Chapter 18

Practical Applications of Immunology

• 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

Modern Developments in Microbiology

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.

– 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

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

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 injection4. 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 VaccineDiphtheria 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

Disease VaccineInfluenza Injected vaccine, inactivated virus

(nasally administered: attenuated virus)

Measles Attenuated virus

Mumps Attenuated virus

Rubella Attenuated virus

Chickenpox Attenuated virus

Poliomyelitis Killed virus

Vaccines Used to Prevent Viral Diseases

Disease VaccineRabies 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

Vaccines Used to Prevent Viral Diseases

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

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 AntibodiesMonoclonal 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)– 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).

Figure 18.7

Patient’s serum, influenza virus and sheep RBCs are mixed in a tubeInfluenza virus agglutinates RBCsWhat happens if the patient has antibodies against influenza virus?

4. Neutralization Reactions • Antibodies prevent hemagglutination

• Antigen-antibody reaction which block the harmful effect of a virus or exotoxin

Figure 18.8b

Serological reactions

• Antibody Titer • Concentration of antibodies against a particular antigen (in this

instance red blood cells)

Figure 18.5

5. Complement Fixation Test

Patient’s serum, Chlamydia, guinea pig complement, sheep RBCs, and anti-sheep RBCs Ab are mixed in a tubeWhat happens if the patient has antibodies against Chlamydia?

Serological reactions

6. Fluorescent-Antibody Techniques (FA)

Figure 18.10a

Direct

Indirect

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

Serological reactions

Enzyme-Linked Immunosorbent Assay 2. Indirect ELISA

Figure 18.12b

• 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)

Serological Tests

Figure 18.13

Serological reactions

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)

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.