Antigens, Epitopes, and Immunogenicity

Chapter Three
Unit 2-Lecture 2
Antigens, Epitopes, and Immunogenicity

• Immunogens/Antigens
▫ Macromolecules capable of triggering an adaptive
immune response by inducing the formation of
antibodies or sensitized T cells in an
immunocompetent host.
 Immunogens can then specifically react with such
antibodies or sensitized T cells.
 This response is actually caused by a combination of
factors.
Nature of Antigens
Immunogenicity – ability to elicit an immune
response
• Characteristics of good immunogens:
▫ Foreignness
▫ Molecular size
▫ Chemical complexity
▫ Susceptibility to recognition, uptake, and degradation
by antigen presenting cells
▫ Indirectly, the method of introduction of the antigen
▫ The presence of certain chemicals that can act as
immune adjuvants.
Foreignness
• The immunogenicity of a molecule depends on
its degree of foreignness
▫ Foreignness is the degree to which antigenic
determinants are recognized as nonself by an
individuals immune system
 The more distant taxonomically the immunogen is
from the host the better it is as a stimulus
Foreignness
• Autoantibodies
▫ Self antigens of the host
 Not immunogenic normally
• Alloantibodies
▫ Antigens from same species as host
 May be immunogenic
• Heteroantigens
▫ Antigens from different species from host
 May be immunogenic or may cross-react
Molecular Weight
• The higher the molecular weight the better the
molecule will function as an antigen
▫ Nonimmunogenic—less than 1000 daltons
▫ Sometimes immunogenic—1000–6000 daltons
▫ Immunogenic—more than 6000 daltons
• Some exceptions immunogenicity and size rule
▫ Small compounds such as glucagon (3400
daltons) can be immunogenic,
▫ Large homopolymers not immunogenic
Size and Complexity
• Haptens
▫ Small compounds
 Too small to stimulate an immune response
 Unless linked to a much larger immunogenic molecule
• Carrier molecule—larger molecule that a hapten
is linked to
Chemical Composition and Molecular Complexity
• The more complex an antigen the greater its
effectiveness
▫ Proteins and polysaccharides are the best immunogens.
▫ Carbohydrates are somewhat less immunogenic than
protein
 The units of sugars are more limited than the number of
amino acids in proteins.
 As immunogens, carbohydrates most often occur in the form
of glycolipids or glycoproteins.
▫ Pure nucleic acids and lipids are not immunogenic by
themselves
Table 3.1
Immunogenicity of Different Types of Antigens
Ability to be Processed and Presented with MHC
Molecules
• For a substance to elicit an immune response, it
must be subject to antigen processing
• Involves enzymatic digestion to create small peptides
that can be complexed to MHC molecules to present to
responsive lymphocytes.
• The particular MHC molecules produced also
determine responsiveness to individual antigens.
▫ Each individual inherits the ability to produce a
certain limited repertoire of MHC molecules.
Nature of Antigens
• Immunization protocols
▫ Influence the immunogenicity of an immunogen
 Particulate antigens elicit responses after
inoculation by intracutaneous or intravenous routes
at a broad range of doses
▫ Soluble antigens may require repeated
immunizations
Nature of Antigens
• Dose and route of administration of the
immunogen is also important
▫ High dose given at one time – can lead to
immune paralysis (anergy)
▫ Low dose given at one time – may be destroyed
too quickly
▫ Optimum – small to medium sized, repeated
doses of antigen
Nature of Antigens
• Very large doses
▫ May result in T- and B-cell tolerance
 Phenomenon that is not well understood.

• The actual amount of immunogen needed to

generate an immune response differs with the
route of inoculation.
▫ These routes include IV, intradermal,
subcutaneous, and oral administration.
Nature of Antigens
• Adjuvant - a substance administered with an
immunogen that increases the immune
response.
 Aluminum salts are the only adjuvants approved for
clinical use in the United States.
Addition of Immune-Enhancing Agents
• Adjuvant – chemicals that enhance response
▫ Increases response to an immunogen
 Stimulates localized inflammatory reaction at site of
injection
▫ Recruits phagocytic and inflammatory cells
 Better uptake of the antigen
▫ Increase the size of the immunogen
 Results in slower antigen release
 More immune cells arrive at the site to participate in
the response.
Antigenic Determinants (Epitopes)
• Immune system recognizes antigens by
immunogenic components
▫ Antigenic determinants-Epitopes
 Restricted portions of a molecule that are involved in
the actual binding with the combining site of a
particular antibody.
 Number of epitopes vary by antigen
 Based on size and chemical complexity.
Nature of Antigens

• Antigenic determinants/epitopes
▫ Only a small part of the immunogen is actually
recognized in the immune response
• Epitopes are molecular shapes or configurations
that are recognized by B or T lymphocytes.
▫ Large molecules may have numerous epitopes
Antigenic Determinants (Epitopes)
• Epitopes
▫ Linear epitopes
 Related to amino acid sequence of an antigen
 Not related to three-dimensional structure
▫ Conformational epitopes
 Depends on antigen's three-dimensional structure
 Depend on antigens secondary and tertiary
conformation
Epitopes
Epitopes
Antigenic Determinants (Epitopes)
• Epitopes
▫ Recognized by and binds to a particular
immunoglobulin or T-cell receptor
▫ An antibody-producing cell clone synthesizes
immunoglobulins that recognize a particular
antigenic determinant
 Epitopes
• Epitopes recognized by surface immunoglobulins on B
cells:
▫ 6–15 amino acids
▫ 2–7 sugars
• Class I MHC molecules can bind peptides of 8–10
amino acids
• Class II can accommodate peptides of 13–18 amino
acids
Nature of Antigens
• B-cell activated by
▫ Anything that is capable of crosslinking surface
immunoglobulin molecules
• If the immunogen is a protein, B cells may
recognize the primary, secondary, tertiary, or
even the quaternary structure.
▫ In polysaccharides, the branch points of branched
chains may contribute most to their recognition.
Nature of Antigens
• T cell Activation
▫ Recognize an epitope only as a part of a complex
formed with MHC proteins on the surface of an
antigen-presenting cell.
 The antigen-presenting cell must process an
immunogen first and degrade it into small peptides
for it to be recognized by T cells.
Cross-Reactivity
• Epitopes may be shared by different antigens
▫ Reaction of an antibody with an antigen other
than the one that induced its formation
• Occurs because
▫ The exact same epitope is on the molecule
 Or a very similar to the epitope
Cross-Reactivity
• Use caution to not misdiagnose patient
▫ FSH, HCG, LH
 Can all cross-react
 Testing must contain specific antibodies to analyte
• Can be helpful for vaccines
▫ Tetanus toxin-harmful
 Tetanus toxoid-non-toxic
 Different biologically and chemically but cross-react
immunologically
▫ Allows for development of an antitoxin vaccine that is non-
toxic
The Exquisite Specificity of the
Immune System
• Haptens
▫ Not immunogenic- can’t elicit a response by
themselves
▫ May become antigenic if conjugated to an
immunogenic carrier
 Acts as a novel antigenic determinant of the carrier
▫ Can be natural (penicillin, poison ivy) or artificial
(vaccines)
The Exquisite Specificity of the
Immune System
• Haptens
▫ Used to study the specificity of antigen–antibody
reactions
 Karl Landsteiner's experiment
 Produced an understanding that the diversity of an
immune response can be immense
The Examples of Haptens Specificity
• Haptens continued
▫ Poison ivy (Rhus radicans) contains chemical
substances called catechols, which are haptens.
 Once in contact with the skin, these can couple with
tissue proteins to form the immunogens that give rise
to contact dermatitis.
▫ Another example of haptens coupling with normal
proteins in the body to provoke an immune response
occurs with certain drug-protein conjugates that can
result in a life-threatening allergic response
 such as that which can occur with penicillin.
• See Figure on next slide
Nature of Antigens
• A genetic predisposition involved that allows
individuals to respond to particular
immunogens.
▫ Major Histocompatibility Complex (MHC)
 Human Leukocyte Antigens (HLA)
 The MHC Complex
• MHC (Major Histocompatibility Complex)
molecules
▫ Found on all nucleated cells in the body
▫ Play a pivotal role in the development of both humoral
and cellular immunity.
• Main function is to bring antigen to the cell surface for
recognition by T cells
▫ T-cell activation will occur only when antigen is
combined with correct MHC molecules.
Major Histocompatibility Complex
(MHC)
• MHC repertoire
▫ Limited by inherited haplotypes
▫ Individuals may respond differently to a particular
antigen
• The polymorphism of the MHC system is
essential to our survival
▫ MHC molecules play a pivotal role in triggering
the immune response to diverse immunogens.
Major Histocompatibility Complex
(MHC)
• MHC repertoire
▫ MHC haplotypes are highly polymorphic,
▫ These alleles are inherited as sets
 One set of alleles from each parent
 Co-dominantly expressed
▫ The products of both sets of genes are expressed on cell
surfaces
 Four different sets that can be inherited
 Reason for different responses to a particular antigen
The MHC Complex
• MHC molecules may be involved in transfusion
reactions, graft rejection, and autoimmune
diseases.
▫ Genes controlling expression of these molecules are
actually a system/cluster of genes known as the
Major Histocompatibility Complex (MHC).
 Genes coding for the MHC molecules in humans
are found on the short arm of chromosome 6 and are
divided into three categories, or classes.
 Genes code for proteins that have a role in immune
recognition.
The MHC Complex
• Class I molecules
▫ Coded for at three different locations, or loci, termed A,
B, and C.
• Class II genes
▫ Encoded in the D region, and there are several different
loci, known as DR, DQ, and DP.
• Class III genes
▫ Situated between the class I and class II regions on
chromosome 6
 Codes for complement proteins and cytokines
 TNF, C2, C4a, C4b and etc.
The MHC Complex
• Class I and II gene products are involved in antigen recognition
and influence the repertoire of antigens to which T cells can
respond.
• Class III proteins are secreted proteins that have an immune
function, but they are not expressed on cell surfaces.
The MHC Complex
• Codominant - all alleles that an individual
inherits, code for products that are expressed on
cells.
▫ MHC genes are described as being codominant
• Haplotype - a set of genes that are located
close together on a chromosome and are
usually inherited as a single unit.
▫ MHC genes are closely linked and are inherited
together as a package.
The MHC Complex

• Each haplotype consists of a package of genes for

A, B, C, DR, DP, and DQ.

• The full genotype would consist of two of each

genes at a particular locus.

• The polymorphism of the MHC system has

implications for organ donation and
paternity testing.
The MHC Complex
• Class I MHC molecules
▫ Glycoprotein dimer,
 Made up of two noncovalently linked polypeptide
chains
• The α chain and a lighter chain associated with it
• Called β2-microglobulin
▫ Expressed on all nucleated cells, although they
differ in the level of expression.
 Highest on lymphocytes
 Low or undetected on hepatocytes, neural cells,
muscle cells, and sperm.
The MHC Complex
• Class I MHC molecules
• >20 genes in the Class I region
▫ Many are pseodogenes – nonfunctional
• Class I has 6 subregions
• Classical Class I molecules:
▫ HLA-A, HLA-B, and HLA-C
 HLA-C antigens expressed at lower levels than HLA-A and HLA-B
antigens.
▫ Directly involved in immune recognition
▫ Thousands of Class I proteins on the cell surface
▫ Tumors and certain viruses (HIV) can suppress Class I gene
transcription
▫ Other molecules can increase affinity of cellular interactions
The MHC Complex
• Class I MHC molecules
▫ Class I proteins on the cell surface bind with T cell
receptors on CD8 (suppressor/cytogenic)
lymphocytes

▫ Serological tests for MHC antigens use

alloantisera and monoclonal antibodies to define
Class I alleles
Major Histocompatibility Complex
(MHC)
The MHC Complex
• Non classical Class I molecules
▫ Another group of class I molecules, are designated
E, F, and G.
▫ Except for G, these are not expressed on cell
surfaces and do not function in antigen
recognition but may play other roles in the
immune response.
 G antigens are expressed on trophoblast cells during
the first trimester of pregnancy.
The MHC Complex
• Class II MHC molecules
▫ Found primarily on antigen-presenting cells
 B lymphocytes
 Activated T-lymphocytes
 Monocytes
 Macrophages
 Dendritic cells.
• The major class II molecules—DP, DQ, and
DR
The MHC Complex
3 main subregions of Class II MHC:
• DR subregion
▫ 1 DRA gene and 9 DRB genes
▫ DRB1 is predominant Class II protein (50%)
 Is highly polymorphic
• DQ subregion – 15-20% of total Class II
▫ 2 DQA and 2 DQB genes
• DP subregion
▫ 2 DPA and 2 DPB genes
The MHC Complex
• Class II region of human MHC spans 1100
kilobases of DNA
▫ Many genes are pseudogenes
▫ Class II molecule is heterodimer
 with α and β chain – both coded for within Class II region
• Class II complex on cell surface presents antigen
to a circulating CD4 (helper) lymphocyte
▫ Antigen recognition by T cell receptor stimulates CD4
lymphs and initiates IR
 T-helper/regulatory
Major Histocompatibility Complex
(MHC)
The MHC Complex
• Nonclassical Class II genes
▫ DO and DM are expressed inside cell
 Assist binding of new Class II molecules to antigen
▫ DM – regulates and stabilizes peptide binding to
DR, DQ, and DP
▫ DO – negatively regulates DM
The MHC Complex
• Class III MHC
▫ Encodes complement components (C2, C4, Bf)
involved in cleaving C3
• Other genes in region not involved in the
immune response:
▫ 21-hydroxylase (Cyp21)
▫ Heat shock protein 70 (Hsp 70)
▫ Tumor necrosis factor (TNF)
The MHC Complex
• Nomenclature
• Historically, numerical designation of Class I
and Class II alleles determined by international
workshops sponsored by World Health
Organization (WHO)
• 1968 – designation of 2 series:
▫ HL-A and HL-B
The MHC Complex
• Nomenclature, Cont.
• 1975 – new nomenclature to accommodate
increasing numbers of new loci
▫ HL replaced by HLA, and a third Class I locus, HLA-C,
was defined
▫ HLA-D described – present only on B cells
• 1984 – HLA-D modified to include DR, DQ, and
DP
Pictorial description of HLA-DR nomenclature. From:
Hurley and Ng, 1994. Used with permission.
The MHC Complex and The Immune
Response (IR)
• The main role of the class I and class II MHC
molecules is to bind peptides within cells and
transport them to the plasma membrane, where
T cells can recognize them in the phenomenon
known as antigen presentation.
The MHC Complex and The Immune
Response (IR)
• First step in IR – binding of peptide fragment
in polymorphic groove of Class I or Class II
molecule
• Class I and II can also bind to self-antigens
▫ Usually not recognized by TCR (tolerance)
 If released into circulation, may lead to
autoimmunity
The MHC Complex and The Immune
Response (IR)
• Most immune responses in transplant patients
generated from presentation of alloepitopes
from transplanted tissue to circulating T
lymphocytes
• 2 types of alloepitopes from transplant:
▫ Private – unique to single gene product
▫ Public – on more than 1 gene product
The MHC Complex and The Immune Response
(IR)
• T cells can only “see” (by way of T-cell Receptors,
(TCR)) and respond to antigens when they are
combined with MHC molecules.

• Class I molecules mainly present peptides that

have been synthesized within the cell to CD8
(cytotoxic) T cells.
▫ Class I molecules are thus the watchdogs of
viral, tumor, and certain parasitic antigens that are
synthesized within the cell.
The MHC Complex and The Immune
Response (IR)
• Class II molecules present antigen to CD4
(helper) T cells.
▫ Class II molecules stimulate CD4 T cells in the
case of bacterial infections or the presence of
other material that is endocytosed by the cell.
The MHC Complex and The Immune
Response (IR)
• In either case, for a T-cell response to be
triggered,
1. Peptides must be available in adequate supply for
MHC molecules to bind,
2. They must be able to be bound effectively
3. They must be recognized by a T-cell receptor.

• The difference in functioning of the two molecules

is tied to the mechanisms by which processed
antigen is transported to the surface.
Antigen Presentation
• Antigens taken up by antigen presenting cells
tend to be strong immunogens
Antigen Presentation
• Activate CD4+ and CD8+ T cells
▫ Accessory or antigen-presenting cell binds a
particular antigen
▫ Internalizes antigen
▫ Processes antigen biochemically
▫ Presents antigen to T cell in Class I or Class II
molecule
Antigen Presentation
• Both class I and class II molecules are
synthesized in the rough endoplasmic
reticulum.
▫ Class I and class II are transported to Golgi
compartment after synthesis
▫ Interaction of peptides with MHC class I and class
II occurs at different sites in the cell
 Class I and class II segregate
 Class I interact with antigen peptides
 Class II are complexed to the invariant chain (Ii)
Table 3.2
MHC Class I and Class II Molecules
Different Roles for MHC Classes I and
II Molecules in an Immune Response
• Class I molecules
▫ Bind antigenic peptides in the endoplasmic
reticulum of the APC
 Processed fragments of proteins from something
infecting the cells
 After processing of antigen and association with class I
components, the MHC-peptide complex is brought to
the surface and recognized by CD8+Tcells.
 CD8 cell is activated
▫ Results in killing the infected antigen presenting cell.
Different Roles for MHC Classes I and
II Molecules in an Immune Response
• Class II molecules
▫ Travel from endoplasmic reticulum to an endosomal
or lysosomal compartment
▫ Interact with antigenic peptides
 Typically phagocytosed antigen brought by APC
proteins and transporters associated with antigen
processing (TAP-1 and TAP-2)
▫ Invariant chain is removed
▫ Antigen presentation follows
 MHC class II-peptide complex transported to surface
Different Roles for MHC Classes I and
II Molecules in an Immune Response
• CD4+ T cell recognizes complex and destruction
takes place.
• The quantity of MHC Class II on monocytes and
macrophages varies
▫ Interferon-g released by activated T cells causes
an increase of MHC Class II molecules
▫ May be related to autoimmunity
Antigen Presentation
The MHC Complex and The Immune
Response (IR)
• TAP1 and TAP2 proteins
▫ Responsible for the ATP-dependent transport of
peptides suitable for binding to class I molecules
from the cytoplasm to the lumen of the
endoplasmic reticulum.
• Once the α chain has bound the peptide, the
MHC I-peptide complex is rapidly
transported to the cell surface. (See Fig on next
slide.)
The MHC Complex
The MHC Complex and The Immune
Response (IR)
• Different class I molecules will have slightly
different binding affinities; these small
differences determine to which particular
antigens one individual will respond.
▫ Display of hundreds of class I molecules
complexed to antigen allows CD8 positive T cells
to continuously check cell surfaces for the
presence of nonself antigens.
The MHC Complex and The Immune
Response (IR)
• Class II molecules must be transported from
the endoplasmic reticulum (ER) to an
endosomal compartment before they can bind
peptides.
▫ Here it encounters peptides derived from
endocytosed, exogenous proteins.
The MHC Complex and The Immune
Response (IR)
• Antigen processing may help to unfold
molecules and uncover functional sites that are
buried deep within the native protein structure.

• The invariant chain is degraded by a protease,

leaving just a small fragment called class II
invariant chain peptide (CLIP) attached to
the peptide-binding cleft.
The MHC Complex and The Immune
Response (IR)
• Once binding has occurred, the class II
protein-peptide complex is stabilized and is
transported to the cell surface. (See Fig)
▫ On the cell surface, class II molecules are
responsible for forming a trimolecular
complex
 Occurs between antigen, the class II molecule, and
an appropriate T-cell receptor.
The MHC Complex and The Immune
Response (IR)
• If binding occurs
with a T-cell receptor
on a CD4-positive
T cell, the T helper
cell recruits and
triggers a B-cell
response, resulting in
antibody formation.