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Introduction and short history of immunology

Immunology is the study of the ways in which the body defends itself from infectious agents
and other foreign substances in its environment.
Immunology began as a branch of microbiology. The latin term ‘immunis’ meaning ‘exempt’ is
the source of the English word ‘immunity’ meaning the state of protection from infectious
The earliest written reference to the phenomenon of immunity lies in the writing of Thucydides
in 430 BC. He wrote that only those who had recovered from plague could nurse the sick
because they would not contract the disease a second time.
The first recorded attempts to induce immunity deliberately were performed by the Chinese
and Turks in the 15th century. The dried crusts derived from small pox pustules were either
inhaled in to the nostrils or inserted in to small cuts in the skin (this technique is known as
variolation). Those who survived the resulting disease were protected from small pox in later
In 1546, Girolamo Fracastoro put forward the concepts of contagion and Germ Theory of
In 1754, it was suggested that inoculation may be tried for rinderpest, a viral disease of cattle.
The process involved soaking a piece of string in the nasal discharge from an animal with
rinderpest and then inserting the string in to an incision in the dewlap of the animal to be
protected. The resulting disease was usually milder than natural infection and the inoculated
animals became resistant to rinderpest.
Ovination involved use of material derived from sheep pox to protect sheep from the disease.
However, similar inoculation procedures to protect animals against canine distemper, hog
cholera, pleuropneumonia etc. ended in failure.
In 1774, Benjamin Jesty, a farmer, inoculated his wife and children with vaccinia virus to protect
her from small pox. However, it was Edward Jenner, an English physician who is considered as
the Father of Immunology, in 1798, received acknowledgement and awards for demonstrating
that material from cowpox lesions could be substituted in variolation to protect against
smallpox. The use of material from cowpox lesions by Jenner was based on his observation that
milkmaids who had contracted cowpox were subsequently immune to small pox.
Between 1879 and 1881, Louis Pasteur developed three attenuated vaccines (after cowpox).
Pasteur had succeeded in growing the bacterium thought to cause fowl cholera (Pasteurella
multocida) in chicken bouillon. He found that under acidic conditions P. multocida becomes
attenuated i.e., reduced in virulence. He used this knowledge to create a partially successful
vaccine (from the Latin word ‘vacca’ meaning ‘cow’ in honour of Jenner’s work with cowpox
inoculation) against fowl cholera..
In 1880, Henry Toussaint, a French Veterinarian, successfully used 3 methods of attenuation to
create the first live anthrax vaccine. Henry Toussaint could not pursue his claim as the originator
of the first anthrax vaccine because of ill health.
In 1881, Louis Pasteur vaccinated sheep with heat attenuated Bacillus anthracis and challenged
them subsequently with the virulent anthrax bacilli. All the vaccinated sheep survived (Pasteur’s
famous experiment at Pouilly-le-Fort).
Pasteur later developed a successful rabies vaccine by drying spinal cords taken from rabies
infected rabbits and then using the dried cords as his vaccine material. These experiments
marked the beginning of the discipline of immunology. [6 July, 1885: first human trial of
Pasteur’s rabies vaccine].
In 1883, Elie Metchnikoff described the phagocytosis of fungal spores by leucocytes and
advanced the idea that immunity was due primarily to WBCs (cell mediated).
1888- Rous and Yersin produced the first defined antigen, the diphtheria toxin.
1888- Nuttall reported the first antibodies (Serum bactericidins/ bactericidal antibodies).
1890- Emil von Behring and Shibasaburo Kitasato produced antitoxins against diphtheria
1894- Jules Bordet discovered complement.
1900- Karl Landsteiner discovered blood group antigens and their corresponding agglutinins.
1902- Richet and Portier coined the term anaphylaxis.

1906- von Pirquet introduced the term allergy.
1917- Karl Landsteiner defined haptens.
1930- Breinl and Haurowitz- template theory of antibody formation.
1936- Grover- MHC antigens.
1939- Tiselius and Kabat showed that antibodies were γ globulins.
1949- Burnet and Fenner proposed the adaptive enzyme theory.
1957- Isaacs and Lindemann discovered interferons.
1957- Burnet: Clonal selection theory
1959- Porter and Edelman- structure of antibodies.
1966- Claman found out cooperation between T and B cells.
1974- Doherty and Zinkernagel- T cell restriction.
1975- Milstein and Kohler produced monoclonal antibodies.
1978- Susumu Tonegawa- immunoglobulin gene rearrangement.
1983- James Allison, Kathryn Haskins et al. isolated the T cell receptor.
1984- Davis et al. and TakMaket al. found out the T cell receptor genes.
1993- NIH- treatment of SCID using genetically altered umbilical cord cells.
Organs and tissues of the immune system
Cells of the immune system move through lymphoid organs dispersed throughout the body.
Primary lymphoid organs regulate the development of immune cells from immature
T lymphocytes originate in bone marrow, but develop in the thymus.
B cells, monocytes, dendritic cells, and granulocytes develop in the bone marrow.
Secondary lymphoid organs facilitate the meeting of antigen with lymphocytes and the
development of antigen specific lymphocytes into effector and memory cells.
Blood vessels and lymphatic systems connect lymphoid organs. Cells of the immune system
travel through blood and lymph between lymphoid organs.

The primary lymphoid organs

Bone marrow

The bone marrow is where self-renewal and differentiation of hematopoietic stem cells (HSCs)
into mature blood cells happen. This process is called haematopoiesis.
The long bones (femur, humerus), ileum, and sternum tend to be the most active sites of
The bone marrow maintains the pool of HSCs throughout the life of a vertebrate.
The adult bone marrow contains several cell types:
Within the bone marrow, the endosteal niche (the area directly surrounding the bone and in
contact with bone-producing osteoblasts) contains quiescent HSCs in close association with
The vascular niche (the area directly surrounding the blood vessels and in contact with
endothelial cells) is occupied by HSCs that have been mobilized to leave the endosteal niche.
The more differentiated a cell is, the farther it is from osteoblasts and the closer it is to the
central regions of the bone.
The bone marrow is also a site to which fully mature myeloid and lymphoid cells can return.
Plasma cells may even take up long-term residence in the bone marrow.
Bursa of Fabricius

It is found only in birds. It is a round sac located just above the cloaca. The bursa of Fabricius
reaches its greatest size in the chick 1-2 weeks after hatching and then shrinks as the bird ages.
Bursa of Fabricius consists of lymphoid follicles among folds of epithelial tissue. Each follicle
consists of a cortex and a medulla.
Cortex contains lymphocytes, plasma cells and macrophages.
Medulla contains medullary epithelial cells, lymphoblasts and lymphocytes.
The bursa is a primary lymphoid organ. Immature B lymphocytes of birds migrate from bone
marrow to the bursa. In bursa they undergo selection processes. If they mature, they exit to
migrate to secondary lymphoid organs.
There is some antigen trapping and antibody synthesis also happening in bursa. There is also a
small focus of T cells near the bursal duct opening. For these reasons, bursa is not considered a
purely primary lymphoid organ.
Peyer's patches
In ruminants, pigs, horses, dogs and humans, 80% to 90% of the Peyer's patches (PPs) are found
in the ileum as a single continuous structure. The ileal PPs regress as the animal ages. Ileal PPs
consist of densely packed lymphoid follicles which contain only B cells. This acts as a primary
lymphoid organ in some species, most especially, the sheep.
There are some PPs in jejunum as well, which contain both B and T cells and which persists for
Other mammals like rabbits, primates and rodents have randomly located PPs in ileum and
jejunum which persists in to old age.
In rabbits, the appendix also plays a role in B cell development.
T cell precursors originate in the bone marrow and travel via blood to the thymus. Immature T
cells, known as thymocytes pass through several developmental stages in specific thymic
microenvironments as described below and mature into functional T cells. This process may be
termed as thymic selection.
a. T cell precursors enter the thymus in blood vessels at the corticomedullary junction (i.e.,
between the thymic cortex and the thymic medulla).
b. These cells settle in thymic cortex and generate unique antigen receptors (T cell receptors, or
c. They are then selected on the basis of their reactivity to self MHC-peptide complexes expressed
on the surface of cortical thymic epithelial cells. Those thymocytes whose T cell receptors bind
self MHC-peptide complexes with too high affinity are induced to die (negative selection).
d. Those thymocytes that bind self MHC-peptides with an intermediate affinity undergopositive
selection (stimulated to grow), resulting in their survival, maturation, and migration to the
thymic medulla.
e. The remaining cells die because they have too low an affinity for the self-antigen- MHC
combinations and they are not positively selected. Approximately 95% of thymocytes die during
these selection processes.
f. Medullary thymic epithelial cells can express proteins found in other organs. They help in
negatively selecting any autoreactive T cells that could not be deleted in the cortex.
g. Mature thymocytes leave the thymus as they entered: via the blood vessels of the
corticomedullary junction.
In secondary lymphoid tissue like spleen and lymph nodes, these new T cells meet antigens
presented to them.

The secondary lymphoid organs

1. Lymph nodes and the spleen are highly organized secondary lymphoid organs (SLOs) and
have capsules.
2. Mucosa-associated lymphoid tissue (MALT) is less well organized and is found associated
with the linings of multiple organ systems. MALT includes:
a. Tonsils
b. Peyer’s patches (in the small intestine)
c. the appendix, and
d. lymphoid follicles within the lamina propria of the intestines and in the mucous
membranes lining the upper airways, bronchi, and genitourinary tract.
All SLOs include distinct regions of T cell and B cell activity, and all develop lymphoid follicles
where development and selection of B cells that produce high-affinity antibodies take place.

Most lymphocytes enter secondary lymphoid organs via blood vessels, and leave via the
lymphatic system.
Foreign antigens that have reached the extracellular fluid in tissues eventually enter the
lymphatic system and reach SLOs. In the SLO, the foreign antigen is trapped by antigen
presenting cells.
Antigen-presenting cells that have processed some antigens from other tissues (like skin) also
reach SLOs through the lymphatic system.
B-lymphocytes in SLOs interact with the trapped antigen, and their activation is started.
T-lymphocytes in SLOs react with antigen-presenting cells and undergo activation.
Most secondary lymphoid tissues are situated along the vessels of the lymphatic system. The
spleen is an exception and is served only by blood vessels. Immune cell trafficking is facilitated
by chemokines.
Lymph nodes
A lymph node can be divided into three roughly concentric regions: the cortex, the paracortex,
and the medulla.

• The cortex, contains lymphocytes (mostly B cells), macrophages, and follicular dendritic
cells (1.b) arranged in follicles.
• Beneath the cortex is the paracortex, which contains mostly T lymphocytes and dendritic
cells that have migrated from tissues.
• The medulla is the innermost layer, and the site where lymphocytes exit the lymph node
through the efferent lymphatics. It contains fewer lymphoid (immature) cells. Plasma cells
that are actively secreting antibody molecules may be seen in the medulla.
T cell activation in lymph node
Afferent lymphatic vessels empty lymph into the subcapsular sinus of the lymph node.
Particulate antigen carried by lymph is trapped by resident antigen-presenting cells. These APCs
process the trapped antigen and present peptides on MHC. Once naïve T cells enter the lymph
node, they browse MHC-peptide antigen complexes on the surfaces of the dendritic cells
present in the paracortex. If the TCRs bind to any of the present antigens, the T cells stop
migrating and start proliferating. Otherwise, they exit the lymph node via the efferent
lymphatics in the medulla of the lymph node.
B cell activation in lymph node
The lymph node is also the site where B cells are activated and differentiate into high affinity
antibody-secreting plasma cells. B cell activation requires:

1. antigen engagement by the B cell receptor (BCR) and

2. direct contact with an activated CD4+Th (T helper) cell.
Like T cells, B cells circulate through the blood and lymph and visit the lymph nodes regularly.
In the lymphoid follicles, they bind any available free antigen. With this, the B cell becomes
partially activated.
B cell can engulf and process the antigens that bind to its BCRs (i.e., they are antigen presenting
cells). Then they move from the follicle to the T cell rich paracortex. Here they present the MHC
II-antigen complexes to CD4+Th cells. If a memory or activated CD4+Th cell recognizes any MHC
II-antigen complex, the partially activated B cell will get T-cell help to become fully activated.
Now they do one of these two things:

• proliferate, and differentiate into plasma cells

• re-enter the follicle to establish a germinal center.
A follicle that develops a germinal center is called a secondary follicle; a follicle without a
germinal center is called a primary follicle.

In the germinal center, the activated B cell proliferates and creates an antigen-specific B cell
clone. Some of these B cells differentiate into plasma cells. They stay at the medulla of the
lymph node and secrete antibodies. Some plasma cells move to the bone marrow, where they
will continue to release antibodies into circulation.
The initial activation of B cells and establishment of the germinal center take place within 4 to 7
days of the initial infection, but germinal centers remain active for 3 weeks or more. Lymph
nodes swell visibly and sometimes painfully, particularly during those first few days after
infection. This swelling is due to an increase in the lymphocyte migration into the node as well
as the proliferation of antigen-specific T and B lymphocytes within the node.
The interactions between Th cells and APCs, and between activated Th cells and activated B cells,
results in the generation of both effector and memory T and B cells.

1. Memory T and B cells can take up residence in SLOs (central memory cells)
2. Memory T and B cells can exit the lymph node and travel to tissues (effector memory

The spleen plays a major role in mounting immune responses to antigens in the bloodstream
and therefore, it is particularly important in the response to systemic infections.
The spleen is not supplied by lymphatic vessels. Only blood-borne antigens and lymphocytes
are carried into the spleen, through the splenic artery and out via the splenic vein. More
recirculating lymphocytes pass daily through the spleen than through all the lymph nodes
Splenectomy (the surgical removal of a spleen) can lead to systemic bacterial infections in
children and an increased vulnerability to blood-borne bacterial infections in adults.
The spleen is surrounded by a capsule from which the trabeculae extend inwards, providing
structural support. The red pulp and white pulp of spleen are separated by the marginal
The splenic red pulp contains red blood cells, macrophages, and some lymphocytes.
The splenic white pulp surrounds the branches of the splenic artery, and consists of the
periarteriolar lymphoid sheath (PALS) populated by T lymphocytes as well as B cell follicles.
As in lymph nodes, germinal centers form within these follicles during an immune response.
The marginal zone is populated by macrophages and B cells. Blood-borne antigens and
lymphocytes reach first at the marginal zone. In the marginal zone, antigen is trapped and
processed by dendritic cells, which travel to the PALS.
The events that initiate the adaptive immune response in the spleen are similar to those that
occur in the lymph node. Circulating naïve B cells encounter antigen in the follicles, and
circulating naïve CD8+ and CD4+ T cells meet antigen in the PALS.
Mucosal membranes are the major sites of entry for most pathogens. These vulnerable surfaces
are defended by mucosa-associated lymphoid tissue (MALT). MALT associated with the
respiratory epithelium is called bronchus-associated lymphoid tissue (BALT) and that associated
with the intestinal epithelium is referred to as gut-associated lymphoid tissue (GALT).
It may occur as loose, barely organized clusters of lymphoid cells in the lamina propria of
intestinal villi or well-organized structures such as the tonsils and adenoids, the appendix, and
PPs. The outer mucosal epithelial layer contains intraepithelial lymphocytes (IELs), many of
which are T cells. The lamina propria contains large numbers of B cells, plasma cells, activated
T cells, and macrophages in loose clusters. Follicles in PPs can develop into secondary follicles
with germinal centers.
MALT contains a large population of antibody-producing plasma cells, whose number exceeds
that of plasma cells in the spleen, lymph nodes, and bone marrow combined.
The epithelial cells of mucous membranes can help delivering foreign antigen from the
respiratory, digestive, and urogenital tracts to the underlying MALT. In the digestive tract,
specialized M cells transport antigen across the epithelium. This antigen might be recognized
by B cells leading to secretion of IgA.
The skin
Langerhans cells are dendritic cells in skin that internalize antigen by phagocytosis or
endocytosis. Following this, the Langerhans cells undergo maturation and migrate from the
epidermis to regional lymph nodes, where they activate naïve T cells.
The epidermis also contains intraepidermal lymphocytes, which are predominantly T cells.
Dermis of the skin also contains scattered lymphocytes, dendritic cells, monocytes,
macrophages, and sometimes, hematopoietic stem cells. Most skin lymphocytes are either
previously activated cells or memory cells.

Tertiary lymphoid tissues

Tissues that are the sites of infection are referred to as tertiary lymphoid tissue. Lymphocytes
activated by antigen in SLO can return to these organs as effector cells.

Cells of the immune system, Types of Immunity

All cells of the immune system arise from the haematopoietic stem cell.
Stem cells are cells that can differentiate into other cell types. They are self-renewing.
An HSC is pleuripotent; i.e., able to differentiate into all types of blood cells.
HSCs differentiate in to either a common lymphoid progenitor cell or a common myeloid
progenitor cell. The types and amounts of growth factors [e. g., colony stimulating factors
(CSFs) or the interleukins (ILs)] in the microenvironment of a particular stem or progenitor cell
control its differentiation.
The common lymphoid progenitor cell gives rise to B, T and the NK cells and some dendritic

The common myeloid progenitor cell generates erythrocytes, the granulocytes, monocytes,
mast cells, dendritic cells and platelets.

Of these, two types of cells are specialized for killing and eating invading microorganisms -
neutrophils and macrophages. Neutrophils are the first line of defense, which eat invading
microorganisms very rapidly, but are incapable of sustained phagocytic effort.

They make up 60-75% of blood leukocytes in carnivores, 50% in horses, 20-30% in ruminants
and laboratory rodents. They remain in circulation for 7-12 hrs and have a lifespan of only a few
days. They are also known as polymorphonuclear leukocytes (PMN) and are the first to reach
the site of inflammation.
Neutrophils contain abundant granules in the cytoplasm which are stores of anti-bacterial
agents and lysosomal enzymes, to kill ingested microorganisms with.
There are two different types of granules:

1. azurophilicor primary granules contain myeloperoxidase as well as numerous

lysosomal enzymes and proteins like defensins, lysozyme, elastase, cathepsins,
proteinase 3 etc.
2. specific or secondary granules lack myeloperoxidase, but contain lysozyme, lactoferrin,
collagenase, gelatinase, β2 macroglobulin, vitamin B12 binding protein etc. as well as
several important membrane bound receptors.
Neutrophils are normally confined to the blood stream. When there is an inflammation, there
are changes in the endothelial cells that line the blood vessel walls near the inflamed tissue and
in the neutrophils themselves. Briefly, capillary endothelial cells and neutrophils express
adhesion molecules which facilitate attachment of neutrophils to the blood vessel walls.
The neutrophils emigrate into the surrounding tissues under the influence of chemoattractants.
They squeeze between the endothelial cells and the basement membrane in a process called
diapedesis or transmigration. They then crawl towards any invading microbes. Once they
reach sites of microbial invasion, they try to destroy foreign particles such as invading bacteria.
Antimicrobial mechanisms of neutrophils
I. Phagocytosis
Phatocytosis involves discreet stages like activation, chemotaxis, adherence, ingestion and
destruction. Neutrophils are activated by attachment to blood vessel walls and cytokines.
Activated neutrophils move towards the invading organisms and damaged cells in a
process called chemotaxis. Chemoattractants include C5a, fibrinopeptide B, chemokines,
lipids and some bacterial peptides. These are generally present at a higher concentration
near the invading organism or damaged cells and so by following the concentration
gradient, neutrophils reach the site of invasion. Both neutrophils and bacteria suspendend
in body fluids usually have a negative charge (zeta potential) and so repel each other. The
charge must be neutralized, which requires that the bacteria be coated with positively
charged molecules. Molecules that improve the adherence of bacteria to a phagocyte in
this way are called opsonins. This word is derived from the Greek word for ‘sauce’, implying
that they make the bacterium ‘tastier’ for neutrophils. Examples of opsonins include
mannose-binding lectin, complement components and antibodies. CD32 (FcγRII, [Fc
gamma receptor 2]. This is a receptor for IgG) is an example of an antibody receptor on a
neutrophil. Antibody mediated phagocytosis is called type I phagocytosis. Complement
mediated phagocytosis is termed type II phagocytosis [CD35 (CR1 or complement
receptor 1) is found on neutrophils, other granulocytes, monocytes etc. and binds C3b
coated particles]. Antibodies are the most effective opsonins.
Hydrophobic bacteria such as Mycobacterium tuberculosis are readily ingested, but
bacteria with a hydrophilic capsule, such as Streptococcus pneumoniae is poorly
phagocytosed unless it is made hydrophobic by opsonisation.
When a bacterium is eventually drawn in to the neutrophil, it becomes enclosed in a
vacuole called a phagosome.
Destruction of the ingested bacterium occurs through two distinct processes: the
respiratory burst (generation of reactive oxygen species and other potent oxidants) and
release of lytic enzymes and antimicrobial peptides from intracellular granules.
II. The respiratory burst
Within seconds of binding to a bacterium, neutrophils increase their oxygen consumption
nearly 100-fold. This increase is a result of activation of enzymes such as NADPH oxidase

(NOX), superoxide dismutase and myeloperoxidase. These enzymes work in sequence to
produce hypohalides such as OCl-, OBr-, etc inside the neutrophil. This process is known as
the respiratory burst. OCl- (hypochloride) is the major product of neutrophil oxidative
metabolism. It kills bacteria by oxidizing bacterial proteins and lipids.
III. Antimicrobial molecules
a. Lytic enzymes: Once a bacterium is attached to the neutrophil membrane, the granules (or
lysosomes) migrate through the cytoplasm and fuse with the phagosome. The enzymes in
phagolysosome (elastase, cathepsin G, lysozyme, proteases, acid hydrolases and
myeloperoxidase) digest bacterial cell walls and kill most microbes.
Some bacteria such as Brucella abortus and Listeria monocytogenes can continue to grow
inside phagocytic cells after preventing formation of phagolysosome.
b. Peptides: they are pore forming peptides. The hydrophobic regions of these peptides
insert themselves into the lipid-rich membranes of bacteria, whereas the hydrophilic
regions can form channel-like pores. This results in membrane disruption and microbial
death. These peptides can kill most species of bacteria, some fungi, protozoa, enveloped
viruses and tumour cells. The two major classes of neutrophil anti-bacterial peptides are
defensins and cathelicidins. Serprocidins, bactericidal permeability-increasing protein and
calprotectin are other important antibacterial proteins seen in neutrophils.
c. Lysozyme: Cleaves the bond between N-acetyl muramic acid and N-acetyl glucosamine
and thus destroys the peptidoglycans of bacteria. It is found in all body fluids except
cerebrospinal fluid and urine. It is not found in bovine neutrophils and tears. It is found in
high concentration in the tears of other mammals and in egg white. It is found in high
concentration in some neutrophil granules. It is a potent innate opsonin for bacteria.
d. Lectins: They are proteins that bind carbohydrates. There are P-, S- and C-type lectins. C-
reactive protein (CRP) and serum amyloid P (SAP) are P-type lectins. They are also called
acute phase proteins, because their blood levels increase during infections or following
trauma. They function by activating the complement system and stimulating leukocytes.
They also function as opsonins. C-type lectins include collectins that link bacteria and
phagocytes or complement components.
e. Iron binding proteins: Many pathogenic bacteria require large amounts of iron for
growth. Following recognition of bacteria, milk neutrophils can release their stores of
lactoferrin, which binds free iron and make it unavailable to bacteria. In turn, bacteria may
produce iron binding proteins (siderophores) that can claim iron from mammalian iron
storage proteins.
f. Complement components: The complement system help in bacterial killing and
phagocytosis. Neutrophils store large quantities of C6 and C7 in their granules and make
them available at sites of bacterial invasion.
g. Cytokines: Under the influence of bacterial products such as LPS, neutrophils can secrete
many different cytokines such as interleukin-1α (IL-1α), IL-1β, tumour necrosis factor-α
(TNF-α) etc. These cytokines help augment the immune response.

The mononuclear phagocytic system consists of cells called monocytes in blood and
macrophages in tissues.

• Macrophages have a single round nucleus.

• Macrophages are capable of repeated phagocytosis.
• They are involved in triggering of inflammation
• They are involved in tissue repair
• They are involved in priming the adaptive immune system by presenting antigen to T
helper (Th) lymphocytes.
Depending upon the tissue into which the monocyte has migrated, the macrophage that
descends from it is called a:
• histiocyte (connective tissue)
• Kupffer cell (liver)
• microglia (brain)
• alveolar macrophages (lung alveoli)
• pulmonary intravascular macrophages (lung capillaries)
• mesangial cells (kidneys)
• osteoclasts (bone)
• peritoneal macrophages (peritoneum).
Macrophages possess PRRs (pattern recognition receptors) to detect invading bacteria and
viruses. They respond by producing cytokines like IL-1, IL-12, IL-23 and TNF-α.

Phagocytosis: monocytes exit blood vessels much the same way as neutrophils do. Several
hours after neutrophils have entered an inflammatory site, the macrophages begin to arrive.
Macrophages are attracted to inflammatory sites by:

• monocyte chemoattractant protein-1 (CCL2) produced by activated neutrophils and

endothelial cells
• bacterial products
• complement components such as C5a
• defensins
Macrophages secrete collagenases, elastases and other proteases. These proteases destroy
connective tissue and permit more effective penetration of the damaged tissue.
Macrophages also phagocytose apoptotic neutrophils and their granules and use these
granules in killing phagocytosed bacteria.
There are M1 macrophages and M2 macrophages.
M1 cells are usually concerned with host defense.
M2 cells promote blood vessel formation, tissue remodeling and tissue repair.
M1 cells are produced early in the inflammatory process.
M2 cells appear late when healing is required.
M1 macrophages generate reactive nitrogen species such as nitric oxide (·NO) with the help of
the enzyme inducible nitric oxide synthase (iNOS or NOS2).·NO reacts with superoxide anion to
produce highly reactive oxidants such as peroxynitrite and nitrogen dioxide radical. These are
used to kill bacteria, fungi, protozoa, some helminths and tumour cells. M1 macrophages can
activate natural killer (NK) cells through cytokines.
M2 macrophages do not produce NO. They work to reduce inflammation and produce
cytokines that suppress immune responses.
Antigen presentation: Macrophages are one of the three professional antigen presenting
cells (APCs). They can present fragments of peptides derived from ingested bacteria and other
particles to T helper lymphocytes (Th cells) through a surface molecule called the class II MHC
molecule. Such peptides may be recognized by the Th cell. Then further ‘co-stimulation’ by the
antigen presenting macrophage activates the Th cell. Macrophage thus acts as a bridge
between the innate immune system and the adaptive immune system.
Eosinophils are mainly recruited to sites of parasitic infestation by mast cells and IgE. They have
low affinity IgE receptors. Eosinophils contribute significantly to Type I hypersensitivity. They
1. Small primary granules containing arylsulfatase, peroxidase and acid phosphatase.
2. Large crystalloid granules containing major basic protein (MBP), eosinophil cationic protein,
eosinophil peroxidase (EPO) and eosinophil-derived neurotoxin.
Eosinophils are majorly concerned with anti-parasite immunity. They attack the parasites by
releasing the granular contents in to the fluid surrounding the parasite.

Basophils and Mast cells

They are important in Type I hypersensitivity reactions. Mast cells are the major cells that trigger
Type I hypersensitivity reactions (which are acute inflammatory responses mediated by
recognition of a specific parasitic antigen or allergen by IgE bound to its high affinity receptor
(FcεRI) on mast cells)
(see or
Basophils also have FcεRI. Mast cell cytoplasmic granules contain histamine, heparin, TNF-α and
other inflammatory mediators.

Dendritic cells (DCs)

In order to trigger acquired immunity, a sample of foreign material must be captured,
processed and presented in the correct fashion to T cells. The cells that carry out this function
are called the antigen presenting cells (APCs). Macrophages, B lymphocytes and dendritic cells
(DCs) are the professional APCs. Of the three types, DCs, which form the third class of
phagocytic cells of the immune system, is the most important type of professional APCs,
because they are the only APCs that can activate naïve T cells (T cells that have never previously
encountered an antigen in the periphery) and thus are essential for initiating primary immune

They take up particulate matter by phagocytosis and also continually ingest large amounts of
the extracellular fluid and its contents by a process known as macropinocytosis. Their main
role in the immune system is antigen presentation. Antigen presenting ensures that an adaptive
immune response is initiated against the invading organism, and that memory cells are
DCs are progenies of bone marrow stem cells. These immature DCs migrate throughout the
body and form lattice-like networks in every tissue except the brain, parts of the eye and the
testes. DCs are classified in many different ways:
1. Myeloid DCs and Plasmacytoid DCs:
Myeloid DCs are derived from blood monocytes. They are seen in tissues.
a. Langerhans cells are a population of myeloid DCs found in epidermis and concerned with
development of skin immune responses such as delayed type hypersensitivity. They contain
characteristic cytoplasmic granules called Birbeck granules.
b. Follicular dendritic cells are specialized myeloid DCs found in lymphoid follicles. They trigger B
cell activation by transferring antigen to B cells as round-beaded structures called exosomes. B
cells take up these exosomes and after processing the antigen, present it to antigen-sensitive T
Plasmacytoid DCs are derived from lymphoid precursors. They are found in blood and
lymphoid organs.
2. DC1 and DC2 cells: DC1 cells secrete IL-12. They tend to stimulate Th1 (T helper subset 1) cells
(controls cell-mediated immune responses). DC2 cells secrete IL-4 and stimulate Th2 (T helper
subset 2) cells (concerned with humoral and anti-parasite immune responses).
3. The more important classification of DCs is based on their level of maturity.
a. Immature DCs are highly efficient antigen-trapping and processing cells. They are seen in
tissues. They have high levels of Fc receptors (for efficient antigen uptake).
b. Mature DCs - efficient antigen-presenting cells. They are seen in lymphoid organs. They have
low levels of FcRs, but have high levels of surface MHC-II, CD40, CD80, CD86 and IL-12 (because
they are more concerned with antigen presentation rather than antigen uptake/processing).
Immature DCs carry the captured and processed antigen to lymph nodes where they mature.
Mature DCs have MHC molecules on their surfaces and they also express the co-stimulatory
molecules. These changes help them activate T cells. One DC may activate as many as 3000 T

Antigen processing and presentation

Antigen processing involves breaking protein antigen molecules into small peptides. Some of
these peptides are then attached to specialized antigen-presenting receptors called major
histocompatibility complex (MHC) molecules.
There are two different classes of MHC molecules, MHC I and MHC II. Each binds a different
type of antigen.
Antigenic peptides bound to MHC are displayed on the cell surface. Acquired immunity is
triggered when these MHC-bound antigen peptides bind to antigen receptors on T
Different organisms trigger acquired immunity in different ways
Organisms such as bacteria and fungi which invade the body and grow in tissues and
extracellular fluids are phagocytosed by professional antigen-processing cells (APCs). Their
antigenic peptides are called exogenous antigens. These antigens are presented coupled to
MHC II molecules on the APC surface.
Pathogens that grow inside host cells, such as viruses, and some bacteria are safe from
phagocytes. It is the infected host cell itself that presents antigenic peptides derived from these
pathogens. This is possible because intracellular pathogens utilize the host cell machinery for
synthesis of their proteins. These proteins are called endogenous antigens. Some of these
proteins as degraded inside the host cell to generate small peptides. Some of these peptides
are presented coupled to MHC I molecules on the host cell surface.

Processing of exogenous antigen

Helper T lymphocytes (Th cells) are the major orchestrators of an acquired immune response.
They can recognize and respond only to an antigen bound to an MHC II molecule. Following
trapping and phagocytosis by DCs, the antigens are broken down by lysosomal proteases into
peptides of varying length. The endosomes containing these peptide fragments then fuse with
other endosomes carrying newly synthesized MHC class II molecules. Newly synthesized MHC

class II molecules are seen associated with a peptide called the invariant chain. This prevents
them from binding other peptides present in endoplasmic reticulum and helps the assembly
and movement of the MHC II molecules to the endosomal compartment containing antigen-
derived peptides. Part of the invariant chain occupies the antigen-binding groove of the MHC II
molecule. This part remains in place while the rest of the chain is digested away in the low pH
endosome. This fragment is called CLIP (for Class II associated Invariant chain peptide).
Molecules such as the HLA-DM (in humans) facilitate removal of CLIP from the peptide binding
groove of the MHC II molecule and binding of antigen-derived peptides to the now empty
groove. An MHC II antigen binding site can hold a peptide of 12 to 24 amino acids. Once the
antigen fragment has been loaded, the vesicles move toward the cell surface where they fuse
with the cell membrane. The MHC-peptide complex that was anchored on the vesicular
membrane is thus presented on the cell surface.
It has been calculated that an APC carries about 2 X 105 MHC class II molecules that can present
antigen fragments to T cells. If co-stimulation is provided, a T cell can be activated by exposure
to 200-300 peptide-MHC complexes.

Processing of endogenous antigen

There is a system in place in normal living cells for presentation of endogenous peptides on
MHC class Ia molecules on their surfaces. Cells routinely break down and recycle proteins. For
this, proteins are first attached to several molecules of ubiquitin, a 76 amino acids polypeptide.
Ubiquitinated proteins are recognized by a large enzyme complex called a proteasome, which
breaks down these proteins into fragments of 8-15 amino acids. A few of these peptides are
rescued from further breakdown by attachment to transporter proteins, TAP1 and TAP2 (for
transporter for antigen processing 1 and 2) which transports them into endosomes. Here they
are shortened one amino acid at a time until they are completely degraded, unless an
intermediate, 9 amino acid long peptide precisely fits the binding site on an empty MHC class
Ia molecule. The MHC I- peptide complexes are carried to the cell surface and displayed there
for many hours. This process of endogenous antigen presentation happens routinely
irrespective of the presence of viruses inside the cell.
Viruses take over the protein-synthesizing machinery of the cell and use it to make new viral
proteins. When a cell is infected by a virus, some of the newly synthesized viral proteins are
processed in this manner, leading to display of MHC I- viral peptide complexes on the cell
surface. These antigen fragments are recognized by cytotoxic T cells (Tc cells) which become
activated. A cell can express about 106 MHC-peptide complexes and about 200 of them
displaying the same viral peptide are required to activate a Tc cell.

Cross-presentation and autophagy

Under some circumstances exogenous antigens may enter the cytoplasm of some DCs, join the
endogenous antigen presentation pathway, and be presented on MHC class I molecules.
Peptides from extracellular sources-such as viruses, bacteria, and phagocytosed dying cells
infected with cytosolic pathogens-can be presented on MHC class I molecules. This is called
cross-presentation or cross-priming and is important in immunity to viruses, because it is
through cross-presentation that these DCs first activate naïve Tccells. On the other hand,
cytosolic antigens may be presented on MHC class II molecules following autophagy, the
natural process of protein turnover, in which damaged organelles and cytosolic proteins are
delivered to lysosomes for degradation.

Macrophages and B cells as APCs

Macrophages and B cells cannot undertake prolonged interactions with T cells. Therefore, they
can only activate memory Th cells and consequently are more important in secondary immune
responses than in primary immune responses. Moreover, antigen processing by macrophages is
inefficient, since much of the ingested antigen is destroyed by lysosomal proteases and
oxidants. Antigen persistence is more in DCs and B cells whose phagosomes generally have a
more alkaline pH.

There are three major types of lymphocytes: natural killer (NK) cells, T cells and B cells.
Lymphocytes are small, round cells, 7-15 µm in diameter, containing a large round nucleus
surrounded by a thin rim of cytoplasm.
NK cells are usually larger and contain cytoplasmic granules. They have been called large
granular lymphocytes.
Lymphocytes are found in lymphoid organs, blood and under mucosal surfaces. Each type and
their subsets are identified by their characteristic cell surface molecules (surface markers) and

their behavior. The pattern of cell surface molecules expressed on a cell is called its
T lymphocytes arise in bone marrow and migrate to thymus where they mature (T for thymus-
derived) and undergo the selection processes. After they leave the thymus, they accumulate in
the paracortex of lymph nodes, the PALS of the spleen and the interfollicular areas of the
Peyer's patches. T cells account for 60-80% of the lymphocytes in blood.
B lymphocytes originate in bone marrow and mature within the bone marrow, bursa or Peyer's
patches before migrating to the secondary lymphoid organs. B cells predominate in the cortex
of lymph nodes, in follicles within the Peyer's patches and spleen, and in the marginal zone of
the white pulp of the spleen. B cells account for 10-40% of blood lymphocytes.
NK cells originate in the bone marrow from the same stem cells as T and B cells but do not
undergo development similar to thymic processing. They are widely distributed throughout the
lymphoid organs and account for 5-10% of blood lymphocytes.

Lymphocyte surface molecules (markers)

In the discipline of Immunology, most molecules associated with immune functions is given a
functional or chemical name as well as a cluster of differentiation (CD) designation. In some
cases, the molecule's common name is more frequently used instead of its CD designation.
The most important surface molecules of lymphocytes are the antigen receptors. These are
abbreviated TCR (T cell antigen receptor) or BCR (B cell antigen receptor). TCR and BCR are not
simple proteins. They are complex structures containing many different proteins. Some of these
proteins act as the antigen binding component, while others transduce signal to the interior of
the cell.
The BCR has a membrane immunologlobulin as their antigen-binding component and
glycoprotein heterodimers formed by pairing Ig-α and Ig-β as signal transducing component.
TCRs are of two types: one formed of α and β peptide chains and the other formed of γ and δ
peptide chains. A given T cell contains only one type of TCR. These chains form the antigen
binding component of the TCR. The signal transducing component of TCRs is called CD3, which
is found on all T cells.
CD4 is found on T cells that recognize processed exogenous antigens, and is a receptor for
MHC II molecules on APCs. CD4+ T cells usually function as T helper cells.
CD8 is only expressed on T cells that recognize processed endogenous antigens and that
further attack and kill abnormal cells. It is a receptor for MHC I molecules. CD8+ T cells usually
function as T cytotoxic cells.
Neither CD4 nor CD8 is expressed on NK and B cells.
NK cells do not even have antigen receptors. Instead, they express receptors for surface
molecules expressed on healthy normal cells but absent from diseased, abnormal cells. NK cells
kill target cells that fail to express these surface molecules.
Apart from these, lymphocytes have receptors for cytokines, antibody and complement, and
adherence molecules such as integrins, selectins and members of the immunoglobulin
(The immunoglobulin superfamily is the large group of proteins that express the
immunoglobulin fold [domain] structure).

T cells, B cells and development of adaptive immune

B lymphocytes and two sets of T lymphocytes are the principal cell types that control acquired
immune responses.
B cells are capable of mass-producing and secreting their BCRs – as antibodies – which then
bind the antigen and help in its elimination. This is called a humoral immune response and is
usually dependent on T cell help.
T cytotoxic (Tc) cells are evolved to recognize and destroy the body's own cells that have been
infected by intracellular pathogens. This is called cell mediated immunity and also is dependent
on T cell help.
T helper cells (Th cells) are the cells that provide the T cell help. T cell help means secretion of
cytokines that will aid the immune response under development by helping the activation of
the effector cells (B or Tc cells).

He lpe r T ce lls

Each T cell has about 30,000 antigen receptors on its surface. TCRs recognize antigenic peptides
complexed with an MHC molecule. All TCRs on a T cell has the same specificity. TCRs, like BCRs
(and antibodies) have variable and constant domains, disulphide bonds, CDRs, and framework

regions. When a TCR is engaged by an antigenic peptide- MHC complex, a signal is transduced
to the interior of the T cell by the CD3 complex, the signal transduction component of TCR.
Th cells generally have CD4 molecules associated with TCRs. Tc cells usually have CD8 molecules
in place of CD4. CD4 binds MHC II and CD8 binds MHC Ia molecules. CD4 and CD8 enhance
TCR signal transduction 100-fold when they bind to an MHC molecule on an antigen presenting
cell/target cell.
T cells do not become activated when only their TCRs are engaged. Usually, they require co-
stimulation (additional activation signals) in order to become activated. There are three types of
additional signals received by the T cell:
1. Ligands such as CD40 on APCs bind receptors on T cells.
2. Cytokines secreted by APCs stimulate T cells.
3. Adhesion molecules bind APCs and T cells together to provide a prolonged, strong signaling
between the cells.
The activated Th cell produce cytokines that trigger the next stages of the immune response.
The sequential events in Th cell activation are as follows (each additional signal amplifies the
original signal generated by TCR-antigen binding):
1. A DC engulfs some bacteria and processes its antigens.
2. The DC migrates to a nearby lymph node and presents antigenic peptides on MHC II
molecules to a CD4+ T cell. The TCRs of the Th cell binds the antigen-MHC complexes.
3. CD4 of the Th cell binds MHC II of the DC.
4. Costimulatory molecules on the DC bind ligands on Th cell.
5. DC secretes multiple cytokines.
6. The activated Th cell produces and secretes other cytokines.
7. About 48 to 72 hours after activation, Th cell is inactivated by inhibitory signals from the DC.
The Th cells activated by DC1 cells are called Th1 cells and they secrete IL-2, IFN-γ, TNF-α and
lymphotoxin (TNF-β). These cytokines together stimulate inflammation and cell mediated
immune responses against intracellular organisms such as the mycobacteria and to viruses.
The Th cells activated by DC2 cells are calledTh2 cells which secrete IL-4, IL-5, IL-10 and IL-13.
These cytokines stimulate B cell proliferation and immunoglobulin secretion, and enhance IgE
production. Th2 responses are therefore associated with humoral immune responses and
immunity to parasites.
Some microbial molecules induce DCs to secrete IL-6 and IL-23. These cytokines stimulate a
subset of Th cells known as Th17 cells, which secrete IL-17 and promote inflammatory reactions
in autoimmune and chronic inflammatory diseases. They help in clearing extracellular bacteria
and fungi.
While there are defined Th1 and Th2 subsets, some Th cells secrete a cytokine mixture that is a
mixture of both Th1 and Th2. These cells are called Th0 cells.
When a T cell and an APC come into contact, the cytoskeleton in each cell is rearranged so that
the TCR-peptide-MHC complexes and the co-stimulatory receptors cluster together to form a
specialized structure in the area of contact called an immunological synapse. T cells may
initially form synapses with multiple APCs, but later polarize towards the cell providing the
strongest stimulus (the antigen that binds with highest affinity).
Naïve T cells must receive a sustained signal for up to 10 hours in the presence of co-
stimulation or for up to 30 hours in its absence. Dendritic cells are able to sustain interactions
for such long periods, while macrophages and B cells can only briefly trigger a TCR. Therefore,
macrophages and B cells fail to activate naïve T cells, but can activate a memory T cell which
needs to be triggered for about an hour to become activated.
In the absence of effective co-stimulation, a T cell will undergo abortive activation. It does not
divide or produce cytokines but either becomes unresponsive to antigen (anergic) or
undergoes apoptosis and dies.
γ/δ T cells
T cells having γ/δ TCRs are in minority in humans and mice (only 5-15% of blood lymphocytes),
but important in young ruminants (up to 60% of blood lymphocytes). In young ruminants and
pigs γ/δ T cells are the major circulating lymphocyte population.
Memory T cells
These do not secrete effector cytokines and are long lived. There are two types of memory T
cells. The central memory T cells remain in secondary lymphoid organs awaiting re-entry of
invaders. The effector memory T cells are found in inflamed tissues.

B ce lls a nd hum ora l im mune re sponse

B cell requires antigen binding and co-stimulation to become fully activated.


Co-stimulation can come from the Th cells.This means that both B cell and T cell activation has
to happen for a humoral immune response to develop.
If it is a memory response (anamnestic response, secondary response), then the B cell itself
could act as the APC that activates the specific Th cell and receive co-stimulation from the same
T cell. (more explanation is given under “T dependent antigens”).
Co-stimulation is received as cytokines and signaling through membrane receptors.
Cytokines: Th2 cells secrete IL-4, IL-5, IL-6 and IL-13 to 'help' B cells. These cytokines generally
stimulate antibody production and B cell differentiation. IL-4 is responsible for B cells switching
class to IgE. IFN-γ from Th1 cells acts in the opposite way i.e., IgE synthesis is inhibited and IgG
production is favoured.
Co-stimulation through other receptors
♦ Signals from CD40 and CD80/86.
♦ Signals transmitted through complement receptor 2.
♦ Stimulation through TLRs.
Following activation, B cell enlarges and proliferates. Some of the progeny differentiate into
cells rich in rough endoplasmic reticulum with a nucleus that has a cartwheel appearance. These
are the plasma cells. They secrete large quantities of IgM. Within a few days, the cell switches
from making IgM to IgG, IgA or IgE although the specificity of the antibody does not change.
Plasma cells can make and secrete up to 10,000 molecules of immunoglobulin per second.
These cells generally live for about 1-2 weeks (there is also a plasma cell population that has a
half-life of 8-15 years). Memory cells persist, however, in germinal centers. Memory cells look
like other lymphocytes, live long (in humans, memory B cell levels appear to be stable for up to
60 years postvaccination) and, following a second exposure, clonally expand to 8 to 10 times
the number of plasma cells seen in a primary response. Predictably, secondary responses are
faster and greater. IgG is produced in preference to IgM in secondary response.
The B cell FcγRIIb (IgG receptor 2b) is a negative regulator of B cell function. When an IgG
molecule binds to it and cross-links to a BCR through antigen, it inhibits antibody formation.
This helps put an end to an ongoing immune response once enough IgG molecules are
available. Because of this possibility, passively transferring antibody to a naïve recipient can be
used to inhibit naïve B cell responses to that same antigen. This phenomenon has been put to
practical use to prevent Rh- mothers from making an immune response to a Rh+ fetus, which
can result in hemolytic disease of the newborn. If anti-Rh antibody is given to the mother
before she is first exposed to her child's Rh+ red blood cells, her immune response will be

T c cells a nd cell me dia te d effe ctor f unctions

Antibodies are ineffective against intracellular pathogens. When a cell is infected, Tc cells are
activated by presentation of endogenous antigens on MHC I molecules. These Tc cells either kill
the infected cell to prevent growth of the intracellular organism (as happens in case of viruses).
Tc cells in lymphoid organs are activated by three key signals:
1. A subset of DCs present endogenous antigen (either from their own cytosol if they are infected,
or gathered from dying infected cells) on MHC I molecules to the Tc cells.
2. The DCs also secrete IL-12.
3. The third signal comes from IL-2 and IFN-γ secreted by Th1 cells.
Once activated, Tc cells are called cytotoxic T lymphocytes (CTLs). They proliferate, leave the
lymphoid organs and seek out infected cells. When a CTL finds a host cell carrying a MHC I-
specific antigen complex on its surface (called a target cell), its TCR binds that antigen and the
target cell is killed by induction of apoptosis. CTLs are also serial killers that can disengage from
a dying cell and move on to other target cells within minutes. CTLs kill target cells through any
of the two pathways given below.
a. The perforin pathway: This is by secretion of proteins called perforins and granzymes from
secretory lysosomes. This kills target cells through intrinsic apoptotic mechanisms.
b. CD95 death receptor engagement: This kills cells through extrinsic apoptotic mechanisms.
The perforin pathway
The adhesion phase: The CD8-TCR complex on a CTL binds to MHC I molecules on the target
cell surface and an immunological synapse forms around the contact area.
The lethal hit: The cytoplasmic granules migrate to the immunological synapse and their
contents are emptied on to target cell surface. Perforins are membrane perturbing
glycoproteins. They insert themselves into the target cell membrane. Between 12-18 monomers
aggregate to form a membrane attack complex (MAC) [as the complement component C9
does], permitting granzymes to enter target cells. Granzyme A causes nuclear damage in the
target cell and granzyme B sets off apoptosis. Granulysin is an antibacterial peptide found in

the granules. It kills any intracellular bacteria released during apoptosis. TNF-β secreted by
some CTLs binds to its receptors on target cells and so triggers their apoptosis.
The death receptor pathway
CD95L (CD95 ligand, Fas-ligand) is a protein on CTL surface that can bind a death receptor by
the name of CD95 (Fas) on target cell. This causes formation of a death inducing signaling
complex (DISC) in the target cell that triggers the apoptosis cascade.
Following recovery, most of the CTLs are cleared by induction of apoptosis through CD95. The
surviving cells (5-10% of the peak number of CTLs) become memory cells.
Cell activation
Bacteria such as Listeria monocytogenes, Mycobacterium tuberculosis and Brucellaabortus and
protozoa such as Toxoplasma gondii survive and multiply inside normal macrophages. Th1 cells
help these macrophages kill these pathogens by activating them through secretion of IFN-γ.
Innate pathways involving TLRs on macrophages and TNF-α activated NK cells also help
activate the macrophages.

NK cells
NK cells are non-phagocytic lymphocytes.
They kill tumours, virus infected cells, xenografted cells, some bacteria, protozoa and fungi.
They can be activated by interferons from virus infected cells and by IL-12 from macrophages.
They participate in innate defenses long before antigen-specific T or B cells are generated.
NK cells recognize their targets using inhibitory and activating receptors. MHC I molecules
present on host cell surface binds the KIR (killer cell immunoglobulin-like receptor) thus
providing an inhibitory signal to the NK cell. If a target cell does not express any MHC I
molecules (some metastatic tumours and host cells infected with some viruses), the NK cell will
not receive the necessary inhibitory signal and the target cell is killed.
A second pathway that triggers NK cell cytotoxicity involves the use of an activating receptor
called NKG2D which recognizes proteins expressed by tumour cells and virus infected cells,
such as MICA and MICB. When engaged, NKG2D overrides the inhibitory effect of MHC I
molecules and enables the NK cell to kill their target.
The third pathway of NK cell mediated cytotoxicity is called ADCC (see antibody dependent
cell-mediated cytotoxicity).

NK cells kill target cells in a manner similar to cytotoxic T lymphocytes: through intrinsic
pathway involving perforins and granulysin and NK-lysin as well as through the extrinsic
pathway involving CD95L.
NK cells have been used in tumour therapy after incubating them in the presence of IL-2 for 4
days. They then develop cytotoxic properties and are called lymphokine activated killer (LAK)

Types of immunity
Immunity is the state of protection from infectious diseases.
The body’s defences against an infection can be divided in to three overlapping layers: the
physical barriers, innate immunity and adaptive immunity.

Physical barriers to infection

The outer epidermal layer of the skin consists of dead cells and is filled with a water proofing protein
called keratin. Sebum produced by the sebaceous glands consists of lactic acid and fatty acids which
maintain the pH of the skin between 3 and 5, which inhibits the growth of most microbes. The acidity of
the stomach contents and of the vagina poses a further barrier to organisms, which are unable to grow
in low pH conditions. The conjunctivae, the alimentary, respiratory and urogenital tracts are lined by
mucous membranes. Mucus secreted by epithelial cells of mucous membranes entraps microbes. In the
lower respiratory tract the mucous membranes are covered by cilia, the synchronous movement of
which propels the mucus-entrapped microorganisms outward, coughing and sneezing aiding this
process. The normal flora, which resides in the mucous membranes, generally out-competes pathogens
for attachment sites on the epithelial cells and also for nutrients. Chicken have innate immunity towards
anthrax because their high body temperature inhibits the growth of the bacillus. The natural flushing
action of tears, saliva, urine etc. helps in expelling the invading microbes.
However, pathogens have adaptations, such as adhesion molecules for attachment to cell surfaces. They
also gain entry when there is any breach in the continuity of skin or mucous membrane. Some are
introduced by arthropod vectors or animal bites. Burns effectively remove the protection offered by skin
and may lead to rampant bacterial and fungal infections.

Innate immunity
Innate immunity employs rapidly responding cells and pre-formed soluble molecules to fight
Pathogens need to be recognized, and pathogen recognition involves engagement of a
recognition molecule expressed by host cells. These recognition molecules may be membrane
bound receptors, soluble receptors or secreted molecules.
Innate immunity recognizes microbes based on molecular patterns. These patterns are called
pathogen associated molecular patterns [PAMPs]. Their receptors on cells of innate immune
system are called pattern recognition receptors [PRRs].
PAMPs and PRRs
The presence of invading microbes is detected by ‘sentinel cells’ such as macrophages,
dendritic cells (DCs) and mast cells. Their PRRs recognize molecules that are found only in
The most famous PRRs are the Toll-Like Receptors (TLRs).
PRRs are expressed on the surfaces or inside the cytoplasm of various cells, including
phagocytes. PRR engagement causes phagocytosis of the pathogen or release of cytokines (IL-
1, IL-6, TNF-α etc.). Depending upon the type of PRR engaged, different mixtures of cytokines
are released (TLRs that recognize bacterial molecules trigger the production of cytokines that
help combat bacteria).
Some of the cytokines produced stimulate the adaptive immune system. Macrophages also
present antigenic peptides of the microbe to T helper lymphocytes. Thus innate immune system
presents bridges that link itself to the more sophisticated adaptive immune system.
Innate immune system can also recognize molecules released by damaged tissues or secreted
by stimulated sentinel cells. These molecules are called alarmins or damage associated
molecular patterns (DAMPs). They include antimicrobial proteins, heparan sulphate, some
intracellular proteins, chemokines and cytokines.
Innate immune system also recruits the complement system, a group of serum proteins
capable of killing or opsonizing microbes.
In general, it can be said that acute inflammation is the process by which innate immune
system responds to invaders, putting in to use preexisting immune mechanisms.

Adaptive immunity
Adaptive immunity (acquired immunity) is the most specialized form of immunity an organism
can develop. Identification of the pathogen is achieved through antigen recognition using
unique antigen receptors. A lymphocyte of the adaptive immune system recognizes a 3-
dimensional shape [an epitope on an antigen]. Such an epitope is likely to be unique to the
invading microorganism. Recognition of an epitope results in an immune response. The
immune response generates specialized protein molecules that bind with the antigen

[antibodies] and/or specialized lymphocytes that eliminate the host cells infected with the
microbe which carries the antigen [effector T lymphocytes]. This is called a primary response.
As a part of primary response, a group of responding cells is preserved as memory cells.
A subsequent encounter of these memory cells with the same epitope generally consists of an
immune response that is more rapid, greater in magnitude, and composed of antibodies that
bind to the antigen with greater affinity and are more effective in clearing the microbe from the
body (or a more rapid and more effective T cell response).This is called a secondary response.
The acquired immune response is very specific. It is also able to recognize very large numbers
of diverse molecules. It is able to mount an immune response against almost any antigen it is
presented with, as long as it is ‘non-self’ (originated outside the body of the host). Lymphocytes
are tolerant to self-antigens. Taken together, adaptive immunity displays four characteristic
1. Antigenic specificity- ability of the products of the immune response to recognize only the
specific antigen that initiated it.
2. Diversity- ability to mount an immune response against almost any antigen.
3. Self/non-self recognition- ability to attack only non-self-antigens and leave self-antigens alone.
4. Immunologic memory- ability to mount a secondary immune response.
The extracellular pathogens, like some bacteria, fungi and some parasites, are mainly dealt with
by one branch of adaptive immune system called humoralimmunity. This is mediated by
proteins called antibodies, which have access to most of the humors of the body (body fluids).
Another branch of adaptive immunity known as cell-mediated immunity is specialized to
destroy pathogens not accessible to antibodies, such as intracellular viruses and bacteria. Cell
mediated immunity is carried out by cytotoxic T lymphocytes (CTLs) which destroy the host cells
that harbour the pathogen.
Acquired immunity may be active or passive.
Active immunity means the immune response arises from the body of the host following an
infection, transplantation or vaccination.
Passive immunity implies that the products of an immune response mounted by another
individual are transferred to the host to offer it protection. e.g.- transfer of antibodies from
mother to offspring via placenta or colostrum, administration of hyperimmune serum or
antiserum, immunoglobulin etc.
Transfer of antigen primed lymphocytes confers a different kind of passive immunity upon the
recipient, because the transferred cells could continue to perform their functions inside the
body of the recipient. This is called an adoptive transfer and is usually done in case of

Antigens, Mitogens and Adjuvants
Agents that induce proliferation of cells are called mitogens. LPS is a B cell mitogen, whereas
phytohaemagglutinin (PHA) and concanavalin A (Con-A) are T cell mitogens. Pokeweed
mitogen is a mitogen for both B and T cells. Mitogens can activate lymphocytes regardless of
the specificity of the lymphocyte. Therefore they are polyclonal activators of B and T cells (refer
theories of antibody production).
Substances capable of inducing a specific immune response are commonly called as antigen.
Both mitogen (non-specific manner) and antigen (specific manner) can induce proliferation of
lymphocytes or cytokine production by lymphocytes in vitro.
Antigens can react with the products of that response; that is, with specific antibody or
specifically sensitized T lymphocytes, or both.
Immunogenicity- is the ability to induce a humoraland/or cell mediated immune response.
Antigenicity- is the ability to be recognized by the final products of the humoral and/or cell
mediated immune response (antibody and/or specifically sensitized T lymphocytes).
Antigenic Determinants / Epitopesare the immunologically active regions of an antigen that
binds to antigen receptors on lymphocytes (BCR, TCR)
Paratope:Combining region of antibody molecule that corresponds to epitope is called
Immunodominant epitopes:some epitopes induce a pronounced immune response than other
epitopes in a particular animal. Such epitopes are called immunodominant epitopes.

Haptens, cross reactivity, specificity

All molecules possessing the property of immunogenicity also possess the property of
antigenicity. However, the reverse is not true. Haptens are small molecules that are antigenic
but not immunogenic (e.g., penicillin, aspirin). Haptens possess the property of antigenicity but
are not capable by themselves of inducing a specific immune response. Haptens are also known
as partial antigens because of this nature.
Haptens have low molecular weight (less than 1000Da which is too small to be processed &
presented to the immune system) and that is the reason why they are not immunogenic.
However, if chemically linked to a large protein molecule (carrier protein) and the complex
(hapten-carrier conjugate/conjugated antigen/azoprotein) is injected into an animal, an
immune response is triggered.
Penicillin is a non-immunogenic molecule. But once degraded it forms a very reactive
‘Penicilloyl group’ which can bind to serum proteins (albumin) to form penicilloyl albumin
complexes which are immunogenic. This is how penicillin allergy is caused.
Immune recognition is remarkable for its specificity. The immune system is able to recognize
subtle chemical differences that distinguish one foreign molecule from another. A lymphocyte
expresses membrane antigen receptors that are specific for a distinct antigen. Antibodies
(which are secrected receptors) can distinguish between two protein molecules that differ in
only a single amino acid. This is called the antigenic specificity of the immune system.
While studying the hapten-carrier system for the first time, Karl Landsteiner observed that
antibodies against a hapten could sometimes bind to other haptens having a slightly different
chemical structure. He called this cross-reaction. Today, cross-reaction is considered to be a
property of not just haptens, but also of antigens and antibodies.

Factors Determining Immunogenicity

1. molecular size
2. chemical nature
3. foreignness of the antigen
4. structural stability
5. chemical complexity
6. shape
7. susceptibility to antigen processing
8. genotype of the recipient
9. dosage & route of administration
Molecular size
Greater than 100 kDa means better antigenic property.e.g.- serum albumin (MW 69kDa),
hemocyanin(MW 670kDa).
Substances less than 5kDa -10 kDa are poor immunogens.
Chemical nature

Proteins are better immunogens as they induce T-dependent humoral and cell mediated
responses. Polysaccharides are poorer immunogens, but are still better than lipids.
Lipids and nucleic acids of infectious agents are generally not immunogens, unless coupled to
protein or polysaccharides.
Antigens are foreign substances. Antigen from the same species is usually less antigenic than
antigen from other species. Likewise, antigen from related species is less antigenic than antigen
from unrelated species. The greater the phylogenetic distance between two species, the greater
the response. e.g., bovine serum albumin is not immunogenic to cow but is highly
immunogenic to rabbits.
Structural stability
Cell surface receptors of the immune system must recognize its shape, and highly flexible
molecules are poor antigens.
e.g., Gelatin
Antigen should be stable & rigid. Polysaccharides like starch & glycogen are poor antigen
because they are rapidly degraded.
Lipids are also poor antigens because of relative simplicity, structural instability & rapid
Mammalian nucleic acids are poor antigens because of simplicity & high flexibility.
Chemical complexity
Substances that are composed of repeating units of single amino acid lack immunogenicity but
substances composed of repeating units of two or more different amino acids are
Addition of aromatic amino acids like tyrosine or phenyl alanine seems to improve
Susceptibility to antigen processing
Macromolecules that cannot be degraded & presented with MHC molecules are poor antigens.
Degradative enzymes within APCs degrade proteins containing L-amino acids, but polymers of
D-amino acids cannot be processed and therefore are poor antigens.
Large insoluble macromolecules (particles) are more immunogenic than soluble ones because
they are readily phagocytosed & processed.
Genotype of the Recipient
Genetic control of the immune response is confined to the genes within MHC. MHC gene
diversity of the host is important.
Antigenicity of an antigen is also influenced by the genes encoding B cells & T cells receptors as
well as genes encoding various proteins involved in immune regulatory mechanism of the host.
Immunogen dosage & route of administration
Insufficient dose will not stimulate an immune response. Excessively high dose also fail to
induce a response.
Both very low dose and very high dose of antigen may cause lymphocytes to enter a non-
responsive state (anergy).

Types of antigens based on antigenic specificity

Species-Specific Antigen: An antigen specific to a species.
Organ-specific antigen:The antigen is specific to a particular organ in a species.
Tumor-specific antigen: Antigens found only in tumor cells.
Alloantigens (Isoantigens):allogeneic antigen occurring in some but not all individuals of the same
species e.g., Major histocompatibility (MHC) antigens & human blood antigens. All individuals do not
possess all the MHC antigens or blood group antigens.
Syngeneic antigen:Antigen present in individuals of the same genetic makeup, e.g., monozygotic
twins(two offsprings from a single fertilized ovum). Such twins will be antigenically very closely related
and therefore, transplantation from one twin to the other is less likely to be rejected.
Autoantigen: self-antigen is an autoantigen, a normal constituent of the body against which antibodies
are formed during autoimmune responses (autoantibodies).
The autoantigens may be those sequestered in tissues like lens of the eye and thyroid and therefore, not
recognized as self. Should such tissue be exposed to the lymphoreticular system during adult life, an
autoimmune response would be elicited.

Super antigens
Antigens that can crosslink MHC-II molecules of APCs and TCR of T cells by binding outside the antigen
binding grooves of both molecules. Superantigens can activate several T cells because they are specific

for a variable region sequence of the TCR, instead of a hypervariable region as normal antigens are.
Although less than 0.01% of T cells respond to a given conventional antigen, 5% or more of the T-cell
population can respond to a given superantigen. Superantigens have been implicated in bacterial toxic
shock and food poisoning.
Heterophile antigen
Antigens of identical nature present in the cells of some bacterial species & in the tissues of different
animals. Antibody formed against one antigen will cross-react with other antigen. Cross- reactivity
occurs if two different antigens share an identical or similar epitope.
An example is Forssman antigen. It is present in the cell wall of many bacterial species such as
Streptococcus & also on the surface of RBC of horses, sheep, cats & mice.
Weil-Felix reaction is an agglutination test used to detect antibody in human beings against some
Rickettsial organisms using Proteus antigen.
A heterophile antigen is found in some strains of P. vulgaris and also in certain Rickettsial spps. (e.g.,
Rickettsia rickettsii). During rickettsial infections in humans, antibodies are formed, which could be
detected using agglutination test using one or more strains of P. vulgaris. This forms the basis of Weil-
Felix reaction.

Types of antigens based on T cell response

1. T-dependent antigen- An antigen that requires helper T-cell help to generate a humoral immune
response by a B cell. Most antigens are T-dependent. When a B cell has its BCR engaged by a soluble
antigen, the B cell can engulf that antigen, process it and present its peptides on MHC-II to any available
activated or memory Th cells specific for the same antigen. The Th cell would then release the necessary
cytokines that would activate the B cell completely and induce formation of memory and plasma cells
from that B cell.
2. T-independent antigen- antigen that elicits an IgM response without the participation of T
lymphocytes. T independent antigens are of 2 types.
a. TI-1 antigens cause activation of several B cells independent of the specificity of their BCRs. E.g., LPS,
Salmonella flagellin and pneumococcal polysaccharide. LPS, at high doses, acts as a mitogen of B cells
by binding to TLR4 (Toll-Like Receptor 4). This might result in a huge polyclonal response and is seen
associated with septic shock. The antibodies secreted are not specific for the TI-1 antigen.
At lower doses, TI-1 antigens work by cross linking PRRs and BCRs of the few B cells that would
recognize these antigens. Antibody responses are specific for the TI-1 antigen.
b. TI-2 antigens work in a different way (not as mitogens). They can cross link large numbers of BCR on
the same B cell. They can indirectly bind complement receptors on the B cell as well. These interactions,
together with some stimulation from macrophages and some T cells result in activation of the B cell and
secretion of antibodies.

Blood group antigens

Antigens found on the surface of red blood cells are called blood group antigens or erythrocyte
Blood groups
1. Humans- ABO system contains 4 blood groups & determined based on the presence or absence of 2
distinct antigens (A & B).
RBCs of group A carry antigen A.
RBCs of group B carry antigen B.
RBCs of group AB carry antigen A and antigen B.

Species Blood group system Serological test for

detecting blood group

Bovine EAA, B, C, F, J, L, M, R, S, Z, I Hemolytic test

Sheep EAA, B, C, D, M, R Hemolytic test, Agglutination test(D Only)

pigs EAA, Hemolytic test, Agglutination test, Antiglobulin

B,C,D,E,F,G,H,I,J,K,L,M,N,O,P test

Horses EAA,C,D,K,P,Q,U Hemolytic test,Agglutination test

Dogs DEA 1.1, 1.2, 3, 4, 5, 6, 7 & 8 Hemolytic test, Agglutination test

Cat AB (1 system)- A, B, or AB Hemolytic test, Agglutination test

RBCs of group O have neither A or B antigen.

A group has anti-B antibody.
B group has anti-A antibody.
AB group has neither anti-A antibody or anti-B antibody.

O group has both anti-A antibody & anti- B antibody.
If RBCs are mixed with serum containing cognate antibody, agglutination occurs.
O group has neither A or B antigen. RBCs from this group of people are not agglutinated by serum of
any other blood groups. Thus O groups are used as universal donor.
Serum from O group contain both anti- A &anti- B antibodies and agglutinate erythrocytes of all
other groups i.e., they can receive blood from group O only.
AB group do not have either anti-A or anti-B antibodies and are considered as universal recipient.

Blood transfusion
If red cells of the donor are identical to those of the recipient, no immune response results.
If however, the recipient possesses preexisting antibodies to donor RBC antigens, they will be attacked
immediately. These preexisting antibodies are usually of IgM class. These antibodies agglutination of
RBCs or haemolysis or stimulate opsonization & phagocytosis of the transfused cells.
If the recipient does not possess preexisting antibodies to donor RBC antigen, nothing happens until the
red cells stimulate an immune response in the recipient. As antibodies are produced, the donor cells are
A second transfusion with identical foreign cells results in their immediate destruction.
Rapid destruction of large numbers of foreign red cells will lead to hypersensitivity reactions & even
Transfusion reaction can be prevented by prior testing of the recipient and the donor by blood typing or
cross-matching. Cross-matching will detect incompatibilities between the donor and recipient that will
not be evident on blood typing. There are two types of cross-matches: Major cross-match and Minor
The major crossmatch involves testing the patient’s serum with donor cells to determine whether the
patient has an antibody which may cause a hemolytic transfusion reaction or decreased cell survival of
donor cells. This is the most important cross-match.
The minor crossmatch involves testing the patient’s cells with donor plasma to determine whether
there is an antibody in the donor’s plasma directed against an antigen on the patient’s cells.
Any evidence of agglutination or haemolysis is taken as incompatibility.
Immunization is the process of eliciting immunity against a disease-causing pathogen. Vaccination is a
method of immunization. Ideally, vaccination should result in production of both humoral and cell
mediated immune responses and formation of memory cells.
Adjuvants are defined as substances that enhance the immunogenicity of antigens. Adjuvants are
administered along with vaccines (immunogens) in order to improve the speed and magnitude of the
host immune response. Adjuvants help reduce the dose of immunogen and are essential for generation
of long term immunity against poor antigens such as killed vaccines, soluble antigens etc.
For example, tetanus toxoid is not immunogenic in the absence of adjuvants, and so tetanus toxoid
vaccines contain alum in the form of noncrystalline gels. Pertussis toxin has adjuvant properties in its
own right and, when given as DTP, not only protects against whooping cough but also acts as an
additional adjuvant for the other two toxoids.
Depending on their mode of action, adjuvants are classified in to 4 groups as follows:

Depot adjuvants
They help delay the elimination of antigens and thus permit the immune response to last longer.
Examples include aluminium salts (Al. hydroxide, Al. phosphate and Al. potassium sulphate a.k.a. alum),
calcium phosphate and Freund's incomplete adjuvant (FIA or IFA). Aluminium salts induce formation of a
macrophage-rich granuloma from within which the antigen slowly leaks out and thus persists for long
duration. Alum induces production of uric acid, which is a potent DAMP. Alum also activates
inflammatory reactions. Therefore, it is not just a depot forming adjuvant. FIA contains light mineral oil,
with which, the antigen is mixed to form an emulsion, which is injected. The light mineral oil stimulates
inflammation and granuloma formation. The antigen slowly leaches from the aqueous phase of the

Particulate adjuvants
The immune system traps and processes particulate antigens more efficiently than soluble antigens.
Particulate adjuvants incorporate soluble antigens in to readily phagocytosable particles. They include
emulsions, microparticles, immune stimulating complexes (ISCOMs) and liposomes (lipid based

Immunostimulatory adjuvants
They promote cytokine production. PAMPs that activate DCs and macrophages act as adjuvants.

Saponins are detergents derived from the bark of the soapbark tree (Quillajasaponaria). Quil A is a
mixture of saponins used as immunostimulatoryadjuvant.
Cytokines like IL-12 may be used as a Th1 cell stimulator.
Many adjuvants seem to work by triggering the innate viral and bacterial sensor pathways, via TLRs and
Monophosphoryl lipid Ais an LPS derivative that retains the adjuvant effects but has much lower toxicity
than LPS.
In natural infections, some bacterial proteins (cholera toxin, E. coli heat-labile enterotoxin, and pertussis
toxin) act as adjuvants to stimulate mucosal immune responses.

Combined adjuvant
Very powerful adjuvants can be constructed by combining a particulate or depot adjuvant with an
immunostimulatory agent. ISCOMs, which contain saponin, are a combination of particulate and
immunostimulatory adjuvants. FIA with added killed Mycobacterium tuberculosis is termed Freund's
complete adjuvant (FCA or CFA). It can form a depot and the tubercle bacilli contain muramyl dipeptide
which activates macrophages and Dcs.
B cell receptors are immunoglobulins. If a B cell encounters an antigen that can bind to its receptors, it
will, with appropriate co-stimulation, start secretingantibodies. An antibody is a secreted B cell receptor
which has the ability to bind specifically with the antigen that triggered its secretion.
During an immune response, a second selection process occurs in which B cell receptors are modified by
random somatic mutation so that the specificity of antibodies increases with time.
When serum was subjected to electrophoresisby Tiselius and Kabat (1939), the proteins formed 4
fractions. The one with the highest electrophoretic mobility (most negatively charged) was identified as
albumin and the next three were called α, β and γ globulins in the decreasing order of electrophoretic
mobility. Most immunoglobulins are found in the γ globulin fraction with the exception of IgM, which
migrates among the β globulins.

Structure of antibodies
Immunoglobulins are glycoproteins. Carbohydrates account for up to one-fifths of an antibody’s mass.
However, their biological properties are determined by their polypeptide components.
Each immunoglobulin molecule (monomer) is made up of two different types of polypeptides. The larger
heavy (H) chains are roughly twice as large as the smaller light (L) chains. Every immunoglobulin
monomer contains two heavy chains and two light chains and therefore, immunoglobulinscan be
represented by the general formula (H2L2)n. The chains are held together by inter and intra-chain
disulphide bonds and noncovalent forces to form a bilaterally symmetrical structure.
The heavy and light polypeptide chains are both composed of folded globular domains. There are only
two types of light chains: the kappa (κ) and the lambda (λ). An immunoglobulin molecule will have
either the κ chain or the λ chain, but never both. Each light chain has two domains only.

Heavy chains may have a variable number of domains. Heavy chains are named mu (µ), delta (δ),
gamma (γ), alpha (α) and epsilon (ε). The immunoglobulin molecule is named after the type of heavy
chain it contains (as a given immunoglobulin molecule will contain only one type of heavy chain). Thus
(Ig standing for Immunoglobulin) we have IgM (µ heavy chain), IgD (δ), IgG (γ), IgA (α) and IgE (ε).
These are called the different classes (isotypes) of immunoglobulins. Class switching is the change in
the antibody class produced by the B cell [B cells produce IgM initially and later start producing IgG or
IgA or IgE of the same antigenic specificity. Only the heavy chain changes. This is class switching].
Delta, α and γ heavy chains have 4 domains each with an extended region in between the 2nd and 3rd
domains. This region is about 12 amino acids long and contains many hydrophilic and proline residues.
This region imparts great flexibility to the molecule and hence is called the hinge region. The µ chains
of IgM do not possess a hinge region, but have an additional domain. The ε chain also has 5 domains.

The antibody molecule is shaped like the letter Y. The tail of the Y (called the Fc region) is formed from
paired heavy chains. The arms of the Y (called the Fab regions) are formed by paired light and heavy
chains and they bind antigens.The antigen binding sites are formed by the grooves between light and
heavy chains. Thus each antibody has two identical antigen binding sites.
The N-terminal domains of each chain are called the variable (V) domain, because of the sequence
variability shown by these domains between individual antibody molecules. This variability is due to
the difference in antigen specificity. In other words, this variability is the reason for diversity in
antigenic specificity. The other domains (one each in light chains, 3 each in δ, α and γ chains and 4 each
in µ and ε chains) are called the constant (C) domains. Hence each light chain has a VL and a CL domain;
heavy chains have VH and CH1, CH2, CH3 and depending upon the class, CH4 domains, plus one hinge
region (absent in µ chain). [CH1 is the second from N-terminal; CH3 and CH4 are the ones nearest the
cell membrane].
Papain digestion of immunoglobulin molecules generates three fragments, because papain digests the
heavy chains at the N-terminal side of the disulphide bonds. Two of these fragments are identical and
bind the same antigen. They are called the Fab(Fragment- antigen binding) fragments and represent
the arms of the ‘Y’. The third fragment was found to crystallize on storage, so was named the Fc
(Fragment-crystallizable) fragment and represents the tail of the ‘Y’. The Fc corresponds to the paired
CH2 and CH3 (and CH4 in IgM and IgE) domains and is the part of the antibody that carries out its
biological functions. Pepsin cuts on the C-terminal side of the disulphide bonds and produces only one
fragment, called the F(ab’)2 fragment.
When the amino acid sequences of a large number of V domains from light and heavy chains are
examined in detail, it becomes apparent that their sequence variation is largely confined to three small
regions, each consisting of 6 to 10 amino acids, within the variable domain. These regions are said to be
hypervariable. Between the hypervariable regions are relatively constant sequences of amino acids
called framework regions. The three hypervariable regions on each chain determine the shape of the
antigen binding site and so determine the specificity of antigen binding. Since the shape of the antibody
binding site is complementary to the conformation of the antigenic determinant (the epitope), the
hypervariable sequences are also called complementarity determining regions (CDRs).


Different classes of immunoglobulins

IgG: IgG is made and secreted by plasma cells in the spleen, lymph nodes and bone marrow. It is the
immunoglobulin found in the highest concentration in the blood and for this reason plays the major role
in antibody mediated defense mechanisms. It has a molecular weight of about 180 kDa with two
identical light chains and two identical γ heavy chains. Because it is the smallest of the immunoglobulin
molecules, IgG can escape from blood vessels more easily than can the others. Hence, it is present at
sites of inflammation. IgG can bind to specific antigens on bacterial surfaces and cause clumping and
opsonisation. IgG activates the classical complement pathway with its CH2 domain, but only when
sufficient molecules have accumulated in a correct configuration on the antigenic surface. It binds to its
Fc receptors also through CH2 domain. IgG has 4 subclasses namely, IgG1, IgG2, IgG3 and IgG4.
IgM:IgM is produced in spleen, lymph nodes and bone marrow. It occurs in the second highest
concentration after IgG in serum. It acts as a BCR on a B cell surface in its monomeric form (180 kDa).
When secreted, it is seen as 5 or 6 monomers linked by disulphide bonds in a circular fashion. Its total
molecular weight is 900 kDa. A small polypeptide chain of 15 kDa (J chain) joins two of the units to
complete the circle. Each IgM monomer contains two κ or λ light chains and two µ heavy chains. The µ
chains have an additional constant domain (CH4) and an additional 20 amino acid segment on their C-
terminus, but no hinge region. The complement activation site is located on the CH4 domain. IgM is the
major immunoglobulin produced during a primary immune response. It is also produced in secondary
responses, but at a lower level than IgG. IgM is more efficient on a molar basis than IgG at complement
activation, opsonisation, virus neutralization and agglutination. Because they are very large, IgM
molecules rarely enter tissue fluids.
IgA: IgA is secreted by plasma cells located under body surfaces and mucous membranes, like in the
walls of the intestine, respiratory tract, urinary system, skin and mammary gland. Its serum concentration
in most mammals is usually lower than that of IgM. IgA monomers have a molecular weight of 150 kDa,
but are normally secreted as dimers. In dimeric IgA two molecules are joined by a J chain. IgA produced
in body surfaces passes through epithelial cells into external secretions. The epithelial cells help in
transport of IgA by binding it with a surface receptor called the polymeric immunoglobulin receptor
(pIgR). A part of this receptor stays with the IgA molecule and protects it from destruction by proteases.
This part is called the secretory component and the complex is called secretory IgA. Secretory IgA is
the major immunoglobulin in the external secretions of nonruminants. IgA does not activate the classical
complement pathway, nor does it act as opsonin. It can agglutinate particulate antigens and neutralize
viruses. Most animal species have 2 IgA subclasses.
IgE: IgE is also made by plasma cells located beneath body surfaces. It has 4 constant domains in its ε
heavy chains and a molecular weight of 190 kDa. It is present in extremely low concentration in serum. It
triggers acute inflammation by acting as a signal transducing molecule as explained below. IgE
molecules are seen bound to FcεRI on mast cells and basophils. When antigen cross links this bound IgE,
it triggers the rapid release of inflammatory molecules from the cell. The resulting acute inflammation
enhances local defenses and helps eliminate the invader. IgE mediates Type I hypersensitivity reactions
and is largely responsible for immunity to parasitic worms. It has the shortest half-life of all
immunoglobulins and is readily destroyed by mild heat treatment.

IgD: IgD is a BCR mainly found attached to B cells and very little is secreted into the blood. IgD is
evolutionarily labile and shows many variations in structure. It has a molecular weight of about 170 kDa.
When an antibody is considered as an antigen, several levels of variation come to our notice.
The antigenic variation between antibodies due to difference in isotypes is called isotypic variation.
In addition to this, individual animals show inherited variations in immunoglobulin amino acid
sequences due to existence of different alleles. These variations are called allotypes.
The third group of structural variants found in immunoglobulins result from the variations in the amino
acid sequences within the variable domains on light and heavy chains. These variants are called
idiotopes. The collection of idiotopes on an immunoglobulin is called its idiotype.

Antigen-antibody binding
When an antigen and antibody combine, they interact through the chemical groups on the surface of the antigen and on the
CDRs antibody. The bonds are exclusively noncovalent, and hence form only when the two molecules approach each other very
closely. So the strongest binding occurs when the shape of the antigen and the shape of the receptor conform to each other.
The major bonds formed between an antigen and its receptor, are hydrophobic. When antigen and antibody molecules come
together, they exclude water molecules from the area of contact (the bond can be likened to two wet glass microscope slides
stuck together). A second type of binding between the antigen and antibody is mediated by hydrogen bonds. When a
hydrogen atom covalently bound to one electronegative atom (e.g., an –OH group) approaches another electronegative atom
(e.g., an O=C- group), the hydrogen is shared between the two electronegative atoms. This is called a hydrogen bond. The
major hydrogen bonds formed in antigen-receptor interaction are O-H-O, N-H-N and O-H-N. Electrostatic bonds formed
between oppositely charged amino acids may also contribute to antigen-receptor binding. In addition to these forces, a
nonspecific attractive force, called a van der Waals force, that becomes operative when two atoms approach very closely, also
contributes to the strength of antigen-antibody binding. Each bond is relatively weak in itself, but collectively the bonds may
have a significant binding strength. Antigens can bind to receptors when they fit less than perfectly, but the strength of binding
will be reduced.

Antibody effector functions

1. Neutralization: neutralization of a toxin involves binding of the antibody to the component of the toxin
that attaches to a cellular receptor. This prevents attachment of the toxin to the receptor and the toxin
becomes ineffective. Likewise, cell entry of a virus may be prevented by an antibody that by binding to
its proteins. Consequently, the virus becomes unable to attach to a receptor on its target cell and thus
viral infectivity is lost.
In the situations depicted here, the toxin and the virus have been neutralized.
2. Prevention of bacterial adherence: Many bacteria have cell surface adhesins that enable them to bind
to host cell surfaces (e.g., pilin). Antibodies against these adhesins can inhibit this adhesive reaction and
prevent infection. IgA antibodies on mucosal surfaces and IgG in tissues function in this manner.
3. Complement activation: Antibody-antigen complexes can activate the complement system through the
classical pathway, causing the formation of a membrane attack complex on the antigenic surface. This
causes damage to the bacterial cell wall. Complement component C3b further acts as opsonin to
promote phagocytosis of the bacteria.
4. Opsonisation: Phagocytes such as macrophages, neutrophils and dendritic cells have Fc receptors and
can capture and ingest particles and miroorganisms that have been coated with antibodies.
5. Antibody-dependent cell-mediated cytotoxicity (ADCC): NK cells express FcγRIII (IgG receptor 3) and
therefore capable of recognizing cells coated with IgG molecules (e.g., a cell infected with an enveloped
virus that buds from the plasma membrane may be targeted for ADCC by antibodies directed against
envelope proteins of that virus) and destroying them. ADCC represents a mechanism by which an
antibody can direct an antigen-specific attack by an effector cell that lacks antigen specificity.
6. Resistance against parasites: Acute inflammation caused by mast cell degranulation and further
recruitment of basophils and eosinophils is mediated by IgE. It is the mainstay of anti-parasite
(helminthes, protozoa and some ticks) immune responses. Mast cells express FcεRI which binds free
monomeric IgE. When a specific antigen (or even an allergen) cross-links the receptor bound IgE
molecules, mast cell degranulation is triggered.

Theories of antibody formation

Numerous interesting theories have been formulated to explain the production of antibodies. The vast
majority of them have been found to be erroneous.

1. Side chain theory (Paul Ehrlich, 1900): According to this theory, cells possessed on their surfaces a wide
variety of side chains that were used to bring nutrients into the cell. When toxic substances blocked one
of these side chains through an accidental affinity, the cell responded by making large number of that
particular side chains. Some of these side chains spilled into the blood and functioned as circulating
2. Template theory (Breinl and Haurowitz, 1930): This theory viewed antibodies as being formed directly on
antigens which functioned as templates.
3. Clonal selection theory (Niels Jerne, David Talmadge, and Frank MacFarlane Burnet, 1950s): According
to this theory, an individual lymphocyte expresses membrane receptors that are specific for a distinct
antigen. This unique receptor specificity is determined before the lymphocyte is exposed to the antigen.
Binding of antigen to its specific receptor activates the cell, causing it to proliferate into a clone of cells
that have the same immunologic specificity as the parent cell.
The clonal selection theory has been further refined and is now accepted as the underlying paradigm of
modern immunology.

Monoclonal antibodies and hybridoma technology
Most antigens possess multiple epitopes and therefore induce proliferation and differentiation of a
variety of B-cell clones, each derived from a B cell that recognizes a particular epitope. The resulting
serum antibodies are heterogeneous, comprising a mixture of antibodies, each specific for one epitope.
Such a polyclonal antibody response facilitates the localization, phagocytosis, and complement-
mediated lysis of antigen; it thus has clear advantages for the organism in vivo. However, for most
research, diagnostic, and therapeutic purposes, monoclonal antibodies (mAbs), derived from a single
clone and thus specific for a single epitope, are preferable.
Direct biochemical purification of a monoclonal antibody from a polyclonal antibody preparation is not
feasible. In 1975, Georges Köhler and Cesar Milstein devised a method for preparing monoclonal
antibody, which quickly became one of immunology’s key technologies. By fusing a normal activated,
antibody-producing B cell with a myeloma cell (a cancerous plasma cell), they were able to generate a
hybrid cell, called a hybridomathat possessed the immortality of the myeloma cell and secreted the
antibody produced by the B cell. The resulting clones of hybridoma cells, which secrete large quantities
of monoclonal antibody, can be cultured indefinitely. The significance of the work by Köhler and Milstein
was acknowledged when each was awarded a Nobel Prize.

How to make a hybridoma…?

The idea is to selectively grow hybridoma cells from the mixture of hybridoma, unfused myeloma and
plasma cells.

1. Fusion of myeloma and plasma cells using polyethylene glycol or Sendai virus:
a. Mice are given multiple doses of the immunogen against which we want a monoclonal
b. Kill the mouse and take spleen. Isolate lymphocytes.
c. Fuse lymphocytes with cells from a myeloma cell line that does not synthesize an
2. Selection:
• The mixture of cells is grown in a medium containing hypoxanthine, aminopterin and
thymidine (HAT medium).
• Hypoxanthine is a purine analogue and thymidine is a pyrimidine. Aminopterin blocks de
novo DNA synthesis in the cells grown in HAT medium.
• Cells such as plasma cells and hybridoma cells possess HGPRT and Thymidine Kinase
enzymes. These enzymes help them survive by synthesizing DNA through an alternate
pathway, called the salvage pathway.
• Myeloma cells used in hybridoma production lack these enzymes. Consequently, they are
unable to grow in HAT medium.
• Plasma cells, though able to use salvage pathway, die off in a short time since they have
a limited life-span.
• Only hybridoma cells survive a
• There will be a screening process to identify cells that produce mAbs specific for the
original immunogen.

Uses of mAbs:
a. In diagnostic reagents- such as pregnancy detection kits, measuring blood levels of drugs, etc.
b. Labeled mAbs can be used to locate specific types of cells, tissues or antigens. e.g., tumours, subcellular
components etc. (refer immunofluorescence)
c. Immunotoxins are anti-tumour mAbs coupled with toxins such as ricin, shigella toxin, diphtheria toxin
etc. The mAbs will specifically bind tumour cells and the immunotoxin will be taken up by endocytosis.
Once inside the cell, the toxin will be released leading to the death of the cell.
d. Abzymes are mAbs that are raised against some substrates. They can catalyse some reactions of those
substrates (antibody+enzyme = abzyme).
e. mAbs may be used to deplete specific cell types in vivo, e.g., Th cells for preventing graft rejection.
f. mAbs may be used to stimulate certain cells through their receptors in vitro.
a. Production of chimeric antibodies is through hybridoma technology. [When mAbs raised in mice are
used to treat humans, anti-mouse antibodies are formed in the recipient. Further treatment with the
same mAbs might cause type III hypersensitivity (serum sickness). The variable regions encoding the
antigen-recognition determinants from mouse antibody can be spliced onto the constant and Fc regions
of human IgG by gene manipulation. Antibodies of this type are called chimeric antibodies. They are
considerably less immunogenic when administered in humans].
Newer methods are aimed at generating fully human monoclonal antibodies directly from human cells
through the use of viral transformation of human primary B-cell lines or antibody-secreting
plasmablasts, or by generating human B-cell hybridomas.
Xenohybridomas are those in which antibody producing cells from a species other than mouse are
fused with murine myeloma cells.

Serology is the science of measuring antibody or antigen inbody fluids.
Three groups of immunological techniques are used to detect and measure antigen- antibody reaction;
these are:
1. Primary binding tests
2. Secondary binding tests and
3. Tertiary binding tests

Primary binding tests

Primary binding tests are tests that directly measure the binding of antigen and antibody (i.e.; directly
measure or visualize the immune complex). They are the most sensitivetechniques in terms of the
amount of detectable antigen or antibody.
In order to measure these reactions, one of the reactants (either antigen or antibody) must be labelled
using radioisotopes, fluorescent dyes, colloidal metals or enzymes.

Radioimmunoassay (RIA) and enzyme-linked

immunosorbent assay (ELISA
Radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) work on the same principle,
but the means of detecting specific binding is different. Radioimmunoassays are commonly used to
measure the levels of hormones in blood and tissue fluids, while ELISA assays are frequently used in viral
diagnostics. For both these methods we need a pure preparation of a known antigen or antibody.

General procedure of direct RIA/ELISA:

The antigen is attached to a solid support (the wells of a plastic multiwell plate, e.g.) which will adsorb a
certain amount of any protein. Following this, further nonspecific adsorption is blocked. The labeled
antibody is allowed to bind to the antigen for some time after which, all unbound antibody is washed
away. The bound label is quantified.

General procedure of indirect tests

The known, pure antigen is coated in wells and further nonspecific adsorption of proteins is blocked. The
test serum dilution is allowed to bind to the antigen for some time. If specific antibody (unlabeled
primary antibody) is present in the test serum, it will bind to the antigen at this stage. Any unbound
antibody is washed away. Labeled secondary antibody (anti-immunoglobulin that binds to the primary
antibody) is added, allowed to bind, and unbound antibody is washed away. The bound label is
The use of a secondary antibody also amplifies the signal, because at least two molecules of the
secondary antibody are able to bind to each primary antibody.
In RIA for detection of an antigen, a pure antibody against that antigen is radioactively labeled, usually
with 125I. RIA are extremely sensitive, but are expensive, hazardous and poses disposal probelms.
Radioallergosorbent test (RAST): This test measures specific IgE in the serum of allergic animals.
Antigen-impregnated cellulose disks are immersed in test serum to facilitate antibody binding. After
washing to remove unbound antibody, the disk is immersed in a solution containing radiolabeled anti-
IgE. After further washing, the amount of radioactivity bound to the disk is measured.
Competitive RIA: Based on the principle that unlabeled antigen will displace radiolabeled antigen from
immune complexes. In this test, a radiolabeled antigen is allowed to bind to the specific antibody and
the resulting immune complexes removed as precipitates. Any radioactivity remaining in solution is due
to unbound labeled antigen. This radioactivity would increase if some unlabeled antigen was mixed with
the labeled antigen prior to addition of antibody. This is because the unlabeled antigen competes with
the labeled antigen for the antibody. Using known quantities of unlabeled antigen, a standard curve of
residual radioactivity is first constructed. The amount of antigen in a test sample may be measured by
reference to this standard curve.
The competitive binding assay: used for detecting antibody in test serum. This is done as follows:

1. Test sample is mixed with a labeled specific antibody to the antigen of interest.
2. The antigen is coated on a surface and reacted with this mixture.
3. After washing, the labeled antibody is measured.
4. If test serum contains antibody specific to the antigen of interest, there will be less bound label
5. Therefore, quantification of bound label will indicate quantity of antibody in test serum
For ELISA, instead of a radioisotope, an enzyme is linked chemically to the pure antibody. As a result of
the action of the enzyme, a colourless substrate is converted into a coloured or luminescent product.
The change can be read directly in the reaction tray. Alkaline phosphatase (ALP) and Horseradish
peroxidase (HRP) are widely used enzymes for ELISA. Substrates of HRP include TMB, ABTS, DAB
(chromogenic), luminal, luciferin (chemiluminescent) etc. ALP substrates most commonly used are
pNPP, 4-MUP (chromogenic) and AMPPD (chemiluminescent). ELISA is preferred over RIA because data
collection is easier and also there is no radioactivity hazard.

ELISA can also be carried out with unlabeled antibody stuck to the plates and labeled antigen added.
A capture or sandwich ELISA is used to detect secreted products such as cytokines:

• Antigen-specific antibodies are bound to the plate.

• These “capture antibodies” bind antigen even when antigen is present in very low concentrations.
• A separate labeled antibody (detection antibody) that recognizes a different epitope of the
same antigen is then used to detect the bound antigen.
Labeled-antigen ELISA: the antigen is bound to the microwell before testing. Then the test serum is
added, followed by labeled antigen. If antibodies are present in the serum, the labeled antigen is bound
and the label can be measured.
Competitive ELISA: microwell is coated with specific antibody. Test sample and enzyme labeled antigen
are then placed in the well together, where the antigens compete for the antibody binding sites. The
amount of labeled antigen bound to the microwell is inversely related to the concentration of antigen in
the test sample.

Western blotting
It is a primary binding test performed to identify protein antigens in a complex mixture.

• It involves electrophoretic separation of individual proteins. The proteins separate as

individual bands in a polyacrylamide gel.
• These bands are blotted (transferred electrophoretically) on to a nitrocellulose membrane.
• The membrane is first incubated in specific antiserum.
• The membrane is washed
• Enzyme-labeled antiglobulin solution is added.
• After further washing, substrate is added
• A colour develops in the bands of target protein.
• if isotope labeled antiglobulin is used, an autoradiograph is made.
Dot blot: a variant of Western blot. Test samples that may contain the antigen are blotted on to a
nitrocellulose membrane as dots. After treatment with antibody and substrate, the dots that develop a
colour are considered as positive samples.
Antibody microarray: many different monoclonal antibodies are blotted as dots on a single sheet of
nitrocellulose. This is then exposed to a complex labeled antigen mixture and after washing and
developing, the relative concentration of many different antigens can be visualized.
This involves the use of enzyme conjugated immunoglobulins or antiglobulins for location of specific
antigens in tissue sections. Horseradish peroxidase (HRP) is the most widely used label. In the direct test,
the tissue is treated with the enzyme labeled antibody. After washing, the tissue is incubated in a
solution of the appropriate substrate. Bound antibody is detected by the development of a brown stain
at the site of antibody binding (where the substrate has been converted to a brown insoluble stain).

It is the collective term for primary binding tests that utilizes fluorescent dyes as labels. FITC (fluorescein
isothiocyanate), PE (phycoerythrin), rhodamine and AlexaFlour dyes are some of the most commonly
used fluorophores. FITC, e.g., absorbs light at 495 nm wavelength and emits green fluorescence (519
nm). This fluorescence is observed under a fluorescence microscope or is measured using a flow

Direct Fluorescent Antibody Test (Direct FAT)

Used to identify the presence of an antigen in a tissue sample or cell suspension. Antibody directed
against a specific antigen is directly labeled with a fluorophore. This antibody is used to stain a tissue
section, smear or cell suspension, followed by washing. Any bound fluorescence detected and is
indicative of the presence of the test antigen. Bacterial, viral or other antigens may be detected by direct
FAT (e.g., detection of rabies virus in the brains of infected animals).

Indirect FAT
This is used to measure antibodies in serum or to identify specific antigens in tissues or cell cultures.
Antigen is taken as a smear on a slide. This is incubated in serum suspected of containing antibodies to
that antigen. The serum is then washed off, leaving any specific antibody bound to the antigen. These
bound antibodies are visualized by incubating the smear in fluorophore-labeled antiglobulin. After
washing off unbound labeled antiglobulin, the slide is examined for bound fluorescence which would
indicate the presence of antibody of interest in test serum. Indirect FAT has the advantage that since
several labeled antiglobulin molecules will bind to each antibody, the fluorescence will be considerably
Other primary binding tests include immunofiltration and immunochromatography.

Secondary binding tests
The reactions between antigens and antibodies are followed by a secondary reaction. This include
precipitation, agglutination and complement mediated lysis of RBCs. Serological assays making use of
these secondary reactions are called secondary binding tests.

Precipitation reactions
The precipitation/precipitin reaction is affected by the valence of the antibody and the valence of the
antigen (the number of binding sites that each antibody has for antigen; the maximum number of
antibodies that can be bound by an antigen molecule or particle at any one time.). The valence of both
the antibodies and the antigen must be two or greater for any precipitation to occur. When large
numbers of antigens are cross linked by antibodies, the resulting complex becomes so large that it falls
out of solution as a precipitate. This precipitate can be centrifuged and the antigen separated from the
precipitating antibodies by biochemical means.
1. To purify antigenic molecules from a heterogeneous mixture of soluble molecules.
2. To remove particular antigens from a solution.
3. To identify an antigen using known antibody, or identify an antibody using known antigen as part of
disease diagnosis.
Immunoprecipitation occurs only when the antibody and antigen concentrations are more or less
equivalent (in optimal proportions). At either antigen or antibody excess, precipitation is low.

When antigen and antibody diffuse toward one another in a gel matrix, it is called immunodiffusion. It
is rapid, easy to perform and surprisingly accurate. In the Ouchterlony method, the most frequently
employed variation of gel immunodiffusion, both antigen and antibody diffuse radially from wells,
thereby establishing a concentration gradient. Where optimal concentrations of antibody and antigen
are reached (equivalence), precipitin line forms in the gel. However, more sensitive serological
methods are available.

Rocket Immunoelectrophoresis
In this test, antigen is placed in wells punched out of an agar gel to which antibody has been
incorporated. The antigen is electrophoresed into the gel. Precipitin arcs are produced which resemble
rockets (The test is done at a pH at which the antibody molecules do not move appreciably). The height
of the rocket is proportional to the concentration of the antigen, since when the concentration of
antigen is low it will be all immobilized by the antib
antibody before it moves too far. In contrast when the
amount of antigen is high, it will move farther
before getting precipitated.
A standard curve can be generated using known
concentrations of antigen, and from this curve the
concentration of the given antigen
igen in an unknown
sample can be calculated. This test is
quantitative. It is commonly used for the
quantification of various serum proteins.

Agglutination Reactions
The cross-linking
linking that occurs between di
di-or multivalent antibodies and multivalent bacterial
bacteri or other
particulateantigens can result in visible clumping of the cells. This clumping reaction is called
agglutination,, and antibodies that produce such reactions are called agglutinins.
agglutinins Agglutination
reactions are identical in principle to precipitat
ion reactions; the only difference is that the cross linked
product is visible to the naked eye because of the larger size of the antigens.

When antibodies bind antigens on the surface of RBCs, the resultant clumping reaction is referred to as
haemagglutination.. In the absence of any agglutinating antibody, the RBCs settle into a tight button in
the bottom of the well. This tight button represents a negativeresult
result in a haemagglutination assay. Anti-
RBC antibodies induce cross-linking
linking of th
thee RBCs, so that they form a large clump and do not fall down
to the bottom of the well. Instead of a tight button, a diffuse shading (or spread pattern) of RBCs is seen.
This represents a positiveinteraction
interaction between the antibodies and the RBC surface antigens.
Haemagglutination reactions are routinely performed to type RBCs.

Passive haemagglutination
In passive haemagglutination, a soluble antigen is adsorbed on red blood cells. These RBC can then be
clumped by test serum containing antibody specific to th
e adsorbed antigento give an agglutination reaction.

Bacterial agglutination
A bacterial infection often causes the production of antibacterial antibodies specific for surface antigens
on the bacterial cells. Bacterial agglutination can be used to detect tthese
hese antibodies in a patient's serum
(e.g., tube agglutination test and plate agglutination test for diagnosis of brucellosis).

Clump formation is noted in plate tests and settling of antigen (bacterial cells) is noted in tube tests.
When performed semi-quantitatively, the test serum is serially diluted and the same amount of antigen
is added to each serum dilution.The last well in which agglutination is visible tells us the agglutinin
titerof the patient, defined as the reciprocal of the greatest serum dilution that elicits a positive
agglutination reaction. The agglutinin titer of an antiserum can be used to diagnose a bacterial infection
(the titre will be high during convalescence).
Agglutination reactions also provide a way to type bacteria (e.g., Salmonella).

Latex agglutination test

If latex particles are coated with antibodies, they can form clumps with soluble specific antigens.
Likewise, if latex particles are coated with soluble antigens, specific antibodies can agglutinate them.

Other secondary binding tests

To determine whether a patient has antibodies to a
particular haemagglutinating virus (a virus that can
cause clumping of RBCs by virtue of its
inhibition test haemagglutinin [HA] protein), a technician would
perform a serial dilution of the patient’s serum in a
microtitre plate and then add fixed amounts of the relevant virus and RBCs to each well. If the patient’s
antiserum has anti-haemagglutinin antibodies (as in the case of a recent infection) that bind the
particular virus being tested, the antibodies will attach to the HA molecules on the surface of the virus,
thereby inhibiting haemagglutination.
Complement fixation is the consumption or binding
of complement (a group of serum proteins recruited
Complement fixation
by bound antibody) by antigen–antibody complexes.
test This serves as the basis for a serologic assay known as
complement fixation test (CFT). In the first phase of
this test, the antigen of interest (e.g., Mycoplasma capricolum subsp. capripneumoniae) is mixed with a
test serum (from an animal suspected of contagious caprine pleuropneumonia). A measured amount of
complement is then added, which is fixed if the test serum contains specific antibody (i.e., if the animal
was infected withMycoplasma capricolum subsp. capripneumoniae). In the second phase of the test,
sheep RBCs sensitized (coated) with anti-sheep RBC antibody are added as an indicator system. Thus
there are two sets of antigen and antibody, of which only one antibody (the test antibody) is of
uncertain status. Failure of the sensitized sheep RBCs to lyse indicates a positive test since all
complement was used up in the first phase. However, sheep RBC lysis indicates that complement was
not consumed during the first phase of the reaction, implying that specific antibody to
Mycoplasmacapricolum subsp. capripneumoniae was not present in the serum, and complement had
remained free to lyse the sheep RBCs sensitized with anti-sheep RBC antibody. Haemolysis, therefore,
indicates a negative reaction. or
The sensitivity of the complement fixation test is between that of agglutination and precipitation. Use of
51 51
RBCs pre-labeled with Cr would increase the sensitivity of the test (release of Cr is measured to
determine haemolysis).
IgG and IgM fix complement by the classical pathway. However, non-antibody mechanisms and IgA
could fix complement by the alternate pathway. This could interfere with CFT results.
It is a variant of the precipitin reaction. Flocculation
requires a greater amount of antigen (In the precipitin
reaction, precipitate is developed with even minute
quantities of antigen). Toxin-antitoxin reaction is of
the flocculation type.
Antitoxin is an antibody against a given toxin. If toxin is added to antitoxin in divided doses, a greater
amount of antitoxin is required for neutralization than is required if the toxin is added all at once. This
form of reaction has been called the Danysz phenomenon or Danysz effect. The addition of one
fraction of toxin to excess antitoxin leads to maximal binding of antitoxin by toxin molecules. When a
second fraction of toxin is added, insufficient antitoxin is available to bring about neutralization.
Therefore, the mixture is toxic due to uncombined excess toxin.

Tertiary binding tests

Tertiary binding tests measure the consequences of immuneresponses in vivo. These tests are much more complex
thanprimary and secondary tests, but their results reflect thepractical significance of the immune response.
E.g.measurement of the protective effects of antibody.
Neutralization is the inactivation of a microbial product such as a toxin by antibody or counteraction of
the infectivity of a microorganism, as in the case of viruses.
Viruses are obligate intracellular parasites. A specific immune response against viruses may consist of
cytolytic T lymphocytes that destroy the virus-infected cell and/or antibodies from B cells. Before host
cell invasion, specific antibodies can neutralize virions.Neutralizing antibodies bind to viral antigens
important in cell invasion thereby blocking the virus from binding to and infecting a host cell.
The neutralization test is an assay based on the ability of antibody to inactivate the biological effects of
an antigen or of a microorganism expressing it. Neutralization applies especially to inactivation of virus

infectivity or of the biological activity of a microbial toxin. So there are virus neutralization tests and
toxin neutralization tests.

Toxin neutralization
Antitoxin is an antibody specific for exotoxins produced by certain microorganisms such as the causative
agents of diphtheria and tetanus. Prior to the antibiotic era, antitoxins were the treatment of choice for
diseases like diphtheria and tetanus. Neutralization does not destroy the reacting toxin.
Antitoxins are believed to act by binding toxins before they have the opportunity to combine with
specific tissue cell receptors.
Major Histocompatibility Complex
The major histocompatibility complex (MHC) is a gene cluster that was first identified by its powerful
effects on the immune response to transplanted tissues. Ithas three classes of gene loci which encodes
three classes of proteins.Some of these proteins are antigen presenting receptors. Antigen fragments
can trigger an immune response only when they can bind TCRs and, they can bind TCRs only when they
are mounted on MHC molecules.The fact, that a peptide antigen can only be recognized by a given T
cell if it is bound to a particular self MHC molecule, is called MHC restriction.
Class I loci code for MHC molecules expressed on most nucleated cells. Class Ia loci are highly
polymorphic (structural variation due to sequence variation) whereas class Ib, Ic or Id loci are hardly so.
Class II loci encode polymorphic MHC molecules found only on professional APCs. Class III loci code for
proteins with diverse functions, some of which are linked to innate immunity (e.g.- some complement
proteins). MHC proteins are called human leukocyte antigens (HLAs), dog leukocyte antigens (DLAs),
rabbit leukocyte antigens (RLAs), bovine leukocyte antigens (BoLA), equine leukocyte antigens (ELA),
swine leukocyte antigens (SLA) etc. depending on the species. In mice, the MHC is termed H-2 and in
chickens it is called B. The complete set of alleles found within an animal's MHC is called its MHC

MHC class Ia molecules:

These are expressed on most nucleated cells. They are not usually present on mammalian red blood
cells, gametes, neurons or trophoblast cells. Skeletal and cardiac muscle cells may express very few
class Ia molecules. Class Ia molecules consist of an α chain (45 kDa) and a smaller chain called β2-
microglobulin (12 kDa). The α chain is inserted in the cell membrane and has 3 extracellular domains:
α1 to α3. The antigen binding site is formed by the α1 and α2 domains. β2 microglobulin consists of a
single domain and serves to stabilize the structure. In humans, the functional polymorphic loci are
called A, B and C (HLA-A, HLA-B and HLA-C). In mice they are called K and D (H-2K and H-2D).
Some of the class Ia loci encode very large numbers of alleles. These allelic differences cause variations
in the amino acid sequences of the α1 and α2 domains. This variation is called polymorphism. Extreme
polymorphism is restricted to three to four small regions within the α1 and α2 domains. The α1 and α2
domains fold together to form an open ended groove, the shape of which determines which peptides
can be bound in it.

MHC class II molecules:

They consist of three paired loci: DP, DQ and DR in primates. Each encode for two protein chains called
α and β, each chain having two extracellular domains each. The antigen binding groove is formed by

the α1 and β1 domains. Polymorphism results from variations in the amino acids forming the sides of
the groove. The transporter proteins TAP1 and TAP2 and some proteasome components are also
encoded by class II region.

MHC molecules and disease:

Since T cells are MHC restricted, it can be said that MHC genes regulate immune responses. A foreign
molecule that cannot bind to the groove of at least one MHC molecule will not stimulate acquired
immunity. In order to escape from the host immune system, the pathogen only needs to mutate its
antigens so that none of the peptides derived from them can be presented on the host MHC molecules.
Two separate properties of the major histocompatibility complex (MHC) make it difficult for pathogens
to evade immune responses in this way. First, the MHC is polygenic:it contains several different MHC
class I and MHC class II genes, so that every individual possesses a set of MHC molecules with different
ranges of peptide-binding specificities. Second, the MHC is highly polymorphic; that is, there are
multiple variants, or alleles, of each gene within the population as a whole. The MHC genes are, in fact,
the most polymorphic genes known. The antigen binding of an MHC molecule is very non-specific (or
degenerate). An average MHC molecule can bind about 2500 different peptides. This means only one or
two peptides from an average antigenic protein can bind to any given MHC molecule. However, each
MHC allele will bind a different set of antigenic peptides. Therefore, the more variety in an animal's
MHC, the more antigens it can respond to. Thus, an MHC heterozygous animal will express more alleles
and can bind a greater variety of antigenic peptides than can a homozygous animal. Therefore, MHC
heterozygotes are at an advantage because they can respond to more antigens and so are best fitted to
survive infectious diseases. Some MHC class Ia loci contain highly polymorphic genes that code for very
large numbers of alleles. Since there can never be more than two alleles/locus in an individual, it appears
that this number of alleles is needed to maximize polymorphism in the population. When a new
infectious disease strikes a population, it is likely that at least some individuals will have MHC molecules
that can bind the new microbial antigens and trigger immunity. Low level of MHC polymorphism is a
feature of low density solitary species such as the Asiatic lions. In cheetahs, there is minimal
polymorphism due to recent population bottlenecks. Because of this lack of MHC diversity, these species
are at risk of extinction due to introduction of new infectious diseases.
Structure of BCR and TCR
The structure of the B cell receptor (BCR) is identical to that of the immunoglobulin, with an
exception: The C-terminal portion of the heavy chain has a transmembrane domain, which is
The cytoplasmic tail of the BCR heavy chain is extremely short—only three amino acids. Such a
short cytoplasmic tail could not pass a signal into the cytoplasm.
Each BCR molecule is noncovalently associated with a heterodimer, Igα/Igβ, that is responsible
for transducing the antigen signal into the interior of the cell. Igα/Igβ chains contain ITAMs,
which include tyrosine residues that become phosphorylated on activation through the
receptor, and serve as docking residues for downstream signaling components.
The BCR is therefore structurally and functionally divided into two components:a recognition
component (the immunoglobulin) and a signal transduction component (Igα/Igβ).
Structure of TCR
T cells bind peptide antigens located in the groove of membrane bound MHC proteins.
When the T-cell receptor (TCR) binds the MHC-peptide antigen on the surface of an APC, the
two cell membranes are brought close to each other. This means, there are several possible
interactions between the APC and T cell. B cell signalling does not involve this kind of
There are two types of T-cell receptors : αβ TCR and γδ TCR. αβ TCR is the more prevalent form.
The TCR proteins are members of the immunoglobulin superfamilyof proteins and therefore
thedomain structures ofTCR heterodimers are similar to those of the immunoglobulins.
Theαchain has a molecular weight of 40–50 kDa, and the βchain is 40–45 kDa. Like the antibody
light chains, theTCR chains have two immunoglobulin-like domains, eachof which contains an
intrachaindisulfide bond spanning 60to 75 amino acids. The amino-terminal(variable) domain in
both chains are variable (exhibits marked sequencevariation), but the sequences of the
remainder of each chainare conserved (constant). Each of the TCR variable domainshas three
hypervariable regions, which appear to be equivalentto the complementarity-determining
regions (CDRs)inimmunoglobulin light and heavy chains.
With the C-terminal end of the constant domain, eachTCR chain sustains a disulfide link with
the otherchain of the heterodimer. C-terminal to this disulfide is atransmembrane region of 21
or 22 amino acids, whichanchors each chain in the plasma membrane. The

transmembranedomains of the TCR α and β chains contain positively charged amino
acidresidues that promote interaction with correspondingnegatively charged residues on the
chains of the signaltransducingCD3complex.
The T-cell signal transduction complex
Signalingthrough theTCR depends on a complex of proteins referred to collectivelyas CD3. The
CD3 complex is made up of threedimers: a δε(delta epsilon) pair, a γε(gamma epsilon) pair,
and a third pair that is made up either of two CD3ζ (zeta) molecules or a ζη(zeta, eta)
The cytoplasmic tails of the CD3molecules are studded with ITAM sequences that help co-
ordinate signal transduction. Each of the CD3 dimerscontains negatively charged amino acids in
its transmembranedomain that form ionic bonds with the positively chargedresidues on
thetransmembraneregions of the T-cell receptor.
The T-cell receptor is noncovalently associated withCD4 orCD8. CD4 is a 55 kDa monomeric
membrane glycoproteinthat contains a long cytoplasmic tail. CD8 is a disulfide-linked dimer.
Each chain consists of a a cytoplasmictail containing 25 to 27 residues.
The extracellular domains of CD4 and CD8 bind to conservedregions of MHC class II and MHC
class I molecules respectively. This co-engagement of a singleMHC molecule by both the TCR
and its CD4 or CD8 coreceptorenhances the avidity of T-cell binding to its target and also helps
toinitiate the activation cascade.

Complement system

Complement is a collection of soluble proteins present in blood and other body fluids. It was
discovered in the 1890s by Jules Bordet as a heat-labile component of normal plasma that
augmented the opsonization and killing of bacteria by antibodies, and so this activity was said
to 'complement' the actions of antibodies. We now understand that it originally evolved as part
of the innate immune system and that it still provides protection early in infection, in the
absence of antibodies, through more ancient pathways of complement activation.
The complement system is composed of more than 30 different plasma proteins, which are
produced mainly by the liver. In the absence of infection, these proteins circulate in an inactive
form. In the presence of pathogens or of antibody bound to pathogens, the complement
system becomes activated. Particular complement proteins interact with each other to form
several different pathways of complement activation, all of which have the final outcome of
killing the pathogen and inducing inflammatory responses that help to fight infection. There are
three pathways of complement activation. As the antibody-triggered pathway of complement
activation was discovered first, this became known as the classical pathway of complement
activation. The next to be discovered was called the alternative pathway, which can be
activated by the presence of the pathogen alone, and the most recently discovered is the lectin
pathway, which is activated by lectin-type proteins that recognize and bind to carbohydrates
on pathogen surfaces.
The lectin pathway
The lectin pathway can be triggered mannose binding lectin (MBL) and the ficolins circulating
in blood and extracellular fluids that recognize carbohydrates on microbial surfaces. MBL and
ficolins in plasma form complexes with the MBL-associated serine proteases MASP-1 and
MASP-2, which bind MBL as inactive zymogens. When MBL binds to a pathogen surface, a
conformational change occurs in MASP-2 that enables it to cleave and activate a second MASP-
2 molecule in the same MBL complex. Activated MASP-2 can then cleave complement
components C4 and C2. C4 contains a buried thioester bond. When MASP-2 cleaves C4 it
releases C4a, allowing a conformational change in C4b that exposes the reactive thioester. C4b
bonds covalently via this thioester to the microbial surface nearby, where it then binds one
molecule of C2. C2 is cleaved by MASP-2, producing C2a, an active serine protease, which
remains bound to C4b, forming C4b2a, which is the C3 convertase of the lectin pathway.
C4b2a now cleaves many molecules of C3 into C3a and C3b. The C3b fragments bond

covalently to the nearby pathogen surface, and the released C3a initiates a local inflammatory
The classical pathway
The classical pathway is similar to the lectin pathway, except that it uses a pathogen sensor
known as the C1. C1q can attach itself to the surface of pathogens by binding directly to surface
components on some bacteria, by binding to C-reactive protein (an acute-phase protein in
human plasma) or by binding to the constant regions of antibodies (the Fc regions) that have
bound pathogens via their antigen-binding sites. Because C1 interacts directly with some
pathogens but can also interact with antibodies, C1 allows the classical pathway to function
both in innate immunity, and in adaptive immunity. Like the MBL-MASP complex, the C1
complex is composed of a large subunit (C1q), which acts as the pathogen sensor, and two
serine proteases (C1r and C1s), initially in their inactive form. C1q molecule has six globular
heads held together by their collagen-like tails, giving it the appearance of a six headed whip.
C1r and C1s are closely related to MASP-2. When two or more of the globular heads of C1q
interact with a ligand, this causes a conformational change in the C1r:C1s complex, which leads
to the activation of an autocatalytic enzymatic activity in C1r; the active form of C1r then
cleaves its associated C1s to generate an active serine protease. The activated C1s acts on the
next two components of the classical pathway, C4 and C2. C1s cleaves C4 to produce C4b,
which binds covalently to the pathogen surface as described earlier for the lectin pathway. C4b
then binds one molecule of C2, which is cleaved by C1s to produce the serine protease C2a.
This produces the active C3 convertaseC4b2a, which is the C3 convertase of both the lectin and
the classical pathways. Because it was first discovered as part of the classical pathway it is often
known as the classical C3 convertase.

Natural antibodies are antibodies produced by the immune system in the apparent
absence of any infection. These antibodies have a low affinity for many microbial
pathogens and are highly cross-reactive. Most natural antibody is of the class known as
IgM, and represents a considerable amount of the total IgM circulating in humans. IgM is
the class of antibody most efficient at binding C1q, making natural antibodies an effective
means of activating complement on microbial surfaces immediately after infection.
The alternative pathway
Although probably the most ancient of the complement pathways, the alternative pathway is so
named because it was discovered as an 'alternative,' pathway for complement activation after
the classical pathway had been defined. The alternative pathway can be activated in two
different ways. The first is by the action of the lectin or classical pathway. C3b generated by
either of these pathways and covalently linked to a microbial surface can bind factor B. This
alters the conformation of factor B, enabling a plasma protease called factor D to cleave it into
Ba and Bb. Bb remains stably associated with C3b, forming the C3 convertase, C3bBb. The
second way of activating the alternative pathway involves the spontaneous hydrolysis (known
as 'tickover') of the thioester bond in C3 to form C3(H20) (which happens at a steady low level).
This C3(H20) can bind factor B, which is then cleaved by factor D, producing a short-lived fluid-
phase C3 convertase, C3(H2O)Bb. Fluid-phase C3(H20)Bb can cleave many molecules of C3 to
C3a and C3b. Some of this C3b attaches covalently via its thioester bond to the surfaces of any
microbes present. The alternative pathway C3 convertases are very short –lived. They are,
however, stabilized by binding the plasma protein properdin (factor P), which binds to C3b or
The formation of C3 convertases is the point at which the three pathways of complement
activation converge. The convertases cleave C3 to C3b and C3a. C3b binds covalently through
its thioester bond to adjacent molecules on the pathogen surface; otherwise it is inactivated by
hydrolysis. C3 is the most abundant complement protein in plasma, occurring at a
concentration of 1.2 mg/ml, and up to 1000 molecules of C3b can bind in the vicinity of a single
active C3 convertase. Thus, the main effect of complement activation is to deposit large
quantities of C3b on the surface of the infecting pathogen.
The next step is the generation of the C5 convertases which cleave C5 into C5a and C5b
fragments, each of which exerts specific downstream actions that are important in propagating
the complement cascade. In the classical and the lectin pathways, a C5 convertase is formed by
the binding of C3b to C4b2a to yield C4b2a3b. The C5 convertase of the alternative pathway is
formed by the binding of C3b to the C3bBb convertase to form C3b2Bb. A C5 is captured by
these C5 convertase complexes through binding to an acceptor site on C3b, and is cleaved by
C2a or Bb.

The terminal pathway
One of the important effects of complement activation is the assembly of the terminal
components of complement to form a membrane-attack complex (MAC) which creates a
pore in the lipid bilayer membrane that destroys membrane integrity. The cleavage of C5 by a
C5 convertase releases C5b. This C5b initiates the assembly of the later complement
components and their insertion into the cell membrane. First, one molecule of C5b binds one
molecule of C6, and the C5b6 complex then binds one molecule of C7. This reaction leads to a
conformational change in the constituent molecules, with the exposure of a hydrophobic site
on C7, which inserts into the lipid bilayer. Similar hydrophobic sites are exposed on the later
components C8 and C9 when they are bound to the complex, allowing these proteins also to
insert into the lipid bilayer. C8 is a complex of two proteins, C8β and C8α-γ. The C8β protein
binds to C5b, and C8α-γ inserts itself into the lipid bilayer. Thereafter, C8 α-γ induces the
polymerization of 10-16 molecules of C9 into a pore-forming structure called the membrane-
attack complex. The MAC has a hydrophobic external face, allowing it to associate with the lipid
bilayer, but a hydrophilic internal channel. The diameter of this channel is about 100 A⁰,
allowing the free passage of solutes and water across the lipid bilayer. The disruption of the
lipid bilayer leads to the loss of cellular homeostasis, the disruption of the proton gradient
across the membrane, the penetration of enzymes such as lysozyme into the cell, and the
eventual destruction of the pathogen.

Regulation of complement
The conseqeuences of complement activation are so significant and potentially dangerous that
all of the activation pathways must be carefully controlled by regulatory proteins.

Other consequences of complement activation

Although the effect of the MAC is very dramatic, the significance of this in host defense seems
to be quite limited. The opsonizing and inflammatory actions of the earlier components of
the complement cascade are more important for host defense against infection. Formation of
the MAC seems to be important only for the killing of a few pathogens, although, it has a major
role in immunopathology.
Opsonization: C3b coated organisms will bind strongly to phagocytic cells possessing
receptors such as CR1 (complement receptor 1) and CRIg. Thus C3b acts as an opsonin.
However, if these organisms cannot be ingested, neutrophils may secrete their lysosomal
enzymes and oxidants in to the surrounding tissue fluid, causing inflammation and tissue
damage- a reaction classified as type III hypersensitivity.
Chemotaxis: C5a and C5b67 are potent chemotactic peptides for neutrophils, eosinophils,
macrophages and basophils. C5a also stimulates the respiratory burst and upregulates CR1 and
integrin expression of neutrophils.
Inflammation: C3a and C5a cause acute inflammation when injected into the skin. These
molecules are called anaphylatoxins because they cause degranulation of mast cells and
stimulation of platelets to release the vasoactive histamine and serotonin. C3a is also an
antibacterial peptide that disrupts membranes of bacteria such as E. coli, and Streptococcus
Immune regulation: when an antigen molecule binds to a B cell receptor, any C3d (a
breakdown fragment of C3) on its surface will bind to CD21 (CR2) on the B cell surface. This
sends a signal that significantly potentiates B cell receptor signaling and is considered an
important co-stimulation for B cells. C3d also facilitates antigen uptake by DCs with the help of

Molecules that communicate among cells of the immune system are referred to as cytokines.
In general, cytokines are soluble molecules, although some also exist in membrane-bound
forms. The interaction of a cytokine with its receptor on a target cell can cause changes in the
expression of adhesion molecules and chemokine receptors on the target membrane, alter and
enhance its effector functions, or instruct a cell when to survive or die. Cytokines are widely
used in experimental systems and diagnostic procedures.
Cytokines act predominantly in a paracrine or autocrine fashion, but some exhibit endocrine
action as well. Each cytokine has multiple biological actions. This property is called
pleiotropism, and it provides diversity of actions but may limit clinical utility of cytokines
because of side-effects. On the other hand, multiple cytokines may share the same or similar
biological activities. This property is known as redundancy. This means that blocking any one
cytokine may not achieve a desired effect.

Major types
Cytokines are relatively small proteins and generally have a molecular mass of less than 30 kDa.
Cytokines characterized so far belong to one of six groups: the interleukin 1 (IL-1) family, the
haematopoietin (class I cytokine) family, the interferon (class II cytokine) family, the tumor
necrosis factor (TNF) family, the interleukin 17 (IL-17) family, and the chemokine family.

IL-1 family
Cytokines of the IL-1 family are typically secreted very early in the immune response by DCs
and macrophages. IL-1 secretion is stimulated by recognition of viral, parasitic, or bacterial
antigens by innate immune receptors. IL-1 family members are generally proinflammatory;
they help increase capillary permeability, and promote leukocyte migration into the infected
tissues. IL-1 further signals the liver to produce acute phase proteinssuch as the Type I
interferons (IFNs αand β), IL-6 etc.. These proteins further induce multiple protective effects,
including the destruction of viral RNA and the generation of a systemic fever response. IL-1 also
activates both T and B cells at the induction of the adaptive immune response. Members of the
IL-1 family are synthesized as precursors which are cleaved by caspases in to active forms. Two
members of the family, IL-1Ra (for Receptor antagonist) and IL-18 BP act as natural inhibitors of
IL-1 family cytokine function.
Haematopoietin (class I) family
They are small, soluble cytokines with diverse cellular origins, target cells and effector functions.
Significant homology in the three-dimensional structure of haematopoietin family cytokines

Family name Examples Receptors Comments

IL-1α, IL-1β, IL-1Ra, IL-18, IL- Type I IL-1R IL-1 was the first noninterferon cytokine to be identified. Members
IL-1 family
33 Type II IL-1R of this family include important inflammatory mediators.

IL-2R subfamily
Haematopoietin IL-2 to IL-7, IL-12, IL-13, (γ)
This large family of small cytokine molecules exhibits striking
(Class I GM–CSF, G-CSF, Growth GM-CSF
sequence and functional diversity.
cytokine) family hormone, subfamily (β)
haematopoietin gp130 subfamily
IFN- α, IFN- β, IFN-γ, IL-10,
While the IFNs have important roles in anti-viral responses,
Interferon (Class II IL-19, IL-20,
all are important modulators of immune responses.
cytokine) family IL-22, IL-24

TNF- α, TNF- β, CD40L, Fas Members of this family may be either soluble or membrane
TNF family (CD95), BAFF, bound; they are involved in immune system development,
APRIL, LT β effector functions, and homeostasis.

This is the most recently discovered family; members

Interleukin 17 IL-17 (IL17-A), IL17B, C, D, function to promote neutrophil accumulation and activation,
family and F and are proinflammatory.


CCL2 (MCP-1),
Chemokines All serve chemoattractant function
CCL3 (MIP-1 α)

defines them as members of a single protein family. The defining structural feature of this class
of cytokines is a four-helix bundle motif, organized into four anti-parallel helices.

Interferon (class II) cytokine family

There are type I interferons and type II interferons. Type I interferonsare composed of
Interferon- α and interferon-β, which are secreted by activated macrophages and dendritic cells.
Interferonsαand βare also secreted by virally infected cells after recognition of viral components
by pattern recognition receptors (PRRs). The secreted Type I interferons then interact in turn
with membrane-bound interferon receptors on the surfaces of many different cell types

resulting in the induction of ribonucleases that destroy viral (and cellular) RNA, and the
cessation of cellular protein synthesis. Thus, interferons prevent virally infected cells from
replicating and from making new viral particles. However, they simultaneously inhibit normal
cellular functions and destroy virally infected cells so that the infection cannot spread. Type I
interferons are used in the treatment of a variety of human diseases, most notably hepatitis
Type II interferon, otherwise known as interferon-γ, is produced by activated T and NK.
Interferon-γis a powerful modulator of the adaptive immune response, inducing cell mediated
immune responses, resulting in destruction of any intracellular pathogens and the
differentiation of cytotoxic T cells. All three interferons increase the expression of MHC complex
proteins on the surface of cells, thus enhancing their antigen-presentation capabilities.
Interferon-γis used medically to bias the adaptive immune system toward a cytotoxic response
in diseases such as leprosy and toxoplasmosis, in which antibody responses are less effective
than those that destroy infected cells. Other members of the Interferon family of cytokines
include IL-10 which shares structural similarities with interferon-γ, and these similarities enable
it to bind to the same class of receptors. In addition, there is a third class of interferons, the
interferon-λ, or type III Interferon family. The Type III interferons up-regulate the expression of
genes controlling viral replication and host cell proliferation.

The tumour necrosis factor family

The TNF family of cytokines regulates the development, effector function, and homeostasis of
cells participating in the skeletal, neuronal, and immune systems, among others. Members of
this family may be membrane-bound or soluble. The TNF family includes TNF-α and TNF-β
among others. TNF-α is a proinflammatory cytokine. TNF-β promotes cell activation and
increased expression of MHC and adhesion molecules. Lymphotoxin-β, CD40L and FasL are
some of the other important TNF family members.

IL-17 family
Th e most recently described family of cytokines, the IL-17 family, includes interleukins 17 A to
F. Most members of this family are pro-inflammatory.

Chemokinesare a structurally related family of small cytokines that bind to cell-surface
receptors and induce the movement of leukocytes up a concentration gradient and toward the
chemokine source (chemotaxis). Therefore, they are chemoattractants. Chemokines are
relatively low in molecular weight (7.5–12.5 kDa) and structurally homologous.

Classification And Mechanism Of Induction

In an immune response, generally the effector molecules induce a localized inflammatory

response that eliminates antigen without much damage to the host’s tissues. But in some
circumstances, this inflammatory response can have deleterious effects. This inappropriate
immune response is termed as hypersensitivity. Hypersensitivity reactions may develop in
course of either humoral or cell mediated responses. Reactions within the humoral branch
initiated by antibody or antigen – antibody complexes are manifested within minutes or hours
after a sensitized recipient encounters an antigen. In delayed type hypersensitivity or cell
mediated hypersensitivity the symptoms appear only after days of exposure to antigen.
For every hypersensitivity reaction there should be sensitization (primary exposure) and shock
(secondary exposure). The effects are expressed only after these two processes.
P.G.H. Gell and R.R.A Coombs classified hypersensitivity into 4:
a) Type I hypersensitivity – IgE mediated (occur within the humoral branch)
b) Type II hypersensitivity – IgG or IgM mediated (occur within the humoral branch)
c) Type III hypersensitivity – immune complex mediated (occur within the humoral branch)
d) Type IV hypersensitivity – cell mediated or delayed type hypersensitivity

Type I or ‘Immediate’ Hypersensitivity

Type I hypersensitivity are acute inflammatory reactions mediated by IgE bound to mast cells
and basophils. It is induced by certain types of antigens referred to as allergens and is similar
to a normal humoral response (The term allergen refers to non parasitic antigens capable of
stimulating Type I hypersensitivity responses in allergic individuals). What distinguishes a Type I
hypersensitivity response from a normal humoral response is that the animals mount an
exaggerated Th2 response and the plasma cells secrete IgE. Though these reactions causes
discomfort to the individual, it performs at least two beneficial effects: (i) it helps antigen
elimination (ii) plays an important role in resistance to parasitic worms.
In normal individuals, an IgE response can be elicited by certain carefully designed
immunization procedure. But some individuals make IgE continuously and excessively. This
condition is called atopy and the individuals are said to be atopic. The response is seen mostly
against common environmental (innocuous) antigens and has a hereditary correlation (atopy
may be seen in offspring of parents who suffer from the condition). Breed predisposition is also
recognized (e.g., atopic dermatitis is more common in terriers, Dalmatians and Irish Setters).
An allergen on primary exposure induces a humoral antibody response with formation of IgE
from plasma cells. This IgE gets distributed in the body and binds with high affinity FcRs (Fc
receptors) on tissue mast cells and basophils. Mast cells and basophils coated by IgE are said to
be sensitized.
A later exposure to the same allergen, causes two distinct inflammatory responses: (i) an
immediate acute inflammatory response that occurs within 10-20 minutes as a result of mast
cell degranulation (ii) a late phase reaction several hours later that peak at 6-12 hours and then
gradually fades.

The immediate reaction

The allergen cross links the membrane bound IgE on sensitised mast cells and basophils
causing degranulation of these cells. The combining of allergen with IgE on the surface of mast
cells also provokes the formation of vasoactive molecules. Some IgG subclasses (e.g., IgG4 in
dogs) may also bind mast cell receptors with much less affinity than IgE and mediate Type I
hypersensitivity. These vasoactive molecules, which are either released preformed from the
granules or newly synthesized, generate the characteristic lesions of Type I hypersensitivity.
Mast cell granules contain histamine and, in some species, serotonin. Histamine causes smooth
muscle contraction in the bronchi, GI tract, uterus and bladder. It increases vascular
permeability causing fluid accumulation leading to wheal formation. It stimulates mucus
secretion, lacrimation and salivation.
Serotonin causes vasoconstriction resulting in the rise in blood pressure. In rats and mice it
induces wheal and flare reaction.
Trypsin or chymotrypsin like neutral proteases released can destroy nearby cells and activate
the complement C3 and C5 to generate anaphylatoxins. Activation of the cycloxygenase

pathway and lipoxygenase pathway lead to formation of prostaglandins and leukotrienes
Mast cell granules release eosinophil chemotactic factor – A (ECF-A) which accounts for
eosinophilia characteristic of Type I hypersensitivity including helminthic infection.
Various cytokines and heparin are also released due to degranulation. Due to release of
heparin, blood from animals experiencing anaphylaxis and dogs with mast cell tumors fail to
coagulate. The cytokines secreted include IL-4 (which is most important), IL-5, IL-6, IL-13 and IL-
16, chitinases, TNF-alpha and the chemokine CCL3. These cytokines are either proinflammatory
or promote Th2 responses or both. The production of chitinases support the hypothesis that
allergic reaction may have evolved to combat these invading insects, fungi and helminthes (all
these have chitin in them).

The late phase reaction

It is characterized by redness, edema, and pruritis. This may be due to release of inflammatory
mediators by eosinophils and neutrophils attracted to the site by mast cell derived chemotactic
Role of eosinophils
Eosinophils are considered as the terminal effector cells of the allergic reponse. They are
attracted to sites of mast cell degranulation and can degranulate releasing their own mediators.
Allergy is indicated by eosinophils. Slight increase in the number of eosinophils is viewed
seriously as it indicates allergic reaction. Eosinophils have a soothing action because they
contain enzymes which nullify histamines, serotonin etc. Hence eosinophilia is a favorable

Clinical Type I hypersensitivity

Acute anaphylaxis
Clinical signs of acute anaphylaxis vary in different species. In ruminants, chicken and humans,
the shock organ is the lungs (lung edema and emphysema); in horse, swine and cats it is
respiratory tract and intestine and in dogs it is the hepatic veins.
Specific allergic conditions
Milk allergy: Jersey cattle become allergic to α casein of their own milk. If milk is removed
promptly, there is no allergy. But in allergic animals, delayed milking forces this protein into the
blood stream leading to symptoms ranging from mild discomfort with urticarial skin lesion to
acute anaphylaxis and death.
Treatment – milk promptly; go for several lactations without drying.
Food allergy: Especially noticed in dogs. May be due to allergy to ingested food, food
idiosyncracy or intolerance or food poisoning. Lesion is popular, erythematous and is pruritic.
There isself inflicted trauma and bacterial and yeast infection.
Diagnosis: by intradermal testing, feeding hypoallergenic diet, radioallergosorbent test (RAST)
Treatment: avoid allergic food
Allergic inhalant dermatitis: In dogs and cats – leads to atopic dermatitis. Terriers and
Dalmatians are predisposed. Moulds, pollen, dust mites, wool etc. may cause this condition.
Nasolacrimal urticaria (hay fever) is an uncommon manifestation of respiratory allergy in dogs
and cats.
Diagnosis: by skin testing
Treatment: in canines corticosteroids or hyposensitization therapy, antihistaminics and NSAIDs
(non steroidalanti inflammatorydrugs) help for sometime.
Atopic dermatitis: It is a chronic multifactorial syndrome characterized by chronically inflamed
and itchy skin. Common in dogs and has been recognized in cats, horses and goats. It is
provoked by environmental, food and respiratory allergens that enter the animals by oral,
respiratory or percutaneous routes.
Treatment: Desensitisation therapy, emollient shampoos, antihsitaminics (limited effect),
glucocorticoids (as last resort; that too orally), prostaglandin analogues, immunosuppressice
drugs etc.
Allergies to vaccines or drugs: Allergies have been recorded to killed foot and mouth disease,
rabies and contagious bovine pleuropneumonia (CBPP) vaccines
Allergies to parasites: Allergies reported to tapeworms, fly bites, mites etc. But flea saliva may
cause Type IV hypersensitivity.
The eosinophil granuloma complex: It is a group of clinical conditions associated with various
types of skin lesions (ulcer, plaque, granuloma) in cats. The cause is unknown and these

conditions have been associated with flea or food allergies or inhalant dermatitis. It has been
suggested that they are an allergic response to a feline autoantigen.
Charcot – Leyden crystals:
Hexagonal bipyramidal crystals of eosinophil granule protein found in sputum of asthmatics. It
is a useful clinical marker in eosinophil mediated airwayreactions.
Diagnosis of Type I hypersensitivity
a) Direct skin testing with dilute aqueous solution intradermally. Site examined for local
inflammatory reaction
b) Passive cutaneous anaphylaxis - Dilutions of test serum injected at different sites into skin of
a normal animal. After 24-48 hours, antigen solution is administered intravenously. In positive
cases, injection site shows an immediate inflammatory reaction.
c) Serology – RAST, Western blot and ELISA
a) avoid the allergen
b) desensitization therapy – small amounts of dilute aqueous solutions of allergen (called as
“allergy shots”) are repeatedly administered. Alternatively, SLIT (sublingual allergen
immunotherapy) may be practiced. This promotes IgG production rather than IgE and reduce
recruitment of inflammatory cells. This also induces a shift in the dormant helper cell response
from Th2 to Th1with an accompanying increase in IFN - γ, which blocks the stimulation of IgE
antibody synthesis by IL-4 from Th2 cells. Desensitisation may downregulateIgE synthesis and
activate CD8+ T cells and regulatory T cells, all of which help in curbing the allergic response.
First injection contains only a small quantity of allergen. Over a period of weeks, dose is
increased. Dogs and cats respond well to this therapy but horses do not.
c) Inhaled corticosteroids- commonly used to reduce irritation and inflammation. Useful in
asthma. Inhibits production of prostaglandins and leukotrienes and is useful in treatment of
long term Type I hypersensitivity reactions.
d) Antibodies that bind the CH3 domain of IgE can reduce its circulating levels as well as the
expression of IgE receptor on mast cells.
e) Agents such as isoprenaline and sodium cromoglycate render mast cells resistant to triggering.
f) Epinephrine, long acting β2 agonists, antihistaminics (H1 – receptor antagonists), leukotriene
receptor antagonists are also used.

Antibody Mediated Cytotoxic (Type II) Hypersensitivity

Type II hypersensitive reactions involve antibody mediated destruction of cells. Antibody can
activate complement and lyse cells or it can destroy cells by ADCC (Antibody Dependent Cell
Mediated Cytotoxicity). It can also function as opsonin thereby facilitating phagocytosis.

Transfusion Reactions are Type II Reactions

The antigens found on the surface of red blood cells are called blood group antigens. Most of
the blood group antigens are cell membrane components. But there are also soluble molecules
passively adsorbed into red cell surfaces.
Animals can make antibodies against foreign blood group antigens even though they may
never have been exposed to foreign red cells. These natural antibodies are derived not from
prior contact with foreign red cells but from exposure to similar or identical epitopes
(heterophile antigen) seen in nature. Many blood group antigens are also common structural
components of a wide range of microorganisms, protozoa, helminthes etc.
During blood transfusion, there will not be any immune response if the donor RBCs are
identical to that of the recipient. But if the recipient possesses natural antibodies (of the IgM
type) to donor red cell antigens, they will be attacked immediately. This may cause
agglutination, hemolysis or opsonisation and phagocytosis. Agglutination of erythrocytes
caused by haemagglutin (antibodies) from another individual of the same species is called
isohaemagglutination. These antibodies are called as isohaemagglutinins and are usually of
the IgM type.
In the absence of natural antibodies, foreign antigen on the transfused RBCs will stimulate an
immune response in the recipient. The transfused cells then circulate for a period before
antibodies (of the IgG type) are produced and immune elimination occurs. A 2nd transfusion
with identical foreign cells results in immediate destruction. The rapid destruction of large
number of foreign RBCs can lead to serious pathological reaction – the Type II hypersensitive
The severity of the reaction depends on the volume of blood transfused. There will be
hemolysis, complement activation, anaphylatoxin release, mast cell degranulation and release of
vasoactive agents. The animals show signs of sympathetic activity – sweating, salivation,

lacrimation, diarrhea and vomiting. In the 2nd stage - hypertension, cardiac arrhythmias and
increased heart and respiratory rate.
Transfusion reactions can be prevented by cross matching. Donor RBCs are mixed with
recipient serum and incubated at 37C for 30 minutes. If the red cells are lysed or agglutinated
by the recipient’s serum, then no transfusion is to be attempted with those cells.
Hemolytic Disease of Newborn (HDN)
Female animals may become sensitized to fetal RBCs leaked into their blood stream through
the placenta during pregnancy. In such females, these anti-red cell antibodies may then be
concentrated in their colostrum. When the newborn suckles, these colostral antibodies are
absorbed through the intestinal wall and so reach its circulation. These antibodies directed
against the blood group antigens of the newborn cause rapid destruction of its red cells. The
resulting disease is called hemolytic disease of newborn or neonatal isoerythrolysis.
For HDN to occur, 4 conditions should be satisfied. (i) the young animal must inherit a red cell
antigen from its sire that is not present in its mother. (ii) the mother must be sensitized to this
antigen (iii) the mother’s response to this antigen may be boosted by transplacental
hemorrhage in late gestation (iv) the young animal must ingest colostrum containing high
titred antibody to its red cells.
Cattle - 11 blood group systems. B and J are important. HDN is rare in calves. It may result
from vaccination against anaplasmosis or babesiosis. If it occurs death is due to disseminated
intravascular coagulation (DIC).
Sheep - 6 blood group systems.
Pigs – 16 systems, HDN can occur due to use of hog cholera vaccine containing pig blood;
horses – 7 systems, HDN only a problem in foals born to mares that had had many foals
Dogs – 8 systems, DEA (Dog Erythrocyte Antigen) 1.1, 1.2, 3, 4 and so on; cats – only one
blood group system. HDN is rare. Chicken – 12 blood group systems.

Immune complexes and type III hypersensitivity

Mononuclear phagocytes are most efficient at binding and removing complexes formed at
optimal proportions and in antibody excess. Small immune complexes formed in antigen excess
are poorly removed by phagocytic cells but are deposited on vessel walls and in glomeruli.
When complement activating immune complexes are deposited in tissues, they generate
chemotactic peptides that attract neutrophils. The neutrophils release free radicals and enzymes
into tissues and so cause inflammation and tissue destruction. Lesions generated in this way are
classified as type III or immune complex mediated hypersensitivity reactions.

Two major forms of reaction are recognized
(a) local:- occur when immune complexes are deposited with in tissues and can be induced
in any tissue to which antigen can gain access.
(b) generalized:- results when large quantities of immune complexes are formed within the
circulation. eg:- when an antigen is administered intravenously to an immune recipient.

Arthus reaction:-
If an antigen is injected subcutaneously into an animal that already has precipitating antibodies,
then acute inflammation will develop at the injection site within several hours. This is called
Arthus reaction. It starts as an erythematous, edematous swelling, eventually local hemorrhage
and thrombosis occur and, if severe, end in local tissue destruction. The antibodies involved in
Arthus reaction must be both precipitating and complement activating and are usually of the
IgG class. A reversed Arthus reaction can be produced if antibodies are given intradermally to
an animal with a high level of circulating antigen. Eosinophil infiltration is not a significant
feature of this type of hypersensitivity. The immune complexes are deposited between and
beneath vascular endothelial cells.

Natural Local Type III Hypersensitivity Reactions

(a) Blue eye:
Condition seen in a small proportion of dogs that have been either infected or vaccinated with
live canine adenovirus type I. Lesion is an anterior uveitis leading to corneal edema and opacity.
The cornea is infilterated by neutrophils and virus-antibody complexes can be detected.
Develops 1-3 weeks after onset of infection and usually resolves spontaneously.
(b) Hypersensitivity pneumonitis:
Type III hypersensitivity reactions may occur in lungs when sensitized animals inhale antigens.
Eg:- cattle fed moldy hay for long periods can become sensitized to spores of
Micropolysporafaeni. Eventually, when spores are inhaled, antibodies formed against spore

antigen will form immune complexes resulting in complement activation and interstitial
A hypersensitivity pneumonitis seen in farmer’s chronically exposed to M. faeni spores is called
farmer’s lung (Hay sickness is a condition seen in horses which is similar to farmer’s lung). A
similar condition arising from exposure to dust from pigeon faeces is called as pigeon
breeder’s lung. Other conditions are mushroom grower’s lung, librarian’s lung etc.
(c) Staphylococcal hypersensitivty
It is a pustular dermatitis of dogs. Type I, II and IV hypersensitivity may be involved. Type III
reaction predominate in some cases.


(i) When large amounts of antigen enter blood and bind to antibody, circulating immune
complexes are formed. If the Antigen is in excess, small complexes form which are not cleared
from the system. They are deposited in the walls of blood vessels, especially medium sized
arteries and in vessels where there is a physiological outflow of fluid such as gomeruli, synovia
and the choroids plexus and can cause tissue damaging Type III reactions. Such kind of reaction
were observed in individuals administered with large doses of hyperimmune serum (eg:
antitetanus serum) from a foreign species and is called as serum sickness. The symptoms are
generalized vasculitis with erythema, edema and urticaria of the skin, neutropenia, lymph node
enlargement, joint swelling and proteinuria. The reaction is of short duration subsiding in a few
days time.
(ii) Glomerulonephritis: When immune complexes are deposited in glomeruli, they cause
basement membrane thickening and stimulate glomerular cells to proliferate. The lesion is
therefore called membranoproliferative glomerulonephritis (MPGN).

Type IV Hypersensitivity (delayed type hypersensitivity)

DTH reactions are classified as Type IV hypersensitivity and results from interaction between the
injected antigen, antigen presenting cells and T cells (cell mediated). When some
subpopulations of activated Th cells encounter certain types of antigens, they secrete cytokines
that induce a localized inflammatory reaction called DTH. The development of the DTH
response begins with an initial sensitization phase of 1-2 weeks after primary contact with
antigen. During this period, Th cells are activated and clonally expanded by antigen presented
together with the requisite class II MHC molecules on an appropriate APC. A subsequent
exposure to the antigen induces the effector phase of the DTH response. The Th1 cells secrete
a variety of cytokines that recruit and activate macrophages and other nonspecific inflammatory
cells. A DTH response normally does not become apparent until an average 24 hours after
second exposure with antigen and peaks 48-72 hours after second contact. The time delay is for
the cytokines to induce localized influx of macrophages and their activation. Macrophages are
the principal effector cells of the DTH response. Activated macrophages bring about nonspecific
destruction of cells and thus of the intracellular pathogen.
E.g., tuberculin reaction – the skin reaction in an animal with tuberculosis that results from an
intradermal injection of tuberculin which is an antigenic extract from the tubercle bacillus.
Tuberculin which is used to test/identify animals
The Tuberculin reaction with tuberculosis is the extract of
Mycobacterium tuberculosis, M. bovisor M.
avium. Several types of tuberculin are used. The most predominant is purified protein derivative
(PPD) tuberculin prepared by growing organism in synthetic medium, killing them with steam
and filtering. The PPD tuberculin is precipitated from this filtrate with trichloracetic acid, washed
and finally resuspended in buffer ready for use. Its antigenic component is thought to be heat
shock protein 65 (HSP 65). Many of the proteins in PPD are shared among different
mycobacterial species and hence is relatively non-specific. But specificity can be increased by
using a defined mycobacterial protein viz. early secretory antigenic target-6 (ESAT-6).
When tuberculin is injected intradermally into a sensitized animal (eg: animal infected with M. bovis),
a red indurated (hard) swelling slowly develops at the injection site. The inflammation begins
between 12 & 24 hours, reaches its greatest intensity by 24 to 72 hours and may persist for several
weeks before gradually fading.
T cells mediate the tuberculin reaction. When an animal is invaded by M. tuberculosis, the
organisms are readily phagocytosed by macrophages. Some of this antigen is presented to Th1
cells, triggers an immune response and generates memory cells. These memory T cells are able
to respond to mycobacterial antigen entering the body by any route and are long lived.
On intradermal injection of tuberculin, it is taken up by Langerhans cells which then migrate to the
draining lymph node where they present it to memory T cells. Memory T cells respond by
generating Th1 effector cells. The circulating Th1 cells recognize the antigen when they encounter it
in the skin and accumulate around the antigen (tuberculin) deposit. By 12 hours in cattle, the
infection site is infiltrated with T cells. The T cells recruit other Th1 lymphocytes and macrophages to
the site. The Th1 cells secrete IFN - γ, IL-2, and IL-16. IFN - γ and IL-2 increase expression of adhesion

molecules. IL-2 stimulates production of chemokine CXCL8, CCL5 and XCL1 which attracts and
activate more T cells. IL-16 attracts CD4+ T cells. Macrophages also release serotonin and CXCL1 and
CCL2 which attracts basophils. Serotonin or histamine (depending on the animal species) from
basophils cause yet more inflammation and enhances migration of mononuclear cells into the
lesion. CCL2 and CCL3 from T cells induce mast cell degranulation and some CD4+ cells activate
mast cells through MHC II bound antigen. T cells derived chemokines cause inflammation and
attract even more T cells. Most of these new T cells are not sensitized to the inducing antigen (only
5% of lymphocytes seen in DTH reaction are specific for antigen). Macrophages accumulate in the
lesion attracted by CXCL8 and may be activated by IFN - γ. Some of the tissue damage in intense
DTH reaction are due to proteases and oxidants from these activated macrophages. The
macrophages ingest and eventually destroy the antigen.

Tuberculin reactions in cattle

Skin testing for identifying animals that have or have had tuberculosis can be done by:
(a) Single intradermal testing (SID): 0.1 ml of PPD tuberculin from Mycobacterium
tuberculosis or M. bovisis injected intradermally into one caudal fold. After 72 to 96 hours a
comparison is made between injected and uninjected folds and a positive reaction consists of a
diffuse hard lump. The injection can also be given into the mucocutaneous junction of the vulva
and side of neck. In latter case, it is more sensitive but restraint is difficult.
Advantage: simple test. Disadvantage: (i) cannot distinguish infection by other mycobacteria
such as M. aviumor M. paratuberculosis or Nocardia. (ii) false positive results may be due to
exposure to M. phlei (iii) false negative in advanced tuberculosis, early infection, in animals
calved within the preceding 4-6 weeks, very old cows and in animals tested within the
preceding 1-10 weeks.
The lack of reaction (anergy) in advanced cases is due to the presence of IgG that prevents T
cells from reacting with antigen. Regulatory T cells are also involved. In repeated testing,
desensitization happens associated with elevated IL-10 levels and reduced IL-1β levels (IFN-γ
levels not affected).
(b) Comparative test: Injection of both avian and bovine tuberculin at side of neck at
separate sites. Examined after 72 hours. If avian tuberculin site shows the greatest reaction,
animal is considered to be infected with M. avium or M. paratuberculosis. If M. bovissite is
showing more reaction, animal infected with M. tuberculosis or M. bovis. This test done when
avian tuberculosis or Johne’s Disease is prevalent. PPD from M. bovis more specific in cattle as it
gives less cross reaction with M. avium.
Advantage: More specific than SID (>99% specificity, <1% false positive cases); 90% sensitivity).
Disadvantage: more complex
(c) Short thermal test: large volume of tuberculin solution is given subcutaneously and
animal is examined for rise in temperature between 4 & 8 hours later (tuberculin acts on T cells
that then release cytokines that stimulate macrophages to release IL-1). Used in post partum
animals and infected animals.
Advantage: High efficiency. Disadvantage: time consuming, anaphylaxis
(d) Stormont test: Performed by giving two injections on the same site 7 days apart. Relies
on the increased sensitivity of a test site that occurs after a single injection. Used in post partum
animals and advanced cases.
Advantage: Very sensitive and accurate Disadvantage: 3 visits required, may sensitize the
In other animals, tuberculin testing is not widely employed.
Pigs & cats positive for a short period only after infection-hence unreliable. In pigs & dogs, the
SID performed in the skin behind the ear. In cats, short thermal test is the best. Sheep & goats
– test done in the anal fold- unreliable. Horses- are unusually sensitive to tuberculin so a lesser
dose is used. But there is no correlation between test results and actual condition. In birds,
good reaction is obtained. Test done in the wattle or wing web.

Johnin reaction
Animals infected with M. paratuberculosis may develop a delayed hypersensitivity reaction
following intradermal inoculation of an extract – johnin. But negative results are obtained in
clinical infection. In such cases, intravenous johnin test is done. Johnin given intravenously and
temperature noted at intervals. A rise in temperature of 1C or a neutrophilia after 6 hours is
considered positive. Used for identification of infected herds.

Other skin tests

The DTH skin reaction may be obtained in any infectious disease in which cell mediate immune
response has a significant role.

For diagnosis of brucellosis (B. abortus) – brucellin- a filtrate of a 20 day broth culture and
brucellergen – a nucleoprotein extract.
Mallein– used for diagnosis of Glanders caused by Burkholderia mallei in horses. Short thermal
test or ophthalmic test can be done. Antigen dropped to eye - transient conjunctivitis in
positive cases. In the intrapalpebral test, antigen injected into skin of lower eyelid – in reactors,
swelling and ophthalmia.
Skin testing also used for detection of histoplasmosis (using histoplasmin), coccidioidomycosis
(using coccidioidin) and toxoplasmosis (using toxoplasmin).
Type IV hypersensitivity reaction that results due
Contact dermatitis to exposure of skin to some chemicals, poison ivy
plant, insecticides etc. The substances bind to
skin proteins and the complexes are processed by Langerhans cells in the dermis which present
it to T cells in the draining lymph node. In the process, Th1 and Th17 cells are activated. In
sensitized animals, following antigen response, macrophages and lymphocytes infiltrate dermis
by 24 hours. The cytotoxic T cells kill the altered cells, resulting in the development of
intraepithelial vesicles. This inflammatory reaction causes an intensely pruritic skin disease
called allergic contact dermatitis (NK cells are especially important in allergic contact
E.g., Chemicals like formaldehyde, picric acid, aniline dyes, plant resins and oils (urushiol of
poison ivy plant), organo-phosphorous compounds, neomycin, nickel and beryllium salts,
dichlorvos (in flea collars), calcium cyanamide etc.
Carpet dyes and deodorizers causes lesions in ventral aspect of abdomen.
In cattle: exposure to rubber in milking machines
In dogs in muzzle region due to contact with plastic food bowls
Lesion include mild erythema to severe erythematous vesiculation. Histologically mononuclear
infiltration and vacuolation of skin cells due to cytotoxic T cell attack.
Diagnosis done by removal of suspected allergen and by patch testing.
Treatment: avoid allergen, steroids used in acute cases with antibiotics to control secondary
infection. Hyposensitization therapy is not effective.
Autoimmunity, immunotolerance
If the immune system mounts a response to self antigens, it is called autoimmunity.
Any immune response directed against self antigens and antigens associated with normal
commensal microbiota in the gut is autoimmunity.
Both B and T lymphocytes can mediate autoimmunity. Autoimmunity is possible because the
antigen binding receptors of lymphocytes are generated randomly. About 20-50% of these
TCRs and BCRs are self-reactive (able to bind with high affinity to self antigens).

Tolerance is the name given to the situation where the immune system will not respond to a
specific antigen. Tolerance is primarily directed towards self antigens. The self-reactive B and T
lymphocytes are usually eliminated by selection processes in primary lymphoid organs. This is
known as central tolerance. Further protection from autoimmunity is achieved by induction of
peripheral tolerance. In peripheral tolerance, mature lymphocytes that encounter self antigens
die, are turned off (induction of anergy) or are suppressed by regulatory T cells (Tregs).
Central T cell tolerance: as T cells mature within the thymus, the cells whose receptors bind
too strongly to self antigens are killed (T cells are even given a chance to edit their TCRs (by
V(D)J recombination) and generate a non-self-reactive TCR and thus survive). For this purpose,
the thymic medullary epithelial cells express antigens usually limited to other tissues.
Macrophages carry normal tissue antigens into thymus as well, for this same purpose. However,
many self antigens (e.g.- antigens in the eye, testis and brain) are not processed in this manner
and so central tolerance to these antigens does not develop.
Peripheral T cell tolerance: Low-affinity self-reactive T cells that leave thymus are suppressed
by peripheral tolerance. One form of peripheral tolerance is clonal anergy. Dendritic cells
present self antigen-derived peptides to T cells (after apoptosis, the dead cells are removed by
phagocytes) without any co-stimulation. Any T cell that recognizes the self antigen derived
peptide becomes anergic. Similarly, very high doses of an antigen can induce a form of anergy
called immune paralysis. The high doses of the antigen bypass the APCs, reach Th cells and
engage their TCRs. In the absence of co-stimulation, the cells become anergic. Anergy is

B cell tolerance: Very immature B cells within bone marrow are killed when they bind self
antigen strongly (there is receptor editing as well). Peripheral B cell tolerance is induced by
apoptosis, clonal anergy (antigenic stimulation without co-stimulation), clonal exhaustion
(repeated exhaustive antigenic stimulation that results in absence of memory cells) and
blockage of BCRs (by some antigens). B cells recover from anergy faster than T cells do.
Other mechanisms of immune regulation
Antigen regulation of immune responses: T cells do not respond to antigens that are
continuously present in lymphoid organs. There will be no immune response against antigens
that does not reach lymphoid organs either.
Antibody regulation of immune responses: Antibodies suppress immune responses.
Inhibitory receptors: FcγRIIb on B cells and CTLA4 on T cells.
Regulatory cells: Regulatory T cells (Tregs), some macrophages, Th17 cells etc. regulate
immunity actively.
Nervous system: Regulates immune system through release of hormones such as
corticotrophin-releasing factor. There is immunosuppression during stress.

How does Autoimmunity develop?

Autoimmunity can result from a normal immune response to an unusual or abnormal antigen,
or from an abnormal immune response to a normal antigen.
Normal immune responses
1. Immune responses to a previously hidden antigen: Some autoimmune responses have
physiological functions. For example, red blood cells must be removed from the blood once
they reach the end of their life span. This process is accomplished by autoantibodies. As red
cells age, an anion transport protein called CD233 (or band 3 protein) is cleaved and a new
epitope is exposed. Autoantibodies bind to this epitope and trigger phagocytosis of RBCs by
macrophages. Injury to testes may lead to formation of autoantibodies against testicular
antigens. Following a heart attack, autoantibodies against the mitochondria of cardiac muscle
cells may be detectable. In diseases such as trypanosomiasis or tuberculosis, there is
widespread tissue damage, and autoantibodies to many different tissue antigens may be
detected in the serum.
2. Antigens generated by molecular changes: The production of autoantibodies may be triggered
by the development of completely new epitopes on normal proteins. Rheumatoid factors are
autoantibodies directed against other immunoglobulins. They develop because when an
antibody binds to an antigen, the shape of the immunoglobulin molecule changes and new
epitopes are exposed on its Fc region. In diseases where large amounts of immune complexes
are generated (e.g.- systemic lupus erythematosus (SLE)), rheumatoid factor formation against
the exposed epitopes on the Fc regions happen. Immunoconglutinins (IKs, after the German
spelling) are autoantibodies directed against the complement components C2, C4 and
especially C3. They may enhance complement-mediated opsonization.
3. Molecular mimicry: Sharing of epitopes between an infectious agent and an autoantigen may
cause autoimmunity. Examples of molecular mimicry include:
a. Nervous system and heart disease caused by auto-reactive antibodies formed against
Trypanosomacruzi antigens.
b. Heart lesions or rheumatic fever in children caused by antibodies to the cell wall M protein of
group A streptococci which cross reacts with cardiac myosin.
c. Epstein-Barr virus DNA polymerase- antibodies cross-react with myelin basic protein and may
be involved in the induction of multiple sclerosis.
d. Poliovirus capsid protein VP2- antibodies cross react with acetylcholine receptor- cause
myasthenia gravis.
4. Epitope spreading: immune response against an exogenous antigen subsequently 'spreads' to
recognize self antigens. e.g.- thyrotoxicosis and diabetes.
Abnormal immune responses
1. Failure of normal immune regulation: Mice having mutation in CD95 (lpr strain) or CD95L (gld
mutation) gene are not able to eliminate self-reactive lymphocytes in thymus. Consequently, lpr
and gld mice develop multiple autoimmune diseases.
2. Virus induced autoimmunity: probably through molecular mimicry or bystander activation
(cytokines produced against viral infection may activate previously dormant T cells). SLE of dogs
and humans has been associated with either a type C retrovirus or paramyxovirus infection.
3. Microchimerism: During pregnancy mothers and fetuses may exchange cells. These cells persist
and may cause autoimmune disease especially in humans. This is called foetalmicrochimerism.
Scleroderma is a form of graft-versus-host disease in these women caused by foetal cells.

Immunity to microbes
In order for a pathogen to establish aninfection in a susceptible host, it must first breach
physical andchemical barriers.
This means that most pathogens never gain productiveentry into the host.
When the basic barriers to infection are breached, innate immune responses come into play at
or near the site of infection. These early responses usemolecular pattern
Some bacteria produceendotoxins (LPS), which stimulatemacrophages or endothelial cells to
produce cytokines, suchas IL-1, IL-6, and TNF-α. These cytokines can activate nearbyinnate cells,
encouraging phagocytosis of the bacteria.
The cellwalls of many gram-positive bacteria contain a peptidoglycanthat activates the
alternative complement pathway, leading toopsonization and phagocytosis or lysis.
Viruses commonly induce the production of interferons, which can inhibit viral replication by
inducing an antiviralresponse inneighbouring cells.
Viruses are also controlled by NK cells.
In many cases,these innate responses can lead to the resolution of infection.
Cells responding via innate immunity at the infectionsite help coordinate the subsequently
more specific adaptive immune response. During this stage of the immune response, final
eradication of the foreign invader often occurs, typically leaving an immunological memory.
However, just as immunity in vertebrates has evolvedover many millennia, pathogens have
evolved a variety ofstrategies to escape destruction by the adaptive immuneresponse.
Some pathogens reduce their own antigenicityeither by growing within host cells, where they
are sequesteredfrom immune attack, or byshedding their membraneantigens.
Other pathogen strategies include camouflage (expressing molecules with amino acid
sequences similar to those of host cell membrane molecules or acquiring a covering of host
membrane molecules); suppressing the immuneresponse selectively or misdirecting it; and
continual variationin surface antigens.
Immunity to viruses
A virus enters a cell via a cell-surface receptorfor which it has affinity and preempts cell
biosynthetic machineryto replicate itself. Large numbers of new virions are produced, many of
them mutants with individual survival advantages.
A virus is more likely to thrive if it does not kill its host or if it is capable of spreading to new
hosts rapidly. Other survival strategies include long latencyperiod before severe illness(during
which time the host maypass the virus to others unknowingly), and facile transmission,such as
with influenzavirus, whereinfection is efficiently transferred during even a short acute illness.
Some viruses have reservoirs.
A number of specificand nonspecific defense mechanisms prevent oreliminate most viral
Virus generally needs to crossthe mucosa of the respiratory, genitourinary, or
gastrointestinaltract.Entrance of the virus may also occur through brokenskin, usually because
of an insect bite or puncture wound.

1. The innate immune response to viral infection begins with the recognition of PAMPs.
E.g.,dsRNA moleculesare detected by aPRR, inducing the expression of type Iinterferons and the
activation of NK cells.Type I interferons can induce an antiviral response or resistanceto viral
replication. The binding oftype I interferon and IL-12 to NK cells induces lytic activity,
makingthem very effective in killing virally infected cells.
2. Antibodies specific for viral surface antigens can neutralize viruses, especially if they are
localized at the site of viral entry into the body. e.g., the attenuated oral polio vaccineinduces
productionof secretory IgA, which effectively blocks attachment ofpoliovirus to epithelial cells
lining the gastrointestinal tract.
3. Viral neutralization by antibody sometimes involves blocking of fusion of the viral envelopewith
the plasma membrane, orlysis of enveloped virions by complement activation. Antibody or
complement can also agglutinate viralparticles and function as an opsonizing agents.
4. Once an infection is established, cell-mediated immune mechanismsare most important in host
defense. In general, both CD8+ Tc cells and CD4+ Th1 cells are required forcell-mediated antiviral
defense. Activated Th1 cells producea number of cytokines, which defend against viruses
eitherdirectly or indirectly. IFN- γacts directly by inducing anantiviral state in nearby cells. IL-2
acts indirectly by assistingthe development of CTL precursors into an effector population. Both
IL-2 and IFN-γactivate NK cells.In most viral infections, specific CTL activity arises within 3 to 4

days after infection, peaks by 7 to 10 days, andthen declines. Within 7 to 10 days of primary
infection, mostvirionswill have been eliminated. CTLs specific for the virus eliminate virus-
infectedhost cells and thuseliminate potential sources of new virus.

Immune evasion by virus

Some virusesencode proteins that interfere with innate and adaptive defense.

1. Blocking the antiviral effect of the interferons.

2. Inhibitionof antigen presentation by infected host cells.
3. Causing reduction in the surface expression of class I MHCmolecules
4. Causing reduction in the surface expression of class II MHC molecules
5. Secreting proteins that binds to complement components, inhibiting complement activation
6. Constantlychanging their surface antigens.
7. Causing generalized or specific immunosuppression.
It is interesting to note how recurrence of flu happens in humans. Flu is caused by Influenza
viruses, which show antigenic shift and antigenic drift. Whenever there is a reinfection, the
memory cells respond quickly. This also means that new naïve B cells fail to get activated even if
there is slight antigenic variation. This concept is referred to as original antigenic sin, or the
tendencyto focus an immune attack on those structures thatwere present during the original, or
primary, encounter witha pathogen and for which we have established memory. Thismeans that
our immune systems effectively ignore the subtlechanges occurring each year in pathogens
that drift antigenically. Once the organism has drifted sufficiently that there are only “new”
epitopes, or insufficient numbers of old epitopes, a new primaryresponse is mounted. In such a
year, we experience a badcase of influenza.

Immunity to bacterial infections

Immunity to bacterial infections is achieved by means ofantibody unless the bacterium is
capable of intracellulargrowth, in which case delayed- type hypersensitivity (DTH)has an
important role.
Bacteria enter the body either througha number of natural routes (e.g., the respiratory,
gastrointestinal,and genitourinary tracts) or through wounds. Ifthe inoculum size and the
virulence are both low, then localizedtissue phagocytes may be able to eliminate the bacteriavia
innate defenses. Larger inocula or organismswith greater virulence tend to induce antigen-
specificadaptive immune responses.
In some bacterial infections, disease symptoms are causedby the immune response itself.
Pathogen-stimulated overproductionof cytokines leads to the symptoms associated with
bacterial septic shock, food poisoning, and toxic shock syndrome.

Infection by extracellular bacteria:

1. Antibodies secreted by plasma cells inregional lymph nodes and the submucosa of the
respiratory and gastrointestinal tracts is the mainstay of immunity against extracellular bacteria.
2. Antibodies cause removal of the bacteria (through phagocytosis, complement activation and
inflammation) and inactivation of bacterial toxins.

Infection by intracellular bacteria:

1. NK cellsmay be activated, providing an early defense.
2. Cell-mediated immune response follows.

Evasion of immune responses by bacteria

There are four primary steps in bacterial infection:
1. Attachment to host cells
2. Proliferation
3. Invasion of host tissue
4. Toxin-induced damage to host cells
Host-defense mechanisms act at each of these steps, andmany bacteria have evolved ways to
circumvent some ofthem.

1. Expression ofadhesion molecules –pili

2. Secreting proteases that cleave secretory IgA atthe hinge region
3. Changing surface antigens
4. Inhibitingphagocytosis – capsule, M protein, protective fibrin coat
5. Interfering with the complement system
6. Through the ability to survive within phagocytic cells - escaping from the phagolysosometo the
cytoplasm or blockinglysosomal fusion with the phagosomeor resist the oxidative attack that
takes place within the phagolysosome

Active immunization with various types of vaccines has played an important role in the
reduction of deaths from infectious diseases, especially among children. In India, vaccination
of children begins at birth. Since passively acquired antibodies interfere with development of
protective active immunity in the young one, the vaccines may have to be repeated several
times in order to achieve stable protection. Multiple immunizations are also required for
viruses like the poliovirus which has multiple strains.
The widespread use of vaccines for common, life-threatening diseases has led to a dramatic
decrease in the incidence of these diseases. As long as these immunization programs are
maintained, especially in young children, the incidence of these diseases typically remains low.
However, the occurrence or even the suggestion of side effects to a vaccine, as well as general
trends toward reduced vaccination in children, can cause a drop in vaccination rates that leads
to re-emergence of that disease.
For example, rare but significant side effects from the original pertussis attenuated bacterial
vaccine included seizures, encephalitis, brain damage, and even death. Decreased usage of the
vaccine led to an increase in the incidence of whooping cough, before the development of an
acellular pertussis vaccine.
Despite the beneficial effects of vaccines on human life all over the world, some parents still
elect not to vaccinate their children. Similar trends are observed among livestock as well,
where large numbers of farmers and owners of animals continue to be unaware of the
importance of vaccinating their stocks.
Vaccination of adults:
Recommendations for vaccination of adults depend on the risk group.
o Vaccines for meningitis, pneumonia, and influenza are often given to groups living in close
quarters (e.g., military recruits and incoming college students) or to individuals with reduced
o Depending on their destination, international travelers are also routinely immunized against
endemic diseases such as cholera, yellow fever, plague, typhoid, hepatitis, meningitis, typhus,
and polio.
o Immunization against anthrax for workers coming into close contact with infected animals or
animal products.
Vaccination is not 100% effective. With any vaccine, a small percentage of recipients will
respond poorly and therefore will not be adequately protected. This is not a serious problem if
the majority of the population is immune to an infectious agent, significantly reducing the
pathogen reservoir. In this case, the chance of a susceptible individual contacting an infected
individual is very low. This phenomenon is known as herd immunity.
The common vaccines currently in use consist of live but attenuated organisms, inactivated
(killed) bacterial cells or viral particles, as well as protein or carbohydrate fragments (subunits)
of the target organism. Several new types of vaccine candidates provide potential advantages
in protection, production, or delivery to those in need.
Live, attenuated vaccines
In some cases, microorganisms can be attenuated or disabled so that they lose their ability to
cause significant disease but retain their capacity for transient growth within an inoculated
host. e.g., spore vaccine of anthrax, the Sabin form of the polio vaccine.
Some microorganisms are naturally attenuated because of their inability to cause disease in a
given host, although they can immunize these individuals. e.g., vaccinia virus inoculation of
humans confers immunity to smallpox.
Attenuation can often be achieved by growing a pathogenic bacterium or virus for prolonged
periods under abnormal culture conditions. This selects mutants that are better suited for
growth in the abnormal culture conditions than in the natural host. e.g., an attenuated strain
of Mycobacterium bovis called Bacillus Calmette- Guérin (BCG) was developed by growing M.
bovison a medium containing increasing concentrations of bile. After 13 years, this strain had
adapted to growth in strong bile and had become sufficiently attenuated that it was suitable
as a vaccine for tuberculosis.
Attenuated vaccines have advantages and disadvantages.
Because of their capacity for transient growth, such vaccines provide prolonged immune
system exposure to the individual epitopes on the attenuated organisms and resemble the
virulent pathogen in growth patterns. Thus, these vaccines often require only a single
They do not usually require adjuvants.
The ability of many attenuated vaccines to replicate within host cells makes them suitable for
inducing cell-mediated responses.

A disadvantage of attenuated vaccines is that these live forms may mutate and revert to
virulent forms in vivo. The rate of reversion of the oral polio vaccine is about 1 case in 2.4
million doses of vaccine.
Attenuated vaccines also may be associated with complications similar to those seen in the
natural disease although the risk of vaccine-related complications is still much lower than risks
from infection.
Inactivated or “Killed” Vaccines
Another common means to make a pathogen safe for use in a vaccine is by treatment with
heat or chemicals. This kills the pathogen, making it incapable of replication, but still allows it
to induce an immune response to at least some of the antigens contained within the
organism. Bacterins are vaccines containing killed bacteria.
It is critically important to maintain the structure of epitopes on surface antigens during
Heat inactivation is often unsatisfactory because it causes extensive denaturation of proteins
and consequent alteration of epitopes.
Chemical inactivation with:
♠ a fixative: e.g., formaldehyde
♠ alkylating agents e.g., beta propiolactone, acetylethyleneimine, ethylethyleneimine
♠ detergents, protein denaturing agents
♠ quaternary ammonium compounds e.g., benzalkonium chloride
Killed vaccines often require repeated boosters to achieve a protective immune status.
They do not replicate in the host and therefore they typically induce a predominantly humoral
immune response and are less effective than attenuated vaccines in inducing cell-mediated
immunity or in eliciting a secretory IgA response.
Pathogenicity of the agent: Production of inactivated pathogen requires handling of the
pathogen while it is infectious. This is risky for the handlers (Some inactivated vaccines have
failed because the inactivating agent did not quite inactivate the pathogen).
In general, the safety of inactivated vaccines is greater than that of live attenuated vaccines.
The advantages of inactivated vaccines include stability and ease of storage and transport.
Modern vaccines
Many of the risks associated with attenuated or killed whole organism vaccines can be
avoided with a strategy that uses only specific, purified macromolecules derived from the
pathogen. The three most common applications of this strategy, referred to as a subunit
vaccine, are inactivated exotoxins or toxoids, capsular polysaccharides or surface
glycoproteins, and key recombinant protein antigens.
e.g., diphtheria toxoid vaccine. Tetanus toxoid vaccine.
The virulence of some pathogenic bacteria depends on the antiphagocytic properties of their
hydrophilic polysaccharide capsule. Coating the capsule with opsonins such as antibodies
and/or complement helps macrophages and neutrophils to eliminate such pathogens. Hence,
we have vaccines consisting of purified capsular polysaccharides. e.g., PCV13 contains 13
antigenically distinct capsular polysaccharides of Streptococcus pneumoniae.
The gene encoding any immunogenic protein of a pathogen can be cloned and expressed in
cultured cells using recombinant DNA technology. This strategy is used to produce toxins as
well, which are converted to toxoids. Such proteins and toxoids are used as immunogens. e.g.,
HBsAg as Hepatitis B vaccine.
One limitation of some subunit vaccines, especially polysaccharide vaccines, is their inability to
activate Th cells. Instead, they activate B cells in a T-independent manner. There will be IgM
production, but class switching, affinity maturation, and development of memory cells mostly
do not happen. However, vaccines that conjugate a polysaccharide antigen to a protein carrier
can alleviate this problem by inducing Th cell responses.

Genetically attenuated vaccines

Genetic engineering provides a way to attenuate a virus irreversibly, by selectively removing
genes that are necessary for virulence or for growth in the vaccinated animal. This has been
done with a herpesvirus vaccine for pigs, in which the thymidine kinase gene was removed.
Because thymidine kinase is required for the virus to grow in certain types of cells (e.g.,
neurons), removal of this gene rendered the virus incapable of causing disease.
This is also how marker Vaccines / DIVA vaccine (Differentiate infected animals from
vaccinated animals) could be made. Organism in the vaccine would be lacking some of the
non-essential proteins of the field strains. Antibodies against the missing proteins would
indicate a natural infection.

Recombinant Vector Vaccines
Recall that live attenuated vaccines prolong antigen delivery and encourage cell-mediated
responses, but have the disadvantage that they can sometimes revert to pathogenic forms.
Recombinant vectors maintain the advantages of live attenuated vaccines while avoiding
Genes that encode antigens of pathogens can be introduced into attenuated viruses or
bacteria. The attenuated organism serves as a vector, replicating within the vaccinated host
and expressing the gene product of the pathogen.
A number of organisms have been used as the vector in such preparations, including vaccinia
virus, the canarypox virus, attenuated poliovirus, adenoviruses, attenuated strains of
Salmonella, the BCG strain of Mycobacterium bovis, and certain strains of Streptococcus.
Vaccinia virus, the attenuated vaccine used to eradicate smallpox, has been widely employed
as a vector for the design of new vaccines. This large, complex virus, with a genome of about
200 genes, can be engineered to carry several dozen foreign genes without impairing its
capacity to infect host cells and replicate. Genetically engineered vaccinia vector vaccines can
be administered simply by scratching the skin.

DNA Vaccines (polynucleotide vaccines)

Plasmid DNA encoding antigenic proteins are injected directly into the muscle of the
recipient. Host cells take up the DNA and produce the immunogenic protein, thus directing
the antigen through endogenous MHC class I presentation pathways, helping to activate
better CTL responses. The addition of a follow-up booster shot with protein antigen (called a
DNA prime and protein boost strategy), or inclusion of CpG DNA motif found in some
pathogens, may enhance the immune response.

o The antigen looks exactly as it is expressed by the pathogen, and induces both humoral and
cell-mediated immunity.
o Induces prolonged expression of the antigen, enhancing the induction of immunological
o DNA vaccines present important practical advantages. No refrigeration of the plasmid DNA is
o Simultaneous manufacture of a variety of DNA vaccines for different pathogens is possible.
o If gene gun is available, plasmid DNA coated on gold particles can be administered to more
animals in less time without needles.

Conjugate or Multivalent Vaccines

Involves fusing of a highly immunogenic protein to a weak vaccine immunogen (a conjugate)
or mixing in extraneous proteins (multivalent) to enhance or supplement immunity to the
pathogen. e.g., the vaccine against Haemophilusinfluenzae type b (Hib), is a conjugate
formulation consisting of type b capsular polysaccharide covalently linked to a protein carrier,
tetanus toxoid. The polysaccharide-protein conjugate is far more immunogenic than the
polysaccharide alone.
One means of producing a multivalent vaccine is to incorporate antigens into protein micelles,
lipid vesicles (called liposomes), or immunostimulating complexes. Membrane proteins from
various pathogens, including influenza virus, measles virus, hepatitis B virus, and HIV, have
been incorporated into micelles, liposomes, and ISCOMs and are being assessed as potential
vaccines. Liposomes and ISCOMs appear to fuse with the plasma membrane to deliver the
antigen intracellularly, where it can be processed by the endogenous pathway, leading to CTL

Quality control of vaccines

Vaccine assessment
Three key factors must be kept in mind in the development of a successful vaccine:
the vaccine must be safe,
it must be effective in preventing infection, and
the strategy should be reasonably achievable given the population in question.
Critical for success is the branch of the immune system that is activated.
Protection must also reach the relevant site of infection; mucosal surfaces are the prime
An additional factor is the development of long-term immunologic memory.
All vaccines are tested for safety and efficacy
Safety tests include confirmation of the purity and identity of the organism used and tests for
toxicity and sterility. Vaccines may contain excess of antigen in order to make up for the loss
in integrity suffered over long periods of storage.

The ability of a vaccine to prevent clinical infection in a vaccinated herd is called its efficacy. To
assess the efficacy of a vaccine, animals must be first vaccinated and then challenged. The
degree of protection desired must be ascertained. As far as prevention of death due to illness
is concerned, the true efficacy of a vaccine, called the preventable fraction (PF), is calculated as

(% of controls dying - % of vaccinates dying)

PF= X 100
% of controls dying

A good vaccine should have a PF of at least 80%.

For example, if a vaccine protects 73% of animals, while 89% of the unvaccinated animals die
following challenge with a virulent virus:
PF = (89-27)÷(89) = 69.7%. The vaccine is not very effective, but may be used if no
better vaccines are available.
When animal studies in suitable animals prove fruitful, follow-up clinical trials in humans can
be initiated for human vaccines.
Phase I clinical trials assess human safety; a small number of volunteers are monitored closely
for adverse side effects.
Only once this hurdle has been successfully passed can a trial move on to phase II, where
effectiveness against the pathogen in question is evaluated. In this case, development of a
measurable immune response to the immunogen is tested. Many vaccines fail at this stage.
Phase III clinical trials are run in expanded volunteer populations, where protection against
natural infection is the desired outcome, and where safety, the evaluation of several
measurable immune markers, and the incidence of wild-type infections with the relevant
pathogen are all monitored carefully over time.
The National Institute of Biologicals, Noida, Uttarpradesh, is the institute, under the Ministry of Health and Family
Welfare, Government of India, which has the mandate to develop and lay down standards for quality control
testing procedures for biologicals and immunobiological products in India, related to human health. NIB follows
ISO/IEC 17025:2005 which specifies the general requirements for the competence to carry out tests and/or

The Division of Biological Standardization, Indian Veterinary Research Institute, Izatnagar, Uttarpradesh, under
ICAR, is the recognized analytical laboratory to the Government of India for quality control of veterinary vaccines,
diagnostic antigens and sera, and for testing the standards of the biologicals received from the Drugs Controller
Authority of the country. Quality testing of veterinary vaccines, diagnostics and antigens are done under the
provisions laid down in the Schedule F1 of the Drug and Cosmetics Act (1940).

Failures in vaccination
A vaccine may fail to confer protective immunity in an animal due to the following reasons:
I. Incorrect administration
a. Due to inappropriate route of administration
b. Insufficient quantity of administration, especially when vaccinating large flocks through
drinking water or aerosol
c. Improperly preserved live vaccine might die and become useless
d. Use of antibiotics in conjunction with live bacterial vaccines might defeat the purpose of
e. Chemicals used to sterilize syringes might kill the live organism
f. Excessive use of alcohol while swabbing skin might interfere with viability of injected
II. Failure of animal to respond following correct administration
a. Problems with vaccine:
i. Late vaccination, the animal already having infected
ii. The vaccine may not be protective against the prevalent strain in field conditions
iii. The antigens used for immunization might be non-protective or insufficient
iv. The vaccine may be of low quality
b. Problems with animal:
i. The animal might be carrying passively received antibodies that interfere with vaccine
ii. The animal might be immunosuppressed- fever, stress including pregnancy, fatigue,
malnutrition and extremes of climate
iii. The animal is normal except that it doesn't respond to that particular vaccine –
biological variation that depends on breed, size, age etc.
Adverse consequences of vaccination

I. Due to errors in manufacture or administration: may result in clinical disease, foetal
death etc.
a. Contamination with bacteria or virus
b. Abnormal toxicity
c. Residual virulence- vaccine may cause infection, immunosuppression etc.
II. Inappropriate responses:
a. Hypersensitivities
b. Neurological reactions
c. Osteodystrophy in Weimaraner pups receiving live canine distemper vaccine
d. Foreign body reactions
e. Vaccine associated autoimmune diseases (e.g. Guillain-Barré syndrome triggered by
administration of influenza vaccine)
f. Injection site-associated sarcomas
III. Normal toxicity: Normal symptoms of inflammation associated with vaccine delivery.