Antigens and Antibodies

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Antigens and Antibodies
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Introduction
Antigen Substances capable of inducing a
specific immune response
Immunologic Properties Of Antigens
Antigens can be defined on the basis of four
immunologic Properties
1 Immuogenicity
2 Antigenicity
3 Allerogenicity
4 Tolerogenicity
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1 Immunogenicity
Ability to induce humoral and/or cell-mediated
immune response
B cells Antigen ? Plasma cells Memory Cells
T cells Antigen MHC ? T effector cells
Memory Cells
In this context, we can refer to an antigen as an
immunogen
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2 Antigenicity
Ability to combine specifically with the final
product of the above responses (i.e antibodies
and/or cell surface receptors)
All molecules immunogenic are antigenic
Reverse is not true
Example Haptens molecules that can bind to
antibodies or surface receptors (antigenic).
However, they cannot induce specific immune
response alone (non immunogenic)
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3 Allerogenicity
Ability to induce various types of allergic
reactions
Allergens Immunogen that specifically activates
humoral and/or cell-mediated responses with
allergic manifestations
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4 Tolerogenicity
Capacity to induce specific immunologic
nonresponsiveness in either humoral or
cell-mediated responses
Tolerance induction is learned or acquired
It is induced more rapidly in immature
lymphocytes than in mature ones.
Maintenance of tolerance depends on persistence
of antigen
Both B and T cells display tolerance
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They differ in quantity of antigen needed to
induce tolerance and duration of tolerance state
Experimental models ? tolerance more effectively
maintained in T cells compared to B cells
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Factors Influencing Immunogenicity
Immune system protects against bacterial, viral,
fungal and parasitic infections.
It recognises macromolecules in the structure of
these infectious agents (Proteins/polysaccharides
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Proteins are most potent immunogens with
polysaccharides ranking second
Lipids and nucleic acids do not serve as
immunogens
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In cell-mediated immunity, ONLY proteins serve as
immunogens
Must be processed to small peptides in
association with MHC molecule
Immunogenicity is not intrinsic property
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Immunogen Contribution To Immunogenicity
Immunogenicity is determined by five properties
of immunogen
1) Foreignness
2) Molecular weight
3) Chemical Composition
4) Complexity
5) Degradability
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1) Foreignness
Ability of lymphocyte to recognise self antigen
occurs during MATURATION
Any molecule not exposed to immature lymphocytes
during this critical period nonself or foreign

Degree of immunogenicity depends on degree of
foreignness
The greater the phylogenetic distance between two
species, the greater the genetic and antigenic
disparity between them
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Bovine serum albumin (BSA) more immunogenic in
chicken than goat
Exceptions
_at_ Some macromolecules that are conserved cross
species
_at_ Sperm/corneal tissue are very sequestered from
immune system
_at_ If sperm or corneal tissue were to be injected
into the same animal from which they originated
? Immunogenic
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2) Molecular Weight
Molecular weight of 100,000 Da (100 kDa) The
best
Molecular weight lt 500-10,000 Da are poor
3) Chemical Composition Heterogeneity
Synthetic homopolymers (single amino acid or
sugar) lack immunogenicity regardless of
molecular weight.
Copolymers composed of different amino acids
are immunogenic
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Addition of aromatic amino acids (e.g. tyrosine
or phenylalanine) increases immunogenicity
Primary, secondary, tertiary and quaternary
structures of proteins affect immunogenicity
4) Degradability
Macromolecules that cannot be degraded and
presented by APC are poor immunogens
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Polymers of D amino acids cannot b degraded by
macrophage enzymes ? therefore poor immunogens
Large, insoluble macromolecules more immunogenic
than small soluble ones
Why ? More readily phagocytosed and processed
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Contribution of Biological Systems To
Immunogenicity
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1 Genotype Of The Recipient Animal
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1 Genotype Of The Recipient Animal. Continued
..
Immune responsiveness was then mapped to a
subregion of MHC
Proteins encoded by MHC play a central role in
determining degree of immune responsiveness to
antigen
Immune response influenced by genes encoding
a) B and T cell receptors
b) Proteins involved in immune regulatory
mechanisms
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2 Immunogen Dosage and Route Of
Administration
Low dose ? failure to activate enough lymphocytes
or induces nonresponsiveness
High dose ? lymphocytes enter nonresposnsivess
state
Single dose usually not enough to induce reaction
Require repeated doses over a period of weeks to
induce a strong immune response
Repeated dosing BOOSTER increases clonal
proliferation of antigen-specific B or T cells.
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Common administration routes intravenous,
intradermal, Subcutaneous, intramuscular and
intraperitoneal.
Route of administration determines which immune
organ and cell population will be deployed
Intravenous administration ? carried first to
spleen
Subcutaneous Move first to lymph nodes
This generated differences due to differences in
residing populations of cells
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Adjuvants
Substances that enhance immunogenicity of
antigen when mixed and injected with it
e.g. Antibody response to influenza vaccine is
higher and prolonged when vaccine is
administered with an adjuvant
Adjuvants used to boost immune response when

• Antigen has low immunogenicity

2) Small amounts of antigen are available
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Mechanisms Of Action Of Adjuvants
A) Prolong Antigen Persistence
Mixing antigen with aluminium potassium sulphate
(alum) ? precipitate antigen
Antigen released slower from site of injection
Effective time of antigen exposure increases from
several days to several weeks
Increased size of antigen precipitate may
contribute to the alum Adjuvant action by
increasing likelihood of phagocytosis
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Freunds incomplete adjuvant ? surround antigen
with oil droplets ? Delay release of antigen
Freunds complete adjuvant contains heat-killed
mycobacteria
The muramyl dipeptide of cell wall activates
macrophages
Macrophages secrete interleukin 1
Interleukin 1 activates TH cells
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B) Stimulate Lymphocytes Non Specifically
Bacterial lipopolysaccharide (LPS) stimulate
nonspecific lymphocyte proliferation
Therefore increase likelihood of antigen-induced
clonal selection of lymphocytes
C) Induce Granuloma Formation
Some adjuvants stimulate local, chronic
inflammatory response ? Increase phagocytic cells
and lymphocytes
This results in a macrophage rich mass of cells
called granuloma (Alum and Freunds incomplete
adjuvants cause granulomas)
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D) Enhance Co-Stimulatory Signal
TH cells, recognising antigen on the surface of
APC in association with MHC require a
co-stimulatory signal
Co-stimulatory signal provided by interaction of
B7 membrane molecule present on macrophages and
CD28 on TH cells
Freunds complete adjuvant increases expression
of B7 membrane molecule
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Epitopes
Lymphocytes recognise discrete sites on the
antigen.
Epitopes Immunologically active region of
antigen able to bind to specific membrane
receptors or to secreted antibodies
Primary, secondary, tertiary or quaternary
structures of proteins may form the epitope
In carbohydrates, extensive side-chain branching
via glycosidic bonds affects the three
dimensional conformation of epitopes
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B cells bind soluble antigen
Therefore, epitopes recognised by B cells are
highly accessible sites on surface of immunogen
The epitopes contain hydrophilic amino acids
T cells only recognise antigen in the context of
MHCI/II
Therefore, T cell epitopes cannot be considered
apart from their associated MHC molecules
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B Cell Epitopes
a Size of B cell epitopes is determined by the
size of the antigen binding site on the antibody
molecule displayed on B cells
Interaction between antibody and epitope occurs
by non covalent bonds (weak)
Therefore, COMPLEMENTARITY is essential
Size of epitope recognised by B cells determined
by size, shape and amino acid residues of the
antibody binding site
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X-ray crystallography studies showed interaction
between antibody and smaller ligands such as
nuclei acids, peptides and carbohydrates to take
place in deep concave pocket
More complex proteins ? antibody makes contact
with the protein antigen across a large planar
face
Protrusions on epitopes or antibodies matched by
depressions on respective antibodies or epitopes
Approximately 15-22 amino acids on the surface of
protein antigens make contact with same number
of amino acids in the antibody binding site
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b B cell epitopes are hydrophilic amino acids
on the surface that are topographically
accessible to antibodies (Free or bound)
c B cell epitopes may contain sequential or
nonsequential amino acids
Epitopes either composed of sequential residues
along polypeptide chain
Alternatively, epitopes may be composed of
nonsequential amino acids from segments of the
chain brought about by tertiary structure folding
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The latter nonsequential epitopes also known as
Conformational Determinants
Main difference is if you breakdown protein
(e.g. sperm whale myoglobin), sequential
epitopes can be maintained
Nonsequential ones cannot as breaking down
proteins will disrupt the disulphide bonds and
the tertiary structure along with it.
d B cell epitopes are located in flexible
regions of an Immunogen and display site
mobility
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e Complex proteins contain multiple overlapping
B cell epitopes
Example 64 Monoclonal antibodies to BSA
recognise 25 epitopes on BSA
However, the subset of antigenic sites on a given
protein in an individual is much smaller than the
potential antigenic repertoire.
Some epitopes induce more pronounced immune
response compared to others in a particular
animal.
These are referred to as IMMUNODOMINANT
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T Cell Epitopes
Primary and secondary immunization occurred with
native protein, then this evoked antibody
production
No antibody production occurred when denatured
protein was used for secondary immunization
In contrast, T cell response was induced by
denatured protein even when primary immunization
was done with the native protein
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a Oligomeric peptides function as T cell
epitopes
Antigen-binding cleft of MHC molecule determines
nature and size of the peptide it binds, and
therefore the maximal size of the peptide it binds
Nonamers and peptides 11-17 amino acids in size
bind most strongly to MHCI and MHCII molecules
respectively
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b Antigenic peptides recognised by T cells form
trimolecular complexes with T cell receptor and
MHC molecule
Antigens that are to be recognised by T cell
receptor must have two components
1) Epitope Interacts with T cell receptor
2) Agretope Interacts with MHC molecule
Binding of epitope to MHC not as specific as
binding to antibody
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MHC molecules can select a variety of peptides
This meant that agretopes may share common
structural features enabling them to bind to the
same MHC molecule
c Antigen processing is required to generate
peptides that specifically interact with MHC
molecules
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d Antigens contain amphipathic peptides
Processing may unfold protein antigens and expose
amphipathic regions
Amphipathic Possess hydrophilic and hydrophobic
amino acid residues
Hydrophobic and hydrophilic residues may act as
agretopes and epitopes respectively
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e Immunodominance determined by MHC molecule
expression
Analyse sequence of immunogeic proteins
Found 3/14 peptides able to activate TH cells
The same three were shown to bind MHCII molecule
Example Transfect L cells mouse fibroblast (non
APC) with two types of MHCII molecules
Different clones of these cells could present
different peptide fragments of lysozyme
One lysozyme T cell epitope is immunodominant in
one clone
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Viral Antigens
Virus nucleic acid protein
Protein subunit Capsomers
Complete viral particle Virion
It is these proteins that are recognised as
antigen by humoral and cell-mediated pathways
Therefore, surface sequence generated by tertiary
conformation of viral proteins function as
immunodominant B cell epitopes
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During viral infection, antibody levels increase.
Antibodies clear virus by
1) Opsonization (coat virus to help its
phagocytosis)
2) Activate complement cascade (lysis of virus)
3) Bind to virus surface ? prevent further
infection
Viruses cause minor changes to their amino acid
sequence (antigenic drift) or major one
(antigenic shift) that allows them to escape
immune response. (e.g. influenza and aids)
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Bacterial Antigens
Bacteria simple prokaryotes
Immunogen is mainly epitope on surface
Gram positive bacteria composed largely of
peptidoglycans
Peptidoglycans Polysaccharide cross-linked by
short peptide chains
Structural differences in these ? unique epitopes
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Gram negative bacteria thin peptidoglycan layer
covered with protein and LPS.
LPS major antigenic component in gram negative
bacteria
Bacterial capsule loose polysaccharide or
polypeptide layer
Associated with virulence as it interferes with
phagocytosis
However, repeating sequence of high molecular
weight sugars allow generation of a significant
humoral antibody response
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Immunoglobulin Structure And Function
Immunoglobulins functions as antibodies that bind
antigen.
Immunoglobulins (Ig) found in serum
1939, Tiselius and Kabat immunized rabbits with
ovallbumin
Divided serum into two fractions
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Immunoglobulin Structure And Function
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Compared proteins by electrophoresis
? Globulin was reduced in the fraction that had
been precipitated with ovalbumin
Conclusion antibodies in ? portion of serum
Named immunoglobulins to distinguish them from
other proteins in the ? globulin fraction
Immunoglubulins consist of two Fab-like fragments
F(ab)2 and one Fc fragment.
F(ab)2 Region for antigen binding
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F(ab)2 region consists of two 50,000 Da molecular
weight polypeptide chains (heavy chains) and two
25,000 Da chains (light chains)
Immunoglobulin Subtypes
Five different Ig subtypes
IgM
5-10 of total serum immunoglobulins
Monomeric IgM is membrane-bound antibody
Secreted IgM is pentamer (I.e. five monomeric
subunits held together by disulphide bonds)
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First Ig produced in response to an antigen
First Ig synthesized by neonates
Can bind multidimensional antigens such as viral
particles and RBC
IgM causes clumping of RBC AGGLUTINATION
More potent at this and at activation of
complement system than other Ig
Large size hinders diffusion into intercellular
fluids
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IgA
10-15 of Ig in serum
Predominant Ig in external secretions such as
breast milk, saliva, tears, mucus of bronchial,
genitourinary and digestive tracts.
In serum IgA Monomer
IgA of external secretion called secretory IgA.
IgA binds to poly Ig receptor on mucosal and
glandular epithelium
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Transported by receptor-mediated endocytosis into
coated pits
Therefore, IgA serves an important effector
function at mucous membranes (main entry sites
for pathogens)
IgG
Most abundant in serum (80)
Monomer
Four IgG subclasses IgG 1-4
IgG1, 3 and 4 Cross the placenta and protect
developing foetus
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IgG activates complement system
IgG Opsonins by binding Fc receptors on
phagocytic cells
IgE
Very low serum concentration
Mediate hypersensitivity reactions responsible
for hay fever, asthma and anaphylactic shock
IgE binds to Fc receptors on membranes of
basophils and mast cells.
Cross linking of IgE molecules by allergens ?
degranulates basophils and mast cells
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IgD
0.2 of total Ig
Unknown function
Membrane-bound Ig
Expressed on mature B cells
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Immunoglobulin Superfamily
One or more proteins shown to be homologous to
immunoglobulins
e.g. ?2 microglobulin, T cell receptor and
adhesion molecules
Proteins whose corresponding genes derived from
a common primordial gene encoding the basic
domain structure
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Cross Reactivity
Antibody elicited by one antigen can cross react
with an unrelated antigen
1 Two different antigens share identical epitope
2 Antibodies specific for one epitope also
binds to an unrelated epitope possessing similar
chemical properties
e.g. Streptococcus Pyogenes express cell wall
proteins called M antigens
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Antibodies produced to M antigen cross-react with
myocardial and skeletal muscle proteins
This might explain heart and kidney damage
following streptococcal infection
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Reading
Immunology. Janis Kuby. Chapters 4 and 5