MICR 201 Microbiology for Health Related Sciences

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MICR 201 Microbiology for Health Related Sciences

• Lecture 11 Practical applications of immunology
  vaccinations
• Edith Porter, M.D.

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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

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Immunological memory

• Once lymphocytes have encountered their specific
  antigen they undergo clonal expansion
• 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 effector function
• 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

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Example Primary and secondary immune responses
to an antigen
Primary Response
Secondary 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

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

• Artificially acquired
• Active immunity
• Injection of Ag (vaccination)
• Passive immunity
• Injection of Ab

• Naturally acquired
• Active immunity
• Resulting from infection
• Passive immunity
• Transplacental
• Colostrum

Long Lasting
Short Lasting
Vaccines toxoid, killed microorganisms, live
attenuated microorganisms
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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.

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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

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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

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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

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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
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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
• Freunds complete adjuvants
• Mycobacterium extract

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Bacterial vaccines used in the US

• Diphtheria Purified diphtheria toxoid
• Pertussis Acellular fragments of B. pertussis or
  antigenic acellular fragments
• Tetanus Purified tetanus toxoid
• Meningococcal meningitis Purified polysaccharide
  from N. meningitidis
• Haemophilus influenzae type b meningitis
  Polysaccharides conjugated with protein
• Pneumococcal conjugate vaccine S. pneumoniae
  antigens conjugated with protein

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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
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Schedule of childhood immunizations
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Booster

• Re-administration of vaccine to boost immune
  defense ( )
• Increased antibody production
• Increased memory cell development

Ab Titer
Time
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Success of vaccines
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Vaccines urgently needed

• HIV
• Malaria
• Tuberculosis
• Cholera

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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
• Bordetella pertussis

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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

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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

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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

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Production of monoclonal antibodies
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Selected serological tests

• Precipitation
• Agglutination
• Hemagglutination
• Fluorescent-antibody technique
• ELISA

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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

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Precipitin ring test
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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

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Agglutination assay to measure antibody titer

• Relates to the concentration of antibodies
  against a particular antigen
• Gives a guide to how active the patients immune
  response is.

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Note Viral hemagglutination is not an
antigenantibody reaction
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Viral hemagglutination inhibition for antibody
detection
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Fluorescent antibody techniques (direct)
Labeled Specific Antibody
To detect difficult to grow pathogens (e.g.
Legionella)
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Fluorescent antibody techniques (indirect)
Specific Primary Antibody
Labeled Secondary Antibody
Figure 18.10b
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Enzyme-inked Immuno-Sorbent Assay (ELISA)
To detect Ag
To detect Ab
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Pregnancy test sticks Mini ELISA
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Important to remember

• Vaccination (against disease) and immunizations
  (for antibody production) require administration
  of attenuated or inactivated antigen
• AbAg 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

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Check your understanding

• 4) In an agglutination test, eight serial
  dilutions to determine antibody titer were set
  up tube 1 contained a 12 dilution tube 2, a
  14, etc. If tube 6 is the last tube showing
  agglutination, what is the antibody titer?
• A) 6
• B) 16
• C) 64
• D) 132
• E) 32
• 16) A test used to identify antibodies against
  Treponema pallidum 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) Toxoid vaccine
• 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