A NTHONY D E F RANCO

S ECTION 8

INNATE IMMUNITY TO
VIRUSES AND ANTIGEN
RECOGNITION

C ONTACT I NFORMATION

Anthony DeFranco, PhD (Email)

R EADING
Basic Immunology: Functions and Disorders
of the Immune System. Abbas, Abul K., and Andrew H. Lichtman. -- Chapter 2 and Chapter 4 (pp. 6776)

O BJECTIVES
Describe the role of RIG-I-like receptors (RLRs) in
the induction of type 1 interferon by virus-infected
cells

Describe in general terms how type 1 interferons

provide anti-viral innate immunity.
Describe how natural killer cells can recognize virusinfected cells and kill them.
Understand the basic functional features of antibody
molecules, including their domain structure, the
location of variable and constant domains, the
location of hypervariable regions within the variable
domains, the different types of heavy and light
chains, and the identity of those parts that confer
antigen binding and effector functions (Note:
effector functions will be covered in detail in a
subsequent lecture).
Explain the difference between affinity and
avidity. Describe how these related concepts apply
to the different classes of antibodies and how
antibodies change during an immune response in this
regard (the latter issue will be covered in more detail
in one of Dr. Cysters lectures).
Enumerate the main ways in which antibodies are
used in medicine.

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Explain the difference between polyclonal antibodies
and monoclonal antibodies, and describe the main
advantages of monoclonal antibodies.
Appreciate that constant parts of antibody molecules
can be incorporated into protein-based therapeutic
agents and describe the advantage conferred by doing
so.
Describe how antibodies are used in laboratory
testing (RIA, hemagglutination, ELISA, and flow
cytometry. NOTE: antibody tests are the subject of
the Immunology laboratory on 9/3).
Describe how the B cell antigen receptors differ from
secreted antibody molecules.
Describe the T cell antigen receptor (TCR) subunit
structure. Explain the role of the CD3 component of
the TCR. Compare and contrast the structure of the
TCR / chains to the structure of antibodies.

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K EY W ORDS :

INTERFERON
NATURAL KILLER (NK) CELL
IMMUNOGLOBULIN/ANTIBODY
T CELL RECEPTOR FOR ANTIGEN (TCR)
AVIDITY
AFFINITY
ANTIBODY STRUCTURE
MONOCLONAL ANTIBODY
ELISA

M AIN I DEAS :
Virus infection is typically recognized by intracellular
sensors in the cytoplasm of cells that recognize the nucleic acids of the replicating virus, such as doublestranded (ds) RNA and induce synthesis of type 1 interferons. The two sensors of viral RNA are RIG-I and
Mda5, which collectively are called RIG-I-like receptors (RLRs). They have different fine specificity for viral RNA molecules. There are also sensors for DNA virus infection that induce production of type 1 interferon, but their molecular identities are in the process
of being characterized at this time. Type 1 interferons
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are secreted from the infected cell and act on neighboring cells to limit the subsequent replication of virus.
This mechanism represents a second distinct type of
cellular innate response to infection, in addition to the
induction of inflammation.
A key mechanism for fighting most virus infections is
the killing of infected cells that are producing virus.
This role is provided early after infection by the natural
killer (NK) cell, a lymphocyte that is part of innate immunity. Later in infection, cytotoxic T cells perform
this function; NK and cytotoxic T cells use the same basic molecular mechanism for killing infected cells.
B cells and T cells use very similar molecules for recognizing antigens, but the nature of the antigen recognized is very different (free antigen in native [unprocessed] form vs. peptide-MHC complexes [generated by
processing of protein antigens]).
A membrane-bound form of the antibody molecule is
expressed on B lymphocytes and serves as a receptor
for antigen, allowing the immune system to selectively
activate those B cells that make antibodies able to bind
to parts of an infecting pathogen.

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The structures of antibodies are well tailored to their
functions. Antibodies have an antigen-binding region,
which exists in a great many variations, and a constant region which is responsible for many of the effector functions of the antibody and which exists in 5 different types, corresponding to the five isotypes
(classes) of antibodies: IgM, IgD, IgG, IgA, and IgE. In
addition, IgG has four subtypes called IgG1, IgG2,
IgG3, and IgG4, each encoded separately in the genome. They differ somewhat in effector function, with
IgG1 and IgG3 being especially good at promoting
phagocytosis and killing of microbes and being more inflammatory in their action.
Antibodies are useful as therapeutics. Polyclonal antibodies pooled from many donors (intravenous immune
globulin IVIG) are used to treat certain immunodeficiencies and also have efficacy for some autoimmune
diseases (Note: the mechanism of action in autoimmune diseases is not well established). Therapies
based on a single antibody (monoclonal antibodies),
are useful for treating an increasing number of conditions (especially cancers and inflammatory diseases) .
Monoclonal antibodies are also frequently useful for diagnostic and research purposes given their reproduci-

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bility and unlimited availability, in contrast to polyclonal antibodies.
There is a specific transmembrane Fc receptor, called
FcRn, that is used by the body for maintaining IgG in
the bloodstream and for transmitting IgG from mother
to fetus. The latter function is critical for immune protection in newborn infants. In addition, some therapeutic molecules are designed to be able to bind to FcRn in
order to increase their half-life in the blood and make
them work better.

O VERVIEW AND A DDITIONAL COMMENTS BEYOND THE

TEXTBOOK

Innate Immunity to Viruses

Induction of interferon by dsRNA in the cytoplasm: Mammalian cells have two related molecules
(called RIG-I and Mda5; collectively these are referred
to as Rig-I-like receptors or RLRs) that recognize viral RNA and/or replication intermediates (doublestranded RNA molecules) in the cytoplasm. These
molecules signal to induce production of interferon-
and interferon-, which signal to neighboring cells via

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the interferon- receptor (the receptor for both
interferon- and interferon-). Note that there is also
an intracellular sensor for viral DNA genomes in the
cell, which also induces interferon production by a
similar pathway.
Anti-viral effects of type 1 interferons
(interferon- and -): Interferon-induced genes
that participate in limiting virus replication are many,
and include the dsRNA-dependent protein kinase
(PKR), which blocks cellular protein synthesis in infected cells, oligo-A synthetase, which in infected cells
makes oligo-A, which activates an RNase to degrade
mRNAs, again inhibiting protein synthesis, and the Mx
proteins, which are less well understood, but are
thought to inhibit virus assembly and/or virus transcription (synthesis of mRNAs).
ANTIGEN RECOGNITION
In general, this material is covered well in the textbook
and you should start there. The following is designed
to cover aspects of the lecture that go beyond the textbook material.
Antibody structure: An editorial comment: it is
hard to overstate how important it is for you to mas43

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ter the basics of antibody structure. The understanding of immunology and the practical use of it build on
these basic concepts in many ways. For example, many
of the effector functions of antibodies make use of recognition of the constant parts of antibody molecules by
cell surface receptors of macrophages, neutrophils,
mast cells, NK cells, etc. and these receptors are called
Fc receptors, which makes sense if you recall which
part of the antibody molecule is the Fc part. Novel
protein-based therapeutics are starting to come into
use that employ Fc parts of antibodies in order to give
the therapeutic protein a longer half-life in the blood.
The differences between IgM and IgG are critical for understanding the consequences of blood group incompatibilities, etc. etc.

from one individual (or even from some animal species) to another person, which is referred to as passive
immunity, since the persons own immune system
did not make the protective immune response. Antibody from an immune individual (or animal) is used
for protection against some infectious diseases, for
treatment against tetanus exposure of an unvaccinated
individual, and for treatment of poison snake bites.
Pooled antibodies from many individuals (intravenous
immune globulin IVIG) are used to treat immunodeficiencies in which antibody production is defective, and
increasingly have been found in be useful in some
other disease states (autoimmune diseases in particular). Increasingly, monoclonal antibodies are being
used both as diagnostic reagents and as therapies.

Antibodies in medicine: Antibodies have been important for medical practice for many years. The success of vaccination relies almost entirely on the production of protective antibodies. The understanding of the
ABO blood groups (which we will describe in further detail later in I-3) and their detection using antibodies
was essential to making blood transfusions practical.
Antibodies are used to aid in diagnosis in many ways.
We shall examine some examples of this in an upcoming laboratory session. Antibodies can be transferred

Polyclonal antibodies vs. monoclonal antibodies: The antibodies produced in a normal immune response result from the activation of many B cells, each
of which may recognize different parts of the antigen
(different epitopes), and each of which may have a different affinity for antigen. Such antibodies are called
polyclonal, because they result from the combined actions of multiple B cells each activated to multiply and
produce a clone of progeny that go on to secrete antibody. In contrast, monoclonal antibodies are the result
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of a single immortalized B cell, so all the antibodies
have the same amino acid sequence, recognize the
same epitope and have the same affinity. Monoclonal
antibodies can be produced in essentially unlimited
quantities. Whereas polyclonal antibodies are by their
nature biological products which vary from batch to
batch, monoclonal antibodies are chemically defined
and always the same. Monoclonal antibodies are used
frequently in diagnostic procedures, including ELISA
and flow cytometry, which we will describe in detail in
an upcoming lab session. In recent years, they are becoming increasingly successful as therapeutic agents as
well.
Humanized monoclonal antibodies. An important limitation of the first generation of monoclonal antibodies, dating back to the 1980s, is that they were
made in rodents and injection of most such monoclonal antibodies into people led to an immune response. Often patients made antibodies against the
parts of the rodent antibodies that were different in
amino acid sequence from human antibodies. Once
that happened, the therapeutic antibody was rapidly
cleared from the body and lost efficacy. In addition,
the formation of large amounts of immune complexes
can be deleterious in several ways. To solve this prob-

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lem, there are several approaches to making monoclonal antibodies less immunogenic and reducing the
fraction of patients that make enough antibody against
them to be a problem. In addition, there is a standardized nomenclature that attaches a suffix to the name of
the therapeutic to indicate which approach was used,
so that the physician can readily know which therapeutic is of which type. The approaches range from gene
splicing methods of swapping out the constant parts of
the monoclonal antibody for their human equivalents
(chimeric monoclonal antibody, -ximab), to additionally replacing the framework regions of the V regions
of the heavy and light chains (humanized, -zumab)
to starting with a fully human monoclonal antibody
(-mumab). A fully mouse monoclonal antibody therapeutic has the suffix omab, which can be fine for a
single use product.
More recent monoclonal antibody therapeutics are
mostly of the humanized or fully human types, whereas
the chimeric antibodies tend to have come into medicine earlier, as they were easier to create. While the
more human the therapeutic, the less immunogenic it
will be on average, this is not a perfect correlation, as
other factors influence the immunogenicity, including
how unusual the V regions are and details of the formu45

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lation, such as how carefully aggregated monoclonal antibody is excluded from the product, since aggregated
proteins are more immunogenic than soluble unaggregated forms. Currently, regulatory agencies require
that the degree of immunogenicity be tested and reported in the product literature for monoclonal antibody therapeutics, but this is insufficiently standardized at this point to be particularly useful. Until this
changes, there should be skepticism about claims of
relative degree of immunogenicity of one product versus another, unless the comparison is based on very
similar methodology.
Immunogenicity is also a potential complication of
other types of therapeutic proteins. For example, older
versions of type 1 interferons, which are used to treat
certain cancers and hepatitis C virus infections, are considerably more immunogenic than some newer formulations, probably due to changes in the formulation itself. As a clinician, if you have a patient who has been
getting some efficacy from a protein therapeutic but
the therapeutic stops working, one possible explanation is that the patient made antibodies against the
therapeutic.

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Anti-TNF therapeutics: When we get to the Rheumatology lectures of I-3, we shall hear about the successful use of anti-TNF therapeutics to control certain
inflammatory diseases. There are currently two types
of TNF-blocking protein therapeutics on the market,
and they represent two strategies that are used more
widely in other therapeutics against other targets. Infliximab (Remicade) and Adalimumab (Humira) are
monoclonal antibodies that are specific for the proinflammatory cytokine TNF (and two additional monoclonal anti-TNF therapies have been approved).
Etanercept (Enbrel), in contrast is a chimeric protein
in which the extracellular domain of a TNF receptor
(TNFR2) is used as the part that binds to and inhibits
TNF. By itself, this receptor fragment turned out to
have a very short half-life in the blood, making its use
for prolonged periods of time impractical. By adding
to the end of this molecule the Fc portion of a human
IgG molecule, the long half-life of IgG in the blood was
conferred upon the chimeric protein. This long halflife is due to the actions of an Fc receptor (called FcRn)
expressed by endothelial cells and some other cell
types. FcRn decreases loss of IgG from the blood, possibly by decreasing its catabolism. FcRn is also responsible for transmission of IgG between mother and fetus.
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T cell antigen receptor: The basic structures of the

TCR and the BCR are similar, although note that only B
cells secrete their antigen-binding molecule. This is because whereas antibody is a soluble recognition element that acts all over the body by coupling to effector
mechanisms of innate immunity (complement activation, phagocytosis, etc.), T cells generally act locally on
cells in a lymph node or at the site of infection. Hence
recognition needs to occur where the T cell is and be
coupled to activation of the T cell to secrete effector
molecules such as cytokines or, in the case of cytotoxic
T cells, molecules involved in killing the antigenexpressing cell.

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