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VACCINES

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Title: VACCINES


1
VACCINES
N7-2006 L. Duroux Slides assembled from diverse
sources
2
Lecture Plan
  1. Introduction
  2. The Immune System
  3. Subunit Peptide Vaccines
  4. Attenuated Vaccines
  5. Vector Vaccines

3
1. INTRODUCTION
4
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

5
Chicken !
BITCH !
6
2. The Immune System
7
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

8
The Invaders . . .
  • Bacteria
  • Viruses
  • parasites
    such as fungi,

    protista, worms

9
Our 1st Line of Defense...
  • The Integumentary System
  • Skin
  • Mucous membranes
  • Mucous
  • provides a physical barrier preventing microbial
    access

10
Other mechanisms of Defense...
  • Physiological variables
  • pH of our environment
  • temperature of our environment
  • chemical defenses
  • nitric oxide, enzymes, proteins
  • AND the IMMUNE SYSTEM

11
Overview of the Immune System
Interactions between the two systems
12
Comparison of Innate and Adaptive Immunity
  • No time lag
  • A lag period
  • Not antigen specific
  • Antigen specific
  • No memory
  • Development
  • of memory

13
Cells of the Immune System
14
Development of the Immune System
Monocyte
Granulocyte

myeloid
B-Cells
15
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

16
What are macrophages ?
  • Phagocytic cells - able to ingest small
    foreign invaders
  • neutrophils
  • monocyte
  • they release cytokines
    that enhance the
    immune response

17
Macrophages
  • Mast cells /basophils
  • release histamine that dilates blood vessels
  • causes redness erythrema, swelling edema, and
    heat fever

18
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

19
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

20
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
21
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!
22
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

23
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

24
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
25
B cells differentiate into . . .
  • Antibody producing cells attack mode
  • Memory cells remembers future protection

Antigen T-helper cell
antibodies
memory
26
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

27
3. Principles of Vaccination
28
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.

29
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

30
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
31
Recombinant Vaccines
1. Subunit Vaccines peptide vaccines Genetic
immunization 3. Attenuated Vaccines 4. Vector
Vaccines 5. Bacterial Antigen Delivery Systems
32
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.

33
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
34
New Generation of Vaccines
  • There are three types of vaccines we will be
    discussing
  • Subunit (protein) vaccines
  • Attenuated vaccines
  • Vector vaccines

35
Vaccine Technology
36
4. Subunit / Peptide Vaccines
37
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


38
Structure of a Virus particle
39
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.

40
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.

41
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.

42
Selection delivery of vaccine peptides
  • Antigenic determinants epitopes on envelope
    proteins
  • Inert carrier hemocyanin from keyhole limpet
  • Highly immunogenic carrier Hepatitis B core prot.

43
5. Attenuated Vaccines
44
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.

45
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.

46
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.

47
Production of a Live Cholera Vaccine
48
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.

49
Production of a Live Cholera Vaccine
50
6. Vector Vaccines
51
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.

52
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.

53
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.

54
Insertion of antigen gene into vaccinia virus
genomeby recombination
55
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.
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