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Chapter 18

Chapter 18. Practical Applications of Immunology. Vaccines. 18-1 Define vaccine . 18-2 Explain why vaccination works. 18-3 Differentiate the following, and provide an example of each: attenuated, inactivated, toxoid, subunit, and conjugated vaccines.

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Chapter 18

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  1. Chapter 18 Practical Applications of Immunology

  2. Vaccines • 18-1 Define vaccine. • 18-2 Explain why vaccination works. • 18-3 Differentiate the following, and provide an example of each: attenuated, inactivated, toxoid, subunit, and conjugated vaccines. • 18-4 Contrast subunit vaccines and nucleic acid vaccines.

  3. Vaccines • 18-5 Compare and contrast the production of whole-agent vaccines, recombinant vaccines, and DNA vaccines. • 18-6 Define adjuvant. • 18-7 Explain the value of vaccines, and discuss acceptable risks for vaccines.

  4. History of Vaccines • Variolation: inoculation of smallpox into skin (eighteenth century) • Vaccination: • Inoculation of cowpox virus into skin (Jenner) • Inoculation with rabies virus (Pasteur)

  5. Chapter 18, unnumbered figure B, page 510, Reported numbers of measles cases in the United States, 1960–2010. (CDC, 2010) 140 120 Vaccine licensed 100 500,000 80 Reported number of cases 60 450,000 40 400,000 20 350,000 0 300,000 04 2000 01 02 03 05 06 07 08 09 10 Reported number of cases Year 250,000 200,000 150,000 100,000 50,000 0 1960 1965 1975 1985 2000 2010 1970 1990 1995 2005 1980 Year

  6. Chapter 18, unnumbered figure C, page 510, Countries with the highest measles mortality.

  7. Vaccines Used to Prevent Bacterial Diseases

  8. Vaccines Used to Prevent Viral Diseases

  9. Vaccines Used to Prevent Viral Diseases

  10. Vaccines for Persons Aged 0–6 Years • Hepatitis B • Rotavirus • DTaP • Haemophilus influenzae type b • Pneumococcal • Inactivated poliovirus • Influenza

  11. Vaccines for Persons Aged 0–6 Years • MMR • Varicella • Hepatitis A • Meningococcal ANIMATION Vaccines: Function

  12. What is the etymology (origin) of the word vaccine? 18-1 • Vaccination is often the only feasible way to control most viral diseases; why is this? 18-2

  13. Types of Vaccines • Attenuated whole-agent vaccines • MMR vaccine • Inactivated whole-agent vaccines • Salk polio vaccine • Toxoids • Tetanus vaccine

  14. Types of Vaccines • Subunit vaccines • Acellular pertussis • Recombinant hepatitis B • Nucleic acid (DNA) vaccines • West Nile (for horses) ANIMATION Vaccines: Types

  15. Figure 18.1 Influenza viruses are grown in embryonated eggs.

  16. Figure 13.7 Inoculation of an embryonated egg. Amniotic cavity Chlorioallantoic membrane Shell Chlorioallantoic membrane innoculation Air sac Amniotic innoculation Yolk sac Allantoic innoculation Shell membrane Yolk sac innoculation Albumin Allantoic cavity

  17. The Development of New Vaccines • Culture pathogen • rDNA techniques • In plants • Adjuvants • Deliver in combination

  18. Safety of Vaccines • Therapeutic index • Risk-versus-benefit calculation

  19. Experience has shown that attenuated vaccines tend to be more effective than inactivated vaccines. Why? 18-3 • Which is more likely to be useful in preventing a disease caused by an encapsulated bacterium such as the pneumococcus: a subunit vaccine or a nucleic acid vaccine? 18-4

  20. Which type of vaccine did Louis Pasteur develop, whole-agent, recombinant, or DNA? 18-5 • What is the derivation of the word adjuvant? 18-6 • What is the name of a currently used oral vaccine that occasionally causes the disease it is intended to prevent? 18-7

  21. Diagnostic Immunology 18-8Differentiate sensitivity from specificity in a diagnostic test. 18-9Define monoclonal antibodies, and identify their advantage over conventional antibody production. 18-10Explain how precipitation reactions and immunodiffusion tests work. 18-11Differentiate direct from indirect agglutination tests.

  22. Diagnostic Immunology 18-12Differentiate agglutination from precipitation tests. 18-13Define hemagglutination. 18-14Explain how a neutralization test works. 18-15Differentiate precipitation from neutralization tests. 18-16Explain the basis for the complement-fixation test.

  23. Diagnostic Immunology 18-17Compare and contrast direct and indirect fluorescent-antibody tests. 18-18Explain how direct and indirect ELISA tests work. 18-19Explain how Western blotting works. 18-20Explain the importance of monoclonal antibodies.

  24. Diagnostic Immunology • Sensitivity: probability that the test is reactive if the specimen is a true positive • Specificity: probability that a positive test will not be reactive if a specimen is a true negative • Immunologic-based tests • Guinea pigs with TB injected with Mycobacterium tuberculosis: site became red and slightly swollen

  25. Monoclonal Antibodies (Mabs) • Hybridoma: “immortal” cancerous B cell fused with an antibody-producing normal B cell • Produces monoclonal antibodies

  26. Figure 18.2.1-2 The Production of Monoclonal Antibodies. Antigen A mouse is injected with a specific antigen that will induce production of antibodies against that antigen. 1 The spleen of the mouse is removed and homogenized into a cell suspension. The suspension includes B cells that produce antibodies against the injected antigen. 2 Spleen

  27. Figure 18.2.3-4 The Production of Monoclonal Antibodies. Suspension of spleen cells The spleen cells are then mixed with myeloma cells that are capable of continuous growth in culture but have lost the ability to produce antibodies. Some of the antibody-producing spleen cells and myeloma cells fuse to form hybrid cells. These hybrid cells are now capable of growing continuously in culture while producing antibodies. 3 Spleen cells Myeloma cells Suspension of myeloma cells Cultured myeloma cells (cancerous B cells) Hybrid cells Hybrid cell Myeloma cell Spleen cell The mixture of cells is placed in a selective medium that allows only hybrid cells to grow. 4

  28. Figure 18.2.5-6 The Production of Monoclonal Antibodies. Hybridomas Hybrid cells proliferate into clones called hybridomas. The hybridomasare screened for production of the desired antibody. 5 Desired monoclonal antibodies 6 The selected hybridomas are then cultured to produce large quantities of monoclonal antibodies. Isolated antibodies are used for treating and diagnosing disease.

  29. Monoclonal Antibodies (Mabs) • Muromonab-CD3: for kidney transplant • Infliximab: for Crohn’s disease • Comalizumab: for allergic asthma • Rituximab: rheumatoid arthritis • Trastuzumab: Herceptin for breast cancer

  30. Monoclonal Antibodies • Chimeric Mabs: genetically modified mice that produce Ab with a human constant region • Humanized Mabs: Mabs that are mostly human, except for mouse antigen-binding sites • Fully human antibodies: Mabs produced from a human gene on a mouse

  31. What property of the immune system suggested its use as an aid for diagnosing disease: specificity or sensitivity? 18-8 • The blood of an infected cow would have a considerable amount of antibodies against the infectious pathogen in its blood. How would an equivalent amount of monoclonal antibodies be more useful? 18-9

  32. Figure 18.4 The precipitin ring test. Antigens (soluble) Zone of equivalence: visible precipitate Precipitation band Antibodies

  33. Figure 18.3 A precipitation curve. Antigen Antibody Zone of antibody excess Precipitate formed Zone of equivalence Antibody in precipitate Zone of antigen excess Antigen added

  34. Figure 10.12 The Western blot. If Lyme disease is suspected in a patient: Electrophoresis is used to separate Borrelia burgdorferi proteins in the serum. Proteins move at different rates based on their charge and size when the gel is exposed to an electric current. Lysed bacteria Polyacrylamide gel Proteins Larger Paper towels Smaller The bands are transferred to a nitrocellulose filter by blotting. Each band consists of many molecules of a particular protein (antigen). The bands are not visible at this point. Sponge Salt solution Gel Nitrocellulose filter The proteins (antigens) are positioned on the filter exactly as they were on the gel. The filter is then washed with patient’s serum followed by anti-human antibodies tagged with an enzyme. The patient antibodies that combine with their specific antigen are visible (shown here in red) when the enzyme’s substrate is added. The test is read. If the tagged antibodies stick to the filter, evidence of the presence of the microorganism in question—in this case, B. burgdorferi—has been found in the patient’s serum.

  35. Figure 18.5 An agglutination reaction. IgM Epitopes Bacterium

  36. Figure 18.7 Reactions in indirect agglutination tests. Latex bead Antigen attached to bead Latex bead Latex bead Latex bead Latex bead Latex bead Latex bead Latex bead Latex bead Latex bead Insert Fig 18.7 Latex bead Latex bead Antibody attached to bead Latex bead Bacterial antigen IgM antibody Reaction in a positive indirect test for antigens. When particles are coated with monoclonal antibodies, agglutination indicates the presence of antigens. Reaction in a positive indirect test for antibodies. When particles (latex beads here) are coated with antigens, agglutination indicates the presence of antibodies, such as the IgM shown here.

  37. Figure 18.6 Measuring antibody titer with the direct agglutination test. 1:640 Control 1:320 1:40 1:80 1:160 1:20 (a) Each well in this microtiter plate contains, from left to right, only half the concentration of serum that is contained in the preceding well. Each well contains the same concentration of particulate antigens, in this instance red blood cells. Top view of wells Enlarged photo of wells (b) In a positive (agglutinated) reaction, sufficient antibodies are present in the serum to link the antigens together, forming a mat of antigen–antibody complexes on the bottom of the well. Side view of wells (c) In a negative (nonagglutinated) reaction, not enough antibodies are present to cause the linking of antigens. The particulate antigens roll down the sloping sides of the well, forming a pellet at the bottom. In this example, the antibody titer is 160 because the well with a 1:160 concentration is the most dilute concentration that produces a positive reaction. Agglutinated Nonagglutinated

  38. Hemagglutination • Hemagglutination involves agglutination of RBCs • Some viruses agglutinate RBCs in vitro • Hemagglutination inhibition:antibodies prevent hemagglutination

  39. Figure 18.8 Viral hemagglutination. Red blood cells Viruses Hemagglutination

  40. Figure 18.9b Reactions in neutralization tests. Viruses neutralized and hemagglutination inhibited Red blood cells Antiviral antibodies from serum Viruses Viral hemagglutination test to detect antibodies to a virus. These viruses will normally cause hemagglutination when mixed with red blood cells. If antibodies to the virus are present, as shown here, they neutralize and inhibit hemagglutination.

  41. Figure 18.9a Reactions in neutralization tests. Toxin molecules Cell Cell damaged by toxin Toxin molecules Antibodies to toxin (antitoxin) Neutralized toxin and undamaged cell Cell The effects of a toxin on a susceptible cell and neutralization of the toxin by antitoxin

  42. Why does the reaction of a precipitation test become visible only in a narrow range? 18-10 • Why wouldn’t a direct agglutination test work very well with viruses? 18-11 • Which test detects soluble antigens, agglutination or precipitation? 18-12 • Certain diagnostic tests require red blood cells that clump visibly. What are these tests called? 18-13

  43. In what way is there a connection between hemagglutination and certain viruses? 18-14 • Which of these tests is an antigen–antibody reaction: precipitation or viral hemagglutination inhibition? 18-15

  44. Figure 18.10 The complement-fixation test. Antigen Antigen Complement Complement Serum with antibody against antigen Serum without antibody Complement-fixation stage No complement fixation Complement fixation Sheep RBC Sheep RBC Antibody to sheep RBC Antibody to sheep RBC Indicator stage Hemolysis (uncombined complement available) No hemolysis (complement tied up in antigen–antibody reaction) (a) Positive test. All available complement is fixed by the antigen–antibody reaction; no hemolysis occurs, so the test is positive for the presence of antibodies. (b) Negative test. No antigen–antibody reaction occurs. The complement remains, and the red blood cells are lysed in the indicator stage, so the test is negative.

  45. Figure 18.11a Fluorescent-antibody (FA) techniques. Group A streptococci from patient’s throat Fluorescent dye–labeled antibodies to group A streptococci Fluorescent streptococci Reactions in a positive direct fluorescent-antibody test

  46. Figure 18.11b Fluorescent-antibody (FA) techniques. T. pallidumfrom laboratory stock Specific antibodies in serum of patient Antibodies binding to T. pallidum Fluorescent dye–labeled anti-human immune serum globulin (will react with any immunoglobulin) Fluorescent spirochetes (see Figure 3.6b) Reactions in a positive indirect fluorescent-antibody test

  47. Figure 18.12 The fluorescence-activated cell sorter (FACS). 1 A mixture of cells is treated to label cells that have certain antigens with fluorescent-antibody markers. 2 Cell mixture leaves nozzle in droplets. Fluorescently labeled cells 3 Laser beam strikes each droplet. Laser beam Detector of scattered light Laser 4 Fluorescence detector identifies fluorescent cells by fluorescent light emitted by cell. Electrode Fluorescence detector 5 Electrode gives positive charge to identified cells. Electrically charged metal plates 6 As cells drop between electrically charged plates, the cells with a positive charge move closer to the negative plate. 7 The separated cells fall into different collection tubes. 6 Collection tubes

  48. Enzyme-Linked Immunosorbent Assay • Also called ELISA • Enzyme linked to Ab is the indicator

  49. Figure 18.14a The ELISA method. 1 Antibody is adsorbed to well. 2 Patient sample is added; complementary antigen binds to antibody. 3 Enzyme-linked antibody specific for test antigen is added and binds to antigen, forming sandwich. 4 Enzyme's substrate ( ) is added, and reaction produces a product that causes a visible color change ( ). A positive direct ELISA to detect antigens

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