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VACCINESVACCINES
N7-2006N7-2006L. DurouxL. Duroux
Slides assembled from diverse sourcesSlides assembled from diverse sources
Lecture PlanLecture Plan
1.1. IntroductionIntroduction
2.2. The Immune SystemThe Immune System
3.3. Subunit & Peptide VaccinesSubunit & Peptide Vaccines
4.4. Attenuated VaccinesAttenuated Vaccines
5.5. Vector VaccinesVector Vaccines
1. INTRODUCTION1. INTRODUCTION
Discovery of VaccinationDiscovery of Vaccination
Discovered in 1796 by Dr. Edward JennerDiscovered in 1796 by Dr. Edward Jenner
Tested empirical knowledge: mild cattle Tested empirical knowledge: mild cattle disease cowpox protects against deadly disease cowpox protects against deadly human disease smallpoxhuman disease smallpox
Inoculated 8-years-old boy with exudate Inoculated 8-years-old boy with exudate from cowpox pustule: full protection from cowpox pustule: full protection against smallpoxagainst smallpox
Chicken !Chicken !
BITCH !
2. The Immune System2. The Immune System
Function of the Immune SystemFunction of the Immune System(Self/Non-self Discrimination)(Self/Non-self Discrimination)
To protect from pathogensTo protect from pathogens• Intracellular (Intracellular (e.g.e.g. viruses and some bacteria viruses and some bacteria
and parasites)and parasites)• Extracellular (Extracellular (e.g. e.g. most bacteria, fungi and most bacteria, fungi and
parasites)parasites)
To eliminate modified or altered selfTo eliminate modified or altered self
The The InvadersInvaders . . . . . .
BacteriaBacteria
VirusesViruses
parasites parasites such such as fungi, as fungi, protista, & protista, & wormsworms
worm trichura.jpg
http://www.hhs.gov/asphep/presentation/images/bacteria.jpg
http://www.sdnhm.org/exhibits/epidemic/teachers/background.html
http://www.skidmore.edu/academics/biology/plant_bio/lab13.FUNGI.html
Our 1Our 1stst Line of Defense... Line of Defense...
The Integumentary System…The Integumentary System… SkinSkin Mucous membranesMucous membranes MucousMucous
provides a physical barrier preventing provides a physical barrier preventing microbial access microbial access
Other mechanisms of Other mechanisms of Defense...Defense...
Physiological variablesPhysiological variables pH of our environmentpH of our environment temperature of our environmenttemperature of our environment
chemical defenseschemical defenses nitric oxide, enzymes, proteins…nitric oxide, enzymes, proteins…
AND the IMMUNE SYSTEM…AND the IMMUNE SYSTEM…
Overview of the Immune SystemOverview of the Immune System
Immune System
Innate(Nonspecific)
Adaptive(Specific)
Protects/re-exposure
Cellular Components Humoral Components Cellular Components Humoral Components
Interactions between the two systemsInteractions between the two systems
Innate Immunity Adaptive Immunity
Comparison of Innate and Comparison of Innate and Adaptive ImmunityAdaptive Immunity
• No memoryNo memory
• No time lag
• Not antigen specific
• A lag period
• Antigen specific
• Development
of memory
Cells of the Immune SystemCells of the Immune System
Immune System
Myeloid Cells Lymphoid Cells
Granulocytic Monocytic T cells B cells
NeutrophilsBasophils
Eosinophils
MacrophagesKupffer cells
Dendritic cells
Helper cellsSuppressor cellsCytotoxic cells
Plasma cells
Natural Killer cells
Development of the Immune Development of the Immune SystemSystem
myeloid
Granulocyte
lymphoid
nk
thymus
CD8+
CD4+
CTL
TH2
TH1
Monocyte
B-Cells
What Happens during an infection?What Happens during an infection?
Innate Immunity -Innate Immunity - the troops are called to battle… the troops are called to battle…
injury & infectioninjury & infection
macrophages slip between cells macrophages slip between cells [extravasation][extravasation] to arrive to arrive
cytokine chemicals attract other “troops” cytokine chemicals attract other “troops” [chemotaxis][chemotaxis]
histamine chemicals dilate blood vessels for easier access histamine chemicals dilate blood vessels for easier access to injury to injury [vasodilatation][vasodilatation]
What are macrophages ?What are macrophages ?
Phagocytic cells - Phagocytic cells - able to ingest small able to ingest small foreign invadersforeign invaders neutrophilsneutrophils monocytemonocyte
they release they release cytokines that cytokines that enhance the enhance the immune responseimmune response
MacrophagesMacrophages
Mast cells /basophils Mast cells /basophils release histamine that release histamine that
dilates blood vesselsdilates blood vessels
causes redness causes redness [erythrema],[erythrema], swelling swelling [edema],[edema], and heat and heat [fever][fever]
http://www.hhs.gov/asphep/presentation/images/wound.jpg
Summary on MacrophagesSummary on Macrophages
Macrophages are able to launch the first Macrophages are able to launch the first strike…strike…
more help is needed to overcome rapidly more help is needed to overcome rapidly reproducing invaders…reproducing invaders…
Help from the ADAPTIVE IMMUNE Help from the ADAPTIVE IMMUNE System results in a coordinated successful System results in a coordinated successful defense !defense !
Major players . . . the B lymphocytesMajor players . . . the B lymphocytes
The Adaptative Immune SystemThe Adaptative Immune System
There are 2 types of lymphocytes:There are 2 types of lymphocytes:
T lymphocytes T lymphocytes [ T - Helper cells ][ T - Helper cells ] - help - help signal immune cells into actionsignal immune cells into action
B lymphocytes B lymphocytes [ B cells ][ B cells ] - make special - make special proteins called antibodiesproteins called antibodies
T-lymphocytes migrate to the T-lymphocytes migrate to the thymus gland ...thymus gland ...
TheseThese Lymphocytes Lymphocytes are sorted are sorted intointo 2 types 2 types
Identification tag is a protein Identification tag is a protein calledcalled Major Histocompatability Major Histocompatability Complex Complex [MHC][MHC]
Self- IDForeign
& in the thymus gland . . .& in the thymus gland . . .
All diversely varying MHC lymphocytes will wait for a call to All diversely varying MHC lymphocytes will wait for a call to action . . .action . . .
These Lymphocytes will mature intoThese Lymphocytes will mature into T-Helper cells T-Helper cells
They function toThey function to stimulate B cells stimulate B cells to activate their attack to activate their attack against the invadersagainst the invaders
Self- IDForeign
Saved to be educated… in body defense
Dropped out!
Adaptive Immune SystemAdaptive Immune System
TThe 2nd type of lymphocyte is:he 2nd type of lymphocyte is:
B lymphocytes B lymphocytes [ B cells][ B cells] - start in the bone - start in the bone marrow and circulate through the bodymarrow and circulate through the body
they are called into action when stimulated by they are called into action when stimulated by a foreign antigen. . . a foreign antigen. . . [ usually a protein [ usually a protein from the invader]from the invader]
When an invader attacks. . .When an invader attacks. . .
An antigen is phagocytized by the B cellAn antigen is phagocytized by the B cell is broken into non-infective piecesis broken into non-infective pieces & attached to the cell’s MHC when processed & attached to the cell’s MHC when processed
through the cell’s machinerythrough the cell’s machinery MHC-antigen complex is placed on the cell MHC-antigen complex is placed on the cell
membrane surface membrane surface where it is recognized by the T Helper cellwhere it is recognized by the T Helper cell
When help arrives . . .When help arrives . . .
The T-helper cell receptor “docks” with the The T-helper cell receptor “docks” with the B cell’s MHComplexB cell’s MHComplex
B cells proliferate . . . B cells proliferate . . .
Antigen & T-helper cell
Proliferation of cell line
Naïve cell
B cells differentiate into . . .B cells differentiate into . . .
Antibody producing cells Antibody producing cells [attack mode][attack mode] Memory cells Memory cells [remembers & future [remembers & future
protection]protection]Antigen & T-helper cell
memory
antibodies
The RESULT . . . The RESULT . . .
The Antibody producing B cells The Antibody producing B cells mounts a successful attack against the mounts a successful attack against the invaderinvader
the memory B cells save the “recognition the memory B cells save the “recognition ID” for many years in preparation for future ID” for many years in preparation for future invasioninvasion
3. Principles of 3. Principles of VaccinationVaccination
Principles of VaccinationPrinciples of Vaccination A A vaccine vaccine renders the recipient renders the recipient resistant to resistant to
infectioninfection.. During vaccination a vaccine is injected or given During vaccination a vaccine is injected or given
orally.orally. The host produces antibodies for a particular The host produces antibodies for a particular
pathogen.pathogen. Upon further exposure the pathogen is Upon further exposure the pathogen is
inactivated by the antibodies and disease state inactivated by the antibodies and disease state prevented.prevented.
Generally to produce a vaccine the pathogen is Generally to produce a vaccine the pathogen is grown in culture and grown in culture and inactivated or nonvirulentinactivated or nonvirulent forms are used for vaccination.forms are used for vaccination.
Old Technology:Old Technology:
Grow in animals (vaccinia Grow in animals (vaccinia in calves for smallpox; in calves for smallpox; rabbit brains for rabies)rabbit brains for rabies)
Simple bacterial culture Simple bacterial culture ((Cholera vibrioCholera vibrio) then ) then inactivationinactivation
Grow in eggs (influenza, Grow in eggs (influenza, vaccinia) then inactivatevaccinia) then inactivate
>100 million eggs used for influenza in the USA every year
Vaccine TechnologyVaccine Technology
Limitations To Traditional VaccinesLimitations To Traditional Vaccines
1. can’t 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
Recombinant VaccinesRecombinant Vaccines
1. Subunit Vaccinespeptide vaccinesGenetic immunization
3. Attenuated Vaccines
4. Vector Vaccines
5. Bacterial Antigen Delivery Systems
New Generation of VaccinesNew Generation of Vaccines
Recombinant DNA technology is being used to produce a Recombinant DNA technology is being used to produce a new generation of vaccinesnew generation of vaccines..
Virulence genes are deletedVirulence genes are deleted and organism is still and organism is still able to stimulate an immune response.able to stimulate an immune response.
Live nonpathogenic strainsLive nonpathogenic strains can carry antigenic can carry antigenic determinants from pathogenic strains.determinants from pathogenic strains.
If the agent cannot be maintained in culture, If the agent cannot be maintained in culture, genes genes of proteinsof proteins for antigenic determinants can be for antigenic determinants can be cloned and expressed in an alternative host e.g. cloned and expressed in an alternative host e.g. E. E. colicoli..
Recombinant VaccinesRecombinant 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. Subunit2. Peptide
New Generation of VaccinesNew Generation of Vaccines
There are three types of vaccines we will There are three types of vaccines we will be discussing:be discussing:
Subunit (protein) vaccinesSubunit (protein) vaccines
Attenuated vaccinesAttenuated vaccines
Vector vaccinesVector vaccines
Vaccine TechnologyVaccine Technology
4. Subunit / Peptide 4. Subunit / Peptide VaccinesVaccines
Subunit vaccinesSubunit 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
Structure of a Virus particle
Subunit vaccines born from Subunit vaccines born from following observationfollowing observation
It has been showed that the It has been showed that the capsid or envelope proteinscapsid or envelope proteins are enough to illicit an immune response:are enough to illicit an immune response: Herpes simplex virus envelop glycoprotein O.Herpes simplex virus envelop glycoprotein O. Foot and mouth disease virus capsid protein (VP1)Foot and mouth disease virus capsid protein (VP1) Extracellular proteins produced by Extracellular proteins produced by Mycobacterium Mycobacterium
tuberculosistuberculosis..
Subunit VaccinesSubunit Vaccines Antibodies usually bind to Antibodies usually bind to surface proteinssurface proteins of the pathogen of the pathogen
or proteins generated after the disruption of the pathogen.or proteins generated after the disruption of the pathogen. Binding of antibodies to these proteins will stimulate an Binding of antibodies to these proteins will stimulate an
immune responseimmune response.. Therefore proteins can be use to stimulate an immune Therefore proteins can be use to stimulate an immune
response.response.
A Subunit Vaccine for A Subunit Vaccine for M. tuberculosisM. tuberculosis
TuberculosisTuberculosis is caused by is caused by Mycobacterium Mycobacterium tuberculosistuberculosis..
The bacterium form lesions in the tissues and The bacterium form lesions in the tissues and organs causing cell death. Often the lung is organs causing cell death. Often the lung is affected.affected.
About About 2 billion people are infected2 billion people are infected and there and there are 3 million deaths/year.are 3 million deaths/year.
Currently tuberculosis is controlled by a vaccine Currently tuberculosis is controlled by a vaccine called called BCG BCG (Bacillus Calmette-Guerin) which is (Bacillus Calmette-Guerin) which is a strain of a strain of M. bovisM. bovis..
M. bovisM. bovis often responds to diagnostic test for often responds to diagnostic test for M. M. tuberculosistuberculosis..
A Subunit Vaccine for A Subunit Vaccine for M. M. tuberculosistuberculosis
Six Six extracellular proteinsextracellular proteins of of M. M. tuberculosistuberculosis were purified. were purified.
Separately and in combinations these Separately and in combinations these proteins were used to immunized guinea proteins were used to immunized guinea pigs.pigs.
These animals were then challenged with These animals were then challenged with M. tuberculosisM. tuberculosis..
After 9-10 weeks examination showed that After 9-10 weeks examination showed that some combinations of the purified proteins some combinations of the purified proteins provided the same level of protection as the provided the same level of protection as the BCG vaccine.BCG vaccine.
Selection & delivery of vaccine Selection & delivery of vaccine peptides peptides
Antigenic determinants = Antigenic determinants = epitopes on envelope epitopes on envelope proteinsproteins
Inert carrier: hemocyanin Inert carrier: hemocyanin from keyhole limpetfrom keyhole limpet
Highly immunogenic Highly immunogenic carrier: Hepatitis B core carrier: Hepatitis B core prot.prot.
5. Attenuated Vaccines5. Attenuated Vaccines
Attenuated VaccinesAttenuated Vaccines
Attenuated vaccines often consists of a Attenuated vaccines often consists of a pathogenic strains in which the virulent genes are pathogenic strains in which the virulent genes are deleted or modifieddeleted or modified..
Live vaccines are more effective than a killed or Live vaccines are more effective than a killed or subunit (protein) vaccines.subunit (protein) vaccines.
A Live Cholera VaccineA Live Cholera Vaccine
The causal agent of cholera is The causal agent of cholera is Vibrio choleraeVibrio cholerae and is and is transmitted through contaminated water.transmitted through contaminated water.
V. choleraeV. cholerae produces a enterotoxin with an produces a enterotoxin with an A subunit A subunit and 5 B subunits.and 5 B subunits.
Presently the cholera vaccine consist of a phenol-killed Presently the cholera vaccine consist of a phenol-killed V. choleraeV. cholerae and it only last 3-6 months. and it only last 3-6 months.
A live vaccine would be more effective.A live vaccine would be more effective.
In the sequence of the A peptide a In the sequence of the A peptide a tetracycline tetracycline resistanceresistance gene is inserted. gene is inserted.
A Live Cholera VaccineA Live Cholera Vaccine
A plasmid with A peptide was digested with 2 A plasmid with A peptide was digested with 2 restriction enzymes restriction enzymes ClaCla1 and 1 and XbaXba11..
This removes 550 bases of A peptide.This removes 550 bases of A peptide. A A XbaXba1 linker1 linker was added and T4 ligase used was added and T4 ligase used
to ligate the DNA. This plasmid was mixed to ligate the DNA. This plasmid was mixed with with V. choleraeV. cholerae with tetracycline resistant with tetracycline resistant gene.gene.
By By conjugation conjugation the plasmid was transferred the plasmid was transferred to the strain with the tetto the strain with the tetRR gene inserted into gene inserted into it’s chromosomal DNA.it’s chromosomal DNA.
Production of a Live Cholera VaccineProduction of a Live Cholera Vaccine
A Live Cholera VaccineA Live Cholera Vaccine
By recombination the A peptide with the By recombination the A peptide with the tettetRR gene was replaced by the deleted A gene was replaced by the deleted A peptide.peptide.
The final result is The final result is V. choleraeV. cholerae with a 550 with a 550 bp of the A peptide deleted.bp of the A peptide deleted.
If this can be used as a vaccine is being If this can be used as a vaccine is being tested.tested.
Production of a Live Cholera VaccineProduction of a Live Cholera Vaccine
6. Vector Vaccines6. Vector Vaccines
Vector VaccineVector Vaccine
A vector vaccine is a vaccine which is A vector vaccine is a vaccine which is introduced by a vector e.g. introduced by a vector e.g. vacciniavaccinia virus virus..
The The vacciniavaccinia virus as a live vaccine led to the virus as a live vaccine led to the globally eradication of the globally eradication of the smallpox virussmallpox virus..
The genome of the The genome of the vacciniavaccinia virus has been virus has been completely sequenced.completely sequenced.
The virus replicates in the cytoplasm rather The virus replicates in the cytoplasm rather than in the nucleus.than in the nucleus.
The The vacciniavaccinia virus is generally nonpathogenic. virus is generally nonpathogenic.
Vector VaccineVector Vaccine
These characteristics makes the These characteristics makes the vacciniavaccinia virus a good candidate for a virus vector to virus a good candidate for a virus vector to carry gene for antigenic determinants form carry gene for antigenic determinants form other pathogens.other pathogens.
The procedure involves:The procedure involves: The DNA sequence for the specific antigen The DNA sequence for the specific antigen
is inserted into a plasmid beside the is inserted into a plasmid beside the vaccinia virus promoter in the middle of a vaccinia virus promoter in the middle of a non-essential gene e.g. non-essential gene e.g. thymidine kinasethymidine kinase..
Vector VaccineVector Vaccine
The plasmid is used to transform The plasmid is used to transform thymdine thymdine kinase negative cellskinase negative cells which were which were previously infected with the vaccinia virus.previously infected with the vaccinia virus.
RecombinationRecombination between the plasmid and between the plasmid and vaccinia virus chromosomal DNA results in vaccinia virus chromosomal DNA results in transfer of antigen gene from the transfer of antigen gene from the recombinant plasmid to the vaccinia virus.recombinant plasmid to the vaccinia virus.
Thus virus can now be used as a vaccine Thus virus can now be used as a vaccine for the specific antigen.for the specific antigen.
Insertion of antigen Insertion of antigen gene into gene into vacciniavaccinia
virus genomevirus genomeby recombinationby recombination
Vector VaccineVector Vaccine
A number of antigen genes have been A number of antigen genes have been inserted into the inserted into the vacciniavaccinia virus genome e.g. virus genome e.g.
Rabies virus G proteinRabies virus G protein Hepatitis B surface antigenHepatitis B surface antigen Influenza virus NP and HA proteins.Influenza virus NP and HA proteins. A recombinant A recombinant vacciniavaccinia virus vaccine for virus vaccine for
rabies is able to elicit neutralizing rabies is able to elicit neutralizing antibodies in foxes which is a major carrier antibodies in foxes which is a major carrier of the disease.of the disease.