Applications of Immunology

1. Applications of Immunology Talaro Chapter 16

2. 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

4. Vaccines Why did the vaccinia virus work? • 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 Immunization using a closely related, less pathogenic organism to give protection against a more pathogenic one.

5. Smallpox www.cdc.gov Cowpox on human forearm www.cdc.gov

7. www.who.int/immunization_safety/en/ www.cdc.gov • 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 Vaccination Success news.bbc.co.uk

8. Polio Eradication

9. www.cdc.gov • 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 childrencontracted Hib • Incidence has decreased 99% since the vaccine was introduced 20,000 cases per year in the early 1980’s 1,247 in 2000

10. www.who.int/immunization_safety/aefi/immunization_misconceptions/en/index.htmlwww.who.int/immunization_safety/aefi/immunization_misconceptions/en/index.html Vaccines cause disease Disease is no longer a threat in my country Cost of vaccination Difficulty reaching vaccination center Not recommended by my physician Safety concerns Side effects SIDS Autism Depress the immune system Mercury Poisoning Religious beliefs Herd Immunity Protection 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.

11. Polio Outbreak Occurs Among Amish Families In Minnesota by David Brown Washington Post Staff WriterOctober 14, 2005 The 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. HAMILTON STATE 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

12. irradiation hypovirulent

15. Abs Proteins Clotting factors Hormones Nutrients Ions Serology Clear fluid from clotted blood leukocytes • 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. Isolate Ab from serum

16. Isolate Abs from patient Agglutination Test • Ab and Ag from a whole cell crosslink • Forming complexes that settle out and from visible clumps in the test chamber

17. Contains patient’Abs Agglutination

18. Contains Abs for the specific pathogen Agglutination

19. Precipitation Tests • Soluble Ag is precipitated • Cloudy or opaque zone • Many variations are used to maximize this technique • Measure optical density

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

21. Detect unknown Ag or Ab A positive result is visualized when a colored product is released by an enzyme-substrate reaction Enzyme-linked immunosorbent assay (ELISA) Anti-immunoglobulin Ab complexed to an enzyme conjugate

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

23. Radioimmunoassay Ag or Abs are labeled with radioactive isotopes and traced

24. ELISAwww.biology.arizona.edu Positive Rxn Negative Rxn + is 0.5 or greater Indeterminate 0.300 – 0.495 - is 0.300 or less

25. Western Blot for a HIV on page 487 Western Blot • 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

26. Western Blot for HIVwww.biology.arizona.edu 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.

27. 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 Patient A is – Patient B is – Patient C is +

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

30. 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

31. 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 • Immunize animal • Harvest spleen • Fuse B & myeloma • Hybridoma • Immortal • Screen hybridomas for Abs • directed against antigen of • interest

32. 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. Mylotarg Acute myeloid leukemia (AML) Rituxan Treat a variety of non-Hodgkin's lymphomas. Target and destroy non-Hodgkin's lymphoma cells. Remicade Auto-immune disorders like Crohn's disease and rheumatoid arthritis Target is tumor necrosis factor Zenapax Prevent rejection in organ transplantation, especially in kidney transplants. Additional information at www.fda.gov