Chapter 18 – Applications of Immunology
Vaccines
A vaccine is a substance that when injected causes the production of specific antibodies or
activated T-cells.
Herd immunity results when most of a population is immune to a disease. The purpose of
vaccination is to establish herd immunity when it does not already exist.
Types of vaccines:
Attenuated whole-agent vaccines consist of attenuated (weakened) microorganisms:
attenuated virus vaccines generally provide lifelong immunity.
Inactivated whole-agent vaccines consist of killed bacteria or viruses.
Toxoids are inactivated toxins.
Subunit vaccines consist of antigenic fragments of a microorganism; these include
recombinant vaccines and acellular vaccines.
Conjugated vaccines combine the desired antigen with a protein that boosts the
immune response.
Nucleic acid vaccines, or DNA vaccines, are being developed. These cause the
recipient to make the antigenic protein associated with class I MHC (HLA).
Adjuvants improve the effectiveness of some antigens.
Viruses for vaccines may be grown in animals, cell cultures, or chick embryos.
Vaccines are the safest and most effective means of controlling infectious diseases.
Complications of Vaccines
 Problems in administration
 Administration to pregnant women
 Infection
 Local reactions at the injection site
 Fever
 Allergies
 Development of disease
 Mutation to a virulent strain
Diagnostic Immunology
Serologic tests to determine the presence of antibodies or antigens in a patient are based on the fact
that antibodies bind to specific antigens.
These tests require 2 things:
A source of specific antibodies
A way to visualize the antigen-antibody interaction
Monoclonal Antibodies
Hybridomas - produced by the fusion of malignant cells and plasma cells. The resulting population of
cells is immortal and able to produce large amounts of a specific antibody.
Uses:
 Serologic identification
 Prevention of tissue rejections
 Cancer research
 Immunotoxins can be produced by combining monoclonal antibody with toxin
o Immunotoxins are targeted to react with specific antigens
o
Precipitation Reactions
The interaction of soluble antigens with IgG or IgM antibodies leads to precipitation reactions.
Precipitation reactions depend on the formation of lattices and occur best when antigen and antibody
are present in optimal proportions.
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The precipitin ring test is performed in a small tube.
Antibodies from the bottom of the tube and antigens from the top of the tube diffuse
toward each other and when the optimal antigen-antibody ratio is reached a visible ring
(precipitate) appears in the tube.
Immunodiffusion procedures are precipitation reactions carried out in an agar gel medium.
Antibody and antigen are loaded in different wells and diffuse through the medium.
When the optimal antigen-antibody ratio is reached a visible band appears in the gel.
Agglutination Reactions
The interaction of particulate antigens (cells that carry antigens) with antibodies leads to agglutination
reactions.
Diseases can be diagnosed by a rising titer (antibody concentration in serum) or seroconversion
(from no antibodies to the presence of antibodies).
Direct agglutination reactions test patient serum against large, cellular antigens to screen for the
presence of antibodies. Antibodies cause agglutination of the cells.
Indirect agglutination reactions can be used to test patient serum for the presence of antibodies
against soluble antigens.
Serum is mixed with latex spheres with the soluble antigens attached. Antibodies will then cause
visible agglutination of the latex spheres with the soluble antigens attached.
Alternatively, antibodies may be attached to the latex spheres to test for the presence of soluble
antigens in patient serum.
Hemagglutination reactions involve agglutination reactions using red blood cells.
Neutralization Reactions
In a toxin neutralization test, the presence of antibodies against a toxin can be detected by the
antibodies’ ability to prevent toxic effects in cells.
In a virus neutralization test, the presence of antibodies against a virus can be detected by the
antibodies’ ability to prevent cytopathic effects of viruses in cell cultures.
Complement-Fixation Reactions
There are two steps, the complement fixation step and the indicator step.
Complement Fixation Step
Add antigen and complement to serum. If the serum contains antibodies against the antigen they will
bind to the antigen and fix the complement.
This ties up all the free complement so it can't participate in the next step, the indicator step.
Indicator Step
Add sheep red blood cells and anti-sheep red blood cell antibodies to the serum. Antibodies to the
sheep red blood cells bind and can fix complement, if any is available.
If complement is available it will be fixed by the sheep red blood cell antigen-antibody complex and
the sheep red blood cells will be lysed. This indicates that the serum did not contain antibodies
against the antigen added in the complement fixation step and complement remained free.
If no complement is available the sheep red blood cells will not be lysed. This indicates there were
antibodies against the antigen added in the complement fixation step and all the complement was tied
up when it was fixed by the original antigen-antibody complex.
Fluorescent-Antibody Techniques
Direct fluorescent-antibody tests are used to identify specific microorganisms/antigens in patient
samples.
Antibodies directed against antigens on the surface of a specific microorganism are labeled
with fluorescent dye.
Fluorescent antibodies are incubated with the patient’s sample and antigen-specific binding
allowed to occur.
The sample is viewed with a fluorescence microscope or plate reader or fluorescenceactivated flow cytometer.
Indirect fluorescent-antibody tests are used to demonstrate the presence of antibody in serum.
Antigen or the microorganism itself is incubated with the patient's serum and any antibodies
against the antigen or organism that are present in the patient’s serum allowed to bind.
Fluorescent anti-human immunoglobulin antibodies are then added and will bind to any patient
antibodies present. (Inject human immunoglobulins into another species and it will produce
anti-human immunoglobulin antibodies).
The sample is viewed with a fluorescence microscope or plate reader or fluorescenceactivated flow cytometer.
Enzyme-Linked Immunosorbent Assay (ELISA)
ELISA techniques use antibodies linked to an enzyme, such as horseradish peroxidase or alkaline
phosphatase.
Antigen – antibody reactions are detected by enzyme activity. The substrate for the enzyme is
converted to a chromogenic product in the indicator step.
The direct ELISA is used to detect specific antigens in a patient's serum.
Coat the bottom of a test well with an antibody against the antigen. Then add patient serum. If
the antigen is present in the patient's serum it will bind to the antibody that is attached to the
bottom of the well (the capture antibody).
Next add enzyme-linked antibody, which is also specific for the antigen – it’s the same
antibody that coated the bottom of the well except it has an enzyme linked to it. If the antigen is
present you'll end up with an antibody-antigen-enzyme-linked antibody sandwich.
Add substrate. If there is any enzyme-linked antibody present (and it should only be there if it
is bound to antigen) a product will be formed that causes the color change.
The indirect ELISA is used to detect antibodies in patient serum against a specific antigen.
Coat the bottom of the well with the antigen that the antibody would be specific for. Then add
patient serum. Any antibodies specific for the antigen will bind.
Next add enzyme-linked anti-human immunoglobulin antibody.
If the patient's serum had antibodies against the antigen coatin the bottom of the well you'll end
up with an antigen-antibody-enzyme-linked anti-human immunoglobulin antibody sandwich.
Add substrate and look for the color change just like in the direct ELISA.
Chapter 19 - Disorders Associated With the Immune System
Hypersensitivities are altered immune reactions (in response to an antigen) leading to tissue damage,
pretty much the same thing as an allergy.
An allergen is an antigen that stimulates a hypersensitivity response.
Immediate hypersensitivities are based on humoral immunity and include Types I, II, and III.
Delayed is based on cell-mediated immunity, Type IV.
Type I (Anaphylaxis) Reactions
Anaphylaxis reactions occur when antigen stimulates IgE production. (This is sensitization.)
IgE antibodies bind to basophils and/or mast cells by their stem region, leaving the antigen
binding sites free.
When adjacent IgE antibodies bound to basophils and/or mast cells are cross-linked by binding
to antigen it results in the release of mediators (histamine, leukotrienes and prostaglandins)
that cause inflammation.
There are two basic kinds of reactions: systemic anaphylaxis and localized reactions.
Anaphylactic reactions can be prevented by determination of the specific allergens that a
patient is sensitive to and injecting small amounts of the allergens over an extended period of
time (desensitization). This causes the production of blocking antibodies, which are IgG.
Type II (Cytotoxic) Reactions
The antibodies are directed toward cellular antigens on foreign cells or foreign antigens on
host cells.
The antigen-antibody complexes cause complement fixation resulting in cell lysis and
phagocytosis. Examples include incompatible blood transfusions, Rh incompatibility, and
drug-induced cytotoxic reactions.
Type III (Immune Complex) Reactions
Antigens involved are not part of host cells but soluble antigens.
The antigens are bound by IgM or IgG antibodies and the antigen-antibody complexes
precipitate and lodge in basement membranes of blood vessels.
Complement fixation leads to inflammation and cell lysis. An examples is glomerulonephritis.
Type IV (Cell Mediated) Reactions
Delayed-type hypersensitivity (TDTH) T-cells are involved.
Sensitized T-cells secrete lymphokines in response to antigen. Lymphokines attract
macrophages and initiate tissue damage. Examples include the tuberculin skin test and allergic
contact dermatitis.
Autoimmune Diseases
Due to loss of self-tolerance (tolerance is the ability to recognize self proteins as self and not
as foreign).
Antibodies to infectious agents may cross react with self-proteins on host cells and cause
damage to the cells. Examples include autoimmune and rheumatic fever.
Antibodies may bind to host cell surface antigens without cell destruction. Examples include
Graves’ disease and myasthenia gravis.
Antibodies may bind to soluble host antigens and the antigen-antibody complexes precipitate
and fix complement. Examples include systemic lupus erythematosus, and rheumatoid
arthritis.
Cell mediated autoimmunity involves activation of cytotoxic T-cells by infectious agents
followed by cross-reactivity with normal host cells. Examples include multiple sclerosis,
Hashimoto’s autoimmune thyroiditis, and insulin-dependent diabetes mellitus.
Immune Deficiencies
Immunodeficiencies may be congenital or acquired.
Congenial deficiencies are due to defective or absent genes.
Acquired immune deficiencies can caused by drugs, cancers, and infectious diseases.
Acquired Immunodeficiency Syndrome (AIDS)
AIDS is the final stage of HIV infections.
HIV is a retrovirus with a phospholipid envelope with gp 120 spikes which attach to CD4
receptors and coreceptors on host cells (helper T cells, macrophages, and dendritic cells).
HIV infection is categorized by symptoms:
 Category A –asymptomatic
 Category B – selected symptoms
 Category C – AIDS indicator conditions, reported as AIDS
Also categorized by CD4 T cell numbers: below 200/mm3 is reported as AIDS (true for
Category A and B also).
Progression from HIV infection to AIDS takes about 10 years.
Transmission is by sexual contact, breast milk, contaminated needles, transplacental infection,
artificial insemination, and blood transfusion although blood transfusions are not a likely source
of infection in developed countries.
In the U.S., Canada, western Europe, Australia, northern Africa, and parts of South America
transmission has been by injecting drug use (IDU) and male-to-male sexual contact.
Heterosexual transmission is increasing.
In sub-Saharan Africa transmission is primarily heterosexual contact.
In Eastern Europe and Asia transmission is by IDU and heterosexual contact.
Worldwide the primary means of transmission is through unprotected (heterosexual)
sex.
Chapter 20 - Antimicrobial Drugs
Antimicrobial drugs should:
1. Have selective toxicity – that is, should be toxic to the microbe not the host
2. Not provoke hypersensitivity
3. Be soluble in body fluids so that they can get into the areas where the infection exists
4. Should be cleared from the blood fast enough that toxic situations do not occur, but not so
fast that therapeutic doses cannot be reached.
5. Have a long shelf life – this makes them less expensive, more readily available and
available for use in rural areas.
6. Not provoke resistance
Paul Ehrlich developed the concept of chemotherapy to treat microbial diseases: he developed
salvarsan to treat syphilis in 1910.
Alexander Fleming discovered the first antibiotic, penicillin, in 1928.
The Spectrum of Antimicrobial Activity
Antibacterial drugs affect many targets in a prokaryotic cell.
Fungal, protozoan, and helminthic infections are more difficult to treat without harming the host
because these organisms have eukaryotic cells.
Narrow –spectrum drugs affect only a select group of microbes—gram positive cells for example;
broad-spectrum drugs affect a wider range of different types of organisms.
The Action of Antimicrobial Drugs
Inhibit cell wall synthesis
Inhibit protein synthesis
Cause injury to plasma membranes
Inhibit nucleic acid synthesis
Inhibit enzyme activity
Antibacterial Antibiotics: Inhibitors of Cell Wall Synthesis
Penicillin
Penicillin inhibits cell wall synthesis in bacteria.
This of course requires that the bacteria are actively growing.
Since human cells do not have peptidoglycan cell walls penicillin has low toxicity; the primary
concern is for allergy, which only occurs in a low percentage of the population, making penicillin
the antibiotic with the fewest side effects.
All penicillins contain a B-lactam ring.
Natural Penicillins
Natural penicillins (Penicillin G, Penicillin V) produced by Penicillium are effective against grampositive cocci and spirochetes.
Natural penicillins have a narrow spectrum activity and are susceptible to penicillinases (or Blactimases) - bacterial enzymes that destroy natural penicillins.
Semisynthetic Penicillins
Semisynthetic penicillins are made in the laboratory by adding different side chains onto the B-lactam
ring after it is synthesized by a fungus.
Semisynthetic penicillins (oxacillin, ampicillin, amoxicillin, aztreonam, imipenem) are resistant to
penicillinases and have a broader spectrum of activity than natural penicillins.
The first penicillinase-resistant semisynthetic penicillin was methicillin. We've got so much methicillin
resistance that methicillin use has been discontinued in the U.S.
Vancomycin inhibits cell wall synthesis and may be used to kill penicillinase-producing
staphylococci.
Used primarily against methicillin-resistant S. aureus.
Very toxic
Streptogramins are bactericidal agents that inhibit protein synthesis and may be used to kill
vancomycin-resistant bacteria.
Syncercid - combination of quinupristin and dalfopristin, blocks protein synthesis.
Effective against a broad range of gram-positive organisms but expensive and has a lot of side
effects.
Useful for treating VRSA but not Enterococcus faecalis (which is likely to be VRE, especially in
clinical settings).
Oxazolidinones are also useful against vancomycin resistance, especially enterococci that are
resistant to syncercid.
Zyvox - totally synthetic, may slow development of of resistance. There are some toxicity
issues and the course of treatment is 28 days.
Daptomycin, a new drug (the first lipopeptide to be released) produced by Streptomyces, binds to
bacterial cell membranes and causes rapid depolarization, the depolarized cell can't synthesize
nucleic acids and proteins and dies.
Effective only against gram positive organisms because it can't penetrate the outer membrane
of gram-negative organisms.
More rapidly bactericidal than vancomycin against Staphylococcus aureus and Enterococcus
(including MRSA and VRE).
Daptomycin has no identifiable transferable mechanism of resistance, however you may see
the statement "resistance has been seen" - 2 isolates out of more than 1000 courses of
therapy in clinical trials (1 was S. aureus and the other was E. faecalis)
Cephalosporins
Cephalosporins inhibit cell wall synthesis like penicillins and have more activity against gramnegative organisms.
They are used against penicillin resistant strains but are susceptible to a different class of Blactamases and also are contraindicated in people with penicillin allergy.
Antimycobacterial Antibiotics
Isoniazid (INH) inhibits mycolic acid synthesis in mycobacteria and is used to treat
tuberculosis. INH is administered with rifampin or ethambutol to avoid resistance.
Tetracyclines
Tetracyclines are said to have the broadest spectrum of all antibiotics; effective against both
gram-positive and gram-negative bacteria as well as against rickettsias, mycoplasmas and
chlamydias. Often used to treat urinary tract infections.
Can suppress the normal flora leading to superinfections of Candida albicans.
May cause discoloration of the teeth in children and liver damage in pregnant women.
Semisynthetic tetracylines (doxycycline and minocycline) have longer retention in the body
than the natural tetracylcines.
Macrolides – (Example: Erythromycin) Spectrum similar to that of penicillin G; good alternative to
penicillin; Drug of choice for legionellosis and mycoplasmal pneumonia.
Newer macrolides include azithromycin and clarithromycin (Biaxin) - broader spectrum and
penetrate tissues better (good against Chlamydia).
Even newer are ketolides, developed to deal with resistance. Example: telithromycin (Ketek)
Quinolones – are a group of antibiotics that inhibits DNA synthesis by effecting DNA gyrase, which is
needed for replication.
Use is limited to urinary tract infections.
Fluoroquinolones - have a broad spectrum of activity, penetrate tissues well, and are safe for
adults.
They are not recommended for children, adolescents, and pregnant women because they may
adversely affect cartilage development.
Norfloxacin, ciprofloxacin (Cipro), and levofloxacin (Levoquin) are the most commonly used.
Sulfonamides (sulfa drugs) – are bacteriostatic antibiotics.
The most widely used sulfa drug is trimethoprim-sulfamethoxazole (Cotrimoxazole, Septra),
which interferes with folic acid metabolism.
It is very effective in penetrating brain tissue and cerebrospinal fluid.
Tests To Guide Chemotherapy
These tests are used to determine which chemotherapeutic agent is most likely to combat a
specific pathogen.
These tests are used when susceptibility cannot be predicted or when drug resistance arises.
The Diffusion Methods
In the Kirby-Bauer test a bacterial culture is inoculated on an agar medium, and filter paper
disks impregnated with chemotherapeutic agents are overlaid on the culture.
After incubation, the absence of microbial growth around a disk is called a zone of inhibition.
The diameter of the zone of inhibition, when compared with a standardized reference table, is
used to determine whether the organism is sensitive, intermediate, or resistant to the drug.
MIC (minimal inhibitory concentration) is the lowest concentration of drug capable of
preventing microbial growth; MIC can be estimated using the E test.
A plastic coated strip contains a gradient of antibiotic concentrations and the minimal inhibitory
concentration is read from a scale printed on the strip.
Broth DilutionTests
In a broth dilution test, the microorganism is grown in a liquid media containing different
concentrations of a chemotherapeutic agent.
The lowest concentration of a chemotherapeutic agent that kills bacteria is called the
minimum bactericidal concentration (MBC).