The document presents an overview of the immune system, detailing the distinction between innate and adaptive immunity and focusing on the roles of B-cells and T-cells in immune response. It further explains the concepts of epitopes, haptens, adjuvants, and superantigens, elaborating on their functions and importance in immunology. Various methods for epitope mapping and the impact of adjuvants on enhancing immune responses are also discussed.

1. THASNEEM MUTHIRA J
21PW28212
I-MSc BIOTECHNOLOGY
IMMUNOLOGY
&IMMUNOTECHNOLOGY

2. INTRODUCTION:
 The immune system is typically divided into two categories, innate
and adaptive.
 Innate immunity involves nonspecific defense mechanisms that act
immediately or within hours after a microbe appearance in the body.
All multicellular beings exhibit some kind of innate immunity.
 In contrast, adaptive immunity is only present in vertebrates and it is
highly specific. In fact, the adaptive immune system is able to
recognize and destroy invading pathogens individually.
 Adaptive immunity is articulated by lymphocytes, more specifically
by B- and T-cells, which are responsible for the humoral and cell-
mediated immunity.
 B- and T-cells do not recognize pathogens as a whole, but molecular
components known as antigens. These antigens are recognized by
specific receptors present in the cell surface of B- and T-cells.

3. EPITOPE:
The small site on an antigen to which a complementary antibody may specifically bind
is called an epitope or antigenic determinant. This is usually one to six monosaccharides
or five to eight amino acid residues on the surface of the antigen.
TWO TYPE OF EPITOPE
Antigen molecules exist in space, the epitope recognized by an antibody may be
dependent upon the presence of a specific three dimensional antigenic conformation
(e.g., a unique site formed by the interaction of two native protein loops or subunits).
This is known as a conformational epitope. The epitope may also correspond to a
simple linear sequence of amino acids and such epitopes are known as linear epitopes.
CHARACTERISTICS OF EPITOPE:
The range of possible binding sites on a target molecule (antigen) is enormous, with
each potential binding site having its own structural properties derived from covalent
bonds, ionic bonds, hydrophilic, and hydrophobic interactions. Indeed, this has
important ramifications for antibody choice and performance.

4.  For efficient interaction to occur between the target antigen and the antibody, the epitope must
be readily available for binding.
If the target molecule is denatured, e.g., through fixation, reduction, pH changes, or during
preparation for gel electrophoresis, the epitope may be altered and this may affect its ability to
interact with an antibody.
 For example, some antibodies are ineffective in Western blotting (WB) but are suitable for
immunohistochemistry (IHC) applications, because, in the IHC procedure, a complex
antigenic site might be maintained in the tissue, whereas in the WB procedure, the process of
sample preparation alters the protein conformation sufficiently to destroy the antigenic site,
and hence eliminates antibody binding.
 In a denatured protein, only the linear epitope may be recognized. Hence, in protocols where a
denatured protein is used, such as in Western blotting, an antibody that recognizes a linear
epitope is preferred. Sometimes an epitope is on the interior of a folded protein.
 The epitope is then inaccessible to the antibody in a non-denaturing protocol, such as
immunoprecipitation. A conformational epitope, by definition, is on the outside of the folded
protein. An antibody that recognizes the conformational epitope is suitable for mild, non-
denaturing procedures, such as immunoprecipitation or flow cytometry.

5. B CELL EPITOPE AND ITS MAPPING:
B-cells recognize solvent-exposed antigens through antigen receptors, named as B-cell
receptors (BCR), consisting of membrane-bound immunoglobulins, as shown in Figure
Upon activation, B-cells differentiate and secrete soluble forms of the
immunoglobulins, also known as antibodies, which mediate humoral adaptive
immunity. Antibodies released by B-cells can have different functions that are
triggered upon binding their cognate antigens. These functions include neutralizing
toxins and pathogens and labeling them for destruction .

6.  A B-cell epitope is the antigen portion binding to the immunoglobulin or antibody.
These epitopes recognized by B-cells may constitute any exposed solvent region in
the antigen and can be of different chemical nature. However, most antigens are
proteins and those are the subjects for epitope prediction methods.
 B cell epitopes are mainly conformational. There are additional epitope types when
the quaternary structure is considered.
 Epitopes that are masked when protein subunits aggregate are called
cryptotopes.Neotopes are epitopes that are only recognized while in a specific
quaternary structure and the residues of the epitope can span multiple protein
subunit.
MAPPING:
There are two main methods of epitope mapping: either structural or functional studies.
Methods for structurally mapping epitopes include X-ray crystallography, nuclear
magnetic resonance, and electron microscopy. X-ray crystallography of Ag-Ab
complexes is considered an accurate way to structurally map epitopes. Nuclear
magnetic resonance can be used to map epitopes by using data about the Ag-Ab
complex. This method does not require crystal formation but can only work on small
peptides and proteins..

7. Electron microscopy is a low-resolution method that can localize epitopes on larger antigens like
virus particles
Methods for functionally mapping epitopes often use binding assays such as western blot, dot
blot, and/or ELISA to determine antibody binding. Competition methods look to determine if two
monoclonal antibodies (mABs) can bind to an antigen at the same time or compete with each
other to bind at the same site.
T-CELL EPITOPE AND ITS MAPPING :
T-cells present on their surface a specific receptor known as T-cell receptor (TCR) that
enables the recognition of antigens when they are displayed on the surface of antigen-
presenting cells (APCs) bound to major histocompatibility complex (MHC) molecules.
T cell epitopes presented by MHC class I molecules are typically peptides between 8
and 11 amino acids in length, whereas MHC class II molecules present longer peptides,
13–17 amino acids in length,and non-classical MHC molecules also present non-
peptidic epitopes such as glycolipids.
T-cell epitopes are presented by class I (MHC I) and II (MHC II) MHC molecules that
are recognized by two distinct subsets of T-cells, CD8 and CD4 T-cells, respectively .

8. Subsequently, there are CD8 and CD4 T-cell epitopes. CD8 T-cells become cytotoxic T
lymphocytes (CTL) following T CD8 epitope recognition.
Meanwhile, primed CD4 T-cells become helper (Th) or regulatory (Treg) T-cells .
 Th cells amplify the immune response, and there are three main subclasses: Th1
(cell-mediated immunity against intracellular pathogens), Th2 (antibody-mediated
immunity), and Th17 (inflammatory response and defense against extracellular
bacteria)
MAPPING:
MHC class I and II epitopes can be reliably predicted by computational means alone,
although not all in-silico T cell epitope prediction algorithms are equivalent in their
accuracy.There are two main methods of predicting peptide-MHC binding: data-driven
and structure-based.Structure based methods model the peptide-MHC structure and
require great computational power.Data-driven methods have higher predictive
performance than structure-based methods.Data-driven methods predict peptide-MHC
binding based on peptide sequences that bind MHC molecules.By identifying T-cell
epitopes, scientists can track, phenotype, and stimulate T-cells.

10. HAPTEN:
Hapten is a molecule that reacts with specific antibody but is not immunogenic by
itself, it can be made immunogenic by conjugation to a suitable carrier. Many drugs
like penicillins are haptens. A hapten is essentially an incomplete antigen. These small
molecules can elicit an immune response only when attached to a large carrier such as
a protein; the carrier typically does not elicit an immune response by itself.
Hapten-carrier protein conjugate induces the production of three types of antibodies i)
Antibody against the unaltered epitope of carrier protein, ii) antibody against a new
epitope formed by the combination of hapten and carrier protein, and iii) antibody
against the epitope of hapten.

11.  The term hapten is derived from the Greek haptein, meaning “to fasten.”
 Haptens can become tightly fastened to a carrier molecule, most often a protein, by a
covalent bond.
 The hapten-carrier complex stimulates the production of antibodies, which the
unbound hapten cannot do, and becomes immunogenic (capable of eliciting an
immune response).
 The hapten then reacts specifically with the antibodies generated against it to
produce an immune or allergic response. Thus, although the hapten cannot elicit an
antibody response on its own, it can bind with antibodies and act as an antigen.
 In the early part of the 20th century, immunologist Karl Landsteiner exploited the
antigenic quality of synthetic haptens to study the highly specific way in which
antibodies bind to antigens.
 For example,
Many drugs that cause allergic reactions, such as penicillin, act as haptens.
When injected or ingested, penicillin reacts chemically with proteins in the body to
form a hapten-carrier complex that can lead to the life-threatening syndrome called
anaphylaxis. Other haptens include synthetic substances, such as the organic
compounds benzene arsonate or trinitrophenol, and naturally occurring polysaccharides,
such as lactose.

12. ADJUVANTS:
 An adjuvant is a pharmacological or immunological agent that modifies the effect
of other agents.
 Directly immunizing most antigens will lead to a poor immune response and rapid
removal of the antigen from the body. To prevent this, the antigen is first combined
with an adjuvant, which is a material that helps stimulate and enhance the immune
response against the antigen through the creation of a depot effect.
 Adjuvants can act in various ways in presenting an antigen to the immune system.
They can act as a depot for the antigen, presenting the antigen over a longer period
of time, thus maximizing the immune response before the body clears the antigen.
ADJUVANT IMMUNOBIOLOGICAL FUNCTION:
 Improve the immunogenicity of highly purified or recombinant antigens
(protein or peptide).
 Increase the innate immune response to antigen by interacting with pattern
recognition receptors (PRRs) on or within accessory cells.
 Provide physical protection to antigens which grants the antigen a prolonged
delivery.

13.  Increase the capacity to cause local reactions at the injection site (during
vaccination), inducing greater release of danger signals by chemokine releasing cells
such as helper T cells and mast cells.
 Help in the translocation of antigens to the lymph nodes where they can be
recognized by T cells.
How do adjuvants work?
 When adjuvants are added into a vaccine they work in four distinct ways to boost
the immune response. The first of these pathways is the activation of antigen-
presenting cells to signal to the immune system’s T cells that foreign substances
have infiltrated.
 To do this adjuvants boost the activation of antigen-presenting cells, cells of the
immune system that encompass foreign substances and break them up, presenting
the resulting particles to the immune system’s T cells. This activates the T cells,
which has the impact of activating the antibody-producing B cells.
 The second way that adjuvants work is by activating T cells indirectly by
discharging phagosomes that attach themselves to the T cells. Following this
binding, the T cells are induced to release cytokines that switch on the antibody-
producing B cells.

14. ADJUVANTS APPROVED FOR HUMAN USE
 The next process involves the targeting of antigens at specific locations. The
location where an adjuvant is injected can induce immune system activity
localized to that specific area. This activation incites T cells to travel through the
bloodstream to that specific location.
 Finally, adjuvants can induce the slow release of an antigen. The depot effect
refers to the process by which adjuvants can regulate the rate of antigen release
into the bloodstream. To achieve this, the adjuvant is enclosed within a polymer
along with an antigen. This has the impact of reducing the rate at which both the
chemicals and antigens are released into the tissue and bloodstream.

15. SUPERANTIGEN:
 Superantigens (SAgs) are a class of antigens that result in excessive activation
of the immune system. Specifically it causes non-specific activation of T-cells
resulting in massive cytokine release.
 The large number of activated T-cells secrete large amounts of cytokines, the
most important of which is Interferon gamma. This excess amount of IFN-
gamma in turn activates the macrophages. The activated macrophages, in
turn, over-produce proinflammatory cytokines such as IL-1, IL-6 and TNF-
alpha. TNF-alpha is particularly important as a part of the body's inflammatory
response. In normal circumstances it is released locally in low levels and helps
the immune system defeat pathogens. However, when it is systemically
released in the blood and in high levels (due to mass T-cell activation resulting
from the SAg binding), it can cause severe and life-threatening symptoms,
including shock and multiple organ failure.
 For example,
Toxic shock syndrome toxin-1 (TSST-1), produced by some strains of
Staphylococcus aureus. This exotoxin causes toxic shock syndrome (TSS).
Excessive cytokine production leads to fever, rash, and shock.

16. STRUCTURE OF SUPER ANTIGEN:
Crystal structures of the enterotoxins reveals that they are compact, ellipsoidal proteins sharing a
characteristic two-domain folding pattern comprising an NH2-terminal β barrel globular domain
known as the oligosaccharide / oligonucleotide fold, a long α-helix that diagonally spans the
center of the molecule, and a COOH terminal globular domain.
The domains have binding regions for the major histocompatibility complex class II (MHC
class II) and the T-cell receptor (TCR), respectively.
BINDING NATURE AND ITS EFFECT:
Superantigens, however, bind directly to the outside of MHC-II molecules and activate
large numbers of T4-lymphocytes (Figure 16.6.3 ). This activation of very large
numbers of T4-lymphocytes results in the secretion of excessive amounts of a cytokine
called interleukin-2 (IL-2) as well as the activation of self-reactive T-lymphocytes. The
normal response to a conventional antigen results in the activation of maybe 1 in 10,000
T-lymphocytes; superantigens can activate as many as 1 in 5 T-lymphocytes.

17.  Production of high levels of IL-2 can result in circulation of IL-2 in the blood
leading to symptoms such as fever, nausea, vomiting, diarrhea, and malaise.
However, excess stimulation of IL-2 secretion can also lead to production of
inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha),
interleukin-1 (IL-1), inflammatory chemokines such as IL-8, and platelet-
activating factor (PAF), and can lead to the same endothelial damage, acute
respiratory distress syndrome, disseminated intravascular coagulation, shock, and
multiple organ system failure seen above with LPS and other bacterial cell wall
factors. Activation of self-reactive T-lymphocytes can also lead to autoimmune
attack.
Binding of Superantigens. Conventional
antigens are only recognized by specific T4-
lymphocytes having a TCR with a shape that
corresponds to a peptide of that antigen bound
to MHC-II molecules. Superantigens, on the
other hand, bind directly to the outside of
MHC-II molecules and the TCRs and activate
many T4-lymphocytes. A specific TCR is not
required for activation.

18. REFERENCE:
1. Janis Kuby,Immunology,W.H.Freeman& company, New York. 1997 9TH
EDITION.
2. P. M. Latha. A Text Book of Immunology, 1st Edition, S.Chand& Company
Ltd, New Delhi,2003
3. Jose L. Sanchez-Trincado ,1Marta Gomez-Perosanz and Pedro A. Reche
,Fundamentals and Methods for T- and B-Cell Epitope Prediction ,Journal
of Immunology Research, vol. 2017, Article ID 2680160, 14 pages, 2017.
https://doi.org/10.1155/2017/2680160