Shingles

1
Shingles

• Caused by reactivation of Herpes Zoster Virus
• Infection of the nerve roots
• After recover from chicken pox, virus is dormant
  in nerve roots -- usually dormant forever
• Virus can be reactivated by stress, aging, or a
  weakened immune system
• Reactivated virus causes shingles, not chicken
  pox

Response to a question in class on Friday May 8
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Figure 11-4
Example of reactivation by a herpes virus
-- Herpes Simplex virus and cold sores

• A virus in a latent state is not being
  replicated.
• Cant be detected by the immune system (not
  producing viral proteins that can be displayed as
  peptides on MHC proteins).
• Infection by herpes simplex virus is cleared by
  the immune system, but a residual infection
  persists in sensory neurons that innervate the
  lips.
• Neurons express very low levels of class I MHC
  molecules, so hard for CD8 T cells to recognize
  and attack infected neurons.
• If virus is reactivated (stress or alteration in
  immune status), area served by the infected nerve
  is reinfected and you get a new cold sore.

3
Antibodies -- serum proteins that combat pathogens
10 nm (100 Å)
Immunoglobulin G (IgG)

• Typical mammal (e.g., you, a mouse) has the
  capacity to make gt1016 different types of
  antibodies.

4
Arrangement of CDRs in Ab combining sites CDR3s
always in center, CDR1 and CDR2 always on sides
CDR2 (L)
CDR3 (H)
CDR1 (H)
CDR2 (H)
CDR3 (L)
CDR1 (L)
Branden and Tooze, Fig. 15.13
5
Janeway et al. (2005) Immunobiology The Immune
system in health and disease Garland Publishing,
6th edition, figure 3-8
6
Enyzme-linked immunosorbent assay (ELISA) to
detect anti-HIV-1 p24 antibodies
Microtitre well coated with p24
Serum added
Anti-human antibody labeled with enzyme added
Enzyme substrate added
Note False positives can result from
cross-reactions.
See Figure 49.15 in Freeman 3/E
7
Western blot to detect anti-HIV-1 antibodies
Lyse (break open) laboratory strain ofHIV-1 in
detergent
Separate HIV proteins by SDS-PAGE
Transfer proteins to membrane (blot)
Cut membrane and incubate in blood sample from
individual being tested
30
6
Days after HIV-1 infection
Detect bound antibodies
SDS is a detergent that binds to and unfolds
proteins PAGE polyacrylamide gel electrophoresis
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Clicker question

• In SDS-polyacrylamide gel electrophoresis
  (SDS-PAGE), proteins migrate towards the positive
  electrode because

1) The polypeptide backbone is negatively
charged. 2) The sidechains are negatively
charged. 3) SDS is negatively charged. 4) Migratio
n is random half of proteins migrate towards
the positive electrode.
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What antibodies do

• Dont directly kill anything
• Can block entry of a virus into a host cell or
  prevent virus from replicating (neutralizing
  antibodies)

gp41
gp120
IgG
CD4
CCR5
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What antibodies do

• Can tag invaders for destruction (first of three
  ways)
• By complement -- binding of IgM or IgG to
  repeated epitope on invader surface triggers
  classical pathway (movie in extra material at
  end of Antibodies Lecture 1)

Electron micrographs of 100 Å diameter membrane
attack complex channels that are one of the end
results of complement activation.
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What antibodies do

• Can tag invaders for destruction (2nd of three
  ways)
• By macrophages antibodies opsinize (decorate the
  surface of) invaders -- Fc receptors on
  macrophages bind to exposed Fcs to increase
  phagocytosis

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What antibodies do

• Can tag invaders for destruction (3rd of three
  ways)
• By Natural Killer (NK) cells Fc receptors on NK
  cells bind to exposed Fcs to activate
  Antibody-Dependent Cellular Cytotoxicity (ADCC)

• NK cells bear surface Fc receptors (CD16). If Fc
  regions of IgG are clustered or aggregated by
  antigen on a target cell, they bind to CD16.
• Binding to CD16 causes contents of granules
  inside cells to be released --gt lysis of target
  cells.

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Antibodies can bind to different epitopes on the
same antigen -- The immune response to any
antigen is polyclonal
Protein antigens
Note that these three Fabs bind to different
regions of the model antigen lysozyme
Figure courtesy of Ian Wilson, Scripps Institute
Haptens
What if you want a single, chemically-homogeneous
antibody against an antigen? Answer you make a
monoclonal antibody -- see next slide and link to
Köhler and Milsteins 1984 Nobel Prize lecture on
Bi1 website.
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Figure A-14 part 1 of 2
B cell hybridomas secrete monoclonal antibodies

• Polyclonal B cells secreting antibodies against
  antigen A cannot be grown in tissue culture, so
  cant produce a clone secreting a single type of
  antibody.
• Fuse B cells with myeloma (malignant tumor)
  cells. These cells have been immortalized (can be
  grown in tissue culture).
• Use myeloma cells that lack the enzyme
  hypoxanthineguanine phosphoribosyl transferase
  (HGPRT) enzyme.
• Resulting hybrid cells (hybridomas) secrete
  antibody and can be grown in tissue culture.
• Make clones of single hybridomas.

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Monoclonal antibodies have many uses in biology,
biotechnology, medicine

• Used to detect presence and/or quantity of an
  antigen e.g., Western blot, ELISA,
  immunofluorescence microscopy, immunoelectron
  microscopy, flow cytometry.
• Used to purify antigens e.g., immunoprecipitation
  (e.g., CHiP), immunoaffinity chromatograpy.
• Used for medical applications, especially for the
  treatment of cancer. 160 different monoclonal
  antibodies in clinical trials or awaiting FDA
  approval (August 2006).

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Examples Therapeutic uses of monoclonal
antibodies

• Rituximab (Genentech) -- against CD20 antigen on
  surface of normal and malignant B cells. Used to
  treat non-Hodgkins lymphomas (B cell lymphomas).
  
• Herceptin (Genentech) -- against HER2 antigen.
  Given to patients with metastatic breast cancer
  whose tumors overexpress the HER2 protein (growth
  factor receptor). (HER2-positive breast cancers
  are more aggressive than HER2-negative breast
  cancers.)

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Figure 14-17
Different ways monoclonal antibodies are used to
eliminate tumors
e.g., ricin or Pseudomonas toxin
Can also link antibody to a chemotherapy drug
(e.g., adriamycin)
(ADCC)
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Labeling techniques for immunofluorescence
microscopy and flow cytometry
Kuby, Kindt, Goldsby, Osborne Immunology Textbook
19
Flow cytometry --Fluorescence Activated Cell
Sorting (FACS)
A two-color FACS analysis
Kuby, Kindt, Goldsby, Osborne Immunology Textbook
20
Stanford used flow cytometry to screen blood
before HIV tests were available

• Reduced ratio of CD4 to CD8 T cells in AIDS
  patients
• July 1983 to June 1985, Stanford Blood Center
  used flow cytometry to test donated units for
  CD4CD8 ratio
• Did not transfuse blood from donors with CD4CD8
  ratio lt 0.85
• Most other blood banks did no screening
• 10,000 cases of transfusion-transmitted AIDS in
  US before HIV test available in 1985

Galel et al., 1995, Prevention of AIDS
transmission through screening of the blood
supply Annu. Rev. Immunol. 201-227
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Laboratory use of complement(Low tech version
of FACS)

• Example
• Have mixture CD4 T cells and CD8 T cells
• Want only CD8 T cells
• Add anti-CD4 antibody to mixture of T cells. It
  binds. Now add complement, and CD4 T cells will
  be killed, leaving you with CD8 T cells only.

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Another way to sort cells
Kuby, Kindt, Goldsby, Osborne Immunology Textbook
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Figure A-15
In vitro selection to produce human monoclonal
antibodies or increase affinity of existing
monoclonal antibody
Clone into a phage so that each phage expresses
one VH-VL surface fusion protein.
Multiply phage display library in bacteria, bind
phage to surface coated with antigen. Wash away
unbound phage.
Repeat procedure (multiply recovered phage, bind
to antigen, wash away unbound phage) for several
cycles. Recover specific high-affinity antigen
binding VH-VL regions.
Generate library of heavy and light chain
variable regions using spleen DNA. Or introduce
random mutations into variable regions genes of a
specific antibody.
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Clicker question

• Epitope CDR
• 1) HV region Ag
• 2) Ab Ig
• 3) Fab Fc
• 4) Ag Ab

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Clicker question

• After an egg is fertilized, the DNA in the egg is
  copied. Copies are passed to daughter cells,
  copied again, passed to new daughter cells, etc.
  etc.
• With the exception of errors arising during
  copying (mutations), all somatic cells end up
  with same DNA as the fertilized egg.
• somatic not a germline cell i.e., not sperm
  or egg
• 1) True?
• 2) False?

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Clicker question

• If you sequenced the receptors and proteins of
  the innate immune system from identical twins,
  they would be identical, regardless of
  differences in immunological experience.
• If you sequenced the receptors and proteins of
  the adaptive immune system from identical twins,
  they would be identical, regardless of
  differences in immunological experience.
• 1) True, True
• False, False
• True, False
• False, True

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Extra slides for reference
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Figure 1-18
The diversity of antigen receptors in both B and
T cells is generated by rearrangements of gene
segments
Antibodies (and T cell receptors) are encoded by
sets of gene segments. During development of a B
(or T) cell, gene segments are joined randomly by
DNA recombination (irreversible). Juxtaposed
gene segments encode variable part of the
antibody (or T cell receptor). Different cells
join gene segments differently, so receptors are
unique. Each B cell bears many copies of its
unique receptor (membrane-bound antibody). Each
T cell bears many copies of its unique receptor
(T cell receptor TCR).
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Variability within antibody V domains clusters in
three regions
Wu and Kabat index of variability aa that
occur at that position / frequency of most common
aa at that position
3 hypervariable (HV) regions (CDRs)
Light Heavy
24-34 31-35 50-56
50-65 89-97 95-102
Janeway et al. Immunobiology Figure 3.6
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V regions encoded by gt1 gene segment (light
chains)
Important point Rearrangement for antibody genes
is at the DNA level -- different from RNA
splicing, which occurs in many genes
Kuby Immunology textbook
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V regions encoded by gt1 gene segment (heavy
chains)
Kuby Immunology textbook