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05-01-12 Applications of the Immune Response
Principals of immunization
Vaccines
Immunizations
Monoclonal antibodies
Immunological tests
17-1
Principals of immunization
• Active immunity – immune response in an individual
upon exposure to antigen; naturally from active
infection or artificially via vaccination
• Passive immunity – occurs during pregnancy (natural) and
when Abs are supplied from another individual or
animal (artificial)
17-2
17-3
Active and passive immunity (18.2)
Antitoxin – antibody preparation against a specific toxin
Antiserum – a preparation of serum containing protective antibodies
Immune serum globulin – passive immune preparation containing
IgG (gamma globulin)
pooled blood serum from many donors
variety of Abs
given to travelers and immunosuppressed individuals
Hyperimmune globulin – sera from donors with high levels of
specific Abs
eg anti tetanus, rabies, hepatitis A and hepatitis B
given during disease incubation period to prevent
disease development
Herd immunity – inability of a pathogen to spread; no hosts
17-4
Vaccination History:
The first known vaccination procedures were performed by the Chinese during
the Sung dynasty (A.D. 960-1280) against smallpox.
“Variolation” used small amounts of powdered crusts from smallpox pustules
which were inhaled or placed in small cuts in the skin.
A mild disease was usually produced, followed by immunity to smallpox.
Variolation was practiced in Europe, but was expensive and sometimes disease
resulted (1 in 100 died), so many people were not treated.
Edward Jenner, in 1796, deliberately introduced material from a cowpox lesion on
a milkmaid to a scratch on the arm of a young boy (our word dairy comes from
“dey-ery” the room in which the “dey”, or woman servant, made milk into butter).
Jenner subsequently exposed the boy to the pus of a smallpox victim;
the boy did not develop smallpox.
Pasteur
coined the word “vaccination” to describe any type of protective
17-5
inoculation.
Figure 12-6
Increase in measles in the UK compared
with uptake of MMR vaccine (P14.6)
(MMR = Measles, Mumps, Rubella)
17-6
New York Times on Measles
http://www.nytimes.com/packages/khtml/2006/04/28/health/20060430_BRINK_AUDIOSS.html
17-7
Vaccines
• Toxoids – inactivated toxins
• Protein subunit vaccines (and recombinant vaccines)
contain key protein antigens
reduced unwanted side effects
• Polysaccharide vaccines – T-independent antigens
conjugate vaccine – polysaccharide plus protein =
T-dependent vaccine – protection for children
against H. influenza type b (meningitis)
and S. pneumoniae (variety of infections)
• Adjuvant – enhances immune response to antigens, provide
“danger signals”
17-8
Vaccines (cont.)
• Attenuated - weakened form of the disease-causing agent
Agent replicates, may cause mild disease
Mimics wild type strain, controls infection
Longer antigen exposure than inactivated vaccines
Can cause disease in immunocompromised people
eg Sabin polio vaccine
• Inactivated - unable to replicate; retains immunogenicity
cannot cause infections or revert to dangerous form
no amplification of dose in vivo; boosters required
Inactivated whole agent vaccines – killed microorganisms
eg Salk vaccine is a mixture of inactivated forms
of the three types of poliovirus
17-9
Viral vaccines are made from whole viruses or
viral components
“Varioloation” – dried pustules from people with milder
forms of small pox administered intranasally or
intradermally – sometimes caused disease and death
Cowpox virus (“vaccinia”) was used by Jenner to produce
immunity to smallpox; eradicated smallpox
Killed or inactivated vaccines are used for immunity against
viruses for which no natural safe counterpart exists
Subunit vaccines contain only antigenic viral surface proteins
17-10
Viral vaccine development is hampered by evasion of the host
immune system (HIV), multiple genetic strains (flu)
and weak immune responses (common cold)
Figure 12-1
Vaccination with cowpox protects against smallpox
(P14.1)
17-11
Figure 12-2 part 1 of 2
Attenuated viruses are selected by growing
human viruses in non-human cells (P14.2)
17-12
Figure 12-2 part 2 of 2
Attenuated viruses are selected by growing
human viruses in non-human cells (P14.2)
17-13
Bacterial vaccines are made from whole bacteria,
capsular polysaccharides or secreted toxins
17-14
Toxoid – purified toxin inactivated with formalin, effective
against diphtheria and tetanus
DTP combination vaccine active against diphtheria, tetanus
and pertussis (whooping cough)
Pertussis bacteria act as adjuvant
DTP replaced by DTaP (acellular pertussis)
Polysaccharide vaccines for encapsulated bacteria designed
to elicit complement fixing Abs that bind strain-specific
and pathogenicity-causing surface polysaccharides
Effective against pneumococcus, salmonellae,
meningococci, H. influenzae, E. coli,
Klebsiella pneumoniae, B. fragilis
T-independent antigen is converted to T-dependent antigen
by coupling to carrier protein that binds T cells
“conjugate vaccines”
Adjuvants nonspecifically enhance the immune
responses
Vaccination must create a state of inflammation created by
bacterial products that activate macrophages
Purified proteins do not elicit a strong immune response
The response can be enhanced by adding substances that
induce inflammation called adjuvants (“helpers”)
Freund’s complete adjuvant is an emulsion of killed
mycobacteria and mineral oil
Adjuvants cause soluble protein antigens to aggregate and
precipitate to facilitate phagocytosis by APCs
17-15
Vaccination can inadvertently cause disease
Live attenuated viruses simulate actual infections and
elicit the best immune stimulation
Live attenuated viruses can revert to a pathogenic form
Sabin polio vaccine induces polio and paralysis in 3
people per million vaccinated
One of the three polio virus strains in Sabin vaccine differs
from natural polio virus by only 10 nucleotide
substitutions; can mutate to the natural strain
and cause disease
17-16
Table 18.1 - Vaccines
17-17
Table 18.1 - Vaccines
17-18
Table 18.2 Characteristics of Attenuated
and Inactivated Vaccines
17-19
Table 18.3 - Effectiveness of immunizations
17-20
Table 17.5 - Future immunizations
17-21
Figure 12-8
17-22
(P14.8)
Immunizations
•
•
•
•
17-23
Paralytic poliomyelitis
Effectiveness of immunizations
Recommended immunizations
Future immunizations
Paralytic poliomyelitis
• 1950 – Salk vaccine (inactivated virus)
no herd immunity – immunized people can be carriers
requires multiple injections over time
• 1960 – Sabin vaccine (attenuated virus)
• Salk vaccine is safe, but wild type virus can replicate and
spread
• Sabin vaccine provides herd immunity
given orally, induces mucosal immunity
stops spread, gives herd immunity
can cause vaccine-related polio in some individuals
17-24
New York Times on polio
http://video.on.nytimes.com/index.jsp?fr_story=77549ceb1b1ae1777e43d4eedfaf7bcff47b0a31
17-25
Immunological testing
• Monoclonal antibodies
• Serology
• Quantifying antigen – antibody reactions
17-26
Therapeutic MAbs:
1895 – French physicians use antiserum from dogs to treat cancer
some patients improved but had immunogenicity problems
nobody cured
2005 – 100 therapeutic MAbs in clinical trials
18 MAbs approved for use in the US
$5-6 billion in revenues in 2003
may triple in next five years
possibly 32% of biotech market by 2008
Genentech produces three anticancer MAbs:
Herceptin
Avastin
Rituxan
$2.7 billion in revenues in 2004
17-27
Perspective 18.1
Monoclonal Antibodies
Movie on Monoclonals
17-28
Problems and solutions encountered during MAb drug development:
1. Removal of inappropriate Abs from antisera
solution: MAbs (Milstein and Kohler, 1984)
produce single mouse Ab
2. Mouse MAbs were immunogenic in humans
solution: “humanization” of mouse MAbs
Mouse variable regions fused to human constant
regions = chimeric MAb reduced HAMA
17-29
3. Chimeric drugs Rituxin (rituximab; binds CD-20 on B cell
to combat non-Hodgkin lymphoma); Remicade (infliximab)
have been associated with serious allergic reactions
solution: insert mouse complementary-determining
regions (CDRs) into human Ab = humanized MAbs
90-95% human
fewer HAMA responses
4. Humanized MAbs technically demanding, reduced Ag affinities
solution: fully human MAbs to evade human immune response
a. change the mouse: Xenomouse, HuMab-Mouse are
transgenic animals with human Ab genes
11 xenomouse MAbs in clinical trials
eg panitumumab – targets EGF receptor
to combat metastatic colorectal cancer
150 HuMab-Mouse MAbs in development
eg MDX-010 – anti-CTLA-4 for
metastatic melanoma in clinical trials
binding of CTLA-4 blocks
inhibition of T-cell proliferation
effector T cells eliminate tumors
17-30
4. Humanized MAbs technically demanding, reduced Ag affinities
solution: fully human MAbs to evade human immune response
b. skip the mouse: in vitro phage display technology
used to produce human MAbs
libraries of human MAbs contain more than
100 billion different phage antibodies
screen to select for strong binding to specific Ag
17-31
17-32
17-33
Principles of Immunological Testing
• Terms
– Seronegative
• Person not yet exposed to antigen and has no
specific antibodies
– Seropositive
• Person with exposure and actively producing
antibody
– Titer
• Concentration of antibody in serum
• Indicates previous exposure
17-34
Using Labeled Antibodies to
Detect Interactions
• Detectable markers can be attached to specific antibodies
– Marked antibodies used to detect presence of given antigen
• Tests include
– Fluorescent Antibody (FA) test
– Enzyme-Linked Immunosorbant Assay (ELISA)
– Western blotting
– Fluorescence Activates Cell Sorter (FACS)
17-35
Using Labeled Antibodies to
Detect Interactions
• Fluorescent antibody test
– Relies on fluorescent microscopy to locate labeled antibodies
fixed to a microscope slide
– Fluorescent polarized immunoassay uses beam of polarized
light to rate spin of labeled antibodies
• Works under principle that bound antibodies are heavier
then unbound and will spin more slowly
17-36
17-37
Fluorescent Antibody (FA) tests (18.10)
Using Labeled Antibodies to
Detect Interactions
• Enzyme-Linked Immunosorbant Assay
– Employs antibody that has been labeled with detectable
enzyme
• Commonly horseradish peroxidase
– Labels antibody bonds to antigen
• Binding can be direct or indirect
– Antigen location is determined using colormetric assay
17-38
Using Labeled Antibodies to
Detect Interactions
• Enzyme-Linked Immunosorbant Assay
– Direct ELISA
• Looks for specific antigen
– Specimen placed in wells of microtiter plate
» Wells treated with antibody for antigen
– Indirect ELISA
• Looks for antibody in patient serum
– Human IgG
– Wells of plate treated with known antigen
17-39
Movie on ELISA
(18.11)
17-40
Detects human chorionic gonadotropin
Present only in pregnant women
17-41
Direct ELISA test for pregnancy (18.12)
Western blot for HIV (18.13)
gp 160
gp 120
Ags separated by
electorphoresis
Transferred to membrane
p66
p55
gp41
p39
p31
Probed with specific Abs
Abs detected indirectly
using anti-HGG
p24
p17
17-42
31
26
30
Serum
control
a
b
c
(a) Strong reactive control
(b) Weak reactive control
(c) Non-reactive control
Courtesy of Genelabs Diagnostics