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Applications of Immunology

TalaroChapter 16

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Acquired Immunity• Natural Immunity

– Acquired as part of normal life experiences

• Artificial Immunity – Acquired through vaccination

• Active Immunity – Results when a person is challenged with Ag that stimulates production of Ab– Memory

• Passive Immunity – Preformed Ab are donated to an individual– Acts immediately but is short term– No memory

• Breast feeding• Gamma globulin• Monoclonal antibodies

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Vaccines• Provide an antigenic stimulus that does not cause disease

– Attenuated strain• Tissue culture or unnatural / unusual host• Hypovirulent

– Dead whole cells or inactivated viruses• Heat, formalin, UV irradiation

– Purified antigen subunits from cells or viruses– Surface antigens produce via rDNA technology– DNA vaccines

• Produces long lasting protective immunity• Edward Jenner (page 476)

– www.sc.edu/library/spcoll/nathist/jenner2.html– Cowpox – Smallpox

• Variola – Controlled experiments

• Vaccinia virus– Cultured cow pox virus for many years

• Small pox eradicated in 1973

Why did the vaccinia viruswork?

Immunization using a closely related, less pathogenic organism to give protection against a more pathogenic one.

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Cowpox on human forearmwww.cdc.gov

Smallpoxwww.cdc.gov

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Vaccination Success•Small pox

• 2 million people a year died from small pox until 1967 • The World Health Organization initiated an immunization campaign that eradicated small pox in 12 years

•Poliomyelitis (polio)

• This virus attacks the motor neurons of the brain and spinal cord• Causes paralysis and death• Immunization campaigns since the 1950s have virtually eradicated polio in developed countries

www.who.int/immunization_safety/en/www.cdc.gov

news.bbc.co.uk

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Polio Eradication

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• Haemophilus influenzae type b – Mistakenly believed to have

caused influenza – Type b strains accounted for

majority of bacterial meningitis

• Meningitis, pneumonia, and epiglottitis

• Was the leading cause of bacterial meningitis of children 5 years or younger– 1/200 children contracted Hib– Incidence has decreased 99%

since the vaccine was introduced

www.cdc.gov

20,000 cases per year in the early 1980’s

1,247 in 2000

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Vaccines cause diseaseDisease is no longer a threat in my countryCost of vaccination Difficulty reaching vaccination centerNot recommended by my physicianSafety concerns

Side effectsSIDS

Autism Depress the immune system

Mercury PoisoningReligious beliefs

www.who.int/immunization_safety/aefi/immunization_misconceptions/en/index.html

Herd ImmunityProtection from a disease among unvaccinated individuals occurs when 90% of a population is immunized.

% depends on the disease & vaccine

Content and Design Attributes of Antivaccination Web Sites Robert M. Wolfe, MD; Lisa K. Sharp, PhD; Martin S. Lipsky, MD

JAMA 2002 287:3245-3248.

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Polio Outbreak Occurs Among Amish Families In Minnesotaby David BrownWashington Post Staff WriterOctober 14, 2005The first outbreak of polio in the United States in 26 years occurred earlier this fall in an Amish community in central Minnesota, state and federal health officials reported yesterday. Four children have been infected with the virus, although none has become paralyzed. The Amish typically decline to vaccinate their children. The last large outbreak of polio occurred in numerous Amish communities in several states in 1979.Fears Rising Over Measles Outbreaks by ROBERT A. HAMILTONSTATE health officials are preparing for what they fear could be ''a major outbreak'' of measles when students who attend Boston University return home next week for spring break. Last week, a measles case was reported in Fairfield, an 18-year-old Boston University freshman who returned home eight days ago with cold symptoms and by Tuesday was in the hospital. ''We expect a lot more students will be returning to Connecticut next week, for the spring break,'' the program director of the state immunization program, Dr. Charles H. Alexander, .

March 3, 1985 The New York Times 

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irradiation

hypovirulent

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Serology• A part of immunology

that attempts to detect signs of infection in a patient’s serum – Use Abs that

specifically bind to Ag• Ag-Ab reactions are

visible by– Clumps– Precipitates– Color changes – Release of radioactivity

• The most effective tests have high specificity and sensitivity.

leukocytes

AbsProteinsClotting factorsHormonesNutrientsIons

Clear fluidfrom clottedblood

Isolate Ab from serum

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Agglutination Test

• Ab and Ag from a whole cell crosslink

– Forming complexes that settle out and from visible clumps in the test chamber

Isolate Abs frompatient

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Agglutination

Contains patient’Abs

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Agglutination

Contains Abs for the specific pathogen

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Precipitation Tests

• Soluble Ag is precipitated– Cloudy or opaque

zone– Many variations are

used to maximize this technique

– Measure optical density

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Calculate titer The highest dilution of serum that shows a positive result

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Enzyme-linked immunosorbent assay (ELISA)

• Detect unknown Ag or Ab

• A positive result is visualized when a colored product is released by an enzyme-substrate reaction

Anti-immunoglobulin Ab complexed to an enzyme

conjugate

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Perform a virtual ELISA courtesy of the Howard Hughes Medical Institute www.hhmi.org/biointeractive/vlabs/

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Radioimmunoassay

Ag or Abs are labeled with radioactive isotopes and traced

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ELISAwww.biology.arizona.edu

Positive Rxn Negative Rxn Positive

Control Negative

ControlPatient A Patient B Patient C

Assay Control

1.689 0.153 0.055 0.412 1.999 0.123

+ is 0.5 or greater

Indeterminate0.300 – 0.495

- is 0.300 or less

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• More sensitive than ELSIA– Less chance of a false +

• Detects specific Ag or Ab proteins in serum• Separates proteins into bands via gel electrophoresis• Protein bands are transferred to a membrane• Specific Abs are used to “probe” the membrane

– Primary Ab are from patient– Secondary Ab

• Anti-immunoglobulin Ab complex to an enzyme – Colorless substrate

• Colormetric

Western Blot Western Blot for a HIV on page 487

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Western Blot for HIVwww.biology.arizona.edu

gp160 viral envelope precursor (env)

gp120 viral envelope protein (env) binds to CD4

p24 viral core protein (gag)

p31 reverse transcriptase (pol)

Serum proteins (virus) separated via electrophoresis.Transferred to a membrane.

The primary Abs from the serum of a patient is added and will react to the HIV proteins (above).

The secondary Ab is an anti-immunoglobulin conjugated to an enzyme. This Ab is specific for the

primary Abs.

A colorless substrate is cleaved if the anti-immunoglobulin conjugated to the primary Ab.

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Band Pattern Interpretation Lane 1, HIV+ serum (positive control) Lane 2, HIV- serum (negative control) Lane A, Patient A Lane B, Patient B Lane C, Patient C

No bands present - Bands at either p31

OR p24 AND bands present at either gp160 OR gp120

+

Bands present, but pattern does not meet criteria for positivity

Indeterminate

Patient A is –Patient B is –Patient C is +

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• Fluorescent Ab (FAbs) either directly or indirectly to visualize cells or cell aggregates

Immunofluorescence

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Monoclonal Antibodies (Mab)• Single specificity antibodies

formed by fusing a mouse B cell with a myeloma cell– A malignant tumor formed by the

cells of the bone marrow

• Used in diagnosis of disease, identification of microbes and therapy

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• Immunize animal• Harvest spleen• Fuse B & myeloma • Hybridoma

• Immortal• Screen hybridomas for Abs directed against antigen of interest

• Hybridomas produce antibody that recognize single epitope• Produce uniform, highly specific Ab in large supply

• Multiple practical applications • Diagnostic tests

• ELISA & Western Blots• Immunosuppressive therapy for transplants

• Prevent action of TC

• Anticancer drugs• Antitoxins• Identify a pathogen• Purification of an important protein

• Precipitate

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ZenapaxPrevent rejection in organ transplantation, especially in kidney transplants.

MylotargAcute myeloid leukemia (AML)

RemicadeAuto-immune disorders like Crohn's disease and rheumatoid arthritis

Target is tumor necrosis factor

RituxanTreat a variety of non-Hodgkin's lymphomas.

Target and destroy non-Hodgkin's lymphoma cells.

Monoclonals on the Market

Herceptin Blocks the effects of the growth factor protein HER2, which transmits growth signals to breast cancer cells.

Causes tumor shrinkage.

Additional information at www.fda.gov