between the two systems
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Comparison of Innate and Adaptive Immunity

• No time lag

• A lag period

• Not antigen specific

• Antigen specific

• No memory

• Development
• of memory

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Cells of the Immune System
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Development of the Immune System
Monocyte
Granulocyte

myeloid
B-Cells
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What Happens during an infection?

• Innate Immunity -
  the troops are called to
  battle
• injury infection
• 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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What are macrophages ?

• Phagocytic cells - able to ingest small
  foreign invaders
• neutrophils
• monocyte
• they release cytokines
  that enhance the
  immune response

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Macrophages

• Mast cells /basophils
• release histamine that dilates blood vessels
• causes redness erythrema, swelling edema, and
  heat fever

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Summary on Macrophages

• Macrophages are able to launch the first strike
• more help is needed to overcome rapidly
  reproducing invaders
• Help from the ADAPTIVE IMMUNE System results in a
  coordinated successful defense !
• Major players . . . the B lymphocytes

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The Adaptative Immune System

• There are 2 types of lymphocytes
• T lymphocytes T - Helper cells - help
  signal immune cells into action
• B lymphocytes B cells - make special
  proteins called antibodies

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T-lymphocytes migrate to the thymus gland ...

• These Lymphocytes are sorted into 2 types
• Identification tag is a protein called Major
  Histocompatability Complex MHC

Self- ID
Foreign
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in the thymus gland . . .

• All diversely varying MHC lymphocytes will wait
  for a call to action . . .
• These Lymphocytes will mature into T-Helper cells
  
• They function to stimulate B cells to activate
  their attack against the invaders

Foreign
Self- ID
Saved to be educated in body defense
Dropped out!
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Adaptive Immune System

• The 2nd type of lymphocyte is
• B lymphocytes B cells - start in the bone
  marrow and circulate through the body
• they are called into action when stimulated by a
  foreign antigen. . . usually a protein from the
  invader

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When an invader attacks. . .

• An antigen is phagocytized by the B cell
• is broken into non-infective pieces
• attached to the cells MHC when processed
  through the cells machinery
• MHC-antigen complex is placed on the cell
  membrane surface
• where it is recognized by the T Helper cell

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When help arrives . . .

• The T-helper cell receptor docks with the B
  cells MHComplex
• B cells proliferate . . .

Antigen T-helper cell
Proliferation of cell line
Naïve cell
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B cells differentiate into . . .

• Antibody producing cells attack mode
• Memory cells remembers future protection

Antigen T-helper cell
antibodies
memory
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The RESULT . . .

• The Antibody producing B cells mounts
  a successful attack against the invader
• the memory B cells save the recognition ID for
  many years in preparation for future invasion

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3. Principles of Vaccination
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Principles of Vaccination

• 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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Vaccine Technology

• Old Technology
• 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

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Limitations To Traditional Vaccines
1. cant grow all organisms in culture 2. safety
to lab personnel 3. Expense 4. insufficient
attentuation 5. reversion to infectious
state 6. need refrigeration 7. do not work for
all infectious agents 8. infants/children
receive them immature immunity
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Recombinant Vaccines
1. Subunit Vaccines peptide vaccines Genetic
immunization 3. Attenuated Vaccines 4. Vector
Vaccines 5. Bacterial Antigen Delivery Systems
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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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Recombinant Vaccines
1. Delete Virulence Genes (can not revert) V/B
as Vaccine 2. Clone gene for pathogenic antigen
into non-pathogenic virus or bacteria V/B as
Vaccine 3. Clone pathogenic antigen gene into
expression vector A. Vaccinate with
protein 1. Subunit 2. Peptide
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New Generation of Vaccines

• There are three types of vaccines we will be
  discussing
• Subunit (protein) vaccines
• Attenuated vaccines
• Vector vaccines

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Vaccine Technology
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4. Subunit / Peptide Vaccines
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Subunit vaccines

• Do NOT use entire virus or bacteria (pathogenic
  agent)
• Use components of pathogenic organism instead of
  whole organism
• Advantage no extraneous pathogenic particles ie
  DNA
• Disadvantage Is protein same as in situ?
• Cost

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Structure of a Virus particle
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Subunit vaccines born from following observation

• It has been showed that the capsid or envelope
  proteins are enough to illicit 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.

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A Subunit Vaccine for M. tuberculosis

• Tuberculosis is caused by Mycobacterium
  tuberculosis.
• The bacterium form lesions 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.

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A Subunit Vaccine 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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Selection delivery of vaccine peptides

• Antigenic determinants epitopes on envelope
  proteins
• Inert carrier hemocyanin from keyhole limpet
• Highly immunogenic carrier Hepatitis B core prot.

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5. Attenuated Vaccines
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Attenuated 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.

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A Live Cholera Vaccine

• The causal agent of cholera is Vibrio cholerae
  and is transmitted through contaminated water.
• V. cholerae produces a enterotoxin with an A
  subunit and 5 B subunits.
• 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.
• In the sequence of the A peptide a tetracycline
  resistance gene is inserted.

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A Live Cholera Vaccine

• A plasmid with A peptide was digested with 2
  restriction enzymes Cla1 and Xba1.
• This removes 550 bases of A peptide.
• A Xba1 linker was added and T4 ligase used to
  ligate the DNA. This plasmid was mixed with V.
  cholerae with tetracycline resistant gene.
• By conjugation the plasmid was transferred to the
  strain with the tetR gene inserted into its
  chromosomal DNA.

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Production of a Live Cholera Vaccine
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A Live Cholera Vaccine

• By recombination the A peptide with the tetR gene
  was replaced by the deleted A peptide.
• The final result is V. cholerae with a 550 bp of
  the A peptide deleted.
• If this can be used as a vaccine is being tested.

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Production of a Live Cholera Vaccine
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6. Vector Vaccines
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Vector Vaccine

• A vector vaccine is a vaccine which is introduced
  by a vector e.g. vaccinia virus.
• The vaccinia virus as a live vaccine led to the
  globally eradication of the smallpox virus.
• The genome of the vaccinia virus has been
  completely sequenced.
• The virus replicates in the cytoplasm rather than
  in the nucleus.
• The vaccinia virus is generally nonpathogenic.

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Vector Vaccine

• These characteristics makes the vaccinia virus a
  good candidate for a virus vector to carry gene
  for antigenic determinants form other pathogens.
• 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 Vaccine

• 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.

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Insertion of antigen gene into vaccinia virus
genomeby recombination
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Vector Vaccine

• 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.