UNIVERSITY OF FORT HARE

DEPARTMENT OF BIOCHEMISTRY AND MICROBIOLOGY

MICROBIOLOGY III

IMMUNOLOGY, VIROLOGY AND ANTIMICROBIAL CHEMOTHERAPY

(MIC 312)

Lecturer: Dr. N Nontongana

COURSE OUTLINE

SECTION 1: IMMUNOLOGY

I INTRODUCTION

1.1 Immunology and immunity

1.2 Overview of frequently used terms and concepts

II CHARACTERISTICS OF THE IMMUNE SYSTEM

2.1 Antigens (Immunogens)

2.2 Antibodies (Immunoglobulins)
2.3 Cells and tissues of immune system

III NATURE OF THE IMMUNE SYSTEM

3.1 Types of immune systems

3.2 Properties of immune response
3.3 Factors that modify immune response

IV THE SYNTHESIS OF ANTIBODY

4.1 Roles of the small lymphocytes

4.2 Cellular cooperation in the immune response
4.3 Generation of antibody diversity
4.4 Development of immunological tolerance
4.5 Theory of antibody synthesis
4.6 Monoclonal antibody and development

V INTERACTION OF ANTIGEN AND ANTIBODY

5.1. Nature of antigen-antibody reactions

5.2. Affinity and avidity
 5.3. Specificity and cross reactivity
5.4. Antigen-Antibody reactions (in vitro)
5.5 Antigen-Antibody reactions (in vivo)

VI IMMUNOLOGICAL DISORDERS

6.1 Overview of immunological disorders

6.2 Hypersensitivity
6.3 Autoimmune disorders
6.4 Transplantation
6.5 Drug reaction
6.6 Immunodeficiency

VII IMMUNITY TO INFECTION

7.1 The Immediate defence systems

7.2 Different immune effectors protect against different pathogens
7.3 Tumor immunity

SECTION 2: ANTIMICROBIAL CHEMOTHERAPY

8.1 Introduction

8.2 Characteristics of Antibiotics

8.3 Kinds of Antimicrobial Agents and their Primary Modes of Action

8.4 Antimicrobial Agents Used in the Treatment of Infectious Disease

8.4.1 Antibacterial agents
8.4.2 Antifungal agents
8.4.3 Antiviral agents
8.4.4 Antiprotozoan and antihelmintic agents

8.5 Microorganisms that Produce Antibiotics

8.6 Microbial Resistance to Antibiotics

8.7 The Genetic Basis of Bacterial Resistance to Antibiotics

8.8 The Medical Problem of Bacterial Resistance

8.9 Laboratory studies on antibiotic action

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SECTION 3: VIROLOGY

I GENERAL CONCEPT OF VIROLOGY

1.1 Historical development of the science of virology

1.2 Nature of viruses
1.3 General terminology
1.4 The variety of virus infections
1.5 The virus infection cycle
1.6 Introduction to the taxonomy of viruses

II VIRUSES WITH RNA GENOMES

III VIRUSES WITH DNA GENOMES

1.1 Viruses with small DNA genomes

1.2 Viruses with medium and large DNA genomes

IV CONSEQUENCES OF VIRUS INFECTION

1.1 Epidemiology of virus infections

1.2 Pathogenicity and virulence of viruses
1.3 Immunological responses to virus infection
1.4 Interferon
1.5 Persistent and low-level virus infections
1.6 Formation of tumors
1.7 Plant response to virus infection

Reading:
1. Lindquester, Gary J. (2006) Introduction to the History of disease. Disease and
Immunity, Rhodes College.
2. Silverstein, Arthur M. (1989) History of Immunology (Hardcover) Academic Press.
3. Prescott et al. (1995). Microbiology, 2nd edition. WC Brown Publishers.
4. Blair, E., Derby, G., Gough, G., Littler, E., Rowland, D., Antiviral Therapy, Bios
Scientific, 1998.
5. Williams, R.A.D., Lambert, P.A., and Singleton, P., Antimicrobial Drug Action,
Oxford Centre for Staff Development, 1996.
6. Fields, B.N., and Knipe, D.M., eds. Fundamental Virology, 3rd
edn. New York: Raven Press, 1996.
7. Fields, B.N., and Knipe, D.M., eds. Virology, 4th edn. New York:
Raven Press, 2002.

COURSE DESCRIPTION AND OBJECTIVES

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This course is designed to present the basic principles and concepts of immunology, virology
and antimicrobial chemotherapy. The immunology section covers topics such as
characteristics and nature of the immune system, synthesis of antibody, antigen-antibody
reaction and the molecular mechanisms used by the immune system to protect organisms
from disease are discussed in detail. The Virology section presents an overview of the
subject of virology from the perspective of its history, to its diversity and implications in
infection. Emphasis is placed on the general concepts of virology, the broad classification of
viruses on the basis of nucleic acid make-up and the consequences of virus infection. The
section on Antimicrobial chemotherapy focuses on the different types of antimicrobial agents
from the perspective of general principles and mechanisms of action, to treatments of
infections, as well as the problems of microbial resistance.

At the end of this course, students will have a very good understanding of the different types
of immune response; their properties and characteristics e.g antigens, antibodies and cells and
tissues of the immune response, as well as the processes involved antibody synthesis. The
students will also have become well informed on the interactions between antibody and
antigens; immunological disorders and immune responses to specific pathogens. The
antimicrobial chemotherapy aspect will afford students familiarity with the mode of action,
spectrum of activity and constraints to the use of antibiotics and properties that distinguish
members of antibiotic groups; Antimicrobial agents that are available for the treatment of
bacterial, fungal, viral, protozoal and helminthes infections; and important issue of resistance
to antimicrobials. At the end of the virology section, the student should have a basic appreciation of
viruses, their historical development, properties, pathogenesis and therapy. They should be able to
describe the different classes of viruses and relate the aforementioned characteristics with the
consequences of viral infection.

CHAPTER I

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 INTRODUCTION

1.1 Immunology and immunity

Immunology (from immunis, Latin for "exempt") is the study of the organs, cells, and
chemical components of the immune system. The immune system creates both innate and
adaptive immune responses. The innate response exists in many lower species, all the way up
the evolutionary ladder to humans, and it acts through relatively crude means against large
classes of pathogens. The adaptive response is unique to vertebrates, reacting to foreign
invaders with specificity and selectivity.
Immunity is describes a state of having sufficient biological defenses to avoid infectious
disease, or other unwanted biological invasion. Immunity involves both specific and non-
specific components. The non-specific components act either as barriers or as eliminators of
pathogens to stop infection by micro-organisms before they can cause disease. Other
components of the immune system adapt themselves to each new disease encountered and
are able to generate pathogen-specific immunity. Specific or adaptive immunity is often
sub-divided into two major types depending on how the immunity was introduced. Natural
immunity occurs through contact with a disease causing agent, when the contact was not
deliberate, whereas artificial immunity develops only through deliberate actions. Both
natural and artificial immunity can be further subdivided, depending on the amount of time
the protection lasts. Passive immunity is short lived, and usually lasts only a few months,
whereas protection via active immunity lasts much longer, and is sometimes life-long. The
diagram below summarizes these divisions of immunity.

A further subdivision of adaptive immunity is characterized by the cells involved; humoral

immunity is the aspect of immunity that is mediated by secreted antibodies, whereas the
protection provided by cell mediated immunity involves T-lymphocytes alone. Humoral
immunity is active when the organism generates its own antibodies and passive when
antibodies are transferred between individuals. Similarly, cell mediated immunity is active
when the organisms’ own T-cells are stimulated and passive when T cells come from another
organism.
The immune system must maintain a delicate balance, with potent defensive responses
capable of destroying large numbers of foreign cells and viruses while refraining from undue
destruction of the host's body. When the immune system cannot mount a sufficient defense of

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the host, there is an immune deficiency; this is seen in HIV infection and SCID (Severe
Combined ImmunoDeficiency). If, on the other hand, the immune system acts too vigorously
and begins to attack the host, we have autoimmunity. This is a defiance of the integral
immune system property of self/nonself recognition. That is, the immune system begins
attacking or forming antibodies against the host's own body tissues. Common forms of
autoimmunity include Graves' disease, Hashimoto's Thyroiditis, and systemic lupus
erythematosus.

1.2 Overview of frequently used terms and concepts

Agglutination: The aggregation of particulate antigen by antibodies. Agglutination applies

to red blood cells as well as to bacteria and inert particles covered with antigen.
Allergen: An antigen responsible for producing allergic reactions by inducing IgE formation.
Allergy: A term covering immune reactions to non-pathogenic antigens, which lead to
inflammation and deleterious effects in the host.
Allograft: A tissue transplant (graft) between two genetically nonidentical members of a
species.
Allotypes: Antigenic determinants that are present in allelic (alternate) forms. When used in
association with immunoglobulin, allotypes describe allelic variants of immunoglobulins
detected by antibodies raised between members of the same species.
Alternate (Alternative) pathway: The mechanism of complement activation that does not
involve activation of the C1, C4, C2 pathway by antigen-antibody complexes, and begins
with the activation of C3.
Anaphylatoxin: Substance capable of releasing histamine from mast cells.
Anaphylaxis: Immediate hypersensitivity response to antigenic challenge, mediated by IgE
and mast cells. It is a life-threatening allergic reaction, caused by the release of
pharmacologically active agents.
Antibody: Serum protein formed in response to immunization; antibodies are generally
defined in terms of their specific binding to the immunizing antigen.
Antibody-dependent, cell-mediated cytotoxicity (ADCC): A phenomenon in which target
cells, coated with antibody, are destroyed by specialized killer cells (NK cells and
macrophages), which bear receptors for the Fc portion of the coating antibody (Fc receptors).
These receptors allow the killer cells to bind to the anti-body-coated target.
Antigen: Any foreign material that is specifically bound by specific antibody or specific
lymphocytes; also used loosely to describe materials used for immunization. Antigens may
also be immunogens if they are able to trigger an immune response, or haptens if not.
Antigen-binding site: The part of an immunoglobulin molecule that binds antigen
specifically.
Antigen-presenting cell (APC): A specialized type of cell, bearing cell surface class II
MHC (major histocompatibility complex) molecules, involved in processing and presentation
of antigen to inducer, or helper , T cells. Examples: macrophage, dendritic cells.
Antigen receptor: The specific antigen-binding receptor on T or B lymphocytes; these
receptors are transcribed and translated from rearrangements of V genes.
Antigenic determinant: A single antigenic site or epitope on a complex antigenic molecule
or particle.

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Antigen processing: Large molecules are broken down (processed) within macrophages into
peptides and presented within the groove of MHC molecules.
Autograft: A tissue transplant from one area to another on a single individual.
Autoimmunity (autoallergy): An immune response to "self" tissues or components. Such an
immune response may have pathological consequences leading to autoimmune diseases.
B lymphocyte (B cell): The precursors of antibody-forming plasma cells; these cells carry
immunoglobulin and class II MHC (major histocompatibility complex) antigens on their
surfaces.
Basophil: A polymorphonuclear leukocyte., whose basophils granules contain heparin,
histamine and other vasoactive amines. Within tissues, these cells are known as mast cells
q.v.
Carcinoembryonic antigen (CEA): Antigen present during embryonic development which
normally disappears but reappears in malignant tissue.
Carrier: A large immunogenic molecule or particle to which an antigenic determinant is
attached, allowing the determinant to become immunogenic.
Cell-mediated cytotoxicity (CMC): Killing (lysis) of a target cell by an effector
lymphocyte.
Cell-mediated immunity (CMI): Immune reaction mediated by T cells; in contrast to
humoral immunity, which is antibody mediated. Also referred to as delayed-type
hypersensitivity.
Chemotaxis: Migration of cells along a concentration gradient of an attractant.
Classical pathway: The mechanism of complement activation initiated by antigen-antibody
aggregates and proceeding by way of C1, C4 and C2.
Clonal deletion: The loss of lymphocytes of a particular specificity due to contact with
either "self" or artificially introduced antigen.
Clonal selection theory: The prevalent concept that specificity and diversity of an immune
response are the result of selection by antigen of specifically reactive clones from a large
repertoire of preformed lymphocytes, each with individual specificities.
Complement: A series of serum proteins involved in the mediation of immune reactions.
The complement cascade is triggered classically by the interaction of antibody with specific
antigen.
Coombs' test: A test named for its originator, R.R.A. Coombs, used to detect non-
agglutinating antibodies on red blood cells by addition of an anti-immunoglobulin antibody.
Cross-reactivity: The ability of an antibody, specific for one antigen, to react with a second
antigen; a measure of relatedness between two different antigenic substances.
Cytokines: Soluble substances secreted by cells, which have a variety of effects on other
cells, e.g. Interleukin 1 (Il-1).
Cytotoxic (Cytolytic) T cell: Cell that kills target cells bearing appropriate antigen within
the groove of an MHC class I molecule that is identical to that of the T cell.
Delayed type hypersensitivity (DTH): A T cell-mediated reaction to antigen, which takes
24-48 hours to develop fully, and which involves release of lymphokines and recruitment of
monocytes and macrophages. Also called c cell-mediated immunity.
Determinant: Part of the antigen molecule which binds to an antibody-combining site or to a
receptor on T cells (see hapten and epitope).

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Enzyme-linked immunosorbent assay (ELISA): An assay in which an enzyme is linked to
an antibody and a coloured substrate is used to measure the activity of bound enzyme and,
hence, the amount of bound antibody.
Epitope: An alternative term for antigenic determinant.
Graft versus host reaction (GVH): The pathologic consequences of a response initiated by
transplanted immunocompetent T lymphocytes into an allogeneic, immunologically
incompetent host. The host is unable to reject the grafted T cells and becomes their target.
HLA complex: See 'Major histocompatibility complex'.
Hapten: A compound, usually of low molecular weight, that is not itself immunogenic but
that, after conjugation to a carrier protein or cells, becomes immunogenic and induces
antibody, which can bind the hapten alone in the absence of carrier.
Helper T cells: A class of T cells which help trigger B cells to make antibody against
thymus-dependent antigens. Helper T cells also help generate cytotoxic T cells.
Histocompatibility: Literally, the ability of tissues to get along; in immunology, it means
identity in all transplantation antigens. These antigens, in turn, are collectively referred to as
histocompatibility antigens.
Humoral immunity: Any immune reaction that can be transferred with immune serum is
termed humoral immunity (as opposed to cell-mediated immunity). In general, this term
refers to resistance that result from the presence of specific antibody.
Hybridoma: A hybrid cell that results from the fusion of an antibody-secreting cell with a
malignant cell; the progeny secrete antibody without stimulation and proliferate continuously
both in vivo and in vitro.
Hypersensitivity: State of reactivity to antigen that is greater than normal for the antigenic
challenge; hypersensitivity is the same as allergy and denotes a deleterious outcome rather
than a protective one.
Immediate-type hypersensitivity: Hypersensitivity tissue reaction occurring within minutes
after the interaction of antigen and antibody.
Immune adherence: The adherence of particulate antigen coated with C3b to tissue having
cells with C3b receptors.
Immune complex: Antigen bound to antibody.
Immune modulators: Substances that control the expression of the immune response.
Immunogen: A substance capable of inducing an immune response (as well as reacting with
the products of an immune response). Compare with antigen.
Immunoglobulin (Ig): A general term for all antibody molecules. Each Ig unit is made up of
two heavy chains and two light chains and has two antigen- binding sites.
Interferon: A group of proteins having antiviral activity and capable of enhancing and
modifying the immune response.
Interleukins: Glycoproteins secreted by a variety of leukocytes which have effects on other
leukocytes.
Isograft: A tissue transplanted between two genetically identical individuals.
Killer T cell: A T cell with a particular immune specificity and an endogenously produced
receptor for antigen, capable of specifically killing its target cell after attachment to the target
cell by this receptor. Also called cytotoxic T cell.
Lymphocyte: Small cell with virtually no cytoplasm, found in blood, in all tissue, and in
lymphoid organs, such as lymph nodes, spleen, and Peyer's patches, and bears antigen-
specific receptors.

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Lymphokines: Soluble substances secreted by lymphocytes, which have a variety of effects
on lymphocytes and other cell types.
Macrophage: A large phagocytic cell of the mononuclear series found within tissues.
Properties include phagocytosis, and antigen presentation to T cells.
Macrophage-activating factor (MAF): Actually several lymphokines, including interferon,
released by activated T cells, which together induce activation of macrophages, making them
more efficient in phagocytosis and cytotoxicity.
Major histocompatibility complex (MHC): A cluster of genes on chromosome 6 in
humans, encoding cell surface molecules that are polymorphic and that code for antigens
which lead to rapid graft rejection between members of a single species which differ at these
loci. Several classes of protein such as MHC class I and II proteins are encoded in this
region. These in humans, are known as 'Human leukocyte antigens' (HLA).
Mast cell: Tissue located cell probably derived from basophils. Possesses receptor for Fc of
IgE. Participates in 'Immediate hypersensitivity' reactions.
Memory: In the immune system, memory denotes an active state of immunity to a specific
antigen, such that a second encounter with that antigen leads to a larger and more rapid
response.
Migration inhibition factor (MIF): A lymphokine that inhibits the motility of macrophages
in culture.
Minor histocompatibility antigens: These antigens, encoded outside the MHC, are
numerous, but do not generate rapid graft rejection or primary responses of T cells in vitro.
They do not serve as restricting elements in cell interactions.
Mitogen: A substance that stimulates the proliferation of many different clones of
lymphocytes.
Monoclonal: Literally, coming from a single clone. A clone is the progeny of a single cell.
In immunology, monoclonal generally describes a preparation of antibody that is
monogenous, or cells of a single specificity.
Monocyte: Large circulating white cell, 2-10% of total white cells, phagocytic, indented
nucleus. Migrates to tissues, where it is known as a macrophage.
Monokines: Soluble substances secreted by monocytes, which have a variety of effects on
other cells.
Myeloma: A tumour of plasma cells, generally secreting a single species of immunoglobulin.
NK cell: Naturally occurring, large, granular, lymphocyte-like killer cells that kill various
tumour cells; they may play a role in resistance to tumours. Also, they participate in ADCC.
They do not exhibit antigenic specificity, and their number does not increase by
immunization.
Opsonin: A substance, usually antibody or complement component, which coats a particle
such as a bacterium and enhances phagocytosis by phagocytic cells.
Opsonization: Literally means "preparation for eating". The coating of a bacterium with
antibody and/or complement that leads to enhanced phagocytosis of the bacterium by
phagocytic cells.
Passive immunization: Immunization by the administration of preformed antibody into a
nonimmune individual.
Phagocytosis: The engulfment of a particle or a microorganism by leukocytes.
Pinocytosis: Ingestion of liquid or very small particles by vesicle formation in a cell.
Plasma cell: End-stage differentiation of a B cell to an antibody-producing cell.

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Polymorphonuclear leukocyte: White cell, granular cytoplasm. Neutral staining
(neutrophil) - most frequent, phagocytic. Basophilic staining - basophil q.v. Eosinophilic
staining - eosinophil q.v.
Primary responses: The immune response to a first encounter with antigen. The primary
response is generally small, has a long induction phase or lag period, consists primarily of
IgM antibodies, and generates immunologic memory.
Prophylaxis: Protection.
Radioimmunoassay (RIA): A widely used technique for measurement of primary antigen-
antibody interactions, and for the determination of the level of important biological
substances in mixed samples. It takes advantage of the specificity of the antigen-antibody
interaction and the sensitivity that derives from measurement of radioactively labelled
materials.
Rheumatoid factor: An autoantibody (usually IgM) which reacts with the individual's own
IgG. Present in rheumatoid arthritis.
Second set rejection: Accelerated rejection of an allograft in an already immune recipient.
Serum sickness: A hypersensitivity reaction consisting of fever, rashes, joint pain and
glomerulonephritis, resulting from localization of circulating, soluble, antigen-antibody
complexes, which induce inflammatory reactions. Serum sickness was originally induced
following therapy with large doses of antibody from a foreign source - e.g. horse serum.
T cell: A lymphocyte which undergoes a developmental stage in the thymus.
T-dependent antigen: An immunogen that is able to induce antibody synthesis only in the
presence of lymphokines released by helper T cells.
T-independent antigen: An immunogen which induces antibody synthesis in the absence of
lymphokines released by T cells; the antibodies are generally only of the IgM isotype.
Vaccination: Originally referred to immunization against smallpox with the less virulent
cowpox (vaccinia) virus; more loosely used for any immunization against a pathogen.

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 CHAPTER II

CHARACTERISTICS OF THE IMMUNE SYSTEM

2.1 Antigens (immunogens)

Antigens were originally defined as non-self molecules which bound specifically to
antibodies. In practice, the term antigen is used to mean any molecule recognized by the
immune system. Antigens which induce adaptive immunity are called immunogens. All
immunogens are antigens, and are usually called antigens unless their ability to induce an
immune response is being discussed. Some antigens, called haptens, are not immunogenic
unless they are covalently linked to immunogenic carriers (usually proteins). Haptens can
bind antibodies once the antibodies are produced, but haptens will not induce antibody
synthesis on their own. Small non-protein organic molecules, for example the antibiotic
penicillin, are haptens.
Immunogenicity is influenced by
- the chemical nature of the antigen.
- the antigen's size.
- the antigen's usual presence in the body.
- antigen dose and route and timing of administration.
- whether the antigen is easily phagocytosed.
- whether antigen is efficiently presented to T cells on MHC.
- the maturity of the immune system and specific lymphocytes.
Note that immunogenicity depends on the immune system as well as on the antigen.
Immunogenicity is generally higher for proteins than for other organic molecules. Protein
antigens, which are the predominant sort that activate helper T cells, consequently induce
more B cell antibody production, synthesis of IgG or IgA, and generation of memory B and T
cells. T-independent antigens, which activate only B cells, induce IgM synthesis but not
other adaptive immune responses.

Immunogenicity increases with molecular size and complexity. Haptens are generally non-
proteins or they are too small to be immunogenic unless they are covalently attached to a
carrier protein molecule. Some carbohydrate antigens can stimulate B cells to secrete IgM in
the absence of T cell help but do not elicit IgG or memory cells. Human infants make poor
responses to carbohydrate antigens, so early vaccinations with bacterial polysaccharide
antigens employ protein carriers. Aggregated proteins are easily phagocytosed and more
immunogenic than soluble proteins. Adjuvants, including bacterial products, alum, or oil
emulsification of antigen, attract and activate antigen-presenting cells (dendritic cells and
macrophages) and slow antigen release to prolong exposure and improve immunogenicity.
Each part of the antigen bound by a unique antibody is called an epitope or antigenic
determinant site. Most proteins have several epitopes that are recognized by different B
cells and induce a polyclonal antibody response. In a polyclonal response, several clones of
B cells each make different antibodies, all able to bind to the same antigen but at different
epitopes. Epitopes may be shared by closely related antigens (cross-reactivity), so that
antibody made to tetanus toxoid binds tetanus toxin. A protein epitope may be a linear
sequence of amino acids or it may be assembled by protein folding. Epitopes with definite
three-dimensional shapes and charged amino acids are particularly well recognized by

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antibodies. External membrane and cell wall molecules, often present in many copies on the
pathogen, are common B cell antigens. A small peptide of 4-6 amino acids could fit into an
antibody binding site, but larger proteins have more extended epitopes across their surfaces.

2.2 Antibodies (immunoglobulin)

An antibody or immunoglobulin is a large Y-shaped protein used by the immune system to
identify and neutralize foreign objects like bacteria and viruses. Each antibody recognizes a
specific antigen unique to its target. This is because the two tips of the "Y" of the antibody
contain a paratope (a structure analogous to a lock) that is specific for one particular epitope
(analogous to a key) on an antigen, allowing these two structures to precisely bind together.
This precise binding mechanism allows an antibody to tag a microbe or an infected cell for
attack by other parts of the immune system, or to directly neutralize its target (i.e. by
blocking a part of a microbe that is essential for its invasion and survival). The production of
antibodies is the main function of the humoral immune system.
Antibodies are soluble glycoproteins of the immunoglobulin superfamily. The terms
antibody and immunoglobulin are often used interchangeably. When attached to the surface
of the B cell, the membrane-bound form of the immunoglobulin is sometimes referred to as
the B cell receptor (BCR). Soluble antibodies are found in the blood and tissue fluids, as well
as many secretions. In structure, they are globulins (in the γ-region of protein
electrophoresis). They are synthesized and secreted by plasma cells that are derived from the
B cells of the immune system. Membrane-bound immunoglobulins are only found on the
surface of B lymphocytes and facilitate the activation of these cells following binding of their
specific antigen, and their subsequently differentiation into plasma cells for antibody
generation, or memory cells that will remember the foreign antigen during future exposure.
In most cases, interaction of the B cell with a T helper cell is necessary to produce full
activation of the B cell and, therefore, antibody generation following antigen binding.
Structure of the antibody

Immunoglobulins are heavy plasma proteins, often with sugar chains added to amino acid
residues by N-linked glycosylation (all antibodies) and occasionally O-linked glycosylation
(e.g. IgA1 and IgD). In other words, they are glycoproteins. The basic unit of each antibody
is a monomer (one Ig unit) but the secreted antibody can also be dimeric (with two Ig units as
with IgA), tetrameric (with four Ig units, like teleost fish IgM), or pentameric (with five Ig
units, like mammalian IgM). The monomer is a "Y"-shaped molecule that consists of four
polypeptide chains; two identical heavy chains and two identical light chains connected by
disulfide bonds.

2.2.1 Structure of immunoglobulin

The basic structure of all immunoglobulin molecules consists of two identical L chains and
two identical H chains (Fig. 2-1). The antibody molecule consists of three major domains
connected by a hinge region. As shown in Fig. 2-1, digestion of antibody molecule with a
proteolytic enzyme-papain results in the separation of these three domains. Two domains are
identical and are called fragment antigen binding (Fab), and the third domain is called
fraction crystallizable (Fc). However, treatment with proteolytic enzyme pepsin results in a
fragment that contains both antigen binding arms (Fab')2 and several pieces of the Fc
fragment. Fab interact with the antigen, and Fc bind to Fc-receptors on different cells.

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Figure 2-1. Prototypic structure of immunoglobulins. The complementarity regions (e.g.,
antigen receptor sites that make specific contact with ligands) sites of the V region are shown
in the insert.

Various forms of immunoglobulins such as IgG and IgE are found as monomers, secreted
IgA as dimers and IgM as pentamers (Fig. 2-2). Consequently, two distinct regions of the
assembled immunoglobulin occur: the first, which binds to an antigenic determinant and the
second, which has other functions, such as binding to special cells and the first component of
complement. The two H chains and each H chain and L chain are linked by disulfide bonds.
Each chain is divided into two regions: The C region at the carboxyl-terminus and the
variable region at the amino-terminus. The C region of each L chain consists of about 107
amino acids and has an invariant structure except for isotypic features (kappa or lambda) and
allotypic variants (e.g., molecular structures that are individually inherited). V and C regions
of H chains are further divided into domains characterized by folding of the polypeptide
chain into 110 amino acid loops. V regions of H and L chains display great variability in the
sequence of amino acids. Localized areas of these hypervariable regions of H and L chains
interact to form antigen binding sites (i.e., CD1, CD2 and CD3; Fig. 2-1). In contrast, C
regions of H chains dictate other functions of immunoglobulins, including binding to cell
surface receptors. Eight immunoglobulin isotypes, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2,
IgM, IgD, and IgE, are produced by B cells as a result of rearrangements of V genes for H
chains (VH), D genes for H chains (DH), J genes for H chains (JH), V genes for L chains
(VL), J genes for L chains (JL), and C region genes (vide infra) The special properties of
each immunoglobulin class are as follows (Table 1-3).

Figure 2-2. Diagram of various forms of immunoglobins; IgG and IgE are found as
monomers, secreted IgA as dimers and IgM as pentamers. Dimers and pentamers are held
together by the J chain.

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IgG: IgG is a monomeric, four-chain structure consisting of two gamma heavy chains and
two kappa or lambda light chains. The C region of the H chain of the molecule consists of
three domains. Inter-chain disulfide linkages between the Cgamma1 and Cgamma2 domains
stabilize the structure and define the hinge region of the molecule. IgG is the dominant
immunoglobulin in extracellular fluids and is the only immunoglobulin transported across the
placenta, and directly acts as an opsonin. There are four subclasses of IgG, each of which
displays unique antigenic determinants on the C region of the H chains. The approximate
proportion of each subclass in blood is IgG1, 70%; IgG2, 20%; IgG3, 8%; and IgG4, 2%.
The antibody specificities are distributed in somewhat specific patterns in each subclass.
Neutralizing antibodies to protein toxins are mostly found in IgG1, antibodies to
polysaccharides in IgG2, and antibodies to viruses in IgG3.
IgM: IgM is a pentamer of 4-chain units that are bound to a separate peptide called the J
chain. IgM molecules consist of µ H chains and kappa or lambda L chains. Monomeric IgM
is the principal antigen receptor on B cells. IgM is found principally in blood, but also occurs
in external secretions. It binds most efficiently the C1q subunit of the first component of
complement (vide infra), and is the first immunoglobulin expressed in B cell development.
IgA: IgA consists of a heavy chains and kappa or lambda light chains. There are two
principal molecular forms of IgA, monomers whose basic structure and numbers of domains
are similar to IgG and dimers that bind to J chains. Monomeric IgA, the second most
common immunoglobulin in adult serum, is primarily produced by plasma cells in the bone
marrow, whereas dimeric IgA, the dominant immunoglobulin in external secretions, is
produced by plasma cells at mucosal sites. Dimeric IgA is complexed and transported with a
secretory component to form secretory IgA (FigURE 2-3). Dimeric IgA binds to polymeric
immunoglobulin receptors (secretory component) on the basolateral membranes of epithelial
cells; the complex is internalized and transported across the cells in an endocytic vesicle to
the apical pole of the cell where it is secreted as secretory IgA. The addition of a secretory

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component not only facilitates the transport of dimeric IgA, but protects the molecule from
proteolysis.

Figure 2-3. Assembly and secretion of secretory IgA.

There are two subclasses of IgA, IgA1 and IgA2. IgA1 predominates in the blood; there is an
equal distribution of the two subclasses in external secretions. IgA2 is more resistant than
IgA1 to bacterial IgA proteases that attack the hinge region of the molecule.
IgD: IgD is a monomeric four-chain polypeptide structure that is similar to IgG but its heavy
chain (delta) is unique. Although this protein is expressed along with monomeric IgM on
mature B cells, only small amounts of it are found in extracellular fluids.
IgE: IgE is also a four-chain polypeptide structure that is similar to IgG, but its heavy chain
(epsilon) is distinct. Only trace amounts of this immunoglobulin are found in serum. IgE
binds avidly to circulating blood basophils and mast cells in the submucosal sites and the
skin. Cell-bound IgE antibodies defend against tissue parasites and initiate the pathogenesis
of immediate hypersensitivity by triggering the release of low-molecular weight vasoactive
compounds, including histamine, leukotrienes, and platelet-activating factor and certain
proinflammatory cytokines such as TNF-alpha and IL-5, once they are cross-linked by
antigens.

2.2.2 Sequence of Antibody Formation

Initial exposure to an antigen results in the production of low affinity antibodies, but
continued exposure to antigen leads to the production of high affinity antibodies. In the
primary antibody response (the first immunization), B cells are activated to produce IgM
antibody. By 3-5 days, specific antibodies, mainly of the IgM isotype, appear in the serum
and the concentration (titer) increases until a peak is reached in 10-14 days (Fig. 2-4).
Antibody titers then fall to preimmunization levels after some weeks. Upon reimmunization,
there is a more rapid and extensive development of antibody-producing cells in regional
lymph nodes, and many of them undergo an isotype switch to produce IgG or other
immunoglobulin classes of specific antibodies. As a result, in most cases following re-
immunization, serum antibodies are primarily IgG and have a greater affinity for antigens;
also, the antibody titers are higher and persist for much longer periods.

15
Figure 2-4. Isotypes of serum antibodies in primary and secondary immunization.

2.3 Cells and tissues of immune system

Immune cells
A: LYMPHOCYTES. These cells have receptors for antigen and confer specificity on an
immune response. Lymphocytes express receptors with varying affinity for the antigen in
question. The cell with the highest affinity for the most abundant antigen will have growth
advantage and will preferentially generate progeny of itself. This process is called clonal
expansion and is antigen driven.
B lymphocytes produce antibodies and some soluble mediators called cytokines. They arise
in the bone marrow in adult mammals.
T lymphocytes arise in bone marrow but mature in the thymus. They do not produce
antibody molecules but have surface receptors structurally related to Ig. T cells see antigen in
a different way to B cells. They recognise peptide fragments of antigen complexed with cell
surface MHC glycoproteins on neighbouring cells. The cell surface glycoproteins encoded by
genes in the Major Histocompatibility Complex(MHC) bind fragments of antigen after it
has been subjected to antigen processing.

There are two sub-types of T cell defined on the basis of function, accessory molecule
expression and the type of MHC protein presenting antigen to them. This can be summarised
as follows:

16
*Different cells of the immune system express complex arrays of cell surface proteins which
can be distinguished with monoclonal antibodies. These surface markers have been given
standardised names CD1 etc (up to 247 at present)
Natural killer (NK) cells are large granular lymphocytes that are cytotoxic in the absence of
prior stimulation. NK cells represent a first line of defence to infections, tumour growth and
other pathogenic alterations of tissue homoeostasis. NK cells do not express antibodies or T
cell receptors at their cell surface. They produce cytokines and express receptors for
immunoglobulin. They also possess other receptor molecules which allow them to detect
some infected host cells, including tumour cells, virus, or intracellular bacteria-infected cells.

B: MONONUCLEAR PHAGOCYTES. If you inject "vital" dyes into experimental animals

they will be taken up by various cell types including macrophages (mf), microglial cells in
the CNS, endothelial cells of vascular sinusoids and reticular cells of lymphoid organs. These
are the cells of the Reticulo-Endothelial System (RES). These cells all take up dye by
pinocytosis. Only cells of the monocyte-macrophage lineage take up large particulate
antigens, pieces of tissue, senescent cells, bacteria etc. by phagocytosis.
These cells have important properties:
- they express a myeloid receptor (CD14) which serves as a recognition molecule for a wide
variety of bacterial envelope molecules, such as LPS from Gram -ve organismsand
components of Mycobacterial and Gram +ve cell walls. Ligation of this receptor leads to
macrophage activation.
- they can act as antigen presenting cells (APC) for T cells.
- they are activated by T cell derived cytokines leading to increased phagocytosis and
microbicidal activity (increased activity of degradative enzymes, nitrogen and oxygen free
radical production and prostaglandins etc.).
- they express receptors for antibody and complement which means that they bind immune
complexes, especially if the antibody involved has complement components bound to it (if
the antibody has fixed complement), and endocytose/phagocytose these rapidly.
- they act as scavengers for cell debris and senescent cells (Kupffer cells in the liver bind
"old" erythrocytes).

17
NOTE: T cell derived cytokines increase the antigen presenting activity of macrophages
which, in turn, are able to present antigen to T cells. This cycle will continue as a positive
feedback loop until the antigen is eliminated.

C: DENDRITIC CELLS. There are two cell types with similar names but different functions.
Cells of the dendritic cell (DC) lineage are bone marrow derived. In the skin they are known
as Langerhans Cells (LC). These cells efficiently process antigen but cannot present it to T
cells. LC have been shown to pick up antigen in skin and carry it via afferent lymphatic
vessels to lymph nodes. Dendritic cells in lymph are known as "veiled" cells. In lymph nodes
the cells, now known as tissue dendritic cells or interdigitating cells, may efficiently present
antigen if they encounter the right T cell. In fact these are the best APC...............far fewer
DC are required to initiate an immune response than any other APC.

Follicular dendritic cells (FDC) are found in lymphoid follicles. They are called dendritic
because of their morphology rather than any lineage relationship with DC. In fact, there is
considerable uncertainty about their developmental origin [some evidence suggests they are
long-lived bone marrow derived cells, other data that they are of epithelial origin]. FDC have
receptors for immunoglobulin and complement and are able to trap antigen at their cell
surface, in the form of antigen/antibody/C3d complexes, for long periods of time. They
cannot present antigen to T cells but are important in developing responses by B cells.

D: GRANULOCYTES. There are three types of granulocyte distinguished according to their

histological staining patterns.
Neutrophils, also known as polymophonuclear leukocytes, express receptors for
immunoglobulin and complement and are involved in the acute inflammatory response.
Eosinophils carry receptors for IgE, are involved in the destruction of IgE coated parasites,
such as helminths, and contribute to the response to allergens.
Basophils are the circulating counterpart of tissue mast cells. They express high affinity
receptors for IgE and are stimulated to secrete the chemicals responsible for immediate
hypersensitivity following antigen induced aggregation of these receptors.

Lymphoid Tissue
Lymphoid tissue is conveniently divided into the central or primary and peripheral or
secondary organs. Central organs include the bone marrow and thymus. Lymphocytes,
monocytes and granulocytes derive from precursor stem cells in the bone marrow. B
lymphocytes migrate directly from marrow to the peripheral lymphoid tissue whereas T
lymphocytes undergo further maturation in the thymus. The bone marrow and thymus are
involved in generating precursor lymphocytes rather than immune responses.
Once released from the bone marrow and thymus lymphocytes begin a life of patrol and
respond. Some 2.5 x 1010 lymphocytes pass through a lymph node per day with 1-2% of the
lymphoid pool traversing the whole lymphoid system every hour. This degree of patrol
allows rapid response to infectious agents.

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 The lymph nodes (see diagram below) and spleen are designed to optimise interaction
between APC and T and B lymphocytes. The lymphatic system is a series of vessels which
drain and filter the tissue fluids . Lymph fluid enters the node via afferent lymphatics, passes
through the sinuses lined with macrophages and leaves via the efferent lymphatic (ultimately
all drain into the portal vein). Lymphocytes enter the node primarily from the blood via
specialised endothelia also within the T areas and leave via the efferent lymphatics. Dendritic
cells migrating from the tissues enter the node into the T cell areas. B cells entering nodes
from the blood must cross the T rich area in transit to the B cell rich areas thus optimising the
chance of T-B co-operation. The B cell rich areas contain mature, resting B cells organised
into structures around follicular dendritic cells (primary follicles).

The spleen is fed by a single artery and does not receive afferent lymphatic drainage. Small
splenic arterioles are surrounded by periarteriolar lymphoid sheaths mainly consisting of
CD4 with some CD8 +ve T cells. The sheaths are associated with lymphoid follicles of
similar organisation to the lymph node.
The largest concentration of lymphoid tissue is, however, in the gut and other mucosal areas.
Here aggregates of lymphoid tissue, similar to lymph nodes in organisation, constitute
Peyer's Patches in the lamina propria of the small intestine, tonsils in the pharynx, and
submucosal lymphoid follicles in the appendix and throughout the upper airways.

19
 CHAPTER III

NATURE OF THE IMMUNE SYSTEM

3.1 Types of the immune system

3.1.1 Innate immune system

Innate (Nonspecific) Immunity - The term, innate immunity, refers to the basic resistance to
disease that a species possesses - the first line of defense against infection.
The characteristics of the innate immune response include the following:
- Responses are Broad-Spectrum (non-specific)
- There is no memory or lasting protective immunity
- There is a limited repertoire of recognition molecules
- The responses are phylogenetically ancient
- Potential pathogens are encountered routinely, but only rarely cause disease. The vast
majority of microorganisms are destroyed within minutes or hours by innate defenses. The
acquired specific immune response comes into play only if these innate defenses are
breached.

Anatomic Barriers
Skin (physical barrier, low pH due to lactic and fatty acids)
epidermis - thin outer layer containing tightly packed epidermal cells and keratin (water-
proofing) completely renewed every 15-30 days.
dermis - thicker inner layer contains sebaceous glands associated with hair follicles -
produce sebum which consists of lactic and fatty acids maintaining a pH 3-5.
Mucous membranes (ciliated epithelial cells; saliva, tears and mucous secretions) - GI,
urogenital, respiratory tracts - collectively represents a huge surface area.

Physiologic Barriers
Temperature - normal body temperature inhibits growth of most microorganisms.
Elevated body temperature (fever) can have a direct effect on pathogenic microorganisms.
pH - low pH of stomach, skin, & vagina (inhibits microbial growth)
Oxygen tension
Huge number of chemical factors (a few examples given below):
 Fatty acids, lactic acid
 Pepsin (digestive enzyme which hydrolyzes proteins)
 Lysozyme -hydrolytic enzyme found in mucous secretions - able to cleave the
petidoglycan layer of the bacterial cell wall.
 Anti-microbial substances which directly destroy microorganims: cryptidins and a-
defensins (produced in base of crypts of small intestine - damage cell membranes) b-
defensins (produced within skin, respiratory tract - also damages cell membranes)
surfactant proteins A & D (present in lungs - function as opsonins which enhance the
efficiency of phagocytosis)
 Interferons - group of proteins produced by cells following viral infection. Secreted
by the cells, and then binds to nearby cells and induces mechanisms which inhibit
viral replication.

20
  Complement - a group of serum proteins that circulate in an inactive proenzyme state.
These proteins can be activated by a variety of specific and nonspecific immunologic
mechanisms that convert the inactive proenzymes into active enzymes.ssss The
activated complement components participate in a controlled enzymatic cascade that
results in membrane-damaging reactions which destroy pathogenic organisms by
formation of a membrane attack comples (MAC).

Endocytic and Phagocytic Barriers Endocytosis - Process by which macromolecules

contained within the extracellular tissue fluid are internalized by cells. Internalization occurs
as small regions of the plasma membrane invaginate, or fold inward, forming small endocytic
vesicles known as endosomes. Occurs through pinocytosis or receptor-mediated endocytosis.
Pinocytosis - nonspecific membrane invagination Receptor-mediated endocytosis -
specific, macromolecules are selectively internalized after binding to specific membrane
receptors.
Following internalization, the endosomes fuse with primary lysosmes. Lysosomes contain
large numbers of degradative enzymes (> 20 different hydrolytic enzymes including
proteases, nucleases, lipases, etc). The ingested macromolecules are subsequently digested
into small breakdown products. Products not utilized by the cell are released through the
process known as exocytosis.
Phagocytosis Involves the ingestion of particulate material including whole pathogenic
microorganisms. The plasma membrane expands around the particulate material to form
large vesicles called phagosomes (10-20times larger than endosome). Only specialized cells
are capable of phagocytosis, whereas endocytosis is carried out by virtually all cells. Once
particulate matter is ingested into phagosomes, the phagosomes fuse with lysosomes and the
ingested material is then digested by a process similar to that seen in endocytosis.
The so-called "professional phagocytes" include: monocytes & macrophages, neutrophils,
and dendritic cells
There are a few other cells which can be induced to become phagocytic under certain
circumstances (i.e. during intense inflammation) Both fibroblasts and epithelial cells are
known as "non-professional" phagocytes.

PATHOGEN-ASSOCIATED MOLECULAR PATTERNS (PAMPs), PATTERN-

RECOGNITION RECEPTORS (PRRs), AND CYTOKINES IMPORTANT IN
INNATE IMMUNITY

Pathogen-Associated Molecular Patterns (PAMPs)

The overall purpose of this Learning Object is:

1) to learn how the innate immune system is able to detect conserved microbial molecules
(pathogen-associated molecular patterns or PAMPs) in order to detect microbial invasion
and initiate innate immune defenses; and
2) to learn examples of microbial molecules that function as pathogen-associated
molecular patterns (PAMPs).

21
 

Innate immunity is antigen-nonspecific defense mechanisms that a host uses

immediately or within several hours after exposure to almost any microbe. This is the
immunity one is born with and is the initial response by the body to eliminate microbes
and prevent infection.

Unlike adaptive immunity, innate immunity does not recognize every possible antigen.
Instead, it is designed to recognize molecules shared by groups of related microbes that
are essential for the survival of those organisms and are not found associated with
mammalian cells.

These unique microbial molecules are called pathogen-associated molecular patterns or

PAMPS and include LPS from the gram-negative cell wall, peptidoglycan and lipotechoic
acids from the gram-positive cell wall, the sugar mannose (a terminal sugar common in
microbial glycolipids and glycoproteins but rare in those of humans), bacterial and viral
unmethylated CpG DNA, bacterial flagellin, the amino acid N-formylmethionine found in
bacterial proteins, double-stranded and single-stranded RNA from viruses, and glucans from
fungal cell walls.

In addition, unique molecules displayed on stressed, injured, infected, or transformed human

cells also act as PAMPS. (Because all microbes, not just pathogenic microbes, possess
PAMPs, pathogen-associated molecular patterns are sometimes referred to as microbe-
associated molecular patterns or MAMPs.)

Most body defense cells have pattern-recognition receptors for these common
PAMPSand so there is an immediate response against the invading microorganism.

Pathogen-associated molecular patterns can also be recognized by a series of soluble pattern-

recognition receptors in the blood that function as opsonins and initiate the complement
pathways.

In all, the innate immune system is thought to recognize approximately 103 of these
microbial molecular patterns.

The innate immune responses do not improve with repeated exposure to a given infection and
involve the following:

 phagocytic cells (neutrophils, monocytes, and macrophages);

 cells that release inflammatory mediators (basophils, mast cells, and eosinophils);
 natural killer cells (NK cells); and
 molecules such as complement proteins, acute phase proteins, and cytokines.

Examples of innate immunity include anatomical barriers, mechanical removal, bacterial

antagonism, pattern-recognition receptors, antigen-nonspecific defense chemicals, the
complement pathways, phagocytosis, inflammation, and fever.

22
We will now take a closer look at pathogen-associated molecular patterns (PAMPs).

Pathogen-Associated Molecular Patterns (PAMPs)

In order to protect against infection, one of the first things the body must do is detect the
presence of microorganisms.

The body initially does this by recognizing molecules unique to groups of related
microorganisms and are not associated with human cells.

These unique microbial molecules are called pathogen-associated molecular patterns or

PAMPs.

In addition, unique molecules displayed on stressed, injured, infected, or transformed

human cells also act as PAMPs. In all, the innate immune system is thought to recognize
approximately 103 molecular patterns.

Examples of microbial-associated PAMPs include:

a. lipopolysaccharide (LPS) from the outer membrane of the gram-negative cell wall

b. bacterial lipoproteins and lipopeptides

c. porins in the outer membrane of the gram-negative cell wall;

d. peptidoglycan found abundantly in the gram-positive cell wall and to a lesser degree in
the gram-negative cell wall;

e. lipoteichoic acids found in the gram-positive cell wall;

f. lipoarabinomannan found in acid-fast cell walls

g. mannose-rich glycans (short carbohydrate chains with the sugar mannose or fructose as
the terminal sugar). These are common in microbial glycoproteins and glycolipids but rare
in those of humans.

h. flagellin found in bacterial flagella;

i. bacterial and viral nucleic acid. Bacterial and viral genomes contain a high frequency of
unmethylated cytosine-guanine dinucleotide or CpG sequences (a cytosine lacking a methyl
or CH3 group and located adjacent to a guanine). Mammalian DNA has a low frequency of
CpG sequences and most are methylated which may mask recognition by pattern-recognition
receptors. Also, human DNA and RNA does not normally enter cellular endosomes where
the pattern-recognition receptors for microbial DNA and RNA are located;

23
j. N-formylmethionine, an amino acid common to bacterial proteins;

k. double-stranded viral RNA unique to many viruses in some stage of their replication;

l. single-stranded viral RNA from many` viruses having an RNA genome;

m. lipoteichoic acids, glycolipids, and zymosan from yeast cell walls; and

n. phosphorylcholine and other lipids common to microbial membranes.

Examples of PAMPs associated with stressed, injured, infected, or transformed host

cells and not found on normal cells include:

a. heat-shock proteins

b. altered membrane phospholipids

To recognize PAMPs such as those listed above, various body cells have a variety of
corresponding receptors called pattern-recognition receptors or PRRs capable of binding
specifically to conserved portions of these molecules. Cells that typically have pattern
recognition receptors include macrophages, dendritic cells, endothelial cells, mucosal
epithelial cells, and lymphocytes.

Cytokines Important in Innate Immunity

The overall purpose of this Learning Object is:

1) to learn introduce how cytokines function to regulate innate immune defenses; and
2) to describe how type I interferons are able to block viral replication within infected
cells.

Innate immunity is antigen-nonspecific defense mechanisms that a host uses

immediately or within several hours after exposure to almost any microbe. This is the
immunity one is born with and is the initial response by the body to eliminate microbes
and prevent infection.

Unlike adaptive immunity, innate immunity does not recognize every possible antigen.
Instead, it is designed to recognize molecules shared by groups of related microbes that
are essential for the survival of those organisms and are not found associated with
mammalian cells. These unique microbial molecules are called pathogen-associated
molecular patterns or PAMPS and include LPS from the gram-negative cell wall,
peptidoglycan and lipotechoic acids from the gram-positive cell wall, the sugar mannose (a
terminal sugar common in microbial glycolipids and glycoproteins but rare in those of

24
humans), bacterial and viral unmethylated CpG DNA, bacterial flagellin, the amino acid N-
formylmethionine found in bacterial proteins, double-stranded and single-stranded RNA
from viruses, and glucans from fungal cell walls. In addition, unique molecules displayed on
stressed, injured, infected, or transformed human cells also act as PAMPS. (Because all
microbes, not just pathogenic microbes, possess PAMPs, pathogen-associated molecular
patterns are sometimes referred to as microbe-associated molecular patterns or
MAMPs.)

Most body defense cells have pattern-recognition receptors for these common
PAMPSand so there is an immediate response against the invading microorganism.
Pathogen-associated molecular patterns can also be recognized by a series of soluble pattern-
recognition receptors in the blood that function as opsonins and initiate the complement
pathways. In all, the innate immune system is thought to recognize approximately 103 of
these microbial molecular patterns.

The innate immune responses do not improve with repeated exposure to a given infection and
involve the following:

 phagocytic cells (neutrophils, monocytes, and macrophages);

 cells that release inflammatory mediators (basophils, mast cells, and eosinophils);
 natural killer cells (NK cells); and
 molecules such as complement proteins, acute phase proteins, and cytokines.

Examples of innate immunity include anatomical barriers, mechanical removal, bacterial

antagonism, pattern-recognition receptors, antigen-nonspecific defense chemicals, the
complement pathways, phagocytosis, inflammation, and fever.

We will now take a closer look at the cytokines involved in innate immunity.

Cytokines are low molecular weight, soluble proteins that are produced in response to
an antigen and function as chemical messengers for regulating the innate and adaptive
immune systems. They are produced by virtually all cells involved in innate and adaptive
immunity, but especially by T- helper (Th) lymphocytes. The activation of cytokine-
producing cells triggers them to synthesize and secrete their cytokines. The cytokines, in
turn, are then able to bind to specific cytokine receptors on other cells of the immune system
and influence their activity in some manner.

Cytokines are pleiotropic, redundant, and multifunctional.

 Pleiotropic means that a particular cytokine can act on a number of different types of
cells rather than a single cell type.
 Redundant refers to to the ability of a number of different cytokines to carry out the
same function.

25
  Multifunctional means the same cytokine is able to regulate a number of different
functions.

Some cytokines are antagonistic in that one cytokine stimulates a particular defense function
while another cytokine inhibits that function. Other cytokines are synergistic wherein two
different cytokines have a greater effect in combination than either of the two would by
themselves.

There are three functional categories of cytokines:

1. cytokines that regulate innate immune responses,

2. cytokines that regulate adaptive Immune responses, and
3. cytokines that stimulate hematopoiesis.

Cytokines that regulate innate immunity are produced primarily by mononuclear

phagocytes such as macrophages and dendritic cells , although they can also be produced by
T-lymphocytes, NK cells , endothelial cells , and mucosal epithelial cells. They are
produced primarily in response to pathogen-associated molecular patterns (PAMPs)
such as LPS, peptidoglycan monomers, teichoic acids, unmethylated cytosine-guanine
dinucleotide or CpG sequences in bacterial and viral genomes, and double-stranded viral
RNA. Cytokines produced in response to PRRs on cell surfaces, such as the inflammatory
cytokines IL-1, IL-6, IL-8, and TNF-alpha, mainly act on leukocytes and the endothelial
cells that form blood vessels in order to promote and control early inflammatory responses .
Cytokines produced in response to PRRs that recognize viral nucleic acids, such as type I
interferons, primarily block viral replication within infected host cells.

Examples include:

a. Tumor necrosis factor-alpha (TNF-alpha)

TNF-alpha is the principle cytokine that mediates acute inflammation. In excessive

amounts it also is the principal cause of systemic complications such as the shock
cascade. Functions include acting on endothelial cells to stimulate inflammation and the
coagulation pathway; stimulating endothelial cells to produce selectins and ligands for
leukocyte integrins during diapedesis ; stimulating endothelial cells and macrophages to
produce chemokines that contribute to diapedesis, chemotaxis , and the recruitment of
leukocytes; stimulating macrophages to secrete interleukin-1 (IL-1) for redundancy;
activating neutrophils and promoting extracellular killing by neutrophils; stimulating the
liver to produce acute phase proteins , and acting on muscles and fat to stimulate catabolism
for energy conversion. In addition, TNF is cytotoxic for some tumor cells; interacts with the
hypothalamus to induce fever and sleep; stimulates the synthesis of collagen and collagenase
for scar tissue formation; and activates macrophages. TNF is produced by monocytes,
macrophages, dendritic cells, Th1 cells, and other cells.

b. Interleukin-1 (IL-1)

26
IL-1 function similarly to TNF in that it mediates acute inflammatory responses. It also
works synergistically with TNF to enhance inflammation. Functions of IL-1 include
promoting inflammation ; activating the coagulation pathway, stimulating the liver to
produce acute phase proteins , catabolism of fat for energy conversion, inducing fever and
sleep; stimulates the synthesis of collagen and collagenase for scar tissue formation;
stimulates the synthesis of adhesion factors on endothelial cells and leukocytes for
diapedesis ; and activates macrophages. IL-1 is produced primarily by monocytes,
macrophages, dendritic cells, endothelial cells, and some epithelial cell.

c. Chemokines

Chemokines are a group of cytokines that enable the migration of leukocytes from the
blood to the tissues at the site of inflammation. They increase the affinity of integrins on
leukocytes for ligands on the vascular wall during diapedesis , regulate the polymerization
and depolymerization of actin in leukocytes for movement and migration, and function as
chemoattractants for leukocytes. In addition, they trigger some WBCs to release their killing
agents for extracellular killing and induce some WBCs to ingest the remains of damaged
tissue. Chemokines also regulate the movement of B-lymphocytes T-lymphocytes , and
dendritic cells through the lymph nodes and the spleen. When produced in excess amounts,
chemokines can lead to damage of healthy tissue as seen in such disorders as rheumatoid
arthritis, pneumonia, asthma, adult respiratory distress syndrome (ARDS), and septic shock.
Examples of chemokines include IL-8, MIP-1a, MIP-1b, MCP-1, MCP-2, MCP-3, GRO-a,
GRO-b, GRO-g, RANTES, and eotaxin. Chemokines are produced by many cells including
leukocytes, endothelial cells, epithelial cells, and fibroblasts.

d. Interleukin-12 (IL-12)

IL-12 is a primary mediator of early innate immune responses to intracellular

microbes. It is also an inducer of cell-mediated immunity. It functions to stimulate the
synthesis of interferon-gamma by T-lymphocytes and NK cells ; increases the killing
activity of cytotoxic T-lymphocytes and NK cells; and stimulates the differentiation of naive
T4-lymphocytes into interferon-gamma producing Th1 cells. It is produced mainly by
macrophages and dendritic cells.

e. Type I Interferons

Interferons modulate the activity of virtually every component of the immune system. Type I
interferons include 13 subtypes of interferon-alpha, interferon-beta, interferon omega,
interferon-kappa, and interferon tau. (There is only one type II interferon, interferon-
gamma, which is involved in the inflammatory response.)

The most powerful stimulus for type I interferons is the binding of viral DNA or RNA to
toll-like receptors TLR-3, TLR-7, and TLR-9 in endosomal membranes .

a. TLR-3 - binds double-stranded viral RNA;

b. TLR-7 - binds single-stranded viral RNA, such as in HIV, rich in guanine/uracil nucleotide

27
pairs;
c. TLR-9 - binds unmethylated cytosine-guanine dinucleotide sequences (CpG DNA) found
in bacterial and viral genomes but uncommom or masked in human DNA and RNA.

Signaling pattern recognition receptors located in the cytoplasm of cells such as RIG-1
and MDA-5 also signal synthesis and secretion of type-I interferons.

Type I interferons, produced by virtually any virus-infected cell, provides an early innate
immune response against viruses. Interferons induce uninfected cells to produce
enzymes capable of degrading mRNA. These enzymes remain inactive until the uninfected
cell becomes infected with a virus. At this point, the enzymes are activated and begin to
degrade both viral and cellular mRNA. This not only blocks viral protein synthesis, it
also eventually kills the infected cell . In addition, type I interferons also cause infected
cells to produce enzymes that interfere with transcription of viral RNA or DNA. They
also promote body defenses by enhancing the activities of CTLs, macrophages, dendritic
cells, NK cells, and antibody-producing cells.

Type I interferons also induce MHC-I antigen expression needed for recognition of antigens
by cytotoxic T-lymphocytes macrophage , NK cell , cytotoxic T-lymphocytes, and B-
lymphocyte activity; and induce fever. Interferon-alpha is produced by T-lymphocytes , B-
lymphocytes, NK cells, monocytes/macrophages; interferon-beta by virus-infected cells,
fibroblasts, macrophages, epithelial cells, and endothelial cells.

f. Interleukin-6 (IL-6)

IL-6 functions to stimulate the liver to produce acute phase proteins ; stimulates the
proliferation of B-lymphocytes and increases neutrophil production. IL-6 is produced
by many cells including T-lymphocytes, macrophages, monocytes, endothelial cells, and
fibroblasts.

g. Interleukin-10 (IL-10)

IL-10 is an inhibitor of activated macrophages and dendritic cells and as such, regulates
innate immunity and cell-mediated immunity. IL-10 inhibits their production of IL-12, co-
stimulator molecules, and MHC-II molecules , all of which are needed for cell-mediated
immunity . IL-10 is produced mainly by macrophages, and Th2 cells.

h. Interleukin 15 (IL-15)

IL-15 stimulates NK cell proliferation and proliferation of memory T8-lymphocytes .

IL-15 is produced by various cells including macrophages.

i. Interleukin-18 (IL-18)

IL-18 stimulates the production of interferon-gamma by NK cells and T-lymphocytes

and thus induces cell-mediated immunity . It is produced mainly by macrophages.

28
 

3.1.2 Adaptive or acquired immune system

The adaptive immune system is composed of highly specialized, systemic cells and
processes that eliminate pathogenic challenges. Thought to have arisen in the first jawed
vertebrates, the adaptive or "specific" immune system is activated by the “non-specific” and
evolutionarily older innate immune system (which is the major system of host defense
against pathogens in nearly all other living things). It is the adaptive immune response that
provides the vertebrate immune system with the ability to recognize and remember specific
pathogens (to generate immunity), and to mount stronger attacks each time the pathogen is
encountered. This is adaptive immunity because the body's immune system prepares itself for
future challenges.

Figure 3-1. A scanning electron microscope (SEM) image of a single human lymphocyte.

The adaptability of the system is achieved by localized somatic mutations and an irreversible
recombination of antigen receptor gene segments. This mechanism allows a small number of
genes to generate a vast number of different antigen receptors, which are then uniquely
expressed on each indiviual lymphocyte. Because the gene rearrangement leads to an
irreversible change in the DNA of each cell, all of the progeny of that cell will then inherit
genes encoding the same receptor specificity, including the B and T memory cells that are the
keys to long-lived specific immunity.

Functions
Adaptive immunity is triggered in vertebrates when a pathogen evades the innate immune
system and generates a threshold level of antigen.
The major functions of the adaptive immune system include:
 The recognition of specific “non-self” antigens in the presence of “self”, during
the process of antigen presentation.
 The generation of responses that are tailored to maximally eliminate specific
pathogens or pathogen infected cells.

29
  The development of immunological memory, in which each pathogen is
“remembered” by a signature antigen. These memory cells can be called upon to
quickly eliminate a pathogen should subsequent infections occur.

Effector cells
The cells of the adaptive immune system are a type of leukocyte, called a lymphocyte. B
cells and T cells are the major types of lymphocytes. The human body has about 2 trillion
lymphocytes, constituting 20–40% of the body’s WBCs; their total mass is about the same as
the brain or liver. The peripheral blood contains 20–50% of circulating lymphocytes; the rest
move within the lymphatic system.
B cells and T cells are derived from the same pluripotential hemopoietic stem cells, and are
indistinguishable from one another until after they are activated. B cells play a large role in
the humoral immune response, whereas T-cells are intimately involved in cell-mediated
immune responses. B-cells may be named for the bursa of Fabricius, an organ unique to
birds, where the cells were first found to develop. However, in nearly all other vertebrates, B
cells (and T-cells) are produced by stem cells in the bone marrow. T-cells travel to and
develop in the thymus, from which they derive their name. In humans, approximately 1-2%
of the lymphocyte pool recirculates each hour to optimize the opportunities for antigen-
specific lymphocytes to find their specific antigen within the secondary lymphoid tissues.
In an adult animal, the peripheral lymphoid organs contain a mixture of B- and T cells in at
least three stages of differentiation:
 naive cells that have matured, left the bone marrow or thymus, have entered the
lymphatic system, but that have yet to encounter their cognate antigen.
 effector cells that have been activated by their cognate antigen, and are actively involved
in eliminating a pathogen and,
 memory cells – the long-lived survivors of past infections.

Humoral immunity
Humoral immunity (HIR) is the aspect of immunity that is mediated by secreted antibodies,
produced in the cells of the B lymphocyte lineage (B cell). Secreted antibodies bind to
antigens on the surfaces of invading microbes (such as viruses or bacteria), which flags them
for destruction. Humoral immunity is called as such, because it involves substances found in
the humours, or body fluids. The study of the molecular and cellular components that
comprise the immune system, including their function and interaction, is the central science
of immunology. The immune system is divided into a more primitive innate immune system,
and acquired or adaptive immune system of vertebrates, the latter of which is further divided
into humoral and cellular components. Humoral immunity refers to antibody production, and
the accessory processes that accompany it, including: Th2 activation and cytokine
production, germinal center formation and isotype switching, affinity maturation and
memory cell generation. It also refers to the effector functions of antibody, which include
pathogen and toxin neutralization, classical complement activation, and opsonin promotion
of phagocytosis and pathogen elimination.

Cell-mediated immunity

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Cell-mediated immunity is an immune response that does not involve antibodies but rather
involves the activation of macrophages and natural killer cells (NK), the production of
antigen-specific cytotoxic T-lymphocytes, and the release of various cytokines in response to
an antigen. Historically, the immune system was separated into two branches; 1. Humoral
immunity, for which the protective function of immunization could be found in the humor
(cell-free bodily fluid or serum), 2. Cellular immunity, for which the protective function of
immunization was associated with cells. Cellular immunity protects the body by:
 activating antigen-specific cytotoxic T-lymphocytes that are able to lyse body
cells displaying epitopes of foreign antigen on their surface, such as virus-infected
cells, cells with intracellular bacteria, and cancer cells displaying tumor antigens;
 activating macrophages and natural killer cells, enabling them to destroy
intracellular pathogens; and
 stimulating cells to secrete a variety of cytokines that influence the function of
other cells involved in adaptive immune responses and innate immune responses.

Cell-mediated immunity is directed primarily at microbes that survive in phagocytes and

microbes that infect non-phagocytic cells as a result of the ineffectiveness of antibodies due
to inability to penetrate cells. It is most effective in removing virus-infected cells, but also
participates in defending against fungi, protozoans, cancers, and intracellular bacteria. It also
plays a major role in transplant rejection.

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Schematic representation of Humoral and Cell-mediated immune reponses.

Antigen presentation
Adaptive immunity relies on the capacity of immune cells to distinguish between the body's
own cells and unwanted invaders.

Figure 3-2. Association between TCR and MHC class I or MHC class II

The host’s cells express “self” antigens. These antigens are different from those on the
surface of bacteria ("non-self" antigens) or on the surface of virally infected host cells
(“missing-self”). The adaptive response is triggered by recognizing non-self and missing-self
antigens. With the exception of non-nucleated cells (including erythrocytes), all cells are
capable of presenting antigen and of activating the adaptive response. Some cells are
specially equipped to present antigen, and to prime naive T cells. Dendritic cells and B-cells
(and to a lesser extent macrophages) are equipped with special immunostimulatory receptors
that allow for enhanced activation of T cells, and are termed professional antigen presenting
cells (APC). Several T cells subgroups can be activated by professional APCs, and each type
of T cell is specially equipped to deal with each unique toxin or bacterial and viral pathogen.
The type of T cell activated, and the type of response generated, depends in part, on the
context in which the APC first encountered the antigen.

Exogenous antigens

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Figure 3-3. Antigen presentation stimulates T cells to become either "cytotoxic" CD8+ cells
or "helper" CD4+ cells.

Dendritic cells engulf exogenous pathogens, such as bacteria, parasites or toxins in the
tissues and then migrate, via chemotactic signals, to the T cell enriched lymph nodes. During
migration, DCs undergo a process of maturation in which they lose most of their ability to
engulf other pathogens and develop an ability to communicate with T-cells. The DC uses
enzymes to chop the pathogen into smaller pieces, called antigens. In the lymph node, the DC
will display these "non-self" antigens on its surface by coupling them to a "self"-receptor
called the Major histocompatibility complex, or MHC (also known in humans as Human
leukocyte antigen (HLA)). This MHC: antigen complex is recognized by T-cells passing
through the lymph node. Exogenous antigens are usually displayed on MHC Class II
molecules, which activate CD4+ helper T-cells.

Endogenous antigens
Endogenous antigens are produced by viruses replicating within a host cell. The host cell use
enzymes to digest virally associated proteins, and displays these pieces on its surface to T-
cells by coupling them to MHC. Endogenous antigens are typically displayed on MHC Class
I molecules, and activate CD8+ cytotoxic T-cells. With the exception of non-nucleated cells
(including erythrocytes), Class I MHC is expressed by all host cells.

CD8+ T lymphocytes and cytotoxicity

Cytotoxic T cells (also known as TC, killer T cell, or cytotoxic T-lymphocyte (CTL)) are a
sub-group of T cells which induce the death of cells that are infected with viruses (and other
pathogens), or are otherwise damaged or dysfunctional.

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Figure 3-4. Killer T cells—also called cytotoxic T lymphocytes or CTL-directly attack other
cells carrying certain foreign or abnormal molecules on their surfaces.
Naive cytotoxic T cells are activated when their T-cell receptor (TCR) strongly interacts with
a peptide-bound MHC class I molecule. This affinity depends on the type and orientation of
the antigen/MHC complex, and is what keeps the CTL and infected cell bound together.
Once activated the CTL undergoes a process called clonal expansion in which it gains
functionality, and divides rapidly, to produce an army “armed”-effector cells. Activated CTL
will then travel throughout the body in search of cells bearing that unique MHC Class I +
peptide.
When exposed to these infected or dysfunctional somatic cells, effector CTL release perforin
and granulysin: cytotoxins which form pores in the target cell's plasma membrane, allowing
ions and water to flow into the infected cell, and causing it to burst or lyse. CTL release
granzyme, a serine protease that enters cells via pores to induce apoptosis (cell death). To
limit extensive tissue damage during an infection, CTL activation is tightly controlled and
generally requires a very strong MHC/antigen activation signal, or additional activation
signals provided by "helper" T-cells (see below). Upon resolution of the infection, most of
the effector cells will die and be cleared away by phagocytes, but a few of these cells will be
retained as memory cells. Upon a later encounter with the same antigen, these memory cells
quickly differentiate into effector cells, dramatically shortening the time required to mount an
effective response.

CD4+ “helper” T-cells

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Figure 3-5. The T lymphocyte activation pathway. T cells contribute to immune defenses in
two major ways: some direct and regulate immune responses; others directly attack infected
or cancerous cells.

CD4+ Lymphocytes, or helper T cells, are immune response mediators, and play an
important role in establishing and maximizing the capabilities of the adaptive immune
response. These cells have no cytotoxic or phagocytic activity; and cannot kill infected cells
or clear pathogens, but, in essence "manage" the immune response, by directing other cells to
perform these tasks. Helper T cells express T-cell receptors (TCR) that recognize antigen
bound to Class II MHC molecules. The activation of a naive helper T-cell causes it to release
cytokines, which influences the activity of many cell types, including the APC that activated
it. Helper T-cells require a much milder activation stimulus than cytotoxic T-cells. Helper T-
cells can provide extra signals that "help" activate cytotoxic cells.

Th1 and Th2: helper T cell responses

Two types of effector CD4+ T helper cell responses can be induced by a professional APC,
designated Th1 and Th2, each designed to eliminate different types of pathogens. The factors
that dictate whether an infection will trigger a Th1 or Th2 type response are not fully
understood, but the response generated does play an important role in the clearance of
different pathogens. The Th1 response is characterized by the production of Interferon-
gamma, which activates the bactericidal activities of macrophages, and induces B-cells to
make opsonizing (coating) antibodies, and leads to "cell-mediated immunity". The Th2
response is characterized by the release of Interleukin 4, which results in the activation of B-
cells to make neutralizing (killing) antibodies, leading to "humoral immunity". Generally,
Th1 responses are more effecitve against intracellular pathogens (viruses and bacteria that are
inside host cells), while Th2 responses are more effective against extracellular bacteria,
parasites and toxins. Like cytotoxic T-cells, most of the CD4+ helper cells will die upon
resolution of infection, with a few remaining as CD4+ memory cells. HIV is able to subvert

35
the immune system by attacking the CD4+ T cells, precisely the cells that could drive the
destruction of the virus, but also the cells that drive immunity against all other pathogens
encountered during an organisms' lifetime. A third type of T lymphocyte, the regulatory T
cells (Treg), limits and suppresses the immune system, and may control aberrant immune
responses to self-antigens; an important mechanism in controlling the development of
autoimmune diseases.

Table: Contrasting between innate and adaptive immunity with regards to response time, specificity and repeat
infection.
  Innate Adaptive
Response Time hours Days
highly diverse, improves during the course
Specificity limited, fixed
of immune response
Response to Repeat identical to primary
much more rapid than primary response
Infection response

Immunization (Artificial)
Historically, infectious disease has been the leading cause of death in the human population.
Over the last century, two important factors have been developed to combat their spread;
sanitation and immunization. Immunization (commonly referred to as vaccination) is the
deliberate induction of an immune response, and represents the single most effective
manipulation of the immune system mankind has developed. Immunizations are successful
because they utilize the immune system's natural specificity as well as its inducibility. The
principle behind immunization is to introduce an antigen, derived from a disease causing
organism, that stimulates the immune system to develop protective immunity against that
organism, but which does not itself cause the pathogenic effects of that organism. An antigen
(short for antibody generator), is defined as any substance that binds to a specific antibody
and elicits an adaptive immune response.
Most viral vaccines are based on live attenuated viruses, while many bacterial vaccines are
based on acellular components of micro-organisms, including harmless toxincomponents.
Many antigens derived from acellular vaccines do not strongly induce an adaptive response,
and most bacterial vaccines require the addition of adjuvants that activate the antigen
presenting cells of the innate immune system to enhance immunogenicity.

Immunological diversity
Most large molecules, including virtually all proteins and many polysaccharides, can serve as
antigens. The parts of an antigen that interact with an antibody molecule or a lymphocyte
receptor, are called epitopes. Most antigens contain a variety of epitopes and can stimulate
the production of antibodies, specific T cell responses, or both.

36
Figure 3-6. An antibody is made up of two heavy chains and two light chains. The unique
variable region allows an antibody to recognize its matching antigen.
A very small proportion (less than 0.01%) of the total lymphocytes are able to bind to a
particular antigen, which suggests that only a few cells will respond to each antigen. For the
adaptive response to "remember" and eliminate a large number of pathogens the immune
system must be able to distinguish between many different antigens, and the receptors that
recognize antigens must be produced in a huge variety of configurations, essentially one
receptor for each different pathogen that might ever be encountered. Even in the absence of
antigen stimulation, a human is capable of producing more than 1 trillion different antibody
molecules. Millions of genes would be required to store the genetic information used to
produce these receptors, but, the entire human genome contains fewer than 50,000 genes. So,
how are so many antibodies and antigen receptors produced?
These myriads of receptors are produced through a process known as clonal selection.
According to the clonal selection theory, at birth, an animal will randomly generate a vast
diversity of lymphocytes (each bearing a unique antigen receptor) from information encoded
in a small family of genes. In order to generate each unique antigen receptor, these genes will
have undergone a process called combinatorial diversification, in which one gene segment
recombines with other gene segments to form a single unique gene. It is this assembly
process that generates the enormous diversity of receptors and antibodies, before the body
ever encounters antigens, and enables the immune system to respond to an almost unlimited
diversity of antigens. Throughout the lifetime of an animal, those lymphocytes that can react
against the antigens an animal actually encounters will be selected for action, directed against
anything that expresses that antigen.
It is important to note that the innate and adaptive portions of the immune system work
together and not in spite of each other. The adaptive arm, B and T cells, would be unable to
function without the input of the innate system. T cells are useless without antigen-presenting
cells to activate them, and B cells are crippled without T-cell help. On the other hand, the
innate system would likely be overrun with pathogens without the specialized action of the
adaptive immune response.

Adaptive immunity during pregnancy

The cornerstone of the immune system is the recognition of “self” versus “non-self”.
Therefore, the mechanisms which protect the human fetus (which is clearly not “self”) from
attack by the immune system, are particularly interesting. Although no comprehensive
explanation has emerged to explain this mysterious, and often repeated, lack of rejection, two

37
classical reasons may explain how the fetus is tolerated. The first is that the fetus occupies a
portion of the body protected by a non-immunological barrier, the uterus, which the immune
system does not routinely patrol. The second is that the fetus itself may promote local
immunosuppression in the mother, perhaps by a process of active nutrient depletion. A more
modern explanation for this induction of tolerance is that specific glycoproteins expressed in
the uterus during pregnancy suppress the uterine immune response.

3.2 Properties of immune response

There are four main common attributes of both humoral and cell-mediated immune reponses
viz.
(i) Recognition of self vs non-self
The immune system has evolved to distinguish between self and non-self antigens and to
largely eliminate self-reactive lymphocytes. Because the repertoire of immune specificities is
vast and largely random, it is not surprising that many nascent lymphocytes possess receptors
for self-antigens. The mechanism of intrauterine tolerance is not well understood, but much
has been learned about the mechanisms for excluding or inactivating self-reactive
lymphocytes, particularly by using the model of experimentally induced immune tolerance to
foreign antigens. When an antigen is introduced into immunologically immature newborn
animals, they may, upon reaching maturity, become unresponsive to immunization with that
antigen (neonatal tolerance). This immunological tolerance is characterized by the absence of
both antibody and cell-mediated responses, and it is specific for the original antigen.
Subsequent experiments revealed that the induction of antigen-specific tolerance is not
always restricted to immature organisms. Unresponsiveness can also be induced in adults by
using relatively higher doses of soluble antigen (high dose tolerance). The induced state of
unresponsiveness to the antigen is sometimes accompanied by the appearance of suppressor
T cells that actively and specifically inhibit the responses of B and T cells. Recent studies
also reveal that IgM+IgD- B cells and mature T lymphocytes may be directly inactivated by
small doses of antigen in vitro (low dose tolerance). In that model, short exposure of
lymphocytes to the antigen, either at a critical concentration or in a certain modality, leads to
an inactivation rather than a stimulation of the cells.

Collectively, the experiments on tolerance induction demonstrate that the unresponsiveness

to self is likely to be achieved at several levels. During normal development, the self-reactive
lymphocyte clones may be inactivated or deleted by exposure to self macromolecules during
the early stages of maturation in the thymus (Fig. 1-6). The autoselection is dependent upon
MHC class I molecules for CD8+ T cells and class II molecules for CD4+ T cells. Those cells
that are not eliminated and reach their full immunological potential may be inactivated, when
self molecules are presented to these cells at high concentrations or in a form that is
tolerogenic rather than immunogenic. Also, it is possible that some self-reactive lymphocytes
are suppressed by other regulatory cells, such as CD8+ suppressor T cells.
The failure of any of the mechanisms involved in self recognition and elimination or down
regulation of self-reactive clones may result in autoimmunity. Autoimmune disorders in
genetically prone individuals may be generated by a) changes in the expression of self
macromolecules or alterations in their presentation to lymphocytes, b) release of sequestered
self-antigens into the circulation, or access of immunogens to normally immunologically
privileged sites, and c) alterations in lymphocyte maturation and immune regulation. In

38
addition, foreign antigens such as bacteria and viruses that cross-react with self antigens may
augment or initiate any of the above mechanisms.

(ii) Immunological memory

During responses to foreign substances some of the proliferated lymphocytes become
memory cells. The immune system can recognize substances it has previously encountered.
Immunological memory allows the immune system to respond rapidly to defend the body
against an antigen to which it has previously reacted. In addition to producing antibodies
during the first reaction to0 the antigen, the immune system also make memory cells which
stand ready for years or decades to initiate antibody production or lymphocyte production
quickly. Memory cells are primed to act with speed and vigor the next time the same foreign
agent in encountered. This prompt response due to recall by memory cells is called an
anamnestic (secondary) response. The existence of this memory capacity makes active
immunization (vaccination) feasible. The first time a foreign agent is encountered, there is a
short lag time before the immune system can generate enough immune products to overcome,
for example, an infectious agent. Memory cells make it possible for the immune system to
remember and to be alerted to specific foreign agents that have been previously encountered
– for example through infection or immunization.

(iii) Heterogeneity
In addition to the above properties, the immune system possesses another attributes, i.e
heterogeneity. The ability of the immune system to respond in a specific way allows it to
attack particular antigens. But in a lifetime, the human body encounters hundreds of different
foreign antigens. The property of heterogeneity (versatility or diversity) refers to the ability
of the immune system to produce many different kinds of antibodies, each of which react
with a different epitope. When a bacterium or other foreign agent has more than one kind of
epitope, the immune system may make a different antibody against each. And the immune
system is even capable of producing antibodies against molecules it has never before
encountered.

(iv) Specificity
The exquisite specificity of the immune system allows it to selectively recognize billions of
different foreign antigens, while maintaining tolerance to an equally diverse panel of self
antigens However, cross-reaction can occur. Cross-reaction also occur between strains of
microorganisms. Failure of this system results in increased susceptibility to disease, or
autoimmune disorders.

3.3 Factors that modify immune response

Host defenses in healthy adult in an unpolluted environment prevent nearly all infectious
diseases. Individuals with reduced resistance are called compromised host. Factors that
reduce host resistance include very young or old age, stress, seasonal patterns, poor nutrition,
traumatic injury, pollution, and excessive exposure to ultraviolet radiation. Complement
deficiencies, immunosuppressants, infections such as HIV(!?), and genetic defects impair
immune system functions.

CHAPTER IV

39
 THE SYNTHESIS OF ANTIBODY

Plasma cells (also called plasma B cells or plasmocytes) are cells of the immune system
that secrete large amounts of antibodies. They differentiate from B cells upon stimulation by
CD4+ lymphocytes. The B cell acts as an antigen presenting cell (APC), consuming an
offending pathogen. That pathogen gets taken up by the B cell by phagocytosis, and broken
down within phagosomes after fusion with lysosomes releasing proteolytic enzymes onto the
pathogen. Once the enzymes break down the pathogen, pieces of the pathogen (which are
now known as antigenic peptides) are loaded onto MHC II molecules, and presented on its
extracellular surface. Once on the extracellular surface, the CD4+ T-helper lymphocyte will
bind to the MHC II/Antigen molecule and cause activation of the B cell, which includes
differentiation into a plasma cell, and subsequent generation of antibody against the
consumed pathogen.

4.1 Roles of small lymphocytes

Two different types of immunological reactions may occur when the antigen enters the body:
the humoral immunity and cell-mediated immunity. The central importance of the
lymphocyte for both types of immune response was established largely by the work of
Gowans. By labeling the lymphocytes with radioisotope and following their fate in the body
it could be shown that there is a pool of recirculating lymphocytes which pass from the blood
into the lymph nodes, spleen and other tissues and back to the blood by the major channels
such as the thoracic duct. From this, two immune responses may be noted.

4.1.1 Primary antibody response

Induction of a primary immune response begins when an antigen penetrates epithelial
surfaces. It will eventually come into contact with macrophages or certain other classes of
Antigen Presenting cells (APCs), which include B cells, monocytes, dendritic cells,
Langerhans cells and endothelial cells. Antigens, such as bacterial cells, are internalized by
endocytosis and "processed" by the APC, then "presented" to immunocompetent
lymphocytes to initiate the early steps of the immunological response. Processing by a
macrophage (for example) results in attaching antigenic materials to the surface of the
membrane in association with MHC II molecules on the surface of the cell . The antigen-
class II MHC complex is presented to a T-helper (TH2) cell which is able to recognize
processed antigen associated with a class II MHC molecule on the membrane of the
macrophage. This interaction, together with stimulation by Interleukin 1 (IL-1), produced
by the macrophage, will activate the TH2 cell. Activation of the TH2 cell causes that cell to
begin to produce Interleukin 2 (IL-2), and to express a membrane receptor for IL-2. The
secreted IL-2 autostimulates proliferation of the TH2 cells. Stimulated TH2 cells produce a
variety of lymphokines including IL-2, IL-4, IL-6, and gamma Interferon which mediate
various aspects of the immune response. For example, IL-2 binds to IL-2 receptors on other
T cells (which have bound the Ag) and stimulates their proliferation, while IL-4 causes B
cells to proliferate and differentiate into antibody-secreting plasma cells and memory B
cells. IL-4 activates only B cells in the vicinity which themselves have bound the antigen,
and not others, so as to sustain the specificity of the immune response.

40
As previously mentioned, B cells themselves behave as APCs. Cross-linked antigens bound
to antibody receptors on the surface of a B cell cause internalization of some of the antigen
and expression on the B cell membrane together with MHC II molecules. The T H2 cell
recognizes the antigen together with the Class II MHC molecules, and secretes the various
lymphokines that activate the B cells to become antibody-secreting plasma cells and memory
B cells. Even if the antigen cannot cross-link the receptor, it may be endocytosed by the B
cell, processed, and returned to the surface in association with MHC II where it can be
recognized by specific TH2 cells which will become activated to initiate B cell differentiation
and proliferation. In any case, the overall B-cell response leads to antibody-mediated
immunity (AMI).
The antigen receptors on B cell surfaces are thought to be the specific types of antibodies
that they are genetically-programmed to produce. Hence, there are thousands of sub-
populations of B cells distinguished only by their ability to produce a unique (reactive) type
of antibody molecule. A B cell can also react with a homologous antigen on the surface of
the macrophage, or with soluble antigens. When a B-cell is bound to Ag, and simultaneously
is stimulated by IL-4 produced by a nearby TH2 cell, the B cell is stimulated to grow and
divide to form a clone of identical B cells, each capable of producing identical antibody
molecules. The activated B cells further differentiate into plasma cells which synthesize and
secrete large amounts of antibody, and into a special form of B cells called memory B cells.
The antibodies produced and secreted by the plasma cells will react specifically with the
homologous antigen that induced their formation. Many of these reactions lead to host
defense and to prevention of reinfection by pathogens. Memory cells play a role in
secondary immune responses. Plasma cells are relatively short-lived (about one week) but
produce large amounts of antibody during this period. Memory cells, on the other hand, are
relatively long-lived and upon subsequent exposure to Ag they become quickly transformed
into Ab-producing plasma cells.

Generation of cell mediated immunity (CMI) begins when (for example) a TC cell
recognizes a processed antigen associated with MHC I on the membrane of a cell (usually
an altered self cell, but possibly a transplanted tissue cell or a eukaryotic parasite). Under
stimulation by IL-2 produced by TH2 cells the TC cell becomes activated to become a
cytotoxic T lymphocyte (CTL) capable of lysing the cell which is showing the new
(foreign) antigen on its surface, a primary manifestation of CMI. The interaction between an
antigen-presenting macrophage and a TH cell stimulates the macrophage to produce and
secrete a cytokine called Interleukin-1 (IL-1) that acts locally on the T H cell. The IL-1
stimulates the TH-cell to differentiate and produce its own cytokines (which in this case might
be called lymphokines because they arise from a lymphocyte). These lymphokines have
various functions. Interleukin-4 has an immediate effect on nearby B-cells. Interleukin-2 has
an immediate effect on T cells as described above.

Time is required before a primary immune response is effective as a host defense. Antigens
have to be recognized, taken up, digested, processed, and presented by APCs; a few select
TH cells must react with Ag and respond; preexisting B or T lymphocytes must encounter the
Ag and proliferate and differentiate into effector cells (plasma cells or CTLs). In the case of
AMI, antibody level has to build up to an effective physiological concentration to render its
host resistant. It may take several days or weeks to reach a level of effective immunity, even

41
though this immunity may persist for many months, or years, or even a lifetime, due to the
presence of the antibodies. In natural infections, the inoculum is small, and even though the
antigenic stimulus increases during microbial replication, only small amounts of antibody are
formed within the first few days, and circulating antibody is not detectable until about a week
after infection.

4.1.2 Secondary antibody response

On re-exposure to microbial antigens (secondary exposure to antigen), there is an accelerated
immunological response, the secondary or memory response. Larger amounts of antibodies
are formed in only 1-2 days. This is due to the activities of specific memory B cells or
memory T cells which were formed during the primary immune response. These memory
cells, when stimulated by homologous Ag, "remember" having previously seen the Ag, and
are able to rapidly divide and differentiate into effector cells. Stimulating memory cells to
rapidly produce very high (effective) levels of persistent circulating antibodies is the basis for
giving "booster"-type vaccinations to humans and pets. The secondary response is often
called the memory, anamnestic (not forgetting), or booster response.

Figure 4-1. Primary and Secondary Immune Responses. Following the first exposure to an antigen the
immune response (as evidenced by following the concentration of specific antibody in the serum) develops
gradually over a period of days, reaches a low plateau within 2-3 weeks, and usually begins to decline in a
relatively short period of time. When the antigen is encountered a second time, a secondary (memory)
response causes a rapid rise in the concentration of antibody, reaching a much higher level in the serum,
which may persist for a relatively long period of time. This is not to say that a protective level of antibody
may not be reached by primary exposure alone, but usually to ensure a high level of protective antibody
that persists over a long period of time, it is necessary to have repeated antigenic stimulation of the
immune system.
 

4.2 Cellular cooperation in the immune response

42
In order to infect, the organism must first penetrate the physical barriers of the skin or
mucous membranes. After an organism has gained entry, the first line of defence is the non-
specific effector mechanism of the host, in particular the phagocytic cells such as neutrophils.
Macrophages also play a role in phagocytosis, but resting macrophages have relatively low
levels of phagocytic activity, and require activation through local cytokine release in order to
become fully active. In addition to the role of phagocytic cells, some infecting organisms will
activate the complement system. Binding of activated complement components to these
organisms may act as an opsonin, enhancing phagocytosis of the organism. Activation of
complement will also promote local inflammation at the site of infection, through the release
of chemoattractant and vasoactive components. In the case of viral infections, infected cells
may synthesise interferons and/or be recognised and lysed by natural killer (NK) cells. The
non-specific immune mechanisms are particularly important early in infection, as the
antigen-specific response takes several days to develop, but the non-specific mechanisms
continue to play a role in the immune response right through to resolution of the infection
and healing of tissue damage. The importance of non-specific immune mechanisms in
infection is emphasised by the susceptibility of individuals who have deficiencies in
phagocytosis or in the complement system to various types of infection.
In addition to triggering these non-specific mechanisms, infection also triggers the antigen-
specific adaptive immune response, but this takes several days to develop, due to the need for
clonal selection, differentiation and expansion in order to generate antigen-specific effectors.
The adaptive immune response to infection involves both the T and B cell mediated
compartments of the immune system. The integrity of the adaptive immune system is based
on the ability of lymphocytes to recirculate between the blood, somatic tissues and lymphoid
tissues. The response can be separated into several distinct phases:

1) The induction phase occurs principally in the lymphoid tissues. Antigen either finds it
own way to lymphoid tissues (eg antigen in the circulation passing through the spleen) or it is
transported to lymphoid tissues by migratory dendritic cells. Different areas of lymphoid
tissues are involved in the induction of different effector arms of the response; most B cell
activation occurs within germinal centres, whilst activation of T cells occurs in the T
dependent areas (paracortex of lymph nodes or periarteriolar zones of the spleen). In both
cases, specialist antigen presenting cells are involved in the initiation of the adaptive immune
response - interdigitating follicular dendritic cells in the case of B cell activation, and
professional antigen presenting cells - activated macrophages and, particularly, dendritic cells
in the case of T cell activation. In addition to the interaction between the antigen receptor and
antigen, other signals are required for efficient activation of naive lymphocytes. These
additional signals are delivered by receptor: ligand interactions between the lymphocyte and
the antigen presenting cell (the cell surface molecules involved in these interactions are
called "costimulatory" molecules), and interactions between cytokines and their receptors on
the lymphocyte cell surface. These requirements for controlling the initiation of the adaptive
immune response within the lymphoid tissues regulate its activation, and reduce the chances
of uncontrolled or inappropriate immunological activation.

2) Following activation of antigen-specific lymphocytes in the lymphoid tissues, these cells

undergo a process of clonal expansion and differentiation. Some of these cells become
activated effector lymphocytes (helper T cells, cytotoxic T cells, antibody-secreting plasma

43
cells), whilst other cells remain in a semi-activated and recirculate as memory cells. Memory
cells have less stringent requirements for activation on re-contact with antigen in terms of
additional signals than naive lymphocytes, and respond more quickly to subsequent exposure
to antigen. During the phase of clonal expansion and differentiation, cell cooperation within
the immune response plays a major role in determining the outcome of the response. For
example (as described earlier in the course) the balance between Th1 and Th2 cells may bias
the outcome towards a pro-inflammatory or a pro-allergic type of response.

3) The final phase of activation of the immune response involves the activated effector cells.
The different sorts of effector cells, and their roles in responses to different types of infection
have been discussed in detail in earlier sessions, and include the production of antibody by
plasma cells, the generation of cytotoxic T lymphocytes and the induction of delayed type
hyperrsensitivity reactions and macrophage activation. This stage of the immune response
also requires considerable cooperation between different cell types in the immune response,
including interactions between antigen-specific and non-specific effector cells, in clearing the
infection. As described earlier, the CD4+ T cells play a central role in coordinating the
antigen-specific immune response, by providing "help", through cytokine release, for B cells
in isotype switching and affinity maturation of antibody responses, for CD8+ T cells in the
induction of effector CTL, for enhancement of NK cell function, and for macrophage
activation in delayed type hypersensitivity reactions and bacterial and fungal killing.
Ultimately it is left to the non-specific phagocytic cells to clear up the mess, and the
macrophages and fibroblasts to resolve any damage caused by the infection and to promote
healing. The importance of each component of the immue response in combating infection is
demonstrated by the predisposition to infections with particular groups of organisms of
individuals with deficiencies in one or more component of the immune response. Thus, in
order to work effectively in response to infections, the different components and cells of the
immune system must act cooperatively to eliminate the infecting micro-organism.

4.3 Generation of antibody diversity

Antibody diversity is generated by the following mechanisms.

4.3.1 Immunoglobulin Gene Rearrangements

1) The joining of various V, D and J genes is entirely random that results in ~ 50,000
different possible combinations for VDJ(H) and ~ 1,000 for VJ(L). Subsequent random
pairing of H and L chains brings the total number of antibody specificities to ~107
possibilities.
2) Diversity is further increased by the imprecise joining of different genetic segments.
3) Rearrangements occur on both DNA strands, but only one strand is transcribed (allelic
exclusion).
4) Only one rearrangement occurs in the life of a B cell because of irreversible deletions in
DNA. Consequently, each mature B cell maintains one immunologic specificity and is
maintained in the progeny or clone. This constitutes the molecular basis of the clonal
selection; i.e., each antigenic determinant triggers the response of the pre-existing clone of B
lymphocytes bearing the specific receptor molecule. It also follows that deletion of the B cell
clone results in immunologic unresponsiveness to the antigen.

44
4.3.2 Somatic Mutations
This mechanism leads to a fine-tuning of the antibody specificity after immunization.
Rearranged VDJH, and VJL genes in the B cells are uniquely susceptible to point
mutagenesis by enzymes that become activated following stimulation of the cell by antigen.
The clonal progeny of an antigen-driven B cell thus produce antibodies that may differ in one
or more amino acid positions in the regions of the protein that are responsible for antigen
binding. Cells producing the mutant antibody with highest affinity for the antigen are
preferentially stimulated and thus eventually dominate the response. Therefore, antibodies
produced after repeated immunization commonly display numerous point mutations (derived
by somatic mutations in B cells found in peripheral lymphoid organs) and have higher
affinities for antigens (affinity maturation), as compared to antibodies produced in the
primary immune response.

4.3.3 Antibody Function

Antibody molecules perform a number of important functions that are necessary for
mounting an effective immune response against microbial pathogens. CH region genes
encode the biological functions of immunoglobulins (see Table below). For example, IgM
and IgG bind to the C1q subunit of C1, IgG crosses the placental barrier to the fetal
circulation, and polymeric immunoglobulins, particularly dimeric IgA, are transported across
epithelial cells into mucosal secretions.
To accomplish these functions, B cells switch their immunoglobulin isotype. The VDJ genes
which are associated with Cm or Cd, which are the original constant genes expressed in
mature B cells, become associated with another C gene (Fig. 1-11). This has been termed the
isotype switch, because the C gene determines the antibody isotype. The switch is
accomplished by genetic recombination, whereby the VDJ gene segment is transferred from
the Cm/Cd junction onto another C region gene downstream (Fig. 1-11). Because the Cm/Cd
and other interposed genes are deleted, the switch is irreversible. The new antibody maintains
the same L chain and the same V H region (encoded by VDJ), but has new properties
determined by the acquired C gene. The isotype switch mechanism is promoted by physical
interactions between T and B cells (for example, the binding of CD40 on B cells to its ligand
on T cells) and by specific cytokines from T cells (for example, IL-5 and IL-10 promote IgA
production; IL-4 promotes IgE production).
Furthermore, each antibody molecule may exist in either a membrane-bound or secreted
form. Every C gene contains a 3 sequence encoding the hydrophobic cytoplasmic tail of the
H chain, so that the immunoglobulin molecule produced by the B cell is inserted in the
surface membrane to function as the receptor for antigen. When the B cell differentiates into
a plasma cell, an enzyme is activated that modifies the RNA transcript. Consequently, the
translated protein ends with a hydrophilic peptide and is secreted from the cell.

45
4.3.4 Some characteristics of the secondary antibody response:

1. The anamnestic response is not the result of sudden release of preformed antibody
that has been stored: it is the result of the bulk synthesis of new antibody.
2. The anamnestic response may be induced at almost anytime after the primary
response. Even many years after the primary response, when the primary titre has
dropped to zero, the anamnestic response is inducible. It may not be quite as striking
as a booster nearer in time to the initial response, but the true secondary response will
develop.

3. The secondary response is repeatable many times until physiologic limit of particular
animal to particular antigen is reached.

4. Cross-reactive antigen will induce the response. In this case, the degree of anamnesis
will be correlated with the sameness of the two antigens: the more they are alike, the
better the response.

5. Nonspecific anamnesis may occur. Antibodies originate in lymphoid tissue. Any

treatment of the immunized animal that causes lysis or hyperplasia of the antibody-
forming cell will cause a minimal anamnesis response.

46
6. The decrease in antibody titre after the secondary response is more gradual than the
decrease after the primary response. One reason is that more cells are involved in
antibody production in the secondary response. If some of these cells are relatively
long lived and continue functioning, then antibody will be formed over a longer
period after the secondary response than after the primary response. Another reason is
that the antiserum seen in the secondary response is qualitatively different from that
seen after the initial response. It contains relatively more IgG, which has a half life of
25 to 35 d. The primary antiserum is relatively rich in IgM which has a half-life of 8
to 10 days. The IgM secondary response is only one-tenth that of IgG; for this reason,
hyperimmune sera are predominantly IgG in nature. IgG has been referred to as a
memory component implying that IgM anamnesis does not occur. This is true in a
relative sense only; since secondary IgM levels are ion fact somewhat greater than
primary levels.

7. Reactivation of Ig response is not entirely without danger, especially when soluble

antigens or autocoupling haptens are employed. IgE formed in response to the
primary injection of antigen is distributed through the blood stream from which it
enters into the tissues where it absorbs to the surface of mast cells. Since soluble
antigens given in the booster inject can also diffuse readily into tissues, this antigen
can combine with the mast cell-bound IgE, with disastrous consequences. This effect
is due to the release of pharmacologic agents from the mast cell, as a result of the
serologic reaction on its surface. If sufficient concentrations of the reagent are
achieved, the antigen may die of anaphylactic shock.

8. The anamnestic response and the primary response are blended when adjuvant is
combined with antigen in the primary immunization. Antibody will appear after about
the same latent period and will rise at about the same rate, but will continue to rise
over an extended period compared with the usual primary response. Enhancing the
immune response by incorporating the antigen into the adjuvant-antigen mixture is
very a practical way to spare expensive antigens, since small quantities of the mixture
will ensure high titre antisera. The application of the adjuvant-antigen mixture is
immunization against infectious diseases creates a better and longer-lasting immunity.

In comparison with the primary immune response, the secondary response is

characterized by:

i) a shorter lag period between the encounter with antigen and the appearance with
antibodies.
ii) Antibody production that has a higher rate and is more persistent.

iii) Higher antibody titre at the peak of the response.

iv) A predominance of IgG molecule and

v) Antibodies with higher affinity for Ag than those produced in the primary
response.

47
 These differences are accounted for by the number of responding B cells. In the primary
response only a few of the B cells that are present have the specificity that permits a
particular antigen to trigger their development into antibody-secreting B cells (plasma-
cell). The initial antigenic stimulus also results in the proliferation and the formation of a
larger number of secondary or memory B cells, capable of responding to the same
antigens. Thus at the time of the secondary stimulus many more B cells can respond to
the antigen. In the absence of over-antigenic stimulation, memory cells seem to persist
for prolonged.

The duration of the primary and secondary response varies with the dose and mode of
administration of antigen. If the antigen is administrated in solution and is rapidly
eliminated, the primary response is short-lived and another injection of antigen is
required to elicit the secondary response. If however, the antigen is retained for a long
period as when given in a water-in-oil emulsion, the resulting antibody production
continues at a high level for a prolonged period, and the transition from the production of
the IgM to IgG molecules, and from low-to high-affinity antibody molecules, occurs
gradually and without the need for another injection. When the prolonged response
finally subsides, after many months, a subsequent injection of the same antigen promptly
elicits the formation of IgG antibody molecules of the same affinity as those of made at
the end of the response.

4.3.5 Genetic Basis of Antibody Diversity

Specific antibodies are generated as a consequence of immunoglobulin gene rearrangement,

i.e., recombination of V, D, J, and C genes (Figure 4-2). The immune system generates
millions of different antibody molecules from the pool of V genes. Separate sets of V genes
encode the variable domains of immunoglobulin H and L chains. The two chains are
produced separately, but the mechanisms by which their diversity is achieved are similar in
principle.

Light Chain Formation: Most antibody molecules use the kappa light chain. The kappa
gene cluster consists of several hundred (~300) VL genes; a few J genes (~4) and one C
gene. These germline genes are tandemly arranged on the chromosome and are
transcriptionally inactive. As the B cell matures, genes are arranged (recombined) so that one
V gene is joined to a J gene, and the rearranged VJ segment together with the C gene is
transcribed. The portion of DNA between the joined segments is deleted, and the transcripts
are processed by splicing to produce the messenger RNA for the L chain. kappa chains are
encoded by a separate cluster of V, J and C genes, but the rearrangement and transcription
are similar to that of the lambda chain. Any given B cell uses only one type of L chain to
produce the immunoglobulin molecule. The L chain combines with the H chain during their
transport from polyribosomes to the membrane.

Heavy Chain Formation: The H chain gene system has a design that is similar to that of
light chain, but is slightly more complex (Fig. 1-14). In addition to ~ 1,000 VH genes, there
are > 10 D genes and ~ 4 J genes. Furthermore, this genetic cluster has nine C genes that
encode different immunoglobulin isotypes. The mature B cell (Fig. 1-14) rearranges its
immunoglobulin genes, joins them together, and deletes the DNA between the joined

48
segments. The rearranged VDJ gene segment is transcribed together with a Cµ or Cdelta
gene, and this long transcript is spliced into VDJCµ or VDJCdelta messages resulting in the
expression of IgM and IgD, respectively, on the B cell surface. Both immunoglobulin
molecules use the same VDJ segment and, therefore, possess the same immunological
specificity. The B cell is now ready to bind to a specific antigen and become further
differentiated.

Figure 4-2. Antibody diversity is principally generated by immunoglobulin gene

rearrangement. H-chain gene rearrangement is depicted.

4.4 Development of immunological tolerance

Immune or immunological tolerance is the process by which the immune system does not
attack an antigen. It occurs in two forms: innate tolerance and acquired tolerance.
Innate tolerance is the body's tolerance for its own antigens and proteins. When naturals
tolerance fails, or when the body does not properly recognize itself, an autoimmune disorder
results. Acquired or induced tolerance is the immune system's tolerance for external antigens.
It is created through some form of manipulation, such as medication. One of the most
important natural kinds of acquired tolerance occurs during pregnancy where the fetus must
be tolerated by the maternal immune system. One model for the induction of tolerance during

49
the very early stages of pregnancy is the eutherian fetoembryonic defense system (eu-FEDS)
hypothesis. However, another model suggests that the induction of tolerance primarily
requires the participation of regulatory T cells. In clinical practice, acquired immunity is
important in organ transplantation, when the body must be forced to accept an organ from
another individual. The failure of the body to accept an organ is known as transplant
rejection. To prevent rejection, a variety of medicines are used to produce induced tolerance.

4.4.1 Eutherian fetoembryonic defense system (eu-FEDS) hypothesis

The Eutherian Fetoembryonic Defense System (eu-FEDS) is a hypothetical model describing
a method by which immune systems are capable of recognizing additional states of
relatedness like "own species" such as is observed in maternal tolerance of a related fetus.
The model includes descriptions of the proposed signaling mechanism and several proposed
examples of exploitation of this signaling in disease states.
The concept of immunity refers to an organism's ability to respond to various foreign
intrusions (as occurs in infection). A basic requirement in such a system is the ability to
avoid self harm through some mechanism of recognizing "self". In classic immunity several
types of molecules label the organism's own cells as "self". Cells lableled in this manner are
tolerated and not damaged by the various defense mechanisms employed to protect against
infection. Dysregulation of this system is responsible for several types of disease states
known collectively as autoimmune disorders.
The term Eutheria is a taxon describing placental organisms such as mammals. The sister
group of Eutheria is Metatheria, which includes marsupials and their extinct relatives.
The term eu-FEDS was first described in 1997 by Gary F. Clark and co-workers as "the
human fetoembryonic defense system", and later renamed to apply more broadly to all
members of the taxon Eutheria. In 1949 Frank Burnet, and later in 1953 Peter Medawar,
observed that the developing fetus was, in fact, similar to a transplanted "foreign" organ,
because of the father's contribution to its genome.In 1960, Medawar and Burnet were
awarded the Nobel Prize in part for their early contibutions and discoveries related to
understanding the necessity for the development of tolerance to the developing eutherian. It
is now apparent that a human fetus is tolerated by its birth mother, even when it is completely
unrelated. These observations were made following the introduction of modern assisted
reproduction technologies involving unrelated donor eggs and the use of in vitro fertilization
(IVF). The eu-FEDS hypothesis was itself proposed to describe the precise immunological
mechanisms that mediate protection of the developing eutherian fetus from the immune
responses of its mother.

4.4.1.1 Hypothesis
The basic premise of the eu-FEDS hypothesis is that both soluble and cell surface associated
glycoproteins, present in the reproductive system and expressed on gametes, suppress any
potential immune responses, and inhibit rejection of the fetus. The eu-FEDS model further
suggests that specific carbohydrate sequences (oligosaccharides) are covalently linked to
these immunosuppressive glycoproteins and act as “functional groups” that suppress the
immune response. The major uterine and fetal glycoproteins that are associated with the eu-
FEDS model in the human include alpha-fetoprotein,CA125, and glycodelin-A (also known
as placental protein 14 (PP14)).

50
Normally, a low level of these glycoproteins is detected in the maternal serum during the
early stages of pregnancy. It appears that the effects of these eu-FEDS associated
glycoproteins are manifested only during implantation and the very early development of the
embryo. In humans, the expression of such glycoproteins greatly decreases toward the end of
the first trimester. Therefore, more highly targeted mechanisms of immune suppression (such
as the expression of the enzyme indoleamine dioxygenase (IDO)) are likely employed by the
fetus during the subsequent stages of development. One potential reason for early
inactivation of the system is that the immunosuppressive effect of these glycoproteins may be
so complete that their continued leakage into the circulatory system could lead to a global
suppression of the maternal immune response, compromising the mother's ability to carry the
fetus to term.

4.4.1.2 Implications of the hypothesis

Human sperm and eggs also lack molecules for the immune recognition of "self". These
immune markers are also known as major histocompatibility (MHC) antigens or more
specifically in humans as human leukocyte antigens (HLA). Therefore a major question is
how are human gametes recognized by immune effector cells. Specifically, their lack of
MHC recognition markers should trigger the immune system, resulting in lysis of both sperm
and eggs by leukocytes known as natural killer, or NK cells. These cells target and kill other
cells lacking such MHC markers, a concept known as “missing self”. One distinct possibility
is that sperm and eggs are recognized via oligosaccharides expressed on their surfaces. For
example, human gametes are coated with carbohydrate sequences that have been implicated
in the suppression of NK cell mediated responses.
One of the major corollaries of the eu-FEDS hypothesis is that persistent pathogens and
aggressive tumor cells are able to either mimic or acquire the same carbohydrate functional
groups used to suppress any immune response that could interfere with the reproductive
imperative, thus enabling them to similarly resist the human immune response. These
pathogens include HIV-1, helminthic parasites such as schistosomes, and Helicobacter
pylori, the bacteria that causes stomach ulcers.
There are some notable examples of this mimicry or acquisition of the same carbohydrate
sequences implicated in this protective system by pathogens and aggressive tumor cells. The
major carbohydrate sequence linked to glycodelin-A also profusely coats the surface of
schistosomes . The profile of the major oligosaccharides linked to CA125 and the major
surface glycoprotein of HIV-1 (gp120) almost perfectly overlap. More persistent pathogens
linked to the eu-FEDS model may be identified as mass spectrometry methods for
sequencing oligosaccharides become more sensitive.

4.4.2 Other experimental models

Several other models have been developed that seek to address this hypothetical system for
immune tolerance, including the depletion of tryptophan via the enzyme indoleamine
dioxygenase (IDO) and the expression of the nonclassical MHC class I molecule designated
HLA-G. However, genetic deletion of IDO in female mice does not lead to the rejection of
their foreign fetal offsrping, indicating that a redundant system for the suppression of the
mother's immune response exists in the uterus during pregnancy. In addition, HLA-G
expresses oligosaccharides that are very different from those linked to other HLA class I
molecules, so the possibility exists that HLA-G at the fetomaternal interface is itself

51
employing its unusual carbohydrate sequences as functional groups to suppress the mother's
immune response.

4.4.3 Maternal Immunologic Agents Transferred to the Recipient Infant

The mother transmits immune factors to the offspring both through the placenta and milk.
Large quantities of IgG are transmitted via the placenta, whereas other immunoglobulin
isotypes are not. Consequently, virtually all IgG in neonatal blood is of maternal origin, the
concentration of IgG in umbilical cord blood is somewhat higher than in adults, and the
levels of other immunoglobulin isotypes are exceptionally low. Low concentrations of some
factors such as IgG and secretory IgA antibodies are also transmitted via amniotic fluid, but
little is known about their in vivo effects upon the fetal mucosal immune system.
An array of host resistance factors are transmitted to the infant in human milk, including
leukocytes, secretory IgA, lactoferrin, lysozyme, and oligosaccharides and glycoconjugates
that are receptor analogs for microbial adhesins and toxins. In addition to those antimicrobial
factors, anti-inflammatory agents, and immunomodulating agents including TNF-alpha,
TGF-beta, IL-1beta, IL-6, IL-8, IL-10, G-CSF, and M-CSF. These factors are designed to act
at mucosal sites and to protect by noninflammatory mechanisms. Since the endogenous
production of these agents is incompletely developed in early infancy and they are scarce in
cow's milk or other substitute feedings, it is not surprising that breastfeeding increases
resistance to gastrointestinal and respiratory infections, allergic diseases, and certain
inflammatory diseases that occur much later in childhood.

4.5 Theory of antibody synthesis

4.4.1 Selective theory
The selection theory of antibody synthesis assumes that the information required for the
synthesis of the different antibodies is already present in the genetic apparatus. The gene
which codes for a specific antibody is selected and “switched on” by contact of antigen with
the cell, and through transcription and translation of the appropriate messenger RNA,
immunoglobulin peptide chains with corresponding individual primary amino acid sequences
are synthesized based on the sequence. These chains then fold spontaneously to a preferred
globular configuration which possesses the specific antigen-combining site.
The clonal selection theory (CST) has become a widely accepted model for how the
immune system responds to infection and how certain types of B and T lymphocytes are
selected for destruction of specific antigens invading the body.
Frank Macfarlane Burnet published the clonal selection theory with the following core
hypotheses:
(i) The entire immunological repertoire develops spontaneously in the host (that is, there is
no information furnished by antigen).
(ii) Each [antibody] pattern is the specific product of a cell and that product is presented on
the cell surface (as an Ehrlich-type receptor).
(iii) Antigen reacts with any cells carrying its specific receptor to signal cell proliferation and
differentiation.
(iv) Some of these daughter cells differentiate (become plasmacytoid) to form clones of
antibodies, whereas others survive as clones of [undifferentiated] memory cells.
This theory was elaborated as in the figure below. It is a theory of selection (hypotheses
i−iii), involving the selective interaction of antigen with preformed antibody, and of clonality

52
(hypotheses iii and iv), involving the cellular dynamics of proliferation and differentiation to
yield clones of cells and clones of their product.

Clonal selection of lymphocytes: 1) A hematopoietic stem cell undergoes differentiation and

genetic rearrangement to produce 2) immature lymphocytes with many different antigen
receptors. Those that bind to 3) antigens from the body's own tissues are destroyed, while the
rest mature into 4) inactive lymphocytes. Most of these will never encounter a matching 5)
foreign antigen, but those that do are activated and produce 6) many clones of themselves.

4.4.4.1 Evidence of selective theory

(a) Absence of antigen from plasma
Using autoradiography to visualize highly radioactive antigens combined with
immunoflurescence to identify cells making specific antibody, Nossall has showed that
nearly all cases which contain intracellular antibody do not have demonstrable antigen
molecules. This is clearlym at variance with the idea of antigen acting as template
(b) Unfolding experiment
Reduction of disulphide bonds in IgG or its Fab fragment followed by treatment with high
concentrations of guanine effectively destroys any organized structure. However, removal of
the guanine from unfolded molecules by dialysis and reoxidation restores significant specific
antigen-binding activity. This is consistent with the instructive view (which requires the
presence of antigen for the formation of a specific antibody) and indicates that the
information held in the primary amino acid sequences is sufficient to allow the correct
tertiary structure to be formed by spontaneous refolding.
(c) Amino acid sequence of antibodies
Purified antibodies show differences in amino acid sequences. Myeloma proteins
representing individual immunoglobulin molecules show considerable variability in the
sequences of the N-terminal part of both L and H chains. Indeed of the many human
myeloma L chains so far sequenced, none have proved to have identical structure. These
differences in amino acid sequence reflect differences in DNA nucleotide sequences strongly
implicating genetic control of specificity.

53
(d) Genetic studies
Immune responsiveness to certain defined antigen has indeed been associated with genetic
constitution, not only with respect to major histocompatibility complex (MHC)-linked genes
controlling the synthesis of antigen specific Ia molecules concerned in T-cell regulation of
the antibody response but in particular with the Ig-allotype linked genes encoding certain
antibody clones and idiotypes which provides strong evidence for the view that the capacity
to from particular antibodies is inherited through the possession of Ig V-region genes.
Conclusion
Based on these evidences, it has been concluded that antigen does not act as a template for
antibody production: the complete information for antibody synthesis is already in the
genome. Folding of the antibody molecule and hence specificity, depends upon the primary
amino acid structure and the differences in amino acid sequences between different
antibodies reflect differences in DNA nucleotide sequence. Immune responsiveness is
genetically controlled.

4.4.2 Instructive theory

The ability of animals to synthesize antibodies directed against antigenic determinants such
as dinitrobenzene and sulfanilic acid, which were so unlikely to occur in nature, made it
difficult to accept the idea based on the Ehrlich’s earlier views that the body has preformed
antibodies whose production is further stimulated by the entry of antigen. Instead, it turned to
theories in which the antigen acted instructively as a template around which a standard
unfolded y-globulin chain could be molded to provide the appropriate complementary shape.
The molecule would be stabilized in this configuration by disulfide linkages, hydrogen bonds
and so forth; on separation from the template the molecule would now have a specific
combining site for antigen.

4.6 Monoclonal antibody and development

Monoclonal antibodies (mAb) are antibodies that are identical because they were produced
by one type of immune cell and are all clones of a single parent cell. Given (almost) any
substance, it is possible to create monoclonal antibodies that specifically bind to that
substance; they can then serve to detect or purify that substance. This has become an
important tool in biochemistry, molecular biology and medicine. When used as medications,
the generic name ends in -mab (see "Nomenclature of monoclonal antibodies").

4.6.1 Discovery
The idea of a "magic bullet" was first proposed by Paul Ehrlich who at the beginning of the
20th century figured that if a compound could be made that selectively targeted a disease-
causing organism, then a toxin for that organism could be delivered along with the agent of
selectivity. In the 1970s the B-cell cancer myeloma was known, and it was understood that
these cancerous B-cells all produce a single type of antibody (a paraprotein). This was used
to study the structure of antibodies, but it was not yet possible to produce identical antibodies
specific to a given antigen. The process of producing monoclonal antibodies described above
was invented by Georges Köhler, César Milstein, and Niels Kaj Jerne in 1975; they shared
the Nobel Prize in Physiology or Medicine in 1984 for the discovery. The key idea was to use

54
a line of myeloma cells that had lost their ability to secrete antibodies, come up with a
technique to fuse these cells with healthy antibody producing B-cells, and be able to select
for the successfully fused cells. In 1988 Greg Winter and his team pioneered the techniques
to humanise monoclonal antibodies, removing the reactions that many monoclonal antibodies
caused in some patients.

4.6.2 Production
If a foreign substance (an antigen) is injected into a vertebrate such as a mouse or a human,
some of the immune system's B-cells will turn into plasma cells and start to produce
antibodies that recognize that antigen. Each B-cell produces only one kind of antibody, but
different B-cells will produce structurally different antibodies that bind to different parts
("epitopes") of the antigen. This natural mixture of antibodies found in serum is known as
polyclonal antibodies. To produce monoclonal antibodies, one removes B-cells from the
spleen or lymph nodes of an animal that has been challenged several times with the antigen
of interest. These B-cells are then fused with myeloma tumor cells that can grow indefinitely
in culture (myeloma is a B-cell cancer) and that have lost the ability to produce antibodies.
This fusion is done by making the cell membranes more permeable by the use of
polyethylene glycol, electroporation or, of historical importance, infection with some virus.
The fused hybrid cells (called hybridomas), being cancer cells, will multiply rapidly and
indefinitely. Large amounts of antibodies can therefore be produced. The hybridomas are
sufficiently diluted to ensure clonality and grown. The antibodies from the different clones
are then tested for their ability to bind to the antigen (for example with a test such as ELISA)
or immuno-dot blot, and the most sensitive one is picked out.

In the above process, one uses myeloma cell lines that have lost their ability to produce their
own antibodies or antibody chain, so as to not contaminate the target antibody. Furthermore,
one employs only myeloma cells that have lost a specific enzyme (hypoxanthine-guanine
phosphoribosyltransferase, HGPRT) and therefore cannot grow under certain conditions
(namely in the presence of HAT medium). These cells are preselected by the use of 8-
azaguanine media prior to the fusion. Cells that possess the HGPRT enzyme will be killed by
the 8-azaguanine. During the fusion process many cells can fuse. Myeloma with myeloma,
spleen cell with spleen cell, 3 cells of different types etc... The desired fusions are between
healthy B-cells producing antibodies against the antigen of interest and myeloma cells. These
are relatively rare, but when one succeeds, then the healthy partner supplies the needed
enzyme and the fused cell can survive in HAT medium. This is the trick to detect the
successfully fused cells. The medium must be enriched during selection to favour hybridoma
growth. This can be achieved by the use of a layer of feeder cells or supplement media such
as briclone.
Monoclonal antibodies can be produced in cell culture or in live animals. When the
hybridoma cells are injected in mice (in the peritoneal cavity, the gut), they produce tumors
containing an antibody-rich fluid called ascites fluid. Production in cell culture is usually
preferred as the ascites technique may be very painful to the animal and if replacement
techniques exist, may be considered unethical. Fermentation chambers have been used to
produce antibodies on a larger scale. Nowadays, bioengineering allow production of
antibodies in plants.

55
4.6.3 Applications
Once monoclonal antibodies for a given substance have been produced, they can be used to
detect the presence and quantity of this substance, for instance in a Western blot test (to
detect a protein on a membrane) or an immunofluorescence test (to detect a substance in a
cell). They are also very useful in immunohistochemistry which detect antigen in fixed tissue
sections. Monoclonal antibodies can also be used to purify a substance with techniques called
immunoprecipitation and affinity chromatography.

4.6.4 Monoclonal antibodies for cancer treatment

One possible treatment for cancer involves monoclonal antibodies that bind only to cancer
cell-specific antigens and induce an immunological response against the target cancer cell.
Such mAb could also be modified for delivery of a toxin, radioisotope, cytokine or other
active conjugate; it is also possible to design bispecific antibodies that can bind with their
Fab regions both to target antigen and to a conjugate or effector cell. In fact, every intact
antibody can bind to cell receptors or other proteins with its Fc region. The illustration below
shows all these possibilities:

Monoclonal antibodies for cancer. ADEPT, antibody directed enzyme prodrug therapy;
ADCC, antibody dependent cell-mediated cytotoxicity; CDC, complement dependent
cytotoxicity; MAb, monoclonal antibody; scFv, single-chain Fv fragment.
Chimeric and humanized antibodies

One problem in medical applications is that the standard procedure of producing monoclonal
antibodies yields mouse antibodies. Although murine antibodies are very similar to human
ones there are differences. The human immune system hence recognizes mouse antibodies as
foreign, rapidly removing them from circulation and causing systemic inflammatory effects.
A solution to this problem would be to generate human antibodies directly from humans.
However, this is not easy primarily because it is clearly not ethical to challenge humans with
antigen in order to produce antibody. Furthermore, it is not easy to generate human
antibodies against human tissues.

56
Various approaches using recombinant DNA technology to overcome this problem have been
tried since the late 1980s. In one approach, one takes the DNA that encodes the binding
portion of monoclonal mouse antibodies and merges it with human antibody producing
DNA. One then uses mammalian cell cultures to express this DNA and produce these half-
mouse and half-human antibodies. (Bacteria cannot be used for this purpose, since they
cannot produce this kind of glycoprotein.) Depending on how big a part of the mouse
antibody is used, one talks about chimeric antibodies or humanized antibodies. Another
approach involves mice genetically engineered to produce more human-like antibodies.
Monoclonal antibodies have been generated and approved to treat;cancer, cardiovascular
disease, inflammatory diseases, macular degeneration, transplant rejection, and viral
infection (see monoclonal antibody therapy). In August 2006 the Pharmaceutical Research
and Manufacturers of America reported that U.S. companies had 160 different monoclonal
antibodies in clinical trials or awaiting approval by the Food and Drug Administration.

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 CHAPTER V

INTERACTION BETWEEN ANTIGEN AND ANTIBODY

5.1. NATURE OF ANTIGEN-ANTIBODY REACTIONS

A. Lock and Key Concept - The combining site of an antibody is located in the Fab portion
of the molecule and is constructed from the hypervariable regions of the heavy and light
chains. X-Ray crystallography studies of antigens and antibodies interacting show that the
antigenic determinant nestles in a cleft formed by the combining site of the antibody. Thus, a
concept of Ag-Ab reactions is one of a key (i.e. the Ag) which fits into a lock (i.e. the Ab).
B. Non-covalent Bonds - The bonds that hold the Ag in the antibody combining site are all
non-covalent in nature. These include hydrogen bonds, electrostatic bonds, Van der
Waals forces and hydrophobic bonds. Multiple bonding between the Ag and the Ab
ensures that the Ag will be bound tightly to the Ab.
C. Reversible - Since Ag-Ab reactions occur via non-covalent bonds, they are by their nature
reversible.

5.2. AFFINITY AND AVIDITY

A. Affinity - Antibody affinity is the strength of the reaction between a single antigenic
determinant and a single combining site on the antibody. It is the sum of the attractive and
repulsive forces operating between the antigenic determinant and the combining site of the
antibody as illustrated in Figure 2.

Figure 2.

Affinity is the equilibrium constant that describes the Ag-Ab reaction as illustrated in Figure
3. Most antibodies have a high affinity for their antigens.

Figure 3
B. Avidity - Avidity is a measure of the overall strength of binding of an antigen with many
antigenic determinants and multivalent antibodies. Affinity refers to the strength of binding
between a single antigenic determinant and an individual antibody combining site whereas
avidity refers to the overall strength of binding between multivalent antigens and antibodies.

58
Avidity is influenced by both the valence of the antibody and the valence of the antigen.
Avidity is more than the sum of the individual affinities. This is illustrated in Figure 4.

Figure 4

5.3. SPECIFICITY AND CROSS REACTIVITY

A. Specificity - Specificity refers to the 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. In general, 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.
B. Cross reactivity - Cross reactivity refers to 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. Figure 5 illustrates
how cross reactions can arise. Cross reactions arise because the cross reacting antigen shares
an epitope in common with the immunizing antigen or because it has an epitope which is
structurally similar to one on the immunizing antigen (multispecificity).

Figure 5

5.4. ANTIGEN-ANTIBODY REACTIONS (in vitro)

A. Factors affecting measurement of Ag/Ab reactions - The only way that one knows that
an antigen-antibody reaction has occurred is to have some means of directly or indirectly
detecting the complexes formed between the antigen and antibody. The ease with which one
can detect antigen-antibody reactions will depend on a number of factors.
1. Affinity - The higher the affinity of the antibody for the antigen, the more stable will be
the interaction. Thus, the ease with which one can detect the interaction is enhanced.
2. Avidity - Reactions between multivalent antigens and multivalent antibodies are more
stable and thus easier to detect.

59
3. 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. This is depicted in Figure 6.
4. 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 complexes.

Figure 6
B. Agglutination Tests
1. Agglutination/Hemagglutination - When the antigen is particulate the reaction of an
antibody with the antigen can be detected by agglutination (clumping) of the antigen. When
the antigen is an erythrocyte the term hemagglutination is used. The term agglutinin is used
to describe antibodies that agglutinate particulate antigens. When the antigen is an
erythrocyte the term hemagglutinin is often used. All antibodies can theoretically agglutinate
particulate antigens but IgM due to its high valence is particularly good agglutinin and one
sometimes infers that an antibody may be of the IgM class if it is a good agglutinating
antibody.
a) Qualitative agglutination test - Agglutination tests can be used in a qualitative manner to
assay for the presence of an antigen or an antibody. The antibody is mixed with the
particulate antigen and a positive test is indicated by the agglutination of the particulate
antigen. (Figure 7).

Figure 7
For example, a patient's red blood cells can be mixed with antibody to a blood group antigen
to determine a person's blood type. In a second example, a patient's serum is mixed with red
blood cells of a known blood type to assay for the presence of antibodies to that blood type in
the patient's serum.

60
b) Quantitative agglutination test - Agglutination tests can also be used to quantitate the level
of antibodies to particulate antigens. In this test one makes serial dilutions of a sample to be
tested for antibody and then adds a fixed number of red blood cells or bacteria or other such
particulate antigen and determines the maximum dilution which gives agglutination. The
maximum dilution that gives visible agglutination is called the titer. The results are reported
as the reciprocal of the maximal dilution that gives visible agglutination. Figure 8 illustrates a
quantitative hemagglutination test.
Prozone effect - Occasionally, one observes that when the concentration of antibody is high
(i.e. lower dilutions), there is no agglutination and then as the sample is diluted agglutination
occurs (See Patient 6 in Figure 8). The lack of agglutination at high concentrations of
antibodies is called the prozone effect. Lack of agglutination in the prozone is due to
antibody excess resulting in very small complexes which do not clump to form visible
agglutination.

Figure 8
Prozone effect - Occasionally, one observes that when the concentration of antibody is high
(i.e. lower dilutions), there is no agglutination and then as the sample is diluted agglutination
occurs (See Patient 6 in Figure 8). The lack of agglutination at high concentrations of
antibodies is called the prozone effect. Lack of agglutination in the prozone is due to
antibody excess resulting in very small complexes which do not clump to form visible
agglutination.
c) Applications of agglutination tests
1) Determination of blood types or antibodies to blood group antigens.
2) To assess bacterial infections
e.g. A rise in titer to a particular bacterium indicates an infection with that bacterial type.
N.B. a fourfold rise in titer is generally taken as a significant rise in antibody titer.
d) Practical considerations - Although the test is easy to perform, it is only semi-quantitative.
2. Passive hemagglutination - The agglutination test only works with particulate antigens.
However, it is possible to coat erythrocytes with a soluble antigen (e.g. viral antigen, a
polysaccharide or a hapten) and used the coated red blood cells in an agglutination test for
antibody to the soluble antigen (Figure 9). This is called passive hemagglutination. The test is
performed just like the agglutination test. Applications include detection of antibodies to
soluble antigens and detection of antibodies to viral antigens.

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 Figure 9
3. Coomb's Test (Antiglobulin Test)
a) Direct Coomb's Test - When antibodies bind to erythrocytes, they do not always result in
agglutination. This can result from the Ag/Ab ratio being in antigen excess or antibody
excess or in some cases electrical charges on the red blood cells preventing the effective
cross linking of the cells. These antibodies that bind to but do not cause agglutination of red
blood cells are sometimes referred to as incomplete antibodies. In no way is this meant to
indicate that the antibodies are different in their structure, although this was once thought to
be the case. Rather, it is a functional definition only. In order to detect the presence of non-
agglutinating antibodies on red blood cells, one simply adds a second antibody directed
against the immunoglobulin (Ab) coating the red cells. This anti-immunoglobulin can now
cross link the red blood cells and result in agglutination. This test is illustrated in Figure 10
and is known as the Direct Coomb's test. 

Figure 10
b) Indirect Coomb's Test - If it is necessary to know whether a serum sample has antibodies
directed against a particular red blood cell and you want to be sure that you also detect
potential non- agglutinating antibodies in the sample, an Indirect Coomb's test is performed
(Figure 11). This test is done by incubating the red blood cells with the serum sample,
washing out any unbound antibodies and then adding a second anti-immunoglobulin reagent
to cross link the cells.

Figure 11

c) Applications - These include detection of anti-Rh antibodies. Antibodies to the Rh factor

generally do not agglutinate red blood cells. Thus, red cells from Rh + children born to Rh-
mothers, who have anti-Rh antibodies, may be coated with these antibodies. To check for this

62
a direct Coombs test is performed. To see if the mother has anti-Rh antibodies in her serum
an Indirect Coombs test is performed. 

4. Hemagglutination Inhibition - The agglutination test can be modified to be used for the
measurement of soluble antigens. This test is called hemagglutination inhibition. It is called
hemagglutination inhibition because one measures the ability of soluble antigen to inhibit the
agglutination of antigen-coated red blood cells by antibodies. In this test a fixed amount of
antibodies to the antigen in question is mixed with a fixed amount of red blood cells coated
with the antigen (see passive hemagglutination above). Also included in the mixture are
different amounts of the sample to be analyzed for the presence of the antigen. If the sample
contains the antigen, the soluble antigen will compete with the antigen coated on the RBC for
binding to the antibodies, thereby inhibiting the agglutination of the RBC as illustrated in
Figure 12.
By serially diluting the sample, you can quantitate the amount of antigen in your unknown
sample by its titer. This test is generally used to quantitate soluble antigens and is subject to
the same practical considerations as the agglutination test.

Figure 12

C. Precipitation tests
1. Radial Immunodiffusion (Mancini) - In radial immunodiffusion antibody is incorporated
into the agar gel as it is poured and different dilutions of the antigen are placed in holes
punched into the agar. As the antigen diffuses into the gel it reacts with the antibody and
when the equivalence point is reached a ring of precipitation is formed as illustrated in Figure
13.
The diameter of the ring is proportional to the log of the concentration of antigen since the
amount of antibody is constant. Thus, by running different concentrations of a standard
antigen one can generate a standard cure from which one can quantitate the amount of an
antigen in an unknown sample. Thus, this is a quantitative test. If more than one ring appears
in the test, more than one antigen/antibody reaction has occurred. This could be due to a
mixture of antigens or antibodies. This test is commonly used in the clinical laboratory for
the determination of immunoglobulin levels in patient samples.

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 Figure 13
2. Immunoelectrophoresis - In immunoelectrophoresis a complex mixture of antigens is
placed in a well punched out of an agar gel and the antigens are electrophoresed so that the
antigen are separated according to their charge. After electrophoresis a trough is cut in the gel
and antibodies are added. As the antibodies diffuse into the agar, precipitin lines are
produced in the equivalence zone when an Ag/Ab reaction occurs as illustrated in Figure 14.
This tests is used for the qualitative analysis of complex mixtures of antigens, although a
crude measure of quantity (thickness of the line) can be obtained. This test is commonly used
for the analysis of components in a patient' serum. Serum is placed in the well and antibody
to whole serum in the trough. By comparisons to normal serum one can determine whether
there are deficiencies on one or more serum components or whether there is an
overabundance of some serum component (thickness of the line). This test can also be used
to evaluate purity of isolated serum proteins.

Figure 14
3. Countercurrent electrophoresis - In this test the antigen and antibody are placed in wells
punched out of an agar gel and the antigen and antibody are electrophoresed into each other
where they form a precipitation line as illustrated in Figure 15. This test only works if
conditions can be found where the antigen and antibody have opposite charges. This test is
primarily qualitative, although from the thickness of the band you can get some measure of
quantity. It's major advantage is its speed.

Figure 15
D. Radioimmunoassay (RIA)/Enzyme Linked Immunosorbent Assay (ELISA)
Radioimmunoassays (RIA) are assays which are based on the measurement of radioactivity
associated with immune complexes. In any particular test, the label may be on either the

64
antigen or the antibody. Enzyme Linked Immunosorbent assays (ELISA) are those that are
based on the measurement of an enzymatic reaction associated with immune complexes. In
any particular assay the enzyme may be linked to either the antigen or the antibody.
1. Competitive RIA/ELISA for Ag Detection - The method and principle of RIA and ELISA
for the measurement of antigen is shown in Figure 16. By using known amounts of a
standard unlabeled antigen one can generate a standard curve relating cpm (Enzyme) bound
vs amount of antigen. From this standard curve one can determine the amount of an antigen
in an unknown sample.
The key to the assay is the separation of the immune complexes from the remainder of the
components. This has been accomplished in many different ways and serves as the basis for
the names given to the assay:
1) Precipitation with ammonium sulphate - Ammonium sulphate (33-50% final
concentration) will precipitate immunoglobulins but not many antigen. Thus, this can be used
to separate the immune complexes from free antigen. This has been called the Farr
Technique
2) Anti-immunoglobulin antibody - The addition of a second antibody directed against the
first antibody can result in the precipitation of the immune complexes and thus the separation
of the complexes from free antigen.

Figure 16
3) Immobilization of the Antibody - The antibody can be immobilized onto the surface of a
plastic bead or coated onto the surface of a plastic plate and thus the immune complexes can
easily be separated from the other components by simply washing the beads or plate (Figure
17). This is the most common method used today and is referred to as Solid phase RIA or
ELISA. In the clinical laboratory competitive RIA and ELISA are commonly used to
quantitate serum proteins, hormones, drugs metabolites.

2. Noncompetitive RIA/ELISA for Ag or Ab - Noncompetitive RIA and ELISAs are also

used for the measurement of antigens and antibodies. In Figure 18 the bead is coated with the
antigen and is used for the detection of antibody in the unknown sample. The amount of
labeled second antibody bound is related to the amount of antibody in the unknown sample.
This assay is commonly employed for the measurement of antibodies of the IgE class
directed against particular allergens by using a known allergen as antigen and anti-IgE
antibodies as the labeled reagent. It is called the RAST test (radioallergosorbent test). In
Figure 19 the bead is coated with antibody and is used to measure an unknown antigen. The
amount of labeled second antibody that binds is proportional to the amount of antigen that
bound to the first antibody.

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 Figure 18

Figure 19

E. Tests for Cell Associated Antigens

1. Immunofluorescence - Immunofluorescence is a technique whereby an antibody labeled
with a fluorescent molecule (fluorescein or rhodamine) is used to detect the presence of an
antigen in or on a cell or tissue by the fluorescence emitted by the bound antibody.
a) Direct Immunofluorescence - In direct immunofluorescence the antibody specific to the
antigen is directly tagged with the fluorochrome (Figure 20).

Figure 20
b) Indirect Immunofluorescence - In indirect immunofluorescence the antibody specific for
the antigen is unlabeled and a second anti-immunoglobulin antibody directed toward the first
antibody is tagged with the fluorochrome (Figure 21). Indirect fluorescence is more sensitive
than direct immunofluorescence since there is amplification of the signal.

Figure 21

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c) Flow Cytometry - Flow cytometry is commonly used in the clinical laboratory to identify
and enumerate cells bearing a particular antigen. Cells in suspension are labeled with a
fluorescent tag by either direct or indirect immunofluorescence. The cells are then analyzed
on the flow cytometer.
Figure 22 illustrates the principle of flow cytometry. In a flow cytometer the cells exit a flow
cell and are illuminated with a laser beam. The amount of laser light that is scattered off the
cells as they passes through the laser can be measured, which gives information concerning
the size of the cells. In addition, the laser can excite the fluorochrome on the cells and the
fluorescent light emitted by the cells can be measured by one or more detectors

Figure 22
The type of data that is obtained from the flow cytometer is shown in Figure 23. In a one
parameter histogram, increasing amounts of fluorescence (e.g. green fluorescence) is plotted
on the x axis and the number of cells exhibiting that amount of fluorescence is plotted on the
y axis. The fraction of cells that are fluorescent can be determined by integrating the area
under the curve. In a 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 in
cells is indicated by the contour and the intensity of the color.

Figure 23
F. Complement Fixation - Antigen/Antibody complexes can also be measured by their
ability to fix complement because an Ag/Ab complex will "consume" complement if it is
present whereas free Ag's or Ab's do not. Tests for Ag/Ab complexes that rely on the
consumption of complement are termed complement fixation tests and are used to
quantitate Ag/Ab reactions. This test will only work with complement fixing antibodies (IgG,
IgM best).
The principle of the complement fixation test is illustrated in Figure 24. Antigen is mixed
with the test serum to be assayed for antibody and Ag/Ab complexes are allowed to form. A
control tube in which no Ag is added is also prepared. If no Ag/Ab complexes are present in
the tube, none of the complement will be fixed. However, if Ag/Ab complexes are present,
they will fix complement and thereby reduce the amount of complement in the tube. After
allowing for complement fixation by any Ag/Ab complexes, a standard amount of red blood

67
cells, which have been pre-coated with anti-erythrocyte antibodies is added. The amount of
antibody-coated RBC is predetermine to be just enough to completely use up all the
complement initially added if it were still there. If all the complement was still present (i.e.
no Ag/Ab complexes formed between the Ag and Ab in question), all the RBC will be lysed.
If Ag/Ab complexes are formed between that Ag and Ab in question, some of the
complement will be consumed and thus when the antibody-coated RBC's are added not all of
them will lyse. By simply measuring the amount of RBC lysis by measuring the release of
hemoglobin into the medium, one can indirectly quantitate Ag/Ab complexes in the tube.
Complement fixation tests are most commonly used to assay for antibody in a test sample but
they can be modified to measure antigen.

Figure 23

5.5 ANTIGEN-ANTIBODY REACTIONS (in vivo)

For developing resistancee to specific viruses, microorganisms, macromolecules, foreign
agents and cancer, vertebrates possess a highly defensive immunologic response mechanism.
This immunologic response is triggered by specific antigen-antibody reactions. This section
describes those reactions occurring in the animal body such as the complement system,
adherence inhibition antibodies, antibody-dependent cell-mediated cytotoxicity, opsonization
and inflammation and immune complex formation.

5.5.1 The complement system

The complement system consists of a group of glycoproteins in the extracellular space that
can be stimulated in a cascading fashion to produce biologically active fragments that either
directly attack foreign substances or enhance the functions of certain types of inflammatory
leukocytes. The complement system consists of two recognition-stimulation pathways that
are designated as the classical and alternative pathways, either of which may lead to the
formation of a cell membrane attack complex (Fig. 1-20).

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Figure 5-1. The complement system. Activation of either wing of the system leads to the
formation of peptide fragments that function on leukocytes and forms the membrane attack
complex.

The Classical Pathway

The classical pathway of the complement system may be activated by antigen-antibody
complexes of the IgG, IgG3, or IgM isotypes by their binding to the C1q subunit of the first
component of complement (Fig. 5-1). Consequently, the C1qrs subunits of C1 form an
esterase that cleaves the next component, C4, to two fragments, the larger of which, C4b,
binds covalently to hydroxyl or amino groups on cellular membranes. The next component,
C2, after binding to C4b is partially digested by C1s esterase to form C2b. The resultant
membrane-bound complex, C4b2a, is an enzyme (C3 convertase) that cleaves C3 into two
biologically active fragments, C3a and C3b.

The Alternative Pathway

The alternative pathway of the complement system is activated independently of antigen-
antibody complexes (Fig. 5-1). The major exogenous activators of the pathway are microbial
agents and their products. The major components of the pathway are the serum protein
factors B, D, and P (properdin). A small amount of C3 in the fluid phase, which normally is
spontaneously activated, interacts with factor B to form C3Bb, which cleaves other C3
molecules to form C3b. C3b in turn attaches to surfaces and binds factor B. The resultant
C3bB is then cleaved by factor D to form C3bBb, the C3 convertase of the alternative
pathway. That enzyme is distinct from the one generated from the classical pathway but
serves the same purpose. This complex then is stabilized by factor P.
The binding of C3 to factor B is prevented, particularly in the fluid phase, by a regulatory
molecule, factor H. The more vigorous activation of this pathway occurs when the host is
exposed to microorganisms that are poor in sialic acid. In those circumstances, the binding of
factor B to C3 is favored, and the activation of the alternative pathway is not readily inhibited
by factor H. Therefore, more C3b is generated and a positive amplification loop that
generates more C3bBb (C3 convertase) is created. In contrast, sialic acid-rich encapsulated
microorganisms such as Streptococcus pneumoniae, Haemophilus influenzae, and Niesseria
meningitides are incapable of activating the alternative pathway and require binding to
specific IgG or IgM antibodies to activate the classical pathway and generate the C3b for
phagocytosis and the formation of the membrane attack complex. The receptors for activated
complement fragments are 1) CR1, principally on phagocytic cells for C3b; 2) CR2,
principally on B cells for a fragment called C3d (receptor for EBV); and CR3 (Mac-1), on
phagocytic and NK cells for inactivated C3b (C3bi) and C3d-g fragments.

5.5.2 Toxin and viral neutralization

Immunity to disease like diphtheria depends on the production of specific antobodies that
inactivate the toxins produced by the bacteria. This process is termed toxin neutralization.
In toxin neutralization, antibodies react with toxins, preventing either the B fragment of the
toxin from binding to the target cell or the A fragment from entereing the cytosol of the cell.
IgG, IgM and IgA antibodies can bind to some viruses during their extracellular phase and
inactivate them. This antibody-mediated viral inactivation is called viral neutralization.
Fixation of classical pathway complement component C4b to the virus aids the neutralization

69
process. Viral neutralization prevents a viral infection due to the inability of virus to bind its
target cell.

Many of the Ag-Ab reactions occurring in vivo can also take place in vitro under controlled
laboratory condition and are used in diagnostic testing. The utilization of the in vitro reaction
between antigen and serum antibodies is termed serology. An example of the use of serology
for identification and classification of antigens is the serotyping of various microorganisms
by the use of specific antisera. Details of general types of Ag-Ab reaction tests have been
discussed earlier.

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 CHAPTER VI

IMMUNOLOGICAL DISORDERS

6.1 Overview of immunological disorders

An immunological disorder results from an inappropriate (exaggerated) or an inadequate
immune response. Most inappropriate responses involve some types of hypersensitivity,
whereas inadequate responses are due to an immunodeficiency. Detail descriptions of some
of these disorders are here given.

6.2 Hypersensitivity
Hypersensitivities are inappropriate immune responses to foreign material that is either
within or in contact with the body. Essentially, the body mounts a sometimes dramatic
immune response against an otherwise harmless or at least less-harmful substance, thereby
doing more harm to the body in the course of the immune response than might have the
original allergen. Hypersensitivities may be divided into four types:

(i) Immediate hypersensitivity (type I hypersensitivity) or anaphylactic hypersensitivity

Immediate hypersensitivity occurs following the production of IgE antibodies against
typically otherwise-harmless foreign antigens (which are known as allergens; an antigen that
evokes hypersensitivity response). Type I sensitivities are allergies. Allegies to pollen, foods
and insect stings are examples of immediate hypersensitivity. Other allergens include
household dust, moulds, and particles from hair, feathers or skin. The first exposure to the
allergen produces no visible signs or symptoms. Hence, immediate hypersensitivity is
characterized by allergic reaction occurring immediately following an individual’s second
contact with the responsible antigen. Non allergic persons do not respond to such antigens.
The signs and symptoms of immediate hypersensitivity are a consequence of the release of
histamine and other chemical mediators from body cells. In the case of histamine, release
occurs when IgE antibodies bound to basophils or mast cells bind to allergens. Histamine is
found intracellularly within vesicles (the granules within these cells) and degranulation is
the term used to describe the release of histamine via the fusion of these vesicles with the
basophil or mast-cell plasma membranes. In addition to histamine, prostoglandins and
leukotrienes are reaction mediators that play important roles in mediating airway
constriction. Anaphylaxis is a general term used to describe the detrimental effect(s)
associated with hypersensitivities. Anaphylaxis may be localized (annoying but not life
threatening) or generalized (systemic and life threatening). Anaphylactic shock is a
generalized anaphylaxis characterized by a significant, life-threatening drop in blood
pressure.

Generalized Anaphylaxis: A shock-like and often fatal state first seen in dogs in the mid
1800s. In, 1909 two French physicians observed a similar phenomenon while on a cruise on
the yacht of the Prince of Monaco. They were asked to develop an antitoxin to the sting of
the jellyfish, Portuguese man-of-war. They experimented with sea anenome extracts given to
dogs and observed a rapid onset of symptoms including vomiting, bloody diarrhea, asphyxia,
unconsciousness and death in some dogs after secondary challege several weeks after. This
response was given the term anaphylaxis; one of the physicians was awarded a Nobel prize

71
14 years later. The symptoms differ among species due to differences in distribution of mast
cells and the types of chemical mediators released in the response. The best model for human
systemic anaphylaxis is the guinea pig. Within 1 minute of secondary challenge with the
appropriate antigen, the animal becomes restless, breathing is labored and blood pressure
drops. Smooth muscles of gastrointestinal and urinary tract contract resulting in defecation
and urination. Smooth muscle contraction of respiratory system leads to bronchiole
constriction; that and mucus secretion result in death by asphyxiation within 2-4 minutes of
the injection. In humans, susceptible people may respond with anaphylaxis to antigens
including bee, wasp, and ant stings, drugs such as penicillin and insulin, and foods such as
seafood and nuts. Without prompt treatment, usually in the form of injection of epinephrine
to counter the histamine's effects, anaphylactic shock can result in death.

Localized Anaphylaxis: Reactions limited to specific target organ or tissue, usually involving
epithelial tissues at sight of exposure - skin, nasal mucosa, and conjunctiva. It is also called
atopic (out of place) allergy. Most common are allergies to pollen, molds, and animal hair.
Symptoms range from asthma - constriction of bronchioles and difficulty breathing to hives
(wheal and flare - swelling and redness) to mild inflammation. Most symptoms occur within
a few minutes of exposure. But often, a late-phase reaction occurs several hours after early
reaction and persists for a couple of days, mainly due to the attraction and activation of
eosinophils. Their degranulation has many of the same effects of mast cell degranulation but
it occurs in a delayed fashion and results in extensive tissue damage.

Mechanism of immediate hypersensitivity: Ag/allergen stimulate release of IL-4, which

promote B-cells specific for that Ag to differentiate into IgE producing plasma cells.
Circulating IgE binds to Fc receptors on mast cells and basophils Eliciting a transduction
event to release mediators (amines, proteins, peptides and proteoglycans ) stored in granules
(Degranulation). Immediate hypersensitivity response (5-10 minutes).

Treatment of allergies: One approach to dealing with allergies is to avoid contact with the
specific allergen. Desensitization (hyposensitization) is the only current available treatment
intended to cure an allergy. This procedure consists of a series of allergen doses injected
beneath the skin to stimulate the production of IgG antibodies rather than IgE antibodies. The
circulating IgG antibodies can then act as blocking antibodies to intercept and neutralize

72
allergens before they have time to react with mast cell-bound IgE. Recent evidence suggests
that suppressor T-cell activity may also cause a decrease in IgE synthesis.

(ii) Type II: Cytotoxic hypersensitivity

The term cytotoxic in cytotoxic hypersensitivity refers to host-cell damage caused by an
over-zealous immune response. The antibodies involved are of the IgM or IgG classes and
cause cell destruction by Fc dependent mechanisms either directly or by recruiting
complement via the classical pathway. Recall that a normal aspect of both specific and non-
specific immune responses is extracellular killing, particularly the killing of host cells that
are thought to be pathogen-infected. Cytotoxic hypersensitivities are mediated by the binding
of antibody's to body tissues which leads to the lysis of cells (either via ADCC or via the
activation of complement). The negative consequences of not correctly matching blood types
(ABO compatibility reaction) for transfusions are examples of the damaging effects of
cytotoxic hypersensitivities (erythroblastosis fetalis is a related, additional example of a
cytotoxic hypersensitivity). Hence, type II hypersensitivity reactions are usually only seen in
blood transfusion recipients and patients with certain autoimmune diseases. Treatment
involves anti-inflammatory and immunosuppressive agents.

Examples of Cytotoxic hypersensitivity include:

(a) Blood transfusion reaction

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(b) Heamolytic disease of the newborn

(iii) Type III: Immune complex hypersensitivity

One role of phagocytic cells (macrophages) is the removal of debris from body tissues (e.g.,
blood) and one kind of debris that results from specific immune reactions (specifically
humoral immunity) are large complexes of antibody and antigen. These complexes form as a
consequence of the multivalent nature of both antibodies and antigens (i.e., an individual
antibody molecule can bind to more than one epitope and thus, potentially, more than one
antigen, while a large antigen or organism can display large numbers of individual epitopes).
The phrase immune complex as in immune complex hypersensitivity refers to these antigen-
antibody complexes, and type III hypersensitivity refers to an immune response that produces
an excess of these immune complexes, particularly faster than macrophages (and the liver)
can remove them. The accumulation of these immune complexes can result in their
depositing in otherwise healthy tissues followed by a damaging hypersensitivity immune
response in those tissues to the not-engulfed immune complexes. Certain autoimmune
diseases e.g rheumatoid arthritis, systematic lupus erythhematosus, serum sickness and
Arthus reaction.
Serum sickness occurs when foreign (sometimes animal) antigens in sera combine with
antibody to form immune complexes, which are deposited in various tissues.
The Arthus reaction is local immune response to an antigenic substance (usually an injected
substance) that causes edema and haemorrhage.

(iv)  Type IV: Cell-mediated Hypersensitivity (Delayed Hypersensitivity)

Cell-mediated hypersensitivity is mediated by T lymphocytes (rather than by antibodies). It is
also known as delayed hypersensitivity because the time between exposure to the eliciting
antigen and the occurrence of symptoms can take many hours. A common example of type
IV hypersensitivity is poison ivy sensitivity (where, of course, the rash appears only after
many hours—e.g., next day—following exposure to the poison ivy urushiol, the triggering
oil).

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 Examples of Type IV hypersensitivity include the follwing:
(i) Allergic contact dermatitis is caused by haptens that combine with proteins in the skin to
form the allergen that elicits the immune response. The haptens are the antigenic
determinants, and the skin proteins are the carrier molecules for the haptens. Examples of the
haptens include cosmetic, plant materials, topical chemotherapeutic agent, metals and
jewelry (that contain nickel). Contact dermatitis occurs after second contact with allergens
such as oils from poison ivy, rubber, certain metals, dyes, soaps, cosmetics, some plastics,
topical medications and other substances, and usually appears as eczema. One way to
minimize a reaction to poison ivy is to wash exposed areas thoroughly with strong soap or
detergent within minutes of contact.
(ii) Tuberculin hypersensitivity occurs in sensitive individuals when they6 are exposed to
tuberculin, from the tubercle bacillus Mycobacterium tuberculosis.
(iii) Granulomatous hypersensitivity occurs when macrophages engulf pathogens but failed
to kill them. Inside the macrophages the protected pathogens survive and sometimes continue
to divide. TH1 cells sensitized to an antigen of the pathogen elicit the hypersensitivity
reaction, attracting several; cell types to the skin or lung. A granuloma in the skin (leproma)
or lungs (tubercle) develops.
A summary of the classification of the hypersensitivity reactions is as shown below.
TYPE DESCRIPTIVE INITIATION MECHANISM EXAMPLES
NAME TIME
Ag induces cross-linking
Systemic anaphylaxis,
IgE-mediated of IgE bound to mast
I 2-30 mins Local anaphylaxis, Hay
hypersensitivity cells with release of
fever, Asthma, Eczema
vasoactive mediators
Ab directed against cell- Blood transfusion
Antibody-mediated surface antigens reactions, Haemolytic
II cytotoxic 5-8hrs mediates cell destruction disease of the newborn,
hypersensitivity via ADCC or Autoimmune Haemolytic
complement anaemia
Ag-Ab complexes
deposited at various sites Arthus reaction
Immune-complex induces mast cell (Localised); Systemic
III mediated 2-8hrs degranulation via reactions disseminated
hypersensitivity FcgammaRIII, PMN rash, arthritis,
degranulation damages glomerulonephritis
tissue
Memory TH1 cells
cell-mediated release cytokines that Contact dermatitis,
IV 24-72hrs
hypersensitivity recruit and activate Tubercular lesions
macrophages

6.3 Autoimmune disorder

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Although the immune system has an elaborate system of checks and balances to ensure self
tolerance, occasionally this system breaks down. When the immune system attacks host
components causing pathological change, this is called autoimmunity. Many people
experience an autoimmune reaction during their lifetime. Mostly these are short-lived, self-
resolving sequelae of infection. However in some 5% of individuals the reaction is chronic,
debilitating and even (rarely) life-threatening. It is these latter conditions where serious
immunopathology occurs which are usually considered autoimmune disease. We shall
consider the following aspects: (i) The characteristics of autoimmune diseases; (ii)Which
immune mechanisms are involved in bringing about the pathogenic change?; and (iii) What
factors initiate the autoreactivity?
Autoimmune diseases form a spectrum ranging from organ-specific conditions in which one
organ only is affected to systemic diseases in which the pathology is diffused throughout the
body. The extremes of this spectrum result from quite distinct underlying mechanisms, but
there are many conditions in which there are components of both organ-specific and systemic
damage.

Autoantibodies - cause or effect?: Almost all patients presenting with autoimmune

conditions have some autoantibodies present in their serum. However they also have
autoreactive T cells present (though these are far harder to demonstrate experimentally). It is
not always known whether the autoantibodies play an important role in the disease or are a
secondary result of the tissue damage which has been caused by the disease process itself.
This problem is particularly difficult in many organ-specific conditions.

Examples of autoimmune disease

Hashimoto's Thyroiditis: This disease is characterized by an intense mononuclear cellular

infiltrate into the thyroid and by the presence of autoantibodies primarily directed at
thyroglobulin and thyroid peroxidase. There are a number of theories about the mechanism of
pathogenic damage to the tissue.

 Autoreactive T cells (TH1) may cause tissue damage by release of cytokines, either
directly (eg TNF) or by recruiting and activating macrophages, which subsequently
mediate tissue destruction.
 Autoreactive antibodies, whose production requires the help of autoreactive T cells,
may be directly responsible for the pathology, by for example interfering with iodine
uptake and binding by thyroglobulin.
 Inflammation may cause tissue damage by triggering apoptosis in thyrocytes by
inducing expression of a 'death' receptor (Fas, a molecule which triggers apoptotic
death). Unusually the ligand for this 'death' receptor appears to be constitutively
expressed by thyrocytes. It is also expressed by activated but not resting T cells.

Overall, while it is clear that activation of autospecific T cells is a prerequisite for this
disease it is far from clear what the significance of the autoantibodies is. Other examples of
autoimmune disease are as listed below:

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Genetics implications in autoimmune disease

Genetic susceptibility plays an important role in almost all autoimmune conditions. The most
significant influence is that of the MHC. The linkage between particular MHC allotypes and
specific diseases has already been mentioned, in almost all cases these diseases have a strong
autoimmune component (and those that don't are likely to be due to non-MHC genes in close
linkage). With the advent of genome wide linkage maps of polymorphic DNA markers it has
been possible to examine the influence of genes on the susceptibility to disease in a
comprehensive way. Studies of this type have shown that within the human population there
are multiple genetic loci which are involved in susceptibility to common autoimmune
conditions such as diabetes (IDDM) and rheumatoid arthritis. It is likely that most
autoimmune conditions have some genetic component. It is important to distinguish these
kind of genetic susceptibilities from traditional inherited disease. No single predisposing
allele has to be present for the disease to occur, rather the presence of combinations of
susceptibility alleles significantly increases the probability of that individual developing the
specific disease. Demonstrating the existence and approximate location of such susceptibility
genes is now routine (given sufficient material) but conclusively identifying which of the
many genes in the identified genetic interval is actually responsible is much harder and time-
consuming. Apart from the MHC very few such loci have been definitely identified.

Endocrine factors in autoimmune disease

Most autoimmune disease do not occur with equal frequency in males and females. For
example Graves' and Hashimoto's are 4-5 times, and SLE 10 times, more common in females
while Ankylosing Spondylitis is 3-4 × more frequent in males. These differences are believed
to be the result of hormonal influences. A second well documented hormonal effect is the
marked reduction in disease severity seen in many autoimmune conditions during pregnancy.

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Rheumatoid arthritis is perhaps the classic example of this effect. In some cases there is also
a rapid exacerbation (rebound) after giving birth.

Environment factors in autoimmune disease

However, it is clear that environmental factors also play a role in autoimmune disease. If you
examine how frequently identical twins both develop a disease (the concordance rate), it is
only about 20-40% for common autoimmune diseases such as diabetes, SLE and rheumatoid
arthritis. This makes it highly likely that environmental factors must also be important. While
we might expect factors such as diet to play a role, we can postulate that infectious organisms
are the most significant environmental factor. In a few cases we have evidence for a direct
link between a specific infection and an autoimmune disease. The classical example is that of
rheumatic fever following Streptococcal infection. More recently, persuasive evidence
implicates infection with a variety of organisms (Yersinia, Shigella ,Chlamydia) and reactive
arthritis (NB not rheumatoid). Nevertheless a causal link has been elusive in many other
conditions for which the environmental factors must exist but remain unidentified (eg.
diabetes).

Mechanisms of autoimmune disorder

Although in many cases the precise combination of pathogenetic mechanisms is not

understood, there are three general types of mechanism which it is important to distinguish.

 Direct antibody mediated effects

 T cell mediated effects (cellular immune)
 Immune complex mediated effects

Direct antibody mediated disorder: We have already seen one example in Graves' disease
where stimulatory antibodies dysregulate thyroid function. A counter effect can also be seen
as in Myasthenia Gravis. In this disease, autoantibodies to the Acetylcholine receptor block
neuromuscular transmission from cholinergic neurons by blocking the binding of
acetylcholine and by causing downregulation (degradation) of its' receptor. Rheumatic fever
is an example of direct tissue pathology following antibody binding.

T cell mediated damage: This term implies that the recognition of autoantigen by T cells
leads to tissue destruction without requiring the production of autoantibody. There are a
number of ways this can come about:

1. Direct T cell cytotoxicity via CD8+ CTL

2. Self-destruction of tissue cells induced by cytokines, eg, TNFa
3. Recruitment and activation of macrophages leading to bystander tissue destruction
4. Induction of target tissue apoptosis by the T cell membrane protein FasL

In most cases we do not know what the relative contribution of these factors is, though
mechanism 4 would have to be confined to those tissues which constitutively express the

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'death' protein Fas or which express it as a consequence of inflammation. See 'Animal
model's of autoimmunity' (below) for further discussion.

Immune-complex mediated disorder: Immune-complexes are frequently implicated in

autoimmune pathology. Systemic diseases such as SLE and vasculitis almost certainly result
from autoantibody-antigen complexes and their consequences. Certain organs are especially
sensitive to immune complex deposition particularly the kidney. SLE patients possess a wide
variety of autoantibodies to both cytoplasmic and nuclear antigens. The presence of IgG anti
double- stranded DNA is characteristic of this condition (Note: IgM anti-ds DNA is NOT
pathogenic). Two significant facts point to the role of immune complexes in SLE. First,
patients demonstrate significant depletion of complement (C3) and neutrophils resulting from
activation by the complexes. Second, complement deficiencies which impair IC clearance
(C1,C2 or C4, see lecture 10) are very strong predisposing factors for SLE.

How are autoimmune reactions initiated?

Release of sequestered antigen:In the last lecture we mentioned that some antigens are
hidden away from the immune system in immunologically privileged sites. When trauma or
other events cause damage to the barriers which protect such special sites this can lead to the
release of novel autoantigens and the production of autoantibodies. In the case of
autoimmune sympathetic opthalmia, damage to one eye leads to subsequent autoimmune
attack on the contralateral eye.

T cell bypass: This concept embodies the idea that autoimmunity can arise as a result of T
cell tolerance being bypassed. This might occur in a number of ways

 modification. This can happen when a small molecule (eg a drug) binds to a protein
and alters an MHC- binding peptide so that it becomes a neoantigen recognised by T
cells. This provides T cell help, through linked recognition, for antibody production
which need not be (and usually is not) directed against a neodeterminant.
 inflammation. During an inflammatory response an immunostimulatory environment
is created by the release of cytokines which recruit and activate professional antigen
presenting cells and provide support for T cell activation rather than anergy. As a
result autoreactive T cells which were anergic or ignorant may become activated.
This concept is central to Matzinger's Danger Hypothesis (see lec 11).
 molecular mimicry is a rather specialised version of the above in which an epitope
of an invading microorganism cross-reacts with a self protein. The T cell help
provided by the other microbial antigens permits the activation of B cells which make
an crossreactive antibody which either escaped tolerance or which acquires sufficient
self reactivity through somatic mutation and selection driven by the cross-reactive
antigen.

Animal models of autoimmunity: Many key aspects of autoimmunity have been uncovered
by the use of animal models of autoimmune disease. These models may be divided into two
distinct types, spontaneous and induced.

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Spontaneous autoimmune disease models exist as a result of deliberate inbreeding of
strains of animal for particular characteristics including the incidence of autoimmune disease.
Such inbred strains are therefore genetically susceptible and individuals spontaneously
develop disease. Examples of this type of model are the obese strain chicken (thyroiditis) and
the NOD mouse (diabetes). Generally the cumulative incidence is less than 100%, in some
cases much less.

Induced autoimmune disease models require some treatment of the animal to trigger the
disease. They also generally require the presence of some genetic susceptibility factors. For
example the injection mice with spinal chord extract and powerful adjuvants will trigger an
autoimmune encephalomyelitis (EAE) but only in a few inbred strains. This disease
resembles multiple sclerosis.

Disease mechanisms: Mechanistic studies are virtually impossible in humans so most of our
understanding comes from the development and analysis of animal models. The NOD mouse
model of diabetes (IDDM) has shown that the disease process involves CD8+ and CD4+ T
cells and macrophages. It has also demonstrated the requirement for a specific MHC class II
susceptibility allele and is proving useful in examining the role of the other dispersed
susceptibility loci (of which there are 15 mapped). It has also shown that inducing tolerance
to candidate antigens of which insulin is only one, will prevent the development of disease.
Induced models such as EAE have an advantage in that the triggering stimulus can be studied
and the autoantigen is usually known. EAE can be transferred with a single clone of T cells.

Immunoregulation: A number of induced autoimmune diseases resolve themselves. This

has revealed the potential for the immune system to return to a tolerant state by some form of
regulation. The regulatory cells are CD4+ T cells and can protect naive mice in transfer
studies as shown below.

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6.4 Transplantation
Transplantation is the introduction of biological material - organs, tissue, cells, fluids - into
an organism. We can distinguish 3 critical relationships between the transplanted material
and the recipient.
(i) syngeneic transplants - from genetically identical individuals, usually the same
individual (these are similar to grafts between identical twins or isogenic strains of
experimental animals).
(ii) allogeneic transplants - from one individual to another of the same species
(iii) xenogeneic transplants - between individuals of different species.
Unsurprisingly, syngeneic transplants do not usually generate any immunological problems,
but allogeneic and xenogeneic transplants are almost always destroyed by immunological
processes unless some action is taken to impair the immunological process. Basically
therefore transplantation presents 2 key problems.
(1) Genetic variation between donor and recipient.
(2) Immunological recognition of the variation.

1. Genetic variation between donor and recipient.

Genetic variation between individuals that results in protein sequence differences is at the
heart of the transplant problem. In an essentially outbred species like Homo sapiens the
extent of this allelic polymorphism is considerable. We do not yet have an accurate estimate
of the average number of proteins whose sequence varies from one individual to another but
it must be greater than a few hundred, possibly as high as several thousand. Obviously the
variation is even greater between individuals from different species.

2. Immunological recognition of the variation.

However genetic differences between donor and recipient are only of significance in
transplantation if they cause incompatibility. Almost ubiquitous in allogeneic transplants is
immunological rejection. Early experimental work on allotransplantation in mice identified a
very clear distinction between one chromosomal region and the remainder of the genome.
Non-identity at this special region always led to very rapid rejection of the transplanted
tissue, even if this was the only genetic difference between the donor and recipient. This
region was therefore termed the Major Histocompatibility Complex (MHC). We now
know that this region exists in all vertebrates and that it is highly polymorphic, so that in
outbred populations 2 individuals will almost certainly differ at this region unless they are
monozygotic. However the corollary is NOT true, that is even if identical at the MHC,
transplants between individuals are likely to be rejected due to minor histocompatibility
loci. In the mouse there are about 50 such loci, in humans probably more. The key distinction
is that individually these minor H loci are less 'strong' and in particular the strength varies
between allelic differences. For example, mice differing only at the locus H-1 will reject skin
grafts in c.25 days if H-1b skin is transplanted onto H-1c recipients but in c.100 days if the
transplant is done in the reverse direction (by comparison MHC-disparate grafts will be
rejected in c. 11 days in all cases).

Recognition and rejection mechanisms: There are 3 basic types of 'recognition' which
allows the host to know that the transplanted tissue is foreign.
(a) recognition by Antibody

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 (b) recognition of foreign MHC by T cells (direct recognition)
(c) recognition of minor H loci by T cells (Indirect recognition)
As implied above these 3 recognitions may lead to very different time scales of destruction of
the transplanted cells/tissue and trigger distinct effector mechanisms. We distinguish 3 types
of rejection: (i) Hyperacute rejection; (ii) Acute rejection; and (iii) Chronic rejection

1. Hyperacute rejection
This type of rejection occurs very rapidly, resulting in necrosis of the transplanted tissue
within minutes or a few hours of contact. It always results from the reactivity of the donor
cells with pre-existing antibody. The most common situation in which this occurs is in ABO
blood group incompatible transplants, therefore we will briefly review the ABO system.

Blood Groups
Blood groups arise from genetic variations within a species. Blood transfusion is the oldest
form of transplantation and today we should be careful to distinguish what component of
blood we are transplanting; what we call blood groups are genetically variable (polymorphic)
structures present on red blood cells. It is important to note that some of these structures are
only present on red blood cells and thus incompatibility at these loci only affects the
transplantation of RBC (blood transfusion), whereas other are present on many tissue cells
and therefore affect other types of transplantation. ABO is special in that it is the only
histocompatibility alloantigen for which PRE-existing antibody is present in naive,
previously untransplanted (untransfused) recipients.

Genetics of the ABO system.

ABO antigens are carbohydrate structures present on most, perhaps all, cells and in most
people on soluble molecules in serum and secretions.
The vast majority of humans make a basic structure known as the H antigen (sometimes also
referred to as O antigen). The acceptor polysaccharide for this carbohydrate can be one of
several structures but the antigenic epitope is composed of the terminal 2 sugars shown
below.

The ability to make H antigen is controlled by the H and Se loci. Both genes are tightly
linked and both encode alpha 1→2 L-fucosyltransferases.
The H gene product (FUT1) is expressed in haemopoietic cells and vascular endothelial cells;
the Se (FUT2) in some other cells, including secretory cells.
In very rare individuals the ability to make this substance is missing (genotype h/h);such
individuals do not express H (or A,B) on their red cells. They will express H (and A, B if

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produced) in their body fluids if they are Se/se or Se/Se.

The H substance is modified by the products of the ABO locus.

* The O allele is a null.
* The A allele adds a terminal N-Acetylgalactoseamine [alpha 1→3 D- N-
Acetylgalactoseaminyltransferase].
* The B allele adds a terminal Galactose [alpha 1→3 D- Galactosyltransferase].

Immunobiology of the ABO system

Expression of the ABO locus is co-dominant

The modification of carbohydrates is competitive and incomplete. Thus an individual of
genotype A/B H/H Se/se will express the A, B and H structures on their red blood cells as
well as on their tissue cells - crucially including vascular endothelium - and on soluble
molecules present in their bodily fluids. Such an individual cannot make antibody against the
A, B or H antigens because the relevant B cells are deleted via central tolerance. BUT an
individual of genotype O/O H/H Se/se will develop anti-A and anti-B because these
structures (or cross-reacting ones) are absorbed from the gut (derived from food and/or
commensal microorganisms). The naturally occurring anti-A, anti-B are almost all IgM.

Compatibility for transplantation (including red cell transfusions)

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In practice red cell transfusions are usually ABO identical.

Non-ABO antibodies can also cause hyperacute rejection

These could be from previous tranplants (transfusions); Particular problem in xenotransplants
e.g * humans lack Gal alpha 1→3 Gal structures and make natural antibody to this (both IgG
and IgM)§; * many species do make express this structure in abundance on their cell surfaces
(eg Pigs); and * Transplants from one species to another where there is natural antibody are
termed discordant.
The actual mecghanism of hyperacute rejection involves
(i) complement activation
(ii) secondary activation of coagulation

2. Acute Graft Rejection

This is the main immunological barrier to allotransplantation. It is caused by T cell
recognition of the transplanted tissue. It is not a significant problem in red cell transfusion
because (i) the cells survive only short periods; and (ii) human rbc do not express MHC
antigens. There are 2 quite distinct modes of recognition

A. Direct recognition of allo MHC.

As referred to earlier, experimental transplantation showed that a single major gene cluster,
the MHC, had a dominant role in histocompatibility. In the context of transplantation the key
attributes of the MHC are: (i) rapid rejection of MHC non-identical transplants; and (ii)
reproducible, consistent rejection rates.

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B. Indirect recognition of minor transplantation antigens

MHC identical grafts are still rejected acutely by T cell dependent mechanisms.
Minor transplantation antigens are proteins which vary in sequence and where one (at least)
of the allomorphs is found in a peptide which binds to the MHC of the recipient.
In the case of indirect recognition, MHC sharing between donor and recipient increases the
reactivity as
 donor dendritic cells can prime recipient T cells for minor H peptides bound to
shared MHC
 activated T cells can be triggered by donor tissue cells (usually class I expressing)

Properties of minor antigens

(i) rejection is slower than for MHC
(ii) it is additive, however, thus many minor differences combine to give rapid rejection
(iii) rejection times (in experimental grafts) are more variable and strength is allele specific

An example of minor antigens

In experimental animals even grafts between animals of different sex within the same
isogenic strain can be rejected where the donor is male. In human transplantation recognition
of male minor antigens (given the generic designation H-Y) can play an important part in
haemopoietic stem cell transplants (see on). The actual MHC-peptide complexes which
form clinally significant minor antigens have now been identified in some cases; examples
are given below.

3. Chronic rejection

In stark contrast to the considerable progress in the management of acute rejection essentially
no improvement in treatment in past 25 years has made a significant impact on the long-term
loss of transplanted organs through chronic rejection. Possibly in part this is because the
mechanism(s) of chronic rejection are still obscure.

Particular transplant situations

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Privileged sites
Transplants at certain anatomical sites are generally accepted without any immune rejection.
The most important of these is the cornea. The absence of lymphatic drainage is probably the
critical common factor (some sites also lack vascularisation).

Vascularised solid organs

This includes the kidney, lung, liver, heart and pancreas combined.

Haemopoietic stem cell transplants

Often previously called bone marrow transplants, now renamed as source is frequently blood.
Three sources of stem cells are used, listed in order of decreasing mature T cell
contamination viz. peripheral blood (enriched by cytokine administration); bone marrow; and
cord blood.

A high percentage of stem cell transplants are autologous, given to restore haemopoiesis after
lethal, high-dose chaemo- or radiotherapy. These do not give rise to immunological
problems. Due to the preconditioning of the recipients which greatly impairs their
immunological function, the major immunological problem associated with allogeneic stem
cell transplants is Graft-versus-Host disease (GvH), the converse to the usual graft rejection
problem. This is because the mature T cells (and also NK cells) of the transplanted
innoculum are immunocompetent, reactive cells. In fact the GvH may be clinically useful in
leukaemic patients, where it has been found to help eliminate residual (host) tumour cells.
Thus while removing T cells from the innoculum reduces GvHD, it increases tumour relapse
in leukaemic patients; to the point where some centers are adding back measured doses of T
cells to T cell depleted stem cells (typically from marrow).

Influence of HLA-matching on allograft survival

The influence varies significantly according to tissue transplanted e.g, it if very important
in Haemopoietic stem cell; significant in kidney and heart; and of no effecting liver
transplant.
For Haemopoietic stem cell (HSC) transplants, the degree of HLA (=human MHC) matching
is critical in determining the probability of Graft-versus-Host disease (GvHD). This is the
major form of rejection in clinical HSC transplants. Most centres will only transplant across
1 MHC class I (HLA-A or B locus) difference. The following graph shows the probability of
serious GvHD (so-called grade III or IV) versus time for different degrees of mismatch.

Immunosuppression
Immunosuppression is essential to clinical transplantation, and "standard" regime varies for
different tissue/organs. For kidney transplants, this involves combination therapy with 3
different sorts of drug
* Steroids - given for systemic immunosuppressive effects, complex mechanism of action
e.g. prednisolone.
* Cytotoxic - lead to cell death, esp. on entry into cell cycle e.g. azathioprine.
* Immunosuppressive - targeting cell signalling pathways in lymphocytes
e.g. cyclosporin A; others include FK506, rapamycin.

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The immunosuppressive drug (3) needs to be maintained indefinitely. Also, many novel
treatments are in development, particularly antibodies blocking surface molecules critical in
T cell activation e.g B7-1,2 (CTLA4-Ig) ; anti-CD40L. Currently licensed biotherapeutic
treatments used to treat graft rejection include: (i) anti-CD3 monoclonal antibodies (CD3 is a
component of the T cell receptor); and (ii) anti-CD25 monoclonal antibodies (CD25 is a
component of the receptor for IL2, a major growth factor for T cells). A major future goal in
transplant research is donor specific tolerance. Because tolerance induction requires a signal
from the T cell receptor (and possibly other co-receptors), this conflicts with the use of some
forms of immunosuppression. For example the major immunosuppressant cyclosporin A is
known to block tolerance induction, as shown below.

6.5 Drug reaction

The immune response can be manipulated to suppress unwanted responses resulting from
autoimmunity, allergy, and transplant rejection, and to stimulate protective responses against
pathogens that largely elude the immune system. Immunosuppressive drugs are used to
control autoimmune disorders or inflammation when excessive tissue damage occurs, and to
prevent transplant rejection after an organ transplant.
Anti-inflammatory drugs are often used to control the effects of inflammation. The
corticosteroids are the most powerful of these drugs; however, these drugs can have many
toxic side-effects and their use must be tightly controlled. Therefore, lower doses of anti-
inflammatory drugs are often used in conjunction with cytotoxic or immunosuppressive
drugs such as methotrexate or azathioprine. Cytotoxic drugs inhibit the immune response by
killing dividing cells such as activated T cells. However, the killing is indiscriminate and
other organs and cell types are affected, which causes toxic side effects. Immunosuppressive
drugs such as cyclosporin prevent T cells from responding to signals correctly by inhibiting
signal transduction pathways.
Larger drugs (>500 Da) can provoke a neutralizing immune response, particularly if the
drugs are administered repeatedly, or in larger doses. This limits the effectiveness of drugs
based on larger peptides and proteins (which are typically larger than 6000 Da). In some
cases, the drug itself is not immunogenic, but may be co-administered with an immunogenic
compound, as is sometimes the case for taxol. Computational methods have been developed
to predict the immunogenicity of peptides and proteins, which are particularly useful in
designing therapeutic antibodies, assessing likely virulence of mutations in viral coat
particles, and validation of proposed peptide-based drug treatments. Early techniques relied
mainly on the observation that hydrophilic amino acids are overrepresented in epitope
regions than hydrophobic amino acids; however, more recent developments rely on machine
learning techniques using databases of existing known epitopes, usually on well-studied virus
proteins, as a training set. A publicly accessible database has been established for the
cataloging of epitopes from pathogens known to be recognizable by B cells. The emerging
field of bioinfomatics-based studies of immunogenicity is referred to as immunoinformatics.

6.6 Immunodeficiencies
Immunodeficiencies occur when one or more of the components of the immune system are
inactive. The ability of the immune system to respond to pathogens is diminished in both the
young and the elderly, with immune responses beginning to decline at around 50 years of

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age. In developed countries, obesity, alcoholism, and illegal drug abuse are common
causes of poor immune function. However, malnutrition is the most common cause of
immunodeficiency in developing countries. Diets lacking sufficient protein are associated
with impaired cell-mediated immunity, complement activity, phagocyte function, IgA
antibody concentrations, and cytokine production. Deficiency of single nutrients such as
zinc; selenium; iron; copper; vitamins A, C, E, and B6; and folic acid (vitamin B9) also
reduces immune responses. Immunodeficiencies can also be inherited or 'acquired'. Chronic
granulomatous disease, in which phagocytes have trouble destroying pathogens, is an
example of an inherited, or congenital, immunodeficiency. AIDS and some types of cancer
cause acquired immunodeficiency. Immunodeficiency diseases arise from an absence of
active lynmphocytes or phagocytes, the presence of defective lymphocytes or phagocytes, or
the destruction of lymphocytes. Immunodeficiency disorder are frequently divided into two
major categories: 1) Primary immunodeficiency diseases, which may be hereditary or
acquired, in which the immune deficiency is the cause of a disease, and 2) secondary
immunodeficiency disorders, in which the immunodeficiency is a result of other disease (s).

6.6.1 Primary immunodeficiency disease

Agammaglobulinemia or B cell deficiency leads to a lack of humoral immunity. It occurs
primarily in male infants in which B cells, and therefore antibodies, are absent. After
maternal antibodies are lost by about 9 months of age, affected infants develop severe
infections because they cannot produce IgM, IgA, IgD and IgE antibodies and produce only
small amount of IgG. Agammaglobuline is treated with massive doses of immune serum
(gamma) globulin to replace missing antibodies and with antibiotics to prevent infections.
DiGeorge syndrome or T cell deficiency, leads to a lack of cell mediated or humoral
immunity. T cell deficiency is probably cause by a congenital defect in the thymus during
embryological development. Humoral immunity is affected because there are no functional
TH2 cells.
Severe combined immunodeficiency disease (SCID) or deficiency of both B and T cells leads
to a lack of both humoral and cell-mediated immunity. SCID can have several genetic
origins. For example, stem cell in the bone marrow that normally gives rise to lymphocytes
fail to develop properly because of a defective gene for IL-2, for the enzyme adenosine
diamine (ADA), or for MHC molecules. People with SCID have neither functional B, nor T
lymphocyte. They have no immunological response. Any infection can be fatal.

6.6.2 Secondary immunodeficiency.

Immunodeficiency diseases are not always inherited; sometimes they are acquired through
infections, malignancies, autoimmune diseases or other conditions. For example, congenital
rubella infections can decrease T cell function and antibody production to the extent that
infants fail to respond vaccines. Once patients develop immunodeficiencies, they may suffer
from chronic or frequent recurrent infections.
Among malignant diseases that produce immunodeficiencies, those of the lymphoid tissues
suppress T cell function, and those of bone marrow suppress both T cell function and
antibody production. Autoimmune diseases, some kidney disorders, severe burns,
malnutrition or starvation, and anaesthesia also can cause temporary or permanent
immunodeficiencies.

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Acquired immune deficiency syndrome (AIDS) is the most well known secondary
immunodeficiency disease, an infectious disease cause (questionably though) by the human
immunodeficiency virus (HIV). The purported virus gradually but relentlessly destroys the
patients immune system. The lack of functional immune system leaves the body open to a
variety of malignancies and opportunistic infections, most of which are rarely seen among
people who are not suffering from advanced HIV disease or AIDS. According to the
HIV=AIDS establishment, the virus destroys TH cells, macrophages, dendritic cells and
Langerhans cells, and eventually impairs all immune functions. All these immune cells bear
on their surface an antigen called the CD4 marker, to which HIV binds. Infection by HIV is
indicted by the presence of anti-HIV antibodies. Without activated T H cells and macrophages,
the immune system cannot “see infectious microbes. Because TH cells are greatly diminished
in number, B cells are not stimulated to form plasma cells, which produce antibodies top
combat infections. The anti-HIV antibodies detected early in the course of infection are made
before TH cell population becomes too depleted to stimulate B cells. Similarly, cytokines are
produced in amounts insufficient to activate macrophages and TH cells. A drop in T H cell
count can be used to predict the onset of disease symptoms (A normal T H count is 800 to
1200 per microlitre of blood).
Progression of HIV disease and AIDS – HIV-infected individuals progress through a series
of stages that lead to AIDS. Figure 6-1 below illustrates different groups. Group 4 individuals
suffer from opportunistic infections and malignancies such as Kaposis sarcoma (a tumor of
blood vessels).

89
Table 6-1. Infections frequently found in AIDS patients

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 CHAPTER VII

IMMUNITY TO INFECTION

Obviously protecting the host from infection is the raison d'être of the immune system. We
have spent the past weeks looking at the molecules and cells of the immune system, in this
last lecture I want to try to look at the immune system as an integrated defence mechanism.
The first thing to remember is that most microorganisms arriving at the external surfaces of
the body fail to establish a colony. Just as in military strategy, the hardest thing for an
invader to do is to establish a beachhead. So one cardinal property which distinguishes those
species of microorganism which are capable of colonising human hosts from the much larger
number which are not is their ability to overcome the physical and physiological barriers
which protect the surfaces of the body. Even then the majority of colonising organisms do
not penetrate the body's surface and indeed the presence of these harmless commensal
bacteria makes a significant contribution to keeping out undesirables. Pathogens, then are
that rather select group which can both colonise and invade the body - there are a very small
number of exceptions to this (almost all gut bacteria) which can cause disease by secreting
toxins which damage the host without the organisms themselves penetrating the body
surface. We will consider the problem in two ways. First temporally, looking at the different
phases of infection and response. Second we will look at the different classes of pathogen
and the importance of different kinds of immunity.

7.1 The Immediate defence systems

The immediate defence of the body against an invasion must be in the hands of preformed
molecules, already present constitutively. Unless the specific microorganism has been
encountered previously (of which more later) these molecules must be part of the innate
immune system. The very earliest events of host defence against a new pathogen are those of
acute inflammation.

Complement
The most important immediate defender is the C3 component of complement. The
alternative pathway initiator is spontaneously activated C3, which is continually generated at
a low rate (C3 'tickover'). Normally this reactive species is shortlived but it can covalently
attach to a protein or carbohydrate surface, recruit the serum factor B which is a substrate for
the protease factor D, generating the active C3 convertase (C3*Bb). If this is a host cell, then
regulators of the complement system rapidly inactivate the convertase. However a pathogen
lacks the host regulatory proteins and thus the C3 convertase rapidly amplifies itself. In
addition some microbes catalyse the binding of another serum component, P (properdin)
which significantly stabilises the convertase and prevents its' inactivation by the soluble
inhibitor factor H. The conditions which favour properdin binding are not fully understood -
possibly low sialic acid (=less -ve charge). Complement alone is able to destroy some
pathogens, primarily gram +ve bacteria by activation of the terminal complement
components and assembly of the membrane attack complex.
Complement also serves to activate the acute inflammatory response. The C5a fragment is a
potent chaemoattractant for neutrophils and activates vascular endothelium directly,

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furthermore C5a also activates mast cells which amplify the inflammatory signals by
releasing their preformed vasoactive mediators.

Phagocytes
Macrophages are resident in almost all tissues and are found in particularly large numbers in
mucosal tissues. Neutrophils are present in the blood in very large numbers, they can be
rapidly recruited to any site which activates complement (see above). Both types of
phagocyte possess receptors which enable them to bind and phagocytose microbial
organisms. These receptors recognise carbohydrate structures which are not present on host
cells including certain mannose linkages (Mannose-fucose receptor) and lipopolysaccharide
on gram -ve bacteria (CD14). Both type of phagocyte also possess receptors for iC3b which
potently stimulate phagocytosis. Once organisms are engulfed, they are subject to a battery of
chemical and enzymatic attacks which in many cases destroys them.

'Natural' antibody
Even when an organism is encountered for the first time there may be some IgM antibody
which may bind to its' surface structures. This is called 'natural' antibody. It is a matter of
some dispute what role environmental stimuli play in the development of this normal serum
antibody. However it should be recognised that even very low affinity reactions with IgM
can produce binding and classical pathway complement activation when the target is a
bacterial carbohydrate due to the density of epitopes on the microbial surface.

The Host-Pathogen Interplay

Despite this armoury of ready-made weapons, the fact that specific immune antibody is
required to prevent some infections shows that they are not always sufficient . Often this is
because the invading organism has evolved some mechanisms for escaping destruction;
examples of this include the capsule of certain bacteria which prevents innate recognition.
Some species of bacteria and protozoa actually welcome phagocytosis, having devised means
of surviving inside the phagosome or cytoplasm of the cell, phagocytosis helps hide them
from other immune mechanisms. Because of their short generation times pathogens will
always hold an advantage in the cat and mouse game of immunity.

7.1.1 Early Immune responses (4-96 hrs)

The immediate defences are available instantly or within an hour or so of invasion. The
recognition events set in motion of a second wave of defence primarily triggered by the de
novo synthesis of cytokines.

The role of macrophages

The secondary effects of recognition of microbial pathogens via either the innate
carbohydrate receptors or the complement receptors is to active macrophages to synthesise
cytokines. In particular TNFalpha, IL12 and IL1 play an important role in the second phase
response. TNFalpha is critical in activating local vascular endothelium. This achieves several
things:
* increased vascular permeability leads to supply of complement (and other serum effector
proteins when present) and increased fluid drainage to the lymph node
* recruits polymorphs and macrophages

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* triggers platelet activation and clotting
The effect of local vessel clotting is important to prevent spread of the pathogen into the
blood stream - antibody neutralisation of TNF allows blood sepsis in experimental bacterial
infections.
TNFalpha also 'primes' neutrophils, causing them to activate oxygen-dependent intracellular
killing mechanisms and making them more effective.
In synergy with IL1, TNF stimulates the acute phase response which triggers massive
increases in the serum concentration of Mannose Binding Protein (MBP), and C Reactive
protein (CRP). These molecules provide additional means to recognise the invaders. Both
bind simple chemical structures on microbial cells, both have specific receptors on
phagocytes and both are capable of activating the complement system mimicking C1q and
IgM respectively.

Natural Killer (NK) cells

These lymphocytes lack the clonally variable antigen specific receptors of T and B cells.
They are part of the innate immune system and we know they play an important role in viral
infections. They are activated by the cytokines IL12 and IFNalpha/beta.

Interferon
Interferon alpha/beta are produced by a variety of cells in response to viral infection. They
have an important role in limiting viral infection in the early phase (before specific immunity
is available). They do this both directly and indirectly. They act on a wide variety of cell
types to induce the synthesis of a series of proteins which interfere with viral replication both
by degrading RNA and by inhibiting protein synthesis. They also potently activate NK cells.

7.1.2 Late Immune responses

After about 4 days the specific immune response begins to 'kick in'. During the early phase
antigen presenting cells have carried peptides resulting from the degradation of the proteins
from the infecting microorganism to the local lymph node and presented them in MHC
bound form to T cells. This process allows the rare (~1 per 10 6) antigen specific T cells to
encounter the presented peptide-MHC complex as the T cells traffic through the lymph node.
One of the early responses to this specific recognition is 'shut down', inhibition of exit of
lymphocytes from the lymph node via the efferent lymph which allows accumulation of cells
in the local lymph node draining a site of infection.
Antigen-specific B cells acquire antigen via their surface IgM, process and present this
antigen via their MHC class II molecules. The innate response also plays a part here, it has
been shown that activation of the antibody response is orders of magnitude more efficient if
the antigen is bound to C3d. The presented antigen-MHC complex is available for activated
T cells to recognise as the B cells traffic through the T cell areas which surround their portal
of entry from the draining afferent lymphatics.
The activation of T cells per se leads to the clonal proliferation of antigen-specific cells and
to the production of effector T cells, such as TH1 cells and cytotoxic T cells (CTL). The
interaction of specific TH cells with B cells leads to the generation first of a primary IgM-led
antibody response and later to a shift to IgG, IgA and/or IgE production and to higher affinity
antibodies (affinity maturation).

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Specific Antibody
Specific antibody clearly plays an important role in clearing many primary infections. The
presence of IgM begins to be detectable at about 5 days after antigen entry and peaks
between 2-3 weeks. The IgG response is delayed by about 4-5 days and persists much longer.

IgM primarily acts as an activator of the complement system. IgG can also do this but in
addition it can call into play further effector mechanisms via the Fc receptors on phagocytes,
eosinophils and mast cells.

Effector T cells
Essentially two types of effector T cells are vital in the clearance of different sorts of
infection. TH1 cell recruit and activate macrophages by secreting appropriate cytokines. CTL
are able to recognise cells harbouring intracellular pathogens which are out of reach of
humoral immunity.

Summary of the phases of the immune response

Immediate Early Late
0-4hrs 4-96hrs >96hrs

Type Innate Innate (inducible) Specific

Key molecules Complement Complement IgM and IgG antibody

Histamine etc IL-1,TNFalpha,IL12 IL2,IL4,IL12,IFNgamma
IFNalpha/beta
MBP, CRP

Key cells Macrophages Macrophages T cells

Mast cells Neutrophils B cells
Neutrophils NK cells Macrophages

7.2 Different immune effectors protect against different pathogens

The immune system has to cope with a spectrum of pathogens which have distinct lifestyles
and obviously different arms of the immune system are needed in different situations. On top
of this many pathogens have evolved specific counter measures which limit or inhibit the
effectiveness of the immune response. We will consider both the type of immunity required

94
to eliminate an first infection and the protection developed against subsequent challenge. We
can divide the type of pathogen up as follows:

Extracellular organisms
Bacteria. These are probably the simplest type of organism to combat and in many cases the
innate immune system may be able to clear an infection using complement and phagocytosis.
Specific antibody is highly effective, both by directing complement lysis and inducing
opsonisation and phagocytosis. Some bacteria have evolved capsules which prevent
recognition by innate mechanisms and require both antibody and complement opsonisation to
promote efficient clearance by phagocytes. Where toxins are produced antibody is of course
vital.
Protective immunity is essentially humoral, with IgA playing an important role in organisms
that infect mucosal surfaces (respiratory tract, gut, genito-urinary tract).
Parasites. Large, multicellular parasites present a special problem to the immune system and
indeed are rather poorly eliminated. The mechanisms deployed include antibody directed
complement attack and ADCC, in particular by eosinophils. Innate immunity is generally
ineffective. The parasites employ many evasion strategies including complement inhibitors,
release of large quantities of soluble antigen (decoy) and acquisition of host proteins.

Intracellular organisms
Bacteria and Protozoa: Many bacteria have evolved resistance to the constitutive killing
mechanisms used by phagocytes. These pathogens actively replicate inside bacteria, either in
the phagosome or, in some cases where a specific adaptive mechanism has been acquired, in
the cytoplasm. This type of bacteria cannot be eliminated by immediate immune
mechanisms. Sometimes innate immunity can be effective, for example Listeria
Monocytogenes can be eliminated by macrophages activated by NK cells, TNFalpha and
IFNgamma are required for this (probably also IL12 but I am not aware this has been
shown). More frequently T cell activation is required and a TH1 response necessary for
clearance of the organism. Antibody is generally ineffective in eliminating a first infection.
Cytotoxic T cells also play a role in clearing many intracellular organisms.
Memory T cells are the key players in protecting against most intracellular bacteria and
parasites. Antibody is occasionally though rarely protective.

Viruses: Viruses are a very diverse group of obligate intracellular pathogens. Almost every
form of immunity comes into play against some type of virus. Enveloped viruses can be
damaged by complement attack, and some directly bind C1q or homologous collectins.
Phagocytes can take up and destroy antibody and complement coated viruses. However the
key players in antiviral immunity are interferon, NK cells, antibody, CTL and TH1 cells taken
in temporal order in a first encounter.
Protection against subsequent challenge varies with the behaviour of the virus but antibody is
highly effective in preventing reinfection if it is of the right type and against the appropriate
epitope. Antibody which prevents infection of a susceptible host cell by a virus is termed
neutralising. This property requires that the antibody either prevent binding of the virus to
its' receptor or blocks some reaction necessary for viral entry. Because of the slower response
time, T cell memory is rarely able to prevent the establishment of a secondary infection but is
of considerable importance in limiting its' spread.

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Vaccination
There is insufficient space for a proper discussion of this topic. It is important to note
however that immunisation is immunology's most significant contribution to health. You will
recognise from the discussion above that developing novel effective vaccines will require an
understanding of the immune mechanisms that protect us against different pathogens. We
will also need to know how to stimulate the immune system to produce the required response
safely.

7.3 Tumor Immunity

7.3.1 Comparison of tumor cells and normal cells
Before considering immune responses to tumor cells some properties of these cells will be
received:
1) Tumors are characterized by unrestricted growth in the animal and in culture: tumor cells
usually respond poorly, if at all, to the signals that regulates the growth of normal cells.

96
2) Tumor cells of the more malignant variety are invasive: as they grow in vivo they
penetrate normal membranes and cell layers and spread by direct extension into neighboring
tissues.
3) Tumors also spread by metastasis: some cells separate from the main tumor mass, aere4
carried in the blood and lymph to distant organs and lymph nodes, and there invade normal
tissues and establish satellite or secondary tumor masses (metastasis). Death from cancer is
usually due more to widespread metastasis than to the primary tumor mass, which can often
be excised.
4) Rapid growth and metastatic spread are not unique ti tumor cells: stem cells in the normal
epidermis, intestine, and bone marrow divide more rapidly than most tumor cells; and the
migration of cells from one part of the body to another is characteristic of normal fetal
development (as of the thymus). What sets tumor apart is a failure to respond to the
regulatory signals that are responsible for the orderly and balanced growth patterns of normal
cells. The identification of these signals, and the basis for their inability to control the growth
of tumor cells, are key problems in tumor biology.
5) The individual tumor is usually a single clone: it is derived from a single progenitor cell.
6) Tumors, nevertheless are usually heterogynous: within a single tumor, the cells often
differ in shape, surface antigens, an the tendency to spread by metastasis, and some tumors
also have both self-renewing tumor “stem” cells and more differentiated tumor cells, derived
from the stem cells.
7) Normal cells can be converted into “transformed” or tumor cells by various agents
(carcinogens). Physical and chemical carcinogens are mutagens and probably cause
mutations in critical but unidentical cellular genes, whereas in viral oncogenesis, the
transforming gene is part of the viral genome.
8) Transformed and untransformed cells from the same clone of cultured cells shoe major
differences in surface glycoproteins, in surface antigens, and in growth behavior. In cultured
cells the division of normal fibroblasts is inhibited by contact inhibition: their continual
proliferation results in heaped up multilayered cell masses, with growth ceasing only when
nutrient are exhausted. When suspended in gel, normal fibroblasts do not proliferate, because
their division requires attachment to a solid surface. Transformed fibroblasts, however, are
anchorage-independent and readily form colonies when suspended in a gel.
9) Tumor cells often express genes that are silent in the corresponding normal cells. For
instance tumors arising in an adult can produce fetal antigens; and non endocrine tumors
(e.g. the bronchus) can produce hormones (such as insulin or parathyroid hormone) that are
ordinarily made by special endocrine cells. These aberrations reflect abnormal regulatory
processes in tumor cells, and they are useful for the early detection of some form of cancers.

7.3.2 Immune response to tumor cells

Cancer cells are the progeny of a single transformed cell that undergo unregulated cell
proliferation. Solid tumors are collections of attached cancer cells which can metastasize
(spread) from their original site. "Liquid" tumors are leukocyte tumors that circulate in the
blood and may also form masses elsewhere in the body. Cancer is thought to be the result of
several sequential events, including genetic predisposition, transformation by viruses or
environmental mutagens such as radiation and chemicals, and tumor promoters. The theory
of immune surveillance says that the immune system continually recognizes and eliminates
tumor cells; when a tumor escapes immune surveillance and grows too large for the immune

97
system to kill, cancer is the result. Immune surveillance is most likely to be successful
against virus-induced tumors which express foreign peptides. Tumors vary greatly in their
immunogenicity, and even tumors with antigens which can be recognized by the host
immune system can evade immune elimination. Lack of tumor rejection by intact immune
systems is not always due to the absence of antigens which can be recognized or to the
absence of T cells which can recognize those antigens. Tumor-specific lymphocytes can be
found in the blood, draining lymph nodes, and the tumor itself of patients with actively
growing tumors. These lymphocytes can kill tumor cells in vitro but fail to do so in vivo. The
presence of these cells has allowed immunologists to identify antigens on some tumor cells.

Some tumors have tumor-specific antigens (TSA, also called tumor-specific

transplantation antigens, TSTA, or tumor rejection antigens, TRA) on their surface. TSA
are not present on non-tumor cells. TSA usually appear when an infecting virus has caused
the cell to become immortal and to express virus antigens. TSAs not induced by viruses are
the idiotypes of BCR on B cell lymphomas or TCR on T cell lymphomas. Tumor-associated
antigens (TAA) are more common. TAA are found on tumor cells and on normal cells
during fetal life (onco-fetal antigens), after birth in selected organs, or in many cells but at
much lower concentration than on tumor cells. Immune responses to TAA may be suppressed
because they are considered "self". The table below illustrates some known tumor antigens.

Oncogenes were discovered in cancer-causing viruses. Most oncogenes were actually

present in the host cell, where they functioned in regulated cell growth. The host cell gene
was called a proto-oncogene. When transduced by the virus and expressed under the control
of a viral promotor, the gene product contributes to the unregulated growth of the tumor cell.
Since proteins encoded by proto-oncogenes are expressed by normal cells, their over-
expression on tumor cells would qualify them as tumor-associated antigens.
If NK cells and tumor-specific CTL can be induced to kill tumor cells in vitro, why don't
these responses occur in tumor-bearing mice? One difficulty may be intrinsic lack of
immunogenicity. Tumor cells may present only self peptides or downregulate Class I MHC
expression. Tumor cells often lack co-stimulatory molecules like B7 or adhesion molecules
that are necessary for them to interact with CD8 T cells. Tumors also shed their tumor
antigens or change their structure spontaneously (antigenic variation) to avoid immune
system elimination. Antibodies to tumor surface antigens may promote tumor survival
(enhancing antibodies) if they bind without being cytotoxic, hiding the tumor antigens from
T cells and inducing the tumor to downregulate tumor antigen expression. Some tumors
actively suppress the immune response by producing TGFb, a suppressive cytokine that
inhibits cellular immunity. Some tumors, including myeloma and HTLV-1 T cell leukemia,
also produce cytokines that stimulate their own proliferation.

Tumors can be detected by using radioisotopically-labeled mouse monoclonal antibodies to

tumor-specific and tumor-associated antigens. These antibodies are also useful for assessing
the success of tumor therapy by testing for shrinkage of the tumor. Immunotherapy of tumors
is usually not done until conventional therapies like surgery, chemotherapy and radiation
have been tried for practical and ethical reasons. Debulking the tumor is essential for success
of immunotherapy, but surgery, radiation and chemotherapy all suppress immune function.

98
Attempts have been made to use tumor-specific antibodies to kill tumor cells by linking the
antibodies to toxins, anti-tumor drugs, or very energetic radioisotopes. Problems with these
therapies include the necessity of making a unique antibody for each tumor, which is time-
consuming and expensive, lack of antibody access to the tumor center, and production of a
human-anti-mouse-antibody (HAMA) response to xenogeneic monoclonal antibodies.
Production of human monoclonals has proved difficult, but chimeric antibodies and
humanized antibodies can be made which are less immunogenic. Some mAb have been able
to induce tumor remission or elimination in a few patients.

Tumor   Antigens
Antigen Antigen   Function Expressed   On
Cyclin-dependent kinase 4 Cell cycle regulator Melanoma
b-catenin Signal transduction Melanoma
Caspase-8 Apoptosis regulator Squamous cell carcinoma
MAGE-1
Normal testicular proteins Melanoma, breast, glioma tumors;
MAGE-3
Tyrosinase Melanin synthesis Melanoma
Surface Ig idiotype BCR Lymphoma
Her-2/neu Receptor tyrosine kinase Breast and ovarian cancer
MUC-1 Underglycosylated mucin Breast and pancreatic tumors
HPV E6 and E7 Viral gene products Cervical carcinoma
Adapted from Janeway et al. Immunobiology (5th ed.). Garland Press,New York, 2001.

Tumor   Antigens   Targeted   by   mAb

Antigen   Type Specific   Antigen Tumor   Type
CD5 T cell lymphoma
Differentiation Idiotype B cell lymphoma
CAMPATH-1 T and B cell lymphomas
B cell signaling CD20 Non-Hodgkin's B cell lymphoma
Epithelial tumors (breast, colon,
Cell surface glycoprotein CEA, mucin-1
lung)
Lewisx Epithelial tumors
Cell surface carbohydrate
CA-125 Ovarian carcinoma
Epidermal growth factor Lung, breast, head, and neck
receptor tumors
Growth factor receptor
p185HER2 Breast, ovarian tumors
IL-2R T and B cell tumors
FAP-a Epithelial tumors
Stromal extracellular
Tenascin Glioblastoma multiforme
antigen
Metalloproteinases Epithelial tumors
Adapted from Janeway et al. Immunobiology (5th ed.). Garland Press, New York, 2001.

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Cytokines are used clinically to treat tumors. IL-2, IFNa, and TNFa are used in vivo, and IL-2
is used in vitro to expand populations of lymphokine-activated killer (LAK) cells and
tumor-infiltrating lymphocytes (TILs) so they can be returned to the patient to kill tumor
cells. Limitations on therapeutic use of cytokines include the need for locally high
concentrations, their short biological half lives, and side effects such as vascular shock and
psychoses. Introduction of irradiated tumor cells transfected with genes for cytokines that
stimulate immunity or antisense genes for inhibitory cytokines are undergoing clinical trials.
For example, a GM-CSF gene transfected into tumor cells recruits hematopoietic precursors
and induces them to become dendritic cells which can take up and present tumor antigens to
T cells.

Other immunotherapies in human trials include melanoma vaccines. These vaccines employ
the patient's tumor cells (irradiated to prevent growth) along with adjuvant (BCG or
Corynebacterium parvum). Melanoma vaccines have induced tumor remission in some
patients. Whole protein, peptide, and recombinant vaccines are also being developed.
Irradiated tumor cells transfected with B7 are being injected in the hopes that they can
activate specific T cells. Heat Shock Proteins (HSP) from tumor cells are being tested as
possible vaccines. HSP are peptide chaperones; their concentration increases in damaged
cells from many species (including prokaryotes). It has been discovered that professional
APC bind HSP and deliver their peptides to the Class I MHC presentation pathway. Finally,
tumor antigen-pulsed autologous dendritic cells are being tested for their ability to stimulate
a tumor-specific immune response.

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 CHAPTER VIII

ANTIMICROBIAL CHEMOTHERAPY

8.1 Introduction
Most microbiologists distinguish two groups of antimicrobial agents used in the treatment of
infectious disease: antibiotics, which are natural substances produced by certain groups of
microorganisms, and chemotherapeutic agents, which are chemically synthesized. A hybrid
substance is a semisynthetic antibiotic, wherein a molecular version produced by the
microbe is subsequently modified by the chemist to achieve desired properties. Furthermore,
some antimicrobial compounds, originally discovered as products of microorganisms, can be
synthesized entirely by chemical means. They might be referred to as synthetic antibiotics to
distinguish them from the chemotherapeutic agents. The modern era of antimicrobial
chemotherapy began in 1929 with Fleming's discovery of the powerful bactericidal substance
penicillin, and Domagk's discovery in 1935 of synthetic chemicals (sulfonamides) with broad
antimicrobial activity.

In the early 1940's, spurred partially by the need for antibacterial agents in WW II, penicillin
was isolated, purified and injected into experimental animals, where it was found to not only
cure infections but also to possess incredibly low toxicity for the animals. This fact ushered
into being the age of antibiotic chemotherapy and an intense search for similar antimicrobial
agents of low toxicity to animals that might prove useful in the treatment of infectious
disease. The rapid isolation of streptomycin, chloramphenicol and tetracycline soon
followed, and by the 1950's, these and several other antibiotics were in clinical usage. The
most important property of an antimicrobial agent, from a host point of view, is its selective
toxicity, i.e., that the agent acts in some way that inhibits or kills bacterial pathogens but has
little or no toxic effect on the host. This implies that the biochemical processes in the bacteria
are in some way different from those in the animal cells, and that the advantage of this
difference can be taken in chemotherapy.

8.2 Characteristics of Antibiotics

Antibiotics are low-molecular weight substances that are produced as secondary metabolites
by certain groups of microorganisms, especially Streptomyces, Bacillus, and a few molds
(Penicillium and Cephalosporium) that are inhabitants of soils (Table 1). Antibiotics may
have a cidal (killing) effect or a static (inhibitory) effect on a range of microbes. The range of
bacteria or other microorganisms that is affected by a certain antibiotic is expressed as its
spectrum of action. Antibiotics effective against procaryotes which kill or inhibit a wide
range of Gram-positive and Gram-negative bacteria are said to be broad spectrum. If
effective mainly against Gram-positive or Gram-negative bacteria, they are narrow
spectrum. If effective against a single organism or disease, they are referred to as limited
spectrum.

Characteristics of a clinically useful antibiotic:

These should include the following:
(i) It should have a wide spectrum of activity with the ability to destroy or inhibit many
different species of pathogenic organisms.

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(ii) It should be nontoxic to the host and without undesirable side effects.
(iii) It should be nonallergenic to the host.
(iv) It should not eliminate the normal flora of the host.
(v) It should be able to reach the part of the human body where the infection is occurring.
(vi) It should be inexpensive and easy to produce.
(vii) It should be chemically-stable (have a long shelf-life).
(viii) Microbial resistance is uncommon and unlikely to develop.

8.3 Kinds of Antimicrobial Agents and their Primary Modes of Action

The table below is a summary of the classes of antibiotics and their properties including their
biological source and mode of action.

Table 1. Classes of antibiotics and their properties.

Spectrum (effective
Chemical class Examples Biological source Mode of action
against)
Inhibits steps in
Penicillium
Beta-lactams cell wall
Penicillin G, notatum and Gram-positive
(penicillins and (peptidoglycan)
Cephalothin Cephalosporium bacteria
cephalosporins) synthesis and
species 
murein assembly
Inhibits steps in
Gram-positive and cell wall
Semisynthetic Ampicillin,
Gram-negative (peptidoglycan)
penicillin Amoxycillin
bacteria synthesis and
murein assembly
Clavamox is Gram-positive and
Streptomyces "Suicide" inhibitor
Clavulanic Acid clavulanic acid Gram-negative
clavuligerus of beta-lactamases
plus amoxycillin bacteria
Inhibits steps in
Gram-positive and cell wall
Chromobacter
Monobactams Aztreonam Gram-negative (peptidoglycan)
violaceum
bacteria synthesis and
murein assembly
Inhibits steps in
Gram-positive and cell wall
Streptomyces
Carboxypenems Imipenem Gram-negative (peptidoglycan)
cattleya
bacteria synthesis and
murein assembly
Gram-positive and
Streptomyces Inhibit translation
Aminoglycosides Streptomycin Gram-negative
griseus (protein synthesis)
bacteria
Gram-positive and
Micromonospora Gram-negative Inhibit translation
Gentamicin
species bacteria esp. (protein synthesis)
Pseudomonas
Glycopeptides Vancomycin Streptomyces Gram-positive Inhibits steps in
orientales bacteria, esp. murein
Staphylococcus (peptidoglycan)

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 biosynthesis and
aureus
assembly
Gram-positive and
Gram-negative
Streptomyces Inhibits translation
Lincomycins Clindamycin bacteria esp.
lincolnensis (protein synthesis)
anaerobic
Bacteroides
Gram-positive
bacteria, Gram-
Streptomyces negative bacteria not Inhibits translation
Macrolides Erythromycin
erythreus enterics, Neisseria, (protein synthesis)
Legionella,
Mycoplasma
Damages
Gram-negative
Polypeptides Polymyxin Bacillus polymyxa cytoplasmic
bacteria
membranes
Inhibits steps in
murein
Gram-positive
Bacitracin Bacillus subtilis (peptidoglycan)
bacteria
biosynthesis and
assembly
Inactivate
Streptomyces
Polyenes Amphotericin Fungi membranes
nodosus
containing sterols
Inactivate
Streptomyces
Nystatin Fungi (Candida) membranes
noursei
containing sterols
Gram-positive and
Inhibits
Gram-negative
Streptomyces transcription
Rifamycins Rifampicin bacteria,
mediterranei (bacterial RNA
Mycobacterium
polymerase)
tuberculosis
Gram-positive and
Streptomyces Inhibit translation
Tetracyclines Tetracycline Gram-negative
species (protein synthesis)
bacteria, Rickettsias
Gram-positive and
Semisynthetic Gram-negative Inhibit translation
Doxycycline
tetracycline bacteria, Rickettsias (protein synthesis)
Ehrlichia, Borrelia
Gram-positive and
Streptomyces Inhibits translation
Chloramphenicol Chloramphenicol Gram-negative
venezuelae (protein synthesis)
bacteria

8.4 Antimicrobial Agents Used in the Treatment of Infectious Disease

8.4.1 Antibacterial agents

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8.4.1.1 Cell wall synthesis inhibitors
Cell wall synthesis inhibitors generally inhibit some step in the synthesis of bacterial
peptidoglycan. Generally they exert their selective toxicity against eubacteria because human
cells lack cell walls.
Beta lactam antibiotics. Chemically, these antibiotics contain a 4-membered beta lactam
ring.They are the products of two groups of fungi, Penicillium and Cephalosporium molds,
and are correspondingly represented by the penicillins and cephalosporins.
The beta lactam antibiotics are stereochemically related to D-alanyl-D-alanine  which is a
substrate for the last step in peptidoglycan synthesis, the final cross-linking between between
peptide side chains. Penicillins bind to and inhibit the carboxypeptidase and transpeptidase
enzymes that are required for this step in peptidoglycan biosynthesis. Beta lactam antibiotics
are normally bactericidal and require that cells be actively growing in order to exert their
toxicity. Different beta lactams differ in their spectrum of activity and their effect on Gram-
negative rods, as well as their toxicity, stability in the human body, rate of clearance from
blood, whether they can be taken orally, ability to cross the blood-brain barrier, and
susceptibility to bacterial beta-lactamases.

Natural penicillins, such as Penicillin G or Penicillin V, are produced by fermentation of

Penicillium chrysogenum. They are effective against streptococcus, gonococcus and
staphylococcus, except where resistance has developed. They are considered narrow
spectrum since they are not effective against Gram-negative rods.

Semisynthetic penicillins first appeared in 1959. A mold produces the main part of the
molecule (6-aminopenicillanic acid) which can be modified chemically by the addition of
side shains. Many of these compounds have been developed to have distinct benefits or
advantages over penicillin G, such as increased spectrum of activity (effectiveness against
Gram-negative rods), resistance to penicillinase, effectiveness when administered orally, etc.
Amoxycillin and Ampicillin have broadened spectra against Gram-negatives and are
effective orally; Methicillin is penicillinase-resistant.

Clavulanic acid is a chemical sometimes added to a semisynthetic penicillin preparation.

Thus, amoxycillin plus clavulanate is clavamox or augmentin. The clavulanate is not an
antimicrobial agent. It inhibits beta lactamase enzymes and has given extended life to
penicillinase-sensitive beta lactams.
Although nontoxic, penicillins occasionally cause death when administered to persons who
are allergic to them. In the U.S. there are 300 - 500 deaths annually due to penicillin allergy.
In allergic individuals the beta lactam molecule attaches to a serum protein which initiates an
IgE-mediated inflammatory response.

Cephalolsporins are beta lactam antibiotics with a similar mode of action to penicillins that
are produced by species of Cephalosporium. The have a low toxicity and a somewhat broader
spectrum than natural penicillins. They are often used as penicillin substitutes, against Gram-
negative bacteria, and in surgical prophylaxis. They are subject to degradation by some
bacterial beta lactamases, but they tend to be resistant to beta-lactamases from S. aureus.

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Two other classes of beta lactams are the carbapenems and monobactams. The latter are
particularly useful for the treatment of allergic individuals. A person who becomes allergic to
penicillin usually becomes allergic to the cephalosporins and the carbapenems as well. Such
individuals can still be treated with the monobactams, which are structurally different so as
not to induce allergy.

Bacitracin is a polypeptide antibiotic produced by Bacillus species. It prevents cell wall

growth by inhibiting the release of the muropeptide subunits of peptidoglycan from the lipid
carrier molecule that carries the subunit to the outside of the membrane.Teichoic acid
synthesis, which requires the same carrier, is also inhibited. Bacitracin has a high toxicity
which precludes its systemic use. It is present in many topical antibiotic preparations, and
since it is not absorbed by the gut, it is given to "sterilize" the bowel prior to surgery.

Cycloserine inhibits the early stages of murein synthesis where D-alanyl-D-alanine is added
to the growing peptide side chain. The antibiotic resembles D-alanine in spatial structure, and
it competitively inhibits the racemase reaction that converts L-alanine to D-alanine and the
synthetase reaction that joins two D-alanine molecules. The affinity of cycloserine for these
enzymes is about a hundred times greater than that of D-alanine. Cycloserine enters bacterial
cells by means of an active transport system for glycine and can reach a relatively high
intracellular concentration. This concentrating effect, along with its high affinity for
susceptible enzymes, enables cycloserine to function as a very effective antimicrobial agent.
However, it is fairly toxic and has limited use as a secondary drug for tuberculosis.
Glycopeptides, such as the antibiotic vancomycin, appear to inhibit both transglycosylation
and transpeptidation reactions during peptidoglycan assembly. They bind to the muropeptide
subunit as it is transferred out of the cell cytoplasm and inhibit subsequent polymerization
reactions. Vancomycin is not effective against Gram-negative bacteria because it cannot
penetrate their outer membrane. However, it has become important in clinical usage for
treatment of infections by strains of Staphylococcus aureus that are resistant to virtually all
other antibiotics.

8.4.1.2 Cell membrane inhibitors

These antibiotics disorganize the structure or inhibit the function of bacterial membranes.
The integrity of the cytoplasmic and outer membranes is vital to bacteria, and compounds
that disorganize the membranes rapidly kill the cells. However, due to the similarities in
phospholipids in eubacterial and eukaryotic membranes, this action is rarely specific enough
to permit these compounds to be used systemically. The only antibacterial antibiotic of
clinical importance that acts by this mechanism is polymyxin,  produced by Bacillus
polymyxis. Polymyxin is effective mainly against Gram-negative bacteria and is usually
limited to topical usage. Polymyxin binds to membrane phospholipids and thereby interferes
with membrane function. Polymyxin is occasionally given for urinary tract infections caused
by Pseudomonas strains that are gentamicin, carbenicillin and tobramycin resistant. The
balance between effectiveness and damage to the kidney and other organs is dangerously
close, and the drug should only be given under close supervision in the hospital.

8.4.1.3 Protein synthesis inhibitors

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Many therapeutically useful antibiotics owe their action to inhibition of some step in the
complex process of protein synthesis. Their attack is always at one of the events occurring on
the ribosome and never at the stage of amino acid activation or attachment to a particular
tRNA. Most have an affinity or specificity for 70S (as opposed to 80S) ribosomes, and they
achieve their selective toxicity in this manner. The most important antibiotics with this mode
of action are the tetracyclines, chloramphenicol, the macrolides (e.g. erythromycin) and
the aminoglycosides (e.g. streptomycin).

The aminoglycosides are products of Streptomyces species and are represented by

streptomycin, kanamycin, tobramycin and gentamicin. These antibiotics exert their activity
by binding to bacterial ribosomes and preventing the initiation of protein synthesis.

Streptomycin binds to 30S subunit of the bacterial ribosome, specifically to the S12 protein
which is involved in the initiation of protein synthesis. Experimentally, streptomycin has
been shown to prevent the initiation of protein synthesis by blocking the binding of initiator
N-formylmethionine tRNA to the ribosome. It also prevents the normal dissociation of
ribosomes into their subunits, leaving them mainly in their 70S form and preventing the
formation of polysomes. The overall effect of streptomycin seems to be one of distorting the
ribosome so that it no longer can carry out its normal functions. This evidently accounts for
its antibacterial activity but does not explain its bactericidal effects, which distinguishes
streptomycin and other aminoglycosides from most other protein synthesis inhibitors.

Kanamycin and tobramycin have been reported to bind to the ribosomal 30S subunit and to
prevent it from joining to the 50S subunit during protein synthesis. They may have a
bactericidal effect because this leads to cytoplasmic accumulation of dissociated 30S
subunits, which is apparently lethal to the cells.

Aminoglycosides have been used against a wide variety of bacterial infections caused by
Gram-positive and Gram-negative bacteria. Streptomycin has been used extensively as a
primary drug in the treatment of tuberculosis. Gentamicin (a mixture of 3 components) is
active against many strains of Gram-positive and Gram-negative bacteria, including some
strains of Pseudomonas aeruginosa. Kanamycin (a complex of three antibiotics, A, B and C)
is active at low concentrations against many Gram-positive bacteria, including penicillin-
resistant staphylococci. Gentamicin and Tobramycin are mainstays for treatment of
Pseudomonas infections. An unfortunate side effect of aminoglycosides has tended to restrict
their usage: prolonged use is known to impair kidney function and cause damage to the
auditory nerves leading to deafness.

The tetracyclines consist of eight related antibiotics which are all natural products of
Streptomyces, although some can now be produced semisynthetically or synthetically.
Tetracycline, chlortetracycline and doxycycline are the best known. The tetracyclines are
broad-spectrum antibiotics with a wide range of activity against both Gram-positive and
Gram-negative bacteria. Pseudomonas aeruginosa is less sensitive but is generally
susceptible to tetracycline concentrations that are obtainable in the bladder. The tetracyclines
act by blocking the binding of aminoacyl tRNA to the A site on the ribosome. Tetracyclines
inhibit protein synthesis on isolated 70S or 80S (eukaryotic) ribosomes, and in both cases,

106
their effect is on the small ribosomal subunit. However, most bacteria possess an active
transport system for tetracycline that will allow intracellular accumulation of the antibiotic at
concentrations 50 times as great as that in the medium. This greatly enhances its antibacterial
effectiveness and accounts for its specificity of action, since an effective concentration
cannot be accumulated in animal cells. Thus a blood level of tetracycline which is harmless
to animal tissues can halt protein synthesis in invading bacteria.
The tetracyclines have a remarkably low toxicity and minimal side effects when taken by
animals. The combination of their broad spectrum and low toxicity has led to their overuse
and misuse by the medical community and the wide-spread development of resistance has
reduced their effectiveness. Nonetheless, tetracyclines still have some important uses, such as
the use of doxycycline in the treatment of Lyme disease.
Some newly discovered members of the tetracycline family (e.g. chelocardin) have been
shown to act by inserting into the bacterial membrane, not by inhibiting protein synthesis.

Chloramphenicol is a protein synthesis inhibitor has a broad spectrum of activity but it

exerts a bacteriostatic effect. It is effective against intracellular parasites such as the
rickettsiae. Unfortunately, aplastic anemia, which is dose-related develops in a small
proportion (1/50,000) of patients. Chloramphenicol was originally discovered and purified
from the fermentation of a Streptomyces, but currently it is produced entirely by chemical
synthesis. Chloramphenicol inhibits the bacterial enzyme peptidyl transferase, thereby
preventing the growth of the polypeptide chain during protein synthesis.
Chloramphenicol is entirely selective for 70S ribosomes and does not affect 80S ribosomes.
Its unfortunate toxicity towards the small proportion of patients who receive it is in no way
related to its effect on bacterial protein synthesis. However, since mitochondria probably
originated from procaryotic cells and have 70S ribosomes, they are subject to inhibition by
some of the protein synthesis inhibitors including chloroamphenicol. This likely explains the
toxicity of chloramphenicol. The eukaryotic cells most likely to be inhibited by
chloramphenicol are those undergoing rapid multiplication, thereby rapidly synthesizing
mitochondria. Such cells include the blood forming cells of the bone marrow, the inhibition
of which could present as aplastic anemia. Chloramphenicol was once a highly prescribed
antibiotic and a number of deaths from anemia occurred before its use was curtailed. Now it
is seldom used in human medicine except in life-threatening situations (e.g. typhoid fever).
The macrolide family of antibiotics is characterized by structures that contain large lactone
rings linked through glycoside bonds with amino sugars. The most important members of the
group are erythromycin and oleandomycin. Erythromycin is active against most Gram-
positive bacteria, Neisseria, Legionella and Haemophilus, but not against the
Enterobacteriaceae. Macrolides inhibit bacterial protein synthesis by binding to the 50S
ribosomal subunit. Binding inhibits elongation of the protein by peptidyl transferase or
prevents translocation of the ribosome or both. Macrolides are bacteriostatic for most
bacteria but are cidal for a few Gram-positive bacteria.

Lincomycin and clindamycin are a miscellaneous group of protein synthesis inhibitors with
activity similar to the macrolides. Lincomycin has activity against Gram-positive bacteria
and some Gram-negative bacteria (Neisseria, H. influenzae). Clindamycin is a derivative of
lincomycin, with the same range of antimicrobial activity, but it is considered more effective.

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It is frequently used as a penicillin substitute and is effective against Gram-negative
anaerobes (e.g. Bacteroides).

8.4.1.4 Effects on Nucleic Acids

Some antibiotics and chemotherapeutic agents affect the synthesis of DNA or RNA, or can
bind to DNA or RNA so that their messages cannot be read. Either case, of course, can block
the growth of cells. The majority of these drugs are unselective, however, and affect animal
cells and bacterial cells alike and therefore have no therapeutic application. Two nucleic acid
synthesis inhibitors which have selective activity against procaryotes and some medical
utility are the quinolones and rifamycins.

Nalidixic acid is a synthetic chemotherapeutic agent which has activity mainly against
Gram-negative bacteria. Nalidixic acid belongs to a group of compounds called quinolones.
Nalidixic acid is a bactericidal agent that binds to the DNA gyrase enzyme (topoisomerase)
which is essential for DNA replication and allows supercoils to be relaxed and reformed.
Binding of the drug inhibits DNA gyrase activity.
Some quinolones penetrate macrophages and neutrophils better than most antibiotics and are
thus useful in treatment of infections caused by intracellular parasites. However, the main use
of nalidixic acid is in treatment of lower urinary tract infections (UTI). The compound is
unusual in that it is effective against several types of Gram-negative bacteria such as E. coli,
Enterobacter aerogenes, K. pneumoniae and Proteus species which are common causes of
UTI. It is not usually effective against Pseudomonas aeruginosa, and Gram-positive bacteria
are resistant.

Some quinolones have a broadened spectrum against Gram-positive bacteria. The

fluoroquinolone, Cipro. (ciprofloxacin) was recently touted as the drug of choice for
treatment and prophylaxis of anthrax, which is caused by a Gram-positive bacillus.

The rifamycins are a comparatively new group of antibiotics, also the products of
Streptomyces. Rifampicin  is a semisynthetic derivative of rifamycin that is active against
Gram-positive bacteria (including Mycobacterium tuberculosis) and some Gram-negative
bacteria. Rifampicin acts quite specifically on the bacterial RNA polymerase and is inactive
towards DNA polymerase or RNA polymerase from animal cells. The antibiotic binds to the
beta subunit of the polymerase and apparently blocks the entry of the first nucleotide which
is necessary to activate the polymerase, thereby blocking mRNA synthesis. It has been found
to have greater bactericidal effect against M .tuberculosis than other anti-tuberculosis drugs,
and it has largely replaced isoniazid as one of the front-line drugs used to treat the disease,
especially when isoniazid resistance is indicated. It is effective orally and penetrates the
cerebrospinal fluid so it is useful for treatment of bacterial meningitis.

8.4.1.5 Competitive Inhibitors

Many of the synthetic chemotherapeutic agents are competitive inhibitors of essential
metabolites or growth factors that are needed in bacterial metabolism.  Hence, these types of
antimicrobial agents are sometimes referred to as anti-metabolites or growth factor analogs,
since they are designed to specifically inhibit an essential metabolic pathway in the bacterial
pathogen. At a chemical level, competitive inhibitors are structurally similar to a bacterial

108
growth factor or metabolite, but they do not fulfill their metabolic function in the cell. Some
are bacteriostatic and some are bactericidal. Their selective toxicity is based on the premise
that the bacterial pathway does not occur in the host.
Sulfonamides were introduced as chemotherapeutic agents by Domagk in 1935, who showed
that one of these compounds (prontosil) had the effect of curing mice with infections caused
by beta-hemolytic streptococci. Chemical modifications of the compound sulfanilamide gave
compounds with even higher and broader antibacterial activity. The resulting sulfonamides 
have broadly similar antibacterial activity, but differ widely in their pharmacological actions.
Bacteria which are almost always sensitive to the sulfonamides include Streptococcus
pneumoniae, beta-hemolytic streptococci and E. coli. The sulfonamides have been extremely
useful in the treatment of uncomplicated UTI caused by E. coli, and in the treatment of
meningococcal meningitis (because they cross the blood-brain barrier).
The sulfonamides (e.g. Gantrisin) and Trimethoprim are inhibitors of the bacterial enzymes
required for the synthesis of tetrahydofolic acid (THF), the vitamin form of folic acid
essential for 1-carbon transfer reactions. Sulfonamides are structurally similar to para
aminobenzoic acid (PABA), the substrate for the first enzyme in the THF pathway, and they
competitively inhibit that step. Trimethoprim is structurally similar to dihydrofolate (DHF)
and competitively inhibits the second step in THF synthesis mediated by the DHF reductase.
Animal cells do not synthesize their own folic acid but obtain it in a preformed fashion as a
vitamin. Since animals do not make folic acid, they are not affected by these drugs, which
achieve their selective toxicity for bacteria on this basis.
Three additional synthetic chemotherapeutic agents have been used in the treatment of
tuberculosis:  (INH), paraaminosalicylic acid (PAS), and ethambutol. The usual strategy in
the treatment of tuberculosis has been to administer a single antibiotic (historically
streptomycin, but now, most commonly, rifampicin is given) in conjunction with INH and
ethambutol. Since the tubercle bacillus rapidly develops resistance to the antibiotic,
ethambutol and INH are given to prevent outgrowth of a resistant strain. It must also be
pointed out that the tubercle bacillus rapidly develops resistance to ethambutol and INH if
either drug is used alone. Ethambutol inhibits incorporation of mycolic acids into the
mycobacterial cell wall. Isoniazid has been reported to inhibit mycolic acid synthesis in
mycobacteria and since it is an analog of pyridoxine (Vitamin B6) it may inhibit pyridoxine-
catalyzed reactions as well. Isoniazid is activated by a mycobacterial peroxidase enzyme and
destroys several targets in the cell. PAS is an anti-folate, similar in activity to the
sulfonamides. PAS was once a primary anti-tuberculosis drug, but now it is a secondary
agent, having been largely replaced by ethambutol.

8.4.3 Antifungal agents

Fungal infections are caused by eukaryotic organisms and for that reason they generally
present more difficult therapeutic problems than do bacterial infections. There are relatively
few agents that can be used to treat fungal infections. The fungal cell wall may be considered
to be a prime target for selectively toxic antifungal agents because of its chitin structure,
absent from human cells. No clinically available inhibitor of chitin synthesis analogous to the
b-lactams exists at present, even though much effort is being directed towards developing
such agents. Other targets are currently being exploited.

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8.4.2.1 Polyene antibiotics
Polyene antibiotics bind to sterols within the fungal membrane, disrupting its integrity. This
makes the membrane leaky, leading to a loss of small molecules from the fungal cell.
Polyene antibiotics include nystatin, used topically for candida infections and amphotericin
B.

Chemical structure of nystatin

Chemical structure of amphotericin B

The antifungal drug Amphotericin B is administered parenterally and is widely used to treat
systemic mycoses. It is most often given intravenously in a bile salt suspension and diluted
with 5% dextrose. It penetrates poorly into cerebrospinal fluid and when used to treat
meningitis it may be delivered directly into the brain ventricles.
Amphotericin B is a very successful and widely used antifungal drug but its use is beset with
problems of toxicity. It can cause unpleasant side effects including chills, fever and a
lowering of blood pressure. It may also cause kidney damage. The side effects of
amphotericin B therapy can mimic the clinical appearance of serious systemic infection,
complicating patient management. The severity of side effects may cause interruption of
antifungal infection.
Amphotericin B remains the drug of choice for life-threatening fungal infections. It may
often be administered together with flucytosine since in combination a lower dose may be
used, reducing the risk of therapeutic complications.

8.4.2.2 The imidazoles and triazoles

These are an emerging group of antifungal agents that act to inhibit synthesis of ergosterol, a
component of fungal membranes. These drugs, like the polyene antibiotics, may cause
leakage of small molecules out of fungal cells. Drugs in this class include miconazole and

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ketoconazole. They have a broad-spectrum of antifungal activity although there is some
variation of activity between the various compounds. Some azoles are also active against
Gram-positive bacteria.

Chemical structure of myconazole

Chemical structure of ketoconazole

Azoles are known to inhibit the fungal cytochrome P450 enzyme. Some members of the
azole group can also affect the human equivalent and are thus toxic to humans. The majority
of azoles can only be used topically but some are used to treat systemic infections. Some may
be taken orally whereas others must be delivered parenterally. Ketoconazole is administered
orally; miconazole is given intravenously.
The azoles fluconazole and itraconazole may be delivered either orally or parenterally. These
drugs are being evaluated in the treatment of systemic mycoses and have met with variable
success. Fluconazole is excreted through the kidneys. It may accumulate within this tissue,
leading to renal damage. Resistance to fluconazole has emerged during therapy. Candida
glabrata quickly becomes resistant to this drug. Certain other species, such as Candida
krusei, are not sensitive to this compound.

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 Chemical structure of fluconazole

Chemical structure of itraconazole

Terbinafine is a synthetic antifungal agent introduced into the UK in 1991 and is used to treat
skin and nail infections. It inhibits ergosterol biosynthesis.

Chemical structure of terbinafine

8.4.2.3 Griseofulvin
The drug griseofulvin is a naturally occurring compound and so is a true antibiotic with
antifungal properties. It binds to the proteins involved in microtubule formation and prevents
separation of chromosomes at mitosis. Why griseofulvin does not affect human cells is not
known. It is used in the treatment of ringworm and other fungal infections of the skin or
nails.

Chemical structure of griseofulvin

8.4.2.4 5-flucytosine
The synthetic pyrimidine 5-flucytosine interferes with the nucleic acid metabolism in fungi.
Because it is an analogue of naturally occurring nucleotide, it is actively taken up by cells
where it is metabolised to 5-fluorouracil. It is a drug used primarily to treat systemic candida
infections and it may be given orally or parenterally. It can be used as a single agent or in
combination with other antifungal drugs including amphotericin B. 5-flucytosine achieves
good penetration into body fluids, including cerebrospinal fluid. It is excreted through the
kidneys and its dosage must be modified in patients with renal problems. It derives its
selective toxicity from the inability of human cells to convert it to 5-fluorouracil. Its major
drawback is the ease with which resistance develops. Susceptibility testing of fungal isolates

112
is necessary during treatment so that resistant variants may be detected early and alternative
therapy may be initiated.

Chemical structure of 5-flucytosine

8.4.3 Antiviral agents

Because of the nature of viruses as intracellular parasites, very few clinical useful agents to
treat virus infections have been produced. Development of drugs that are active against
viruses is one of the most challenging areas in antimicrobial chemotherapy.

Acyclovir
The purine acyclovir, or acycloguanosine, inhibits the thymidine kinase of herpes viruses. It
is particularly active against herpes simplex virus types 1 and 2, is less active against
varicella zoster virus and has significantly reduced activity against cytomegalovirus. It has
prevented significant mortality associated with herpes simplex encephalitis and is of value in
treating varicella zoster infections in immunocompromised patients.
Acyclovir is also used for the treatment of severe cases of genital herpes. Resistance to
acyclovir because of mutations in the thymidine kinase gene limits the use of this drug for
more trivial conditions. It does not eradicate latent virus but acyclovir does shorten the
duration of clinical symptoms. Gancyclovir is an analogue of acyclovir that is active against
cytomegalovirus as well as other herpes viruses.

Chemical structure of acyclovir

Chemical structure of ganclovir

Amantidine

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This agent is used to treat shingles, particularly in elderly or debilitated patients. It is also
used for the treatment of influenza A virus infections. Its mode of action has not been fully
elucidated but it is thought to interfere with the uncoating of the virus. During epidemics of
influenza A virus infection, it may be used as a prophylactic agent to prevent disease in the
most vulnerable patients.

Chemical structure of amantidine

Ribavirin
This is a broad-spectrum drug active against both RNA and DNA viruses. In the Developed
World, its principal use is in treating severe respiratory syncytial virus infections in infants.
The virus infects the bronchioles, causing bronchiolitis and the drug may be inhaled. Its
mode of action is unclear.

Chemical structure of ribavirin

Zidovudine
This was formerly called azidothymidine and is also known as AZT. It is an anti-retrovirus
agent that is phosphorylated to form a triphosphate derivative inside infected and uninfected
calls. Zidovudine triphosphate is a competitive inhibitor of the retrovirus reverse
transcriptase, the enzyme that produces provirus DNA from the virus RNA template. Its
principal use is in the management of patients with advanced human immunodeficiency virus
disease (AIDS).

Chemical structure of zidovudine

Interferon

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This is probably the most known antiviral agent. These small proteins, produced by the host
in response to viral infection, inhibit virus replication and may be clinically useful in the
treatment of influenza, hepatitis, herpes, and colds.
8.4.4 Antiprotozoan and antihelmintic agents
A wide variety of antibacterial agents are available for the treatment of infectious diseases.
However, because the metabolism of parasitic eukaryotes is very similar to that of humans,
only a few a protozoal drugs have been effective in clinical practice. Although many
protozoa are free living organisms, a few are parasitic in humans. The parasite that causes
malaria invades red blood cells and causes the patient to suffer alternating fever and chills.
Other parasites cause intestinal or urinary tract infections. Several antiprotozoan agents have
been found that are successful in controlling or even curing most protozoan infection, but
some have rather unpleasant effects. Among these antiprotozoan agents, we have: quinine
(from cinchona tree), chloroguine and primaquine. A new prophylactic agent, mefoquine
proved effective against resistant strains of protozoa. The synthetic imidazole metronidazole
is effective in treating Trichomonas infections and intestinal infections caused by parasitic
amoebas and Giardia. Metronidazole does not prevent overgrowth of Candida yeast strains.
Various helnithes can infect humans and animals. A variety of antihelmintic agents are
available to help rid the body of those unwelcome parasites. These agents include
niclosamide, mebendazole, piperazine, and ivermectin.
Niclosamide interferes with carbohydrate metabolism, thereby a parasite to release a large
quantity of lactic agent. This drug may also inactivate products made by the worm to resist
digestion by host proteolytic enzymes. It is effective mainly in treatment of tape worm
infections.
Mebendazole (Vermox) block uptake of glucose by parasitic roundworms. It is useful in
treating shipworm, pinworm, and hookworm infections. Piperazine is a powerful neurotoxin
that paralyzes body and wall muscles of roundworms and is useful in treating Ascaris and
pinworm infections. Ivermectin is effective against Oncocerca volvulus, in human infections
with this roundworm causing a progressive loss of sight known as onchocerciasis, or river
blindness.

8.5 Microorganisms that Produce Antibiotics

The molds, Penicillium and Cephalosporium, produce Beta-lactam antibiotics, i.e., penicillin,
cephalosporin, and their relatives. Actinomycetes, mainly Streptomyces species: produce
tetracyclines, aminoglycosides (streptomycin and its relatives), macrolides (erythromycin and
its relatives), chloramphenicol, ivermectin, rifamycins, and most other clinically-useful
antibiotics that are not beta-lactams. Bacillus species, such as B. polymyxa and Bacillus
subtilis produce polypeptide antibiotics (e.g. polymyxin and bacitracin). These organisms all
have in common that they live in a soil habitat and they form some sort of a spore or resting
structure. It is not known why these microorganisms produce antibiotics. It may rest in the
obvious, i.e., the antibiotics afford the microbes some nutritional advantage in their habitat
by antagonism against the competition. However, it may rest on the subtle: i.e., the
antibiotics act as some sort of hormone or signal molecule associated with sporulation or
dormancy or germination.
Antibiotics are secondary metabolites of microorganisms and they are produced at the same
time that the cells begin sporulation processes. Antibiotics tend to be rather large,
complicated, organic molecules and may require as many as 30 separate enzymatic steps to

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synthesize. The maintenance of a substantial component of the bacterial genome devoted
solely to the synthesis of an antibiotic leads one to the conclusion that the process (or
molecule) is important, if not essential, to the survival of these organisms in their natural
habitat. Most of the microorganisms that produce antibiotics are resistant to the action of
their own antibiotic, although the organisms are affected by other antibiotics, and their
antibiotic may be effective against closely-related strains. Generally speaking, how or why
bacteria are resistant to their own antibiotics is also unknown, but the mechanisms may be
similar to resistance that develops in medically-important bacteria.

8.6 Microbial Resistance to Antibiotics

The first antibiotic, penicillin, was discovered in 1929 by Sir Alexander Fleming who
observed inhibition of staphylococci on an agar plate contaminated by a Penicillium mold.
World War II (and the inevitable bacterial infections that occurred in war-related wounds)
was an important impetus to study the chemotherapeutic value of penicillin. Penicillin
became generally available for treatment of bacterial infections, especially those caused by
staphylococci and streptococci, about 1946. Initially, the antibiotic was effective against all
sorts of infections caused by these two Gram-positive bacteria. It is important to note that a
significant fraction of all human infections are caused by these two bacteria (i.e., strep throat,
pneumonia, septicemia, skin infections, wound infections, scarlet fever, toxic shock
syndrome). Penicillin had unbelievable ability to kill these bacterial pathogens without
harming the host that harbored them.
Resistance to penicillin in some strains of staphylococci was recognized almost immediately
after introduction of the drug. Resistance to penicillin today occurs in as many as 80% of all
strains of Staphylococcus aureus and some strains of S. aureus have been isolated that are
resistant to virtually all clinically-available antibiotics. Surprisingly, Streptococcus pyogenes
(Group A strep) has never fully developed resistance to penicillin, and it remains a
reasonable choice antibiotic for many types of streptococcal infections. Interestingly,
penicillin has never been effective against most Gram-negative pathogens (e.g. Salmonella,
Shigella, Bordetella pertussis, Yersinia pestis, Pseudomonas) with the notable exception of
Neisseria gonorrhoeae. Gram-negative bacteria are inherently resistant to penicillin because
their vulnerable cell wall is protected by an outer membrane that prevents permeation of the
penicillin molecule.
The period of the late 1940s and early 1950s saw the discovery and introduction of
streptomycin, chloramphenicol, and tetracycline, and the age of antibiotic chemotherapy
came into full being. These antibiotics were effective against the full array of bacterial
pathogens including Gram-positive and Gram-negative bacteria, intracellular parasites, and
the tuberculosis bacillus. However, by 1953, during a Shigella outbreak in Japan, a strain of
the dysentery bacillus was isolated which was multiple drug resistant, exhibiting resistance to
chloramphenicol, tetracycline, streptomycin, and the sulfanilamides. There was also evidence
mounting that bacteria could pass genes for multiple drug resistance between strains and
even between species. It was also apparent that Mycobacterium tuberculosis was capable of
rapid development of resistance to streptomycin which had become a mainstay in
tuberculosis therapy. Today, drug-resistant strains of M. tuberculosis are threatening to break
through in one of the world's most prevalent infectious diseases.

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8.7 The Genetic Basis of Bacterial Resistance to Antibiotics
Inherent (Natural) Resistance. Bacteria may be inherently resistant to an antibiotic. For
example, a streptomycete has some gene that is responsible for resistance to its own
antibiotic; or a Gram-negative bacterium has an outer membrane that establishes a
permeability barrier against the antibiotic; or an organism lacks a transport system for the
antibiotic; or it lacks the target or reaction that is hit by the antibiotic.
Acquired Resistance. Bacteria can develop resistance to antibiotics, e.g. bacterial populations
previously-sensitive to antibiotics become resistant. This type of resistance results from
changes in the bacterial genome. Acquired resistance is driven by two genetic processes in
bacteria: (1) mutation and selection (sometimes referred to as vertical evolution); (2)
exchange of genes between strains and species (sometimes called horizontal evolution).
Vertical evolution: is strictly a matter of Darwinian evolution driven by principles of natural
selection: a spontaneous mutation in the bacterial chromosome imparts resistance to a
member of the bacterial population. In the selective environment of the antibiotic, the wild
type (non mutants) are killed and the resistant mutant is allowed to grow and flourish. The
mutation rate for most bacterial genes is approximately 10 -8. This means that if a bacterial
population doubles from 108 cells to 2 x 108 cells, there is likely to be a mutant present for
any given gene. Since bacteria grow to reach population densities far in excess of 10 9 cells,
such a mutant could develop from a single generation during 15 minutes of growth.
Horizontal evolution is the acquisition of genes for resistance from another organism. For
example, a streptomycete has a gene for resistance to streptomycin (its own antibiotic), but
somehow that gene escapes and gets into E. coli or Shigella. Or, more likely, a bacterium like
E. coli develops genetic resistance through the process of mutation and selection and then
donates these genes to some other bacterium through one of several processes for genetic
exchange that exist in bacteria (below).
Bacteria are able to exchange genes in nature by three processes: conjugation, transduction
and transformation. Conjugation involves cell-to-cell contact as DNA crosses a sex pilus
from donor to recipient. During transduction, a virus transfers the genes between mating
bacteria. In transformation, DNA is acquired directly from the environment, having been
released from another cell. Genetic recombination can follow the transfer of DNA from one
cell to another leading to the emergence of a new genotype (recombinant). It is common for
DNA to be transferred as plasmids between mating bacteria. Since bacteria usually develop
their genes for drug resistance on plasmids (called resistance transfer factors, or RTFs), they
are able to spread drug resistance to other strains and species during genetic exchange
processes.
The combined effects of fast growth rates, high concentrations of cells, genetic processes of
mutation and selection, and the ability to exchange genes, account for the extraordinary rates
of adaptation and evolution that can be observed in the bacteria. For these reasons bacterial
adaptation (resistance) to the antibiotic environment seems to take place very rapidly in
evolutionary time. Bacteria evolve fast!

8.8 The Medical Problem of Bacterial Resistance

Obviously, if a bacterial pathogen is able to develop or acquire resistance to an antibiotic,
then that substance becomes useless in the treatment of infectious disease caused by that
pathogen (unless the resistance can somehow be overcome with secondary measures). So as
pathogens develop resistance, humanity must find new (different) antibiotics to fill the place

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of the old ones in treatment regimes. Hence, natural penicillins have become useless against
staphylococci and must be replaced by other antibiotics; tetracycline, having been so widely
used and misused for decades, has become worthless for many of the infections that once
designated it as a "wonder drug".
Not only is there a problem in finding new antibiotics to fight old diseases (because resistant
strains of bacteria have emerged), there is a parallel problem to find new antibiotics to fight
new diseases. In the past two decades, many "new" bacterial diseases have been discovered
(Legionnaire's disease, gastric ulcers, Lyme disease, toxic shock syndrome, "skin-eating"
streptococci). Broad patterns of resistance exist in these pathogens, and it seems likely that
new antibiotics will soon be needed to replace the handful that are effective now against
these bacteria, especially as resistance begins to emerge among them in the selective
environment antibiotic chemotherapy.
It is said that the discovery and use of antibiotics and immunization procedures against
infectious disease are two developments in the field of microbiology that have contributed
about twenty years to the average life span of humans in developed countries where these
practices are employed. While the greater part of this span in time is probably due to
vaccination, most of us are either still alive or have family members who are still alive
because an antibiotic conquered an infectious disease that otherwise would have killed the
individual. If we want to retain this medical luxury in our society, we must be vigilant and
proactive: we must fully understand how and why antimicrobial agents work, and why they
don't work, and realize that we must maintain a stride ahead of microbial pathogens that can
only be contained by antibiotic chemotherapy.

8.9 Laboratory studies on antibiotic action

In the laboratory, susceptibility is most often measured using a disk diffusion test. Antibiotic
solutions of particular concentrations are dried onto filter paper disks. These are then applied
to a lawn of the microbe under examination which has previously been inoculated onto an
appropriate solid medium. In the Stokes controlled sensitivity test, a control organism is
inoculated on part of a plate and the test organism is plated on the remainder. Disks are
placed at the interface and the zones of inhibition are compared. The use of a sensitive
control shows that the antibiotic is active, so that if the test organism grows up to the disk it
may safely be assumed that the test organism is resistant to that drug.

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 Stokes' sensitivity test

The bacterium in the diagram is susceptible to drug "x" but resistant to drug "y". The disc
containing drug "y" contains active antibiotic as shown by the zone of inhibition it causes in
the control bacterium. An alternative measure of susceptibility is to determine the Minimum
Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC)
of a drug. A series of broths are mixed with serially diluted antibiotic solutions and a
standard inoculum is applied. After incubation, the MIC is the first broth in which growth of
the organism has been inhibited. The more resistant an organism is, then the higher will be
the MIC.

The MBC is measured by inoculating the broths used for MIC determinations onto drug-free
medium. The MBC is the first dilution at which no growth is observed. Cidal drugs have
MBC values that are close to the MIC value for particular organisms. With static agents,
the MIC is much lower than the MBC.

The MIC/MBC test of a moderately resistant bacteriostatic drug.

Note that once the bacteria are removed from the drug they can grow on drug free medium at
most concentrations.

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 The MIC/MBC test of a moderately resistant bactericidal drug.
Note that once the bacteria are removed from the drug they cannot grow on drug free
medium apart from the tube representing the MIC of the antibiotic.
One tube difference is allowed in this test.

Most antibiotics are given as single agents. There are, however, occasions when two or more
drugs are used in combination. Antimicrobial agents may affect each other when used in
combination. The effect may simply be additive. In some cases the activity of one drug
enhances that of a second drug. This is referred to as synergy. Alternatively, drugs may
interfere with each other - antagonism. Penicillins and bacteriostatic drugs such as
tetracyclines are antagonistic, since penicillins require actively growing cells and static drugs
prevent cell growth. In contrast, aminoglycosides are synergistic when used in combination
with penicillins. This is important when considering antimicrobial therapy, for example for
endocarditis. In this condition, it is essential that the antimicrobial regime is bactericidal,
since the bacteria become walled off inside vegetations. Synergistic combinations are
typically used to treat this condition.

The effects of combining antimicrobial agents

Although, as illustrated above, laboratory tests in vitro can test for synergy or antagonism
this effect is not necessarily apparent when combinations are used in vivo. Sulphonamides
and trimethorpim both act on folic acid metabolism and show synergistic activity against
bacteria in vitro it is difficult to achieve a synergistic ratio of these drugs in humans.

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 CHAPTER IX

VIROLOGY

CLASSIFICATION OF VIRUSES

The overall purpose of this Learning Object is to introduce how viruses are
classified by the type of nucleic acid they have, the shape of their capsid, and
whether or not they are enveloped.

Viruses are obligate intracellular infectious agents (Parasites) with both living and
nonliving characteristics.

The vast majority of viruses possess either DNA or RNA but not both.

Classification of Viruses

Viruses can store their genetic information in six different types of nucleic acid which
are named based on how that nucleic acid eventually becomes transcribed to the
viral mRNA capable of binding to host cell ribosomes and being translated into viral
proteins.

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In the diagrams below, (+) and (-) represent complementary strands of nucleic acid.
Copying of a (+) strand by complementary base pairing forms a (-) strand. Only a (+)
viral mRNA strand can be translated into viral protein. Regarding the enzymes
involved, the "dependent" part of the name tells what type of nucleic acid is being
copied. The "polymerase" part of the name tells what type of nucleic acid is being
synthesized, eg, DNA-dependent RNA-polymerase would synthesize a strand of
RNA complementary to a strand of DNA. These six forms of viral nucleic acid are:

a. (+/-) double-stranded DNA . To replicate the viral genome, DNA-dependent

DNA polymerase enzymes copy both the (+) and (-) DNA strands producing
dsDNA viral genomes. To produce viral mRNA molecules. DNA-dependent
RNA polymerase enzymes copy the (-) DNA strand into (+) viral mRNA. The
(+) viral mRNA can then be transtated into viral proteins by host cell
ribosomes. Examples include most bacteriophages, Papovaviruses,
Adenoviruses, and Herpesviruses.

b. (+) single-stranded DNA . To replicate the viral genome, DNA-dependent DNA

polymerase enzymes copy the (+) DNA strand of the genome producing a dsDNA
intermediate. DNA-dependent DNA polymerase enzymes then copy the (-) DNA
strand into ss (+) DNA genomes. To produce viral mRNA molecules. DNA-

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dependent RNA polymerase enzymes copy the (-) DNA strand into (+) viral mRNA.
The (+) viral mRNA can then be transtated into viral proteins by host cell ribosomes.
Examples include Phage M13 and Parvoviruses.

c. (+/-) double-stranded RNA . To replicate the viral genome, RNA-dependent RNA

polymerase enzymes copy both the (+) RNA and (-) RNA strands of the genome
producing a dsRNA genomes. To produce viral mRNA molecules. RNA-dependent
RNA polymerase enzymes copy the (-) RNA strand into (+) viral mRNA. The (+) viral
mRNA can then be transtated into viral proteins by host cell ribosomes. Reoviruses
are an example.

d. (-) RNA To replicate the viral genome, RNA-dependent RNA polymerase

enzymes copy the (-) RNA genome producing ss (+) RNA. RNA-dependent RNA
polymerase enzymes then copy the (+) RNA strands producing ss (-) RNA viral
genome. The (+) mRNA strands also function as viral mRNA and can then be
transtated into viral proteins by host cell ribosomes. Examples include
Orthomyxoviruses, Paramyxoviruses, Rhabdoviruses.

e. (+) RNA. To replicate the viral genome, RNA-dependent RNA polymerase

enzymes copy the (+) RNA genome producing ss (-) RNA. RNA-dependent RNA
polymerase enzymes then copy the (-) RNA strands producing ss (+) RNA viral
genome. To produce viral mRNA molecules. RNA-dependent RNA polymerase
enzymes copy the (-) RNA strand into (+) viral mRNA. The (+) viral mRNA can then
be transtated into viral proteins by host cell ribosomes. Examples include
Picornaviruses, Togaviruses, and Coronaviruses.

f. (+) RNA Retroviruses . To replicate the viral genome, reverse transcriptase

enzymes (RNA-dependent DNA polymerases) copy the (+) RNA genome producing
ss (-) DNA strands. DNA-dependent DNA polymerase enzymes then copy the (-)
DNA strands to produce a dsDNA intermediate. DNA-dependent RNA polymerase
enzymes then copy the (-) DNA strands to produce ss (+) RNA genomes. To
produce viral mRNA molecules. DNA-dependent RNA polymerase enzymes copy
the (-) DNA strand into (+) viral mRNA. The (+) viral mRNA can then be transtated
into viral proteins by host cell ribosomes. Retroviruses, such as HIV-1, HIV-2, and
HTLV-1 are examples.

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The Table below describes some of the medically important viruses.

Classification of Viruses

1. single-stranded DNA; naked; polyhedral capsid

 Viral family: Parvoviridae

 Size: 18-25nm
 Examples and diseases: parvoviruses (roseola, fetal death, gastroenteritis; some depend
on coinfection with adenoviruses)

2. double-stranded, DNA; naked; polyhedral capsid

 Viral family: Papovaviridae; circular dsDNA
 Size: 40-57nm
 Examples and diseases: human papilloma viruses (HPV; benign warts and genital warts;
genital and rectal cancers)
 Viral family: Adenoviridae; dsDNA
 Size: 70-90nm
 Examples and diseases: adenoviruses (respiratory infections, gastroenteritis, infectious
pinkeye, rashes, meningoencephalitis)

3. double-stranded, circular DNA; enveloped; complex

 Viral family: Poxviridae
 Size: 200-350nm
 Examples and diseases: smallpox virus (smallpox), vaccinia virus (cowpox), molluscipox
virus (molluscum contagiosum-wartlike skin lesions)

4. double-stranded DNA; enveloped; polyhedral capsid

 Viral family: Herpesviridae
 Size: 150-200nm
 Examples and diseases: herpes simplex 1 virus (HSV-1; most oral herpes; herpes simplex
2 virus (HSV-2; most genital herpes), herpes simplex 6 virus (HSV-6; roseola), varicella-
zoster virus (VZV; chickenpox and shingles), Epstein-Barr virus (EBV; infectious
mononucleosis and lymphomas), cytomegalovirus (CMV; birth defects and infections of a
variety of body systems in immunosuppressed individuals)
 Viral family: Hepadnaviridae
 Size: 42nm
 Examples and diseases: hepatitis B virus (HBV; hepatitis B and liver cancer)

5. (+)single-stranded RNA; naked; polyhedral capsid

 Viral family: picornaviridae
 Size: 28-30nm
 Examples and diseases: enteroviruses (poliomyelitis), rhinoviruses (most frequent cause of
the common cold), Norwalk virus (gastroenteritis), echoviruses (meningitis), hepatitis A virus
(HAV; hepatitis A)

6. (+)single-stranded RNA; enveloped; usually a polyhedral capsid

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  Viral family: Togaviridae
 Size: 60-70nm
 Examples and diseases: arboviruses (eastern equine encephalitis, western equine
encephalitis), rubella virus (German measles)
 Viral family: Flaviviridae
 Size: 40-50nm
 Examples and diseases: flaviviruses (yellow fever, dengue fever, St. Louis encephalitis),
hepatitis C virus (HCV; hepatitis C)
 Viral family: Coronaviridae
 Size: 80-160nm
 Examples and diseases: coronaviruses (upper respiratory infections and the common cold;
SARS;

7. (-)single-stranded RNA; enveloped; pleomorphic

 Viral family: Rhabdoviridae; bullet-shaped
 Size: 70-189nm
 Examples and diseases: rabies virus (rabies)
 Viral family: Filoviridae; long and filamentous
 Size: 80-14,000nm
 Examples and diseases: Ebola virus, Marburg virus (hemorrhagic fevers)
 Viral family: Paramyxoviridae; pleomorphic
 Size: 150-300nm
 Examples and diseases: paramyxoviruses (parainfluenza, mumps); measles virus
(measles)

8. (-) strand; multiple strands of RNA; enveloped

 Viral family: Orthomyxoviridae
 Size: 80-200nm
 Examples and diseases: influenza viruses A, B, and C (influenza)
 Viral family: Bunyaviridae
 Size: 90-120nm
 Examples and diseases: California encephalitis virus (encephalitis); hantaviruses
(Hantavirus pulmonary syndrome, Korean hemorrhagic fever;
 Viral family: Arenaviridae
 Size: 50-300nm
 Examples and diseases: arenaviruses (lymphocytic choriomeningitis, hemorrhagic fevers)

9. produce DNA from (+) single-stranded RNA using reverse transcriptase; enveloped; bullet-
shaped or polyhedral capsid
 Viral family: Retroviridae
 Size: 100-120nm
 Examples and diseases: HIV-1 and HIV-2 (HIV infection/AIDS; HTLV-1 and HTLV-2 (T-cell
leukemia)

10. dsRNA; naked; polyhedral capsid

 Viral family: Reoviridae
 Size: 60-80nm
 Examples and diseases: reoviruses (mild respiratory infections, infant gastroenteritis);
Colorado tick fever virus (Colorado tick fever)

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CHARACTERISTICS OF VIRUSES

The overall purpose of this Learning Object is to introduce the general

characteristice of viruses and how they may be cultivated in the laboratory.

General Characteristics of Viruses

Viruses are infectious agents with both living and nonliving characteristics. They can
infect animals, plants, and even other microorganisms. Viruses that infect only
bacteria are called bacteriophages and those that infect only fungi are termed
mycophages .

1. Living characteristics of viruses

a. They reproduce at a fantastic rate, but only in living host cells.

b. They can mutate.

2. Nonliving characteristics of viruses

a. They are acellular, that is, they contain no cytoplasm or cellular organelles.

b. They carry out no metabolism on their own and must replicate using the
host cell's metabolic machinery. In other words, viruses don't grow and divide.
Instead, new viral components are synthesized and assembled within the infected
host cell.

c. The vast majority of viruses possess either DNA or RNA but not both.

3. Criteria used to define a virus

a. The vast majority of viruses contain only one type of nucleic acid: DNA or RNA,
but not both.

b. They are totally dependent on a host cell for replication. (They are strict
intracellular parasites.)

c. Viral components must assemble into complete viruses (virions) to go from one
host cell to another.

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4. Laboratory cultivation of viruses

Since viruses lack metabolic machinery of their own and are totally dependent on
their host cell for replication, they cannot be grown in synthetic culture media. Animal
viruses are normally grown in animals, embryonated eggs, or in cell cultures where
in animal host cells are grown in a synthetic medium and the viruses are then grown
in these cells.

Virus Structure

At the simplest level, the function of the outer shells (CAPSID) of a virus particle is to
protect the fragile nucleic acid genome from:
 Physical damage - Shearing by mechanical forces.
 Chemical damage- UV irradiation (from sunlight) leading to chemical
modification.
 Enzymatic damage - Nucleases derived from dead or leaky cells or deliberately
secreted by vertebrates as defence against infection.

The protein subunits in a virus capsid are multiply redundant, i.e. present in many copies
per particle. Damage to one or more subunits may render that particular subunit non-
functional, but does not destroy the infectivity of the whole particle.
Furthermore, the outer surface of the virus is responsible for recognition of the host cell.
Initially, this takes the form of binding of a specific virus-attachment protein to a
cellular receptor molecule. However, the capsid also has a role to play in initiating
infection by delivering the genome from its protective shell in a form in which it can
interact with the host cell.
To form an infectious particle, a virus must overcome two fundamental problems:
1. To assemble the particle utilizing only the information available from the components
which make up the particle itself (capsid + genome).
2. Virus particles form regular geometric shapes, even though the proteins from which
they are made are irregularly shaped.

How do these simple organisms solve these difficulties? The information to answer this
problem lie in the rules of symmetry.

In 1957, Fraenkel-Conrat & Williams showed that when mixtures of purified tobacco
mosaic virus (TMV) RNA & coat protein were incubated together, virus particles formed.
The discovery that virus particles could form spontaneously from purified subunits without

127
any extraneous information indicated that the particle was in the free energy minimum state
& was therefore the favoured structure of the components. This stability is an important
feature of the virus particle.
Although some viruses are very fragile & are essentially unable to survive outside the
protected host cell environment, many are able to persist for long periods, in some cases for
years.

Helical capsids

Tobacco mosaic virus (TMV) is representative of one of the two major structural classes
seen in viruses of all types, those with helical symmetry. The simplest way to arrange
multiple, identical protein subunits is to use rotational symmetry & to arrange the irregularly
shaped proteins around the circumference of a circle to form a disc.
Multiple discs can then be stacked on top of one another to form a cylinder, with the virus
genome coated by the protein shell or contained in the hollow centre of the cylinder.
Closer examination of the TMV particle by X-ray crystallography reveals that the structure
of the capsid actually consists of a helix rather than a pile of stacked disks.
A helix can be defined mathematically by two parameters:
 amplitude (diameter)   &
 pitch (the distance covered by each complete turn of the helix)

Helices are rather simple structures formed by stacking repeated components with a constant
relationship (amplitude & pitch) to one another - note that if this simple constraint is broken a
spiral forms rather than a helix - unsuitable for containing a virus genome.

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 TMV particles are rigid, rod-like structures, but some
helical viruses demonstrate considerable flexibility &
longer helical virus particles are often seen to be
curved or bent. Flexibility is important attribute since
long helical particles are subject to damage from shear
forces & the ability to bend reduces the chance of
breakage.

The fact that helical symmetry is a useful way of arranging a single protein subunit to form a
particle is confirmed by the large number of different types of virus which have evolved with
this capsid arrangement.

Icosahedral (isometric) capsids:

An alternative way of building a virus capsid is to arrange protein subunits in the form of a
hollow quasi-spherical structure, enclosing the genome within. The criteria for arranging
subunits on the surface of a solid are more complex than those for building a helix.
In the 1950s, Brenner & Horne (among others) developed
sophisticated techniques which enabled them to use electron
microscopy to reveal many of the fine details of the structure of virus
particles. One of the most useful techniques proved to be the use of
electron-dense dyes such as phosphotungstic acid or uranyl acetate to
examine virus particles by negative staining. The small metal ions in
such dyes are able to penetrate the minute crevices between the
protein subunits in a viral capsid to reveal the fine structure of the
particle.
Francis Crick & James Watson (1956), were the first to suggest that virus capsids are
composed of numerous identical protein sub-units arranged either in helical or cubic
(=icosahedral) symmetry.
In order to construct a capsid from repeated subunits, a virus must 'know the rules' which
dictate how these are arranged. For an icosahedron, the rules are based on the rotational
symmetry of the solid, which is known as 2-3-5 symmetry:
 An axis of two-fold rotational
symmetry through the centre of
each edge
 An axis of three-fold
rotational symmetry through
the centre of each face

 An axis of five-fold rotational

symmetry through the centre of

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 each corner (vertex)
To help you understand this further, you can build your own icosahedral virus particle.
The simplest icosahedral capsids are built up by using 3 identical subunits to form each
triangular face, thereby requiring 60 identical subunits to form a complete capsid. A few
simple virus particles are constructed in this way, e.g. bacteriophage ØX174:

In most cases, analysis reveals that icosahedral virus capsids contain more than 60 subunits,
for the reasons of genetic economy given above. The capsids of picornaviruses provide a
good illustration of the construction of icosahedral virus particles (e.g. polioviruses, foot-and-
mouth disease virus, rhinoviruses).

Poliovirus Foot & Mouth Disease Virus (FMDV)

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 Enveloped viruses

'Naked' virus particles, i.e. those in which the capsid proteins

are exposed to the external environment are produced from
infected cells at the end of the replicative cycle when the cell
dies, breaks down & lyses, releasing the virions which have
been built up internally. This simple strategy has drawbacks. In
some circumstances it is wasteful, resulting in the premature
death of the cell.

Many viruses have devised strategies to exit the infected cell without its total destruction.
This presents a difficulty in that all living cells are covered by a membrane composed of a
lipid bilayer. The viability of the cell depends on the integrity of this membrane. Viruses
leaving the cell must therefore allow this membrane to remain intact & this is achieved by
extrusion (budding) of the particle through the membrane, during which process the particle
becomes coated in a lipid envelope derived from the host cell membrane & with a similar
composition:

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The structure underlying the envelope may be based on helical or icosahedral symmetry &
may be formed before or as the virus leaves the cell. In the majority of cases, enveloped
viruses use cellular membranes as sites allowing them to direct assembly. The formation of
the particle inside the cell, maturation & release are in many cases a continuous process.
The site of assembly varies for different viruses. Not all enveloped viruses bud from the cell
surface membrane, many viruses use cytoplasmic membranes such as the golgi complex,
others such as herpesviruses which replicate in the nucleus may utilize the nuclear
membrane. In these cases, the virus is usually extruded into some form of vacuole, in which
it is transported to the cell surface & subsequently released.

Envelope Proteins:

If the virus particle became covered in a smooth, unbroken lipid bilayer, this would be its
undoing. Such a coating is effectively inert, & though effective as a protective layer
preventing desiccation of or enzymatic damage to the particle, would not permit recognition
of receptor molecules on the host cell. Therefore, viruses modify their lipid envelopes by the

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synthesis of several classes of proteins which are associated in one of three ways with the
envelope:

 Matrix Proteins: These are internal virion proteins whose function is effectively to
link the internal nucleocapsid assembly
 Glycoproteins: These are transmembrane proteins, anchored to the membrane by a
hydrophobic domain & can be subdivided into two types, by their function:
o External Glycoproteins - Anchored in the envelope by a single
transmembrane domain. Most of the structure of the protein is on the outside
of the membrane, with a relatively short internal tail. Often individual
monomers associate to form the 'spikes' visible on the surface of many
enveloped viruses in the electron microscope. Such proteins are the major
antigens of enveloped viruses.
o Transport Channels - This class of proteins contains multiple hydrophobic
transmembrane domains, forming a protein-lined channel through the
envelope, which enables the virus to alter the permeability of the membrane,
e.g. ion-channels.

Complex Virus Structures

The majority of viruses can be fitted into one of the three structural classes outlined above,
i.e. those with helical symmetry, icosahedral symmetry or enveloped viruses based on
either of these two.
However, there are many viruses whose structure is more complex. In these cases, although
the general principles of symmetry already described are often used to build part of the virus
shell (this term being appropriate here since such viruses often consist of several layers of
protein & lipid), the larger & more complex viruses cannot be simply defined by a
mathematical equation as can a simple helix or icosahedron.
Because of the complexity of some of these viruses, they have defied attempts to determine
detailed atomic structures using the techniques described earlier.

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Poxviruses:

An example of such a group & the problems of

complexity is shown by the members of the
poxvirus family. These viruses have oval or 'brick-
shaped' particles 200-400nm long. In fact, these
particles are so large that they were first observed
using high resolution optical microscopes in 1886
& thought at that time to be 'the spores of
micrococci'.

The external surface of the virion is ridged in parallel rows, sometimes arranged helically.
The particles are extremely complex & have been shown to contain more than 100 different
proteins.
Antigenically, poxviruses are very complex, inducing both specific & cross-reacting
antibodies - hence ability to vaccinate against one disease with another virus (i.e. the use of
vaccinia virus to immunize against smallpox (variola) virus).
Poxviruses & a number of other complex viruses also emphasise the true complexity of some
virus - there are at least ten enzymes present in poxvirus particles, mostly concerned with
nucleic acid metabolism/genome replication.

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Tailed Phages:

In other cases, the particle structure of

complex viruses has been much more
completely investigated. One of the
prime examples of such a group are the
tailed phages of Escherichia coli.
These viruses have been extensively
studied for excellent reasons - they are
easy to propagate in bacterial cells & can
be obtained in high titres & are easily
purified, facilitating biochemical &
structural studies.

The head of the particle consists essentially of an icosahedral shell attached via a collar to a
contractile, helical tail. At the end of the tail is a plate which functions in attachment to the
bacterial host & also in penetration of the bacterial cell wall by virtue of lysozyme-like
enzymes associated with the plate. In addition to these structures, thin protein fibres are
attached to the plate, which along with the tail plate, are involved in binding to the receptor
molecules in the wall of the host cell.
In detail, the structure of these phages is rather more complex than this simple picture, for
example, there are several internal proteins & polyamines associated with the genomic DNA
in the head & an internal tube structure inside the outer sheath of the helical tail. In the
infected bacterial cell, there are separate assembly pathways for the head & tail sections of
the particle, which come together at a late stage to make up the virion.
 

Protein-nucleic acid interactions & genome packaging

In most cases, the linear virus genome when stretched out in solution is at least an order of
magnitude longer than the diameter of the capsid.
Folding the genome in order to stuff it into such a confined space is quite a feat of topology,
but is compounded by repulsion by the cumulative negative electrostatic charges on the
phosphate groups of the nucleotide backbone resulting in the genome resisting being
crammed into a small space. Viruses overcome this difficulty by packaging along with the
genome a number of positively-charged molecules in order to counteract this negative
charge repulsion. These include:

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  small positively charged ions (Na, Mg, K, etc)
 polyamines
 various nucleic acid-binding proteins

Some of these latter proteins are virus-encoded & contain amino acids with basic side-
chains such as arginine & lysine which interact with the genome.
Many viruses with double-stranded DNA genomes have basic histone-like molecules closely
associated with the DNA. Some of these are virus-encoded, in other cases, the virus may
utilize cellular proteins, e.g. the polyomavirus genome assumes a chromatin-like structure in
association with four cellular histone proteins, similar to that of the host cell genome.
The second problem the virus must overcome is to achieve the specificity required to select
& encapsidate the virus genome from the large background of cellular nucleic acids. In
most cases, by the late stages of virus infection when assembly of virus particles occurs,
transcription of cellular genes has been reduced & a large pool of virus genomes has
accumulated. The overproduction of viral nucleic acids eases but does not eliminate the
problem of specific genome packaging. Therefore, a specific virus-encoded capsid or
nucleocapsid protein is required to achieve this end. Hence many viruses, even those with
relatively short, compact genomes such as retroviruses & rhabdoviruses encode this type of
protein.
The other sides of the packaging equation are the specific nucleotide sequences in the
genome (the packaging signal) which permit the virus to select genomic nucleic acids from
the cellular background. The packaging signal from a number of virus genomes has been
identified.
Viruses with segmented genomes face further problems. Not only must they encapsidate
only viral nucleic acid & exclude host cell molecules, but they must also attempt to package
one of each of the required genome segments. Like many other aspects of virus assembly, the
way in which packaging is controlled is not well understood, but the key must lie in the
specific molecular interactions between the genome & the capsid. In general, the physical
structure of virus genomes within virus particles has been poorly studied.

Virus Receptors - Recognition & Binding

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 Cellular receptor molecules used by a number of different
viruses from diverse taxonomic groups have now been
identified. This is achieved by binding of a specific virus-
attachment protein on the outer surface of the virion to a
cellular receptor molecule on the outer surface of the host
cell. The interaction of viruses with a cellular receptors is a
major event in determining the subsequent events in
replication & the outcome of infections. It is this interaction
which 'activates' extracellular virus particles & initiates the
replication cycle.

Other interactions of the virus capsid with the host cell:

The function of the virus capsid is not only to protect the genome, but also to deliver it to a
suitable host cell & more specifically, the appropriate compartment of the host cell (in
the case of eukaryote hosts) to allow replication to proceed.
An example are the nucleocapsid proteins of viruses which replicate in the nucleus of the
host cell. These molecules contain within their primary amino acid sequences 'nuclear
localization signals' which are responsible for the migration of the virus genome plus its
associated proteins into the nucleus where replication can occur.
Virions are not inert structures. Many virus particles contain one or more enzymatic
activities, although in most cases these are usually not active outside the biochemical
environment of the host cell.
All viruses with negative-sense RNA genomes must carry with them a virus-specific RNA-
dependent RNA polymerase since uninfected cells have no mechanism for RNA-dependent
RNA polymerization & therefore, genome replication could not occur if this enzyme were
not included in the virus particle. The more complex DNA viruses (e.g. herpesviruses &
poxviruses) carry a multiplicity of enzymes in the particles, mostly concerned with nucleic
acid metabolism in some way.

SUMMARY

1. To protect the genome

2. To deliver the genome to the appropriate site in the host cell so that it can be
replicated.
3. A number of repeated structural motifs found in many different virus groups are
evident. The most obvious is the division of many virus structures into those based on
helical or icosahedral symmetry.
4. Virus particles are not inert. Many are armed with a variety of enzymes which carry
out a range of complex reactions, most frequently concerned with the replication of
the genome.

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 PATHOGENICITY OF ANIMAL VIRUSES

The overall purpose of this Learning Object is to introduce various

mechanisms by which animal viruses harm their host.

Pathogenicity of Animal Viruses

A. Damaging infected host cells.

Animal viruses may cause cytopathic effect or CPE that damages infected host
cells in a variety of means, including:

1. Inhibiting normal host cell DNA, RNA, or protein synthesis. This can cause
structural or functional defects in the infected host cell leading to cytolysis or altered
cell functions;

2. Causing nicks or breaks in the host cell's chromosomes, as seen in congenital

rubella syndrome;

3. viral proteins and glycoproteins changing the antigenic surface of the host cell's
cytoplasmic membrane resulting in its being recognized as foreign and destroyed by
the body's immune defenses

4. Depleting the host cell of cellular materials essential for life or normal function;

5. Stimulating body cells to release inflammatory cytokines and chemokines;

6. Stimulating body cells to release inflammatory vasoactive peptides, bradykinins,

histamines, etc. resulting in vasodilation and increased mucous secretion;

7. Inducing adjacent host cells to fuse together forming giant multinucleated cells or
syncytias as seen with cytomegalovirus (CMV), varicella-zoster virus (VZV), and
HIV.

8. Playing a role in normal cells becoming malignant (cell transformation by

oncogenic viruses; and

9. Causing cytolysis of the infected host cell.

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B. Evading Host Immune Defenses

1. As will be seen in Unit 5, one of the major defenses against free viruses is the
immune defenses' production of antibody molecules against the virus. The "tips"
of the antibody (the Fab portion; have shapes that have a complementary shape to
portions of viral attachment proteins and glycoproteins called epitopes found on the
viral surface. When antibodies react with these attachment proteins, they block viral
adsorption to host cell receptors and, therefore, block viral replication.

In addition, Antibodies such as IgG function as opsonins and stick viruses to

phagocytes.

♦The influenza viruses undergo what is called antigenic drift and antigenic shift.

♦ With antigenic drift, mutations cause a gradual change in the hemagglutinin

antigen that adsorbs to receptors on host cells.

♦Antigenic shift is caused by a human influenza virus acquiring a new genome

segment from an influenza virus capable of infecting other animals such as a ducks
or swine. This new genome segment causes a major change in the hemagglutinin
antigen.

♦Antibodies made against the original human influenza virus can no longer
bind to the new strain of virus or stick the virus to phagocytes .

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♦Likewise HIV, because of its high rate of mutation and its intracellular
recombination with other strains of HIV, as mentioned earlier in this unit, prouces
altered gp120 to which antibodies made against the earlier strains of HIV can no
longer bind.

♦The hepatitis C virus (HCV) frequently through mutation produces viral varients
("escape mutants") to resist antibodies.

2. Another major defense against viruses, is the killing of virus-infected host cells by
cytotoxic T-lymphocytes (CTLs) . Virus-infected host cells naturally bind viral
epitopes to a host molecule called MHC-I and place the MHC-1 with bound
viral epitope on the surface of the infected cell where they can be recognized
by T-cell receptors on the surface of the CTLs. In this way the CTL can kill the
infected cell by apoptosis, a programmed cell suicide, or by cytolysis.

♦Epstein-Barr virus (EBV) and cytomegalovirus (CMV) inhibit proteasomal activity

so that viral proteins are not degraded into viral peptides.

♦Herpes simplex viruses (HSV) can block the TAP transport of peptides into the
endoplasmic reticulum.

♦Numerous viruses, such as the cytomegalovirus (CMV) and adenoviruses can

block the formation of MHC-I molecules by the infected cell. As a result, no
viral peptide is displayed on the infected cell and the CTLs are no longer able
to recognize that the cell is infected and kill it

♦Epstein-Barr virus (EBV) down regulates several host proteins involved in

attaching viral epitopes to MHC-I molecules and displaying them on the host
cell's surface.

♦Adenoviruses and Epstein-Barr Virus (EBV) code for proteins that blocks
apoptosis , the programmed cell suicide mechanism triggered by various defense
mechanisms in order to destroy virus-infected cells.

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3. Another defense cell that is able to kill virus-infected cells is the NK cell. NK cells
recognize infected cells displaying stressed-induced proteins and not
displaying MHC-I molecules on their surface and kill these cells.

♦The cytomegalovirus (CMV) can also trigger its host cell to produce altered MHC-I
molecules that are unable to bind viral epitopes, and, therefore, are not
recognized by CTLs. However, NK cells are also unable to kill this infected cell
because it is still displaying "MHC-I molecules" on its surface.

♦CMV also produces microRNAs (miRNAs), small non-coding RNA molecules that
down-regulates the production of stress-induced proteins that the killer-
activating receptor of NK cells first binds. Without this binding there is no kill signal
by the NK cell .

4. Some viruses cause infected host cells to secrete molecules that bind and tie
up cytokines, preventing them from binding to normal cytokine receptors on host
cells.

♦Poxviruses cause infected host cells to secrete molecules that bind interleukin-1
(IL-1) and interferon-gamma (IFN-gamma).

♦Cytomegaloviruses (CMV) cause infected host cells to secrete molecules that bind
chemokines .

5. Some viruses suppress immunocompetent cells.

♦Epstein-Barr virus (EBV) produces a protein that is homologous to the

cytokine interleukin-10 (IL-10). IL-10 inhibits the activation of dendritic cells
and macrophages antigen-presenting cells that are needed to present antigens to
T-lymphocytes for their activation. EBV also produces microRNAs (miRNAs), small
non-coding RNA molecules that inhibit an interferon response by infected cells.

♦The human immunodeficiency virus (HIV) infects immunocompetent dendritic

cells and T4-lymphocytes leading to their death or disfunction.

6. Some viruses block apoptosis of infected host cells enabling the infected host
cell to survive and produce new viruses.

♦Cytomegalovirus (CMV) and herpes simplex type 1 virus (HSV-1) produce

microRNAs (miRNAs), small non-coding RNA molecules that block protein
involved in apoptosis, a programmed cell suicide.

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 ANIMAL VIRUS LIFE CYCLES

1. The Productive Life Cycle

The overall purpose of this Learning Object is to learn how animal viruses
reproduce within their host cells.

Viruses that infect animal cells replicate by means of what is called the productive
life cycle .Some viruses, such as HIV and the herpes viruses are able to become
latent in certain cell types. A few viruses increase the risk of certain cancers.

We will now look at the life cycles of viruses that infect animal cells.

The Productive Life Cycle of Animal Viruses

For many animal viruses, the details of each step in their life cycle have not yet been
fully characterized, and among the viruses that have been well studied there is great
deal of variation. What follows is a generalized productive life cycle for animal
viruses consisting of the following steps: adsorption, viral entry, and viral movement
to the site of replication and release of the viral genome from the remainder of the
virus, viral replication, viral assembly, and viral release.

1. Viral Attachment or Adsorption to the Host Cell

Adsorption involves the binding of attachment sites on the viral surface with
receptor sites on the host cell cytoplasmic membrane.. The first diagram is an
enveloped virus while the other is naked.

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143
For a virus to infect a host cell, that cell must have receptors for the virus on its
surface and also be capable of supporting viral replication. These host cell receptors
are normal surface molecules involved in routine cellular function, but since a portion
of a molecule on the viral surface resembles the chemical shape of the body's
molecule that would normally bind to the receptor, the virus is able to attach to the
host cell's surface.

For example:

 Most human rhinoviruses that cause the common cold bind to

intercellular adhesion molecules (ICAM-1) found on cells of the
nasal epithelium. These ICAM-1 molecules are used normally
for the recruitment of leukocytes into the respiratory tract.
 The human immunodeficiency viruses (HIV) adsorbs to first
CD4 molecules and then chemokine receptors found on the
surface of human T4-lymphocytes and macrophages. CD4
molecules are normally involved in immune recognition while
chemokine receptors play a role in initiating inflammation and
recruiting leukocytes.
 Human cytomegaloviruses (CMV) adsorb to MHC-I molecules.
MHC-I molecules on human cells enable T8-lymphocytes to
recognize antigens during adaptive immunity.
 The hepatitis B virus (HBV) adsorbs to IgA receptors on human
cells. These receptors normally bind the antibody isotype IgA for
transport across cells.

2. Viral Entry into the Host Cell

a. Enveloped viruses

Enveloped viruses enter the host cell in one of two ways:

1. In some cases, the viral envelope may fuse with the host cell cytoplasmic
membrane and the nucleocapsid is released into the cytoplasm

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145
2. Usually they enter by endocytosis whereby the host cell cytoplasmic membrane
invaginates and pinches off, placing the virus in an endocytic vesicle 

b. Naked viruses

Naked viruses enter the cell in one of two ways:

1. In some cases, interaction between the viral capsid and the host cell cytoplasmic
membrane causes a rearrangement of capsid proteins allowing the viral nucleic
acid to pass through the membrane into the cytoplasm

2. Most naked viruses enter by receptor-mediated endocytosis whereby the host

cell cytoplasmic membrane invaginates and pinches off, placing the virus in an
endocytic vesicle 

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3. Viral Movement to the Site of Replication within the Host Cell and Release of
the Viral Genome from the Remainder of the Virus.

In the case of viruses that enter by endocytosis, the endocytic vesicles containing
the virus move within the host cell. During this process the pH of the endocytic
vesicle typically decreases and this enables the virus to leave the endocytic vesicle.
Viruses exit the endocytic vesicle through a variety of mechanisms, including:

a. Fusion of the viral envelope with the membrane of the endocytic vesicle
enabling the viral nucleocapsid to enter the cytoplasm of the host cell

b. Lysis of the endocytic vesicle releasing the viral nucleocapsid into the
cytoplasm of the host cell 

c. The viral capsid undergoing conformational changes that forms pores in the
endocytic vesicle enabling the virial genome to enter the cytoplasm of the host
cell 

Before viruses can replicate within the infected host cell, the viral genome needs to
release from the remainder of the virus. This process is sometimes referred to as
uncoating.

In the case of most viruses with an RNA genome, the viral RNA genome is
released from the capsid and enters the cytoplasm of the host cell, where
replication generally occurs.

In the case of most viruses with a DNA genome, the viral genome enters the nucleus
of the host cell through one the mechanisms shown below. Larger DNA viruses use
either a or b to enter the nucleus. Method c is used by some very small DNA whose
capsid is small enough to be carried through the nuclear pores.

a. The viral DNA genome is released from the capsid, enters the cytoplasm of
the host cell, and subsequently enters the nucleus of the host cell through the
pores in the nuclear membrane

b. The capsid of the viruses interacts with the nuclear membrane of the host cell
enabling the viral DNA genome to enter the nucleus of the host cell via the
pores in the nuclear membrane

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c. The nucleocapsid of a small DNA virus enters the nucleus of the host cell and
the capsid is subsequently removed releasing the viral DNA genome into the
nucleoplasm

This uncoating begins the eclipse period the period during which no intact virions
can be detected within the cell. After uncoating and during the replication stage the
virus is not infectious.

4. Viral Replication within the Host Cell

The viral genome directs the host cell's metabolic machinery (ribosomes, tRNA,
nutrients, energy, enzymes, etc.) to synthesize viral enzymes and viral parts. The
viral genome has to both replicate itself and become transcribed into viral
mRNA molecules. The viral mRNA can then be transcribed by the host cell into
viral structural components and enzymes need for replication and assembly of
the virus.

As mentioned earlier under Viral Classification, viruses can store their genetic
information in six different types of nucleic acid which are named based on how that
nucleic acid eventually becomes transcribed to the viral mRNA:

a. (+/-) double-stranded DNA. To replicate the viral genome, DNA-dependent DNA

polymerase enzymes (usually provided by the cell) copy both the (+) and (-) DNA
strands producing dsDNA viral genomes. To produce viral mRNA molecules, host
cell-DNA-dependent RNA polymerase enzymes copy the (-) DNA strand into (+) viral
mRNA. The (+) viral mRNA can then be translated into viral proteins by host cell
ribosomes. Examples include most bacteriophages, Papovaviruses, Adenoviruses,
and Herpesviruses.

b. (+) single-stranded DNA. To replicate the viral genome, DNA-dependent DNA

polymerase enzymes (usually provided by the cell) copy the (+) DNA strand of the
genome producing a dsDNA intermediate. DNA-dependent DNA polymerase
enzymes (again, usually provided by the cell) then copy the (-) DNA strand into ss
(+) DNA genomes. To produce viral mRNA molecules, host cell-DNA-dependent
RNA polymerase enzymes copy the (-) DNA strand into (+) viral mRNA. The (+) viral
mRNA can then be translated into viral proteins by host cell ribosomes. Examples
include Phage M13 and Parvoviruses.

c. (+/-) double-stranded RNA . To replicate the viral genome, viral RNA-dependent

RNA polymerase enzymes (replicase) copy both the (+) RNA and (-) RNA strands of
the genome producing a dsRNA genomes. To produce viral mRNA molecules, viral
RNA-dependent RNA polymerase enzymes (transcriptase) copy the (-) RNA strand

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into (+) viral mRNA. The (+) viral mRNA can then be translated into viral proteins by
host cell ribosomes. Reoviruses are an example.

d. (-) RNA To replicate the viral genome, viral RNA-dependent RNA polymerase
enzymes (transcriptase) copy the (-) RNA genome producing ss (+) RNA.
Transcriptase must be carried into the cell with the virion.  Viral RNA-dependent
RNA polymerase enzymes (replicase) then copy the (+) RNA strands producing ss
(-) RNA viral genome. The (+) mRNA strands also function as viral mRNA and can
then be translated into viral proteins by host cell ribosomes. Examples include
Orthomyxoviruses, Paramyxoviruses, Rhabdoviruses.

e. (+) RNA To replicate the viral genome, viral RNA-dependent RNA polymerase
enzymes (replicase) copy the (+) RNA genome producing ss (-) RNA. Viral RNA-
dependent RNA polymerase enzymes (replicase) then copy the (-) RNA strands
producing ss (+) RNA viral genome. To produce viral mRNA molecules. RNA-
dependent RNA polymerase enzymes (replicase) copy the (-) RNA strand into (+)
viral mRNA. The (+) viral mRNA can then be translated into viral proteins by host cell
ribosomes. Examples include Picornaviruses, Togaviruses, and Coronaviruses.

f. (+) RNA Retroviruses To replicate the viral genome, viral reverse transcriptase
enzymes (RNA-dependent DNA polymerases) copy the (+) RNA genome producing
ss (-) DNA strands. Viral reverse transcriptase can also function as a DNA-
dependent DNA polymerase enzyme and will copy the (-) DNA strands to produce a
dsDNA intermediate. Reverse transcriptase must be carried into the cell with the
viron.  The viral DNA will move to the nucleus where it integrates into the cell’s DNA
using the viral enzyme integrase which also must be carried into the host cell with
the virion. Once in the host cell’s DNA, host cell DNA-dependent RNA polymerase
enzymes then copy the ds (-) DNA strands to produce ss (+) RNA genomes. To
produce viral mRNA molecules, host cell DNA-dependent RNA polymerase
enzymes copy the ds (-) DNA strand into (+) viral mRNA. The (+) viral mRNA can
then be translated into viral proteins by host cell ribosomes. Retroviruses, such as
HIV-1, HIV-2, and HTLV-1 are examples.

As the host cell's ribosomes attach to the viral mRNA molecules, the mRNAs are
translated into viral structural proteins and viral enzymes. During the early phase of
replication, proteins needed for the replication of the viral genome are made and the
genome makes thousands of replicas of itself. During the late phase of replication,
viral structural proteins (capsid and matrix proteins, envelope glycoproteins, etc.)
and the enzymes involved in maturation are produced. Some viruses translate
mRNA molecules that are transcripts of several genes into one or more large
polyproteins. These polyproteins are subsequently cut into individual functional
proteins by viral enzymes called proteases. Other viruses produce monocistronic
mRNA molecules, each coding for a separate functional protein.

In the case of most RNA viruses, replication and assembly occurs in the host
cell's cytoplasm. With DNA viruses, most replication and assembly occurs in the

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nucleus of the host cell. The viral genome enters the nucleus of the host cell and
here is transcribed into viral mRNA. The viral mRNA molecules then leave the
nucleus through the pores in the nuclear membrane and are translated into viral
proteins by the host cell's ribosomes in the cytoplasm. Most of these viral proteins
then re-enter the nucleus where the virus assembles around the replicated
genomes.

Also during replication, viral envelope proteins and glycoproteins coded by the
viral genome are incorporated into the host cell's cytoplasmic membrane or
nuclear membrane.

Whether a virus has RNA or a DNA genome is significant when it comes to

developing antiviral agents to control viruses. In the case of RNA viruses, all of the
enzymes used in genome replication and transcription are viral encoded enzymes
different from those of the host cell so these enzymes can potentially be targeted.
On the other hand, DNA viruses use the host cell's RNA transcription machinery and
DNA replication machinery so these enzymes, shared by the virus and the host cell,
cannot be targeted without killing the host cell. Since all viruses use the host cell's
translation machinery regardless of genome type, translation can not be targeted in
any viruses.

5. Viral Assembly or Maturation within the Host Cell

During maturation, the capsid is assembled around the viral genome.

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6. Viral Release from the Host Cell

a. Naked viruses

Naked viruses are predominantly released by host cell lysis While some viruses
are cytolytic and lyse the host cell more or less directly, in many cases it is the
body's immune defenses that lyse the infected cell.

b. Enveloped viruses

With enveloped viruses, the host cell may or may not be lysed. The viruses obtain
their envelopes from host cell membranes by budding. As mentioned above,
prior to budding, viral proteins and glycoproteins are incorporated into the host cell's
membranes. During budding the host cell membrane with incorporated viral proteins
and glycoproteins evaginates and pinches off to form the viral envelope. Budding
occurs either at the outer cytoplasmic membrane, the nuclear membrane, or at the
membranes of the Golgi apparatus.

1. Viruses obtaining their envelope from the cytoplasmic membrane are

released during the budding process

2. Viruses obtaining their envelopes from the membranes of the nucleus, the
endoplasmic reticulum, or the Golgi apparatus are then released by
exocytosis via transport vesicles

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Some viruses, capable of causing cell fusion, may be transported from one cell to
adjacent cells without being released, that is, they are transmitted by cell-to-cell
contact whereby an infected cell fuses with an uninfected cell

7. Reinfection

As many as 10,000 to 50,000 animal viruses may be produced by a single infected

host cell.

2. The Productive Life Cycle with Possible Latency

The overall purpose of this Learning Object is to learn how some animal
viruses can become latent within their hosts.

Latent Life Cycle of Animal Viruses (Productive Life Cycle with

Possible Latency)

Some double-stranded DNA animal viruses such as the herpes viruses and a
group of viruses known as the retroviruses are able to remain latent within
infected host cells for long periods of time without replicating or causing
harm. Some of these viruses remain latent within the cytoplasm of the host cell
while others are able to insert or integrate their DNA into the host cell's
chromosomes. When the viral DNA is incorporated into the host cell's DNA, it is
called a provirus.

Viral latency is thought to result primarily from the lack of production of specific
host cell proteins that are required for the activation of the viral genes
responsible for turning on viral replication. As long as these specific host cell
proteins are not being made by the host cell, the virus can't replicate. However,
because the virus is inside the infected cell, it also can't be removed by the body's
immune responses and the person carries the virus throughout their life.

Subsequent activation of the host cell's DNA in response to extracellular stimuli,

however, can lead to synthesis of the specific host cell proteins required by
the virus and these proteins now activate the viral genes leading to a burst of viral
replication via the productive life cycle (def).

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Herpes viruses, for example, are often latent in some cell types but productive in
others. Herpes viruses include herpes simplex type 1 (HSV-1) which usually causes
fever blisters or oral herpes, herpes simplex type 2 (HSV-2) which usually causes
genital herpes, Epstein-Barr virus (EBV) which causes infectious mononucleosis and
plays a role in certain cancers, varicella-zoster virus (VZV) which causes chickenpox
and shingles, and cytomegalovirus (CMV) which causes a variety of infections in
immunosuppressed persons and is also a leading cause of birth defects.

In the case of HSV-1, HSV-2, and VZV, primary infection causes the virus to
replicate within epithelial cells. However, some of the viruses enter and migrate
down neurons where they become latent in the body of neurons. Subsequent
activation of the latently infected neurons by a variety of extracellular stimuli enables
the viruses to migrate back up the nerve cell and replicate again in the epithelial
cells.

With EBV, the virus is productive in epithelial cells but latent in B-lymphocytes)

In the case of HIV, the viral genome eventually becomes a provirus. The provirus
can directly proceed into the productive life cycle and produce more virions or, when
the specific host cell proteins required for turning on the viral genes are not being
produced by the host cell, it may remain latent in the host cell's chromosomes.
Subsequent activation of the host cell by extracellular stimuli, however, causes the
needed proteins to be made and the virus replicates via the productive life cycle.

HIV has an envelope derived from host cell membranes during replication.
Associated with the envelope are two HIV-encoded glycoproteins, gp120 and gp41.
Underneath the envelope is a protein matrix composed of p17. Inside the virus is a
capsid or core made of the protein p24. The nucleocapsid also contains p6, p7,
reverse transcriptase (p66/p51), integrase (p32), protease (p10), and 2 molecules of
single-stranded RNA, the viral genome 

TYPES OF VIRAL INFECTIONS

The overall purpose of this Learning Object is to learn some of the common
terms used to describe the various types of viral infections in humans.

Viral Infections of Humans

Most viruses that infect humans, such as those that cause routine respiratory
infections (e.g., cold viruses, influenza viruses) and gastrointestinal infections (e.g.,
Rotaviruses, Norwalk virus), cause acute infections.

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Acute infections are of relatively short duration with rapid recovery.

In persistent infections, the viruses are continually present in the body. Some
persistent infections are late complications following an acute infection and
include subacute sclerosing panencephalitis (SSPE) that can follow an acute
measles infection and progressive encephalitis that can follow rubella.

Other persistent infections are known as latent viral infection. In a latent viral
infection the virus remains in equilibrium with the host for long periods of time before
symptoms again appear, but the actual viruses cannot be detected until reactivation
of the disease occurs. Examples include infections caused by HSV-1 (fever blisters),
HSV-2 (genital herpes), and VZV (chickenpox-shingles).

In the case of chronic virus infections, the virus can be demonstrated in the body
at all times and the disease may be present or absent for an extended period of
time. Examples include hepatitis B (caused by HBV) and hepatitis C (caused by
HCV).

Slow infections are ones in which the infectious agents gradually increase in
number over a very long period of time during which no significant symptoms are
seen. Examples include AIDS (caused by HIV-1 and HIV-2) and certain lentiviruses
that cause tumors in animals. Although not viruses, prions also cause slow
infections.

The Role of Viruses in Tumor Production

The overall purpose of this Learning Object is to understand the role a few
viruses play in increasing the risk for certain cancers.

Some viruses can also play a role in converting normal host cells into tumor cells.
These viruses are capable of viral transformation, that is, they transform normal
cells into malignant cells.

In fact, five viruses, hepatitis B virus (HBV), hepatitis C virus (HCV), human
papilloma virus (HPV), Epstein-Barr virus (EBV), and human T-lymphotropic virus
type I (HTLV-I) are thought to contribute to over 15% of the world's cancers. Up to
80% of these human viral-associated cancers are cervical cancer (associated with
HPV) and liver cancer (associated with HBV and HCV).

The hepatitis B virus (HBV) is a DNA virus that may potentially cause chronic
hepatitis in those infected. There is a strong link between chronic infection with HBV

154
and hepatocellular carcinoma, which typically appears after 30-50 years of chronic
liver damage and liver cell replacement.

Chronic carriers of HBV have a 300 times greater risk of eventually developing liver
cancer. Around 90% of individuals infected at birth and 10% of individuals infected
as adults become chronic carriers of HBV.

There are about one million chronic carriers of HBV in the US. Worldwide, HBV is
responsible for 60% of all liver cancer cases.

The hepatitis C virus (HCV) is a RNA virus that may also cause chronic hepatitis in
those infected. As with HBV, there is a strong link between chronic infection with
HCV and liver cancer, typically appearing after 30-50 years of chronic liver damage
and liver cell replacement.

Around 85% of individuals infected with HCV become chronic carriers and there are
approximately four million chronic carriers of HCV in the US. Worldwide, HCV is
responsible for 22 % of all liver cancer cases.

The human papilloma viruses (HPV) are responsible for warts. While warts are
generally considered as benign tumors, some sexually-transmitted strains of HPV
(HPV-16 and 18 are definitely carcinogenic in humans; HPV-31 and 33 are probably
carcinogenic), have been implicated in cervical and vulvar cancer, rectal cancer, and
squamous cell carcinoma of the penis.

In these tumor cells the viral DNA is usually found integrated in host cell
chromosomes. In the US, HPVs are associated with 82% of the deaths due to
cervical cancer each year, as well as a million precancerous lesions.

The Epstein-Barr virus (EBV), a herpes virus, normally causes benign

proliferations such as infectious mononucleosis and hairy leukoplakia of the tongue.

However, it can contribute to nonHodgkin's lymphoma in AIDS patients and post-

transplantation lymphoproliferative diseases, appears to be an essential factor for
posterior nasopharyngeal cancer in some individuals, can be a co-factor for Burkitt's
lymphoma, and contributes to smooth-muscle tumors in immunosuppressed
children.

The retrovirus human T-lymphotropic virus type I (HTLV-I) can induce a rare adult
T-lymphocyte leukemia-lymphoma.

The development of tumors is a multistep process depending on the

accumulation of mutations altering a number of genes. The altered genes then
function collectively to cause malignant growth.

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Proliferation of normal cells is regulated by growth-promoting proto-oncogenes
and counterbalanced by growth-restricting tumor suppressor genes ( .
Mutations that increase the activities of proto-oncogenes to create oncogenes
and/or decrease the activities of tumor suppressor genes can lead to growth
of tumors. It is now known that many tumors require both activation of oncogenes
from proto-oncogenes and inactivation of tumor suppressor genes for their
development.

Viruses are thought to play a role in cancer development both indirectly and directly.
Indirectly, the viruses may induce immunosuppression so that cancer cells are not
removed by immune responses, as in the case of HIV/AIDS, or they may cause long
term damage to tissues resulting in large scale cell regeneration which increases the
chances of natural mutation in proto-oncogenes and tumor suppressor genes, as in
the case of HBV and HCV. Directly, by integrating into the host cell's chromosomes,
some viruses may alter the normal function of the proto-oncogenes and tumor
suppressor genes, as is seen with HPV and HBV.

However, most virus-associated cancers have long latency periods of several

decades and only a small percentage of the people infected with the virus actually
develop the cancer. This indicates other factors promoting changes in cellular
genes are also involved. For example, in the case of cervical cancer and HPV, two
varients of a tumor suppressor gene known as p53 are known. One form of the p53
gene produces a suppressor protein that is much more susceptible to degradation
by an oncoprotein called E6 which is produced by carcinogenic strains of HPV.

  

CONTROL OF VIRUSES

The overall purpose of this Learning Object is:

1) to learn how our antiviral control agents affect viruses; and
2) to introduce a number of chemical agents used to control certain viral
infections.

 Since viruses lack the structures and metabolic processes that are altered by
common antibiotics, antibiotics are virtually useless in treating viral infections.

To date, only a few chemotherapeutic agents have been found to be somewhat

effective against just a few limited viruses. Antivirals used for viruses other than HIV
include:

1. amantadine (Symmetrel): used prophylactically against influenza A in high-risk

individuals.

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2. rimantidine (Flumadine): used for treatment and prophylaxis of influenza A.

3. zanamivir (Relenza): used to limit the duration of influenza A and B infections.

4. oseltamivir (Tamiflu): used limit the duration of influenza infections.

5. acyclovir (Zovirax): used against herpes simplex viruses (HSV) to treat genital
herpes, mucocutaneous herpes in the immunosuppressed, HSV encephalitis
neonatal herpes, and to reduce the rate of recurrences of genital herpes. It is also
used against varicella zoster viruses (VZV) to treat shingles).

6. trifluridine (Viroptic): used to treat eye infection (keratitis and conjunctivitis)

caused by HSV.

7. famciclovir (Famvir): used to treat HSV and VZV infections.

8. valacyclovir (Valtrex): used to treat HSV and VZV infections.

9. penciclovir (Denavir): used in treating HSV infections.

10. gancyclovir (Cytovene; Vitrasert): used in treating severe cytomegalovirus

(CMV) infections such as retinitis

11. valganciclovir (Valcyte): used in treating severe CMV infections such as

retinitis).

12. foscarnet (Foscavir): used in treating severe CMV infections such as retinitis

13. cidofovir (Vistide): used in treating CMV retinitis.

14. fomivirsen (Vitravene): used in treating CMV retinitis.

15. ribavirin (Copegus; Rebetol; Virazole): used in treating respiratory syncytial

virus (RSV) infections and hepatitis C virus (HCV).

16. lamivudine (Epivir-HBV): used in treating chronic hepatitis B.

17. adefovir dipivoxil (Hepsera): used in treating hepatitis B.

Amantadine and rimantidine are drugs that prevent influenza A viruses from the
uncoating step necessary for viral replication. Zanamivir and oseltamivir are
inhibitors of the influenza virus surface enzyme called neuraminidase that is
needed for release of newly formed influenza viruses from the infected cell.

Fomivirsen inhibits cytomegalovirus (CMV) replication through an antisense

mechanism. The nucleotide sequence of fomivirsen is complementary to a

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sequence in mRNA transcripts that encodes several proteins responsible for
regulation of viral gene expression that are essential for production of infectious
CMV. Binding of fomivirsen to the target mRNA results in inhibition of protein
synthesis, subsequently inhibiting virus replication.

Most antiviral agents, however, work by inhibiting viral DNA synthesis. These
drugs chemically resemble normal DNA nucleosides, molecules containing
deoxyribose and either adenine, guanine, cytosine, or thymine. Viral enzymes then
add phosphate groups to these nucleoside analogs to form DNA nucleotide analogs.
The DNA nucleotide analogs are then inserted into the growing viral DNA
strand in place of a normal nucleotide. Once inserted, however, new nucleotides
can't attach and DNA synthesis is stopped. They are selectively toxic because viral
polymerases are more prone to incorporate nucleotide analogs into their nucleic acid
than are host cell polymerases.

Current anti-HIV drugs include the following (classified by their action):

1. HIV nucleoside-analog reverse transcriptase inhibitors

In order to replicate, HIV uses the enzyme reverse transcriptase to make a DNA
copy of its RNA genome. A complimentary copy of this DNA is then made to
produce a double-stranded DNA intermediate which is able to insert into host cell
chromosomes to form a provirus

Most reverse transcriptase inhibitors are nucleoside analogs. A nucleoside is part

of the building block of DNA, consisting of a nitrogenous base bound to the sugar
deoxyribose but no phosphate group. A nucleoside analog chemically resembles a
normal nucleoside.

Once phosphate groups are added by either viral or host cell enzymes, the drugs
now chemically resemble normal DNA nucleotides, the building block
molecules for DNA synthesis. The nucleotide analog binds to the active site of the
reverse transcriptase which, in turn, inserts it into the growing DNA strand in place of
a normal nucleotide. Once inserted, however, new DNA nucleotides are unable to
attach to the drug and DNA synthesis is stopped. This results in an incomplete
provirus. For example, zidovudine (AZT, ZDV, Retrovir) resembles the
deoxyribonucleotide containing the base thymine. Once zidovudine is inserted into
the growing DNA strand being transcribed from the viral RNA by reverse
transcriptase, no further nucleotides can be attached Examples of nucleoside
reverse transcriptase inhibitors include:

a. zidovudine (AZT; ZDV; Retrovir)

b. didanosine (ddI; dideoxyinosine; Videx)

c. zalcitabine (ddC;dideoxycytosine; HIV-ID)

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d. stavudine(d4T; Zerit)

e. lamivudine (3TC; Epivir)

f. abacavir (ABC; Ziagen)

g. emtricitabine (FTC; Emtriva)

 2. Nucleotide Reverse Transcriptase Inhibitors (NtRTIs)

A NtRTI inhibitor ia a nucleotide analog. A nucleotide is the building block of DNA,

consisting of a nitrogenous base bound to the sugar deoxyribose, and a phosphate
group. A nucleotide analog chemically resembles a normal nucleotide. The
nucleotide analog binds to the active site of the reverse transcriptase which, in turn,
inserts it into the growing DNA strand in place of a normal nucleotide. Once inserted,
however, new DNA nucleotides are unable to attach to the drug and DNA synthesis
is stopped. This results in an incomplete provirus. An example of nucleoside reverse
transcriptase inhibitor is tenofovir (TDF;Viread).

3. HIV Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs)

These drugs do not resemble regular DNA building blocks. They bind to an
allosteric site that regulates reverse transcriptase activity rather than to the
enzyme's active site itself as do the above nucleoside analogues. This also prevents
HIV provirus formation.

a. nevirapine (NVP; Viramune)

b. delavirdine (Rescriptor)

c. efavirenz (EFV; Sustiva)

  

4. HIV Protease Inhibitors (PIs)

In order for maturation of HIV to occur, a HIV enzyme termed a protease has to
cleave a long HIV-encoded gag-pol polyprotein to produce reverse transcriptase
and integrase (coded by the HIV pol gene) and gag polyprotein (coded by the HIV
gag gene). The HIV protease then cleaves the gag polyprotein into capsid protein
p17, matrix protein p24, and nucleocapsid protein p7, as well as proteins p6, p2, and
p1 whose functions are not yet fully understood. Proteases also cleave the env-
polyprotein (coded by the HIV env gene) into the envelope glycprroteins gp120 and

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gp41. Protease inhibitors are drugs that bind to the active site of this HIV-encoded
protease and prevent it from cleaving the long gag-pol polyprotein and the gag
polyprotein into essential proteins essential to the structure of HIV and to RNA
packaging within its nucleocapsid. As a result, viral maturation does not occur and
noninfectious viral particles are produced.

Protease inhibitors include:

a. saquinavir (Inverase; Fortovase)

b. ritonavir RTV; (Norvir)

c. idinavir (Crixivan)

d. nelfinavir (Viracept)

e. amprenavir (Agenerase)

f. atazanavir (Reyataz)

g. fosamprenavir (908; Lexiva)

h. ritonavir RTV; (Norvir)

i. lopinavir + ritonavir (Kaletra)

5. Entry Inhibitors (EIs)

EIs are agents interfering with the entry of HIV-1 into cells. During the
adsorption and penetration stages of the life cycle of HIV, a portion or domain of the
HIV surface glycoprotein gp120 binds to a CD4 molecule on the host cell. This
induces a change in shape that brings the chemokine receptor binding domains of
the gp120 into proximity with the host cell chemokine receptor. This brings about
another conformational change that exposes a previously buried portion of the
transmembrane glycoprotein gp41 that enables the viral envelope to fuse with the
host cell membrane. EIs interfere with various stages of this process.

a. Agents that block the binding of gp120 to host CD4 molecules.

The first step in adsorption of HIV is the binding of gp120 in the viral envelope to
CD4 molecules on the host cell.

TNX-355 is an example of a CD4 binding blocker.

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b. Agents that block the binding of gp120 to host chemokine receptor 5 (CCR5).

After the gp120 on the envelope of HIV binds to a CD4 molecule on the host cell, it
must then also bind to a co-receptor - a chemokine receptor. CCR5-tropic strains of
HIV bind to the chemokine receptor CCR5. (An estimated 50%-60% of people
having previously received HIV medication have circulating CCR5-tropic HIV.)

maraviroc (Selzentry) is a chemokine receptor binding blocker.

c. Agents that block the fusion of the viral envelope with the cytoplasmic
membrane of the host cell.

enfuvirtide (ENF; Fuzeon) binds a gp41 subunit of the viral envelope glycoprotein
and prevents the conformational changes required for the fusion of the viral
envelope with the cellular cytoplasmic membrane.

Many clinical trials of various combinations of these drugs are currently underway.
Best results to date have occurred when using a protease inhibitor in combination
with one or two reverse transcriptase inhibitors. An example is a combination of
abacavir lamivudine, and zidovudine (Trizivir)

Certain cytokines have now been produced by recombinant DNA technology and
several are showing some success for viral infections. These include:

1. Recombinant interferon alfa-2a (Roferon-A): a cytokene used to treat Kaposi's

sarcoma, chronic myelogenous leukemia, and hairy cell leukemia.

2. peginterferon alfa-2a (Pegasys) : used to treat hepatitis C (HCV).

3. recombinant interferon-alpha 2b (Intron A): a cytokine produced by

recombinant DNA technology and used to treat Hepatitis B; malignant melanoma,
Kaposi's sarcoma, follicular lymphoma, hairy cell leukemia, warts, and Hepatitis C.

4. peginterferon alfa-2b (PEG-Intron; PEG-Intron Redipen): used to treat hepatitis

C (HCV).

5. Recombinant Interferon alfa-2b plus the antiviral drug ribavirin (Rebetron):

used to treat hepatitis C (HCV).

6. Recombinant interferon-alpha n3 (Alferon N): used to treat warts.

7. Recombinant iInterferon alfacon-1 (Infergen) : used to treat hepatitis C (HCV).

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None of the current antiviral agents kill and eliminate the viruses; they simply inhibit
their replication and decrease the severity of the disease. In the case of some drugs,
resistant virus strains are starting to emerge.

Since there are no antiviral drugs for the vast majority of viral infections and the few
drugs that are available are only partially effective against limited types of viruses, to
control viruses, we must rely on the body's immune responses. As will be seen in
detail in Units 4 and 5, the immune responses include innate immunity as well as
adaptive immunity (antibody production and cell-mediated immunity). Adaptive
immunity can be either naturally acquired or, in some cases, artificially acquired.

VIROIDS AND PRIONS

The overall purpose of this Learning Object is to introduce viroids and prions
and several diseases they may cause.

Viroids and Prions

Viroids are even simpler than viruses. They are small, circular, single-stranded
molecules of infectious RNA lacking even a protein coat. They are the cause of a
few plant diseases such as potato spindle-tuber disease,cucumber pale fruit, citrus
exocortis disease, and cadang-cadang (coconuts).

Prions are infectious protein particles thought to be responsible for a group of

transmissible and/or inherited neurodegenerative diseases including
Creutzfeldt-Jakob disease, kuru, and Gerstmann-Straussler- syndrome in humans
as well as scrapie in sheep and goats. Most evidence indicates that the infectious
prion proteins are modified forms of normal proteins coded for by a host gene in the
brain. In the case of the disease scrapie, the normal prion protein in an animal
without the disease has alpha-helices in the proteins secondary structure while the
scrapie prion protein in diseased animals has beta-sheets for the secondary
structure. When the scrapie prion protein contacts the normal protein it causes it to
change its configuration to the scrapie beta-sheet form. This suggests that the
conversion of a normal prion protein into an infectious prion protein may be
catalyzed by the prion protein itself upon entering the brain. Inherited forms may be
a result of point mutations that make the prion protein more susceptible to a change
in its protein structure.

  

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