PowerPoint Lecture Presentations prepared by …structure2.konkuk.ac.kr/Microbiology/weekly_materials/W...PowerPoint ® Lecture Presentations prepared by Bradley W. Christian, McLennan

PowerPoint® Lecture

Presentations prepared by

Bradley W. Christian,

McLennan Community

College

C H A P T E R

© 2016 Pearson Education, Ltd.

Practical

Applications of

Immunology

18


Vaccines

• Variolation: inoculation of smallpox into the skin

• Jenner inoculated cowpox to prevent smallpox

• Termed vaccination by Pasteur

• vacca = cow

• Vaccine: suspension of organisms or fractions of

organisms that induce immunity


PLAY Animation: Vaccines: Function

Vaccines: Function

vaccines_function.html


Principles and Effects of Vaccination

• Provokes a primary immune response

• Leads to the formation of antibodies and memory cells

• Produces a rapid, intense secondary response

• Herd immunity: immunity in most of the

population

• Outbreaks are sporadic due to the lack of susceptible

individuals


Table 18.1 Principal Vaccines Used in the United States to Prevent Bacterial Diseases in Humans


Table 18.2 Principal Vaccines Used in the United States to Prevent Viral Diseases in Humans (1 of 2)


Table 18.2 Principal Vaccines Used in the United States to Prevent Viral Diseases in Humans (2 of 2)


Types of Vaccines and Their Characteristics

• Live attenuated vaccines

• Weakened pathogen

• Closely mimic an actual infection

• Confers lifelong cellular and humoral immunity

• Inactivated killed vaccines

• Safer than live vaccines

• Require repeated booster doses

• Induce mostly humoral immunity



• Subunit vaccines use antigenic fragments to

stimulate an immune response

• Recombinant vaccines: subunit vaccines produced by

genetic modification

• Virus-like particle (VLP) vaccines: resemble intact

viruses but do not contain viral genetic material

• Toxoids: inactivated toxins

• Antitoxins: serums containing antibodies against the

toxin



• Conjugated vaccines

• Used for diseases in children with poor immune

response to capsular polysaccharides

• Nucleic acid (DNA) vaccines

• Injected naked DNA produces the protein antigen

encoded in the DNA

• Protein antigens carried to the red bone marrow

stimulate humoral and cellular immunity


PLAY Animation: Vaccines: Types

Vaccines: Types

vaccines_types.html


The Development of New Vaccines

• Less profitable than medicines

• Develop vaccines without the use of animals

• Use of plants as source for vaccines

• More oral vaccines

• Vaccines for chronic diseases

• "Reverse vaccinology" for the development of

cellular immunity


Vaccination Technologies

• Nanopatch: delivers a dry formulation of a vaccine

to the skin

• Skin contains high numbers of APCs

• Requires no refrigeration

• Multiple-combination vaccines


Adjuvants

• Adjuvants are chemical additives added to

vaccines to improve effectiveness

• Alum is the only approved adjuvant in the United States

• Improve the innate immune response


Safety of Vaccines

• On rare occasions, vaccines can cause the

disease

• No medical or scientific proof of MMR vaccines

being linked to autism

• Safest and most effective means of preventing

infectious disease in children


Diagnostic Immunology

• Sensitivity: probability that the test is reactive if

the specimen is a true positive

• Specificity: probability that a positive test will not

be reactive if a specimen is a true negative

• Immunologic-based diagnostic tests

• Interactions of humoral antibodies with antigens

• Known antibody can identify an unknown pathogen

• Known pathogen can identify an unknown antibody


Monoclonal Antibodies

• Hybridoma: "immortal" cancerous B cell

(myeloma) combined with an antibody-producing

normal B cell

• Hybridoma produces monoclonal antibodies (Mabs)



• Mabs are uniform, highly specific, and produced in

large quantities

• Used in diagnostic tools

• Used in human therapy

• Neutralize TNF with rheumatoid arthritis

• Treat allergic asthma by preventing the binding of IgE on

mast cells and basophils

• Often derived from mouse cells, leading to side effects



• Chimeric monoclonal antibodies: Mabs with a

mouse variable region and human constant region

• Humanized antibodies: Mabs that are mostly

human, except for mouse antigen-binding sites

• Fully human antibodies: Mabs produced from a

human gene on a mouse


Figure 18.2 The Production of Monoclonal Antibodies.

A mouse is injected with a specific antigen that

will induce production of antibodies against

that antigen.

The spleen of the mouse is removed and

homogenized into a cell suspension. The

suspension includes B cells that produce

antibodies against the injected antigen.

The spleen cells are then mixed with myeloma

cells that are capable of continuous growth in

culture but have lost the ability to produce

antibodies. Some of the antibody-producing

spleen cells and myeloma cells fuse to form

hybrid cells. These hybrid cells are now

capable of growing continuously in culture

while producing antibodies.

Cultured myeloma cells

(cancerous B cells)

Suspension of

myeloma cells

Spleen cells

Myeloma cells

Hybrid cells

Suspension of

spleen cells

Spleen

Antigen

The mixture of cells is placed in a

selective medium that allows only hybrid

cells to grow.

Hybrid cell Myeloma cell Spleen cell

Hybridomas

Hybrid cells proliferate into clones called

hybridomas.The hybridomas are

screened for production of the desired

antibody.

The selected hybridomas are then

cultured to produce large quantities of

monoclonal antibodies. Isolated

antibodies are used for treating and

diagnosing disease.

Desired

monoclonal

antibodies

•

•

•

The fusion of cultured myeloma cells

(cancerous B cells) with antibody-

producing spleen cells forms a

hybridoma.

Hybridomas can be cultured to produce

large quantities of identical antibodies,

called monoclonal antibodies

Monoclonal antibody production is an

important advancement in medicine

and also is integral to common

diagnostic and therapeutic tools. A

monoclonal antibody can attach to a

target cell while carrying a diagnostic

marker or an anticellular toxin.

KEY CONCEPTS


Precipitation Reactions

• Reaction of soluble antigens with antibodies to

form large, interlocking aggregates called lattices

• Antigen-antibody complex forms, followed by the

formation of a lattice that precipitates from solution

• Precipitin ring test: a cloudy line forms where

there is the optimal ratio of antigen and antibody


Figure 18.3 A precipitation curve.

AntibodyAntigen

Zone of

antibody

excess

Zone of

equivalence

Precipitate

formed

Zone of

antigen

excess


Figure 18.4 The precipitin ring test.

Antigens

(soluble)

Zone of equivalence:

visible precipitate

AntibodiesPrecipitation

band


Precipitation Reactions

• Immunodiffusion tests are precipitation reactions

carried out in an agar gel medium

• Precipitate develops at the point where the optimal

antigen-antibody ratio occurs

• Immunoelectrophoresis combines

electrophoresis with immunodiffusion

• Separates proteins in human serum


Agglutination Reactions

• Particulate antigens binding to antibodies to form

visible aggregates

• Direct agglutination tests

• Detect antibodies against large cellular antigens

• Measure concentration of serum antibody (known as

titer)

• Rise in titer indicates a greater immunity to disease

• Seroconversion is a significant change in titer as a

disease progresses


Figure 18.5 An agglutination reaction.

Epitopes

IgM

Bacterium


Figure 18.6 Measuring antibody titer with the direct agglutination test.


Agglutination Reactions

• Indirect (passive) agglutination tests

• Antibody reacts with the soluble antigen adhering to the

particles or vice versa

• Hemagglutination

• Agglutination of RBC surface antigens and

complementary antibodies; used in blood typing

• Viral hemagglutination occurs when viruses

agglutinate RBCs without an antigen-antibody reaction

• Mumps, measles, influenza


Figure 18.7 Reactions in indirect agglutination tests.

Antigen attached

to beadLatex

bead

Latex

bead

Latex

bead

Latex

bead

Latex

bead

Latex

bead

Latex

bead

Latex

bead

Latex

bead

Latex

bead

Latex

bead

Latex

bead

Latex

beadIgM antibodyBacterial

antigen

Reaction in a positive indirect test for

antibodies. When particles (latex beads

here) are coated with antigens, agglutination

indicates the presence of antibodies,

such as the IgM shown here.

Reaction in a positive indirect test for antigens.

When particles are coated with monoclonal

antibodies, agglutination indicates the presence

of antigens.

Antibody

attached to bead


Figure 18.8 Viral hemagglutination.


Neutralization Reactions

• Antigen-antibody reaction where the harmful

effects of an exotoxin or a virus are blocked by

antibodies to the toxin (antitoxin)

• Viral hemagglutination inhibition test is used for

subtyping viruses

• Viruses and RBCs are mixed with the patient's serum

• If the serum contains antibodies to a virus, they neutralize

the virus and inhibit hemagglutination


Figure 18.9 Reactions in neutralization tests.


Complement-Fixation Reactions

• Complement fixation: complement serum protein

binds to and is fixed to the antigen–antibody

complex

• Detects small amount of antibody

• Works for antibodies that do not work with precipitation

or agglutination reactions


Figure 18.10 The complement-fixation test.


Fluorescent-Antibody Techniques

• Combine fluorescent dyes with antibodies

• Direct FA tests

• Identify a microorganism in a clinical specimen

• Indirect FA tests

• Detect a specific antibody in serum

• Anti-human immune serum globulin (anti-HISG) is

added and will react with any antibody in serum if the

result is positive


Figure 18.11 Fluorescent-antibody (FA) techniques.


Fluorescent-Antibody Techniques

• Fluorescence-activated cell sorter (FACS)

• Laser beam strikes a droplet containing a cell

• Detector determines size and fluorescence of surface

molecules

• Imparts a charge to the cell, separating cells


Figure 18.12 The fluorescence-activated cell sorter (FACS).

Fluorescently

labeled cells

Laser beam

Laser

Fluorescence

detector

Electrically

charged

metal plates

Collection

tubes

The separated cells

fall into different

collection tubes.

As cells drop between

electrically charged

plates, the cells with

a positive charge

move closer to the

negative plate.

Electrode gives

positive charge to

identified cells.

Detector of

scattered light

Laser beam strikes

each droplet.

Cell mixture leaves

nozzle in droplets.

A mixture of cells is

treated to label cells

that have certain

antigens with

fluorescent-antibody

markers.

ElectrodeFluorescence detector

identifies fluorescent

cells by fluorescent

light emitted by cell.


Enzyme-Linked Immunosorbent Assay (ELISA)

• Direct ELISA

• Detects antigens

• Sample containing antigens is mixed with antibody

• Enzyme-linked antibodies react with the antigen

• Detected by adding a substrate for the linked enzyme; a

color is produced

• Indirect ELISA

• Detects antibodies

• Western blotting

• Identifies proteins via electrophoresis and a blotter


Figure 18.13 The use of monoclonal antibodies in a home pregnancy test.

Control

windows

Test

windows

Not pregnant Pregnant

Free monoclonal antibody specific

for hCG, a hormone produced during

pregnancy.

Capture monoclonal antibody bound

to substrate.

Sandwich formed by combination of

capture antibody and free antibody when

hCG is present, creating a color change.


Figure 18.14 The ELISA method.


The Future of Diagnostic and Therapeutic

Immunology

• Tests that require less human judgment and

personnel

• PCR and DNA probes

• Inexpensive and simple tests for developing

countries

• Disease prevention and therapy

Documents

PowerPoint Lecture Presentations prepared by …structure2.konkuk.ac.kr/Microbiology/weekly_materials/W...PowerPoint ® Lecture Presentations prepared by Bradley W. Christian, McLennan