MICR 201 Microbiology for Health Related Sciences

1. Lecture 11: Practical applications of immunology ; vaccinations Edith Porter, M.D. MICR 201 Microbiology for Health Related Sciences

2. Lecture outline • Major applications of immunology • Vaccines • Immunological memory • Types of adaptive immunity • Types of vaccines • Development of new vaccines • Safety of vaccines • Generation of antibodies for research and diagnostic • Anti-sera • Monoclonal antibodies • Diagnostic immunology • Precipitation and agglutination reactions • Complement fixation • ELISA, western blot, immunofluorescence

3. Immunological memory • Once lymphocytes have encountered their specific antigen they undergo clonalexpansion • Some of these cells develop further into memory cells • Can circulate for many years • Upon re-contact with the same antigen they quickly proliferate and resume effectorfunction • B cells: antibody production • T cells: cytokine production (TH, Treg) and cytotoxicity (CTL) • Instead of requiring 10 – 14 days for a full response measurable effects appear within 2 days

4. Example: Primary and secondary immune responses to an antigen Secondary Response Primary Response • IgM is always the first antibody • IgG follows IgM • IgG level does not go back to baseline • Re-exposure to the same antigen will lead to an augmented and accelerated immune response, increased IgG response and with higher residual antibody levels • Presence of antibodies is not equivalent to sickness

5. Types of adaptive immunity Vaccines do not always target B cells and antibodies but some target T cells.

6. Naturally acquired Active immunity Resulting from infection Passive immunity Transplacental Colostrum Artificially acquired Active immunity Injection of Ag (vaccination) Passive immunity Injection of Ab Adaptive Immunity Long Lasting Short Lasting Vaccines: toxoid, killed microorganisms, live attenuated microorganisms

7. Check your understanding • 1) What type of immunity results from vaccination? • A) Innate immunity • B) Naturally acquired active immunity • C) Naturally acquired passive immunity • D) Artificially acquired active immunity • E) Artificially acquired passive immunity • 3) What type of immunity results from recovery from mumps? • A) Innate immunity • B) Naturally acquired active immunity • C) Naturally acquired passive immunity • D) Artificially acquired active immunity • E) Artificially acquired passive immunity • 15) The antibodies found in mucus, saliva, and tears are • A) IgG. • B) IgM. • C) IgA. • D) IgD. • E) IgE. • 26) The best definition antibody is • A) A serum protein. • B) A protein that inactivates or kills an antigen. • C) A protein made in response to an antigen that can combine with that antigen. • D) An immunoglobulin. • E) A protein that combines with a protein or carbohydrate.

8. Principles of Vaccination • Induce active immune response with a related or inactivated agent that does not cause major disease • Induce cross-reactive antibodies or T cells • Herd immunity is sufficient to prevent epidemic diseases • Must weigh the benefit of protection versus the danger of vaccine associated disease

9. Types of Vaccines (1) • Attenuated whole agent vaccines • Live, weakened microbes • Generated through long term culture • Mimic most closely actual infection • Caution: danger of backmutation to a virulent form • Dangerous for immunocompromised patients! • Inactivated whole agent vaccines • Toxoid

10. Types of Vaccines (2) • Subunit or acellular vaccines • Use of antigenic subunit that triggers an immune response. • Inherent safer as it cannot reproduce in the host • Conjugated vaccine • Combine as antigen poorly immunogenic material (e.g. capsules) with an immunogenic protein • Nucleic acid vaccines (DNA vaccines) • Introduce genes for protein targets into the host • Host will express the protein and mount an immune response to this foreign protein

11. Where to get the vaccine from? • Classical • Bacterial culture • (Animal extract) • Cell culture • Embryonated eggs • New Developments • Recombinant vaccines • Plants Influenza virus grown in embryonated eggs

12. Adjuvants • Improve effectiveness of vaccines/immunizations by providing co-stimulatory signals for T and B cells • In humans • Alum • Oil based substance MF59 and virosomes • In animals • Freund’s complete adjuvants • Mycobacterium extract

13. Bacterial vaccines used in the US • Diphtheria: Purified diphtheria toxoid • Pertussis: Acellular fragments of B. pertussis or antigenic acellular fragments • Tetanus: Purified tetanus toxoid • Meningococcalmeningitis: Purified polysaccharide from N. meningitidis • Haemophilusinfluenzae type b meningitis: Polysaccharides conjugated with protein • Pneumococcal conjugate vaccine: S. pneumoniae antigens conjugated with protein

14. Viral vaccines used in the US • Influenza: Inactivated or attenuated virus • Measles: Attenuated virus • Mumps: Attenuated virus • Rubella: Attenuated virus • Chickenpox: Attenuated virus • Poliomyelitis: Inactivated virus • Hepatitis B: Antigenic fragments (recombinant) • Smallpox: Live vaccinia virus • Rabies: Inactivated virus • Hepatitis A: Inactivated virus • Human papilloma virus: Antigenic fragments Selected patients

15. Schedule of childhood immunizations

16. Booster • Re-administration of vaccine to boost immune defense ( ) • Increased antibody production • Increased memory cell development Ab Titer Time

17. Success of vaccines

18. Vaccines urgently needed • HIV • Malaria • Tuberculosis • Cholera

19. Concerns with vaccines • Safety • Sometimes illness follows vaccination • Risk benefit analysis • Autism had been linked to vaccination • Most recent studies conclude that there is no link but instead a genetic disposition • Weaning protection • Bordetellapertussis

20. Use of antibodies as a diagnostic Tool • Patients antibodies • indicate that patient had contact with the agent before • “serology” • Commercial antibodies • used to detect patient antigen • In research • Detection of antigen • Purification

21. Serology • Study of reactions between antibodies and antigens • Globulins • Serum proteins • Gamma () globulin • Serum fraction containing Antibodies (immunoglobulin) • Antiserum • Generic term for serum when it contains specific Ab • Polyclonal • Monoclonal antibodies

22. Production of antiserum • Prepare antigen • Inoculate animal with antigen and adjuvants • Administer several boosts • Draw blood and let it coagulate • Remove cells by centrifugation and collect supernatant = serum with high titers of specific antibodies

23. Production of monoclonal antibodies

24. Selected serological tests • Precipitation • Agglutination • Hemagglutination • Fluorescent-antibody technique • ELISA

25. Precipitation reactions • Involve soluble antigens and antibodies • Upon cross linking a visible interlocking molecular aggregate is formed (lattice) • Only in equivalence zone precipitates are formed • Example • Ouchterlony

26. Precipitin ring test

27. Agglutination reactions • Involve particulate antigens and antibodies • IgM are most efficient • Direct agglutination • Antibodies against large cellular antigens • Indirect agglutination • Antibodies against soluble antigen adsorbed to a particle or erythrocytes

28. Agglutination assay to measure antibody titer • Relates to the concentration of antibodies against a particular antigen • Gives a guide to how active the patient’s immune response is.

29. Note: Viral hemagglutinationis not an antigen:antibody reaction

30. Viral hemagglutinationinhibitionfor antibody detection

31. Fluorescent antibody techniques (direct) Labeled Specific Antibody To detect difficult to grow pathogens (e.g. Legionella)

32. Fluorescent antibody techniques (indirect) Specific Primary Antibody Labeled Secondary Antibody Figure 18.10b

33. Enzyme-inked Immuno-Sorbent Assay (ELISA) To detect Ag To detect Ab

34. Pregnancy test sticks: Mini ELISA:

35. Important to remember • Vaccination (against disease) and immunizations (for antibody production) require administration of attenuated or inactivated antigen • Ab:Ag reaction is the fundamental reaction in immunology • Precipitation: ab--soluble ag • Agglutination: ab--particulate ag • Presence of antibodies does not mean illness • High titer means high concentration of ab

36. Check your understanding 4) In an agglutination test, eight serial dilutions to determine antibody titer were set up: tube #1 contained a 1:2 dilution; tube #2, a 1:4, etc. If tube #6 is the last tube showing agglutination, what is the antibody titer? A) 6 B) 1:6 C) 64 D) 1:32 E) 32 16) A test used to identify antibodies against Treponemapallidum in a patient is the A) Direct fluorescent-antibody test. B) Indirect fluorescent-antibody test. C) Direct agglutination test. D) Direct ELISA test. E) Hemagglutination-inhibition test. 15) What type of vaccine is live measles virus? A) Conjugated vaccine B) Subunit vaccine C) Nucleic acid vaccine D) Attenuated whole-agent vaccine E) Toxoidvaccine 13) What type of vaccine involves host synthesis of viral antigens? A) Conjugated vaccine B) Subunit vaccine C) Nucleic acid vaccine D) Attenuated whole-agent vaccine E) Toxoid vaccine