Practical Applications of Immunology Chapter 18 Practical Applications of Immunology

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

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 450,000 60 40 400,000 20 350,000 300,000 2000 01 02 03 04 05 06 07 08 09 10 Reported number of cases Year 250,000 200,000 150,000 100,000 50,000 1960 1965 1970 1975 1980 1985 1990 1995 2000 2005 2010 Year

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

Vaccines Used to Prevent Bacterial Diseases Diphtheria Purified diphtheria toxoid Meningococcal meningitis Purified polysaccharide from Neisseria meningitidis Pertussis (whooping cough) Inactivated toxin plus acellular fragments of Bordetella pertussis Pneumococcal pneumonia Purified polysaccharide from seven strains of Streptococcus pneumoniae Tetanus Purified tetanus toxoid Haemophilus influenzae type b meningitis Polysaccharide from Haemophilus influenzae type b conjugated with protein to enhance effectiveness

Vaccines Used to Prevent Viral Diseases Influenza Injected vaccine, inactivated virus (nasally administered: attenuated virus) Measles Attenuated virus Mumps Rubella Chickenpox Poliomyelitis Killed virus

Vaccines Used to Prevent Viral Diseases Rabies Killed virus Hepatitis B Antigenic fragments of virus Hepatitis A Inactivated virus Smallpox Live vaccinia virus Herpes zoster Attenuated virus Human papillomavirus

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

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

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

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

Figure 18.1 Influenza viruses are grown in embryonated eggs.

Chlorioallantoic membrane innoculation Air sac 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

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

Safety of Vaccines Therapeutic index Risk-versus-benefit calculation

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

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

Figure 18.2.1-2 The Production of Monoclonal Antibodies. Antigen 1 A mouse is injected with a specific antigen that will induce production of antibodies against that antigen. 2 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. Spleen

Hybrid cell Myeloma cell Figure 18.2.3-4 The Production of Monoclonal Antibodies. Suspension of spleen cells 3 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. Spleen cells Myeloma cells Cultured myeloma cells (cancerous B cells) Suspension of myeloma cells Hybrid cells Hybrid cell Myeloma cell Spleen cell 4 The mixture of cells is placed in a selective medium that allows only hybrid cells to grow.

Figure 18.2.5-6 The Production of Monoclonal Antibodies. Hybridomas 5 Hybrid cells proliferate into clones called hybridomas. The hybridomas are screened for production of the desired antibody. 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.

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

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

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

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

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.

Figure 18.5 An agglutination reaction. IgM Epitopes Bacterium

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

Figure 18.6 Measuring antibody titer with the direct agglutination test. 1:20 1:40 1:80 1:160 1:320 1:640 Control (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

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

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

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.

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

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 No hemolysis (complement tied up in antigen–antibody reaction) Hemolysis (uncombined complement available) (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.

Fluorescent dye–labeled 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

Fluorescent dye–labeled Fluorescent spirochetes Figure 18.11b Fluorescent-antibody (FA) techniques. T. pallidum from 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

Figure 18.12 The fluorescence-activated cell sorter (FACS). 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 Electrode 4 Fluorescence detector identifies fluorescent cells by fluorescent light emitted by cell. 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. Collection tubes 6

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

Figure 18.14a The ELISA method. 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

Figure 18.14b The ELISA method. Antigen is adsorbed to well. 2 Patient serum is added; complementary antibody binds to antigen. 3 Enzyme-linked anti-HISG (see page 518) is added and binds to bound antibody. 4 Enzyme's substrate ( ) is added, and reaction produces a product that causes a visible color change ( ). A positive indirect ELISA to detect antibodies

Figure 18.13 The use of monoclonal antibodies in a home pregnancy test. Control windows 1 Free monoclonal antibody specific for hCG, a hormone produced during pregnancy. Test windows 2 Capture monoclonal antibody bound to substrate. 3 Sandwich formed by combination of capture antibody and free antibody when hCG is present, creating a color change. Not pregnant Pregnant

Serological Tests Direct tests detect antigens (from patient sample) Indirect tests detect antibodies (in patient’s serum)

Serological Tests Precipitation: soluble antigens Agglutination: particulate antigens Hemagglutination: agglutination of RBCs Neutralization: inactivates toxin or virus Fluorescent-antibody technique: antibodies linked to fluorescent dye Complement fixation: RBCs are indicator ELISA: peroxidase enzyme is the indicator

Question 1 Patient’s serum, influenza virus, sheep RBCs, and anti-sheep RBCs are mixed in a tube Influenza virus agglutinates RBCs What happens if the patient has antibodies against influenza virus?

Question 2 Patient’s serum, Chlamydia, guinea pig complement, sheep RBCs, and anti-sheep RBCs are mixed in a tube What happens if the patient has antibodies against Chlamydia?