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

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.