Immunization and Immune Testing 17 Immunization and Immune Testing

Immunization Two Artificial Methods of Immunity Active immunization Administration of antigens so patient actively mounts an adaptive immune response Passive immunotherapy Individual acquires immunity through the transfer of antibodies formed by immune individual or animal

Immunization Brief History of Immunization Chinese noticed children who recovered from smallpox did not contract the disease again They infected children with material from a smallpox scab to induce immunity This process known as variolation Variolation spread to England and America but was stopped due to risk of death

Immunization Brief History of Immunization 1796 — Edward Jenner discovered process of vaccination 1879 — Louis Pasteur developed a vaccine against Pasteurella multocida Antibody transfer developed when it was discovered vaccines protected through the action of antibodies

Figure 17.1 The effect of immunization in reducing the prevalence of two infectious diseases in the United States. 50 Introduction of two different vaccines Polio 40 30 Number of reported cases (thousands) 20 10 1950 1960 1970 1980 1990 2000 2010 Year 800 Introduction of vaccine Measles 600 Number of reported cases (thousands) 400 200 1950 1960 1970 1980 1990 2000 2010 Year

Immunization Brief History of Immunization Many developing nations do not receive vaccines Effective vaccines not developed for some pathogens Vaccine-associated risks discourage investment in developing new vaccines

Immunization Active Immunization Vaccine Types Attenuated (modified live) vaccines Use pathogens with reduced virulence Process of reducing virulence called attenuation Can result in mild infections Active microbes stimulate a strong immune response Can provide contact immunity Modified microbes may retain enough residual virulence to cause disease in susceptible individuals

Immunization Active Immunization Vaccine Types Inactivated (killed) vaccines Safer than live vaccines Whole agent vaccines Inactivate but whole microbes Subunit vaccines Antigenic fragments of microbes Often require multiple doses to achieve full immunity Often contain adjuvants Chemicals added to increase effective antigenicity

Immunization Active Immunization Vaccine Types Toxoid vaccines Chemically or thermally modified toxins used to stimulate active immunity Useful for some bacterial diseases Stimulate antibody-mediated immunity Require multiple doses because toxoids possess few antigenic determinants

Immunization Active Immunization Vaccine Types Combination vaccines Simultaneous administration of antigens from several pathogens Vaccines using recombinant gene technology Research attempts to make vaccines more effective, cheaper, and safer Recombinant DNA techniques used to improve vaccines

Figure 17.2 Some uses of recombinant DNA technology for making improved vaccines. Production of vaccine Components of vaccine Virulence gene is deleted Virulent pathogen with antigens Harmless, attenuated pathogen Antigen Cell Insertion of DNA into a cell Cell releases antigenic protein Antigenic protein molecules Isolated DNA coding for antigenic protein Antigen Insertion of DNA into virus or cell Virus or cell presenting pathogen's antigen Isolated DNA coding for antigen Antigen Recombinant plasmids produced Insertion of DNA into plasmid vector Isolated DNA coding for antigen Plasmid in body's cell, which then synthesizes pathogen's antigen 11

Vaccines: Function PLAY Vaccines: Function

Vaccines: Types PLAY Vaccines: Types

Immunization Active Immunization Vaccine Manufacture Mass-produce many vaccines by growing microbes in culture vessels Viruses are cultured inside chicken eggs Individuals with egg allergies must avoid some vaccines

Catch-up immunization Figure 17.3 The CDC's recommended immunization schedule for the general population. CDC-Recommended Immunization Schedule—United States, 2015 Childhood Adolescent Adult 1 mo 2 mos 4 mos 6 mos 12 mos 15 mos 18 mos 19–23 mos 2–3 yrs 4–6 yrs 7–10 yrs 11–12 yrs 13–15 yrs 16 yrs 17–18 yrs 19–49 yrs 50–64 yrs ≥65 yrs Vaccine Birth Hepatitis B (Hep B) Dose 1 Dose 2 Dose 3 Catch-up immunization Rotavirus 1 2 Diphtheria, tetanus, pertussis 1 2 3 4 5 6 Tdap once, and Td every 10 yrsa Haemophilus influenzae type b (Hib) 1 2 3 4 Pneumococcal (PCV) 1 2 3 4 5 Inactivated polio (IPV) 1 2 3 4 Influenza Annually Measles, mumps, rubella (MMR) 1 2 1 or 2 Varicella-zoster 1 2 1 2 Hepatitis A 2 Doses Human papillomavirus (HPV) 1 2 3 3 Doses Meningococcal 1 2 Range of recommended ages for immunization aTdap and Td, used for adult boosters, are slightly different vaccines than the childhood vaccine, DTaP. Range for catch-up immunization 15

Table 17.1 Principal Vaccines to Prevent Human Diseases (1 of 2) 16

Table 17.1 Principal Vaccines to Prevent Human Diseases (2 of 2)

Immunization Active Immunization Vaccine Safety Problems associated with immunization Mild toxicity Risk of anaphylactic shock Residual virulence from attenuated viruses Allegations certain vaccines cause autism, diabetes, and asthma Research has not substantiated these allegations

Immunization Passive Immunotherapy Administration of antiserum that contains preformed antibodies Provides immediate protection against a recent infection or ongoing disease Antisera have several limitations: Can trigger allergic reactions called serum sickness Antibodies of antisera are degraded relatively quickly Individual not protected from subsequent infections Limitations are overcome through development of hybridomas

Figure 17.4 The production of hybridomas. Mouse is injected with antigen. Long-lived myeloma cell lines are grown in culture. Plasma cells, which secrete antibodies, are removed. Antibodies Hybridomas are formed by mixing and fusing plasma cells and myeloma cells; hybridomas are long lived and produce antibodies. Hybridoma Hybridomas are placed individually in small wells, and their antibodies are tested for reactivity against the antigen. A hybridoma that makes antibodies that react with the antigen is cloned. Monoclonal antibodies Hybridoma clone 20

Antibody (IgG, IgM) concentration (titer) Figure 17.5 The characteristics of immunity produced by active immunization (red) and passive immunotherapy (green). Passive immunotherapy Injection Active immunization Antibody (IgG, IgM) concentration (titer) Boosters Initial inoculation Time 21

Serological Tests That Use Antigens and Corresponding Antibodies Serology is the determination of the presence of specific antigens or antibodies in blood serum Serological tests available to identify a variety of antigens and antibodies in serum Serological tests have several uses: Monitor the spread of infection within a population Establish diagnosis of disease

Serological Tests That Use Antigens and Corresponding Antibodies Precipitation Tests One of the simplest of serological tests Antigens and antibody mixed in the proper proportion form large complexes called precipitates Antigen-antibody complexes also called immune complexes Immunodiffusion is a common precipitation technique

Figure 17.6 Characteristics of precipitation reactions. Precipitate Amount of antibody precipitated Increasing amount of antigen Antibody Antigen Antibody excess Optimal proportions Antigen excess 24

Figure 17.7 Immunodiffusion, a type of precipitation reaction. Well containing antigen molecules Line of immune precipitation Well containing antibodies against the antigen Agar Zone of antigen excess Zone of optimal precipitation Zone of antibody excess Well containing four different antigens Lines of immune precipitation Well containing a mixture of antibodies, each reacting to a different antigen 25

Serological Tests That Use Antigens and Corresponding Antibodies Turbidimetric and Nephelometric Tests Turbidimetry and nephelometry measure the cloudiness of a solution Turbidimetry measures the light passing through a solution Nephelometry measures the light reflected from a solution Can be used to quantify the amounts of proteins in serum

Serological Tests That Use Antigens and Corresponding Antibodies Agglutination Tests Agglutination occurs due to the cross-linking of antibodies with particulate antigens Agglutination is the clumping of insoluble particles Precipitation involves the aggregation of soluble molecules Reactions can be easy to see and interpret with the unaided eye Hemagglutination Agglutination of red blood cells Can be used to determine blood type

Figure 17.8 The use of hemagglutination to determine blood types in humans. Anti-A antibody added Anti-B antibody added Blood sample A B A B Negative result: no agglutination of blood cells Positive result: agglutination of blood cells 28

Serological Tests That Use Antigens and Corresponding Antibodies Agglutination Tests Titration Method to measure antibody levels in blood sera Serum being tested is serially diluted and tested for agglutinating activity Highest dilution of serum giving a positive reaction is the titer

Serum added in increasing dilutions Figure 17.9 Titration, the use of agglutination to quantify the amount of antibody in a serum sample. Serum added in increasing dilutions Control (no specimen added) 1:1 1:10 1:100 1:1,000 1:10,000 Antigen (identical in each well) ++++ Very strong agglutination +++ ++ + – No agglutination Control 30

Serological Tests That Use Antigens and Corresponding Antibodies Neutralization Tests Viral Neutralization Cytopathic effect Viruses introduced into appropriate cell cultures will kill the cells Ability of virus to kill culture cells is neutralized when virus is first mixed with antibodies against it Viral neutralization test Mixture of virus and serum added to cell culture Absence of cytopathic effect indicates presence of antibodies against the virus in the serum Identifies whether individual exposed to a particular virus

Serological Tests That Use Antigens and Corresponding Antibodies Neutralization Tests Viral Hemagglutination Inhibition Test Useful for viruses that aren't cytopathic Based on viral hemagglutination Some viral surface proteins can clump red blood cells A serum sample that contains antibodies against a specific virus will inhibit viral hemagglutination Commonly used to detect antibodies against influenza, measles, and mumps

Serological Tests That Use Antigens and Corresponding Antibodies The Complement Fixation Test Based on generation of membrane attack complexes during complement activation Used to detect the presence of specific antibodies in an individual's serum Can detect antibody amounts too small to detect by agglutination Replaced by other serological methods

Serological Tests That Use Antigens and Corresponding Antibodies Labeled Antibody Tests Uses antibody molecules linked to some "label" that enables them to be easily detected Used to detect either antigens or antibodies

Serological Tests That Use Antigens and Corresponding Antibodies Labeled Antibody Tests Fluorescent Antibody Tests Use fluorescent dyes as labels Fluorescently labeled antibodies used in two types of tests: Direct fluorescent antibody tests Indirect fluorescent antibody tests

Figure 17.10 A direct fluorescent antibody test. 36

Figure 17.11 The indirect fluorescent antibody test. Cells with antigen are attached to slide. Antigen on cell bound to slide Slide is flooded with patient's serum. IgG from patient's serum Fluorescent-labeled anti-Ig antiglobulin is added. Fluorescent label Anti-IgG (antiglobulin, anti-antibody) 37

Serological Tests That Use Antigens and Corresponding Antibodies Labeled Antibody Tests ELISAs (EIAs) Stands for enzyme-linked immunosorbent assay (ELISA) or enzyme immunoassay (EIA) Uses an enzyme as the label Reaction of enzyme with its substrate produces a colored product indicating a positive test Commonly used to detect the presence of serum antibodies

Figure 17.12 Enzyme-linked immunosorbent assay (ELISA), also known as enzyme immunoassay (EIA). Antigen is attached to well in plate. A protein such as gelatin is added to block the uncoated surface. Patient serum is added; complementary antibody binds to antigen. Enzyme Anti-antibody Enzyme-linked anti-antibody is added and binds to bound antibody. Substrate Colored product Enzyme's substrate is added, and reaction produces a visible color change. 39

Serological Tests That Use Antigens and Corresponding Antibodies Labeled Antibody Test ELISAs (EIAs) Advantages of the ELISA: Can detect either antibody or antigen Sensitive Can quantify amounts of antigen or antibody Easy to perform and can test many samples quickly Relatively inexpensive and easy to automate Plates coated with antigen can be stored for later testing

Serological Tests That Use Antigens and Corresponding Antibodies Labeled Antibody Test ELISAs (EIAs) Antibody sandwich ELISA Modification of the ELISA technique Commonly used to detect antigen Antigen being tested for is "sandwiched" between two antibody molecules

Figure 17.13 An antibody sandwich ELISA. Colored product Gelatin Enyme-linked antibody Substrate Antigen in patient's serum Antibody bound to microwell 42

The Nature of Infectious Disease Dr. Bauman's Microbiology Video Tutor For more information, listen to Dr. Bauman explain when an ELISA is used and how it is performed.

Serological Tests That Use Antigens and Corresponding Antibodies Labeled Antibody Test Immunoblots Also called a western blot Technique to detect antibodies against multiple antigens Used to confirm the presence of proteins Three steps: Electrophoresis separates proteins in a solution Blotting transfers protein to nitrocellulose membrane Detects color where antibody has bound to proteins

Figure 17.14 Immunoblotting (western blotting). Wells containing proteins Polyacrylamide gel Electrophoresis (not shown) Polyacrylamide gel Blotting Filter paper Nitrocellulose membrane Patient Polyacrylamide gel 1 Absorbant paper 2 3 Nitrocellulose is removed from blotting stack. 4 5 Nitrocellulose is incubated with a test solution. 6 Positive control Negative control Polypeptides 45

Serological Tests That Use Antigens and Corresponding Antibodies Point-of-Care Testing Simple immunoassays that give results in minutes Useful in determining a quick diagnosis Common tests: Immunofiltration assay Immunochromatography assay

Serological Tests That Use Antigens and Corresponding Antibodies Point-of-Care Testing Immunofiltration Rapid ELISA that uses antibodies bound to membrane filters rather than plates Reduced time to complete the assay Due to the large surface area of the membrane filter

Serological Tests That Use Antigens and Corresponding Antibodies Recent Developments in Immune Testing Immunochromatography Very rapid and easy-to-read ELISAs Antigen solution flows through a porous strip Encounters labeled antibody Visible line produced when antigen-antibody immune complexes encounter antibody against them Used in pregnancy testing and for rapid identification of some infections

Figure 17.15 Immunochromatographic dipstick. Zone of antibodies linked to colloidal metal, color too diffuse to see Line of fixed anti-antibody Strep A Dip Strep A Anti-antibodies stop movement of antibody- antigen complexes. Color becomes visible because of density of complexes. Dip Movement of fluid containing complexes of antibodies bound to antigen Prepared antigen extract from patient's nasal sample 49

Table 17.2 Antibody-Antigen Immunological Tests and Some of Their Uses 50