1. Practical Applications of Immunology
Chapter 18
Practical Applications of Immunology

2. 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

3. 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.

4. Types of Vaccines and Their Characteristics
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.
Induce killer T-cell (TC) responses, helper T-cell (TH) responses and antibody immunity
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:
Measles (rubeola), Sabin polio vaccine, Mump, Tuberculosis
Orally administrated thiphoid vaccine

5. 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 antibody-mediated 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

6. 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.

7. 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.

8. Vaccines Used to Prevent Bacterial Diseases
Diphtheria
Purified diphtheria toxoid
Meningococcal meningitis
Purified polysaccharide from Neisseria meningitidis
Pertussis (whooping cough)
Killed whole or acellular fragments of Bordetella pertussis
Pneumococcal pneumonia
Purified polysaccharide from 7 strains of Streptococcus pneumoniae, or conjugated vaccine
Tetanus
Purified tetanus toxoid
Haemophilus influenzae type b meningitis
Polysaccharide from Haemophilus influenzae type b conjugated with protein to enhance effectiveness

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

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

11. Vaccines for Persons Aged 0–6 Years
Hepatitis B
Rotavirus
DTP
Haemophilus influenzae b
Pneumococcal
Inactivated poliovirus
Influenza
MMR (measles, mumps and rubella )
Varicella
Hepatitis A
Meningococcal

12. 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

13. 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.

14. Conventional 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.

15. Monoclonal Antibodies
Figure 18.2

16. 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.

17. Serological reactions –
1. Precipitation Reactions
When an antibody ( IgG or IgM) binds to the soluble antigen to form large molecular aggregates ( insoluble fraction, lattices)
Involve soluble antigens with antibodies
Immunodiffusion
Carried out in a solution
Agar gel medium
a petri plate
a microscope slide.
Figure 18.3

18. 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

19. 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

20. Serological reactions
4. Neutralization Reactions
Antibodies prevent hemagglutination
Antigen-antibody reaction which block the harmful effect of a virus or exotoxin
Figure 18.8b

21. Antibody Titer
Concentration of antibodies against a particular antigen (in this instance red blood cells)
Figure 18.5

22. 5. Complement Fixation Test
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?

23. 6. Fluorescent-Antibody Techniques (FA)
Serological reactions
6. Fluorescent-Antibody Techniques (FA)
Direct
Indirect
Figure 18.10a

24. 7. Enzyme-Linked Immunosorbent Assay Direct ELISA
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

25. 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

26. Serological reactions
Serological Tests
Figure 18.13

27. 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: Enzyme-substrate reaction is the indicator
Direct tests detect antigens (from patient sample)
Indirect tests detect antibodies (in patient’s serum)

28. Learning objectives 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.