Chapter 18
Practical Applications of
Immunology
Modern Developments in Microbiology
• Immunology is the study of our protection from foreign
macromolecules or invading organisms and our responses to them.
– The invaders - viruses, bacteria, protozoa or even larger parasites.
– Immune responses
• Against invaders
• Against our own proteins (and other molecules) in autoimmunity and against our own
aberrant cells in tumor immunity.
• The use of immunology to identify some bacteria according to
serotypes (variants within a species) was proposed by Rebecca
Lancefield in 1933.
• Vaccines and interferons are being investigated to prevent and
cure viral diseases.
• Development of a variety of diagnostic techniques
• Immunotherapy - the use of immune system components to treat
a disease
Vaccines
• Variolation: Inoculation of smallpox into skin (18th century)
• Vaccination: Edward Jenner developed the modern practice of
vaccination when he inoculated people with cowpox virus to protect
them against smallpox.
• Vaccination (immunization) is the administration of antigenic
material (a vaccine) to stimulate the immune system of an individual
to develop adaptive immunity to a disease.
– artificial induction of immunity,
• 'priming' the immune system with an 'immunogen'.
• Antibodies and long term memory cells are formed
• In general, vaccination is considered to be the most effective method
of preventing infectious diseases.
• Herd immunity results when most of a population is immune to a
disease.
Types of Vaccines and Their Characteristics
1.
•
Attenuated whole-agent vaccines - life microbes
–
Living but attenuated (weakened) microorganisms or attenuated virus
vaccines
–
Usually derived from mutations accumulated during long-term
artificial culture.
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Induce killer T-cell (TC) responses, helper T-cell (TH) responses and
antibody immunity
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Can result in mild infections but no disease
–
Generally provide lifelong immunity.
–
Vaccinated individuals can infect those around them, providing herd
immunity
Problems
–
Attenuated microbes may retain enough virulence to cause disease,
especially in immunosuppressed individuals
–
Pregnant women should not receive live vaccines due to the risk of the
modified pathogen crossing the placenta
–
Life microbes can back mutate to a virulent form.
•
Examples:
–
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Measles (rubeola), Sabin polio vaccine, Mump, Tuberculosis
Orally administrated thiphoid vaccine
Types of Vaccines and Their Characteristics
2. Inactivated whole-agent
– Killed bacteria or viruses ( usually by formalin or phenol).
– Killed vaccines cannot revert to a virulent form.
– Recognized as exogenous antigens and stimulate an antibodymediated immunity
– They cannot generate specific killer T cell (TC) responses
– May not work at all for some diseases.
• Examples:
– Rabies, influenza, polio
– Pneumococcal pneumonia
– Cholera
Types of Vaccines and Their Characteristics
3. Toxoids
– Bacterial toxins (usually an exotoxin) who toxicity has been weakened or
suppressed either by chemical (formalin) or heat treatment, while other
properties, typically immunogenicity, are maintained.
– Useful for some bacterial diseases
– Stimulate antibody-mediated immunity
– Often require multiple doses because they possess few antigenic
determinants
• Tetanus and diphtheria toxoids
– require series of injection
4. Subunit vaccines consist of antigenic fragments of a
microorganism;
– Acellular vaccines (fraction of disrupted bacterial cell).
– Recombinant vaccines
• They are able to generate TH and antibody responses,
• but not killer T cell responses.
Types of Vaccines and Their Characteristics
5. Conjugated vaccines combine the desired antigen with a
protein that boosts the immune response.
– Polysaccharide is combined with the proteins
6. Nucleic acid vaccines, or DNA vaccines, are being developed.
– DNA vaccines are third generation vaccines, and are made up of a a
plasmid that has been genetically engineered to produce one or two
specific proteins (antigens) from a pathogen.
– Introducing the DNA cause the recipient to make the antigenic
protein associated with MHC class I.
– DNA remains active only until it is degraded.
Vaccines Used to Prevent Bacterial Diseases
Disease
Diphtheria
Meningococcal
meningitis
Pertussis (whooping
cough)
Vaccine
Purified diphtheria toxoid
Purified polysaccharide from
Neisseria meningitidis
Killed whole or acellular fragments
of Bordetella pertussis
Purified polysaccharide from 7
Pneumococcal
strains of Streptococcus
pneumonia
pneumoniae, or conjugated vaccine
Tetanus
Purified tetanus toxoid
Haemophilus influenzae Polysaccharide from Haemophilus
type b meningitis
influenzae type b conjugated with
protein to enhance effectiveness
Vaccines Used to Prevent Viral Diseases
Disease
Influenza
Measles
Vaccine
Injected vaccine, inactivated virus
(nasally administered: attenuated
virus)
Attenuated virus
Mumps
Rubella
Attenuated virus
Attenuated virus
Chickenpox
Poliomyelitis
Attenuated virus
Killed virus
Vaccines Used to Prevent Viral Diseases
Disease
Rabies
Vaccine
Killed virus
Hepatitis B
Antigenic fragments of virus
(recombinant vaccine)
Hepatitis A
Inactivated virus
Smallpox
Live vaccinia virus
Herpes zoster
Attenuated virus
Antigenic fragments of virus
Human papillomavirus
Vaccines for Persons Aged 0–6 Years
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Hepatitis B
Rotavirus
DTP
Haemophilus influenzae b
Pneumococcal
• Inactivated poliovirus
• Influenza
• MMR (measles, mumps and
rubella )
• Varicella
• Hepatitis A
• Meningococcal
The Development of New Vaccines
• Culture pathogen
– Viruses for vaccines may be grown in animals, cell cultures, or chick embryos.
• rDNA techniques
– Recombinant vaccines and nucleic acid vaccines do not need to be grown in cells or
animals.
– Genetically modified plants may some day provide edible vaccines
• Adjuvants
– Adjuvants are generally used with soluble protein antigens to increase antibody titers
and induce a prolonged response with accompanying memory
– Only alum (a white crystalline double sulfate of aluminum) has been approved for
human use
• Deliver in combination
Diagnostic Immunology
• Serology is the science that deals with the properties and reactions
of serums, especially blood serum.
– The characteristics of a disease or organism shown by study of
blood serums (presence of antigens and antibodies)
• Many tests based have been developed to determine the presence
of antibodies or antigens in a patient
– The test sensitivity
• determined by the percentage of positive samples it
correctly detects;
– The test specificity
• determined by the percentage of false positive results it
gives.
Conventional antibody production
• Antibody production
– The primary goal is to obtain high titer, high affinity antiserum
for use in experimentation or diagnostic tests.
– Use of laboratory animals
• Polyclonal antibodies are antibodies that are derived from different
B-cell lines.
– They are a mixture of immunoglobulin molecules secreted
against a specific antigen, each recognizing a different epitope.
• Monoclonal antibodies are antibodies that are derived from a single
B-cell clone
– Immunoglobulin molecules secreted against a specific antigen,
each recognizing a the same epitope.
Monoclonal Antibodies
Figure 18.2
Monoclonal Antibodies
Monoclonal antibodies are used in:
– Serological identification ( diagnostic) tests
– To prevent tissue rejections
– To make immunotoxins to treat cancer.
• Immunotoxins can be made by combining a monoclonal
antibody and a toxin;
– The antibody localize the target (antigen)
– The toxin will then kill a specific antigen.
Serological reactions –
1. Precipitation Reactions
• When an antibody ( IgG or IgM) binds to the soluble antigen
to form large molecular aggregates ( insoluble fraction,
lattices)
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–
–
–
Involve soluble antigens with antibodies
Immunodiffusion
Carried out in a solution
Agar gel medium
• a petri plate
• a microscope slide.
Figure 18.3
Serological reactions
2. Agglutination Reactions - involve particulate antigens and
antibodies
– Direct agglutination – antigens on a cell surface
• test that uses whole organisms as a means of looking for serum antibodies
– Indirect or passive agglutination - attached to latex spheres
Figure 18.4
Serological reactions
3. Hemagglutination
• A specific form of agglutination that involves red blood cells.
• Some viruses agglutinate RBCs in vitro.
• It has two common uses in the laboratory:
– blood typing
– quantification of virus dilutions (Many viruses attach to
molecules present on the surface of red blood cells).
Patient’s serum, influenza virus and sheep RBCs are mixed in a tube
Influenza virus agglutinates RBCs
What happens if the patient has antibodies against influenza virus?
Figure 18.7
Serological reactions
4. Neutralization Reactions
• Antibodies prevent hemagglutination
• Antigen-antibody reaction which block the harmful effect of a
virus or exotoxin
Figure 18.8b
• Antibody Titer
• Concentration of antibodies against a particular antigen (in this
instance red blood cells)
Figure 18.5
Serological reactions
5. Complement Fixation Test
Patient’s serum, Chlamydia, guinea
pig complement, sheep RBCs, and
anti-sheep RBCs Ab are mixed in a
tube
What happens if the patient has
antibodies against Chlamydia?
Serological reactions
6. Fluorescent-Antibody Techniques (FA)
Direct
Indirect
Figure 18.10a
Serological reactions
7. Enzyme-Linked Immunosorbent Assay
Direct ELISA
• Detect the antigen
– Sandwich ELISA - two specific antibodies against the
antigen (each one binds to a different epitope)
– Enzyme-substrate reaction is the indicator. (Peroxidase,
Alkaline phosphatase enzyme linked to the second antibody)
Figure 18.12a
Enzyme-Linked Immunosorbent Assay
2. Indirect ELISA
• Detect antibodies
– One specific antibodies against the antigen
– Enzyme-substrate reaction is the indicator. (Peroxidase,
Alkaline phosphatase enzyme linked to a secondary antibody
that binds to the primary one)
Figure 18.12b
Serological reactions
Serological Tests
Figure 18.13
Serological Tests
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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: Enzyme-substrate reaction is the indicator
• Direct tests detect antigens (from patient sample)
• Indirect tests detect antibodies (in patient’s serum)
Learning objectives
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Define vaccine
Explain why vaccination works.
Differentiate between the following, and provide an example of each: attenuated,
inactivated, toxoid, subunit, and conjugated vaccines.
Compare and contrast the production of whole-agent vaccines, recombinant vaccines,
and DNA vaccines.
Define adjuvant.
Explain the value of vaccines, and discuss acceptable risks for vaccines.
Explain how antibodies are used to diagnose diseases.
Define monoclonal antibodies, and identify their advantage over conventional antibody
production.
Explain how precipitation and immunodiffusion tests work.
Differentiate direct from indirect agglutination tests.
Differentiate agglutination from precipitation tests.
Define hemagglutination.
Differentiate precipitation from neutralization tests.
Explain the basis for the complement-fixation test.
Compare and contrast direct and indirect fluorescent-antibody tests.
Explain how direct and indirect ELISA tests work.