The Immune system

• Role: protect body against pathogens

• Pathogens; bacteria, bacterial toxin, viruses, parasites, and fungi

• Immunity can be defined as the way in which the body protects

itself from invasion by pathogenic microorganisms and provides a
defense against their harmful effect
• Immunology is defined as the study of the molecules, cells,
organs, and systems responsible for the recognition and disposal
of foreign material
• Immune system is a network of cells and organs that extend
through out the body and function as a defense against infection
Immune system: Importance
 Outline
• Anatomy of the Immune system
• Organization of the body’s defenses
• Humoral immunity
• Cell-mediated immunity
• Immune responses in Health and diseases
Anatomy
• Leukocytes
- Phagocytes (phagocytosis):
neutrophils, eosinophils,
monocytes, macrophages,
dendritic cells (skin)
- Lymphocytes: Bs and Ts,
natural killer cells
- Mast cells

• Lymphoid tissues
- primary lymphoid tissues
(bone marrow and thymus)
- secondary lymphoid tissues

• Immune system: Lymphoid

system; main cells
lymphocytes
Lymphoid Tissue
Lymphocytes
• Lymphocytes arise from progenitor cells in yolk sac and
liver
• Later in fetal development and throughout the life cycle, the
bone morrow becomes the main provider of undifferentiated
progenitor cells, which develop into lymphoblasts
• Lymphoid precursors develop and proliferate as they travel
to primary and secondary lymphoid organs via the
lymphatic vessels or lymphatics. These vessels carry a
fluid called lymph
The Primary Lymphoid Organs
• Suitable conditions for initial production of lymphocytes
from progenitor cells
• Thymus (gland situated in front of heart & behind sternum)
• Progenitor cells from bone marrow migrate to thymus &
proliferate and differentiate here
• Hormone thymosin
• Thymus-derived -T cells
• Age-related involution during lifetime: decreased
synthesis of thymosin and inability to differentiate
immature cells
• Increase in immature lymphocytes in thymus and
peripheral blood T cells
• Bone marrow source of progenitor cells which
differentiate into lymphocytes, granulocytes, erythrocytes
• Role in the differentiation of progenitor cells into B
lymphocytes
• Bursa of fabricius-primary lymphoid organ in birds
• Bone marrow functions as the bursa equivalent in
humans
• B lymphocyte differentiations occur through the life time
• Resting state (mitotically inactive): potential to divide
and conduct immunological functions
• Non-stimulated
• Naïve or virgin lymphocytes
The Secondary Lymphoid Organs
Lymph nodes: lymphoid filters, respond to antigens introduced
distantly and routed to them by afferent lymphatic, generalized
lymph node reactivity following systemic antigen challenge
Spleen: lymphatic filter with in the blood vascular tree, important
site of antibody production in response to intravenous particulate
antigens, clearance of particles
Gut – associated lymphoid tissue (GALT): Peyer’s patch and liver;
lymphocyte circulation i.e. pre- B cells develop in Peyer’s patches,
after meeting antigen from the gut, they enter to the general
circulation and then return back to the gut
Tonsils: nodular aggregates of lymphoid tissue, detect and respond
to pathogens in the respiratory secretion
Blood: important lymphoid organ and immunological effector tissue;
has enough mature T-cells to produce graft vs host reactions
Approximate percentages of lymphocytes in
lymphoid organs
• Naïve lymphocytes: short half
life, die within a few days after
leaving the marrow or thymus
• Upon receiving antigenic signals,
undergo activation: Many cell
divisions over several days
• Some resulting progeny cells:
resting state: memory
lymphocytes; survive for many
years
• Rest differentiate in to effector
cells: survive for few days to
carry out specific defensive
activities against the foreign
pathogen
 The lymphocytes

• One of the classes of white blood cells, capable of

responding to antigens

• Morphologically homogenous

• T, B and NK cells

• T & B cells: split into subsets defined by many criteria

• Each subset-network of clones, each expressing a

specific receptor for a different antigenic eptiope

Clonal selection: Burnet (1950s)
• Once a T, B or killer
lymphocytes have made
contact with their specific
antigen, they are triggered into
action but they also divide and
form a large amount of identical
cells, called a clone
• Receptor proteins expressed by
cells in a given clone are
identical
• Clonal selection determines
speed and intensity of response
to a given antigen
• Larger the specific clone, the
more lymphocytes are available
to participate in immune
reaction
 Clonal deletion
• T cells and B cells, have proteins on their surface that allow them to
recognize foreign antigens and attack them
• Protein epitopes can vary from cell to cell, allowing them to react to a
variety of antigens
• Sometimes, T cells and B cells are produced that react to proteins that
are expressed by the body's own cells, called autoantigens
• Clonal deletion is a process that allows these cells to be neutralized
before they are released into the body, where they could potentially
begin attacking healthy tissue
• In early foetal life, self-reactive lymphocytes that are part of developing
immune system are deleted when exposed to self-antigens; deletion by
apoptosis by macrophages and thymic dendritic cells
• Thymic dendritic cells contain surface proteins usually found elsewhere
• Clonal deletion occurs with immune cells that would normally react to
these proteins
Binding to self
molecules in
bone marrow
can lead to the
death or
inactivation of
immature B cells
 Outline
• Anatomy of the Immune system
• Organization of the body’s defenses
• Humoral immunity
• Cell-mediated immunity
• Immune responses in Health and diseases
Organization of the body’s defenses: types of
immunity
• Non-specific immunity (natural/innate): no need to decipher
pathogen’s identity. Always present in the body, first line of
defense against the invading pathogens
- Physical and biochemical barriers
- Inflammation
- Interferons
- Natural cell killers (NK cells)
- Complement system

• Specific immunity: Based on recognition of the pathogen’s

identity
– Humoral immunity
– Cell-mediated immunity
 Non-specific defenses
Physical/mechanical barriers
 Skin (low moisture, low pH, presence of secreted inhibitory
substances)
 Mucus (epithelial and connective tissue) digestive, respiratory,
reproductive, urinary
 Stream of tears or rapid flow of urine
 Inflammation

Biochemical barriers
 Keratin: dryness of the skin, very little water
 Acidic pH (stomach-HCl, bile salts of liver, vagina)
 Enzymes (stomach, tears-lysozyme:kills microbes by hydrolyzing
the peptidoglycan layer)
 Interferons
 Complement system
Inflammation
Vascular and cellular reaction to the presence
of invading microorganisms or injury

Symptoms of inflammation:
- Redness
- Heat
- Swelling
- Pain

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Steps of inflammation

Initiation/ tissue damage

Leukocyte response

Tissue response
Prevention of leakage, confinement

Vasodilatation and increased

permeability of capillaries influx of
fluids and blood cells

Tissue repair & cure

 Cellular mechanisms: Macrophage & phagocytosis
• Alveolar macrophages like
neutrophils and NK cells:
remove particles and organisms
from alveoli
• Neutrophils: first phagocytes of
infected area, non-specifically
phagocytize some microbes
• Natural killer cells: large
lymphocytes: kill tumor cells and
virus infected cells
 Natural Killer cells = NK cells

• Cytotoxic T cells, lacking markers

• Able to recognize a wide range of
pathogens without prior exposure
• When recognition and binding occur,
the NK cells destroy the pathogen
through cell lysis

• Granules containing perforin and granzymes (proteases)

• Perforins create holes in cell membrane
• Granzymes: apoptosis/osmotic cell lysis
Interferon
• Protein secreted by cells infected by a virus
• Interferons prevent the neighboring cells from being
attacked by the virus
• The neighboring cells synthesize proteins that block the
virus from hijacking the cell DNA replication machinery
Complement system
• About 20, heat-labile serum proteins that are activated
either by direct contact with a pathogen or by contact with a
bound antibody.
• In an activated state, they form a complex which creates a
hole in the pathogen’s cell membrane [membrane attack
complex] bacterial lysis; complement system also
promotes inflammation
 Normal flora/ Commensals

• Microorganisms, mainly bacteria: harmless and non-disease

causing
• Stop the growth of potential pathogens by occupying attachment
sites and by producing substance against pathogens, compete
for essential nutrients for growth
– Propionibacterium acnes: Skin
– Escherichia coli : Intestine
– Candida and lactobacilli found in the vaginal tract
 Specific immunity
(Acquired/Adaptive immunity)

• Triggered in response to microbes or their antigens

delivered to the lymphoid organs
• Generates mechanisms that are specialized to combat
different types of infections
• Often uses cells, cytokines and molecules of the innate
immune system to eliminate microbes
• It allows the body to recognize, remember and respond
to a specific antigenic stimulus
• Types: Humoral and Cell-mediated
 Specific immunity

• Specificity: based on shape

recognition of cell surface
antigens
• Diversity: Any shape that can be
recognized by a B or T-
lymphocytes can trigger an
immune reaction
• Memory: once a pathogen has
activated the immune system,
memory cells remain and will
protect against a secondary
infection
• Self-tolerance: the immune
system does not attack itself
 Specific immunity : the players

• Macrophages (antigen presenting cell = APC): phagocytize

pathogens and present antigens to helper-T lymphocytes
• Helper-T lymphocytes: secrete lymphokines and activate B and
killer T lymphocytes
• B-lymphocytes: multiply and specialize into plasma cells 
secrete antibodies (acquired resistance)
• Killer-T lymphocytes: kill (through lysis) infected or cancerous
cells
 Types of Specific immunity

Passive immunity Active immunity

Antibodies (Abs) produced Abs by individual’s immune system
elsewhere given to the individual in response to a foreign antigen

Naturally acquired Naturally acquired

Abs transferred from mother Immunity that comes from
to fetus across the placenta & infections encountered in daily life
in colostrums and breast milk

Artificially acquired Artificially acquired

Abs formed by an animal or a Stimulated by exposure to specific

human given to an individual to foreign macromolecules through
prevent or treat infection vaccines to establish a state of
artificial immunity
Factors Associated With Immunologic
Disease
• Age: non-specific and specific body defense are present in
the unborn and newborn infants, but underdeveloped; Older
adults may have weakened immunity due to changes in
natural barriers like skin and weakened lungs & cough reflex
• Nutrition: Every constituent of the body defense is influenced
by nutritional intake; healthy diet is important for maximum
immune response
• Genetic factors: possession of certain genes linked to
immune disorders; lead to a deficiency in the production of
neutrophilis & complement e.g Sickle cell disease
predisposes to Haemophilus influenza & E. coli infection
 Outline
• Anatomy of the Immune system
• Organization of the body’s defenses
• Humoral immunity
• Cell-mediated immunity
• Immune responses in Health and diseases
 Humoral/Antibody-mediated immunity (AMI)
• Works to eliminate extracellular
• Stimulation of B-cells to produce antibodies
B-lymphocytes
• < 15% of circulating lymphocytes
• Derived from progenitor cells through an Ag independent
maturation process in the bone marrow and GALT
• Responsible for Humoral Response: primary host defense
against microbes
• Accomplished by their stimulation in to plasma cells, with
subsequent synthesis and secretion of immunogobulins
• Requires interaction between macrophages, T-cells and B-
cells

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• Macrophages (APCs) phagocytize a
pathogen and present an antigen to
a matching helper-T cell
• The T Cell with antigen on its
surface binds with specific antigen
receptor of B-cell (B Cell receptor)
• B-cell recognize its specific antigen
and become activated
• Produce clones and differentiate into
plasma cells, the latter produce Abs
• Some from clones: fight current
infection: effectors
• Others: memory for future infections
• Plasma cells are not found normally
in the circulating blood, end stage of
B cell differentiation
 Humoral Response
Primary
Abs during a primary immune
response are produced when
a person first encounters a
particular antigen
first Ab: IgM
Detectable production 1-2
weeks
Produced Abs decline rapidly
Secondary
Initiated by memory B-cells
when there is second exposure
with similar antigen
Major Ab: IgG
Time for production is short
Abs persist for years to months
Higher affinity than primary
response because of memory
cells
 What are antibodies?
• Glycoproteins, which are sensitized and secreted by
plasma cells in response to antigenic stimulation
• Immunoglobulins; 20% of plasma protein
• Found in blood plasma or serum & in many body fluids
such as tears, saliva and colostrums
• Primary function: combine with antigens to neutralize
bacterial toxins or some viruses
• Secondary interaction with compliment system is
required to dispose off larger Ags like bacteria
 Antibodies

Complete Incomplete

1. Heat resistant 1. Heat Labile

2. Combine with specific 2. Known as blocking Abs
Ag;produce immunologic 3. No immunologic reaction
reaction on combining with Ags
3. Capable of passing the 4. Not capable of passing
transplacental barrier the transplacental barrier
 Classification: Role of the antibodies
• Antitoxins: block activity of pathogens by neutralizing the
antigens

• Lysines: together with complement dissolve Ag

• Agglutinins: aggregation of multiple pathogens by antibodies,
first immobilize motile bacteria and aggregate cells forming
clumps
• Opsonins: antibodies, which combine with surface components
of microbes so that they are more readily phagocytized

• Precipitins : form precipitates with soluble Ags

• Complement activation: antibodies bound to pathogens activate
complement system resulting in cell lysis

• Antibody-Dependent Cellular Cytotoxicity (ADCC): Antibody-

bound abnormal body cells are recognised by NK cells and
elminated
 Basic Structure
• Four polypeptide chains held together by disulphide bond
• Each half: heavy (long) chain and one light (short) chain
• Heavy (H) chain is twice as large as the light (L) chain
• Every Ab contains equal number of H & L chains: (H2L2)n
• All H & L in any single Ab are identical
A schematic of bivalent
antibody molecule, composed
of four polypeptide chains—
two identical heavy chains
and two identical light chains.
The two antigen-binding sites
are identical, each formed by
the N-terminal region of a
light chain and the N-terminal
region of a heavy chain. The
two heavy chains also form
both the tail and hinge region
of the antibody
 Basic Structure
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• Two main regions:

– the upper region is highly variable and bind to a specific
shape (the antigen): N terminal region (VH and VL)
– The base region is constant for all antibodies  this
region activates the complement system when the
antibody is bound to its antigen (CH and CL)
– Light chain polypeptide contain only a single CL domain
but heavy chain CH regions comprise 3 or more domain
which are numbered sequentially (CH1, CH2, CH3) from
the domain closest to VH
– Hinge region confers flexibility, enabling the two arms to
move relatively freely
Types of light chains (mol. wt 23000)
• Kappa (κ) and lambda (λ ), based on their CL regions
• A given Ab contain either, never both
• Also, a given B cell lineage produces only one type of light chain
• Functionally same, proportion in an individual 2:1

Types of heavy chains (mol wt. 50000-70000)

• Classes of heavy chains: μ , δ , α , γ and Σ; difference in C H
physical and biological properties
• IgM, IgD, IgA, IgG & IgE
• γ1,2,3,4, α1 and α2 based on minor differences in CH regions
• IgG, IgG2 , IgG3 , IgG4 , IgA1 & IgA2
 Functions of regions of Abs
• Ab chains cleaved by papain, a proteolytic enzyme
• Two identical fragments: L chain, VH, CH domains of one H
chain
• They contain Ag binding site of Ab: Fab fragments (antigen
binding fragment)
• Third fragment: Carboxy terminal portion of both H chains held
together by disulfide linkage  crystallizable/ Fc
• Fc structure: common for many Abs
• Fc sequence: complement activation
• Binds to Fc receptors on many types of cells
Classes of antibodies
• IgG Most abundant (75%),
• Mostly in blood, lymph, able to cross the placenta
• MOA: Diffuses easily into extra vascular spaces, direct protection by
neutralizing viruses and toxins, immobilizing motile organisms, preventing
the adherence of microbes to cell surfaces, and agglutinating/precipitating
antigens
• IgG complexes with Ag: complement activation, phagocytosis, ADCC
• Two identical Ag binding sites: Divalent
• Protection of the newborn during the first months of life
• IgG1 , IgG2 , IgG3 & IgG4 depending on heavy chain composition
• IgA  15-20%, Found in tears, milk, blood, lymph; largely synthesized
by plasma cells on body surface, if produced by intestinal cell passes
into intestinal lumen/diffuse into blood circulation,
• After transportation through intestinal cells or hepatocytes, combines
with glycoprotein, secretory piece (complex formation: secretory IgA) 
protects IgA from GI proteolytic enzymes, protection of body surfaces
against invading microorganisms microbes
• Two subclasses with identical functions; IgA1:IgA2 = 5:1
• do not activate complement, do not cross placenta
• IgM  10%, First Ab to be secreted. Found in blood, lymph. Unable to
cross placenta, pentamer (five monomers connected by J chain), ten
antigen binding sites, intravascular localization because of large size,
effective in agglutination and cytolysis reactions
• IgD  monomer, < 1%,
• Found in blood, lymph, primarily on cell membrane of B cells in
association with IgM
• Very susceptible to proteolysis
• IgE  monomer, 0.004%,
• Important as involved in hypersensitivity reactions, generally
responsible for immunity against invading parasites,
• binds to receptors on mast cells and basophils, mediates release of
histamine, heparin from these cells together with Ags
Properties of human immunoglobulins
 Complement Activation
• About 20, heat-labile plasma proteins
• Normally inactive, activated directly by pathogen or Pathogen-Ab
complex
• Classic and alternative pathways of activation

Classic pathway is activated by complexing of Ag to specific IgM or IgG

Composed of three stages:
1. Recognition
2. Enzymatic activation
3. Membrane attack leading to cellular destruction
• Recognition unit of the complement system is the C1 complex
C1q, C1r & C1s: an interlocking enzyme system
• C1q binds to single IgM or atleast a pair of IgGs
• C1r & C1s do not bind to Ab, but are involved in subsequent
activation of the classic pathway
• C1r/s, activate each other splitting of C4 & C2, each into two
fragments
• One fragments each of C4 and C2  form C3 convertase
(C4b2a) & activate C3  fragments
• C3a, inflammation and C3b, opsonization
• C3b + C4b2a  C5 convertase  C5a + C5b (chemotactic
factors)
• C5b binds to C6 and C7  complex  inserted into membrane
bilayer
• C8 + C5b/C6/C7 complex  polymerization of upto 16 C9
molecules  membrane attack complex that causes cytolysis
Alternate pathway
• Non-antibody initiated pathway
• Activated by microbial and mammalian cells surface in
absence of Ag-Ab complex
• C3 activated by complement proteins: factor B, which
resembles C2; factor D, similar to C1 and Properdin (factor
P)
• Properdin (activator) catalyzes the activation of C3
• Uptake of factor B on to C3b when latter bound to an
activator surface C3bB  converted to active C3
convertase C3b, Bb
• Loss of Ba fragment through action of enzyme factor D
Alternate pathway (contd)
• C3b, Bb complex is able to convert more C3 to C3b, which
binds more factor B
• C3b, Bb decays due to loss of Bb, with half life 5 mins
• If properdin (P) binds to C3b, Bb  C3b, Bbp  half-life is
extended to 30 minutes
• association of many C3b units, factor Bb and properdin on
an aggregate of protein on the surface of a microorganism
 has a potent activity as a C5 convertase with the
cleavage of C5
Properdin
Activated Complement & Immune Response
• Physiologic consequence: blood vessel dilation and increased
vascular permeability
• Cellular consequences:
– production of inflammatory mediators eg. C3a, C4a & C5a
can cause degranulation of mast cell with release of
inflammatory mediators
– Cytolysis or haemolysis by membrane insertion of C5b6789
complex leads to lysis of many cell types, incl. erythrocytes
– Opsonization: C3b complement is a powerful opsonin, which
renders cells vulnerable to phagocytosis
Properties of Complement
• Inactivation by heating at 56℃ for 30 min

• Aging, especially at room temperature leads to almost complete

deterioration within a day or two and even in the refrigerator 
considerable loss of activity occurs in 3-4 days
• Destroyed by violet shaking for 20-25 min, by some acids and
alkalis, ether and soaps
• Glassware must be chemically cleaned
• Long term preservation as lyophilised product
 Antigens
• Substances recognized by a particular immunoglobulin or T-cell
receptor
• Capable of inducing an immune response

• Antigenic determinant or epitope: site of recognition and binding

by Ab/T-cell receptor
• Not all are immunogenic
• Multivalent (many epitopes) / monovalent (one epitope)

• Multivalent antigens produce a stronger immune response

because of range of Abs are produced against diverse eptiopes
 Adjuvants
• Enhance response to Ag
– By prolonging retention of the immunogen,
– By increasing the effective size of the immunogen,
– By stimulating the local influx of macrophages and/or other
immune cell types to the injection site and promoting their
subsequent activities
• Alum: alumnium hydroxide  most common
• Ag adsorbed onto alum increases effective size; so
promotes its ingestion & presentation by macrophages
 Criteria for immunogenic Ags
• Chemical composition: Large macromolecular proteins are
potent, Polysaccharides and short polypeptides can be
immunogenic, Pure lipids & nucleic acids: antigenic but not
immunogenic
• Molecular size: generally substances with mol weight
<10,000 are non- or weakly immunogenic, proteins with
>100,000 are strong immunogens
• Chemical Complexity: certain degree of chemical
complexity; polypeptides that contain tyrosine are better
immunogens than those without tyrosine
• Foreignness: discrimination between self and non-self; only
molecules that are foreign to the host are immunogenic
• Method of administration: dose and mode of administration
 Outline
• Anatomy of the Immune system
• Organization of the body’s defenses
• Humoral immunity
• Cell-mediated immunity
• Immune responses in Health and diseases
 Cell-mediated immunity
• Moderated by the link between T-lymphocytes and phagocytic
cells
• T-lymphocytes recognize the Ag only when present on the
surface of an antigen- presenting cell, the macrophages
• Lymphocytes are activated through direct contact and by the
production of soluble factors (lymphokines)
• Cell mediated immunity is responsible for the following
immunologic events:
– Immunity to intracellular organisms
– Rejection of foreign tissue grafts
– Immunity to viral and fungal antigens
– Delayed hypersensitivity
 T cells
• 60-80% of total circulating lymphocytes
• Responsible for cellular responses & regulation of Ab-reactions
either by helping or suppressing the activation of B-lymphocytes

Classification based on functions

• Cytotoxic or effector T cells: found in the peripheral blood, kill other
cells, can destroy virally infected cells
• Helper or regulatory T cells: secret a variety of substances that
help B cells in Ab response, stimulate activated T cells to proliferate
and activate macrophages, control many B cell functions including
proliferation & differentiation
• Suppressor T cells: <10% of T-cells, inhibit the response of helper T
cells, suppress T cell functions (cytotoxic response); B cell
functions (antibody synthesis by plasma cells)
• All T-cells have Ag receptor protein (T cell receptor)  binds to Ag
• Ag binding site recognizes self and foreign Ags

Cytotoxic T Cells Lyse Infected Cells

The cytotoxic T cell (TC) releases perforin and proteolytic enzymes onto
the surface of an infected target cell by localized exocytosis. The high
concentration of Ca2+ in the extracellular fluid causes the perforin to
assemble into transmembrane channels in the target cell plasma
membrane. The channels are thought to allow the proteolytic enzymes to
enter the target cell cytosol. Granzyme B, triggers release of cytochrome
c from mitochondria to initiate a caspase cascade leading to apoptosis
• Fas ligand on the surface of the cytotoxic T cell binds to and
activates the Fas protein on the surface of a target cell
• The cytosolic tail of Fas contains a death domain, which, when
activated, binds to an adaptor protein, which in turn recruits a
specific procaspase (procaspase-8)
• Clustered procaspase-8 molecules become activated and initiate
a proteolytic caspase cascade leading to apoptosis
(A) Electron micrograph showing an effector cytotoxic T cell binding to a
target cell. The cytotoxic T cells were obtained from mice immunized with
the target cells, which are foreign tumor cells. (B) Electron micrograph
showing a cytotoxic T cell and a tumor cell that the T cell has killed.
 Central Role of Helper T Cells

• TH1 cells are mainly involved in immunity to intracellular microbes 

activate macrophages, cytotoxic T cells, and B cells
• TH2 cells are mainly involved in immunity to extracellular pathogens 
activate B cells to make antibodies against the pathogen
 T cell Ag recognition process
• A foreign antigen is taken up and processed by the APCs
• The processed antigen is combined with a self antigen to
form antigen complex
• The antigen-binding site of a T-cell simultaneously
recognizes and binds to both foreign and self-antigens
• Self-Antigen: Major histocompatibility complex (MHC)
• Class I MHC: binds to Ags present on all nucleated cells;
recognized by only cytotoxic T cells
• Class II MHC: produced by only specialized cell types
(dendritic cells, B cells, and macrophages), recognized by
onlyT-helper cells/regulatory T cells
• First cell to be activated by immune response: T helper cells
• Activation enhanced by some factors produced by macrophages
• Activated T helper cells proliferate and stimulate cytotoxic T cells
• Recognize Ag (peptide fragments) presented with MHC I Ags
• Activated T cells may also regulate immune response by
intensifying it (T helper cells) or by lowering it (T suppresser
cells)
• Helper and suppressor T cells are the principal regulators of
immune responses
• Protect the human body against infection by mediating
intracellular pathogens ie bacteria, viruses and protozoa
• Responsible for chronic rejection in organ transplantation
• Affinity of TCRs for peptide–MHC complexes is usually too low
by itself
• T cells normally require accessory receptors to help stabilize the
interaction by increasing the overall strength of the cell–cell
adhesion
• CD4 and CD8 proteins, both of which are single-pass
transmembrane proteins
• CD4 is expressed on both helper T cells and regulatory T cells
and binds to class II MHC proteins
• CD8 is expressed on cytotoxic T cells and binds to class I MHC
proteins
First and second exposures to a pathogen
 Delayed Type of Hypersensitivity
• Cell-mediated, involves antigen sensitized T cells, which
respond directly or by the release of lymphokines to exhibit
contact dermatitis and allergies of infection
• One of cell mediated immunologic events
• e.g. Tuberculin test

Auto Immune Disease

• Inability of the immune system to discriminate between self and
non-self
• Characterized by the persistent activation of immunologic
effectors
• Alteration in the functions and integrity of individual cells and
organs
 Autoimmune Diseases (contd…)
• Immunoglobulins (autoantibodies) or cytotoxic T cells display specificity for
self-antigens or auto antigens
• Contribute to the pathogenesis of the disease
• Potential present in everyone because lymphocytes that are potentially
reactive with self- antigens exist in the body
Factors affecting are:
• Genetic factors: tendency for familial aggregates to occur, some human
leukocyte antigen increases risk; more frequently in women than in men
• Age: incidence of autoantibodies increase steadily with age, maximum at
60-70 years
• Exogenous factors: Ultraviolet radiation, drugs, viruses, and chronic
infectious disease, abnormal functioning of immunolgic regulatory
mechanism; these factors alter antigens, which the body then perceives
as non-self e.g. Rheumatoid arthritis, Systemic Lupus erythematosus,
Addison’s disease
 Antigen-Antibody interactions
• Non-covalent interaction
• Known antigen suspension or antiserum is used to detect
and measure unknown antibody or microbial antigen
• Does not lead to irreversible alteration of Ag or Ab
• This exact and specific interaction has led to many
immunological assays used to:
• detect and quantify Ag or Ab
• diagnose disease
• measure magnitude of humoral IR
• identify molecules of biomedical interest
 Nature of Antigen-Antibody interactions

1. Lock and Key Concept

• Combining site of Ab: Fab region
• Highly variable regions of the heavy
and light chains
• The antigenic determinant nestles in a
cleft formed by the combining site of
the antibody
• X-Ray crystallography studies
• Key (i.e. the Ag) which fits into a lock
(i.e. the Ab)
2. Non-covalent Bonds
 Hydrogen
 Ionic
 Hydrophobic interactions
 Van der Waals forces

• Each bond is weak; multiple

bonding ensures tight binding
• To “hold” they require high
complementarity

3. Reversible nature
• Non-covalent and hence
reversible
 Characteristics of Ag-Ab Reactions

Affinity
• Affinity of Ab is the strength of the reaction between a single
antigenic determinant and a single combining site on the
antibody
• Sum of the attractive and repulsive forces operating between the
antigenic determinant and the combining site of the antibody
• Affinity is the equilibrium constant that describes the Ag-Ab
reaction
• Most antibodies have a high affinity for their antigens
Calculation for Affinity
 Avidity
• Avidity is a measure of the overall strength of binding of an
antigen with many antigenic determinants and multivalent
antibodies
• Avidity refers to the overall strength of binding between
multivalent Ag's and Ab’s, additional considerations such as the
structural arrangement of both molecules
• Influenced by both the valence of the antibody and the valence
of the antigen. Avidity is more than the sum of the individual
affinities
 Specificity
• Ability of an individual antibody combining site to react with only
one antigenic determinant or the ability of a population of
antibody molecules to react with only one antigen
• Generally, there is a high degree of specificity in Ag-Ab
reactions.
• Antibodies can distinguish differences in 1) the primary structure
of an antigen, 2) isomeric forms of an antigen, and 3) secondary
and tertiary structure of an antigen.
 Cross reactivity
• The ability of an individual antibody combining site to react with
more than one antigenic determinant or the ability of a
population of antibody molecules to react with more than one
antigen
• Cross reacting Ags shares an epitope in common with the
immunizing Ag or because it has an epitope which is structurally
similar to that on the immunizing antigen (multispecificity)
 Factors Affecting Ag-Ab reactions
• Affinity - The higher the affinity of the antibody for the antigen,
the more stable will be the interaction. Thus, the sensitivity/ ease
with which one can detect the interaction is enhanced
• Avidity - Reactions between multivalent antigens and multivalent
antibodies are more stable and thus easier to detect
• Ag:Ab ratio - The ratio between the antigen and antibody
influences the detection of Ag/Ab complexes because the sizes
of the complexes formed is related to the concentration of the
antigen and antibody
• Physical form of the antigen -
The physical form of the
antigen influences how one
detects its reaction with an
antibody. If the antigen is a
particulate, one generally looks
for agglutination of the antigen
by the antibody. If the antigen
is soluble one generally looks
for the precipitation of the
antigen after the production of
large insoluble Ag/Ab Ag:Ab ratio
complexes
 Types of Ag-Ab Reactions

• Precipitation reaction
• Agglutination reaction
• Complement fixation reaction
• Enzyme Immuno Assay (EIA)
• Radio Immuno Assay (RIA)
 Precipitation Reaction
• Precipitins can be produced against most proteins and some
carbohydrates and carbohydrates- lipid complexes
• Precipitation tests are performed in semisolid media such as agar or
agarose, or non-gel support medium such as cellulose acetate, Agar:
interference for migration of charged particles, Agarose: transparent,
colorless, neutral gel
• Reaction involves formation of lattice which requires: 1) polyvalent Ab’s
and 2) Ag must be bivalent, polyvalent
• Immunodiffusion & electroimmunodiffusion
 Immunodiffusion
• Single radial immunodiffusion (Mancini): identification &
quantitation of a number of proteins found in serum & other body
fluids
• Principle: specific antibody is added to a buffered agarose
medium
• Serum containing the test Ag at different concentrations/dilutions
is placed in wells centered in the agarose
• Diameter of the resulting precipitin zone is related to the
concentration of antigen
• Greater the antigen concentration the larger the circle of
precipitation
Radial Immunodiffusion (Mancini)
• Amount of Ab constant; the diameter of the ring is
proportional to the log of the concentration of Ag
• a standard curve using known concentrations of
standard Ag
• Quantification of unknown Ag in sample
Double diffusion: the Ouchterlony method
• Principle: Antibody dilutions and specific soluble antigens are
placed in adjacent wells
• If the well size and shape, distance between wells, temperature,
and incubation time are optimal  solutions diffuse out cross-
link and form a visible precipitate at the point of equivalence
perpendicular to the axis line between the wells
• Precipitation bands will be compared with a standard Ag
• Location of band: Conc and rate of diffusion of Ag or Ab
• Two Ags: two precipitin bands form independently
• Detection of Abs associated with rheumatoid arthritis and
systemic lupus erythematosus
Double diffusion: the Ouchterlony method
• To test the similarity between antigens
• Ags from different species are loaded into two wells, Abs in a well slightly
below
Identity Reaction
• Precipitin band forms a single smooth area
• Ab is precipitating identical Ag specificities in each preparation
• Ag identical so far as the Ab can distinguish them

Non-Identity Reaction
• Precipitation lines cross each other
• The samples contain no antigenic determinants in common
Partial Identity Reaction
• Precipitation lines merge with spur formation
• Ags are non identical but possess common determinants
 Electro-immunodiffusion (EID)
• Variation of double-diffusion, using electric current to enhance
mobility and movement of Ags and Abs towards each other
• Antibody is placed in the well favoring its migration towards the
cathode
• Antigens that tend to be more negatively charged and placed in the
well, that favors migration towards the anode
• Precipitin bands in a shorter periods of time
• When voltage is applied:
• Immuno-double diffusion: counter current immunoelectrophoresis
(CIE)
• Radial immunodiffusion (RID): electro immunoassay (EIA)
 Countercurrent immunoelectrophoresis (CIE)
• The Ag and Ab are placed in wells in agar gel
• They are electrophoresed into each other where they form a
precipitation
• This test only works if conditions can be found where Ag and Ab
have opposite charges (types of gel, amount of current,
concentration of Ag/Ab)
• Qualitative, although from the thickness of the band you can get
some measure of quantity
• Advantages: Quick and 10-20 times more sensitive than immuno-
double diffusion
• Disadvantage: More expensive than immunodiffusion
Immunoelectrophoresis (electro immunoassay/EIA)

• Complex mixture of Ags placed in a well in agar gel

• Ags are electrophoresed to separate them according to their
charge
• After electrophoresis, a trough is cut in the gel and Abs are added
• Abs diffuse into the agar, precipitin lines are produced in the
equivalence zone
• Qualitative analysis of complex mixtures of antigens, although a
crude measure of quantity (thickness of the line) can be obtained
• Analysis of components in a patient' serum: Ab to whole serum in
the trough
• Determines deficiency/overabundance of one or more serum
components
 Agglutination Reaction

• Agglutination of particles to which soluble antigen has been

absorbed/particulate antigen  agglutination (clumping) of Ag
• Artificial carriers: latex particles and colloidal charcoal
• Biological carriers: Cells (erythrocytes) coated with foreign antigen
• Whole bacterial cells contain Ags  bind Abs  produced in
response to that Ag in hosts
• Easy and sensitive but only semi-quantitative
• Clinical diagnosis of non-infectious immune disorders and infectious
diseases
• When the antigen is an erythrocyte the term hemagglutination
• IgM due to its high valence is particularly good agglutinin
• Qualitative: assay presence of an antigen or an antibody
• e.g. A patients red blood cells mixed with antibody to a blood group
antigen to determine a persons blood type
• Quantitative: quantitate the level of antibodies to particulate antigens
• Serial dilutions of a sample to be tested for Ab concentration + a fixed
number of RBCs/bacteria/other particulate Ags and determines the
maximum dilution which gives visible agglutination: Titer
• Results are reported as the reciprocal of the maximal dilution that gives
visible agglutination
• Prozone effect
• Condition where very high concentration of Ab gives no agglutination
• Upon dilution agglutination observed
• Ab excess  very small complexes which do not clump  no visible
reaction

Applications of agglutination tests

• Determination of blood types or antibodies to blood group antigens.
• To assess bacterial infections
• A fourfold rise in titer is generally taken as a significant rise in Ab titre
Types
• Latex agglutination: Many Abs bound
to latex beads  increase in
potential no of exposed binding sites
• Ag from test sample will bind to the
combining sites of the Ab exposed
on the beads
• Visible cross-linked aggregates of
latex beads and antigen
• In some systems, Ags on latex
particles
• In the presence of serum Abs, these
particles agglutinate in to large
visible clumps
• Used for detecting IgG and IgM
rheumatoid factors
• Direct bacterial agglutination: Direct whole pathogens can be
used to detect antibodies directed against pathogens
• Basic tests measure Abs produced by the host to determinants
on the surface of a bacterial agent, in response to infection with
that bacterium
• Thick suspension of bacteria  Abs bind to Ag on bacterial
surface  clumping of bacteria  visible aggregates
• Tube testing allows more time for Ag-Ab reaction  more
sensitive than slide testing
• Indirect or passive hemagglutination: erythrocytes are coated
with soluble antigen
• Coated with extracts of bacterial cells, protozoa, viral Ag or
purified polysaccharides or proteins/haptens
• Agglutination of red blood cells for Ab detection
• Erythrocyte of animals such as sheep or rabbits, or from group
“O” humans
• Passive because Ag is not that of erythrocytes but the passively
attached from other source
• Rubella antibody test
Coombs Test (Antiglobulin Test) : Direct
• When antibodies bind to erythrocytes, they do not always result in
agglutination
• Ag/Ab excess or electrical charges on RBCs prevent effective cross
linking of the cells
• Abs that bind to but do not cause agglutination of red blood cells are
sometimes referred to as incomplete Abs
• Certain diseases: IgG/IgM Abs that can specifically bind to the RBC
surface/C3 bound to RBC
• These RBCs are detected by adding a second Ab directed against the
immunoglobulin (Ab) coating the red cells
• This anti-immunoglobulin (antihuman globulin or "Coombs reagent")
can now cross link the red blood cells and result in agglutination
• Used in hemolytic anemia (antibody-mediated destruction of RBCs)
• Indirect Coombs Test :
• Indirect Coombs test, is used to determine the presence of antibody in
the serum or plasma
• Non-agglutinating antibodies in the serum sample have to be detected
• IgG/IgM or both, IgG clinically significant
• RBCs are incubated with the serum sample  wash off unbound Abs
second Ab reagent to cross link the cells
• Screen pregnant women for antibodies that may cause hemolytic
disease of the newborn-destruction of RBCs of the fetus
• Woman with Rh-negative blood is pregnant with a baby (fetus) with
Rh-positive blood, Rh sensitization may occur
• Sensitization happens when the baby's blood mixes with the mother's
blood
• Mother’s blood makes Abs against the baby's red blood cells in future
pregnancies
Hemagglutination Inhibition technique
• Used for measurement of soluble antigens
• Measures the ability of soluble antigen to inhibit the agglutination
of antigen-coated red blood cells by antibodies
• A fixed amount of antibodies to the antigen in question is mixed
with a fixed amount of red blood cells coated with the antigen
• Also included in the mixture are different amounts of the sample
to be analyzed for the presence of soluble antigen
• If the sample contains the soluble antigen, it will compete with
the antigen coated on the RBC for binding to the antibodies,
thereby inhibiting the agglutination of the RBC
https://www.jove.com/embed/player?id=55833&access=d8mk5q2vns&t=1&s=1&fpv=1
• Used to detect some viral Abs, for example, rubella
• Known quantity of rubella viral Ags is mixed with dilutions of the
patient’s serum, to which red blood cells are added
• In absence of serum Ab: Virus + RBCs  agglutination
• If Abs are present: Virus + Abs  inhibit agglutination
• The serum is therefore positive for the antibodies
• The highest dilution of serum that totally inhibits agglutination of
red cells determines the antibody titer of the serum
• Disadvantage: time consuming & subjective bias in the
interpretation for results, false negative results can occur from a
low titer of antibody, Nonspecific inhibitors can cause false
positive results
• Complement Fixation Reaction: classic method for demonstrating
the presence of antibody (only complement fixing; IgG, IgM) in
serum, ability of Ag/Ab complex will "consume" complement
• Components:
• I: an indicator system consisting of a combination of sheep red
cells, complement fixing antibody produced against the sheep red
cells in another animal, and an exogenous source of complement,
usually guinea pig serum
• When combined in an optimal concentration, the anti-sheep Ab,
hemolysin, can bind to the surface of the red cells
• Complement subsequently bind to this Ag-Ab complex and cause
cell lysis
• II: a known antigen and patient serum
• II is added to sheep erythrocytes, hemolysin, and a complement
• Patient serum is first added to the known antigen, and
complement is added to the solution
• If the serum contains Ab to Ag, Ag-Ab complexes bind to the
complement
• + Sheep red cells and hemolysin
• If complement is bound  no heamolysis
• If no Ab, complement is free  heamolysis
• Immunofluorescence Test (IFT): Advantage: Specific and
sensitive
• Labeling Ab with fluorescein isothiocyantate, which has affinity
for proteins to form a complex, conjugate
• Direct Assay: Fluorescein- conjugated antibody is used in Ag-Ab
reactions
• Fluorescent microscope is used to observe the production of
yellow- colour
• Used detection of hepatitis B virus & chlamydia
• Indirect Assay: Ag to the specific Ab being tested is fixed to the
surface of a microscopic slide
• Patient’s serum is diluted and placed on the slide to cover the
antigen source
• If Ab is present in the serum, it will bind to its specific Ag;
unbound Abs are then removed by washing the slide
• + antihuman globulin conjugated to a fluorescent substance
which fluoresces in UV light
• Marker of human Ab will bind to Abs bound to Ag
• Flow Cytometry (Fluorescence Activated Cell Sorting; FACS):
identify and enumerate cells bearing a particular antigen
• Cells in suspension are labeled with fluorescent tags by either
direct or indirect immunofluorescence
• The cells are then analyzed on the flow cytometer
• In a flow cytometer the cells exit a flow cell and are illuminated
with a laser beam
• Amount of laser light scattered off the cells as they passes
through the laser can be measured, gives information
concerning the size of the cells
• Laser can excite the fluorochrome on the cells and the
fluorescent light emitted by the cells can be measured by one or
more detectors
• One parameter histogram: increasing amounts of fluorescence
(e.g. green fluorescence) is plotted on x axis and no. of cells
exhibiting that amount of fluorescence is plotted on the y axis
• Fraction of fluorescent cells: integrating the area under the curve
• Two parameter histogram: the x axis is one parameter (e.g. red
fluorescence) and the y axis is the second parameter (e.g. green
fluorescence)
• The number of cells is indicated by the contour and the intensity
of the color
• Enzyme Immuno Assay (EIA)/Enzyme linked Immunosorbent
Assay (ELISA): enzyme labeled Ab or Ag used in immunologic
assays for detection of Ags or Abs
• E.g. HIV antibody, HIV antigen, hepatitis A antibody
• Most commonly used enzymes are peroxidase ((3,3',5,5'-
tetramethylbenzidine) and alkaline phosphatase (p-Nitrophenyl
Phosphate)
• Plastic beads or plastic plate is coated with Ag which reacts with
Abs from patient’s serum
• Beads or plates are then incubated with an enzyme labeled
antibody conjugate
• Enzyme activity is measured spectrophotometrically after the
addition of the specific chromogneic substrate
• Results of patient’s serum compared with control serum
https://www.jove.com/embed/player?id=10496&access=wrq5ny02gf&t=1&s=1&fpv=1
Competitive ELISA
• Primary antibody (unlabeled) is incubated with sample
antigen (patients serum)
• Antibody-antigen complexes are then added to 96-well
plates which are pre-coated with the same antigen
• The more antigen in the sample (serum), the less free
antibodies will be able to bind to the antigen in the well,
hence "competition”
• Unbound antibody removed by washing
• The secondary antibody that is specific to the primary
antibody and conjugated with an enzyme is added
• A substrate is added, and enzymes elicit a chromogenic
or fluorescent signal
Advantages
• High specificity, since two antibodies are used the
antigen/analyte is specifically captured and detected
• Suitable for complex samples, since the antigen does not
require purification prior to measurement
• Significantly less sensitive to sample dilution and sample
matrix effects
• Greater precision, accuracy, and reproducibility than
sandwich
• Flexibility and sensitivity, since both direct and indirect
detection methods can be used
• Radio Immunoassay (RIA): used to measure the concentration
of antigen by use of antibodies
• If Ag concentration is being measured, radioactively labeled Ag
competes with unlabeled Ag from patient’s serum for binding
sites on a known amount of Ab
• Advantage: extreme sensitivity and ability to detect trace
amounts
• A large number of tests can be performed in a relatively short
time period
• Disadvantage: hazards and instability of isotopes, expensive
and specialized instrument, precautions and license to handle
radioactive substance
Isotopes of iodine
such as 125-I

Cold Ag

• The samples to be assayed (the

unknowns) are run in parallel
• After determining the ratio of bound
to free antigen ("cpm Bound/cpm
Free") in each unknown, the
antigen concentrations can be read
directly from the standard curve
 Applications of Immune Responses

• Naturally acquired immunity is acquisition of adaptive

immunity through natural events
• Immunization mimics these events by inducing artificially
acquired immunity
• Natural or artificial immunity can be divided into
– Active immunity
– Passive immunity
 Principles of Immunization
• Protection from infectious disease
• Usually indicated by the presence of antibody
• Very specific to a single organism
• Self vs. nonself

Active immunity
• Results from immune response upon exposure to an
antigen
• Active immunity can develop naturally following illness
• Or artificially after immunization
Passive Immunity

• Occurs naturally during pregnancy

• IgG from mother crosses placenta
• Infers protection to the baby
• Occurs naturally as result of breast feeding
• IgA antibodies in breast milk given to child
• Artificial passive immunity involves transfer of antibodies
produced by another person or animal
• Can be used to prevent disease before or after likely
exposure
Acquired Natural
Active
Passive
 Vaccines and Immunization
Classification of Vaccines
• Live attenuated: Viral and Bacterial
• Inactivated
• Whole: viruses and bacteria
• Fractional
• protein-based: toxoid and subunit
• polysaccharide-based: pure and conjugate
Principle of Vaccination
General Rule: The more similar a vaccine is to the disease-
causing form of the organism, the better the immune response
to the vaccine
Attenuated vaccines
• Weakened form of pathogen
• Generally unable to cause disease
• Strain replicates in vaccine recipient
• Causes infection with undetectable or mild symptoms
• Results in long lasting immunity
Advantages
• Single dose usually sufficient to induce long-lasting
immunity
• Due to multiplication of microbe in body continued
stimulation of immune system
• Vaccine has added potential for being spread
• “Disease” after immunization could be spread to un-
immunized individuals inadvertently
• Immune response similar to natural infection
Disadvantages
• Have potential to cause disease in immunocompromised
individuals
• Pregnant women should also avoid immunization with
attenuated vaccine
• Severe reactions possible
• Interference from circulating antibody
• Fragile – must be stored and handled carefully

Attenuated vaccines in use include

Viral: Measles, mumps, rubella, yellow fever, varicella/zoster,
rotavirus, influenza, polio; given orally or intranasally

Bacterial: BCG, oral typhoid

Inactivated vaccines
• Unable to replicate in vaccinated individual
• Retains immunogenicity of infectious agent: Immunogenic
not pathogenic
• Generally not as effective as live vaccines
• Less interference from circulating antibody than live
vaccines
• Generally require 3-5 doses
• Immune response mostly humoral
• Antibody titer may diminish with time
Inactivated vaccines fall into two categories
• Whole agents
• Contain killed organisms or inactivated organisms
(viruses)
• Does not change epitopes
• Viral: polio, hepatitis A, rabies, influenza
• Bacterial: pertussis, typhoid, cholera, plague
• Fragments/ fractional vaccines
• Portions of organisms or agents including toxins, proteins
and cell wall components
• Subunit: hepatitis B, influenza, pertussis, human
papillomavirus, anthrax
• Toxoid: diphtheria, tetanus
Polysaccharide vaccines
• Long chains of sugar molecules that make up the surface
capsule of certain bacteria
• E.g: pneumococcal, meningococcal, and Salmonella typhi
• Able to stimulate B-cells without the assistance of T-helper
cells
• Not consistently immunogenic in children younger than 2
years of age
• Immaturity of the immune system
Polysaccharide vaccines
• No booster response
• IgM, and little IgG is produced
• Immunogenicity improved by conjugation to carrier protein
from same organism: T-cell independent to T-cell
dependent
• Conjugate polysaccharide
• Haemophilus influenzae type B, pneumococcal,
meningococcal
Principles of Vaccination
• Inactivated vaccines are generally not affected by
circulating antibody to the antigen
• Live attenuated vaccines may be affected by circulating
antibody to the antigen
• Vaccine doses should not be administered at intervals less
than the minimum intervals or earlier than the minimum
age
• It is not necessary to restart the series or add doses
because of an extended interval between doses
Principles of action of vaccines
• Antigen dissociates from the adjuvant
• Cells of the non-specific immune system (i.e.
macrophages and dendritic cells) recognize the antigen as
foreign and engulf it
• Antigen presentation to T cells and B cells
• Adjuvant and preservative, if present, are absorbed into
the blood where they circulate and are excreted in the
stools and urine
Vaccine Adverse Reactions
• Local: pain, swelling, redness at site of injection
• common with inactivated vaccines
• usually mild and self-limited
• Systemic: fever, malaise, headache
• non-specific
• may be unrelated to vaccine
•Allergic
• due to vaccine or vaccine component
• rare, risk minimized by screening
Serological testing
Important terms:
Contraindication: A condition in a recipient that greatly
increases the chance of a serious adverse reaction e.g.
severe allergic reaction, encephalopathy: pertusis vaccine
Precaution: A condition in a recipient that might increase the
chance or severity of an adverse reaction, or might
compromise the ability of the vaccine to produce immunity
Important terms:
Seronegative
• Person not yet exposed to antigen and has no specific
antibodies
Seropositive
• Person with exposure and actively producing antibody

Titer
• Concentration of antibody in serum---Indicates previous
exposure
Vaccination of Pregnant Women
• Live vaccines should not be administered
• Inactivated vaccines may be administered as indicated

Vaccination to Immunosuppressed Persons

• Live vaccines should not be administered
• Inactivated vaccines are safe to use but the response to
the vaccine may be decreased
Vaccination During Acute Illness
• No evidence of reduced vaccine efficacy or increased
adverse reactions
• Vaccines should be delayed until the illness has improved
• Mild illness: not a contraindication
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