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 B booster • 15-16 YEARS• TETANUS booster

** Taken from the Scientist;17(2004)

…..

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Progression of Vaccine Development **

Function of Immune System is PROTECTION against:

1. Bacteria2. Virus3. Fungus/ multicellular parasites4. Cancer5. Toxins 6. ( 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

•Appendix, tonsils, intestines

Cell Types1. Lymphocytes: derived in bone marrow from

stem cells include both T cells and B cells. 1012

A) T cells: stored & mature in thymus-migrate throughout the body

-Killer Cells Perform lysis (infected cells)Cell mediated immune response

-Helper CellsEnhance T killer or B cell activity

-Suppressor CellsReduce/suppress immune activityMay 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)

Lymphocytes (cont.)

2. Neutrophils- found throughout body, in blood-phagocytosis of Ab-Ag CX

3. Macrophages- throughout body, blood, lymphatics-phagocytose non-specifically (non Ab coated Ag)-phagocytose specifically Ab-Ag 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

Bacterial Infection

Macrophage

Bacteria

ComplementSeries 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 IgIgG: 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 B- cell

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

• LIVER• (4 Weeks)

• BONE MARROW• (4-5 Weeks )

• THYMUS• (7-10 Weeks)

• BLOOD LYMPH• (14 Weeks)

• SPLEEN

• (16 Weeks)

• T-cells migrate and appear in tissues with development and increase in number

throughout Gestation

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 Agents Which Affect the Immune ResponseImmune Response

Rheumatoid ArthritisRheumatoid ArthritisDisease that leads to

inflammation of the joints and surrounding tissues

Can affect organsThe 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/uesc_01_img0050.jpg

Systemic Lupus Systemic Lupus ErythematosusErythematosus

Autoimmune diseaseSymptoms:

◦ 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 Other Immunological DiseasesDiseases

• Type I diabetes mellitus• Multiple sclerosis• Asthma• Allergies• SCID

Treatment StrategiesTreatment 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 – Immunosuppression – GlucocorticoidsGlucocorticoids

• Usually co-administered with other suppressive agents to treat auto-immune 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 – Immunosuppression – GlucocorticoidsGlucocorticoids

• Side Effects– Toxic– Causes increased infection risk– Poor wound healing– Hyperglycemia– Hypertension

http://img.medscape.com/article/588/548/588548-fig3.jpg

Immunosuppression – Immunosuppression – GlucocorticoidsGlucocorticoids

Prednisone

Dexamethasone

Cortisol

Immunosuppression – Immunosuppression – Calcineurin InhibitorsCalcineurin 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 – Immunosuppression – Calcineurin InhibitorsCalcineurin Inhibitors

Tacrolimusa.k.a. FK-506

Cyclosporin A

http://drtedwilliams.net/cop/753/753CalcineurinInhibitors.GIF

Immunosuppression – Immunosuppression – Anti-proliferative and Anti-Metabolic Anti-proliferative and Anti-Metabolic

DrugsDrugs– Azathioprine

• Purine anti-metabolite

Tioguanine

Azathioprine Mercaptopurine

Guanine

Immunosuppression – Immunosuppression – Anti-proliferative and Anti-Metabolic Anti-proliferative and Anti-Metabolic

DrugsDrugs– 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 – Immunosuppression – Monoclonal AntibodiesMonoclonal Antibodies

http://www.facetbiotech.com/images/moa_illustrations/FACET_MoA_ELOTUZUMAB.jpg

ToleranceTolerance

• 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

ImmunizationImmunization

• 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 life’s 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 gene3. An abnormal gene repaired through selective

reverse mutation4. 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 patient’s target cell

• The target cells become infected with the viral vector

• The vector’s 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 gene’s 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 hasn’t approved any human gene therapy

product for saleReasons:• 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, Alzheimer’s, 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 (adeno-associated 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 DevelopmentsRecent Developments

• Genes get into brain using liposomes coated in polymer call polyethylene glycol– potential for treating Parkinson’s disease

• RNA interference or gene silencing to treat Huntington’s– 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 THERAPYVIRAL VECTORS IN GENE THERAPY   

Gene TherapyGene TherapyThe introduction of nucleic acids into cells for the purpose of altering the course of medical condition or disease.   AdministrationAdministration

• 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 TransferThe Ideal Vector for Gene Transfer• High concentration of virus allowing many cells to be infected or transducedHigh concentration of virus allowing many cells to be infected or transduced

• Convenience and reproducibility of productionConvenience and reproducibility of production

• Ability to transduce dividing and non-dividing cellsAbility to transduce dividing and non-dividing cells

• Ability to integrate into a site-specific location in the host chromosome, or to be Ability to integrate into a site-specific location in the host chromosome, or to be successfully maintained as stable episomesuccessfully maintained as stable episome

• A transcriptional unit that can respond to manipulation of its regulatory elementsA transcriptional unit that can respond to manipulation of its regulatory elements

• Ability to target the desired type of cellAbility to target the desired type of cell

• No components that elicit an immune responseNo components that elicit an immune response

Introduction of Genes Into AnimalsIntroduction of Genes Into Animals

METHODS METHODS MAJOR LIMITATIONS MAJOR LIMITATIONS

Calcium PhosphateCalcium Phosphate

DEAE DextranDEAE Dextran Low EfficiencyLow Efficiency

Cationic Lipids, LiposomesCationic Lipids, Liposomes

Direct DNA InjectionsDirect DNA Injections Low EfficiencyLow Efficiency

ElectroporationElectroporation Transient expressionTransient expression

Introduction of Genes Into AnimalsIntroduction of Genes Into Animals

VIRAL VECTORSVIRAL VECTORS MAJOR LIMITATIONSMAJOR 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

Genetic DiseasesGenetic Diseases

Cystic FibrosisCystic Fibrosis

Blood DisordersBlood Disorders

Muscular DystrophyMuscular Dystrophy

DiabetesDiabetes

Acquired DiseasesAcquired Diseases

CancerCancer

CardiovascularCardiovascular

Neurological DisordersNeurological Disorders

Infectious Disease (AIDS)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: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: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 can’t replicate.

• 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. 

Adenoviral vectors- LimitationsAdenoviral vectors- Limitations

Adeno-associated viral vectors: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: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 imm

une responses that ultimately lead to the

neutralization of the adenovirus as well as to the elim

ination of the transduced cells.