DEVELOPMENT OF IMMUNE

SYSTEM
- GESTATIONAL TOLERANCE

(PREVENTING REJECTION)
- FETAL/NEONATAL PROTECTION
- VACCINATION/IMMUNIZATION

VACCINATIONS

BIRTH
BCG (BACILLUS CALMETTE-GUERIN)
ORAL POLIO
HEPATITIS
6 WEEKS
DPT (DIPHTHERIA, TETANUS,
PERTUSSIS
ORAL POLIO 2ND DOSE
HEPATITIS 2ND

10 WEEKS
DPT (DIPHTHERIA, TETANUS,
PERTUSSIS)
ORAL POLIO 3RD

14 WEEKS
DPT 3RD
ORAL POLIO 4TH

6-9 MONTHS
ORAL POLIO 5TH
HEPATITIS B

9 MONTHS
MEASLES

15-18 MONTHS
MMR (MEASLES, MUMPS, RUBELLA)
DPT booster dose
ORAL POLIO 6TH

5 YEARS
DPT 2ND booster
ORAL POLIO 7TH

10 YEARS
TT (TETANUS) 3RD booster
HEPATITIS Bbooster

15-16 YEARS
TETANUS booster

Progression of Vaccine Development **

..

..
** Taken from the Scientist;17(2004)

Function of Immune System is

PROTECTION against:
1.
2.
3.
4.
5.
6.

Bacteria
Virus
Fungus/ multicellular parasites
Cancer
Toxins
( 5,000 daltons-protein/lipid/CHO/nucleic acids)

Tissues and Organs Important for

Immune Function

Cells derived from stem cells: liver,

bone marrow
Cells are stored, multiply, interact, and
mature in: thymus, spleen, lymph nodes,
blood
Transport: lymphatic vessels
Accessory Organs

Cell Types
1. Lymphocytes: derived in bone marrow from
stem cells include both T cells and B cells. 10 12

Lymphocytes (cont.)
A) T cells: stored & mature in thymus-migrate
throughout the body
-Killer Cells
Perform lysis (infected cells)
Cell mediated immune response
-Helper Cells
Enhance T killer or B cell activity
-Suppressor Cells
Reduce/suppress immune activity
May help prevent auto immune
disease

Lymphocytes (cont.)
B) B-Cells: stored and mature in spleen
secrete highly specific Ab to bind foreign
substance (antigen: Ag), form Ab-Ag
complex
responsible for humoral response
perform antigen processing and
presentation
differentiate into plasma cells (large Ab
secretion)

2. Neutrophilsfoundthroughoutbody,inblood
phagocytosisofAbAgCX

3. Macrophages- throughout
body, blood, lymphatics
-phagocytose non-specifically
(non Ab coated Ag)
-phagocytose specifically AbAg CX
-have large number of
lysosomes (degradative
enzyme)
-perform Ag processing and
presentation
-present Ag to T helper cell
-secrete lymphokines/
cytokines to stimulate T
helper
cells and immune activity

4. Natural Killer Cells-in blood throughout body

-destroy cancer cells
-stimulated by interferons

Macrophage
Bacteria

Bacterial
Infection

Complement
Series of enzymes which are sequentially
activated and result in lysis of cell
membrane of infected cell at bacterium
Permeabilizes membrane leaky

Complement binding and

activation
~35 enzymes and factors
involved in cascade

CDC involves: 1) recognition, 2) attachment of complement-fixing antibodies to tumor

specific surface antigens, 3) complement activation, 4) formation of MAC resulting in
transmembrane pores (perforins) that disrupt the osmotic barrier of the membrane and
lead to osmotic lysis.

5 classes of Ig
IgG: 150,000 m.w.
most abundant in blood, cross placental
barrier,
fix complement, induce macrophage
engulfment
IgA: associated with mucus and secretory glands,
respiratory tract, intestines, saliva,
tears, milk
variable size
IgM: 900,000 m.w.
2nd most abundant , fix complement,
induce macrophage engulfment, primary
immune response

5 Classes of Ig
IgD: Low level in blood, surface receptor on
Bcell
IgE: Binds receptor on mast cells (basophils)
secretes histamine, role in allergic
reactions
Increased histamine leads to vasodilation,
which leads to increase blood vessel
permeability. This induces lymphocyte
immigration swelling and redness.

Thymus
Involution

Repertoire of lymphocytes shift with aging

(membrane components shift)

ORGAN AND T-CELL DEVELOPMENT

YOLK SAC

BONE MARROW
(4-5 Weeks )

LIVER
(4 Weeks)

THYMUS
(7-10 Weeks)

BLOOD LYMPH
(14 Weeks)

SPLEEN
(16 Weeks)

T-cells migrate and appear in tissues

with development and increase in

B-CELLS

FIRST appear in immature state - Liver at 7 weeks

LATER appear mature by 14-20 weeks

CAN DIFFERENTIATE INTO IMMUNOLOGICALLY COMPETENT

ANTIBODY-PRODUCING PLASMA CELLS

NATURAL KILLER CELLS

FIRST APPEAR IN FETAL BONE MARROW
AROUND 13 WEEKS GESTATION
FOUND THROUGHOUT BODY
NK CELLS HAVE DIMINISHED ACTIVITY BEFORE
BIRTH COMPARED TO ADULT
STIMULATED BY INTERFERON AFTER 27 WEEKS

COMPLEMENT PROTEINS

ARISE FROM LIVER

FIRST DETECTED 5-6 WEEKS GESTATION
INCREASE GRADUALLY IN CONCENTRATION
AT ABOUT 28 WEEKS COMPLEMENT PROTEINS
ARE AROUND 2/3 THAT OF ADULT
CONCENTRATIONS

INDIVIDUAL VARIATION

SEVERE COMBINED
IMMUNODEFICIENCY DISEASE
(SCID)
CHARACTERISTICS:
GENERALLY CAUSED BY DEFECT OF SINGLE GENE NEEDED
FOR T-CELL AND B-CELL FUNCTION
SUBJECT EXHIBITS NO CELL MEDIATED RESPONSE
SUBJECT CANNOT MAKE ANTIBODIES
ABOUT 25% OF CASES INVOLVES DEFECTIVE GENE FOR
THE ENZYME ADENOSINE DEAMINASE
(REQUIRED FOR PURINE BREAKDOWN)

SEVERE COMBINED IMMUNODEFICIENCY

DISEASE (SCID)
TREATMENT OPTIONS:

GERM FREE ENVIRONMENT

BONE MARROW TRANSPLANT
ROUTINE INJECTIONS OF ADENOSINE DEAMINASE ENZYME
(ADA)

GENE THERAPY USING SUBJECTS OWN

CELLS
(RETROVIRUS CONTAINING ADA TO
INFECT

SUBJECTS BONE MARROW STEM CELLS)

Agents Which Affect the

Immune Response

Rheumatoid Arthritis
Disease that leads to
inflammation of the
joints and surrounding
tissues
Can affect organs
The immune system
confuses healthy
tissue with foreign and
begins to attack itself
Occurs at any age,
usually affects women
more than men
Affects joints on both
sides equally
Wrists, fingers, knees,
feet, ankles

http://www.scienceclarified.com/images/ues
c_01_img0050.jpg

Systemic Lupus
Erythematosus
Autoimmune disease
Symptoms:

Chest pain, fatigue,

fever, general
discomfort, hair loss,
mouth sores, sensitivity
to sunlight, skin rash,
swollen lymph nodes,
arrhythmias, blood in
urine, abdominal pain,
coughing up blood,
patchy skin colors

Other form: lupus

nephrititis
Can cause kidney
failure and lead to
dialysis

http://www.taconichills.k12.ny.us/webquests
/noncomdisease/lupuspic.jpg

Other Immunological
Diseases

Type I diabetes mellitus

Multiple sclerosis
Asthma
Allergies
SCID

Treatment Strategies
1. Immunosuppression involves
downregulating immune system
activity
2. Tolerance the idea that a body can
be taught not to reject somthing
3. Immunostimulation involves
upregulating immune system activity
4. Immunization active or passive

Immunosuppression
Glucocorticoids
Usually co-administered with other
suppressive agents to treat autoimmune disorders or treatment of
transplant rejection
Exact mechanism not elucidated
Very broad anti-inflammatory effects
Downregulate IL-1 and IL-6
Cause apoptosis in activated cells

Immunosuppression
Glucocorticoids
Side Effects
Toxic
Causes increased infection risk
Poor wound healing
Hyperglycemia
Hypertension

http://img.medscape.com/article/588/548/5
88548-fig3.jpg

Immunosuppression
Glucocorticoids

Prednisone
Dexamethasone

Cortisol

Immunosuppression
Calcineurin Inhibitors
Calcineurin protein phosphatase that
activates T Cells by dephosphorylating
transcription factors, including NFAT
(nuclear factor of activated T cells).
Blocks T Cell proliferation
Decreased immune response

Immunosuppression
Calcineurin Inhibitors

Tacrolimus
a.k.a. FK-506
Cyclosporin A

http://drtedwilliams.net/cop/753/753Calcine
urinInhibitors.GIF

Immunosuppression
Anti-proliferative and Anti-Metabolic
Drugs
Azathioprine
Purine anti-metabolite

Tioguanine
Azathioprine

Mercaptopurine

Guanine

Immunosuppression
Anti-proliferative and Anti-Metabolic
Drugs
Mycophenolate Mofentil (CellCept)
Hydrolyzed to mycophenolic acid
IMPDH inhibitor (inosine monophosphate
dehydrogenase enzyme
Important in biosynthesis of guanine
Good alternative to azathioprine when
toxicity is an issue

Mycophenolic acid

Immunosuppression
Monoclonal Antibodies

http://www.facetbiotech.com/images/moa_illustrations/FACET_Mo
A_ELOTUZUMAB.jpg

Tolerance
Strategy is to induce and maintain
tolerance
Useful strategy for organ transplantation
Very much the target of research today
Would represent a true cure for
autoimmune conditions without side
effects of immunosuppressive agents
Holy Grail of immunomodulation

Immunization
Active or passive
Active stimulation with antigen to develop
antigens for future prevention
Passive administration of antibodies to
individual already exposed or about to be
exposed to antigens

Vaccines active; administration whole,

killed organism, live organism, or specific
peptide from organism
Immune Globulin used in passive
immunization; used in individuals
deficient in antibodies

Genes

Are carried on a chromosome

The basic unit of heredity

Encode how to make a protein

DNARNA proteins

Proteins carry out most of lifes function.

When altered causes dysfunction of a protein

When there is a mutation in the gene, then it will change the

codon, which will change which amino acid is called for which will
change the conformation of the protein which will change the
function of the protein. Genetic disorders result from mutations in
the genome.

Picture of a Chromosome

http://www.accessexcellence.org/RC/VL/GG/genes.html

What is Gene Therapy

It is a technique for correcting
defective genes that are responsible
for disease development
There are four approaches:
1. A normal gene inserted to compensate
for a nonfunctional gene.
2. An abnormal gene traded for a normal
gene
3. An abnormal gene repaired through
selective reverse mutation
4. Change the regulation of gene pairs

The Beginning
In the 1980s, Scientists began to look
into gene therapy.
They would insert human genes into a
bacteria cell.
Then the bacteria cell would transcribe
and translate the information into a
protein
Then they would introduce the protein
into human cells

The First Case

The first gene therapy was
performed on September
14th, 1990
Ashanti DeSilva was
treated for SCID
Sever combined
immunodeficiency

Doctors removed her white

blood cells, inserted the
missing gene into the WBC,
and then put them back
into her blood stream.
This strengthened her
immune system
Only worked for a few
months

How It Works
A vector delivers the therapeutic
gene into a patients target cell
The target cells become infected with
the viral vector
The vectors genetic material is
inserted into the target cell
Functional proteins are created from
the therapeutic gene causing the cell
to return to a normal state

Principle

http://encarta.msn.com/media_461561269/Gene_Therapy.html

Viruses
Replicate by inserting their DNA into
a host cell
Gene therapy can use this to insert
genes that encode for a desired
protein to create the desired trait
Four different types

Retroviruses

Created double stranded DNA copies from

RNA genome
The retrovirus goes through reverse
transcription using reverse transcriptase
and RNA
the double stranded viral genome
integrates into the human genome using
integrase
integrase inserts the gene anywhere
because it has no specific site
May cause insertional mutagenesis
One gene disrupts another genes
code (disrupted cell division
causes cancer from uncontrolled
cell division)
vectors used are derived from the
human immunodeficiency virus (HIV)
and are being evaluated for safety

Adenoviruses
Are double stranded DNA genome that
cause respiratory, intestinal, and eye
infections in humans
The inserted DNA is not incorporate
into genome
Not replicated though
Has to be reinserted when more cells
divide

Ex. Common cold

Adenovirus cont.

http://en.wikipedia.org/wiki/Gene_therapy

Adeno-associated Viruses

Adeno-associated Virus- small, single stranded DNA that insert genetic

material at a specific point on chromosome 19
From parvovirus family- causes no known disease and doesn't trigger
patient immune response.
Low information capacity
gene is always "on" so the protein is always being expressed, possibly
even in instances when it isn't needed.
hemophilia treatments, for example, a gene-carrying vector could be
injected into a muscle, prompting the muscle cells to produce Factor IX
and thus prevent bleeding.

Study by Wilson and Kathy High (University of Pennsylvania), patients

have not needed Factor IX injections for more than a year

Herpes Simplex Viruses

Double stranded DNA viruses that
infect neurons
Ex. Herpes simplex virus type 1

http://www.ucmp.berkeley.edu/alllife/virus.html

Non-viral Options

Direct introduction of therapeutic DNA

But only with certain tissue
Requires a lot of DNA
Creation of artificial lipid sphere with aqueous core, liposome
Carries therapeutic DNA through membrane
Chemically linking DNA to molecule that will bind to special
cell receptors
DNA is engulfed by cell membrane
Less effective
Trying to introduce a 47th chromosome
Exist alongside the 46 others
Could carry a lot of information
But how to get the big molecule through membranes?

Current Status
FDA hasnt approved any human gene
therapy product for sale
Reasons:
In 1999, 18-year-old Jesse Gelsinger
died from multiple organ failure 4 days
after treatment for omithine
transcarboxylase deficiency.
Death was triggered by severe immune
response to adenovirus carrier

January 2003, halt to using retrovirus

vectors in blood stem cells because
children developed leukemia-like
condition after successful treatment for
X-linked severe combined
immunodeficiency disease

Problems with Gene

Therapy

Short Lived
Hard to rapidly integrate therapeutic DNA into genome and
rapidly dividing nature of cells prevent gene therapy from long
time
Would have to have multiple rounds of therapy
Immune Response
new things introduced leads to immune response
increased response when a repeat offender enters
Viral Vectors
patient could have toxic, immune, inflammatory response
also may cause disease once inside
Multigene Disorders
Heart disease, high blood pressure, Alzheimers, arthritis and
diabetes are hard to treat because you need to introduce more
than one gene
May induce a tumor if integrated in a tumor suppressor gene
because insertional mutagenesis

Unsuccessful Gene
therapies
Jesse Gelsinger, a gene therapy patient who lacked ornithine
transcarbamylase activity, died in 1999.
Within hours after doctors shot the normal OTC gene attached
to a therapeutic virus into his liver, Jesse developed a high
fever. His immune system began raging out of control, his
blood began clotting, ammonia levels climbed, his liver
hemorrhaged and a flood of white blood cells shut down his
lungs.
One problem with gene therapy is that one does not have
control over where the gene will be inserted into the genome.
The location of a gene in the genome is of importance for the
degree of expression of the gene and for the regulation of the
gene (the so-called "position effect"), and thus the gene
regulatory aspects are always uncertain after gene therapy

Successful Gene Therapy for Severe

Combine Immunodeficiency
Infants with severe combined
immunodeficiency are unable to
mount an adaptive immune
response, because they have a
profound deficiency of lymphocytes.
severe combined immunodeficiency
is inherited as an X-linked recessive
disease, which for all practical
purposes affects only boys.
In the other half of the patients with
severe combined
immunodeficiency, the inheritance
is autosomal recessive and there
are several abnormalities in the
immune system when the defective
gene is encoded on an autosome.

Severe Combine Immunodeficiency

Continued
A previous attempt at gene therapy for
immunodeficiency was successful in
children with severe combined
immunodeficiency due to a deficiency of
adenosine deaminase.
In these patients, peripheral T cells were
transduced with a vector bearing the gene
for adenosine deaminase.
The experiment was extremely labor
intensive, because mature peripheral-blood
T cells were modified rather than stem
cells, and the procedure therefore had to
be repeated many times to achieve
success.

Successful One Year Gene

Therapy Trial For Parkinson's
Disease

Neurologix a biotech company

announced that they have
successfully completed its
landmark Phase I trial of gene
therapy for Parkinson's Disease.

This was a 12 patient study with

four patients in each of three dose
escalating cohorts. All procedures
were performed under local
anesthesia and all 12 patients
were discharged from the hospital
within 48 hours of the procedure,
and followed for 12 months.
Primary outcomes of the study
design, safety and tolerability,
were successfully met. There were
no adverse events reported
relating to the treatment.

Parkinson's Disease Cont.

The gene transfer procedure
utilized the AAV (adenoassociated virus) vector, a
virus that has been used
safely in a variety of clinical
gene therapy trials, and the
vehicle that will be used in
all of the company's first
generation products,
including epilepsy and
Huntington's disease. In its
Parkinson's disease trial,
Neurologix used its gene
transfer technology.

Recent Developments
Genes get into brain using liposomes coated
in polymer call polyethylene glycol
potential for treating Parkinsons disease

RNA interference or gene silencing to treat

Huntingtons
siRNAs used to degrade RNA of particular sequence
abnormal protein wont be produced

Create tiny liposomes that can carry therapeutic

DNA through pores of nuclear membrane
Sickle cell successfully treated in mice

http://www.wellesley.edu/Biology/Courses/219/Gen_news/i3_Gene_Therapy.jpg

VIRAL VECTORS IN GENE THERAPY

Gene Therapy
The introduction of nucleic acids into cells for the
purpose of altering the course of medical condition or
disease.
Administration
ex vivo- cells removed, genetically modified,
transplanted back into a patient
in vivo- direct transfer of genetic material into
patient

The Ideal Vector for Gene Transfer

High concentration of virus allowing many cells to be infected or transduced

Convenience and reproducibility of production
Ability to transduce dividing and non-dividing cells
Ability to integrate into a site-specific location in the host chromosome, or to be
successfully maintained as stable episome
A transcriptional unit that can respond to manipulation of its regulatory elements
Ability to target the desired type of cell
No components that elicit an immune response

Introduction of Genes Into Animals

METHODS

MAJOR LIMITATIONS

Calcium Phosphate
DEAE Dextran

Low Efficiency

Cationic Lipids, Liposomes

Direct DNA Injections

Low Efficiency

Electroporation

Transient expression

Introduction of Genes Into Animals

VIRAL VECTORS

MAJOR LIMITATIONS

Papova (SV40, Polyoma)

Size; Host range

Papilloma (BPV)

Size; Integration, Transformation

Adeno associated (AAV)

Size; production

Adeno

Size; antigenicity, episomal DNA, toxicity

Herpes/Vaccinia

Pathogenic, cytotoxic, lytic

Retroviruses

Inability to infect post-mitotic cells

Lentiviruses

Safety, integration

GeneticDiseases
Cystic Fibrosis
Blood Disorders
Muscular Dystrophy
Diabetes

AcquiredDiseases
Cancer
Cardiovascular
Neurological Disorders
Infectious Disease (AIDS)

Genetic Defects that are Candidates for Gene Therapy

Recombinant Vaccinia virus

Expression Vector:

Construction of an infectious vaccinia virus

expressing the influenza virus HA gene

Adenoviral vectors:
Advantages:
Higher titer
Efficient transduction of nondividing cells
in vitro and in vivo
Disadvantages:
Toxicity
Immunological response
Prior exposure

Adenovirus particle structure:

Nonenveloped particle
Contains linear double
stranded DNA
Does not integrate into the
host genome
Replicates as an episomal
element in the nucleus

Adenoviral vectors:
Double-stranded DNA viruses, usually cause benign respiratory disease; serotypes
2 and 5 are used as vectors.
Can infect dividing and non-dividing cells, can be produced at high titers.
Replication-deficient adenovirus vectors can be generated by replacing the E1 or E3
gene, which is essential for replication.
The recombinant vectors are then replicated in cells that express the products of the
E1 or E3 gene and can be generated in very high concentrations.
Cells infected with recombinant adenovirus can express the therapeutic gene, but
because essential genes for replication are deleted, the vector cant replicate.

Adenoviral vectors- Limitations

Adenoviral vectors can infect cells in vivo, causing them to express high levels of the
transgene. However, expression lasts for only a short time (5-10 days post-infection).
Immune response is the reason behind the short-term expression.
Immune reaction is potent, eliciting both the cell-killing cellular response and the
antibody producing humoral response.
Humoral response results in generation of antibodies to adenoviral proteins and
prevents any subsequent infection if a second injection of the recombinant adenovirus is
given.

Adeno-associated viral vectors:

AAV is a simple, non-pathogenic, single stranded DNA virus dependent on the
helper virus (usually adenovirus) to replicate.
It has two genes (cap and rep), sandwiched between inverted terminal repeats that
define the beginning and the end of the virus and contain the packaging sequence.
The cap gene encodes viral capsid proteins and the rep gene product is involved in
viral replication and integration.
It can infect a variety of cell types and in the presence of the rep gene product, the
viral DNA can integrate preferentially into human chromosome 19.

AAV vectors:

Gene therapy
Gene therapy:
to correct a genetic defect by transferring of a
functional normal copy of the gene into cells
Examples of diseases caused by genetic defect
Ornithine transcarbamylase (OTC deficiency)
Hemophilia (blood coagulation factors VIII or IX)
SCID( severe combined immunodeficiency)
Muscular dystrophy
Cystic fibrosis
Sickle cell anemia

Application of gene therapy

Genetic disorder (deficiency): OTC
Cancer
Genetic predisposition
Mutation in oncogene or tumor suppressor gene

Autoimmunity diseases: rheumatoid

arthritis
Delivery of counteracting gene

Diseases involve several genes and the

environmental interact: diabetes

Factors to be considered in Gene therapy

How to deliver genes to specific cells, tissue
and whole animals? (methods of delivery)
How much and how long the introduced gene
will be expressed?
The site and dose of gene delivery
Is there any adverse immunological
consequence of both delivery vehicle (Virus)
and the gene in animals?
Is there any toxic effects?

Methods of gene delivery

Viral Vectors:

Adenovirus
Retrovirus
Lentivirus
Adeno-associated virus (AAV)
Herpes simplex virus (HSV)

Non-viral vector based

Naked DNA (plasmid DNA): injection or genegun
Liposomes (cationic lipids): mix with genes

Ex-vivo
In vivo

Why use viral vectors

Virus are obligate intracellular
parasites
Very efficient at transferring viral
DNA into host cells
Specific target cells: depending on
the viral attachment proteins (capsid
or glycoproteins)
Gene replacement: non-essential
genes of virus are deleted and
exogenous genes are inserted

The application of an adenoviral vector leads to innate and

adaptive immune responses that ultimately lead to the
neutralization of the adenovirus as well as to the elimination of the
transduced cells.