Download Chapter. 6 Immune Technology

Chapter. 6 Immune Technology
吳彰哲 (Chang-Jer Wu)
Department of Food Science
National Taiwan Ocean University
Review of immunology
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Dual Nature of Adaptive Immunity
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Antibody structure and function
FIGURE 6.1 Foreign Antigens Are Recognized by Antibodies
(A) Antibodies are Y-shaped molecules produced by the immune system in vertebrates. These bind to
specific proteins, or antigens, of any invading pathogen.
Antibody structure and function
FIGURE 6.2 Pre-Designed Antibodies Are Ready for Foreign Antigens
Long before an attack by a pathogen, an army of B cells produces a large
repertoire of antibodies (A). When one of the antibodies binds to an antigen (B),
that particular B cell starts dividing and expanding (C). The majority of the B
cells refine the antibody so that the antigen/antibody complex binds more tightly,
and they fight the pathogens (D). A small subset of B cells become memory cells
that never die, waiting for another attack by the same pathogen (E).
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Antibody structure and function
Antibodies, antigens and epitopes
Antigen (Ag): A substance that causes the body to produce specific antibodies or
sensitized T cells.
Antibody (Ab): Proteins made in response to an Ag; can combine with that Ag.
Antibodies (Ab) interact with epitopes or antigenic determinants
FIGURE 6.3 Surface Antigens of
Microorganisms
The surfaces of bacteria and
viruses are coated with
glycoprotein and lipoproteins
that are recognized by antibodies
in the host organism.
FIGURE 6.4 Antibodies Bind to
Epitopes on an Antigen
Antibodies only recognize a small
ridge or dimple on the surface of
a protein. The region of the
antigen that binds to the
antibody is called an epitope.
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The great diversity of antibodies
FIGURE 6.5 Modular Gene Assembly
Linking different segments of genes creates
exponential numbers of unique combinations.
Antibody structure
FIGURE 6.6 Structure of an Antibody
Y-shaped antibodies consist of two light chains and two
heavy chains. Each consists of segments: CH1, CH2, and
CH3 are heavy-chain constant regions; CL is the lightchain constant region; VH is the heavy-chain variable
region; and VL is the light-chain variable region.
Antigens bind to the variable regions.
FIGURE 6.7 Fab Fragments and Fc
Fragment of an Antibody
Antibodies can be spit into two Fab
fragments and one Fc fragment by
breaking the molecule at the hinge region.
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Structure and function of immunoglobulins
TAB 6.1 Different types and
functions of human antibodies
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Monoclonal antibodies for clinical use
FIGURE 6.8 Principle of the Hybridoma
Monoclonal antibodies derive from a single antibody-producing B cell. The
antigen is first injected into a mouse to provoke an immune response. The spleen
is harvested because it harbors many activated B cells. The spleen cells are
short-lived in culture, so they are fused to immortal myeloma cells. The
hybridoma cells are cultured and isolated so each hybrid is separate from the
other. Each hybrid clone can then be screened for the best antibody to the target
protein.
Monoclonal Antibodies
31 2 4
抗 原
BA
1
3
2
4
LB /
c
免 疫
1
2
3
31 2 4
傳統抗血清
4
m
m
m
m
1
2 3
4
1
2
3
取出脾細胞
+
骨髓瘤細胞
細胞融合
4
單株抗體
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Humanization of monoclonal antibodies
™
Chimeric mabs: Genetically modified mice that
produce Ab with a human constant region.
™
Humanized mabs: Mabs that are mostly human,
except for mouse antigen-binding.
™
Fully human antibodies: Mabs produced from
a human gene on a mouse.
FIGURE 6.9 Humanization of Monoclonal Antibodies
Antibodies from a mouse can be altered to become more like a
human antibody. (A) The entire constant region of the heavy
and light chain can be replaced with constant regions from a
human. (B) Antibodies have six CDRs that determine the
actual antigen binding site. The entire antibody except the
CDR region can be replaced with human sequence.
Humanized antibodies in clinical applications
™Immunotoxins:
Mabs conjugated with a toxin to target cancer cells.
FIGURE 6.10 Herceptin Helps Kill Cancer Cells with HER2
Herceptin is a humanized monoclonal antibody that recognizes the HER2 receptor on breast cancer cells.
When the antibody binds to the receptor, the immune system helps destroy the cancer cell, and the cancer
cell becomes more sensitive to chemotherapeutic treatments.
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Humanized antibodies in clinical applications
FIGURE 6.11 Humanized Antibodies to S. aureus Prevent Colonization
Antibodies to the cell surface protein, ClfA, prevent the bacteria from binding to the extracellular matrix
protein, fibrinogen. If S. aureus cannot bind to the extracellular matrix, the bacteria cannot colonize and
therefore do not cause infections.
Antibodies engineering
FIGURE 6.12 Fab and Fv Antibody Fragments
Fab fragments are produced by protease digestion of the hinge region. A disulfide bond holds the heavy and
light chains together. To make an antibody fragment without any constant region, the genes for the VH
domain and the VL domain are expressed on a bacterial plasmid. This structure is unstable because of a
lack of disulfide bonds. Therefore, disulfide bonds are engineered into the two halves (dsFv fragment), or a
linker is added to hold the VH and VL domains together (scFv fragment).
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Diabodies and bispecific antibody constructs
FIGURE 6.13 Engineered Diabody Constructs
(A) Engineering a diabody construct begins by
genetically fusing the variable domains of the
heavy and light chain (VH and VL) with a linker.
The long linker allows a single polypeptide to
form into a single antibody binding domain. The
short linker allows two polypeptides to complex
into a diabody with two antibody binding domains.
The construct is expressed in bacteria using a
bacterial promoter and RBS (ribosome binding
site). The signal sequence tells the bacteria to
secrete the engineered protein. (B) Instead of
identical Fv units, two different Fv chains can be
coexpressed in the bacterial cell. The two
different Fv chains will unite into a diabody with
two different antibody binding domains, a
different one on each side. (C) Bispecific
antibodies can be made as one single transcript
with a linker between VHA and VLB, a linker
between the two halves, and finally a linker
between VHB and VLA.
Diabodies and bispecific antibody constructs
FIGURE 6.14 Engineered Bispecific Antibody Constructs
Instead of genetic linkers to hold diabodies, various proteins can also hold scFv fragments together.
Proteins with a leucine zipper domain dimerize; therefore, when scFv genes are genetically fused to these,
the scFv domains come together as dimers. Proteins such as streptavidin or proteins with four helix
bundle domains can be genetically fused to scFv domains. When expressed, there are four scFv domains
on the outside, providing four different antibody binding sites.
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ELISA assay
Enzyme-linked immunosorbent assay (ELISA)
™
A group of enzyme immunoassay (EIA)
™
Two basic methods: direct and indirect ELISA
™
Advantages: little interpretive skill required, clear
positive or negative result presented
FIGURE 6.15 Principle of the ELISA
ELISA detects and quantifies the amount of a particular
protein bound to the well of a microtiter dish. Anti-A
antibody is linked to an enzyme such as alkaline
phosphatase. The antibody recognizes only the orange
protein, and not the green protein (A). After the antibody
binds to its target, the unbound antibody is washed from the
dish (B). A colorimetric substrate of alkaline phosphatase is
added to each well (C), and wherever there is antibody, the
substrate is cleaved to form its colorful product (D). The
amount of color is proportional to the amount of protein.
ELISA assay
Direct ELISA
Indirect ELISA
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ELISA assay
Indirect ELISA for detection of antibodies in serum
Substrate
Substrate
Coloured product
E
E
E
E
E
E
Antiglobulin linked to
enzyme
Serum from animal
(+/- antibodies)
Antigen
Antibody-negative animal
Antibody-positive animal
ELISA assay
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ELISA assay
Direct ELISA (Sandwich ELISA) for detection of antigen
E
E
E
E
Substrate
Second specific antibody
linked to enzyme
E
Coloured product
E
Substrate
Sample (+/- antigen)
Specific antibody
Antigen-positive
Antigen-negative
The ELISA as a diagnostic tool
FIGURE 6.16 Home Pregnancy Tests Are an ELISA
Diagnostic Tool
The pregnancy test shown here has four important
areas along the paper wick. The urine or blood is
applied on the far left and wicks to the right. The
anti-hCG antibodies loosely attached to the paper
are next. If the urine has hGC this binds to its
antibody and travels along the paper as a complex.
In the next area, a secondary antibody that only
recognizes the hCG–primary antibody complex is
firmly attached in a plus pattern. When the hCG
complex binds to the secondary antibody, the
detection system turns blue. The final spot is a
different secondary antibody that recognizes the
primary antibody without any hCG. This is a
positive control, to ensure that the antibody was
released and wicked up the paper with the urine.
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Visualizing cell components using antibodies
Direct: to identify a microorganism in a clinical specimen.
Indirect: to detect the presence of a specific antibody in serum following
exposure to a microorganism. (often more sensitive than direct)
Visualizing cell components using antibodies
Immunostaining
* Direct tests used to detect antigen in tissue sections
Coloured
product
Antibody labelled with
fluorescein
E
antibody linked
to enzyme
Tissue section +/- viral antigen
Direct immunofluorescence
Direct immunoperoxidase
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Visualizing cell components using antibodies
* Indirect immunostaining tests used to detect antigen in tissue
sections or antibody in serum
Anti-Ig labelled with fluorescein
Antibody against antigen
Tissue section + antigen
Indirect immunofluorescence for detection of antibody
Visualizing cell components using antibodies
FIGURE 6.17 Fluorescent Antibody Staining
Colocalization of Streptococcus pneumoniae and membrane antigens in mouse brain sections as seen by
confocal microscopy at 63-fold magnification. The bacteria are expressing GFP and appear green. All
three antigens were visualized separately by a red fluorescent stain. Yellow regions indicate colocalization
of bacteria with antigens. At left, wild-type mice and at right mice lacking Toll-like receptor 2. (A)
GLT1v-stained plexus choroideus epithelial cells. (B) Third ventricle with infiltrating Gr1-stained
granulocytes (insert: 3D picture of bacteria taken up by granulocytes). (C) Third ventricle and GFAPstained astrocytes. From: Echchannaoui et al.(2005). Regulation of Streptococcus pneumoniae
distribution by Toll-like receptor 2 in vivo. Immunobiology210, 229–236. Reprinted with permission.
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Fluorescence activated cell sorting (FACS)
FIGURE 6.18 FACS Separates CD4+ and CD8+ Cells
FACS machines can separate fluorescently labeled cells into
different compartments. A mixture of CD4+, CD8+, and unlabeled
cells is separated based on their fluorescence. When the
fluorescence detector notes green, the charged metal plates pull that
drop to the left or minus plate, allowing those cells to collect into
the left tube. If no fluorescence is detected, the drop stays neutral
and is collected in the middle tube. If the drop fluoresces red, the
charged plates pull the drop to the plus side, and it collects in the
right tube.
Fluorescence activated cell sorting (FACS)
FIGURE 6.19 Example of Flow Cytometry Data
Expression of proteins on the surface of white blood cells. White blood cells were stained with an MHCclass I tetramer and anti-CD107 labeled with APC (allophycocyanin, blue) and treated as follows: (Left
panel) Stained with anti-CD3 labeled with phycoerythrin (red) and anti-CD8 labeled with peridinin
chlorophyll protein (green), and analyzed without further incubation. (Right panel) Stained after
stimulation with the cognate peptide (NLVPMVATV). From: Betts et al. (2003). Sensitive and viable
identification of antigen-specific CD8+ T cells by a flow cytometric assay for degranulation. J Immunol
Methods281, 65–78. Reprinted with permission.
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History of Vaccines
™
Variolation: Inoculation of smallpox into skin
(18th century)
™
Vaccination:
Z Inoculation of cowpox virus into skin (Jenner)
Z Inoculation with rabies virus (Pasteur)
™
Vaccine: A suspension of organisms or fractions of organisms
that is used to induce immunity
™
If most of the population is immune called herd immunity.
Principles and effects of vaccination
™
Provoke a primary immune response in the recipients leading
to the formation of antibodies and long-term memory cells.
™
When recipient encounter antigen, the memory cells are
stimulated to produce a rapid intense secondary immune
response.
™
Herd immunity results when most of a population is immune
to a disease.
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Immune memory and vaccination
免疫反應的兩個階段
抗原
初級反應
抗原
次級反應
Primary
response
Secondary
response
初次入侵
再次入侵
免
疫
反
應
時間
Adapted from Roitt et al (1985) Immunology. 1.9
免疫增幅
抗 原
初次入侵
初級反應
作用細胞
記憶細胞
抗 原
再次入侵
次級反應
作用細胞
記憶細胞
Adapted from Roitt et al (1985) Immunology. 2.5
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Types of Vaccines
™
™
™
™
™
™
Attenuated whole-agent vaccines
MMR
Inactivated whole-agent vaccines
Salk polio
Toxoids
Tetanus
Subunit vaccines
Cellular pertussis
Recombinant hepatitis B
Conjugated vaccines
Nucleic acid (DNA) vaccines
West Nile (for horses)
Creating a vaccine
FIGURE 6.20 Whole Vaccines Include Killed or Attenuated Pathogens
(A) High heat or chemical treatment kills pathogens, but leaves enough antigens intact to elicit an
immune response. Once exposed to a dead virus or bacterium, memory B cells are established and
prevent the live pathogen from making the person sick. (B) Attenuated viruses or bacteria have been
mutated or genetically engineered to remove the genes that cause illness. The immune system generates
antibodies to kill the attenuated pathogen and establishes memory B cells that prevent future attack.
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Creating a vaccine
FIGURE 6.21 Subunit Vaccines Rely on a
Single Antigen
A single antigenic protein from a pathogen is
isolated and its gene is cloned into an
expression vector. The gene is expressed in
cultured mammalian cells (such as Chinese
hamster ovary [CHO] cells), isolated, purified,
and used as a vaccine.
Creating a vaccine
FIGURE 6.22 Peptide Vaccines Are Conjugated to Carrier Proteins
Peptide vaccines are small regions of an antigenic protein from a
pathogen. The peptide is often an epitope that elicits a strong immune
response. Because the peptide is small, multiple peptides are conjugated
to a carrier protein to prevent degradation, and to stimulate the immune
system.
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Making vector vaccines using homologous
recombination
FIGURE 6.23 Homologous Recombination Adds New Genes to the Vaccinia Genome
The plasmid contains two regions homologous to the virus thymidine kinase gene and flanking the cloned
antigen gene. When the plasmid aligns with the vaccinia genome, the regions of homology elicit a
recombination event. The recombinant vaccinia will gain the cloned antigen gene and lose the gene for
thymidine kinase.
Reverse vaccinology
FIGURE 6.24 Reverse Vaccinology
Reverse vaccinology uses the genes
identified in the genome of pathogenic
agents. First, the genes are cloned into
expression vectors and expressed to give
proteins. Each protein is tested in mice
for an immune response.
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Identifying new antigens for vaccines
FIGURE 6.25 Differential Fluorescence Induction (DFI)
First, genes from the pathogen of interest are cloned in frame with the GFP gene. The fusion proteins are
then expressed in bacteria. First the entire bacterial population is exposed to low pH. Those bacteria that
are expressing GFP are isolated. These clones either express the GFP protein constitutively or were
induced by the low pH. To isolate the clones that are expressed only at low pH, the green cells are shifted
to neutral pH, and this time, only the colorless cells are kept. Repeating this procedure will ensure a pure
set of genes that are induced only under low pH.
Identifying new antigens for vaccines
FIGURE 6.26 In Vivo Induced Antigen Technology (IVIAT)
Finding novel antigens to make a new vaccine relies on identifying proteins that elicit an immune
response. IVIAT identifies antigens directly from patients who have been exposed to the pathogenic
organism. First, an expression library is established that includes each of the genes from the pathogen of
interest. Next, serum from infected patients is collected and preabsorbed to the infectious organism
(grown in culture) to remove the antibodies that recognize surface proteins. The remaining antibodies are
used to screen the expression library. When an antibody recognizes a cloned protein, the specific DNA
clone is sequenced to identify the gene product.
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DNA vaccines bypass the need to purify antigens
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DNA vaccines bypass the need to purify antigens
FIGURE 6.27 DNA-Coated Microbeads
Microbeads are coated with plasmid DNA encoding an antigen gene and injected into a patient. Once
inside the cells, the plasmid DNA is slowly released and the protein antigen is expressed over a period of
time. The expressed protein elicits an immune response without causing disease, thereby vaccinating the
person against future exposures to the pathogen.
Edible vaccines
• A pathogen transgene protein gene is cloned
• Gene is inserted into the DNA of plant (potato, banana, tomato)
• Humans eat the plant
• The body produces antibodies against pathogen protein
• Human are “immunized” against the pathogen
• Examples:
9Diarrhea
9Hepatitis B
9Measles
Advantages:
Administered Directly
no purification required
no hazards assoc. w/injections
Production
may be grown locally, where needed most
no transportation costs
Naturally stored
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Edible vaccines
Edible vaccines
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Thank you
Tel: 2462-2192 ext 5137
E-mail: [email protected]
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